Rational Construction of a Mitochondrial Targeting, Fluorescent Self-Reporting Drug-Delivery Platform for Combined Enhancement of Endogenous ROS Responsiveness

Abstract

To maximize the utilization and response to the high oxidative stress environment of tumor sites while avoiding the dilemma of enhancing reactive oxygen species (ROS) response in a single way, mitochondrial targeting combined with fluorescent self-reporting polymeric nanocarriers (<b>1K-TPP</b> and <b>2K-TPP</b>) with grafted structures were synthesized via a chemoenzymatic method in a high yield to simultaneously enhance the drug delivery of endogenous ROS responses. <b>1K-TPP</b> and <b>2K-TPP</b> loaded doxorubicin (DOX) at a high content over 12% and formed homogeneous spherical micelles. <i>In vitro</i>, both of them showed promising high sensitivity (detection limit below 200 nM H₂O₂), fast response, and ratiometric fluorescent self-reporting properties (fluorescent enhancement more than 200 times) to ROS and excellent stability under physiological conditions, while achieving a rapid release of the DOX in response to 1 mM H₂O₂. Cell co-localization experiments exhibited that they had favorable mitochondrial targeting, and mitochondrial isolation experiments also confirmed that the TPP-modified <b>1K-TPP</b> selectively accumulated nearly three times in mitochondria than that in total cells. The internalization of <b>1K-TPP</b> and <b>2K-TPP</b> into cancer cells was greatly improved by nearly 200% compared to that of unmodified control (<b>1K-OH</b> and <b>2K-OH</b>) and also explored a unique energy-dependent endocytosis. Furthermore, stimulation of endogenous ROS enhanced the green fluorescence intensity (up to 51.4%) of the linked probe so as to destroy the internal structure of the nanocarriers, achieving self-reporting of the drug's intracellular release and tracking of the intracellular location of nanocarriers. The cytotoxicity of DOX-loaded <b>1K-TPP</b> and <b>2K-TPP</b> in tumor cells with a higher ROS content showed statistical superiority to that of <b>1K-OH</b> and <b>2K-OH</b>, benefiting from the extremely good endogenous ROS response sensitivity leading to the differential selective release of drugs. These results demonstrate the potential of <b>1K-TPP</b> and <b>2K-TPP</b>, especially for <b>1K-TPP</b>, as mitochondria-targeted, fluorescent self-reporting nanocarriers for combined enhancement of endogenous ROS responsiveness.