Ultrasound Microbubbles Associated With Liposome Enhance PNOGO-R SHRNA Delivery To Neural Stem Cells In Vivo

Objective: To investigate the parameters of ultrasound-mediated microbubble destruction of pGPU6/Neo plasmid transfected into NSCs of rats by ultrasound.Methods: The plasmid was used to transfect into NSCs by ultrasound-mediated microbubble destruction. The NSCs were divided into 5 groups: blank control, pGPU6/Neo plasmid, pGPU6/Neo plasmid-microbubble, pGPU6/Neo plasmid-ultrasound and pGPU6/Neo plasmid-microbubble-ultrasound. The last group was further divided into subgroups according to different conditions of ultrasound. After 48 h, the expression of GFP was detected by fluorescent microscope. The cell vitality was measured by typan-blue stainning.Results: The transfection efficiency was the highest and the cell vitality was not different from other subgroups when ultrasound was radiated at frequency of 300 kHz and sound intensity of 1.0 W/cm2 for 30 s. The transfection efficiency of plasmid-microbubble-ultrasound group was statistically higher than those of other groups (P<0.05).Conclusions: Under sound intensity 1.0 W/cm~2 and exposure time 30 s, ultrasound-mediated microbubble destruction could enhance gene transfection in NSCs.Objective: To explore the feasibility and efficiency of transfection into NSCs with pNogo-R shRNA by ultrasound-mediated microbubble destruction (UMMD), and to assess synergistic effect of UMMD together with liposome -plasmid DNA in increasing transfection efficiency. Methods: NSCs was sterilely obtained from neonatal Sprague Dawley rats. The third passaged cells were treated transgene following by different transfection conditions with (A) blank, (B) negative control+ultrasound microbubble, (C) pNogo-R shRNA+ ultrasound microbubble, (D) pNogo-R shRNA+Liposome, and (E) pNogo-R shRNA+Liposome+ultrasound microbubble, respectively.Results: In vitro cell experiments, no significant difference between C and D with fluorescence microscope was found, while transfection efficiency in group E was the highest compared with those of the other groups. More importantly, Liposome+ultrasound microbubble-mediated group did not affect the cell vitality comparing with the other groups by trypan-blue staining. In addition, Nogo-R mRNA and protein expression was dramatically decreased in group E (P<0.05), compared with other groups after 24 h transfection.Conclusion: Taken together, the present data demonstrates that UM+Lip-mediated transtection could be an efficiently noninvasive mean in transfection of NSCs. Ultrasound-microbubble technique could increase Nogo-R gene transfer into NSCs together with liposome, suggesting that the combination transfection provides a new method for transgene therapy of NSCs...