Microencapsulation Methods for Delivery of Protein Drugs

Biotechnol. Bioprocess Eng. 2001, 6: 213-230
Microencapsulation Methods for Delivery of Protein Drugs
Yoon Yeo, Namjin Baek, and Kinam Park*
Departments of Pharmaceutics and Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA
INTRODUCTION
Recent advances in recombinant DNA technology have resulted in development of
Abstract
new protein drugs. Due to the unique properties of protein drugs, they have to be delivered
many
parenteral injection. Although delivery of protein drugs by other routes, such as pulmonary
by
nasal routes, has shown some promises, to date most protein drugs are administered by par-
and
routs. For long-term delivery of protein drugs by parenteral administration, they have been
enteral
into biodegradable microspheres. A number of microencapsulation methods have been
formulated
and the currently used microencapsulation methods are reviewed here. The microen-
developed,
methods have been divided based on the method used. They are: solvent evaporacapsulation
phase separation (coacervation); spray drying; ionotropic gelation/polyelectrolyte
tion/extraction;
interfacial polymerization; and supercritical fluid precipitation. Each method is de-
complexation;
scribed for its applications, advantages, and limitations.
Keywords: protein, peptide, drug delivery, microparticle, microencapsulation
advances in biotechnology have brought
Significant
availability of recombinant peptide pro-
ever-increasing
drugs in large quantities. The successful completein
of the human genome project will undoubtedly
tion
to the explosion of new protein drugs with exqui-
lead
bioactivities. Since protein drugs carry out biologisite
processes and reactions with high specificity and
cal
protein drugs will continue to be drugs of
potency,
for treating various diseases. Formulating pro-
choices
drug delivery systems, however, poses several probtein
Due to extremely low bioavailability of protein
lems.
by oral administration, which is the most conven-
drugs
mode of drug delivery, protein drugs are usually
ient
by parenteral route. One way of minimizing
administered
and improving patient compliance is to pro-
discomfort
sustained-release formulations that deliver protein
duce
continuously over long periods of time. Another
drugs
in protein drug delivery is to maintain the terti-
challenge
protein structure, which is essential to bioactivity. Exary
of protein drugs to unfavorable conditions during
posure
tends to reduce their bioactivities.
formulation
widely used approach for long-term delivery of
Most
drugs has been parenteral administration of
protein
drugs in microspheres made of biodegradable
protein
Preparation of protein drug-containing mi-
polymers.
without reducing bioactivity has been the
crospheres
of microencapsulation methods. A number of mi-
goal
methods have been developed through
croencapsulation
the years, but none of the methods has been ideal for
Corresponding author
*
+1-765-494-7759 Fax: +1-765-496-1903
Tel:
e-mail: kpark@purdue.edu
protein drugs. Each method has its own advan-
loading
as well as limitations. For further improvement in
tages
technologies, it is important to un-
microencapsulation
the strengths and drawbacks of each method.
derstand
1 lists examples of microencapsulation processes
Table
in the literature. Microencapsulation processes
found
been frequently classified either chemical or me-
have
[1]. Chemical processes refer to methods that
chanical
the change of solvent property, chemical reaction
utilize
monomers, or complexation of polyelectro-
between
In mechanical processes, a gas phase is utilized at
lytes.
stage to provide force to break up materials to
some
particles. There are many situations, however,
small
such a distinction is not clear.
where
MICROENCAPSULATION METHODS
SOLVENT EVAPORATION/EXTRACTION
evaporation/extraction methods have been
Solvent
used to prepare microspheres loaded with vari-
widely
drugs, especially hydrophobic drugs. For the encapous
of peptide and protein drugs, oil/water (o/w),
sulation
(o/o) and water/oil/water (w/o/w) emulsifica-
oil/oil
methods have been used. Depending on the numtion
of emulsions produced during the preparation of
ber
solvent evaporation/extraction can be
microspheres,
into two methods, single emulsion and double
divided
(Fig. 1).
emulsion
Methods
Emulsion (o/o or o/w) Methods
Single
single emulsion methods, peptides/proteins are pre-
In

214 Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4
Table 1. Examples of microencapsulation methods
Chemical processes
1.
evaporation and extraction
Solvent
solvent extraction
Crygenic
separation (Coacervation)
Phase
addition
Non-solvent
change
Temperature
polymer or salt addition
Incompatible
interaction (complex coacervation)
Polymer-polymer
complexation
Polyelectrolyte
polymerization
Interfacial
Mechanical processes
2.
drying
Spray
chilling
Spray
desolvation
Spray
Supercritical fluid precipitation
Single Emulsion Double Emulsion
suspension/solution
Protein
polymer solution (oil)
in
Emulsion (o/o or o/w)
solution
Protein
water in
oil or water oil
removal
Solvent
drying &
Hardened microsphere
Primary emulsion (w/o)
water
Secondary emulsion (w/o/w)
removal
Solvent
drying &
Hardened microsphere
1. Single emulsion (left) and double emulsion (right) sol-
Fig.
evaporation methods for microencapsulation of proteins.
vent
in a dispersed phase, which is a polymer solution in
sent
solvent such as dichloromethane or ethyl ace-
organic
Polylactic acid (PLA) and poly(lactide-co-glycolide)
tate.
are the most widely used biodegradable syn-
(PLGA)
polymers for sustained-release preparations. The
thetic
kinetics of active components can be controlled
release
changing molecular weight and/or copolymer ratio
by
those polymers. Drugs can be dispersed as solid parti-
of
or dissolved in polymer solution. The drug solution
cles
suspension is added into a continuous phase, which
or
be mineral oil (o/o) or aqueous solution (o/w) con-
can
emulsifiers. Emulsification is carried out by agitaining
homogenization, or sonication. Emulsifiers in
tation,
continuous phase stabilize o/o or o/w emulsions
the
produced. Emulsifiers such as Span 85 [2], sorbi-
being
sesquioleate [3], aluminium tristearate [4] have
tan
used for o/o interface, and Carbopol been ® [2], 951
cellulose [5], polyvinyl alcohol (PVA) [6] have
methyl
used for o/w interface.
been
solvent in dispersed phase is removed by sol-
Organic
evaporation or by solvent extraction. In solvent
vent
process, hardening of emulsion occurs
evaporation
volatile organic solvent in dispersed phase leaches
when
continuous phase and evaporates from continuous
into
at atmospheric pressure. Using vacuum or a mod-
phase
increase in temperature can accelerate the evapoerate
of organic solvent. In solvent extraction process,
ration
emulsion is transferred to a large amount of water
the
other quenching medium, and the extraction of or-
or
solvent occurs faster than in solvent evaporation
ganic
Thus, microspheres produced by solvent ex-
process.
process are more porous than the ones protraction
by solvent evaporation. The porous structure
duced
results in faster release of peptide/protein drugs.
usually
long-term sustained release, solvent evaporation is
For
The prepared microspheres are collected by
preferred.
centrifugation or filtration, and freeze-dried.
Emulsion (w/o/w) Methods
Double
double emulsion methods, an aqueous drug solu-
In
is first emulsified in a polymer-dissolved organic
tion
The w/o emulsion is then added into an aque-
solvent.
phase that contains emulsifier, thereby forming w/
ous
emulsion. Then, the organic solvent is removed by
o/w
extracting into external aqueous phase and evaporation.
Applications
Emulsion
Single
emulsion solvent evaporation method has been
Single
to prepare microspheres/nanospheres containing
used
peptide and protein drugs. Insulin solution in
various
solvent was used in emulsification to prepare
organic
[7]. Other researchers used solid particles
nanospheres
bovine serum albumin (BSA) [2] or ovalbumin (OVA)
of
instead of protein solution, in a hope that protein
[4],
can be preserved in organic solvent. High en-
activity
efficiency was obtained using protein particapsulation
The prepared microparticles tend to show initial
cles.
release profiles. Table 2 lists some examples of
burst
preparation using protein particles. A
microparticle
approach of protein particle preparation was used
novel
the encapsulation of bovine superoxide dismutase
for
into PLGA/PLA microspheres [5]. Protein
(bSOD)
particles were prepared by lyophilization of
spherical
glycol (PEG) aqueous mixture.
protein-polyethylene
approach resulted in high encapsulation efficiency
This
and high activity of the loaded enzyme (95%). In
(88%)

Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4 215
2. Encapsulation of protein particles by single emul-
Table
evaporation methods
sion/solvent
Protein
particles
Spray-dried
BSA
Micronized
OVA
Lyophilized
bSOD/PEG
)
(% 75
S
O
D
o
f b
e
50
le
a
s
e
re
u
la
tiv
Q
u
m
100
25
Polymer Type
PLGA o/o
o/w
Encapsulation
efficiency
>90%
20-50%
Initial
in release
1 day

216 Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4
100
)
e
(%
m
75 z
y
o
s
o
f ly
e
50
le
a
s
e
re
u
la
tiv
Q
u
m
25
0
3. Release of lysozyme from PEG-PBT microspheres. Mo-
Fig.
weight of PEG segment was 600. Polyvinyl alcohol
lecular
was used as a stabilizer in the external aqueous phase.
(4%)
percentage of PBT in PEG-PBT block copolymer
Increasing
resulted in slower release of lysozyme. From reference [16].
of glycolide in PLGA polymers result in rapid deg-
tents
of the polymer, resulting in faster release of the
radation
drugs. The release profile can also be controlled
loaded
the processing factors, such as shear forces, since
by
shear forces tend to produce smaller microparticles
high
release drugs at a faster rate due to higher surface
which
area and shorter diffusion path length.
Limitations
0 10 20 30 40 50
Time (days)
23%
45%
solvent evaporation/extraction methods have
While
used widely in preparing microparticles for pep-
been
delivery, the methods are still not ideal and
tide/protein
many aspects to be improved. First, the drug
have
efficiency into microspheres is not high.
encapsulation
protein drugs can be produced in larger scale
Although
to development of genetic engineering technique,
due
cost is still high. Thus, increasing the encapsulation
the
is still quite important for preparing micro-
efficiency
Second, the solvent evaporation/extraction
spheres.
require use of toxic organic solvents, such as
methods
and ethyl acetate, as a solvent for dis-
dichloromethane
biodegradable polymers. To meet the regulatory
solving
regarding the residual solvent in the final
requirement
the use of toxic organic solvent should be
products,
or avoided. Third, in most situations, the
minimized
proteins are released with the initial burst release,
loaded
in many situations the loaded proteins are not re-
and
completely. The initial burst release may cause
leased
response in patients due to abnormally high
abnormal
concentration in blood. Incomplete release of the
drug
proteins is often related to protein stability. Pro-
loaded
have to maintain their 3-dimensional conformateins
to keep their bioactivity. Proteins, however, are
tion
prone to be denatured and to form aggregates by vari-
factors, including exposure to large interface beous
organic and aqueous phases, shear force in the
tween
steps (e.g., homogenization, and sonication),
processing
protein-polymer interaction. Some reports showed
or
the loss of protein activity or denaturation/ aggre-
that
of protein could be minimized by using stabilizgation
such as poloxamer, BSA or hydroxypropyl-βers
[13,14,18]. Fourth, although continuous
cyclodextrin
of peptide/protein drugs is desirable, there are
release
where pulsatile release is preferred as in the
situations
of insulin delivery. Furthermore, the timing and the
case
amount of the released insulin need to be con-
exact
carefully. This type of release can not be obtrolled
by biodegradable microspheres prepared by soltained
vent evaporation/extraction methods.
Modifications of Solvent Extraction
an effort to protect proteins from denaturation
In
microencapsulation processes, alternative methods
during
been developed to minimize exposure of protein
have
to denaturing conditions, such as high tempera-
drugs
ture and water-oil interfaces [19].
Spray-desolvation
involves spraying a polymer solu-
Spray-desolvation
onto desolvating liquid. For example, microdroplets
tion
made by spraying PVA aqueous solution onto ace-
were
bath, where the polymer solvent (water) was extone
into acetone and PVA precipitated to form solid
tracted
[20]. This method was also applied to
microparticles
peptides and BSA as model drugs in PLGA.
encapsulate
micronized drug was suspended in a PLGA-acetone
The
and atomized ultrasonically into ethanol bath,
solution
where microspheres were precipitated [21].
Solvent Extraction
Cryogenic
is dissolved in an organic solvent such as di-
PLGA
together with protein powders. As shown
chloromethane,
Fig. 4, the polymer/protein mixture is atomized over
in
bed of frozen ethanol overlaid with liquid nitrogen, at
a
temperature below the freezing point of the poly-
a
solution. The microdroplets freeze upon
mer/protein
the liquid nitrogen, then sink onto the fro-
contacting
ethanol layer. As the ethanol layer thaws, the mizen
which are still frozen sink into the ethanol.
croparticles
the solvent in the microparticles
Dichloromethane,
thaws and is slowly extracted into ethanol, result-
then
in hardened microparticles containing proteins and
ing
matrix.
polymer
process was used to produce a microsphere for-
This
for human growth hormone (hGH) [23-26].
mulation
to the encapsulation process, hGH is formulated
Prior
zinc to produce insoluble Zn:hGH complex. The
with
of this complex contributed to stabilize
encapsulation
during the fabrication process and within the mi-
hGH
after hydration [23,24]. An in vivo study of
crospheres
formulation demonstrated a lower Cmax and an ex-
this
serum level for weeks, but a similar bioavailabiltended
as compared with the protein in solution [26]. The
ity,

Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4 217
suspension
Protein
polymer solution
in
Liquid nitrogen
Frozen
microspheres
4. Schematic diagram of the ProLease Fig. ® encapsulation
From reference [22].
process.
formulation using this method (referred to as
hGH
ProLease ® was approved by the FDA in December 1999,
)
an injectable suspension for once- or twice-a-month
as
under a brand name of Nutropin depot adminitration, ® .
this process, all manipulations are performed at
In
temperatures, therefore thermal denaturation of
low
drugs can be prevented. There are no aqueous
protein
thus the protein is not subjected to oil-water
phases,
where some proteins may denature. In addi-
interfaces
the process utilizes solvents in which most protion,
are insoluble (dichloromethane as a polymer solteins
ethanol as an extraction solvent), therefore a high
vent,
efficiency can be achieved [22,27]. How-
encapsulation
this system is not easy to apply to various protein
ever,
The use of liquid nitrogen also makes scale-up
drugs.
difficult. The system still utilizes toxic organic
rather
and thus it has similar problems associated
solvents,
solvent evaporation/extraction methods.
with
PHASE SEPARATION (COACERVATION)
Methods
Nozzle
Extraction solvent
Atomized
droplets
of solvent
Extraction
microspheres
from
Drug particle
Release modifier
PLG microsphere
by phase separation is basically a
Microencapsulation
process: (1) phase separation of the coating
three-step
to form coacervate droplets; (2) adsorption of
polymer
coacervate droplets onto the drug surface; and (3)
the
of the microcapsules [28], as shown in Fig.
solidification
Phase separation techniques can be classified accord-
5.
to the method to induce phase separation; noning
addition, temperature change, incompatible
solvent
or salt addition, and polymer-polymer interac-
polymer
[29]. tion
Addition
Non-solvent
polymer to be coated is dissolved in a good sol-
The
and drug may be dissolved or suspended or emulvent,
in the polymer solution. Then the first nonsified
(also called ‘coacervating agent’ or ‘phase insolvent
which is miscible with the good solvent but
ducer’)
not dissolve the polymer is slowly added to the
does
solution system. The polymer is concen-
polymer-drug
as the solvent for the polymer is slowly extracted
trated
the first non-solvent. The polymer is then induced
into
by
Induced
Non-solvent
1.
Temperature change
2.
Salt / Incompatible polymer
3.
4. Polymer-polymer interaction
separation
Phase
the coating polymer
of
Drug particle
of
Adsorption
droplets
coacervate
of
Solidification
microcapsules
the
5. Schematic diagram of the formation of a coacervate
Fig.
From reference [30].
microcapsule.
phase separate and form coacervate droplets that
to
the drug (Fig. 6(A)). The coacervate droplet size
contain
be controlled by adjusting the stirring speed. At this
can
of the process, the coacervates are usually too soft
point
be collected, and thus they are usually transferred to
to
large body of a second non-solvent (also called ‘hard-
a
agent’) to harden the microparticles [31].
ening
polyesters, such as PLA and PLGA, are used,
When
widely used solvents are dichloromethane, ethyl
most
and acetonitrile. The first non-solvents for the
acetate,
are low-molecular weight liquid polybutadi-
polymers
low-molecular weight liquid methacrylic polymers,
ene,
oil, vegetable oil, and light liquid paraffine oils.
silicone
hydrocarbons, such as heptane, hexane, and
Aliphatic
ether, have been used as the second non-
petroleum
solvent.
Change
Temperature
system comprised of a polymer and a solvent exists
A
a single phase at all points above the phase boundary.
as
is dispersed in the polymer solution with stirring.
Drug
the temperature is decreased below the curve (Fig.
As
phase separation of the dissolved polymer occurs
6(B)),
the form of immiscible liquid and the polymer coa-
in
around the dispersed drug particles, thus forming
lesces
Further cooling accomplishes gelation
microcapsules.
solidification of the coating. The microcapsules are
and
from the solvent by filtration, decantation, or
collected
techniques [32].
centrifugation
Polymer Addition or Salt Addition
Incompatible
two chemically different polymers dissolved in
When
common solvent are incompatible and do not mix in
a
phase separation takes place. This phenome-
solution,
is well described by the phase diagram of a ternary
non
consisting of a solvent, and two polymers, X
system
Y (Fig. 6(C)). A drug is dispersed in a solution of
and
Y (point a in Fig. 6(C)) and polymer X is added
polymer
the system, denoted by the arrowed line. When the
to
boundary is passed with the further addition of
phase
X, polymer rich phase begins to separate to
polymer
immiscible droplets containing the drug, and coa,-
form

218 Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4
( A )
1 0 0 %
0 0 % 1 0 0 %
1
o n s o l v e n t P o l y m e r
N
( C )
S o l v e n t
S o l v e n t
2 p h a s e s
1 0 0 %
2 p h a s e s
1 0 0 %
1 0 0 %
o l y m e r Y P X r e m y l o P
6. General phase diagrams of phase separation techniques based on solvent (A), temperature (B), incompatible polymers (C),
Fig.
interpolymer interactions (D). From reference [32].
and
to make microcapsules [32]. In a similar manner
lesce
salts can be added to aqueous solution of wa-
inorganic
ter-soluble polymers to cause phase separation.
Interaction (Complex Coacervation)
Polymer-polymer
interaction of oppositely charged polyelectro-
The
can result in the formation of a complex with relytes
solubility leading to phase separation (Fig. 6(D)).
duced
method is often called complex coacervation [1].
This
method differs from incompatible polymer addi-
The
in that both polymers become part of the final caption
wall. Gelatin is normally used as a cationic polymer
sule
pH below its isoelectric point. A variety of natural
at
synthetic anionic polymers are available for interac-
and
with gelatin to form complex coacervates. A well
tion
example of natural polyanion for making com-
known
coacervates with gelatin is gum arabic. Aqueous
plex
of gum arabic and gelatin are adjusted to pH
solutions
and warmed to 40-45°C. The pI of gelatin is 8.9. The
4.5
charged macromolecules interact to undergo
oppositely
separation, to which a water insoluble liquid core
phase
is added and emulsified to yield the desired
material
size. The mixture is then slowly cooled for an
droplet
During the cooling cycle, phase separation is
hour.
enhanced, resulting in the microcapsules formed
further
the core material with thin film of coacervate [32].
on
harden the microcapsules, the capsules are normally
To
further and treated with glutaraldehyde. This
cooled
a
( B )
( D )
1 0 0 %
1 0 0 %
- e P + e P
is mainly for water insoluble materials, include-
method
inks for carbonless paper, perfumes for advertising
ing
inserts, and liquid crystals for display devices.
Applications
2 p h a s e s
P O L Y M E R C O N C E N T R A T I O N - w t %
W a t e r
1 0 0 %
2 p h a s e s
the non-solvent addition method does not in-
Since
an aqueous continuous phase as in w/o/w double
clude
methods, the water-soluble drugs may be pro-
emulsion
from partitioning into the water phase. This
tected
the non-solvent addition method one of the
makes
widely used techniques for encapsulating water-
most
compounds [28,33-41]. Triptoreline (luteinizing
soluble
releasing hormone analogue)-containing micro-
hormone
were prepared by the non-solvent addition
capsules
and the effect of the physicochemical nature of
method,
polymer on the stability of the coacervate droplets
the
examined [34,35]. Triptoreline was suspended in
was
solution, and silicone oil was
PLGA-dichloromethane
added to the suspension as the first non-solvent.
then
embryonic microspheres were further hardened in
The
the second non-solvent. It was found that as
n-heptane,
copolymer became more hydrophobic, it dissolved
the
in dichloromethane and thus more silicone oil
better
necessary to desolvate the copolymer. The average
was
of the microspheres increased with the volume
diameter
silicone oil, thereby lowering the initial burst effect.
of
was also observed from in vivo experiments. With op-
It

Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4 219
d
s
e
a
R
e
le
e
%
u
la
tiv
C
u
m
100
75
50
25
0
0 2 4 6 8
Time (days)
100/0
80/20
0/100
7. Release of GM-CSF from microspheres prepared from
Fig.
PLA (MW 6,100) (0/100), 80%/20% mixture of PLGA
100%
40,400)/PLA (80/20), and 100% PLGA (100/0). From
(MW
[41].
reference
microspheres, the in vivo peptide release reached
timized
peak level on day 15 and the effect lasted for 1
the
month.
colony-stimulating factor
Granulocyte-macrophage
was encapsulated in different blends of
(GM-CSF)
and low molecular weight PLA by the non-
PLGA
addition method, and the prepared microsolvent
were characterized both in vitro and in vivo [41].
spheres
microspheres with high encapsulation efficiency
The
obtained within a size range between 20 µm and
were
µm. The in vitro release kinetics was dependent on
80
ratio of blended polymers. Steady release of GM-
the
could be achieved over a period of one week with-
CSF
significant burst effect using blend of low molecular
out
PLA and high molecular weight PLGA (Fig. 7).
weight
released from the microspheres was found to
GM-CSF
physically intact and biologically active both in vitro
be
in vivo. and
avoid the aggregation of microparticles often en-
To
in non-solvent addition, a modified method
countered
developed [42]. Instead of adding non-solvent to a
was
mixture, the polymer solution was added
polymer-drug
an emulsion of aqueous ovalbumin in the non-
to
silicon oil. The obtained microspheres appeared
solvent
and remained individual, and displayed a small
smooth
release of entrapped ovalbumin over the first 12
initial
with the subsequent release profile close to a
hours,
order kinetics for a month. Another modification
zero
the non-solvent addition method was introduced to
of
protein entities from denaturation at the or-
protect
interface or unfolding and/or aggregaganic/aqueous
within hydrophobic polymer matrix [40,43,44].
tion
hydrophilic nanoparticles were pre-
Drug-containing
prior to encapsulation with hydrophobic polymer.
pared
[40], agarose [43], and poly(vinyl alcohol) (PVA)
Gelatin
were used as nanoparticle matrices for BSA or insu-
[44]
Nanoparticles were suspended in PLGA-dichlorolin.
solution. Microparticles embedded with nanomethane
were made by phase separation method using
particles
oil as the first non-solvent and heptane as the
silicone
non-solvent. The average diameter of the hydro-
second
nanoparticle-hydrophobic microsphere composi-
philic
was 150-180 µm. The protein release from the mi-
tes
was prolonged to nearly two months, withcrospheres
degradation nor aggregation of the protein as
out
by size exclusion chromatograms [44].
shown
separation by salt addition was adopted in
Phase
of ionotropic hydrogel in an attempt to
preparation
the size distribution of microspheres [45]. Prior
control
cross-linking with calcium ions, salts of monovalent
to
(e.g., sodium chloride) were added to aqueous solu-
ions
of poly [di(carboxylatophenoxy) phosphazene]
tion
to form coacervate microdroplets with a size in
(PCPP)
range 1-10 µm. Upon addition of calcium chloride,
the
microdroplets were stabilized via cross-linking be-
the
PCPP and calcium salts without changing the
tween
and shape. The microsphere size increased linearly
size
the sodium chloride concentration, incubation
with
and polymer concentration. This method avoided
time,
use of organic solvents, heat, and complicated
the
equipment. Gelatin/Chondroitin 6-sulfate
manufacturing
microspheres were prepared to encapsulate pro-
(CS6)
using complex coacervation for the joint therapy
teins
Gelatin (a polycation) and CS6 (a polyanion) solu-
[46].
containing model proteins were mixed and vortions
at 37°C, pH 5.5 to form coacervates. The resulting
texed
microspheres were crosslinked with glu-
coacervate
It was claimed that proteins could be entaraldehyde.
with high encapsulation efficiency, retaining
capsulated
bioactivity. The release of protein depended on the
high
of human matrix metalloprotease (MMP), since
level
coacervate was degraded by its gelatinase
gelatin/CS6
The MMP concentration is one of the factors
activity.
for the joint pain, and thus the MMP-
responsible
release property is of distinctive advantage
responsive
the delivery of MMP inhibitors or the receptors for
for
stimulating the synthesis of MMP to diseased
cytokines
Gelatin/CS6 showed no significant cytotoxic
joints.
effect in vitro.
Advantages
non-solvent addition method is preferred in en-
The
of water-soluble drugs, such as proteins,
capsulation
and vaccines, since drug-polymer mixtures are
peptides,
exposed to the continuous aqueous phase. This
not
the loss of water-soluble drugs to the water
minimizes
and results in the high encapsulation efficiency.
phase
advantage of the phase separation method is
Another
it enables efficient control of the particle size with
that
narrower size distribution by simply varying the
a
variables, such as concentration of added
component
[45] or the viscosity and amount of the non-
salts
and/or molecular weight of polymer used [47].
solvent

220 Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4
Limitations
many successful phase separation systems
Although
been demonstrated, each method has several limi-
have
The microspheres tend to aggregate and the
tations.
for mass production is difficult [48]. Agglom-
scale-up
occurs when the coacervate droplets are sticky
eration
adhere to each other before the solvent is com-
and
removed or before the droplets are hardened [31].
pletely
produce well-defined microspheres, wide ‘stability
To
is required. The stability window is defined as
window’
step where addition of non-solvent does not induce
a
but forms stable coacervates [34]. The
agglomeration
window is affected by the physicochemical
stability
of polymers used and/or the viscosity of the
nature
added. The presence of residual solvents is
non-solvent
concern in the non-solvent addition method. The
a
includes at least three different solvents, i.e.,
method
polymer solvent and two polymer non-solvents. In
the
the polymer solvents are toxic compounds,
general,
as halogenated hydrocarbons (e.g., dichloro-
such
for which the exposure limits are regulated
methane),
the authority. For example, the concentration limit
by
dichloromethane is 600 ppm [49]. In addition to the
for
solvent, two non-solvents are generally known
polymer
remain in a substantial quantity. A study showed
to
depending on the type of solvent and polymer used,
that
residual solvents could be minimized by appropriate
the
methods [38]. The solvents, however, were in
drying
hard to remove. High levels of residues may al-
general
the microsphere morphology due to their plasticizter
effect. Moreover, it may cause a toxicity problem
ing
influence crucial properties such as release profiles
and
drug stability. Undoubtedly, the search for toxico-
and
and technologically acceptable solvents and
logically
should continue for the non-solvent addi-
non-solvents
method to be more useful.
tion
a polymer-polymer interaction (complex coacerva-
In
the limited pH range and the use of cross-linking
tion),
can be potential problems. Since the technique is
agent
on electrostatic interactions between two polye-
based
pH of the medium is an important factor.
lectrolytes,
example, in a gelatin-acacia system, the pH should
For
below the isoelectric point of gelatin (pH 8.9) to
be
it positively charged. The pH range suitable for
make
gelatin-acacia system is between 2.6 and 5.5 [30].
the
condition, however, is not appropriate for encapsu-
This
of acid-labile drug entities. The pH range could
lation
extended by the addition of water-soluble nonionic
be
such as polyethylene glycol [50,51]. Glutaral-
polymers,
and formaldehyde are commonly used as crossdehyde
agents to harden the microcapsules in this techlinking
A condensation reaction occurs between the
nique.
groups of the protein (e.g., gelatin) and the alde-
amino
The cross-linking agents may diffuse into the
hydes.
and also react with drug entities, therefore this
core
may not be appropriate for the protein drugs.
method
toxicity problems arise as well [30].
Potential
SPRAY DRYING
drying has been widely used in the pharmaceu-
Spray
food, and biochemical industries. The main applitical,
in the pharmaceutical field include drying proccations
granulation, preparation of solid dispersions, alteraess,
of polymorphism of a drug, preparation of dry
tion
for aerosols, and encapsulation of drugs for
powders
masking and protection from oxidation [52]. Since
taste
late 1970s, the spray drying technique has been
the
for making microparticulate systems for controlled
used
delivery.
drug
Methods
hydrophilic or hydrophobic polymer is dis-
Either
in a suitable solvent. Drug can be dissolved or
solved
in the solvent. Alternatively, drug solution
suspended
be emulsified in the polymer solution. The mixture
can
then sprayed through a nozzle of a spray dryer,
is
in solid microspheres that precipitated into
resulting
bottom collector [53]. Sometimes, the process
the
the use of plasticizers to form microcapsules of
include
spherical shape and smooth-surface by reducing
regular
rigidity of polymer chain [54].
the
Alternative Technique
(congealing): Spray congealing includes
Spray-chilling
dissolution or dispersion of the drug in a melted
the
without using solvent. This mixture fluid is then
carrier
into a cold air stream to make small droplets
sprayed
solidified by cooling at the temperature lower than
and
melting point of the carrier. This method was used
the
encapsulate bovine somatotropin (bST) in fat or wax
to
bST was dispersed in molten fat or wax and
[55].
through an air/liquid spray nozzle. The micro-
sprayed
were formed as the molten droplets and were
spheres
on sieves in the desired size range (45-180 µm).
collected
was demonstrated that the microspheres were effec-
It
in maintaining increased level of bST in the blood
tive
treated animals for 4 weeks, increasing weight gains
of
and milk production.
Applications
spray-drying is relatively simple, fast and easy
Since
scale-up, it has been tried as an attractive alternative
to
the conventional methods, such as coacervation and
to
method to encapsulate protein drugs [48,56-
emulsion
The spray drying technique was applied to produce
65].
microparticles containing thyrotropin releasing
PLGA
(TRH) [48]. TRH in water and PLGA in ace-
hormone
were mixed to form a clear solution and then
tonitrile
Since the microparticles produced by conven-
sprayed.
spray dryer tended to agglomerate and adhere to
tional
surface of the chamber, they designed a double noz-
the
system such that additional nozzle might spray a
zle
solution as an anti-adherent simultaneously
mannitol
8). Microparticles with mean particle size of 20 µm
(Fig.

Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4 221
Raw
materials
8. Schematic diagram of a spray dryer used for prepara-
Fig.
of PLGA microparticles containing TRH. From reference
tion
[48].
obtained, from which zero order release of TRH
were
for one month with a minimal initial burst.
continued
4 lists some examples of microspheres prepared by
Table
drying.
spray
Advantages
of the major advantages of spray drying is its
One
applicability. Both hydrophilic and hydrophobic
general
can be used with proper selection of the sol-
polymer
[52]. Spray drying is useful for encapsulating even
vent
drugs, such as proteins or peptides, be-
heat-sensitive
it involves mild temperatures [52]. Although
cause
drying includes hot air stream, the temperature of
spray
droplets may be maintained below the drying air
the
due to rapid evaporation of the solvent.
temperature
effective temperature applied to the drug itself is
The
enough to be used for proteins. Moreover, spray
mild
is time effective. Spray drying can yield results
drying
to those of conventional methods in terms
equivalent
size distribution, particle morphology, and release
of
yet with the advantage of high encapsulation
kinetics,
and the short duration of the preparation
efficiency
[53]. The spray drying equipment is easily
procedure
at the manufacturing site. Also, as a one-stage
available
process, spray drying is ideal for production of
closed
materials and good manufacturing practice
sterile
(GMP) [30].
Limitations
TRH-PLGA
solution
Air filter
Pump
air
Double
nozzle
Heater
Drying
chamber
Mannitol
solution
Spray
nozzle
Blower
Cyclone
Receiver
During spray drying, considerable amounts of the
4. Examples of microencapsulation of proteins by spray
Table
drying
Polymer Solvent Protein Particle
size
β-glucuronidase
Bovine
somatotropin
Human
erythropoietin
Albumin/
with
Acacia
as PVP
coacervate
stabilizer
anhy- Poly
dride
PLGA
Distilled
water
5 µm
Dichloro-
1-5 µm
methane
Dichloro-
10 µm
methane
Release kinetics Ref.
initial burst
Biphasic:
within 6h followed
(31%)
zero order release for 2
by
weeks
release for 6 h due to
90%
fast degradation of
the
and small parti-
polymer
size cle
burst during 24 h
Initial
no further release for
and
months, likely due to
2
interac-
protein-polymer
and insoluble protein
tion
aggregates
can be lost during the process due to sticking
material
the microparticles to the wall of the drying chamber.
of
drying process can often lead to change of poly-
Spray
of the spray dried drugs [66,67]. For exammorphism
progesterone crystal in its original alpha form (m.p.
ples,
turned into beta form (m.p. 395K) when it was
402K)
dried in combination with PLA. Fiber formation
spray
be another major problem in spray drying PLA [67].
can
are formed due to insufficient forces present to
Fibers
up the liquid filament into droplets. It depends on
break
the type of polymer and, to a lesser extent, the
both
of the spray solution. For example, ethyl cellu-
viscosity
solutions made spherical droplets at relatively high
lose
however PLA solutions of considerably
concentrations,
concentration and viscosity resulted in fibers.
low
are a number of process variables that should be
There
for drug encapsulation. They include feed
optimized
properties, such as viscosity, uniformity, and
material
of drug and polymer mixtures, feed rate,
concentration
of atomization, and the inlet and outlet tem-
method
[32]. The dependence on so many variables
peratures
become a problem in terms of reproducibility and a
may
process. In addition, the amount of polymer
scale-up
drugs for encapsulation may be limited, since
and/or
fluid of high viscosity cannot be sprayed.
GELATION /
IONOTROPIC
COMPLEXATION
POLYELECTROLYTE
gelation (IG) is based on the ability of
Ionotropic
to crosslink in the presence of counter
polyelectrolytes
to form hydrogels. Since the use of alginate for cell
ions
[68], ionotropic gelation has been widely
encapsulation
used for both cell and drug encapsulation.
Method
is one of the most widely used polyanion for
Alginate
Alginate is composed of 1,4-linked
microencapsulation.
beta-D-mannuronic acid and alpha-D-guluronic acid
[60]
[56]
[62]

222 Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4
(A)
(B)
HO HO
-OOC
OHO
9. Monomer units (A) found in alginate and the alginate
Fig.
structure (B). From reference [69].
polymer
Divalent and trivalent cations induce gelation
residues.
binding mainly to the guluronic blocks (Fig. 9). The
by
and macro-spheres are produced by dropping a
microor
drug-loaded alginate solution into aqueous cal-
cell-
chloride solution. Calcium ions diffuse into the
cium
drops, forming a three dimensional lattice of
alginate
crosslinked alginate. Polyelectrolyte complexa-
ionically
(PEC) with oppositely charged polyelectrolytes
tion
be added to increase the mechanical strength of
may
hydrogel and/or to provide a permeability barrier.
the
instance, addition of polycation (usually poly-L-
For
allows a membrane of polyelectrolyte complex
lysine)
form on the surface of alginate beads [69]. A number
to
natural and synthetic polyelectrolytes have been in-
of
Examples are listed in Table 5.
vestigated.
most of the droplets have been made with sy-
Since
needles, the particle sizes have been relatively
ringe
Smaller droplets can be formed using a vibration
large.
or air atomization method to extrude the algi-
system
solution (Fig. 10) [30]. The latter involves a Turbonate
air-atomizer. Pressurized air is fed to mix with the
tak
alginate solution, forcing tiny liquid droplets
sodium
through the orifice of a nozzle. The calcium ions
out
the droplets of sodium alginate on contact to
crosslink
microgel droplets, which were further crosslinked
form
poly-L-lysine to form a membrane on the droplets.
by
obtained using this method were within
Microparticles
size range 5-15 µm [91].
the
Applications
OH
Mannuronate (M)
OH
O -OOC
O
OH
-OOC OHO
O HO
O
HO
-OOC
OHO
-OOC
has been widely used in microcapsule sys-
IG/PEC
for cell encapsulation [82,87-89,92-94] and drug
tems
systems [76,77,83,85,86,95,96]. Due to mild
delivery
conditions, IG/PEC has recently attracted a new
process
for an application to protein delivery [71,78-
attention
Calcium alginate hydrogels have been
81,84,90,97,98].
for delivery of vascular endothelial growth factor
used
[71]. The alginate beads demonstrated the abil-
(VEGF)
to incorporate VEGF with efficiency between 30%
ity
67% and constant release (5%/day) of VEGF for up
and
O
OH
O
OH
OH
OH
(G)
Guluronate
-OOC
OH
OH
O
OH
O -OOC
G M M G G
O
OH
O
5. Examples of ionotropic gelation (IG) and polyelec-
Table
complexation (PEC)
trolyte
Polyelectrolytes IG with PEC with References
Alginate (-) Calcium (+) - [70-72]
Alginate (-) Calcium (+) Poly-L-Lysine (+) [73]
Alginate (-) Calcium (+) Chitosan (+) [74, 75]
Chitosan (+)
Carboxymethyl
cellulose (-)
Tripolyphosphate
(TPP)(-)
- [76-81]
Aluminium (+) Chitosan (+) [82]
κ-carrageenan (-) Potassium (+) Chitosan (+) [83]
ι-carrageenan (-) Amines (+) - [84]
Pectin (-) Calcium (+) - [85]
Gelan gum (-) Calcium (+) - [86]
Alginate and
cellulose sulfate (-)
Calcium (+)
Poly (methylene-
co-guanidine) (+)
[87, 88]
Polyphosphazene (-) Calcium (+) Poly-L-Lysine (+) [89, 90]
Sodium
alginate
10. Schematic diagram of the preparation of alginate-
Fig.
microcapsules.
polylysine
14 days. The ionic interaction between VEGF and
to
contributed to maintaining the biological activ-
alginate
ity of VEGF.
Advantages
main advantage of IG/PEC is the mild formula-
The
conditions for maintaining cell viability and protion
integrity. All of the polyelectrolytes are watertein
and thus proteins can be encapsulated without
soluble,
of organic solvents or elevated temperatures, both
use
which may damage protein integrity. Also, this sys-
of
tem is simple, fast and cost-effective.
Limitations
Turbotak
atomizer
chloride
Calcium
solution
Polylysine
added
Pressurized
gas
Microcapsule
biggest problem in applications of IG/PEC for
The
protein delivery, in spite of the great advan-
controlled
of mild conditions, is that the matrix and memtage
formed by IG/PEC is not capable of controlling
brane
release rate for a long period of time. It does not
the
in cell encapsulation, where the membrane
matter
should provide sufficient permeability for the cell prod-

Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4 223
i.e., a therapeutic protein. However, for IG/PEC to
uct,
used for controlled delivery of protein entities, a cer-
be
type of dense membrane (e.g., biodegradable polytain
preferably capable of controlling the release rate,
mers),
be incorporated or alternative combinations of
should
should be explored to control the
polyelectrolytes
of the membrane, so that the release rate
permeability
be controlled over the desired span. Thus far, the
may
rate of the protein is likely to depend on the
release
properties of the model protein or the
physicochemical
medium in which the microparticles are placed
release
In case of alginate-poly-L-lysine system, all para-
[71].
are tied to a single chemical complex, therefore
meters
is not easy to optimize the capsule condition. For this
it
new combinations of polyelectrolytes were
reason,
to allow independent modification of capsule
proposed
including size, wall thickness, mechanical
parameters
permeability, and surface characteristics [99].
strength,
issue in IG/PEC is that some polyelectrolytes
Another
have biocompatibility problem. There have been
might
arguments concerning the biocompatibil-
contradictory
of alginates [100-102]. A host foreign-body reaction
ity
occurred on account of the implanted algi-
(fibrosis)
particles and mannuronic acid block was
nate-based
to be responsible for the fibrotic response, as a
found
stimulator of IL-1 and TNF-α production. Use of
potent
of high guluronic acid content was suggested
alginates
minimize the cytokine response. On the other hand,
to
study indicated that alginate particles with
another
guluronic acid contents provoked stronger re-
high
than the high mannuronic acid alginate particles
sponse
In general, polyionic hydrogels obtained by
[102].
have rather low mechanical strength. A few
IG/PEC
have suggested ways to overcome this problem
papers
exploring alternative combinations of polyanions
by
polycations [87,99], introducing a different process
and
[103], or applying additional coating [82,98].
INTERFACIAL POLYMERIZATION
polymerization features in situ polymeri-
Interfacial
of reactive monomers on the surface of a droplet
zation
or particles.
Method
reactive monomers (typically dichloride and
Two
are respectively dissolved in immiscible sol-
diamine)
and mixed to form o/w emulsion (dichloride in oil
vents
and diamine in water phase). The monomers dif-
phase
to the o/w interface where they react to form a
fuse
membrane (Fig. 11). A typical example of this
polymeric
is making nylon microcapsules [104]. Nona-
method
phase (chloroform/cyclohexane) containing surqueous
(e.g., sorbitan trioleate) and aqueous buffer confactant
drugs to be incorporated (enzymes or proteins),
taining
protein (e.g., BSA or hemoglobin) and dia-
protective
are prepared respectively. Two phases are mixed
mine
and stirred to make an w/o emulsion in an ice bath un-
11. Microencapsulation by interfacial polymerization
Fig.
From reference [104].
method.
the desired droplet size is reached. Another nonaque-
til
phase containing acid chloride is added to the emulous
for interfacial polymerization. Polymerization is
sion
by addition of excess nonaqueous phase.
quenched
are allowed to sediment and collected
Microcapsules
then washed in saline several times to remove or-
and
solvents and byproducts.
ganic
combinations of monomers can be used to
Various
a range of polymer membranes [104]. Sebacoyl
obtain
and 1,6-hexane diamine can be used to form
chloride
polyamide nylon 6, 10. The microcapsules using
the
system, however, tend to be fragile and difficult to
this
Terephthaloyl chloride and 1,6-hexane diamine
handle.
yield polyester membrane. Alternatively tere-
can
chloride can be used in combination with Lphthaloyl
to yield poly(terephthaloyl L-lysine). Typically
lysine
chlorides and diamines are reactive monomers;
acid
isocyanates can be used instead of or as a par-
however,
substitute for acid chloride [105]. Alternatively, the
tial
composed of only one type of monomer can be
polymer
on the interface (Interfaicial addition polymeri-
formed
[104]. Polyalkylcyanoacrylate belongs to this
zation)
Aqueous drug solution and oil phase containing
type.
monomer are mixed to form an w/o
cyanoacrylate
The polymerization is initiated by water in
emulsion.
aqeous phase with cyanoacrylate dissolved in the oil
the
phase.
Application
Monomer B
Monomer A
Polymer membrane
insulin nanoparticles were prepared using
Recently
addition polymerization techniques [106].
interfacial
were prepared by addition of ethyl 2-
Nanoparticles
to a stirred w/o microemulsion consistcyanoacrylate
of aqueous insulin solution, oil and surfactant. To
ing
exhaustive washing steps after polymerization
avoid
biocompatible oils (caprylic/capric triglyc-
reaction,
and mono-/diglycerides) and surfactants (polyerides
80 and sorbitan monooleate) were used to forsorbate
the microemulsions. The obtained poly(ethyl 2mulate
is known to be biodegradable. Since incyanoacrylate)
was confined to the aqueous phase in w/o emulsulin
sions, a high encapsulation efficiency (86%) could be

224 Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4
Insulin was released with initial burst over
achieved.
first 30 minutes followed by a constant release up
the
to 3 h, after which the release rate declined.
Limitations
this method was initially proposed for en-
Although
encapsulations, it has some serious problems in
zyme
First, large w/o interface is produced during
practice.
reaction, where proteins or enzymes are likely to be
the
Second, large amounts of proteins may par-
inactivated.
in the polymerization reaction changing its bioticipate
activity. Third, it is often hard to control the
logical
reaction. The yield and quality of the
polymerization
obtained by interfacial polymerization may
membrane
controlled by a number of factors, such as chemical
be
of reactive monomers, and reaction conditions.
natures
monomer concentrations, temperature, the mixing
The
and the reaction time are likely to be important
rate,
[105]. Fourth, exhaustive washing steps are
parameters
to remove monomers, byproducts, organic sol-
required
and surfactants. At the same time, several washvents,
steps may lead to further loss of water soluble drugs.
ing
pH change due to HCl byproduct formed by reac-
Fifth,
of an acid chloride and an amine can damage the
tion
labile drugs.
acid
SUPERCRITICAL FLUID PRECIPITATION
fluid is defined as a fluid of which tem-
Supercritical
and pressure are simultaneously higher than at
perature
critical point, i.e. critical temperature Tc and critical
the
Pc, at which the density of gas is equal to that
pressure
the remaining liquid and the surface between the
of
phases disappears (Fig. 12). In practice, the term is
two
used to describe a fluid in the relative vicinity of
often
critical point, where a gas can be highly compressed
the
a small change of pressure into a fluid which is not
with
liquid but almost close to liquid in density. These two
a
features of supercritical fluids (i.e., a high
distinctive
and a liquid-like density) enabled super-
compressibility
fluids to attract considerable interest recently as
critical
for microparticle production.
vehicles
Methods
are two main routes to particle formation with
There
fluids: the rapid expansion of supercritical
supercritical
(RESS); and supercritical antisolvent crystal-
solutions
(SAS) routes [108]. RESS exploits the liquid-like
lization
power of the supercritical fluids while SAS uses
solvent
fluid as an antisolvent. Carbon dioxide is
supercritical
commonly used since the critical conditions are
most
attainable, i.e., Tc=31°C and Pc=73.8 bar. CO2 is
easily
benign, relatively non-toxic, non-
environmentally
inexpensive, and has a reasonably high
inflammable,
dissolving power. [109]
RESS(Rapid expansion of supercritical solutions): In
12. Pressure-temperature projection of the phase diagram
Fig.
a pure component. From reference [107].
for
the drug and the polymer are dissolved in a su-
RESS,
fluid at high pressure and precipitated by
percritical
the solvent’s density through a rapid decom-
reducing
(expansion) [110]. The actual solubility of the
pression
in supercritical fluids can be higher than the
solutes
calculated assuming ideal gas behavior at the
value
temperature and pressure by factors of up to 10 same 6
Rapid expansion of supercritical solutions, there-
[107].
can lead to very high supersaturation ratios. Since
fore,
supercritical fluid is a gas after expansion, the solid
the
product is recovered in pure, solvent-free form.
antisolvent crystallization): The
SAS(supercritical
is also called as ASES (aerosol solvent extrac-
method
system) or PCA (precipitation with a compressed
tion
antisolvent). In SAS, a supercritical fluid is used as
fluid
anti-solvent that causes precipitation of solids. The
an
are dissolved in a suitable organic phase. At high
solutes
anti-solvent enters into the solution and low-
pressures,
the solvent power, thereby precipitating the solutes.
ers
precipitation is followed by a large volume expan-
The
and the solid product is collected. SAS process is
sion
in Fig. 13. As a modification of the SAS, the
illustrated
phase can be sprayed into a supercritical fluid,
organic
causes the solutes to precipitate as fine particles.
which
method is also called gas-antisolvent (GAS)
This
SAS is useful for processing of solids which are
method.
to solubilize in supercritical fluids, such as pep-
difficult
tides and proteins.
Applications
Solid Liquid
Gas
TEMPERATURE
supercritical
region
fluid
Critical point
offers distinctive advantages over the conven-
RESS
methods, such as no requirements of surfactants,
tional
a solvent-free product, and moderate process
yielding
[107]. However, the very low solvent power
conditions

Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4 225
Carbon
dioxide
Pump
Precipitation
vessel
13. Schematic diagram of the SAS method. From refer-
Fig.
[111]. ence
common supercritical solvents toward the high mo-
of
weight polymers and the solutes of therapeutic
lecular
such as proteins has restricted RESS to a
importance
low molecular weight polymers and a limited range
few
drugs, e.g., lovastatin [107,112]. Lovastatin was dis-
of
in supercritical CO2 together with DL-PLA and
solved
subjected to RESS. The morphology of the copre-
then
product was found to be very sensitive to the
cipitation
amounts of drug and polymer, ranging from
relative
networks at high drug loading to drug
bicontinuous
encapsulated within polymer at low drug load-
needles
ings.
has been applied to protein drugs for size reduc-
SAS
[113] and bioerodible polymers for microencapsulation
of pharmaceuticals in a polymer matrix [114]. Retion
this technique was used to produce PLGA microcently,
containing lysozyme [115]. PLGA in dichlorospheres
solution with suspended lysozyme was
methane
into a CO2 vapor phase through a capillary
sprayed
to form droplets which solidified after falling
nozzle
a CO2 liquid phase. In this study, several problems
into
encountered in SAS process were overcome.
previously
size (70 µm) large enough to encapsulate pro-
Particle
particles was achieved by delayed precipitation ustein
CO2 vapor phase above a CO2 liquid phase. Aggloing
due to plasticization of the polymer by CO2 meration
minimized at the optimum temperature of –20 was oC. many proteins are insoluble in organic solvents
Since
are less likely to be denatured when suspended, this
and
can be extended to other proteins.
method
Advantages and Limitations
Pump
Expansion
vessel
Polymer
solution
Vent
Solvent
fluids, particularly CO2 offers various
Supercritical
when compared with widely used organic
advantages
It is relatively non-toxic, environmentally ac-
solvents.
non flammable, and inexpensive. In general,
ceptable,
with narrow size distribution can be achieved
particles
supercritical fluid methods. Potential advantages
using
include mild process temperatures, the potential for
also
and the possibility for aseptic preparation of
scale-up
6. Examples of micorencapsulation of maromolecular
Table
by IG/PEC
drugs
Protein
Salmon
calcitonin
IG/PEC
system
Encapsulation
efficiency
Particle
size
Chitosan-TPP 54-59% 0.9 mm
Insulin Chitosan-TPP > 87%
Bovine
albu-
serum
min
Dextran
Horseradish
preoxidase
Chitosan/
PEO/PPO-
TPP
Alginate-
Calcium-
Chitosan
carrageenan
ι
amines -
with Varies
formulations
49-89%
(depend-
1-72%
on amine
ing
employed)
300-400
nm
200-1000
nm
-1.07 0.91
mm
50 µm
Release kinetics Ref.
burst (

226 Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4
effect and incomplete release behavior. As an ideal
burst
technique, one should satisfy the follow-
encapsulation
conditions. First, the manufacturing conditions
ing
be mild enough to conserve the protein stability
should
the process. Oil-water interface, shear forces, or
during
may be major causes of protein denaturation and
heat
Second, high encapsulation efficiency is
aggregation.
Even if the progress of genetic engineering
important.
many therapeutic proteins available at a lower
made
than before, protein drugs are in general much
price
expensive than other low molecular drugs. Third,
more
is preferable to minimize the exposure to toxic or-
it
solvents. Fourth, the manufacturing process
ganic
be reproducible and easy to scale up to the
should
scale. Also, aseptic manufacturing should
commercial
available. Fifth, in order to ensure product quality as
be
injectable formulation, uniform size distribution of
an
microparticles should be achievable.
the
numerous novel protein drugs coming
Considering
the genome projects, new microencapsulation sys-
from
that minimizes use of toxic organic solvents and
tems
denaturating conditions will be invaluable tools
protein
the future, and this is an exciting time to be involved
in
the microencapsulation technology.
in
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[Received May 22, 2001; accepted July 24, 2001]