A cation-free platform based on azocalixarene poly(disulfide)s for direct cytosolic protein delivery

Protein therapeutics face intracellular delivery barriers due to poor membrane permeability and lysosomal entrapment. While carriers incorporating cationic motifs enable direct cytosolic delivery, their inherent systemic toxicity and propensity for nonspecific adsorption of biological interferents redirect the internalization pathway to inefficient endocytosis. Herein, we report a cation-free motif, lipoic acid-modified carboxylated azocalix[4]arene (LC4A), which was polymerized into carboxylated azocalix[4]arene poly(disulfide)s (CAPS) for direct intracellular proteins delivery. CAPS leverages the recognition of azocalixarenes and the multivalent interactions of its polymers to enable multiple protein binding and structural stabilization. Critically, the cation-free nature of CAPS minimizes nonspecific adsorption and ensures a consistent direct cytosolic delivery. Moreover, the hypoxia responsiveness of azocalixarenes and the glutathione cleavable of poly(disulfide)s enable controllable intracellular proteins activation. The cation-free LC4A-based platform in the screen library exhibits high efficiency in the delivery of diverse proteins and shows significant antitumor effects for Ribonuclease A (RNase A) delivery in vivo.