Studies On TNF-BP-Loaded Sustained-Release Microspheres And TNF-BP-Loaded Long-Circulating Nanoparticles

Tumor Necrosis Factor alpha blocking peptide (TNF-BP), a cycle-seven-peptide, which was shown to be capable to block the biological activities of TNF-α, can protect the joint from inflammol/Latory damage induced by rheumatoid arthritis and reduce the damage from Acute Lung Trauma induced by Endotoxic Shock. The major problem for the application of TNF-BP is that it is a small peptide with molecular weight about 1 kDa so that its half-life time in blood circulation is very short. These often results in low bioavailability. In order to overcome the problems, drug carriers are needed to sustained-release, control delivery and extend half-life of TNF-BP. Two novel drug carriers as sustained-release microspheres and long-circulating nanoparticles would be suitable for our purpose. Poly (lactide-co–glycolic acid) (PLGA), as a biocompatible and slowly degradable polymer, has been targeting, widely used in drug delivery systems, especially in drug-controlled release.The dissertation consists of two parts. In partⅠ, the subject was to explore the methods of coupling TNF-BP to PLGA microspheres based on double emulsion process, and provide references for the study of targeting, sustained and controlled release delivery systems of TNF-BP. In order to gain the best parameters for preparing PLGA microspheres, the influences of different factors on average drug loading and average entrapment efficiency of PLGA microspheres were evaluated by orthogonal-designing method using L16(45) table. The physicochemical properties of PLGA microspheres were investigated in detail by using optical microscope, scanning electronic microscope (SEM) and laser dynamic scattering. The average particle size of PLGA microspheres was (10.23±0.026)μm. The average drug loading and the average entrapment efficiency were (13.39±0.286)%, (83.54±0.241) % respectively.The lyophilized powders of TNF-BP-loaded PLGA microspheres were obtained in order to enhance the stability. Glucose, sucrose and mannitol were used as cryoprotectants in the freeze-drying process. Mannitol, concentration in 3%, was selected as the optimum cryoprotectant due to the minimum change of physicochemical properties of PLGA microspheres before and after freeze-drying process.In vitro releasing property of TNF-BP PLGA microspheres was studied and the drug releasing performance was good. TNF-BP releasing from PLGA microspheres in vitro could be described by double phase dynamic model and could be described by the following equation: Q = ?0. 010+38.856·(1-e^-t/0.496)+27.96·(1-e^-t/50.004).In partⅡ, the subject was to explore the methods of coupling TNF-BP to polyethyleneglycol-modified poly ( d,l-lactide-co-glycolide ) ( PEG-PLGA ) nanoparticles based on solvent evaporation process, and provide references for the study of targeting, sustained and controlled release delivery systems of TNF-BP. In order to gain the best parameters for preparing PEG-PLGA nanoparticles, the influences of some factors on average particle size and polydispersity index (PDI) of PEG-PLGA nanoparticles such as the type of polymer, the polymer concentration, the type of emulsifiers and the emulsifier concentration were studied. The size, morphology and zeta potential of PEG-PLGA nanoparticles were investigated in detail by using transmission electronic microscope (TEM), Zeta potential analysis and laser dynamic scattering. The average particle size and the average PDI of PEG-PLGA nanoparticles were(80.8±0.6) nm, (0.073±0.034). The average drug loading and the average entrapment efficiency were (1.22±0.15) %, (56.10±7.49) % respectively. And the average zeta potential was (-17.90±1.00) mV.In vitro releasing property of PEG-PLGA nanoparticles was studied and the release of PEG-PLGA nanoparticles was 71.85% for 24 h. TNF-BP releasing from PEG-PLGA nanoparticles in vitro could be described by double phase dynamic model and could be described by the following equation: Q = 0. 008+36.710·(1-e^-t/0.134)+34.705·(1-e^-t/2.580). The in vitro release results showed that the peptide released fast which was probably attributed to the TNF-BP adsorbed to the surface of nanoparticles by electrostatic interactions. The pattern of the fast release may be useful for treatment of acute diseases, such as endotoxic shock and multi-organ function failure.In vitro uptake of PEG-PLGA nanoparticles by murine peritoneal macrophages (MPM) was analyzed by a flow cytometry. The results indicated that the nanoparticles modified with PEG could reduce the uptake by MPM. Western-blot was used to detect the content of IκB in order to assess the ability of the protection to TNF-BP by the PEG-PLGA nanoparticles. The results showed that PEG-PLGA nanoparticles give high protection to TNF-BP.