Designing and enhancing the antifungal activity of corneal specific cell penetrating peptide using gelatin hydrogel delivery system

Abstract

BackgroundFungal 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.PurposeIn 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 MethodsUsing 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.ResultsVRF005 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.ConclusionWe have designed corneal specific cell penetrating peptides using subtractive proteomic approach. Bioavailability and delivery of peptide was enhanced using gelatin hydrogel system.

Enzymatic characterization and molecular mechanism of a novel aspartokinase mutant M372I/T379W fromCorynebacterium pekinense

“…In addition, Ramachandran plot was used to further verify the accuracy of the structure. 25,26 The plot showed that more than 97% of residues were located in spatially allowed region ( Fig. S2 †).…”

“…The optimum temperature of the enzyme was determined by incubation of the reaction mixture (the enzymatic-activity assay system) at 15,20,25,26,28,30,35,40,45, or 50 C. The optimum pH of the enzyme was determined using 100 mM Tris-HCl with adjustment of buffer pH from 6 to 10 in 0.5-unit steps. The maximum enzymatic activity, which was achieved at the optimum temperature and pH, was set to 100%.…”

Gelatin Nanoparticles-HPMC Hybrid System for Effective Ocular Topical Administration of Antihypertensive Agents

“…Significant differences have been observed in all the cases (p < 0.05). The aforementioned results could be explained by the mucoadhesive properties of gelatin, leading to a higher retention time on the ocular surface [48] and higher corneal penetration of the nanoparticulate system [47,51,83]. These data demonstrate that timolol loaded gelatin nanoparticles can result in a very promising strategy for ocular delivery of hypotensive compounds and that, interestingly, target pressure can be modulated employing different concentrations of TM-GNP.…”

Deciphering the mechanoresponsive role of β-catenin in keratoconus epithelium

“…50 mg/ml of gelatin concentration with 0.8 µg/mL of glutaraldehyde as crosslinker were used for preparing hydrogel and incubated at 4 °C for 4 h for gelation and cross-linking. The cross-linked gelatin hydrogels were immersed in a 50 mM glycine aqueous solution under agitation for 1 h to block the residual aldehyde groups of glutaraldehyde, followed by two washes with double-distilled water for 1 h 43 . The stiffness of the hydrogels were measured using Microsquischer (M/s.Cell Scale) instrument.…”