Formulation aspects of biodegradable polymeric microspheres for antigen delivery

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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.


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-


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]


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


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


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


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


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,


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


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


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


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).


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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Formulation aspects of biodegradable polymeric microspheres for antigen delivery

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