Ultrasound-mediated microbubble delivery of pigment epithelium-derived factor gene into retina inhibits choroidal neovascularization.
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 PEDF gene 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.
Zhou
Xi-yuan
XY
Department of Ophthalmology, Second Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing, China. zhouxiyuan2002@yahoo.com.cn
Liao
Qing
Q
Pu
Yi-min
YM
Tang
Yong-qiang
YQ
Gong
Xiao
X
Li
Jia
J
Xu
Yan
Y
Wang
Zhi-gang
ZG
D000818:Animals; D002478:Cells, Cultured; D020256:Choroidal Neovascularization / Q000517:prevention & control*; D005136:Eye Proteins / Q000235:genetics*; D005260:Female; D015316:Genetic Therapy; D006801:Humans; D045423:Microbubbles; D009414:Nerve Growth Factors / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D020318:Rats, Long-Evans; D012160:Retina / Q000378:metabolism*; D015843:Serpins / Q000235:genetics*; D014162:Transfection; D014465:Ultrasonics; D000099174:Pigment Epithelium-Derived Factor
D005136:Eye Proteins; D009414:Nerve Growth Factors; D012333:RNA, Messenger; D015843:Serpins; D000099174:Pigment Epithelium-Derived Factor