Injectable Adhesive Hydrogel for Sustained Local Delivery of CRISPR-RNP Loaded Lipid Nanoparticles

Abstract

CRISPR-Cas9 therapeutics hold great promise for the treatment of solid tumors but are limited by inefficient and short-lived delivery of gene-editing components, particularly at the viable tumor rim and adjacent hypoxic regions. Lipid nanoparticles (LNPs) are widely used as non-viral carriers, yet both systemic administration and conventional intratumoral injections result in rapid dispersion, off-target biodistribution, and poor intratumoral retention. This study presents a bio-adhesive, surface-adherent alginate hydrogel as an in situ-forming local depot for the sustained delivery of CRISPR-Cas9 ribonucleoprotein (RNP)-loaded LNPs

The C12-200 based LNP platform was formulated and characterized to ensure consistent encapsulation and physicochemical properties suitable for CRISPR-RNP delivery. Alginate was functionalized with N-hydroxysuccinimide (Alg-NHS) and ionically crosslinked with calcium ions to generate an adhesive hydrogel exhibiting an interconnected porous microstructure, strong tissue adhesion, and stable gelation under physiological conditions. Release studies using LNP-loaded hydrogels demonstrated diffusion- controlled, sustained release that could be tuned by polymer and crosslinking density. Fully crosslinked Alg-NHS hydrogels showed negligible intrinsic cytotoxicity, and prolonged, lower-intensity exposure to LNPs via hydrogel release mitigated non-specific formulation-related toxicity while preserving potent CRISPR-RNP mediated tumor cell killing in vitro.

In vivo, subcutaneously injected Alg-NHS hydrogels formed stable depots that adhered to surrounding tissues and were retained for extended periods. In a subcutaneous HCT116 xenograft model, local administration of CRISPR-RNP (MT50) loaded alginate adhesive hydrogels around the tumor suppressed tumor growth without apparent systemic toxicity. Further optimization of hydrogel composition and payload dose enhanced the antitumor efficacy. Overall, these findings indicate that bio-adhesive, surface-adherent alginate hydrogels can function as a long-acting local depot for CRISPR-LNP formulations and offer a conceptually distinct framework for designing sustained, localized gene-editing therapies in solid tumors.