EGFR and CD44 Dual-Targeted Multifunctional Hyaluronic Acid Nanogels Boost Protein Delivery to Ovarian and Breast Cancers In Vitro and In Vivo

Protein drugs with intracellular targets like Granzyme B (GrB) have demonstrated great proliferative inhibition activity in cancer cells. Their clinical translation, however, relies on the development of safe, efficient, and selective protein-delivery vehicles. Here, we report that epidermal growth factor receptor (EGFR) and CD44 dual-targeted multifunctional hyaluronic acid nanogels (EGFR/CD44-NGs) boost protein delivery to ovarian and breast cancers in vitro and in vivo. EGFR/CD44-NGs obtained via nanoprecipitation and photoclick chemistry from hyaluronic acid derivatives with tetrazole, GE11 peptide/tetrazole, and cystamine methacrylate groups had nearly quantitative loading of therapeutic proteins like cytochrome C (CC) and GrB, a small size of ca. 165 nm, excellent stability in serum, and fast protein release under a reductive condition. Flow cytometry assays showed that EGFR/CD44-NGs exhibited over 6-fold better uptake in CD44 and EGFR-positive SKOV-3 ovarian cancer cells than CD44-NGs. In accordance, GrB-loaded EGFR/CD44-NGs (GrB-EGFR/CD44-NGs) displayed enhanced caspase activity and growth inhibition in SKOV-3 cells as compared to GrB-loaded CD44-NGs (GrB-CD44-NGs) control. Intriguingly, the therapeutic studies in SKOV-3 human ovarian carcinoma and MDA-MB-231 human breast tumor xenografted in nude mice revealed that GrB-EGFR/CD44-NGs at a low dose of 3.85 nmol GrB equiv/kg induced nearly complete growth suppression of both tumors, which was obviously more effective than GrB-CD44-NGs, without causing any adverse effects. EGFR and CD44 dual-targeted multifunctional hyaluronic acid nanogels have appeared as a safe and efficacious platform for cancer protein therapy.