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Dung Nguyen, Sergio Caballero, Sharon Matthews, Jill Verlander, Michael Boulton, Peixuan Guo, Maria Grant; pRNA Nanoparticles for Intraviteal Delivery of siRNA in Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4704.
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Considerable progress has been made in the delivery methods of small interfering RNA (siRNA) through intravitreal injection. However, naked siRNA lack the ability for targeting specific cells. Phi 29 phage RNA (pRNA) DNA packaging motor can be modified for nanotechnological applications. The domains allow for the conjugation of different siRNA combinations onto a nanoparticle. Additionally, one junction of the domain can be used for intracellular targeting for more efficient delivery, e.g., into proliferating endothelial cells in neovascularization. We sought to characterize the properties of pRNA nanoparticles in the mouse retina as an improved platform for siRNA delivery.
AlexaFlour 647-labeled pRNA nanoparticles containing either three- or four-way junctions were injected intravitreally into mouse eyes. Twenty four hours following injections mice were euthanized. Select eyes were fixed in 4% paraformaldehyde (PFA), the retinas dissected, and then sectioned for examination by laser confocal microscopy. A separate proband of eyes were fixed for electron microscopy (EM) by in vivo perfusion with 4% PFA in Tyrode’s buffer and 0.25% glutaraldehyde. Retina sections from these eyes were incubated with primary mouse anti-AlexaFlour 647 followed by gold conjugated anti-mouse antibody prior to EM capture.
Imaging analyses revealed robust penetration of pRNA nanoparticles across retinal layers capable of reaching the retinal pigment epithelium (RPE) at 24 hours post-injection by both detection techniques. pRNA nanoparticles were observed in the choroidal region, suggesting they can traverse the Bruch’s membrane. Nanoparticles conjugated to folate for intracellular uptake showed an association to cellular surfaces. No morphological abnormalities were observed at 24 hours post-injection. No differences in penetration were observed in the different pRNA nanoparticle formulations.
pRNA nanoparticles can migrate efficiently toward the outer regions of the retina by 24 hours following intravitreal injection and showed no toxicity based on morphology. Each formulation we tested with different structural properties crossed the retina making pRNA nanoparticles useful as a platform for siRNA delivery. With specific cellular targeting, pRNA nanoparticles can potentially deliver combinations of therapeutics to sites of choroidal or pre-retinal neovascularization without compromising normal homeostatic processes.
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