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Abstract P‐glycoprotein (P‐gp, ABCB1) is a well‐researched ATP‐binding cassette (ABC) drug efflux transporter linked to the development of cancer multidrug resistance (MDR). Despite extensive studies, approved therapies to safely inhibit P‐gp in clinical settings are lacking, necessitating innovative strategies beyond conventional inhibitors or antibodies to reverse MDR. Photodynamic therapy is a globally approved cancer treatment that uses targeted, harmless red light to activate non‐toxic photosensitizers, confining its cytotoxic photochemical effects to disease sites while sparing healthy tissues. This study demonstrates that photodynamic priming (PDP), a sub‐cytotoxic photodynamic therapy process, can inhibit P‐gp function by modulating cellular respiration and ATP levels in light accessible regions. Using chemoresistant (VBL‐MDA‐MB‐231) and chemosensitive (MDA‐MB‐231) triple‐negative breast cancer cell lines, we showed that PDP decreases mitochondrial membrane potential by 54.4% ± 30.4 and reduces mitochondrial ATP production rates by 94.9% ± 3.46. Flow cytometry studies showed PDP can effectively improve the retention of P‐gp substrates (calcein) by up to 228.4% ± 156.3 in chemoresistant VBL‐MDA‐MB‐231 cells, but not in chemosensitive MDA‐MB‐231 cells. Further analysis revealed that PDP did not alter the cell surface expression level of P‐gp in VBL‐MDA‐MB‐231 cells. These findings indicate that PDP can reduce cellular ATP below the levels that is required for the function of P‐gp and improve intracellular substrate retention. We propose that PDP in combination with chemotherapy drugs, might improve the efficacy of chemotherapy and overcome cancer MDR. 

Abstract Accurate detection of ATP-binding cassette drug transporter ABCB1 expression is imperative for precise identification of drug-resistant tumors. Existing detection methods fail to provide the necessary molecular details regarding the functional state of the transporter. Photoimmunoconjugates are a unique class of antibody–dye conjugates for molecular diagnosis and therapeutic treatment. However, conjugating hydrophobic photosensitizers to hydrophilic antibodies is quite challenging. Here, we devise a photoimmunoconjugate that combines a clinically approved benzoporphyrin derivative (BPD) photosensitizer and the conformational-sensitive UIC2 monoclonal antibody to target functionally active human ABCB1 (i.e., ABCB1 in the inward-open conformation). We show that PEGylation of UIC2 enhances the BPD conjugation efficiency and reduces the amount of non-covalently conjugated BPD molecules by 17%. Size exclusion chromatography effectively separates the different molecular weight species found in the UIC2–BPD sample. The binding of UIC2–BPD to ABCB1 was demonstrated in lipidic nanodiscs and ABCB1-overexpressing triple negative breast cancer (TNBC) cells. UIC2–BPD was found to retain the conformation sensitivity of UIC2, as the addition of ABCB1 modulators increases the antibody reactivity in vitro . Thus, the inherent fluorescence capability of BPD can be used to label ABCB1-overexpressing TNBC cells using UIC2–BPD. Our findings provide insight into conjugation of hydrophobic photosensitizers to conformation-sensitive antibodies to target proteins expressed on the surface of cancer cells.