Advanced Drug Delivery Reviews 57 (2005) 357 – 376 www.elsevier.com/locate/addr Formulation aspects of biodegradable polymeric microspheres for antigen delivery Harjit Tambera,b, P3l Johansena,c, Hans P. Merklea, Bruno Gandera,* a Institute of Pharmaceutical Sciences, ETH Zurich, ETH-Hoenggerberg, HCI, 8093 Zurich, Switzerland Napp Pharmaceuticals Research Ltd., Cambridge Science Park, Milton Road, Cambridge, CB4 0GW, U.K. c Department Dermatology, University Hospital of Zurich, Gloriastrasse 31, 8091 Zurich, Switzerland b 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. 2. 3. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biodegradable polymers and methods for antigen microencapsulation . 2.1. PLGA as biodegradable matrix material . . . . . . . . . . . . 2.2. Commonly used microencapsulation techniques . . . . . . . . The challenges of antigen release testing and stability . . . . . . . . . 3.1. Antigen release from microspheres . . . . . . . . . . . . . . . 3.2. Antigen stability . . . . . . . . . . . . . . . . . . . . . . . . * Corresponding author. Tel.: +41 44 633 7312; fax: +41 44 633 1314. E-mail address: bruno.gander@pharma.ethz.ch (B. Gander). 0169-409X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2004.09.002 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358 359 359 359 360 360 361 358 H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376 4. Improving antigen stability during microencapsulation . . . . . . 4.1. Increasing the antigen concentration . . . . . . . . . . . . 4.2. Addition of several antigens or nonantigenic proteins . . . 4.3. Addition of surfactants. . . . . . . . . . . . . . . . . . . 4.4. Addition of osmolytes . . . . . . . . . . . . . . . . . . . 4.5. Addition of other stabilising excipients . . . . . . . . . . 4.6. Selection of polymer solvents . . . . . . . . . . . . . . . 4.7. Use of antigen powders . . . . . . . . . . . . . . . . . . 4.8. Use of hydrophobic ion pairing . . . . . . . . . . . . . . 5. Maintaining antigen stability during in vitro release testing . . . 5.1. Use of additives . . . . . . . . . . . . . . . . . . . . . . 5.2. Use of pH modifiers . . . . . . . . . . . . . . . . . . . . 5.3. Insoluble metal complexes. . . . . . . . . . . . . . . . . 5.4. Chemical modification . . . . . . . . . . . . . . . . . . . 5.5. Other methods . . . . . . . . . . . . . . . . . . . . . . . 6. Trends towards using more appropriate polymers. . . . . . . . . 6.1. PLA/PLGA blends. . . . . . . . . . . . . . . . . . . . . 6.2. Modified PLA/PLGA and new polymers . . . . . . . . . 7. Trends towards using more appropriate technologies . . . . . . . 7.1. Modifications of conventional methods . . . . . . . . . . 7.2. Atomisation using gases in the supercritical state . . . . . 7.3. ProLeaseR technology . . . . . . . . . . . . . . . . . . . 7.4. Ultrasonic atomisation . . . . . . . . . . . . . . . . . . . 7.5. Formation of semisolid microglobules . . . . . . . . . . . 7.6. Surface adsorption of antigens on preformed microspheres 8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . with ionic surface charge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362 362 362 363 363 364 364 364 365 365 365 365 366 366 366 367 367 367 368 368 369 370 370 370 371 371 371 371 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 stimulating the immune system [12]. H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376 A major problem hindering the progression of MSbased 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 (M w of approximately 10–100 kDa) and end-group chemistry (free carboxylic acid or esterified carboxylic acid). These three parameters largely determine the hydrophobicity (water swelling) and degradation kinetics of the materials, and thereby, the microencapsulation efficiency and release rate of drugs and antigens. When used as materials for MS, the PLGA hydrophobicity will also affect interactions of the MS with phagocytosing 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 359 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 codissolved 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 hardening 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 evaporation 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 compound 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 physically 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 360 H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376 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 investigations 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 singleinjection 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 incubating MS in an aqueous medium, antigen located at or near the particle surface is dissolved by the penetrat- 361 H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376 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 microencapsulation, different schedules for pulsatile antigen release are achievable [9,10]. 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 instability often develops through conformational changes leading to denaturation, surface adsorption, aggregation or precipitation of the antigen and is considered critical in microencapsulation technology [40]. Naturally, 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 freezedrying stage [42]. The storage stability of microencapsulated antigens should be increased over that of fluid vaccines, as antigen stability in the dry state is generally greater than in solution. Yet, residual Table 1 Causes of physical and chemical antigen instability Mechanism of antigen instability Reference W1/O emulsion formation Increased aqueous phase surface area and new W1/O interface: Antigen adsorption, unfolding and exposure of hydrophobic domains to organic front Protein unfolding due to high shear forces during emulsification Chemical degradation at W1/O interface [43,52,57] [15] [130] Freeze-drying of microspheres Poorly developed drying method resulting in instability or aggregation of insufficiently stabilised antigen [49] Storage Residual solvents and moisture absorption: Solvent/moisture induced aggregation Change in PLGA characteristics, such as T g and hydrolytic resistance, affecting antigen stability and release [15,49,88,147,148] [148] Incubation in simulated/physiological environment at 37 8C: Protein aggregation during rehydration in aqueous environment Chemical reactions: thiol-disulfide exchange, deamidation, oxidation, acylation and hydrolysis Protein adsorption at polymer/liquid interfaces Instability and degradation due to acid-catalysed reactions in acidic microenvironment created during polymer hydrolysis [42] [149,150] [42,151] [45,152–154] 362 H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376 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 consequentially 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 irrecoverable 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 concentration 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 irreversibly adsorbed to the interface; at higher concentrations, they behave as bself-protectantsQ. 4.2. Addition of several antigens or nonantigenic proteins Protein excipients with significant interfacial activity, 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 individual 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 aggregating 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 following 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 microencapsulation, 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 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 additional 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(propylene oxide)-b-poly(ethylene oxide) (PEO-PPOPEO) block copolymer, partially reduced Ttxd aggregation during emulsification of aqueous Ttxd solutions with DCM [57]. Limited stabilising activity of poloxamer 188 was found for Ttxd at a W1/O interface, 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 chromatography (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 poloxamer, 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 363 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 proteins 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 (osmolyte 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 mannitol, 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 (M w) were investigated for stabilising EPO [49] and rhGH [16]. Microencapsulated EPO aggregates were only marginally reduced with dextran (40 kDa M w; 5%, w/w), whereas rhGH stability was adversely affected (70 kDa 364 H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376 M w; 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 kDa M w; 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 interactions, 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 Stransferase 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 ghydroxypropyl-cyclodextrin effectively increasing Ttxd encapsulation. g-HPCD also inhibited EPO aggregation during microencapsulation [49]. Although the precise mechanism is unclear, interactions between amino acids and the hydrophobic inner cavity of cyclodextrins may play a role [71]. Further examples of additives investigated include carboxymethyl cellulose [16], hydrophobic compounds such as ethyl stearate, sodium acetate and sodium glutamate [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 BSA was encapsulated 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 emulsions, 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, typically using dried antigen powders. The dry state offers increased stability owing to the reduced conformational flexibility and, hence, less potential for structural 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 encapsulation 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 coacervation method [75], reduction of a-helical content H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376 and increases in h-sheet and random structure were less pronounced when trehalose was added for sprayfreeze-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 cryoprotectant 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 nanoparticles 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 365 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, coencapsulation 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 recombinant 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 peptide fragments comparable to those seen in aqueous solutions of pHb3 stored for up to 12 days. Noncovalent aggregates were diminished when Mg(OH)2 or 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 366 H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376 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. Generally, 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 substantial amounts of dimerised and aggregated protein was released from MSrhGH. Release of purely monomeric and bioactive rhGH from MSrhGH: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 microencapsulated 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-linking, 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 carboxymethylated 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 succinylation 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 liposome-entrapped antigens, such as influenza hemagglutinin (HA), has been realised as a means of improving antigen release [90]. Release of ELISAreactive 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 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 exploitation [95]. 6. Trends towards using more appropriate polymers Numerous issues are associated with PLA/PLGA MS such as their low glassy-to-rubbery-state transition temperature (T g), the relatively hydrophobic interface they offer to proteins, and the production of acidic degradation products. While the low T g 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 considered 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 hydrophobic, crystalline polymers with hydrophilic, amorphous 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 188 367 (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 unaltered 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 microenvironment 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 minimised 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-PEGPLA block polymer MS as compared to PLA or PLGA MS [107]. Again, this was attributed to the 368 H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376 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 polymers 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 moistureinduced 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 T g or T m of the end polymer [110]. For this purpose, 10–20 kDa stearyl-poly(l-lactide)-stearate and oleylpoly(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 degradation and erosion times can vary between days and months. Among the various POE classes, class IV PEO are the most hydrophobic and contain backboneintegrated 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 synthesised 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 zeroorder 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 polyelectrolytes 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 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, recombinant human insulin-like growth factor I (rhIGF-I) was encapsulated into PLGA MS 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. Without 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, respectively [123]. The acetonitrile mediated partial mixing of the W and O1 phases and subsequent protein/peptide precipitation, which was a prerequisite for microencapsulation. 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 reservoirtype 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. 369 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, transported separately through the channels, are discharged 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 scaleup, methodological robustness, continuous production 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-supercritical 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 supercritical 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 oxidation 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 avoidance of oxygen exposure during atomisation, with 370 H. Tamber et al. / Advanced Drug Delivery Reviews 57 (2005) 357–376 both parameters being potentially important to antigen stability. gation and degradation. In view of these studies, ProLeaseR technology appears to have potential for sustaining antigen stability and release from MS. 7.3. ProLeaseR technology 7.4. Ultrasonic atomisation 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 releasecontrolling 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 atomised 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 evaporates, the frozen ethanol liquefies (T m 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 ProLeaseR system has been successfully used, e.g., for encapsulation 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 encapsulating 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-freezedrying, 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 aggregation 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- 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 manufacturing 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 components were extracted and protein containing matrixtype 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 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(ethyleneimine); stearlyamine] or an anionic emulsifier (sodium dioctyl-sulfosuccintate; sodium dodecylsulfate). 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 microencapsulation, 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 investigations. 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 immunogenicity can occur and accentuate during manufacture, storage and application. Instability arises primarily from the innate physicochemical properties of anti- 371 gens, polymers and excipients used, as well as unavoidable processing and environmental conditions. 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. 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