Anticancer drug delivery strategies are designed to take advantage of the differential chemical environment in solid tumors independently, or to high levels of reactive oxygen species (ROS) or to low pH, compared to healthy tissue. Here, the design and thorough characterization of two functionalizable "AND gate" multiresponsive (MR) block amphiphilic copolymers are reported, aimed to take full advantage of the coexistence of two chemical cues-ROS and low pH-present in the tumor microenvironment. The hydrophobic blocks contain masked pH-responsive side chains, which are exposed exclusively in response to ROS. Hence, the hydrophobic polymer side chains will undergo a charge shift in a very relevant pH window present in the extracellular milieu in most solid tumors (pH 5.6-7.2) after demasking by ROS. Doxorubicin (DOX)-loaded nanosized "AND gate" MR polymersomes (MRPs) are fabricated via microfluidic self-assembly. Chemical characterization reveals ROS-dependent pH sensitivity and accelerated DOX release under influence of both ROS and low pH. Treatment of tumor-bearing mice with DOX-loaded nonresponsive and "AND gate" MRPs dramatically decreases cardiac toxicity. The most optimal "AND gate" MRPs outperform free DOX in terms of tumor growth inhibition and survival, shedding light on chemical requirements for successful cancer nanomedicine.

Center for Advanced Preclinical Imaging 1st Faculty of Medicine Charles University Salmovská 3 Prague 120 00 Czech Republic

Department of Tumor Immunology Radboud Institute for Molecular Life Sciences Radboud University Medical Center Geert Grooteplein 26 Nijmegen 6525 GA The Netherlands

Institute for Chemical Immunology Geert Grooteplein Zuid 26 Nijmegen 6525 GA The Netherlands

Institute of Biophysics and Informatics 1st Faculty of Medicine Charles University Salmovska 1 Prague 120 00 Czech Republic

Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Heyrovsky Sq 2 Prague 162 06 Czech Republic

Oncode Institute Geert Grooteplein Zuid 26 Nijmegen 6525 GA The Netherlands

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