Organelle-Level\nTrafficking and Metabolism Kinetics\nfor Redox-Responsive Paclitaxel Prodrug Nanoparticles Characterized\nby Experimental and Modeling Analysis

Redox-responsive self-assembled prodrug nanoparticles\nhave received\nextensive attention for their high loading efficiency and environmentally\nresponsive properties. However, the intracellular metabolism and transportation\nkinetics were poorly understood, which limited the rational design\nand development of this delivery system. Herein, tetraphenylporphyrin-paclitaxel\n(TxP) prodrugs with thioether, disulfide, and dicarbon linkers (TsP,\nTssP, and TccP) were synthesized and self-assembled as nanoparticles.\nThe redox responsiveness was investigated both in the simulated medium\nand in tumor cells via mass spectrometry. TxP NP and PTX concentrations\nin 4T1 whole cells, endosomal systems, and cytoplasm over time were\nquantified by UPLC–MS/MS and modeled using the nonlinear mixed\neffect (NLME) approach. Cytotoxicity was studied in 4T1 and MCF-7\ncell lines, and antitumor efficacy was analyzed in 4T1 tumor-bearing\nmice. Mass spectrometry identified both oxidative and reductive metabolites\nin redox simulants for TssP NPs and TsP NPs. In 4T1 cells, only reductive\nmetabolites for TssP NPs were detected, while both oxidative and reductive\nmetabolites for TsP NPs were detected. The developed subcellular pharmacokinetic\nmodel suggested that the estimated metabolism rates of TxP NPs in\nendosomal systems were 10 to 27 times of the rates in cytoplasm, indicating\nthat endosomal systems were the dominant place for intracellular metabolism.\nThese rates were numerically higher for TsP NPs than TssP NPs in endosomal\nsystems (1.7-fold) and the cytoplasm (2.5-fold). The internalization\nof nanoparticles was identified to be slow (<i>k</i><sub>max,int</sub>, 0.015 h<sup>–1</sup>) and saturable. The transportation\nrate constant across the endosomal membranes was fast for PTX (27.1\nh<sup>–1</sup>) and slow for TxP NPs (0.098 h<sup>–1</sup>). TsP and TssP NPs had comparable <i>in vivo</i> antitumor\nefficacy, which was higher than that of TccP NPs. This study quantified\nthe organelle-level transportation and metabolism kinetics for three\nprodrug nanoparticles with redox-responsive or inert linkers using\na combined experimental and modeling approach. These findings and\nthe modeling framework might inform future studies for redox-responsive\nprodrug design and drug delivery systems.