Study Of Long-acting Sustained-release Microspheres Loading Erythropoietin With Closely Zero-release Behavior

Recombinant erythropoietin is a kind of pleiotropic cytokine to treat anemia caused by renal failure or heart failure,cardiac trauma by anemia and so on.Sustained-release therapeutics can reduce the frequency of injection and decrease the concentration peak.However,it may bring the problem of aggregation,denature,immunoreaction and the risk of failure of sustained release preparation.In the past decades,there were many research about EPO-loaded microspheres.But none of the problems have been conquered.None of the products have been launched in the market successfully yet.Our lab have developed some delivery systems of biomolecules to protect the advanced structure of proteins which is fragile and changeable.A lot of innovative and practical technologies have been studied by us to solve the problems comprehensively,for example,aqueous-aqueous emulsion method,SPG membrane emulsification method,phase orientation microencapsulation method and so on.The sustained release preparation of EPO can protect their natural structure,prevent adverse antibodies and reduce the burst release.On the basis of the technologies we had,long-acting sustained-release microspheres loading erythropoietin with closely zero-release behavior have been studied in this article to treat the new indication,heart failure by insufficient of oxygen.High performance liquid chromatography has been used to study the stability of EPO in citric acid solution.As a release medium,citric acid can protect EPO at least for half a month in 4℃,25℃,and 37℃.Dextran particles loaded with EPO were prepared by phase separation method before encapsulated into PLGA microspheres in case of denature during encapsulation and release.A better prescription was selected according to the release curve.Then SPG membrane method was used to produce uniform and spherical microspheres without small-sized particles which would have adverse effect on release kinetics.Some controlled release technologies were applied to solve the particular problems of microspheres’ release.In order to decrease the burst release,annealing heal was used to make the microspheres smooth so as to make the pores in the surface of the microspheres closed.The theory of annealing is that PLGA can restore by itself when the temperature gets its Tg.To modify the incomplete release of microspheres especially in later period,magnesium hydroxide was added into the prescription to neutralize the degradation products of PLGA and the content was studied to make the release more sustained.Formulation optimization including magnesium hydroxide,annealing combined with SPG membrane method,long-acting sustained-release microspheres loading erythropoietin with closely zero-release behavior can be achieved.In the last part of the thesis,the pharmacokinetic was studied by mice with subcutaneous injection.An in-vivo release curve with low initial burst,smoothly sustained released over 30 days and released more than 80% of the total loading amount.