title: 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.
authors.affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Hai-ke Rd, Shanghai 201203, China; University of Chinese Academy of Sciences, 19A Yuquan Rd, Beijing 100049, China.
authors.forename: Yingzhi
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Hai-ke Rd, Shanghai 201203, China; University of Chinese Academy of Sciences, 19A Yuquan Rd, Beijing 100049, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Hai-ke Rd, Shanghai 201203, China.
authors.forename: Hongyue
authors.initials: H
authors.lastname: Jin
authors.affiliation: Guangzhou University of Chinese Medicine Tropical Medical Institute, 12 Ji-chang Rd, Guangzhou 510405, China.
authors.forename: Yisi
authors.initials: Y
authors.lastname: Tang
authors.affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Hai-ke Rd, Shanghai 201203, China.
authors.forename: Huiyuan
authors.initials: H
authors.lastname: Wang
authors.affiliation: Guangzhou University of Chinese Medicine Tropical Medical Institute, 12 Ji-chang Rd, Guangzhou 510405, China.
authors.forename: Qin
authors.initials: Q
authors.lastname: Xu
authors.affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Hai-ke Rd, Shanghai 201203, China.
authors.forename: Yaping
authors.initials: Y
authors.lastname: Li
authors.affiliation: Hampton University School of Pharmacy, Kittrell Hall RM216, Hampton, VA 23668, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Li
authors.affiliation: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Hai-ke Rd, Shanghai 201203, China. Electronic address: yzhuang@simm.ac.cn.
authors.forename: Yongzhuo
authors.initials: Y
authors.lastname: Huang
mesh_terms: D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000008:administration & dosage; D045744:Cell Line, Tumor; D016503:Drug Delivery Systems / Q000379:methods*; D018093:Gelatinases / Q000737:chemistry*; D047668:Inteins; D051379:Mice; D009374:Neoplasms, Experimental / Q000737:chemistry* / Q000188:drug therapy* / Q000473:pathology; D011092:Polyethylene Glycols / Q000737:chemistry*; D011355:Prodrugs / Q000008:administration & dosage / Q000737:chemistry; D016896:Treatment Outcome; D015978:Trichosanthin / Q000008:administration & dosage*
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D011355:Prodrugs; D011092:Polyethylene Glycols; D015978:Trichosanthin; D018093:Gelatinases
keywords: Cell-penetrating peptide; Gelatinase; Intein-mediated protein ligation (IPL); Protein toxin; Site-specific PEGylation; Trichosanthin