Microencapsulation Methods for Delivery of Protein Drugs

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