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Send Orders for Reprints to [email protected] 6340 Current Pharmaceutical Design, 2013, 19, 6340-6352 Microspheres and Microcapsules for Protein Delivery: Strategies of Drug Activity Retention Lianyan Wang1,*, Yuan Liu1,2,§, Weifeng Zhang1,2,§, Xiaoming Chen1,2,§, Tingyuan Yang1 and Guanghui Ma1 1National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, P.R. China; 2Graduate University of Chinese Academy of Sciences, Beijing, 100049, P.R. China Abstract: With the recent progress in biotechnology and genetic engineering, a variety of proteins have formed a very important class of therapeutic agents. However, most proteins have short half-lives in vivo requiring multiple treatments to provide efficacy. In order to overcome this limitation, sustained release systems as hydrophilic microspheres and hydrophobic microcapsules have received extensive attention in recent years. As therapeutic proteins delivery systems, it is necessary to maintain protein bioactivity during microspheres or microcapsules formation as much as possible. This paper reviews different influencing factors that are closely involved in protein denatu- ration during the preparation of hydrophilic polymer microspheres and hydrophobic polymer microcapsules. The various strategies usu- ally employed for overcoming these obstacles are described in detail. Both processing and formulation parameters can be modified for improving protein stability. The maximum or full protein stability retention within the microspheres or microcapsules might be achieved by individual or combined optimized strategies. In addition, the common techniques for proteins stability determination are also briefly reviewed. Keywords: Hydrophilic polymer microspheres, hydrophobic polymer microcapsules, protein delivery, activity retention, strategies. 1. INTRODUCTION profile could be obtained by designing the size and inner structure of microspheres and microcapsules; (iv) targeting molecules could The advent of recombinant DNA technology has made possible the commercial production of proteins for pharmaceutical applica- be conjugated onto the surface of microspheres or microcapsules, tions from the early 1980s [1]. These proteins generally display and the proteins could be delivered into definite location. therapeutically favorable properties, such as higher target specific- ity and pharmacological potency when compared to traditional Injections small molecule drugs [2]. Therefore, they become a very important Microspheres Formulations n cfolar sds ioafb ethtiecr appaetuietinct ,a gTeunmtso irn Nreeccernots iyse aFrasc, tfoorr (eTxNamF)p laen, din sInutleinrf uersoend atio Effective Plasma Concentration for cancer, and also vaccine and mono-antibodies for therapy. tr n However, most protein drugs have short half-lives in vivo requiring e multiple treatments to provide efficacy. Until now, most of thera- nc peutic proteins are presently delivered by injections. In some cases, o C multiple injections in one day are necessary in order to maintain a effective plasma concentration, which results in poor compliance of m patients. At the same time, proteins are also unstable in biological s fluids, can be easily digested by enzyme, and not fully absorbed a Pl from the gastrointestinal tract because of their relatively high mo- lecular weights, which lead to low bioavailability in vivo. Time In order to overcome above limitations, sustained release sys- tems for therapeutic proteins have received extensive attention in Fig. (1). Schematic illustration of drug levels in blood with traditional re- recent years. These systems can not only effectively prevent degra- peated dosing and microspheres/microcapsules formulations. dation of proteins in the body,but also improve the stability and achieve sustained-release or controlled-release profile in vivo [3-5]. Until now, many delivery systems for proteins are developed, such It is well known that proteins as large macromolecules consist as liposome, micelle, degradable polymer microspheres and micro- of a sequence of amino acids and highly assemble into complex capsules [6-9]. Among them, polymer microspheres and microcap- structures, which is closely related with their biologically active sules show great potentials in delivery of proteins, because they state and corresponding physiological functions [14, 15]. Chemical own many advantages as follows [10-13]: (i) they can protect pro- stability of proteins typically involves the integrity of the amino teins against rapid degradation and clearance after administration; acid sequence (primary structure) and the reactivity of the side (ii) desired therapeutic effect over a long period of time can be chains. As usual, the activity of proteins depends on the secondary, achieved,while for traditional injections, the schematic illustration tertiary and even quaternary structures, which is crucial for the of drug levels in the blood rise after administration and then de- biological functions. Although encapsulation of biomacromolecules crease until the next one as shown in (Fig. 1); (iii) suitable release in microspheres or microcapsules is now a routine procedure in many laboratories for achieving controlled and targeted delivery strategies, there are many process parameters during encapsulation *Address correspondence to this author at the National Key Laboratory of operations can affect the activity and stability of proteins [16], Biochemical Engineering, Institute of Process Engineering, Chinese Acad- which will lead to denaturation of proteins, and this will further emy of Sciences, Beijing, 100190, P. R. China; Tel:/Fax: 86-10-82544931; result in therapeutic inactivity and unpredictable side effects, such E-mail: [email protected] §The authors have the same contributions as immunogenicity or toxicity [17]. Therefore, it is significantly 1873-4286/13 $58.00+.00 © 2013 Bentham Science Publishers Microspheres and Microcapsules for Protein Delivery Current Pharmaceutical Design, 2013, Vol. 19, No. 35 6341 important to retain the stability and activity of proteins during en- specific, folded, three-dimensional structure (conformation) during capsulation process. encapsulation process. Loss of biological activity and change in Different microspheres and microcapsules, both hydrophilic immunogenicity as a result of protein aggregation or denaturation will result in serious side effects. It is the most important challenges and hydrophobic counterparts, were developed for encapsulation of proteins, such as chitosan microspheres and poly (lactic acid) to maintain the complexity of protein structure for delivery of bio- (PLA) microcapsules. Microspheres and microcapsules are spheri- logically native one during encapsulation [38]. Studies showed that many manufacturing methods for microspheres can damage com- cal microparticles with small diameter in range of micrometer or nanometer, which are usually made of a biodegradable or resorb- plex structures of proteins by exposing them to potentially damag- able plastic polymer and filled with a substance (as a drug or anti- ing conditions, such as aqueous/organic interfaces, elevated tem- peratures, vigorous agitation, detergents and so on [14]. Therefore, body) for sustained release. There is a little difference between them. Microspheres are matrix type and the encapsulated substance the protein activity must be maintained for different preparation and polymer are uniform mixture, while microcapsules are reservior methods of microspheres. type and the encapsulated substance disperses into polymer cavity Table 2. Preparation Methods of Hydrophilic Polymer Micro- by agglomerates. spheres for Protein Delivery During microencapsulation process, protein stability and activ- ity may be hampered at various stressful stages, such as the genera- Factors for influencing tion of the aqueous/organic interface (W/O emulsion), formation of Methods References 1 protein stability protein-protein conjugate or protein-material conjugate, and the production of protein aggregates [18]. In this review, imaginative water-oil interface, shear- approaches for protein stability and activity retention during fabri- Emulsion cross-linking ing stress, harsh cross- [21, 39] cation of both hydrophilic and hydrophobic polymer-based micro- spheres and microcapsules will be described, discussed, and pros- linking reaction pected. Coacervation/precipitation agglomeration [40] 2. MICROSPHERES BASED HYDROPHILIC POLYMERS high temperature, shear- Spray-drying [1, 41] 2.1. Type of Polymers for Protein Delivery ing stress Many hydrophilic polymers were employed to form micro- Ionic gelation polymer [42, 43] spheres for protein encapsulation as shown in Table 1, including chitosan [19-23], alginate [24-26], dextran [27-30], hyaluronic acid Template-assembly [31-34], starch [35-37] and so on. As protein delivery system, these polymer [44] method hydrophilic polymers demonstrate lots of advantages such as excel- lent biocompatibility and biodegradability, non-immunogenicity and low toxicity. Furthermore, the hydrophilic polymers micro- 2.2.1. Emulsion Cross-linking spheres can easily facilitate water uptake, which is favorable for the Proteins could be entrapped into water soluble polymer micro- continuous release of protein in vivo [1]. And this is necessary to spheres like chitosan, collagen and gelatin by emulsification com- maintain stable plasma concentration for therapeutic proteins. Dur- bined with cross-linking method. The detailed preparation process ing encapsulation of proteins, the bioactivity retention depends on is shown as (Fig. 2) [45]. Protein is dissolved or dispersed into preparation methods and operation parameters. Therefore, the cor- polymer solution as water phase (W), and then this solution is responding methods and factors influencing protein stability will be emulsified into oil phase (O) to form W/O emulsion by mechanical elaborated in detail in the following sections. stirring, ultrasonication technique or homogenization. Finally, the Table 1. Type of Hydrophilic Polymers for Protein Delivery cross-linker agent is dropped into the system to solidify the emul- sion into microspheres. The environment of water phase such as pH values and additives will not only exert important influence on drug Polymers Encapsulated proteins References activity, but also have impact on microsphere formation, morphol- ogy and structure, which will further affect the encapsulation effi- Insulin, Bovine Serum Albumin [19-23] ciency and release profile in vivo. Cross-linking process includes Chitosan (BSA), Ovalbumin (OVA), Enzymes, physical and chemical cross-linking methods. Physical cross- Growth factors, Salmon calcitonin linking can usually maintain high activity of proteins, while the lower encapsulation efficiency and fast release can be obtained due Growth factors, hemoglobin, OVA, [24-26] Alginate to weak solidification for microspheres. However, the higher en- Insulin capsulation efficiency and lower release profile can be achieved when the microspheres are solidified by chemical cross-linking lysozyme, BSA, Immunoglobin G [27-30] Dextran agents. Therefore, the suitable water phase and cross-linking proc- (IgG), Insulin ess should be chosen during preparation of protein loaded micro- Hyaluronic Growth factors, Insulin [31-34] spheres by W/O emulsion method. For example, Jose et al. em- acid ployed this method to prepare chitosan microspheres containing insulin, and formulation parameters as chitosan concentration, glu- Starch Growth factors, Insulin [35-37] taraldehyde concentration and cross-linking time were optimized to develop microspheres with suitable performance in vivo. The results revealed that there was a significant reduction in blood glucose 2.2. Preparation Methods of Microspheres for Protein Delivery level after administration of the optimized formulation, in compari- son to a subcutaneous injection of insulin solution [46]. Sivakumar Until now, there are many methods developed for preparation et al. also used the same technique to encapsulate hepatitis B sur- of hydrophilic polymer microspheres for protein delivery as shown face antigen (HBsAg) into chitosan. It was found that the chitosan in Table 2, including emulsion cross-linking, coacervation/precipi- microspheres induced significantly higher immunoglobulin levels tation, spray-drying, ionic gelation and template-assembly method. comparison with the conventional alum-adsorbed system [47]. For different methods, the encapsulated proteins must maintain 6342 Current Pharmaceutical Design, 2013, Vol. 19, No. 35 Wang et al. Polymer solution Crosslinking Protein drug Polymer microspheres W/O emulsion containing drug Fig. (2). The schematic process of hydrophilic polymer microspheres containing protein. Although the protein could be successfully encapsulated into This technique shows some important advantages over other hydrophilic polymer microspheres, there still existed a few draw- encapsulation techniques. The preparation process is rapid, conven- backs. The proteins easily aggregate in the interface between water ient and easy to scale-up. Furthermore, this process is suitable for and oil phase during emulsifying process, which result in their ac- most of proteins because it doesn’t depend on the solubility pa- tivity reduction. Moreover, the intense shearing strength brought by rameter of proteins. This process are easily to induce aggregation a high-speed homogenizer or a sonicator also led to protein denatu- and denaturation of sensitive proteins due to thermal and shearing ration and aggregation. In addition, the cross-linking reactions will stresses, so stability of microencapsulated proteins during prepara- happen between proteins and/or between protein and polymer, lead- tion becomes a major concern. ing to activity reduction of proteins. Furthermore, complete re- 2.2.4. Ionic Gelation moval of the unreacted cross-linking agent may also be a big chal- In recent year, the method of ionic gelation for preparation of lenge. hydrophilic polymer microspheres containing proteins has attracted 2.2.2. Coacervation/Precipitation much attention because the process is very simple and mild, which This process consists of dispersing the proteins into solution of is helpful to retain bioactivity of loaded proteins. In this method, the polymer and decreasing the solubility of the polymer by addition of reversible physical cross-linking by electrostatic interaction is em- a third component to the system [48-51]. At a particular point, the ployed for microspheres formation. In detail, proteins are premixed process yields two phases (phase separation): the polymer contain- with hydrophilic polymer solution, which is then dropped into ionic ing coacervate phase and the supernatant phase depleted in poly- solution under constant stirring. Bodmeier et al. reported that the mer. The coacervation process includes the following three steps: tripolyphosphate (TPP) -chitosan microspheres were obtained by (i) phase separation of the coating polymer solution, (ii) adsorption dropping chitosan solution containing proteins into a TPP solution of the coacervate around the protein particles, and (iii) solidification [27], and many researchers have explored its potential for pharma- of the microspheres. ceutical usage [42, 43, 57]. The gelation of alginate can also be The coacervation process is mainly used to encapsulate water- carried out under an extremely mild environment. Alginate micro- spheres can be prepared by extruding a solution of sodium alginate soluble proteins. Ozbas-Turan S. et al employed chitosan to encap- containing proteins as droplets into a divalent cross-linking solution sulate interleulin-2 by this process. They found that the addition rate of non-solvent (sodium sulfate) should match with slow extrac- such as Ca2+, Sr2+, or Ba2+. The native activity of loaded proteins by this method could be well retained. tion of chitosan, so that the chitosan has sufficient time to deposit and evenly coat on the protein particle surface during the coacerva- 2.2.5. Template-assembly Method tion process [40, 52]. Moreover, the concentration of the polymer is In this process, a suitable template should be chosen at first, and also very important, since too higher concentrations would result in then protein drugs were adsorbed on the surface of template mi- rapid phase separation and non-uniform coating of the polymer on croparticles, and next the polyelectrolyte were assembled on the the protein particles. Due to absence of any emulsion stabilizer in core template by layer-by-layer technology, and finally the template the coacervation process, agglomeration is a common problem in was removed by certain method [44, 58]. The activity of proteins this method [40]. The coacervate droplets are extremely sticky and could be highly maintained due to the mild preparation conditions, adhere to each other before the complete phase separation or the and the whole process was conducted in aqueous phase avoiding hardening stages. Adjusting the stirring rate, temperature, or the oil-water interface and intense shear force. addition of an additive is known to overcome this problem [40]. This process avoids the use of toxic organic solvents and cross- 2.3. Factors Influencing Protein Stability linking agent, which is favorable for activity retention of proteins. The protein can be dissolved in the hydrophilic polymer solu- 2.2.3. Spray-drying tion and be further entrapped in the microspheres. Until now, it is Spray drying is a well-established method commonly used in identified that there are some process parameters to affect protein the pharmaceutical industry for producing a dry powder from a stability during preparation, including water-oil interface, intense liquid phase. The method is based on drying of atomized droplets in shearing stress, high temperature and harsh cross-linking reaction. a stream of hot air. In principle, hydrophilic polymer is dissolved in The factors influencing protein stability vary in preparation meth- aqueous solution, and the drug is dispersed or dissolved in polymer ods and processes. solution. This solution or dispersion is then atomized in a stream of The water-oil interface is a well-known factor to cause denatu- hot air that leads to the formation of small droplets, from which ration of proteins. As a usual, proteins become especially prone to solvent instantaneously evaporates leading to the microspheres in migrate and adsorb at the interface to form aggregation. They un- typical size ranges from 1 to 100 (cid:2)m depending on atomizing con- dergo unfolding by presenting their hydrophobic area to the organic ditions. To date, many hydrophilic polymer microspheres loading solvent, and this area is usually buried in the molecular structure proteins were successfully prepared by this method, such as chito- when protein in active state [1]. This influence is often happened in san, mannitol, hydroxypropyl methylcellulose and carbopol micro- the emulsion cross-linking method, and the formation of w/o emul- spheres [53-56]. sion is mainly responsible for protein denaturation. Microspheres and Microcapsules for Protein Delivery Current Pharmaceutical Design, 2013, Vol. 19, No. 35 6343 Protein degradation might also take place upon mechanical such as sugars, amino acids, amines, glycerol and so on. These shearing and exposure to ultrasound due to intense shearing force. additives can increase stability of proteins through various mecha- For ultrasound emulsification technique, there is a serious drawback nisms. The protective effect can be mediated by the formation of that ultrasound cavitation could create drastic conditions inside the new hydrogen bonds and polar interactions at the surface of the medium for an extremely short time. According to the hot spot protein, which can increase the free energy of protein denaturation theory, high temperatures and high pressures inside the collapsing and changes in water [65]. On the other hand, proteins can be stabi- cavitation bubbles could be produced during ultrasound emulsifica- lized by electrostatic interactions between the additive substance tion [59, 60]. Moreover, there exist strong physical forces as shear- and protein because the tertiary structure of protein can be well ing forces, disturbance force and shock waves during broken of maintained by charged macromolecules [66]. droplets [61]. This inertial cavitation also induces the formation of For example, Chou et al. prepared biocompatible microparticles free radical via the thermal dissociation of water [62]. Under these for intranasal protein delivery by lyophilization [67]. Hydroxypro- extreme physical conditions, sonication could cause the breakdown pyl-(cid:3)-cyclodextrin (HP-(cid:3)-CD) and d-alpha-tocopheryl poly (ethyl- of the hydrogen bonding and Van der Waals interactions in the ene glycol 1000) succinate (TPGS 1000) were used as additives. polypeptide chains, leading to change of the secondary and tertiary The results showed that the activity of lysozyme was well preserved structure of the protein [63]. The bioactivities were lost with the by HP-(cid:3)-CD during the preparation process [68]. The stabilizing alteration of protein secondary and tertiary structure. It is also con- effect of HP-(cid:3)-CD is contributed by the fact that the aromatic siderable that shearing augments the chances of soluble proteins phenylalanine, tyrosine and tryptophan residues in proteins could adsorption onto air/water and water/oil interfaces [1], which further incorporate in the hydrophobic internal cavity of CD [69]. Wang et promotes hydrophobic interactions between proteins and interface al. prepared chitsoan microspheres for encapsulation of insulin by leading to aggregation. emulsion cross-linking method. Different additives were added into High temperature is also an important parameter affecting pro- the aqueous phase to improve stability of insulin. It was shown that tein stability. During the temperature-induced denaturing transition, gelatin could greatly improve insulin stability. The possible reason a protein changes from a rather well-organized structure into a ran- is that chemical reaction between amine groups of gelatin and the dom coil-like structure, which results in the contact between hydro- cross-linker protect insulin against chemical modification during phobic amino acids and water [64]. For spay drying, one of the the preparation process [20]. major issues is the maintenance of protein activity during the proc- 2.4.2. Optimization of Microsphere Formation ess of high temperature. Although microspheres can be obtained by emulsifica- In emulsion cross-linking method, the use of harsh cross- tion/solidification technique, the emulsion is usually prepared by linking agents might possibly induce chemical reactions between high-energy instruments or methods with intense shearing force, the active proteins and/or between proteins and polymers, which which may result in loss of protein activity. To overcome this dis- will also lead to denaturation of proteins. For example, chitosan advantage, the membrane emulsification technique was applied to microspheres are solidified by reaction between primary amine prepare hydrophilic microspheres with narrow size distribution. groups of chitosan and aldehyde group of glutaraldehyde. And this This technique could improve protein activity due to mild emulsifi- reaction could be also occurred between proteins and glutaralde- cation without intense shearing force [20, 21]. hyde due to abundant of primary amine groups in proteins, which Another useful method for avoiding the denaturation of protein finally induce the denaturation of proteins. is to modify or optimize solidification process of microspheres. For Sometimes, the polymers may also affect proteins stability by example, in order to reduce chemical reaction between insulin and themselves due to the interaction between polymers and the pro- cross-linking agent of glutaraldehyde and improve the encapsulated teins in the solid formulation. For example, Yang et al. prepared insulin activity during solidification, Wang et al. employed a step- chitosan-based microspheres containing protein of salmon calci- wise cross-linking method to greatly improve the stability of protein tonin (sCT) by spray drying process using mannitol as cryoprotect- [20]. First, a w/o emulsion of chitosan/insulin in paraffin/petroleum ing agent [22], and the effect of chitosan on the physicochemical ether mixture was prepared, and then TPP was added as an ionic stability of the protein was investigated. The results demonstrated cross-linker to form gel network structure, and finally glutaralde- that addition of chitosan decreased the recovery of sCT from the hyde was introduce for further solidification of microspheres as spray-dried powders, because the interaction between chitosan and shown in (Fig. 3). In this method, the gel network could effectively sCT was strengthened by dehydration during the spray-drying, and inhibit the interaction between insulin and glutaraldehyde, so the this further led to some irreversible complex formation. It was native insulin was maintained in maximum extent as shown in (Fig. found that chitosan had slight effect on secondary structure of sCT 4d). in an aqueous formulation, but it was not detrimental to chemical In addition, the stability of encapsulated protein could be also integrity of sCT. improved by different loading ways. For example, Wang et al. In summary, there are many parameters and conditions to affect compared the integrity of loaded insulin between adsorption and protein stability during microspheres preparation. In order to realize direct encapsulation [21]. As shown in (Fig. 4), no loss of insulin successful encapsulation of active macromolecules as proteins into activity was found in the case of adsorption method (Fig. 4b), while hydrophilic polymer microspheres, the above factors influencing serious activity loss was found in direct encapsulation method due protein stability must be considered. to reaction between insulin and cross-linker of glutaraldehyde (Fig. 4c). Zhang et al. prepared uniform-sized alginate-chitosan micro- 2.4. Strategies for Improving Protein Stability During Microen- spheres and employed three ways for insulin loading as shown in capsulation (Fig. 5) [25]. It was found that the remarkable activity maintenance As therapeutic proteins delivery systems, it is necessary to (99.4%) was obtained when the insulin was loaded during the chito- maintain protein bioactivity during microspheres formation as much san solidification process (Method B) as shown in (Fig. 6). In case as possible. In order to achieve this aim, many strategies are devel- of activity retention, the denaturation of the insulin in Method A oped in recent years, which will be elaborated in detail as follows. (insulin was loaded during the first solidification liquid) was proba- 2.4.1. Additives bly caused by the ultrasonic oscillation during the preparation of mini-emulsion. While Method B is a mild process and could effec- For stabilization of proteins, the use of additives during the tively maintain the activity of insulin. Method C was the combina- encapsulation process is the most widely used and common strat- tion of Method A and Method B, so the loading efficiency and ac- egy. Until now, many additives were attempted and developed, tivity retention decreased. 6344 Current Pharmaceutical Design, 2013, Vol. 19, No. 35 Wang et al. Dropping ionic Dropping chemical solidifier solidifier Membrane Ionic chemical Washing emulsification gelation crosslinking Chitosan solution Chitosan microspheres Fig. (3). Preparation process of chitosan microspheres by step-wise solidification. Reprinted from Wang et al. (2006), with permission from ELSEVIER Publisher. 0.06 a 80 0.04 Loading efficiency 100 ABS0.02 70 Activity retention 0.00 0 5 10 15 Time (min) %)60 80%) ABS0000....00004026 0 5 10 b15 Time (min) ading efficiency (52430000 6400ctivity retention ( o A 0.06 c L 20 0.04 10 ABS0.02 0.00 0 0 Method A Method B Method C 0 5 10 15 Time (min) Fig. (6). Insulin loading efficiency and activity retention obtained with 0.06 d different insulin loading procedures (A, B, C). Results are expressed as 0.04 mean±SD (n=3). Statistical significant differences (p<0.05) exist between ABS0.02 all the series. Reprinted from Zhang et al. (2011), with permission from 0.00 ELSEVIER Publisher. 0 5 10 15 Time (min) were developed for protein delivery. For example, Li et al. modi- Fig. (4). The integrity of insulin released from chitosan microspheres by different insulin-loading methods. (a) Standard insulin, (b) Adsorption fied chitosan into glycol chitosan-5(cid:3)-cholanic acid conjugates method, (c) Crosslinked by glutaraldehyde, and (d) TPP ionic gelation com- (HGC) as a novel protein delivery system. It was found that the bined with glutaraldehyde crosslinking. Reprinted from Wang etal. (2006), biological activities of encapsulated Noggin are not impaired by with permission from ELSEVIER Publisher. encapsulation in the HGC carrier compared with chitosan [70]. In addition, N-trimethyl chitosan chloride (TMC) was also employed CaCl2 Solution + Oil Phase for microspheres formation as protein delivery, which could pro- Method A Ultrasonication vide maximum stability of the protein due to its good water solubil- ity over a wide pH range [71, 72]. Mini-emulsion of CaCl2 Solution 3. MICROCAPSULES BASED HYDROPHOBIC POLYMERS Method B 3.1. Type of Polymers for Protein Delivery Chitosan Alginate Solution The prerequisites for polymers used as proteins delivery sys- tems include biocompatibility, controllable biodegradability, ab- Alginate Emulsion Alginate Gel ALG-CHI Microspheres sorbability, low toxicity of the degradation products, and controlled and sustained release potential [73]. Microcapsules based hydro- SPG Membrane A+B=Method C phobic polymers have been prepared from polyesters such as poly(d, l-lactide-co-glycolide) (PLGA), poly(lactic acid) (PLA), Fig. (5). Schematic presentation of the preparation process of alginate- chitosan microspheres with three ways for insulin loading. Reprinted from poly(glycolic acid) (PGA), and monomethoxypoly(ethylene gly- Zhang etal. (2011), with permission from ELSEVIER Publisher. col)-b-poly-d,l-lactide (PELA), polyanhydrides as 1,6-bis(p- carboxyphenoxy) hexane (CPH), poly(1,8-bis(p-carboxyphenoxy)- 3,6-dioxa-octane) (poly(CPTEG)), and poly 1,3-bis-(p-carboxy- 2.4.3. Modification of Hydrophilic Polymers phenoxy) propane-co-sebacic acid (p(CPP:SA)) copolymer, and To prevent the instability of protein during encapsulation, poly(orthoesters) [10, 74-76]. which induced by the interaction between polymers and the pro- Among these polymers, PLGA has been most extensively in- teins, an effective strategy of modification for hydrophilic polymers vestigated for proteins delivery, owing to its advantages such as has been developed. Chitosan is only soluble in its protonated form biocompatibility, non-toxic degradation products, approval by US in acidic environments, which may affect stability of protein. In Food and Drug Administration (FDA), well-defined protein encap- order to overcome this adverse environment, different derivatives Microspheres and Microcapsules for Protein Delivery Current Pharmaceutical Design, 2013, Vol. 19, No. 35 6345 sulation and long-acting profiles [77]. The drawback of PLGA for Solvent-evaporation method was usually chosen for solidification protein delivery is its deleterious effect on protein stability and of microcapsules when methylene chloride as solvent due to its low activity. PLGA is a bulk-eroding polymer, so protein encapsulated melting point [10, 75, 92], and the solvent could be removed in PLGA carriers is exposed to elevated moisture environment through evaporation at atmosphere or at a reduced pressure [10, 93, which may cause aggregation of proteins [75, 78, 79]. Another 94]. In some literatures, methylene chloride could also be removed issue is that some proteins may become denatured and/or irreversi- by solvent-extraction method using aqueous solution containing 6% bly aggregated in the acid microenvironment (pH 2-3) caused by isopropanol [10, 95]. The microcapsules could be solidified by PLGA degradation products [74, 78, 79]. solvent-extraction method when ethyl acetate as solvent due to its Polyanhydrides have also been investigated for proteins deliv- high solubility in water (8.7%, w/v) [10, 96]. In detail, the water-in- oil-in-water double emulsion was poured into a large volume of ery application. Their surface erosion characteristics dedicates the superiority with near zero-order release rate, and the protein stabil- aqueous solution, and the solvent of ethyl acetate was extracted to ity is also improved by minimizing water interaction with drug allow the solidification of microcapsules [10, 96]. prior to release [74, 75, 78]. The microenvironmental pH resulting 3.2.2. Spray Drying Method from polyanhyride degradation is higher than that caused by PLGA, Spray drying is an attractive and relatively simple method to which is less detrimental to biologically active proteins [74, 79]. prepare hydrophobic polymer microcapsules for protein delivery What’s more, the degradation rates of polyanhydrides can be easily [18]. As shown in (Fig. 7) (2), W/O emulsion is atomized in a flow tailored by adjusting the polymer chemistry [74, 75, 78]. of drying air at higher temperature, and microcapsules are collected with rapid vaporization of organic solvent [18]. This method is not 3.2. Preparation Methods of Microcapsules for Protein Delivery suitable for encapsulation of thermally sensitive proteins due to the Various techniques have been utilized to fabricate hydrophobic application of high temperature during preparation process. In order polymer microcapsules, including hot melt, emulsification-solvent to overcome this limitation, the novel spraying method with low- removal, spray drying, organic phase separation, cryogenic atomi- temperature was reported by several groups [18]. For example, zation, supercritical fluid and so on [4, 10, 74, 76, 78]. The com- Lopac et al. had fabricated polyanhydride-based microspheres us- mon methods for proteins encapsulation are summarized in Table 3. ing solid-in-oil-in-oil double emulsion combined with cryogenic Table 3. Preparation Methods of Hydrophobic Polymer Micro- atomization [74]. In detail, the polymer and lyophilized OVA dis- capsules for Protein Delivery solved in methylene chloride were pumped into ethanol which was cooled by liquid nitrogen. The mixture was then atomized by an ultrasonic atomizing nozzle, which was placed in a (cid:2)80(cid:2) freezer Factors for influencing pro- for three days to further remove liquid nitrogen, ethanol, and meth- Methods References tein stability ylene chloride, and finally the microcapsules were collected and dried. The results indicated that protein released from polyanhy- Emulsification- water-oil interface, shearing dride-based microcapsules remained integral structure. [82, 84, 90] solvent removal stress There are many advantages for spray drying including rapidity, convenience, ease to scale-up, mild preparation conditions, less high temperature, shearing Spray-drying [74] dependence on the solubility parameter of drug and the polymer stress [48]. Organic phase sepa- agglomeration [18, 48] 3.2.3. Organic Phase Separation Method ration Organic phase separation is also called coacervation. As shown in (Fig. 7) (3), microcapsules fabrication using this technique con- sists of two steps: (i) phase separation induced by adding an organic 3.2.1. Emulsification-solvent Removal Method non-solvent for polymer and protein to water-in-oil (W/O) emul- For the hydrophobic microcapsules as proteins delivery system, sion; (ii) solidification of the microcapsules by transferring the two- the method of emulsification-solvent removal technique has been phase system into a large volume of an organic hardening agent widely used and extensively investigated. The emulsions can be miscible only with the polymer solvent and non-solvent [18, 48]. prepared by emulsifying a concentrated aqueous solution of protein The drawbacks of organic phase separation method include or the freeze-dried solid in organic solvent, followed by secondary tendency to produce agglomerated particles, difficulty in mass pro- emulsification in aqueous continuous phase or dispersing the emul- duction, requirement of large quantities of organic solvent, and sion into another oil phase. The former is called water-in-oil-in- difficulty to remove residual solvents from the final microsphere water double emulsion (W/O/W) as shown in (Fig. 7) (1), and the product [48]. latter is solid-in-oil-in-water emulsion (S/O/W) [3, 10, 74, 77, 80- 87]. When W/O/W method was employed, the water-oil interface 3.3. Factors Influencing Protein Stability between internal aqueous phase and organic phase in the formation Although hydrophobic polymer microcapsules containing pro- of the primary emulsions, was regarded as the major cause for pro- teins could be successfully prepared by above methods, which tein instability during proteins microencapsulation [83, 88, 89]. To could also solve the limitation of therapeutic proteins in some de- overcome this limitation, a primary emulsion-free method, solid-in- gree such as poor stability, low bioavailability, short in vivo half- oil-in-water emulsion (S/O/W), was developed [3, 82, 84, 85, 90, lives, the microencapsulation process may lead to protein instability 91], which could effectively maintain protein stability due to thor- including denaturation, aggregation, unfolding, deamidation [17, oughly avoiding the presence of water-oil interface in the whole 73, 88, 89]. The factors resulting in protein instability include wa- process [73, 74]. In addition, some excipients as poly(ethylene gly- ter-oil interface [3, 17, 73, 75, 80, 81, 88, 93, 97, 98], shear forces col) (PEG), dextran, and cetyltrimethylammonium bromide [10, 17, 73, 88], heat [3, 73] and so on. (CTAB), water-soluble proteins were also employed for keeping protein activity retention [3, 82, 84, 85, 90, 91]. During the microcapsules preparation process by W/O/W, the water-oil interface has been regarded as the major reason for pro- The organic solvents most frequently used in this technique are tein instability [83, 88, 89]. During formation of primary emulsion methylene chloride and ethyl acetate. The solvent removal tech- of water in oil by sonication or homogenization, there appear inter- nique may be divided into solvent-evaporation and solvent- face with large area. Protein may adsorb at the interface, unfold, extraction according to different characteristics of solvents [10]. and subsequently aggregate, resulting in inactivation and denatura- 6346 Current Pharmaceutical Design, 2013, Vol. 19, No. 35 Wang et al. Protein solution (W) 1 Polymer solution (O) Homogenization or sonification W/O emulsion 1 Transfer into external aqueous phase (W) 2 Homogenization or sonification Non-solvent Transfer into hardening agent 1. Solvent evaporation 2. Spray drying 3. Phase separation or solvent extraction Fig. (7). Techniques available to fabricate microcapsules containing proteins. W/O emulsion is prepared by emulsifying aqueous solution of proteins in or- ganic solvent, further processed by the specific methods to obtain microcapsules containing proteins: (1) Solvent evaporation or extraction; (2) Spray drying; (3) Organic phase separation. The schematic illustration is modified from ref. [18]. Reprinted from Tamber etal. (2005), with permission from ELSEVIER Publisher. tion [3, 17, 73, 75, 80, 81, 83, 88, 89, 97, 98]. When protein accu- Stabilizer could play the function of stabilizing proteins by mulates at the interface, they may interact with hydrophobic poly- various ways. Firstly, the stabilizer could effectively maintain pro- mers, which further leads to protein instability [74, 97]. During tein stability via competitive adsorption on the oil-water interface. preparation of double emulsion of water-in-oil-in-water, the forma- He et al. employed PVA as a stabilizer to prepare the PLGA micro- tion of oil-water interface may also destroy protein stability. capsules loaded protein of staphylokinase variant K35R (DGR) [106]. It was found that the aggregation of DGR could be effec- Cavitation stress or heat production provoked by homogeniza- tion or ultrasonification during primary and double emulsion forma- tively inhibited by addition of PVA into inner water phase, and the tion will also result in activity loss of proteins [73]. Moreover, active DGR recovery reached to more than 90%, while approxi- mately 84% DGR aggregation was found in control (without PVA). physical stresses or heat during spray-drying or spray-freeze-drying are also crucial factors leading to protein instability [3, 73]. Secondly, some stabilizer could protect proteins stability by form- ing a hydration layer. Jeffrey et al. prepared PLGA microcapsules In addition, accumulation of acidic monomers and subsequent loaded recombinant human growth hormone (rhGH) or recombinant generation of acidic microenvironment inside microcapsules with human interferon-(cid:1) (rhIFN-(cid:1)) by emulsion solvent evaporation degradation of hydrophobic polymers would also result in protein method, using Tween 20, Tween 80, trehalose and mannitol to sta- instability [74, 78, 80, 81, 98, 99]. Furthermore, moisture in bulk- bilize encapsulated proteins [107]. The results showed that sugars eroding polymer-based microcapsules is elevated during protein of trehalose and mannitol demonstrated much better stabilizing release due to the degradation of polymers, which will lead to de- effect on proteins comparison with surfactants of Tween 20 and naturation of proteins [17, 81, 98-100]. Tween 80. Thirdly, the formation of polyelectrolyte complex be- tween therapeutic protein and polymer could also maintain protein 3.4. Strategies for Improving Stability of Proteins stability during encapsulation. Chondroitin sulfate (CsA) is an Microencapsulation processes usually involve harsh conditions acidic mucopolysaccharide, which is able to form ionic complexes leading to protein instability and denaturation as mentioned in with positively charged proteins. CsA was used as a polymeric above section. Denatured proteins will further result in therapeutic additive to form the ionic complex with insulin (InS) [108], and inactivity and unpredictable side effects, such as immunogenicity or then this complex was encapsulated into PLGA microcapsules by toxicity [101]. Therefore, it is significantly important that the stabil- emulsion-solvent evaporation technique. The results of reverse ity and activity of proteins must be retained during encapsulation phase high-performance liquid chromatography (RP-HPLC) dem- process. With the development of protein encapsulation, many at- onstrated that the retention time of insulin in complex was almost tempts are made for improvement of protein stability, which would identical to native protein comparison with free insulin as shown in be elaborated in details as follows. (Fig. 8). 3.4.1. Stabilizer Ajay et al. synthesized a poly-(ethylene glycol)-b-poly(L- To prevent protein aggregation in water-oil interface during histidine) diblock copolymer (PEG-polyHis) and then incubated microcapsule formation, the addition of stabilizer into the systems with insulin to form protein-PGE-polyHis complex. This complex is well developed strategies. Until now, there are various stabilizers greatly reduced aggregation of insulin during exposing water-oil to be verified useful for improving protein stability, including poly- interface [109]. Ionic interactions between insulin and PEG-polyHis vinyl alcohol (PVA), amino acids, proteins, sugars, surfactants and could prevent contacts of insulin with the oil phase as shown in inorganic salts [97, 102-105]. (Fig. 9). Microspheres and Microcapsules for Protein Delivery Current Pharmaceutical Design, 2013, Vol. 19, No. 35 6347 physical modification, the protein and modifier was co-lyophilized 1000 (a) to form microparticles, which further encapsulated into PLGA mi- 800 Control crocapsules. For example, He mixed recombinant human erythro- 600 poietin (rhEPO) with serum albumin (HSA) to fabricate microparti- mV400 organic/water solution cTlhees, raensdu ltthse no ff uinrttheegrr ietyn cmapesausluarteedm ienntto fPoLr GrhAE mPOic rsohcoawpseudl etsh [a1t 1t2h]e. 200 native structure of protein was successfully maintained during the 0 encapsulation process. The co-lyophilization of (cid:4)-chymotrypsin and poly (ethylene-glycol) (PEG) also greatly reduced aggregation of -200 proteins in interface [113]. In the method of chemical modification, 300 (b) Control the protein is chemically conjugated with PEG, and then further 250 encapsulated into hydrophobic microcapsules. In general, PEGyla- 200 organic/water solution tion of proteins could improves stability due to prevention protein V150 from adsorbing on water-oil interface [114]. Ingrid et al. prepared m 100 methoxy polyethylene glycol (mPEG, MW 5000) conjugation of 50 lysozyme, and further encapsulated into PLGA microcasules by W/O/W process [113]. The results demonstrated that Pegylated 0 lysozyme exhibited much better stability than native lysozyme dur- -50 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 ing exposure to organic solvent (dichloromethane) and homogeni- Time(Min) zation. Manish et al. prepared mPEG conjugated IFN-(cid:2) and then encapsulated it into PLGA microcapsules [115]. Most of the pegy- Fig. (8). CsA/InS complex stability in the O/W interface (RP-HPLC Chro- lated proteins maintained their native structure during exposure to matogram): (a) InS only and (b) CsA/InS complex. Reprinted from Park et harsh microencapsulation process. al. (2009), with permission from ELSEVIER Publisher. 3.4.4. Modification of Polymers The modification of hydrophobic polymers such as PLA and I II III IV V PLGA with either hydrophilic or hydrophobic segments is another potential strategy for improving protein stability during encapsula- tion. The modified polymer could effectively reduce protein adsorp- tion on water-oil interface, which further decreases the aggregation of proteins. For example, Bouillot et al. synthesized a PLA-PEG- PLA tri-blocks co-polymer, which was employed to encapsulate BSA by double-emulsion method [116]. Results suggested that protein adsorption and denaturation had been minimized through introducing hydrophilic PEG into PLA chains. Wan et al. obtained block polymer of POE-PEG-POE by conjugating PEG with hydro- Water Native insulin PEG-polyHis Insulin/PEG- philic Poly (ortho esters) (POE), which was further used to encap- polyHis sulate BSA [117]. It was demonstrated that the protein integrity was MC Denatured insulin PLGA complex well maintained for up to 8 weeks, which suggested that the conju- gation of PEG could well protect protein activity. Fig. (9). The mechanism of ionic interactions between insulin and PEG- 3.4.5. Modification of Preparation Process polyHis in preventing contacts of insulin with methylene chloride. Reprinted A charming strategy for avoiding the denaturation of protein is from Taluja et al. (2007), with permission from AERICAN CHEMICAL to modify or optimize preparation process of microcapsules. In SOCIETY Publisher. order to prevent the protein adsorption on water/oil interface, the solid protein was directly dispersed into oil phase containing poly- 3.4.2. Increase of Protein Concentrations mer, and then further encapsulated into microcapsules by suitable Studies have demonstrated that interfacial denaturation of pro- process. For example, Yuan et al. developed a novel multi- teins closely associates with their concentrations [18, 110]. The emulsion method of S/O1/O2/W for protein encapsulation [118]. protein amounts for adsorption and denaturation at the water-oil The model proteins of Horse myoglobin and Bovine serum albumin interface are always considered to be fixed. As usual, the proportion were encapsulated in the dextran glassy microparticles, which were of irrecoverable protein may be especially high in case of low further dispersed in the PLGA oil phase. The use of dextran glassy amounts of proteins. From this point of view, increasing the protein microparticles could effectively stabilize proteins in the PLGA concentrations is another useful strategy for improving protein sta- matrix. Ingrid et al. also employed S/O/W method to stabilize en- bility. Sah et al. prepared PLGA microcapsules loaded ribonuclease capsulated protein of (cid:4)-chymotrypsin. In these processes, protein A (RNase) by emulsion-solvent evaporation using different RNase conformational mobility is reduced due to the absence of water, concentrations from 0.2 to 1.5 mg/mL, and the results exhibited that which is significantly favorable for maintaining stability of proteins the active proportion of recoverable protein increased from 78% to during encapsulation [113]. Lee et al. compared the stability of 93% with increase of protein concentrations [111]. Similar results encapsulated lysozyme by conventional W/O/W and S/O/W meth- also obtained in encapsulation of human growth hormone (rhGH) ods [119]. The results demonstrated that the method of S/O/W of- into PLGA microcapsules, and the recoverable soluble monomer of fered an effective strategy to preserve stability and bioactivity of rhGH increased from 53% to 86% with its concentrations raising the encapsulated proteins. from 10 to 100 mg/mL. Therefore, the increase of protein concen- 3.4.6. Mitigation of Acidic Environment trations is favorable for improvement of its stability. The acid environment could be produced during degradation of 3.4.3. Modification of Proteins PLGA microcapsules, which had significant influence on protein The third attractive strategy for improving proteins stability stability and activity [120]. The acidic microenvironment could be during encapsulation is modification of proteins. The proteins could improved by adding basic additives during microcapsule prepara- be modified by physical or chemical method. In the method of tion. For example, Zhu et al. prepared PLGA microcapsule contain- 6348 Current Pharmaceutical Design, 2013, Vol. 19, No. 35 Wang et al. ing bovine serum albumin (BSA) [121]. The basic additive of The chemical stability of proteins could be analyzed by using Mg(OH) was added into inner water phase for neutralization of high performance liquid chromatography (HPLC) [73, 84, 86, 92, 2 acid. The results exhibited that the BSA structural loss and aggrega- 99, 125], high performance liquid chromatography-mass spec- tion was inhibited for over one month. Anne et al. also co- trometry (HPLC-MS) [99], or matrix-assisted laser desorp- encapsulated Mg(OH) into PLGA microcapsule to improve the tion/ionization time-of-flight mass spectrometry (MALDI-TOF 2 stability of lysozyme, which demonstrated better lysozyme stability MS) [75]. Aggregation of proteins could be evaluated by using size comparison with the absence of the Mg(OH) [114]. exclusion chromatography (SEC) [75, 88, 92, 125-127], capillary 2 In actual encapsulation processes, the suitable individual or electrophoresis [128], and western blotting [90, 112]. combined strategies could be employed for improvement of protein Differential scanning calorimetry (DSC) is an ideal method to stability. The maximum or full protein activity could be achieved study protein thermal stability in solution, which is mainly used to by optimizing processing and operation parameters according to measure the change of enthalpy ((cid:3)H) and the midpoint temperature real systems. (T ) of the denaturation for proteins [73, 88, 126]. The antigenicity m activity of proteins could be determined by protein-specific en- 4. METHODS FOR EVALUATION OF PROTEIN STABIL- zyme-linked immunosorbent assay (ELISA) [73, 88, 127]. ITY The stability of some specific proteins can be measured accord- Various techniques available for evaluation proteins stability ing to their intrinsic characteristics. The stability of enzyme is usu- are summarized in Table 4. These techniques can be employed to ally determined by measuring their bioactivity. Several groups have detect the primary structure, secondary structure, tertiary structure, evaluated the activity of lysozyme via enzymatic activity assay, by chemical stability and thermal stability of proteins. digesting bacterial cell walls of M. lysodeikticus with lysozyme and The primary structure is usually measured by sodium dodecyl measuring the turbidity of the suspension [79, 83, 88]. Castellanos sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [73, 75, et al. detected the activity of (cid:2)-chymotrypsin and PEG-(cid:2)- 78, 88]. The secondary structure could be assessed by Fourier trans- chymotrypsin using succinyl-Ala-Ala-Pro-Phe -p-nitroanilide as the form infrared spectroscopy (FTIR) [78, 85, 87, 89] and circular substrate [82, 84]. Meng et al. determined the oxygen-loading ac- dichroism [73, 75, 79, 87, 88, 122-124]. Fluorescence spectroscopy tivity of bovine hemoglobin to evaluate its molecular quaternary was usually employed to detect changes in tertiary structure of pro- structure [10]. Pean et al. evaluated the stability of nerve growth tein according to the emission (300-500 nm) from tryptophan and factor (NGF) by measurement of bioactive NGF using a PC 12 cell- tyrosine residues in proteins under excitation wavelength of 280 nm based bioassay [129]. [79, 85, 124]. Table 4. Representative List of Techniques Available to Evaluate the Stability of Proteins Common techniques Protein structure/stability Reference SDS-PAGE Primary structure [73, 75, 78, 88] CD Secondary structure (proteins released from micro- [73, 75, 79, 87, 88, 122-124] spheres/micro- capsules) FTIR Secondary structure (proteins in microspheres/microcapsules) [78, 85, 87, 89] Fluorescence spectroscopy Tertiary structure [79, 85, 124] HPLC Chemical stability [73, 84, 86, 92, 99, 125] HPLC-MS Chemical stability [99] MALDI-TOF MS Chemical stability [75] SEC Aggregation of proteins [75, 88, 92, 125-127] CE Aggregation of proteins [128] WB Aggregation of proteins [90, 112] DSC Thermal stability [73, 88, 126] ELISA Antigenicity activity [73, 88, 127] Specific techniques Enzymatic activity assay Bioactivity [79, 82-84, 88] Oxygen-loading activity Quaternary structure of BHb [10] PC 12 cell-based bioassay Bioactivity of NGF [129] In vivo biological analysis Bioactivity of insulin and rhEPO [7575, 112] Abbreviations: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; CD, circular dichroism; FTIR, Fourier transform infrared spectroscopy; HPLC, high per- formance liquid chromatography; PHLC-MS, high performance liquid chromatography-mass spectrometry; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of- flight mass spectrometry; SEC, size exclusion chromatography; CE, capillary electrophoresis; WB, western blotting; DSC, differential scanning calorimetry; ELISA, enzyme-linked immunosorbent assay; BHb, bovine hemoglobin; NGF, nerve growth factor; rhEPO, recombinant human erythropoitin. Microspheres and Microcapsules for Protein Delivery Current Pharmaceutical Design, 2013, Vol. 19, No. 35 6349 The bioactivity of some proteins could be evaluated using in [10] Meng FT, Ma GH, Liu YD, et al. Microencapsulation of bovine vivo biological analysis. For example, Manoharan et al. studied the hemoglobin with high bio-activity and high entrapment efficiency bioactivity of insulin from microspheres in male Sprague-Dawley using a W/O/W double emulsion technique. Colloid Suface B rats. The microspheres containing insulin were subcutaneously 2004; 33: 177-83. [11] Sood A, Panchagnula R. Peroral route: An opportunity for protein injected at neck region of diabetic rats, and then blood glucose con- and peptide drug delivery. Chem Rev 2001; 101: 3275-303. centrations and serum insulin concentrations were determined using [12] del Valle EMM, Galan MA, and Carbonell RG. Drug Delivery glucose kits and the radioimmunoassay kits [75]. He et al. deter- Technologies: The Way Forward in the New Decade. Ind Eng mined the biological activity of recombinant human erythropoietin Chem Res 2009; 48: 2475-86. (rhEPO) by detecting the number of reticulocytes in blood from [13] Kokai LE, Tan HP, Jhunjhunwala S, et al. Protein bioactivity and BALB/c mice after subcutaneous administration of a single dose of polymer orientation is affected by stabilizer incorporation for rhEPO. The activity of rhEPO could be calculated according to the double-walled microspheres. J Control Release 2010; 141: 168-76. correlation between reticulocyte percentage and the rhEPO activity [14] Fu K, Klibanov AM, Langer R. Protein stability in controlled- [112]. release systems. Nat Biotechnol 2000; 18: 24-5. [15] Rathore N, Rajan RS. Current perspectives on stability of protein 5. CONCLUSION drug products during formulation, fill and finish operations. Biotechnol Progr 2008; 24: 504-14. As therapeutic proteins delivery systems, it is necessary to [16] van der Walle CF, Sharma G, Kumar MNVR. Current approaches maintain protein bioactivity during microspheres or microcapsules to stabilising and analysing proteins during microen-capsulation in formation as much as possible. Process operating conditions as well PLGA. Expert Opin Drug Del 2009; 6: 177-86. as the various surfaces that come in contact with proteins can have [17] van de Weert M, Hennink WE, and Jiskoot W. Protein Instability in a direct or indirect influence on their integrity. Until now, the pro- Poly(Lactic-co-Glycolic Acid) Microparticles. Pharm Res 2000; 17: 1159-67. duction of hydrophilic polymer microspheres and hydrophobic [18] Tamber H, Johansen P, Merkle HP, et al. Formulation aspects of polymer microcapsules containing stable therapeutic proteins still biodegradable polymeric microspheres for antigen delivery. Adv remains a major challenge. 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operations can affect the activity and stability of proteins [16], which will lead to .. dom coil-like structure, which results in the contact between hydro- phobic amino . Among these polymers, PLGA has been most extensively in- vestigated for
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