Study Of Preparation Of High-adherence-rate Lymphatic Targeted Microbubbles Of Breast Cancer

Objective:① Optimize the preparation of high-adherence-rate lymph vessel-targeted phospholipid-coated microbubbles of antibody, namely targeted microbubbles carrying antibody VEGFR-3 and Podoplanin, and conduct relevant evaluation on the physicochemical properties. ② Observe their targeting effects in experiments in vitro. ③ Investigate the effective parameters of ultrasonic radiation force that promoting the displacement of ultrasonic contrast agent in experiments in vitro.Methods:Apply biotin-avidin bridge method to prepare targeted phospholipid-coated microbubbles carrying antibody VEGFR-3 and Podoplanin, optimize the preparation technology, improve the production process, adjust the proportions of lipid, biotin and avidin, and adopt immunofluorescence for relevant evaluations. ② Carry out grouping and controlled study and observe the targeting effects with direct and indirect method. Direct method:add regular microbubbles and targeted microbubbles respectively to lymph vessel cell culture plates. Conduct incubation for 10min and washing for 3 times, and observe the conditions in these two groups under a microscope. Use image analysis software IPP for off-line analysis, and use SPSS software package for the statistical analysis of the amount of microbubbles combined with cells; indirect method:take advantage of the weight difference between microbubbles and cells, and adopt grouping incubation and centrifugation method to observe the effect of targeted combination in an intuitive manner. ③ When the lumen is at a deep position, explore the optimal radiation force parameters of promoting microbubble displacement to the contralateral wall under the conditions of static fluid and simulative lymph fluid flow respectively. Then use high-speed camera to evaluate its efficacy of achieving promoting microbubble displacement.Results:① Targeted microbubbles with high adherence rate were prepared successfully:the mean particle size was 0.99μm and the mean antibody fixation rate was demonstrated by FCM to be up to 81.3%. ② The off-line analysis conducted with image analysis software IPP indicated that the amount of combined microbubbles with the endothelial cells of lymph vessel in the targeted group was significantly prior to that in the general group; under a high-power microscope (×100), there were (151±20) targeted adherent microbubbles in each field in the targeted group and (12±4) general adherent microbubbles in each field in the general group (t=19.19, P<0.01 and the result had statistical significance); in comparison with general microbubbles, targeted microbubbles combined with large amount of cells and were capable of maintaining firm connection even under 400g centrifugal force. ③ Under static condition, peak negative pressure 530kPa, duty ratio 0.031, pulse repetition frequency 0.97kHz, sound intensity 0.29W/cm2 and mechanical index 0.35 can exactly make the microbubbles adhered to the contralateral wall; when flew as the flow velocity of lymph, approximately 1/2 microbubbles flew away through the lumen but not adhered to the wall. If irradiation parameters are increased, although the amount of microbubbles adhered to the wall is increased,75% will be broken by high sound pressure; the radiation force acts on shallow lumen first and the microbubbles in deep lumen will not be affected by acoustic radiation force until the microbubbles in shallow lumen are completely blocked or separated.Conclusion:① The technology, process, reagent formula and other aspects of preparing targeted microbubbles were optimized on the basis of biotin-avidin bridge method. High-adherence-rate lymph vessel-targeted microbubbles of antibody VEGFR-3 and Podoplanin with small and uniform particle size were prepared successfully and verified in targeting trials in vitro; ② the successful preparation of antibody high-adherence-rate targeted contrast medium and its good in vitro targeting performance have provided an experimental basis for the targeted development of lymph angiogenesis of tumor in vivo. Hopefully the contrast medium can aggregate and maintain for a long time at the targeted area of tumor lymph angiogenesis so that the effect of contrast imaging can be enhanced and the targeted area can be recognized more easily in comparison with the soon-subsided non-targeted area. In other words, this type of antibody high-adherence-rate targeted microbubble can be expected to display the lymph angiogenesis of tumor in a precise manner and achieve rapid identification on tumor character under ultrasonic tracking as a kind of molecular probe that specifically recognize the tumor cells of lymphatic system; ③Radiation force can promote more microbubbles in lumen to adhere to the wall. However, the required radiation force parameters and their effects under various flow states and positional conditions are different.