Department of Nanobiotechnology, Vision Research Foundation, Sankara Nethralaya campus, Chennai, Tamil Nadu, India,
School of Chemical and Biotechnology, SASTRA University, Tanjore, Tamil Nadu, India.
Int J Nanomedicine. 2019 Jan 15;14:605-622. doi: 10.2147/IJN.S184911. eCollection 2019.
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.
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.
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. 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.
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 and . 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.
We have designed corneal specific cell penetrating peptides using subtractive proteomic approach. Bioavailability and delivery of peptide was enhanced using gelatin hydrogel system.
真菌性角膜炎是导致角膜盲的主要原因，占微生物学证实病例的三分之一以上。真菌性角膜炎的治疗方法是单独使用或联合手术治疗局部或全身抗真菌药物。由于穿透角膜上皮的能力差，纳他霉素和伏立康唑等局部药物的应用存在很大挑战。为了解决这个问题，人们探索了各种载体分子，如纳米颗粒、脂质体和细胞穿透肽。但非特异性和生物利用度低等主要缺点仍然存在。
本研究采用消减蛋白质组学方法从已发表的文献中设计角膜特异性细胞穿透肽，并尝试通过明胶水凝胶递药系统提高其生物利用度。
采用消减蛋白质组学方法，根据溶解度、细胞通透性和两亲性，从文献中鉴定出两种肽 VRF005 和 VRF007。对肽进行三维结构建模并模拟其跨膜渗透。用圆二色性光谱法、动态光散射和天然聚丙烯酰胺凝胶电泳对肽进行表征。进一步在原代角膜上皮细胞上进行摄取研究，并在角膜上皮组织裂解液中分析稳定性。对肽的预测表明其具有抗真菌活性，并用集落形成试验和时间杀伤动力学进一步验证。通过持续递药提高明胶水凝胶中肽的抗真菌活性持续时间。
VRF005 和 VRF007 显示出 α-螺旋结构，且位于 Ramachandran 图的允许区域内。模拟研究表明它们可以穿透细胞膜。研究发现，肽的摄取具有角膜上皮细胞特异性，并在 和 中显示出细胞内定位。当与角膜上皮组织裂解液孵育时，肽在 2 小时内保持稳定。动态光散射和天然聚丙烯酰胺凝胶电泳显示肽聚集。VRF007 显示出长达 24 小时的抗真菌活性，而 VRF005 显示出长达 4 小时的活性。因此，使用明胶水凝胶递药系统来提高其活性。角膜上皮细胞的肌动蛋白染色显示细胞附着在明胶水凝胶上。
我们采用消减蛋白质组学方法设计了角膜特异性细胞穿透肽。使用明胶水凝胶系统提高了肽的生物利用度和递送。
