Intratumoral Distribution and pH-Dependent Drug Release of High Molecular Weight HPMA Copolymer Drug Conjugates Strongly Depend on Specific Tumor Substructure and Microenvironment.

Stimulus-sensitive polymer drug conjugates based on high molecular weight N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers carrying doxorubicin via a pH-dependent cleavable bond (pHPMA-Dox) were previously shown to be able to overcome multi-drug resistance. Nevertheless, a tumor type dependent differential response was observed. Although an improved and more selective tumor accumulation of pHPMA-Dox is generally achieved due to the enhanced permeability and retention (EPR) effect, little is known about the fate of these conjugates upon entering the tumor tissue, which could explain the different responses. In this study, we compared in vitro and in vivo accumulation and Dox-activation of pHPMA-Dox in three cancer cell line models (1411HP, A2780cis, HT29) and derived xenograft tumors using a near-infrared fluorescence-labeled pHPMA-Dox conjugate. Firstly, cytotoxicity assays using different pH conditions proved a stepwise, pH-dependent increase in cytotoxic activity and revealed comparable sensitivity among the cell lines. Using multispectral fluorescence microscopy, we were able to track the distribution of drug and polymeric carrier simultaneously on cellular and histological levels. Microscopic analyses of cell monolayers confirmed the assumed mechanism of cell internalization of the whole conjugate followed by intracellular cleavage and nuclear accumulation of Dox in all three cell lines. In contrast, intratumoral distribution and drug release in xenograft tumors were completely different and were associated with different tissue substructures and microenvironments analyzed by Azan- and Hypoxisense