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Page 14 of 18 Wang et al. Cancer Drug Resist. 2026;9:8

approach, we established an icotinib-resistant HCC827 cell line (HCC827/IR), in which resistance is
primarily mediated by the epidermal growth factor receptor (EGFR) T790M mutation. As shown in
Supplementary Figure 13, pretreatment with PTTP-DC6 under either dark or light conditions did not
re-sensitize these cells to icotinib.

Given the challenges in establishing reliable animal models of MCF-7/ADR tumors, we utilized
three-dimensional (3D) cell spheroids to better mimic tumors in vivo and evaluate the drug
sensitivity-restoring effect of PTTP-DC6. Compared with traditional two-dimensional (2D) monolayers, 3D
tumor spheroids are better models to reflect drug efficacy in vivo, achieving more efficient and cost-effective
preclinical screening of anticancer drugs [40,41] . The 3D cell spheroids were formed by 4 days’ culturing in
Akura™ PLUS Hanging Drop Plate. 1 day after transferring to Akura™ 96 Spheroid Microplate, cell spheroids
were treated with blank cell medium or 5 μM PTTP-DC6 for 24 h, following by light irradiation or dark
incubation for 30 min. Notably, the fluorescence emitted by PTTP-DC6 and DOX in these spheroids
significantly interfered with the detection of conventional fluorescence dyes such as calcein and propidium
iodide (PI), hindering the accurate assessment of MCF-7/ADR cell survival using live-dead staining assays.
Therefore, a viability assay using CellTiter-Glo® kit was applied to determine the survival rates after another
48-hour incubation with 100 μg·mL DOX. Consistent with the trend in 2D monolayers [Figure 6A and B],
-1
PTTP-DC6-treated cell spheroids were more fragile to DOX compared to untreated cell spheroids under
dark. Light irradiation could further decrease the cell viability by 2 orders to almost total death compared to
cells spheroids treated with DOX or PTTP-DC6 only [Figure 6C]. The morphology of cell spheroids was
further examined on a high-content imaging system and a scanning electron microscope (SEM). Notably, the
loss of apparent microtissue morphology was not evident for all the groups, except the group treated with
PTTP-DC6, DOX, and light irradiation, which exhibited obvious cellular debris dispersion [Figure 6D and
Supplementary Figure 14]. The details of 3D cell spheroids were imaged using SEM. The cell spheroids
treated with DOX or PTTP-DC6 only slightly increased the surface roughness as compared to the controls,
while small portion of debris can be observed on the cell spheroid treated with both DOX and PTTP-DC6.
Notably, the introduction of PTTP-DC6 together with light irradiation could violently trigger the cell lysis to
small debris in the presence of DOX [Figure 6E and Supplementary Figure 15].

DISCUSSION
In this study, we developed a series of MICOE-based photosensitizers (PTTP-DCns) with varying side chain
lengths to overcome lysosomal sequestration-mediated MDR via a dual-mode mechanism. All PTTP-DCns
display significantly increased zeta potentials, enhanced fluorescence and moderate photosensitizing abilities
upon insertion into lipid bilayers. These findings indicate that PTTP-DCns in the membrane are capable of
generating moderate O upon light excitation, enabling the light-regulation of membrane permeabilization
1
2
as proposed in Scheme 1C.

A key finding of our work is the significant impact of side-chain length on cellular interaction and
therapeutic efficacy. Studies on the interaction of PTTP-DCns with MCF-7/ADR cells reveal that PTTP-DC6
exhibits the highest lysosomal membrane assembly efficiency, which is likely attributable to its optimal side
chain length (C6) that balances hydrophobic insertion and electrostatic interaction with the lipid bilayer.
This structure-dependent behavior underscores the critical role of molecular design in the assembly of
MICOEs with lipid membranes. Furthermore, the endocytosis mechanism studies reveal that the
internalization of PTTP-DC6 is mainly mediated by clathrin-related endocytosis and affected obviously by
membrane fluidity, providing insights into the uptake pathway of such amphiphilic molecules.

The core mechanism by which PTTP-DC6 overcomes lysosomal sequestration-mediated MDR involves the
disruption of lysosomal membrane through a dual-mode action. We observed that PTTP-DC6 alone could

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