Research On Targeted Microbubbles Combined With Ultrasound Thrombolysis In Prevention Of Peripheral Microcirculation

Objective: To prepare the Arg-Gly-Asp-Ser(RGDS)-urokinase-loading targeting ultrasound contrast agent using the avidin-biotin system(BAS)and to investigate its physicochemical properties and thrombus-targeting affinity.To study the thrombolytic effect of low-frequency ultrasound combined with targeted urokinase-containing microbubble contrast agents on treatment of thrombosis in rabbit femoral artery;and to determine the optimal combination of parameters for achieving thrombolysis.Methods: A biotinylated-avidin method was used to prepare microbubble contrast agents carrying urokinase and RGDS peptides,and the products were then tested the binding rate and stability by flow cytometry.Following femoral artery thrombosis in New Zealand white rabbits,microbubble contrast agents were injected intravenously,and ultrasonic exposure was applied.A 3 × 2 × 2 factorial table was applied to categorize the experimental animals based on different levels of combination of ultrasonic frequencies(Factor A: 1.6 MHz,2.2 MHz,2.8 MHz),doses of urokinase(Factor B: 1.5mg/Kg,3.0mg /Kg)and ultrasound exposure time(Factor C: 30 min,60 min).A total of 72 experimental animals were randomly divided into 12 groups(n= 6/group).Doppler techniques were used to assess blood flow in the distal end of the thrombotic femoral artery during the 120 minutes thrombolysis experiment.The rate of recanalization following thrombolysis was calculated,and thrombolytic efficacy was evaluated and compared.With the rabbit blood tests 6-Keto-PGF1 a,TXB2,P/T,SP index,this research observes the general situations of blood vessels,microcirculation,and embolism from HE staining confirmed presence.Results: The results of flow cytometry revealed that the Targestar SA microbubbles successfully carried RGDS and urokinase,the binding rate was more than 98%,and the stability was not affected by the rinsing.The thrombolytic recanalization rate for all experimental subjects after thrombolytic therapy was 68.1%.The optimal parameters for thrombolysis were determined to be 1)an ultrasound frequency of 2.2 MHz and 2)a 1.5mg /kg dose of urokinase.Ultrasound exposure time(30 min vs.60 min)had no significant effect on the thrombolytic effects.The other groups of rabbits’ blood vassels are not dissolved completely,and 11 rabbits had different degree of microcirculation and embolism.The group without microembolism had an increased 6-Keto-PGF1 compared with the group of microembolism group(P<0.05).Conclusions: Targestar SA microbubbles prepared by BAS successfully carried RGDS and urokinase,and had high binding rate and exhibited targeting affinity towards thrombus in vivo.The combination of local low-frequency ultrasound radiation,targeted microbubbles,and thrombolytic urokinase induced thrombolysis of femoral artery thrombosis in a rabbit model.The ultrasonic frequency of 2.2 MHz and urokinase dose of 1.5mg /kg induced optimal thrombolytic effects,while the application of either 30 min or 60 min of ultrasound exposure had similar effects.There may be correlation between the decrease of 6-Keto-PGF1 a and the formation of microcirculation of thrombus.