Study On The Preparation Of PELA-Microspheres For GST Vaccine Prolonged Release

In recent years, the potential of gutathione-S-transferase(GST) has been extensively considered as an anti-schitosome vaccine. Via recombinant DNA technology GST has been cloned and expressed, which can therefore, unlike some other traditional vaccines(TVs), solve the problems such as the shortage of schitosome-source for development, the side-effect or toxicity caused by TVs. But GST vaccine can be degraded and cleared rapidly in the body and the immune activity is poor while used only, which undoubtedly limit its clinical application. Biodegradable microspheres(MS) as a novel drug delivery system has drawn more and more attentions in modern pharmaceutical sciences. MS loading antigens can control or prolong the release of antigen over a period up to several months, protect vaccines from being degraded rapidly, and even, can simulate effectively the multiple recall immunization of traditional vaccines, And further decrease the antigen dose, increase the compliance of patients, and lower the cost of drugs. The thesis presented here focuses mainly on the preparation of Poly-D, L-lactide-co-polyethylene glycol (PELA) microspheres and the quality evaluation in vitro, which may be divided into four parts: review, preparation of PELA microspheres using albumin as model drug, quality evaluation, preparation and evaluation in vitro of GST microspheres.Part one: ReviewThe advanced progresses of controlled release microspheres(CR-MS) for biopharmaceuticals, especially for protein/peptide and vaccines are described, including the carrier materials, preparation methods, release characteristics and the stability of the sustained release microspheres, which of course, may be regarded as a basis for our succeeding research.Part two: Preparation of the PELA microspheresIn the study of pre-formulation of PELA microspheres, we chose bovine serumalbumin(BSA) as a model drug to optimize the preparation technique. The PELA microspheres were prepared by a modified W/O/W double emulsion-solvent diffusion method using ethyl acetate (EA) as organic solvent. We investigated the influences of different factors on the drug loading (DL) and the encapsulation efficiency (EE), including solvent type, carrier materials, stabilizer, concentration of polyvinyl alcohol (PVA) and the inner W/O volume ratio. The morphology of each formulation was also observed. Based on the single factor experiment, we optimized the fabrication of microspheres following an orthogonal experiment design with three factors, the ratio of drugs to carriers, concentration of PELA and the amount of PVA. The optimal condition is, EA as organic solvent; PELA as carrier material at a concentration of 50 mg ? mL"1; 2%(w/v), 20mL PVA as stabilizer; the inner W/O volume ratio is 1/10; and the weight ratio of BSA/PELA is 1/5.Part three: Quality evaluation of the PELA microspheres in vitroIn this part, the morphology, particle size and distribution, the DL and EE, release profile of the PELA microspheres were evaluated. After microspheres digested with 0.3 mol ? L''NaOH-SDS ( 2% ) , BSA was determined with UV-VIS spectrophotometer. The average DL and EE of the BSA-PELA microspheres is 12.44% and 86.31%, respectively. The shape of microspheres was investigated with scanning electron microcope, and the size distribution was measured with laser particle analyzer. The results showed that the microspheres are almost spherical and average diameter is 6.585 u m. The release of BSA from microspheres may follow two equations, in the beginning of three days, as Higuchi equation, Q=0.2681t1/2-0.0064 (r=0.9983), 4-28d, as zero-order kinetics, at a almost constant release speed, Q=0.0051t+0.5023 (r=0.9949). On the 28th day, the time of accumulative release of BSA is 64.83%, which show a remarkable sustained release characteristic.Part four: Preparation and quality evaluation of GST microspheresOn the basis of above research, we tried to make PELA microspheres containing GST, which were also evaluated with the indexes described above, such as themorphology, particle size, DL and EE, release behavior in vitro, in addition, assessed with the integrity of GST and the residual amount of organic solvent after the microspheres formation. BCA kits were used to measure the DL and EE of GST in microspheres and the average DL and EE of GST-PELA microspheres is 5.12% and 75.25%, respectively. Scanning electron micrographs showed the GST microspheres are almost spherical. Laser particle analyzer told us the average diameter is 5.387 u m. The integrity experiment via SDS-PAGE sugested that GST is stable after being encapsulated in PELA microspheres. In vitro release data may be best described with the following kinetic models: 0-3d as Higuchi equation, Q=0.2537tl/2-0.0005, r=0.9977, 4-28d with zero-order kinetic, Q=0.0071t+0.4875, r=0.9922. In addition, the release behavior indicated that PELA microspheres can obviously prolong the GST release and the accumulation at 28d is 69.10%. Capillary gas chromatography revealed the residues of EA in microspheres was lower than the permissible maximum limits.