Design of functional dendritic polymers for application as drug and ...

Gene Therapy and Molecular Biology Vol 10, page 71
Gene Ther Mol Biol Vol 10, 71-94, 2006
Design of functional dendritic polymers for
application as drug and gene delivery systems
Review Article
Zili Sideratou, Leto-Aikaterini Tziveleka, Christina Kontoyianni, Dimitris
Tsiourvas, Constantinos M. Paleos*
Institute of Physical Chemistry, NCSR “Demokritos”, 15310 Aghia Paraskevi, Attiki, Greece
__________________________________________________________________________________
*Correspondence: Constantinos M. Paleos, Institute of Physical Chemistry, NCSR "Demokritos"; 15310 Aghia Paraskevi, Attiki,
Greece; Tel.: +30 210 6503666; Fax: +30 210 6529792; e-mail: paleos@chem.demokritos.gr
Key words: Dendrimers, Hyperbranched Polymers, Dendritic Polymers, Nanocarriers, Drug Delivery System, Gene Delivery
Abbreviations: Adriamycin, (ADR); arginine-grafted-PAMAM dendrimer, (PAMAM-Arg); Asialo-glycoprotein, (ASGP);
betamethasone dipropionate, (BD); betamethasone valerate, (BV); Boron Neutron Capture Therapy, (BNCT); diaminobutane
poly(propylene imine) dendrimer, (DAB); diaminobutane poly(propylene imine) fourth generation dendrimer functionalized with 6
guanidinium groups, (DAB-G6 ); diaminobutane poly(propylene imine) fourth generation dendrimer functionalized with 12 guanidinium
groups, (DAB-G12); Dynamic Light Scattering, (DLS); epidermal growth factor, (EGF); green fluorescent protein, (GFP); injected dose,
(ID); hyperbranched poly(ethylene imine), (PEI); hyperbranched polyglycerol, (PG); Isothermal Titration Calorimety, (ITC); L-lysine
grafted-PAMAM dendrimer, (PAMAM-Lys); methotrexate, (MTX); methoxypoly(ethylene glycol)-isocyanate, (PEG-isocyanate);
PEGylated diaminobutane poly(propylene imine) dendrimer with 4 PEG chains, (DAB-4PEG); PEGylated diaminobutane
poly(propylene imine) dendrimer with 8 PEG chains, (DAB-8PEG); PEGylated polyglycerol, (PG-PEG); PEGylated-Folate
polyglycerol, (PG-PEG-Folate); Phosphate Buffer Saline, (PBS); poly(amidoamine) dendrimer, (PAMAM); poly(amidoamine)
dendrimer with terminal hydroxyl groups, (PAMAM-OH); poly(ethylene imine)-poly(ethylene glycol)-folate, (PEI-PEG-FOL);
poly(ethylene glycol), (PEG); poly(ethylene glycol) monomethyl ether, (M-PEG); poly(propylene imine) dendrimer, (PPI); primaquine
phosphate, (PP); pyrene, (PY); quaternized poly(amidoamine) dendrimer with terminal hydroxyl groups, (QPAMAM-OH); tamoxifen,
(TAM)
Received: 28 November 2005; Accepted: 10 February 2006; electronically published: March 2006
Summary
The present review deals with the design and preparation of functional and multifunctional dendrimeric and
hyperbranched polymers (dendritic polymers), in order to be employed as drug and gene delivery systems. In
particular, using as starting materials known and well-characterized basic dendritic polymers, the review discusses
the kind of structural modifications that these polymers were subjected for preparing nanocarriers of low toxicity,
high encapsulating capacity, specificity to certain biological cells and transport ability through their membranes.
Due to the great number of external groups of dendritic polymers either functionalization or multifunctionalization
can occur, providing products that fulfill one or more of the requirements that an effective drug carrier should
exhibit. A common feature of these dendritic polymers is the exhibition of the so-called polyvalent interactions,
while for the multifunctional derivatives a number of targeting ligands determines specificity, other groups secure
stability in biological milieu, while others facilitate their transport through cell membranes. In addition, for gene
delivery applications these multifunctional systems should be or become cationic in the biological environment for
the formation of complexes with the negatively charged genetic material.
I. Introduction
Dendrimers are prepared by tedious synthetic
procedures (Bosman et al, 1999; Schlüter and Rabe, 2000;
Fréchet and Tomalia, 2001; Newkome et al, 2001) and
they are nanometer-sized, highly branched and
monodisperse macromolecules with symmetrical
architecture. They consist of a central core, branching
units and terminal functional groups. The core and the
71
internal units determine the environment of the
nanocavities and consequently their solubilizing or
encapsulating properties, whereas, the external groups
their solubility and chemical behaviour. On the other hand,
hyperbranched polymers (Inoue, 2000), including the
extensively investigated hyperbranched polyether polyols
or polyglycerols (Sunder et al, 1999a, b; 2000a,b; Haag,
2001; Frey and Haag, 2002; Siegers et al, 2004) are
conveniently prepared. Hyperbranched polymers are non-

Sideratou et al: Dendritic polymers for application as drug and gene delivery systems
symmetrical, highly branched and polydispersed
macromolecules, while their main structural feature, also
common to dendrimers, is that they exhibit nanocavities.
These two types of polymers are called dendritic polymers
the nanocavities of which, depending on their polarity, can
encapsulate various molecules, including active drug
ingredients. The external groups of dendritic polymers can
be modified providing a diversity of functional materials
(Vögtle et al, 2000) that can be employed for various
applications.
Within this context, commercially available or
custom-made dendrimeric or hyperbranched polymers can
be functionalized for being used as effective systems for
drug (Liu and Fréchet, 1999; De Jes-s et al, 2002; Stiriba
et al, 2002; Beezer et al, 2003; Gillies and Fréchet, 2005)
and gene (Bielinska et al, 1999; Luo et al, 2002; Ohsaki et
al, 2002) delivery. Since more than one type of groups can
be introduced at the surface of the dendritic polymers,
these systems are characterized as multifunctional as
shown in Figure 1. Each type of groups plays a specific
role in the application of multifunctional dendritic
polymers as drug delivery systems. Thus, specificity for
certain cells can be accomplished by attaching targeting
ligands at the surface of dendritic polymers, while
enhanced solubility, decreased toxicity, biocompatibity,
stability and protection in the biological milieu can be
achieved by the functionalization of the end groups of
dendritic polymers, for instance, with poly(ethylene
glycol) chains (PEG). The function of PEG-chains is
crucial for modifying the behaviour of drug themselves or
of their carriers (Noppl-Simson and Needham, 1996;
Ishiwata et al, 1997; Liu et al, 1999; Liu et al, 2000;
Veronese, 2001; Roberts et al, 2002; Pantos et al, 2004;
Vandermeulen and Klok, 2004).
Targeting ligands are complementary to cell
receptors (Cooper, 1997; Lodish et al, 2000) and induce
the attachment of the nanocarrier to the cell surface. This
Figure 1. Schematic representation of a multifunctional dendrimer.
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
72
binding is further enhanced due to the so-called polyvalent
interactions (Mammen et al, 1998; Kitov and Bundle,
2003) attributed to the close proximity of the recognizable
ligands on the limited surface area of the dendritic
molecules. On the other hand, as it has long been
established with liposomes (Lasic and Needham, 1995;
Crosasso et al, 2000; Needham and Kim, 2000; Silvander
et al, 2000), PEG-chains may prolong the circulation of
liposomes in biological milieu. Transport through the cell
membrane can also be facilitated by the introduction of
appropriate moieties at the surface of the dendritic
polymers. In addition, modification of the internal groups
of dendrimers affects their solubilizing character, making,
therefore, possible the encapsulation of a diversity of
drugs. In this connection, cationization of dendrimers, and
particularly of their external groups, facilitates their
application as gene transfer agents (Bielinska et al, 1999;
Luo et al, 2002; Ohsaki et al, 2002) due to formation of
DNA-Dendritic Polymer complexes.
Monofunctional dendritic drug carriers do not
simultaneously show the desired properties that
multifunctional derivatives exhibit. Thus, in this review,
starting from selected monofunctional systems and
specifically from the dendrimeric compounds
poly(amidoamine), PAMAM, and diaminobutane
poly(propylene imine), DAB, and also from the
hyperbranched polymers polyglycerol, PG and
poly(ethylene imine), PEI, (Figure 2) a stepwise design of
multifunctional systems will be discussed, aiming at
obtaining appropriate nanocarriers for drug delivery and
gene transfection. This review is by no means exhaustive
and only selected examples will be discussed highlighting
on work performed recently in our laboratory. The
objective of this review is to illustrate the effectiveness of
the strategy of molecular engineering, applied on dendritic
surfaces, to prepare drug carriers with desired properties.
= Branching point
=
=
=
Terminal group
Recognizable group
Protective coating

NH H 2 2N
Gene Therapy and Molecular Biology Vol 10, page 73
NH2 H2N NH2 H N
H
N H H
H2N N O O
N
O N
H
N
N
H
O
N
O
O
H
N O
H
N N
N N
N
H
O
H
N
O
O
N
N N
H
H
O N
O
N
H
N O
O
N
H
N
O
N
H
H
H
H H
2N
N
O
H2N N O
H
H
N N
H2N O
H2N O
N
H N
H
N
O
H2N H
O
2N
N
H
H
N O
H
H2N N
NH2 O
N
H N
O
N
N O
H
H
N N
O
O
N
H
N
H
O N
O
N
H
N
H
N O
N
O
O
N
N
H
N
O
H NH2 N
N H
H
O
H
N
2N
O
N
O N H
N O
H
H
N
N NH2 N
O
O
NH2 O
N
H
N H
N
O NH2 N
O
O
N N
H H
H
N O
H2N H2N O N
O
N
H
O
N N
H N
H
H
N O
O N
N
NH2 H H
O
2N H N
N
O
O N
N
H
H
N
O
O N
N H NH2 H
O NH2 N
N H
H
N
O
O NH2 N N NH2 O
N
N
H
O
N H
H2N H
H
O N
NH2 NH2 Core
HO
HO OH
~
O
~
O
OH
O
O
O
O
OH
NH 2
H 2N
PAMAM
O O
Figure 2. Chemical structure of dendrimeric compounds.
II. Drug carriers: from
monofunctional to multifunctional
dendrimers
In an example on molecular engineering of PAMAM
surface, poly(ethylene glycol) monomethyl ether (M-
PEG), having an average molecular weight of 550 or 2000,
was attached at the terminal amino groups of the third and
fourth generation polymers as shown in Figure 3. Inside
the nanocavities of the so-prepared PEGylated dendrimers,
Adriamycin, ADR, or Methotrexate, MTX, anticancer
drugs (Figure 4) were encapsulated (Kojima et al, 2000).
As the amount of ADR employed for encapsulation inside
these PEGylated dendrimers increased, the number of
ADR molecules associated with the dendrimer increased
OH
O
O
OH
O
O
O
O
OH
O
O
OH
OH
OH
O
OH
OH
O
O
OH
OH
OH
OH
OH
H 2N
H 2N
H 2N
H 2N
H 2N
H 2N
H 2N
H 2N
H 2N
N
N
N
N
H 2N
~
H 2N
N
N
N
H 2N
N
NH 2
~
N
HN
N
NH 2 NH2
N
NH 2
N
N
N
N
N
N
NH 2
N
N
N
N
NH 2
N
N
NH 2
NH 2
DAB
PG PEI
73
N
N
N
N
N
N
N
NH2
N
N
H
N
N
N
N
N
HN
N
N
N
NH2 NH2 N
N
N
N
NH2 NH2 and finally reached a plateau. Depending on the
generation, the maximum number of ADR molecules
encapsulated per dendrimer i.e. by the M-PEG(550)-G3,
M-PEG(2000)-G3, M-PEG(550)-G4, and M-PEG(2000)-
G4 dendrimeric derivatives are ca. 1.2, 2.3, 1.6, and 6.5,
respectively, as shown in Figure 5. Thus, the
encapsulation ability varied for these PEG-dendrimers and
it was found to depend on the molecular weight of PEGchains
and also on dendrimers’ generation.
PAMAM has a basic interior and, therefore, it is
possible to encapsulate MTX, which is acidic, since it
bears two carboxyl groups. The number of MTX
molecules associated with one dendrimer molecule, as a
function of the MTX/dendrimer ratio during loading is
shown in Figure 6. As it was observed in the
N
NH 2
NH 2
NH 2
NH 2
N
H
NH 2
N
H
H
N
NH 2
NH 2
N
NH 2
NH 2
NH 2
NH 2
N
H
NH 2
NH 2
NH 2
N
NH 2
N
N
N
NH 2
NH 2
NH 2
NH 2
NH 2
NH 2
NH 2
NH 2
NH 2
NH 2
NH 2

Sideratou et al: Dendritic polymers for application as drug and gene delivery systems
M-PEG
(MW 550, 2000)
O
OH + Cl C O NO2O C O NO2 NH2 H2N NH2H2N H N
H
H
N H
NH2 H2N N
O O
N
H N
O
O
O
N
N
H
N
O
H
H
NH2 H2N N
NH2
H
N
N
N
H
N H
NH2 N
O
N
O
O H
O
H N
N
O
H2N
H
O
O
N
H
N
N
N
O
H
H H
O N
N N
N NH2 NH N
2
O
O
O
N O
H
N
O
N
NH2 N O
H
H
H
O
N
N
H
N
H
O
N
N
H
N
H
N
N
O
O
O
H2N N
N H
O
H
O
H
N O
H2N H
N
O
N
N N
N N
N
O NH
N
2
H
H
O
O
N
H
O
H
NH2
N
O
N
O
O
N
H
N
H O
N
N
N H
N
H
N
N
O
H
H
H
H2N N
N
N
O
H
O
H
O
2N
O
O N
O
N
O
NH2 N
H
N N
NH2
H H
N N
H
N O
NH2 H2N O
N
N
H
H
N O
O
N
N
H O
H N
O
N
N
O
O
N
H
O
N H
N
N
H
N
O O
N
H
H
H
O N
NH2 NH2 N
NH2 H2N
H
O O
N
H N
H
N H
NH2 H2N NH2 H2N PAMAM
A
O
M-PEG 4-nitrophenyl carbonate
(A)
NH HN
O
CO
CO
NH
HN
H N
N
H
H
H
NH
HN
N
O O
N
O
CO
O
CO
O
O O
CO CO
NH HN
H N
O
OOCHN
H
N
O N
NHCOO
H N
O O
N
H
N
H
N H
N
O
N
O
O
N
H
N
O
N H
N
O
CO
H
N
NH
N H
NH
N
O H
O
N
O
O
N
H
N
H
H
N
N
N
NH
O
O
N O
H
N
N O
H
H
H
N N
N
O H
N N
O
O
OOCHN
N
O
H
O
H
OOCHN
O
N
N N
N N
N
H
H
O
H
O
N
N
O
H
N
H O
N
N
N
H
O
OOCHN
H
N
O
N
O
OOCHN
O
O
N
N
H
N N
H H
H
O N
N
N
O
O
O
N
H
O O
N
NHCOOHN
N
N
H N
N
H
H
H
O
O N
NH
H
O
H
N
N
H
N
O
N O
N H
H
N
N
O NH
H
O NH
N
O
O
N
N H
N
N
H
H
N
H
O
O N
O
NH
NH
N N
H
H
O N
O
N
NH
N NH
H
N O
O N
N
O
H
O
N H
NH
HN
N
H O O
N
H N
H
N H
NH HN
NH HN
O
O O
CO
CO
CO O
CO O
CO
O
CO O
CO O
CO O
CO O
CO O
CO O
CO O
CO
O CO
O
CO
O
CO
O
CO
O
CO
O
M-PEG-PAMAM
Figure 3. Preparation and structure of M-PEG PAMAM dendrimer of the third generation. Reproduced from Kojima et al, 2000 with
kind permission from the authors and American Chemical Society.
OCH 3
O
O
OH
OH
H3C H2N OH
O
CH2OH OH
O
H 2N
ADR MTX
Figure 4. Chemical structure of the anticancer drugs adriamycin, ADR, and Methotrexate, MTX
NH 2
74
N
N
N
CH 3
O COOH
N
H
COOH
Figure 5. Encapsulation of ADR
by M-PEG(550)-attached (open
symbols) or M-PEG(2000)attached
(closed symbols)
PAMAM G3 (�,�) and G4
(�,▲) dendrimers. The number of
ADR encapsulated per dendrimer
is shown as a function of the
ADR/dendrimer molar ratio during
loading. Reproduced from Kojima
et al, 2000 with kind permission
from American Chemical Society.

encapsulation of ADR, the number of MTX molecules
associated with the modified dendrimer increased with
increasing amount of MTX employed during loading, and
finally reached a constant value. The maximum numbers
of MTX molecules associated with the M-PEG(550)-G3,
M-PEG(2000)-G3, M-PEG(550)-G4, and M-PEG(2000)-
G4 dendrimers are approximately 10, 13, 20, and 26
mol/mol of dendrimer, respectively. Apparently, the
number of the encapsulated drugs by the PEGylated
dendrimers increased when MTX was used instead of
ADR. Since these drugs have similar molecular weights,
this result suggests that the electrostatic interaction from
the acid-base interaction between the dendrimer and MTX
molecules results in an enhanced encapsulation of MTX
by these dendrimers. As it was the case with ADR
encapsulation, the number of MTX encapsulated by the
dendrimer was affected both by generation of the
PAMAM and by the chain length of the M-PEG.
Release experiments performed in PBS buffer
(Phosphate Buffer Saline) showed that ADR was readily
released from the modified dendrimers. Apparently,
hydrophobic interaction between ADR and the dendrimer
is not strong enough to retain the drug in the interior of the
PAMAM dendrimeric moiety. The release of MTX from
the M-PEG-functionalized dendrimers was also
investigated by the same method. The time dependency of
MTX concentration in the outer phase during the dialysis
is shown in Figure 7. Apparently, the MTX concentration
in the outer phase increased at a slower rate when MTX
was encapsulated in the M-PEG-attached dendrimer than
in the case of free MTX. This indicates that MTX was
gradually released from the modified dendrimer. As
mentioned above, MTX was electrostatically bound to the
dendrimeric interior and, therefore, dissociation of MTX
from the dendrimer was suppressed to some extent.
However, when the dialysis was performed in the presence
of 150 mM NaCl, no difference in the release rate was
Gene Therapy and Molecular Biology Vol 10, page 75
75
Figure 6. Encapsulation of MTX
by M-PEG(550)-attached (open
symbols) or M-PEG(2000)attached
(closed symbols)
PAMAM G3 (�,�) and G4 (�,
▲) dendrimers. The number of
MTX encapsulated per dendrimer
is shown as a function of the
MTX/dendrimer molar ratio during
loading. Reproduced from Kojima
et al, 2000 with kind permission
from American Chemical Society.
observed between MTX encapsulated in the M-PEGattached
dendrimer and free MTX. In this case, MTX can
dissociate readily from the dendrimer because the
electrostatic interaction is weakened by the shielding
effect of Na + and Cl - (Kojima et al, 2000).
Effective solubilization of hydrophobic drugs was,
however, achieved with another PEGylated dendrimeric
system (Sideratou et al, 2001), which is analogous to the
one previously discussed. PEGylation of dendrimers was
performed under facile experimental conditions by the
interaction of methoxypoly(ethylene glycol)-isocyanate
(PEG-isocyanate) with the external primary amino groups
of DAB dendrimers of fifth generation, as shown in
Figure 8. Two different PEGylated dendrimeric
derivatives were prepared i.e. the DAB-4PEG (weakly
PEGylated) and DAB-8PEG (densely PEGylated). In this
manner, the role of PEG-coating on encapsulation and
release properties was possible to be assessed.
Comparison of solubilizing ability of the parent and
PEGylated DAB dendrimers is shown in Table 1. For this
purpose, betamethasone valerate, BV, and betamethasone
dipropionate, BD, were used as active drug ingredients
(Figure 9). These anti-inflammatory corticosteroids are
practically water insoluble and it is, therefore, necessary to
encapsulate these compounds in a water-soluble carrier for
facilitating their use as drugs. The concentration of
encapsulated betamethasone derivatives was significantly
increased in PEGylated dendrimers. Thus, for DAB-8PEG
the loading was 13 and 7 wt.% for BV and BD, while for
DAB-4PEG was 6 and 4 wt.%, respectively. The observed
solubility increase was attributed to an additional
solubilization of the compounds in PEG-chains by which
the dendrimers are coated. This is also verified by the fact
that upon protonation they remain solubilized in PEGchains
environment. As expected, by increasing dendrimer
concentration, solubilization of drugs analogously
increases to a certain limit.

DAB64
Sideratou et al: Dendritic polymers for application as drug and gene delivery systems
NH 2
64
O C NH
NH 2 60 or 56
M-PEG-isocyanate
(MW 5000) HN NH
O
DAB64-PEG
Figure 7. Release of MTX from
the M-PEG(2000)-attached G4
dendrimer. The MTX-loaded M-
PEG(2000)-G4 dendrimer (�,
�) or free MTX (�, ▲)
dissolved in 1 mM Tris-HClbuffered
solution (pH 7.4)
containing (open symbols) or not
containing (closed symbols) 150
mM NaCl and dialyzed against
the same solution. The time
course of MTX concentration in
the outer phase during the
dialysis is shown in the figure.
Reproduced from Kojima et al,
2000 with kind permission from
American Chemical Society.
Figure 8. Preparation and structure of PEGylated DAB dendrimer of the fifth generation functionalized with 4 or 8 PEG chains.
For a detailed investigation of the solubilization site
and release properties of these PEGylated dendrimers the
hydrophobic pyrene was employed. This is a very
sensitive probe and it is used as a model compound when
drugs cannot offer this type of information. By employing
the well-known I1/I3 fluorescence intensity ratio, which
probes the polarity of the medium (Thomas, 1980), it was
found that pyrene is solubilized both in the core and in
PEG-chains. In addition, upon protonation of the loaded
PEGylated dendrimer, pyrene is not released in the bulk
aqueous phase as judged again by the I1/I3 ratio and
fluorescence intensity (F/F 0 ) results. This is attributed to
the fact that as pyrene is leaving the core it is possible to
be solubilized inside PEG-chains, as shown schematically
in Figure 10. The results of I1/I3 fluorescence intensity
ratio indicate that pyrene is neither solubilized in the bulk
water phase nor in the interior of the dendrimer. Normally,
one would expect release of pyrene in water, since, due to
protonation, the environment of the nanocavities becomes
polar and, therefore, the hydrophobic pyrene cannot
remain solubilized. In addition, protonated tertiary amino
groups of the core do not exhibit anymore the property to
form charge-transfer complexes with pyrene (Sideratou et
76
4 or 8
al, 2000) and, therefore, encapsulation of the pyrene is no
longer favoured. It should, however, be noted that
complete release of pyrene can be achieved upon
exhaustive dilution of the PEGylated dendrimer. The same
behaviour was observed for the hydrophobic drugs BV
and BD. In conclusion, the enhanced solubilization of
these drugs in PEGylated dendrimers secures their
application as promising controlled release drug delivery
systems.
In another recent report, extending the previous
work, a novel multifunctional dendrimeric carrier was
designed (Paleos et al, 2004) based on diaminobutane
poly(propylene imine) dendrimer of the fifth generation.
The synthetic procedure of this derivative is shown in
Figure 11. This carrier is intended to simultaneously
address issues such as stability in the biological milieu,
targeting and very possibly transport through cell
membranes. For this purpose, in addition to surface
protective poly(ethylene glycol) chains, guanidinium
moieties were introduced as targeting ligands. In addition,
the accumulation of guanidinium groups at the surface of
the dendrimer may also facilitate its transport ability. The
functional groups were covalently attached at the

dendrimeric surface and it was possible to secure, in
principle, desired drug delivery properties due to: a.
Protection of the carrier because of the coverage of the
dendrimeric surface with poly(ethylene glycol) chains, b.
Recognition ability towards complementary moieties;
surface guanidinium groups secure the facile interaction
with acidic receptors including the biologically significant
carboxylate and phosphate groups. Combined electrostatic
forces and hydrogen bonding are exercised making this
interaction thermodynamically favorable (Hirst et al,
Gene Therapy and Molecular Biology Vol 10, page 77
1992), c. Possibility of encapsulation and release of active
drug ingredients from the nanocavities, which can be
tuned by environmental changes (Sideratou et al, 2001), d.
Complexation with DNA for gene therapy applications, e.
The occurrence of polyvalency interactions, associated
with enhanced binding, due to the accumulation of
recognizable moieties on the limited surface area of the
dendrimer as schematically illustrated in Figure 12, f. The
expected decrease of toxicity due to the facile
modification of the toxic amino groups (Malik et al, 2000).
Table 1. Comparative solubility of pyrene (PY), betamethasone valerate (BV) and betamethasone dipropionate (BD) in
parent DAB and PEGylated derivatives. Reproduced from Sideratou et al, 2001 with kind permission from Elsevier.
O
Compound [dendrimer]/M [PY]/M [BV]/M [BD]/M
DAB 5 x 10 -5
2.15 x 10 -6
2.95 x 10 -5
1.84 x 10 -5
DAB-8PEG 5 x 10 -5
5.40 x 10 -5
3.85 x 10 -4
2.56 x 10 -4
DAB-4PEG 5 x 10 -5
2.14 x 10 -5
2.05 x 10 -4
1.25 x 10 -4
DAB-8PEG 5 x 10 -4
8.75 x 10 -5
3.65 x 10 -3
1.87 x 10 -3
DAB-4PEG 5 x 10 -4
5.25 x 10 -5
1.70 x 10 -3
1.09 x 10 -3
Me
H
HO
F
H
BV
Me
H
C O C4H9 Figure 9. Chemical structure of betamethasone valerate, BV, and betamethasone dipropionate, BD
OH
O
H
Me
O
O
Me
H
HO
F
H
Me
H
C O C2H4 Figure 10. Schematic representation of the solubilization of pyrene in PEGylated dendrimers. Reproduced from Sideratou et al, 2001
with kind permission from Elsevier BV.
77
BD
O
O
H
Me
C 2H 4
O
O

N
N
N N
N
N
N N
N
N N N
N N
N
N N N
N
N N N
N N
N
N
N
N
N
N
where:
Sideratou et al: Dendritic polymers for application as drug and gene delivery systems
= NH 2
= PEG
O C N
= NHCNHCH2CH3 O
= NHC
NH2 Cl -
NH 2 +
N
N
N N
N
N
N
N
N
N
N N
N N
N
N N N
N
N N N
N N
N
N
N
N
N
N
O C NCH 2CH 3
N
N
N N
N
N
N
N
N
N N N
N N
N
N N N
N
N N N
N N
N
N
N
N
N
N
N N
+
NH2 - NH2 Cl
N
N
N N
N
N
N
N
N
N
N N
N N
N
N N N
N
N N N
N N
N
N
N
N
N
N
Figure 11. Reaction scheme for the synthesis of a multifunctional dendrimeric derivative. Reproduced from Paleos et al, 2004 with kind
permission from American Chemical Society.
For evaluating the loading capacity and release
properties of the above multifunctional dendrimer, pyrene
(PY) and betamethasone valerate (BV), were used as
model compounds. The dendrimeric derivative
encapsulated significantly higher concentrations of the
above compounds compared to the parent dendrimer, as
determined by UV spectroscopy and shown in Table 2.
This is particularly significant for betamethasone valerate,
of which seven molecules are solubilized per dendrimeric
molecule. As previously mentioned (Sideratou et al,
2001), this was attributed to the presence PEG-chains.
Additionally, in the case of betamethasone valerate the
loading capacity is 11 wt% for the multifunctional
dendrimer, i.e. almost double compared to the loading
capacity of the simply PEGylated dendrimer (6 wt%) and
more than five times compared to the loading capacity of
the parent dendrimeric solution (1.7 wt %) (Sideratou et al,
2001). This is quite beneficial for its use as drug delivery
system and it can only be attributed to the other two
functional groups introduced at the surface of the
multifunctional derivative. As it will be discussed below,
78
Figure 12. Schematic representation
of a dendrimer exhibiting polyvalent
properties
they may act synergistically enhancing solubilization of
betamethasone valerate.
Pyrene, as in the previous work (Sideratou et al,
2001), was in first place employed as model hydrophobic
compound for probing the solubilization properties of the
multifunctional dendrimer. For this reason fluorescence
intensity (F/F 0 ) changes and I1/I3 ratio were monitored,
which are sensitive parameters and their values depend on
the medium of solubilization of the probe. These
parameters were monitored by a titration-like addition of
the dendrimer to an aqueous pyrene solution. A significant
quenching of fluorescence intensity (F/F 0 ) was observed
and the I1/I3 ratio decreased (Figure 13). Fluorescence
quenching was attributed (Sideratou et al, 2001) to the
formation of a charge-transfer complex between pyrene
and tertiary amino groups, as evidenced by the appearance
of a weak exciplex fluorescence centered at approximately
485 nm (Lakowicz, 1983). As the concentration of the
dendrimer increases, the I1/I3 ratio decreases to a value of
about 0.90, which is close to the one observed in the
hydrophobic environment usually encountered in the

conventional micelles. Thus, pyrene is mainly
incorporated inside the nanocavities of the dendrimer, in
order to avoid contact with the hydrophilic external
groups.
The release of the active ingredient from the
dendrimer when it reaches the target site enhances its
bioavailability and efficacy. In addition, drug release from
endosomal compartment appears a limiting factor for
several targeted drug delivery formulations (Boomer et al,
2003). These requirements impose the need for developing
drug delivery systems in which the release of drug can be
triggered by appropriate stimulus. For this purpose pHtriggered,
enzymatic, thermal and photochemically
induced processes have been reported (Boomer et al,
2003). For instance low pH within endosomal and
ischemic tissue environments renders acid triggerable
delivery systems attractive for controlled release.
The multifunctional poly(propylene imine)
dendrimers prepared, due to the presence of tertiary amino
groups in their core fulfill at least one of these
requirements, i.e. being pH responsive (Sideratou et al,
2000; Sideratou et al, 2001; Paleos et al, 2004). As found
in the previous experiment pyrene is solubilized in the
interior of dendrimer and also within PEG chains, while
upon protonation of tertiary amines of the nanocavities
pyrene is repositioned in the PEG coat.
For achieving the release of the encapsulated pyrene
Gene Therapy and Molecular Biology Vol 10, page 79
from the PEG protective coat another method has,
therefore, to be explored. We were prompted to use
aqueous sodium chloride solution for triggering pyrene
release since, as it has been established in independent
studies (Wang et al, 2000; Bogan and Agnes, 2002), ions
of alkali metals cationize poly(ethylene glycol) moieties
through complexation. The designed multifunctional
dendrimer, due to the attachment of PEG chains at its
surface, is susceptible to analogous interactions and,
therefore, it could be possible for metal cations to replace
solubilized pyrene releasing it to the bulk aqueous phase.
Indeed, by titrating dendrimeric solutions with sodium
chloride solution, pyrene was released and dispersed in the
bulk solution in the form of crystallites. The isolated
crystallites were indentified by 1 H NMR and proved to be
pure pyrene.
The two-step triggered release from the
multifunctional dendrimer was also investigated using the
lipophilic drug betamethasone valerate. Release of the
drug with hydrochloric acid has not been observed since
betamethasone valerate remained solubilized within the
dendrimeric environment and preferably within the
poly(ethylene glycol) chains. Betamethasone valerate
encapsulated in the multifunctional dendrimer was
completely released upon addition of sodium chloride as
shown in Figure 14. However, within the concentration
Table 2. Comparative solubility of pyrene (PY) and betamethasone valerate (BV) in the parent fifth generation DAB and
multifunctional dendrimer. Reproduced from Paleos et al, 2004 with kind permission from American Chemical Society.
Compound
[dendrimer]
/M
DAB 1.0 x 10 -3
Multifunctional
Dendrimer 2.5 x 10 -4
1.6
1.4
1.2
I 1 /I 3
1.0
0.8
[PY] /M
2.1±0.2 x 10 -5
1.9±0.08 x 10 -5
1 2 3 4 5
[dendrimer] x 10 -4 M
PY/Dendrimer
molar ratio
0.021±0.002
0.076±0.002
F/F 0
1.0
0.8
0.6
0.4
0.2
[BV] /M BV/Dendrimer
molar ratio
2.5±0.4 x 10 -4
1.80±0.4 x 10 -3
0.25±0.04
7.20±0.03
0.0
0 1 2 3 4 5
[dendrimer] x 10 -4 M
Figure 13. Plot of F/F 0 and I 1/I 3 ratio as a function of the concentration of the multifunctional dendrimer. F 0 is the total fluorescence
intensity of 6.81 x 10 -7 M aqueous solution of pyrene and F is the measured fluorescence intensity at various dendrimer concentrations.
Reproduced from Paleos et al, 2004 with kind permission from American Chemical Society.
79

Sideratou et al: Dendritic polymers for application as drug and gene delivery systems
range of the sodium cation present in extracellular fluids,
i.e. 0.142 M, (Guyton and Hall, 2000) the betamethasone
valerate was released in relatively small quantities. The
gradually released betamethasone valerate from the
multifunctional dendrimer formed crystallites in the
aqueous medium as determined by light scattering. The
precipitated material was analyzed with 1 H NMR and its
spectrum corresponded to that of betamethasone valerate.
This finding should be considered when PEGylated
dendrimers are used as drug delivery systems in
experiments in vitro and in vivo, since sodium chloride in
extracellular fluids and potassium chloride in intracellular
environment can be complexed with PEG chains (Wang et
al, 2000; Bogan and Agnes, 2002) affecting the overall
release profile of the drug. Thus, the possibility of
triggering drug release in the extracellular fluid, i.e. before
endocytosis to the target-cells, should be taken into
account when designing a targeted PEGylated drug
delivery system.
The drug delivery effectiveness of analogous
multifunctional dendrimers was modeled by investigating
their interaction with multilamellar liposomes consisting
of phosphatidylcholine/cholesterol/dihexadecyl phosphate
(19:9.5:1) and dispersed in aqueous or phosphate buffer
solutions (Pantos et al, 2005). The multilamellar liposomes
bear the phosphate moiety as recognizable group.
They were used as simple models before one resorts
to the use of cells; after all liposomes are considered as the
closest analogues to cells. On the other hand,
poly(propylene imine) fourth generation dendrimers were
functionalized with 6 (DAB-G6 ) or 12 (DAB-G12)
guanidinium groups as targeting ligands, while the
remaining toxic, external primary amino groups of the
dendrimers were allowed to interact with propylene oxide
affording the corresponding hydroxylated derivatives. The
scheme of the reactions modifying the dendrimeric surface
[BTV] x10 -4 M
1.6
1.4
1.2
1.0
0.8
0.6
0.4
is shown in Figure 15. DAB-G0 dendrimer, which does
not contain any guanidinium group was used as a
reference compound. The so-prepared dendrimers were
loaded with corticosteroid drugs, i.e. betamethasone
dipropionate and betamethasone valerate for investigating
their transfer to liposomes.
Microscopic, ζ-potential, and Dynamic Light
Scattering (DLS) techniques have shown that liposomesdendrimers
molecular recognition occurs leading to the
formation of large aggregates at dendrimer/dihexadecyl
phosphate molar ratios higher than 1:30, as visually
observed with phase contrast optical microscopy. Calcein
liposomal entrapment experiments demonstrate a limited
leakage, i.e. less than 13%, following liposomes
interaction with the modified dendrimers at 1:25
dendrimer/ dihexadecyl phosphate molar ratio. This
indicates that the membrane of the liposomes remains
almost intact during their molecular recognition with these
dendrimers. Isothermal Titration Calorimety (ITC)
indicates that the enthalpy of the interaction is dependent
on the number of the guanidinium groups present at the
dendrimeric surface. Furthermore, the process is reversible
and redispersion of the aggregates occurs by adding
concentrated phosphate buffer.
The interaction between these drug-loaded
dendrimers and multilamellar liposomes results in the
transport of drugs from the dendrimeric derivatives to the
‘empty’ liposomes as summarized in Table 3. The
experiments demonstrate that about 25% of BD or BV is
present in the precipitated aggregates when DAB-G0 was
used. When the guanidinylated dendrimers DAB-G6 and
DAB-G12 were used, the amount of drugs in the
precipitate increases substantially becoming about 60%
and 80%, respectively.
0.2
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
[NaCl] / M
Figure 14. Plot of the concentration of betamethasone valerate in a 2.50 x 10 -5 M dendrimeric solution as a function of added NaCl.
Reproduced from Paleos et al, 2004 with kind permission from American Chemical Society.
80

These significant differences observed in the
transport of drugs between guanidinylated and nonguanidinylated
dendrimers can be attributed to the
functionalization of the dendrimeric molecules. The
presence of guanidinium groups at the external surface of
the dendrimers results in an effective adhesion to the
multilamellar liposomes as the ITC and DLS experiments
demonstrated. As expected, when the interaction is taking
place in 10mM phosphate buffer the drug present in the
aggregates decreases slightly. In this case, the decrease of
drug transport can be rationalized by the competitive
interaction of the phosphate groups in bulk with the
guanidinium dendrimeric groups leading to less effective
adhesion with the multilamellar liposomes.
Upon the addition of concentrated phosphate buffer
followed by the redispersion of the aggregates in the
medium and the separation of the no-longer interacting
dendrimers, drugs are still present in the obtained
DAB
(NH 2) 32
O
CH 3
Gene Therapy and Molecular Biology Vol 10, page 81
(NHCH 2CH(CH 3)OH) 32
(NH 2) 6
(NHCH 2CH(CH 3)OH) 26
(NH 2) 12
DAB-G0
DAB-N6
(NHCH 2CH(CH 3)OH) 20
DAB-N12
multilamellar liposomes. Determination of BD or BV in
the multilamellar liposomes indicates that, in all cases, ca.
50% (Table 3) of the amount of drugs found in the
aggregates before redispersion is still present, suggesting
that they are located in the lipid bilayer, since their
solubility in water is extremely low. Drug transport is
induced by the use of guanidinylated dendrimers since
drug transport values of about 40-45% were obtained in
the case of DAB-G12, while only 12-15% was observed in
the case of the non-guanidinylated derivative.
Carbohydrates, in general, being targeting ligands for
selectins can be introduced at the external surface of
dendrimers leading to the formation of targeted drug
delivery systems. In a recent study (Bhadra et al, 2005),
galactose surface-coated poly(propylene imine) (PPI)
dendrimeric derivatives were prepared and loaded with
primaquine phosphate (PP) (Figure 16), which is an
antimalarial drug. Galactose functionalization was carried
N
N
NH 2
+ H2N Cl -
DIEA
N
N
NH 2
+ H2N Cl -
DIEA
NH
+
NH2 Cl
NH2 -
(NHCH2CH(CH3)OH) 26
DAB-G6
NH
NH 2 +
NH 2
(NHCH2CH(CH3)OH) 20
DAB-G12
Figure 15. Functionalization of poly(propylene imine) dendrimer of the fourth generation including guanidinylation at the final step.
Reproduced from Pantos et al, 2005 with kind permission from American Chemical Society.
Table 3. Drug transfer (%) from dendrimers to multilamellar liposomes in a) aggregates obtained after their interaction in
water or in 10 mM phosphate buffer (pH 7.4) and b) multilamellar liposomes obtained following redispersion of the
aggregates. Reproduced from Paleos et al, 2005 with kind permission from American Chemical Society.
Drug transfer (%) in aggregates Drug transfer (%)after redispersion
Drug Dendrimer Water Phosphate Buffer Water Phosphate Buffer
DAB-G0 24.4±2.4 19.8±1.2 15.8±0.9 12.1±1.1
BD DAB-G6 62.5±1.9 48.5±1.6 28.1±1.7 24.5±1.3
DAB-G12 84.5±2.1 68.4±1.5 45.1±1.8 40.0±1.4
DAB-G0 32.9±2.0 27.1±1.0 15.9±1.2 14.1±0.9
BV DAB-G6 59.0±1.5 39.5±2.1 29.0±1.0 26.1±1.5
DAB-G12 78.1±2.3 57.5±2.0 42.0±1.5 38.2±1.2
81
Cl -
6
12

Sideratou et al: Dendritic polymers for application as drug and gene delivery systems
H 3CO
HN
CH 3
PP
out by a ring opening reaction followed by Schiff’s base
reaction and reduction to secondary amine in sodium
acetate buffer as shown in Figure 17.
Galactose had been shown to be a promising ligand
for hepatocyte (liver parenchymal cells) targeting because
liver cells possess a large number of the Asialoglycoprotein
(ASGP) receptors that can recognize the
galactose units on the oligosaccharide chains of
glycoproteins, or on chemically galactosylated drug
carriers (Ashwell and Harford, 1982). The receptor-ligand
interaction was known to exhibit a significant ‘cluster
PPI
NH2 n
n=16, 32, 64
NH 2 . H 3PO 4
Figure 16. Chemical structure of
primaquine phosphate.
effect’ in which a polyvalent interaction results in
extremely strong binding of ligands to the receptors.
The results obtained indicated that galactose coating
of PPI systems increases the drug entrapment efficiency
by 5-15 times depending upon dendrimers’ generation.
Also galactose coating prolonged release up to 5–6 days as
compared to 1-2 days for uncoated PPI. The hemolytic
toxicity, blood level and hematological studies proved that
these carriers are safer and suitable for sustained drug
delivery. Blood level studies proved the suitability of the
carriers in prolonging the circulations and delivery of PP
to liver.
OH
CH2OH
O
HO
OH
OH
HOH
HOH
2C
2C
H HO H CH2OH HO
H HO H HO
H
HO
OH H OH HO
H
H
H HO H H
OH HO CH2OH HO
H
HO
CH 2C
H HO H
2
NH
CH H
2 H HO
HN
HO
CH2OH OH HO
NH
H H H H
2C HO H
OH
N
NH H2C H HO
HHO
CH2OH N
NH HO
H
H
HN C
N
OH
H H
2
N
HO H HO OH
H
CH2OH N C
N N
H2 H OH H H
N
H H OH H OH OH
2
N C CH2OH N
H H OHH
H
sodium acetate buffer
N OH H OH
C
OH
pH 4.0
H
N
2
N N N
H
CH
HO
2OH
H
H H H
2 OH
N C H
OH
OH
H HO
N
N
H
H CH2OH 2 OH H
N C H
OH
H
OH
H
HO CH2OH N
H2 OH
H
H
N
N C H
H
OH
HN H
OH
CH HO
2 H CH2OH NH NH
OH H
H2C HN
H
H2C H H
H
CH2 HO
HO
OH OH
OH
HO
H OH
H
H
H
OH
H
H HO H
H OH
H
OH
H
H OH CH2OH OH
HOH2C OH
H OH
HOH2C CH2OH ~
Figure 17. Galactosylation of poly(propylene imine) dendrimer of the third to fifth generation.
~
82

Proceeding with further functionalization and
employing a third generation PAMAM dendrimer as a
starting compound, multifunctional dendrimers were
prepared (Shukla et al, 2003). These derivatives, in
addition to the protective PEG-chains they also bear the
folate moiety at the end of poly(ethylene glycol) chain
which can induce endocytosis into folate receptor-bearing
cells (Sudimack and Lee, 2000; Hofland et al, 2002;
Antony, 2004; Sabharanjak and Mayor, 2004). The folate
receptor is known to be significantly overexpressed over a
wide variety of human cancers and, therefore, folatemediated
targeting has been widely applied with
liposomes (Lee and Low, 1995; Lee and Huang, 1996;
Gabizon et al, 2004), dendrimers (Kono et al, 1999; Konda
et al, 2001; Shukla et al, 2003), various polymers and
particles (Dauty et al, 2002; Dubé et al, 2002; Aronov et
al, 2003; Zuber et al, 2003; Yoo and Park, 2004; Kim et al,
2005b; Licciardi et al, 2005; Wang and Hsiue, 2005) when
used as drug delivery systems. In addition, in the
previously functionalized dendrimer 12 to 15 decaborate
clusters were covalently attached, which can be used for
the treatment of cancer in Boron Neutron Capture
Therapy(BNCT) requiring the selective delivery of 10 B to
cancerous cells within a tumor. Varying number of PEG
chains of varying length were linked to these boronated
dendrimers to reduce hepatic uptake. Among all prepared
combinations, boronated dendrimers with 1-1.5 PEG2000
units exhibited the lowest hepatic uptake in C57BL/6 mice
(7.2-7.7% injected dose (ID)/g liver).
Two folate receptor-targeted boronated third
generation poly(amidoamine) dendrimers were prepared,
the one shown in Figure 18, one containing ~15
decaborate clusters and ~1 PEG2000 unit with a folic acid
moiety attached to the distal end, while the other was
containing ~13 decaborate clusters, ~1 PEG2000 unit and
~1 PEG800 unit with folic acid attached to the distal end. In
vitro studies using folate receptor (+) KB cells
demonstrated receptor-dependent uptake of the latter folic
acid-functionalized derivative. Biodistribution studies with
this derivative in C57BL/6 mice bearing folate receptor
(+) murine 24JK-FBP sarcomas resulted in selective tumor
uptake (6.0% ID/g tumor), but also high hepatic (38.8%
ID/g) and renal (62.8% ID/g) uptake (Table 4), indicating
that attachment of a second PEG unit and/or folic acid
may adversely affect the pharmacodynamics of this
conjugate.
In conclusion, the optimal modification of Boronated
dendrimers as well as of dendrimers in general with PEG
chains for reducing reticuloendothelial system affinity
appears to be a highly complex process that depends on a
variety of factors requiring extensive evaluation. The folic
acid functionalized PEGylated G3-Boronated Dendrimer
showed significantly increased tumor selectivity compared
with non-PEGylated Boronated Dendrimeric-antibody and
Boronated Dendrimer-EGF (Epidermal growth factor)
conjugates previously evaluated for potential application
in BNCT (Barth et al, 1994; Yang et al, 1997). However,
the hepatic and renal uptake of this conjugate was very
high.
Gene Therapy and Molecular Biology Vol 10, page 83
83
III. Drug carriers: from
monofunctional to multifunctional
hyperbranched polymers
Based on polyglycerol (PG) and also on
poly(ethylene imine) (PEI), pH-responsive molecular
carriers were prepared (Krämer et al, 2002) through
appropriate functionalization. The concept of pHresponsive
carriers may have potential application for
selective drug delivery in tissues of a lower pH value (for
example, infected or tumor tissue). Polyglycerol and
poly(ethylene imine), which are commercially available,
are randomly branched but have defined dendritic
structures with a degree of branching 60 to 75%.
Functionalization of these dendritic polymers was
achieved through a facile condensation reaction between
the 1,2-diol or NH2 moieties at their external surface and
various carbonyl compounds as shown in Figure 19.
Several dendritic structures originating from these
reactions have been prepared, differing in the following: a.
the type and molecular weight of the core polymer, b. the
structure of the attached peripheral shell and c. the degree
of alkylation.
The loading capacities (number of encapsulated
congo red per polymeric nanocarrier) of dendritic
polymers together with their structural features are shown
in Table 5. It was found that a minimum core size (ca.
3000 gmol -1 ) and a highly branched architecture are
required for successful encapsulation of the guest
molecules. For efficient encapsulation the degree of
alkylation should be about 45-50% and the alkyl chains
should have a minimum length (>C10). For example, the
conversion of the terminal groups in polyglylcerol, PG (21
000 gmol -1 ) with a C16 aldehyde (PGa) containing one
alkyl chain per diol unit results in an effective degree of
alkyl functionalization of 25% (Table 5) and a poor
encapsulation capacity (0.15 congo red molecules). With
the same PG core (21 000 gmol -1 ), the ketal functionalized
carrier, PGb, with two alkyl chains per diol unit and 45%
effective alkyl functionalization (Table 5) can encapsulate
up to 13 congo red molecules. A higher degree of ketal
functionalization (PGc: 55%, Table 5) indicates an
optimal shell density of 45-50%. The exact determination
of the encapsulation capacities for the amine based
poly(ethylene imine) carriers was complicated because of
the hydrolytic sensitivity of the imine-bound peripheral
shell in the PEI-based systems, for instance in PEIb
(Table 5). To avoid hydrolysis the dye was directly
encapsulated from the solid/organic solution interface.
The complexation of an antitumor drug,
mercaptopurine, several oligonucleotides, as well as
bacteriostatic silver compounds (for example, AgI salts
and Ag 0 nanoparticles) (Haag et al, 2002) have been
studied for the potential use of these carriers in drug and
gene delivery. Successful encapsulation was observed in
all cases by the PEI-based carriers while complexation
was not observed with the PG-based carriers for the same
guest molecules.
The objective to develop a pH-sensitive carrier was
tested using several buffer solutions for both the acetal-
and imine-bound shells. The encapsulated congo red in the

Sideratou et al: Dendritic polymers for application as drug and gene delivery systems
carrier PGb was stable for several months at neutral and
basic pH values (pH>7). However, an immediate release
of the guest molecules occurred in acidic media (pH

Gene Therapy and Molecular Biology Vol 10, page 85
Table 5. Encapsulation capacities of congo red in dendritic nanocarriers based on PG and PEI. Reproduced from Krämer et
al, 2002 with kind permission from Wiley-VCH.
Structure
Mn core
[gmol -1 ]
Shell
Degree of
alkylation
Encapsulation
capacity
PG 21 000 - - -
PGa 21 000
PGb 21 000
PGc 21 000
H 31C 15
H 33C 16
H 33C 16
O
O
O
C 16H 33
C 16H 33
25% 0.15±0.05
45% 13±4
55% 2±0.5
PEI 25 000 - - 0.02±0.005
PEIa 25 000
PEIb 25 000
H 31C 15
H 11C 5
In another recent study, the same as above
polyglycerol exhibiting low toxicity and biocompatibility,
was functionalized for developing drug nanocarriers
whose drug release can be salt-triggered. The outmost
objective of this hyperbranched polymer functionalization
is, as it is the case with dendrimers, to simultaneously
address the main issues encountered with drugs
themselves, as well as with their carriers, i.e., water
solubility, stability in biological milieu and targeting.
O
O
c 5H 11
85
33% 0.6±0.1
53% 0.2±0.05
Figure 20. Time-dependence of
the shell cleavage of PEI-imine 4b
on a KBr plate. Insert: The IR band
of the imine peak at 1655 cm -1
decreases because of cleavage,
while the N-H out- of- plane
vibration at 1565 cm -1 increases.
Reproduced from Krämer et al,
2002 with kind permission from
the authors and Wiley-VCH.
In this context, PEGylated and PEGylated-Folate
functional derivatives of polyglycerol, i.e. PG-PEG and
multifunctional PG-PEG-Folate (Figure 21) were
prepared and investigated as potential drug delivery
systems (Tziveleka et al, 2006). For investigating this
possibility, experiments have been performed employing
PY and Tamoxifen (TAM), (Figure 22), an anti-cancer
hydrophobic drug, for studying their encapsulation and
release properties.

Sideratou et al: Dendritic polymers for application as drug and gene delivery systems
O
O
O
*
O
H
N PEG
O
H
N
C
O
O
H
N CH 2
PG-PEG
HOOC
O
N
H
PG-PEG-Folate
CH 2 O CH 3
n
Figure 21. Functionalized PG-PEG and PG-PEG-Folate hyperbranched dendritic polymers.
TAM
Based on promising results on pyrene encapsulation
and release, the loading capacity and release properties of
the polyglycerol derivatives for TAM were also
investigated. TAM is a non-steroidal antiestrogen drug,
which is widely used in the treatment and prevention of
breast cancer (Wiseman, 1994; Mocanu and Harrison,
2004). Its encapsulation and release was comparatively
investigated for the parent polyglycerol, PG, PG-PEG and
the multifunctional PG-PEG-Folate derivative. The
solubility of TAM in water was found to be 1.9 x 10 -6 M.
Its solubility, however, increases by a factor of 5 when
solubilized in PG solution (Table 6). The solubility of
TAM is considerably further enhanced by a factor of 65 in
the presence of PG-PEG. This significant solubility
increase indicates that TAM is not only solubilized inside
the hyperbranched interior but also inside the covalently
bound poly(ethylene glycol) chains. This is in line with
previous results employing PEGylated dendrimeric
derivatives (Sideratou et al, 2001; Paleos et al., 2004) and
other hydrophobic drugs, establishing that the introduction
of the poly(ethylene glycol) chains in general enhances the
solubilization efficiency of dendritic polymers. It is
interesting to note that for PG-PEG-Folate a ~1300-fold
increase of TAM solubility was observed.
For triggering the release of TAM from PG and its
derivatives, increasing concentrations of NaCl solutions
were used in analogy with the experiments with
PEGylated dendrimeric derivatives (Paleos et al., 2004).
O
CH 3
N
CH 3
86
N
H
2
N
N
OH
N
N
H 2N
3
Figure 22. Chemical structure of
Tamoxifen (TAM).
Solubilized molecules can be replaced by the metal ion
and it is, therefore, necessary to investigate whether
sodium cation complexation can cause premature release
of the drug in the extracellular fluids, before the
nanocarrier loaded with the drug reaches the target cell.
By titrating TAM loaded polymeric solutions with sodium
chloride solution, the drug was released and suspended in
the bulk aqueous phase. In the presence of 0.142 M NaCl
solution, 39 % and 24 % of the solubilized TAM in PG
and PG-PEG (Figure 23) were released respectively in the
aqueous media. Under the same conditions and in the
presence of PG-PEG-Folate, only 6 % of the solubilized
TAM was released (Figure 23). It should, therefore, be
noted that for the most elaborated derivative prepared in
this study, i.e. the multifunctional PG-PEG-Folate, most of
TAM remains encapsulated in the polymer and it is not
released in the extracellular fluid at a concentration of
0.142 M NaCl solution. Therefore, this nanocarrier can
reach target cells appreciably loaded with TAM.
These results have to be taken into consideration
before PEGylated polyglycerols are to be applied as drug
delivery systems in experiments in vitro and in vivo.
Sodium cation, in extracellular fluids can form complexes
with PEG chains affecting the overall release profile of the
drug. It is therefore required, for designed PEGylated drug
delivery systems, to investigate whether drug release
occurs in the extracellular fluid and before entering the
target cells.

Gene Therapy and Molecular Biology Vol 10, page 87
Table 6. Solubility of Tamoxifen in PG, PG-PEG and PG-PEG-Folate aqueous solutions. Reproduced from Tziveleka et
al, 2006 with kind permission from Wiley-VCH.
Hyperbranched Polymer
PG
CPolymer [M]
1.0 x 10
CTamoxifen [M]
-3
9.6 x 10 -6
PG-PEG 1.0 x 10 -3
1.23 x 10 -4
PG-PEG-Folate 1.0 x 10 -3
2.48 x 10 -3
Tamoxifen (10 -5 M)
15
10
5
1.0
0.5
PG-PEG-Folate
PG-PEG
PG
0.0
0.0 0.2 0.4 0.6
NaCl (M)
Figure 23. Release of Tamoxifen from PG (�), PG-PEG (�) and PG-PEG-Folate (▲) aqueous solutions (1x 10 -3 M) as a function of
added NaCl solutions. Reproduced from Tziveleka et al, 2006 with kind permission from Wiley-VCH.
IV. Gene carriers: from
monofunctional dendrimers to
multifunctional dendritic polymers
Numerous gene delivery systems based on viral
(Verma and Somia, 1997; Lotze and Kost, 2002) and nonviral
(Li and Huang, 2000; Brown et al, 2001; Nishikawa
and Huang, 2001) vectors have been developed and tested
so far. Recently, several recurring issues about safety of
viral vectors have led to a careful reconsideration of their
application in human clinical trials and prompted the use
of synthetic systems. Moreover, viral vectors experience
significant limitation in large-scale production and the
available size of DNA they can carry. For addressing these
problems, non-viral gene delivery systems such as cationic
polymers or cationic lipids, liposomes or cationic
dendrimers have attracted great attention for achieving a
breakthrough in the development of an effective gene
carrier. Specifically, synthetic non-viral carriers of genetic
material present insignificant risks of genetic
recombinations in the genome. Transfection with synthetic
vectors, through appropriate tailoring, may exhibit low cell
toxicity, high reproducibility and ease of application.
However, the currently known synthetic vectors present
disadvantages, which are due to their generally low
effectiveness compared to viral vectors and to their
inability for targeted gene expression. For an effective
gene expression, genes must be transferred in the interior
of the nucleus of the cell and this procedure has to
87
250
225
200
175
150
circumvent a series of endo- and exo-cell obstacles.
Among these obstacles are included cell targeting,
effective transport of the carriers together with attached
genetic material through cell membranes and the need of
carriers release from the endosome following endocytosis.
For the synthetic carriers that have been described in the
literature, some or all of these difficulties have been
addressed, without, however, completely achieving this
objective yet.
The strategy employed for the delivery of the
conventional drugs through the preparation of functional
dendritic polymers can also be applied for the delivery of
genetic material. Specifically, the method involves
molecular engineering of dendritic surface and/or the core
aiming at obtaining polymers, which should be positively
charged, biologically stable, non-toxic, exhibit targeting
ability, and have the ability to be effectively transported
through cell membranes. In addition, the dendrimer-DNA
complex should have the possibility of being released
from the endosome following endocytosis.
Dendrimers and hyperbranched polymers are stable
nanoparticles in contrast to liposomes, which, as a rule, are
unstable. Additionally, the dependence of dendrimers’ size
on their generation can affect transfection efficiency.
Several studies (Boas and Heegaard, 2004) have reported
the use of unmodified amino-terminated PAMAM or DAB
dendrimers as non-viral gene transfer agents, enhancing
the transfection of DNA into the cell nucleus. The exact
structure of these host–guest binding motifs has not been

Sideratou et al: Dendritic polymers for application as drug and gene delivery systems
determined in detail, but it is presumably based on
acid-base interactions between the anionic phosphate
moieties on the DNA backbone and the primary and
tertiary amines of the dendrimers, which are positively
charged under physiological conditions. It has also been
found (Tang et al, 1996) that partially degraded (or
fragmented) dendrimers, are more appropriate for gene
delivery than the intact dendrimers and a fragmentation (or
activation step) consisting of hydrolytic cleavage of the
amide bonds is needed to enhance the transfection. These
dendrimers are characterized as activated and are shown in
Figure 24. It has been concluded from several
investigations that the spherical shape of dendrimers is not
advantageous in gene delivery. This agrees with earlier
work, where ‘fragmented’ PAMAM dendrimers show
NH2 H2N O N
H
N
N
H
O
N
O
O
H
N O
H
N N
N N
N
H
O
H
N
O
O
N
N N
H
H
O N
O
H
N
N O
O
N
H
N
O
N
H
H
H
NH H 2 2N H
N
H N
O
N H H
O N
NH2 H2N H
N O O
O
N
N
O
H
H
H
N
2N
H NH2 NH
N
2
N
O
N
N H
H
O H
N
2N
O
H N
N
O
H H
N
2N
O
N O
H
N
H
H
O N
N
N NH2 H2N N
O
N O
O
NH2 H
N O
H
H
O
N
H
N N
H
N N
O
O
H N
N H
2N
O
H
N
H N
NH
2N O
O
2
N
H
H N
O N
O
H
N
H
N
N
O
H H
2N
N O
N
H O
2N
O O
N
H
N
N N
H H
H
O N
N
N
O
O
O
N
H O
H
O N
2N H2N N N
H N
N H
H
H
H
N O
O N
N
O
N
NH O O
2 H H
N
2N H N N H NH2 H
O NH2 N
N H
H
O
N
O NH2 N N NH2 H
H
O N
O
N
NH2 N NH2 H
O
N H
H2N NH2 H2N Figure 24. Structural features of intact vs activated PAMAM Dendrimers.
superior transfection efficacy in comparison with the
spherical ‘intact’ dendrimers (Boas and Heegaard, 2004).
In comparison to the intact dendrimers, the partially
degraded dendrimers have a more flexible structure (fewer
amide bonds) and form a more compact complex with
DNA, which is preferable for gene delivery by the
endocytotic pathway (Dennig and Duncan, 2002). In
addition, it is generally found that the maximum
transfection efficiency (Figure 25) is obtained with an
excess of primary amines to DNA phosphates, yielding a
positive net charge of the complexes The more flexible
higher generation DAB dendrimers (containing no amides)
are found to be too cytotoxic for use as non-viral gene
vectors, however, the lower generation dendrimers are
well-suited for gene delivery (Zinselmeyer et al, 2002).
H 2N
H 2N
H 2N
O
N
H
HO
H 2N
HO
N
O
N O
H N
O
O
N
H
N
H
N
O
H
HO
N O
O
HO
H
O
N
O
O
N
H
O
N
HO
N
O
N
N
H
HO
N O
O
N
H
N
H
N
O
O
HO
O
O
N
H H
N N
H N
O
H
N
N
O
O
N
N
H
O
N H
O
N
N
H
N
O
H N
N
O
H N
NH 2
H 2N
N H
O
N H
O H
N
O
O
OH
N
N
OH
O
O
O
N
OH
O
N
O
N
H
N
H
N
O
Figure 25. Transfection efficacy of poly(propylene imine) dendrimers of various generations (G1-G5) relative to N-[1-(2,3dioleoyloxy)propyl]-N,N,N-trimethylammonium
methylsulphate (DOTAP) in the A431 cell line studied in 96-well plates. DAB G1 and
DAB G2 were dosed at a dendrimer: DNA ratio of 5:1, and a DNA dose of 20 µg per well was used. DAB G3 was also dosed at a
dendrimer: DNA ratio of 5:1, but a DNA dose of 5 µg per well DNA was used; DAB G4 and DAB G5 were dosed at a dendrimer: DNA
ratio of 3:1 and a DNA dose of 20 µg per well. DOTAP was dosed at a DOTAP:DNA ratio of 5:1, and a DNA dose of 20 µg per well.
Data represented as the mean ±SD of at least 3 replicates. Reproduced from Zinselmeyer et al., 2002 with kind permission from
Springer.
88
N
H
N
O
OH
H
N
O
O
OH
H
O N
NH 2
N
O
N
N
H
H
O N
O N
H
NH 2
O
NH 2
NH 2

In this connection, PAMAM-OH dendrimers, which
are structurally similar to PAMAM, except that surface
amino groups have been replaced by hydroxyl groups (Lee
et al, 2003) have been prepared. Absence of surface
primary amino groups in PAMAM-OH renders this
polymer nearly neutral, which might be advantageous in
terms of cytotoxicity. However, PAMAM-OH is nearly
unable to form DNA polyplex because of the low pKa of
interior tertiary amino groups (Tomalia et al, 1985). For
this purpose, the synthesis and characterization of
internally quaternized PAMAM-OH has been reported, as
shown in the Figure 26. The internal quaternary
ammonium groups of QPAMAM-OH will interact with
negatively charged DNA, while preserving a neutral
polymer and/or a polyplex surface.
It was found that QPAMAM-OH/DNA polyplexes
were round-shaped with the more compact and small
OH
HO
HO
OH
H H
H H
N HO
N
N N
O
OH
O O
O
N H
N
H OH
O
N
N
N
HO
H
O
N H
H
H N
O
H N
O N
HO HO N
N H O
O N
H
O
N
O
H
O
N
N
N OH
O N
N
H
H N
N
O
H N
O H
O N
N
O
O
H
HO O N
H
H
O OH
H
N
H
H
N
N
N
N N N
N
H
HO O
O
N
O N
N O
N H
O
N
H
H
H N O
O N
N
O
H
N
O
HO
~
~
N
N
O H
N
~
PAMAM-OH
Figure 26. Quaternization of PAMAM-OH dendrimer.
Gene Therapy and Molecular Biology Vol 10, page 89
OH
CH 3I
2M NaCl dialysis
89
particles formed as the charge ratio increased. Although
the transfection efficiency of functional QPAMAM-OH
derivatives was lower by one order of magnitude than
parent PAMAM (Figure 27), the QPAMAM-OH/DNA
particles exhibited reduced cytoxicity compared with
PAMAM and PEI. Shielding of the interior positive
charges by surface hydroxyl may be possibly the reason
for this behaviour.
As already mentioned, one of the major problems
with non-viral gene delivery systems is their lower
efficiency compared to viral vectors. Many methods have
tried to overcome such problems, including linking or
conjugating cell-targeting ligands or cell penetrating
peptides as efficient vectors for intracellular delivery of
bioactive molecules (Futaki, 2005). Arginine-rich peptides
have exhibited enhanced translocational ability, which was
N
N
N
O
N H
N
N
+
OH
N
N
H
O
H
H N
HO N
N H O
O
N
O N
H N
O
O
N
H
H
N
H N
H
N
O
O
O
+
HO
OH
H
H
N N
O O
N
HO O
N
H N
H
N
HO
O
+
HO
N H
H
O N
HO
O N
H
O
H
O
N
N
N OH
H
N
H
O
N
O
H
O N
N
H
HO O
H
N
H
N N N
O
N O
N
O
H
+
N
O
N
H
H
N
O
OH
OH
+
HO
OH
H H
N
N
O
O
N +
HO
N H H OH
O N
O
Cl- Cl- Cl -
Cl- Cl -
Cl -
~
~ ~
QPAMAM-OH
Figure 27. Transfection efficiency
of PEI, PAMAM and QPAMAM-
OH dendrimers with 52, 78 and
97% quaternization degrees in
293T cell at charge ratio (+/-) = 6.
Data are expressed as a RLU
(Relative light unit) per µg protein.
Reproduced from Lee et al., 2003
with kind permission from
American Chemical Society.

Sideratou et al: Dendritic polymers for application as drug and gene delivery systems
attributed to the presence of the guanidinium moiety
(Vives et al, 1997; Rothbard et al, 2000; Wender et al,
2000; Futaki et al, 2001; Kirschberg et al, 2003), a
structural feature of L-arginine, which is capable of
hydrogen-bonding and electrostatic interactions (Onda et
al, 1996) with phosphate or carboxylic group located at the
surface of cell membranes. In a recent study (Choi et al,
2004), it has been reported a new three dimensional
artificial protein, L-arginine-grafted-PAMAM dendrimer
(PAMAM-Arg), as a novel non-viral gene delivery vector,
which consisted of a PAMAM scaffold the surface of
which is covered with L-arginine residues (Figure 28).
By the introduction of arginine moieties on the
PAMAM surface, gene delivery efficiency is greatly
increased in comparison to that of starting PAMAM
(Figure 29). It was comparable to PEI for HepG2 and
primary rat vascular smooth muscle cells, and was more
efficient in the case of Neuro 2A cells than PEI and
Lipofectamine. As a control, L-lysine grafted-PAMAM
(PAMAM-Lys) was prepared and tested showing slightly
better transfection efficiency in HepG2 cells than that of
basic PAMAM, while increased effect was not observed in
primary cells. In conclusion, a polyvalent arginine
functionalized PAMAM is easily prepared, which
possesses outstanding transfection efficiency with
relatively low cytotoxicity. These properties would make
PAMAM-Arg a promising non-viral vector for both in
vitro and in vivo use. Potentially, PAMAM-Arg could be
used as a dendritic nanocarrier encapsulating or
incorporating small molecules, peptides, proteins,
oligonucleotides, and plasmids that are deficient in cellpenetrating.
The structural features set forth for a successful
dendritic gene vector are possibly satisfied in a PEI-
PAMAM
NH2 64
HOBt, HBTU
Fmoc-Arg(pbf)-OH
in DMF
30% piperidine
in DMF(v/v)
TFA/triisopropylsilane/H 2O
(95:2.5:2.5, v/v)
Figure 28. Introduction of L-Arginine at the external surface of PAMAM.
poly(ethylene glycol)-folate (PEI-PEG-FOL) derivative
which was recently synthesized (Figure 30) and its
efficiency as gene carrier was tested (Kim et al, 2005a).
This multifunctionalization of PEI aimed at the
preparation of a nanocarrier that would simultaneously
combine protective and targeting properties. In this study,
the PEI-PEG-FOL nanocarrier, was tested for its capacity
to complex with plasmid DNA and be transfected to folate
receptor overexpressing cells that produce exogenous
green fluorescent protein, GFP, (GFP-KB cells). A special
plasmid system (pSUPER-siGFP) was prepared, that
carried a siRNA-expressing sequence, used for inhibiting
the expression of exogenous GFP in mammalian cells. The
pSUPER-siGFP/PEI-PEG-FOL complexes inhibited GFP
expression of KB cells more effectively than pSUPERsiGFP/PEI
(Figure 31). These results indicated that folate
receptor-mediated endocytotosis is a major pathway in the
process of cellular uptake.
V. Concluding remarks
Molecular engineering of basic dendrimeric and
hyperbranched polymer scaffolds resulted in the
preparation of nanocarriers of low toxicity, with
significant encapsulating capacity, specificity to certain
biological cells and transport ability through their
membranes. Depending on the degree and type of
functionalization, products that fulfill one or more of the
above characteristics were prepared. The exhibition of
these properties is further induced by the so-called
polyvalent interactions attributed to the placement of the
functional groups in close proximity on the external
surface of the dendritic polymers.
H O
N
O N
O
N
H
N
N
N
H
O H
O
N
N
H O
H
N N
N
N
N O
H
H
N
H
O
H
O N
HN
N O
N
O
NH
N H
HN
H
NH2 H O HN
H2N N
+ H2N NH2 H2N NH2 +
H2N NH
HN
HN
H H
N N
N
H
O O O N H
O
N
H NH2
N N
N H N
H
H
O
N
O
HN +
O
NH2 H2N NH2 O
N
HN + NH2 H2N O
H2N + HN NH2 NH2 O
NH + H2N
+ H2N NH2 NH
NH
2
NHH2N
O
HN
+ H2N
+
+ H2N
NH2 H2N NH2 NH NH
2
NH
N
H2N H O
O
NH2 O
NH
O
2
NH
+ NH2 2
NH2 H H
N N
+ H2N NH2 O
NH O O
NH
N
N
H
O
O
H
H N
H2N N N
H
O
N
H
N
+H2N NH2 O
O N
NH
N O
H
H2N NH
N
O O
H
N
N
H
O
O
H2N N
N N
H
+ NH N
H
H2N O
O H
N
N O
H
H2N O
N
H H2N H
H N N
N
2N
H
+ H2N O H
O
N
H2N NH O NH
+ H2N
N
90
NH 2
~
~
~
PAMAM-Arg

Gene Therapy and Molecular Biology Vol 10, page 91
Figure 29. Transfection efficiency for Neuro 2A cell lines (1x10 5 cells/well). DNA amount per well was 0.2 µg (black) and 1.0 µg
(gray). Values in parentheses are representing the charge ratio (N/P) of dendrimer/plasmid DNA complexes. The luciferase expression
mediated by reagents was measured at each optimum condition. Results are expressed as mean ±SD of 3 replicates. Reproduced from
Choi et al, 2004 with kind permission from Elsevier.
H
N
N
H
Figure 30. Chemical structure of PEI-PEG-FOL
O
PEG
H
N
C
O
HOOC
O
N
H
PEI-PEG-FOL
91
N
H
N
N
OH
N
N
H 2N
m
Figure 31. GFP gene inhibition
efficiency of pSUPER-siGFP/PEI
and pSUPER-siGFP/PEI-PEG-
FOL complexes as a function of
N/P ratio against GFP-KB cells.
Reproduced from Kim et al, 2005a
with kind permission from
Elsevier.

Sideratou et al: Dendritic polymers for application as drug and gene delivery systems
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