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J. Amaral, R.N. Fariss, M. Campos, W.G. Robison Jr, H. Kim, R. Lutz, S.P. Becerra; Subconjuntival Delivery of Pigment Epithelium–derived Factor (PEDF) Protein to the Choroid and Retina: Clinical Implications . Invest. Ophthalmol. Vis. Sci. 2004;45(13):5056.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: PEDF, an extracelullar glycoprotein of 50–kDa secreted by the retinal pigment epithelium (RPE), has neurotrophic and antiangiogenic properties for the eye. Its down–regulation has been associated with diseases like age–related macular degeneration, retinopathy of prematurity and diabetic retinopathy, in which the development of choroidal or retinal neovascularization leads to severe visual loss. Intravitreal, subretinal or systemic delivery of PEDF has neurotrophic and antiangiogenic effects in animal models of neurodegenerative and angiogenic eye diseases. To establish novel routes for PEDF delivery to the choroid and retina, we evaluated protein diffusion through the sclera–RPE. Methods: Purified recombinant human PEDF was conjugated with fluorescein 5–EX succinimidyl ester. Ovalbumin (ova), a serpin protein of 45–kDa with homology to PEDF, was fluoresceinated and used as control. Fluorescein was detected by immunobloting with anti–fluorescein antiserum, fluorometry, epifluorescent and confocal microscopy. Diffusion through scleral tissue was evaluated in vitro with a Ussing–type chamber with dissected sclera from porcine eyes. Polymer implants were prepared as a blend of Fl–ova in 10% (weight) solubilized polyvinyl alcohol. Delivery of Fl–ova or Fl–PEDF from the subconjuntiva was performed using implants or by injection in rats. Results: Fl–ova diffused through the scleral tissue at a constant rate from the orbital to the uveal side of the chamber. Western blots of choroid/RPE or retinal homogenates from rats injected with Fl–PEDF and Fl–ova showed presence of both proteins in the choroid/RPE and retina. Maximal diffusion was observed at 1 hour post injection with a gradual decrease during the next 12 hours. In vitro release rate studies of implants showed an initial rapid bolus delivery of Fl–ova which declined within 2 to 4 hours to a slower release rate thereafter. Fl–ova implants located in the subconjuntiva revealed diffusion of fluorescein into the choroid/RPE and retina as early as 1 hour post–implantation with the highest signal by 24 hours. Conclusions: These results demonstrate that PEDF and ovalbumin can diffuse through the sclera to reach the choroid/RPE and retina, and suggest that delivery of proteins through implants in the subconjunctiva may allow for sustained diffusion. Thus, subconjuntival implants provide a more direct and less invasive route for sustained delivery of PEDF to the choroid and retina than systemic, intravitreal or subretinal routes.
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