Experimental Study Of AAV-9 Mediated MiR-1 Nanobubbles Combined With UTMD To Promote The Differentiation Of Induced Pluripotent Stem Cells Into Cardiomyocytes

Objective:To investigate the effect of AAV-9/miRNA-1 T-NBs combined ultrasound targeted microbubble destruction(UTMD)to promote differentiation of cardiomyocytes from human induced pluripotent stem cells(hiPSC).Methods:AAV-9/miRNA-1 gene complexes were linked to NBs to form AAV-9/miRNA-1 T-NBs by "mechanical oscillation" "membrane hydration" and "streptavidin-biotin bridging".To detect the morphology,particle size,potential and dispersion of AAV-9/miRNA-1 T-NBs.The hiPSC activity was assessed at different concentrations(10~6/ml,10~7/ml,10~8/ml),intervention time(24h,48h,72h)and intervention methods(NBs,NBs+AAV-9/miRNA-1,NBs+AAV-9/miRNA-1+UTMD).The binding efficiency of NBs+AAV-9/miRNA-1+UTMD and hiPSC was examined.According to the different intervention methods,the experimental groups were divided into 6 groups:group A(NBs),group B(NBs+5μl AAV-9),group C(NBs+5μl AAV-9+UTMD),group D(NBs+5μl AAV-9/miRNA-1),group E(NBs+5μlAAV-9/miRNA-1+UTMD)and group F(NBs+5μl AAV-9/miRNA-1+UTMD).Cx43 expression was detected after 14 days of co-culture with hiPSC,suggesting the degree of cardiomyocyte differentiation.Results:AAV-9/miRNA-1 T-NBs concentration(405.75±26.08)×10~8/ml,particle size(437.17±27.28)nm,potential(-8.43±2.40)m V,dispersion0.39±0.08,milky white and opaque suspension,uniform in size,spherical in shape and uniform in distribution under optical microscope,have no obvious toxic and side effects on the viability of hiPCS(P>0.05).NBs+AAV-9/miRNA-1+UTMD showed the highest binding efficiency with hiPCS at 97.43±3.25%,with statistical difference(P<0.05).Compared with group A,Cx43 expression in other 5 groups increased,which was statistically significant(P<0.05).The pairwise comparison between groups showed that Cx43 expression in group B and E,C and E(P<0.05)but not between group B and C(P>0.05);Cx43 expression between D and E,E and F(P<0.05).Conclusion:AAV-9/miRNA-1 T-NBs in this study have no obvious toxic and side effects on hiPCS,and could increase Cx43 protein expression and promote hiPCS differentiation to cardiomyocytes.