Ultrasound-mediated microbubble delivery of pigment epithelium-derived factor gene into retina inhibits choroidal neovascularization

• Published in: Chinese medical journal Vol. 122; no. 22; pp. 2711 - 2717
• Main Authors: Zhou, Xi-yuan, Liao, Qing, Pu, Yi-min, Tang, Yong-qiang, Gong, Xiao, Li, Jia, Xu, Yan, Wang, Zhi-gang
• Format: Journal Article
• Language: English
• Published: China Department of Ophthalmology, Second Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 400010, China%Institute of Ultrasonic Imaging, Second Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 400010, China 20.11.2009
• Subjects: Animals; Cells, Cultured; Choroidal Neovascularization - prevention & control; Eye Proteins - genetics; Female; Genetic Therapy; Humans; Microbubbles; Nerve Growth Factors - genetics; Rats; Rats, Long-Evans; Retina - metabolism; RNA, Messenger - analysis; Serpins - genetics; Transfection; Ultrasonics; 眼科学; 视网膜; microbubble; gene therapy; pigment epithelium-derived factor; choroidal neovascularization; ultrasound
• ISSN: 0366-6999; 2542-5641
• DOI: 10.3760/cma.j.issn.0366-6999.2009.22.007

Background Many studies have suggested that the imbalance of angiogenic factor and anti-angiogenic factor expression contributes significantly to the development of choroidal neovascularization （CNV）, and ultrasound microbubble combination system can increase the gene transfection efficiency successfully. This study was designed to investigate whether ultrasound-mediated microbubble destruction could effectively deliver therapeutic plasmid into the retina of rat, and whether gene transfer of pigment epithelium-derived factor （PEDF） could inhibit CNV. Methods Human retinal pigment epithelial cells were isolated and treated either with ultrasound or plasmid alone, or with a combination of plasmid, ultrasound and microbubbles to approach feasibility of microbubble-enhanced ultrasound enhance PEDFgene expression; For in vivo animal studies, CNV was induced by argon lasgon laser in rats. These rats were randomly assigned to five groups and were treated by infusing microbubbles attached with the naked plasmid DNA of PEDF into the vitreous of rats followed by immediate ultrasound exposure （intravitreal injection）; infusing liposomes with the naked plasmid DNA of PEDF into the vitreous （lipofectamine ＋ PEDF）; infusing microbubbles attached with PEDF into the orbit of rats with ultrasound irradiation immediately （retrobular injection）; infusing microbubbles attached with PEDF into the femoral vein of rats with exposed to ultrasound immediately （vein injection）. The CNV rats without any treatment served as control. Rats were sacrificed and eyes were enucleated at 7, 14, and 28 days after treatment. Gene and protein expression of PEDF was detected by quantitative real-time RT-PCR, Western blotting and immunofluorescence staining, respectively. The effect of PEDF gene transfer on CNV was examined by fluorescein fundus angiography. Results In vitro cell experiments showed that microbubbles with ultrasound irradiation could significantly enhance PEDF delivery as compared with microbubbles or ultrasound alone. In the rat CNV model, transfection efficiency mediated by ultrasound/microbubbles was significantly higher than that by lipofectamine-mediated gene transfer at 28 days after treatment. The study also showed that with the administration of ultrasound-mediated microbubbles destruction, the CNV of rats was inhibited effectively. Conclusions Ultrasound-microbubble technique could increase PEDF gene transfer into rats＇ retina and chorioid, in association with a significant inhibition of the development of CNV, suggesting that this noninvasive gene transfer method may provide a useful tool for clinical gene therapy.