| Thymosin alpha-1 (Ta1) was the first thymic hormone purified homogeneity and sequenced from partially purified thymosin fraction 5 (TF5). Tal is an acidic polypeptide (PI 3.8) consisting of 28 amino acids with complexed biological activities . In addition to inducing the expression of T-cell markers, Ta 1 influences primarily T-cells and stimulates the production of lymphokins such as Y -interferon and interleukin 2 receptors. In the past few years, numerous reports have demonstrated that lower Tal level in patients have relationship with acquired immunodeficiency syndrome (AIDS), Hepatitis B and Hepatitis C, whereas higher Tal level was shown on some pathological cells in the condition of tumor. Therefore, Ta 1 might play a certain roles in the pathogenesis of some diseases.Clinically, Tal is used to assist the treatment of chronic virus infection such as HBV and HCV infection, tumor such as melanoma, lung cancer, leukemia, squamous epithelial cell carcinoma, colon carcinoma, and immune deficiency patients and get good results. Ta 1 can also used as vaccine adjuvant. Recently, the studies of Ta 1 mainly focus on its biologic function and application which have made a great progress, but the research of pharmaceutical dosage form is seldom reported, the research about sustained release of Ta 1 has not ever been reported.Nevertheless, a major obstacle to the realization of the possible therapeutic use of Ta 1 is its rapid inactivation (half-life is less than 2h ), Ta 1 has to be injected by iv or im administration continuously for maintaining therapeutic effect, which may bring inconvenience to patients. Our purpose of this study is designed to encapsulate Ta 1 with poly(lactic-co glycolic acid) as a biodegradable carrier so as to control their release properties of Ta 1 to get the purpose of one month after single administration.Double emulsion (w/o/w) technique was used first to prepare Ta 1 PLGA microspheres . Single-factor experiment was designed to investigate the factors influencing microspheres size and encapsulation efficiency. Influential factors which have effect on characteristics of microspheresinclude stirring rate, polyvinyl alcohol(PVA) concentration, PLGA concentration, the ratio of inner and outer water phase, the osmotic pressure of the outer water phase. It is suggested that stirring rate, PVA concentration and PLGA concentration have significant effects on the appearance and particle size of microspheres, the appearance, particle size are important factors to affect the drug release from microspheres. The smaller of microspheres, the more significant burst effect of microspheres was observed. The encapsulation efficiency can be improved (more than 80%) through enhancing the osmotic pressure of outer water phase. Other factors did not show significant effects on the size distribution of microspheres with exception of PLGA concentration and PVA concentration in our test. PLGA with lower molecular weight has also been found to signally increase the degradation rate of microspheres as well as enhancing the cumulated release of Tod. When the concentration of NaCl in outer water phase was 5%, the morphology of microspheres was compact, the cumulated release can reach more than 80% and release kinetics fitted the zero order mode. When the concentration of Glucose in outer water phase was 10%, the cumulated release also reach more than 80% and the release kinetics fitted the Riger-Peppasr mode.Due to the water/oil interface formed during double emulsion formation, drug may be damaged and inactivated. Therefore, solid in oil in oil(s/o/o) methods were try to be used to prepare Tal loaded PLGA microspheres. The key process of this techniques is to micronize peptide powder. To dissolve the Tal and PEG6000 in aqueous solution, frozen and lyophilized, we can gain the microparticles after washed PEG with CH2CI2. In order to decrease the burst effect, zinc ion was added to form Zinc-Tal complex. Because of no aqueous solution and less drug solubility in the outer oil phase with s/o/o method, the encapsulation efficiency is higher than that with other method. In vitro release tests suggested that the release behavior of microspheres made by PLGA with molecular weight of 34,000 consisted of typical three stages: burst release phase, plateau phase and slow release phase. Usually, the plateau phase was relatively longer(15~20days) after burst release. This may be caused by the insoluableaggregation and slow degradation rate of polymeric material. When using PLGA with molecular weight of 14,000, there was almost no plateau phase while the cumulated release of slow release phase increased significantly. Total drug release with this polymer in the slow release phase was able to reach 40%. This suggested that PLGA molecular weight was the major influential factor of plateau phase and slow release stages. After optimization of prescription and manufacture technique of PLGA microspheres, the cumulated release of Tal reached approximately 80%, burst effect was less than 25% in 24 hours, the release kinetics fitted the zero order equation.Tal can accelerate T cell mature and differentiate, the bioactivity of Tal microsphere in vivo was analyzed by the CCK-8 method. The optical density (OD) of each well was measured at 490 nm and as the reference wavelength at 630nm with the ELISA microtiter plate reader, The number of live cell can be calculated. The results showed that the preparation technique can keep the bioactivity of Tal, but the bioactivities of Tal was much lower with method of W/O/W(Nacl) and S/O/O than that with W/O/W(glucose)The CD4+/CD8+ levels in the blood of immune inhibition SD rats was determined with flow cytometry after administration of Tal injection, blank microsphere and Tal microsphere. The CD4+/CD8+ levels significantly increased with Tal MS group (pO.Ol) and Tal injection group(p<0.05), compared to that with blank microsphere group.Conclusion: Tal can be encapsulated in injectable microspheres to yield one-month continuous release when using biodegradable polymers PLGA as carrier material. |
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