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Advanced Drug Delivery Rev
Formulation aspects of biodegradable polymeric microspheres
for antigen delivery
Harjit Tambera,b, P3l Johansena,c, Hans P. Merklea, Bruno Gandera,*
aInstitute of Pharmaceutical Sciences, ETH Zurich, ETH-Hoenggerberg, HCI, 8093 Zurich, SwitzerlandbNapp Pharmaceuticals Research Ltd., Cambridge Science Park, Milton Road, Cambridge, CB4 0GW, U.K.
cDepartment Dermatology, University Hospital of Zurich, Gloriastrasse 31, 8091 Zurich, Switzerland
Received 31 March 2004; accepted 1 September 2004
Available online 30 September 2004
Abstract
Biodegradable microspheres (MS) have proven to be very useful antigen delivery systems that are ingested by
immunocompetent cells and provide prolonged antigen release and lasting immunity thanks to sustained release of the
microencapsulated material. This review provides an applicable summary of different formulation routes for the purpose of
producing safe, qualified and efficacious products of microencapsulated peptide and protein antigens. We have brought to
attention, with case examples, not only the most common means of improving the quality of microsphere formulations, i.e., the
use of stabilising additives, but also less commonly known and applied approaches, e.g., ion pairing, novel polymer systems,
solid-state and other innovative microencapsulation methods.
D 2004 Elsevier B.V. All rights reserved.
Keywords: PLGA microspheres; Antigen stability; Antigen microencapsulation; Antigen release
Contents
1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
2. Biodegradable polymers and methods for antigen microencapsulation . . . . . . . . . . . . . . . . . . . . . . . 359
2.1. PLGA as biodegradable matrix material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
2.2. Commonly used microencapsulation techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359
3. The challenges of antigen release testing and stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
3.1. Antigen release from microspheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360
3.2. Antigen stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361
0169-409X/$ - s
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ding author. Tel.: +41 44 633 7312; fax: +41 44 633 1314.
ess: [email protected] (B. Gander).
H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376358
4. Improving antigen stability during microencapsulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
4.1. Increasing the antigen concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
4.2. Addition of several antigens or nonantigenic proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362
4.3. Addition of surfactants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
4.4. Addition of osmolytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
4.5. Addition of other stabilising excipients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
4.6. Selection of polymer solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
4.7. Use of antigen powders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
4.8. Use of hydrophobic ion pairing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
5. Maintaining antigen stability during in vitro release testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
5.1. Use of additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
5.2. Use of pH modifiers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
5.3. Insoluble metal complexes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
5.4. Chemical modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
5.5. Other methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
6. Trends towards using more appropriate polymers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
6.1. PLA/PLGA blends. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
6.2. Modified PLA/PLGA and new polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 367
7. Trends towards using more appropriate technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368
7.1. Modifications of conventional methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 368
7.2. Atomisation using gases in the supercritical state . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
7.3. ProLeaseR technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
7.4. Ultrasonic atomisation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
7.5. Formation of semisolid microglobules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
7.6. Surface adsorption of antigens on preformed microspheres with ionic surface charge . . . . . . . . . . . 371
8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
1. Introduction
New vaccine formulations have to satisfy detailed
physicochemical quality control criteria to guarantee
the highest possible quality, safety and efficacy
standards. This implies that all components of the
formulation must be well chemically specified and
characterised. An important answer to this demand is
the use of specific antigen epitopes (so-called subunit
antigens), recombinant proteins or DNA. These
compounds can be readily purified and generally
offer greater safety than live attenuated or killed
pathogens. However, they require the presence of
adjuvants and, mostly, repeated dosing to boost and
maintain immune responses [1–3].
Until recently, hydroxide and phosphate salts of
aluminium and calcium were the only adjuvants
licensed for human use [4]. Although antigens
adsorbed to the hydrated aluminium salts are released
slowly [5], repeated injections are generally required
to mount a long-lasting immune response. As an
alternative, biodegradable polymeric microspheres
(MS) have been intensively studied for their feasibility
in single-injection vaccine formulations, i.e., vaccines
with priming and boosting doses in one formulation
[6–8]. The MS have mostly been made from various
types of poly(d,l-lactide-co-glycolide) (PLGA), as
such polymers are already commercialised for the
delivery of protein and peptide drugs.
PLGA MS can provide antigen release over weeks
and months following continuous or pulsatile kinetics
[9,10]. It was hoped that the pulsatile antigen release
would mimic the booster doses necessary with most
other nonlive vaccines [6] by controlling polymer
properties [10,11] and due to the fact that PLGA MS
are readily recognised and ingested by macrophages
and dendritic cells, an important property for stim-
ulating the immune system [12].
H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376 359
A major problem hindering the progression of MS-
based vaccine formulations for human use is the issue
of antigen stability during microencapsulation, storage
and release [13–17]. Nonetheless, means to retain and
maintain antigen stability and immunogenicity have
been proposed [18–20]. Consequently, this review
will focus on in vitro antigen stability and release
issues, with an attempt to elaborate on some of the
different approaches and strategies employed to
overcome these limiting factors.
2. Biodegradable polymers and methods for
antigen microencapsulation
2.1. PLGA as biodegradable matrix material
PLGA-types and related poly(hydroxyalkanoates)
have a long and successful history of medical and
pharmaceutical use in fields as diverse as sutures,
bone fixatives, artificial skins and cartilages, dental
materials, materials for bone regeneration, drug
delivery and many others, as well reviewed recently
by Ueda and Tabata [21]. For drug and antigen
delivery, mainly amorphous d,l-PLGA is used,
whose types differ in LA:GA monomer ratio (50:50
up to 100:0), molecular mass (Mw of approximately
10–100 kDa) and end-group chemistry (free carbox-
ylic acid or esterified carboxylic acid). These three
parameters largely determine the hydrophobicity
(water swelling) and degradation kinetics of the
materials, and thereby, the microencapsulation effi-
ciency and release rate of drugs and antigens. When
used as materials for MS, the PLGA hydrophobicity
will also affect interactions of the MS with phagocy-
tosing cells, such as macrophages and dendritic cells
[22]. These interactions are of crucial importance for
use of such MS in vaccine formulations.
For PLGA, the term biodegradable refers to a
nonenzymatic, hydrolytic cleavage upon contact of
any PLGA device with artificial or biological fluids.
PLGA-hydrolysis produces lactic and glycolic acids,
which are metabolised in the Krebs cycle to CO2 and
water [23,24]. When used as matrix material for MS,
PLGA degradation proceeds in two stages [25]. The
first involves the hydrolytic scission of the ester bonds
(degradation), generating oligomers and monomers
and a general decrease in the polymer molecular
weight. In the second stage (erosion), the MS lose
mass and the rate of polymer chain scission may
increase due to autocatalysis in the presence of acidic
degradation products [26,27].
2.2. Commonly used microencapsulation techniques
The most commonly used methods of antigen
microencapsulation encompass solvent extraction or
evaporation from a W1/O/W2-dispersion, coacervation
and spray-drying [28,29]. Each of these methods
employs a similar first step, where an aqueous antigen
solution is emulsified in an organic polymer solution
to form a water-in-oil dispersion (W1/O) (Fig. 1). If
appropriate, the antigen may also be dispersed as solid
powder in the organic polymer solution, or codis-
solved in a common solvent with the polymer. The
solution or dispersion is then processed according to
one of the mentioned microencapsulation methods.
In solvent extraction or evaporation, the antigen
solution or W1/O emulsion is further dispersed, in one
or two steps, into a larger aqueous volume containing
a suitable emulsifier, commonly poly(vinyl alcohol) to
form a double emulsion (W1/O/W2). Polymer hard-
ening and MS formation is induced by solvent
extraction into the W2-phase. Solvent extraction may
be facilitated either by the use of a cosolvent in the W2
phase, such as an alcohol or acetone, or by evapo-
ration of the solvent under atmospheric or reduced
pressure. At the end of the procedure, the solidified
particles are harvested, washed and dried.
Coacervation, also called polymer phase separation,
involves several stages of polymer desolvation and
hardening during which the solid MS are formed. To
the antigen solution or W1/O emulsion, an organic
nonsolvent for the polymer and proteinaceous com-
pound is added. The nonsolvent induces polymer
phase separation into a coacervate phase, engulfing the
proteinaceous compound, and a continuous phase. The
polymer solvent is then gradually extracted from the
coacervate phase, yielding polymer-rich and physi-
cally quite stable coacervate droplets. The two-phase
system is then transferred into a large volume of an
organic hardening agent (e.g., alkanes) miscible only
with the polymer solvent and nonsolvent. Here, the
solid MS are formed by rapid and efficient extraction
of the remaining polymer solvent from the coacervate
droplets. The MS are harvested, washed with a suitable
Fig. 1. Conventional microencapsulation methods. An aqueous antigen solution is dispersed into an organic polymer solution by ultrasonication
or homogenisation (W1/O emulsion). The W1/O emulsion is processed further by the specific methods to prepare antigen containing MS: (1)
Solvent extraction or evaporation; (2) Spray-drying; (3) Polymer phase separation. In the final stages before drying and storage, the MS are
collected and washed with water to remove nonencapsulated antigen.
H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376360
volatile nonsolvent for the polymer to remove residual
coacervation liquids, and dried [10,30,31].
Spray-drying offers an attractive and relatively
simple alternative to the previous two methods. Here,
the antigen solution or W1/O emulsion is atomised in a
flow of drying air at slightly elevated temperature. The
organic solvent is rapidly vaporised leaving behind
solid MS that are separated from the drying air in a
cyclone and collected in a deposition chamber [32,33].
3. The challenges of antigen release testing and
stability
3.1. Antigen release from microspheres
Single-injection vaccine formulations should be
capable of evoking immune responses similar to those
elicited after multiple immunisations with current
vaccines. Hence, the focus of the majority of inves-
tigations has been towards developing MS providing
pulsatile antigen release. By mixing MS types with
different degradation and pulsatile release kinetics,
multiple discrete booster doses of microencapsulated
hepatitis B surface antigen (HBsAg) was provided
after a single administration of the formulation [34].
Similarly, a regime has been proposed for a single-
injection tetanus vaccine, where after the priming dose,
booster doses of the toxoid would be delivered at
approximately 1–2 and 6–12 months [35]. However,
the concept of continuous antigen release should not
be disregarded, since continuous exposure to low
quantities of antigen may also be useful for inducing
and maintaining protective immunity [36,37].
Antigen release from MS essentially occurs
through diffusion and polymer erosion. Upon incubat-
ing MS in an aqueous medium, antigen located at or
near the particle surface is dissolved by the penetrat-
H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376 361
ing waterfront and diffuses out into the surrounding
medium within a very short time (burst release).
Release after this initial burst depends on MS porosity
and hydrophilicity, as well as molecular interaction
forces between polymer and antigen [32,33]. In
porous and hydrophilic MS or if there is little affinity
between antigen and polymer, water penetration into
the MS and antigen dissolution/diffusion out of the
matrix are facilitated. In this case, a second phase of
continuous release may succeed the burst, resulting in
final antigen release before MS erosion reaches an
advanced stage (total of two release phases). When
MS possess a dense core structure or the antigen
interacts strongly with the polymer, a lag phase with
minimal antigen release may be observed. A lag phase
may also be seen if polymer hydrophobicity restricts
water uptake into the core or when MS swelling
causes pores and channels to collapse and block
further antigen release. The duration of the lag phase
depends on the polymer degradation kinetics. During
the final stage of MS erosion, antigen diffuses out of
the eroding matrix through expanded pores and
channels (total of three release phases). Therefore,
by selecting specific polymers for microencapsula-
tion, different schedules for pulsatile antigen release
are achievable [9,10].
Table 1
Causes of physical and chemical antigen instability
Mechanism of antigen instability
W1/O emulsion formation
Increased aqueous phase surface area and new W1/O interface:
Antigen adsorption, unfolding and exposure of hydrophobic domains to o
Protein unfolding due to high shear forces during emulsification
Chemical degradation at W1/O interface
Freeze-drying of microspheres
Poorly developed drying method resulting in instability or aggregation of
Storage
Residual solvents and moisture absorption:
Solvent/moisture induced aggregation
Change in PLGA characteristics, such as Tg and hydrolytic resistance, aff
Incubation in simulated/physiological environment at 37 8C:Protein aggregation during rehydration in aqueous environment
Chemical reactions: thiol-disulfide exchange, deamidation, oxidation, acyl
Protein adsorption at polymer/liquid interfaces
Instability and degradation due to acid-catalysed reactions in acidic micro
hydrolysis
3.2. Antigen stability
The uttermost criterion for delivery systems is the
capacity to deliver the entrapped material in a
bioactive form, i.e., a fully immunogenic form for
antigens. Antigen instability is, however, one of the
major obstacles in the development of MS vaccines.
Instability arises through the various stages of
processing, storage and application [38]. Therefore,
it is of vital importance to scrutinise the causes of
antigen instability (Table 1), which may be of
chemical or physical nature [15,39]. Physical insta-
bility often develops through conformational changes
leading to denaturation, surface adsorption, aggrega-
tion or precipitation of the antigen and is considered
critical in microencapsulation technology [40]. Natu-
rally, the extent of chemical and physical instability
affects the immunogenicity of embedded and released
antigen [41]. Antigen stability may be hampered at
various stressful stages, such as the generation of the
aqueous/organic interface (W1/O emulsion) in the
microencapsulation process or in the final freeze-
drying stage [42]. The storage stability of micro-
encapsulated antigens should be increased over that of
fluid vaccines, as antigen stability in the dry state is
generally greater than in solution. Yet, residual
Reference
rganic front [43,52,57]
[15]
[130]
insufficiently stabilised antigen [49]
[15,49,88,147,148]
ecting antigen stability and release [148]
[42]
ation and hydrolysis [149,150]
[42,151]
environment created during polymer [45,152–154]
H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376362
solvents in the MS or imbibed moisture can have
deleterious effects on both the antigen and the PLGA
characteristics. Rehydration of the MS in simulated or
physiological fluids also introduces further conse-
quentially harmful conditions which lead to antigen
instability.
4. Improving antigen stability during
microencapsulation
Issues of antigen instability may be resolved
through coencapsulation of stabilising additives,
solid-state microencapsulation or physicochemical
stabilisation of the antigen itself, as well as through
improving encapsulation conditions or polymeric
materials. The principal aim remains at minimising
reversible unfolding and preventing irreversible
aggregation and chemical degradation.
4.1. Increasing the antigen concentration
Antigen adsorbed and denatured at the W1/O
interface is often considered as a fixed loss. With
low amounts of antigen, the proportion of irrecover-
able antigen may be quite high, resulting in only
modest microencapsulation efficiency. Studies have
shown that interfacial denaturation depends on the
antigen concentration. When aqueous solutions of
ribonuclease A (RNase) were emulsified with
dichloromethane (DCM), the amount of recoverable
protein increased from 78% to 93% when its concen-
tration was raised from 0.2 to 1.5 mg ml�1 [43].
Similarly, recovery of soluble monomers of human
growth hormone (rhGH), after emulsification with
DCM, improved from 53% to 86% after raising its
concentration from 10 to 100 mg ml�1 [16]. These
data indicate only limited amounts of protein irrever-
sibly adsorbed to the interface; at higher concen-
trations, they behave as bself-protectantsQ.
4.2. Addition of several antigens or nonantigenic
proteins
Protein excipients with significant interfacial activ-
ity, e.g., serum albumins, have been widely used as
stabilisers for various proteins. As an example, RNase
recovery from a W1/O system was maximised after
addition of human serum albumin (HSA) at a
concentration largely exceeding that of RNase [43].
This was ascribed to a greater rate of HSA transfer
from the bulk to the W1/O interface, thus restricting
RNase adsorption and aggregation at the interface. In
our own studies, precipitation of aqueous diphtheria
toxoid (Dtxd) during emulsification with solutions of
stearyl-poly(l-lactide)-stearate in DCM was inhibited
upon addition of 2% bovine serum albumin (BSA) to
the aqueous phase [44]. BSA (1–5%) also improved
the encapsulation of ELISA-reactive tetanus toxoid
(Ttxd) of different qualities into PLGA MS by a factor
of 3 or N100 [45]. Recently, microencapsulation
efficiencies of Haemophilus influenzae b antigen
(Hib), Ttxd, Dtxd and pertussis toxoid (Ptxd) were
increased to 60–75% for all antigens, when several
antigens were coencapsulated rather than the individ-
ual ones. These microencapsulation efficiencies of
ELISA-reactive antigens was further improved to
N80% when BSA was coencapsulated and resulted
in strong immune responses for all antigens [46,47].
Similarly, HSA, BSA and rat serum albumin (RSA)
stabilised aqueous Ttxd in contact with DCM,
increasing the ELISA reactivity from b10% without
albumin to 70–80% in the presence of 2% protein
[48]. Erythropoietin (EPO), another readily aggregat-
ing protein, was successfully encapsulated into PLGA
MS only in the presence of BSA, which increased the
entrapment of soluble EPO monomers and lowered
the proportion of insoluble EPO aggregates from 5%
(no BSA) to below 1% (with BSA) [49].
On a precautionary note, the use of albumins and
other stabilising proteins raises safety issues [50]. The
immunogenicity of microencapsulated proteins is
generally altered so that new immunogenic epitopes
on the stabilising protein may be revealed, resulting
from exposure to the solvent or coating polymer. This
could eventually lead to autoimmune reactions fol-
lowing protein release from MS. Therefore, Chang
and Gupta [50] selected porcine gelatine type A over
HSA, for safety reasons, to stabilise Ttxd in PLGA
MS. Although microencapsulation of Ttxd protein
decreased with gelatine, the fraction of antigenic Ttxd
released in vitro was improved. Gelatines play a
double role as stabilisers in antigen microencapsula-
tion, i.e., as viscofiers to increase protein/peptide
encapsulation efficiency [51], and as protectants to
restrict antigen exposure to interfaces. Another study
H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376 363
highlighted the importance of gelatine type and pH
towards hepatitis B core antigen (HBcAg) stability
[52]. Aqueous HBcAg solutions (1–25 Ag ml�1)
exposed to DCM for several hours retained complete
ELISA reactivity in the presence of 4–8% (w/w) of a
10–150 kDa gelatine and a pH of 6. Conversely, lower
molecular weight fractions of gelatine or pH values
below 6 were less stabilising. Excellent HBcAg
stability in the presence of gelatine was further
reflected by the high encapsulation efficiency in
PLA MS (61%). Gelatine has also been used for
microencapsulating Dtxd [53,54].
4.3. Addition of surfactants
Protection offered by surfactants is primarily a
function of their surface activity. Unlike proteins,
which reduce antigen loss by inhibiting unfolding and
aggregation at interfaces, surfactants provide addi-
tional protection against irreversible aggregation of
partially denatured antigens [55]. However, surfactant
use should be limited to the minimum level required
to avoid possible toxic and hypersensitivity reactions
[56].
Poloxamer 188, a poly(ethylene oxide)-b-poly(pro-
pylene oxide)-b-poly(ethylene oxide) (PEO-PPO-
PEO) block copolymer, partially reduced Ttxd aggre-
gation during emulsification of aqueous Ttxd solutions
with DCM [57]. Limited stabilising activity of
poloxamer 188 was found for Ttxd at a W1/O inter-
face, i.e., maximal 15% ELISA reactivity as the
surfactant concentration was increased from 0.1% to
1% [48]. The limited stabilising properties of the
polymeric surfactant in the presence of DCM may
partly be attributable to its solubility in the organic
solvent.
Poloxamer 188 lowered BSA encapsulation into
PLGA MS by up to 20%, as assessed by chromatog-
raphy (BSAmonomer) and spectrophotometry (BSAtotal)
[58]. Interestingly, however, poloxamer reduced the
percentage of BSA aggregates from 31% to 5%, as
estimated from the difference between BSAtotal and
BSAmonomer. Complex interactions between polox-
amer, BSA and PLGA were believed to have
influenced BSA microencapsulation [59]. Other
PEO-PPO-PEO block copolymers have also exhibited
stabilising properties. EPO aggregates in PLGA MS
decreased when the poloxamer 407 was incorporated
at a level of 10% (w/w) [49]. Moreover, the
bioactivity of urease in PLGA MS improved with
poloxamer 407 from 63% to 89% [60].
Nonionic surfactants can interact with both pro-
teins and organic solvents [61]. The balance of these
interactions determines whether a surfactant is useful
or not for stabilising proteins at a W1/O interface. The
addition of 1–10 mg ml�1 of either polysorbate 20 or
polysorbate 80 to an aqueous solution of rhGH (10
mg ml�1) increased the recovery of native rhGH by
11–25% [16]. Conversely, the surfactant’s stabilising
properties diminished at high protein concentrations
(~100 mg ml�1), and recovery of native protein was
reduced by 16–27%, possibly due to a partially
denatured form of rhGH, stabilised by the surfactant.
Exchange of polysorbate 20 for a less hydrophobic
surfactant, PEG 3350 (2–10 mg ml�1), provided
almost complete rhGH recovery irrespective of
protein concentration. However, an opposing trend
was seen with EPO encapsulation in PLGA MS [49].
Encapsulated protein aggregates increased (~15%)
with different PEG types (0.4–10%, w/w) codissolved
in the W1 phase.
4.4. Addition of osmolytes
Osmolytes, such as polyols, carbohydrates and
amino acids are frequently used as protein stabilisers
in parenteral formulations [56,62]. One of their
stabilising properties is by strengthening the water
structure, which favours the compact native form and
inhibits unfolding of proteins [63]. Osmolytes also
substitute for water during drying, whereby hydrogen
bonds play an important role [64].
In MS technology, trehalose and mannitol (osmo-
lyte concentration of 50 mg ml�1) preserved the
stability of aqueous rhGH following emulsification
with DCM [16], while the stability of rhIFN-g was
only slightly improved (~63% recovered) with man-
nitol, but fully preserved with trehalose. Trehalose also
improved the encapsulation of the malaria antigen
TBV25H [14] and of ELISA-reactive Ttxd in PLGA
MS [18], although this was not the case for Dtxd [44].
Dextrans of different molecular weights (Mw) were
investigated for stabilising EPO [49] and rhGH [16].
Microencapsulated EPO aggregates were only margin-
ally reduced with dextran (40 kDa Mw; 5%, w/w),
whereas rhGH stability was adversely affected (70 kDa
H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376364
Mw; 50 mg ml�1). The recovery of water-soluble rhGH
decreased by over 40%. In addition, arginine (0.2–
4.8%, w/w) reduced EPO aggregation, although not in
combination with dextran (40 kDaMw; 5%, w/w) [65].
From the above studies, the stabilising properties
of osmolytes appear to be balanced between their
binding to (deteriorating effect) and exclusion from
(stabilising effect) the antigen surface. As binding or
exclusion predominantly results from hydrophobic
interactions, hydrogen bonding and electrostatic inter-
actions, the sum of the various interaction parameters
are dissimilar for different antigens. Therefore, it
becomes crucial to examine the individual nature of
the additive towards each individual antigen and to
assess whether it will offer either a stabilising or
destabilising effect [64,66].
4.5. Addition of other stabilising excipients
Numerous other types of additives have been used
for stabilising antigens during microencapsulation.
Both poly(vinyl alcohol) (PVA) and methylcellulose
(0.4–3%, w/w) were used in coencapsulating F1 and
V subunit antigens of Yersinia pestis into PLA MS
[67,68]; the content of ELISA-reactive antigens
improved 14- and 30-fold. PVA was also used as a
steric barrier between the W1/O interface to preserve
the integrity of the recombinant 28 kDa glutathione S-
transferase of Schistosoma mansoni (rSm28GST)
[69]. A known feature of such hydrogel forming
polymers is their capacity to stabilise emulsions
through increased solution viscosity [70]. Here, this
function may have been important in reducing the
mass transfer rate of antigen to the W1/O interface,
thus lowering encapsulation of interface-denatured
antigen.
Cyclodextrins (a, h and g) were examined for
encapsulating Ttxd in PLGA MS [18], with g-
hydroxypropyl-cyclodextrin effectively increasing
Ttxd encapsulation. g-HPCD also inhibited EPO
aggregation during microencapsulation [49].
Although the precise mechanism is unclear, interac-
tions between amino acids and the hydrophobic inner
cavity of cyclodextrins may play a role [71]. Further
examples of additives investigated include carboxy-
methyl cellulose [16], hydrophobic compounds such
as ethyl stearate, sodium acetate and sodium gluta-
mate [18,57], sorbitol [72], and others [13,70].
4.6. Selection of polymer solvents
Solvent properties are known to influence antigen
microencapsulation [32,33]. When BSAwas encapsu-
lated into PLA MS utilising different polymer
solvents, BSA contents were comparable when
DCM and ethyl acetate were used as polymer solvents
(~100%), whereas water miscible solvents lowered
the ELISA-reactive fraction (b60%) [73]. DCM and
ethyl acetate had quite distinct effects on rhGH
stability [16]. Protein recovery was good with ethyl
acetate (N93%), but not with DCM (53%), in which
case additives were a prerequisite to maintain rhGH
stability. Similarly, aggregate formation increased and
antigenicity deteriorated following exposure of Ttxd
to DCM [57], whereas ethyl acetate exerted little
effect. Interestingly, the length of protein exposure to
the solvent interface may be a critical factor [19].
Dtxd, on the other hand, showed the reverse
behaviour [44]. During preparation of W1/O emul-
sions, the toxoid precipitated in contact with PLA and
PLGA in ethyl formate, but remained soluble when
replaced with DCM.
4.7. Use of antigen powders
Exposure of antigen to potentially harmful aqueous
conditions or W1/O liquid interfaces can be avoided
by nonaqueous microencapsulation procedures, typi-
cally using dried antigen powders. The dry state offers
increased stability owing to the reduced conforma-
tional flexibility and, hence, less potential for struc-
tural perturbations. Microencapsulation of solid
antigen powders may involve a first step of either
spray-drying or freeze-drying aqueous antigen, or
embedding the aqueous antigen into water-soluble
excipients which act as protective barriers against the
organic solvent; in a second step, the dry antigen
powder or embedded antigen is then dispersed in the
organic polymer solution.
Nonaqueous processing has been successful for
several proteins and peptides. During BSA encapsu-
lation into PLGA MS by a solid-in-oil-in-water (S/O/
W) method [74], the protein secondary structure was
less altered as compared to encapsulation by an
aqueous W1/O/W2 method. When spray-freeze-dried
BSA was microencapsulated by an oil-in-oil coac-
ervation method [75], reduction of a-helical content
H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376 365
and increases in h-sheet and random structure were
less pronounced when trehalose was added for spray-
freeze-drying (1:4 BSA:trehalose). Similarly, rhGH
was also prestabilised with various excipients and
encapsulated as solid particles into PLGA MS [16].
Retention of monomeric protein depended highly on
formulation parameters; freeze-drying with the cry-
oprotectant mannitol or lactose completely eliminated
rhGH aggregation.
Encasing the antigen in a stabilising matrix has
also proved to be effective against denaturation.
HBsAg preembedded into hydroxypropylcellulose
(HPC) (HBsAg:HPC 1:5–1:15) and then further
encapsulated, as solid particles, into PLGA MS
remained 90% antigenic [20]. After dispersing the
HBsAg:HPC particles in various organic solvents,
HBsAg antigenicity dropped to 50–80%, whereas the
uncoated antigen lost almost entirely its antigenicity.
Similarly, preentrapment of horseradish peroxidase
into PEG particles allowed further encapsulation into
PLGA MS without a substantial loss of activity [76].
4.8. Use of hydrophobic ion pairing
Aqueous processing can also be avoided by the use
of protein- or peptide-counter-ion complexes. The role
played by the counter-ion is solely to decrease the
aqueous solubility of the protein or peptide and
enhance its dissolution in nonaqueous media, such
as organic (polymer) solvents. In one example,
lysozyme-oleate was encapsulated into PLGA nano-
particles by an O/W method [77]. After incubation at
80 8C for 60 h, the unprotected lysozyme lost almost
30% of its a-helical content, whereas the hydrophobic
complex, dissolved in dimethylsulphoxide, retained
~95% of a-helix structure. The increased structural
stability was ascribed to restricted chain mobility of
the protein in the complex.
5. Maintaining antigen stability during in vitro
release testing
5.1. Use of additives
The first approach towards improving antigen
integrity and immunogenicity during incubation and
release is generally through the coencapsulation of
additives, as discussed in the previous section.
However, one of the limitations of coencapsulated
water-soluble additives may be their limited residence
time within the hydrated MS. Nevertheless, Ttxd has
been kept antigenic for up to 60 days of pulsatile in
vitro release when additives such as trehalose and
BSA had been coentrapped in the MS [18]. The
promising in vitro data were confirmed by the high
antibody response induced in mice with MS stabilised
with BSA and trehalose [78]. Conversely, coencap-
sulation of other water-soluble additives yielded MS
which released some Ttxd in a moderate burst (~20–
40%), though with virtually no further release of
antigenic protein [19,50]. Here, the additives tested
appeared to confer little stability to the antigen during
in vitro release testing, possibly due to their own early
release.
When PVA was coencapsulated with the recombi-
nant glutathione S-transferase of S. mansoni
(rSm28GST) by spray-drying [69], the produced MS
released the antigen in fully active form during 28
days. It may be conceived that the increased viscosity
and lower acidification of the aqueous medium inside
the microspheres were critical for maintaining antigen
stability over the 28 days (pH 6–8 for 1% PVA
solution).
5.2. Use of pH modifiers
The development of an acidic microclimate within
the MS upon polymer degradation and the continued
exposure to this acidic environment may induce
antigen degradation and aggregation, leading to loss
of antigenicity. As a countermeasure, pH buffering
salts may be incorporated into the polymer matrix to
sustain a more favourable pH environment.
Improvement of antigen stability through pH
moderation was illustrated with PLGA MS, where
salts of differing basicity (ZnCO3bMg(OH)2~Mg
CO3bCa(OH)2) were coencapsulated [79]. Salt-free
MS contained noncovalent BSA aggregates and pep-
tide fragments comparable to those seen in aqueous
solutions of pHb3 stored for up to 12 days. Non-
covalent aggregates were diminished when Mg(OH)2or MgCO3 were coencapsulated. Typically, with the
more soluble MgCO3, the BSA fraction released
increased from 16% to 68% after 51 days, with
noncovalent aggregates being reduced from 24% to
H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376366
1.5%. On the other hand, disulfide-bonded aggregates
formed when the stronger base Ca(OH)2 was used,
suggesting a neutral to alkaline microclimate. Gener-
ally, aggregation was reduced at higher salt content,
although at the cost of faster release rates through
increased water uptake and osmotic effects [80]. The
stabilising effect of Mg(OH)2 was also demonstrated
for the release of the acid-labile basic fibroblast growth
factor and bone morphogenic protein-2 [79]. However,
poorly water-soluble and weakly basic calcium salts
[CaCO3 and Ca3(PO4)2] did not significantly improve
the release of ELISA-responsive Ttxd, despite their
effect on the pH of the in vitro release test medium [18].
5.3. Insoluble metal complexes
Reversible complex formation with metal ions
represents an elegant means of antigen stabilisation.
Physicochemical antigen integrity can be preserved for
prolonged periods of time, until dissociation from the
complex, dissolution and release from the MS, by
encapsulating insoluble complexes with the proteins.
Zn-salts have been successfully used to form stable
insoluble complexes with, e.g., insulin [81], r-hirudin
[82] and hGH [83]. These studies demonstrated retarded
dissolution rates of the protein from the complex.
A major investigation into this approach saw the
encapsulation of a rhGH:Zn complex by a nonaqueous
procedure into PLGA MS [84,85]. The rhGH:Zn
complex was initially formed as a precipitate between
rhGH and zinc acetate (rhGH:Zn 1:6, w/w) in aqueous
media and subsequently microencapsulated. In the
burst release stage (initial 48 h), mostly monomeric
rhGH was released from MSrhGH:Zn, whereas substan-
tial amounts of dimerised and aggregated protein was
released from MSrhGH. Release of purely monomeric
and bioactive rhGH fromMSrhGH:Zn continued over 28
days. Critical to ensuring release of intact rhGH was an
excess of Zn, provided by coencapsulating ZnCO3
(1%, w/w), which may also have offered some
buffering capacity. This approach of encapsulating
protein–metal complexes should be applicable to
many proteins/peptides.
5.4. Chemical modification
The physicochemical instability of microencapsu-
lated antigens, arising when MS become exposed to
aqueous media, is predominantly due to reactive side
chains of amino acids [86]. Chemical modification of
antigens, e.g., by inter- or intramolecular cross-link-
ing, derivation or covalent conjugation, may yield
immunogenically more stable compounds.
With BSA, for which degradation pathways are
well characterised [87] and which tends to aggregate
readily in hydrated MS, the nature of aggregation
typically occurs by thiol-disulfide exchange. With the
free thiol group blocked and the resulting carbox-
ymethylated BSA encapsulated (CM-BSA), no
increases in covalent aggregates were found in MS
incubated for 28 days [42]. Moreover, the release of
protein monomers over 56 days improved from 40%
(BSA) to 80% (CM-BSA). Aggregation of Ttxd and
Dtxd (formalinised toxins) in the presence of moisture
is caused by intramolecular nondisulfide cross-linking
[88] and has also been claimed to be one of the causes
behind incomplete Ttxd release from PLGA MS [18].
Moisture-mediated aggregation of the toxoids can be
prevented by chemical modifications, such as succi-
nylation of free amino groups or reduction of reactive
amine groups [88], which might also improve the
delivery over prolonged periods.
5.5. Other methods
Liposomal entrapment of antigens has been found
useful for retaining antigen stability and enhancing
immunogenicity [89]. Microencapsulation of lipo-
some-entrapped antigens, such as influenza hemag-
glutinin (HA), has been realised as a means of
improving antigen release [90]. Release of ELISA-
reactive HA from such systems in vitro followed a
pulsatile pattern over 50 days. A significant second
pulse of antigenic HA occurred only when it was
preentrapped in liposomes. Similarly, liposomal
preentrapment of BSA prior to encapsulation into
PLGA MS allegedly improved the stability of the
protein prior to and during release [91].
Polyethylene glycol modifications, so-called
PEGylations, often impact favourably on retention
of bioactivity and immunogenicity of peptides and
proteins. PEGylated peptide antigens have shown
prolonged in vitro and in vivo half-lives [92,93]. In
MS, PEGylated lysozyme (PEG-lysozyme) was more
resilient to DCM compared to native lysozyme [94],
and its release from PLGA MS was pulsatile,
H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376 367
proceeding with a small burst (~10%) and followed by
further release (N90%) between days 35 and 83.
Conversely, native lysozyme was only released in
significant quantity within the first few days of
incubation in vitro (50% of dose). PEG-lysozyme
also adsorbed substantially less onto blank MS
compared to lysozyme alone. The improved stability
of PEG-lysozyme was ascribed to steric protection of
lysozyme by PEG, shielding the protein from the
denaturing W1/O interface during microencapsulation
and inhibiting protein–polymer interactions as well as
aggregation. Therefore, PEGylation is a promising
route to improving antigen delivery from MS and one
which would deserve further development and exploi-
tation [95].
6. Trends towards using more appropriate
polymers
Numerous issues are associated with PLA/PLGA
MS such as their low glassy-to-rubbery-state tran-
sition temperature (Tg), the relatively hydrophobic
interface they offer to proteins, and the production of
acidic degradation products. While the low Tg may
cause softening and coalescence of the PLGA MS at
relatively warm environmental temperatures (30–35
8C), the latter two phenomena are detrimental to the
efficient entrapment and release of stable antigens.
Therefore, new and improved biodegradable delivery
systems are desirable. Where entirely new polymers
are not of interest, lateral approaches can be consid-
ered to exploit the properties of available materials.
6.1. PLA/PLGA blends
Physicochemical properties and degradation rates
of specific polymers can be fine-tuned by blending
with different polymer types. The blending of hydro-
phobic, crystalline polymers with hydrophilic, amor-
phous polymers may improve protein and peptide
entrapment in matrices [96] or adjust their release to
suit a particular need [97–99]. Ideally, the polymers
should be miscible to rely on the additivity of
properties [100]. Blending of polymers may also
improve antigen stability and release, as illustrated
with a blend of PLGA and poloxamer for the
entrapment of Ttxd [101,102]. Here, poloxamer 188
(10–50%, w/w) was blended with PLGA to inhibit
allegedly detrimental interactions between Ttxd and
PLGA. While PLGA MS exhibited a fast initial burst
release, blended PLGA/poloxamer MS provided an
improved pulsatile delivery of antigenic Ttxd, with the
pulse occurring between 22 and 50 days. The small
initial burst and extent and duration of the pulse was
dependent on the poloxamer content in the blend.
PEG has also been blended with PLA to improve
BSA delivery from MS [103]. At PEG contents of
below 20% (w/w), water insoluble noncovalent
aggregates formed in the MS, whereas above this
level, encapsulated BSA remained structurally unal-
tered and water soluble. Similarly, aggregation and
degradation of encapsulated insulin and ovalbumin
during incubation in vitro was diminished in blended
PEG/PLA MS [104,105]. Most of the blended PEG/
PLA MS showed a near-constant release rate of
encapsulated protein, which was attributed to the
increased water uptake and porosity of the MS
following rapid dissolution of the hydrophilic PEG.
The fast release of PEG from the MS was ascribed to
its partial miscibility with PLA. This created extensive
porosity that facilitated clearance of acidic polymer
degradation products, possibly balancing the micro-
environment pH and helping to maintain protein
stability prior to release.
6.2. Modified PLA/PLGA and new polymers
Similar to the principle behind blending different
polymer types, PLA and PLGA can be chemically
tailored to suit particular requirements. Attachment of
either hydrophilic or hydrophobic segments to the
polyester can alter its hydrophobicity, thus influencing
antigen microencapsulation, adsorption and stability
as well as polymer degradation kinetics. For example,
antigen adsorption and denaturation has been mini-
mised by introducing PEG into PLA chains to form
PLA-PEG-PLA blocks [106]. According to the
authors, PEG mediated good BSA entrapment (93–
99% efficiency), due to its stabilising properties at the
W1/O interface. Consequently, this reduced BSA
adsorption onto the polymer and increased the amount
of protein available for release. In another study,
glucose oxidase activity was increased in PLA-PEG-
PLA block polymer MS as compared to PLA or
PLGA MS [107]. Again, this was attributed to the
H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376368
hydrophilic environment created by PEG chains.
PLA-PEG-PLA MS also provided pulsatile glucose
oxidase release, with the onset of the pulse arriving
sooner with increasing PEG content (0–30%). PEG
also served to protect the antigen from the degrading
polymer components. PLGA-PEG-PLGA block poly-
mers were also successfully evaluated for the delivery
of other proteins [65,108,109].
Contrary to increasing polymer hydrophilicity,
introduction of hydrophobic groups into the polymer
chain may also serve to maintain antigen stability by
retarding water uptake and subsequent moisture-
induced antigen instability. The introduction of,
e.g., fatty alcohol or acid moieties into PLA or
PLGA is preferably done with low molecular weight,
crystalline l-PLA, which is hydrophobic, slowly
degrading, and can be processed with an adequate
Tg or Tm of the end polymer [110]. For this purpose,
10–20 kDa stearyl-poly(l-lactide)-stearate and oleyl-
poly(l-lactide)-oleate were proposed and processed
by spray-drying or solvent evaporation into MS
[111]. BSA release from such hydrophobic MS was
slow (burstb10%), with little additional release over
15 weeks (20–40%), in agreement with the slow
polymer degradation kinetics [111,112]. Such delayed
release systems, potentially capable of providing
pulses of stable and immunogenic antigen after long
periods of dormancy, might be very appealing for
vaccine delivery [44,113].
Poly(ortho esters) (POE) have been available for
over 30 years, although their potential for antigen
delivery was only illustrated recently. POE degrada-
tion and erosion times can vary between days and
months. Among the various POE classes, class IV
PEO are the most hydrophobic and contain backbone-
integrated lactides or glycolides, which catalyse
polymer hydrolysis and thereby control polymer
erosion and antigen release. Studies with BSA and
rhGH in POE IV matrices showed some correlation
between release and polymer erosion [114]. The
hydrophobic particle surface ensured a low burst,
and the duration of the lag phase was related to the
polymer weight. Other POE modifications were with
PEG 4600, yielding hydrophilic POE-PEG-POE
block polymers [115]. POE hydrophilicity was
increased by raising the PEG content, which improved
the stability of W1/O emulsions during solvent
evaporation and increased BSA encapsulation effi-
ciency from 32% to 90%. BSA release was slightly
pulsatile, and the total amount released attained 60–
70% of the dose [116]. Protein integrity (SDS-PAGE)
was maintained for up to 8 weeks.
Similar to POE, triblock polymers of poly(butylene
terephthalate) and PEG (PBT-PEG-PBT) have shown
prospectives in antigen delivery [117]. The synthes-
ised PBT-PEG-PBT contained multiple sequences of
short-chain segments of PBT-PEG(600–1000), which
should limit the loss of PEG while maintaining a more
hydrophilic structure. Lysozyme encapsulation into
PBT-PEG-PBT MS by solvent evaporation was
improved as PBT-PEG-PBT appeared to stabilise the
W1/O emulsion. Most strikingly, lysozyme release
from MS was almost complete and followed zero-
order kinetics, which contrasts previous lysozyme
release data from PLGA particles [94].
A very interesting and recent approach used
biodegradable polymers carrying cationic or anionic
groups, such as sulfobutylated copolymers [118,119]
and chitosan [120]. MS made from such polyelec-
trolytes exposed surface charges, which were used to
adsorb oppositely charged protein antigens or DNA
onto the polymers. The great advantage of this
approach resides in the mild conditions that prevail
for protein or DNA loading. Provided that the ionic
interaction between the particle surface and the
adsorbate does not hamper the activity and availability
of the bioactive material, such systems should hold
great promise for antigen and DNA delivery (see
Section 7.6).
7. Trends towards using more appropriate
technologies
Conventional microencapsulation methods involve
relatively harsh conditions that are not generally
tolerated by antigens without stabilisation. Therefore,
new and improved processes shielding the antigen
from deleterious conditions have been proposed and
evaluated.
7.1. Modifications of conventional methods
The W1/O/W2 solvent evaporation or extraction is
probably one of the most widely used methods for
peptide and protein microencapsulation [70], despite
H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376 369
its many drawbacks. Improvements and alternatives
have therefore been proposed such as O/W, *O/W
(*including cosolvent) and O1/O2 [121].
Utilising a modified W1/O/W2 method, recombi-
nant human insulin-like growth factor I (rhIGF-I) was
encapsulated into PLGAMS after increasing the pH of
the protein solution from pH 4.5 to a value of pH 5.5–
6.0, where rhIGF-I formed a viscous gel [122]. High
entrapment efficiency of fully bioactive protein was
achieved, and 92–100% of pure, monomeric and
bioactive rhIGF-I was released in vitro over 21 days.
The lowering of the rhIGF-I solubility at pH of 5.5–6.0
probably restricted its conformational flexibility and
changes upon exposure to the polymer solvent. With-
out pH adjustment, approximately 10–32% of rhIGF-I
was lost upon solvent exposure, due to degradation
and aggregation. Elsewhere, a W1/O1/O2 system was
investigated for encapsulating different proteins and
peptides, with the O1 and O2 phases consisting of
acetonitrile/DCM and liquid paraffin/Span 80, respec-
tively [123]. The acetonitrile mediated partial mixing
of the Wand O1 phases and subsequent protein/peptide
precipitation, which was a prerequisite for micro-
encapsulation. The proteins BSA, Ttxd and lysozyme
precipitated at low acetonitrile concentration, resulting
in efficient microencapsulation (N90%), while a
decapeptide and a linear gelatine did not precipitate
so rapidly, resulting in poor entrapment. Ttxd and
lysozyme released during the burst phase (15%)
maintained their bioactivity, although lack of further
release suggested aggregation within the MS.
Another approach consisted of dispersing the
antigen in a mineral oil before encapsulation into
PLGA MS by a O1/O2/W method [124]. The mineral
oil (O1) was intended as a barrier to protect the antigen
during emulsification with the polymer solution and
from exposure to moisture during release. Over 92% of
ELISA-reactive Ttxd was released from the reservoir-
type MS in a pulsatile pattern, proceeding with an
initial burst and followed by a second release pulse
between 14–35 or 35–63 days, depending on the
polymer type used. The latter stage of release was
ascribed to Ttxd diffusion through the oily phase, once
an appreciable loss of polymer mass had occurred. The
authors claimed the mineral oil was the key to protect
the solid antigen during polymer erosion, where acidic
degradants and moisture would otherwise have led to
antigen inactivation.
To improve solvent extraction, a novel method
using a static micromixer was recently presented
where a W1/O dispersion (aqueous BSA in organic
PLGA solution) is fed into an array of microchannels
and the extraction fluid (W2) into a second array of
interdigitated channels [125]. The two fluids, trans-
ported separately through the channels, are dis-
charged through an outlet slit where alternating
fluid lamellae are formed with the W1/O fluid lamella
disintegrating into microdroplets, which harden
quickly to form MS. This process offers easy scale-
up, methodological robustness, continuous produc-
tion and a simple setup, making it ideally suited for
aseptic production, a strongly needed feature for MS
vaccine formulations.
7.2. Atomisation using gases in the supercritical state
Atomisation of PLA and PLGA solutions using
gases, e.g., CO2, in the supercritical or near-super-
critical state has been proposed as an alternative way
to prepare MS. Various parent techniques have been
conceived, such as the so-called gas antisolvent
precipitation (GAS) [126], aerosol solvent extraction
system (ASES) [127] and rapid expansion of super-
critical solution (RESS) [128]. For illustration, ASES
involves spraying an organic polymer solution into
an excess of supercritical CO2 [127,129]. After
atomisation of the polymer solution, the polymer
solvent is extracted into the supercritical fluid
leading to immediate polymer precipitation and
particle formation. For microencapsulation, antigens
are either dispersed as powder in the polymer
solution or codissolved with the polymer in suitable
solvents, hence avoiding aqueous processing. ASES
has been compared with conventional spray-drying
in terms of effects on the stability of the peptide,
tetracosactide [130]. Almost no intact peptide was
recovered from spray-dried PLA particles, whereas
the tetracosactide was well protected against oxida-
tion during ASES (~94% unmodified peptide). A
serious limitation of GAS, ASES and RESS for
producing MS is the need of polymer types that form
discrete crystalline domains upon solidification, such
as l-PLA [131,132]. The advantages these methods
offer, e.g., over spray-drying, are the low critical
temperatures for processing (34 8C) and the avoid-
ance of oxygen exposure during atomisation, with
H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376370
both parameters being potentially important to
antigen stability.
7.3. ProLeaseR technology
The ProLeaseR technology was developed to
ensure optimum stability of proteins or peptides
during and after microencapsulation [133]. The
method relies on the use of stabilising and release-
controlling agents, low processing temperature, and
nonaqueous microencapsulation. Typically, a protein
powder is micronised, possibly with a stabiliser, by
spray-freeze-drying, and then suspended in an
organic polymer solution. The suspension is atom-
ised into a vessel containing liquid N2 underlaid by
frozen ethanol (extraction solvent). The atomised
droplets freeze in the liquid N2 and deposit on the
surface of the frozen ethanol. As liquid N2 evapo-
rates, the frozen ethanol liquefies (Tm approximately
�110 8C) so that the frozen polymeric droplets will
transfer into the ethanol where the polymer solvent is
extracted, yielding solid MS. To date, the ProLeaseRsystem has been successfully used, e.g., for encap-
sulation of rhGH in PLGA MS (N98% encapsulation
efficiency, N99% monomer) [84,85,134]. As a
reference, rhGH was unstable in contact with ethyl
acetate or DCM [16]. In addition, protein released in
vitro over 28 days retained almost complete integrity
(N97% monomer) and bioactivity. Stabilisation with
zinc acetate to form a solid zinc–protein complex,
and coencapsulating ZnCO3, were key to rhGH
stability.
ProLeaseR technology was also used for encapsu-
lating recombinant human vascular endothelial
growth factor (rhVEGF) and insulin-like growth
factor-I (rhIGF-I) [135,136]. Both proteins were
stabilised in aqueous solution, prior to spray-freeze-
drying, and encapsulated (9–20%, w/w) into PLGA
MS. The MS also contained ZnCO3 (3–6%, w/w) as
release modifier. The resistance of rhIGF-I to aggre-
gation and oxidation, determined from in vitro release
studies, hardly changed. Protein, released in an almost
pulsatile fashion over 21 days, was composed of
predominantly monomeric rhIGF-I with only minor
amounts (~6%) of degradants forming towards day
21. Similarly, the integrity of rhVEGF dimer released
over 21 days was good and its bioactivity remained
largely unaffected, regardless of the extent of aggre-
gation and degradation. In view of these studies,
ProLeaseR technology appears to have potential for
sustaining antigen stability and release from MS.
7.4. Ultrasonic atomisation
Ultrasonic atomisation of W1/O dispersions is
presently under investigation for preparing antigen
containing MS. In one setup, the atomised antigen/
polymer dispersion was sprayed into a nonsolvent
where the polymer solvent was extracted, resulting in
MS formation [137]. A comparable technique was
proposed where the antigen or polymer dispersion
was atomised into a reduced pressure atmosphere and
the preformed MS hardened in a collection liquid
[138]. Similarly, PLGA solutions were also atomised
by acoustical excitation and the atomised droplets
transported by an annular stream of a nonsolvent
phase (aqueous PVA) into a vessel containing
aqueous PVA [139]. Solvent evaporation and MS
hardening occurred in the vessel over several hours.
The main advantages of these atomisation techniques
encompass the possibility of easy particle size control
and scale-up, processing at ambient or reduced
temperature, and the suitability for aseptic manufac-
turing in a small containment chamber such as an
isolator.
7.5. Formation of semisolid microglobules
All the encapsulation techniques discussed so far
rely on the preparation of solid MS. However, a
method for preparing a stable dispersion of protein
containing semisolid PLGA microglobules has been
reported [140]. Here, a protein dissolved in PEG 400
was added to a solution of PLGA in triacetin or triethyl
citrate. This mixture, stabilised by Tween 80, was
added dropwise and under stirring to a solution of
MiglyolR 812 or soyabean oil, containing Span 80,
resulting in a stable dispersion of protein inside
semisolid PLGA microglobules. The microglobules
remained in an embryonic state until mixed with an
aqueous medium, so that the water-miscible compo-
nents were extracted and protein containing matrix-
type MS formed. Myoglobin was encapsulated and
found to remain physically unchanged (circular
dichroism analysis) after the process and during
storage of the microglobular dispersion (15 days/4 8C).
H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376 371
7.6. Surface adsorption of antigens on preformed
microspheres with ionic surface charge
An elegant and efficient method for protein antigen
and DNA loading is by surface adsorption of
bioactive materials onto unloaded PLGA MS carrying
a surface charge [119,141–146]. As outlined in the
contribution of Jilek et al. in this issue, this is a very
efficient method for loading negatively charged DNA
onto cationic particles. Similarly, one may take
advantage of the protein’s surface charge, which
depends on its pI and the pH of the medium in which
it is dispersed. PLGA or any other type of MS can be
readily decorated with positive or negative surface
charges by simply preparing the particles by a W1/O/
W2 solvent evaporation/extraction process where the
W2 phase contains a cationic emulsion stabiliser
[hexadecyltrimethylammonium bromide; poly(ethyle-
neimine); stearlyamine] or an anionic emulsifier
(sodium dioctyl-sulfosuccintate; sodium dodecylsul-
fate). Such compounds attach tightly to PLGA
surfaces during preparation and provide the necessary
surface charge for ionic adsorption of counter-ions.
Alternatively, biodegradable polymers carrying ionic
groups may be used to prepare unloaded MS [118–
120]. The use of particles with ionic surface charge
offers several advantages over classical microencap-
sulation, amongst which the mild conditions for
loading is probably the most attractive. PLGA MS
with surface adsorbed protein antigens and DNA have
been highly efficient in inducing strong immune
responses, as recently reviewed by Singh et al.
[144]. Nonetheless, it remains to be shown whether
such particles are also suitable to elicit long-term
immunity after one or two injections.
8. Conclusions
The importance of stable antigen delivery from MS
has been highlighted by a vast number of inves-
tigations. The necessity to understand causes of
destabilisation and developing routes to ensure
maximum stability of the delivered antigen is even
more critical. Destabilisation and loss of immunoge-
nicity can occur and accentuate during manufacture,
storage and application. Instability arises primarily
from the innate physicochemical properties of anti-
gens, polymers and excipients used, as well as
unavoidable processing and environmental condi-
tions. Where these material properties or processes
cannot be altered, additives that shield the antigen or
regulate the local environment prove to be useful in
maintaining antigen stability. On the other hand,
selective modification and design of a new generation
of polymers and polymer systems, as well as
improved manufacturing processes, can be equally
applied to ensure microencapsulation and delivery of
stable antigens. Continued efforts to establish methods
for stable antigen delivery from MS may hopefully
pave the way for future MS-based vaccines.
Acknowledgements
This work was supported in part by the Swiss
National Foundation for Scientific Research, Bern
(No. 31-37440.93) and ETH, Zurich.
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