Chemical Modification of Cytochrome C for Acid-Responsive Intracellular Apoptotic Protein Delivery for Cancer Eradication.

Delivering bioactive proteins into cells without carriers presents significant challenges in biomedical applications due to limited cell membrane permeability and the need for targeted delivery. Here, we introduce a novel carrier-free method that addresses these challenges by chemically modifying proteins with an acid-responsive cell-penetrating peptide (CPP) for selective intracellular delivery within tumours. Cytochrome C, a protein known for inducing apoptosis, served as a model for intracellular delivery of therapeutic proteins for cancer treatment. The CPP was protected with 2,3-dimethyl maleic anhydride (DMA) and chemically conjugated onto the protein surface, creating an acid-responsive protein delivery system. In the acidic tumour microenvironment, DMA deprotects and exposes the positively charged CPP, enabling membrane penetration. Both in vitro and in vivo assays validated the pH-dependent shielding mechanism, demonstrating the modified cytochrome C could induce apoptosis in cancer cells in a pH-selective manner. These findings provide a promising new approach for carrier-free and tumour-targeted intracellular delivery of therapeutic proteins for a wide range of potential applications.