Smart hydrogel for enzyme responsive vaginal delivery of anti-HIV peptide therapeutics

Abstract

In response to an urgent need for advanced formulations for the delivery of anti-retrovirals, a stimuli sensitive hydrogel formulation that intravaginally delivers HIV-1 entry inhibitor upon being exposed to a specific protease was developed. The hydrogel formulation consists of PEG-azide and PEG-DBCO covalently linked to the entry inhibitor peptide, Enfuvirtide via substrate linker. The substrate linker is designed to undergo proteolysis by prostate specific antigen (PSA) present in seminal fluid and release innate Enfuvirtide. Of the tested PSA substrate linkers (HSSKLQYY, GISSFYSSK, AYLMYY and AYLMGRR), HSSKLQ was found to be an ideal candidate for PEG-based hydrogel with kcat/KM of 2.2 M-1 s-1. The PEG-based hydrogel displayed a pseudoplastic, thixotropic behavior with overall viscosity varying between 1516 Pa. s to 2.2 Pa. s, within the biologically relevant shear rates of 0.01-100 s-1. It also exhibited viscoelastic properties appropriate for uniform spreading and being retained in vagina. PEG-based hydrogels were loaded with N3-HSSKLQ-Enfuvirtide (HF42) that is customarily synthesized Enfuvirtide prodrug with its N-terminus connected to HSSKLQ linker. The stimuli sensitive PEG-based hydrogel formulations released 31.3 ± 8.7% of Enfuvirtide upon being exposed to PSA at pH 7.4 and 45.5 ± 6.5 % of Enfuvirtide at pH 8.6 over 24 hr., both of which are significantly greater than its IC50. The PEG-based hydrogel was non-cytotoxic to vaginal epithelial cells (VK2/E6E7) and murine macrophages (RAW 264.7) and did not significantly induce production of nitric oxide, an inflammatory mediator. The PEG-based hydrogel is found to have suitable physicochemical properties for an intravaginal formulation of the PSA substrate linked anti-retrovirals and is safe towards vaginal epithelium. It is capable of delivering Enfuvirtide with effective concentrations and has the potential to be used in clinical setting for effective prevention of HIV-1.