Enzyme-mediated in situ formation of pH-sensitive nanogels for proteins delivery

Abstract

Novel pH-sensitive nanogels based on poly(ethylene glycol)-b-poly(L-glutamate-g-tyramine) (PEG-b-P(LGA-g-Tyr)) copolymer were developed for efficiently delivering and releasing proteins into HeLa cells. The core–shell nanogels were in situ fabricated through the enzyme-catalyzed oxidative coupling of tyramine moieties in the core of the self-assembled PEG-b-P(LGA-g-Tyr) micelles in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂). The stable nanogels have spherical morphology with an average diameter of about 125 nm under physiological condition. The pH-dependent size shrink of the nanogels was observed by dynamic light scattering (DLS). Fluorescein isothiocyanate conjugate bovine serum albumin (FITC-BSA) was in situ incorporated into the nanogels with an entrapment efficiency of 69.9% during the enzyme-catalyzed crosslinking reaction. In vitro protein release profiles at pH 7.4 and pH 6.8 showed a burst effect followed by a continuous release phase. The FITC-BSA loaded nanogels exhibited pH-sensitive protein release. A significantly fast FITC-BSA release was observed at endosomal pH than at physiological pH. The cumulative release of FITC-BSA from the nanogels at pH 7.4 and pH 6.8 were 24.2% and 40.3%, respectively. Cell Counting Kit-8 (CCK-8) assay showed that these nanogels were non-toxic up to a concentration of 2.0 mg mL⁻¹. Confocal laser scanning microscopy (CLSM) studies revealed that FITC-BSA loaded nanogels efficiently delivered and released proteins into HeLa cells. We are convinced that these enzymatically crosslinked nanogels with excellent biocompatibility and pH-responsibility have a promising potential for protein delivery system.