Experimental Study On The Preparation And Physicochemical Properties Of Urokinase-loaded CRGD-modified Liposomes

Background: Thrombotic diseases are a group of diseases of the circulatory system that seriously endanger human health.They are common and frequently-occurring diseases in the elderly and middle-aged groups.They have a high mortality and disability rate,and have become an important disease that threatens human health and life in today’s society.Among the current treatment methods for thrombotic diseases,thrombolytic therapy is the most common and effective method.Urokinase is the most commonly used thrombolytic drug in clinic because of good curative effect,abundant raw materials and low price.However,since urokinase is not thrombo-targeting,thrombolysis with high concentration of urokinase is likely to lead to systemic bleeding complications.A large number of studies have shown that the RGD sequence can specifically bind to the activated platelet membrane glycoprotein IIb/IIIa receptor,has anti-platelet aggregation and thrombus targeting effects,and is one of the targets of thrombosis.Liposome is a drug carrier with passive targeting and slow release.The long-circulation liposome prepared by modifying the surface of liposome with polyethylene glycol(PEG)can prolong the circulation time in the body,making the drug increased amount of accumulation in the lesion.Therefore,PEGlated liposomes were used as carrier,and the surface-modified RGD sequence was used as the thrombus-directing molecule,urokinase was encapsulated inside the liposomes to prepare urokinase-loaded cRGD modified liposomes.It was used to improve the specificity of urokinase and the targeting of thrombosis,so that urokinase can selectively act on the thrombus site and reduce the side effects of bleeding and other adverse reactions.Objective: Firstly,to use film dispersion method to prepare urokinase-loaded cRGD modified liposomes,then to detect physical and chemical properties for preparation for the follow-up animal model of chronic pulmonary hypertension in vivo thrombolytic experiments.Methods: Blank liposomes and urokinase-loaded cRGD modified liposomes were prepared by film dispersion method.A small amount of prepared liposomes were took to observe their morphology and size with an optical microscope.The morphology and size of urokinase-loaded cRGD modified liposomes were observed with transmission electron microscope.The particle size of liposomes was measured with a laser nanoparticle size analyzer.Observed the particle size change of urokinase-loaded cRGD modified liposomes in PBS buffer containing50% fetal bovine serum within a certain period of time to study its stability in serum.The prepared urokinase-loaded cRGD modified liposomes were used for in vitro hemolysis experiments on red blood cells to investigate whether liposomes with different concentration had hemolytic reactions on red blood cells.Human umbilical vein endothelial cells were used to investigate whether urokinase-loaded cRGD modified liposomes at different concentration had toxic effects on the cells.Bradford protein assay kit was used to detect the encapsulation efficiency and drug loading capacity of urokinase-loaded cRGD modified liposomes on urokinase.The release experiment of urokinase-loaded cRGD modified liposomes was simulated in vitro,and calculated the cumulative release rate of urokinase.Under circulating conditions,urokinase-loaded cRGD modified liposomes were used to dissolve thrombus,and observed the thrombolytic effect of liposmes in vitro.Results: 1.The particle size of the blank liposomes was about 143.9nm,and the particle size of the urokinase-loaded cRGD modified liposomes was about176.8nm.The liposomes were not clear under the optical microscope.2.The particle size of urokinase-loaded cRGD modified liposomes in PBS buffer containing 50% fetal bovine serum did not change much within 72 hours,indicating that it can remain stable in serum.3.The hemolysis rate of urokinase-loaded cRGD modified liposomes was less than 5%,indicating that liposomes had no hemolytic effect on red blood cells.4.Cytotoxicity test showed that the cell survival rate of liposomes at all concentrations was greater than 90%,suggesting that urokinase-loaded cRGD modified liposomes had no obvious cytotoxic effect.5.The encapsulation rate of urokinase-loaded cRGD modified liposomes was about 58.83%,and the drug loading was about 40.64%.6.The release experiment results in vitro simulated of urokinase-loaded cRGD modified liposomes showed that the cumulative release rate of urokinase was18.57% at 1h,and by 8h,the cumulative release rate was 56.19% and reached the plateau period,including that the urokinase-loaded cRGD modified liposomes could release most of its encapsulated urokinase within 8 hours.7.The results of thrombolysis experiments in vitro showed that the thrombolysis rate of urokinase-loaded cRGD modified liposomes was 83.14%.Conclusions: The urokinase-loaded cRGD modified liposomes were successfully prepared by thin-film dispersion method,which is a nano preparation,and is stable in serum,and has no hemolytic reaction to red blood cells in vitro,and has no cytotoxic effect,and most of the encapsulated urokinase can be release within 8 hours in vitro.It can be thrombolytic in vitro.