| With the rapid growth in the field of biotechnology, more and more protein drugs such as peptide hormones, enzymes, vaccines and cytokines have been extensively studied for the therapeutic availability. These protein drugs have some disadvantages, including physical and chemical instabilities relate to degradation by pH of the gastrointestinal tract or by the enzyme, low oral bioavailability for their high molecular weights, and frequent injection for their short half-lives in vivo. As a result, high doses should be administered repeatedly, and which will lead to side effects and even toxicity. These difficulties in administration of protein drugs remain a major challenge about how to develop delivery systems by pharmaceutical means to improve their stabilities and retard their releases.Two kinds of biodegradable polymers are commonly used as protein carriers. The natural polymers are mostly hydrophilic such as glutin, dextran, albumin, starch, alginate and chitosan, and the synthetic ones are usually hydrophobic such as polyester, poly (carboxylic anhydride), poly (amino acid) and poly (phosphazene), etc. Hydrophilic alginate and chitosan were firstly selected to entrap bovine serum albumin (BSA) to obtain alginate-chitosan microspheres (ACM) with high protein encapsulation efficiency and extended release characteristics. BSA-loaded alginate microspheres were prepared by an emulsion method and further incubated in chitosan. Many process factors including the concentration and molecular weight of alginate, the concentration and pH of chitosan, and surfactants, etc, were tested. Microspheres were achieved with diameters less than 2μm, high encapsulation efficiency (>80%) and high loading rate (>10% w/w). The results also showed that alginate microspheres coated within 0.2% and 1.0% chitosan solutions of pH 4 by the two-stage procedure present the best sustained releasing more than two weeks.To investigate the retarded release profile of ACM in vivo, interferon-alpha (IFN) was entrapped in ACM, the entrapment efficiency of IFN in ACM was 40% and the stability of IFN solution was improved greatly. The pharmacokinetics of IFN-ACM suspension was studied in ICR mice by IM routes. The pharmacokinetic parameters presented the prolonged serum levels of IFN. Compared with IFN solution, Cmax of IFN-ACM reduced to 43.4%, and time to achievement of maximum serum concentrations (Tmax) was increased 4 folds,the absorption half-life (ty2a) increased to 3.2 h from 1.2 h, and the elimination half-life (ti/2B) varied from 14.09 h to 97.36 h. Meanwhile the area under the concentration-time curve (AUC) was the same as that of solution.On the other hand, hydrophobic PLGA microspheres were prepared in the present study, because that most protein drugs, especially at the case of vaccine, need more prolonged releases in vivo. BSA was encapsulated in PLGA as a model protein by a double emulsion solvent evaporation method and additives were used to change the initial release of BSA from PLGA microspheres. Both the determination methods of BSA and that in PLGA microspheres were established. For activity-protection test, newcastle disease virus (NDV) was entrapped in PLGA microspheres instead.Various methods of determining protein content loaded in PLGA microspheres from the literature were performed in this experiment. All these results about BSA content in the same microspheres were different and some of the differences were obvious. Among these different methods, the hydrolysis method can extract BSA completely, and the two-step hydrolysis method with acetonitrile as a solvent was the most successful one. The total protein amount can be analyzed either by the Coomassie Brilliant Blue (CBB) Staining method or by micro-BCA kits. Herein the BSA concentration was determined by CBB method.The optimized formula of BSA-PLGA microspheres is as following: (a) the volume ratio of methylene chloride to the internal aqueous phase is 20:1, (b) 1%PVA, (c) 16 000 rpm, 3 min, (d) 6% (w/w), (e) the lowest ultrasonic tension to form emulsification.The burst release of BSA at lh from PLGA microspheres is as high as 47.7%, and the cumulative release amount is only 66.2% after the in vitro release of 19 weeks. After the period of 19 weeks, PLGA (50:50) with molecular weight 21 KD should be degraded thoroughly as theoretically.No statistically significant differences of particle sizes and entrapment efficiencies were appeared in PLGA microspheres with different processing parameters or additives. Interestingly, the initial burst releases were markedly changed. The release of BSA was accelerated by hydrophilic additives except for PEG 6000 and retarded by the formulation composed of higher PVA concentration, tween-20 as an emulsifier in the internal aqueous phase, glycerol in the oil phase, inorganic salt in the external aqueous phase, and operated atlow temperature. Scanning electron microscopy showed the more porous and dimpled structure on the surface of the PLGA microspheres, the larger initial burst release.Besides of the burst and incomplete release, another serious problem is the instability of entrapped protein in PLGA microspheres. To investigate the effects of formulation technique of PLGA microspheres on the stability of protein, NDV-PLGA microspheres were prepared by the same w/o/w method. Both the concentration and activity of NDV were determined simultaneously. The virus valences of NDV were estimated by erythrocyte agglutination. The shearing forces such as vortex, high-speed centrifugation and stirring had little influences on the activity of NDV. In contrast, ultrasound had effects at some degree, and organic solvents played the major key in it. The activity of virus can be improved by adding some protectants such as PEG 6000 and HP- ￡ -cyclodextrin.Neither hydrophilic polymer nor hydrophobic polymer is ideal for protein or peptide drug delivery systems when they are used alone. Hydrophilic polymers always make drug release quickly despite of their excellent biocompatibility. PLGA, as a hydrophobic polymer, is an ideal carrier to achieve sustained drug release, but its incompatible property to hydrophilic protein, low entrapment ability and burst release make it unacceptable. Though the protein can be protected with additives, its low entrapment efficiency and burst release profile cannot be improved. As a result, a kind of composite microspheres was designed by combination of both hydrophilic and hydrophobic polymer.The first kind of composite microspheres was only modified by alginate and chitosan. The low level of alginate was added into the internal aqueous phase with protein together, meanwhile calcium chloride and chitosan were put into the external one, the other process kept invariability. Now the protein entrapped in microspheres had a hydrophilic microenvironment, the protein nearby the surface was partly covered by alginate and chitosan that combined with alginate by electrostatic interaction. Therefore the initial release was inhibited markedly. Compare to the simple microspheres, the burst protein amount of composite PLGA microspheres at 0.5 h was reduced to 1/6 and 1/3 when modified with 1.5% and 0.75% alginate solution respectively. Almost all of little pores at the surface of PLGA microspheres had been closed with the modification under SEM. However, entrapment efficiency could not be increased or decreased slightly, and the total burst release at 24 h hadno statistically difference.The second kind of composite microspheres was then constructed. BSA was entrapped into the alginate microspheres by forementioned modified emulsification method in an isopropyl alcohol-washed way. The rapid drug releases were sustained by chitosan coating. To obtain the desired release properties, the alginate-chitosan microspheres were further incorporated in the PLGA to form the composite microspheres. This structure of composite microspheres not only gives protein a stable microenvironment but also makes microspheres have higher EE and lower burst release. The average encapsulation efficiency was 81%~87%, while that of conventional PLGA microspheres was just 61%~65%. Furthermore, the burst releases in composite PLGA (50:50) microspheres at 24 h decreased to 38.89% from 52.83% in PBS, and further to 14.03% in saline. Moreover, the release profiles could be manipulated by regulating the ratios of poly (lactic acid) to poly (glycolic acid) in the composite microspheres.To observe the protective effects of composite microspheres, the PLGA microspheres and the composite alginate-chitosan-PLGA microspheres of SOD were prepared respectively. The SOD activity was measured by xanthine oxidase system. All the effects of various operation factors on SOD activity were evaluated and both the concentrations and activities of SOD entrapped in different microparticles were compared. The SOD was sensitive to organic solvents: methylene chloride > acetonitrile > isooctane > isopropyl alcohol. In vitro release tests demonstrated that the SOD activities in the 50:50 composite microspheres were well protected compared to that in the PLGA microspheres within one week.Hepatitis B vaccine has played a key role in preventing hepatitis B infection, one of the most deadly diseases, from traditional plasma-derived vaccine to recombinant vaccine. New generation of vaccines, such as purified subunits, recombinant proteins, synthetic peptides, and DNA plasmid, will be less reactivity and immunogenic than traditional vaccines and will require better adjuvants and delivery systems to induce optimal immune responses. Biodegradable microspheres-based single-dose vaccines have been the hot topic in the last two decades. In our program, yeast recombinant HBV was entrapped in two kinds of PLGA microspheres and their composite ones. Compared with double HB solution and aluminum (HB-A1) group as a control group, four experimental groups are listed as: HB-PLGA (50:50)and HB-PLGA (70:30) microspheres group, corresponding composite microspheres group, the group of the corresponding composite microspheres and cytokine IFN added, or corresponding composite microspheres and HB-A1 added. 10 pig of HB per mouse was injected subcutaneously on the back of Balb/C mice, the doses of PLGA (50:50) microspheres and PLGA (70:30) microspheres were fixed and the concentration of IFN was 100 IU/ml. The serum antibody levels of IgA+IgG+IgM, IgG, IgGi and IgG2a at the 1st, 2nd, 3rd months after sc injection were determined respectively. The primary results suggested that the composite microspheres are promising carriers for a single-shot vaccine.
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