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Chatterjee Amit,1,2 Shalini Muralikumar,3 Sargunam Janaki,4 Meena Lakshmipathy,5 Kulandai Lily Therese,4 Vetrivel Umashankar,3 Prema Padmanabhan,5 Janakiraman Narayanan1 1Department of Nanobiotechnology, Vision Research Foundation, Sankara Nethralaya campus, Chennai, Tamil Nadu, India; 2School of Chemical and Biotechnology, SASTRA University, Tanjore, Tamil Nadu, India; 3Centre for Bioinformatics, Vision Research Foundation, Sankara Nethralaya campus, Chennai, Tamil Nadu, India; 4L&T Microbiology Research Centre, Vision Research Foundation, Sankara Nethralaya campus, Chennai, Tamil Nadu, India; 5Department of Cornea, Medical Research Foundation, Sankara Nethralaya campus, Chennai, Tamil Nadu, India Background: Fungal keratitis is a major cause of corneal blindness accounting for more than one-third of microbiologically proven cases. The management of fungal keratitis is through topical or systemic antifungal medications alone or in combination with surgical treatment. Topical medications such as natamycin and voriconazole pose major challenges due to poor penetration across the corneal epithelium. To address the issue various carrier molecules like nanoparticles, lipid vesicles, and cell penetrating peptides were explored. But the major drawback such as non-specificity and lack of bioavailability remains.Purpose: In this study, we have attempted to design corneal specific cell penetrating peptide using subtractive proteomic approach from the published literature and tried to improve its bioavailability through gelatin hydrogel delivery system.Material and Methods: Using subtractive proteomic approach two peptides VRF005 and VRF007 were identified on the basis of solubility, cell permeability and amphipathicity. The peptides were modeled for three-dimensional structure and simulated for membrane penetration. The peptides were characterized using circular dichroism spectroscopy, dynamic light scattering and native polyacrylamide gel electrophoresis. Further uptake studies were performed on primary corneal epithelial cells and the stability was analyzed in corneal epithelial tissue lysates. Insilico prediction of peptides showed it to have antifungal activity which was further validated using colony forming assay and time killing kinetics. The duration of antifungal activity of peptide was improved using gelatin hydrogel through sustained delivery.Results: VRF005 and VRF007 showed α-helical structure and was within the allowed region of Ramachandran plot. The simulation study showed their membrane penetration. The peptide uptake was found to be specific to corneal epithelial cells and also showed intracellular localization in Candida albicans and Fusarium solani. Peptides were found to be stable up to 2 hours when incubated with corneal epithelial tissue lysate. Dynamic light scattering, and native polyacrylamide gel electrophoresis revealed aggregation of peptides. VRF007 showed antifungal activity up to 24 hour whereas VRF005 showed activity up to 4 hours. Hence gelatin hydrogel-based delivery system was used to improve the activity. Actin staining of corneal epithelial cells showed that the cells were attached on gelatin hydrogel.Conclusion: We have designed corneal specific cell penetrating peptides using subtractive proteomic approach. Bioavailability and delivery of peptide was enhanced using gelatin hydrogel system. Keywords: cell-penetrating peptide, gelatin hydrogel, CD spectroscopy, subtractive proteomic approach |
