Intein-mediated site-specific synthesis of tumor-targeting protein delivery system: Turning PEG dilemma into prodrug-like feature

Abstract

Poor tumor-targeted and cytoplasmic delivery is a bottleneck for protein toxin-based cancer therapy. Ideally, a protein toxin drug should remain stealthy in circulation for prolonged half-life and reduced side toxicity, but turn activated at tumor. PEGylation is a solution to achieve the first goal, but creates a hurdle for the second because PEG rejects interaction between the drugs and tumor cells therein. Such PEG dilemma is an unsolved problem in protein delivery. Herein proposed is a concept of turning PEG dilemma into prodrug-like feature. A site-selectively PEGylated, gelatinase-triggered cell-penetrating trichosanthin protein delivery system is developed with three specific aims. The first is to develop an intein-based ligation method for achieving site-specific modification of protein toxins. The second is to develop a prodrug feature that renders protein toxins remaining stealthy in blood for reduced side toxicity and improved EPR effect. The third is to develop a gelatinase activatable cell-penetration strategy for enhanced tumor targeting and cytoplasmic delivery. Of note, site-specific modification is a big challenge in protein drug research, especially for such a complicated, multifunctional protein delivery system. We successfully develop a protocol for constructing a macromolecular prodrug system with intein-mediated ligation synthesis. With an on-column process of purification and intein-mediated cleavage, the site-specific PEGylation then can be readily achieved by conjugation with the activated C-terminus, thus constructing a PEG-capped, cell-penetrating trichosanthin system with a gelatinase-cleavable linker that enables tumor-specific activation of cytoplasmic delivery. It provides a promising method to address the PEG dilemma for enhanced protein drug delivery, and importantly, a facile protocol for site-specific modification of such a class of protein drugs for improving their druggability and industrial translation.

Extracellular vesicles as a platform for membrane‐associated therapeutic protein delivery

“…As proteins are widely used to treat many diseases, extensive research has focused on developing techniques for transporting these biomolecules [124–126]. The purpose of a protein drug delivery system is to decrease nonspecific targeting, improve the effect of the drug, and reduce its side effects [127].…”

Nanotechnology-based combination therapy for overcoming multidrug-resistant cancer

“…However, PEGylation can reduce the membrane permeability of proteins. We previously developed a PEGylated, matrix metallopeptidase 2 (MMP2)-activatable cell-penetrating protein toxin trichosanthin (TCS) (termed rTLM-PEG) based on a recombinant intein-mediated site-specific conjugation method 90 . The protein system can dePEGylate in the TME via the enzymatic activation of matrix metalloproteinase to the substrate peptide and thus release the cell penetrating TCS.…”

Glioma Dual-Targeting Nanohybrid Protein Toxin Constructed by Intein-Mediated Site-Specific Ligation for Multistage Booster Delivery

“…We have previously succeeded in developing a method for intein-mediated site-specific PEGylation of protein toxin. 23 , 24 In this work, we applied this strategy to achieve the site-specific modification of rTLM with LF to construct a BBB-penetrating nanohybrid protein toxin for antiglioma treatment. This method could offer a facile protocol for enhanced delivery of protein toxins for improving cancer treatment outcomes.…”