| Interferon- a (IFN- α ) is a protein with extensive antiviral, antiproliferative and immunomodulatory biological activity. It's one of the first cell factor used in clinic. IFN- a is the most widely researched cytokine in basic and clinic. The relative molecular weight of IFN- a is 19KD. Its circulation half life is 4-16 hours. It reaches peak serum concentration in 3-8 hours after hypodermical inject or intramuscular inject. It can not be detected after intravenous inject, hypodermical inject or intramuscular inject 24 hours later. It need to be administered at least 3 times a week, which may restrict the development and utilization of IFN- α.The most commonly used method preparing microspheres is double emulsion technique(w/o/w) . The equipments used in this method is relatively simple. The burst effect of microspheres prepared by w/o/w technique is relatively low. According to previous studies, there were plenty of influential factors when preparing microspheres by double emulsion method, and due to the water/oil interface formed using this technique, protein may be damaged and inactived. Solid in oil in oil (s/o/o) method was studied to prepare IFN-α-2b loading P1GA microspheres. Using this method can obviate the water/oil interface. But acetonitrile must be used in this method and it is difficult to be removed from microspheres. The burst effect of the microspheres prepared by s/o/o method is higher and the cumulated release is less.According to previous studies, we used solid in oil in water (s/o/w) method to prepare the microspheres. The fundamental principle of keeping the bioactivity of macromolecule drug was that the anhydrated protein powder is passive in organic solvent and the conformation change is restricted. First, Zinc-complexed IFN-α-2b microparticles were formulated successfully which can be redissolved and whose electrophoretic and chromatographic behavior were the same as IFN- a -2b original solution. Microparticles of Zinc-complexed IFN- a -2b were prepared by phase separation method which were then encapsulated into microspheres.Single-factor experiment was designed to investigate the influential factors whichaffect the appearance, size distribution and encapsulation efficiency of microspheres. The influential factors include stirring rate, polyvinyl glycolide (PVA) concentration, the volume of PVA solution, the theory protein loading, PLGA concentration and the Mw of PLGA. It suggested that stirring rate, PVA concentration, PLGA concentration and the Mw of PLGA have significant effect on the appearance and particle size of microspheres. We can control the size of microspheres by changing those factors. Other factors didn't have significant effect on the encapsulation efficiency except the theory protein loading, PLGA concentration and PVA concentration. After optimization of prescription and manufacture technique of PLGA microspheres prepared by s/o/w methods, the encapsulation efficiency was 79.27%.IFN- a -2b concentration in release medium was determined by BCA (Bicinchoninic acid) protein determination method and the in vitro release behavior of microspheres was studied. The influential factors include stirring rate, polyvinyl glycolide (PVA) concentration, PLGA concentration and the Mw of PLGA.The biological activity and immunological activity of IFN- a -2b in microsphere and in vitro release medium was determined by cytopathic effect (CPE) and enzyme linked immunosorbent assay (ELISA) method respectively.Due to the particularity of macromolecule drug and PLGA, we used gamma irradiation sterilization. Five different dosages of gamma ray were designed to investigate the influence of microsphere appearance, physico-chemical property of polymer, biological activity of IFN in microspheres and the release characters of microspheres. We desired to find a lowest sterilization dosage that can't influence the characters of microspheres. According to previous studies, y-ray cause microsphere shape changed and made microspheres more fragile. It also cause the glass transition temperature of polymer reduced and the burst effect of microspheres increased. The loss of biological active was too much after y-ray irradiation sterilization, so y-ray irradiation sterilization is not suitable for IFN- a -2b microspheres.Conclusion: IFN- a -2b can be encapsulated in injectable microspheres by partly-nonaqueous method to yield one-month continuous release using biodegradablepolymers PLGA as carrier material, and this technique will have a favorable perspective in the future.
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