Experimental ES-GFP Gene Inhabitation Of Retinal Neovascularization Using Ultrasound Cationic Microbubble Destruction

PART I:Cationic microbubbles (CMB) and neutral microbubbles (NMB) preparation and characterizationObjective To develop neutral microbubbles (NMB) and cationic microbubbles (CMB) for use in gene therapy. Methods Microbubbles were prepared by sonication of relevant lipid mixtures, DC-cholesterol was added to make the microbubbles charge positively. The size distribution and zeta-potential were analyzed and the ability of carrying gene were investigated. Results NMB and CMB was constructed successfully, all of them had a good dispersion with uniform size under light microscope. The surface charge, mean diameter, and concentration were+25.62±4.38 mV, 1.64±0.28μm,4.16±0.18×109/ml, respectively. Cationic microbubbles exhibited a high gene loading capacity owing to the positive surface charge which compared to the neutral microbubbles. The saturated DNA loading capacities of NMB and CMB were 2.01±0.74μg and 19.02±0.76μg, respectively for 5×108 microbubbles. Conclusions The cationic microbubbles could become an efficient gene carrier. Conclusions NMB and CMB was constructed successfully.PART II:Gene transfection into HRECs using ultrasound-targeted cationic microbubble destructionObjective To investigate the inhibition effect of pEZ-M46-ES gene transfection on the growth, migration and angiogenesis of human retinal vascular endothelial cells (HRECs) mediated by UTMD. Methods pEZ-M46-ES plasmids grown in Escherichia coli were purified and suspended in 2.5 mM Tris-HCl (pH 8.5) at a concentration of 1.0μg/μL. HRECs in logarithmic phase were collected and divided into four groups: ① control (no treatment);② pEZ-M46-ES plasmid with NMBs and ultrasound; ③ pEZ-M46-ES plasmids with liposomes; and ④ pEZ-M46-ES plasmids with CMBs and ultrasound. The second and fourth groups of cells were grown in an Opticell for 24 hours before transfection. Twenty four hours later, the expression of the reporter green fluorescent protein (GFP) was observed using fluorescence microscopy and flow cytometry. The cell viability of HRECs was measured using the MTT assay. The apoptosis rate and cell cycle progression were detected via flow cytometry. The transcription and expression of endostatin, VEGF, Bcl-2 and Bcl-xl were measured via qPCR and Western blotting, respectively. Moreover, experiments assessing vascular formation and HREC migration were performed to evaluate the in vitro efficiency of gene therapy. Results After 24 h, numerous cells incubated with CMBs expressed GFP, while few cells incubated with NMBs and liposomes expressed GFP, and almost no GFP expression was observed in the controls using a laser confocal microscope; The transfection efficiencies of the control, NMB, liposome and CMB groups were 1.20±0.21%,28.25 ±0.57%,30.74 ±7.50% and 41.84 ±8.90%, respectively 24 h post-transfection; The percentage of cells that were arrested in G1 phase in the CMB group was higher compared with the other groups 24h and 48h after transfection; MTT test results showed the growth curve of HRECs in CMB group was obvious inhibited than other groups; the number of vascular tube cavities formed by the HRECs were 26.47±3.52,15.17±1.52,13.19±3.43,8.42± 1.02, respectively. Following ultrasound-targeted microbubble destruction (UTMD)-mediated gene therapy, the transcription and expression of endostatin were highest in the CMB group, while the transcription and expression of VEGF, Bcl-2 and Bcl-xl were lowest compared with the other groups. Conclusions Compared to the group of plasmid combining with NMB or liposomes, ultrasound-mediated cationic microbubbles could enhance the transfection efficiency of pEZ-M46-ES gene, which had obvious impacts on the resultant inhibition of the growth process, vascular formation, the migration of HRECs in vitro, while there was almost no difference between the NMB and liposome groups.PART III The effect of pEZ-M46-ES gene transfection mediated by UTMD on retinal neovascularization in miceObjective To investigate the inhibitory effect of pEZ-M46-ES gene transfection mediated by UTMD on retinal neovascularization in mice and explore the relevant mechanisms. Methods pEZ-M46-ES plasmids grown in Escherichia coli were purified and suspended in 2.5 mM Tris-HCl (pH 8.5) at a concentration of 1.0 μg/μL. A mouse model of oxygen-induced retinopathy was employed in the experiments. Sixty mice were randomly divided into three groups, ① pEZ-M46-ES plasmid with NMBs and ultrasound, ② pEZ-M46-ES plasmids with liposomes and ③ pEZ-M46-ES plasmids with CMBs and ultrasound. For each group, a mixture of 1μg of plasmids and 1μl of microbubbles or 1μl Lipofectamine (Invitrogen) was infused into the right eye’s vitreous of the mice. An ultrasound probe was then immediately introduced, with the following parameters being used for ultrasound:continuous wave,1 MHz,2 W/cm2,50% duty cycle,30S and a 10% concentration of microbubbles. The left eye as the control group. The number of retinal neovascularization was quantified by counting preretinal neo vascular cells in HE staining in 5 days after injection. The transcription and expression of endostatin, VEGF, Bcl-2 and Bcl-xl in retinal tissue were measured via qPCR and Western blotting, respectively. Results The number of retinal neovascularization was significantly inhibited in the CMB group compared with the other groups, each group of the nucleus number was:41.7±6.1,26.1±1.4,23.1±3.5,16.4±1.2, respectively. The transcription and expression of endostatin in retinal tissue were highest in the CMB group, while the transcription and expression of VEGF, Bcl-2 and Bcl-xl were lowest compared with the other groups. Conclusions Compared to the group of plasmid combining with NMB or liposomes, ultrasound-mediated cationic microbubbles could significantly inhibited retinal neovascularization in mice. It may become a novel means for the gene therapy of retinal neovascularization based on anti-angiogenesis in the future.