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Wang et al. Cancer Drug Resist. 2026;9:8 Page 15 of 18

induce mild LMP in MCF-7/ADR cells, while light irradiation significantly amplified this effect via ROS
generation. These results demonstrate that even a low dose of PTTP-DC6 under mild conditions (darkness
or weak light) can induce LMP in MDR cells, highlighting its potential to restore the efficacy of DOX by
overcoming lysosomal drug sequestration. The proteomic analysis further confirmed this mechanism. The
coordinated upregulation of antioxidant enzymes (SOD1, CAT, PRDX4) suggests that PTTP-DC6 treatment
induces a state of oxidative stress, which is a well-known upstream inducer of LMP . Moreover, the
[42]
increased expression of LMP-related proteins (LAMP2 and PI4K2A) points to a cellular
compensatory/reparative response to membrane damage [43,44] . These proteomic findings indicate that
treatment with PTTP-DC6, whether in darkness or under low-dose light, can trigger proteomic alterations
associated with enhanced ROS metabolism and lysosomal dysfunction in MDR cancer cells, providing
mechanistic support for its dual-mode action of PTTP-DC6. Specifically, we propose that the mechanism by
which PTTP-DC6 overcomes cancer drug resistance involves: (i) its physical association with lysosomal
membranes increases LMP under both dark and light conditions; (ii) light exposure further enhances LMP
via lipid peroxidation initiated by in situ ROS generation.

Subsequently, the ability of PTTP-DC6 to reverse lysosomal sequestration-mediated drug resistance was
validated through drug resensitization assays. The IC for DOX in PTTP-DC6 pre-treated MCF-7/ADR cells
50
under light decreased significantly, with the RI dropping from 39 to 19. These results reveal a clear side-chain
length–dependent structure–activity relationship. Only the C6 chain achieves the optimal balance between
lysosomal integration and membrane perturbation, enabling dual-mode drug resensitization. In contrast, the
C4 chain is too short for stable anchoring, and the C8 chain is too long to efficiently disrupt the membrane.
When compared to CQ, a classical lysosomal disruptor, PTTP-DC6 combined with light achieved superior
efficacy while maintaining excellent biosafety, as CQ exhibited significant cytotoxicity at effective
concentrations. Additionally, the failure of PTTP-DC6 to re-sensitize icotinib-resistant cells (where
resistance is not lysosome-dependent) confirms the specificity of our strategy towards lysosomal drug
sequestration.

Finally, the efficacy of PTTP-DC6 was confirmed in MDR cancer spheroids, a model that mimics the
physiological environment of solid tumors. The cell viability and morphology results illustrate that
PTTP-DC6 can significantly improve the anticancer efficacy of DOX in a 3D in vitro model of MDR cells,
further validating the dual-mode mechanism in a more physiologically relevant context. Nevertheless, the
absence of in vivo validation remains a key limitation of this study. The translational potential of PTTP-DC6,
therefore, necessitates future evaluation in clinically relevant patient-derived xenograft (PDX) models of
resistant tumors. To enable tumor-targeted delivery in vivo, future efforts could leverage hybrid cell
membrane-coated nanoparticles as a co-delivery system, with DOX encapsulated in the core and
[45]
PTTP-DC6 anchored in the membrane, to achieve synergistic lysosomal disruption. Additionally, clinical
translation of this photodynamic approach would require strategies to overcome the limited tissue
penetration of the green light used here, which could be achieved by developing next-generation MICOE
photosensitizers activatable by longer-wavelength or two-photon excitation.

In summary, this study presents a rational design of side-chain-engineered MICOEs that effectively reverse
MDR by regulating lysosomal membrane functions. The dual-mode action of PTTP-DC6, which combines
physical membrane intercalation in the dark with photochemical ROS generation under light, offers a
promising strategy to enhance chemotherapy outcomes with high specificity and minimal toxicity. Overall,
this work demonstrates the great potential of MICOEs as a versatile molecular platform for cancer therapy.
Building on their unique topological structure and optical property, they establish a novel insight and
strategy for overcoming drug resistance by synergistically regulating subcellular organelle membranes.

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