Preparation And Evaluation Of Long Circulation System Based On Vitamin K1 In Red Blood Cells

Objective: to prepare long cycle carrier of vitamin K1 loaded on erythrocytes based on chitosan nanoparticles containing vitamin K1.The red blood cell carrier can carry nanoparticles to avoid being swallowed by the reticuloendothelial system,so as to carry vitamin K1 in the body for a long cycle,provide new drug options for patients who need long-term treatment of vitamin K1,and increase the compliance of such patients.Methods: chitosan nanoparticles loaded with vitamin K1 were prepared by ion coacervation.The factors influencing the preparation of chitosan nanoparticles were determined by single factor test.The formulation of chitosan nanoparticles was screened by box Behnken effect surface method with the particle size and encapsulation rate as evaluation indexes.The particle size,zeta potential,morphology,entrapment efficiency and drug loading of chitosan nanoparticles were evaluated;the release of chitosan nanoparticles in vitro was investigated by dialysis method;the influencing factors in the process of loading nanoparticles by red blood cells were determined by single factor investigation,and the optimal process conditions were determined.The morphology of drug loaded erythrocytes was determined by scanning electron microscopy,and the effects of nanoparticles on the fluidity and deformability of erythrocytes were investigated by turbulent and osmotic fragility.The adhesion of nanoparticles was studied by shear force simulation in vitro.The long circulation effect of drug loaded erythrocytes was investigated by pharmacokinetic test in rats.Results: The optimal formulation of chitosan nanoparticles was: chitosan 0.5mg/ml,pH = 4.0,adding VK1 3mg,the mass ratio of chitosan to sodium tri-polyphosphate was 3:1.The morphology of chitosan nanoparticles was spherical.The size of chitosan nanoparticles was 353 nm,zeta potential was + 15.3mv,entrapment efficiency was 70%,and drug loading was 3.26%.The cumulative release rate was 80% within 256 hours.The optimal formulation of the prepared red blood cell carrier is: the concentration of chitosan nanoparticles is 1mg / ml,the volume ratio of chitosan nanoparticles to red blood cells is 5:1,the incubation time of nanoparticles to red blood cells is 20 min,and the incubation temperature is 37 ?.The drug loading was 373 ug / ml and the loading rate was 80.17%.Under the scanning electron microscope,the morphology of the drug red blood cells was double concave,and there was no significant change compared with the drug red blood cells.The results of turbulent fragility showed that compared with normal red blood cells,the turbulent fragility and osmotic fragility of drug loaded red blood cells decreased.The shear stress test in vitro showed that chitosan nanoparticles adhered to the surface of red blood cells and could circulate with red blood cells in vivo.The results of pharmacokinetics in rats showed that the average Cmax of carrier red blood cells was 12.141 ng / L,significantly lower than 471.785 ng / L in the injection group and 26.845 ng / ml in the nanoparticles group.Meanwhile,the half-life of vitamin K1 in RBC carrier group was 30.225 h,7.5 times of that in injection group,2.14 times of that in nanoparticle group,and the average residence time was significantly prolonged.Conclusion: chitosan nanoparticles prepared by ion coacervation can successfully adhere to the surface of erythrocyte membrane,avoid being engulfed by reticuloendothelial system,and achieve the goal of long cycle.