The aim of this study was to evaluate the possibility of poly (D, L-lactide-co-glycolide) nanoparticle (NPs) as a gene vector for functional plasmid DNA (pDNA) and to investigate its inhibitory efficacy on experimental choroidal neovascularization (CNV). We developed intravitreal administered, hypoxia-inducible factor 1a (HIF-1a) short hairpin RNA and green fluorescent protein (GFP) co-expressed pDNA-loaded NPs (pshHIF-1a NPs). CNV was induced by laser photocoagulation in 112 rats. The rats were then randomly assigned to be injected intravitreally with phosphate-buffered saline (PBS), blank NPs, naked pDNA, control pDNA NPs and pshHIF-1a NPs, respectively, and non-injection group was set as the control. Immunofluorescence staining, fluorescein fundus angiography and histologic analysis were performed to evaluate the inhibitory efficacy on CNV. The results showed that the expression of GFP preferentially localized in the retinal pigment epithelium cell layer and lasted for 4 weeks. The fluorescein leakage areas of CNV were significantly larger in the PBS, blank NPs, control pDNA NPs, non-injection group and naked pDNA group than in pshHIF-1a NPs group (Po0.01). The mean thickness of the CNV lesions in the intravitreally pshHIF1a NPs-treated group was significantly smaller than other groups (Po0.01). No signs of functional or ultrastructural destruction in retina were detected. Therefore, pshHIF-1a NPs may act as a novel therapeutic option to transfer specific pDNA and inhibit the formation of experimental CNV.

Sindbis virus expression has been used for in vitro investi-50% of transfected cells. Cells were then cotransfected gations of antigen processing, presentation and epitope with DH-BB helper sequences that enabled the recombimapping. The recent development of a replication-deficient nant Sindbis virus RNA packaging. Nonreplicative Sindbis recombinant Sindbis virus expression vector has made in viral stock was collected 24 h after transfection. BHK cells vivo expression possible with minimal pathogenic risk.were then infected with viral stock and histochemistry Advantages of Sindbis virus over other available viral sysanalysis was performed. Again, approximately 50% of the tems include a comparatively smaller genome size making cells expressed ␤-gal. The same viral stock was infused it possible to clone larger inserts, the ability to infect a wide into the nucleus caudatus/putamen and nucleus accumrange of host cell types with reduced pathogenicity for bens septi and histochemical analysis on frozen sections humans. These features suggest the possible utility of from the relevant brain areas confirmed that ␤-gal was Sindbis virus for the in vivo delivery of genes to neural expressed in neurons in a time-dependent manner. ␤-Gal cells. We used the recombinant Sindbis viral expression was detected at 24 h after inoculation and was present for system to target delivery of the lacZ gene to neuronal cells at least 14 days, with maximum expression at 48 h. These of mice by stereotactic surgery. Sindbis viral mRNA results suggest that a nonreplicative Sindbis virus obtained by in vitro transcription was used to transfect baby expression system may be useful for delivery of foreign hamster kidney (BHK) cells. As shown by histochemistry, genes into the central nervous system (CNS). ␤-galactosidase (␤-gal) was expressed in approximately

Purpose To investigate the role of Ca 2 þ in lipofuscin formation in human retinal pigment epithelial (RPE) cells that phagocytize bovine photoreceptor outer segments (POSs). Methods Cultured human RPE cells fed with 2 Â 10 7 per l bovine POS were treated with flunarizine, an antagonist of Ca 2 þ channel, or/and centrophenoxine, a lipofuscin scavenger. The Ca 2 þ changes and lipofuscin formation were measured with fluoresence dye Fluo-3/AM ester, laser scanning confocal microscopy (LSCM) and flow cytometry (FCM). The activity of RPE cells was measured by methyl thiazolyl tetrazolium (MTT) assay and argyrophilic nucleolar organizer regions (AgNORs) assay. Results The Ca 2 þ fluorescence intensity (CFI) of RPE cells fed with POS was significantly increased compared with the controls (165.36±29.92 U). It reached a peak with 777.33±63.86 U (Po0.01) at 12 h, and then decreased but still maintained a high level of 316.90 ± 36.07 U (Po0.01) for 4 days. Flunarizine and centrophenoxine significantly decreased the Ca 2 þ overload to 227.18 ± 14.00 U at 12 h and 211.06 ± 20.45 U at 4 days. FCM confirmed these changes. The drugs also showed an inhibitory effect on the lipofuscin formation. The proliferation rate of the cells fed with POS increased significantly. Both drugs had inhibitory effects on the activity of the cultured cells. This tendency was confirmed by AgNORs assay. Conclusions The Ca 2 þ inflow initiated lipofuscin accumulation in RPE cells fed with POS. Flunarizine and centrophenoxine can decrease Ca 2 þ overload and lipofuscin formation in RPE cells, accompanied by maintaining cellular vitality.