| Background Age-related macular degeneration (AMD) is the major cause of visual loss in patients older than 50 years in developed countries, and choroidal neovascularization (CNV) is responsible for the majority of severe visual loss in AMD. Present therapies, such as thermal laser or photodynamic therapy (PDT), however, have significant shortcomings such as full-thickness retinal damage, central visual loss, limited indications and high recurrence rates after these treatments. As the pathogenesis of CNV is better understood, recent studies have suggested that antiangiogenesis therapy may be beneficial in the treatment of CNV. Several novel therapies for CNV have emerged based on antagonism of vascular endothelial growth factor(VEGF) or the VEGF receptor, such as intravitreal administration of bevacizumab(avastin), ranibizumab (Lucentis) and pegaptanib (Macugen), but a potential drawback of these therapies is that there has been some evidence demonstrating that suppressed expression of VEGF alone is not sufficient to inhibited CNV.Clinical studies have shown that age-related changes in Bruch's membrane lead to outer retina hypoxia, which may be an important driving force of CNV formation, by stimulating VEGF overexpression in the retinal pigment epithelium (RPE) cells. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that functions as a master regulator of oxygen homeostasis. As a"master switch"for mammalian circulation in response to low oxygen tension, HIF-1 regulates the transcription of various genes, which play critical and diverse roles in the hypoxic stage, including the process of CNV. HIF-1 is a heterodimer composed of HIF-1αand HIF-1βsubunits. HIF-1βis constitutively expressed, while HIF-1αis induced by hypoxia. Under hypoxic conditions, HIF-1αis activated prior to the expression of many proangiogenic growth factors, including VEGF, erythropoietin(EPO), and platelet-derived growth factor (PDGF). Knockdown of HIF-1αgene results in the down regulation of VEGF. Thus, the strategy to block some master modulators, such as HIF-1α, will be necessary and has drawn a great deal of attention because it perhaps not only downregulates the VEGF expression, but also adjusts some other uncertain factors to achieve a synergistic effect.Small interference RNA (siRNA) has recently emerged as a very useful therapeutic method that suppresses the gene expression through the introduction of double-stranded RNA. Recently our work demonstrated that VEGF expression could be inhibited by targeting HIF-1αwith siRNA in human RPE cells. Using siRNA directed against VEGF or VEGF receptors in CNV animal models also have showed promising results. Clinical trials involving RNA interference(RNAi) targeting VEGF or its receptor through intravitreal injection delivery are currently underway. However, one of the problems exist in gene therapy is to sustain the expression of transgenes at high levels. Genetic material like siRNA, are degraded quickly in the targeted cells and thus can only transiently inhibit the angiogenesis process, which lead to the regrowth of vessels after these anti-angiogenesis agents are given to patients. In order to achieve a long-term gene expression or pharmacodynamic action in the eye, intravitreal injection must be administered repeatedly to maintain the drug concentrations within a therapeutic range for a long period of time, which sometimes causes certain ocular complications, such as vitreous hemorrhage, retinal detachment, or endophthalmitis. Therefore, the application of drug-delivery systems to facilitate therapeutic efficacy and to minimize side effects may be of primary consideration in our study.An ideal controlled-release formulation should release the entrapped drugs in a continuous manner over a desired time period. Biodegradable, biocompatible nanoparticles(NPs) formulated from poly(D, L-lactide-co-glycolide) (PLGA) and polyvinyl alcohol (PVA) have been extensively investigated for various drug-delivery applications. They are hydrolyzed and metabolized without direct toxic reactions to living tissues. By changing their physicochemical properties, such as molecular weight, hydrophilicity, and the ratio of lactide to glycolide, the degradation rate of PLGA polymer enable the delivery of encapsulated genetic material or drug to be modulated in a slow and extended intracellular release manner. Besides, DNA fragments encapsulated in PLGA matrix can be protected from endonuclease activity. Thus this strategy may provide an efficient, non-viral technology to deliver and express genetic material safely into the posterior segment of the eye.Purposes: (1) To construct and select the most efficient short hairpin RNA(shRNA)-expressing plasmid DNA (pDNA) vector(pshHIF-1α). (2) To construct and evaluate the possibility of PLGA NPs as a gene vector for functional pDNA. (3) To investigate its inhibitory efficacy on experimental CNV. (4) To evaluate the toxicity of pDNA-loaded nanoparticles to the retina tissue.Methods: (1) Four shRNA-expressing sequence targeting HIF-1αwere introduced into pGCsi-U6/neo/GFP/shRNA vector. Real-time PCR was used to observe the effect of RNAi on expression of HIF-1αin IEC6 cells transfected by Lentivirus vector and selected the most efficient shRNA. pGCsi-U6/neo/GFP vector, which produces a non-targeting negative control sequence, was used as a control pDNA throughout the study. Each pDNA was amplified in the DH5αstrain of Escherichia coli and purified.(2) Nanoparticles were prepared by a water-in-oil-in-water (W/O/W) multiple emulsion technique. The loading percent of the nanoparticles and the pDNA content encapsulated in nanoparticles were calculated by ultraviolet spectrophotometer. Blank nanoparticles were prepared by using Tris-EDTA buffer(TE buffer) in the first emulsion. The average particle size, size distribution and zeta potential of the prepared PLGA nanoparticles were measured by laser light scattering. The morphologic characteristics were observed by transmission electron microscopy.In vitro release of PLGA nanoparticles carried out in 0.02% NaN3/phosphate buffered saline (PBS) solution at 37℃under gentle shaking and samples were withdrawn after centrifugation and replaced by fresh PBS at regular time intervals. The tests were carried out in triplicate.(3)Induction of CNV in Brown Norway rats(BN) and intravitreal administration:Laser-induced CNV was performed on BN rats. CNV was induced in 116 rats unilaterally. Intravitreal administration was applied immediately after photocoagulation. PBS, blank NPs(1.89mg/ml), naked pDNA(21.2μg/ml), control pDNA nanoparticles(1.89mg/ml) or pshHIF-1αNPs(1.89mg/ml), in a volume of 10μl(the pDNA content were equal to 21.2μg/ml), were injected into the vitreous respectively.(4)The in vivo kinetics of nanoparticles uptake and their distribution within the eye were observed by immunofluorescence staining at 3, 7, 14 and 28 days after photocoagulation. Cryostat sections at 3 day after laser photocoagulation were chosen for semiquantitative analysis of HIF-1αprotein expression. At 14 day after laser photocoagulation, the area of fluorescein leakage of CNV lesions was studied by fluorescein fundus angiography (FFA), and the CNV with the greatest thickness was measured by light microscopy.(5) Flash electroretinography (f-ERG) was performed before intravitreal injection (at baseline) and 7, 28 days post-injection, amplitudes as well as implicit times of a- and b-wave were recorded. The ultrastructural changes in retina were examined with transmission electron microscope(TEM) at days 14 and 28.Results: (1) siRNA target sequence of rat mRNA encoding HIF-1αgene was as follows: HIF-1α, 5′-CCAGTTGAATCTTCAGATA-3′(Genbank No. NM024359). The plasmid vector produces a shRNA with the loop sequence: TTCAAGAGA. The expression level of GFP in IEC6 cells after transfected by Lentivirus was 70%, and Real-time PCR result showed the inhibitory efficacy of siRNA was 45%.(2) Average size of the pDNA loaded NPs was 284nm and zeta potential was -8.91mV as measured by laser light scattering. The encapsulation efficiency and loading percent was 60.2% and 1.06%, respectively. The in vitro release behavior of pDNA loaded NPs is depicted in the cumulative percentage release. The results showed that about 60%70% of total encapsulated pDNA was released within 5 days followed by a constant and sustained release for 4 weeks. NPs appeared to have a spherical shape and smooth surface as determined by TEM.(3) Confocal images showed that gene expression of GFP in naked NPs-, control pDNA NPs-, and pshHIF-1αNPs-injection eyes was observed within the retina, chorioid, ciliary body and CNV lesions as soon as 3 days after intraocular injection. On day 7, some cells expressing GFP were observed in the inner retinal layers, but most of the expression was localized preferentially within the RPE layer, and the protein expression remained apparent in RPE cells until day 28. Fluorescence for naked pDNA-injection eyes showed a short-term expression, which cannot be detected on day 7. Controls injected either with blank NPs or PBS showed no fluorescence at any time point.(4) By immunofluorescence staining, expression of HIF-1αwas observed increased in CNV lesions 3 days after laser photocoagulation. The expressions of HIF-1αobserved in the CNV specimens of the pshHIF-1αNPs and naked pDNA-treated groups were diminished compared with other groups (P<0.01). Semiquantitative analyses revealed that while PBS-, blank NPs-, control pDNA NPs- and non-injection-groups showed moderate-to-severe fluorescein leakage in CNV, the rats treated with pshHIF-1αNPs showed a statistically significant decreased fluorescein leakage in CNV membranes (P<0.01). Histopathology revealed that comparing to other groups, CNV lesions in pshHIF-1αNPs -treated eyes has a thinner center (P<0.01). The mean thicknesses of CNV in PBS-, blank NPs-, control pDNA NPs-, naked pDNA-, pshHIF-1αNPs- and uninjected-groups were 107.86±11.29μm (n=9), 99.85±10.42μm (n=9), 104.33±11.64μm (n=8), 91.79±8.78μm (n=10), 58.28±9.57μm (n=10) and 102.2±8.3μm (n=10), respectively.(5) At 7 day post-injection, a significant decrease amplitudes and a prolonged implicit times of a- and b-wave from baseline was observed in all injected eyes (PBS, blank NPs, naked pDNA, or pshHIF-1α, P<0.05) by electroretinography. However there were no significant differences among any of the intravitreal injected groups (P>0.05) in the amplitudes or in the implicit times at any time point. By 28 days post-injection, no significant difference in a- and b-wave amplitudes or in implicit times was observed between baseline and intravitreal injected groups (P>0.05).TEM showed that slight derangement of microtubule and microfilament in retina nerve fiber layer and the vacuolization of mitochondria were revealed in pshHIF-1αNPs-injectied group or in PBS group at 14 day. Lots of the NPs were found in the cytoplasm of ganglionic cells and even approaching to the nucleus, suggesting that they can be uptaken by retinal cells. At 28 day, small amounts of similar changes could also be observed, but most of the mitochondria, rough endoplasmic reticulum, Golgi's complex in the ganglionic layer and the mitochondria, microtubule, and microfilament in the nerve fiber layer were recovered. Conclusions:The shRNA-expressing pDNA targeting against rat HIF-1αgene was successfully designed and constructed, and HIF-1αexpression in vivo was dramatically decreased by this approach. This study demonstrated that in addition to offer an increased facility of tissue penetration and intracellular uptake, PLGA NPs"protect"the carried plasmid and obtain a sustained release rate. Application of pshHIF-1αloaded NPs significantly reduced the extent of neovascularization in the murine laser photocoagulation model of CNV. This treatment strategy provides an efficient, non-viral technology to deliver and express genetic material safely.
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