Study On Protein-loaded PCADK/PLGA Microspheres And The Application In RhGH Delivery

Protein drugs have attracted a wide spread attention due to their excellent specificity and activity, but their short biological half-life and poor stability requires multiple injections and high-dose, resulting in patient discomfort and severe side effects. Thus, reducing the injection frequency of protein drugs and constructing biodegradable controlled-release formulations are universally desired.Using biodegradable polymers such as poly(lactic-co-glycolic acid)(PLGA) to construct microspheres system has received tremendous interes and the technology has been successfully applied to small molecule drugs. However, the degradation of PLGA leads to the accumulation of lactic acid(LA) and glycolic acid(GA) inside the microspheres, causing a sharp rise in acidity of the surrounding microenvironment and leading to protein chemical degradation and loss of activity, which bring a lot of trouble to the sustained release of protein drugs. Poly(cyclohexane-1,4-diyl acetone dimethylene ketal)(PCADK) is a kind of polyketals with ketal linkages in their backbones and whose degradation into neutral compounds may prevent the intense drop in p H. At present, PCADK has been used in the preparation of microspheres for intracellular drug delivery and tumor targeting therapy; however, it could not be utilized in common injected microspheres because it does not substantially degrade under physiological p H. In this study, conventional subcutaneous PCADK/PLGA microspheres were prepared for improving release behavious, decreasing the μp H inside the microspheres, and increasing the protein stability.In this dissertation, the following studies were conducted:(1) The synthesis, characterization and biocompatibility of PCADKPCADK was synthesized and characterized by NMR, GPC and DSC.Biocompatibility of PCADK were investigated by acute systemic toxicity test, pyrogen test, cytotoxicity test, hemolysis test and skin irritation test, the results showed that PCADK has good biocompatibility and blood-compatibility.(2) Protein compatibility of PCADK degradation productsProtein drugs(BSA, Ig G and rh GH) incubated in PCADK and PLGA degradation products were investigated using SDS-PAGE, SEC-HPLC, CD and fluorescence spectroscopy. The results showed that the structural stability of BSA was preserved better in the presence of PCADK monomers than PLGA monomers. Protein aggregation and decomposition were significantly reduced and high-level structures were closer to natural conformation. Hpx rats in PCADK group had a weight gain of 12.5 g, while the PLGA group only increased by 5 g. The results indicated that PCADK degradation products could maintain the pharmacological activity of rh GH better. Compared with PLGA, the degradation products of PCADK were more favorable for the structure stability and pharmacological activities of protein. The evaluation of protein structure stability lays a foundation for further work.(3) Preparation and evaluation of BSA-loaded PCADK/PLGA microspheresBSA-loaded PCADK/PLGA microspheres were successfully prepared, and the optimal PCADK/PLGA ratio was selected by inspecting morphology, drug-loading and release behavious. The stability of BSA inside the PCADK/PLGA microspheres were evaluate. Compared with PLGA HIGH microspheres and PLGA 4A microspheres, the aggregation of BSA in PCADK/PLGA HIGH microspheres and PCADK/PLGA 4A microspheres decreased by 50% and 22% respectively, indicating that PCADK/PLGA microspheres reduced the aggregation and improved the stability of BSA significantly. The mechanism of PCADK/PLGA microspheres was investigated by DSC, SEM, CLSM and gravimetric analysis. The addition of PCADK improved the release behavior, decreased the μp H inside the microspheres, and increased the protein stability.(4) Study on rh GH-loaded PCADK/PLGA microspheresAfter the implementation of the PCADK/PLGA microspheres platform, a protein drug-rh GH was encapsulated in PCADK/PLGA drug delivery system. The optimal formulation was determined by investigating morphology, drug-loading and release behavious. The in vivo release of rh GH from PCADK/PLGA microspheres were examined by detecting the content of rh GH in blood of rats. The AUC value of the PCADK/PLGA microspheres was increased 2-fold compared to that of the PLGA microspheres, suggesting that the addition of PCADK effectively increases the bioavailability of rh GH. At last, the pharmacodynamics was studied by detecting IGF-1 level in blood and the body weight of Hpx rats and the results suggested that the PCADK/PLGA microspheres has better pharmacological efficacy than the PLGA microspheres.In summary, PCADK/PLGA microspheres were successfully constructed and the addition of PCADK improved the release behavior, relieved the acid environment inside the microspheres, and increased the protein stability and pharmacological efficacy. The PCADK/PLGA microspheres are uniquely effective carriers for protein drugs, and could potentially have diverse applications in protein delivery systems.