In Vitro And In Vivo Evaluation Of Recombinant Human Erythropoietin Loaded Sustained Release Microspheres

Recombinant human erythropoietin is developed using genetic engineering techniques to stimulate red blood cell proliferation, differentiation and maturation, which are used to treat anemia caused by kidney failure, anemia of AIDS and cancer chemotherapy-induced anemia. However, rhEPO treatment of anemia is a long period, while rhEPO has a very short half-life in vivo (about 8 h or so). This will require long-term frequent injections to maintain their efficacy, but longer treatment period made the patient’s work and life inconvenient. Therefore, finding the right formulation to solve this problem is particularly pressing.To extend the single dose efficacy of rhEPO, reduce dosing frequency and enhance patient medication compliance. In this study, we chose the good biocompatibility of biodegradable synthetic polymers PLGA, prepared rhEPO loaded PLGA sustained release microspheres using S/O/W emulsion solvent evaporation method. In this paper, basic properties of rhEPO loaded PLGA microspheres were investigated, and in-depth study the in vivo pharmacodynamics, pharmacokinetics of rhEPO microspheres, and the toxicology of PLGA microspheres were also studied.First, we investigate the critical factor affecting the properties of PLGA microspheres fabricated by a solid-in-oil-in-water (S/O/W) emulsion technique with BSA as a model protein. Prior to encapsulation, the BSA microparticles were fabricated by a modified freezing-induced phase separation method. The microparticles were subsequently encapsulated into PLGA microspheres by S/O/W emulsion method, then Motic BA200 biological microscope, confocal laser scanning microscope, scanning electron microscope were used to observe the structure of S/O/W emulsion and PLGA microspheres. The protein content extracted or released from BSA microspheres was measured by Bradford protein assay method. It was found that NaCl added in the outer aqueous phase effectively suppressed material exchange between the inner and outer phase of S/O/W emulsion. Then, the structure and permeability of obtained microspheres was influenced. As a result, with the increase of NaCl concentration in the outer aqueous phase, the encapsulation efficiency of microspheres significantly improved from 60% to more than 85%, the burst release of microspheres reduced from 70% to 20%, the particle size decrease from 103μm to 62μm. Furthermore, the rehydration of encapsulated protein was also retarded and then integrity of BSA was successfully protected during encapsulation process. In vitro release test showed that BSA released from PLGA microspheres in a sustained manner for more than 30 days.In the drug prescription design, we applied a large number of human serum albumin (HSA) as a protective agent for rhEPO, prepared rhEPO loaded PLGA sustained release microspheres using S/O/W emulsion solvent evaporation. In this experiment, encapsulation efficiency, drug loading and burst release rate of rhEPO microspheres were investigated by double sandwich ELISA, particle size of microspheres were measured by laser particle size distribution, the stability of rhEPO microspheres was also measured by SEC-HPLC, SDS-PAGE and Western blot, and studied the in vivo bioactivity of rhEPO within microspheres. The encapsulation efficiency of rhEPO microspheres reached to76.2%, drug loading was 0.21%, burst release rate was only 14%, and the mean particle size of rhEPO microspheres was 62.9μm. The stability of rhEPO was well protected by HSA whthin the Microspheres, there was no aggregation and degradation of rhEPO, the in vivo biological activity of rhEPO did not decrease.After a single intramuscular injection of rhEPO loaded PLGA sustained release microspheres to SD rats, blood were collected from medial canthus of the eye at a given time, Studied the pharmacodynamics of rhEPO microspheres by measured the changes of blood values; Serum drug concentration were measured using double sandwich ELISA, and pharmacokinetic parameters were analyzed by pharmacokinetic software 3p87, then the half-life of rhEPO Microsphere were calculated. Results after single injection of rhEPO microspheres in rats the blood values were maintained above the baseline value for up to 25 days; Plasma concentrations maintained at a stable state, its higher than the control value for up to 30 days, half-life t1/2 of rhEPO microspheres was 14 days, there were significantly longer than the half-life t1/2 of rhEPO water injection with 7 hours, show that rhEPO microspheres has a good sustained release effect, and has a long and duration drug efficacy. Study the toxicology of PLGA microspheres using a large number of HSA microspheres injected to rat, then observed acute toxic effects on the body, There was not any response to acute toxicity of microspheres, such as convulsions, shortness of breath, and abnormal changes in food intake and so on; Eventually show that biodegradable PLGA polymers have good biocompatibility. This paper also examined the surface morphology change after in vivo degradation of PLGA microspheres; this study found that microspheres complete degradation after approximately 30 days.