Preparation Of Co-carrying RGDAS/rt-PA Targeted Microbubbles And An Experimental Study Of The Dissolution Of The Simulated Artery Thrombus In Vitro

Objectives: Co-carrying RGDS/rt-PA target microbubbles were prepared by BAS method,then detect the best binding rate,physical properties,drug loading,acoustic development and thrombus targeting.In vitro preparation of quasi arterial thrombi model(PRT),the application of low frequency ultrasound combined with the co-carrying RGDS/rt-PA target microbubbles under the simulated physiological environment of thrombolytic experiment,evaluation of RGDS/rt-PA microbubbles targeting PRT and thrombolysis.Methods: Single loaded microbubbles of RGDS and rt-PA were prepared and detect the binding rate;The co-carrying RGDS/rt-PA target microbubbles were prepared by the optimum combination rate.Then we detected the co-carrying binding rate and physical properties of the double loaded microbubbles,and measured the drug loading concentration.Application of in vitro preparation of quasi artery thrombus(PRT)model,HE staining,Masson staining and scanning electron microscopy to estimate properties of thrombus.The thrombolytic experiment was performed on PRT with the plasma of 37 at constant temperature.According to the different combination of ultrasound,rt-PA,microbubbles were divided into 6 groups:(1)the ultrasound group(US group);(2)the ultrasound + microbubbles group(US+M group);(3)the rt-PA group(rt-PA group);(4)the ultrasound + rt-PA(US+rt-PA group);(5)the ultrasound +rt-PA +microbubbles group(US+M+rt-PA group);(6)the ultrasound + Co-carrying RGDS/rt-PA target microbubbles(US+TRM group).The(6)group PRT after thrombolysis were observed under a fluorescence microscope to clear and definite the targeting combined with microbubble.The change rate of maximum cross section area and lytic rate of PRT before and after thrombolysis were derived.Structural changes of PRT were observed under a scanning electron microscope.Results: The average binding rate of co-carrying RGDS/rt-PA targeted microbubbles reached best when we added microbubbles into themixture of RGDS and rt-PA,which is 68.3%.The average particle size was(2.32 + 0.02)m.The concentration was(2.71 + 0.01)x 109/ml,and there was no significant difference from the naked microbubble(P > 0.05).The concentration of rt-PA protein in co-carrying RGDS/rt-PA targeted microbubbles was 498.5ug/ml.In vitro,the co-carrying RGDS/rt-PA targeted microbubbles showed high-echo dots with uniform distribution,and exhibited thrombolytic activity by targeted combination with PRT.Each group PRT after thrombolytic treatment were dissolved,with the US+M+rt-PA group(rt-PA 60KU/ml)and group US+TRM(rt-PA 30KU/ml)was the best thrombolytic effect,the maximum cross-section area change rate were 52.22 + 8.53 and 51.61 + 9.91,lytic rate were 75.03+ 9.95 and 80.37 + 9.62,the difference with other comparison between the groups were statistically significant(P < 0.05).Compared with before treatment,the low frequency ultrasound combined with co-carrying RGDS/rt-PA targeted microbubbles cause the PRT electron microscope after thrombolysis change into fibrin was significantly thinner,the emergence of a large number of fuse shape and cut shaped fiber filament ends,fiber mesh loose and disordered structure,a large number of cavity formation,thrombosis was dissolved.Conclusion: Co-carrying RGDS/rt-PA targeted microbubbles have stable properties,high binding rate to ligands,good acoustic development and certain PRT targeting.They can efficiently dissolve the simulated artery thrombus in the simulated physiological state of thrombolysis,and effectively reduce the dosage of thrombolytic drugs.