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To construct the ultrasound microbubble co-carrying platelet targeting ligand RGDS and tissue plasminogen activator rt-PA, study the maximum efficiency of carrying RGDS and rt-PA combined with microbubble contrast agent, and definite ultrasound imaging characteristics of RGDS/rt-PA targeted microbubbles in vitro and targeted binding ability.
Microbubbles were combined separately with the recombination of rt-PA and fluorescein isothiocyanate (FITC) as well as RGDS peptide and 5- carboxyl methyl four Luo Danming (5-TAMRA). Combined 1.25mg, 0.25mg, 0.125mg, 0.0625mg, 0.03125mg fluorescent labeling RGDS, rt-PA with 200ul Targestar SA microbubbles respectively via “biotin-streptavidin method”, detecting the maximum efficiency of combination(the best binding rate of single load). The double load RGDS/rt-PA targeted microbubbles were prepared by adding rt-PA and RGDS to 200ul microbubbles with the best binding rate of single load, while in accordance with the different order they were divided into three groups: add RGDS before adding rt-PA (group A);add rt-PA before adding RGDS; add rt-PA, RGDS after mixing (group C). The microbubbles were evaluated though physiochemical properties, fluorescence cytometry and flow cytometry test the binding rate of RGDS and rt-PA in microbubbles were detected. The intravascular thrombus model was prepared in vitro and injected into RGDS/rt-PA targeted microbubbles, observe the characteristics of ultrasonic imaging. Observe the thrombus in the model under the fluorescence microscope to determine the target binding ability of RGDS/rt-PA targeted microbubbles.
The binding rate of RGDS and microbubbles was 93.9%, the average binding rate was, the binding rate of rt-PA and microbubbles was as high as 83.6%, and the average binding rate was up to 82.3%. RGDS, rt-PA and micro bubble Double load combination, a, B, C double load group with different rates (F=18.208, P=0.03, P<0.05, there was a significant difference between the a group), the average binding rate was 67.2%, the average rate was 61.5% with B group, C group with the rate of 68.3%, namely double load the mixing in 0.0625mg RGDS and 0.0625mg rt-PA and 200ul combined with microbubbles can achieve the best combination rate of 68.3%. RGDS/rt-PA target were prepared to ultrasound microbubble morphology under microscope showed no significant difference between the micro bubble, stable performance, the average particle size of 2.32 + 0.03um, 2.71 + 0.01 concentration, pH value of 6.27 + 0.03; red fluorescent green fluorescence under fluorescence microscope and RGDS marker RGDS/rt-PA targeted microbubbles uniformly distributed on the surface of rt-PA markers. Under scanning electron microscope, RGDS/rt-PA targeted microbubbles were observed to be spherical and smooth, with no obvious aggregation. In vitro vascular thrombosis model in RGDS/rt-PA targeted microbubbles showed dense fine hyperechoic in ultrasound image. The results show that the micro bubble has reflected ultrasonic signals good; by agglomeration trend after a slight oscillation directional thrombosis. In the model, the red and green fluorescence was observed under the fluorescent microscope, and it was confirmed that the microbubbles carrying the corresponding fluorescent substances adhered to the surface of thrombus.
RGDS/rt-PA targeted microbubbles can be successfully prepared by BAS method, which has high binding rate and good stability, and can be targeted to thrombus.