Redox-responsive self-assembled prodrug nanoparticles have received extensive attention for their high loading efficiency and environmentally responsive properties. However, the intracellular metabolism and transportation kinetics were poorly understood, which limited the rational design and development of this delivery system. Herein, tetraphenylporphyrin-paclitaxel (TxP) prodrugs with thioether, disulfide, and dicarbon linkers (TsP, TssP, and TccP) were synthesized and self-assembled as nanoparticles. The redox responsiveness was investigated both in the simulated medium and in tumor cells via mass spectrometry. TxP NP and PTX concentrations in 4T1 whole cells, endosomal systems, and cytoplasm over time were quantified by UPLC–MS/MS and modeled using the nonlinear mixed effect (NLME) approach. Cytotoxicity was studied in 4T1 and MCF-7 cell lines, and antitumor efficacy was analyzed in 4T1 tumor-bearing mice. Mass spectrometry identified both oxidative and reductive metabolites in redox simulants for TssP NPs and TsP NPs. In 4T1 cells, only reductive metabolites for TssP NPs were detected, while both oxidative and reductive metabolites for TsP NPs were detected. The developed subcellular pharmacokinetic model suggested that the estimated metabolism rates of TxP NPs in endosomal systems were 10 to 27 times of the rates in cytoplasm, indicating that endosomal systems were the dominant place for intracellular metabolism. These rates were numerically higher for TsP NPs than TssP NPs in endosomal systems (1.7-fold) and the cytoplasm (2.5-fold). The internalization of nanoparticles was identified to be slow (k_max,int, 0.015 h^–1) and saturable. The transportation rate constant across the endosomal membranes was fast for PTX (27.1 h^–1) and slow for TxP NPs (0.098 h^–1). TsP and TssP NPs had comparable in vivo antitumor efficacy, which was higher than that of TccP NPs. This study quantified the organelle-level transportation and metabolism kinetics for three prodrug nanoparticles with redox-responsive or inert linkers using a combined experimental and modeling approach. These findings and the modeling framework might inform future studies for redox-responsive prodrug design and drug delivery systems.

中文翻译：

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通过实验和建模分析表征氧化还原反应性紫杉醇前药纳米颗粒的细胞器水平运输和代谢动力学


氧化还原响应性自组装前药纳米颗粒因其高负载效率和环境响应特性而受到广泛关注。然而，对细胞内代谢和转运动力学知之甚少，这限制了该递送系统的合理设计和开发。在此，合成了具有硫醚、二硫键和二碳接头 （TsP、TssP 和 TccP） 的四苯卟啉-紫杉醇 （TxP） 前药，并自组装为纳米颗粒。通过质谱法在模拟培养基和肿瘤细胞中研究氧化还原反应性。使用 UPLC-MS/MS 定量 4T1 全细胞、内体系统和细胞质中 TxP NP 和 PTX 浓度随时间的变化，并使用非线性混合效应 （NLME） 方法进行建模。在 4T1 和 MCF-7 细胞系中研究细胞毒性，并在 4T1 荷瘤小鼠中分析抗肿瘤疗效。质谱法鉴定了 TssP NP 和 TsP NP 的氧化还原模拟物中的氧化和还原代谢物。在 4T1 细胞中，仅检测到 TssP NPs 的还原代谢物，而检测到 TsP NPs 的氧化和还原代谢物。开发的亚细胞药代动力学模型表明，内体系统中 TxP NPs 的估计代谢速率是细胞质中代谢速率的 10 至 27 倍，表明内体系统是细胞内代谢的主要场所。TsP NPs 的这些比率在数值上高于内体系统 （1.7 倍） 和细胞质 （2.5 倍） 中的 TssP NPs。纳米颗粒的内化被确定为缓慢 （k_max，int， 0.015 ^h-1） 且可饱和。PTX 跨内体膜的转运速率常数很快 （27.1 小时^-1 小时），TxP NP 较慢 （0.098 小时^-1）。TsP 和 TssP NPs 具有相当的体内抗肿瘤疗效，高于 TccP NPs。本研究使用实验和建模相结合的方法量化了三种具有氧化还原响应或惰性接头的前药纳米颗粒的细胞器水平运输和代谢动力学。这些发现和建模框架可能会为氧化还原反应前药设计和药物递送系统的未来研究提供信息。