Calixarene Modification Strategy for Efficient Intracellular Protein Delivery.

Efficient intracellular protein delivery is of great importance for the development of protein-based therapy and modern biotechnologies. However, the hydrophilic and macromolecular nature of proteins greatly hinders their ability to cross cell membranes. Herein, a calixarene modification strategy for the intracellular delivery of protein drugs is developed. The decoration of sulfonate azocalix[4]arene (SAC4A) on proteins results in a nano-multivalent effect between Protein-S and amino acids on the cell surface, leading to efficient intracellular delivery of the protein via the clathrin-mediated endocytic pathway. By using SAC4A as a novel ligand, this calixarene modification strategy efficiently delivers 7 proteins, bovine serum albumin (BSA), trypsin (TRY), horseradish peroxidase (HRP), α-chymotrypsin (α-Chyt), lysozyme (LYZ), cytochrome C (Cyt C) and ribonuclease A (RNase A), into cells and significantly enhances the cytotoxicity of Cyt C and RNase A. Moreover, SAC4A-modified Cyt C demonstrates markedly enhanced antitumor efficacy in 4T1-bearing mice without notable side effects. Considering that these proteins are varied in molecular weight and isoelectric point, this calixarene modification strategy provides a platform technology for intracellular protein delivery and the development of protein drugs targeting intracellular pathways.