Tailorable Trimethyl Chitosans as Adjuvant for ... - TI Pharma

TAILORABLE TRIMETHYL CHITOSANS
AS ADJUVANT FOR INTRANASAL
IMMUNIZATION

The printing of this thesis was financially supported by:
J.E. Jurriaanse Stichting
Utrecht Institute for Pharmaceutical Sciences
ISBN: 978-90-39354292
© 2010 RJ Verheul, Utrecht
Cover: Drawings taken from ‘Poissons, écrevisses et crabs’ by Louis Renard. First
published in 1719, the book describes exotic sea creatures (including a mermaid) from
the Netherlands West-Indies, now Indonesia. Many thanks to Marijn van Hoorn,
curator of the Teylers Museum, Haarlem
The research presented in this thesis was performed under the framework of TI
Pharma project D5-106-1; Vaccine delivery: alternatives for conventional multiple
injection vaccines
Printed by: Vandenberg, Maarn

TAILORABLE TRIMETHYL CHITOSANS AS
ADJUVANT FOR INTRANASAL IMMUNIZATION
Varieerbare Trimethyl Chitosanen als Adjuvans voor Intranasale Vaccinatie
(met een samenvatting in het Nederlands)
Proefschrift
ter verkrijging van de graad van doctor aan de Universiteit Utrecht op
gezag van de rector magnificus, prof. dr. J.C. Stoof, ingevolge het besluit
van het college voor promoties in het openbaar te verdedigen op
maandag 8 november 2010 des middags te 2.30 uur
door
Rudolf Johannus Verheul
geboren op 8 maart 1980 te Oss

Promotoren: Prof. dr. ir. W.E. Hennink
Prof. dr. W. Jiskoot

Voor mijn ouders

Table of Contents
Chapter 1
General introduction
Chapter 2
Synthesis, characterization and in vitro biological properties of O-methyl free
N,N,N,-trimethylated chitosan
Chapter 3
Influence of the degree of acetylation on the enzymatic degradation and in vitro
biological properties of trimethylated chitosans
Chapter 4
Relationship between structure and adjuvanticity of trimethyl chitosan (TMC)
structural variants in a nasal influenza vaccine
Chapter 5A
A step-by-step approach to study the influence of N-acetylation on the
adjuvanticity of N,N,N-trimethyl chitosan (TMC) in an intranasal whole
inactivated influenza virus vaccine
Chapter 5B
Maturation of human monocyte derived dendritic cells by trimethyl chitosan is
correlated with its N-acetyl glucosamine (GlcNAc) content
Chapter 6
Tailorable thiolated trimethyl chitosans for covalently stabilized nanoparticles
Chapter 7
Covalently stabilized trimethyl chitosan-hyaluronic acid nanoparticles for nasal
and intradermal vaccination
Chapter 8
Summary and future perspectives
Appendices
Affiliations of collaborating authors
List of abbreviations
Curriculum vitae
List of publications
Nederlandse samenvatting
Dankwoord
Page
9
25
47
69
91
113
125
149
169
183

CHAPTER 1
GENERAL INTRODUCTION

General Introduction
Nasal Vaccination
Ever since Edward Jenner’s successful inoculations with cowpox to prevent a potentially
lethal smallpox infection in the end of the 18 th century, active vaccination has proven to be the
most (cost) effective tool in the fight against infectious diseases. Active vaccination, or
immunization, involves activation of the immune system by controlled exposure to a
milder/inactivated form of the pathogen causing the disease, or to components derived from it,
thereby inducing (immunological) memory and fortifying the host’s response towards the real
pathogen. Vaccines are usually made of live attenuated or inactivated pathogens (e.g. viruses
or bacteria) or purified immunogenic protein(conjugate)s derived from these pathogens. After
two hundred years, still, most vaccines are administered via parenteral injection due to the
limited absorption and enhanced degradation of these large molecular structures when using
alternative administration routes [1]. There is however a need for vaccines that can be
administered via non-invasive other routes for reasons pointed out below. Table I gives an
overview of the (dis)advantages of the different immunization routes.
Table I. Advantages and disadvantages of different immunization routes (adopted from Slütter et al.
[2]).
Immunization route Advantages Disadvantages
Parenteral
Nasal
Oral
Pulmonary
Dermal
Powerful systemic immune
response
Accurate dosing
Non-invasive
Mucosal and systemic immune
responses
Easily accessible
Little antigen degradation
(compared to oral)
Non-invasive
Mucosal and systemic immune
responses
Large surface area
Non-invasive
Mucosal and systemic immune
responses
Little degradation (compared to
oral)
Non or minimally invasive
Large, easily accessible application
area
High density of immune cells in skin
Mucosal and systemic immune
responses
Invasive
Limited mucosal immune response
Risk of contaminated needles
Need for trained personnel
Mucociliary clearance
Inefficient uptake of antigen
Application device needed
Vaccine/antigen digestion in
stomach and gut
Inefficient uptake of antigen
Mucosal tolerance
Highly variable antigen delivery
Dry powder inhaler or nebulizer
needed
Clearance from lungs
May require invasive technology
(e.g. tattooing, microneedles)
Patch or application device needed
Less established technology
11

Chapter 1
Of these alternatives, nasal vaccination in particular has some interesting advantages. The
nose is easily accessible, commonly used and accepted for drug administration (e.g. nosesprays).
A relatively simple, painless, device can be used without the aid of trained personnel
and the nasal environment is less harsh for vaccine components than the oral route [3]. Also,
high numbers of antigen presenting cells (APCs) are present in the nasal mucosal linings
mediating both mucosal and systemic immune responses against foreign pathogens that try to
invade the human body through the respiratory tract. Importantly, this mucosal immunity,
hardly induced by parenteral immunization, may highly contribute to overall protection
against a foreign pathogen. The excretion of secretory immunoglobulins is not only limited to
the area of antigen-exposure but can occur on mucosal surfaces across the body (e.g. nasal
immunization against sexually transmitted human papilloma virus may thus be an interesting
option) [2, 4, 5].
All these potential benefits taken into consideration, it should be mentioned that currently
only one nasal vaccine is on the market: a live attenuated influenza vaccine administered via a
nasal spray, thereby mimicking the natural route of invasion of the pathogen. Although
effective, immunization with live attenuated viruses is under debate because of their potential
to mutate, thereby escaping the immune system and regaining their pathogenicity. As a
consequence, elderly, small infants and immuno-compromised people (e.g. by AIDS) are
excluded from such vaccines. Subunit vaccines consisting of better defined and characterized
antigenic proteins cannot mutate into pathogenic forms but are generally less immunogenic
and need potent adjuvant(system)s to elicit an adequate immune response [3, 6]. As vaccine
safety is now top priority, a number of hurdles needs to be tackled to make intranasal (i.n.)
vaccination a success-story.
Successful nasal vaccine delivery
After administration of the vaccine to the nose with an appropriate device, several successive
steps can be identified that should lead to an adequate immune response (Figure 1). In short, a
successful i.n. vaccine formulation has to adhere to the mucosal surfaces of the nasal cavity and
provide protection against the proteolytic degradation of the antigen in the nasal environment.
Prolonging nasal residence time may be an important mode of action for adjuvants; normally
rapid mucociliarly clearance of the antigen will limit contact of the antigen with the epithelial
barrier. Next, sufficient uptake and/or transport of antigen through the epithelial barrier
should be achieved. Macromolecules up to a certain size (some suggest 22 kDa [1]) may use
12

General Introduction
paracellular pathways via cellular tight junctions (that can be opened by penetration
enhancers) to overcome the epithelial barrier [1].
Figure 1. Schematic overview of the consecutive steps towards successful nasal vaccine delivery: 1)
muco-adhesion; 2) antigen uptake, by M-cell transport; 3) delivery to and subsequent
activation/maturation of DC; 4) induction of B- and T-cell responses. DC= dendritic cell, M-cell =
microfold cell, Th cell = helper T cell (adopted from Slütter et al. [2]).
Microfold (M) cells play an important role especially for particulate systems, since they are
capable of transporting the antigen by transcytosis to nasal associated lymphoid tissue (NALT)
[2-4]. NALT consists of agglomerates of cells involved in the initiation and execution of an
immune response, like dendritic cells (DCs), T- and B-cells. Alternatively, DCs may establish
contact with the antigen through close interaction with epithelial cells [3]. After transport
through the epithelium, the antigen has to be taken up by antigen presenting cells (APC), most
likely DCs or macrophages, which subsequently should mature, migrate and interact with
helper T (Th) cells. Here, DCs will present a peptide (epitope) of the (degraded) antigen via the
13

Chapter 1
major histocompatibility complex class II (MHC II) to the Th cells. Upon recognition of the MHC
II-peptide complex and co-stimulation of the APC, naïve Th cells differentiate into effector Th
cells, which can be divided in two major subtypes: Th1 and Th2 cells. Th1 cells are mainly
involved in activation and proliferation of the cellular immune system, i.e. stimulate the
cytotoxic T cells (CTL). Th2 cells are involved in stimulation of B-cells to differentiate into
plasma cells and increase the humoral immune responses i.e. the production of
immunoglobulins (IgG) also named antibodies. Interestingly, mucosal B-cells may differentiate
into mucosal plasma cells that secrete protease resistant secretory dimeric IgA (sIgA) into the
lumen to prevent mucosal invasion [7].
Ideally, adaptive immune responses elicited by vaccination should comprise both cellular
and humoral immune responses and should result in long-lived specific T- and B-memory cells,
as well as some readily available circulating antibodies [3]. Importantly, DC or APC signaling
determines the fate of the naïve Th cell and this can be modulated by the use of delivery
systems and/or adjuvants. Thus, a vaccine formulation may not only enhance the antigen
availability or increase the immune response (i.e. “the danger signal”) but can also influence
the type of immune response (i.e. immunomodulation) [2, 3, 8].
Polymeric carrier systems
Polymeric carriers have the advantage over other delivery systems (e.g. liposomes, ISCOMs)
because of their endless potential of chemical modifications thereby allowing fine-tuning of the
physico-chemical properties of the carrier system [6]. Although many polymers have been
studied for nasal immunization [4], most research has been done with the synthetic polymer
poly(lactide-co-glycolide) (PLGA) and chitosan (derivatives) which are obtained from naturally
abundant chitin. Interestingly, chitosan and its derivatives are much more effective in eliciting
immune responses in micro- or nanoparticulate form than as plain polymer solution [9-12],
most likely because of the particle’s resemblance to the original pathogen, their multimeric
antigen presentation and improved protection of the antigen against degradation [7].
Furthermore, particles are better taken up by APCs and can co-deliver antigen and adjuvant to
the same cell (13). While PLGA is FDA approved for several therapeutic applications and is
considered biodegradable and biocompatible in humans, chitosan derivatives are gaining
interest because of their superior efficacy in mucosal antigen delivery [2, 6, 14].
14

General Introduction
Chitosan
Chitin (poly β1→4 N-acetyl D-glucosamine) is the second most abundant natural biopolymer
and is derived from exoskeletons of crustaceans, insects and cell walls of several fungi [15].
Chitosan (Scheme 1) is a polysaccharide consisting of β1→4-D-glucosamine and β1→4 N-
acetyl D-glucosamine units and is obtained from chitin by partial deacetylation, which makes it
water-soluble in an acidic aqueous solution [1]. It can vary in size (molecular weight) and/or
degree of acetylation and is used in many areas of drug delivery and tissue engineering [16]. It
is approved for dietary applications in several countries and the FDA has approved chitosan
for use in wound dressings [16]. Chitosan’s muco-adhesiveness, penetration-enhancing
abilities and its properties allowing the preparation of particles without the use of organic
solvents has led to many investigations on chitosan formulations for mucosal vaccination [1, 5,
17]. Additionally, the functional groups (amines and hydroxyls) allow many modifications
depending on the foreseen application [15]. However, despite many studies on toxicity and
biocompatibility, chitosan is not (yet) considered a GRAS (generally regarded as safe) material
and no approval for use in drug delivery has been given so far. One reason might be that
chitosan’s high chemical variability confuses regulatory authorities [16].
Scheme 1. Chemical structure of chitosan. The degree of acetylation (x) is variable.
Chitosan’s primary amine-groups have a pK a of around 6, resulting in low aqueous solubility
and loss of penetration-enhancing abilities at neutral pH [1]. Carboxylation of the amines
and/or hydroxyl moieties and N-PEGylation have proven to be useful strategies for improving
the aqueous solubility of chitosan at neutral pH [18-20]. Other modifications encompass the
introduction of quaternary amine groups thereby giving chitosan a permanent, pH
independent positive charge and thus aqueous solubility also at neutral pH. While sometimes
15

Chapter 1
quaternary ammonium groups are introduced directly e.g. by coupling 2-diethylaminoethyl
chloride to chitosan [21, 22], mostly quaternization is achieved by partial trimethylation of the
amine resulting in N,N,N-trimethylated chitosan (TMC) [23].
N,N,N,-Trimethylated chitosan (TMC)
TMC (Scheme 2) has been shown to have muco-adhesive properties [27] and is able to open
tight junctions above a degree of quaternization (DQ) of 20% [25-29]. In addition, TMC has
been used to complex and condense DNA to yield polyplexes for gene delivery purposes [30-
32]. TMC is synthesized based on the method first published by Domard and coworkers [33]
and later modified by Sieval et al. [23]. They showed that alkylation of primary amines of
chitosan occurred by reaction of this polymer in strong alkaline conditions with an excess of
iodomethane at elevated temperature (60°C) using N-methyl-2-pyrrolidone (NMP) as solvent.
These relatively vigorous reaction conditions also lead to polymer chain scission [34] as well
as to partial and uncontrolled methylation of the C-3 and C-6 hydroxyl groups of chitosan [35,
36].
Scheme 2. Chemical structure of TMC. TMC can vary in degree of acetylation (x), quaternization (y),
dimethylation (z) and O-methylation (z). The various substitutions are randomly distributed throughout
the polymer; O-methylation (z) may also occur on the quaternized and acetylated units.
Several studies have been performed to determine the optimal DQ for either trans-epithelial
delivery of low molecular weight drug molecules and/or proteins, or to increase the
transfection potential of complexes of TMC with plasmid DNA. A DQ of about 40-50% was
found to be the optimum for transepithelial delivery of both low molecular weight compounds
and proteins [28, 37-40]. Furthermore, TMC has been used successfully for nasal immunization
in mice [41-44]. Amidi et al. showed high levels of serum IgG and HI titers after i.n.
administration of influenza A subunit encapsulated in TMC-tripolyphosphate (TPP)
16

General Introduction
nanoparticles as compared to plain antigen and antigen with TMC in solution [9]. Additionally,
our results and those of others suggest that optimizing the DQ of TMC may lead to further
improvement of the vaccine formulation: Boonyo et al. proposed an optimal DQ of 40% based
upon an i.n. immunization with ovalbumin [41] and we found that TMC with a DQ of 37% may
be superior to TMC with a DQ of 15% in an i.n. vaccination with whole inactivated influenza
virus [44]. Importantly, in these studies the TMCs used also had a variable extent of O-
methylation (DOM), degree of acetylation (DAc) and differences in polymer molecular weights.
While increasing TMC polymer molecular weight leads to an increase in toxicity [45], the
effects of the other polymer compositional variables are currently unknown. In particular the
role of the DAc can be anticipated to be quite substantial: in chitosan the DAc is associated with
its enzymatic degradability [16], penetration-enhancing capability [46] and stimulating effect
on APCs [47-50]. Tailorability of these variables (DQ, DOM and DAc) will allow better
understanding of the contributions of each of those side groups to the physico-chemical and
biological properties of TMC. Additionally, the introduction of novel substitutions such as thiolmoieties
may further broaden the potential pharmaceutical applications of TMC by enhancing
its muco-adhesive potential and allowing further chemical derivatization reactions via
reducible disulfide bridges [51-54].
TMC nanoparticle preparation
As mentioned above, nanoparticles composed of (subunit) antigen and TMC are more
effective in i.n. vaccination than soluble antigen and polymer. In some cases TMC is used as
coating for particulate systems (e.g. PLGA nanoparticles or whole inactivated influenza virus
particle) but more frequently TMC nanoparticles are prepared by ionic crosslinking methods.
The complexation between the positively charged TMC and oppositely charged
(macro)molecules added drop-wise under stirring in low ionic strength buffer results in
spontaneous formation of nanoparticles. This ionic gelation method is simple and mild for
proteins as no chemical crosslinkers, organic solvents or elevated temperatures are required
[1]. It has been reported that is some cases the antigen or therapeutic protein alone as such act
as crosslinker to yield nanoparticles [55], but often an additional crosslinker is needed. Both
small molecules such as tripolyphosphate (TPP) [9, 56-58] and larger anionic macromolecules
like polyglutamic acid [59] and hyaluronic acid [60] have been successfully used for
nanoparticle preparation. However, the physico-chemical stability of these complexes is
dependent on the characteristics of the crosslinker used and they often have a limited stability
17

Chapter 1
in physiological saline [60, 61] or when the pH drops [59, 62]. So, although TMC nanoparticles
are successfully used in nasal vaccination, many variables such as the type of crosslinker used
and particle stability leave room for improvement.
TMC mode of action
Several studies have shown the superiority of positively charged particles above negatively
charged or neutral particles in nasal immunization. E.g. chitosan coated PLGA particles
improved immunogenicity of ‘naked’ PLGA particles [63], TMC particles were superior in i.n.
vaccination with tetanus toxoid as compared to negatively charged particles made from
carboxylated chitosan [19] and TMC coated whole inactivated influenza virus resulted in
protection against a live virus challenge, in contrast with the uncoated virus which was not
effective [44]. Often, muco-adhesion due to charge interactions between polymer and mucus is
suggested as potential mechanism of action for these penetration-enhancing polymers. It has
further been postulated that muco-adhesive polymers increase nasal residence time and
improve interaction of the antigen with the epithelial cell barrier leading to an increased
antigen uptake [1, 24, 43]. Alternatively, the opening of cellular tight junctions (as determined
in a transepithelial electrical resistance (TEER) assay) may result in enhanced antigen/protein
uptake [59].
Chitosan and TMC are known to induce a rearrangement of cytoskeletal F-actin and tight
junction protein ZO-1, leading to enhanced permeability of the epithelium [59, 64]. However,
fully quaternized diethyl aminoethyl dextran was ineffective as absorption enhancer indicating
that solely cationic charge is not sufficient for a polymer to have adjuvant activity [65].
Additionally, nanoparticles are considered too big to pass through these junctions [1] and also
non-TEER reducing TMCs can improve immunogenicity of i.n. vaccine formulations [9, 44].
Also, the direct adjuvant effect of TMC(-particles) on APCs or DCs has hardly been studied, but
as mentioned before, the N-acetyl glucosamine units may interact with C-type lectin receptors
present on these cells [47-50]. Taken together, clearly, the exact mode of action of TMC is not
yet fully understood.
18

General Introduction
Aim and thesis outline
As pointed out in the previous paragraphs, TMCs are promising polymers for use in nasal
immunization. However, many opportunities for optimizing TMC structure and TMC- based
nanoparticles still remain and mechanistic insights in the mode of action of TMC is lacking. The
aim of this thesis is to develop synthetic routes to make TMC structural variants in a
controllable and tailorable manner and introduce substitutions such as thiol-moieties that may
further improve TMC’s properties. In this way, structure-activity relationships can be
investigated in in vitro assays and in in vivo nasal vaccination studies. Also, novel covalently
stabilized TMC-based particles are prepared and investigated in in vivo immunization studies.
Chapter 2 challenges the current synthetic method that introduces several side-reactions
such as O-methylation and chain scission. A novel synthesis route is proposed that allows
tailorability of the DQ without introducing other modifications. These novel TMC polymers are
studied for physico-chemical properties, evaluated for cytotoxicity and the ability to open tight
junctions, and compared with TMCs synthesized via the traditional method.
Chapter 3 describes the synthesis of TMC with a high degree of acetylation (DAc) and
investigates the lysozyme-catalyzed degradability of TMCs with different DAcs and with or
without O-methylated groups, and evaluates these polymers for their physico-chemical
properties, cytotoxicity and ability to open tight junctions.
In Chapter 4 the adjuvant effect of several TMC types is investigated for i.n. administered
whole inactivate influenza virus (WIV). In particular, O-methyl free TMCs with varying DQs,
reacetylated O-methyl free TMC (TMC-RA), and conventional O-methylated TMCs with similar
DQ are compared. The TMC-WIV vaccines are physicochemically characterized and the
immunogenicity and protectivity of the vaccines are assessed in a murine challenge model.
Additionally, the influence of TMC:WIV ratio on the type and extent of humoral immune
responses is investigated.
To understand the differences in adjuvanticity of the different TMCs in Chapter 5A TMC-
WIV, TMC-RA-WIV and WIV formulations are compared at six potentially critical steps in the
induction of an immune response after i.n. administration. In particular, (1) the degradation of
TMC and TMC-RA in nasal washes, (2) nasal residence time, (3) nasal distribution patterns, (4)
cellular uptake and (5) transport through an epithelial (Calu-3) cell line of WIV formulated
19

Chapter 1
without or with TMC(-RA), and (6) the effect of the different formulations on maturation of
murine bone marrow derived dendritic cells (DCs) are studied. In Chapter 5B the effects of
TMC and TMC-RA in solution and in combination with WIV on human monocyte-derived
human DCs are investigated.
Chapter 6 introduces a novel synthetic method to yield thiolated TMCs with a high DQ. The
different thiolated TMCs are physico-chemically characterized, evaluated in in vitro
cytotoxicity assays and used for preparation of covalently stabilized nanoparticles with
thiolated hyaluronic acid.
Physico-chemically characterized covalently stabilized and/or PEGylated TMC-hyaluronic
acid nanoparticles are used in an intranasal and intradermal vaccination study in mice in
Chapter 7. For comparison, non-stabilized particles are used as control. Both the type and the
extent of the immune responses are investigated.
Chapter 8 summarizes the findings and conclusions of this thesis and addresses the future
opportunities for these novel TMCs and TMC-based delivery systems.
20

General Introduction
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Chapter 1
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49. J. Nadesalingam, A. W. Dodds, K. B. M. Reid, and N. Palaniyar. Mannose-binding lectin
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23

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2862 (2004).
24

CHAPTER 2
SYNTHESIS, CHARACTERIZATION AND IN
VITRO BIOLOGICAL PROPERTIES OF O-
METHYL FREE N,N,N-TRIMETHYLATED
CHITOSAN
Rolf J. Verheul, Maryam Amidi, Steffen van der Wal,
Elly van Riet, Wim Jiskoot, Wim E. Hennink.
Biomaterials 2008, 29, 3642-3649

Chapter 2
Abstract
N,N,N-trimethylated chitosan (TMC) with varying degrees of quaternization (DQs) is
currently being investigated in mucosal drug, vaccine and in gene delivery. However, besides
N-methylation, also O-methylation and chain scission occur during the synthesis of this
polymer. Since both side reactions may affect the polymer characteristics, there is a need for
TMCs without O-methylation and disparities in chain lengths while varying the DQ. In this
study, O-methyl free TMC with varying DQs was successfully synthesized by using a two-step
method. First, chitosan was quantitatively dimethylated using formic acid and formaldehyde.
Then, in presence of an excess amount of iodomethane, TMC was obtained with different DQs
by varying reaction time. TMC obtained by this two-step method showed no detectable O-
methylation ( 1 H-NMR) and a slight increase in molecular weight with increasing DQ (GPC),
implying that no chain scission occurred during synthesis. The solubility in aqueous solutions
at pH 7 of O-methyl free TMC with DQ < 22% was less as compared to O-methylated TMC with
the same DQ. On the other hand, O-methyl free TMC with DQ > 30% had a good aqueous
solubility. On Caco-2 cells O-methyl free TMCs demonstrated a larger decrease in transepithelial
electrical resistance (TEER) than O-methylated TMCs. Also, with increasing DQ, an
increase in cytotoxicity (MTT) and membrane permeability (LDH) was observed.
26

Synthesis and Characterization of O-methyl Free TMC
Introduction
Chitosan is a polysaccharide consisting of β1→4-D-glucosamine and β1→4 N-acetyl D-
glucosamine units and is obtained by partial deacetylation of the natural polymer chitin. In
recent years, chitosan has been under investigation for various biomedical and pharmaceutical
applications [1-4]. However, its poor aqueous solubility and loss of penetration-enhancing
activity above pH 6 is a major drawback for its use at physiological conditions [5]. Partial
quaternization of chitosan’s primary amine groups has been used to obtain a chitosan
derivative that is soluble at physiological conditions. N,N,N-trimethylated chitosan (TMC) has
been shown to have muco-adhesive properties and is able to open tight junctions above a
certain degree of quaternization (DQ) [6-10]. In addition, TMC has been used to complex and
condense DNA to yield polyplexes for gene delivery purposes [11, 12].
Without exceptions, TMC is synthesized based on the method first published by Domard and
coworkers [13] and later modified by Sieval et al [7]. They showed the alkylation of primary
amines of chitosan by reaction of this polymer in strong alkaline conditions with an excess of
iodomethane using N-methyl-2-pyrrolidone (NMP) as solvent. The relatively vigorous reaction
conditions lead to polymer chain scission [14] and, importantly, partial and uncontrolled
methylation of the C-3 and C-6 hydroxyl groups of chitosan [15, 16]. Furthermore, the DQ
proved difficult to control and often multiple reaction steps are required to obtain the desired
TMC [17].
Several studies have been performed to determine the optimal DQ for either transepithelial
delivery of low molecular weight drug molecules and/or proteins, or to increase the
transfection potential of complexes of TMC with plasmid DNA. It has been reported that a DQ
of about 40-50% is the optimum for transepithelial delivery of both low molecular weight
compounds [17, 18] and proteins [19]. In these studies the TMCs used also had a variable
extent of O-methylation and disparities in polymer chain lengths. Since O-methylation and
variations in polymer chain length may affect the physicochemical properties and, likely, also
the biological properties of TMC, there is a need for a synthetic method that yields TMC
without O-methylated groups and prevents chain scission.
Muzzarelli and Tanfani reported on the synthesis of TMC using iodomethane and N-dimethyl
chitosan (DMC) obtained by reaction of chitosan with formaldehyde and sodium borohydride
[20]. They showed that up to 60% of the amine groups could be trimethylated by this method,
but no investigations were done on the tailorability of the DQ. Interestingly, this two-step
method likely prevents chain scission and deacetylation of remaining N-acetyl groups, and
might result in TMC without O-methylation. Nevertheless, this was not investigated by these
27

Chapter 2
researchers. Recently, several adjustments to this method were presented by Jia et al. [21] and
Guo et al. [22] introducing sodium hydroxide in the second step to increase the degree of
substitution. However, the use of a strong base when trimethylating DMC will very likely result
in O-methylation. So far, TMC synthesized by these methods has only been studied for its
antibacterial activity [20-22].
The aim of this study was to investigate a two-step method to synthesize TMC with tailorable
DQ avoiding O-methylation and chain scission as side reactions. The synthesized TMC with
different DQs were studied for physico-chemical properties, evaluated for cytotoxicity and the
ability to open tight junctions, and compared with TMC synthesized via the traditional method.
Materials and Methods
Materials. Chitosan with a residual degree of acetylation of 17% (determined by NMR) and
M n of 25 kDa, M w of 42 kDa (determined by Viscotek triple detection system as described
below) was purchased from Primex (Siglufjordur, Iceland). N-methyl-2-pyrrolidone (NMP),
formaldehyde (37% stabilized with methanol), formic acid, thiazolyl blue tetrazolium bromide
(MTT), sodium acetate, acetic acid (anhydrous), sodium hydroxide and hydrochloric acid were
obtained from Sigma-Aldrich Chemical Co. Dulbecco’s Modified Eagle’s Medium (DMEM),
Hank’s balanced salt solution (HBSS), Fetal calf serum (FCS) were obtained from Invitrogen
(Breda, The Netherlands). Sodium dodecyl sulfate (SDS) was ordered from Merck (Darmstadt,
Germany). Iodomethane 99% stabilized with copper was obtained from Acros Organics (Geel,
Belgium). All other chemicals used were of analytical grade.
Synthesis of dimethylated chitosan (DMC). The two-step reaction pathway to synthesize
TMC avoiding O-methylation is depicted in scheme 1 and is based on the method published by
Muzzarelli and Tanfani [20], with some modifications. In detail, a formic acid-formaldehyde
methylation (Eschweiler-Clarke) was used to synthesize N,N-dimethylated chitosan [23].
Instead of sodium borohydride as the reducing agent [20-22], we used formic acid that allows
chitosan to dissolve in the aqueous solution without the use of an acetate buffer. Ten grams of
chitosan was transferred into a 500 ml roundbottom flask. Next, 30 ml of formic acid was
added followed by 40 ml of 37% formaldehyde and 180 ml of distilled water yielding a total
volume of about 250 ml. A reflux condenser was attached and the solution was heated to 70 °C
and stirred using a magnetic stirrer for 118 hours. Then, the slightly yellow, viscous solution
was evaporated under reduced pressure and 1 M NaOH solution was used to increase the pH
28

Synthesis and Characterization of O-methyl Free TMC
to 12 at which gel formation occurred. This gel was washed with deionized water over a glass
filter to remove impurities. Then, the DMC was dissolved in deionized water at pH 4 (adjusted
with 1 M HCl), filtered over a glass filter and dialyzed against deionized water for three days
(changing buffer twice-daily). Finally, the product was filtered through a 0.8 µm filter and
freeze dried.
Synthesis of trimethylated chitosan from DMC. DMC was reacted with iodomethane to
yield TMC following the method described by Muzzarelli, with some modifications (see Scheme
1) [20]. To prevent O-methylation, the reaction of DMC with iodomethane was done in NMP,
without the addition of a base catalyst [21, 22]. In detail, 250 mg of DMC was dissolved in 40
ml deionized water and the pH was adjusted to 11 with NaOH by which gel formation
occurred. This step is performed to ensure deprotonation of the tertiary amino groups of the
DMC. Then, the gel was washed with water and finally three times with acetone. Next, DMC
was suspended in 50 ml NMP and 2 ml iodomethane was added. The dispersion was stirred at
40°C for the desired time and subsequently dropped in 150 ml of an ethanol/diethyl ether
mixture (50/50). The precipitate (TMC) was isolated by centrifugation and washed extensively
with diethyl ether. After drying overnight, the TMC was dissolved in 100 ml of an aqueous 10%
NaCl solution and put on a shaker for a minimum of 18 hours for ion-exchange. Finally, the
TMC was dialyzed against deionized water for 3 days changing buffer twice daily, filtered
through a 0.8 µm filter and freeze dried. Analysis on NMR 500 MHz was performed to
determine the degree of quaternization (DQ) and to confirm the absence of O-methylation.
TMCs with different DQs were synthesized starting with one gram of DMC following the same
procedure but using 3 ml of iodomethane, 100 ml of NMP and a 250 ml round bottom flask and
varying reaction time.
29

Chapter 2
Scheme 1. Two-step synthetic pathway for the preparation of TMC avoiding O-methylation.
Synthesis of O-methylated TMC from chitosan. TMC was synthesized by methylation of
chitosan with iodomethane in presence of an aqueous solution of NaOH essentially as
described previously [7]. In detail, 1 g of chitosan and 2.4 g of sodium iodide were added to a
mixture of 40 ml of NMP and 6 ml of 15% w/v aqueous NaOH solution. Subsequently, the
mixture was heated to 60°C and after stirring for 20 min, 6 ml of methyl iodide was added and
30

Synthesis and Characterization of O-methyl Free TMC
the reaction mixture was refluxed for 60 min. The reaction was stopped by dropping the
mixture in a 200 ml mixture of diethyl ether and ethanol (50/50). The obtained precipitate
was washed extensively with diethyl ether. In this way, TMC with a DQ of about 20% and
comparable O-methylation was obtained (step 1). To synthesize TMC with a DQ of around 40-
50%, before precipitation, 3 ml of 15% NaOH solution and 3 ml of iodomethane were added
and the solution was stirred for another 60 minutes before stopping the reaction as described
above (step 1.5). TMCs with higher DQs (60% to 90%) were synthesized by dissolving the
dried TMC (DQ ~ 20) together with 2.4 g of sodium iodide in 40 ml of NMP at 60°C.
Subsequently, 5.5 ml of an aqueous 15% w/v NaOH solution was added and, after stirring for
20 min 3.5 ml CH 3I was added and the reaction was done for 45 minutes under refluxing to
yield TMC with a DQ of about 60 to 70% (step 2). To obtain TMC with a DQ of about 80 to 90%,
after 45 minutes, 0.6 g NaOH and 1 ml iodomethane were added and the reaction was
continued for 60 min at 60°C (step 2.5). The reaction was terminated by precipitation the
reaction mixture in 200 ml of a mixture of diethyl ether and ethanol (50:50) and the
precipitate was washed extensively with diethyl ether.
Finally, the products were dissolved in 50 ml aqueous 10% w/v NaCl solution and put on a
shaker for a minimum of 18 hours for ion-exchange. The obtained solution was dialyzed at
room temperature against deionized water for 3 days changing buffer twice daily, filtered
through a 0.8 µm filter and freeze dried. Analysis on NMR 500MHz was performed to
determine the DQ and the degree of O-methylation.
31

Chapter 2
Scheme 2. Synthetic pathway for the preparation of TMC according to the method of Sieval et al [7].
Determination of the degrees of dimethylation, quaternization and O-methylation. The
1H-NMR spectra of the DMC and the various TMCs were recorded with a Varian INOVA
500MHz NMR spectrometer (Varian Inc., Palo Alto, Ca, USA) at 80°C in D 2O. The degree of
dimethylation of the DMC was calculated as follows:
DDM = [(CH 3) 2]/[H2-H6] x 100
Here, [(CH 3) 2] is the integral of the hydrogens of the dimethyl amino groups at 2.9 ppm and
[H2-H6] is the integral corresponding the H-2 to H-6 protons between 4.0 and 3.2 ppm.
32

Synthesis and Characterization of O-methyl Free TMC
The DQ, degree of dimethylation (DM) and degree of 3- and 6-O-methylation (DOM-3 and
DOM-6, respectively) of the TMCs were calculated according to previous described methods[7,
15, 24].
DQ = [[(CH 3) 3]/[H] × 1/9] × 100
DM = [[(CH 3) 2]/[H] × 1/6] × 100
DOM = [[CH 3]/[H] × 1/3] × 100
Here, [(CH 3) 3], [(CH 3) 2] and [CH 3] are the integrals of the hydrogens of the trimethylated amino
groups at 3.3 ppm, the dimethylated amino groups at 2.9 ppm and the methylated hydroxyl
groups at either 3.4 (DOM-6) or 3.5 (DOM-3) ppm, respectively. [H] is the integral of the H-1
peaks between 4.7 and 5.7 ppm; the signal related to the hydrogen atoms bound to the C-1’s of
the TMC molecule. For the DMC and TMC synthesized with the mild method and a DQ below
25% addition of 0.05 ml of DCl was needed to dissolve the polymers in D 2O.
Determination of M n and M w of chitosan and TMC. M n and M w of chitosan and the various
TMCs were determined by gel permeation chromatography (GPC) on a Viscotek-triple
detection system using a Shodex OHPak SB-806 column (30 cm) and 0.3 M sodium acetate pH
4.4 (adjusted with acetic acid) as running buffer [25]. To remove residual water, chitosan and
the TMC samples were dried in a vacuum oven at 40°C overnight. Then, the samples were
dissolved overnight in the running buffer at a concentration of 5 mg/ml, filtered through a 0.2
µm filter and injected (50 µl); the flow rate was 0.7 ml/min. Data from the laser photometer (λ
= 670 nm) (right (90 0 ) and low (7 0 ) angle light scattering), refracting index detector and
viscosity detector were integrated using the provided Viscotek-software to calculate the M n,
M w and dn/dc of the different samples. Pullulan (M n = 102 kDa, M w = 106 kDa) obtained from
Viscotek Benelux (Oss, the Netherlands) was used for calibration.
Water solubility of chitosan and various TMCs. Aqueous solubility of the different
polymers was determined at pH 7 at room temperature. First the polymers were dissolved
overnight in a 0.5% acetic acid solution at 2.5 mg/ml. Then the pH was adjusted to 7 using 1 M
NaOH and the transmittance of the solutions at 500 nm was measured on an UV/VIS
spectrophotometer (UV-2450, Shimadzu, Japan). The polymers were considered insoluble
when the transmittance was less than 90% compared to the transmittance of 0.5% acetic acid
solution [26].
33

Chapter 2
Transepithelial electrical resistance (TEER) measurements. Caco-2 cells were seeded at
a density of 2x10 5 cells per well on 12-transwell plates with a microporous membrane. The
cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) for 10 days until a
confluent cell layer was formed. The medium was replaced by Hank’s Balanced Salt Solution
(HBSS) at the basolateral side 10 minutes before the start of the experiments. Then, 0.5 ml
solution of TMC (with various DQs, with or without O-methylation, dissolved (2 mg/ml) in
HBSS, pH adjusted to 7 with 0.1 M NaOH) was applied at the apical site of the cell monolayers.
SDS (10 mg/ml) was used as positive control and HBSS as reference. The resistance measured
of the membrane without cells was used as blank. The TEER of the Caco-2 cells was measured
with a Millicell-ERS (Millipore, Billerica, USA) measuring device at certain time points (0, 15,
30, 45, 60 and 90 min.) after addition of the stimuli. After 90 minutes the cells were washed
with HBSS and incubation of the cells was continued in DMEM for 24 hours at 37°C, CO 2 5% to
determine the recovery of the TEER [24, 27].
MTT cell toxicity assay. Caco-2 cells were seeded in a 96-well plate at a density of 4x10 4
cells per well and incubated for 2 days at 37°C, CO 2 5% in culture medium (DMEM, high
glucose, 10% FCS, L-glutamine, pyruvate, non essential amino acids). The medium was
removed and the cells were incubated for 2.5 hours with 100 µl TMC solutions in HBSS (TMC
concentrations were 0.1, 1 and 10 mg/ml, pH set at 7 with 0.1 M NaOH). SDS (10 mg/ml) was
used as positive control and HBSS as reference for 100% cell viability. Thereafter, the HBSS
was removed and the cells were washed with phosphate buffered saline. One hundred µl of a
freshly prepared solution of 0.5 mg/ml MTT in DMEM, without any additions, was added and
the cells were incubated for 3 hours at 37°C and 5% CO 2. Subsequently, the wells were
emptied, 100 µl of DMSO was used to dissolve the formed formazan crystals and the
absorbance was read at 595 nm [28].
LDH assay. Caco-2 cells were seeded in a 96-well plate at a density of 4x10 4 cells per well
and incubated for 2 days at 37°C, CO 2 5% in culture medium (see section on MTT assay for
composition). The medium was removed and the cells were washed with HBSS and incubated
for 2.5 hours with 100 µl TMC solutions in HBSS (TMC concentrations were 0.1, 1 and 10
mg/ml, pH set at 7 with 0.1 M NaOH). After incubation, the concentration of LDH present in the
supernatant of the samples was determined with the Cytotoxicity Detection Kit-Plus (Roche
Diagnostics, Mannheim, Germany) by measuring absorbance at 490 nm with 650 nm as a
reference wavelength. A calibration curve was made with the lysis buffer provided by the
34

Synthesis and Characterization of O-methyl Free TMC
manufacturer, setting the LDH concentration measured with the undiluted lysis buffer at 100%
LDH release. HBSS was used as a negative control.
Results and discussion
Synthesis and characterization. To synthesize O-methyl free TMC, chitosan was first
converted into DMC using the Eschweiler-Clarke reaction with formaldehyde and formic acid
(Figure 1). 1 H-NMR analysis showed that the obtained polymer had a degree of dimethylation
of 83%. Since the chitosan used in this study had a degree of acetylation of around 17 % it can
be concluded that the free amines were quantitatively dimethylated. In the next step, DMC
dissolved in NMP was converted into TMC using an excess amount of iodomethane. Figure 1
shows that the degree of quaternization can be accurately tailored by varying the reaction time
while keeping reaction temperature and DMC/CH 3I ratio constant. In other published
procedures to synthesize TMC, sodium iodide was added to the reaction mixture of chitosan
and iodomethane [7, 21, 22]. However, the presence of sodium iodide (0.011 M) during the
synthesis of TMC from DMC using CH 3I did not affect the obtained DQ in our studies (data not
shown).
degree of quaternization
(%)
70
60
50
40
30
20
10
0
0 10 20 30 40 50 60 70
reaction time (h)
Figure 1. Effect of reaction time on the DQ of TMC. The reaction temperature was 40°C. Error bars
represent standard deviation of three independent syntheses.
35

Chapter 2
Figure 2. 1 H-NMR-spectra of TMC with a DQ of about 60% synthesized with a) one-step method under
alkaline conditions and b) the two-step method presented in this article. Red oval indicates signals
correlated to O-methylation.
Typical 1 H-NMR spectra of TMC synthesized with the new procedure described in this paper
and with the procedure described by Sieval et al. are shown in Figure 2. From the spectra it can
be concluded that both TMCs have a DQ of about 60%. The spectrum of TMC synthesized
36

Synthesis and Characterization of O-methyl Free TMC
according to the procedure of Sieval et al. (Figure 2a) clearly shows O-methylation of the
hydroxyl groups at the C-3 and C-6 of the glucosamine units (peaks observed at 3.5 and 3.4
ppm, respectively). It was calculated that DOM-6 and DOM-3 are 56 and 44%, respectively. The
higher degree of methylation observed on the C-6 hydroxyl groups is likely because this
hydroxyl group is less sterically hindered than the hydroxyl group on the C-3. An overview of
the 1 H-NMR results of the O-methylated TMCs is presented in Table 1. As also reported by
others [15, 16], both the DQ and the DOM-3 and DOM-6 increased with the number of reaction
steps, but the DQ and DOM proved hard to control. In contrast, 1 H-NMR analysis of TMC with a
DQ of 56% synthesized with the new method described in this paper shows no peaks at 3.4
and 3.5 ppm (Figure 2b), demonstrating the absence of O-methylation.
Table 1. Degree of quaternization (DQ), dimethylation (DM), O-methylation on C-6 (DOM-6) and on
C-3 (DOM-3) of TMCs synthesized according to Sieval et al. [7] as determined by 1 H-NMR analysis.
DQ DM DOM-6 DOM-3
Step 1 22% 61% 18% 12%
Step 1.5 50% 35% 45% 40%
Step 2 61% 25% 56% 44%
Step 2.5 86% 1% 76% 72%
In line with the results of Snyman et al. [14], GPC analysis showed a slightly decreased
molecular weight of TMC synthesized in presence of aqueous NaOH with increasing DQ (Table
2). Under the alkaline reaction conditions hydrolysis of some glycosidic bonds linking the
glucosamine units likely occurs, thus with increasing reaction time, the molecular weight and
the polymer chain length of the O-methylated TMCs decreases. Although acid hydrolysis of
chitosan has been reported [29], we show that the molecular weight increases after converting
chitosan into DMC (Table 2) which demonstrates that no or only limited chain scission occurs
during reaction. The slight increase in molecular weight can be ascribed to the addition of
methyl groups to the molecule. Further, GPC analysis also shows that TMC synthesized from
DMC shows a slight increase in molecular weight with increasing DQ (Table 2). Therefore, as
expected, the trimethylation of DMC with the synthetic method described in this thesis is not
associated with chain scission, and all O-methyl free TMCs will have the same polymer chain
length. This is in contrast with the ‘standard’ method to synthesize TMC as described by Sieval
et al [7] where TMCs with varying DQ will also have discrepancies in polymer chain lengths.
37

Chapter 2
Table 2. Molecular weights and water solubility of various derivatives of chitosan.
M n M w dn/dc Solubility in water
at pH 7
Chitosan 25 kDa 42 kDa 0.18 -
TMC-OM 22% 34 kDa 56 kDa 0.15 +
TMC-OM 50% 32 kDa 49 kDa 0.15 +
TMC-OM 61% 31 kDa 49 kDa 0.14 +
TMC-OM 86% 29 kDa 44 kDa 0.14 +
DMC 28 kDa 57 kDa 0.16 -
TMC 22% 31 kDa 60 kDa 0.16 -
TMC 30% 33 kDa 59 kDa 0.15 +
TMC 43% 36 kDa 75 kDa 0.15 +
TMC 56% 37 kDa 78 kDa 0.15 +
TMC 68% 39 kDa 84 kDa 0.15 +
All TMCs tested (with or without O-methylation) and a DQ >22% were readily soluble in
aqueous solutions at pH 7. As expected, chitosan and DMC became insoluble when the pH was
increased to 7. Remarkably, O-methyl free TMC with a DQ 22% was insoluble at pH 7 at a
concentration of 2.5 mg/ml while TMC with around the same molecular weight and DQ (22%)
and a DOM of 18 and 12% at C-6 and C-3, respectively, was readily dissolved in the same
solvent. This is rather unexpected since O-methylated glucosamine units can only act as
hydrogen-acceptor, whereas non O-methylated units can both accept and donate hydrogen
bridges in interaction with aqueous solvents. Possibly, TMC with low DQs will still partially
resemble the behavior of chitosan, where intra- and intermolecular interactions decrease the
aqueous solubility at pH 7 [1]. Partial O-methylation might reduce these interactions and
therefore result in better aqueous solubility of O-methylated TMCs with a DQ

Synthesis and Characterization of O-methyl Free TMC
50% for penetration enhancing effects [10, 18, 19]. However, the possible contribution of O-
methylation on these effects is unknown. The availability of TMCs with varying DQ but without
O-methylated groups or discrepancies in polymer chain lengths allows a better evaluation of
the effect of DQ on the biological properties of TMC. Figures 3a and 3b show the effect of TMCs
with different DQ with and without (partial) O-methylation on the TEER. The results of the O-
methylated TMCs are in line with those reported by other researchers [6, 10, 18, 24], namely
TMC with a DQ of 22% and DOM-3 and DOM-6 of 12 and 18%, respectively, showed no effect
on the TEER, indicating that the polymer is unable to open the tight junctions. The TMC with a
DQ of 61% and similar O-methylation demonstrates the largest decrease in the TEER and
showed marginal toxicity using the MTT assay (Figure 4a) implying that this polymer induces
opening of tight junctions without exerting major acute toxic effects. However, since the LDH
levels (Figure 5a) are elevated, this TMC apparently induces some membrane damage to cells.
Interestingly, as compared to O-methylated TMC with a DQ of 61%, TMC with the highest DQ
(86%) had similar effect on the TEER while it showed substantial cellular toxicity (MTT assay)
at a concentration of 10 mg/ml. The highly elevated LDH release upon exposure of the Caco-2
cells to this polymer supports the data obtained with the MTT assay. Other groups [10, 18]
reported the highest tight-junction opening activity of O-methylated TMC with a DQ of about
40-50%. However, the molecular weights of the TMCs used in those studies were higher which
may account for the somewhat different optimal DQ found in these studies.
39

Chapter 2
TEER (% HBSS)
A
SDS 1%
100 TMC-OM 22%
TMC-OM 50%
80
TMC-OM 61%
60
TMC-OM 86%
40
20
0
0 15 30 45 60 75 90
Time (min.)
1140
TEER (% HBSS)
B
100 SDS 1%
80
60
40
20
TMC 30%
TMC 43%
TMC 56%
TMC 68%
0
0 15 30 45 60 75 90
Time (min.)
1140
Figure 3. Effect of (a) O-methylated TMC and (b) O-methyl free TMC with various DQs on the TEER of
Caco-2 cells at a TMC concentration of 2 mg/ml. SDS 10 mg/ml was used as a positive control. Error bars
represent the standard deviation of six measurements.
Figure 3 shows that O-methyl free TMC with a DQ of 30% had a similar TEER effect as the O-
methylated TMC with a DQ of 56%, whereas the MTT and LDH assays showed some toxicity
above a concentration of 1 mg/ml (Figures 4b and 5b). Figure 3b also shows that the effect on
the TEER slightly increased (reduction from 35% to 20% remaining resistance compared with
HBSS) with increasing DQ (from 30 to 68%). This might indicate that with increasing DQ the
polymers have greater capacity to open the tight junctions, but the decrease in TEER can also
be the result of acute toxic effects on the Caco-2 cells (see Figures 4b and 5b). The MTT assay
clearly displays a DQ depending cytotoxicity for O-methyl free TMCs and these polymers show
40

Synthesis and Characterization of O-methyl Free TMC
a larger decrease in cell viability than O-methylated TMC. The LDH assay shows a DQdependent
LDH release for both O-methylated and O-methyl free TMCs. Interestingly, although
the O-methyl free TMCs demonstrate a higher LDH release than the O-methylated TMCs, the
difference is much less prominent as with the MTT assay; O-methylated TMC with a DQ of 56%
induces a similar LDH release as O-methyl free TMC with a DQ of 30% and the LDH release of
O-methylated TMC with a DQ of 86% is comparable to the amount of LDH released by
application of O-methyl free TMC with a DQ of 68%. Partial recovery of the TEER was observed
(24 hours after removal of the polymer) for all TMCs (with or without O-methylation), which
indicates that cells were still viable after the removal of the stimulus. Taken together, O-methyl
free TMCs lead to a larger decrease of the TEER than O-methylated TMCs but the penetrationenhancing
effects of the TMCs without O-methylation remain to be established.
Cell viability (%)
A
100
80
60
40
20
TMC-OM 22%
TMC-OM 50%
TMC-OM 61%
TMC-OM 86%
0
0.1 1 10
Conc. (mg/ml)
Cell viability (%)
B
100 TMC 30%
TMC 43%
80
TMC 56%
60
TMC 68%
40
20
0
0.1 1 10
Conc. (mg/ml)
Figure 4. Effect of (a) O-methylated TMC and (b) O-methyl free TMC with various DQs on the viability of
Caco-2 cells (MTT assay) at different concentrations. Error bars represent the standard deviation of six
measurements.
41

Chapter 2
The synthesis method described in this paper provides a better defined TMC compared to
the traditionally synthesized TMC with unavoidable O-methylation [15]. Moreover, the lack of
chain scission during the quaternization of TMC allows studies of TMCs with varying DQ
without discrepancies in polymer chain lengths.
Our studies demonstrate that O-methyl free TMCs have a stronger effect on the Caco-2 cells
in the TEER, MTT and LDH assays than the O-methylated TMCs. Since the molecular weights of
the polymers used in the present study are comparable (Table 2), the results imply a reduction
in toxicity of TMC by the (partial) O-methylation of the polymer. Additionally, TMC without O-
methylation resembles to a larger extent the original chitosan polymer. This may have
beneficial effects on the enzymatic degradability of the polymer.
LDH release (%)
A
30
20
10
HBSS
TMC-OM 22%
TMC-OM 50%
TMC-OM 61%
TMC-OM 86%
0
0.1 1.0 10.0
Conc. (mg/ml)
LDH release (%)
B
30
20
10
HBSS
TMC 30%
TMC 43%
TMC 56%
TMC 68%
0
0.1 1.0 10.0
Conc. (mg/ml)
Figure 5. Effect of (a) O-methylated TMC and (b) O-methyl free TMC with various DQs on the viability of
Caco-2 cells (LDH release) at different concentrations. Error bars represent the standard deviation of
three measurements.
42

Synthesis and Characterization of O-methyl Free TMC
TMCs without O-methylation may improve the penetration-enhancing effects of the
‘traditional’ TMCs, demonstrating a strong (reversible) effect of the opening of tight junctions.
Furthermore, it shows limited toxicity at concentrations used for cell transfection experiments
[11, 12]. Since incorporation into nanoparticles reduces the toxicity of TMC [24], this non O-
methylated TMC may be very suitable for incorporation into nanoparticles for mucosal
vaccination, which is currently under investigation.
Conclusion
This paper shows a straightforward method to synthesize TMC avoiding O-methylation while
preventing chain scission and to tailor the DQ of the TMC by varying the reaction time. O-
methyl free TMCs with a DQ > 30% are readily soluble in aqueous solutions at pH 7. In the
TEER, MTT and LDH assays O-methyl free TMCs have a stronger effect on the Caco-2 cells than
the O-methylated TMCs. In conclusion, these new well-characterized O-methyl free TMCs have
potentially favorable biological characteristics over the traditional TMC and allow to effectively
study the influence of the DQ of TMC in various delivery systems.
Acknowledgement. This research was performed under the framework of TI Pharma
project number D5-106-1; Vaccine delivery: alternatives for conventional multiple injection
vaccines.
43

Chapter 2
References
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chitosan chloride (TMC) as a novel excipient for oral and nasal immunisation against
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9. Snyman D, Hamman JH, Kotze AF. Evaluation of the mucoadhesive properties of N-
trimethyl chitosan chloride. Drug Develop Ind Pharm 29:61-9 (2003).
10. Thanou MM, Kotze AF, Scharringhausen T, Lueßen HL, De Boer AG, Verhoef JC, et al.
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(2000).
11. Thanou M, Florea BI, Geldof M, Junginger HE, Borchard G. Quaternized chitosan oligomers
as novel gene delivery vectors in epithelial cell lines. Biomaterials 23:153-9 (2002).
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gene vector: synthesis and application. Macromol Biosci 7:855-63 (2007).
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Macromol 8:105-7 (1986).
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absolute molecular weight and the degree of quaternisation of N-trimethyl chitosan chloride.
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15. Polnok A, Borchard G, Verhoef JC, Sarisuta N, Junginger HE. Influence of methylation
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16. Curti E, De Britto D, Campana-Filho SP. Methylation of chitosan with iodomethane: effect of
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18. Hamman JH, Stander M, Kotze AF. Effect of the degree of quaternisation of N-trimethyl
chitosan chloride on absorption enhancement: in vivo evaluation in rat nasal epithelia. Int J
Pharm 232:235-42 (2002).
44

Synthesis and Characterization of O-methyl Free TMC
19. Boonyo W, Junginger HE, Waranuch N, Polnok A, Pitaksuteepong T. Chitosan and
Trimethyl chitosan chloride (TMC) as adjuvants for inducing immune responses to ovalbumin
in mice following nasal administration. J Control Release 121:168-75 (2007).
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chitosan. Carbohydr Res 333:1-6 (2001).
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chitosan, N-substituted chitosan and quaternized chitosan. Carbohydr Res 342:1329-32 (2007).
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(2001).
45

CHAPTER 3
INFLUENCE OF THE DEGREE OF
ACETYLATION ON THE ENZYMATIC
DEGRADATION AND IN VITRO BIOLOGICAL
PROPERTIES OF TRIMETHYLATED
CHITOSANS
Rolf J. Verheul, Maryam Amidi, Mies van Steenbergen,
Elly van Riet, Wim Jiskoot, Wim E. Hennink.
Biomaterials 2009, 30, 3129-3135

Chapter 3
Abstract
Chitosan derivatives such as N,N,N-trimethylated chitosan (TMC) are currently being
investigated for the delivery of drugs, vaccines and genes. However, the influence of the extent
of N-acetylation of these polymers on their enzymatic degradability and biological properties
is unknown. In this study, TMCs with a degree of acetylation (DAc) ranging from 11 to 55%
were synthesized by using a three-step method. First, chitosan was partially re-acetylated
using acetic anhydride followed by quantitative dimethylation using formaldehyde and sodium
borohydride. Then, in presence of an excess amount of iodomethane, TMC was synthesized.
The TMCs obtained by this method showed neither detectable O-methylation nor loss in acetyl
groups ( 1 H-NMR) and a slight increase in molecular weight (GPC) with increasing degree of
substitution, implying that no chain scission occurred during synthesis. The extent of
lysozyme-catalyzed degradation of TMC, and that of its precursors chitosan and dimethyl
chitosan, was highly dependent on the DAc and polymers with the highest DAc showed the
largest decrease in molecular weight. On Caco-2 cells, TMCs with a high DAc (~50%), a DQ of
around 44% and with or without O-methylated groups, were not able to open tight junctions in
the trans-epithelial electrical resistance (TEER) assay, in contrast with TMCs (both O-
methylated and O-methyl free; concentration 2.5 mg/ml) with a similar DQ but a lower DAc
which were able to reduce the TEER with 30 and 70%, respectively. Additionally, TMCs with a
high DAc (~50%) demonstrated no cell toxicity (MTT, LDH release) up to a concentration of 10
mg/ml.
48

Influence of Degree of Acetylation on Degradation and Properties of TMC
Introduction
Chitosan is a polysaccharide consisting of β1→4-D-glucosamine and β1→4 N-acetyl D-
glucosamine units and is obtained from the natural polymer chitin (poly β1→4 N-acetyl D-
glucosamine) by partial deacetylation. Chitosan has been under investigation for various
biomedical and pharmaceutical applications due to its biocompatibility, low toxicity and its
muco-adhesive properties [1-4]. It has been reported that chitosan can be degraded in human
tissues by several enzymes (e.g. lysozyme, chitinase), by acid hydrolysis and by oxidativereductive
depolymerization (ORD) reactions [5, 6]. Nordtveit et al. demonstrated that the ORD
of chitosan driven by hydrogen peroxide is independent of the amount of residual N-acetylated
units [7]. However, in vitro studies on the degradation of chitosan by acid hydrolysis [8] and by
enzymes [6, 7, 9-14] revealed a major dependency on the degree of acetylation (DAc).
Completely de-acetylated chitosan shows very limited degradation by (enzyme catalyzed)
hydrolysis while the extent of degradation and degradation kinetics increase with the extent of
acetylation. Lysozyme is widely present in human body fluids (e.g. serum, saliva, tears) and is
actively secreted by macrophages and neutrophils [15], and consequently the enzymatic
degradation of chitosan by lysozyme has been studied in depth [7, 9-13, 16, 17]. Temperature,
pH and ionic strength have been found to influence degradation kinetics [10]. Additionally, in
vivo experiments on the degradability of chitosan have shown that the DAc also plays a key
role in the depolymerization of chitosan in living animals [13, 17]. Furthermore, uptake-,
toxicity- and cell transfection studies with chitosan suggest that the degree of acetylation has
an important influence on biological polymer characteristics as well [18-20]. Finally, partially
acetylated chitosan has been found to have better adjuvant properties for macrophage
stimulation compared to chitosan with a low amount of residual acetylated groups or chitin
[21-23], and recently, it was reported that N-acetylated glucosamines can bind to C-type lectin
receptors on denditric cells thereby working as an adjuvant [24].
In contrast to chitosan, N,N,N,-trimethylated chitosan (TMC), a partially quaternized
derivative of chitosan, is water-soluble at neutral pH. TMC has been widely studied in the
biomedical field as drug, vaccine and gene delivery vehicle [25-31]. It has been shown in
several in vitro and in vivo models that TMC has limited toxicity, possesses muco-adhesive
properties and can increase the uptake of small drug molecules as well as proteins via various
mucosal routes [25-27, 29, 30, 32-35]. Several investigators have studied the optimal degree of
quaternization (DQ) of TMC for mucosal transport and gene delivery [26, 33, 36, 37]. However,
molecular weight [38, 39] and, as demonstrated in chapter 2, O-methylation [40] also have a
major impact on the physical and biological characteristics of TMC. Besides its solubility at pH
49

Chapter 3
7, another often suggested potentially beneficial characteristic of TMC is its biodegradability.
These biodegradability claims, however, are based on degradation studies performed on
chitosan, and no investigations on the enzymatic degradability of TMC have been carried out so
far. The aim of this paper was to investigate the lysozyme-catalyzed degradability of TMCs
with different DAcs and with or without O-methylated groups, and to evaluate these polymers
for their physico-chemical properties, cytotoxicity and ability to open tight junctions.
Materials and Methods
Materials. Chitosan with a residual degree of acetylation of 17% (determined with 1 H-NMR
as described in below) and a number average molecular weight (M n) and weight average
molecular weight (M w) of 28 and 43 kDa, (determined with GPC-TD as described below),
respectively, was purchased from Primex (Siglufjodur, Iceland). Hen-egg white lysozyme
(41800 units per mg solid), acetic anhydride, sodium borohydride, formic acid, formaldehyde
37% (stabilized with methanol), DCl 35% w/w in D 2O, sodium azide, deuterium oxide, sodium
acetate, acetic acid (anhydrous), sodium hydroxide and hydrochloric acid were obtained from
Sigma-Aldrich Chemical Co. Dulbecco’s Modified Eagle’s Medium (DMEM), Hank’s balanced salt
solution (HBSS) and fetal calf serum (FCS) were obtained from Invitrogen (Breda, The
Netherlands). Sodium dodecyl sulfate (SDS) and Sicapent were ordered from Merck
(Darmstadt, Germany). Iodomethane 99% stabilized with copper was obtained from Acros
Organics (Geel, Belgium). All other chemicals used were of analytical grade.
Acetylation of chitosan. Chitosan (degree of acetylation 17%) was used as obtained and
acetylation was carried out according to a previously described method [19]. Briefly, chitosan
(10 g) was dissolved in 1% acetic acid (500 ml). Then, 500 ml methanol and 2.7 ml acetic
anhydride were added. The resulting mixture was stirred overnight at room temperature in a
roundbottom flask. Next, the reacetylated chitosan was precipitated by dropping the reaction
mixture into an aqueous solution of 1 M NaOH, filtrated using a glass filter and washed
extensively with methanol. Then, the precipitate was dissolved in an aqueous acidic solution
(pH 4, adjusted with 1 M HCl) and dialyzed against deionized water for 3 days changing buffer
twice-daily. Finally, the solution was filtered through a 0.8 µm filter and the polymer was
collected after freeze-drying.
50

Influence of Degree of Acetylation on Degradation and Properties of TMC
Synthesis of dimethylated chitosan. Dimethylated chitosan (DMC) with a degree of
acetylation of 17% was synthesized as described previously in chapter 2 [40]. In short,
chitosan (5 g) was dissolved in a mixture of 90 ml deionized water and 15 ml formic acid.
Subsequently, 20 ml of 37% formaldehyde was added, and the solution was heated to 70 °C
and stirred under refluxing for 2 days. The slightly yellow, viscous solution was evaporated
under reduced pressure and the pH was adjusted to 8 using 1 M NaOH resulting in the
formation of a gel, which was extensively washed with deionized water to remove impurities.
Then, the DMC was dissolved in deionized water at pH 4 (adjusted with 1 M HCl), filtered over
a glass filter and dialyzed against deionized water for three days (changing buffer twice-daily).
Finally, the product was filtered through a 0.8 µm filter and collected after freeze-drying.
GPC analysis revealed that under these conditions, some chain scission occurred with
chitosan with a DAc of 55% and therefore less vigorous reaction conditions were applied to
obtain dimethylated chitosan with a high degree of acetylation. Here, sodium borohydride
instead of formic acid was used as a reductor to allow the reaction to take place at room
temperature [41, 42]. In detail, chitosan (1 g) with a degree of acetylation of 55% was
dissolved in 50 ml of 2% acetic acid (v/v). Next, 10 ml of 37% formaldehyde solution was
added, the pH was adjusted to 5 with 1M NaOH and the mixture was stirred for 2 hours at
room temperature. Subsequently, sodium borohydride (2.5 g) was added in portions of 250 mg
over a period of 24 hours, adjusting the pH to 5 with 1M HCl after each addition. After
precipitation with acetone, the product was washed extensively with acetone and dried
overnight.
Synthesis of O-methyl free trimethylated chitosan. DMC with different degrees of
acetylation synthesized from chitosan with a DAc of 55% or 17% as described above, were
reacted with iodomethane to yield trimethylated chitosan (TMC). To prevent O-methylation,
the reaction of DMC with iodomethane was done in NMP, without the addition of a base
catalyst. TMC with a degree of acetylation of 17% and a degree of quaternization of around
45% was obtained by the method described previously [40]. TMC with a degree of acetylation
of 55% and a DQ of 45% was obtained with a slightly modified method. In detail, DMC (500
mg) with a degree of acetylation of 55% was dissolved in 80 ml deionized water and the pH
was adjusted to 11 with a 1 M solution of NaOH, resulting in gel formation. Then, the gel was
washed with water followed by acetone. To remove residual solvents, the DMC was dried
under vacuum for 4 hours. Next, DMC was suspended in 125 ml NMP followed by the addition
of 8 ml iodomethane. The dispersion was stirred at 40°C for 50 hours and subsequently
51

Chapter 3
dropped into 400 ml of an ethanol/diethyl ether mixture (50/50) to precipitate the formed
TMC, which was collected by centrifugation and subsequently extensively washed with diethyl
ether. After drying overnight at room temperature, the obtained TMC was dissolved in 100 ml
of an aqueous 10% NaCl solution for ion-exchange. Finally, the TMC was dialyzed against
deionized water for 3 days changing buffer twice daily, filtered through a 0.8 µm filter and
collected after freeze-drying.
Synthesis of O-methylated TMC from chitosan. O-methylated TMC with a DQ of about 45%
was synthesized by methylation of chitosan with iodomethane in a mixture of an aqueous
solution of NaOH and NMP essentially as described previously [43]. In detail, chitosan (500
mg) with a degree of acetylation of 17 or 55% and sodium iodide (1.2 g) were dispersed in a
mixture of 40 ml of NMP and 5 ml of 15% w/v aqueous NaOH solution. Subsequently, the
mixture was heated to 60°C and after stirring for 20 min, 4 ml of methyl iodide was added and
the reaction mixture was refluxed for 60 min. Then, 2 ml of 15% NaOH solution and 1.5 ml of
iodomethane were added and the solution was stirred for 60 minutes. Next, the reaction
mixture was dropped into 200 ml of a mixture of diethyl ether and ethanol (50/50) to
precipitate the O-methylated TMC, which was subsequently washed extensively with diethyl
ether. Finally, the product was dissolved in 50 ml aqueous 10% w/v NaCl solution, put on a
shaker for 18 hours for ion-exchange and the obtained solution was dialyzed at room
temperature against deionized water for 3 days changing buffer twice daily, filtered through a
0.8 µm filter and freeze dried.
Determination of the degrees of acetylation, dimethylation, and quaternization. The
1H-NMR spectra of the various chitosans, DMC and TMCs were recorded with a Varian INOVA
500MHz NMR spectrometer (Varian Inc., Palo Alto, Ca, USA) at 80°C in D 2O. For the DMC and
the chitosans addition of DCl (35% w/w in D 2O) was needed to dissolve the polymers.
The degree of acetylation of the chitosans, DMC and TMCs was calculated as described
previously [44]:
DAc = [[CH 3]/[H2-H6] x 1/2] x 100
Here, [CH 3] is the integral of the three hydrogens of the acetyl groups at 2.0 ppm and [H2-H6]
is the integral corresponding the six H-2 to H-6 protons between 3.9 and 3.0 ppm.
52

Influence of Degree of Acetylation on Degradation and Properties of TMC
The degree of dimethylation of the DMC was calculated as follows:
DDM = [(CH 3) 2]/[H2-H6] x 100
Here, [(CH 3) 2] is the integral of the hydrogens of the dimethyl amino groups at 2.9 ppm and
[H2-H6] is the integral corresponding the H-2 to H-6 protons between 3.9 and 3.0 ppm.
The DQ and degree of dimethylation (DM) of the TMCs were calculated as previously described
[25, 43, 45].
DQ = [[(CH 3) 3]/[H] × 1/9] × 100
DM = [[(CH 3) 2]/[H] × 1/6] × 100
Here, [(CH 3) 3] and [(CH 3) 2] are the integrals of the hydrogens of the trimethylated amino
groups at 3.3 ppm and the dimethylated amino groups at 2.9 ppm, respectively. [H] is the
integral of the H-1 peaks between 4.7 and 5.7 ppm; the signal related to the hydrogen atoms
bound to the C-1’s of TMC.
Enzymatic degradation of various TMCs. Table 1 gives an overview of the different
chitosans and TMCs used for evaluation of the lysozyme-catalyzed degradation. The different
polymers were placed overnight in a vacuum oven at 40 o C in presence of Sicapent to remove
residual water. Next, the samples were dissolved in 2% acetic acid solution for at least 18
hours. Then, lysozyme dissolved in H 2O, and 1 M NaCl and 1 M NaOH were added to the
polymer solutions to obtain a lysozyme concentration of 38 µg/ml, polymer concentrations of
5 mg/ml, 150 mM NaCl, 1% acetic acid and a pH of 4.5.
The degradation of the different TMC polymers was also studied at physiological pH in a
phosphate-buffered salt (PBS) buffer (8.2 g/l NaCl, 3.1 g/l Na 2HPO 4 12 H 2O, 0.3 g/l NaH 2PO 4 2
H 2O, pH 7.4). In detail, the different polymers were placed overnight in a vacuum oven at 40 o C
in presence of Sicapent to remove residual water. Next, the samples were dissolved in PBS with
0.02% sodium azide for 18 hours. Then, lysozyme dissolved in PBS (also containing 0.02%
sodium azide) was added to the polymer solutions to obtain a lysozyme concentration of 38
µg/ml, polymer concentrations of 5 mg/ml and a pH of 7.4. Additionally, the influence of
lysozyme concentration on polymer degradation was studied. To this end, TMC-RA was
dissolved in PBS with 0.02% sodium azide as described above and incubated with varying
concentrations of lysozyme (9 µg/ml to 38 µg/ml) in PBS with 0.02% sodium azide.
53

Chapter 3
Polymer solutions were incubated at 37 °C and samples were taken at various time points
during 6 days. Polymer molecular weights were determined with a GPC-triple detection
described below. Polymers not exposed to lysozyme were used as controls.
Determination of M n and M w of the different polymers. The number average weight (M n)
and weight average weight (M w) of chitosan and the various TMCs were determined by gel
permeation chromatography (GPC) on a Viscotek-triple detection system using a Shodex
OHPak SB-806 column (15 cm) and 0.3 M sodium acetate pH 4.4 (adjusted with acetic acid) as
running buffer [46]. Data from the laser photometer (λ = 670 nm) (right (90 0 ) and low (7 0 )
angle light scattering), refractive index detector and viscosity detector were integrated using
the provided Omnisec-software to calculate the M n, M w, dn/dc and the intrinsic viscosity ([η])
of the different samples. Pullulan (M n = 102 kDa, M w = 106 kDa) obtained from Viscotek
Benelux (Oss, the Netherlands) was used for calibration.
Transepithelial electrical resistance (TEER) measurements. Caco-2 cells were seeded at
a density of 2x10 5 cells per well on 12-transwell plates with a microporous membrane. The
cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) for 10 days until a
confluent cell layer was formed. The medium was replaced by Hank’s Balanced Salt Solution
(HBSS) at the basolateral side 10 minutes before the start of the experiments. Then, 0.5 ml
solution of TMC (with various DQs, DAcs and with or without O-methylation, dissolved (2.5
mg/ml) in HBSS, pH adjusted to 7 with 0.1 M NaOH) was applied at the apical site of the cell
monolayers. SDS (10 mg/ml) was used as positive control and HBSS as reference. The
resistance measured of the membrane without cells was used as blank. The TEER of the Caco-2
cells at certain time points (0, 15, 30, 45, 60 and 90 min.) after addition of the stimuli was
measured with a Millicell-ERS (Millipore, Billerica, USA) measuring device. After 90 minutes
the cells were washed with HBSS and incubation of the cells was continued in DMEM for 24
hours at 37°C, CO 2 5% to determine the recovery of the TEER [25, 47].
MTT cell toxicity assay. Caco-2 cells were seeded in a 96-well plate at a density of 4x10 4
cells per well and incubated for 2 days at 37°C, CO 2 5% in culture medium (DMEM, high
glucose, 10% FCS, L-glutamine, pyruvate, non essential amino acids). The medium was
removed and the cells were incubated for 2.5 hours with 100 µl TMC solutions in HBSS (TMC
concentrations were 0.1, 1 and 10 mg/ml, pH set at 7 with 0.1 M NaOH). SDS (10 mg/ml) was
used as positive control and HBSS as reference for 100% cell viability. Thereafter, the HBSS
54

Influence of Degree of Acetylation on Degradation and Properties of TMC
was removed and the cells were washed with phosphate buffered saline. One hundred µl of a
freshly prepared solution of 0.5 mg/ml MTT in DMEM (without any additions), was added and
the cells were incubated for 3 hours at 37°C and 5% CO 2. Subsequently, the wells were
emptied, 100 µl of DMSO was used to dissolve the formed formazan crystals and the
absorbance was read at 595 nm [48].
LDH assay. Caco-2 cells were seeded in a 96-well plate at a density of 4x10 4 cells per well
and incubated for 2 days at 37°C, CO 2 5% in culture medium (see section above for
composition). The medium was removed and the cells were washed with HBSS and incubated
for 2.5 hours with 100 µl TMC solutions in HBSS (TMC concentrations were 0.1, 1 and 10
mg/ml, pH set at 7 with 0.1 M NaOH). After incubation, the concentration of LDH present in the
supernatant of the samples was determined with the Cytotoxicity Detection Kit-Plus (Roche
Diagnostics, Mannheim, Germany) by measuring absorbance at 490 nm with 650 nm as a
reference wavelength. A calibration curve was made with the lysis buffer provided by the
manufacturer, setting the LDH concentration measured with the undiluted lysis buffer at 100%
LDH release. HBSS was used as a negative control.
Results and discussion
Synthesis and characterization of different polymers. To investigate the influence of the
degree of acetylation on the biological properties and enzymatic degradability of trimethylated
chitosan (TMC), chitosan (CS, DAc 17%) was partially re-acetylated using acetic anhydride to a
DA of 55% (CS-RA, DAc 55%) as determined by 1 H-NMR analysis. Both CS and CS-RA were
subsequently trimethylated to a degree of about 45% according to the method described by
Sieval [43]. 1 H-NMR analysis (Table 1) showed that this synthesis method also leads to
considerable O-methylation and loss of N-acetylated units, likely due to the alkaline reaction
conditions.
Besides the frequently used method of Sieval et al, CS and CS-RA were also trimethylated to a
degree of 45% via the two-step synthesis route described recently by Verheul et al. [40] (with
some modifications for the synthesis of TMC-RA) which avoids O-methylation and loss of N-
acetylated units (Table 1). Chitosan with a DAc of 17% was quantitatively dimethylated with
formaldehyde using formic acid as reducing agent. However, using these reaction conditions to
dimethylate chitosan with a DAc of 55%, which is more susceptible to acidic hydrolysis than
55

Chapter 3
chitosan with a DAc of 17% [8], resulted in the reduction of molecular weights (data not
shown). When sodium borohydride was used as reducing agent, relatively mild reaction
conditions (pH 4, room temperature) could be applied to quantitively dimethylate chitosan.
Table 1. Characteristics of the chitosans and TMCs. Degree of acetylation (DAc), quaternization (DQ),
dimethylation (DM), O-methylation on C-6 (DOM-6) and on C-3 (DOM-3) of TMCs as determined by 1 H-
NMR analysis. M n, M w and dn/dc were determined by GPC-triple detection.
DAc DQ DM DOM-6 DOM-3 Mn Mw dn/dc
(kDa) (kDa)
CS 17% - - - - 28 43 0.18
TMC 17% 43% 40% - - 39 64 0.16
TMC-OM 11% 45% 44% 25% 16% 35 48 0.16
CS-RA 55% - - - - 35 65 0.16
DMC-RA 55% - 45% - - 42 71 0.16
TMC-RA 55% 44% 1% - - 43 79 0.16
TMC-RA-OM 49% 46% 5% 77% 57% 40 63 0.15
Table 1 shows that particularly in case of the re-acetylated chitosan, these milder reaction
conditions prevented polymer chain scission; the molecular weights of CS, CS-RA, DMC-RA and
TMC-RA slightly increased with increasing extent of substitutions (TMC-RA>DMC-RA>CS-
RA>CS). As reported previously, TMC synthesized according to the method of Sieval et al.
resulted in polymers with a lower molecular weight than the polymers obtained via the
method without the use of a strong base [40]. Likely, the strong basic reaction conditions
caused some chain scission during synthesis [40, 49]. Importantly, the mild two-step synthetic
method described in this paper yields TMC with varying DQs while O-methylation, polymer
chain scission and the hydrolysis of N-acetylated groups do not occur. Previous studies showed
that changes in TMC molecular weight [38, 39] and the presence of O-methyl groups [40]
influence polymer characteristics, such as cytotoxicity and the ability to open tight junctions.
GPC-TD analysis showed that re-acetylated chitosan and TMCs contained some high molecular
weight material (about 3% of the injected amount) which can be likely ascribed to aggregation
[50-52]. However, this high-molecular-weight material had no impact on the degradation
studies due to its relatively small contribution and it was easily separated on the GPC column
from the free polymer.
Enzymatic degradation of various chitosans and TMCs. Figure 1 shows the decrease of
the M n of TMC-RA in presence of various concentrations of hen egg-white lysozyme at pH 7.4 at
37 °C. Clearly, the degradation rate increases with lysozyme concentration while without
lysozyme almost no decrease in molecular weight was observed. The enzymatic degradation
essentially occurred in the first 24 hours and after this time period hardly any decrease of the
56

Influence of Degree of Acetylation on Degradation and Properties of TMC
M n of TMC-RA was detected. Addition of fresh lysozyme after 70 hours did not result in a
further drop of molecular weight (results not shown), implying that arrest of the polymer
degradation is not due to inactivation of lysozyme but that lysozyme is not able to catalyze the
hydrolysis of the remaining glyosidic bonds. As expected, the molecular weight obtained after
prolonged exposure to lysozyme was independent of the concentration of the enzyme. Overall,
in the presence of lysozyme, the M n of TMC-RA dropped from 43 kDa to about 11 kDa
corresponding on average to approximately three cuts per molecule. Figure 2 shows the
changes in M n, M w and intrinsic viscosity ([η]) of TMC-RA in presence of lysozyme (38 µg/ml)
at pH 7.4 at 37 °C as obtained with the GPC-triple detection method. As expected, both the M w
and the [η] followed a similar pattern as the M n of TMC-RA. This was observed for all analyzed
polymers (data not shown).
4.0×10 4 TMC-RA 38 μg/ml lyso
Mn (Da)
3.0×10 4
2.0×10 4
TMC-RA 0μg/ml lyso
TMC-RA 9 μg/ml lyso
TMC-RA 19 μg/ml lyso
1.0×10 4
0
0 5 10
100 200
time (h)
Figure 1. Degradation of TMC-RA by lysozyme (various concentrations) at pH 7.4 and 37°C. Error bars
represent the standard deviation of three measurements.
The lysozyme catalyzed enzymatic degradation of different chitosans and TMCs was
compared as shown in Figure 3. Since CS, CS-RA and DMC-RA are not soluble at physiological
pH, degradation was studied at pH 4.5 and at 37 °C in presence of 38 µg/ml lysozyme. This
figure clearly demonstrates that the extent of lysozyme-catalyzed degradation of the
investigated polymers is highly dependent on the degree of acetylation. Polymers with a DAc ≤
17% (CS, TMC and TMC-OM) are less susceptible to lysozyme-catalyzed degradation than the
re-acetylated polymers with a DAc ≥49%. Although the changes in molecular weights are less
57

Chapter 3
prominent, it is obvious that TMC-OM, with the lowest DAc (DAc 11%), was also to the lowest
extent degraded by lysozyme after 6 days (decrease in M n from 35 kDa to 30 kDa). In contrast,
the M n of TMC-RA with a DAc of 55% dropped from 43 kDa to 10 kDa under the same
conditions. Re-acetylated CS-RA (DAc 55%), in agreement with others [6, 7, 10, 14, 17, 53], was
much more susceptible to lysozyme-catalyzed degradation than CS (DAc 17%), and their M n’s
decreased from 35 kDa to 7 kDa and from 28 kDa to 20 kDa, respectively.
0.4
Mn or Mw (Da)
8.0×10 4 100 200
6.0×10 4
4.0×10 4
2.0×10 4
[η]
Mn
Mw
0.3
0.2
0.1
[η] (dl/g)
0
0 5 10
time (h)
0.0
Figure 2. Changes in the M n, M w and intrinsic viscosity of TMC-RA by lysozyme (38 µg/ml) at pH 7.4 and
37°C. Error bars represent the standard deviation of three measurements.
Nordtveit et al. demonstrated that at least 3 to 4 acetylated units are required in the
hexameric binding pocket for lysozyme to allow enzymatic cleavage [7]. Since these domains
containing the required number of acetylated units are destroyed through cleavage of the
glycosidic bond [7, 16], degradation by lysozyme occurs till a minimum molecular weight
depending on the DAc of the chitosan [11]. As DMC-RA was degraded to practically the same
molecular weight as CS-RA (Figure 3), it can be concluded that methylation of the free NH 2
groups of chitosan did not affect its extent of lysozyme-catalyzed degradation. TMC-RA was
cleaved up to a slightly higher M n than CS-RA (10 kDa vs. 7 kDa, respectively), however, this
can be explained by its higher degree of substitution due to the quaternization of the free
amines thereby increasing the weight per glucosamine-residue. Therefore, it can be stated that
quaternized chitosan synthesized according to the two-step method presented here is
58

Influence of Degree of Acetylation on Degradation and Properties of TMC
degraded by lysozyme to the same extent as the original chitosan. In contrast, chitosan
quaternized using iodomethane and a strong base (TMC-RA-OM) was cleaved less than the
original chitosan (CS-RA) or the O-methyl free TMC-RA, likely due to loss of N-acetylated units
during synthesis. To explain, the M n of TMC-RA-OM with a DAc of 49% decreased from 40 kDa
to 18 kDa. Although TMC-RA-OM has a modestly higher weight per glucosamine unit than CS-
RA, the difference in final M n of 7 kDa of CS-RA and 18 kDa of TMC-RA-OM has to be attributed
to less chain scission by lysozyme for TMC-RA-OM due to its lower DAc as compared to the
other re-acetylated polymers. Interestingly, TMC-RA and, especially DMC-RA, were faster
cleaved by lysozyme than CS-RA implying that enzyme-affinity and/or maximum substrate
conversion rates may be enhanced by the methylation of the free amines.
Mn (Da)
4.0×10 4 CS
CS-RA
DMC-RA
3.0×10 4
TMC-RA
TMC-RA-OM
TMC
2.0×10 4
TMC-OM
1.0×10 4
0
0 5 10
100 200
time (h)
Figure 3. Degradation of various chitosans and TMCs by lysozyme (38 µg/ml) at pH 4.5 and 37°C. Error
bars represent the standard deviation of three measurements.
To investigate the lysozyme-catalyzed degradation of quaternized chitosans under
physiological conditions, changes in molecular weight of TMCs in the presence of lysozyme
were studied in PBS at pH 7.4. The extent of lysozyme-catalyzed degradation of quaternized
chitosans was comparable to the degradation observed at pH 4.5 (Figure 4). In detail, at pH 7.4
59

Chapter 3
the polymer with the lowest DAc of 11% (TMC-OM) showed the smallest decrease in molecular
weight (M n from 34 to 29 kDa) while the TMC with a DAc of 55% (TMC-RA) was readily
degraded (M n from 43 to 11 kDa). At pH 4.5 the lysozyme-catalyzed degradation of the reacetylated
TMC-RA and TMC-RA-OM was slightly faster than at pH 7.4 likely due to higher
enzyme activity at lower pH [9]. Overall, our data show that the degradation of quaternized
chitosans by lysozyme both in terms of kinetics and in final molecular weight of the
degradation products is hardly affected by the pH in the range studied (pH 4.5-7.4). This was
not observed for the degradation of chitosan, where the degradation rate at pH 7.4 is
substantially slower than that in acidic environment [14]. At a pH above 6.5 chitosans are
insoluble and degradation by enzymes decreases due to limited accessibility of the binding
sites for lysozyme. Since TMCs are soluble at physiological pH, solubility does not limit the
lysozyme-catalyzed degradation.
4.0×10 4 TMC
Mn (Da)
3.0×10 4
2.0×10 4
TMC-RA
TMC-OM
TMC-RA-OM
1.0×10 4
0
0 5 10
100 200
time (h)
Figure 4. Degradation of various TMCs by lysozyme (38 µg/ml) at pH 7.4 and 37°C. Error bars represent
the standard deviation of three measurements.
The degradability of TMCs will have important consequences for its pharmaceutical
applications. Onishi and co-workers demonstrated in mice that, after intraperitoneally
injection, chitosan with a DAc of 50% is readily degraded to a molecular weight of less than 10
60

Influence of Degree of Acetylation on Degradation and Properties of TMC
kDa and after 24 hours the full dose was excreted by the kidneys [54]. Since glomerular
filtration by kidneys is size-dependent, our results imply that the degradation products of the
re-acetylated polymers, due to their lower final molecular weight, will be more easily removed
from the body than the chitosans with a low DAc. Also, TMC-carrier systems may be tailored
for its degradation characteristics; ideally, the carrier molecule will degrade after delivery at
the target site thereby releasing its pharmaceutically active compounds.
In conclusion, our data demonstrate that the lysozyme-catalyzed degradation of TMCs is, like
that of chitosan, facilitated for polymers with a high DAc. However, the degradation of TMC is
similar at pH 4.5 and 7.4.
Evaluation of various TMCs on TEER, MTT and LDH assays. TMC is capable to open tight
junctions of epithelial cells and the highest activity was observed with DQs of around 40-50%
[29, 35, 55]. Recently, it was shown that O-methylation substantially reduces the activity of
TMC on transepithelial electrical resistance (TEER) [40]. In this study, TMCs were tested with
only minor variations in DQ (see Table 1), so the charge ratio and the cationic character of
these polymers were considered similar. However, the polymers differed in DAc and in extent
of O-methylation.
TEER (% HBSS)
120
100
80
60
40
20
SDS 1%
TMC
TMC-OM
TMC-RA
TMC-RA-OM
0
0 15 30 45 60 75 90
time (min.)
1140
Figure 5. Effect of TMCs with various DAcs and with or without O-methylated units on the TEER of
Caco-2 cells at a TMC concentration of 2.5 mg/ml. SDS 10 mg/ml was used as a positive control. Error
bars represent the standard deviation of six measurements.
Figure 5 shows the effect of various TMCs on the TEER of a Caco-2 monolayer. In line with
previous data [40], O-methyl free TMC with a DQ of 43% and a DAc of 17% demonstrated the
61

Chapter 3
largest decrease in TEER (about 70% reduction), while the partially O-methylated TMC with a
DQ of 42% and a DAc of 11% reduced the TEER with about 30% compared to the TEER
observed with HBSS. Importantly, the Caco-2 monolayer partially recovered after removal of
these polymers implying that no permanent toxicity was induced on the cells. Fascinatingly,
TMC polymers with a similar DQ but with higher DAcs had no effect on the TEER, indicating
that these polymers, although positively charged, were unable to open tight junctions. Also, the
results of the MTT and LDH assays (Figures 6 and 7) obtained with TMC and TMC-OM were as
expected [40], showing a higher toxicity for O-methyl free TMC. Additionally, even at a
concentration of 10 mg/ml both TMC-RA and TMC-RA-OM clearly show neither a decrease in
cell viability nor LDH release. Studies with chitosan have shown that an increase in DAc lead to
lower penetration through a Caco-2 monolayer [20], a decrease in cytotoxicity [18, 20] and a
reduction in DNA transfection [19]. However, these experiments were carried out in slightly
acidic environment to solubilize the chitosan polymer. In acidic environment the primary
amines will be (partially) protonated and thus become positively charged. A higher DAc will
reduce the number of amines available for protonation thereby decreasing the charge density
of chitosan. Therefore, with chitosan, it was not possible to study the effect of DAc on polymer
characteristics without altering the charge density of the polymer.
cell viability (%)
125
100
75
50
25
TMC
TMC-OM
TMC-RA
TMC-RA-OM
0
0.1 1 10
conc. (mg/ml)
Figure 6. Effect of TMCs with various DAcs and with or without O-methylated units on the viability of
Caco-2 cells (MTT assay) at different concentrations. Error bars represent the standard deviation of six
measurements.
62

Influence of Degree of Acetylation on Degradation and Properties of TMC
LDH release (%)
70
60
50
40
30
20
10
HBSS
TMC-RA-OM
TMC-RA
TMC-OM
TMC
0
0.1 1 10
conc. (mg/ml)
Figure 7. Effect of TMCs with various DAcs and with or without O-methylated units on the viability of
Caco-2 cells (LDH release) at different concentrations. Error bars represent the standard deviation of six
measurements.
Our studies were done at physiological pH and with TMCs with a similar DQ (and thus charge
density) allowing evaluation of the influence of the DAc without changing other polymer
characteristics. Consequently the remarkable decrease in toxicity and ability to open tight
junctions when increasing the DAc has to be contributed to intra- and/or intermolecular
changes induced by the N-acetylated units. Possibly, the N-acetylated glucosamine parts of the
TMC can form hydrophobic domains, thereby shielding the positively charged quaternized
parts of the macromolecule. However, further studies are needed to provide more insight into
the macromolecular changes that occur when increasing the DAc.
Conclusion
This paper presents a method to synthesize TMC with a high DAc without introducing other
alterations of the polymer such as O-methylation, chain scission or loss of N-acetylation. The
enzymatic degradation of TMC by lysozyme was essentially identical to the degradation of
chitosan (thus highly dependent on the degree of acetylation) however, in contrast to chitosan,
this degradation was pH independent. TMCs with a DAc of ~50%, a DQ of around 44% and
with or without O-methylation, were not able to open tight junctions of Caco-2 cells, in contrast
to TMCs (O-methylated or O-methyl free) with a similar DQ but a lower DAc. Additionally,
TMCs with a high DAc (~50%) showed no cell toxicity up to a concentration of 10 mg/ml. In
conclusion, the degree of N-acetylation has great impact on the biological characteristics of
63

Chapter 3
TMC and future research will be done to determine the optimal polymer structure for the
various potential pharmaceutical applications of TMC.
Acknowledgement. This research was performed under the framework of TI Pharma
project number D5-106-1; Vaccine delivery: alternatives for conventional multiple injection
vaccines. The authors thank Sara Riera Rivas for her contributions to the project.
64

Influence of Degree of Acetylation on Degradation and Properties of TMC
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67

CHAPTER 4
RELATIONSHIP BETWEEN STRUCTURE AND
ADJUVANTICITY OF N,N,N-TRIMETHYL
CHITOSAN (TMC) STRUCTURAL VARIANTS
IN A NASAL INFLUENZA VACCINE
Rolf J. Verheul*, Niels Hagenaars*, Imke Mooren, Pascal H.J.L.F. de Jong,
Enrico Mastrobattista, Harrie L. Glansbeek, Jacco G.M. Heldens
Han van den Bosch, Wim E. Hennink, Wim Jiskoot.
*authors contributed equally
Journal of Controlled Release 2009, 140, 126-133

Chapter 4
Abstract
The aim of this study was to assess the influence of structural properties of N,N,N-trimethyl
chitosan (TMC) on its adjuvanticity. Therefore, TMCs with varying degrees of quaternization
(DQ, 22-86%), O-methylation (DOM, 0-76%) and acetylation (DAc 9-54%) were formulated
with whole inactivated influenza virus (WIV). The formulations were characterized
physicochemically and evaluated for their immunogenicity in an intranasal (i.n.)
vaccination/challenge study in mice.
Simple mixing of the TMCs with WIV at a 1:1 (w/w) ratio resulted in comparable positively
charged nanoparticles, indicating coating of WIV with TMC. The amount of free TMC in solution
was comparable for all TMC-WIV formulations. After i.n. immunization of mice with WIV and
TMC-WIV on day 0 and 21, all TMC-WIV formulations induced stronger total IgG, IgG1 and
IgG2a/c responses than WIV alone, except WIV formulated with reacetylated TMC with a DAc
of 54% and a DQ of 44% (TMC-RA44). No significant differences in antibody titers were
observed for TMCs that varied in DQ or DOM, indicating that these structural characteristics
play a minor role in their adjuvant properties. TMC with a DQ of 56% (TMC56) formulated
with WIV at a ratio of 5:1 (w/w) resulted in significantly lower IgG2a/c:IgG1 ratio’s compared
to TMC56 mixed in ratios of 0.2:1 and 1:1, implying a shift towards a Th2 type immune
response. Challenge of vaccinated mice with aerosolized virus demonstrated protection for all
TMC-WIV formulations with the exception of TMC-RA44-WIV.
In conclusion, formulating WIV with TMCs strongly enhances the immunogenicity and
induced protection after i.n. vaccination with WIV. The adjuvant properties of TMCs as i.n.
adjuvant are strongly decreased by reacetylation of TMC, whereas the DQ and DOM hardly
affect the adjuvanticity of TMC.
70

Relationship between Structure and Adjuvanticity of TMC
Introduction
Intranasal (i.n.) vaccination offers several advantages over the intramuscular (i.m.) route,
like simple, needle-free administration without the need for trained personnel, potentially less
adverse effects and the induction of local mucosal immune responses [1]. On the other hand,
vaccines administered via the i.n. route generally induce low systemic immune responses
when compared to i.m. administration likely due to mucociliary clearance and low antigen
uptake. Mucoadhesive polymers have been used to increase the immunogenicity of i.n.
vaccines by increasing nasal residence time and enhancing antigen presentation [1].
Chitosan, a polysaccharide that is obtained by deacetylation of the natural polymer chitin,
has mucoadhesive properties and showed promising results as an adjuvant in nasal vaccines
[2-5]. However, the unfavorable pH-dependent solubility and charge density led to the
synthesis of its quaternized derivative N,N,N-trimethyl chitosan (TMC) (Scheme 1), which is
well soluble in aqueous solution at neutral pH.
Scheme 1. General structure of TMC. Depending on the synthesis route TMCs can be varied in degree of
acetylation (see block ‘x’), quaternization (see block ‘y’), and O-methylation (see block ‘z’). The various
substitutions are randomly distributed throughout the polymer; O-methylation (block ‘z’) may also
occur on the quaternized and acetylated units (blocks ‘x’ and ‘y’).
TMC is traditionally synthesized by reaction of chitosan with excess iodomethane in strong
alkaline conditions with N-methyl-2-pyrrolidone (NMP) as solvent and the degree of
quaternization (DQ) can be varied by varying the number of reaction steps [6]. Besides N-
methylation this synthesis method also introduces substantial O-methylation on the hydroxyl
groups located at the C-3 and C-6 of the glucosamine unit. The degree of O-methylation (DOM)
increases with increasing DQ (up to 80-90%) [7, 8]. Recently, O-methyl free TMC was
synthesized using a novel two-step synthesis procedure, allowing good control of the DQ
without altering other structural properties. Both DQ and DOM were found to influence
toxicity and transepithelial electrical resistance (TEER), an indicator for opening of tight
71

Chapter 4
junctions, in a Caco-2 cell model (Chapter 2). A higher DQ leads to more toxicity and a stronger
TEER effect, with a maximum effect on TEER at a DQ above 60%. Furthermore, O-methyl free
TMC has a much stronger effect on TEER than O-methylated TMC (TMC-OM) and shows more
in vitro cell toxicity [8]. Another characteristic of TMCs is the degree of N-acetylation (DAc).
Partial reacetylation of TMC (from 17 to 54%) decreased the in vitro cell toxicity and effect on
TEER but increased the enzymatic degradability of TMC by lysozyme (Chapter 3) [9].
Little is known about the relationship between the structural characteristics and adjuvant
properties of TMCs in vivo. For TMC-OM solutions in i.n. vaccination with ovalbumin an
optimal DQ of 40% was reported although differences were small [10]. Previously, whole
inactivated influenza virus (WIV) vaccine was formulated with TMC-OM with a DQ of 15% or
37%. This resulted in positively charged nanoparticles with partially bound TMC-OM. These
particles had an intact viral ultrastructure. Strong, protective immune responses were induced
after i.n. vaccination [11]. No significant differences were observed between the two different
TMC-OMs. Most likely, TMC exerts its adjuvant effect by an improved antigen delivery, through
an increased nasal residence time and/or enhanced uptake through the epithelium and by
antigen presenting cells. Besides differences in DQ, the TMC-OMs used in these studies also
differed in DOM and, likely, polymer molecular weight. So, the individual contributions of DQ,
DAc and DOM on the adjuvant effect of TMC are unknown.
In the present study we investigated for i.n. administered WIV the adjuvant properties of O-
methyl free TMCs with varying DQs and reacetylated O-methyl free TMC in comparison to
conventional TMC-OMs with similar DQ. The TMC-WIV vaccines were physicochemically
characterized and the immunogenicity and protectivity of the vaccines were assessed in a
murine challenge model. Additionally, the influence of TMC:WIV ratio on the quality and
quantity of humoral immune responses was investigated.
Materials and Methods
Materials. Chitosan with a DAc of 17% (determined with 1 H-NMR as described in [9]) and a
number average molecular weight (M n) and weight average molecular weight (M w) of 28 and
43 kDa, as determined by gel permeation chromatography (GPC) as described in [8],
respectively, was purchased from Primex (Siglufjordur, Iceland). Acetic anhydride, sodium
borohydrate, formic acid, formaldehyde 37% (stabilized with methanol), deuterium oxide,
sodium acetate, acetic acid (anhydrous), sodium hydroxide and hydrochloric acid were
obtained from Sigma-Aldrich Chemical Co. Iodomethane 99% stabilized with copper was
72

Relationship between Structure and Adjuvanticity of TMC
obtained from Acros Organics (Geel, Belgium). Live, egg-grown, mouse adapted influenza
A/Puerto Rico/8/34 virus (A/PR/8/34) and purified, cell culture-grown (Madin-Darby Canine
Kidney (MDCK) cells), β-propiolacton (BPL)-inactivated A/PR/8/34, as well as polyclonal
rabbit anti-A/PR/8/34 serum were from Nobilon International BV, Boxmeer, The Netherlands.
PO-labeled goat anti mouse -IgG (H+L), -IgG1, -IgG2a/c and -IgA(Fc) were purchased from
Nordic Immunological Laboratories (Tilburg, The Netherlands). All other chemicals used were
of analytical grade.
Synthesis and characterization of methylated chitosans. N,N,N-Trimethylated chitosans
with varying DQ and DAc, and DOM were synthesized from chitosan as described previously
[8, 9]. Briefly, O-methyl free TMCs were made with a two-step method: first quantitative
dimethylation of the free amino-groups of chitosan with formaldehyde and formic acid was
carried out, followed by reaction of the dimethylated chitosan with an excess of iodomethane.
By varying the reaction time the DQ of the TMCs could be tailored [8]. TMC-OMs, with
substantial O-methylation of the hydroxyl groups on the C-3 and C-6 of the glucosamine units,
were synthesized according to the method of Sieval et al. [6, 8]. Here, chitosan was
trimethylated to various extents by reacting with iodomethane in the presence of a strong base
(NaOH) for several times depending on the desired DQ. Finally, to obtain TMC with a high
degree of acetylation, chitosan was first re-acetylated using acetic anhydride [9, 12]. Then, this
re-acetylated chitosan was quantitatively dimethylated with formaldehyde and sodium
borohydrate followed by complete trimethylation of the dimethylated amino groups with
iodomethane [9]. All synthesized TMCs were dissolved in an aqueous 10% w/v NaCl solution,
put on a shaker overnight for ion-exchange and the obtained solution was dialyzed at room
temperature against deionized water for 3 days changing water twice daily, filtered through a
0.8 µm filter and freeze dried.
The DQ, DAc and DOM of the hydroxyl groups on C-3 and C-6 (DOM-3 and DOM-6,
respectively) of the TMCs were determined with 1 H-NMR on a Varian INOVA 500MHz NMR
spectrometer (Varian Inc., Palo Alto, Ca, USA) at 80 °C in D 2O [9]. Furthermore, M n and M w of
the various TMCs were determined, as described previously [9, 13], by GPC on a Viscotek
system detecting refractive index, viscosity and light scattering. A Shodex OHPak SB-806
column (30 cm) was used with 0.3 M sodium acetate pH 4.4 (adjusted with acetic acid) as
running buffer. The structural characteristics of the synthesized TMCs are summarized in
Table 1.
73

Chapter 4
Preparation of TMC-WIV formulations. Purified, cell culture-derived, BPL-inactivated
A/PR/8/34 suspended in a 10 mM phosphate buffered saline solution (150 mM NaCl, pH 7.4)
(PBS) was concentrated by centrifugation at 22,000 x g for 30 min at 4 °C and resuspended in 5
mM HEPES buffer (pH 7.4). The WIV concentration is expressed as mg total protein/ml as
determined by DC protein assay (Bio-Rad, Hercules, CA, USA). The amount of hemagglutinin
(HA) was approximately 35 % of the total protein content, as determined previously [14]. The
TMC-WIV vaccines were prepared by adding equal volumes of TMC solution (in 5 mM HEPES,
pH 7.4) to a WIV dispersion (in 5 mM HEPES pH 7.4) at a 1:1 w/w ratio using a Gilson pipette
while gently mixing for 5 seconds. TMC-WIV was formulated at a final WIV concentration of
1.25 mg/ml, except for the samples used for dynamic light scattering (DLS) and zeta-potential
measurements, which were performed at lower concentrations (62.5 µg/ml) for optimal testconditions.
To study the immunogenicity of TMC-WIV at other ratios, TMC56 was also
formulated with WIV at ratios of 0.2:1 (TMC56-WIV(0.2:1))and 5:1(TMC56-WIV(5:1)), by
varying the TMC concentration added to WIV.
Dynamic light scattering and zeta-potential measurements. WIV and TMC-WIV
formulations were prepared at a final WIV concentration of 62.5 µg/ml in 5 mM HEPES buffer
pH 7.4. Particle size was measured by dynamic light scattering (DLS) using a Malvern ALV CGS-
3 (Malvern Instruments, Malvern, UK). DLS results are given as a z-average particle size
diameter and a polydispersity index (PDI). The PDI can range from 0 (indicating monodisperse
particles) to 1 (a completely heterodisperse system). Zeta-potential was measured using a
Zetasizer Nano (Malvern Instruments, Malvern, UK).
Quantification of unbound TMCs by GPC. The fraction of the various TMCs that was not
bound to WIV was quantified in the supernatant of centrifuged TMC-WIV formulations, using
the GPC method described earlier [11]. TMC-WIV formulations were centrifuged for 40 min at
22,000 x g at 4 °C and the supernatant was collected. Prior to injection, 20 µl GPC running
buffer was added to 100 µl supernatant to adjust the pH of the sample to pH 4.4. The sample
concentration was determined using refractive index detection.
Immunization protocol. Animal experiments were conducted according to the guidelines
provided by the Dutch Animal Protection Act and were approved by a Committee for Animal
Experimentation. For all experiments 6-8 weeks old female C57-BL/6 mice (Charles River)
were used. Mice were housed in groups of 7-11 mice and food and water were provided ad
74

Relationship between Structure and Adjuvanticity of TMC
libitum. Prime and boost immunizations at day 0 and 21, respectively, were performed without
anesthesia. Groups of 11 mice were vaccinated i.n. with the various TMC-WIV formulations at a
dose of 12.5 µg WIV (corresponding to approximately 4.3 µg HA). All TMC-WIV vaccines were
freshly prepared by mixing WIV dispersion with solutions of the various TMCs, as described
above. Additionally, a group of 11 mice were vaccinated with WIV i.n. without TMC. As
negative control groups, one group of 11 mice was treated i.n. with 5 mM HEPES and another
with TMC56 solution (1.25 mg/ml in 5 mM HEPES pH 7.4) without WIV. For i.n. immunization,
mice were held in supine position without anesthesia and the formulations were administered
to the left and right nostril in a total volume of 10 µl. As a reference, one group of mice was
vaccinated i.m. with WIV at a dose of 12.5 µg protein in a volume of 100 μl in the left and right
quadriceps for prime and boost vaccination, respectively.
Blood sampling and nasal washes. Blood samples were collected by orbital puncture in
MINICOLLECT® serum separator tubes coated with SiO 2 (Greiner Bio-One, Alphen a/d Rijn,
the Netherlands) 20 days after prime vaccination and 17 days after boost vaccination.
Coagulated blood samples were centrifuged at 6,500 x g for 8 min at room temperature to
obtain serum samples. Individual serum samples were stored at -20 °C until further analysis.
Seventeen days after boost vaccination, 4 mice from each group were sacrificed by a lethal
intraperitoneal injection of 100 µl sodium pentobarbital (200 mg/ml). The trachea was then
cannulated towards the nasopharyngeal duct with a PVC tube (inner/outer diameter 0.5/1.0
mm). PBS (500 μl) containing complete Mini, EDTA free protease inhibitor (Roche Diagnostics,
Indianapolis, IN, USA) at a concentration of 1 tablet / 7 ml PBS was flushed through the nasal
cavity and collected from the nostrils 3 times. The combined nasal washes were stored at -70
°C until further analysis.
Challenge. Twenty one days after the boost vaccination, mice were challenged with 50 ml (2
x 10 8 x the 50% egg infectious dose (EID50)/ml) aerosolized, egg-grown A/PR/8/34 using a
DeVilbiss Ultra-Neb 2000 ultrasonic nebulizer (Direct Medical Ltd, Lecarrow, Ireland) for 25 min.
After challenge, mice were put back in their cages and any observed signs of illness like
lethargy, standing fur and curved back were recorded. Additionally, their body weight was
monitored daily for 15 days. For comparison of loss in body weight, the average area under the
curve (AUC) was calculated for each group from relative body weight curves of individual mice.
All i.n. groups were compared to the negative control group (PBS i.n.) and the positive control
75

Chapter 4
group (WIV i.m.) by the average (AUC) of individual mice using a one-way ANOVA and
Bonferroni’s correction for multiple comparisons.
Hemagglutination inhibition test. First, 25 μl serum was incubated for 18 h at 37 °C with
75 μl Receptor Destroying Enzyme (RDE) solution (Denka Seiken UK Ltd, Coventry, UK) to
suppress nonspecific hemagglutination inhibition. RDE was then inactivated by incubating the
mixture for 30 min at 56 °C. Next, 150 μl PBS was added to obtain a final 10-fold serum
dilution. Fifty μl diluted serum was transferred in duplicate to V-bottom 96-wells plates
(Greiner, Alphen a/d Rijn, The Netherlands) and serially diluted twofold in PBS. Next, 4
hemagglutination units (HAU) of A/PR/8/34 (in 25 μl PBS) were added and the mixture was
incubated for 40 min at room temperature. Finally, 50 μl 0.5% (v/v) chicken erythrocytes in
PBS was added. Plates were incubated for 1 h at room temperature. The HI titer is expressed as
the reciprocal value of the highest serum dilution capable of completely inhibiting the virusinduced
agglutination of chicken erythrocytes. If no complete inhibition could be detected in
the first lane, serum was arbitrarily scored 5. Comparison between different experimental
groups from the same dose was made by a one-way ANOVA test and the Tukey post test on the
log transformed HI titers.
Antibody assays. Antigen specific serum antibody responses were determined by a
sandwich ELISA. Maxisorp ELISA plates (Nunc, Roskilde, Denmark) were coated overnight
with polyclonal rabbit anti-A/PR/8/34 serum (dilution 1:1620). Plates were washed in
between all prescribed steps with wash buffer (0.64 M NaCl, 3 mM KCl, 0.15% polysorbate 20
in 10 mM phosphate buffer pH 7.2) using a Skanwasher 300 (Molecular Devices, Sunnyvale,
CA, USA). Next, plates were incubated for 1 h at 37 °C with blocking buffer (0.2% (w/w) casein,
4% (w/w) sucrose, 0.05% (w/w) Triton X-100 and 0.01% (w/w) sodium azide in 30 mM TRIS
pH 7.4), followed by incubation for 1 h at 37 °C with egg-grown, BPL-inactivated A/PR/8/34
(25.6 HAU/ml). Plates were then incubated with twofold serially diluted sera (100 μl/well) for
1 h at 37 °C. Next, plates were incubated with 100 μl of a 1:2500 dilution of horseradish
peroxidase linked goat anti mouse -IgG (H+L), -IgG1, -IgG2a/c or -IgA(Fc) (Nordic
Laboratories, Tilburg, the Netherlands) for 30 min, and washed twice. Finally, 100 μl 3,3’,5,5’-
Tetramethylbenzidine (TMB) substrate solution was added and plates were incubated for 15
min at room temperature before enzymatic conversion was stopped by adding 50 μl 2 M
sulfuric acid. Optical density (OD) was then measured at 450 nm using a Tecan Sunrise plate
reader (Tecan Trading AG, Zurich Switzerland). Titers are given as the reciprocal sample
76

Relationship between Structure and Adjuvanticity of TMC
dilution corresponding to 20% of the maximal ELISA signal above background. Seronegative
sera were arbitrarily scored with a titer of 15. Comparison between different experimental
groups was made using the log transformed titers by a one-way ANOVA test and the Tukey
post test. Boost IgG2a/c:IgG1 ratios were calculated and compared using the log transformed
data by a one-way ANOVA test and Bonferroni’s correction for multiple comparison.
Results and discussion
Structural properties of TMCs. The structural properties of the TMCs used in this study are
summarized in Table 1. The DQ of the O-methyl free TMCs ranged between 30 and 68%,
allowing us to selectively study the influence of trimethylation on adjuvant properties of TMC.
With the TMC-OM group (DQ varying from 22 to 86%, O-methylation from 12 to 76% along
with increasing DQ) the combined effect of charge density and O-methylation on adjuvanticity
can be studied. The effect of O-methylation can be evaluated by comparing the O-methylated
TMCs with O-methyl free TMCs. Finally, the re-acetylated TMC with a DAc of 54% and a DQ of
44% can be used to assess the role of N-acetylated units in the adjuvant properties of TMCs.
Especially the comparison of TMC43, TMC-OM45 and TMC-RA44 will provide insight into the
optimal structural properties of TMC for its use in nasal vaccine delivery. Importantly, it is
unlikely that the minor differences in molecular weights between the various TMCs (Table 1)
caused by variation in the degrees of substitutions and/or synthesis methods will affect the
biological properties of the polymers [15].
Table 1. Structural properties of synthesized TMCs. 1)
M n
M w
Abbreviation
(kDa) (kDa)
DQ
(%)
DAc
(%)
DOM-6
(%)
DOM-3
(%)
TMC30 33 59 30 17 - -
TMC43 36 75 43 17 - -
TMC56 37 78 56 17 - -
TMC68 39 84 68 17 - -
TMC-OM22 34 56 22 12 18 12
TMC-OM45 32 49 45 11 25 16
TMC-OM61 31 49 61 10 56 44
TMC-OM86 29 44 86 9 76 72
TMC-RA44 43 83 44 54 - -
1) Degree of acetylation (DAc), quaternization (DQ), O-methylation on C-6 (DOM-6) and on
C-3 (DOM-3) of TMCs were determined by 1 H-NMR analysis. M n, M w were determined by GPC.
77

Chapter 4
Characterization of TMC-WIV formulations. Various TMC-WIV formulations were
prepared by mixing the two components at a 1:1 (w/w) ratio. For comparison, TMC56-WIV
formulations were also prepared at 0.2:1 and 5:1 (w/w) ratios. Particle size and size
distribution were determined and compared with those of plain WIV. Figure 1 shows that plain
WIV had a diameter of approximately 170 nm with a polydispersity index (PDI) of 0.2,
indicating a fairly homogeneous particle size distribution. Formulating the WIV particles with
the various TMCs led to a small increase in particle size (200-220 nm) and comparable size
distributions, independent of the type of TMC used or the TMC/WIV ratio.
Figure 1. Diameter and size distribution (PDI) of TMC-WIV formulations as determined by dynamic
light scattering. Error bars represent standard deviations of three measurements.
Furthermore, the zeta-potential of the TMC-WIV particles was analyzed in 5 mM HEPES pH
7.4. The addition of TMC to negatively charged WIV (-13 mV) in a 1:1 (w/w) ratio resulted in
positively charged particles (+12 to +20 mV) as seen in Figure 2, suggesting that all TMCs
adsorb onto WIV in a similar fashion. The zeta-potential increased slightly with increasing DQ
and the lowest surface charge was observed with TMC-OM22-WIV. As expected, compared to
the 1:1 ratio a lower TMC56:WIV ratio (0.2:1 (w/w)) resulted in a lower surface charge (+12
mV vs. +18 mV), but a higher ratio (5:1 (w/w)) did not result in a higher zeta-potential.
78

Relationship between Structure and Adjuvanticity of TMC
Figure 2. Zeta-potential of TMC-WIV formulations determined in 5 mM HEPES pH 7.4 Error bars
indicate standard deviations of three samples.
The amount of free TMC present in the TMC-WIV formulations was quantified using GPC. The
concentration and relative amount of free TMC are depicted in Table 2. TMC-WIV formulations
at a ratio of 1:1 (w/w) had an average free TMC content between 0.95 and 1.15 mg/ml
(corresponding to 76 and 91% of total TMC in the formulation), independent of the DQ of the
polymers. TMC-RA44 had the highest amount of TMC bound to the WIV particles. In the
TMC56-WIV(0.2:1) mixture about 50% of the TMC56 remained free in solution, whereas in the
TMC56-WIV(5:1) almost 98% of the total TMC56 in the formulation remained unbound. These
results, together with the results of the zeta-potential measurements, indicate that WIV is
likely saturated with TMCs at TMC:WIV ratios 1:1 and 5:1 (w/w).
79

Chapter 4
Table 2. Specifications of TMC in various TMC-WIV formulations.
Total TMC in
WIV
TMC:WIV
Formulation
formulation
(µg)
(w/w) ratio
(µg)
Unbound TMC
(mg/ml) ab
Unbound TMC
(% of total) b
TMC30-WIV 12.5 12.5 1:1 1.13± 0.04 90.0±3.3
TMC43-WIV 12.5 12.5 1:1 1.14± 0.03 91.2±2.6
TMC56-WIV 12.5 12.5 1:1 1.09± 0.01 87.1±0.8
TMC68-WIV 12.5 12.5 1:1 1.08± 0.08 86.1±6.2
TMC-OM22-WIV 12.5 12.5 1:1 1.02± 0.04 81.8±3.4
TMC-OM45-WIV 12.5 12.5 1:1 1.05± 0.06 83.9±4.4
TMC-OM61-WIV 12.5 12.5 1:1 1.01± 0.04 80.7±3.2
TMC-OM86-WIV 12.5 12.5 1:1 1.06± 0.01 85.1±0.9
TMC-RA44-WIV 12.5 12.5 1:1 0.95± 0.00 76.0±0.4
TMC56-WIV (0.2:1) 12.5 2.5 0.2:1 0.10± 0.05 49.3±1.5
TMC56-WIV (5:1) 12.5 62.5 5:1 6.11± 0.07 97.7±1.1
WIV 12.5 - - - -
a Amount of free TMC in the TMC-WIV formulations determined by GPC.
b Values are presented as the average of three samples ± standard deviation.
Summarizing, mixing WIV with various TMCs in a ratio of 1:1 resulted in particles with
similar size, size distribution, surface charge and amount of free TMC, thus allowing a fair
evaluation of the influence of the structural characteristics of the TMCs on their adjuvant
properties. Altering the TMC:WIV ratio mostly changed the amount of free TMC present in the
formulation and here the contribution of free TMC can be assessed.
Serum antigen specific total IgG after prime and boost vaccination. The TMC-WIV
formulations were compared to WIV alone in an intranasal vaccination study. Serum samples
were analyzed for antigen-specific total IgG responses three weeks after prime and boost
vaccination (Figure 3). IgG responses were induced by all formulations containing WIV after
prime vaccination and had increased after boost vaccination. TMCs with a DAc ≤ 17% had a
strong adjuvant effect that is not critically affected by DOM and DQ. All TMC-WIV formulations,
except TMC-RA44-WIV, showed a higher number of responding mice and elicited significantly
higher IgG titers than WIV alone. Interestingly, TMC-RA44-WIV induced significantly lower
total IgG responses than all other TMC-WIV formulations with a TMC:WIV ratio of 1:1.
Additionally, no significant differences were observed between the various TMC56-WIV ratio
formulations, indicating that free TMC does not strongly influence total IgG titers and that the
lowest dose of TMC that was tested already significantly increased immune responses after i.n.
vaccination with WIV.
80

Relationship between Structure and Adjuvanticity of TMC
Figure 3. Geometric mean antigen specific total IgG titers three weeks after prime and boost
vaccination. Error bars indicate 95% confidence intervals (n=11). Non-responding mice were arbitrarily
given a total IgG serum titer of 15. Below the x-axis the number of seropositive mice per group after
boost vaccination are depicted. All titers after boost vaccination were compared using a one-way ANOVA
test and Tukey’s post test. *** p

Chapter 4
the IgG2a/c:IgG1 ratio per individual mouse illustrates that the Th1/Th2 balance of humoral
immune responses shifts towards Th2 with increasing TMC:WIV ratio (Figure 4B).
A
B
Figure 4. A) IgG1 and IgG2a/c titers elicited by WIV formulations with varying TMC56:WIV ratios
after prime and boost vaccination. Error bars indicate 95% confidence intervals (n=11). Indicated
above the bars is the number of mice that developed detectable IgG1 or IgG2a/c titers (e.g. 1/11
indicates one out of eleven mice). ** p

Relationship between Structure and Adjuvanticity of TMC
HI titers. After boost vaccination, HI titers were hardly detectable in any of the i.n.
vaccinated groups (data not shown). Only WIV i.m. induced substantial HI titers (average HI
titer of 160). This is in line with previous results [11, 14]
Antigen-specific secretory IgA (sIgA) in nasal washes. Nasal washes were performed 17
days after boost vaccination to determine whether antigen-specific sIgA were elicited by any of
the various formulations. As shown in Figure 5 only in the TMC-WIV formulations some mice
developed detectable sIgA responses in the nasal cavity. Interestingly, although WIV i.m.
immunization showed the highest serum IgG titers, no sIgA was found in the nasal washes of
any of these mice. Altogether, the determination of sIgA in the nasal mucus showed a relatively
high variation within the formulation-groups (as observed by others [10]) likely due to the
collection and detection methods.
OD 450
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
TMC30-WIV
TMC43-WIV
TM56-WIV
TMC68-WIV
TMC-OM20-WIV
TMC-OM45-WIV
TMC-OM61-WIV
TMC-OM86-WIV
TMC-RA44-WIV
TMC56-WIV (0.2:1)
TMC56-WIV (5:1)
WIV
TMC56 sol
WIV i.m.
HEPES
Figure 5. Antigen-specific secretory IgA levels in nasal washes of four individual mice per formulation
17 days after boost vaccination. Only in groups represented by black dots sIgA positive washes were
found.
Challenge with live, aerosolized virus. To assess the protective effect of the immunization,
seven mice per group were challenged with potentially lethal, homologous, egg-grown
influenza virus and loss of body weight as a measure of illness was monitored. All mice
vaccinated i.n. with O-methyl free or O-methylated TMC-WIV formulations were protected
against the live virus. As representative examples the average body weight over 15 days after
83

Chapter 4
the challenge of the mice immunized with TMC43-WIV and TMC-OM45-WIV formulations are
shown in Figure 6A and B.
A
B
C
D
Figure 6. Average relative body weight curves after challenge of mice (n=7) vaccinated with a) TMC43-
WIV; b) TMC-OM45-WIV; c) TMC-RA44-WIV and d) WIV i.n.. For comparison each panel contains the
curves of WIV i.m. and HEPES i.n.. Error bars indicate the 95% confidence intervals. *** p< 0.001 and **
p< 0.01 indicate that average AUCs are significantly higher than the HEPES i.n. group. ### p< 0.001 and #
p

Relationship between Structure and Adjuvanticity of TMC
Additionally, there were no signs of illness observed in these groups and the AUC of the
average body weight was significantly higher (p

Chapter 4
Furthermore, O-methylated TMCs had similar effects on both antibody titers and protection as
O-methyl free TMCs, indicating that the DOM does not affect the adjuvant properties of TMC.
Interestingly, these findings do not correlate with the large influence of DQ and O-methylation
in vitro on cell toxicity and TEER. This teaches us that DQ and DOM have a much more
pronounced effect on in vitro cell toxicity and TEER assays than on mucosal adjuvant
properties. It should be noted that the DOM and DQ may have an influence on the activity of
TMC as a penetration enhancer in mucosal drug delivery, as suggested by reports on the
influence of DQ [18-20]. As HA likely is too big to pass even fully opened tight junctions, it is
unlikely that opening of tight junctions will directly improve antigen uptake from the nasal
cavity.
The reacetylation of TMC on the other hand induced a strong decrease in adjuvant effect
illustrated by lower antibody titers and poor protection against challenge with live influenza
virus. Since the mucoadhesion of particulate systems can be attributed to positive charge and
hydrophobic effect [3], it is likely that TMC-RA44-WIV has similar or even better
mucoadhesive properties as the other TMC-WIV vaccines. Also, the zeta-potential of the
particles was similar to the other TMC-coated particles, implying that the introduction of a
positive surface charge alone is not sufficient to improve adjuvanticity. Therefore other factors
must be responsible for the decreased adjuvant effect of reacetylated TMC.
The first explanation for the decreased adjuvant effect of TMC-RA44 is a difference in
interaction with cells, illustrated by a much lower in vitro toxicity and TEER effect than most
other TMCs on Caco-2 cells [9]. TMC-OM22, however, hardly induced a TEER effect or cell
toxicity either but showed to be a good mucosal adjuvant. This indicates that TEER and toxicity
studies, as carried out for these TMCs [8, 9] cannot fully explain the loss of adjuvant effect by
reacetylation.
A second explanation for the poor adjuvant properties of TMC-RA44 is its enhanced
enzymatic degradation by lysozyme compared to other TMCs. Previous research showed that
the extent of lysozyme-catalyzed degradation of TMC is highly dependent on the DAc; TMC-
RA44 showed a large decrease in molecular weight while TMCs with DAc of ≤17% were only
slightly degraded in presence of lysozyme [9]. Lysozyme is a strong antibacterial cationic
protein that is excreted in high concentrations in the nasal cavity [21]. This may result in rapid
degradation of the TMC-RA44 after i.n. administration, thereby strongly limiting its adjuvant
effect on the WIV vaccination. Finally, chitin, an insoluble polysaccharide of N-
acetylglucosamine units, which are also present in TMC-RA44, can be recognized by specific
receptors of the innate immune system [22]. It has recently been suggested that chitin induces
86

Relationship between Structure and Adjuvanticity of TMC
pro-inflammatory but also anti-inflammatory signals depending on its size [23]. Further
studies should be done to investigate whether TMC-RA44 also induces anti-inflammatory
signals through interaction with the innate immune system.
In addition to changes in the type of TMC, the influence of free TMC was studied by varying
the TMC concentration in the formulation. An excess of free TMC induced a shift towards
higher IgG1 titers after boost vaccination. Several studies investigated the influence of TMC on
the quality of immune responses [24-26] while other studies determined the effect of adjuvant
dose on the type of responses elicited, using different adjuvants [27, 28]. However, since the
type of adjuvant, animal model used, route of administration and animal model can affect the
quality of immune responses, it is difficult to compare these studies to our data. This is the first
time that the effect of adjuvant dose was studied for TMC and additional studies should be
performed to provide mechanistical insight.
Conclusion
All TMC-WIV formulations had comparable physicochemical properties, and therefore
observed differences in immunogenicity are related to the various chemical structures of the
TMCs. Formulating WIV with TMCs strongly enhances the immunogenicity and protection of
i.n. vaccination with WIV. The adjuvant properties of TMCs as i.n. adjuvant are strongly
decreased by reacetylation of TMC, whereas the DQ and DOM did not significantly affect the
adjuvant effect of TMC.
Acknowledgement. This research was partially performed under the framework of TI
Pharma project number D5-106-1; Vaccine delivery: alternatives for conventional multiple
injection vaccines. The authors acknowledge Frouke Kuijer for making the graphical abstract.
87

Chapter 4
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89

CHAPTER 5A
A STEP-BY-STEP APPROACH
TO STUDY THE INFLUENCE OF N-
ACETYLATION ON THE ADJUVANTICITY OF
N,N,N-TRIMETHYL CHITOSAN (TMC) IN AN
INTRANASAL WHOLE INACTIVATED
INFLUENZA VIRUS VACCINE
Rolf J. Verheul*, Niels Hagenaars*, Thomas van Es, Ethlinn van Gaal,
Pascal H.J.L.F. de Jong, Sven Bruins, Ivo Que, Bram Slütter,
Enrico Mastrobattista, Harrie L. Glansbeek, Jacco G.M. Heldens,
Han van den Bosch, Wim E. Hennink, Wim Jiskoot.
*authors contributed equally
Manuscript submitted

Chapter 5A
Abstract
In a previous study, we observed that reacetylation of N,N,N-trimethyl chitosan (TMC)
reduced the adjuvant effect of TMC in mice after intranasal (i.n.) administration of whole
inactivated influenza virus (WIV) vaccine. The aim of the present study was to elucidate the
reason for the lack of adjuvanticity of reacetylated TMC (TMC-RA) by comparing TMC-RA
(degree of acetylation 54%) with TMC (degree of acetylation 17%) at six potentially critical
steps in the induction of an immune response after i.n. administration in mice: chemical
stability of the polymer in a nasal wash, local i.n. distribution of WIV, nasal residence time of
WIV, cellular uptake of WIV by epithelial cells, transport of WIV by epithelial cells, and capacity
of the formulation to induce maturation of murine bone marrow derived dendritic cells (DCs).
GPC analysis showed that TMC-RA was degraded in a nasal wash to a slightly larger extent
than TMC. The local i.n. distribution and nasal clearance were similar for both TMC types.
Fluorescently labeled WIV was taken up more efficiently by Calu-3 cells when formulated with
TMC-RA compared to TMC and both TMCs significantly reduced transport of WIV over a Calu-3
monolayer. Murine bone-marrow derived dendritic cell activation was similar for plain WIV,
and WIV formulated with TMC-RA or TMC.
In conclusion, the inferior adjuvant effect of TMC-RA over that of TMC might be caused by a
slightly lower stability of TMC-RA-WIV in the nasal cavity, rather than by any of the other
factors studied in this paper.
92

Influence of N-Acetylation on the Adjuvanticity of TMC
Introduction
Intranasal (i.n.) immunization offers several advantages over traditional parenteral routes,
such as painless, needle-free administration without the need for trained personnel and the
potential induction of both systemic and local immune responses [1-3]. Nonetheless, its
success has been limited due to delivery issues associated with the physiology of the nasal
epithelium and the tolerogenic nature of immune cells in the nasal associated lymphoid tissue.
This is illustrated by the strong immune responses that the same vaccines elicit after
intramuscular administration compared to nasal administration [4]. A successful i.n. vaccine
formulation should therefore adhere to the mucosal surfaces of the nasal cavity and provide
protection against the degradation of the antigen in the nasal environment. Next, the
formulation should induce the uptake and/or transport of antigen through the epithelium, to
ensure that a sufficient amount of antigen will reach the antigen presenting cells in the
subepithelial space. Finally, these APCs (most noticeably dendritic cells (DCs)), have to be
activated (matured) either by the antigen or by addition of an adjuvant, in order for them to
migrate to the nearby lymph nodes and elicit the desired type of immune response [5-8].
N,N,N-trimethyl chitosan (TMC), a quaternized, water-soluble derivative of chitosan, has
previously been shown to possess mucoadhesive [9] and absorption enhancing [10-12]
characteristics and when formulated with whole inactivated influenza virus vaccine (WIV)
greatly enhanced the efficacy of the i.n. administered vaccine in mice [13]. To further improve
its adjuvant effect, the structural properties of TMC, like the degree of quaternization (DQ), the
degree of O-methylation (DOM), and the degree of N-acetylation (DAc), can be varied during
synthesis [14, 15]. In a previous study on the influence of these structural properties on the
adjuvant effect of TMC in i.n. WIV vaccines in mice [16], we showed that both the DQ and the
DOM of TMC did not have a significant effect on the immunogenicity of the i.n. WIV vaccines,
whereas the DAc did. A striking loss in adjuvanticity of TMC was observed when WIV was
formulated with re-acetylated TMC (TMC-RA) with a DAc of 54% and a DQ of 44% (compared
to a DAc of 17% for other tested TMCs). Importantly, the physical characteristics of WIV
formulated with TMC-RA or with TMC (both polymers having a DQ of about 45%) were similar
for size, zeta-potential and amount of unbound TMC, indicating that these physical properties
can not be held accountable for the difference in adjuvant effect. In contrast to TMC, TMC-RA is
a substrate for lysozyme, has a lower toxicity profile than TMC and has no effect on the
transepithelial electrical resistance (TEER) of Caco-2 cells [15].
The aim of this study, was to understand why TMC-RA, in contrast to TMC, does not act as an
adjuvant for an i.n. WIV vaccine in mice. This will provide more insight into the mechanism of
93

Chapter 5A
adjuvanticity of TMC and may aid in the rational, structural design of TMC as an adjuvant. TMC-
WIV, TMC-RA-WIV and WIV formulations were compared at six potentially critical steps in the
induction of an immune response after i.n. administration. In particular, we studied (i) the
stability of TMC and TMC-RA in nasal washes, (ii) the effect of both polymers on the nasal
residence time of WIV, (iii) nasal distribution patterns, (iv) cellular uptake and (v) transport of
WIV through an epithelial (Calu-3) cell line and (vi) the effect of the different formulations on
maturation of murine bone-marrow derived dendritic cells (DCs).
Materials and Methods
Materials. Chitosan was purchased from Primex (Siglufjordur, Iceland) and had a DAc of 17%
and a number average molecular weight (M n) and weight average molecular weight (M w) of 28
and 43 kDa, respectively. Acetic anhydride, sodium borohydrate, formic acid, formaldehyde
37% (stabilized with methanol), deuterium oxide, sodium acetate, acetic acid (anhydrous),
sodium hydroxide and hydrochloric acid were obtained from Sigma-Aldrich Chemical Co.
Iodomethane 99% stabilized with copper was obtained from Acros Organics (Geel, Belgium).
Purified, cell culture-grown (Madin-Darby Canine Kidney (MDCK) cells), β-propiolacton (BPL)-
inactivated A/PR/8/34, as well as polyclonal rabbit anti-A/PR/8/34 serum were from Nobilon
International BV (Boxmeer, The Netherlands). Alexa Fluor® 488-labeled goat anti-rabbit IgG,
Alexa Fluor® 488 labeling kit and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) were
purchased from Invitrogen (Breda, the Netherlands). Osteosoft decalcifier (10% EDTA) was
from Merck Serono (Schiphol, the Netherlands). Citric acid, Triton-X, trypsin from bovine
pancreas, normal goat serum and normal rabbit serum were obtained from Sigma
(Zwijndrecht, the Netherlands). Phosphate buffered saline (10 mM phosphate buffer, pH 7.4,
150 mM NaCl; PBS) was purchased from Braun (Melsungen, Germany). IRDye800CW®-labeled
epidermal growth factor (EGF) and the IRDye800CW® protein labeling kit were from LI-COR
Biosciences (Lincoln, NE, USA). Fixative (4% formalin in phosphate buffer pH 7.0) was
obtained from Klinipath (Duiven, the Netherlands). Calu-3 cells were obtained from ATCC
(Manassas, VA, USA). Virally transformed human bronchial epithelial cell line 16HBE14o- was
a kind gift from Dr. D. Gruenert (University of California at San Francisco). Trypsin/EDTA 10x,
Plain DMEM (Dulbecco’s modified Eagle’s medium, with 3.7 g/l sodium bicarbonate, 1 g/l L-
glucose, L-glutamine) and antibiotics/antimycotics (penicillin, streptomycin sulfate,
amphotericin B) were from PAA Laboratories GmbH (Pasching, Austria) and propidium iodide
(PI) and MEM with 0.292g/L L-glutamine, 1 g/L Glucose, 2.2 g/L NaHCO3, was from Invitrogen
94

Influence of N-Acetylation on the Adjuvanticity of TMC
(Breda, The Netherlands). Matrigel ® was acquired from Becton Dickinson (Franklin Lakes, NJ
USA). All other chemicals used were of analytical grade. Animal experiments were conducted
according to the guidelines provided by the Dutch Animal Protection Act and were approved
by a Committee for Animal Experimentation.
Synthesis and characterization of trimethylated chitosans. O-methyl free TMCs with a
DQ of 45% and varying DAc were synthesized from chitosan as described previously [15]. The
DQ and DAc of the TMCs were determined with 1H-NMR on a Varian INOVA 500 MHz NMR
spectrometer (Varian Inc., Palo Alto, Ca, USA) at 80 °C in D 2O. Furthermore, number average
weight (M n) and weight average weight (M w) of the TMCs were determined as described before
[15] by GPC on a Viscotek system detecting refractive index, viscosity and light scattering. A
Shodex OHPak SB-806 column (30 cm) was used with 0.3 M sodium acetate pH 4.4 (adjusted
with acetic acid) as running buffer. The structural characteristics of the synthesized TMCs are
summarized in Table 1.
Preparation and characterization of TMC-WIV formulations. TMC-WIV formulations
were prepared as described previously (16). Briefly, purified, cell culture-grown (Madin-Darby
Canine Kidney (MDCK) cells), β-propiolacton (BPL)-inactivated mouse adapted influenza
A/Puerto Rico/8/34 virus (A/PR/8/34) suspended in a 10 mM phosphate buffered saline (150
mM NaCl, pH 7.4) was concentrated by centrifugation at 22,000xg for 30 min at 4 °C and
resuspended in 5 mM HEPES buffer (pH 7.4). WIV suspension was mixed with equal volumes
of TMC or TMC-RA in 1:1 or 5:1 weight/weight ratio. For dynamic light scattering (DLS)
(Malvern ALV CGS-3, Malvern Instruments, Malvern, UK) and zeta-potential measurements
(Zetasizer Nano, Malvern Instruments, Malvern, UK) a final WIV concentration of 62.5 μg/ml
(expressed as total protein concentration) in 5 mM HEPES pH 7.4 was used. DLS results are
given as z-average particle size diameter and a polydispersity index (PDI). The PDI can vary
from 0 (indicating monodisperse particles) to 1 (a completely heterodisperse particle size
distribution)).
Degradation of TMCs in nasal wash. Six female 6-8 weeks old Balb/c nu/nu mice (Charles
River, L’Arbresle, France) were sacrificed by a lethal intraperitoneal injection of 100 μl sodium
pentobarbital (200 mg/ml). The trachea of each mouse was then cannulated towards the
nasopharyngeal duct with a PVC tube (inner/outer diameter 0.5/1.0 mm). PBS (600 μl) was
flushed through the nasal cavity and collected from the nostrils 3 times; the collected samples
95

Chapter 5A
were pooled. TMC and TMC-RA were dissolved in 5 mM HEPES at 2.5 mg/ml, mixed 1:1 (v/v)
with nasal wash and the mixtures were incubated at 37 °C. Samples were taken after 4 and 24
hours and 5 and 9 days. M n and M w were determined by GPC on a Viscotek system detecting
refractive index, viscosity and light scattering. A Shodex OHPak SB806 column (15 cm) was
used with 0.3 M sodium acetate, pH 4.4 (adjusted with acetic acid), as running buffer [17].
Pullulan (M n 102 kDa, M n 106 kDa) obtained from Viscotek Benelux (Oss, The Netherlands)
was used for calibration and polymer solutions mixed with PBS 1:1 (v/v) were used as
controls. Prior to injection, 30 μl GPC running buffer was added to 120 μl sample to adjust the
pH of the sample to pH 4.4.
In vivo imaging of nasal residence time of TMC-WIV formulations. In vivo imaging of
TMC-WIV formulations was done according to a previous study [18]. Female nude (Balb/c
nu/nu) mice were obtained from Charles River (L’Arbresle, France) and received plain
IRDye800CW®-labeled WIV (n=9) or formulated with TMC (n=9) or TMC-RA (n=3) in a ratio
of 1:1 (w/w) under light anesthesia with isoflurane inhalation. Next, mice were scanned with
an IVIS Spectrum imaging system from Caliper Life Sciences (Hopkinton, MA, USA). Scans were
performed regularly over at least 2 h. Scanned images were analyzed using Living Image 3.1
software from Caliper Life Sciences (Hopkinton, MA, USA). The threshold was set using the
background scan made from each mouse before administration of the IRDye800CW®-labeled
formulations. The excitation wavelength was set at 710 nm and emitted light was measured at
760; 780; 800; 820 and 840 nm. Spectral unmixing was performed to decompose the emitted
light into auto fluorescence and label-specific fluorescence. To compare the fluorescence in
different mice and different groups, the absolute fluorescence was converted to relative
fluorescence (% of the maximal fluorescence in the nasal cavity). The areas under the curve
(AUC) of the relative fluorescence of individual mice were used to compare the fluorescence
over time for the different groups.
Immunostaining of TMC-WIV formulations in nasal cross-sections. Immunostaining of
TMC-WIV formulations was carried out essentially as described before [18]. Six to eight weeks
old female C57-BL/6 mice from Charles River (L’Arbresle, France) were i.n. vaccinated with
TMC-RA-WIV or TMC-WIV in a (w/w) ratio of 1:1 or plain WIV formulations. As negative
controls, mice received PBS or solutions of TMCs and 4 mice were left untreated. After 20
minutes or 1 hour, animals were sacrificed by cervical dislocation and the nasal cavity was
isolated by removing the brains, lower jaw, skin and muscle tissue. The nasal cavities were
96

Influence of N-Acetylation on the Adjuvanticity of TMC
then fixed in 10% formalin, decalcified, embedded in paraffin and 3.5 µm thick cross-sections
were made at different depths of the nasal cavity using a Microm HM 355 S microtome from
Thermo Fisher Scientific (Walldorf, Germany). Cross-sections for immunohistochemistry were
mounted on superfrost plus glass slides from Menzel-Gläser (Braunschweig, Germany). After
deparaffinization, hydration and a heat-induced epitope retrieval (HIER) step with citrate
buffer, cross-sections were washed 3 x 5 min with 0.2% Triton-X in PBS. Subsequently, crosssections
were incubated first with normal goat serum for 15 minutes and then with a 1:500
dilution of polyclonal WIV-specific rabbit antiserum overnight at 4°C. As a negative control,
one of the two paraffin sections was incubated with nonspecific rabbit serum as primary
antibody. After washing the slides 3x for 5 min with 0.2% Triton-X in PBS, a 1:200 dilution of
Alexa Fluor® 488-labeled goat anti-rabbit IgG was applied. The samples were incubated for 45
minutes at room temperature. Next, the slides were washed 3x with PBS and stained with a
1:25000 dilution of DAPI for 6 min at room temperature and washed 3x with PBS again.
Finally, the slides were mounted with Fluorosave from Calbiochem (San Diego, CA, USA). All
slides were examined using a fluorescence microscope (Nikon Eclipse TE-2000 Nikon,
Amstelveen, the Netherlands) equipped with a Digital Sight DS-2Mv camera. Slides were
blindly scored for the presence, location, pattern and intensity of antigen staining and pictures
were taken with fixed camera settings for a fair comparison.
Uptake by Calu-3 epithelial cells of fluorescently labeled TMC-WIV formulations. Calu-
3 were grown in MEM with 0.292g/L L Glutamine, 1g /L Glucose, 2.2g/L NaHCO3 (Invitrogen,
Breda, The Netherlands) supplemented with antibiotics/antimycotics and 10% heatinactivated
fetal calf serum (FCS; Integro, Zaandam, The Netherlands). Cells were cultured in
fibronectin-coated flasks and maintained at 37°C in a 5% CO2 humidified air atmosphere and
split once a week.
Cells were transferred into a flat bottom 24-well plate (500,000 cells/well) and after
maturation for 2 days cells were incubated with 500 μl of plain AlexaFluor-488®-labeled WIV
or formulated with TMC or TMC-RA in PBS at a TMC:WIV (w/w) ratio of 5:1 and diluted as
indicated (50µg WIV/ml diluted 10-160x) for 1 h at 37 ºC or 4 ºC. After incubation, cells were
washed with PBS (400 μl) and detached with 100 μl trypsin/EDTA at 37 ºC. Then, 150 μl MEM
+ 10% FCS was added and the cells were transferred into a 96 well plate. Cells were
centrifuged (250 x g for 5 minutes at 4 ºC) and washed with three times with 200 μl phosphate
buffered albumin (PBA, 1 g albumin per 100 ml PBS) and finally resuspended in 200 μl PBA.
Immediately prior to measurement, 20 μl propidium iodide solution (PI; 1 μg/ml in water) was
97

Chapter 5A
added for live/dead cell discrimination. Flow cytometric analysis was performed on a FACS
Canto (Becton and Dickinson, Mountain View, CA, USA) using a 15 mW 488 nm, air-cooled
argon-ion laser and data were analyzed using FACS Diva software (Becton and Dickinson,
Mountain View, CA, USA). 10,000 cells were recorded per sample to determine bead-uptake
(FL1-channel) and PI-staining (FL3-channel).
Transport of fluorescently labeled TMC-WIV formulations over Calu-3 monolayer.
Calu-3 cells were seeded at a density of 5x10 5 cells per well on 12-transwell plates with
Matrigel ® coated 2 µm microporous membranes. The cells were cultured in Dulbecco’s
Modified Eagle’s Medium (DMEM) for 14 days until a confluent cell layer was formed. The
medium was replaced by Hank’s Balanced Salt Solution (HBSS) at the apical (300 μl) and
basolateral (1.2 ml) sides and after 30 minutes equilibration the transepithelial electrical
resistance (TEER) was measured using a home made dipstick electrode. Then, HBSS was
replaced by 300 μl of plain AlexaFluor-488®-labeled WIV or formulated with TMC or TMC-RA
at a TMC:WIV (w/w) ratio of 5:1 (25µg WIV/ml in HBSS). After incubation for 60 minutes at 37
ºC the TEER was measured again and the amount of fluorescently labeled WIV particles in the
basolateral acceptor compartment was determined by flow cytometry.
Maturation of murine bone marrow derived dendritic cells. The femur and tibia of C57-
BL/6 mice were removed, both ends were cut and bone marrow was flushed with Iscove’s
Modified Dulbecco’s Medium (IMDM; Gibco, CA, USA) using a syringe with a 0.45 mm diameter
needle. The bone marrow suspension was vigorously resuspended and passed over a 100 μm
gauge to obtain a single cell suspension. After washing, cells were seeded 2x10 6 cells per 100
mm petridish (Greiner Bio-One, Alphen aan den Rijn, The Netherlands) in 10 ml IMDM,
supplemented with 2 mM L-glutamine, 50 U/ml penicillin, 50 ug/ml streptomycin and 50 μM
β-mercaptoethanol (Merck, Darmstadt, Germany) and 30 ng/ml recombinant murine GM-CSF
(rmGM-CSF). At day 2, 10 ml medium containing 30 ng/ml rmGM-CSF was added. At day 5
another 30 ng/ml rmGM-CSF was added to each plate. From day 6 onwards, the non-adherent
DCs were harvested and used for subsequent experiments.
These immature DCs were seeded (50,000 cells/well) in a 96-well plate in a total volume of
100 μl RPMI 10% FCS in the presence of TMC (0.001-25 μg/ml), TMC-WIV formulations at a
1:1 or 5:1 (w/w) ratios or lipopolysaccharide (LPS) (0.01-100 ng/ml) as a positive control. DCs
were incubated with the samples for 16 h at 37 °C. DC maturation was determined by
98

Influence of N-Acetylation on the Adjuvanticity of TMC
analyzing cell-surface expression of co-stimulatory molecules (CD40) on CD11 and MHC II
positive cells by flow cytometry [19].
Results
Properties of TMC-WIV formulations. The structural characteristics of the synthesized
TMCs are summarized in Table 1.
Table 1. Structural characteristics of TMC and TMC-RA used in this study.
Polymer M n (kDa) M w (kDa) DQ (%) DAc (%)
TMC 36 75 43 17
TMC-RA 43 83 44 54
As in a previous study [16], WIV was mixed in a 1:1 or 5:1 w/w ratio with TMC or TMC-RA
and analyzed for size, PDI and zeta potential. Figure 1 shows that mixing of WIV with TMC led
to a minor increase in size and PDI, and the zeta potential reversed from -17 mV (plain WIV) to
about +18 mV, independent of type or amount of TMC used for coating. It can therefore be
stated that all formulations were physically similar, except for the amount of free polymer;
formulations with a 5:1 w/w polymer to WIV ratio will very likely contain more free polymer
[16].
Diameter (nm)
A
400
300
200
100
PDI
Diameter
1.0
0.8
0.6
0.4
0.2
PDI
Zeta potential (mV)
B
25
15
5
-5
-15
0
WIV
TMC-WIV 1:1
TMC-WIV 5:1
TMC-RA-WIV 1:1
TMC-RA-WIV 5:1
0.0
-25
WIV
TMC-WIV 1:1
TMC-WIV 5:1
TMC-RA-WIV 1:1
TMC-RA-WIV 5:1
Figure 1. Z-average diameter and PDI (A) and zeta potential (B) of the WIV formulations. Error bars
represent the standard deviation (n=5).
99

Chapter 5A
Biodegradation. An important difference between TMC and TMC-RA is their enzymatic
biodegradability by lysozyme [15], an enzyme which is present in the nasal cavity [20].
Therefore, after i.n. immunization TMC-RA-WIV may be more rapidly degraded in the nasal
cavity. To study whether TMC and TMC-RA are also degraded upon contact with enzymes
present in the nasal cavity, solutions of these TMCs were incubated at 37 °C with murine nasal
washes. At different time points, the solutions were analyzed by GPC to determine the
molecular weight (M n and M w) of the TMCs. As expected, TMCs in PBS that were taken as
controls, did not show decrease in M n/M w. As shown in Figure 2, TMC-RA degraded to a greater
extent than TMC in a nasal wash. However, the degradation of both TMC-RA and TMC was
relatively slow in the nasal wash (compared to the in vitro findings) and up to 24 h of
incubation, no significant differences were observed between the two TMCs.
Figure 2. Biodegradation of TMC (squares) and TMC-RA (dots) in pooled nasal wash of mice. Error bars
indicate the standard deviation (n=3).
In vivo fluorescence imaging. In vivo fluorescence imaging was used to compare the effect
of TMC and TMC-RA on the nasal clearance of WIV. Fluorescently labeled WIV was formulated
with TMC and TMC-RA and the formulations were administered i.n. to mice. Imaging of the
fluorescence in the nasal cavity as a function of time did not reveal a significant difference
between the formulations (p>0.05), as calculated from the AUC (see Figure 3). After an initial
increase in fluorescence, likely due to fluorescence dequenching [18], all formulations showed
a comparable decrease in fluorescence over time. This suggests that the nasal clearance of
plain WIV, TMC-WIV and TMC-RA-WIV is comparable.
100

Influence of N-Acetylation on the Adjuvanticity of TMC
Relative fluorescence
(%)
110
100
90
80
70
60
50
40
30
20
10
0
0 25 50 75 100 125
Time (min)
WIV
TMC-WIV
TMC-RA-WIV
Figure 3. Average relative fluorescence in the nasal cavity over time after intranasal administration of
fluorescently labeled WIV formulations. Error bars indicate standard deviation (n=3 mice).
Local distribution in the nasal cavity of intranasally applied vaccine formulations. In a
previous study it was shown that in the murine nasal cavity, TMC-WIV formulations exhibit a
completely different distribution pattern as plain WIV: TMC coated WIV was located tightly
along the epithelial cells while plain WIV was mainly observed in mucosal blobs [18]. To
investigate whether the distribution pattern is different for TMC-RA-WIV, mice were
administered TMC-RA-WIV and TMC-WIV and nasal sections were stained to visualize the
location of WIV. After 20 min, WIV-specific staining was found on the epithelial surfaces of the
naso- and maxilloturbinates in all mice, for both TMC-WIV and TMC-RA-WIV (see Figure 4 for
representative examples, n=5). This indicates that the degree of acetylation is unlikely to affect
the contact between WIV with the mucosal surfaces. After 1 hour, WIV was still present on the
naso- and maxilloturbinates but the staining was less intense (results not shown). TMC
solutions showed no fluorescence upon excitation nor did staining with non-specific rabbit
serum [18].
101

Chapter 5A
A
B
Figure 4. Representative picture of fluorescently labeled WIV (depicted in green) on the mucosal
surfaces of the nasoturbinates when formulated with TMC-RA (A) and TMC (B) 20 minutes after
administration.
102

Influence of N-Acetylation on the Adjuvanticity of TMC
A
Fluorescence Geo Mean
(arb. units)
2000
1500
1000
500
0
5.00
*
2.50
*
1.25
*
0.62
WIV
TMC-WIV
TMC-RA-WIV
PBS
** **
0.31
B
Fluorescence Geo Mean
(arb. units)
1250
1000
750
500
250
0
**
**
5.00
** ***
2.50
** ** ***
***
1.25
WIV
TMC-WIV
TMC-RA-WIV
PBS
0.62
0.31
Conc. WIV (μg/ml)
Conc. WIV (μg/ml)
Figure 5. Uptake/association of fluorescently labeled WIV, uncoated or coated with TMC or TMC-RA by
Calu-3 cells when incubated with solution of different concentrations for 1 hour at 37°C (A) or 4°C (B). *
p

Chapter 5A
Change in TEER:
% of WIV transported
1.0×10 -2 -1% (±9) -26% (±7) -7% (±4)
7.5×10 -3
5.0×10 -3
2.5×10 -3
0
oo
***
WIV
TMC-WIV
***
TMC-RA-WIV
Figure 6. Transport of fluorescently labeled WIV, uncoated or coated with TMC or TMC-RA, by Calu-3
human bronchial epithelial cells after incubation at 37°C for 1 h. Changes in TEER values (± standard
deviation) after 1 h incubation are depicted above the bars. Error bars indicate standard deviations
(n=3). *** value significantly (p

Influence of N-Acetylation on the Adjuvanticity of TMC
CD40 exp. (MFI)
500
400
300
200
100
medium
0
WIV
TMC-WIV 5:1
TMC-RA-WIV 5:1
LPS 100 ng/ml
TMC 25 μg/ml
TMC-RA 25 μg/ml
0.63μg/ml WIV
0.31
0.16
0.08
0.04
Controls
Figure 7. A representative example of DC maturation (n=2) (monitored by CD40 expression) by plain
WIV, TMC-WIV and TMC-RA-WIV formulations at 5:1 TMC:WIV (w/w) ratio in different concentrations.
Discussion
As pointed out before, adjuvants for nasal immunization can act by enhancing the
immunoavailability of the antigen, or as immune potentiator by providing or increasing a
“danger” signal [1, 2, 5, 6]. In general, TMC is associated with improving antigen delivery
through its muco-adhesiveness [6, 13, 21, 22], thereby increasing nasal residence time and/or
improving antigen-epithelial barrier interactions [18]. Although of importance for mucosal
peptide and protein delivery [6, 12], TMC’s capability to open tight junctions seems not to be of
major relevance for nasal vaccination as several studies showed successful nasal immunization
with TMCs with low DQ (

Chapter 5A
In the present study, we found that TMC-RA is degraded to a larger extent in nasal wash than
TMC, although this degradation appeared to be rather slow. However, note that the
biodegradability of TMC in a nasal wash may not accurately reflect the degradation in the nasal
cavity after i.n. administration. It is likely that the concentration of lysozyme and other
proteases in a nasal wash is much lower than the concentration in the nasal cavity, firstly
because of a strong dilution effect in the wash and secondly because most of the lysozyme is
stored and excreted from submucosal glands [20] and may not be efficiently extracted by a
nasal wash. Thus, although in vitro degradation occurred quite slowly, the intranasal
degradation of TMC-RA during its nasal residence may be significant in vivo. Our results
consequently suggest that TMC-RA is enzymatically degraded more extensively than TMC.
However, both the extent of degradation and the degradation rate in the nasal cavity are
difficult to establish quantitatively.
This reduction of polymer molecular weight may lead to a decrease of cytotoxicity [24],
however, TMC-RA has already a very low toxicity profile showing no reduction of cell viability
or increased lactate dehydrogenase (LDH) release in Caco-2 cells even up to a concentration of
10 mg/ml [15]. Furthermore, both in vivo WIV nasal clearance and local nasal distribution
patterns are similar for TMC and TMC-RA coated WIV, indicating that possible nasal
degradation of TMC-RA does not affect antigen exposure to the epithelial barrier. Therefore,
although nasal degradation of TMC-RA in vivo may occur, its effects on the exposure of the
antigen to the epithelial barrier are likely rather small.
Importantly, our data indicate that enhancing the interaction between the antigen and the
epithelial barrier alone is not sufficient for an adequate immune response and consequently
other factors have to play a role as well. One possibility is that a co-stimulatory ‘danger’ signal
due to toxicity of the TMC is crucial in provoking an adequate immune response [25]. As TMC-
RA showed the lowest in vitro cyto-toxicity of all TMC polymers [15] this polymer may not be
capable of inducing such a danger signal. Interestingly, this difference in toxicity between TMC
and TMC-RA may become even more pronounced due to the higher extent of nasal degradation
of TMC-RA.
The uptake and binding of TMC coated WIV by Calu-3 cells decreased compared to TMC-RA
coated WIV and uncoated WIV. In contrast, in other cell-types (e.g. 16-HBE14o and HeLa cells),
TMC coated WIV was taken up to a higher extent than plain WIV and TMC-RA coated WIV
(results not shown). This implies that the extent of uptake of TMC coated WIV is highly
dependent on the model cell line used. Calu-3 cells are, although derived from human
bronchial epithelium, used extensively for ‘nasal’ transepithelial electrical resistance (TEER)
106

Influence of N-Acetylation on the Adjuvanticity of TMC
studies [26, 27] as they form tight monolayers and also secrete mucus [28]. Several
investigation have shown increased transport of proteins or peptides through Caco-2 and Calu-
3 monolayers by chitosan and TMC [29, 30], however the transport and uptake by Calu-3 cells
of virus particles has not been studied. Strong interactions with mucus (which is washed away
after incubation) may be a reason for the observed reduced uptake and binding of TMC-WIV by
these cells as compared to WIV [31]. Interestingly, this was not observed with the TMC-RA
coated WIV, despite its similar zeta potential compared to TMC-WIV. Perhaps due to steric
hindrance, the electrostatic interactions between TMC-RA and WIV are weaker than those
between TMC and WIV, resulting in better association properties for WIV in these mucusproducing
cells due to loss of the TMC-RA coating. This suggests that the intranasal stability in
the presence of mucus of the TMC-RA-WIV formulation may be inferior compared to the TMC-
WIV particle. This inferior stability for TMC-RA-WIV particles was also observed in saline
conditions [32] and may result in decreased protection against proteolytic enzymes.
Additionally, the results obtained by studying the transport of WIV over the Calu-3
monolayer (WIV>>TMC-RA-WIV>TMC-WIV) support the assumption that WIV particles are
too large to be transported through epithelial tight junctions, since the only formulation that
significantly decreased the TEER values (TMC-WIV) showed the lowest transport rate (Figure
6). As the negatively charged uncoated WIV was transported to the largest extent, interactions
between the coated particles and mucus may play a key role: the positively charged TMC- and
TMC-RA-WIV particles likely adhere to the negatively charged mucus and are therefore unable
to pass through the cell monolayer. Importantly, as in vivo i.n. immunization has shown that
TMC greatly enhances antigen immunogenicity, most likely by improved delivery [6, 22], our
results imply that the uptake by and transport through nasal epithelial cells is not the main
route for improved antigen delivery by TMC. This suggests that the uptake and transport of
TMC based particulate systems is mediated through M-cells [1, 3, 6] or direct DC sampling [2].
Interestingly, influenza viruses and influenza derivated virosomes are known to specifically
interact with M-cells [33] most likely due to recognition of sialic acid and galactose residues by
lectin receptors [34]. Additionally, N-acetyl glucosamine moieties, which are more abundant in
TMC-RA, are known to interact with several lectin receptors present on DCs and/or
macrophages [35-38]. However, the effect of coating of WIV with TMC(-RA) on these
interactions remains to be clarified. More advanced in vitro or ex vivo models [39] should be
developed to further study the transport and uptake of WIV particulate systems over mucosal
epithelial surfaces [40]. Possibly co-culturing Calu-3 cells with microfold (M)-cells may
107

Chapter 5A
improve the predictability of these models [21] for in vivo applications, as was observed for a
Caco-2/M-cell co-culture model [41].
Recently it was shown that TMC, both in particulate form and in solution, can exert
significant immunostimulatory effects on human monocyte derived DCs [41-43]. The
mechanism, however, remains unclear although suggestions were made about residual N-
acetylated glucosamine units capable of interacting with C-type lectin receptors present on
DCs [36, 43]. However, since the differences in adjuvanticity between TMC and TMC-RA coated
WIV were observed in mice, it is more appropriate to investigate the effect of TMC and TMC-RA
coated WIV on the maturation of murine DCs. Since TMC-RA contains a higher number of N-
acetyl glucosamine units, an altered interaction with DCs could be expected [36-38, 44]. Our
data, however, suggest that WIV is the main inducer of DC maturation and that there is no
additive effect of TMC or TMC-RA, or any effect of plain TMC or TMC-RA (Figure 7).
Importantly, murine DCs express different C-type lectin receptors on their surface than human
DCs and therefore adjuvants may have a different effect on murine or human DC-subsets [19].
Further investigations on human DCs with these formulations are therefore appropriate to
clarify these findings.
In summary, we found that TMC-RA was degraded to a higher extent in nasal washes than
TMC. In vivo fluorescence imaging did not reveal a significant difference in nasal clearance of
TMC-WIV and TMC-RA-WIV. In nasal cross-sections both TMC-WIV formulations were
similarly distributed in the nasal cavity, indicating that enhancing the interaction between the
antigen and the epithelial barrier alone is not sufficient for improving the immune response.
The uptake and binding of fluorescent WIV in Calu-3 cells was less pronounced when coated
with TMC than with TMC-RA or uncoated and both TMCs significantly decrease the transport
of WIV over a Calu-3 monolayer. Furthermore, experiments with murine BM-DCs indicated
that WIV is the main inducer of DC maturation, measured as CD40 expression, and that TMC
and TMC-RA do not have an additive effect. Our data suggest that the loss of adjuvanticity by
reacetylation of TMC might be due to the lower stability of TMC-RA-WIV in the nasal cavity,
rather than by any of the other factors studied in this paper.
Acknowledgement. This research was partially performed under the framework of TI
Pharma project number D5-106-1; Vaccine delivery: alternatives for conventional multiple
injection vaccines. E. Kaijzel and C. Löwik are acknowledged for their help with the nasal
residence time experiment.
108

Influence of N-Acetylation on the Adjuvanticity of TMC
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A. Bouwstra. Efficient induction of immune responses through intradermal vaccination with N-
trimethyl chitosan containing antigen formulations. J Control Release 142: 374-383 (2010).
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111

CHAPTER 5B
MATURATION OF HUMAN MONOCYTE
DERIVED DENDRITIC CELLS BY TRIMETHYL
CHITOSAN IS CORRELATED WITH ITS N-
ACETYL GLUCOSAMINE (GLCNAC) CONTENT
Rolf J. Verheul, Niels Hagenaars, Thomas van Es, Sven Bruins,
Bram Slütter, Wim E. Hennink, Wim Jiskoot.
Manuscript submitted

Chapter 5B
Abstract
N-acetylated glucosamine units or GlcNAcs present in trimethyl chitosan (TMC) have been
described to interact with several pathogen associated molecular pattern receptors involved in
the human innate immune response. The aim of this study was to compare the immunomodulatory
effects of TMC with a low (17%) and high (54%, TMC-RA) degree of acetylation or
GlcNAc content on the uptake and maturation of human monocyte derived dendritic cells (MO-
DCs) in vitro, using whole inactivated influenza virus (WIV) as antigen. The GlcNAc content of
TMC had no effect on the uptake of TMC(-RA) coated WIV by MO-DCs. Interestingly, TMC-RA,
either as a solution or when formulated with WIV, induced a much stronger DC maturation
than any of the other formulations, as judged from CD86 expression and cytokine release (IL-
10, TNF-α and IL-12p40 and IL-12p70). Since both IL-10 and IL-12p70 levels were elevated, no
polarization towards a Th1 or Th2 type immune response could be established. In conclusion,
as compared to TMC, TMC-RA has strong immuno-stimulatory effects in vitro on human MO-
DCs. This implies that the degree of N-acetylation or GlcNAc content may be critical for the
adjuvant effect of TMC in humans.
114

Maturation of Human DCs is Correlated with the GlcNAc Content
Introduction
Subunit or inactivated vaccines are generally safer but less effective than live attenuated
vaccines and therefore require potent adjuvants to elicit adequate immune responses [1, 2]. As
few adjuvants are approved for use in humans [3], there is a need for novel compounds. N,N,Ntrimethyl
chitosan (TMC), a quaternized chitosan derivative, has successfully been used for
mucosal and intradermal vaccination [4-7] and the structural properties of TMC like the
degree of quaternization (DQ), the degree of O-methylation (DOM), and the degree of N-
acetylation (DAc) can be varied during synthesis [8, 9]. In vitro evaluation showed that
increasing the DAc or N-acetyl glucosamine (GlcNAc) content of TMC resulted in a decrease in
cell-toxicity and better enzymatic degradation by lysozyme [9]. GlcNAc moieties are, as
pathogen-associated molecular patterns (PAMPs), known to interact with C-type lectins [10],
like DC-SIGN [11, 12], mannose receptors [13, 14], and toll-like receptor type 2 (TLR2) [15]
present on the surface of several antigen presenting cells (APCs), such as DCs and
macrophages [10]. However, after intranasal (i.n.) immunization with whole inactivated
influenza virus (WIV) in mice, a striking loss in adjuvanticity of TMC was observed when WIV
was formulated with reacetylated TMC (TMC-RA; DAc of 54%), compared to all other tested
TMCs with a low DAc (17%) [16]. Since no differences in the physico-chemical properties of
the formulations were observed, this lack of adjuvanticity had to be attributed to the higher
GlcNAc content of TMC-RA, however the precise mechanism still remains to be elucidated.
A recent study by our group showed no significant difference in activation of murine bone
marrow derived DCs after exposure to regular TMC or TMC-RA (Chapter 5A). Interestingly,
human DCs may be different from mouse DCs as murine and human DCs express different
receptors on their surface [11, 17]. Therefore, in this study we further explored the differences
in immuno-stimulatory effect between reacetylated TMC-RA (GlcNAc content 54%) and
conventional TMC (GlcNAc content 17%) using human monocyte derived DCs (MO-DCs).
Materials and Methods
Materials. Chitosan with a DAc of 17% (determined with 1 H-NMR as described in [9] and a
number average molecular weight (M n) and weight average molecular weight (M w) of 28 and
43 kDa, respectively, as determined by gel permeation chromatography (GPC) as described in
[8], was purchased from Primex (Siglufjordur, Iceland). Acetic anhydride, sodium borohydrate,
formic acid, formaldehyde 37% (stabilized with methanol), deuterium oxide, sodium acetate,
115

Chapter 5B
acetic acid (anhydrous), sodium hydroxide and hydrochloric acid were obtained from Sigma-
Aldrich Chemical Co (Zwijndrecht, The Netherlands). Iodomethane 99% stabilized with copper
was obtained from Acros Organics (Geel, Belgium). Purified, cell culture-grown (Madin-Darby
Canine Kidney (MDCK) cells, β-propiolacton (BPL)-inactivated influenza virus, strain
A/PR/8/34 (WIV) was from Nobilon International BV (Boxmeer, The Netherlands). Alexa
Fluor® 488 labeling kit was purchased from Invitrogen (Breda, the Netherlands).
Trypsin/EDTA 10x, Plain DMEM (Dulbecco’s modification of Eagle’s medium, with 3.7 g/l
sodium bicarbonate, 1 g/l L-glucose, L-glutamine) and antibiotics/antimycotics (penicillin,
streptomycin sulphate, amphotericin B) were from PAA Laboratories GmbH (Pasching,
Austria) and MEM with 0.292 g/L L-glutamine, 1 g/L glucose, 2.2 g/L NaHCO 3 was from
(Invitrogen, Breda, The Netherlands). All other chemicals used were of analytical grade.
Synthesis and characterization of methylated chitosans. O-methyl free N,N,Ntrimethylated
chitosans (TMCs) with a DQ of about 45% and varying DAc were synthesized
from chitosan as described previously [9]. The DQ and DAc of the TMCs were determined with
1H NMR on a Varian INOVA 500 MHz NMR spectrometer (Varian Inc., Palo Alto, Ca, USA) at 80
°C in D 2O [9]. Furthermore, M n and M w of the TMCs were determined, as described previously
[8], by GPC on a Viscotek system detecting refractive index, viscosity and light scattering. A
Shodex OHPak SB-806 column (30 cm) was used with 0.3 M sodium acetate pH 4.4 (adjusted
with acetic acid) as running buffer. The structural characteristics of the synthesized TMCs are
summarized in Table 1.
Table 1. Polymer characteristics of TMC and TMC-RA.
Abbreviation M n (kDa) M w (kDa) DQ (%) DAc (%)
TMC 36 75 43 17
TMC-RA 43 83 44 54
In vitro studies on human MO-DCs. Immature DCs were cultured as described before [18].
In short, human blood monocytes were isolated from buffy coats by use of a Ficoll gradient and
subsequently a Percoll gradient. Purified monocytes were differentiated into immature DCs in
the presence of interleukin-4 (IL-4, 500 U/ml) and granulocyte-macrophage colonystimulating
factor (GM-CSF, 800 U/ml).
Immature DCs (day 6) were seeded (50,000 cells/well) in a 96-well plate in a total volume of
100 μl RPMI 10% FCS in the presence of TMC (0.001-30 μg/ml), TMC-WIV formulations at 1:1
and 5:1 (w/w) ratios (0.001-6 μg/ml WIV) or lipopolysaccharide (LPS) (0.01-1000 ng/ml) as a
116

Maturation of Human DCs is Correlated with the GlcNAc Content
positive control. As described elsewhere, TMC-WIV formulations had similar physico-chemical
properties (size and zeta potential) independent of the type or amount of TMC used (Chapter
5A). Therefore, it can be stated that all formulations were physically similar, except for the
amount of free polymer.
Uptake and binding of TMC-coated WIV formulations by human MO-DCs. The uptake
and binding of (TMC coated) WIV by human MO-DCs were studied with AlexaFluor-488®
labeled WIV. WIV was labeled with the AlexaFluor-488® labeling kit using the procedure
provided by Invitrogen. DCs were incubated for 1 h at 37 or 4 °C with the labeled WIV
formulations and flow cytometry was used to determine cell fluorescence (n=2).
Maturation and cytokine production of human MO-DCs. Human MO-DCs were incubated
for 16 h at 37 °C with the various polymers with or without WIV. The cells were washed
extensively with PBS and incubated for 30 min with anti-CD86-APC (BD, Breda, The
Netherlands). DC maturation was determined by analyzing cell-surface expression of costimulatory
molecules (CD86) by flow cytometry. After 16 h culture supernatants were
harvested and frozen at −80 °C until analysis. The supernatants were analyzed for the presence
of the cytokines IL-10 and IL-12p40, IL12p70 and TNFα by ELISA (Biosource International,
CA). For the DC maturation studies, 4 donors were used. For the cytokine experiments, 2
donors were used.
Results
Uptake and binding of (TMC coated) WIV by human MO-DCs. The influence of TMC and
TMC-RA on the uptake and binding of fluorescently labeled WIV by human MO-DCs was
studied. After incubation for 1 h, all formulations showed a concentration dependent uptake
and binding (Figure 1). At 37 °C (Figure 1A) much higher intensities were observed than at 4
°C (Figure 1B), indicating active uptake of WIV. Surprisingly, uncoated WIV showed a slightly
higher binding and uptake than TMC or TMC-RA coated WIV.
117

Chapter 5B
WIV binding/uptake
(MFI)
A
200
150
100
50
WIV
TMC-WIV
TMC-RA-WIV
WIV binding/uptake
(MFI)
B
20
15
10
5
WIV
TMC-WIV
TMC-RA-WIV
0
3.0 1.5 0.8 0.4 0.2
Conc. WIV (μg/ml)
0
3.0 1.5 0.8 0.4 0.2
Conc. WIV (μg/ml)
Figure 1. WIV uptake and/or binding by human MO-DCs after incubation for 1 hour with fluorescently
labeled (TMC or TMC-RA coated at w/w ratio 5:1) WIV at 37 °C (A) and 4°C (B). Results from one
representative donor are shown (n=2). TMC(-RA)-WIV formulations at w/w ratio 1:1 showed a similar
outcome (results not shown).
Maturation and cytokine production by human MO-DCs. The effect of TMC and TMC-RA
on MO-DC maturation was studied by quantifying maturation marker CD86 and production of
cytokines IL-10, TNF-α and IL-12. Incubation of human MO-DCs with TMC solutions and TMC-
WIV formulations ((w/w) ratio 5:1) revealed a striking difference in CD86 expression between
TMC-RA and TMC (Figure 2).
CD 86 expression
(MFI)
A
300
200
100
TMC
TMC-RA
CD 86 expression
(MFI)
B
500
400
300
200
100
WIV
TMC-WIV
TMC-RA-WIV
0
15.0
7.5
3.8
1.9
0.9
Medium
1 μg/ml LPS
0
3.0
1.5
0.8
0.4
0.2
Medium
1 μg/ml LPS
Conc. TMC (μg/ml)
Conc. WIV (μg/ml)
Figure 2. CD 86 expression by human MO-DCs after incubation with TMC and TMC-RA solutions (A) and
by TMC-WIV and TMC-RA-WIV (5:1 w/w ratio) formulations (B). Results from one representative donor
are shown (n=4).
118

Maturation of Human DCs is Correlated with the GlcNAc Content
TMC-RA induced a much higher concentration dependent CD86 expression than TMC, for
both soluble TMC-RA (Figure 2A) and TMC-RA co-formulated with WIV (Figure 2B).
Furthermore, TMC-RA-WIV also stimulated, in a concentration dependent manner, the
secretion of pro-inflammatory cytokines IL-10, TNF-α, IL12p40 and IL-12p70 more strongly
than the other TMC-WIV formulations or naked WIV did (Figure 3). As compared to TMC-RA-
WIV, TMC-RA in solution induced similar cytokine levels (results not shown). Importantly, an
endotoxin dose-effect calibration on DCs verified that the observed effects on DCs cannot be
attributed to the relatively low endotoxin levels in the TMCs (≤0.16 EU/µg TMC), as measured
with a LAL test. As TMC-RA showed very low in vitro cytotoxicity compared to TMC [9], it is
highly unlikely that the maturation of DCs by TMC-RA is attributable to its toxicity. Since both
IL-10 (as marker for a Th2 type response) and IL-12p70 (as marker for a Th1 type response)
were elevated by TMC-RA (Figure 3), no polarization towards a Th1 or Th2 type of immune
response could be established. These results suggest that TMC-RA has much stronger intrinsic
adjuvant effect than conventional TMC due to its higher GlcNAc content and that this enhanced
activation does not seem to be the result of increased uptake of antigen.
Discussion
In general, cationic nanoparticles are better taken up by APCs than their anionic
counterparts [19, 20], likely due to improved interaction with the negatively charged cell
membrane. Despite being anionic, uncoated WIV demonstrated slightly better uptake
compared to the cationic, TMC-coated WIV, indicating that besides electrostatic interactions
other interactions may play a role. It is known that sialic acid and galactose residues on the
surface of influenza viruses can interact with C-type lectin receptors of APCs and enhance
uptake of these particles [21, 22]. The coating of WIV with TMC or TMC-RA may hinder these
interactions. Additionally, the GlcNAc units present in TMC(-RA) may have altered the uptake
mechanism [10, 13]. This obviously resulted in a slightly decreased uptake of TMC(-RA)-coated
WIV compared to plain WIV. This effect was similar for TMC (DAc 17%) and TMC-RA (DAc
54%), indicating that the GlcNAc content of TMC does not critically influence antigen uptake.
In agreement with our present results, in previous studies the extent of antigen uptake did
not appear to be a major determinant for DC maturation [5, 23, 24]. However, previous studies
with TMC with low DAc (

6.0
0.2
3.0
6.0
3.0
6.0
3.0
1.5
0.2
Chapter 5B
(with a low DAc) has only a limited intrinsic adjuvant effect on human MO-DCs compared to
TMC-RA (high DAc). TMC, as coating of WIV, was not capable to induce additional MO-DC
maturation compared to WIV alone. The different effects of TMC on the maturation of MO-DCs
may be explained by the nature of the antigens used for these studies: WIV has a
nanoparticulate structure (appr. 180 nm) and already induces (minor) DC activation without
TMC. Ovalbumin, as a soluble antigen, has no effect on DC maturation and mixing with TMC
will lead to (minor) self-assembly into larger structures due to charge interactions [5, 25]. In
the latter case, DC activation by TMC, either due to changes in the particulate nature of a
formulation [1, 26] or due to a direct effect of the TMC [5, 24], will be more noticeable.
IL-10 (pg/ml)
A
120
100
80
60
40
20
WIV
TMC-WIV
TMC-RA-WIV
1 μg/ml LPS
TNFα (pg/ml)
B
10000
7500
5000
2500
WIV
TMC-WIV
TMC−RA-WIV
1 μg/ml LPS
0
0
0.8
0.4
6.0
1.5
0.8
0.4
0.2
Conc. WIV (μg/ml)
Conc. WIV (μg/ml)
IL-12p40 (pg/ml)
C
10000
7500
5000
2500
WIV
TMC-WIV
TMC-RA-WIV
1 μg/ml LPS
IL-12p70 (pg/ml)
D
50
40
30
20
10
WIV
TMC-WIV
TMC-RA-WIV
1 μg/ml LPS
0
3.0
1.5
0.8
0.4
0
1.5
0.8
0.4
0.2
Conc. WIV (μg/ml)
Conc. WIV (μg/ml)
Figure 3. Expression of IL-10 (A); TNFα (B); IL-12p40 (C) and IL12p70 (D) by human MO-DCs after
incubation with TMC-WIV formulations (w/w ratio 5:1). The figure shows results from one
representative donor (n=2).
TMC-RA, as coating of WIV or solution, lacked immuno-stimmulatory effects in murine bonemarrow
derived DCs (Chapter 5A). In contrast, our present data show that TMC-RA induces
maturation of human monocyte derived DCs. This suggest that TMC-RA may activate human
120

Maturation of Human DCs is Correlated with the GlcNAc Content
DCs by binding to a receptor that is not present on murine DCs. GlcNAc moieties are known to
interact with species-independent C-type lectins, like mannose receptors [10, 13, 14], and tolllike
receptor type 2 (TLR2) [15] present on the surface of APCs. DC-SIGN is a human C-type
lectin not present on murine DCs [11, 17], but experiments with DCs derived from DC-SIGN
transgenic mice showed no immuno-stimulatory effect of TMC-RA (unpublished results). This
suggests that other GlcNAc, human specific, receptors may play a role. Further studies should
be done to investigate the mechanism of human MO-DC activation by TMC-RA in more detail.
Conclusion
As opposed to TMC with a low GlcNAc content, TMC-RA with a high GlcNAc content (54%),
both as solution and as coating of WIV, was shown to be capable of inducing maturation of
human MO-DCs. This maturation was not a result of increased antigen uptake. Both IL-10 and
IL-12p70 levels were elevated after stimulation with TMC-RA, indicating a mixed Th1/Th2
type immune response. These immuno-stimulatory characteristics, together with its low
toxicity profile, make TMC-RA a promising adjuvant for future vaccinations in humans.
Acknowledgement. This research was partially performed under the framework of TI
Pharma project number D5-106-1; Vaccine delivery: alternatives for conventional multiple
injection vaccines. Nobilon International BV (Boxmeer, The Netherlands) is acknowledged for
supplying the WIV.
121

Chapter 5B
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14. Y. Shibata, W. James Metzger, Q.N. Myrvik. Chitin particle-induced cell-mediated immunity is
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9 (2006).
123

CHAPTER 6
TAILORABLE THIOLATED TRIMETHYL
CHITOSANS FOR COVALENTLY STABILIZED
NANOPARTICLES
Rolf J. Verheul, Steffen van der Wal, Wim E. Hennink.
Biomacromolecules 2010, 11, 1965-1971

Chapter 6
Abstract
A novel four-step method is presented to synthesize partially thiolated trimethylated
chitosan (TMC) with tailorable degree of quaternization and thiolation. First, chitosan was
partially N-carboxylated with glyoxilic acid and sodium borohydride. Next, the remaining
amines were quantitatively dimethylated with formaldehyde and sodium borohydride and
then quaternized with iodomethane in NMP. Subsequently, these partially carboxylated TMCs
dissolved in water were reacted with cystamine at pH 5.5 using EDC as coupling agent. After
addition of DTT and dialysis, thiolated TMCs were obtained varying in degree of quaternization
(25-54%) and degree of thiolation (5-7%) as determined with 1 H-NMR and Ellman’s assay. Gel
permeation chromatography with light scattering detection indicated limited intermolecular
crosslinking. All thiolated TMCs showed rapid oxidation to yield disulfide crosslinked TMC at
pH 7.4 while the thiolated polymers were rather stable at pH 4.0. Using Calu-3 cells, XTT and
LDH cell viability tests showed a slight reduction in cytotoxicity for thiolated TMCs as
compared to the non-thiolated polymers with similar DQs. Positively charged nanoparticles
loaded with fluorescently labeled ovalbumin were made from thiolated TMCs and thiolated
hyaluronic acid. The stability of these particles was confirmed in 0.8 M NaCl, in contrast to
particles made from non-thiolated polymers which dissociated under these conditions
demonstrating that the particles were held together by intermolecular disulfide bonds.
Chitosan
+
Thiolated TMC
N(CH 3 ) 3
HS
Thiolated
Hyaluronic
acid
+
―SH
+
+
―SH
+
-
-
--
+
―SH
―SH
+
+
―SH
Covalently stabilized
nanoparticles
126

Thiolated TMC for Covalently Stabilized Nanoparticles
Introduction
Chitosan, a polysaccharide consisting of β1→4-D-glucosamine and β1→4 N-acetyl D-
glucosamine units, is under investigation for various biomedical and pharmaceutical
applications [1-3]. However, its poor aqueous solubility and loss of penetration enhancing
activity above pH 6 is a major drawback for its use at physiological conditions. N,N,N,-
trimethylated chitosan (TMC), a partially quaternized derivative of chitosan, is water-soluble
at physiological pH. TMC has been widely studied in the biomedical field as drug, antigen and
gene delivery vehicle. It has been shown in several in vitro and in vivo studies that TMC has low
toxicity, possesses muco-adhesive properties and can facilitate the uptake of small drug
molecules as well as proteins via various mucosal routes [4-11] Several studies have suggested
an optimal degree of quaternization (DQ) of 40-50% for mucosal transport and gene delivery
[12-16]. In addition, molecular weight [17, 18] and, as recently demonstrated, O-methylation
[19] and degree of N-acetylation [20] also have a major impact on the physical and biological
characteristics of TMC.
Introduction of thiol-moieties will further broaden the potential pharmaceutical applications
of TMC by enhancing its muco-adhesive potential and allowing further chemical derivatization
reactions via reducible disulfide-bridges [11, 21-24]. Yin et al. [25] synthesized thiolated TMC
by coupling cysteine to the remaining free –NH 2 units of the polymer. However, this method is
dependent on available free amines remaining after quaternization, and therefore this
synthetic route is only applicable for TMC with a low DQ (up to 30 %).
Nanoparticulate systems have superior penetration enhancing characteristics in mucosal
protein and/or antigen delivery over polymer solutions [1, 26, 27]. Although often
tripolyphosphate (TPP) is used as a crosslinker to form TMC nanoparticles via ionic gelation,
recent results by Sayın et al. [28] showed that nasal immunization with tetanus toxoid loaded
TMC:mono-N-carboxymethyl chitosan nanoparicles resulted in superior antibody titers
compared to the TMC/TPP particles. The physico-chemical stability of these complexes is
dependent on the characteristics of the polyelectrolytes used and they often have a limited
stability in physiological salt conditions [29, 30] or at low pH [21]. Thiolated chitosans, and
more recently, thiolated TMCs complexed with insulin or using TPP as crosslinker resulted in
nanoparticles stabilized via intracellularly degradable disulfide bridges [21, 25, 31]. Combining
the cationic, thiolated TMC polymer with the muco-adhesive [22], anionic, thiolated hyaluronic
acid to form stabilized nanoparticles may further improve these mucosal delivery vehicles.
Further, it can be anticipated that the charge of the particles can be tuned by the
127

Chapter 6
TMC:hyaluronic acid ratio and that both anionically and cationically charged proteins can be
loaded.
In this paper, a novel synthetic method is described to yield thiolated TMCs with a high DQ.
The different thiolated TMCs were physico-chemically characterized, evaluated in in vitro
cytotoxicity assays and used for preparation of covalently stabilized nanoparticles with
thiolated hyaluronic acid.
Materials and Methods
Materials. Chitosan with a residual degree of acetylation of 17% (determined with 1 H-NMR
as described below) and a number average (M n) and weight average molecular weight (M w) of
28 and 43 kDa, (determined with GPC-TD as below), respectively, was purchased from Primex
(Siglufjodur, Iceland). Sodium borohydride, formaldehyde 37% (stabilized with methanol),
glyoxilic acid monohydrate, cystamine dihydrochloride, dithiotreitol (DTT), 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide HCl (EDC), L-cysteine HCl monohydrate, deuterium oxide,
sodium 3’-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene
sulphonic acid hydrate (XTT), N-methyl dibenzopyrazine methylsulphate (PMS), sodium
acetate, acetic acid (anhydrous), sodium hydroxide and hydrochloric acid were obtained from
Sigma-Aldrich Chemical Co. Fluorescently labeled ovalbumin (OVA-FITC), minimal essential
medium (MEM) and fetal calf serum (FCS) were obtained from Invitrogen (Breda, The
Netherlands). Sicapent was ordered from Merck (Darmstadt, Germany). Iodomethane 99%
stabilized with copper was obtained from Acros Organics (Geel, Belgium). 5,5-dithio-bis-(2-
nitrobenzoic acid) (Ellman’s reagent) was purchased from Pierce (Rockford, IL, USA). Linear
polyethylenimine (PEI) (22kDa) was synthesized according to Thomas et al [32]. TMCs with
DQs of 30% (M n 33 kDa, M w 59 kDa) and 56% (M n 37 kDa, M w 78 kDa) were synthesized and
characterized as described previously [19]. HA-SH with a M n of 39.8 kDa, PDI of 1.26 and a
degree of thiolation of 52% was synthesized as described elsewhere [33]. All other chemicals
used were of analytical grade.
Synthesis of Partially N-Carboxylated Chitosan. Chitosan (degree of acetylation 17%) was
used as obtained and selective, partial N-carboxylation was carried out according to a
previously described method with some adjustments [10, 13]. Briefly, chitosan (10 g) was
dissolved in 1% (v/v) acetic acid (300 ml). Then, 2.3 g glyoxylic acid was added and pH was
raised to 4.5 with 1 M NaOH. Subsequently, 3 g of sodium borohydride dissolved in H 2O (5%
128

Thiolated TMC for Covalently Stabilized Nanoparticles
w/v) was added drop wise over a period of 3 hours in portions of 500 mg re-adjusting the pH
with 1 M HCl to 4.5 after each addition. Finally, the partially carboxylated chitosan (CS-COOH)
was precipitated by dropping the reaction mixture into an ethanol/diethyl ether mixture
(50/50 v/v) and the product was extensively washed with diethyl ether and dried overnight.
Synthesis of Dimethylated, Partially N-Carboxylated Chitosan. N,N,-Dimethylated,
partially N-carboxylated chitosan (DMC-COOH) was synthesized from CS-COOH as described
previously for the synthesis of N,N,-dimethylated chitosan [20, 34]. In short, CS-COOH (10 g)
was dissolved in 500 ml of 1% acetic acid (v/v). Next, 20 ml of 37% formaldehyde solution was
added, the pH was adjusted to 4.5 with 1M NaOH and the mixture was stirred for 30 minutes at
room temperature. Subsequently, sodium borohydride (5 g) was added in portions of 500 mg
over a period of 5 hours, adjusting the pH to 4.5 with 1M HCl after each addition. After
precipitation with acetone, the product was washed extensively with acetone and dried
overnight. To obtain quantitative dimethylation a second methylation step was performed
using the procedure described above.
Synthesis of O-Methyl free, Trimethylated, Partially N-Carboxylated Chitosan. DMC-
COOH was reacted with iodomethane to yield trimethylated, partially N-carboxylated chitosan
(TMC-COOH). To prevent O-methylation, the reaction of DMC with iodomethane was done in
NMP, without the addition of a base catalyst. TMC-COOH with a degree of quaternization of
around 25% or 54% were obtained by the method described previously [19]. In detail, DMC-
COOH (2.5 g) was dissolved in 80 ml deionized water and the pH was adjusted to 11 with a 1 M
solution of NaOH, resulting in gel formation. Then, the gel was washed with water and
subsequently with acetone. To remove residual solvents, the DMC was dried under vacuum for
2 hours. Next, DMC-COOH was suspended in 100 ml NMP followed by the addition of 5 or 15
ml iodomethane. The dispersion was stirred at 40°C for 40 hours and subsequently dropped
into 400 ml of an ethanol/diethyl ether mixture (50/50 v/v) to precipitate the formed TMC-
COOH, which was collected by centrifugation and subsequently extensively washed with
diethyl ether. After drying overnight at room temperature, the obtained TMC-COOH was
dissolved in 100 ml of an aqueous 10% NaCl solution for ion-exchange. Finally, the TMC-COOH
was dialyzed against 1% NaCl in diluted HCl (pH 4) for 2 days followed by dialysis against
diluted HCl (pH 4) for another 2 days (changing buffer twice daily). The polymer solution was
filtered through a 0.8 µm filter and collected after freeze-drying.
129

Chapter 6
Synthesis of O-Methyl Free, Trimethylated, Partially Thiolated Chitosan. Thiolated TMC
was synthesized by first coupling cystamine to the carboxylic acid moieties of TMC-COOH
using EDC as coupling agent followed by reduction of the S-S bonds of the coupled cystamine
groups by dithiothreitol. In detail, TMC-COOH (1 g) was dissolved in deionized water at a
concentration of 100 mg/ml. Then, five hundred milligram of cystamine dihydrochloride was
added (molar ratio COOH:cystamine approx. 1:4). After complete dissolution, EDC was added
to the reaction mixture (final concentration of 200 mM) to activate the carboxylic acid groups
of the polymer and the pH was adjusted to 5.5 with 1 M NaOH. After reacting for 6 hours at
room temperature on a roller bank, pH was raised to 8 with 1 M NaOH and DTT was added in
final concentration of 100 mM. After approximately 2 hours, NaCl was added to the reaction
mixture (final concentration 1% (g/v)), and the pH was adjusted to 4 with 1M HCl. The
resulting solution was dialyzed against 1% NaCl in diluted HCl (pH 4) for 2 days changing
buffer twice daily followed by dialysis against diluted HCl (pH 4) for another 2 days (changing
buffer twice daily). Dialysis was performed at 4°C. Finally, the solution was filtered through a
0.8 µm filter and the polymers were collected after freeze-drying. To improve the reduction of
remaining disulfide bonds, TMC-SH obtained after this first cycle was subjected to a second
reduction cycle with DTT as described above.
Determination of the Degrees of Carboxylation, Dimethylation, Acetylation and
Quaternization. The 1 H-NMR spectra of the various chitosan derivatives were recorded with
a Varian INOVA 300MHz NMR spectrometer (Varian Inc., Palo Alto, Ca, USA) at 25°C in D 2O.
The degree of carboxylation of CS-COOH was calculation as follows:
D Carb = [[CH 2]/[H2-H6] x 6/2] x 100%
Here, [CH 2] is the integral of the two hydrogens of the N-carboxymethyl groups at 3.2 ppm [35,
36] and [H2-H6] is the integral corresponding the six protons bound to the C-2 to C-6 ring
carbons between 3.9 and 3.0 ppm (without the signal observed at 3.2 ppm).
The degree of dimethylation (DDM) of the DMC-COOH was calculated as follows [19]:
DDM = [(CH 3) 2]/[H2-H6] x 100%
130

Thiolated TMC for Covalently Stabilized Nanoparticles
Here, [(CH 3) 2] is the integral of the hydrogens of the dimethyl amino groups at 2.9 ppm and
[H2-H6] is the integral corresponding to the six protons bound to the C-2 to C-6 ring carbons
between 3.9 and 3.0 ppm (without the signal observed at 3.2 ppm).
The 1 H-NMR spectra of the carboxylated and thiolated TMCs were recorded with a Varian
INOVA 500MHz NMR spectrometer (Varian Inc., Palo Alto, Ca, USA) at 80°C in D 2O. The DQ and
degree of dimethylation (DDM) of the TMC-COOHs and TMC-SHs were calculated as previously
described [4, 37, 38]:
DQ = [[(CH 3) 3]/[H] × 1/9] × 100%
DDM = [[(CH 3) 2]/[H] × 1/6] × 100%
Here, [(CH 3) 3] and [(CH 3) 2] are the integrals of the hydrogens of the trimethylated amino
groups at 3.3 ppm and the dimethylated amino groups at 2.9 ppm, respectively. [H] is the
integral of the H-1 peaks between 4.7 and 5.7 ppm; the signal related to the hydrogen atoms
bound to the C-1 ring carbon of TMC.
The degree of acetylation (DAc) of the chitosan derivatives was calculated as described
previously [39]:
DAc = [[CH 3]/[H] x 1/3] x 100
Here, [CH 3] is the integral of the three hydrogens of the acetyl groups at 2.0 ppm and [H] is the
integral of the H-1 peaks between 4.7 and 5.7 ppm; the signal related to the hydrogen atoms
bound to the C-1 ring carbon of TMC.
Determination of Free Thiol Group Content. The degree of thiolation was quantified with
5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB, Ellman’s reagent). DTNB was dissolved in 0.1 M
sodium phosphate, pH 8.0 containing 1 mM EDTA in a final concentration of 100 μg/ml. TMC-
SHs were dissolved in 0.1 M sodium phosphate, pH 8.0 containing 1 mM EDTA in a
concentration of 2.5 mg/ml. Then, 20 μl of this solution was mixed with 180 μl of DTNB
reagent solution in a microplate and after incubating for 15 minutes at room temperature the
absorbance was measured with a microplate reader at a wavelength of 405 nm (Bio-Rad
Novapath, Hercules, Ca, USA). Cysteine standards were used to calculate the amount of free
thiol moieties in the polymer. The degree of thiolation (D thiol) was calculated using the
estimated average molecular weight per glucosamine unit.
131

Chapter 6
Determination of Total Thiol Content. The total thiol content (free thiol moieties and
disulfides) on the polymers was determined according to a slightly modified method described
by Hombach et al. [40]. In detail, TMC-SHs were dissolved in 50 mM phosphate buffer pH 7.0 (1
mg/ml). Subsequently, 400 μl of a freshly prepared 10% (w/v) sodium borohydride solution in
deionized water was added to 500 μl of polymer solution and the mixture was slightly shaken
at 37°C for 60 minutes. Thereafter, 1 ml of 1 M HCl was added to decompose the remaining
sodium borohydride and after 10 minutes the pH was raised to 8 by adding 1 ml of 0.5 M
phosphate buffer pH 8.0. Then, 100 μl of DTNB reagent solution (4 mg/ml in 0.5 M sodium
phosphate, pH 8.0) was added and after incubating for 15 minutes at room temperature, a 200
μl sample was transferred into a microplate. The absorbance was measured with a microplate
reader at a wavelength of 405 nm (Bio-Rad Novapath, Hercules, Ca, USA). Cysteine standards
prepared in the same way as the samples were used to calculate the total amount of thiol
moieties (as free thiols or disulfides) on the polymer.
Thiol Oxidation. TMC-SH was dissolved in 100 mM acetate buffer pH 4.0, 100 mM acetate
buffer pH 5.0, 100 mM phosphate buffer pH 6.2, or 100 mM phosphate buffer pH 7.4, in a final
concentration of 5 mg/ml. Samples were incubated at 37°C under permanent shaking and
aliquots were taken at different time points. Immediately after collecting the aliquots, the
amount of remaining thiol moieties was determined with Ellman’s reagent as described before.
Determination of M n and M w of the Different Polymers. Polymers were placed overnight
in a vacuum oven at 40 o C in the presence of Sicapent to remove residual water and
subsequently dissolved in 0.3 M sodium acetate pH 4.4. The number average weight (M n) and
weight average weight (M w) of chitosan and the various TMCs were determined by gel
permeation chromatography (GPC) on a Viscotek-triple detection system using a Shodex
OHPak SB-806 column (30 cm) and 0.3 M sodium acetate pH 4.4 (adjusted with acetic acid) as
running buffer [41]. Data from the laser photometer (λ = 670 nm) (right (90 0 ) and low (7 0 )
angle light scattering), refractive index detector and viscosity detector were integrated using
the provided Omnisec-software to calculate the M n, M w of the different samples. Pullulan (M n =
102 kDa, M w = 106 kDa) obtained from Viscotek Benelux (Oss, the Netherlands) was used for
calibration.
XTT Cytotoxicity Assay. Calu-3 cells (ATCC, Teddington, UK) were seeded into a 96-well
plate at a density of 2x10 4 cells per well and incubated for 2 days at 37°C, CO 2 5% in culture
132

Thiolated TMC for Covalently Stabilized Nanoparticles
medium (MEM with L-glutamine and sodium pyruvate supplemented with
antibiotics/antimycotics and 10% FCS). The medium was removed and the cells were
incubated for 2.5 hours with 100 µl TMC solutions in MEM (TMC concentrations were 0.01, 0.1,
1 and 10 mg/ml, pH set at 7 with 0.1 M NaOH). Linear PEI (0.1 and 0.01 mg/ml) was used as
positive control and cells incubated with MEM were used as reference. Thereafter, the
solutions were removed and the cells washed MEM. Then, 100 µl of MEM containing 10% FCS
was added to the cells together with 50 µl of a freshly prepared solution of 1 mg/ml XTT in
MEM containing 25 µmol PMS as electron-coupling reagent. Cells were incubated for 1 hour at
37°C in 5% CO 2 and the absorbance was read at 490 nm with 655 nm as reference wavelength.
Obtained values of the samples were related to the mitochondrial activity of Calu-3 cells
incubated with MEM only [42].
LDH Cytotoxicity Assay. Calu-3 cells were seeded into a 96-well plate at a density of 2x10 4
cells per well and incubated for 2 days at 37°C, CO 2 5% in culture medium (MEM with L-
glutamine and sodium pyruvate supplemented with antibiotics/antimycotics and 10% FCS).
The medium was removed and the cells were washed with MEM and incubated for 2.5 hours
with 100 µl TMC solutions in MEM (TMC concentrations were 0.01, 0.1, 1 and 10 mg/ml, pH set
at 7 with 0.1 M NaOH). After incubation, the concentration of lactate dehydrogenase (LDH)
present in the supernatant of the samples was determined with the Cytotoxicity Detection Kit-
Plus (Roche Diagnostics, Mannheim, Germany) by measuring absorbance at 490 nm with 655
nm as a reference wavelength. A calibration curve was made with the lysis buffer provided by
the manufacturer, setting the LDH concentration measured with the undiluted lysis buffer at
100% LDH release. Cells incubated with linear PEI (0.1 and 0.01 mg /ml) were used as control.
One-way ANOVA with Tukey’s post-test was used for comparison.
Preparation and Characterization of Covalently Stabilized Nanoparticles.
Covalently stabilized nanoparticles loaded with fluorescently labeled ovalbumin were
prepared with TMC-SH and thiolated hyaluronic acid (HA-SH). TMC-SH with a DQ 25% and
D thiol 5% or a DQ of 54% and D thiol 6% were dissolved in 10 mM HEPES pH 7.4 at 1 mg/ml and
subsequently mixed with 2.5 mg/ml fluorescently labeled ovalbumin (OVA-FITC) in 10 mM
HEPES pH 7.4 at a weight ratio of 10:1 (TMC-SH:OVA-FITC) under magnetic stirring. Then, HA-
SH (0.5 mg/ml in 10 mM HEPES pH 7.4) was added drop-wise yielding an opalescent
nanoparticle dispersion which was incubated at 37°C for 3 hours to allow disulfide formation.
As a control nanoparticles with non-thiolated TMC (DQ 30% or 56%), OVA-FITC and HA were
133

Chapter 6
prepared in a similar way. After 3 hours of incubation, the nanoparticle dispersions were
mixed with either 10 mM HEPES pH 7.4 or 0.8 M NaCl in 10 mM HEPES pH 7.4 and
subsequently incubated at 37°C for 1 hour.
Particle size was measured by dynamic light scattering (DLS) using a Malvern ALV CGS-3
(Malvern Instruments, Malvern, UK). DLS results are given as a z-average particle size
diameter and a polydispersity index (PDI). The zeta-potential of the nanoparticles was
measured in 10 mM HEPES pH 7.4 using a Zetasizer Nano (Malvern Instruments, Malvern, UK).
The protein loading was determined by assaying the concentration of OVA-FITC in the
supernatant obtained after centrifugation (10 min at 13000 rpm; Biofuge Pico, PP1/97 #3324;
Heraeus Instruments, Osterode, Germany) of the nanoparticle dispersion using a Fluostar
Optima with 488 nm as excitation wavelength and emission was measured at 520 nm (BMG
Labtech, Offenburg, Germany). The OVA-FITC association efficiency (AE%) was calculated
using the following equation:
AE% = (total OVA-FITC ― OVA-FITC in supernatant)/ total OVA-FITC x 100%
134

Thiolated TMC for Covalently Stabilized Nanoparticles
Scheme 1. Synthetic route for thiolated TMCs.
135

Chapter 6
Results and discussion
Synthesis and Characterization of Thiolated TMC. Thiolated TMCs with tailorable degree
of quaternization (DQ) and thiolation (D thiol) were obtained as depicted in Scheme 1. First,
chitosan was selectively N-carboxylated with glyoxylic acid and sodium borohydride to a
degree of carboxylation of 12% as determined with 1 H-NMR. Higher degrees of carboxylation
could be achieved by using larger quantities of glyoxylic acid (results not shown), however, a
further increase in carboxylic acid moieties that ultimately leads to a higher degree of
thiolation was not considered useful for the foreseen applications; about 10-20 thiol groups
per polymer chain is sufficient for intermolecular disulfide-bridge formation. Partially
carboxylated chitosan was subsequently quantitatively dimethylated using formaldehyde and
sodium borohydride, as confirmed with 1H-NMR. Reaction of dimethylated, partially
carboxylated chitosan (DMC-COOH) with different amounts of iodomethane (5 or 15 ml) in
NMP resulted in TMC-COOH with DQs of 25% and 54%, respectively, indicating tailorability of
the DQ (Table 1). 1 H-NMR analysis also demonstrated that neither O-methylation nor loss of
residual N-acetylated groups was observed after quaternization, in contrast to the synthesis
method applied by others using a strong alkaline component to induce quaternization [38].
TMC-COOH was not soluble at pH 7.4 confirming the zwitterionic character of this polymer.
Table 1. Characteristics of chitosan and the synthesized thiolated TMCs.
DQ DAc
Total thiol
amount
Amount of
free –SH D thiol M n (kDa) M w (kDa)
(μmol/g) (μmol/g)
Chitosan - 17% - - - 28 43
TMC-SH
High DQ
54% 17% 529 (±2) 283 (±21) 6% 66 174
TMC-SH
Low DQ
25% 17% 478 (±10) 236 (±13) 5% 62 215
TMC-SH
Low DQ
2 nd cycle
25% 17% 478 (±10) 342 (±4) 7% 60 144
The carboxylic acid groups of TMC-COOH were reacted with cystamine using EDC as
coupling agent. In agreement with previous studies [43] we found that this reaction proceeded
most efficiently at pH 5.5. An excess of cystamine was used to avoid the formation of
crosslinked products. Incubation of cystamine with TMC-COOH without EDC did not result in
polymers with detectable thiol groups. Ellman’s assay showed that significant thiolation of
TMC was achieved with 200 mM EDC (Table 1). This table shows that about 529 μmol/g thiol-
136

Thiolated TMC for Covalently Stabilized Nanoparticles
moieties (as free thiols or disulfides) were introduced into TMC-COOH DQ 54% corresponding
with approx. 12% of glucosamine-units and indicates quantitative substitution of the
carboxylic acids with cystamine. Treatment with DTT resulted in 283 μmol/g free –SH groups
in TMC-SH DQ 54% corresponding to a degree of thiolation (D thiol) of ~6%. For TMC-COOH
with a DQ of 25% the conversion was slightly less efficient (478 μmol/g thiol moieties as free
thiols or disulfide groups, approx. 10% of glucosamine-units) and reduction with DTT resulted
in 236 μmol/g free –SH groups (approx. D thiol of 5%). D thiol increased up to 341 μmol/g polymer
(about 7% degree of thiolation) after a second reduction cycle with DTT. GPC analysis
demonstrated an increase in both M n and M w with increasing the substitution degree of the
TMC-SHs. This suggests that some intermolecular crosslinking had occurred. A second
reduction cycle with DTT resulted in a decrease of M w (215 to 144 kDa) compared to TMC-SH
DQ 25% with only one DTT treatment, indicating that the intermolecular crosslinks were
partly broken.
Remaining thiol content (%)
A
100
80
60
40
20
0
0 1 2 3 4 5 6
time (h)
pH 4.0
pH 5.0
pH 6.2
pH 7.4
Remaining thiol content (%)
B
100
80
60
40
20
0
0 1 2 3 4 5 6
time (h)
TMC-SH DQ 25% D thiol 5%
TMC-SH DQ 54% D thiol 6%
TMC-SH DQ 25% D thiol 7%
Figure 1.Thiol group content of TMC-SH DQ 25%, D thiol 5% in a concentration of 5 mg/ml, at pH 4.0, 5.0,
6.2 and 7.4 at 37°C (A). Thiol group content of TMC-SH DQ 25%, D thiol 5%, TMC-SH DQ 54%, D thiol 6% and
TMC-SH DQ 25%, D thiol 7% in a concentration of 5 mg/ml, at pH 7.4 at 37°C (B). Error bars indicate
standard deviation of three independent samples.
The pH-dependent oxidation of thiol groups of TMC-SH DQ 25%, D thiol 5%, was studied
(Figure 1A) at 37°C. This figure shows that at pH 4.0 no reduction of free thiols was observed
while at pH 7.4 almost quantitative oxidation was achieved after 6 hours. For oxidation the
thiol anion is needed and thus oxidation occurred much faster at higher pH. It was further
observed that an increase in DQ lead to a slight reduction in oxidation rate likely due to
137

Chapter 6
increased charge repulsion [25, 43] but this had no effect on the extent of oxidation: after 6
hours TMC-SH DQ 54%, D thiol 6% was quantitatively oxidized (Figure 1B).
Evaluation of TMC-SH on Cell Viability. Figure 2A shows the effect of TMC-SH on the
mitochondrial activity (XTT) of Calu-3 cells. In agreement with previous data [19], an increase
in DQ of the TMCs resulted in a decrease in mitochondrial activity, especially at 10 mg/ml. The
thiolated TMCs with a DQ of 25% were less cytotoxic at 1 mg/ml (p < 0.001, one-way ANOVA),
as measured with the XTT assay, than the non-thiolated TMC with DQ of 30%. This might be
due to the lower charge density of the TMC-SHs. Importantly, all TMCs were relatively nontoxic
compared to linear PEI at concentrations < 1 mg/ml (p < 0.001). The influence of the
various TMCs on membrane permeability determined with the LDH assay resulted in similar
trends as obtained with the XTT assay (Figure 2B). Here, TMC-SH with a DQ of 54% showed
less LDH release than TMC DQ 56% at 1 mg/ml (p < 0.001). The LDH assay also indicated that
linear PEI was much more cytotoxic than the different TMCs (p < 0.001).
# cells (x 10 3 )
A
25
20
15
10
5
0
#
#
* *
TMC-SH DQ 25% D thiol 5%
TMC-SH DQ 25% D thiol 7%
TMC DQ 30%
TMC-SH DQ 54% D thiol
6%
TMC DQ 56%
linear PEI
MEM
10 mg/ml
1.0 mg/ml
0.1 mg/ml
0.01 mg/ml
LDH release (%)
B
70
60
50
40
30
20
10
0
ο
TMC-SH DQ 25% D thiol
5%
ο
TMC-SH DQ 25% D thiol
10 mg/ml
1.0 mg/ml
0.1 mg/ml
0.01 mg/ml
7%
TMC DQ 30%
TMC-SH DQ 54% D thiol
#
6%
TMC DQ 56%
linear PEI
Figure 2. Effect of TMCs with various DQs and with or without thiolated units on the viability of Calu-3
cells (XTT assay) at different concentrations (A). Asterics (*) indicate that no reliable XTT values were
obtained due to gel-formation of the TMC-SHs with a DQ of 25% at 10 mg/ml and therefore no bars are
depicted. # indicate that significantly (p

Thiolated TMC for Covalently Stabilized Nanoparticles
Recently, Hagenaars et al. demonstrated that nasal administration of TMC with a DQ of 68%
in a concentration of 1.25 mg/ml induced only minimal local toxicity in mice while a solution of
PEI resulted in moderate local toxicity [44].
Covalently Stabilized Nanoparticles. Thiolated and non-thiolated TMCs were mixed with
OVA-FITC and thiolated or non-thiolated hyaluronic acid in low ionic strength buffer (10 mM
HEPES pH 7.4) to yield positively charged nanoparticles. Since the thiolation of hyaluronic acid
leads to a reduction of negatively charged carboxylic acid moieties [33], more HA-SH than HA
was needed for ionic cross-linking to obtain particles with a size around 200 nm. Similarly,
when TMC with a higher DQ was used, larger amounts of negatively charged HA-SH or HA
were needed to obtain nanoparticles (Table 2). The polydispersity index (PDI) was below 0.2
for all formulations indicating fairly narrow size distributions. After formulation, particles
were incubated at 37°C for 3 hours while shaking to allow disulfide-bridge formation. Table 2
shows that the zeta-potential of the particles was dependent on the DQ of the TMC: TMCs with
a DQ of ~55% gave zeta-potentials of around +20 mV while TMCs with DQs of 25-30% resulted
in particles with zeta-potentials of +16 mV. Thiolation of the polymers had no effect on the
zeta-potential.
Interestingly, when non-thiolated particles were dispersed in high ionic strength buffer (0.8
M NaCl), DLS measurements showed a substantial drop in counts indicating that nanoparticles
disintegrated in this high ionic strength buffer. Similar destabilization in high ionic strength
buffer was observed for particles prepared with one thiolated polymer (e.g. TMC-SH or HA-SH)
and one non-thiolated polymer (e.g. TMC or HA). Immediately after preparation, particles
composed of TMC-SH and HA-SH also destabilized after dispersion in 0.8 M NaCl (results not
shown). Importantly, nanoparticles made with TMC-SH and HA-SH and stabilized for 3 hours
at 37°C showed almost no loss of DLS counts when incubated in this high ionic strength buffer
indicating that the particles are held together by the formed covalent disulfide bonds between
TMC-SH and HA-SH. All TMC:HA particles showed considerable association of OVA-FITC in 10
mM HEPES pH 7.4 (AE% up to 30%). The non-thiolated particles lost all their OVA-FITC in 0.8
M NaCl whereas the TMC-SH:HA-SH particles retained significant amounts of OVA-FITC
(approx. 30-40% of originally associated protein) in high ionic strength buffer likely losing
only surface bound protein.
139

Chapter 6
Table 2. Characteristics of thiolated and non-thiolated TMC-HA nanoparticles after incubation for 3
hours at 37°C and dispersing in 10 mM HEPES pH 7.4 or in 0.8 M NaCl 10mM HEPES pH 7.4. Values are
presented as the average of three samples ± standard deviation.
TMC-SH 25%:
HA-SH
TMC:HA
ratio
(w/w)
TMC 30%:HA 10:2
TMC-SH 54%:
HA-SH
Zetapotential
(mV)
HEPES
HEPES
Size
(nm)
+
0.8 M NaCl HEPES
OVA-FITC
Association Efficiency
(%)
+
0.8 M NaCl
10:2.4 + 13 (±0.8) 188 (±7) 192 (±4) 19 (±7) 8 (±2)
+ 16.5
(±0.3)
224 (±11)
Low
scattering
intensity
22 (±9) -0.3 (±1)
10:5.5 + 21 (±0.5) 191 (±3) 178 (±2) 25 (±7) 7 (±3)
TMC 56%:HA 10:4 + 19 (±0.2) 197 (±3)
Conclusion
Low
scattering
intensity
30 (±2) 0 (±3)
In this paper a synthetic method for partial thiolation of TMC is presented resulting in D thiol
up to 7% and DQs varying from 25-54% allowing investigation of thiolated TMCs with a
potentially optimal DQ for mucosal vaccination and/or protein delivery or other applications
such as DNA or siRNA delivery is presented. These thiolated TMCs readily formed disulfides at
pH 7.4 and 37°C. Cell viability assays indicated a minor reduction in cytotoxicity on Calu-3 cells
of TMC-SHs compared to non-thiolated TMCs with a similar DQ. The functionality of the thiol
moieties was confirmed by preparing covalently stabilized nanoparticles with thiolated
hyaluronic acid. The opportunity to modify the remaining free thiols on the surface of the
particles with e.g. PEG and targeting ligands as well as the option to prepare both negatively
and positively charged particles by varying the TMC:HA ratio in the formulation opens up wide
possibilities for future pharmaceutical applications.
Acknowledgement. This research was partially performed under the framework of TI
Pharma project number D5-106-1; Vaccine delivery: alternatives for conventional multiple
injection vaccines. Roberta Censi is acknowledged for the synthesis and characterization of the
thiolated hyaluronic acid.
Supporting Information Available. Copies of 1 H-NMR spectra of TMC-COOH and TMC-SH
for both DQs are depicted in the Supporting Information.
140

Thiolated TMC for Covalently Stabilized Nanoparticles
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143

Chapter 6
144

Thiolated TMC for Covalently Stabilized Nanoparticles
Supporting Information:
TAILORABLE THIOLATED TRIMETHYL CHITOSANS FOR
COVALENTLY STABILIZED NANOPARTICLES
Rolf J. Verheul, Steffen van der Wal, Wim E. Hennink
Contents:
1H-NMR (D 2O, 80°C, 500MHz), TMC-COOH DQ 25%
1H-NMR (D 2O, 80°C, 500MHz), TMC-COOH DQ 54%
1H-NMR (D 2O, 80°C, 500MHz), TMC-SH DQ 25%, D thiol 7%
1H-NMR (D 2O, 80°C, 500MHz), TMC-SH DQ 54%, D thiol 6%
S1
S2
S3
S4
145

Chapter 6
ppm (f1)
5.50
5.00
4.50
S1. 1 H-NMR of TMC-COOH DQ 25%.
4.00
3.50
3.00
2.50
2.00
ppm (f1)
5.50
5.00
4.50
4.00
3.50
3.00
2.50
2.00
S2. 1 H-NMR of TMC-COOH DQ 54%.
146

Thiolated TMC for Covalently Stabilized Nanoparticles
ppm (f1)
5.50
5.00
4.50
4.00
S3. 1 H-NMR of TMC-SH DQ 25%, D thiol 7%.
3.50
3.00
2.50
2.00
ppm (f1)
5.50
5.00
4.50
4.00
S4. 1 H-NMR of TMC-SH DQ 54%, D thiol 6%.
3.50
3.00
2.50
2.00
147

CHAPTER 7
COVALENTLY STABILIZED
TRIMETHYL CHITOSAN-HYALURONIC ACID
NANOPARTICLES FOR NASAL AND
INTRADERMAL VACCINATION
Rolf J. Verheul, Bram Slütter, Suzanne M. Bal,
Joke A. Bouwstra, Wim Jiskoot, Wim E. Hennink.
Manuscript submitted

Chapter 7
Abstract
The physical stability of polyelectrolyte nanocomplexes composed of trimethyl chitosan
(TMC) and hyaluronic acid (HA) is limited in physiological conditions. This may minimize the
favorable adjuvant effects associated with particulate systems for nasal and intradermal
immunization. Therefore, covalently stabilized nanoparticles loaded with ovalbumin (OVA)
were prepared with thiolated TMC and thiolated HA via ionic gelation followed by
spontaneous disulfide formation after incubation at pH 7.4 and 37 °C. Also, maleimide PEG was
coupled to the remaining thiol-moieties on the particles to shield their surface charge.
OVA-loaded TMC/HA nanoparticles had a size of around 250-350 nm, a positive zeta
potential and OVA loading efficiencies up to 60%. Reacting the thiolated particles with
maleimide PEG resulted in a slight reduction of zeta potential (from +7 to +4 mV) and a minor
increase in particle size. Stabilized TMC-S-S-HA particles (PEGylated or not) showed superior
stability in saline solutions compared to non-stabilized particles (composed of nonthiolated
polymers) but readily disintegrated upon incubation in a saline buffer containing 10 mM
dithiothreitol. In both the nasal and intradermal immunization study, OVA loaded stabilized
TMC-S-S-HA particles demonstrated superior immunogenicity compared to non-stabilized
particles (indicated by higher IgG titers). Intranasal, PEGylation completely abolished the
beneficial effects of stabilization and it induced no enhanced immune responses against OVA
after intradermal administration. In conclusion, stabilization of the TMC/HA particulate
system greatly enhances the immunogenicity of OVA in nasal and intradermal vaccination.
+
TMC/HA
+
+
-
+
+
-
+
TMC-S-S-HA
+
TMC-S-S-HA PEG
+
- +
intranasal
- -
intradermal
vaccination
log IgG titres
5
4
3
2
1
0
intranasal
prime
boost
6/8
1/8
1/8
TMC/HA
TMC-S-S-HA
TMC-S-S-HA PEG
log IgG titres
intradermal
5 prime boost
4
3
2
1
0
OVA
TMC/HA
TMC-S-S-HA
TMC-S-S-HA PEG
OVA i.m.
150

Covalently Stabilized Nanoparticles for Nasal and Intradermal Vaccination
Introduction
Most vaccines under development today are subunit vaccines based on highly purified and
well-characterized antigens derived from the respective pathogens against which one wants to
protect. Although favorable because of their safety profile, these purified proteins generally
show reduced immunogenicity compared to inactivated or attenuated pathogens [1-3].
Therefore, subunit vaccines have to be formulated with adjuvants, i.e. delivery systems and/or
immune potentiators that improve the immunogenicity of the antigens to elicit adequate,
protective immune responses [4]. For instance, when co-formulated in micro- or nanoparticles,
foreign proteins are much more effective in eliciting immune responses than as plain protein
solution [5-9], most likely because of the particle’s resemblance to the original pathogen, their
multimeric antigen presentation and improved protection of the antigen against degradation
[10]. Furthermore, particles are better taken up by antigen presenting cells (APCs), they may
prolong the residence time of the antigen at the site of action and can co-deliver antigen and
adjuvant to the same cell [8, 11, 12]. As a consequence, several types of particulate systems
have been studied for vaccine delivery, including liposomes, oil-in-water emulsions, virus like
particles, ISCOMs and polymeric carriers [1, 8, 9, 13].
Alternative vaccine administration routes to conventional intramuscular immunization have
been widely studied [2, 3, 5, 10, 14]. Mucosal vaccination offers several advantages over
invasive (intramuscular or subcutaneous) immunization routes, like needle-free
administration, potentially less adverse effects and the induction of local mucosal immune
responses [15]. However, adequate antigen delivery via the nasal route is challenging, because
of intranasal degradation and poor antigen uptake through the nasal epithelium. As trimethyl
chitosan (TMC, a quaternized, water-soluble derivative of chitosan) and hyaluronic acid (HA)
have muco-adhesive properties [16, 17], both polymers have been investigated in particulate
form in mucosal vaccine delivery [5, 18-23]. In nasal vaccine delivery, TMC nanoparticles have
proven to have excellent adjuvant properties, most likely due to improved antigen delivery [5,
24], but also immunostimulatory effects of TMC on monocyte derived dendritic cells (DCs)
were observed [12, 23]. In these studies tripolyphosphate (TPP) was used as a physical
crosslinker to form TMC nanoparticles via ionic gelation. Interestingly, results by Sayın et al.
[25] showed that nasal immunization with tetanus toxoid loaded TMC:mono-N-carboxymethyl
chitosan nanoparticles resulted in superior antibody titers compared to the TMC/TPP
particles, indicating that combining TMC with an anionic polymer may further improve the
adjuvant activity. However, the physical stability of such polyelectrolyte complexes may be
limited in physiological conditions [26, 27] or at low pH [28].
151

Chapter 7
Intradermal (ID) immunization is another interesting immunization route. While muscular
and subcutaneous tissues contain only limited numbers of APCs [29], skin tissue is abundant in
DCs that play a central role in eliciting an immune response [12]. Recently, TMC nanoparticles
were studied as intradermal vaccine carrier system, showing superior antibody titers against
ovalbumin and diphtheria toxoid (DT) compared to the plain antigens. For DT encapsulated in
TMC nanoparticles comparable immunopotency as subcutaneously administered DT-alum was
observed [12].
Recently, we developed covalently stabilized nanoparticles made from two oppositely
charged, partially thiolated polymers, namely, thiolated trimethyl chitosan (TMC-SH) and
thiolated hyaluronic acid (HA-SH) [30]. Polyelectrolyte complexes prepared with these
polymers had a size of about 200-300 nm, a positive zeta potential and showed antigen
encapsulation capacity up to 30%. The intermolecular disulfide bonds resulted in increased
stability of the TMC-S-S-HA particles in saline as compared to particles made with their
nonthiolated counterparts (TMC/HA particles), which were kept together only by electrostatic
interactions. Importantly, these covalently stabilized particles still allowed simple, aqueous
and low stress preparation conditions as used with the preparation of conventional
polyelectrolyte complexes [20, 27].
It can be expected that both nasal and ID immunization antigen-loaded covalently stabilized
TMC-S-S-HA particles may show enhanced immunogenicity compared to non-stabilized
particles because superior particle integrity in the external environment may result in
improved antigen delivery to, and activation of, APCs.
Furthermore, remaining thiol groups present on the surface of TMC-S-S-HA particles allow
post-particle modifications [31, 32]. Selective PEGylation of the free thiol moieties with PEGmaleimide
may prove an interesting strategy as PEGylation of chitosan led to improved
antibody titers in a nasal vaccination study with diphtheria toxoid possibly due to improved
stability [1]. Furthermore, shielding of cationic charges may be beneficial in ID vaccination by
limiting interactions with the negatively charged extracellular matrix. Therefore, PEGylation
may result in increased mobility and uptake by APCs [33].
In the present study we investigated covalently stabilized TMC-S-S-HA nanoparticles, with
and without PEG coating, as potential nasal and intradermal vaccine delivery systems and
compared them with non-stabilized TMC/HA nanoparticles. The particles contained ovalbumin
as a model antigen. The formulations were physico-chemically characterized and their stability
in buffered saline and in the presence or absence of a reducing agent was studied.
152

Covalently Stabilized Nanoparticles for Nasal and Intradermal Vaccination
Furthermore, the extent and type of immune response elicited after nasal and intradermal
administration of the formulations in mice was determined.
Materials and Methods
Materials. Chitosan with a residual degree of acetylation of 17% (determined with 1 H-NMR)
and a number average (M n) and weight average molecular weight (M w) of 28 and 43 kDa,
(determined with GPC-TD as described below), respectively, was purchased from Primex
(Siglufjodur, Iceland). Sodium borohydride, formaldehyde 37% (stabilized with methanol),
glyoxilic acid monohydrate, cystamine dihydrochloride, dithiotreitol (DTT), 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide HCl (EDC), L-cysteine HCl monohydrate, hen egg-white
ovalbumin (OVA, grade V), deuterium oxide, sodium acetate, acetic acid (anhydrous), sodium
hydroxide and hydrochloric acid were obtained from Sigma-Aldrich Chemical Co.
Fluorescently labeled ovalbumin (OVA-FITC) was obtained from Invitrogen (Breda, The
Netherlands). Horseradish peroxidase (HRP) conjugated goat anti-mouse IgG (γ chain specific),
IgG1 (γ1 chain specific) and IgG2a (γ2a chain specific) were purchased from Southern Biotech
Birmingham, USA). Chromogen 3, 3′, 5, 5′-tetramethylbenzidine (TMB) and the substrate buffer
were purchased from Invitrogen. Iodomethane 99% stabilized with copper was obtained from
Acros Organics (Geel, Belgium). 5,5-dithio-bis-(2-nitrobenzoic acid) (Ellman’s reagent) was
purchased from Pierce (Rockford, IL, USA). Hyaluronic acid (HA, molecular weight 17 kDa as
determined by manufacturer) was obtained from Lifecore (Chaska, USA). Methoxy
polyethylene glycol (mPEG) maleimide (M w 2000) was purchased from JenKem Technology
(Beijing, China). TMCs with DQs of 30% (M n 33 kDa, M w 59 kDa) and 56% (M n 37 kDa, M w 78
kDa) were synthesized and characterized as described previously [34]. Thiolated hyaluronic
acid (HA-SH) with a M n of 15 kDa, PDI of 2.6 and a degree of thiolation of 21% was synthesized
and characterized according to the procedure by Shu et al.[35, 36]. All other chemicals used
were of analytical grade.
Synthesis and characterization of O-methyl free, trimethylated, partially thiolated
chitosan (TMC-SH). Thiolated TMCs with different degrees of quaternization (DQ) were
synthesized as described before [30]. The degree of thiolation and the total thiol content (free
thiol moieties and disulfides) of the TMC-SHs were quantified with 5,5-dithio-bis-(2-
nitrobenzoic acid) (DTNB, Ellman’s reagent) as described elsewhere [30, 37]. The M n and M w of
the TMC-SHs were determined, as described previously [38], by GPC on a Viscotek system
153

Chapter 7
detecting refractive index, viscosity and light scattering. A Shodex OHPak SB-806 column (30
cm) was used with 0.3 M sodium acetate pH 4.4 (adjusted with acetic acid) as running buffer.
Preparation of covalently stabilized nanoparticles with or without post-PEGylation.
Covalently stabilized nanoparticles loaded with ovalbumin (OVA) were prepared with TMC-SH
and HA-SH essentially as described before [30]. TMC-SH with a DQ 25% and D thiol 5% or a DQ
of 54% and D thiol 6% were dissolved in 10 mM HEPES pH 7.4 at 1 mg/ml and subsequently
mixed with 2.5 mg/ml ovalbumin in 10 mM HEPES pH 7.4 at a weight ratio of 10:1 (TMC-
SH:OVA) under magnetic stirring. Then, HA-SH (0.5 mg/ml in 10 mM HEPES pH 7.4) was added
drop-wise yielding an opalescent nanoparticle dispersion which was incubated at 37 °C for 3
hours to allow disulfide formation. In case of PEGylated particles, after 30 minutes of
incubation at 37 °C mPEG maleimide (M w 2000 Da) was added to the TMC-S-S-HA particles in a
TMC:PEG w/w ratio of 2/1 and particles were incubated for an additional 2.5 hours at 37 °C.
Remaining thiol-moieties on the surface of the particles were used to react with the maleimide
group on the PEG at pH 7.4 (post PEGylation of the TMC-S-S-HA particles). Also, nanoparticles
with non-thiolated TMC (DQ 30% or 56%), OVA and hyaluronic acid (HA) were prepared in a
similar way to obtain ‘conventional’ particles only kept together by charge interactions.
After incubation, the nanoparticle dispersions were centrifuged for 10 min at 10000 rpm
(Biofuge Pico, PP1/97 #3324; Heraeus Instruments, Osterode, Germany) to remove the free
polymers and unbound OVA. The obtained nanoparticle pellets were resuspended in 10 mM
HEPES pH 7.4 and diluted to obtain a final OVA concentration of 0.5 mg/ml.
Physical characterization of prepared nanoparticles. Particles were diluted in 10 mM
HEPES until a slightly opalescent dispersion was obtained. Particle size was measured by
dynamic light scattering (DLS) using a Malvern ALV CGS-3 (Malvern Instruments, Malvern,
UK). DLS results are given as a z-average particle size diameter and a polydispersity index
(PDI). The PDI can vary from 0 (indicating monodisperse particles) to 1 (indicating a
completely heterodisperse system). The zeta potential of the nanoparticles was measured in
10 mM HEPES pH 7.4 using a Zetasizer Nano (Malvern Instruments, Malvern, UK).
Determination of remaining thiol moieties on surface of particles. Remaining thiol
groups on the surface of the particles after preparation were assessed with 5,5-dithio-bis-(2-
nitrobenzoic acid) (DTNB, Ellman’s reagent). DTNB was dissolved in 0.1 M sodium phosphate,
pH 8.0 containing 1 mM EDTA in a final concentration of 100 μg/ml. Then, 10 μl of particle
154

Covalently Stabilized Nanoparticles for Nasal and Intradermal Vaccination
solution (corresponding to 0.5 mg/ml OVA) was mixed with 400 μl of DTNB reagent solution.
After incubation at room temperature for 10 min, the mixtures were centrifuged for 10 min at
13000 rpm (Biofuge Pico, PP1/97 #3324; Heraeus Instruments, Osterode, Germany). The
supernatant was transferred in a microplate and the absorbance was measured with a
microplate reader at a wavelength of 405 nm (Bio-Rad Novapath, Hercules, Ca, USA). Cysteine
standards were used to calculate the amount of remaining thiol moieties on the particles and
non-incubated particles were used as a control.
Loading efficiency of prepared nanoparticles. The protein loading was determined by
assaying the concentration of fluorescently labeled OVA (OVA-FITC) in the supernatant
obtained after centrifugation of non-purified nanoparticles prepared in exactly the same way
as OVA loaded particles (10 min at 10000 rpm; Biofuge Pico, PP1/97 #3324; Heraeus
Instruments, Osterode, Germany). After resuspension of the particle pellet in 10 mM HEPES pH
7.4, weakly associated OVA-FITC was determined by centrifuging the purified particles for 10
min 13000 rpm and then measuring the OVA-FITC content in the supernatant. Fluorescence
intensity was determined using a Fluostar Optima with 488 nm as excitation wavelength and
emission was measured at 520 nm (BMG Labtech, Offenburg, Germany). The OVA loading
efficiency (LE%) was calculated using the following equation:
LE% = (total OVA-FITC ― OVA-FITC in supernatant)/ total OVA-FITC x 100%
Stability of nanoparticles To assess the stability of the prepared nanoparticles, changes in
size, PDI and number of nanoparticles (by optical density) were determined by dispersing the
nanoparticles in physiological and high saline solutions buffered with 10 mM HEPES pH 7.4,
with or without a disulfide reducing agent present. In detail, non-stabilized, stabilized and
PEGylated nanoparticles prepared with TMC with high or low DQ as described before were
diluted 50 fold in 10 mM HEPES pH 7.4 and dispersed 1:1 (v/v) in buffer or buffered saline
solutions to final concentrations of 150 or 800 mM NaCl and with or without 10 mM DTT. After
incubation for 30 minutes at room temperature, size and PDI were determined using DLS and
samples were transferred into a 96-well plate to measure the optical density at 450 nm
(microplate reader, Bio-Rad Novapath, Hercules, Ca, USA) to quantify the amount (turbidity) of
nanoparticles in the various (saline) buffers [27]. The optical density of the particles measured
in 10 mM HEPES was set at 100%.
155

Chapter 7
Nasal immunization. Groups of eight female Balb/c mice (Charles River, Boxmeer, The
Netherlands), 6-8 weeks old, received two nasal doses of 10 μg OVA in the different
formulations with an interval of 3 weeks. Injections of 20 μg plain OVA were administered
intramuscularly (i.m.) as control (n=5). For nasal administration, formulations were applied in
a volume of 10 μl 10 mM HEPES pH 7.4, 5 μl per nostril in two sessions. Blood samples were
taken 3 weeks after the prime and booster dose. After sacrificing the animals, spleens were
harvested and nasal washes collected.
Intradermal immunization. The immunogenicity of intradermally (ID) administered OVA
formulations was assessed in female Balb/c mice (Charles River, Boxmeer, The Netherlands),
6-8 weeks old. The mice were vaccinated twice with 3 weeks intervals. Groups of five mice
were injected ID with a Hamilton syringe equipped with a 30-Gauge needle. A total volume of
30 μl containing 2 μg OVA dissolved in PBS or co-formulated with TMC/HA nanoparticles was
injected into the abdominal skin under anaesthesia (by intraperitoneal injection of 150 mg/kg
ketamine and 10 mg/kg xylazine). As a control 20 μg plain OVA was injected i.m.. Blood
samples were collected from the tail vein 3 weeks after the prime dose and three weeks after
boost vaccination the mice were sacrificed. Just before euthanasia total blood was collected
from the femoral artery. Blood samples were collected in MiniCollect® tubes (Greiner Bio-one,
Alphen a/d Rijn, The Netherlands) till clot formation and centrifuged 10 minutes at 10,000 g to
obtain cell-free sera.
Determination of serum IgG, IgG1, IgG2a and secretory IgA. Micro titer plates (Nunc,
Roskilde, Denmark) were coated with OVA, by incubation of 1 μg/ml OVA in 40 mM sodium
carbonate buffer pH 9.6 for 24 hours at 4°C. To reduce aspecific binding, wells were blocked
with 1% (w/v) BSA in PBS for 1 hour at room temperature. After extensive washing with PBS
serial dilutions of serum ranging from 10 to 2*10 6 were applied, whereas nasal washes were
added undiluted. After incubation for 1.5 hours at room temperature and extensive washing,
OVA specific antibodies were detected using HRP conjugated goat anti mouse IgG, IgG1, IgG2a
or IgA (1 hour room temperature) and by incubating with 0.1 mg/ml TMB and 30 μg/ml H 2O 2
in 110 mM sodium acetate buffer pH 5.5 for 15 min at room temperature. Reaction was
stopped with 2 M H 2SO 2 and absorbance was determined at 450 nm with an EL808 microplate
reader (Bio-Tek Instruments, Bad Friedrichshall, Germany).
156

Covalently Stabilized Nanoparticles for Nasal and Intradermal Vaccination
Statistical analysis. Statistical analysis was performed with Prism 5 for Windows
(Graphpad, San Diego, USA). Data are presented as mean ± standard deviation. Statistical
significance was determined by a one way analysis of variance (ANOVA) with a Bonferroni
post-test.
Results and discussion
Characteristics of nanoparticle formulations. The structural characteristics of the
synthesized TMC-SHs are summarized in Table 1.
D thiol M n (kDa) M w (kDa)
Table 1. Characteristics of the synthesized thiolated TMCs.
DQ DA
Total thiol
amount
Amount of
free –SH
(μmol/g) a (μmol/g) a
TMC-SH
High DQ
TMC-SH
Low DQ
54% 17% 529 (±2) 283 (±21) 6% 66 174
25% 17% 478 (±10) 236 (±13) 5% 62 215
a values given as mean (± standard deviation) (n=3).
Thiolated and nonthiolated TMCs with high and low DQ were mixed with OVA and thiolated
or nonthiolated hyaluronic acid in low ionic strength buffer (10 mM HEPES pH 7.4) to yield
positively charged nanoparticles. As was observed before [30], more HA-SH than HA was
needed for ionic cross-linking to obtain particles and, similarly, when TMC with a higher DQ
was used, larger amounts of negatively charged HA-SH or HA were needed to obtain
nanoparticles (Table 2). Additionally, mPEG maleimide was added to thiolated particles after
30 min of incubation at 37 °C to obtain PEGylation via selective maleimide coupling to the
remaining free thiol moieties on the surface of the particle. Importantly, when mPEG
maleimide was added to the thiolated particles without this prior incubation step, complete
disintegration of the particles was observed; likely this is due to shielding of the chargeinteractions
between TMC-SH and HA-SH by PEGylation. Apparently, some disulfide crosslinking
connecting TMC-SH and HA-SH is required to allow PEGylation while maintaining
particle integrity. The particle formulations were incubated at 37 °C for 3 hours while shaking
to allow disulfide-bridge formation. Previously we demonstrated that after 3 hours incubation
at 37 °C, pH 7.4 more than 80% of the thiol groups of TMC-SH polymers were converted into
157

Chapter 7
disulfides [30]. Nanoparticle formulations prepared with TMC with a low DQ resulted in higher
OVA loading (50-60%) than formulations with a high DQ (ca. 30%). However, loading
efficiencies were highly dependent on TMC:crosslinker ratio and the type of TMC used [39], so
this may explain the differences between the formulations. Post PEGylation of the particles had
no effect on the association efficiency.
Table 2. Characteristics of nonthiolated, thiolated and post-PEGylated nanoparticles in 10 mM HEPES
pH 7.4. Values are presented as the average of three samples ± standard deviation.
OVA-FITC
TMC:OVA TMC:HA TMC:mPEG
Zeta
Loading
Size
ratio ratio ratio
potential
Efficiency
(nm)
(w/w) (w/w) (w/w)
(mV)
(%)
TMC-S-S-HA
+ 7.3
10:1 10:3.7 n.a. 58 (±1)
338 (±73)
low DQ
TMC-S-S-HA
low DQ PEG
TMC/HA
low DQ
TMC-S-S-HA
high DQ
TMC-S-S-HA
high DQ PEG
TMC/HA
high DQ
n.a. = not applicable
10:1 10:3.7 10:5 59 (±1)
(±1.2)
+ 4.4
(±1.2)
352 (±27)
10:1 10:2.8 n.a. 52 (±1) + 13 (±1.6) 321 (±39)
10:1 10:9.3 n.a. 30 (±3) + 16 (±0.7) 226 (±16)
10:1 10:9.3 10:5 30 (±2) + 10 (±0.6) 251 (±16)
10:1 10:6 n.a. 29 (±1) + 18 (±0.9) 290 (±26)
Free polymers and unassociated OVA were removed by centrifugation and resuspension of
the obtained particle-pellet in 10 mM HEPES pH 7.4. All particle formulations showed limited
burst-release of associated OVA (< 10%) after resuspension in 10 mM HEPES pH 7.4. The zeta
potential of the non PEGylated particles varied from +18 mV to +7 mV (Table 2) most likely
due to differences in the DQ of TMC and amount of cross-linker used [39]. Importantly,
PEGylation of the thiolated particles reduced the zeta potential for both PEGylated
formulations compared to the non PEGylated TMC-S-S-HA particles (from +7 mV to +4 mV and
+16 mV to +10 mV for TMC-S-S-HA with low and high DQ, respectively. When mPEG maleimide
was added to nonthiolated particles, no reduction in zeta potential was observed, indicating
that available thiol groups are required for PEGylation. Similarly, when mPEG maleimide was
added to TMC-S-S-HA particles after three hours of incubation at 37 °C, no significant
reduction in zeta potential was observed, indicating that almost all thiols were converted into
disulfides. This was confirmed with Ellman’s assay as neglectable amounts of remaining thiol
moieties on the surface of TMC-S-S-HA particles were found (< 4 % of amount of thiol groups
on the surface of non-incubated particles). DLS analysis showed that the size of the formed
158

Covalently Stabilized Nanoparticles for Nasal and Intradermal Vaccination
nanoparticle varied from 226 to 352 nm. TMCs with a low DQ yielded particles with a bigger
size and higher PDI compared those prepared with TMC with a high DQ (Figure 1A). Also,
PEGylation resulted in a slight increase in particle diameter by 15-25 nm.
Particle stability in saline and in presence of a disulfide-reducing agent. In general, the
stability of polyelectrolyte complexes is dependent on the type of polymers used and may be
limited solutions of physiological salt concentrations [26, 27]. We therefore studied the
physical stability of nonthiolated, thiolated and PEGylated nanoparticles in buffers with
different sodium chloride concentrations. Figure 1A shows that non-stabilized TMC/HA high
DQ particles demonstrated a large increase in size in 150 mM NaCl implying aggregation while
an increase in polydispersity is seen in 800 mM NaCl. On the other hand, the particles made of
thiolated polymers, with and without PEG-coating showed that particle size and PDI was
hardly affected by salt in the buffer. The turbidity of particle solutions was measured at 450
nm and was used to quantify the amount of particles remaining in physiological or high saline
conditions and with or without DTT (Figure 1B). After dispersion of the non-stabilized
TMC/HA particles in 150 mM NaCl, a reduction of more than 75% in optical density was
observed which became even more dramatic in 800 mM NaCl. In contrast, stabilized TMC-S-S-
HA particles showed a much less reduction in OD 450 in 800 mM NaCl indicating superior
stability in high ionic strength buffers. However, the TMC-S-S-HA low DQ particles showed less
stability in 800 mM NaCl than was observed in the previous study where almost no reduction
in DLS counts was observed [30]. This may be attributed to the lower degree of thiolation and
lower molecular weight of the HA-SH used in this study (21 vs 52% and M n of 14 vs. 39 kDa).
Both altered characteristics will likely decrease the probability of the formation of disulfidebased
networks between TMC-SH and HA-SH polymers in a nanoparticle. Interestingly, TMC-S-
S-HA high DQ showed less reduction of OD 450 nm in the saline solutions compared to TMC-S-
S-HA low DQ. This higher stability of TMC-S-S-HA high DQ particles may be due to the higher
amount of HA-SH incorporated in the particles (Table 2); more HA-SH present in a particle
increases the chances of TMC-S-S-HA formation which is crucial for the particle stability [30].
When a disulfide-reducing agent (10 mM DTT) was present in a physiological saline solution,
the turbidity of the stabilized particles dramatically decreased and this shows that the
stabilization of the particles is due to disulfide formation. PEGylation of the thiolated
nanoparticles had no effect on the stability in presence of salt and particles were still sensitive
to DTT.
159

Chapter 7
OD 450 nm (% in HEPES)
B
100
75
50
25
0
TMC/HA low DQ
TMC-S-S-HA low DQ
TMC-S-S-HA low DQ PEG
TMC/HA high DQ
TMC-S-S-HA high DQ
TMC-S-S-HA high DQ PEG
HEPES
150 mM NaCl
800 mM NaCl
150 mM NaCl
+10 mM DTT
Figure 1. Physical characteristics and stability of the nanoparticle formulations in presence of
physiological or high saline concentrations after 30 minutes incubation at room temperature; Z-average
diameter (bars) and PDI (dots) (A). Optical density at 450 nm (OD 450 nm) of the nanoparticle
formulations in presence of physiological or high saline concentrations and with or without DTT (B).
Error bars represent the standard deviation (n=3).
160

Covalently Stabilized Nanoparticles for Nasal and Intradermal Vaccination
Immunization studies. Nasal and ID immunization studies were carried out with
nanoparticles prepared with TMC with a low DQ because we previously demonstrated that the
DQ of TMC plays only a minor role in its (nasal) adjuvanticity [22].
Serum antigen-specific antibodies after nasal vaccination. The OVA specific total IgG
antibody titers elicited after nasal administration of the antigen and the various TMC-HA
formulations were determined three weeks after prime and boost vaccination (Figure 2). After
prime vaccination hardly any IgG titers were detected for all nasal formulations. Interestingly,
after the boost vaccination the stabilized particles showed significantly higher total IgG titers
compared to the non-stabilized or the PEGylated system and six out of eight mice had a
detectable IgG titer after intranasal vaccination with the stabilized particles. These results
suggest that stabilization does indeed as anticipated improve the adjuvanticity of the TMC-HA
nanoparticulate system and that PEGylation of these particles abolishes this effect. The
administered dose was apparently too low to (significantly) discriminate between plain OVA
and the TMC-S-S-HA particles and this also led to hardly detectable IgG1 and IgG2a after boost
vaccination and secretory IgA (sIgA) levels in the nasal wash (results not shown).
5
prime
boost
5/5
10 log IgG titres
4
3
2
1
3/8
1/8
6/8
* *
1/8
0
OVA
TMC/HA low DQ
TMC-S-S-HA low DQ
TMC-S-S-HA low DQ PEG
OVA i.m.
Figure 2. Antigen-specific total IgG antibody titers after nasal vaccination with OVA formulations. Error
bars indicate standard deviation (n=8 (nasal) or 5 (i.m.)). * p

Chapter 7
Serum antigen-specific total IgG after intradermal vaccination. The antigen specific total
IgG antibody titers elicited after ID administration of the various OVA formulations are shown
in Figure 3. After prime vaccination the stabilized particles (with or without PEGylation)
showed significantly higher antibody titers compared to plain OVA (up to 75-fold) and the nonstabilized
TMC/HA particles (up to 20-fold). Interestingly, the non-stabilized nanoparticles did
not significantly improve immune responses compared to plain OVA. Also, after boost
vaccination the stabilized systems showed increased total IgG antibody titers compared to
OVA, in contrast to the non-stabilized formulations. The immunogenicity of ID administered
PEGylated stabilized particles was similar to that of the non-PEGylated ones. These results
indicate that stabilization, but not PEGylation of TMC/HA particles is essential for their ID
adjuvanticity.
10 log IgG titres
5
4
3
2
1
prime
boost
** **
OOO
OO
***
***
***
0
OVA
TMC/HA low DQ
TMC-S-S-HA low DQ
TMC-S-S-HA low DQ PEG
OVA i.m.
Figure 3. Antigen-specific total IgG antibody titers after intradermal vaccination with OVA formulations.
Error bars indicate standard deviation (n=5). *** p

Covalently Stabilized Nanoparticles for Nasal and Intradermal Vaccination
immune response. Log IgG1/IgG2a ratio for each individual mouse did not reveal a significant
shifting of the type of immune response compared to plain OVA (results not shown). This is in
line with previous results observed in an ID vaccination study with TMC/OVA/TPP particles
[12].
10 log IgG1/IgG2a titres
4
3
2
1
0
OVA
IgG1 IgG2a
*
*
TMC/HA low DQ
TMC-S-S-HA low DQ
TMC-S-S-HA low DQ PEG
Figure 4. Antigen-specific IgG1 and IgG2a antibody titers after boost intradermal vaccination with OVA
and nanoparticle formulations. Error bars indicate standard deviation (n=5). * p

Chapter 7
2). However, previously we found that for nasal vaccination with TMC-coated whole
inactivated influenza virus, similar differences in zeta potential did not result in altered
adjuvant effects [22]. This indicates that it is unlikely that the lower zeta potential of the
stabilized particles is responsible for the observed beneficial effects in the present nasal
vaccination study. Thus, it can be concluded that the stabilization of TMC-S-S-HA nanoparticles
resulted in improved immunogenicity, however, the exact mechanism(s) need to be
determined.
PEGylation of the stabilized particles inhibited the beneficial effects of stabilization (nasal
administration), while for ID administration the PEGylated particles had similar effects as the
non-PEGylated ones. This indicates that PEGylation of particles decreased nasal antigen
delivery. Although it has been suggested that PEGylation of TMC improves muco-adhesion
[41], possibly, PEGylation will also hinder electrostatic interactions between the cationic
particle and the epithelial barrier thereby reducing particle transport over epithelial cells [42]
or particle uptake by microfold (M)-cells. Van den Berg et al. showed that PEGylation of
cationic polyplexes improves DNA transfection efficiencies after ID tattooing [33]. Although
PEGylation may improve mobility in the intracellular matrix, reduced electrostatic interactions
with DCs may lead to lower uptake [43]. Better understanding of these conflicting effects may
result in optimal use of PEGylation for ID vaccination. Importantly, we showed that post
particle modifications of the stabilized TMC-S-S-HA polyelectrolyte system are possible and
this opens up a variety of options such as specific targeting towards DCs or M-cells.
Conclusion
In this paper we showed that stabilized nanoparticles can be prepared using TMC-SH with
high and low DQ together with HA-SH and that these particles can be post PEGylated. These
particles showed adequate OVA association efficiency, preserved their particle integrity under
saline conditions but readily disintegrated when a disulfide reducing agent was present.
Stabilized particles showed enhanced adjuvanticity in nasal and ID vaccination compared to
non-stabilized particles. PEGylation abolished the beneficial effects of stabilization in nasal
vaccine administration and showed similar immunogenicity as stabilized particles in ID
immunization. Our results imply that the stabilized TMC-S-S-HA nanoparticles form a highly
versatile and promising vaccine carrier system while also offering options for post particle
modifications. Further studies should be performed to elaborate the exact mechanism by
which stabilization results in improved immunogenicity.
164

Covalently Stabilized Nanoparticles for Nasal and Intradermal Vaccination
Acknowledgement. This research was partially performed under the framework of TI
Pharma project number D5-106-1; Vaccine delivery: alternatives for conventional multiple
injection vaccines.
165

Chapter 7
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CHAPTER 8
SUMMARY AND FUTURE PERSPECTIVES

Summary and Future Perspectives
Summary
Active vaccination has proven to be the most (cost) effective tool in the fight against
infectious diseases. Vaccines are usually made of live attenuated or inactivated pathogens (e.g.
viruses or bacteria) or purified immunogenic protein(conjugate)s derived from these
pathogens. Nowadays, most vaccines are administered via parenteral injection however, the
risk for contaminated needles and need for trained personnel have risen interest for
alternative immunization routes.
Chapter 1 discusses the (dis)advantages of intramuscular and alternative administration
routes, in particular intranasal immunization that allows relatively simple, needle-free
administration, reduces the need for trained professionals and may, importantly, elicit both
mucosal and systemic immune responses. On the other hand, antigen degradation and poor
delivery to antigen presenting cells (APCs) are major drawbacks of nasal vaccination. As live
attenuated vaccines raise considerable safety issues, (nasal) vaccine development now mainly
focuses on purified, well-characterized antigenic proteins. However, these subunit vaccines are
generally less immunogenic and need potent adjuvant(system)s to elicit an adequate immune
response. The use of muco-adhesive polymers like N,N,N-trimethylchitosan (TMC), a partially
quaternized, water-soluble chitosan derivative, can enhance the immune responses against
antigens, presumably by increasing the nasal residence time and/or improving the contact
area between the antigen and the mucosal surface. TMC’s chemical structure can vary in the
degree of quaternization (DQ), giving the polymer its cationic charge, but also in extent of O-
methylation (DOM), degree of acetylation (DAc) and polymer molecular weight. Tailorability of
these structural elements will allow better understanding of the contribution of these moieties
to the physico-chemical and biological properties of TMC. Additionally, the introduction of side
groups such as thiol-moieties may further be applied to improve and optimize TMC’s
properties.
Chitosan and its derivatives are much more effective in eliciting immune responses in microor
nanoparticulate form than as plain polymer solution. Ionic gelation based upon electrostatic
interactions is a simple, commonly used method to yield nanoparticulate systems. The
complexation between the positively charged TMC and oppositely charged (macro)molecules
added drop-wise under stirring in low ionic strength buffer results in the spontaneous
formation of nanoparticles. However, the physico-chemical stability and the immunogenicity of
these antigen-loaded complexes are dependent on the characteristics of the crosslinker used,
and the currently applied carrier-systems may be sub-optimal. Furthermore, although TMC’s
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mode of action is usually attributed to its muco-adhesive and penetration enhancing
properties, detailed knowledge of the mechanism behind the favorable adjuvant
characteristics is lacking (e.g., the effect of TMC on APCs is currently unknown). Concluding,
many opportunities for optimizing TMC structure and TMC-based nanoparticles remain and
mechanistic insights into the mode of action of TMC have to be obtained.
The aim of this thesis is to develop synthetic routes to synthesize TMC structural variants in
a controllable and tailorable manner and introduce substitutions such as thiol-moieties that
may further improve TMC’s properties. In this way, structure-activity relationships can be
investigated exploiting in vitro assays and in in vivo (nasal) vaccination studies.
Chapter 2 challenges the current synthetic method to synthesize TMC that is associated with
several side-reactions such as O-methylation and chain scission. Since these side reactions may
affect the polymer characteristics, there is a need for TMCs without O-methylation and
disparities in chain lengths while varying the DQ. In this chapter, O-methyl free TMCs with
varying DQs were successfully synthesized by using a two-step method. First, chitosan was
quantitatively dimethylated using formic acid and formaldehyde. Then, in presence of an
excess amount of iodomethane, TMC was obtained with different DQs (22-68%) by varying the
reaction time. TMC obtained by this two-step method showed no detectable O-methylation
( 1 H-NMR) and a slight increase in molecular weight with increasing DQ (GPC), implying that no
chain scission occurred during synthesis. The solubility in aqueous solutions at pH 7 of O-
methyl free TMC with DQ < 22% was less as compared to O-methylated TMC with the same DQ.
On the other hand, O-methyl free TMC with DQ > 30% had an excellent aqueous solubility. On
Caco-2, cells O-methyl free TMCs demonstrated a larger decrease in trans-epithelial electrical
resistance (TEER) than O-methylated TMCs. Also, with increasing DQ, an increase in
cytotoxicity (MTT) and membrane permeability (LDH) was observed. This chapter clearly
demonstrates that the DQ as well as O-methylation substantially influence the physicochemical
and in vitro biological properties of TMC.
The influence of another structural variant of TMC, the degree of acetylation (DAc), on in
vitro degradation and biological properties was evaluated in Chapter 3. TMCs with a DAc
ranging from 11 to 55% were synthesized by using a three-step method. First, chitosan was
partially re-acetylated using acetic anhydride followed by quantitative dimethylation using
formaldehyde and sodium borohydride. Then, in presence of an excess amount of
iodomethane, TMC was synthesized. The TMCs obtained by this method showed neither
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Summary and Future Perspectives
detectable O-methylation nor loss in acetyl groups ( 1 H-NMR) and a slight increase in molecular
weight (GPC) with increasing degree of substitution, implying that no chain scission occurred
during synthesis. The extent of lysozyme-catalyzed degradation of TMC, and that of its
precursors chitosan and dimethyl chitosan, was highly dependent on the DAc; polymers with
the highest DAc showed the largest decrease in molecular weight. On Caco-2 cells, TMCs with a
high DAc (~50%), a DQ of around 44% and with or without O-methylated groups, were not
able to open tight junctions in the trans-epithelial electrical resistance (TEER) assay. This in
contrast to TMCs (both O-methylated and O-methyl free; concentration 2.5 mg/ml) with a
similar DQ but a lower DAc which were able to reduce the TEER with 30 and 70%,
respectively. Additionally, TMCs with a high DAc (~50%) demonstrated no cell toxicity (MTT,
LDH release) up to a concentration of 10 mg/ml. This chapter shows that the degree of N-
acetylation dramatically influences the enzymatic degradation and in vitro biological
properties of TMC.
The results of Chapter 2 and 3 demonstrate that the DQ, DOM and DAc all influence the in
vitro biological properties of TMC. Therefore we investigated the influence of the structural
properties of TMC on its adjuvanticity in an in vivo intranasal (i.n.) immunization study. In
Chapter 4, TMCs with varying degrees of quaternization (DQ, 22-86%), O-methylation (DOM,
0-76%) and acetylation (DAc 9-54%) were formulated with whole inactivated influenza virus
(WIV). Simple mixing of the TMCs with WIV at a 1:1 (w/w) ratio resulted in comparable
positively charged nanoparticles, indicating coating of the negatively charged WIV with TMC.
The amount of free TMC in solution was comparable for all TMC-WIV formulations. After i.n.
immunization of mice with WIV and TMC-WIV on day 0 and 21, all TMC-WIV formulations
induced stronger total IgG, IgG1 and IgG2a/c responses than WIV alone, except WIV
formulated with re-acetylated TMC with a DAc of 54% and a DQ of 44% (TMC-RA44). No
significant differences in antibody titers were observed for TMCs that varied in DQ or DOM,
indicating that these structural characteristics play a minor role in their adjuvant properties.
TMC with a DQ of 56% (TMC56) formulated with WIV at a ratio of 5:1 (w/w) resulted in
significantly lower IgG2a/c:IgG1 ratio’s compared to TMC56 mixed in ratios of 0.2:1 and 1:1,
implying a shift towards a Th2 type immune response. Challenge of vaccinated mice with
aerosolized virus demonstrated protection for all TMC-WIV formulations with the exception of
TMC-RA44-WIV. This chapter demonstrates that coating of WIV with TMCs strongly enhances
the immunogenicity and induced protection after i.n. vaccination with WIV. The adjuvant
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properties of TMCs as i.n. adjuvant are strongly decreased by re-acetylation of TMC, whereas
the DQ and DOM hardly affect the adjuvanticity of TMC.
The aim of Chapter 5A was to elucidate the reason for the lack of adjuvanticity of reacetylated
TMC (TMC-RA) by comparing TMC-RA (degree of acetylation 54%) with TMC
(degree of acetylation 17%) at six potentially critical steps in the induction of an immune
response after intranasal (i.n.) administration in mice: chemical stability of the polymer in
murine nasal washings, local i.n. distribution of WIV, nasal residence time of WIV, cellular
uptake of WIV by epithelial cells, transport of WIV by epithelial cells, and capacity of the
formulation to induce maturation of murine bone marrow derived dendritic cells (DCs). TMC-
RA was degraded in a nasal wash to a slightly larger extent than TMC. The local i.n. distribution
and nasal clearance were similar for both TMC types. Fluorescently labeled WIV was taken up
more efficiently by Calu-3 cells when formulated with TMC-RA compared to TMC and both
TMCs significantly reduced transport of WIV over a Calu-3 monolayer. Murine bone-marrow
derived dendritic cell activation was similar for plain WIV, as well as for WIV formulated with
TMC-RA or TMC. The inferior adjuvant effect of TMC-RA over that of TMC might be caused by a
slightly lower stability of TMC-RA in the nasal cavity, rather than by any of the other factors
studied in this paper.
Interestingly, N-acetylated glucosamine units or GlcNAcs present in TMC have been
described to bind several human C-type lectins, a family of lectins involved in the human
innate immune response. Therefore the effect of TMC and re-acetylated TMC with a degree of
acetylation of 54% (TMC-RA) on the uptake and maturation of human dendritic cells (DCs) was
assessed in Chapter 5B using whole inactivated influenza virus (WIV) as antigen. Studies on
monocyte-derived human DCs indicated that the uptake of TMC(-RA) coated WIV was slightly
lower than plain WIV. TMC-RA, as a solution or when formulated with WIV, induced much
stronger DC maturation, as measured with CD86 expression, than any of the other
formulations. Also, only TMC-RA(-WIV) induced high IL-10, TNF-α and IL-12p40 and IL-12p70
release by DCs. Since both IL-10 and IL-12p70 levels were elevated, no polarization towards a
Th1 or Th2 type immune response could be established. Concluding, TMC-RA has strong
immuno-stimulatory effects in vitro on human monocyte derived dendritic cells which
indicates that the degree of N-acetylation is critical for the adjuvant effect of TMC on human
DCs, but not on mice DCs.
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Summary and Future Perspectives
Introduction of thiol-moieties will further broaden the potential pharmaceutical applications
of TMC by enhancing its muco-adhesive properties, allowing further chemical derivatization
reactions via reducible disulfide-bridges and opening up possibilities for covalently linked
nanoparticles together with a thiolated anionic polymer. In Chapter 6, a novel four-step
method is presented to synthesize partially thiolated TMC with tailorable degrees of
quaternization and thiolation. First, chitosan was partially N-carboxylated with glyoxylic acid
and sodium borohydride. Next, the remaining amines were quantitatively dimethylated with
formaldehyde and sodium borohydride and then quaternized with iodomethane in N-
methylpyrrolidone. Subsequently, these partially carboxylated TMCs, dissolved in water, were
reacted with cystamine at pH 5.5 using EDC as coupling agent. After addition of dithiothreitol
for disulfide reduction and dialysis, thiolated TMCs were obtained varying in degree of
quaternization (25-54%) and degree of thiolation (5-7%) as determined with 1 H-NMR and
Ellman’s assay. Gel permeation chromatography with light scattering detection indicated
limited intermolecular crosslinking. All thiolated TMCs showed rapid oxidation to yield
disulfide crosslinked TMC at pH 7.4 while the thiolated polymers were rather stable at pH 4.0.
Using Calu-3 cells, XTT and LDH cell viability tests showed a slight reduction in cytotoxicity for
thiolated TMCs as compared to the non-thiolated polymers with similar DQs. Positively
charged nanoparticles loaded with fluorescently labeled ovalbumin were made from thiolated
TMCs and thiolated hyaluronic acid. The stability of these particles was confirmed in 0.8 M
NaCl, in contrast to particles made from non-thiolated polymers which dissociated under these
conditions demonstrating that the particles were held together by intermolecular disulfide
bonds.
As shown in Chapter 6, the physical stability of polyelectrolyte nanoparticles composed of
trimethyl chitosan (TMC) and hyaluronic acid (HA) is limited in physiological conditions. This
may adversely affect the favorable adjuvant effects of particulate systems for nasal and
intradermal immunization. Therefore, in Chapter 7 the effect of covalent stabilization of
antigen-loaded TMC/HA nanoparticles by disulfides on their immunogenicity is evaluated.
Furthermore, remaining thiols on the surface of these particles allow PEGylation which may
result in additional beneficial effects by hindering interactions with the extracellular matrix
(intradermal) and/or enhanced muco-adhesion (intranasal). Ovalbumin (OVA) loaded,
covalently stabilized nanoparticles were prepared with thiolated TMC and thiolated HA via
ionic gelation followed by spontaneous disulfide formation after incubation at pH 7.4 and 37°C.
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Chapter 8
Also, PEG maleimide was coupled to the remaining thiol-moieties on the particles to shield the
surface charge.
TMC/HA nanoparticles had a size of around 250-350 nm, a positive zeta potential and OVA
association efficiencies up to 60%. PEGylation resulted in a slight reduction of zeta potential
and a minor increase in particle size. Stabilized TMC-S-S-HA particles (PEGylated or not)
showed superior stability in saline solutions compared to non-stabilized TMC/HA particles
(composed of nonthiolated polymers), but readily disintegrated when a disulfide reducing
agent was introduced. In both the nasal and intradermal immunization study, OVA-loaded
stabilized TMC-S-S-HA particles demonstrated superior immunogenicity compared to nonstabilized
particles. For intranasal immunization, PEGylation completely abolished the positive
effects of stabilization and it had no additional effect when used for intradermal vaccination. In
conclusion, stabilization of the TMC/HA particulate system enhances its immunogenicity in
nasal and intradermal vaccination, however, PEGylation of these stabilized particles had no
beneficial effects.
The novel synthetic methods to obtain structural variants of TMC presented in this thesis
allowed tailorability of the degrees of quaternization and acetylation and excluded the
introduction of other alterations such as O-methylation and polymer chain scission.
Additionally, thiol-moieties were introduced in a controllable manner. This tailorability of TMC
provided the possibility to properly establish structure-activity relationships in in vitro
biological assays and in in vivo (nasal) vaccination studies. Also, mechanistic insight into the
mode of action of TMC was acquired by investigating several potentially crucial steps in nasal
vaccination. Finally, a promising new, covalently stabilized, polymeric carrier system was
introduced based upon the formation of intracellularly degradable disulfides.
Discussion and Future perspectives
TMC-based particulate systems for nasal vaccine delivery. Although already synthesized
from chitosan in the mid-80s by Muzzarelli [1] and later by Domard [2], trimethyl chitosan
(TMC) has not been used for mucosal vaccination until the beginning of this millennium [3].
Even more recently, in 2007, Amidi et al. demonstrated for the first time the potency of TMCtripolyphosphate
ionically crosslinked nanoparticulate systems in an intranasal vaccination
study with influenza subunit antigen [4]. Although this system showed promising results as
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Summary and Future Perspectives
nasal vaccine delivery system, it was anticipated that much could be gained by optimizing, in a
controllable way, the chemical structure of TMC. In particular the degree of quaternization
(DQ) of TMC seemed important since this is the polymer’s main determinant for charge
density. Also, in vitro and in vivo studies with formulation of TMC and both protein and low
molecular weight compounds suggested an optimal DQ of 40-50% for transepithelial delivery
[5-10].
In this thesis we show that the DQ, but also the exclusion of O-methylation, has no effect on
the immunogenicity of TMC when used as coating on whole inactivated influenza virus (WIV).
Increasing another variable, the degree of acetylation (DAc), resulted even in a reduction of the
beneficial effects of coating WIV with TMC in nasal immunization in mice. So, looking from an
immunological perspective, despite all efforts put in the development of novel synthetic routes
to obtain structural variants of TMC, no improvements in immunological efficacy were
achieved. However, looking from a pharmaceutical perspective, the availability of
reproducible, well-characterized TMCs that can be structurally tailored in a controllable
manner without introducing side reactions, may be considered as a major step forward.
Additionally, these structural variants (DQ, DOM, DAc) also influence others physicochemical
characteristics and superior stability of O-methyl free TMC-WIV formulations was found [11].
The covalently stabilized polyelectrolyte nanocomplexes of TMC-SH and HA-SH offer a novel
vaccine delivery platform with high versatility. To mention, positively charged antigens may be
entrapped using HA-SH for initial complexation and TMC-SH as crosslinker and also covalent
coupling of antigens, adjuvants or targeting ligands is easily realizable on remaining free thiol
moieties (as is already demonstrated for PEG maleimide). Active targeting to lectin receptors
or integrins present on microfold (M)-cells can improve the immunogenicity of a formulation
[12] and thus coupling of sialic acid, galactose residues or specific antibodies (like IgA) on the
particles’ surface may be an attractive next step to enhance binding and uptake of the particles
by antigen presenting cells. Interestingly, these and similar sugar-moieties like mannose and
N-acetyl glucosamine (GlucNAc) are also associated with targeting to dendritic cells (DCs).
Indeed, the activation of DCs via GlucNAc is described in this thesis with TMC-RA (containing
high GlucNAc) showing promising results on human DCs (Chapter 5B). Furthermore the type
of immune response may be steered by incorporation of CpG motifs (usually towards Th1) and
lipopolysaccharides (LPS, usually towards Th2) or other immuno-modulators. Finally, as
thiolation of TMC enhances its muco-adhesiveness [13], the use of TMC-SHs in ‘conventional’
TMC-TPP particles may further increase nasal residence time of the particles resulting in
better antigen delivery compared to nonthiolated TMCs.
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This thesis further shows the limitations of in vitro predictability and/or correlation for in
vivo outcome as large differences were observed in vitro for toxicity and capacity to open
cellular tight junctions for polymers that showed no differences in vivo nasal vaccination
studies. Additionally, extensive investigation in various potentially crucial steps for nasal
vaccination with both in vitro and in vivo models did not reveal major differences between an
effective (TMC-WIV) and ineffective (TMC-RA-WIV) formulation. Apparently, displaying
cationic charges, muco-adhesiveness and improving interaction between the formulation and
the epithelial barrier are not sufficient for effective nasal vaccination. This emphasizes the
need for even more detailed knowledge of TMC’s (and that of other adjuvants) mode of action.
Only then, in a rational way, in vitro models can be developed that adequately predict in vivo
outcome and ultimately minimize the use of animal models.
Another issue that is raised by this thesis is the discrepancy in results between human and
murine in vitro models (as was observed for differences in the immuno-stimulatory effects of
TMC-RA in human and murine DCs). Again, in depth knowledge on the mode of action of TMC
is crucial for the predictability of murine in vivo/in vitro results for human outcome. E.g. if
TMC’s adjuvant effect is achieved through improved muco-adhesion, minor differences may be
anticipated between mice and men. In contrast, M-cells are more abundant in murine nasal
cavity and murine and human M-cells may express different receptors as may antigen
presenting cells [14]; if TMC-based systems accomplish their effect mainly via these
mechanisms, limited predictability can be expected from studies with mice models.
As can be concluded from the above, to choose just one type of TMC(-system) for further
research for nasal vaccine development is not recommended: in particular, the effects of
increasing the GlucNAc content are not fully understood yet and the full potential of the
stabilized TMC-S-S-HA particles needs further investigation. Animal models (such as ferrets or
transgenic mice) that mimic the human immune responses in a better extent may elaborate
whether a future application of TMC for nasal vaccination in man is feasible.
Other potential applications for novel TMCs. Next to mucosal vaccination, TMCs are
frequently studied for mucosal delivery of pharmaceutically active proteins and low molecular
weight compounds. Interestingly, for these applications, good correlations are found between
the capacity of a polymer to open tight junctions (as measured in a transepithelial electrical
resistance (TEER) assay) and in vivo uptake. This implies that, in particular, O-methyl free
TMCs can improve the mucosal uptake compared to ‘conventional’ TMCs as they showed a
better, reversible reduction of the TEER. Further studies should be carried out to confirm this.
178

Summary and Future Perspectives
TMCs have been used for DNA delivery with some encouraging results. However,
introductory studies with O-methyl free TMCs for DNA transfection showed low transfection
efficiencies probably due to poor endosomal escape. Interestingly, thiolation of TMC may not
only enhance the transfection potential of DNA [15] but also gene silencing via delivery of
siRNA into the cytosol (Varhouchi et al, manuscript submitted). Moreover, the stabilized TMC-
S-S-HA carrier system shows excellent properties for DNA/siRNA delivery demonstrating
enhanced stability under physiological conditions but they readily dissociate in a reductive
environment (like in the cytosol). Depending on the target cell or the application route
(systemic or local) PEG or targeting ligands (such as nano- or antibodies) can be attached. This
makes TMC-SH and in particular the TMC-S-S-HA carrier systems highly interesting for
siRNA/DNA delivery and they are currently being evaluated for this purpose in our
Department.
PEGylated nanoparticles (e.g. PEG-liposomes) are widely studied for systemic delivery of
proteins, cytokines and anti-cancer and immunomodulatory drugs to area’s of tumor growth
or inflammation as a result of the enhanced permeability and retention effect in those tissues.
The PEGylated, stabilized TMC-S-S-HA system may be a valuable alternative to currently used
systems because of its simple preparation method and its potentially high loading capacity. As
TMCs with a high DAc are readily degraded by lysozyme, an interesting option may be the
incorporation of this enzymatically-degradable TMC into particles. In environments with high
lysozyme concentrations, for example in inflammated or infectioned tissues (as lysozyme is
excreted by macrophages), these particles should disintegrate and release their contents.
Finally, the thiolated polymers can be used for covalently linked layer-by-layer technologies
[16, 17] and/or the preparation of covalently stabilized hydrogels with acrylated or
methacrylated polymers (based upon Michael addition) [18].
TMC has only been used for a decade or so for mucosal vaccination and other applications
and the last couple of years interest has risen rapidly due to its promising results. However, to
look into the future of TMC much can be learned from its older precursor, chitosan. This
polymer has been investigated for over forty years in various biomedical, nutritional and
technological fields and widely studied for toxicity, biocompatibility and biodegradation. In
contrast to TMC, it has only two (major) variables, the molecular weight and the DAc and their
effects on the physicochemical and biological properties of chitosan have been thoroughly
studied. Despite all this scientific effort, the FDA has not yet approved chitosan as GRAS
(generally regarded as safe) material which hinders its application in (biomedical) products
179

Chapter 8
[19]. Likely, in the past manufacturers disliked the difficulty of patentability for chitosanproducts
and therefore little stress on regulatory authorities was applied. Recently, however,
pressure is build by researchers and clinicians to revise this regulatory perspective as
chitosan-like substances are highly awaited in the various biomedical fields. For instance, a
chitosan-based delivery platform is exploited by Archimedes (UK-based pharmaceutical
company) and this system is currently evaluated in phase I clinical trails for the nasal delivery
of granisetron as anti-emetic (stated on the Archimedes website by December 2009). If
encouraging results are obtained, it can be anticipated that approval by regulatory authorities
will follow. As TMC has superior characteristics to chitosan, TMC may shortly go behind.
Further convincing studies showing TMC’s potential may speed up this process as may the
availability of well-characterized and defined polymers described in this thesis.
180

Summary and Future Perspectives
References
1. R. A. A. Muzzarelli and F. Tanfani. The N-permethylation of chitosan and the preparation of
N-trimethyl chitosan iodide. Carbohydr Polym 5: 297-307 (1985).
2. A. Domard, M. Rinaudo, and C. Terrassin. New method for the quaternization of chitosan. Int J
Bio Macromol 8: 105-107 (1986).
3. I. M. Van der Lubben, J. C. Verhoef, M. M. Fretz, O. Van, I. Mesu, G. Kersten, and H. E.
Junginger. Trimethyl chitosan chloride (TMC) as a novel excipient for oral and nasal
immunisation against diphtheria. S.T.P. Pharma Sciences 12: 235-242 (2002).
4. M. Amidi, S. G. Romeijn, J. C. Verhoef, H. E. Junginger, L. Bungener, A. Huckriede, D. J. A.
Crommelin, and W. Jiskoot. N-Trimethyl chitosan (TMC) nanoparticles loaded with influenza
subunit antigen for intranasal vaccination: Biological properties and immunogenicity in a mouse
model. Vaccine 25: 144-153 (2007).
5. F. Chen, Z. R. Zhang, F. Yuan, X. Qin, M. Wang, and Y. Huang. In vitro and in vivo study of
N-trimethyl chitosan nanoparticles for oral protein delivery. Int J Pharm 349: 226-233 (2008).
6. G. Di Colo, S. Burgalassi, Y. Zambito, D. Monti, and P. Chetoni. Effects of different N-
trimethyl chitosans on in vitro/in vivo ofloxacin transcorneal permeation. J Pharm Sci 93: 2851-
2862 (2004).
7. J. H. Hamman, C. M. Schultz, and A. F. Kotze. N-trimethyl chitosan chloride: Optimum degree
of quaternization for drug absorption enhancement across epithelial cells. Drug Develop Ind
Pharm 29: 161-172 (2003).
8. J. H. Hamman, M. Stander, and A. F. Kotze. Effect of the degree of quaternisation of N-
trimethyl chitosan chloride on absorption enhancement: In vivo evaluation in rat nasal epithelia.
Inter J Pharm 232: 235-242 (2002).
9. A. F. Kotze, M. M. Thanou, H. L. Luessen, A. B. G. De Boer, J. C. Verhoef, and H. E.
Junginger. Effect of the degree of quaternization of N-trimethyl chitosan chloride on the
permeability of intestinal epithelial cells (Caco-2). Eur J Pharm Biopharm 47: 269-274 (1999).
10. M. M. Thanou, A. F. Kotze, T. Scharringhausen, H. L. Lueßen, A. G. De Boer, J. C. Verhoef,
and H. E. Junginger. Effect of degree of quaternization of N-trimethyl chitosan chloride for
enhanced transport of hydrophilic compounds across intestinal Caco-2 cell monolayers. J
Control Release 64: 15-25 (2000).
11. N. Hagenaars. Towards an intranasal influenza vaccine - Based on whole inactivated influenza
virus with N,N,N-trimethylchitosan as adjuvant. Utrecht University, Utrecht (2010).
12. B. Slütter, N. Hagenaars, and W. Jiskoot. Rational design of nasal vaccines. J Drug Target 16: 1-17
(2008).
13. L. Yin, J. Ding, C. He, L. Cui, C. Tang, and C. Yin. Drug permeability and mucoadhesion
properties of thiolated trimethyl chitosan nanoparticles in oral insulin delivery. Biomaterials 30:
5691-5700 (2009).
14. S. K. Singh, J. Stephani, M. Schaefer, H. Kalay, J. J. García-Vallejo, J. den Haan, E. Saeland, T.
Sparwasser, and Y. van Kooyk. Targeting glycan modified OVA to murine DC-SIGN
transgenic dendritic cells enhances MHC class I and II presentation. Mol Immunol 47: 164-174
(2009).
15. X. Zhao, L. Yin, J. Ding, C. Tang, S. Gu, C. Yin, and Y. Mao. Thiolated trimethyl chitosan
nanocomplexes as gene carriers with high in vitro and in vivo transfection efficiency. J Control
Release 144: 46-54 (2010).
16. B. G. De Geest, G. B. Sukhorukov, and H. Möhwald. The pros and cons of polyelectrolyte
capsules in drug delivery. Exp Opin Drug Deliv 6: 613-624 (2009).
17. A. Szarpak, D. Cui, F. Dubreuil, B. G. De Geest, L. J. De Cock, C. Picart, and R. Auzély-Velty.
Designing hyaluronic acid-based layer-by-layer capsules as a carrier for intracellular drug
delivery. Biomacromolecules 11: 713-720 (2010).
18. R. Censi, P. J. Fieten, P. di Martino, W. E. Hennink, and T. Vermonden. In Situ Forming
Hydrogels by Tandem Thermal Gelling and Michael Addition Reaction between
181

Chapter 8
Thermosensitive Triblock Copolymers and Thiolated Hyaluronan. Macromolecules 43: 5771-5778
(2010).
19. T. Kean and M. Thanou. Biodegradation, biodistribution and toxicity of chitosan. Adv Drug
Deliv Rev 62: 3-11 (2010).
182

APPENDICES

Affiliations of Collaborating Authors
Affiliations of collaborating authors:
Maryam Amidi
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht
University, Utrecht, The Netherlands
Suzanne Bal
Division of Drug Delivery Technology, Leiden/Amsterdam Center for Drug Research, Leiden
University, Leiden, The Netherlands
Han van den Bosch
Nobilon, part of Merck, Boxmeer, The Netherlands
Joke Bouwstra
Division of Drug Delivery Technology, Leiden/Amsterdam Center for Drug Research, Leiden
University, Leiden, The Netherlands
Sven Bruins
Department of Molecular Cell Biology and Immunology, Vrije University Medical Center,
Amsterdam, The Netherlands
Thomas van Es
Department of Molecular Cell Biology and Immunology, Vrije University Medical Center,
Amsterdam, The Netherlands
Ethlinn van Gaal
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht
University, Utrecht, The Netherlands
Harrie Glansbeek
Nobilon, part of Merck, Boxmeer, The Netherlands
Niels Hagenaars
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht
University, Utrecht, The Netherlands
Jacco Heldens
Nobilon, part of Merck, Boxmeer, The Netherlands
Wim Hennink
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht
University, Utrecht, The Netherlands
Wim Jiskoot
Division of Drug Delivery Technology, Leiden/Amsterdam Center for Drug Research, Leiden
University, Leiden, The Netherlands
Pascal de Jong
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht
University, Utrecht, The Netherlands
185

Affiliations of Collaborating Authors
Enrico Mastrobattista
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht
University, Utrecht, The Netherlands
Imke Mooren
Animal Service Department, Intervet Animal Health part of Merck, Boxmeer, The Netherlands
Ivo Que
Department of Endocrinology and Metabolic Diseases, Leiden University Medical Center,
Leiden, The Netherlands
Elly van Riet
Division of Drug Delivery Technology, Leiden/Amsterdam Center for Drug Research, Leiden
University, Leiden, The Netherlands
Bram Slütter
Division of Drug Delivery Technology, Leiden/Amsterdam Center for Drug Research, Leiden
University, Leiden, The Netherlands
Mies van Steenbergen
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht
University, Utrecht, The Netherlands
Steffen van der Wal
Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical
Sciences, Utrecht University, Utrecht, The Netherlands
186

List of Abbreviations
List of abbreviations:
APC
CpG
CS
CS-RA
DAc
DC
D carb
DDM
DLS
DMC
DOM
D thiol
DTT
DQ
EDC
ELISA
FACS
FCS
FDA
FITC
GPC
HA
HA-SH
HBSS
HEPES
ID
IgG
IL
i.m.
i.n.
kDa
LDH
antigen presenting cell
cytosine guanine dinucleotide
chitosan
re-acetylated chitosan
degree of acetylation
dendritic cell
degree of carboxylation
degree of dimethylation
dynamic light scattering
dimethyl chitosan
degree of O-methylation
degree of thiolation
dithiothreitol
degree of quaternization
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide)
enzyme-linked immunosorbent assay
fluorescence activated cell sorter
fetal calf serum
food and drug administration
fluorescein isothiocyanate
gel permeation chromatography
hyaluronic acid
thiolated hyaluronic acid
Hank’s balanced salt solution
N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid
intradermal
immunoglobulin G
interleukin
intramuscular
intranasal
kilodalton
lactate dehydrogenase
187

List of Abbreviations
LPS
lipopolysaccharide
MALT
mucosal associated lymphoid tissue
MTT
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
M n
number average molecular weight
M w
weight average molecular weight
NALT
nasal associated lymphoid tissue
NMP
N-methyl-2-pyrrolidone
NMR
nuclear magnetic resonance
OD
optical density
OVA
ovalbumin
PBS
phosphate buffer saline
PDI
polydispersity index
PEG
poly(ethylene glycol)
PEI
polyethylenimine
SDS
sodium dodecyl sulphate
sIgA
secretory immunoglobulin A
siRNA
small interfering ribonucleic acid
TEER
trans epithelial electrical resistance
Th1/Th2 T helper cell type 1/2
TMC
trimethyl chitosan
TMC-COOH
carboxylated trimethyl chitosan
TMC-OM
O-methylated trimethyl chitosan
TMC-RA
re-acetylated trimethyl chitosan
TMC-SH
thiolated trimethyl chitosan
TNF-α
tumor necrosis factor alfa
TPP
tripolyphosphate
WIV
whole inactivated influenza virus
XTT
sodium 3´-[1-(phenylaminocarbonyl)- 3,4-tetrazolium]-
bis (4-methoxy-6-nitro) benzene sulfonicacid hydrate
188

Curriculum Vitae
Curriculum Vitae
Rolf Verheul was born on the 8 th of March 1980 in Oss, The
Netherlands. After finishing pre-university education
(Gymnasium) at the Titus Brandsma Lyceum in Oss in 1998,
he started studying pharmacy at Utrecht University. In 2003
he obtained his Master’s degree in pharmaceutical sciences
cum laude, followed by his pharmacist degree (PharmD) in
2005. During his study, he completed a research traineeship
at the University of British Columbia, Vancouver, Canada under the supervision of prof. dr.
Cullis as well as internships at the National Institute for Public Health and the Environment
(RIVM) and various public and hospital pharmacies. Also, under the framework of the honors
program in pharmacy, he followed a minor in medical anthropology and sociology at the
University of Amsterdam. Thereafter, he worked as a laboratory pharmacist in the hospital
pharmacy of the Academic Medical Center, Amsterdam, for one year. In November 2006 he
started his PhD research program at the department of Pharmaceutics, Utrecht University
under the supervision of prof. dr. ir. W.E. Hennink and prof. dr. W. Jiskoot. The results of his
work are presented in this thesis.
189

List of Publications
List of publications:
RJ Verheul, M Amidi, S van der Wal, E van Riet, W Jiskoot, WE Hennink. Synthesis,
characterization and in vitro biological properties of O-methyl free N,N,N,-trimethylated
chitosan. Biomaterials 29, 3642-3649 (2008).
RJ Verheul, M Amidi, M van Steenbergen, E van Riet, W Jiskoot, WE Hennink. Influence of the
degree of acetylation on the enzymatic degradation and in vitro biological properties of
trimethylated chitosans. Biomaterials 30, 3129-3135 (2009).
N Hagenaars, E Mastrobattista, RJ Verheul, I Mooren, HL Glansbeek, JGM Heldens, H van den
Bosch, W Jiskoot. Physicochemical and immunological characterization of N,N,N-trimethyl
chitosan-coated whole inactivated influenza virus vaccine for intranasal administration.
Pharmaceutical Research 26,1353-1364 (2009).
RJ Verheul, N Hagenaars, I Mooren, PH de Jong, E Mastrobattista, HL Glansbeek, JGM Heldens,
H van den Bosch, WE Hennink, W Jiskoot. Relationship between structure and adjuvanticity
of N,N,N-trimethyl chitosan (TMC) structural variants in a nasal influenza vaccine. Journal of
Controlled Release 140, 126-133 (2009).
B Slütter, PC Soema, Z Ding, R Verheul, W Hennink, W Jiskoot. Conjugation of ovalbumin to
trimethyl chitosan improves immunogenicity of the antigen. Journal of Controlled Release
143, 207-214 (2010).
RJ Verheul, S van der Wal, WE Hennink. Tailorable thiolated trimethyl chitosans for
covalently stabilized nanoparticles. Biomacromolecules 11, 1965-1971 (2010).
RJ Verheul, N Hagenaars, T van Es, E van Gaal, P de Jong, S Bruins, I Que, B Slütter, E
Mastrobattista, HL Glansbeek, JGM Heldens, H van den Bosch, WE Hennink, W Jiskoot. A stepby-step
approach to study the influence of N-acetylation on the adjuvanticity of N,N,Ntrimethyl
chitosan (TMC) in an intranasal whole inactivated influenza virus vaccine.
Submitted.
RJ Verheul¸ N Hagenaars, T van Es, S Bruins, B Slütter, WE Hennink, W Jiskoot. Maturation of
human monocyte derived dendritic cells by trimethyl chitosan is correlated with its N-acetyl
glucosamine (GlcNAc) content. Submitted as short communication.
RJ Verheul, B Slütter, SM Bal, JA Bouwstra, W Jiskoot, WE Hennink. Covalently stabilized
trimethyl chitosan-hyaluronic acid nanoparticles for nasal and intradermal vaccination.
Submitted.
SM Bal, B Slütter, RJ Verheul, JA Bouwstra, W Jiskoot. Adjuvanted, antigen loaded N-trimethyl
chitosan nanoparticles for nasal and intradermal vaccination: adjuvant- and site-dependent
immunogenicity in mice. Submitted.
AK Varkouhi, RJ Verheul, RM Schiffelers, T Lammers, G Storm, WE Hennink. Gene silencing
activity of siRNA polyplexes based on thiolated N,N,N-trimethylated chitosan. Submitted.
190

NEDERLANDSE SAMENVATTING
EN
DANKWOORD

Nederlandse Samenvatting
Nederlandse Samenvatting
Vaccinatie is het actief opwekken van een afweerreactie tegen een bepaalde
lichaamsvreemde stof of eiwit (antigeen) van een ziekteverwekker. Hierdoor zal het
immuunsysteem in het vervolg deze indringer snel herkenen en het kunnen verwijderen
voordat het daadwerkelijk schade kan veroorzaken. Over de jaren heeft vaccinatie bewezen
om het meest (kosten)effectieve instrument te zijn in de strijd tegen besmettelijke ziekten.
Vaccins worden meestal gemaakt van levende, verzwakte of geïnactiveerde ziekteverwekkers
(bijv. virussen of bacteriën) of gezuiverde immunogene eiwitten die afkomstig zijn van deze
pathogenen. Hierdoor zullen vaccins zelf geen ziekte induceren, maar kunnen ze wel het
immuunsysteem activeren. Tegenwoordig worden de meeste vaccins toegediend via een
injectienaald. Echter, vanwege het risico van besmette naalden, met name in derde wereld
landen, en de noodzaak van geschoold personeel voor een dergelijke toediening, is de
belangstelling voor alternatieve vaccinatieroutes toegenomen.
Hoofdstuk 1 bediscussieert de voor- en nadelen van de intramusculaire en alternatieve
toedieningsroutes voor vaccins. In het bijzonder wordt immunisatie via de neus (intranasale
vaccinatie) besproken, waarmee relatief eenvoudig met bijvoorbeeld een neusspray of
neusdruppels, naald-vrij en zonder getraind personeel een vaccin toegediend kan worden.
Bovendien kan intranasale vaccinatie zowel mucosale (op de slijmvliezen) als systemische (in
het lichaam) immuunresponsen veroorzaken wat een verbeterde bescherming zou kunnen
betekenen t.o.v. intramusculaire toediening. Aan de andere kant zijn, vanwege de natuurlijke
barrières in de neus, antigeenafbraak en het bereiken van de antigeen presenterende cellen
(APC's) over de slijm- en cellagen heen, belangrijke nadelen van nasale vaccinatie (Figuur 1).
Aangezien verzwakte, levende vaccins een aanzienlijk veiligheidsrisico met zich meebrengen
voor mensen met een suboptimaal immuun systeem (b.v. kinderen, ouderen, patiënten met
bepaalde ziektes als HIV), richt de ontwikkeling van vaccins zich nu vooral op het gebruik van
gezuiverde, goed gekarakteriseerde antigene eiwitten. Echter, deze subunit vaccins zijn over
het algemeen minder immunogeen en hebben een krachtig adjuvans (systeem) nodig om een
adequate afweerreactie op te roepen. Door een muco-adhesief polymeer (dat ‘plakt’ aan de
slijmlaag), zoals N,N,N-trimethylchitosan (TMC), te gebruiken bij intranasale vaccinatie kan de
immuunrespons tegen een antigeen worden verhoogd.
193

Nederlandse Samenvatting
Figuur 1. Schematische weergave van de cruciale stappen bij intranasale vaccinatie. Na toediening zal
het antigeen in contact komen met de oppervlaktes van het neusslijmvlies (1) waarna het vervolgens
getransporteerd wordt over het nasale epitheel (2) en opgenomen wordt door de antigeen
presenterende cellen (APCs) zoals dendritische cellen (DCs) (3). Deze APCs zullen na activering o.a. tot
de aanmaak van antilichamen overgaan (4).
Vermoedelijk werkt TMC, een in water-oplosbaar chitosan derivaat, door de verblijftijd in de
neus te verlegen en/of het contact tussen het antigeen en het mucosale oppervlak te
verbeteren. TMC (Figuur 2) is eigenlijk een verzamelnaam voor een groep van polymeren met
veel overeenkomsten in de chemische structuur maar met specifieke verschillen (zoals de
ketenlengte, ladingsdichtheid en mate van methylering en acetylering). Het gecontroleerd
kunnen variëren van deze structurele elementen maakt het mogelijk beter inzicht te krijgen in
de afzonderlijke bijdrage van deze groepen op de fysisch-chemische en biologische
eigenschappen van TMC. Ook kunnen nieuwe funtionaliteiten worden geïntroduceerd, zoals
194

Nederlandse Samenvatting
thiolgroepen die kunnen bijdragen aan het verbeteren en optimaliseren van de eigenschappen
van TMC.
Chitosan, en zijn derivaten als TMC, zijn in de vorm van nano- of microdeeltjes effectiever in
het induceren van een immuunrespons dan een ‘gewone’ polymeeroplossing. Nano-gelering,
gebruikmakend van electrostatische interacties, is een eenvoudige, veel gebruikte methode om
nanodeeltjes van TMC te maken. Door negatief geladen (macro)moleculen druppelsgewijs
onder roeren aan de TMC oplossing toe te voegen, vormen zich spontaan nanodeeltjes in een
buffer met lage ionsterkte. Echter, de fysisch-chemische stabiliteit en de immunogeniciteit van
deze met antigeen beladen complexen zijn afhankelijk van de eigenschappen van de gebruikte
componenten. Mogelijk kunnen de momenteel toegepaste systemen nog worden verbeterd.
Bovendien, hoewel het werkingsmechanisme van TMC doorgaans wordt toegeschreven aan de
muco-adhesieve en penetratie verbeterende eigenschappen, ontbreekt er gedetailleerde
kennis over het mechanisme (zo is bijvoorbeeld het directe effect van TMC op APCs niet
bekend). Concluderend kan gesteld worden dat er zijn vele mogelijkheden voor het
optimaliseren van de structuur van TMC en de op TMC gebaseerde nanodeeltjes. Tegelijkertijd
zal een beter inzicht in het werkingsmechanisme van TMC tot een rationeel ontwerp van
dergelijke adjuvantia kunnen leiden.
Figuur 2. Schematische weergave van de structurele variaties van TMC. TMC kan variëren in de mate
van acetylering (blok x), quaternisering (blok y) en O-methylering (blok z). Deze variaties kunnen
willekeurig over het polymeer verdeeld zijn.
Het doel van dit proefschrift is om synthese-routes te ontwikkelen waarmee op een
controleerbare wijze de chemische structuur van TMC kan worden gevarieerd. Verder worden
er nieuwe groepen geïntroduceerd, zoals thiolen, die mogelijk de eigenschappen van TMC nog
195

Nederlandse Samenvatting
verder kunnen verbeteren. Op deze manier kunnen structuur-activiteit-relaties worden
onderzocht in zowel in vitro studies als in in vivo (nasale) vaccinatie studies.
De huidige methode om TMC te synthetiseren veroorzaakt, naast de gewenste trimethylering
van de vrije amines, ook O-methylering op de hydroxylgroepen en ketenbreuk van het
polymer. Omdat deze nevenreacties van invloed kunnen zijn op de eigenschappen van het
polymeer, is er een behoefte aan een syntheseroute waarbij deze ongewenste nevenreacties
niet optreden en waarbij de mate van quaternisering (oftewel het percentage amine groepen
dat getrimethyleerd is (DQ)) vrijelijk gevarieerd en gecontroleerd kan worden.
In Hoofdstuk 2 werden met behulp van een twee-staps methode O-methyl vrije TMCs met
variërende DQs gesynthetiseerd. Eerst werd chitosan kwantitatief gedimethyleerd met behulp
van mierenzuur en formaldehyde. Vervolgens kon TMC worden verkregen door dit
dimethylchitosan te laten reageren met een overmaat aan iodomethaan. Door de reactietijd te
variëren, konden TMCs met verschillende DQs (22-68%) worden gesynthetiseerd. Deze TMCs
bleken geen waarneembare O-methylering (bepaald met 1 H-NMR) te hebben en uit een geringe
verhoging van het molecuulgewicht met toenemende DQ (bepaald met gel permeatie
chromatografie of GPC) kon geconcludeerd worden dat zich geen noemenswaardige
ketenbreuk had voorgedaan tijdens de synthese. De oplosbaarheid in water met een pH van 7
was voor O-methyl vrij TMC met een DQ 30% losten uitstekend op in water met
een pH van 7. O-methyl vrije TMCs gaven een grotere daling in de trans-epitheliale elektrische
weerstand (TEER) van een caco-2 monolaag dan O-gemethyleerde TMCs. Door het meten van
de TEER wordt een idee verkregen in hoeverre een polymeer in staat is de intercellulaire
ruimtes tussen epitheelcellen te openen waardoor het transport van stoffen over deze cellaag
gemakkelijker wordt. Een daling in de TEER geeft aan dat de weerstand afneemt en dus dat de
cellaag permeabeler wordt. Ook werd met de verhoging van de DQ, een stijging van de
cytotoxiciteit (m.b.v. een MTT test) en celmembraan-permeabiliteit (m.b.v. een LDH test)
waargenomen. Dit hoofdstuk toont duidelijk aan dat zowel de DQ als de aan/afwezigheid van
O-methylering een belangrijke invloed hebben op de fysische en in vitro biologische
eigenschappen van TMC.
De invloed van een andere variatie in de moleculaire structuur, de mate van acetylering
(DAc), op de in vitro enzymatische afbraak en biologische eigenschappen van TMC is
onderzocht in hoofdstuk 3. TMCs met een DAc variërend van 11 tot 55% werden
196

Nederlandse Samenvatting
gesynthetiseerd met behulp van een drie-stappen-methode. Eerst werden de amines van
chitosan gedeeltelijk geacetyleerd met azijnzuuranhydride en natrium borohydride, gevolgd
door de kwantitatieve dimethylering van de resterende amines met formaldehyde en natrium
borohydride. Vervolgens werd TMC gesynthetiseerd in aanwezigheid van een overmaat aan
iodomethaan. De TMCs verkregen via deze methode bleken geen O-methylering, noch een
verlies in acetylgroepen ( 1 H-NMR) of ketenbreuk (GPC) te vertonen. De mate van afbraak van
TMC, en dat van haar voorgangers chitosan en dimethyl chitosan, door lysozyme (een
lichaamseigen enzym) was sterk afhankelijk van de DAc; polymeren met de hoogste DAc
vertoonde de grootste daling in het molecuulgewicht. TMCs met een hoge DAc (~ 50%), een
DQ van ongeveer 44% en met of zonder O-gemethyleerde groepen, waren niet in staat om de
intercellulaire ruimtes van een caco-2 monolaag te openen zoals onderzocht in de transepitheliale
elektrische weerstand (TEER) test. Dit in tegenstelling tot TMCs (zowel O-
gemethyleerde en O-methyl vrij; concentratie van 2.5 mg/ml) met een soortgelijke DQ maar
een lagere DAc; deze polymeren waren in staat de TEER tot 70 en 30% te verminderen,
respectievelijk voor de O-methyl vrij en de O-gemethyleerde TMCs. Daarnaast vertoonden
TMCs met een hoge DAc (~ 50%) geen noemenswaardige celtoxiciteit (MTT, LDH afgifte) tot
een concentratie van 10 mg/ml. Dit hoofdstuk toont aan dat de mate van N-acetylering van
TMC een grote invloed heeft op de in vitro enzymatische afbraak en biologische eigenschappen
van TMC.
De resultaten uit hoofdstukken 2 en 3 tonen aan dat de DQ, DAc en mate van O-methylering
van invloed zijn op de in vitro biologische eigenschappen van TMC. Daarom onderzochten we
de effecten van deze structurele variaties van TMC op de werkzaamheid als adjuvans in een
intranasale (i.n.) immunisatie studie in muizen.
In hoofdstuk 4 werden TMCs met variërende mate van quaternisering (DQ, 22-86%), O-
methylering (DOM, 0-76%) en N-acetylering (DAc 9-54%) geformuleerd met geïnactiveerd
influenza virus (WIV). Eenvoudig mengen van het WIV met de TMCs in een 1:1
gewichtsverhouding resulteerde in vergelijkbare, positief geladen nanodeeltjes door coating
van het negatief geladen WIV met het positief geladen TMC. De hoeveelheid vrij TMC in
oplossing was vergelijkbaar voor alle TMC-WIV formuleringen en er is dus een overmaat aan
TMC aanwezig in de formuleringen.
Na intranasale immunisatie van muizen met WIV en TMC-WIV op dag 0 en 21, vertoonden de
TMC-WIV formuleringen een sterkere respons (gemeten als hogere totaal IgG, IgG1 en IgG2a/c
titers) dan WIV zonder TMC, met uitzondering van WIV gecoat met gereacetyleerd TMC (DAc
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54 %, DQ 44%). Er werden geen significante verschillen in de antistof-titers waargenomen
voor TMCs die varieerden in DQ of DOM, wat aangeeft dat deze structurele kenmerken een
ondergeschikte rol spelen in de i.n. adjuvans eigenschappen. TMC met een DQ van 56%
(TMC56) geformuleerd met WIV in een gewichtsverhouding van 5:1 resulteerde in significant
lagere IgG2a/c:IgG1 ratio's ten opzichte van TMC56 gemengd in de verhouding van 0.2:1 en
1:1. De IgG2a/c:IgG1 ratio geeft een indicatie voor het type immuunrespons dat opgewekt
wordt: een hoge IgG1 spiegel wijst op een type 2 (of humorale, met antilichamen,
afweerreactie) terwijl een hoge IgG2a/c titer een type 1 (of cellulaire, met cytotoxische T-
cellen, afweerreactie) indiceert. Deze resultaten impliceren dus een verschuiving naar een
Th2-type immuunrespons bij een hogere TMC dosis. Blootstelling van de gevaccineerde
muizen aan een actief, ziekmakend, virus bewezen dat alle TMC-WIV formuleringen
bescherming boden met uitzondering van WIV gecoat met gereacetyleerd TMC. Dit hoofdstuk
toont aan dat de immunogeniciteit van WIV sterk verbetert door het gebruik van TMC en dat
bescherming tegen een actief virus na i.n. vaccinatie met TMC-WIV kan worden bewerkstelligd.
De werkzaamheid van TMC als adjuvans wordt sterk verminderd door het acetyleren van TMC,
terwijl de DQ en DOM nauwelijks van invloed zijn.
Het doel van hoofdstuk 5A was om de oorzaak te achterhalen voor het gebrek aan
werkzaamheid van gereacetyleerd TMC (TMC-RA) als adjuvans. TMC-RA (mate van acetylering
54%) werd vergeleken met TMC (mate van acetylering 17%) op zes potentieel kritieke
stappen in de inductie van een immuunrespons na intranasale (i.n.) toediening bij muizen:
chemische stabiliteit van het polymeer in een neuswassing, de locatie van WIV in de neus, de
nasale verblijftijd van WIV, de cellulaire opname van WIV door calu-3 epitheelcellen, het
transport van WIV over een monolaag van epitheel cellen, en de capaciteit van de verschillende
formuleringen om activering te induceren van muizen dendritische cellen (DCs).
TMC-RA werd in een grotere mate afgebroken in de neuswassing dan TMC. De locatie van het
antigeen in de neus en de nasale verblijftijd waren hetzelfde voor beide types TMC.
Fluorescent gelabeld WIV associeerde beter met calu-3 cellen indien gecoat met TMC-RA dan
met TMC, en beide TMCs verminderden aanzienlijk het transport van WIV over een calu-3
monolaag. Muizen DCs werden door alle formuleringen in gelijke mate geactiveerd.
Samenvattend, de inferieure werkzaamheid van TMC-RA als adjuvans zou kunnen worden
verklaard door een lagere stabiliteit van de TMC-RA-WIV in de neusholte, en het is in ieder
geval onwaarschijnlijk dat een van de andere cruciale stappen die zijn onderzocht in deze
studie, een rol spelen.
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Het is bekend dat de N-geacetyleerde glucosamine blokken (of GlcNAcs) aanwezig in TMC
kunnen binden aan verschillende humane C-type lectines, een familie van cel-receptoren die
betrokken zijn bij de immuunrespons. Mogelijk is hierdoor het effect van TMC en
gereacetyleerd TMC (met DAc van 54%, TMC-RA) op opname en activering van menselijke
dendritische cellen (DCs) verschillend. Dit werd onderzocht in hoofdstuk 5B met behulp van
het geïnactiveerde influenza virus (WIV) als antigeen. Studies met monocyt-afgeleide humane
DCs lieten zien dat de opname van TMC(-RA) gecoat WIV iets lager was dan ‘gewoon’ WIV.
TMC-RA, als oplossing of als coating van WIV, veroorzaakte een veel sterkere activering van
DCs, zoals gemeten met CD86 expressie, dan de andere formuleringen. Verder induceerde
alleen TMC-RA(-WIV) een hoge afgifte van de cytokines IL-10, TNF- α en IL-12p40 en IL-12p70
door de DCs. Omdat zowel de afgifte van IL-10 en IL-12p70 was verhoogd, konden er geen
uitspaken worden gedaan over het mogelijk induceren van een Th1 of Th2 type
immuunrespons. Concluderend, TMC-RA heeft sterke immuno-stimulerende effecten in vitro
op humane dendritische cellen. Dit impliceert dat de mate van N-acetylering mogelijk van
cruciaal belang is voor het adjuvans effect van TMC in mensen.
Door thiol-groepen te introduceren in de structuur zullen de (farmaceutische) toepassingen
van TMC nog verder verbreed kunnen worden. Thiolen kunnen de muco-adhesieve
eigenschappen verbeteren, vergemakkelijken verdere derivatiseringsreacties via o.m.
intracellulair afbreekbare disulfide-bruggen, en maken het mogelijk om covalent vernette
nanodeeltjes te produceren met een gethioleerd anionisch polymeer als crosslinker. In
Hoofdstuk 6 werd een nieuwe vier-stappen methode gepresenteerd om gedeeltelijk
gethioleerd TMC te synthetiseren waarmee de mate van quaternisering en thiolering vrijelijk
kunnen worden gevarieerd. Eerst werden de amines van chitosan gedeeltelijk gecarboxyleerd
met glyoxylzuur en natrium borohydride. Daarna werden de resterende amines kwantitatief
gedimethyleerd met formaldehyde en natrium borohydride en vervolgens konden
gecarboxyleerde TMCs verkregen worden door dit polymeer te laten reageren met een
overmaat aan iodomethaan in N-methylpyrrolidon. Hierna werden de gedeeltelijk
gecarboxyleerde TMCs opgelost in water en werd cystamine aan de carboxylzuren gekoppeld
bij een pH van 5.5 met behulp van EDC. Na toevoeging van dithiotreïtol disulfide om de
disulfide-bruggen te breken, werden gethioleerde TMCs verkregen die varieerden in mate van
quaternisering (25-54%) en thiolering (5-7%) zoals bepaald met 1 H-NMR en Ellman's test. Gel
permeatie chromatografie toonde aan dat er een beperkt aantal intermoleculaire bindingen
was ontstaan. De thiol-groepen in de TMCs bleken bij pH 7.4 snel te oxideren tot disulfides,
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Nederlandse Samenvatting
terwijl ze bij een pH van 4.0 redelijk intact bleven na 8 uur op 37 graden. Studies met calu-3
cellen lieten zien dat er een lichte daling in cytotoxiciteit is voor de gethioleerde TMCs in
vergelijking met de niet-gethioleerde polymeren met eenzelfde DQ.
Positief geladen nanodeeltjes werden gemaakt van de gethioleerde TMCs en gethioleerd
hyaluronzuur (HA-SH) en hierin werd fluorescent gelabeld ovalbumine als model-eiwit
ingesloten. Deze covalent gestabiliseerde nanodeeltjes bleven stabiel in 0.8 M NaCl, in
tegenstelling tot de deeltjes gemaakt van niet-gethioleerde polymeren die in deze sterk
ionische buffer uit elkaar vielen. Dit toonde aan dat de gestabiliseerde deeltjes bij elkaar
gehouden werden door intermoleculaire zwavelbruggen tussen TMC-SH en HA-SH.
In hoofdstuk 6 werd aangetoond dat de fysische stabiliteit van polyelectroliet nanodeeltjes
van trimethyl chitosan (TMC) en hyaluronzuur (HA) beperkt is onder fysiologische
omstandigheden. Vooral in aanwezigheid van zout kan de stabiliteit verminderen aangezien zij
de ladingsinteracties tussen het positief geladen TMC en het negatief geladen HA geringer
worden. Dit kan de werkzaamheid van het adjuvans effect van de nanodeeltjes voor de
intranasale en mogelijk ook intradermale immunisatie verminderen. Zodoende werd in
hoofdstuk 7 het effect van de stabilisatie van TMC/HA nanodeeltjes door disulfide-bruggen op
hun immunogeniciteit geëvalueerd. Ook werden, als alternatief, de resterende thiol-groepen op
het oppervlak van deze deeltjes gepegyleerd waardoor de positieve lading van deze deeltjes
(enigszins) werd afgeschermd. Mogelijk heeft dit een additioneel gunstig effect aangezien PEG
(poly(ethyleen glycol)) de interacties met de extracellulaire matrix zou kunnen verminderen
(intradermaal) en/of de muco-adhesie zou kunnen verbeteren (intranasaal). Covalent
gestabiliseerde nanodeeltjes beladen met ovalbumine (OVA) werden gemaakt met gethioleerd
TMC en gethioleerd HA via gelering op basis van ladings-interactie gevolgd door spontane
vorming van disulfide-bruggen na een incubatie van 3 uur bij pH 7.4 en 37 °C. Tevens werd
maleïmide PEG gekoppeld aan de resterende thiol-groepen aan het oppervlak van de deeltjes
om de positieve lading af te schermen.
De nanodeeltjes hadden een grootte van circa 250-350 nm, een positieve zeta potentiaal en
OVA ladings-efficiënties tot 60%. Pegylering resulteerde in een kleine verlaging van de zeta
potentiaal en een geringe toename in deeltjesgrootte. Gestabiliseerde TMC-S-S-HA deeltjes
(gepegyleerd of niet) hadden een superieure stabiliteit in zoutoplossingen in vergelijking met
niet-gestabiliseerde TMC/HA deeltjes (bestaande uit niet-gethioleerde polymeren). Echter, de
TMC-S-S-HA deeltjes vielen momentaan uiteen wanneer een reagens werd toegevoegd dat
disulfide-bruggen breekt. In zowel de intranasale als intradermale vaccinatie-studie,
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Nederlandse Samenvatting
vertoonden de gestabiliseerde TMC-S-S-HA deeltjes beladen met OVA superieure
immunogeniciteit in vergelijking met niet-gestabiliseerde deeltjes. Pegylering resulteerde
intranasaal in een volledige tenietdoening van de positieve effecten van stabilisatie en het had
geen additioneel effect wanneer deze gepegyleerde deeltjes werden gebruikt voor
intradermale vaccinatie. Concluderend, de stabilisatie van de TMC/HA deeltjes verbeterde de
immunogeniciteit na intranasale en intradermale vaccinatie, echter pegylering van deze
gestabiliseerde deeltjes had geen gunstig effect.
De nieuwe synthese-routes die in dit proefschrift worden beschreven, maken het mogelijk
voor elke specifieke toepassing de gewenste structurele variant van TMC te produceren. Zo
kunnen de mate van quaternisering en acetylering worden gevarieerd zonder dat er andere
wijzigingen, zoals O-methylering of ketenbreuk, in het polymeer optreden. Daarnaast wordt
een methode beschreven om op een controleerbare manier thiol-groepen in TMC te
introduceren. Het gecontroleerd kunnen variëren van de structurele eigenschappen van TMC
geeft de mogelijkheid om op de juiste manier structuur-activiteit-relaties te onderzoek in in
vitro biologische assays en in vivo (nasale) vaccinatie-studies. Verder werd het inzicht in het
werkingsmechanisme van TMC verdiept door verschillende, potentieel cruciale, stappen in
intranasale vaccinatie op een systematische manier te onderzoeken. Ten slotte werd een
veelbelovend nieuw, covalent gestabiliseerd, drager systeem onderzocht. De bemoedigende
resultaten met deze nieuwe TMC-varianten geven meer dan voldoende aanleiding tot verdere
pre-klinische en eventueel klinische ontwikkeling.
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Dankwoord
Dankwoord
Beste mensen, mijn mooie periode als AIO zit erop! Natuurlijk zijn er wel eens wat
tegenslagen geweest en heb ik af en toe hard moeten werken, maar al met al is het project
soepel verlopen en heb ik me erg vermaakt op het Went. Dit is dan ook naar mijn mening het
grootste compliment voor iedereen met wie ik, op welke manier dan ook, te maken mee heb
gehad de afgelopen jaren. Zonder jullie hulp, enthousiasme en kritische noot zou dit boekje
nooit op deze prettige wijze zijn voltooid. Heel erg bedankt! Een aantal mensen wil ik graag in
het bijzonder bedanken:
In den beginne is er Wim H, mijn promotor: ‘Wat een biologische meuk dat TMC (Thai
Massage Center)! Kun je er geen hydrogel van maken?’ Waarna de voetbaluitslagen van het
weekend werden besproken, vaak met enig leedvermaak aangezien Ajax de afgelopen 4 jaar
niet de beste periode uit haar geschiedenis kende. Beste Wim, bedankt dat je deze PSV-er
zoveel vrijheid en vertrouwen heb gegeven maar ook voor me klaar stond als er even spijkers
met koppen geslagen moesten worden.
Beste Wim J, mijn andere promotor. Hoewel officieel pas op het einde aan boord geklommen,
was je eigenlijk vanaf het begin al de grote coördinator van het TMC (Toxiciteit Misschien
Cruciaal) project. Mooie discussies en een gezonde rivaliteit maakten de tripjes naar Leiden
een leuk vooruitzicht. Dank voor je wetenschappelijke hulp en tekstuele creativiteit!
Dan zijn er natuurlijk nog mijn alter-AIOs uit Leiden, Suzanne en Bram. Toch mooi dat we na
3 jaar vergaderen onze projecten hebben kunnen afsluiten met een gezamenlijk in vivo
experiment! Heel veel succes in de toekomst en bedankt voor de leuke samenwerking. Ook Elly
mag hier niet ontbreken voor alle hulp bij de cel-experimenten en Joke voor de bijdrage op het
gebied van de intradermale vaccinaties.
Mijn studenten, Sara de Madrid, muchas gracias por todo en Francesco di Camarino, grazie
mille! Steffen, jouw vrijdagmiddag-experimentje heeft mij een pikstart bezorgd en ook daarna
heb je me regelmatig de weg gewezen uit het organisch-chemische moeras. Bijna was ik in een
chemicus veranderd maar gelukkig heeft het niet zover hoeven komen, dank. Natuurlijk mag
ook de man die in elk dankwoord staat vermeld ook hier niet ontbreken: Mies, zonder jou ziet
het leven er toch een stuk minder rooskleurig uit, crying at the Viscotek!
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Dankwoord
Thomas en Sven van het VUmc, geweldig dat jullie als immunologen zo enthousiast met onze
polymeren aan de gang wilden gaan. Uiteindelijk heeft het nog mooie resultaten opgeleverd.
De mensen van Nobilon en Intervet, de twee uur durende treinreis was snel vergeten door de
stimulerende en prettige sfeer die er in Boxmeer heerste. Heel veel succes in deze tijd.
Dr. ‘so you think you can dance’ Ethlinn, dat je hebt tijdens je eigen lange weg nog tijd hebt
gevonden om me te helpen met experimenten en tips en trucs te geven, heb ik zeer kunnen
waarderen. Ook anderen die me op de een of andere manier met de experimenten hebben
geholpen mogen natuurlijk niet ontbreken: Maryam, Enrico, Roel, Pascal en Roberta. Dank
voor jullie hulp!
Het sterrenensemble van Z605, Marina (sorry if I teased you too much), Hajar (sorry if I
teased you too much) and Amir V (sorry if I teased you too much), thanks for the good times on
and off the lab. De oude garde Sophie, Marjan, Marion, Frankie, Sabrina, Marcel, Jozef ‘je
gedraagt je wel hè? Ik hoef maar dìt te zien...’ en de rijpe garde Wouter, Joris en Ellen bedankt
voor de gezelligheid en de wijsheden. De jonge hindes van biofarmacie: Inge, Roy, DaMarkus,
Bart, speedy Maria, Alex, Pieter, Emmy, Albert, Melody, Kimberley, Audrie, Luis, Kim, Barbara,
Lydia, Peter en de anderen, heel veel plezier en succes met het afronden!
Gelukkig waren er nog voldoende mensen die me naast het werk bezig hielden. Mijn
teamgenoten van de Ody-zondag, we gaan er weer een frank en vrij seizoen van maken! De
oud-huisgenoten van de pleasuredome, Sindre & Torkel, Japus, de Farmacie-gang, dank voor
jullie vriendschap. Gert, zonder je voedzame maaltijden en de waardevolle gesprekken zou er
slechts een skelet resteren, got to get behind the mule in the morning and plow!
Amir-idjan, alle (oude) wijze mannen komen uit het Oosten. Vaak hebben we Utrecht onveilig
gemaakt en nog vaker heb je me uitgelegd hoe de dingen werkelijk in elkaar zitten. Over vijf
jaar zitten we bij je op de veranda ergens in Toronto met een lekkere whisky maar eerst heb ik
je nog hier even nodig. Merci!
Nelis, met jou iets ondernemen staat garant voor een succesvol avontuur. Met -5 ⁰C bananen
roosteren aan de Sunshine Coast, raven op surrealistisch Roskilde of afreizen naar Boxmeer
om muizen te pesten, het was allemaal enerverend. Je bent een unieke gozer en ik weet zeker
dat wij elkaar blijven zien.
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Dankwoord
Lieve Femke, het avontuur met jou is eigenlijk pas net begonnen maar hoe meer ik van je
ontdek hoe meer ik er van zeker van ben dat we een mooie toekomst voor ons hebben.
En als laatste wil ik nog mijn familie bedanken. Oma, die afspraak van vier jaar geleden
komen we allebei na, geweldig dat je hier bij kan zijn. Lieve Marc en Nienke, nog even wachten
en dan is het jullie dag. Lieve ‘spap en ‘smam, dank voor de liefde, interesse en vertrouwen.
Zonder jullie was dit allemaal niet mogelijk geweest dus geniet ervan.
Tijd voor een nieuw hoofdstuk!
Rolf
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