Microencapsulation Methods for Delivery of Protein Drugs
Microencapsulation Methods for Delivery of Protein Drugs
Microencapsulation Methods for Delivery of Protein Drugs
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Biotechnol. Bioprocess Eng. 2001, 6: 213-230<br />
<strong>Microencapsulation</strong> <strong>Methods</strong> <strong>for</strong> <strong>Delivery</strong> <strong>of</strong> <strong>Protein</strong> <strong>Drugs</strong><br />
Yoon Yeo, Namjin Baek, and Kinam Park*<br />
Departments <strong>of</strong> Pharmaceutics and Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA<br />
INTRODUCTION<br />
Recent advances in recombinant DNA technology have resulted in development <strong>of</strong><br />
Abstract<br />
new protein drugs. Due to the unique properties <strong>of</strong> protein drugs, they have to be delivered<br />
many<br />
parenteral injection. Although delivery <strong>of</strong> protein drugs by other routes, such as pulmonary<br />
by<br />
nasal routes, has shown some promises, to date most protein drugs are administered by par-<br />
and<br />
routs. For long-term delivery <strong>of</strong> protein drugs by parenteral administration, they have been<br />
enteral<br />
into biodegradable microspheres. A number <strong>of</strong> microencapsulation methods have been<br />
<strong>for</strong>mulated<br />
and the currently used microencapsulation methods are reviewed here. The microen-<br />
developed,<br />
methods have been divided based on the method used. They are: solvent evaporacapsulation<br />
phase separation (coacervation); spray drying; ionotropic gelation/polyelectrolyte<br />
tion/extraction;<br />
interfacial polymerization; and supercritical fluid precipitation. Each method is de-<br />
complexation;<br />
scribed <strong>for</strong> its applications, advantages, and limitations.<br />
Keywords: protein, peptide, drug delivery, microparticle, microencapsulation<br />
advances in biotechnology have brought<br />
Significant<br />
availability <strong>of</strong> recombinant peptide pro-<br />
ever-increasing<br />
drugs in large quantities. The successful completein<br />
<strong>of</strong> the human genome project will undoubtedly<br />
tion<br />
to the explosion <strong>of</strong> new protein drugs with exqui-<br />
lead<br />
bioactivities. Since protein drugs carry out biologisite<br />
processes and reactions with high specificity and<br />
cal<br />
protein drugs will continue to be drugs <strong>of</strong><br />
potency,<br />
<strong>for</strong> treating various diseases. Formulating pro-<br />
choices<br />
drug delivery systems, however, poses several probtein<br />
Due to extremely low bioavailability <strong>of</strong> protein<br />
lems.<br />
by oral administration, which is the most conven-<br />
drugs<br />
mode <strong>of</strong> drug delivery, protein drugs are usually<br />
ient<br />
by parenteral route. One way <strong>of</strong> minimizing<br />
administered<br />
and improving patient compliance is to pro-<br />
discom<strong>for</strong>t<br />
sustained-release <strong>for</strong>mulations that deliver protein<br />
duce<br />
continuously over long periods <strong>of</strong> time. Another<br />
drugs<br />
in protein drug delivery is to maintain the terti-<br />
challenge<br />
protein structure, which is essential to bioactivity. Exary<br />
<strong>of</strong> protein drugs to unfavorable conditions during<br />
posure<br />
tends to reduce their bioactivities.<br />
<strong>for</strong>mulation<br />
widely used approach <strong>for</strong> long-term delivery <strong>of</strong><br />
Most<br />
drugs has been parenteral administration <strong>of</strong><br />
protein<br />
drugs in microspheres made <strong>of</strong> biodegradable<br />
protein<br />
Preparation <strong>of</strong> protein drug-containing mi-<br />
polymers.<br />
without reducing bioactivity has been the<br />
crospheres<br />
<strong>of</strong> microencapsulation methods. A number <strong>of</strong> mi-<br />
goal<br />
methods have been developed through<br />
croencapsulation<br />
the years, but none <strong>of</strong> the methods has been ideal <strong>for</strong><br />
Corresponding author<br />
*<br />
+1-765-494-7759 Fax: +1-765-496-1903<br />
Tel:<br />
e-mail: kpark@purdue.edu<br />
protein drugs. Each method has its own advan-<br />
loading<br />
as well as limitations. For further improvement in<br />
tages<br />
technologies, it is important to un-<br />
microencapsulation<br />
the strengths and drawbacks <strong>of</strong> each method.<br />
derstand<br />
1 lists examples <strong>of</strong> microencapsulation processes<br />
Table<br />
in the literature. <strong>Microencapsulation</strong> processes<br />
found<br />
been frequently classified either chemical or me-<br />
have<br />
[1]. Chemical processes refer to methods that<br />
chanical<br />
the change <strong>of</strong> solvent property, chemical reaction<br />
utilize<br />
monomers, or complexation <strong>of</strong> polyelectro-<br />
between<br />
In mechanical processes, a gas phase is utilized at<br />
lytes.<br />
stage to provide <strong>for</strong>ce to break up materials to<br />
some<br />
particles. There are many situations, however,<br />
small<br />
such a distinction is not clear.<br />
where<br />
MICROENCAPSULATION METHODS<br />
SOLVENT EVAPORATION/EXTRACTION<br />
evaporation/extraction methods have been<br />
Solvent<br />
used to prepare microspheres loaded with vari-<br />
widely<br />
drugs, especially hydrophobic drugs. For the encapous<br />
<strong>of</strong> peptide and protein drugs, oil/water (o/w),<br />
sulation<br />
(o/o) and water/oil/water (w/o/w) emulsifica-<br />
oil/oil<br />
methods have been used. Depending on the numtion<br />
<strong>of</strong> emulsions produced during the preparation <strong>of</strong><br />
ber<br />
solvent evaporation/extraction can be<br />
microspheres,<br />
into two methods, single emulsion and double<br />
divided<br />
(Fig. 1).<br />
emulsion<br />
<strong>Methods</strong><br />
Emulsion (o/o or o/w) <strong>Methods</strong><br />
Single<br />
single emulsion methods, peptides/proteins are pre-<br />
In
214 Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4<br />
Table 1. Examples <strong>of</strong> microencapsulation methods<br />
Chemical processes<br />
1.<br />
evaporation and extraction<br />
Solvent<br />
solvent extraction<br />
Crygenic<br />
separation (Coacervation)<br />
Phase<br />
addition<br />
Non-solvent<br />
change<br />
Temperature<br />
polymer or salt addition<br />
Incompatible<br />
interaction (complex coacervation)<br />
Polymer-polymer<br />
complexation<br />
Polyelectrolyte<br />
polymerization<br />
Interfacial<br />
Mechanical processes<br />
2.<br />
drying<br />
Spray<br />
chilling<br />
Spray<br />
desolvation<br />
Spray<br />
Supercritical fluid precipitation<br />
Single Emulsion Double Emulsion<br />
suspension/solution<br />
<strong>Protein</strong><br />
polymer solution (oil)<br />
in<br />
Emulsion (o/o or o/w)<br />
solution<br />
<strong>Protein</strong><br />
water in<br />
oil or water oil<br />
removal<br />
Solvent<br />
drying &<br />
Hardened microsphere<br />
Primary emulsion (w/o)<br />
water<br />
Secondary emulsion (w/o/w)<br />
removal<br />
Solvent<br />
drying &<br />
Hardened microsphere<br />
1. Single emulsion (left) and double emulsion (right) sol-<br />
Fig.<br />
evaporation methods <strong>for</strong> microencapsulation <strong>of</strong> proteins.<br />
vent<br />
in a dispersed phase, which is a polymer solution in<br />
sent<br />
solvent such as dichloromethane or ethyl ace-<br />
organic<br />
Polylactic acid (PLA) and poly(lactide-co-glycolide)<br />
tate.<br />
are the most widely used biodegradable syn-<br />
(PLGA)<br />
polymers <strong>for</strong> sustained-release preparations. The<br />
thetic<br />
kinetics <strong>of</strong> active components can be controlled<br />
release<br />
changing molecular weight and/or copolymer ratio<br />
by<br />
those polymers. <strong>Drugs</strong> can be dispersed as solid parti-<br />
<strong>of</strong><br />
or dissolved in polymer solution. The drug solution<br />
cles<br />
suspension is added into a continuous phase, which<br />
or<br />
be mineral oil (o/o) or aqueous solution (o/w) con-<br />
can<br />
emulsifiers. Emulsification is carried out by agitaining<br />
homogenization, or sonication. Emulsifiers in<br />
tation,<br />
continuous phase stabilize o/o or o/w emulsions<br />
the<br />
produced. Emulsifiers such as Span 85 [2], sorbi-<br />
being<br />
sesquioleate [3], aluminium tristearate [4] have<br />
tan<br />
used <strong>for</strong> o/o interface, and Carbopol been ® [2], 951<br />
cellulose [5], polyvinyl alcohol (PVA) [6] have<br />
methyl<br />
used <strong>for</strong> o/w interface.<br />
been<br />
solvent in dispersed phase is removed by sol-<br />
Organic<br />
evaporation or by solvent extraction. In solvent<br />
vent<br />
process, hardening <strong>of</strong> emulsion occurs<br />
evaporation<br />
volatile organic solvent in dispersed phase leaches<br />
when<br />
continuous phase and evaporates from continuous<br />
into<br />
at atmospheric pressure. Using vacuum or a mod-<br />
phase<br />
increase in temperature can accelerate the evapoerate<br />
<strong>of</strong> organic solvent. In solvent extraction process,<br />
ration<br />
emulsion is transferred to a large amount <strong>of</strong> water<br />
the<br />
other quenching medium, and the extraction <strong>of</strong> or-<br />
or<br />
solvent occurs faster than in solvent evaporation<br />
ganic<br />
Thus, microspheres produced by solvent ex-<br />
process.<br />
process are more porous than the ones protraction<br />
by solvent evaporation. The porous structure<br />
duced<br />
results in faster release <strong>of</strong> peptide/protein drugs.<br />
usually<br />
long-term sustained release, solvent evaporation is<br />
For<br />
The prepared microspheres are collected by<br />
preferred.<br />
centrifugation or filtration, and freeze-dried.<br />
Emulsion (w/o/w) <strong>Methods</strong><br />
Double<br />
double emulsion methods, an aqueous drug solu-<br />
In<br />
is first emulsified in a polymer-dissolved organic<br />
tion<br />
The w/o emulsion is then added into an aque-<br />
solvent.<br />
phase that contains emulsifier, thereby <strong>for</strong>ming w/<br />
ous<br />
emulsion. Then, the organic solvent is removed by<br />
o/w<br />
extracting into external aqueous phase and evaporation.<br />
Applications<br />
Emulsion<br />
Single<br />
emulsion solvent evaporation method has been<br />
Single<br />
to prepare microspheres/nanospheres containing<br />
used<br />
peptide and protein drugs. Insulin solution in<br />
various<br />
solvent was used in emulsification to prepare<br />
organic<br />
[7]. Other researchers used solid particles<br />
nanospheres<br />
bovine serum albumin (BSA) [2] or ovalbumin (OVA)<br />
<strong>of</strong><br />
instead <strong>of</strong> protein solution, in a hope that protein<br />
[4],<br />
can be preserved in organic solvent. High en-<br />
activity<br />
efficiency was obtained using protein particapsulation<br />
The prepared microparticles tend to show initial<br />
cles.<br />
release pr<strong>of</strong>iles. Table 2 lists some examples <strong>of</strong><br />
burst<br />
preparation using protein particles. A<br />
microparticle<br />
approach <strong>of</strong> protein particle preparation was used<br />
novel<br />
the encapsulation <strong>of</strong> bovine superoxide dismutase<br />
<strong>for</strong><br />
into PLGA/PLA microspheres [5]. <strong>Protein</strong><br />
(bSOD)<br />
particles were prepared by lyophilization <strong>of</strong><br />
spherical<br />
glycol (PEG) aqueous mixture.<br />
protein-polyethylene<br />
approach resulted in high encapsulation efficiency<br />
This<br />
and high activity <strong>of</strong> the loaded enzyme (95%). In<br />
(88%)
Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4 215<br />
2. Encapsulation <strong>of</strong> protein particles by single emul-<br />
Table<br />
evaporation methods<br />
sion/solvent<br />
<strong>Protein</strong><br />
particles<br />
Spray-dried<br />
BSA<br />
Micronized<br />
OVA<br />
Lyophilized<br />
bSOD/PEG<br />
)<br />
(% 75<br />
S<br />
O<br />
D<br />
o<br />
f b<br />
e<br />
50<br />
le<br />
a<br />
s<br />
e<br />
re<br />
u<br />
la<br />
tiv<br />
Q<br />
u<br />
m<br />
100<br />
25<br />
Polymer Type<br />
PLGA o/o<br />
o/w<br />
Encapsulation<br />
efficiency<br />
>90%<br />
20-50%<br />
Initial<br />
in release<br />
1 day<br />
216 Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4<br />
100<br />
)<br />
e<br />
(%<br />
m<br />
75 z<br />
y<br />
o<br />
s<br />
o<br />
f ly<br />
e<br />
50<br />
le<br />
a<br />
s<br />
e<br />
re<br />
u<br />
la<br />
tiv<br />
Q<br />
u<br />
m<br />
25<br />
0<br />
3. Release <strong>of</strong> lysozyme from PEG-PBT microspheres. Mo-<br />
Fig.<br />
weight <strong>of</strong> PEG segment was 600. Polyvinyl alcohol<br />
lecular<br />
was used as a stabilizer in the external aqueous phase.<br />
(4%)<br />
percentage <strong>of</strong> PBT in PEG-PBT block copolymer<br />
Increasing<br />
resulted in slower release <strong>of</strong> lysozyme. From reference [16].<br />
<strong>of</strong> glycolide in PLGA polymers result in rapid deg-<br />
tents<br />
<strong>of</strong> the polymer, resulting in faster release <strong>of</strong> the<br />
radation<br />
drugs. The release pr<strong>of</strong>ile can also be controlled<br />
loaded<br />
the processing factors, such as shear <strong>for</strong>ces, since<br />
by<br />
shear <strong>for</strong>ces tend to produce smaller microparticles<br />
high<br />
release drugs at a faster rate due to higher surface<br />
which<br />
area and shorter diffusion path length.<br />
Limitations<br />
0 10 20 30 40 50<br />
Time (days)<br />
23%<br />
45%<br />
solvent evaporation/extraction methods have<br />
While<br />
used widely in preparing microparticles <strong>for</strong> pep-<br />
been<br />
delivery, the methods are still not ideal and<br />
tide/protein<br />
many aspects to be improved. First, the drug<br />
have<br />
efficiency into microspheres is not high.<br />
encapsulation<br />
protein drugs can be produced in larger scale<br />
Although<br />
to development <strong>of</strong> genetic engineering technique,<br />
due<br />
cost is still high. Thus, increasing the encapsulation<br />
the<br />
is still quite important <strong>for</strong> preparing micro-<br />
efficiency<br />
Second, the solvent evaporation/extraction<br />
spheres.<br />
require use <strong>of</strong> toxic organic solvents, such as<br />
methods<br />
and ethyl acetate, as a solvent <strong>for</strong> dis-<br />
dichloromethane<br />
biodegradable polymers. To meet the regulatory<br />
solving<br />
regarding the residual solvent in the final<br />
requirement<br />
the use <strong>of</strong> toxic organic solvent should be<br />
products,<br />
or avoided. Third, in most situations, the<br />
minimized<br />
proteins are released with the initial burst release,<br />
loaded<br />
in many situations the loaded proteins are not re-<br />
and<br />
completely. The initial burst release may cause<br />
leased<br />
response in patients due to abnormally high<br />
abnormal<br />
concentration in blood. Incomplete release <strong>of</strong> the<br />
drug<br />
proteins is <strong>of</strong>ten related to protein stability. Pro-<br />
loaded<br />
have to maintain their 3-dimensional con<strong>for</strong>mateins<br />
to keep their bioactivity. <strong>Protein</strong>s, however, are<br />
tion<br />
prone to be denatured and to <strong>for</strong>m aggregates by vari-<br />
factors, including exposure to large interface beous<br />
organic and aqueous phases, shear <strong>for</strong>ce in the<br />
tween<br />
steps (e.g., homogenization, and sonication),<br />
processing<br />
protein-polymer interaction. Some reports showed<br />
or<br />
the loss <strong>of</strong> protein activity or denaturation/ aggre-<br />
that<br />
<strong>of</strong> protein could be minimized by using stabilizgation<br />
such as poloxamer, BSA or hydroxypropyl-βers<br />
[13,14,18]. Fourth, although continuous<br />
cyclodextrin<br />
<strong>of</strong> peptide/protein drugs is desirable, there are<br />
release<br />
where pulsatile release is preferred as in the<br />
situations<br />
<strong>of</strong> insulin delivery. Furthermore, the timing and the<br />
case<br />
amount <strong>of</strong> the released insulin need to be con-<br />
exact<br />
carefully. This type <strong>of</strong> release can not be obtrolled<br />
by biodegradable microspheres prepared by soltained <br />
vent evaporation/extraction methods.<br />
Modifications <strong>of</strong> Solvent Extraction<br />
an ef<strong>for</strong>t to protect proteins from denaturation<br />
In<br />
microencapsulation processes, alternative methods<br />
during<br />
been developed to minimize exposure <strong>of</strong> protein<br />
have<br />
to denaturing conditions, such as high tempera-<br />
drugs<br />
ture and water-oil interfaces [19].<br />
Spray-desolvation<br />
involves spraying a polymer solu-<br />
Spray-desolvation<br />
onto desolvating liquid. For example, microdroplets<br />
tion<br />
made by spraying PVA aqueous solution onto ace-<br />
were<br />
bath, where the polymer solvent (water) was extone<br />
into acetone and PVA precipitated to <strong>for</strong>m solid<br />
tracted<br />
[20]. This method was also applied to<br />
microparticles<br />
peptides and BSA as model drugs in PLGA.<br />
encapsulate<br />
micronized drug was suspended in a PLGA-acetone<br />
The<br />
and atomized ultrasonically into ethanol bath,<br />
solution<br />
where microspheres were precipitated [21].<br />
Solvent Extraction<br />
Cryogenic<br />
is dissolved in an organic solvent such as di-<br />
PLGA<br />
together with protein powders. As shown<br />
chloromethane,<br />
Fig. 4, the polymer/protein mixture is atomized over<br />
in<br />
bed <strong>of</strong> frozen ethanol overlaid with liquid nitrogen, at<br />
a<br />
temperature below the freezing point <strong>of</strong> the poly-<br />
a<br />
solution. The microdroplets freeze upon<br />
mer/protein<br />
the liquid nitrogen, then sink onto the fro-<br />
contacting<br />
ethanol layer. As the ethanol layer thaws, the mizen<br />
which are still frozen sink into the ethanol.<br />
croparticles<br />
the solvent in the microparticles<br />
Dichloromethane,<br />
thaws and is slowly extracted into ethanol, result-<br />
then<br />
in hardened microparticles containing proteins and<br />
ing<br />
matrix.<br />
polymer<br />
process was used to produce a microsphere <strong>for</strong>-<br />
This<br />
<strong>for</strong> human growth hormone (hGH) [23-26].<br />
mulation<br />
to the encapsulation process, hGH is <strong>for</strong>mulated<br />
Prior<br />
zinc to produce insoluble Zn:hGH complex. The<br />
with<br />
<strong>of</strong> this complex contributed to stabilize<br />
encapsulation<br />
during the fabrication process and within the mi-<br />
hGH<br />
after hydration [23,24]. An in vivo study <strong>of</strong><br />
crospheres<br />
<strong>for</strong>mulation demonstrated a lower Cmax and an ex-<br />
this<br />
serum level <strong>for</strong> weeks, but a similar bioavailabiltended<br />
as compared with the protein in solution [26]. The<br />
ity,
Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4 217<br />
suspension<br />
<strong>Protein</strong><br />
polymer solution<br />
in<br />
Liquid nitrogen<br />
Frozen<br />
microspheres<br />
4. Schematic diagram <strong>of</strong> the ProLease Fig. ® encapsulation<br />
From reference [22].<br />
process.<br />
<strong>for</strong>mulation using this method (referred to as<br />
hGH<br />
ProLease ® was approved by the FDA in December 1999,<br />
)<br />
an injectable suspension <strong>for</strong> once- or twice-a-month<br />
as<br />
under a brand name <strong>of</strong> Nutropin depot adminitration, ® .<br />
this process, all manipulations are per<strong>for</strong>med at<br />
In<br />
temperatures, there<strong>for</strong>e thermal denaturation <strong>of</strong><br />
low<br />
drugs can be prevented. There are no aqueous<br />
protein<br />
thus the protein is not subjected to oil-water<br />
phases,<br />
where some proteins may denature. In addi-<br />
interfaces<br />
the process utilizes solvents in which most protion,<br />
are insoluble (dichloromethane as a polymer solteins<br />
ethanol as an extraction solvent), there<strong>for</strong>e a high<br />
vent,<br />
efficiency can be achieved [22,27]. How-<br />
encapsulation<br />
this system is not easy to apply to various protein<br />
ever,<br />
The use <strong>of</strong> liquid nitrogen also makes scale-up<br />
drugs.<br />
difficult. The system still utilizes toxic organic<br />
rather<br />
and thus it has similar problems associated<br />
solvents,<br />
solvent evaporation/extraction methods.<br />
with<br />
PHASE SEPARATION (COACERVATION)<br />
<strong>Methods</strong><br />
Nozzle<br />
Extraction solvent<br />
Atomized<br />
droplets<br />
<strong>of</strong> solvent<br />
Extraction<br />
microspheres<br />
from<br />
Drug particle<br />
Release modifier<br />
PLG microsphere<br />
by phase separation is basically a<br />
<strong>Microencapsulation</strong><br />
process: (1) phase separation <strong>of</strong> the coating<br />
three-step<br />
to <strong>for</strong>m coacervate droplets; (2) adsorption <strong>of</strong><br />
polymer<br />
coacervate droplets onto the drug surface; and (3)<br />
the<br />
<strong>of</strong> the microcapsules [28], as shown in Fig.<br />
solidification<br />
Phase separation techniques can be classified accord-<br />
5.<br />
to the method to induce phase separation; noning<br />
addition, temperature change, incompatible<br />
solvent<br />
or salt addition, and polymer-polymer interac-<br />
polymer<br />
[29]. tion<br />
Addition<br />
Non-solvent<br />
polymer to be coated is dissolved in a good sol-<br />
The<br />
and drug may be dissolved or suspended or emulvent,<br />
in the polymer solution. Then the first nonsified<br />
(also called ‘coacervating agent’ or ‘phase insolvent<br />
which is miscible with the good solvent but<br />
ducer’)<br />
not dissolve the polymer is slowly added to the<br />
does<br />
solution system. The polymer is concen-<br />
polymer-drug<br />
as the solvent <strong>for</strong> the polymer is slowly extracted<br />
trated<br />
the first non-solvent. The polymer is then induced<br />
into<br />
by<br />
Induced<br />
Non-solvent<br />
1.<br />
Temperature change<br />
2.<br />
Salt / Incompatible polymer<br />
3.<br />
4. Polymer-polymer interaction<br />
separation<br />
Phase<br />
the coating polymer<br />
<strong>of</strong><br />
Drug particle<br />
<strong>of</strong><br />
Adsorption<br />
droplets<br />
coacervate<br />
<strong>of</strong><br />
Solidification<br />
microcapsules<br />
the<br />
5. Schematic diagram <strong>of</strong> the <strong>for</strong>mation <strong>of</strong> a coacervate<br />
Fig.<br />
From reference [30].<br />
microcapsule.<br />
phase separate and <strong>for</strong>m coacervate droplets that<br />
to<br />
the drug (Fig. 6(A)). The coacervate droplet size<br />
contain<br />
be controlled by adjusting the stirring speed. At this<br />
can<br />
<strong>of</strong> the process, the coacervates are usually too s<strong>of</strong>t<br />
point<br />
be collected, and thus they are usually transferred to<br />
to<br />
large body <strong>of</strong> a second non-solvent (also called ‘hard-<br />
a<br />
agent’) to harden the microparticles [31].<br />
ening<br />
polyesters, such as PLA and PLGA, are used,<br />
When<br />
widely used solvents are dichloromethane, ethyl<br />
most<br />
and acetonitrile. The first non-solvents <strong>for</strong> the<br />
acetate,<br />
are low-molecular weight liquid polybutadi-<br />
polymers<br />
low-molecular weight liquid methacrylic polymers,<br />
ene,<br />
oil, vegetable oil, and light liquid paraffine oils.<br />
silicone<br />
hydrocarbons, such as heptane, hexane, and<br />
Aliphatic<br />
ether, have been used as the second non-<br />
petroleum<br />
solvent.<br />
Change<br />
Temperature<br />
system comprised <strong>of</strong> a polymer and a solvent exists<br />
A<br />
a single phase at all points above the phase boundary.<br />
as<br />
is dispersed in the polymer solution with stirring.<br />
Drug<br />
the temperature is decreased below the curve (Fig.<br />
As<br />
phase separation <strong>of</strong> the dissolved polymer occurs<br />
6(B)),<br />
the <strong>for</strong>m <strong>of</strong> immiscible liquid and the polymer coa-<br />
in<br />
around the dispersed drug particles, thus <strong>for</strong>ming<br />
lesces<br />
Further cooling accomplishes gelation<br />
microcapsules.<br />
solidification <strong>of</strong> the coating. The microcapsules are<br />
and<br />
from the solvent by filtration, decantation, or<br />
collected<br />
techniques [32].<br />
centrifugation<br />
Polymer Addition or Salt Addition<br />
Incompatible<br />
two chemically different polymers dissolved in<br />
When<br />
common solvent are incompatible and do not mix in<br />
a<br />
phase separation takes place. This phenome-<br />
solution,<br />
is well described by the phase diagram <strong>of</strong> a ternary<br />
non<br />
consisting <strong>of</strong> a solvent, and two polymers, X<br />
system<br />
Y (Fig. 6(C)). A drug is dispersed in a solution <strong>of</strong><br />
and<br />
Y (point a in Fig. 6(C)) and polymer X is added<br />
polymer<br />
the system, denoted by the arrowed line. When the<br />
to<br />
boundary is passed with the further addition <strong>of</strong><br />
phase<br />
X, polymer rich phase begins to separate to<br />
polymer<br />
immiscible droplets containing the drug, and coa,-<br />
<strong>for</strong>m
218 Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4<br />
( A )<br />
1 0 0 %<br />
0 0 % 1 0 0 %<br />
1<br />
o n s o l v e n t P o l y m e r<br />
N<br />
( C )<br />
S o l v e n t<br />
S o l v e n t<br />
2 p h a s e s<br />
1 0 0 %<br />
2 p h a s e s<br />
1 0 0 %<br />
1 0 0 %<br />
o l y m e r Y P X r e m y l o P<br />
6. General phase diagrams <strong>of</strong> phase separation techniques based on solvent (A), temperature (B), incompatible polymers (C),<br />
Fig.<br />
interpolymer interactions (D). From reference [32].<br />
and<br />
to make microcapsules [32]. In a similar manner<br />
lesce<br />
salts can be added to aqueous solution <strong>of</strong> wa-<br />
inorganic<br />
ter-soluble polymers to cause phase separation.<br />
Interaction (Complex Coacervation)<br />
Polymer-polymer<br />
interaction <strong>of</strong> oppositely charged polyelectro-<br />
The<br />
can result in the <strong>for</strong>mation <strong>of</strong> a complex with relytes<br />
solubility leading to phase separation (Fig. 6(D)).<br />
duced<br />
method is <strong>of</strong>ten called complex coacervation [1].<br />
This<br />
method differs from incompatible polymer addi-<br />
The<br />
in that both polymers become part <strong>of</strong> the final caption<br />
wall. Gelatin is normally used as a cationic polymer<br />
sule<br />
pH below its isoelectric point. A variety <strong>of</strong> natural<br />
at<br />
synthetic anionic polymers are available <strong>for</strong> interac-<br />
and<br />
with gelatin to <strong>for</strong>m complex coacervates. A well<br />
tion<br />
example <strong>of</strong> natural polyanion <strong>for</strong> making com-<br />
known<br />
coacervates with gelatin is gum arabic. Aqueous<br />
plex<br />
<strong>of</strong> gum arabic and gelatin are adjusted to pH<br />
solutions<br />
and warmed to 40-45°C. The pI <strong>of</strong> gelatin is 8.9. The<br />
4.5<br />
charged macromolecules interact to undergo<br />
oppositely<br />
separation, to which a water insoluble liquid core<br />
phase<br />
is added and emulsified to yield the desired<br />
material<br />
size. The mixture is then slowly cooled <strong>for</strong> an<br />
droplet<br />
During the cooling cycle, phase separation is<br />
hour.<br />
enhanced, resulting in the microcapsules <strong>for</strong>med<br />
further<br />
the core material with thin film <strong>of</strong> coacervate [32].<br />
on<br />
harden the microcapsules, the capsules are normally<br />
To<br />
further and treated with glutaraldehyde. This<br />
cooled<br />
a<br />
( B )<br />
( D )<br />
1 0 0 %<br />
1 0 0 %<br />
- e P + e P<br />
is mainly <strong>for</strong> water insoluble materials, include-<br />
method<br />
inks <strong>for</strong> carbonless paper, perfumes <strong>for</strong> advertising<br />
ing<br />
inserts, and liquid crystals <strong>for</strong> display devices.<br />
Applications<br />
2 p h a s e s<br />
P O L Y M E R C O N C E N T R A T I O N - w t %<br />
W a t e r<br />
1 0 0 %<br />
2 p h a s e s<br />
the non-solvent addition method does not in-<br />
Since<br />
an aqueous continuous phase as in w/o/w double<br />
clude<br />
methods, the water-soluble drugs may be pro-<br />
emulsion<br />
from partitioning into the water phase. This<br />
tected<br />
the non-solvent addition method one <strong>of</strong> the<br />
makes<br />
widely used techniques <strong>for</strong> encapsulating water-<br />
most<br />
compounds [28,33-41]. Triptoreline (luteinizing<br />
soluble<br />
releasing hormone analogue)-containing micro-<br />
hormone<br />
were prepared by the non-solvent addition<br />
capsules<br />
and the effect <strong>of</strong> the physicochemical nature <strong>of</strong><br />
method,<br />
polymer on the stability <strong>of</strong> the coacervate droplets<br />
the<br />
examined [34,35]. Triptoreline was suspended in<br />
was<br />
solution, and silicone oil was<br />
PLGA-dichloromethane<br />
added to the suspension as the first non-solvent.<br />
then<br />
embryonic microspheres were further hardened in<br />
The<br />
the second non-solvent. It was found that as<br />
n-heptane,<br />
copolymer became more hydrophobic, it dissolved<br />
the<br />
in dichloromethane and thus more silicone oil<br />
better<br />
necessary to desolvate the copolymer. The average<br />
was<br />
<strong>of</strong> the microspheres increased with the volume<br />
diameter<br />
silicone oil, thereby lowering the initial burst effect.<br />
<strong>of</strong><br />
was also observed from in vivo experiments. With op-<br />
It
Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4 219<br />
d<br />
s<br />
e<br />
a<br />
R<br />
e<br />
le<br />
e<br />
%<br />
u<br />
la<br />
tiv<br />
C<br />
u<br />
m<br />
100<br />
75<br />
50<br />
25<br />
0<br />
0 2 4 6 8<br />
Time (days)<br />
100/0<br />
80/20<br />
0/100<br />
7. Release <strong>of</strong> GM-CSF from microspheres prepared from<br />
Fig.<br />
PLA (MW 6,100) (0/100), 80%/20% mixture <strong>of</strong> PLGA<br />
100%<br />
40,400)/PLA (80/20), and 100% PLGA (100/0). From<br />
(MW<br />
[41].<br />
reference<br />
microspheres, the in vivo peptide release reached<br />
timized<br />
peak level on day 15 and the effect lasted <strong>for</strong> 1<br />
the<br />
month.<br />
colony-stimulating factor<br />
Granulocyte-macrophage<br />
was encapsulated in different blends <strong>of</strong><br />
(GM-CSF)<br />
and low molecular weight PLA by the non-<br />
PLGA<br />
addition method, and the prepared microsolvent<br />
were characterized both in vitro and in vivo [41].<br />
spheres<br />
microspheres with high encapsulation efficiency<br />
The<br />
obtained within a size range between 20 µm and<br />
were<br />
µm. The in vitro release kinetics was dependent on<br />
80<br />
ratio <strong>of</strong> blended polymers. Steady release <strong>of</strong> GM-<br />
the<br />
could be achieved over a period <strong>of</strong> one week with-<br />
CSF<br />
significant burst effect using blend <strong>of</strong> low molecular<br />
out<br />
PLA and high molecular weight PLGA (Fig. 7).<br />
weight<br />
released from the microspheres was found to<br />
GM-CSF<br />
physically intact and biologically active both in vitro<br />
be<br />
in vivo. and<br />
avoid the aggregation <strong>of</strong> microparticles <strong>of</strong>ten en-<br />
To<br />
in non-solvent addition, a modified method<br />
countered<br />
developed [42]. Instead <strong>of</strong> adding non-solvent to a<br />
was<br />
mixture, the polymer solution was added<br />
polymer-drug<br />
an emulsion <strong>of</strong> aqueous ovalbumin in the non-<br />
to<br />
silicon oil. The obtained microspheres appeared<br />
solvent<br />
and remained individual, and displayed a small<br />
smooth<br />
release <strong>of</strong> entrapped ovalbumin over the first 12<br />
initial<br />
with the subsequent release pr<strong>of</strong>ile close to a<br />
hours,<br />
order kinetics <strong>for</strong> a month. Another modification<br />
zero<br />
the non-solvent addition method was introduced to<br />
<strong>of</strong><br />
protein entities from denaturation at the or-<br />
protect<br />
interface or unfolding and/or aggregaganic/aqueous<br />
within hydrophobic polymer matrix [40,43,44].<br />
tion<br />
hydrophilic nanoparticles were pre-<br />
Drug-containing<br />
prior to encapsulation with hydrophobic polymer.<br />
pared<br />
[40], agarose [43], and poly(vinyl alcohol) (PVA)<br />
Gelatin<br />
were used as nanoparticle matrices <strong>for</strong> BSA or insu-<br />
[44]<br />
Nanoparticles were suspended in PLGA-dichlorolin.<br />
solution. Microparticles embedded with nanomethane<br />
were made by phase separation method using<br />
particles<br />
oil as the first non-solvent and heptane as the<br />
silicone<br />
non-solvent. The average diameter <strong>of</strong> the hydro-<br />
second<br />
nanoparticle-hydrophobic microsphere composi-<br />
philic<br />
was 150-180 µm. The protein release from the mi-<br />
tes<br />
was prolonged to nearly two months, withcrospheres<br />
degradation nor aggregation <strong>of</strong> the protein as<br />
out<br />
by size exclusion chromatograms [44].<br />
shown<br />
separation by salt addition was adopted in<br />
Phase<br />
<strong>of</strong> ionotropic hydrogel in an attempt to<br />
preparation<br />
the size distribution <strong>of</strong> microspheres [45]. Prior<br />
control<br />
cross-linking with calcium ions, salts <strong>of</strong> monovalent<br />
to<br />
(e.g., sodium chloride) were added to aqueous solu-<br />
ions<br />
<strong>of</strong> poly [di(carboxylatophenoxy) phosphazene]<br />
tion<br />
to <strong>for</strong>m coacervate microdroplets with a size in<br />
(PCPP)<br />
range 1-10 µm. Upon addition <strong>of</strong> calcium chloride,<br />
the<br />
microdroplets were stabilized via cross-linking be-<br />
the<br />
PCPP and calcium salts without changing the<br />
tween<br />
and shape. The microsphere size increased linearly<br />
size<br />
the sodium chloride concentration, incubation<br />
with<br />
and polymer concentration. This method avoided<br />
time,<br />
use <strong>of</strong> organic solvents, heat, and complicated<br />
the<br />
equipment. Gelatin/Chondroitin 6-sulfate<br />
manufacturing<br />
microspheres were prepared to encapsulate pro-<br />
(CS6)<br />
using complex coacervation <strong>for</strong> the joint therapy<br />
teins<br />
Gelatin (a polycation) and CS6 (a polyanion) solu-<br />
[46].<br />
containing model proteins were mixed and vortions<br />
at 37°C, pH 5.5 to <strong>for</strong>m coacervates. The resulting<br />
texed<br />
microspheres were crosslinked with glu-<br />
coacervate<br />
It was claimed that proteins could be entaraldehyde.<br />
with high encapsulation efficiency, retaining<br />
capsulated<br />
bioactivity. The release <strong>of</strong> protein depended on the<br />
high<br />
<strong>of</strong> human matrix metalloprotease (MMP), since<br />
level<br />
coacervate was degraded by its gelatinase<br />
gelatin/CS6<br />
The MMP concentration is one <strong>of</strong> the factors<br />
activity.<br />
<strong>for</strong> the joint pain, and thus the MMP-<br />
responsible<br />
release property is <strong>of</strong> distinctive advantage<br />
responsive<br />
the delivery <strong>of</strong> MMP inhibitors or the receptors <strong>for</strong><br />
<strong>for</strong><br />
stimulating the synthesis <strong>of</strong> MMP to diseased<br />
cytokines<br />
Gelatin/CS6 showed no significant cytotoxic<br />
joints.<br />
effect in vitro.<br />
Advantages<br />
non-solvent addition method is preferred in en-<br />
The<br />
<strong>of</strong> water-soluble drugs, such as proteins,<br />
capsulation<br />
and vaccines, since drug-polymer mixtures are<br />
peptides,<br />
exposed to the continuous aqueous phase. This<br />
not<br />
the loss <strong>of</strong> water-soluble drugs to the water<br />
minimizes<br />
and results in the high encapsulation efficiency.<br />
phase<br />
advantage <strong>of</strong> the phase separation method is<br />
Another<br />
it enables efficient control <strong>of</strong> the particle size with<br />
that<br />
narrower size distribution by simply varying the<br />
a<br />
variables, such as concentration <strong>of</strong> added<br />
component<br />
[45] or the viscosity and amount <strong>of</strong> the non-<br />
salts<br />
and/or molecular weight <strong>of</strong> polymer used [47].<br />
solvent
220 Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4<br />
Limitations<br />
many successful phase separation systems<br />
Although<br />
been demonstrated, each method has several limi-<br />
have<br />
The microspheres tend to aggregate and the<br />
tations.<br />
<strong>for</strong> mass production is difficult [48]. Agglom-<br />
scale-up<br />
occurs when the coacervate droplets are sticky<br />
eration<br />
adhere to each other be<strong>for</strong>e the solvent is com-<br />
and<br />
removed or be<strong>for</strong>e the droplets are hardened [31].<br />
pletely<br />
produce well-defined microspheres, wide ‘stability<br />
To<br />
is required. The stability window is defined as<br />
window’<br />
step where addition <strong>of</strong> non-solvent does not induce<br />
a<br />
but <strong>for</strong>ms stable coacervates [34]. The<br />
agglomeration<br />
window is affected by the physicochemical<br />
stability<br />
<strong>of</strong> polymers used and/or the viscosity <strong>of</strong> the<br />
nature<br />
added. The presence <strong>of</strong> residual solvents is<br />
non-solvent<br />
concern in the non-solvent addition method. The<br />
a<br />
includes at least three different solvents, i.e.,<br />
method<br />
polymer solvent and two polymer non-solvents. In<br />
the<br />
the polymer solvents are toxic compounds,<br />
general,<br />
as halogenated hydrocarbons (e.g., dichloro-<br />
such<br />
<strong>for</strong> which the exposure limits are regulated<br />
methane),<br />
the authority. For example, the concentration limit<br />
by<br />
dichloromethane is 600 ppm [49]. In addition to the<br />
<strong>for</strong><br />
solvent, two non-solvents are generally known<br />
polymer<br />
remain in a substantial quantity. A study showed<br />
to<br />
depending on the type <strong>of</strong> solvent and polymer used,<br />
that<br />
residual solvents could be minimized by appropriate<br />
the<br />
methods [38]. The solvents, however, were in<br />
drying<br />
hard to remove. High levels <strong>of</strong> residues may al-<br />
general<br />
the microsphere morphology due to their plasticizter<br />
effect. Moreover, it may cause a toxicity problem<br />
ing<br />
influence crucial properties such as release pr<strong>of</strong>iles<br />
and<br />
drug stability. Undoubtedly, the search <strong>for</strong> toxico-<br />
and<br />
and technologically acceptable solvents and<br />
logically<br />
should continue <strong>for</strong> the non-solvent addi-<br />
non-solvents<br />
method to be more useful.<br />
tion<br />
a polymer-polymer interaction (complex coacerva-<br />
In<br />
the limited pH range and the use <strong>of</strong> cross-linking<br />
tion),<br />
can be potential problems. Since the technique is<br />
agent<br />
on electrostatic interactions between two polye-<br />
based<br />
pH <strong>of</strong> the medium is an important factor.<br />
lectrolytes,<br />
example, in a gelatin-acacia system, the pH should<br />
For<br />
below the isoelectric point <strong>of</strong> gelatin (pH 8.9) to<br />
be<br />
it positively charged. The pH range suitable <strong>for</strong><br />
make<br />
gelatin-acacia system is between 2.6 and 5.5 [30].<br />
the<br />
condition, however, is not appropriate <strong>for</strong> encapsu-<br />
This<br />
<strong>of</strong> acid-labile drug entities. The pH range could<br />
lation<br />
extended by the addition <strong>of</strong> water-soluble nonionic<br />
be<br />
such as polyethylene glycol [50,51]. Glutaral-<br />
polymers,<br />
and <strong>for</strong>maldehyde are commonly used as crossdehyde<br />
agents to harden the microcapsules in this techlinking<br />
A condensation reaction occurs between the<br />
nique.<br />
groups <strong>of</strong> the protein (e.g., gelatin) and the alde-<br />
amino<br />
The cross-linking agents may diffuse into the<br />
hydes.<br />
and also react with drug entities, there<strong>for</strong>e this<br />
core<br />
may not be appropriate <strong>for</strong> the protein drugs.<br />
method<br />
toxicity problems arise as well [30].<br />
Potential<br />
SPRAY DRYING<br />
drying has been widely used in the pharmaceu-<br />
Spray<br />
food, and biochemical industries. The main applitical,<br />
in the pharmaceutical field include drying proccations<br />
granulation, preparation <strong>of</strong> solid dispersions, alteraess,<br />
<strong>of</strong> polymorphism <strong>of</strong> a drug, preparation <strong>of</strong> dry<br />
tion<br />
<strong>for</strong> aerosols, and encapsulation <strong>of</strong> drugs <strong>for</strong><br />
powders<br />
masking and protection from oxidation [52]. Since<br />
taste<br />
late 1970s, the spray drying technique has been<br />
the<br />
<strong>for</strong> making microparticulate systems <strong>for</strong> controlled<br />
used<br />
delivery.<br />
drug<br />
<strong>Methods</strong><br />
hydrophilic or hydrophobic polymer is dis-<br />
Either<br />
in a suitable solvent. Drug can be dissolved or<br />
solved<br />
in the solvent. Alternatively, drug solution<br />
suspended<br />
be emulsified in the polymer solution. The mixture<br />
can<br />
then sprayed through a nozzle <strong>of</strong> a spray dryer,<br />
is<br />
in solid microspheres that precipitated into<br />
resulting<br />
bottom collector [53]. Sometimes, the process<br />
the<br />
the use <strong>of</strong> plasticizers to <strong>for</strong>m microcapsules <strong>of</strong><br />
include<br />
spherical shape and smooth-surface by reducing<br />
regular<br />
rigidity <strong>of</strong> polymer chain [54].<br />
the<br />
Alternative Technique<br />
(congealing): Spray congealing includes<br />
Spray-chilling<br />
dissolution or dispersion <strong>of</strong> the drug in a melted<br />
the<br />
without using solvent. This mixture fluid is then<br />
carrier<br />
into a cold air stream to make small droplets<br />
sprayed<br />
solidified by cooling at the temperature lower than<br />
and<br />
melting point <strong>of</strong> the carrier. This method was used<br />
the<br />
encapsulate bovine somatotropin (bST) in fat or wax<br />
to<br />
bST was dispersed in molten fat or wax and<br />
[55].<br />
through an air/liquid spray nozzle. The micro-<br />
sprayed<br />
were <strong>for</strong>med as the molten droplets and were<br />
spheres<br />
on sieves in the desired size range (45-180 µm).<br />
collected<br />
was demonstrated that the microspheres were effec-<br />
It<br />
in maintaining increased level <strong>of</strong> bST in the blood<br />
tive<br />
treated animals <strong>for</strong> 4 weeks, increasing weight gains<br />
<strong>of</strong><br />
and milk production.<br />
Applications<br />
spray-drying is relatively simple, fast and easy<br />
Since<br />
scale-up, it has been tried as an attractive alternative<br />
to<br />
the conventional methods, such as coacervation and<br />
to<br />
method to encapsulate protein drugs [48,56-<br />
emulsion<br />
The spray drying technique was applied to produce<br />
65].<br />
microparticles containing thyrotropin releasing<br />
PLGA<br />
(TRH) [48]. TRH in water and PLGA in ace-<br />
hormone<br />
were mixed to <strong>for</strong>m a clear solution and then<br />
tonitrile<br />
Since the microparticles produced by conven-<br />
sprayed.<br />
spray dryer tended to agglomerate and adhere to<br />
tional<br />
surface <strong>of</strong> the chamber, they designed a double noz-<br />
the<br />
system such that additional nozzle might spray a<br />
zle<br />
solution as an anti-adherent simultaneously<br />
mannitol<br />
8). Microparticles with mean particle size <strong>of</strong> 20 µm<br />
(Fig.
Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4 221<br />
Raw<br />
materials<br />
8. Schematic diagram <strong>of</strong> a spray dryer used <strong>for</strong> prepara-<br />
Fig.<br />
<strong>of</strong> PLGA microparticles containing TRH. From reference<br />
tion<br />
[48].<br />
obtained, from which zero order release <strong>of</strong> TRH<br />
were<br />
<strong>for</strong> one month with a minimal initial burst.<br />
continued<br />
4 lists some examples <strong>of</strong> microspheres prepared by<br />
Table<br />
drying.<br />
spray<br />
Advantages<br />
<strong>of</strong> the major advantages <strong>of</strong> spray drying is its<br />
One<br />
applicability. Both hydrophilic and hydrophobic<br />
general<br />
can be used with proper selection <strong>of</strong> the sol-<br />
polymer<br />
[52]. Spray drying is useful <strong>for</strong> encapsulating even<br />
vent<br />
drugs, such as proteins or peptides, be-<br />
heat-sensitive<br />
it involves mild temperatures [52]. Although<br />
cause<br />
drying includes hot air stream, the temperature <strong>of</strong><br />
spray<br />
droplets may be maintained below the drying air<br />
the<br />
due to rapid evaporation <strong>of</strong> the solvent.<br />
temperature<br />
effective temperature applied to the drug itself is<br />
The<br />
enough to be used <strong>for</strong> proteins. Moreover, spray<br />
mild<br />
is time effective. Spray drying can yield results<br />
drying<br />
to those <strong>of</strong> conventional methods in terms<br />
equivalent<br />
size distribution, particle morphology, and release<br />
<strong>of</strong><br />
yet with the advantage <strong>of</strong> high encapsulation<br />
kinetics,<br />
and the short duration <strong>of</strong> the preparation<br />
efficiency<br />
[53]. The spray drying equipment is easily<br />
procedure<br />
at the manufacturing site. Also, as a one-stage<br />
available<br />
process, spray drying is ideal <strong>for</strong> production <strong>of</strong><br />
closed<br />
materials and good manufacturing practice<br />
sterile<br />
(GMP) [30].<br />
Limitations<br />
TRH-PLGA<br />
solution<br />
Air filter<br />
Pump<br />
air<br />
Double<br />
nozzle<br />
Heater<br />
Drying<br />
chamber<br />
Mannitol<br />
solution<br />
Spray<br />
nozzle<br />
Blower<br />
Cyclone<br />
Receiver<br />
During spray drying, considerable amounts <strong>of</strong> the<br />
4. Examples <strong>of</strong> microencapsulation <strong>of</strong> proteins by spray<br />
Table<br />
drying<br />
Polymer Solvent <strong>Protein</strong> Particle<br />
size<br />
β-glucuronidase<br />
Bovine<br />
somatotropin<br />
Human<br />
erythropoietin<br />
Albumin/<br />
with<br />
Acacia<br />
as PVP<br />
coacervate<br />
stabilizer<br />
anhy- Poly<br />
dride<br />
PLGA<br />
Distilled<br />
water<br />
5 µm<br />
Dichloro-<br />
1-5 µm<br />
methane <br />
Dichloro-<br />
10 µm<br />
methane<br />
Release kinetics Ref.<br />
initial burst<br />
Biphasic:<br />
within 6h followed<br />
(31%)<br />
zero order release <strong>for</strong> 2<br />
by<br />
weeks<br />
release <strong>for</strong> 6 h due to<br />
90%<br />
fast degradation <strong>of</strong><br />
the<br />
and small parti-<br />
polymer<br />
size cle<br />
burst during 24 h<br />
Initial<br />
no further release <strong>for</strong><br />
and<br />
months, likely due to<br />
2<br />
interac-<br />
protein-polymer<br />
and insoluble protein<br />
tion<br />
aggregates<br />
can be lost during the process due to sticking<br />
material<br />
the microparticles to the wall <strong>of</strong> the drying chamber.<br />
<strong>of</strong><br />
drying process can <strong>of</strong>ten lead to change <strong>of</strong> poly-<br />
Spray<br />
<strong>of</strong> the spray dried drugs [66,67]. For exammorphism<br />
progesterone crystal in its original alpha <strong>for</strong>m (m.p.<br />
ples,<br />
turned into beta <strong>for</strong>m (m.p. 395K) when it was<br />
402K)<br />
dried in combination with PLA. Fiber <strong>for</strong>mation<br />
spray<br />
be another major problem in spray drying PLA [67].<br />
can<br />
are <strong>for</strong>med due to insufficient <strong>for</strong>ces present to<br />
Fibers<br />
up the liquid filament into droplets. It depends on<br />
break<br />
the type <strong>of</strong> polymer and, to a lesser extent, the<br />
both<br />
<strong>of</strong> the spray solution. For example, ethyl cellu-<br />
viscosity<br />
solutions made spherical droplets at relatively high<br />
lose<br />
however PLA solutions <strong>of</strong> considerably<br />
concentrations,<br />
concentration and viscosity resulted in fibers.<br />
low<br />
are a number <strong>of</strong> process variables that should be<br />
There<br />
<strong>for</strong> drug encapsulation. They include feed<br />
optimized<br />
properties, such as viscosity, uni<strong>for</strong>mity, and<br />
material<br />
<strong>of</strong> drug and polymer mixtures, feed rate,<br />
concentration<br />
<strong>of</strong> atomization, and the inlet and outlet tem-<br />
method<br />
[32]. The dependence on so many variables<br />
peratures<br />
become a problem in terms <strong>of</strong> reproducibility and a<br />
may<br />
process. In addition, the amount <strong>of</strong> polymer<br />
scale-up<br />
drugs <strong>for</strong> encapsulation may be limited, since<br />
and/or<br />
fluid <strong>of</strong> high viscosity cannot be sprayed.<br />
GELATION /<br />
IONOTROPIC<br />
COMPLEXATION<br />
POLYELECTROLYTE<br />
gelation (IG) is based on the ability <strong>of</strong><br />
Ionotropic<br />
to crosslink in the presence <strong>of</strong> counter<br />
polyelectrolytes<br />
to <strong>for</strong>m hydrogels. Since the use <strong>of</strong> alginate <strong>for</strong> cell<br />
ions<br />
[68], ionotropic gelation has been widely<br />
encapsulation<br />
used <strong>for</strong> both cell and drug encapsulation.<br />
Method<br />
is one <strong>of</strong> the most widely used polyanion <strong>for</strong><br />
Alginate<br />
Alginate is composed <strong>of</strong> 1,4-linked<br />
microencapsulation.<br />
beta-D-mannuronic acid and alpha-D-guluronic acid<br />
[60]<br />
[56]<br />
[62]
222 Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4<br />
(A)<br />
(B)<br />
HO HO<br />
-OOC<br />
OHO<br />
9. Monomer units (A) found in alginate and the alginate<br />
Fig.<br />
structure (B). From reference [69].<br />
polymer<br />
Divalent and trivalent cations induce gelation<br />
residues.<br />
binding mainly to the guluronic blocks (Fig. 9). The<br />
by<br />
and macro-spheres are produced by dropping a<br />
microor<br />
drug-loaded alginate solution into aqueous cal-<br />
cell-<br />
chloride solution. Calcium ions diffuse into the<br />
cium<br />
drops, <strong>for</strong>ming a three dimensional lattice <strong>of</strong><br />
alginate<br />
crosslinked alginate. Polyelectrolyte complexa-<br />
ionically<br />
(PEC) with oppositely charged polyelectrolytes<br />
tion<br />
be added to increase the mechanical strength <strong>of</strong><br />
may<br />
hydrogel and/or to provide a permeability barrier.<br />
the<br />
instance, addition <strong>of</strong> polycation (usually poly-L-<br />
For<br />
allows a membrane <strong>of</strong> polyelectrolyte complex<br />
lysine)<br />
<strong>for</strong>m on the surface <strong>of</strong> alginate beads [69]. A number<br />
to<br />
natural and synthetic polyelectrolytes have been in-<br />
<strong>of</strong><br />
Examples are listed in Table 5.<br />
vestigated.<br />
most <strong>of</strong> the droplets have been made with sy-<br />
Since<br />
needles, the particle sizes have been relatively<br />
ringe<br />
Smaller droplets can be <strong>for</strong>med using a vibration<br />
large.<br />
or air atomization method to extrude the algi-<br />
system<br />
solution (Fig. 10) [30]. The latter involves a Turbonate<br />
air-atomizer. Pressurized air is fed to mix with the<br />
tak<br />
alginate solution, <strong>for</strong>cing tiny liquid droplets<br />
sodium<br />
through the orifice <strong>of</strong> a nozzle. The calcium ions<br />
out<br />
the droplets <strong>of</strong> sodium alginate on contact to<br />
crosslink<br />
microgel droplets, which were further crosslinked<br />
<strong>for</strong>m<br />
poly-L-lysine to <strong>for</strong>m a membrane on the droplets.<br />
by<br />
obtained using this method were within<br />
Microparticles<br />
size range 5-15 µm [91].<br />
the<br />
Applications<br />
OH<br />
Mannuronate (M)<br />
OH<br />
O -OOC<br />
O<br />
OH<br />
-OOC OHO<br />
O HO<br />
O<br />
HO<br />
-OOC<br />
OHO<br />
-OOC<br />
has been widely used in microcapsule sys-<br />
IG/PEC<br />
<strong>for</strong> cell encapsulation [82,87-89,92-94] and drug<br />
tems<br />
systems [76,77,83,85,86,95,96]. Due to mild<br />
delivery<br />
conditions, IG/PEC has recently attracted a new<br />
process<br />
<strong>for</strong> an application to protein delivery [71,78-<br />
attention<br />
Calcium alginate hydrogels have been<br />
81,84,90,97,98].<br />
<strong>for</strong> delivery <strong>of</strong> vascular endothelial growth factor<br />
used<br />
[71]. The alginate beads demonstrated the abil-<br />
(VEGF)<br />
to incorporate VEGF with efficiency between 30%<br />
ity<br />
67% and constant release (5%/day) <strong>of</strong> VEGF <strong>for</strong> up<br />
and<br />
O<br />
OH<br />
O<br />
OH<br />
OH<br />
OH<br />
(G)<br />
Guluronate<br />
-OOC<br />
OH<br />
OH<br />
O<br />
OH<br />
O -OOC<br />
G M M G G<br />
O<br />
OH<br />
O<br />
5. Examples <strong>of</strong> ionotropic gelation (IG) and polyelec-<br />
Table<br />
complexation (PEC)<br />
trolyte<br />
Polyelectrolytes IG with PEC with References<br />
Alginate (-) Calcium (+) - [70-72]<br />
Alginate (-) Calcium (+) Poly-L-Lysine (+) [73]<br />
Alginate (-) Calcium (+) Chitosan (+) [74, 75]<br />
Chitosan (+)<br />
Carboxymethyl<br />
cellulose (-)<br />
Tripolyphosphate<br />
(TPP)(-)<br />
- [76-81]<br />
Aluminium (+) Chitosan (+) [82]<br />
κ-carrageenan (-) Potassium (+) Chitosan (+) [83]<br />
ι-carrageenan (-) Amines (+) - [84]<br />
Pectin (-) Calcium (+) - [85]<br />
Gelan gum (-) Calcium (+) - [86]<br />
Alginate and<br />
cellulose sulfate (-)<br />
Calcium (+)<br />
Poly (methylene-<br />
co-guanidine) (+)<br />
[87, 88]<br />
Polyphosphazene (-) Calcium (+) Poly-L-Lysine (+) [89, 90]<br />
Sodium<br />
alginate<br />
10. Schematic diagram <strong>of</strong> the preparation <strong>of</strong> alginate-<br />
Fig.<br />
microcapsules.<br />
polylysine<br />
14 days. The ionic interaction between VEGF and<br />
to<br />
contributed to maintaining the biological activ-<br />
alginate<br />
ity <strong>of</strong> VEGF.<br />
Advantages<br />
main advantage <strong>of</strong> IG/PEC is the mild <strong>for</strong>mula-<br />
The<br />
conditions <strong>for</strong> maintaining cell viability and protion<br />
integrity. All <strong>of</strong> the polyelectrolytes are watertein<br />
and thus proteins can be encapsulated without<br />
soluble,<br />
<strong>of</strong> organic solvents or elevated temperatures, both<br />
use<br />
which may damage protein integrity. Also, this sys-<br />
<strong>of</strong><br />
tem is simple, fast and cost-effective.<br />
Limitations<br />
Turbotak<br />
atomizer<br />
chloride<br />
Calcium<br />
solution<br />
Polylysine<br />
added<br />
Pressurized<br />
gas<br />
Microcapsule<br />
biggest problem in applications <strong>of</strong> IG/PEC <strong>for</strong><br />
The<br />
protein delivery, in spite <strong>of</strong> the great advan-<br />
controlled<br />
<strong>of</strong> mild conditions, is that the matrix and memtage<br />
<strong>for</strong>med by IG/PEC is not capable <strong>of</strong> controlling<br />
brane<br />
release rate <strong>for</strong> a long period <strong>of</strong> time. It does not<br />
the<br />
in cell encapsulation, where the membrane<br />
matter<br />
should provide sufficient permeability <strong>for</strong> the cell prod-
Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4 223<br />
i.e., a therapeutic protein. However, <strong>for</strong> IG/PEC to<br />
uct,<br />
used <strong>for</strong> controlled delivery <strong>of</strong> protein entities, a cer-<br />
be<br />
type <strong>of</strong> dense membrane (e.g., biodegradable polytain<br />
preferably capable <strong>of</strong> controlling the release rate,<br />
mers),<br />
be incorporated or alternative combinations <strong>of</strong><br />
should<br />
should be explored to control the<br />
polyelectrolytes<br />
<strong>of</strong> the membrane, so that the release rate<br />
permeability<br />
be controlled over the desired span. Thus far, the<br />
may<br />
rate <strong>of</strong> the protein is likely to depend on the<br />
release<br />
properties <strong>of</strong> the model protein or the<br />
physicochemical<br />
medium in which the microparticles are placed<br />
release<br />
In case <strong>of</strong> alginate-poly-L-lysine system, all para-<br />
[71].<br />
are tied to a single chemical complex, there<strong>for</strong>e<br />
meters<br />
is not easy to optimize the capsule condition. For this<br />
it<br />
new combinations <strong>of</strong> polyelectrolytes were<br />
reason,<br />
to allow independent modification <strong>of</strong> capsule<br />
proposed<br />
including size, wall thickness, mechanical<br />
parameters<br />
permeability, and surface characteristics [99].<br />
strength,<br />
issue in IG/PEC is that some polyelectrolytes<br />
Another<br />
have biocompatibility problem. There have been<br />
might<br />
arguments concerning the biocompatibil-<br />
contradictory<br />
<strong>of</strong> alginates [100-102]. A host <strong>for</strong>eign-body reaction<br />
ity<br />
occurred on account <strong>of</strong> the implanted algi-<br />
(fibrosis)<br />
particles and mannuronic acid block was<br />
nate-based<br />
to be responsible <strong>for</strong> the fibrotic response, as a<br />
found<br />
stimulator <strong>of</strong> IL-1 and TNF-α production. Use <strong>of</strong><br />
potent<br />
<strong>of</strong> high guluronic acid content was suggested<br />
alginates<br />
minimize the cytokine response. On the other hand,<br />
to<br />
study indicated that alginate particles with<br />
another<br />
guluronic acid contents provoked stronger re-<br />
high<br />
than the high mannuronic acid alginate particles<br />
sponse<br />
In general, polyionic hydrogels obtained by<br />
[102].<br />
have rather low mechanical strength. A few<br />
IG/PEC<br />
have suggested ways to overcome this problem<br />
papers<br />
exploring alternative combinations <strong>of</strong> polyanions<br />
by<br />
polycations [87,99], introducing a different process<br />
and<br />
[103], or applying additional coating [82,98].<br />
INTERFACIAL POLYMERIZATION<br />
polymerization features in situ polymeri-<br />
Interfacial<br />
<strong>of</strong> reactive monomers on the surface <strong>of</strong> a droplet<br />
zation<br />
or particles.<br />
Method<br />
reactive monomers (typically dichloride and<br />
Two<br />
are respectively dissolved in immiscible sol-<br />
diamine)<br />
and mixed to <strong>for</strong>m o/w emulsion (dichloride in oil<br />
vents<br />
and diamine in water phase). The monomers dif-<br />
phase<br />
to the o/w interface where they react to <strong>for</strong>m a<br />
fuse<br />
membrane (Fig. 11). A typical example <strong>of</strong> this<br />
polymeric<br />
is making nylon microcapsules [104]. Nona-<br />
method<br />
phase (chloro<strong>for</strong>m/cyclohexane) containing surqueous<br />
(e.g., sorbitan trioleate) and aqueous buffer confactant<br />
drugs to be incorporated (enzymes or proteins),<br />
taining<br />
protein (e.g., BSA or hemoglobin) and dia-<br />
protective<br />
are prepared respectively. Two phases are mixed<br />
mine<br />
and stirred to make an w/o emulsion in an ice bath un-<br />
11. <strong>Microencapsulation</strong> by interfacial polymerization<br />
Fig.<br />
From reference [104].<br />
method.<br />
the desired droplet size is reached. Another nonaque-<br />
til<br />
phase containing acid chloride is added to the emulous<br />
<strong>for</strong> interfacial polymerization. Polymerization is<br />
sion<br />
by addition <strong>of</strong> excess nonaqueous phase.<br />
quenched<br />
are allowed to sediment and collected<br />
Microcapsules<br />
then washed in saline several times to remove or-<br />
and<br />
solvents and byproducts.<br />
ganic<br />
combinations <strong>of</strong> monomers can be used to<br />
Various<br />
a range <strong>of</strong> polymer membranes [104]. Sebacoyl<br />
obtain<br />
and 1,6-hexane diamine can be used to <strong>for</strong>m<br />
chloride<br />
polyamide nylon 6, 10. The microcapsules using<br />
the<br />
system, however, tend to be fragile and difficult to<br />
this<br />
Terephthaloyl chloride and 1,6-hexane diamine<br />
handle.<br />
yield polyester membrane. Alternatively tere-<br />
can<br />
chloride can be used in combination with Lphthaloyl<br />
to yield poly(terephthaloyl L-lysine). Typically<br />
lysine<br />
chlorides and diamines are reactive monomers;<br />
acid<br />
isocyanates can be used instead <strong>of</strong> or as a par-<br />
however,<br />
substitute <strong>for</strong> acid chloride [105]. Alternatively, the<br />
tial<br />
composed <strong>of</strong> only one type <strong>of</strong> monomer can be<br />
polymer<br />
on the interface (Interfaicial addition polymeri-<br />
<strong>for</strong>med<br />
[104]. Polyalkylcyanoacrylate belongs to this<br />
zation)<br />
Aqueous drug solution and oil phase containing<br />
type.<br />
monomer are mixed to <strong>for</strong>m an w/o<br />
cyanoacrylate<br />
The polymerization is initiated by water in<br />
emulsion.<br />
aqeous phase with cyanoacrylate dissolved in the oil<br />
the<br />
phase.<br />
Application<br />
Monomer B<br />
Monomer A<br />
Polymer membrane<br />
insulin nanoparticles were prepared using<br />
Recently<br />
addition polymerization techniques [106].<br />
interfacial<br />
were prepared by addition <strong>of</strong> ethyl 2-<br />
Nanoparticles<br />
to a stirred w/o microemulsion consistcyanoacrylate<br />
<strong>of</strong> aqueous insulin solution, oil and surfactant. To<br />
ing<br />
exhaustive washing steps after polymerization<br />
avoid<br />
biocompatible oils (caprylic/capric triglyc-<br />
reaction,<br />
and mono-/diglycerides) and surfactants (polyerides<br />
80 and sorbitan monooleate) were used to <strong>for</strong>sorbate<br />
the microemulsions. The obtained poly(ethyl 2mulate<br />
is known to be biodegradable. Since incyanoacrylate)<br />
was confined to the aqueous phase in w/o emulsulin <br />
sions, a high encapsulation efficiency (86%) could be
224 Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4<br />
Insulin was released with initial burst over<br />
achieved.<br />
first 30 minutes followed by a constant release up<br />
the<br />
to 3 h, after which the release rate declined.<br />
Limitations<br />
this method was initially proposed <strong>for</strong> en-<br />
Although<br />
encapsulations, it has some serious problems in<br />
zyme<br />
First, large w/o interface is produced during<br />
practice.<br />
reaction, where proteins or enzymes are likely to be<br />
the<br />
Second, large amounts <strong>of</strong> proteins may par-<br />
inactivated.<br />
in the polymerization reaction changing its bioticipate<br />
activity. Third, it is <strong>of</strong>ten hard to control the<br />
logical<br />
reaction. The yield and quality <strong>of</strong> the<br />
polymerization<br />
obtained by interfacial polymerization may<br />
membrane<br />
controlled by a number <strong>of</strong> factors, such as chemical<br />
be<br />
<strong>of</strong> reactive monomers, and reaction conditions.<br />
natures<br />
monomer concentrations, temperature, the mixing<br />
The<br />
and the reaction time are likely to be important<br />
rate,<br />
[105]. Fourth, exhaustive washing steps are<br />
parameters<br />
to remove monomers, byproducts, organic sol-<br />
required<br />
and surfactants. At the same time, several washvents,<br />
steps may lead to further loss <strong>of</strong> water soluble drugs.<br />
ing<br />
pH change due to HCl byproduct <strong>for</strong>med by reac-<br />
Fifth,<br />
<strong>of</strong> an acid chloride and an amine can damage the<br />
tion<br />
labile drugs.<br />
acid<br />
SUPERCRITICAL FLUID PRECIPITATION<br />
fluid is defined as a fluid <strong>of</strong> which tem-<br />
Supercritical<br />
and pressure are simultaneously higher than at<br />
perature<br />
critical point, i.e. critical temperature Tc and critical<br />
the<br />
Pc, at which the density <strong>of</strong> gas is equal to that<br />
pressure<br />
the remaining liquid and the surface between the<br />
<strong>of</strong><br />
phases disappears (Fig. 12). In practice, the term is<br />
two<br />
used to describe a fluid in the relative vicinity <strong>of</strong><br />
<strong>of</strong>ten<br />
critical point, where a gas can be highly compressed<br />
the<br />
a small change <strong>of</strong> pressure into a fluid which is not<br />
with<br />
liquid but almost close to liquid in density. These two<br />
a<br />
features <strong>of</strong> supercritical fluids (i.e., a high<br />
distinctive<br />
and a liquid-like density) enabled super-<br />
compressibility<br />
fluids to attract considerable interest recently as<br />
critical<br />
<strong>for</strong> microparticle production.<br />
vehicles<br />
<strong>Methods</strong><br />
are two main routes to particle <strong>for</strong>mation with<br />
There<br />
fluids: the rapid expansion <strong>of</strong> supercritical<br />
supercritical<br />
(RESS); and supercritical antisolvent crystal-<br />
solutions<br />
(SAS) routes [108]. RESS exploits the liquid-like<br />
lization<br />
power <strong>of</strong> the supercritical fluids while SAS uses<br />
solvent<br />
fluid as an antisolvent. Carbon dioxide is<br />
supercritical<br />
commonly used since the critical conditions are<br />
most<br />
attainable, i.e., Tc=31°C and Pc=73.8 bar. CO2 is<br />
easily<br />
benign, relatively non-toxic, non-<br />
environmentally<br />
inexpensive, and has a reasonably high<br />
inflammable,<br />
dissolving power. [109]<br />
RESS(Rapid expansion <strong>of</strong> supercritical solutions): In<br />
12. Pressure-temperature projection <strong>of</strong> the phase diagram<br />
Fig.<br />
a pure component. From reference [107].<br />
<strong>for</strong><br />
the drug and the polymer are dissolved in a su-<br />
RESS,<br />
fluid at high pressure and precipitated by<br />
percritical<br />
the solvent’s density through a rapid decom-<br />
reducing<br />
(expansion) [110]. The actual solubility <strong>of</strong> the<br />
pression<br />
in supercritical fluids can be higher than the<br />
solutes<br />
calculated assuming ideal gas behavior at the<br />
value<br />
temperature and pressure by factors <strong>of</strong> up to 10 same 6<br />
Rapid expansion <strong>of</strong> supercritical solutions, there-<br />
[107].<br />
can lead to very high supersaturation ratios. Since<br />
<strong>for</strong>e,<br />
supercritical fluid is a gas after expansion, the solid<br />
the<br />
product is recovered in pure, solvent-free <strong>for</strong>m.<br />
antisolvent crystallization): The<br />
SAS(supercritical<br />
is also called as ASES (aerosol solvent extrac-<br />
method<br />
system) or PCA (precipitation with a compressed<br />
tion<br />
antisolvent). In SAS, a supercritical fluid is used as<br />
fluid<br />
anti-solvent that causes precipitation <strong>of</strong> solids. The<br />
an<br />
are dissolved in a suitable organic phase. At high<br />
solutes<br />
anti-solvent enters into the solution and low-<br />
pressures,<br />
the solvent power, thereby precipitating the solutes.<br />
ers<br />
precipitation is followed by a large volume expan-<br />
The<br />
and the solid product is collected. SAS process is<br />
sion<br />
in Fig. 13. As a modification <strong>of</strong> the SAS, the<br />
illustrated<br />
phase can be sprayed into a supercritical fluid,<br />
organic<br />
causes the solutes to precipitate as fine particles.<br />
which<br />
method is also called gas-antisolvent (GAS)<br />
This<br />
SAS is useful <strong>for</strong> processing <strong>of</strong> solids which are<br />
method.<br />
to solubilize in supercritical fluids, such as pep-<br />
difficult<br />
tides and proteins.<br />
Applications<br />
Solid Liquid<br />
Gas<br />
TEMPERATURE<br />
supercritical<br />
region<br />
fluid<br />
Critical point<br />
<strong>of</strong>fers distinctive advantages over the conven-<br />
RESS<br />
methods, such as no requirements <strong>of</strong> surfactants,<br />
tional<br />
a solvent-free product, and moderate process<br />
yielding<br />
[107]. However, the very low solvent power<br />
conditions
Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4 225<br />
Carbon<br />
dioxide<br />
Pump<br />
Precipitation<br />
vessel<br />
13. Schematic diagram <strong>of</strong> the SAS method. From refer-<br />
Fig.<br />
[111]. ence<br />
common supercritical solvents toward the high mo-<br />
<strong>of</strong><br />
weight polymers and the solutes <strong>of</strong> therapeutic<br />
lecular<br />
such as proteins has restricted RESS to a<br />
importance<br />
low molecular weight polymers and a limited range<br />
few<br />
drugs, e.g., lovastatin [107,112]. Lovastatin was dis-<br />
<strong>of</strong><br />
in supercritical CO2 together with DL-PLA and<br />
solved<br />
subjected to RESS. The morphology <strong>of</strong> the copre-<br />
then<br />
product was found to be very sensitive to the<br />
cipitation<br />
amounts <strong>of</strong> drug and polymer, ranging from<br />
relative<br />
networks at high drug loading to drug<br />
bicontinuous<br />
encapsulated within polymer at low drug load-<br />
needles<br />
ings.<br />
has been applied to protein drugs <strong>for</strong> size reduc-<br />
SAS<br />
[113] and bioerodible polymers <strong>for</strong> microencapsulation<br />
<strong>of</strong> pharmaceuticals in a polymer matrix [114]. Retion<br />
this technique was used to produce PLGA microcently,<br />
containing lysozyme [115]. PLGA in dichlorospheres<br />
solution with suspended lysozyme was<br />
methane<br />
into a CO2 vapor phase through a capillary<br />
sprayed<br />
to <strong>for</strong>m droplets which solidified after falling<br />
nozzle<br />
a CO2 liquid phase. In this study, several problems<br />
into<br />
encountered in SAS process were overcome.<br />
previously<br />
size (70 µm) large enough to encapsulate pro-<br />
Particle<br />
particles was achieved by delayed precipitation ustein<br />
CO2 vapor phase above a CO2 liquid phase. Aggloing<br />
due to plasticization <strong>of</strong> the polymer by CO2 meration<br />
minimized at the optimum temperature <strong>of</strong> –20 was oC. many proteins are insoluble in organic solvents<br />
Since<br />
are less likely to be denatured when suspended, this<br />
and<br />
can be extended to other proteins.<br />
method<br />
Advantages and Limitations<br />
Pump<br />
Expansion<br />
vessel<br />
Polymer<br />
solution<br />
Vent<br />
Solvent<br />
fluids, particularly CO2 <strong>of</strong>fers various<br />
Supercritical<br />
when compared with widely used organic<br />
advantages<br />
It is relatively non-toxic, environmentally ac-<br />
solvents.<br />
non flammable, and inexpensive. In general,<br />
ceptable,<br />
with narrow size distribution can be achieved<br />
particles<br />
supercritical fluid methods. Potential advantages<br />
using<br />
include mild process temperatures, the potential <strong>for</strong><br />
also<br />
and the possibility <strong>for</strong> aseptic preparation <strong>of</strong><br />
scale-up<br />
6. Examples <strong>of</strong> micorencapsulation <strong>of</strong> maromolecular<br />
Table<br />
by IG/PEC<br />
drugs<br />
<strong>Protein</strong><br />
Salmon<br />
calcitonin<br />
IG/PEC<br />
system<br />
Encapsulation<br />
efficiency<br />
Particle<br />
size<br />
Chitosan-TPP 54-59% 0.9 mm<br />
Insulin Chitosan-TPP > 87%<br />
Bovine<br />
albu-<br />
serum<br />
min<br />
Dextran<br />
Horseradish<br />
preoxidase<br />
Chitosan/<br />
PEO/PPO-<br />
TPP<br />
Alginate-<br />
Calcium-<br />
Chitosan<br />
carrageenan<br />
ι<br />
amines -<br />
with Varies<br />
<strong>for</strong>mulations<br />
49-89%<br />
(depend-<br />
1-72%<br />
on amine<br />
ing<br />
employed)<br />
300-400<br />
nm<br />
200-1000<br />
nm<br />
-1.07 0.91<br />
mm<br />
50 µm<br />
Release kinetics Ref.<br />
burst (
226 Biotechnol. Bioprocess Eng. 2001, Vol. 6, No. 4<br />
effect and incomplete release behavior. As an ideal<br />
burst<br />
technique, one should satisfy the follow-<br />
encapsulation<br />
conditions. First, the manufacturing conditions<br />
ing<br />
be mild enough to conserve the protein stability<br />
should<br />
the process. Oil-water interface, shear <strong>for</strong>ces, or<br />
during<br />
may be major causes <strong>of</strong> protein denaturation and<br />
heat<br />
Second, high encapsulation efficiency is<br />
aggregation.<br />
Even if the progress <strong>of</strong> genetic engineering<br />
important.<br />
many therapeutic proteins available at a lower<br />
made<br />
than be<strong>for</strong>e, protein drugs are in general much<br />
price<br />
expensive than other low molecular drugs. Third,<br />
more<br />
is preferable to minimize the exposure to toxic or-<br />
it<br />
solvents. Fourth, the manufacturing process<br />
ganic<br />
be reproducible and easy to scale up to the<br />
should<br />
scale. Also, aseptic manufacturing should<br />
commercial<br />
available. Fifth, in order to ensure product quality as<br />
be<br />
injectable <strong>for</strong>mulation, uni<strong>for</strong>m size distribution <strong>of</strong><br />
an<br />
microparticles should be achievable.<br />
the<br />
numerous novel protein drugs coming<br />
Considering<br />
the genome projects, new microencapsulation sys-<br />
from<br />
that minimizes use <strong>of</strong> toxic organic solvents and<br />
tems<br />
denaturating conditions will be invaluable tools<br />
protein<br />
the future, and this is an exciting time to be involved<br />
in<br />
the microencapsulation technology.<br />
in<br />
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[Received May 22, 2001; accepted July 24, 2001]