A Study On Sonoporation-based Systematic Optimization And Gene Therapy Of Ultrasound Mediated Targeted Delivery

[Background]Ultrasound mediated targeted delivery,as novel genes or drugs delivery strategy with broad application prospects,has get an attention all over the world with the advantages of safety and ease of operation.The approach achieves targeted controlled release but its accuracy and controllable effect are still far from clinical application and promotion.Sonoporation is the most important mechanism for ultrasound mediated targeted delivery to achieve intracellular transport,but there is lack of a convenient and efficient experiment device to carry out this delivery strategy,which affects the promotion and development of the technology.[Objective]Based on a number of literature analysis regarding to both various ultrasonic devices and multiple parameter optimization,this study going to designe a self-assembly ultrasonic apparatus contains multi-parameter,programmed,and easy-to-operate.Multi-parameter optimization experimental was performed to explore the best in vitro gene delivery conditions and guide the next step of ovarian cancer cell gene therapy research.[Methods]By performing acoustic field distribution scanning,ultrasound power measurement and sound induced cellular membrane perforation experiments on the instrument,it was confirmed that its sound field distribution is uniform and has an efficient efficiency of mediating cell perforation.Furthermore,optimal parameters such as acoustic intensity,duty cycle,exposure time were determined through delivery efficiency of fluorescent molecules,which verified by flow cytometry;CCK8 was used to verify the cell viability after sonoporation treatment.Finally,combining the above mentioned delivery efficiency and vitality value to determind the optimal parameters,tumor cells apoptosis experiments was performed with the same condition;Laser confocal localization of the distribution of therapeutic agent Cy5 labeled mi RNA-34a mimic in cells;flow apoptosis detection to verify cell apoptosis;RT-PCR and Western Blot analysis of mi RNA-34a mediated Notch1 and Bcl-2 cellular apoptosis pathways.[Result]The transport of fluorescent molecules to tumor cells indicated that there is a positive relationship between the acoustic intensity and duty cycle with efficiency of molecule transmembrane transport,which increased by up to 66 times.However,with higher acoustic energy output,the worse cell damaged.When the parameters are under the conditions of acoustic intensity 1.0 W/cm~2,duty cycle 5%and exposure time 20 s,it has the best sonoporation efficiency for overian cancer cells.Under the same optimal condition,further research of ultrasound mediated targeted delivery was carried out for gene therapy.,mi R-34a mimic was introduced into cells and successfully expressed via ultrasound targeted delivery technology.Flow cytometry analyzed cell apoptosis;With the optimal parameter,delivery of mi RNA-34a via ultrasound combine with microbubble can effectively inhibit the growth of tumor cells.The main mechanism has been proven to inhibit the proliferation of tumor cells and induce apoptosis by down-regulating the expression of Notch1 and Bcl-2.[Conclusion]The new sonoporation apparatus combined with optimal parameters can effectively mediate the cell membrane pore formation and enhance the effect of macromolecular drugs and gene delivery and eventually achieve the purpose of tumor treatment.The study provides a safe and effective strategy for in vitro gene therapy,and also for subsequent development ultrasound-mediated sonoporation in vivo research lays the foundation.