Poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazol-4,7-diyl) purchased from RuiXun Technology Co., Ltd. (Xi'an, China). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], lecithin, Cy5-DSPE-PEG₂₀₀₀ and cholesterol were from Xi'an Ruixi Biological Technology Co., Ltd. Calretinin antibody, BiP, p-eIF2α, eIF2α, β-actin and ERp57 antibody were purchased from Abcam (Shanghai, China). Cell Counting Kit-8 assay, RPMI1640 medium, and fetal bovine serum (FBS) were provided by GIBCO Invitrogen Corp. Co., Ltd. (Carlsbad, CA, USA). Paraformaldehyde (PFA) (4%), Calcein-AM/PI Double Stain Kit, and Ca²⁺ Kit were purchased from Beyotime Company Co., Ltd. (Shanghai, China). Annexin V-FITC/PI apoptosis kit was obtained from MultiSciences Co., Ltd. (Hangzhou, China). Roxadustat was obtained from MCE Co., Ltd. (Shanghai, China). Ultrapure water (18.2 MΩ; Millipore Co., USA) was used to prepare all buffers and employed in all experiments. All chemical agents were used directly without further purification.

Briefly, cholesterol, lecithin, DSPE-PEG₂₀₀₀, Roxadustat and PFBT were dissolved in a mixed solution of THF solution, the optimal therapeutic dose ratio of PFBT and Roxadustat in our nanosystem is 2:1, evaporated at 40 ℃ to get a dry lipid film, followed by the addition of 10 mL of aqueous solution with ultrasonic hydration for 10 min.

Cellular uptake was investigated by confocal laser scanning microscopy (CLSM). Colon26 cells were seeded onto a 24-well plate containing cover glass (1 × 10⁴ cells in each well) and incubated overnight for adherence. Then, RPMI 1640 (0.5 mL/well) containing PFBT@Rox Lip (0.2 μg/mL) was added to the plate for 4, 8, 12 and 24 h. Finally, the cells were observed by CLSM.

Colon26 cells were seeded in 96-well plates (Corning) at a density of 8 × 10³ cells per well and incubated overnight. Then cells were treated with PFBT@Rox Lip at different drug concentrations for 24 h, during which light irradiation (40 mW/cm² for 10 min) was treated at 12 h. The cell viability was measured using the Cell Counting Kit-8 assay.

Poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-(benzo[2,1,3]thiadiazol-4,7-diyl)] (PFBT) and Roxadustat were self-assembled into liposomal nanoparticles (PFBT@Rox Lip) via nanoprecipitation using cholesterol, lecithin and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-2000 as surfactants (Fig. 1A). PFBT@Rox Lip nanoparticles had a uniform and transparent appearance in aqueous solutions. Transmission electron microscopy (TEM) analysis demonstrated that PFBT@Rox Lip nanoparticles were round in shape and ∼50 nm in diameter (Fig. 1B). The hydrodynamic diameter of a PFBT@Rox Lip nanoparticle was 56 nm, as measured using dynamic light scattering (Fig. 1C). PFBT@Rox Lip nanoparticles were dispersed in water, phosphate-buffered saline (PBS), and Dulbecco's modified Eagle medium. The stability examination of PFBT@Rox Lip nanoparticles suggested no obvious aggregation was observed in various aqueous solutions within 14 days, implying good stability for PFBT@Rox Lip nanoparticles (Fig. 1D and Supporting Information Fig. S1). The encapsulation rates of Roxadustat and PFBT were calculated to be 70 % and 95 % respectively from established standard curves by measuring absorbance values. The loading rates of Roxadustat and PFBT were determined to be 15% and 64% respectively (Supporting Information Fig. S2). We also investigated the Roxadustat release in vitro from PFBT@Rox Lip nanoparticles. The results showed that abundant PFBT and Roxadustat were released from PFBT@Rox Lip upon incubating PFBT@Rox Lip in PBS containing H₂O₂ (pH 7.4) (Supporting Information Fig. S3). A slower release of drug behavior was observed at the condition without irradiation, suggesting 39% and 19.8% of PFBT and Roxadustat release after 24 h incubation, respectively. However, upon light irradiation for 1h, a marked increase in the release of PFBT and Roxadustat was detected with a cumulative drug release of 98% and 48.8% for PFBT and Roxadustat, respectively after 24 h (Fig. S3). Next, the zeta potential of PFBT@Rox Lip nanoparticles was determined to be −12.3 mV, which is conducive to maintaining nanoparticle stability in the bloodstream (Fig. 1E). In addition, the ultraviolet–visible–near infrared absorption and emission spectra were analyzed and showed that PFBT@Rox Lip nanoparticles had an absorption peak at ∼470 nm and an emission peak at ∼540 nm (Fig. 1F). To gain a deeper understanding of the excitation properties of PFBT-conjugated polymers, we performed theoretical calculations to reveal the process of in situ self-assembling coating and photophysical properties of the obtained nanoparticles. As shown in Fig. 1G and H, the density functional theory calculations demonstrated that the PFBT ΔE_S1-T1 value was 0.26 eV. Such a low ΔE_S1-T1 value is beneficial for the energy transfer from S₁ to T₁ and generates a sensitized T₁ state, and it enables the energy transfer to the adjacent O₂ for ¹O₂ generation efficiency. In addition, the energy gap between the highest occupied molecular orbital and the lowest unoccupied molecular orbital (HOMO–LUMO) for the PFBT-conjugated polymer was calculated to be 3.2 eV. Full separation of the HOMO and LUMO is beneficial for the in situ self-assembling coating and promotes the generation of type II ROS. Because the PFBT-conjugated polymer possesses a π‒electronic backbone structure, the excited PFBT∗ further transfers electrons to the surrounding O₂ or H₂O to produce superoxide radicals (O₂·⁻) and hydroxyl radicals (·OH) (Supporting Information Fig. S4). In addition, PFBT energy levels measured using an ultraviolet photoelectron spectrometer showed an energy tendency similar to the calculated one for the conjugated polymer (Fig. 1G). Total ROS production by PFBT@Rox Lip nanoparticles was determined using the ROS probe 2,7-dichlorofluorescein diacetate (DCFH-DA). In the presence of ROS, DCFH-DA is oxidized to 2,7-dichlorofluorescin (DCF), which emits bright fluorescence at 525 nm. As shown in Fig. 1I, upon white light irradiation with a direct current for 6 min, the fluorescence intensity of DCF increased over time. However, there was no significant change in the fluorescence of DCFH without PFBT@Rox Lip nanoparticles, indicating that the latter sensitized the surrounding oxygen or water molecules to produce ROS. The identities of ROS (¹O₂, ·OH and O₂·⁻) were experimentally evaluated using electron paramagnetic resonance. Established methods typically use 2,2,6,6-tetramethylpiperidine (TEMP) as a probe for ¹O₂, and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin-trap to detect O₂·⁻ and ·OH. The hyperfine splitting constants (aN, aH) and g factor for the spin adducts were used to examine the presence of each ROS. As shown in Fig. 1J, triplet peaks corresponding to typical TEMPO signals were observed for white light irradiation (aN = 16.5 G, g = 2.0058), suggesting the appearance of ¹O₂, in sharp contrast to the silent spectra for the untreated sample. The signals of the DMPO/O₂⁻ adduct were also observed under the experimental conditions (Fig. 1K). In addition, as shown in Fig. 1L, OH formation was also evidenced by the electron paramagnetic resonance signals of the DMPO/·OH adduct (aN = aH = 14.8 G, g = 2.0031). Additionally, the frequently used photosensitizer Hematoporphyrin monomethyl ether (HMME), was also employed as a control to compare its capability of ROS generation with that of PFBT. We found that PFBT showed a more efficient capability of ROS generation when compared with HMME, indicating that PFBT is an excellent photosensitizer. (Supporting Information Fig. S5). Together, these results indicated that PFBT@Rox Lip nanoparticles showed excellent Type I and Type II ROS generation capacities.

Given the capacity of abundant ROS generation by PFBT@Rox Lip nanoparticles, we next evaluated in vitro anti-tumor efficacy of PDT in the Colon26 cell line. The therapeutic efficacy of a nanomedicine is highly dependent on its cellular uptake. The uptake of PFBT@Rox Lip nanoparticles by Colon26 cells was observed using confocal laser scanning microscopy. As shown in Fig. 2A, the intracellular green fluorescence gradually became stronger with the extension of the incubation time from 4 to 24 h, indicating that PFBT@Rox Lip nanoparticles could be internalized into cells, which would facilitate the induction of the photodynamic effect. ROS can induce cell death. To verify the in vitro anti-tumor efficacy of the PFBT@Rox Lip nanosystem that would be achieved by releasing ROS, in vitro ROS production was detected using a DCFH-DA probe, and the cell viability in different treatment groups was examined using the Cell Counting Kit-8 assay. As shown in Fig. 2B and C, PFBT@Rox Lip nanoparticles produced a large amount of ROS after light irradiation, and the cytotoxicity of the combination treatment showed the inhibition of cell viability to the greatest extent. However, Roxadustat and PFBT alone just slightly induced viability suppression compared with PFBT@Rox Lip (Supporting Information Fig. S6). In addition, we also evaluated the PDT effects of PFBT and PFBT@Rox Lip under hypoxic conditions (2%). PFBT@Rox Lip plus irradiation could still significantly repress cancer cell viability (Supporting Information Fig. S7). Subsequently, calcein-AM/propidium iodide staining was performed to observe cell survival. As shown in Fig. 2D, PFBT@Rox Lip-treated cells showed red fluorescence, which was not observed in “blank” and “light alone” groups, indicating that PFBT@Rox Lip nanoparticles alone could induce cancer cell death, possibly resulting from a small quantity of toxicity itself. However, treatment with a combination of PFBT@Rox Lip nanoparticles and light led to the most conspicuous red fluorescence and weak green fluorescence in the cells when compared with other control groups, which further demonstrated that PFBT@Rox Lip nanoparticles can trigger ROS production under white light, eventually killing cancer cells. This observation aligns with the results of the Cell Counting Kit-8 assay. Additionally, cell cycle analysis with the annexin-V/propidium iodide staining showed that the proportion of early apoptotic cells (annexin-V⁺) in the PFBT@Rox Lip alone group was significantly higher than that in the control group, consistent with the results of calcein-AM/propidium iodide assay, and notably, the proportion of late apoptotic cells (annexin-V⁺/PI⁺) in the PFBT@Rox Lip nanoparticles + light (PFBT@Rox Lip + L) group was 41% higher than the apoptotic fraction of cells treated with PFBT@Rox Lip alone and another two control groups (Fig. 2E). This finding further confirmed that PDT had a prominent in vitro killing effect on cancer cells. At the same time, we also examined the cytotoxic effect of PFBT@Rox Lip on NCM460, a normal intestinal epithelial cell with varying concentrations. PFBT@Rox Lip nanoparticles alone did not produce significant viability inhibition to normal intestinal epithelial cells, indicating good safety (Supporting Information Fig. S8). Taken together, these results demonstrate that the PFBT@Rox Lip system potently decreased the viability of cancer cells through ROS generation under irradiation with white light. Given that excessive ROS generation causes the ER stress, we further examined the expression of ER stress-related proteins, such as BiP and phosphorylated elF2α. Our data suggested that BiP and phosphorylated elF2α levels were increased after the combined treatment with PFBT@Rox Lip nanoparticles and light, indicating that PDT caused severe ER stress (Fig. 2F and G). Excess ROS also causes severe oxidative stress in the mitochondria and disrupts calcium homeostasis, eventually leading to the release of calcium from the mitochondria into the cytoplasm via the mitochondrial transition pores. As shown in Fig. 2H and Supporting Information Fig. S9, intracellular calcium levels were higher in the Colon26 cells incubated with PFBT@Rox Lip nanoparticles under white light than in the cells receiving control treatments. Interfering with intra/extracellular ion homeostasis not only induces tumor cell death but also ICD in tumor cells. Additionally, the ICD effect triggered by PDT in tumor cells was assessed by the translocation of CRT, ERp57 and the release of HMGB1. Immunofluorescence images indicated that PFBT@Rox Lip plus light irradiation induced obvious translocation of CRT and ERp57 compared with other groups. Moreover, the release of HMGB1 from Colon26 cancer cells was also increased correspondingly. These results demonstrate that PFBT@Rox Lip plus light irradiation could initiate ICD in cancer cells, thereby potentially provoking anti-tumor immunity (Fig. 2I and J). In addition, given that Roxadustat is an EPO inducer which could possibly alleviate hypoxia, we detected the expression of EPO protein after treatment with PFBT@Rox Lip plus light irradiation. The data clearly showed that EPO levels were increased more after the combined treatment with PFBT@Rox Lip nanoparticles plus light compared with nanoparticles alone and PBS, indicating that light irradiation could accelerate the release of Roxadustat from PFBT@Rox Lip nanoparticles, and the nanoparticles could potentially re-oxygenate the tumor tissues after the release of Roxadustat upon irradiation (Supporting Information Fig. S10). Collectively, these results demonstrated that PFBT@Rox Lip nanoparticles could trigger ICD and increase EPO, potentially initiating strong anti-tumor responses and reshaping the TME in vivo.

To further explore the molecular mechanism of PDT in-depth, RNA-seq was used to explore the related pathways involved in the therapeutic effect of combined treatment with PFBT@Rox Lip nanoparticles and light in Colon26 cells. The principal component analysis showed a significant difference between the treatment and blank control groups, indicating an extensive change in the gene expression pattern after the PFBT@Rox Lip + L treatment (Fig. 3A). Further analysis showed 4172 differentially expressed genes (fold change ≥2, adjusted P < 0.05) after the PFBT@Rox + L treatment (Fig. 3B–E), among which 676 were up-regulated and 3496 were down-regulated (Fig. 3D). Enrichment analysis revealed that several pathways were enriched after the PFBT@Rox Lip + L treatment, such as those related to ER stress-induced apoptosis, ROS production, angiogenesis, and cell cycle (Fig. 3C and E). Notably, almost all genes involved in angiogenesis were down-regulated (Fig. 3F and G). These results suggested that PFBT@Rox Lip nanoparticles under light irradiation could inhibit cancer cells through a complex network probably initiated by ROS production via photodynamic activity.

The efficient accumulation of nanoparticles into tumor tissues is a key factor in realizing therapeutic examination. We subsequently assessed the tumor-targeting ability of PFBT@Rox Lip nanoparticles. Cy5-labeled PFBT@Rox Lip nanoparticles were injected into Colon26 tumor-bearing mice to determine their distribution and tumor-specific enrichment at different time points. As shown in Fig. 4A and B, and Supporting Information Fig. S11, the fluorescence signals for Cy5-PFBT@Rox Lip detected by an in vivo imaging system were mainly localized at the tumor sites 4 h after administration. Cy5-PFBT@Rox Lip could be highly enriched in the tumors after systemic administration in the Colon26 tumor-bearing mice; whereas free Cy5 did not show a good ability for tumor imaging (Fig. S11). The fluorescence signals increased gradually over time and peaked at 48 h after injection, and apparent fluorescence signals were still detectable at the tumor site even at 72 h. These results demonstrated that PFBT@Rox Lip nanoparticles could efficiently target tumors and retain in tumor tissues for at least 72 h, thereby providing a sufficient time window for PDT application. For detailed tissue distribution of PFBT@Rox Lip nanoparticles, ex vivo fluorescence images of isolated organs collected 72 h post-injection disclosed the strongest fluorescence signals associated with Cy5-labeled PFBT@Rox Lip nanoparticles at the tumor site (Fig. 4C and D). This might be because the Colon26 tumors are highly vascularized, facilitating the passive targeting of liposomes to tumor sites. This finding was consistent with the in vivo imaging results, which further confirmed the nanosystem's precise targeting and accumulation properties. Hypoxia, caused by abnormal tumor vasculature and rapid tumor growth, is the main feature of solid tumors and plays an important role in their development and occurrence. Given our observation that Roxadustat in PFBT@Rox Lip nanoparticles could increase EPO levels which potentially elevated the oxygen delivered by increased hemoglobin, we thus examined whether our nanosystem could at least partially decrease the degree of hypoxia in the Colon26 tumor model in vivo. Firstly, we measured EPO levels in solid tumors after two doses of nanoparticle administration, the data showed that PFBT@Rox Lip nanoparticles indeed increased EPO levels. The oxygenation of tumor tissues treated with PFBT@Rox Lip nanoparticles was further analyzed by photoacoustic imaging. According to the different absorbance values by oxygenated hemoglobin (λ = 850 nm) and deoxyhemoglobin (λ = 750 nm), the oxygenated hemoglobin levels at different time points after intravenous injection (i.v.) were recorded by photoacoustic imaging, accompanied with B-mode ultrasound imaging based on the saturated O₂ levels. As shown in Fig. 4E, the saturated O₂ levels gradually increased over time in tumors treated with nanoparticles or Roxadustat. However, it seemed that nanoparticles containing Roxadustat could result in better oxygenation in tumors, indicating that the nanosystem enhances the delivery of Roxadustat and induces stronger oxygenation in tumor tissues. Nevertheless, the saturated O₂ levels gradually decreased after 24 h injection for both nanoparticles or Roxadustat (Fig. 4E). This might be due to the fact that Roxadustat generally reaches its functional peak effect 8–24 h after administration³³. The extent of hypoxia in tumors treated was determined with pimonidazole hydrochloride (hypoxyprobe-1) after treatment with PFBT@Rox Lip nanoparticles; The staining indicated that the untreated control tumors showed strong hypoxia signals, whereas the hypoxia level was significantly reduced in the tumors treated with PFBT@Rox Lip nanoparticles (Fig. 4F and G), suggesting that the latter relieves hypoxia in solid tumors and reoxygenates the hypoxic tumor microenvironment. It has been demonstrated that tumor hypoxia results in abnormal angiogenesis and aberrant vascular structure, which accelerate the tumor development in turn. We wondered whether PFBT@Rox Lip nanoparticles could normalize tumor blood vessels and improve vessel perfusion due to hypoxia alleviation. Subsequently, we assessed tumor vascular perfusion with the staining of vessel marker CD31 (red) for endothelial cells and pre-injected FITC-lectin (green) for perfusion. Compared with the blank group, the PFBT@Rox Lip group displayed better vessel perfusion, which indicated that PFBT@Rox Lip nanoparticles could improve vascular functionality and normalize the tumor blood vessels. These results together demonstrated that PFBT@Rox Lip could efficiently target tumors and normalized tumor vessels by relieving hypoxia via Roxadustat (Fig. 4H and I). The improvement in oxygenation and alleviation of hypoxia strongly indicated that the use of PFBT@Rox Lip nanoparticle showed the greatest promise for in vivo cancer therapy.

Having demonstrated in vivo tumor targeting ability and hypoxia alleviation, we further investigated the half-life of PFBT@Rox Lip nanoparticles after intravenous administration and in vivo anti-tumor effect of the nanosystem in Colon26 tumor-bearing mice. First, the half-life of PFBT@Rox Lip nanoparticles was estimated to be approximately 1.8 h after intravenous administration by examining the intrinsic fluorescence intensity of PFBT@Rox Lip nanoparticles at different time points (Supporting Information Fig. S12). Next, in vivo anti-tumor performance was further explored. The therapeutic schedule for the in vivo assessment was illustrated in Fig. 5A. Mice were randomly divided into four groups: 1) blank (PBS), 2) light alone, 3) PFBT@Rox Lip alone, and 4) PFBT@Rox Lip + L. The PFBT@Rox Lip solution (150 μL, 50 μg/mL) and PBS were administered intravenously to mice on the 1st and 3rd day of the experiment, respectively, when the tumor volume reached about 100 mm³. In 24 h after the solution administration, mice from the light alone and PFBT@Rox Lip + L groups were given 40 mW/cm² white light irradiation for 30 min. Body weight and tumor volume were monitored every 2 days, and the mice were sacrificed on the 12th day. The data indicated that the largest tumor growth inhibition was attained in animals that received the treatment with the PFBT@Rox Lip + L group (Fig. 5B and C, and Supporting Information Fig. S13). This efficacy was likely attributed to the potent suppression of tumor cell viability caused by ROS production, apoptosis, ICD and potential anti-tumor immunity induced by relieving the hypoxia under PDT with PFBT@Rox Lip nanoparticles. No significant influence on body weight, blood parameters and tissue morphology was observed in any group (Fig. 5D, Supporting Information Figs. S15–S17), which implicated the excellent bio-safety of the therapeutic modality. Subsequently, tumor sections from each group were stained with hematoxylin and eosin (H&E), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and antibody against the proliferation marker Ki-67 to assess the effects of combined treatments with PFBT@Rox Lip nanoparticles and irradiation on tumor necrosis, apoptosis, and proliferation. As shown in Fig. 5E, no evident cell death was observed in the two control groups, whereas more apoptotic cells and fewer proliferating cells were observed in the PFBT@Rox Lip group, indicating that PFBT@Rox Lip nanoparticles alone could produce an anti-tumor effect to some extent, consistent with in vitro data shown before. Of note, tumor tissues from mice receiving the PFBT@Rox Lip + L treatment showed the highest percentage of tumor cell apoptosis and the lowest proportion of cell proliferation, confirming the strong anti-tumor effect of the combined treatment, highly consistent with the tumor volume data. Considering that the exposure of the CRT protein on the cell surface following PDT is a crucial event for ICD, CRT immunofluorescence staining in tumor tissues was also performed. Minimal cell-surface CRT exposure (red) was observed in the two control groups and the PFBT@Rox Lip group, whereas the PFBT@Rox Lip + L indicated a higher extent of CRT exposure. This was in accordance with the in vitro results, confirming the induction of ICD by PDT and providing a plausible explanation for the potent tumor inhibition by combination therapy. To exclude the possible influence of Roxadustat and PFBT alone on tumor progression, we also determined the tumor development in tumor-bearing mice treated with Roxadustat and PFBT alone as illustrated in Fig. S14A. Both of them alone did not affect the tumor growth (Fig. S14B). Meanwhile, Roxadustat and PFBT alone did not reshape the tumor microenvironment based on the analysis for infiltrated T cells and macrophage polarization after treatment (Figs. S14C–S14E). Collectively, these results further demonstrate that PDT mediated by PFBT@Rox Lip plus irradiation possesses excellent biocompatibility and exhibits outstanding anti-tumor performance in tumor-bearing mice.

PDT has been reported to stimulate T-cell infiltration by triggering an ICD effect which enhances antigen presentation of APCs (antigen-presenting cells). Next, we explored how PDT affects the microenvironment of Colon26 subcutaneous tumors by analyzing immune cells and cytokines (Fig. 6A). We found that CD8⁺ T cell infiltration was significantly increased in the PFBT@Rox Lip + L group (40%) compared to control groups, indicating that PDT promotes greater immune responses in vivo possibly owing to ICD induction and vessel normalization (Fig. 6B and Supporting Information Fig. S18). The percentage of NK cells in animals receiving the combination treatment (0.45%) was significantly elevated when compared with the Light alone (0.15%) and PFBT@Rox Lip alone (0.22%), which implies that PFBT@Rox Lip nanoparticles significantly promote the recruitment of NK cells under light irradiation (Fig. 6C). About the effect on macrophage polarization, the highest proportion of the M1 macrophages (27%) and the lowest proportion of the M2 macrophages (18%) among all treatment groups were observed in the PFBT@Rox Lip + L group (Fig. 6D–F, and Supporting Information Fig. S19), suggesting that PDT promotes macrophage transition from M2 phenotype towards the M1 phenotype. It has been demonstrated that photodynamic immunotherapy not only directly kills cancer cells but also reprograms TME^34-36. Typically, photosensitizers for PDT can efficiently produce ROS in tumors under localized light irradiation, which induces immunogenic cell death (ICD) besides apoptosis in cancer cells. The dying tumor cells release tumor-associated antigens (TAAs) and immunogenic damage-related molecular patterns (DAMPs) such as ecto-CRT/ERp57, HMGB1 et al., which could interact with tumor-associated macrophage (TAMs), induce more M1 polarization and enhance phagocytosis of macrophages to dying cancer cells, ultimately increasing the infiltration of T cells^37,38. Next, serum levels of cytokines critically important for the immune regulation (TNF-α, IL-1β, and IL-6) were analyzed using ELISA. As shown in Fig. 6G–I, TNF-α, IL-1β, and IL-6 levels in the PFBT@Rox Lip + L group were significantly up-regulated than those in other control groups, further confirming a powerful immune response triggered by PDT in vivo. Overall, these results supported the notion that PFBT@Rox Lip-mediated photo-immunotherapy efficiently increased the infiltration of CD8⁺ T cells, and NK cells, induced the M1 polarization of macrophages and up-regulated inflammatory cytokine secretion. This novel PDT modality not only affords excellent anti-tumor performance but also reprograms the TME and induces anti-tumor immunity.

Considering that PDT induces a powerful immune response, especially more CD8⁺ T cell infiltration, the PFBT@Rox Lip + L treatment combined with checkpoint blockades, such as anti-PD-1 antibody, which is currently one of the preferred immunotherapy regimens, might further amplify anti-tumor efficacy and potentially suppress metastasis and tumor recurrence. A commonly used 4T1-luc cell line was implanted bilaterally into BALB/c mice to simulate primary and metastatic tumors. When the primary tumor volume reached 100 mm³, the mice were randomly divided into five groups: 1) blank (PBS), 2) anti-PD-1 antibody alone, 3) PFBT@Rox Lip, 4) PFBT@Rox Lip + L, and 5) PFBT@Rox Lip + L + anti-PD-1 antibody. According to the therapeutic schedule shown in Fig. 7A, PFBT@Rox Lip nanoparticle suspension (150 μL, 50 μg/mL) and PBS solution were administered intravenously on the 1st and 3rd day, respectively. Animals in the light-exposed groups received irradiation with 40 mW/cm² white light for 30 min on the 2nd and 4th day. Meanwhile, mice in both the anti-PD-1 antibody alone and PFBT@Rox Lip + L + anti-PD-1 antibody groups were intraperitoneally administered an anti-PD-1 antibody (10 mg/kg) on Days 6 and 8. Body weight and bilateral tumor volumes of the mice were measured every 2 days during the treatment period. Tumor images were obtained using an in vivo imaging system on Days 1, 5, 10, and 15. As shown in Fig. 7B–D, imaging and direct bilateral tumor volume consistently showed that primary tumors treated with PBS, PD-1 antibody alone, or PFBT@Rox Lip nanoparticles grew rapidly, reaching almost 600 mm³ on the 13th day. This was consistent with the data observed in the Colon26 subcutaneous tumor model. However, the combined treatment with PFBT@Rox Lip + L + anti-PD-1 antibody effectively repressed the growth of primary tumors. For untreated contralateral tumors, the anti-PD-1 treatment alone was minimally effective against distant tumors, possibly because of the high malignancy of 4T1 tumors which are refractory and less responsive to PD-1 therapy. As expected, distant tumor growth was remarkably suppressed in the PFBT@Rox Lip + L + anti-PD-1 combination group, suggesting that the triple combination treatment induced strong systemic anti-tumor immunity in vivo. Finally, H&E, TUNEL, and Ki-67 staining performed on distal tumor sections showed that the triple combination treatment indicated the strongest tumor necrosis and apoptosis as well as less tumor cell proliferation when compared with other treatment groups (Fig. 7E–H). Moreover, mice treated with PFBT@Rox Lip + L + anti-PD-1 antibody showed the longest survival time among all groups (Fig. 7I), indicating the advantages of the combination therapy in survival extension. Together, these results revealed that PFBT@Rox Lip + L combined with ICB immunotherapy could achieve the best anti-tumor efficacy, suggesting the great potential of the immunosynergistic activity of PDT for cancer treatment and greatly warranting potential translation in clinical settings in the future.