Hypoxia-tolerant polymeric photosensitizer prodrug for cancer photo-immunotherapy

Yu, Jie; Wu, Jiayan; Huang, Jingsheng; Xu, Cheng; Xu, Mengke; Koh, Clarence Zhi Han; Pu, Kanyi; Zhang, Yan

doi:10.1038/s41467-024-55529-8

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Published: 02 January 2025

Hypoxia-tolerant polymeric photosensitizer prodrug for cancer photo-immunotherapy

Nature Communications volume 16, Article number: 153 (2025) Cite this article

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Abstract

Although photodynamic immunotherapy represents a promising therapeutic approach against malignant tumors, its efficacy is often hampered by the hypoxia and immunosuppressive conditions within the tumor microenvironment (TME) following photodynamic therapy (PDT). In this study, we report the design guidelines towards efficient Type-I semiconducting polymer photosensitizer and modify the best-performing polymer into a hypoxia-tolerant polymeric photosensitizer prodrug (HTPS_Niclo) for cancer photo-immunotherapy. HTPS_Niclo not only performs Type-I PDT process to partially overcome the limitation of hypoxic tumors in PDT by recycling oxygen but also specifically releases a Signal Transducer and Activator of Transcription-3 (STAT3) inhibitor (Niclosamide) in response to a cancer biomarker in the TME. Consequently, HTPS_Niclo inhibits the phosphorylation of STAT3, and suppresses the expression of hypoxia-inducible factor-1α. The synergistic effect results in the enhanced activation of immune cells (including mature dendritic cells, cytotoxic T cells) and production of immunostimulatory cytokines compared to Type-I PDT alone. Thus, HTPS_Niclo-mediated photodynamic immunotherapy enhances tumor inhibition rate from 75.53% to 91.23%, prolongs the 100% survival from 39 days to 60 days as compared to Type-I PDT alone. This study not only provides the generic approach towards design of polymer-based Type-I photosensitizers but also uncovers effective strategies to counteract the immunosuppressive TME for enhanced photo-immunotherapy in 4T1 tumor bearing female BALB/c mice.

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Introduction

Combination cancer immunotherapy has emerged as a prevalent strategy for cancer treatment, primarily because single treatment method is usually insufficient to address the challenges posed by tumor heterogeneity and the complex immunosuppressive tumor microenvironment (TME)^1,2. In comparison to surgery, phototherapy³ in particularly, photodynamic therapy (PDT)^4,5 offers a minimal invasive way to ablate tumor⁶ and triggers immunogenic cell death (ICD)⁷ to initiate tumor-specific immune response^8,9,10. Thus, it has evolved as a prominent clinical therapeutic approach in multiple tumor types¹¹. To amplify the antitumor response, photodynamic-immunotherapeutic nanomedicines¹² composed of photosensitizers and immune checkpoint blockade (ICB) molecules¹³, tumor vaccine¹⁴, or cytokines¹⁵ have been developed. These nanomedicines can achieve controllable release of immunotherapeutic agents in the TME¹⁶, offering precise immune modulation¹⁷ with the potential to minimize immune-related adverse events (irAEs)¹⁸.

Currently, photodynamic-immunotherapeutic nano-agents¹⁹ face two main challenges^20,21: tumor hypoxia and the immunosuppressive TME following phototherapy^22,23. Because most photosensitizers are involved in Type-II photodynamic process, which rely on the molecular oxygen (O₂) to generate singlet oxygen (¹O₂)²⁴_; their antitumor efficacy is hindered by hypoxic TME²⁵. Furthermore, O₂ consumption in Type-II PDT often worsens tumor hypoxia and upregulates the hypoxia-inducible factor-1α (HIF-1α), leading to the accumulation of immunosuppressive factors²⁶. For example, elevated regulatory T cells (Treg) activity in immunosuppressive TME hinders immune function and promotes immune tolerance post-PDT²⁷. In contrast, Type-I photosensitizers²⁸ exhibit diminished oxygen-dependent generation of reactive oxygen species, mainly, superoxide anion (O₂^•–) and hydroxyl radical (·OH) via transfer electrons to O₂²⁹. In the process, O₂ could be recycled after O₂^•– transfers electrons to other molecules such as H₂O, lipid, DNA, and protein³⁰. As such, Type-I photo-immunotherapeutic nanoagents hold the potential to mitigate these two shortcomings, which unfortunately most photosensitizers used in this modality are inorganic materials³¹, and such nanoagents need to be better paired to achieve a synergetic effect.

Semiconducting polymer nanoparticles (SPNs)^32,33,34 have been widely employed as efficient photosensitizers and drug carriers^35,36 for cancer photo-immunotherapy due to their structural versatility and controllable photophysical properties³⁷. To reverse the immunosuppressive TME, SPN-based photo-immunotherapeutic nano-agents have been developed by incorporating SPNs with ICB (PD-1/PD-L1, CTLA-4 checkpoint pathway) therapy^38,39, immune-metabolic agents (adenosine deaminase, ADA)⁴⁰ or immune activation agents (Toll-like receptor and STING agonist)⁴¹. Furthermore, to minimize irAEs induced by off-target immunotherapy, their immunomodulation components are often devised to be activated by either external (light or ultrasound)^42,43 or internal stimuli (cancer-overexpressed enzymes)⁴⁴. These activatable SPNs-based photo-immunotherapeutic nano-agents enhance tumor-specific immune responses⁴⁵, leading to precision therapy with reduced off-target effects⁴⁰. However, there are limited SPNs that can exert Type-I PDT process^46,47, and they have been rarely exploited for Type-I photo-immunotherapy.

Herein, we report the development of a hypoxia-tolerant semiconducting polymeric photosensitizer Niclosamide prodrug (HTPS_Niclo) for cancer photo-immunotherapy (Fig. 1). Through screening a library of semiconducting polymers (SPs), we identify an optimal Type-I SPs with high efficiency in generating O₂^•– and •OH (higher cytotoxicity compared with ¹O₂) as the Type-I PDT component in HTPS_Niclo. Furthermore, to alleviate the immunosuppressive TME following PDT, a Signal Transducer and Activator of Transcription 3 (STAT3) inhibitor (Niclosamide) is conjugated to HTPS via Cathepsin B (CatB)-cleavable linker to afford HTPS_Niclo. HTPS_Niclo can efficiently produce O₂^•– in an oxygen-recyclable manner with reduced O₂ consumption, which is beneficial for relieving hypoxic TME. Moreover, HTPS_Niclo can specifically release Niclosamide in response to the overexpressed CatB in the TME to inhibit the phosphorylation of STAT3, which not only increase the secretion of immunostimulatory cytokines such as IL-12 and IFN-γ but also decrease the expression of HIF-1α⁴⁸. Due to the synergistic effect of Type-I PDT and immune modulation, HTPS_Niclo transforms the immunosuppressive TME into an active phenotype, resulting in tumor suppression, lung metastasis prevention and prolonged survival rate in 4T1 tumor-bearing mice.

**Fig. 1: Schematic illustration of hypoxia-tolerant polymeric photosensitizer prodrug for photo-immunotherapy.**

Results

Screening polymeric photosensitizers for Type-I PDT

A library of potential Type-I polymeric photosensitizers was constructed by copolymerization of a non-fullerene oligomer acceptor 2,2′-((2Z,2′Z)-((12,13-bis(2-octyldodecyl)-3,9-diundecyl-12,13-dihydro-[1,2,5]thiadiazolo[3,4-e]thieno[2″,3″:4’,5’]thieno[2′,3′:4,5]pyrrolo-[3,2-g]thieno[2′,3′:4,5]thieno[3,2-b]indole-2,10-diyl)bis(methanylylidene))bis(6-bromo-3-oxo-2,3-dihydro-1H-indene-2,1-diylidene))dimalononitrile (BTP-2Br-OD) with four types of donors including 4,7-Bis(5-(trimethylstannyl)thiophen-2-yl)benzo[c][1,2,5]thiadiazole (BDT), 2,5‐ bis(triMethylstannyl)thieno[3,2‐b]thiophene (TT), 4,8-bis((2-ethylhexyl)oxy)benzo[1,2b:4,5-b′]dithiophene-2,6-diyl)bis(trimethylstannane) (OT), 2,5-Bis(2-ethylhexyl)-3,6-bis(5-(trimethylstannyl)thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione (DPP) via Stille coupling, denoted as P1-P4 (Fig. 2a). To screen the most efficient photosensitizer for generating Type-I PDT ROS, P1-P4 were assembled with the assistance of amphiphilic polymer poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) (PEO–PPO–PEO, F127)⁴⁹ into aqueous solution to form nanoparticles (denoted as PN1-PN4), showing absorption maxima at 766, 775, 770, and 775 nm, respectively (Fig. 2b). Under same concentration, PN3 exhibited the highest fluorescence emission at 819 nm (Fig. 2c) followed by PN2 and PN1; while PN4 had negligible fluorescence peak above 800 nm. The photodynamic properties of PN1-PN4 were studied. The generation of ¹O₂, O₂^•- and •OH for PN1-PN4 under NIR irradiation were detected by singlet oxygen sensor green (SOSG), dihydrorhodamine 123 (DHR123) and hydroxyphenyl fluorescein (HPF), respectively. The fluorescence intensity of SOSG gradually increased for PN1-PN4 under continuous NIR irradiation, proving the generation of ¹O₂. Among them, PN2 produced the highest amount of ¹O₂, increasing the fluorescence signal of SOSG by 21.41-fold, followed by PN1 with a 12.46-fold increase (Fig. 2d). Different from the trend in the Type-II PDT ROS generation, PN1 exhibited the highest Type-I PDT ROS generation capability among all nanoparticles (Fig. 2e, f), showing 22.83-fold and 12.98-fold fluorescence increases for DHR123 (for O₂^•- determination) and HPF (for •OH determination), respectively. This is significantly higher than PN2-PN3 with the DHR123 fluorescence enhancements ranging from 8.13 to 15.90-fold and the HPF enhancements ranging from 4.71 to 12.34-fold.

**Fig. 2: Synthesis and characterization of 4 kinds of hypoxia-tolerant polymeric photosensitizer (P1-P4).**

The mechanism of Type-I ROS generation was investigated by determining the HOMO and LUMO energy levels of P1-P4 using cyclic voltammetry (CV) (Fig. 2g, Supplementary Fig. 1). The LUMO energy levels of P1, P2, P3, and P4 were −4.29 eV, −4.30 eV, −4.23 eV and −4.21 eV, respectively. Given that the LUMO levels of P1 and P2 are closer to the redox potentials of O₂/O₂^•− (−4.30 eV)⁵⁰ compared to those of P3 and P4, electrons can more easily transfer from excited PN1 and PN2 to O₂, resulting in more efficient generation of O₂^•−. However, the HOMO energy levels of P1-P4 (−5.03 eV, −5.02 eV, −5.37 eV, and −5.25 eV) were higher than E_H of H₂O/•OH (−6.70 eV), which hindered •OH generation by direct oxidation of H₂O. Thus, •OH generated from PN1 was likely derived from the transfer of electrons in O₂^•– to H₂O³¹. The mechanism for the efficient Type-I ROS generation by PN was proposed in Fig. 2h. Under NIR irradiation, the electrons of PN are excited from ground states (S₀) to higher energy excited states (S_n) to obtain singlet excitons, which can be efficiently populated from the triplet states (T_n) to the triplet excitons at the lowest level (T₁) state via intersystem crossing. Subsequently, electrons can be transferred to O₂ to generate O₂^•-, and then further transferred to H₂O to produce •OH. Due to PN1 had the highest O₂^•- and •OH generation ability among all nanoparticles, P1 was selected as the optimal Type-I photosensitizer backbone for construction of hypoxia-tolerant polymeric photosensitizer (HTPS) in the following studies.

Synthesis and characterization of polymeric photosensitizer prodrug

Based on the best-performing polymer P1, the BDT-BTP-BDT-DPP-Br with Type-I PDT ability was first synthesized via Stille polycondensation of 3 monomers, including BDT, BTP-2Br-OD, and DPP (Fig. 3a). BDT-BTP-BDT-DPP-Br was then reacted with sodium azide to afford BDT-BTP-BDT-DPP-N₃ for post-functionalization. To obtain CatB-responsive Niclosamide prodrug, the alkynyl modified CatB cleavable peptide (Alk-Val-Cit) was synthesized and conjugated with Niclosamide through a self-immolative linker to get Alk-Val-Cit-PAB-Niclo. Then, Alk-Val-Cit-PAB-Niclo was further modified into NH₂-Val-Cit-PAB-Niclo and reacted with the carboxyl group on polyethylene glycol (PEG, Mw=2000), affording Niclosamide prodrug-modified alkyne-PEG (Alk-PEG_2k-Niclo), with 85.7% grafting efficiency base on NMR analysis (Supplementary Figs. 2–8). Thereafter, BTP-BDT-DPP-N₃ was reacted with Alk-PEG_2k-Niclo through click reaction and then self-assembled into HTPS_Niclo in aqueous solution. The control nanoparticle HTPS without Niclosamide prodrug was synthesized through the click reaction between BTP-BDT-DPP-N₃ and Alk-m PEG_2k (Supplementary Fig. 9).

**Fig. 3: Synthesis and characterization of Niclosamide conjugated hypoxia-tolerant Type-I polymeric photosensitizer (HTPS_Niclo).**

As shown in Transmission Electron Microscopy (TEM) images, both HTPS and HTPS_Niclo exhibited spherical morphology with uniform size distribution. Dynamic Light Scattering measurements revealed that hydrodynamic diameters of HTPS and HTPS_Niclo ranged from 19 nm to 26 nm with good dispersity in PBS (Fig. 3b). The stabilities of HTPS and HTPS_Niclo were evaluated by measuring their hydrodynamic size in presence of PBS and FBS. Both of them exhibited minimal hydrodynamic size changes up to 72 h, indicating their good biostability (Supplementary Fig. 10). A slightly red-shifted fluorescence emission for HTPS_Niclo (838 nm) was observed compared to HTPS (833 nm) (Fig. 3c). In addition, hematological analysis was performed by incubation both HTPS and HTPS_Niclo with red blood cells (RBCs) for 24 h, and no hemolysis were detected, indicating their good biosafety. (Supplementary Fig. 11). The cellular uptake of HTPS and HTPS_Niclo was evaluated in 4T1 cells using confocal laser scanning microscopy. The obvious fluorescent signals of HTPS and HTPS_Niclo were detected, which were similarly 11-fold higher than PBS group. Moreover, the signals of nanoparticles colocalized well with lysosomal tracker, suggesting that both HTPS and HTPS_Niclo underwent endocytosis through the lysosomal pathway (Fig. 3d and Supplementary Fig. 12). Then, photodynamic ROS generation properties of HTPS and HTPS_Niclo were evaluated. After NIR irradiation (808 nm, 0.2 W/cm²) for 3 min, the fluorescence intensity of DHR123 in HTPS and HTPS_Niclo solutions increased by ~5.65-fold and 5.05-fold, respectively. Similarly, the HPF fluorescence intensity in HTPS and HTPS_Niclo solutions increased by about 6.24-fold and 5.98-fold, respectively. In contrast, the SOSG fluorescence intensity increased slightly, which was 1.75-fold and 1.65-fold for HTPS and HTPS_Niclo, respectively (Fig. 3e–g). These data demonstrated that HTPS and HTPS_Niclo exhibited effective Type-I rather than Type-II photodynamic properties.

Afterward, HTPS and HTPS_Niclo photodynamic cytotoxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay under normoxic and hypoxic conditions. Both nanoparticles only exhibited strong cytotoxicities after NIR irradiation (808 nm, 0.2 W/cm²) for 5 min. Under normoxic conditions, HTPS and HTPS_Niclo showed significant photodynamic cytotoxicity to 4T1 cells in a concentration-dependent manner, with cell viabilities of 24.78% and 19.20%, respectively, at a concentration of 20 μg/mL. More importantly, both HTPS and HTPS_Niclo induced obvious photodynamic cytotoxicity under hypoxic conditions, demonstrating their excellent hypoxia tolerance in PDT (Fig. 3h, i). The cytotoxic effect of Niclosamide on 4T1 cells was evaluated. Niclosamide exhibited slight cytotoxicity towards 4T1 cells at the concentrations up to 2 μM (Supplementary Fig. 14).

To investigate the mechanism for the cellular cytotoxicity, the intracellular photodynamic ROS and O₂^•- generation were evaluated by turn on probes 2′,7′-dichlorodihydrofluorescein (H₂DCFDA) and DHR123 under normoxic and hypoxic conditions (Fig. 3j–m). Under normoxic conditions, the mean fluorescence intensities (MFI) of DCF in HTPS and HTPS_Niclo + NIR group were 4.07-fold and 3.80-fold higher than the PBS group, respectively. In addition, MFI of DHR123 in HTPS and HTPS_Niclo + NIR group were 6.58-fold and 6.29-fold stronger than the PBS group, respectively. Under hypoxic conditions, the photodynamic total ROS and O₂^•- generation abilities of HTPS and HTPS_Niclo were comparable to their performance under normoxic conditions. Similar results could be got from the flow cytometry analysis (Supplementary Fig. 13), where significant DCF and DHR123 emission was detected, indicating that both HTPS and HTPS_Niclo-treated 4T1 cells could generate photodynamic total ROS and O₂^•- after NIR irradiation (808 nm, 0.2 W/cm²) for 5 min. The hypoxia tolerance of HTPS and HTPS_Niclo mediated Type-I PDT can be explained by the recycling of O₂, leading to less O₂ consumption than Type-II PDT.

In vitro photo-immunotherapy

To confirm the CatB-specific drug release behavior of HTPS_Niclo, high performance liquid chromatography (HPLC) analysis was conducted to monitor the Niclosamide release after incubating HTPS_Niclo with CatB. A new elution peak at 11.91 min, attributed to free Niclosamide, gradually became higher with increased incubation time (Fig. 4a). It was calculated that 96.47% Niclosamide was released from HTPS_Niclo after incubation with CatB for 5 h (Fig. 4b). In contrast, no Niclosamide peak was detected in the absence of CatB (Supplementary Fig. 15). To determine whether CatB was overexpressed in the 4T1 cell line, immunofluorescence (IF) staining and western immunoblot analysis of expression levels of CatB was performed. The results clearly showed that the expression of CatB in 4T1 cancer cells was significantly higher compared to normal 3T3 cells (Fig. 4c and Supplementary Fig. 16). Then, the cellular STAT3 inhibition ability of HTPS_Niclo was studied. After incubating HTPS_Niclo with 4T1 cells for 24 h, the phosphorylation of STAT3 (p-STAT3) was 11.67-fold lower compared to PBS group, as shown in Fig. 4d. This can be attributed to the successful release of free Niclosamide in CatB-overexpressed 4T1 cells. To further confirm this, CA-074, a CatB inhibitor, was used to inhibit its cleavage activity⁵¹. As quantified in the western blotting data, the p-STAT3 expression in HTPS_Niclo + CA-074 group exhibited 76.0-fold higher compared to the group treated with HTPS_Niclo alone (Fig. 4d and Supplementary Fig. 17).

**Fig. 4: HTPS_Niclo-mediated photo-immunotherapy.**

To study the ICD of 4T1 cells after HTPS_Niclo-mediated Type-I PDT, the calreticulin (CRT) and high-mobility group box 1 (HMGB1) were then evaluated (Fig. 4e, f and Supplementary Figs. 18, 19). In contrast to the PBS group, cells treated by HTPS and HTPS_Niclo-mediated PDT showed a 6.12-fold and 6.52-fold higher MFI of CRT signals on the plasma membrane, respectively. A more quantitative evaluation of CRT exposure by flow cytometry confirmed a 4.92-fold and 4.32-fold higher proportion of 4T1 cells undergoing ICD after HTPS and HTPS_Niclo-mediated PDT treatment, respectively, compared to PBS treatment. In addition, the nuclear HMGB-1 signals after HTPS and HTPS_Niclo-mediated PDT were 7.65-fold and 5.16-fold lower compared to PBS group, respectively. These results indicated that potent ICD was induced by HTPS_Niclo-mediated Type-I PDT. To study the immune activation effect of ICD and immune modulation ability of HTPS_Niclo, bone marrow derived dendritic cells (BMDCs) and macrophages were incubated with 4T1 cells’ supernatants that underwent different treatments. As shown in Fig. 4g, TNF-α and IL-12 secretion in BMDCs treated with 4T1 tumor supernatants underwent HTPS_Niclo-mediated PDT (HTPS_Niclo + NIR) was 1.36-fold and 1.69-fold higher than that in HTPS + NIR group, respectively. This could be attributed to p-STAT3 inhibition by the released Niclosamide from HTPS_Niclo. The flow cytometry results showed that the matured DCs in HTPS + NIR group and HTPS_Niclo + NIR group was 3.39-fold and 2.61-fold higher than the untreated group, respectively (Fig. 4h and Supplementary Fig. 20a, b). Moreover, HTPS_Niclo + NIR treatment group induced the lowest percentage of M2 macrophages, which was 8.22-fold lower than HTPS + NIR group (Fig. 4i and Supplementary Fig. 20c). The immune activation of Niclosamide towards DC maturation and downregulation of M2 macrophage was also investigated as shown in Supplementary Fig. 21. After 24 h of Niclosamide treatment, the proportion of mature DCs increased, while M2 macrophages decreased in a concentration-dependent manner. In summary, the synergistic effects of ICD and p-STAT3 inhibition effectively induced DC maturation and downregulated M2 macrophage percentage, with the potential to reverse the immunosuppressive TME and enhance the tumor suppression.

In vivo photodynamic immunotherapy

The pharmacokinetics of HTPS and HTPS_Niclo were investigated in Supplementary Fig. 22, indicating that HTPS could be metabolized normally in vivo, demonstrating their good biosafety. The potential off-target effects and best timepoint for in vivo photo-irradiation study of HTPS and HTPS_Niclo were first evaluated by evaluating the accumulation of nanoparticles in tumor tissues using NIR fluorescence (NIRF) imaging in 4T1 tumor-bearing mice. The NIRF intensity in tumors of HTPS_Niclo- and HTPS-injected mice gradually increased, reaching similar maximum levels at 12 h post-injection, showing 15.33-fold and 15.42-fold increase compared to the background, respectively (Fig. 5a, b). Ex vivo fluorescence imaging at 48 h post-injection demonstrated that the highest nanoparticle accumulation was in tumors, followed by spleen and liver (Supplementary Fig. 23). To verify the photodynamic activity of HTPS_Niclo and HTPS in vivo, DHR123 was used to measure the generation of O₂^•- in tumors. The fluorescence signals of DHR123 in tumors treated with HTPS_Niclo + NIR and HTPS + NIR group were 5.03-fold and 5.05-fold higher compared to saline group, respectively (Fig. 5c and Supplementary Fig. 24).

**Fig. 5: In vivo fluorescence imaging and tumor inhibition of HTPS_Niclo-mediated photo-immunotherapy.**

The therapeutic efficacy of HTPS_Niclo-mediated Type-I photo-immunotherapy was then evaluated using a bilateral animal model (Fig. 5d). NIR irradiation (808 nm, 0.2 W/cm², 5 min) was locally conducted on primary tumor 12 h post HTPS_Niclo injection. The growth of both primary and distant tumors in different treatment groups, as well as the survival of mice, were monitored. In the HTPS + NIR group, the growth of primary tumors was inhibited until Day 10, but the tumor growth accelerated afterwards. In contrast, the tumors in the HTPS_Niclo + NIR group remained well-inhibited throughout the experiment. The inhibition rate of primary tumor growth in the HTPS_Niclo + NIR and HTPS + NIR group was 91.23% and 75.53%, respectively (Fig. 5e). A more pronounced difference was observed in the distant tumor growth curve. The HTPS_Niclo + NIR group shown an inhibition rate of 88.22%, much higher than the 37.99% observed in the HTPS + NIR group (Fig. 5f). To further confirm the inhibition of tumor metastasis by HTPS_Niclo-mediated photo-immunotherapy, the hematoxylin and eosin (H&E) staining of lung tissues was conducted. No lung metastasis lesions were observed in lung tissue from the mice in HTPS_Niclo + NIR group. In contrast, the lung metastasis area was 64.52%, 13.41% and 14.17% in the saline group, HTPS _Niclo group, and HTPS + NIR group, respectively (Fig. 5g, h). In the HTPS + NIR group, 100% survival was observed up to Day 39. By Day 60, only 60% of the mice remained alive (Fig. 5i). In contrast, all mice in the HTPS_Niclo + NIR group survived through Day 60. The data demonstrated that HTPS_Niclo-based photo-immunotherapy not only more effectively inhibited the growth of both primary and distant tumors but also significantly extended mice survival, compared to HTPS-based single PDT. The therapeutic effects were further confirmed by IF staining of caspase-3 and H&E staining analysis. Significant tumor cell apoptosis was observed in both primary and distant tumors in the HTPS_Niclo + NIR group. In contrast, no apoptosis was observed in the distant tumors in the HTPS + NIR group (Supplementary Fig. 25).

To further investigate the long-term anti-tumor recurrence and anti-metastasis ability of HTPS_Niclo-mediated photo-immunotherapy, a 4T1 tumor rechallenge model was established (Fig. 5j). The growth of rechallenged tumors in HTPS_Niclo + NIR group was greatly suppressed compared to that of the saline group (Fig. 5k and Supplementary Fig. 26). At the end of rechallenged tumor monitoring, the lungs of mice were stained with India ink and the number of lung metastasis nodules was counted. Negligible tumor metastasis was found in the lungs of mice treated with HTPS_Niclo + NIR on Day 57, while more than 40 white nodules were found in saline group (Fig. 5l, m). These results further demonstrated that HTPS_Niclo-mediated photo-immunotherapy can effectively prevent tumor recurrence and lung metastasis. During the treatment process, the body weight of HTPS_Niclo- and HTPS-injected mice did not change significantly compared to saline group. The immune response over time after HTPS_Niclo injection was evaluated by the detection of IL-12, TNF-α, IL-10, IFN-γ in the serum. These cytokines were upregulated (IL-12, TNF-α, IFN-γ) or downregulated (IL-10) on Day 3 compared to Day 0 and returned to normal levels by Day 7. In addition, H&E staining of the major organs (heart, liver, spleen, kidney) in HTPS_Niclo- and HTPS-injected mice showed similar physiological morphologies compared to those in saline group, indicating the good biocompatibilities of HTPS_Niclo and HTPS. Pharmacokinetics, biodistribution, H&E staining of the major organs and immune response over time have proven the long-term Safety of HTPS_Niclo (Supplementary Figs. 26–29).

Mechanistic study of photodynamic immunotherapy

To gain insight into the mechanism of HTPS_Niclo-mediated photo-immunotherapy, immunological characterizations including tumor ICD, immune cell populations, immunostimulatory cytokines levels, angiogenesis level and p-STAT3 inhibition in nanoparticle-injected tumor-bearing mice were analyzed. First, IF staining of HMGB1 and CRT was used to visualized ICD in primary tumors. The MFI of HMGB1 in the tumor tissues of HTPS + NIR and HTPS_Niclo + NIR group was 4.90- and 5.19-fold higher compared to the saline group, respectively (Fig. 6a and Supplementary Fig. 30). Similarly, MFI of CRT in HTPS + NIR and HTPS_Niclo + NIR group was 3.07- and 3.11-fold higher than that in saline group, respectively (Fig. 6b and Supplementary Fig. 31). Then, flow cytometry was used to analyze changes in immune cell populations in tumor-draining lymph nodes (TDLNs) and tumors after HTPS_Niclo-mediated photo-immunotherapy (Supplementary Fig. 32). The mature DC ratio in TDLNs of HTPS_Niclo + NIR group was 4.29-fold higher than that in the saline group and 1.51-fold higher than that in the HTPS + NIR group, respectively. (Fig. 6c and Supplementary Fig. 33). Moreover, the immunosuppressive M2 macrophages in tumors of HTPS_Niclo + NIR group was 14.4-fold lower compared to saline-treated mice and 14.1-fold lower compared to HTPS + NIR group, respectively (Fig. 6d and Supplementary Fig. 33). Considering the essential role of tumor-infiltrating T lymphocytes in combating tumors, the CD8⁺ T cells, CD4⁺ T cells and regulatory T cells in primary and distant tumors were further evaluated. The HTPS_Niclo-mediated photo-immunotherapy induced the highest CD8⁺/Treg and CD4⁺/Treg ratio in both primary and distant tumors (Fig. 6e, f and Supplementary Figs. 34, 35). In detail, the CD8⁺/Treg ratio in the primary and distant tumor after HTPS + NIR treatment was 2.32-fold and 2.17-fold higher relative to that in saline group, respectively. In contrast, HTPS-mediated PDT could not upregulate the ratio of CD8⁺/Treg and CD4⁺/Treg in distant tumor. These results demonstrated that HTPS_Niclo-mediated photo-immunotherapy promoted DC maturation, downregulated M2 macrophage percentage, and induced a robust T cell response in both primary and distant tumors.

**Fig. 6: Immune activation studies after HTPS_Niclo-mediated photo-immunotherapy.**

Next, immunostimulatory cytokines including IL-12, IFN-γ, TNF-α and immunosuppressive cytokine IL-10 in the serum of mice were analyzed by enzyme-linked immunosorbent assay (ELISA) after various treatments. The serum concentration of TNF-α, IL-12, and IFN-γ in HTPS_Niclo + NIR treated mice were 1.40-, 3.42- and 1.40-fold higher than that in saline treated mice, respectively. In addition, the IL-10 level in HTPS_Niclo + NIR group was 3.45-fold lower compared with saline treated group (Fig. 6g). In general, the oxygen consumption and damaged vessels after PDT are two main factors for hypoxia condition, leading to the expression of HIF-1α and further angiogenesis in TME. Therefore, the HIF-1α and angiogenesis level in immunosuppressive TME after HTPS_Niclo-mediated photo-immunotherapy were further evaluated. The expression of HIF-1α in tumor tissues after HTPS + NIR treatment was 1.26-fold higher compared to the saline group, indicating that single-mode HTPS-mediated PDT could not completely overcome the hypoxic TME (Fig. 6h and Supplementary Fig. 36). In contrast, HTPS_Niclo-mediated photo-immunotherapy resulted in 3.28-fold lower HIF-1α expression compared to the saline group. Considering that HIF-1α is involved in tumor angiogenesis, CD31, the indicator of newly generated vessels, was stained in the tumors after different treatment. As expected, the new vascular area in HTPS + NIR and HTPS_Niclo + NIR treated mice were 1.84-fold larger and 4.14-fold smaller compared to saline group, respectively (Fig. 6h and Supplementary Fig. 37), indicating the higher angiogenesis level in single-mode HTPS-mediated PDT treated mice compared to that of HTPS_Niclo-mediated photo-immunotherapy. To further evaluate HTPS_Niclo could inhibit HIF-1α and angiogenesis level through STAT3 pathway, western blotting analysis on the p-STAT3 in tumor lysis was conducted. In both the NIR irradiation and non-irradiation group, HTPS_Niclo treatment significantly downregulated the p-STAT3 level, which can be attributed to the release of STAT3 inhibitor Niclosamide (Fig. 6i and Supplementary Fig. 38). The immune memory effect induced by HTPS_Niclo-mediated photo-immunotherapy was further evaluated. As shown in Fig. 6j, k, the proportion of effector memory T cells (CD8⁺CD44⁺CD62L^-) in HTPS_Niclo + NIR group was 1.49-fold higher than that in the saline group.

Collectively, the synergistic effects of Type-I PDT and p-STAT3 inhibition in HTPS_Niclo-mediated photo-immunotherapy was summarized in Fig. 6l. In a traditional Type-II PDT, O₂ was largely consumed to generate ¹O₂. This consumption leads to hypoxia environment and HIF-1α upregulation, resulting in tumor angiogenesis. Although ICD of tumor cells induces a tumor-specific immune response, the immune tolerance after PDT leads to an immunosuppressive TME. This dilemma was effectively solved by HTPS_Niclo-mediated photo-immunotherapy. This approach first generates Type-I ROS in an O₂ recyclable manner, enhancing hypoxia-tolerance. Additionally, it releases Niclosamide through CatB cleavage, which inhibits pSTAT-3, leading to a reduction in HIF-1α, immunosuppressive cells and cytokines. Simultaneously, it promotes the secretion of immunostimulatory cytokines, enhances DCs maturation and T cell activation. These effects collectively reverse the immunosuppressive TME and improve tumor treatment efficacy.

Discussion

By tuning the structure units, we synthesized a series of photodynamic semiconducting polymers (SPs) and evaluated their efficiency in exerting the Type-I PDT process to generate O₂^•– and •OH, which were more toxic than ¹O₂. The mechanism for the difference in generating O₂^•– and •OH by SPs can be explained by how their HOMO/LUMO energy levels align with E_H of O₂/ O₂^•– (−4.3 eV) and E_H of H₂O/•OH (−6.7 eV). Although the LUMO energy levels of the four SPs all exceeded −4.3 eV, the LUMO of P1 was the closest to −4.3 eV among all SPs (Fig. 2g). It is thus the easiest for electrons to be transferred from excited P1 to O₂, resulting in the most efficient production of O₂^•– from PN1. Since the HOMO energy levels of SPs were higher than E_H of H₂O/•OH (−6.7 eV), it was theoretically impossible for them to directly oxidize H₂O into •OH. Thus, •OH was most likely to be derived from O₂^•– through its electron transfer to H₂O. Given its highest efficiency in generating O₂^•– and •OH, P1 was further constructed into the hypoxia-tolerant polymeric photosensitizer (HTPS) for tumor-specific cancer photo-immunotherapy.

By virtue of the advantages of hypoxia tolerant capability of Type-I PDT, HTPS-based single therapy had already enhanced tumor inhibition efficiency (from 50% to 75.53%) and extended the 100% survival rate (from 25 days to 39 days) in 4T1 tumor-bearing mice compared to reported Type-II PDT⁵². Yet, it failed to effectively inhibit the growth of distant tumors (Fig. 5f). Furthermore, lung metastasis was observed due to the HIF-1α upregulation after HTPS-based single-mode PDT (Fig. 5g). In contrast, HTPS_Niclo-mediated photo-immunotherapy released Niclosamide in the TME, inhibiting the phosphorylation of STAT3 and downregulating HIF-α expression by 7.63-fold compared to HTPS-based PDT. In addition, HTPS_Niclo-based therapy could polarize the immunosuppressive TME into an active phenotype, resulting in higher DC maturation, CD8⁺/Treg and CD4⁺/Treg ratios in both primary and distant tumor compared to HTPS-based therapy (Fig. 6c–f). Furthermore, the levels of immunostimulatory cytokines TNF-α, IL-12, and IFN-γ after HTPS_Niclo-mediated photo-immunotherapy were 1.40-, 3.42- and 1.40-fold higher compared to HTPS-based PDT, respectively (Fig. 6g). As a result of synergetic effect, HTPS_Niclo-mediated photo-immunotherapy significantly improved tumor inhibition rate (91.23%) and reduced lung metastasis area (1.56%) compared to HTPS-based PDT, which showed 75.53% tumor inhibition rate and 14.17% lung metastasis area. Finally, HTPS_Niclo-mediated photo-immunotherapy significantly prolonged the 100% survival rate into 60 days, compared to 39 days in HTPS-based single-mode PDT.

In summary, we developed a TME-activatable hypoxia-tolerant polymeric photosensitizer prodrug HTPS_Niclo that overcame the limitations of PDT. We revealed the importance of aligning HOMO/LUMO energy levels of polymeric photosensitizer with the redox potentials of (O₂/ O₂^•–) to optimize the generation of Type-I photodynamic ROS generation and identified various Type-I polymeric photosensitizers. Coupled with the immune modulator Niclosamide to inhibit the phosphoralation of STAT3, HTPS_Niclo-based photo-immunotherapy upregulated the level of immunostimulatory cytokines and downregulated HIF-1α, further mitigating the immunosuppressive TME following PDT. This study not only develops a set of polymer-based Type-I photosensitizers but also uncovers strategies to counteract the immunosuppressive TME for enhanced immunotherapeutic efficacy. Despite its effectiveness, PDT faces limitations such as its dependency on oxygen and challenges posed by the immunosuppressive TME, which can reduce therapeutic efficacy. However, advancements in the development of Type-I photosensitizers to address oxygen dependency, along with the integration of PDT with other treatments, particularly immunotherapies, present promising avenues for overcoming these challenges and improving cancer treatment outcomes.

Methods

Materials

All chemicals and reagents were purchased from Sigma-Aldrich unless otherwise stated. Y6-OD was purchased from derthon optoelectronics materials science technology co ltd. 4,7-bis(5-trimethylstannyl-2-thienyl)-2,1,3-benzothiadiazole, 2,5-bis(triMethylstannyl)th ieno[3,2‐b]thiophene, 1,1′-[4,8-Bis[(2-ethylhexyl)oxy]benzo[1,2-b:4,5-b′]dithiophene-2,6-diyl]bis[1,1,1-trimethylstannane] and 2,5-Bis(2-ethylhexyl)-3,6-bis(5-(trimethylstannyl)thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione, 2,5-Bis(6-bromohexyl)-3,6-bis(5-bromothiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H, 5H)-dione were purchased from BLD Pharmatech Ltd. PEG (Mw=2000) was purchased from Biopharma PEG Scientific Inc. Agarose powder (Certified Molecular Biology Agarose) was purchased from Bio-Rad (Hercules, CA, USA). SOSG was purchased from Molecular Probes Inc. (Carlsbad, CA, USA). 3-(4,5-Dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) solution was purchased from Promega Corp. (Madison, WI, USA). Dulbecco’s modified eagle medium (DMEM), Trypsin–EDTA (0.05%), penicillin–streptomycin (10,000 U/mL), fetal bovine serum (FBS), ACK lysis, type I collagenase, and type IV collagenase were purchased from Gibco. Anti-HMGB1 and anti-caspase 3 were purchased from Abcam Inc. (Cambridge, CA, USA). ELISA kits for TNF-α, IFN-γ, IL-10, and IL-12 detection, anti-STAT3, anti-p-STAT3, anti-CD16/32, anti-CD8a, anti-CD3, anti-CD4, anti-CD80, anti-CD86, and anti-CD11c antibodies were purchased from BioLegend (USA). BCA protein assay kit was purchased from Thermo Fisher Scientific. All antibodies were purchased from Biolegend (USA) unless otherwise indicated. Antibody against CD16/32 (93, 1:50) was used to block the non-specific binding. Antibodies against CD11b (M1/70, 1:200), F4/80 (BM8, 1:20) and CD206 (C068C2, 1:100) were used for M2 macrophage staining. Antibodies against CD3 (17A2, 1:100) and CD8 (53-6.7, 1:100) were used for cytotoxic T cell staining. Antibodies against CD4 (RM4-5, 1:100) and FOXP3 (MF-14, 1:200) were used for Treg cell staining. Antibodies against CD62L (MEL-14, 1:100) and CD44 (IM7, 1:20) were used for memory T cell staining. Antibodies against CD11c (N418, 1:100), CD80 (16-10A1, 1:50), and CD86 (A17199A, 1:50) were used for mature dendritic cell staining. Tumour slices were stained with antibodies against high mobility group box 1 (HMGB-1) (3E3, Alexa Fluor 488-labeled, 1:200, Biolegend), Caspase-3 (#9664, rabbit anti-mouse, 1:400, Cell Signalling Technology), CD31 (MEC13.3, rat anti-mouse, 1:200, Biolegend), HIF-1A (mgc3, 1:200, Thermo Fisher Scientific) and rabbit anti-mouse calreticulin (PA3-900, 1:200, Thermo Fisher Scientific). Alexa Fluor 488-conjugated secondary antibody (goat anti-rabbit, 1:500, Thermo Fisher Scientific) was used in caspase-3 and calreticulin staining.

Instrumentation

Proton nuclear magnetic resonance (¹H NMR) spectra were recorded on a Bruker AVIII 400 MHz NMR system (Bruker Physik AG, Germany). Electrospray ionization-mass spectrometry (ESI-MS) spectra were recorded by Thermo Finnigan Polaris Q quadrupole ion trap mass spectrometer. UV-Vis spectra were recorded on a Shimadzu UV-2450 spectrophotometer. Fluorescence measurements were performed on a Fluorolog 3TCSPC spectrofluorometer (Horiba Jobin Yvon). Diameter size and zeta potential were measured by a Malvern Nano-ZS Particle Sizer. The absorbance in 96-well plates was measured using a SpectraMax M5 microplate reader (Molecular Devices). TEM images were captured from a JEM 1400 transmission microscope with accelerating voltage ranging from 40 to 120 kV. Fluorescence imaging was performed on an IVIS imaging system (IVIS-CT machine, PerkinElmer). Fluorescence images of cells were acquired using a Laser Scanning Microscope LSM800 (Zeiss). Tissue sectioning was performed using a crystat (Leica). Western blotting images were obtained from iBright CL750 Imaging system.

Synthesis of P1-P4

For P1, Y6-OD-2Br (50.00 mg, 0.027 mmol), 4,7-bis(5-trimethylstannyl-2-thienyl)-2,1,3-benzothiadiazole (16.70 mg, 0.027 mmol), Tris(dibenzylideneacetone)dipalladium(0) (5 mg, 0.005 mmol) and Tri(o-tolyl)phosphine (10 mg, 0.0329 mmol) were dissolved in anhydrous chlorobenzene. The reaction mixture was stirred at 120 °C after being degassed by freeze-thaw cycles three times. The reaction was monitored by UV-Vis absorption. The product was precipitated in cold methanol, collected through centrifugation at 1000 × g for 10 min, washed thrice with methanol and dried under vacuum to obtain P1. P2-P4 was synthesized using 2,5-bis(triMethylstannyl)thieno[3,2‐b]thiophene (P2), 1,1′-[4,8-Bis[(2-ethylhexyl)oxy]benzo[1,2-b:4,5-b′]dithiophene-2,6-diyl]bis[1,1,1-trimethylstannane] (P3), and 2,5-Bis(2-ethylhexyl)-3,6-bis(5-(trimethylstannyl)thiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione (P4) under similar conditions.

In vitro generation of ¹O₂, O₂ ^•–and •OH

The generation of ¹O₂ by P1-P4 was measured by adding 1 µL SOSG probe (500 µM) to 1 mL nanoparticle solutions ([BTP-OD] = 5 μg/mL), followed by NIR irradiation (808 nm, 0.2 W/cm²) for 5 min. The fluorescence intensity at 520 nm was recorded every 30 s. The generation of ¹O₂ was assessed as F/F₀, where F₀ represents the fluorescence intensity of each sample before NIR irradiation. DHR123 and HPF were used to measure the generation of O₂^•–and •OH respectively, with a method like that of the SOSG probe.

Electrochemical cyclic voltammetry (CV) tests and energy of conduction band and valence band calculation

The cyclic CV tests were conducted according to previously reported literature⁵³. 0.1 mol/L tetrabutylammonium hexafluorophosphate (Bu4NPF6) dichlorobenzene/acetonitrile (5:1, v/v) solution as the supporting electrolyte with a scan speed at 0.05 V/s. Pt wire, glassy carbon discs, and Ag/AgCl were used as the counter, working electrode, and reference electrodes, respectively. To estimate whether the synthesized polymer (P1-P4) can generate reactive oxygen species (ROS), the energy levels of HOMO and LUMO were calculated according to CV tests.

Synthesis of amino-modified Val-Cit-PAB-Niclo (NH₂-Val-Cit-PAB-Niclo)

Val-Cit-PAB-OH (200 mg, 0.53 mmol), 4-pentynoic acid (77 mg, 0.78 mmol), HOBT (105.3 mg, 0.78 mmol), HBTU (295.8 mg, 0.78 mmol) and DIPEA (185 μL, 1.06 mmol) were dissolved in anhydrous DMF (15 mL) and stirred under room temperature for 4 h to get Alk-Val-Cit-PAB-OH. Then, the Alk-Val-Cit-PAB-OH was purified by HPLC. ¹H NMR (400 MHz, DMSO-d6) δ 9.89 (s, 1H), 8.12–8.10 (d, 1H), 7.95–7.93 (d, 1H), 7.56–7.54 (d, 2H), 7.25–7.23 (d, 2H), 4.43 (s, 2H), 4.39–4.35 (t, 1H), 4.25–4.21 (t, 1H), 3.01–2.96 (m, 2H), 2.75 (s, 1H), 2.38–2.36 (m, 4H), 2.00–1.95 (m, 1H), 1.73–1.70 (m, 1H), 1.62–1.58 (m, 1H), 1.44–1.36 (m, 2H), 0.87–0.84 (m, 6H). ¹³C NMR (400 MHz, DMSO-d₆) δ 171.58, 171.07, 170.83, 159.37, 137.95, 137.86, 127.39, 119.32, 84.25, 71.70, 63.05, 58.14, 53.59, 39.08, 34.48, 30.93, 29.76, 27.26, 19.66, 18.61, 14.77. LCMS (ESI) m/z calcd for C₂₃H₃₃N₅O₅ [M + H]⁺ 460.25, found 460.30.

Then, Alk-Val-Cit-PAB-OH (36 mg, 0.078 mmol) was dissolved in 10 mL anhydrous THF. Then, phosphorus tribromide (37 μL, 0.39 mmol) was added dropwise into the reaction mixture under ice bath. Afterward, the reaction system was stirred for 3 h under ice bath and argon protection. The THF was removed by rotary evaporation, and saturated NaHCO₃ (40 mL) was added to neutralize phosphorus tribromide. Subsequently, the product was extracted with ethyl acetate (EA, 150 mL, three times) and washed three times with NaHCO₃ (30 mL). After drying by anhydrous sodium sulfate, the EA was removed by rotary evaporation. Then, Niclosamide (STAT3 inhibitor) (4.238 mg, 0.013 mmol), K₂CO₃ (1.1 mg, 0.0078 mmol), Cs₂CO₃ (2.5 mg, 0.0078 mmol) were added into the system. Anhydrous acetonitrile (ACN, 10 mL) was used to dissolve reactants, and the reaction was stirred under room temperature for 3 h. Alk-Val-Cit-PAB-Niclo (9.2 mg, 0.012 mmol) and 2-azidoethan-1-amine (5.2 mg, 0.06 mmol) were dissolved in dimethyl sulfoxide (DMSO, 2 mL). Then, CuSO₄·5H₂O (3 mg, 0.012 mmol) and L-ascorbic acid sodium (4.8 mg, 0.024 mmol) were dissolved in water (200 μL) and added into the DMSO solution quickly. After stirring at room temperature for 3 h, the NH₂-Val-Cit-PAB-Niclo was purified by HPLC. ¹H NMR (400 MHz, DMSO-d6) δ 10.70 (s, 1H), 10.00 (s, 1H), 8.72 (d, 1H), 8.35 (d, 1H), 8.30–8.27 (m, 1H), 8.19 (d, 1H), 8.02 (d, 1H), 7.88 (s, 1H), 7.69–7.63 (m, 3H), 7.48–7.45 (d, 3H), 6.03 (s, 1H), 5.44 (s, 2H), 4.56–4.53 (m, 2H), 4.42–4.37 (m, 1H), 4.23–4.19 (m, 1H), 3.05–3.02 (m, 3H), 2.99 (m, 1H), 2.89–2.85 (m, 2H), 2.01–1.96 (m, 1H), 1.75–1.70 (m, 1H), 1.66–1.61 (m, 2H), 1.58–1.54 (m, 1H), 1.49–1.45 (m, 1H), 1.41–1.36 (m, 1H), 0.84 (m, 6H). ¹³C NMR (400 MHz, DMSO-d₆) δ 171.14, 171.09, 159.39, 155.64, 141.61, 141.20, 134.52, 134.21, 131.16, 130.53, 130.35, 129.88, 125.87, 125.28, 125.10, 124.36, 124.27, 124.09, 123.52, 122.93, 122.87, 121.97, 121.30, 119.92, 119.64, 119.55, 116.96, 84.24, 71.80, 71.69, 58.13, 53.62, 40.45, 39.60, 34.47, 30.93, 29.63, 27.28, 19.65, 18.61, 14.76. LCMS (ESI) m/z calcd for C₃₆H₃₉Cl₂N₇O₈ [M + H]⁺ 768.22, found 768.07.

Synthesis of alkynyl-modified PEG_2k prodrug

NH₂-Val-Cit-PAB-Niclo (9.383 mg, 0.011 mmol) and Alk-PEG_2k-COOH (22 mg, 0.011 mmol) were dissolved in 6 mL anhydrous DMF. Then, HOBT (2.2 mg, 0.0165 mmol), HBTU (6.3 mg, 0.0165 mmol) and DIPEA (4 μL, 0.022 mmol) were added into the DMF solution. The mixture was stirred at room temperature for 12 h. Then, PEG_2k prodrug was purified using dialysis bag (molecular weight cutoff 2000). After lyophilization, PEG_2k prodrug powder was collected and stored at 4 °C until use. ¹H NMR (400 MHz, DMSO-d6) δ 10.71 (s, 1H), 9.98 (d, 1H), 8.72 (d, 1H), 8.34 (s, 1H), 8.29–8.26 (m, 1H), 8.18–8.13 (m, 1H), 8.02 (d, 1H), 7.96–7.93 (m, 1H), 7.89–7.86 (m, 1H), 7.78 (s, 1H), 7.69–7.62 (m, 3H), 7.48–7.45 (m, 3H), 6.00–5.97 (m, 1H), 5.42 (d, 4H), 4.40–4.37 (m, 2H), 4.25–4.17 (m, 1H), 4.15 (d, 1H), 3.86 (s, 2H), 3.51 (m, 212H), 3.06–3.01 (m, 1H), 2.98–2.91 (m, 1H), 2.86–2.73 (m, 2H), 2.39–2.34 (m, 1H), 2.03–1.96 (m, 1H), 1.74–1.69 (m, 1H), 1.63–1.56 (m, 1H), 1.49–1.36 (m, 4H), 0.88–0.81 (m, 6H).

Synthesis of P1-DPP co-polymer

BTP-2Br-OD (50.00 mg, 0.027 mmol), 2,5-Bis(6-bromohexyl)-3,6-bis(5-bromothiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H, 5H)-dione (20.9 mg, 0.027 mmol), 4,7-bis(5-trimethylstannyl-2-thienyl)-2,1,3-benzothiadiazole (33.40 mg, 0.054 mmol), Tris(dibenzylideneacetone)dipalladium(0) (5 mg, 0.005 mmol) and Tri(o-tolyl)phosphine (10 mg, 0.0329 mmol) were dissolved in anhydrous chlorobenzene. The reaction mixture was stirred at 120 °C after being degassed by freeze-thaw cycles three times. The reaction was monitored by UV-Vis absorption. The product was precipitated in cold methanol, collected through centrifugation at 1000 × g, washed thrice with methanol and dried under vacuum to obtain P1-DPP co-polymer.

Niclosamide modified hypoxia-tolerance polymeric Type-I PDT photosensitizer (HTPS_Niclo)

P1-DPP co-polymer modification with niclosamide: P1-DPP co-polymer (5 mg, 0.007 mmol), CuBr (5 mg, 0.035 mmol), N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (36 μL, 0.17 mmol) were dissolved in 6 mL THF. Then, alkynyl modified mPEG_2k (30 mg, 0.015 mmol) or alkynyl modified PEG_2k prodrug (43.8 mg, 0.015 mmol) was added into the mixture. The mixture was stirred at room temperature for 48 h. Then, dialysis bag (molecular weight cutoff 3500) was used to purify the HTPS and HTPS_Niclo.

Nilclosamide release measurement

The niclosamide release profile of PEG_2k-Niclo and was measured after incubation with CatB for different times. For PEG_2k-Niclo, the released profile was measured by HPLC directly. For HTPS_Niclo, the samples were centrifuged at 1000 × g for 50 min using 3500 MWCO centrifugal concentrator (ThermoFisher). The released niclosamide in the bottom tubes was measured by HPLC.

Cell culture and in vitro cellular uptake assay

4T1 breast cancer cell line purchased from ATCC (American Type Culture Collection) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin and streptomycin) in an atmosphere containing 5% CO₂ and 95% humidified air at 37 °C. 4T1 cancer cells were seeded into confocal cell culture dish at a density of 5 × 10⁴ cells/dish and cultured overnight. Then the cells were treated with HTPS or HTPS_Niclo ([BTP-OD] = 1 μg/mL) for 24 h for cellular uptake. The treated cells were washed with PBS and stained with Hoechst 33342 (NucBlue Live ReadyProbesReagent, Thermo Fisher) for nuclei staining and lysosome tracker green. Fluorescence images of the live cells were acquired on Laser Scanning Microscope LSM800 (Zeiss).

In vitro cytotoxicity assay

4T1 cancer cells were seeded in 96-well plates (6000 cells in 200 μL DMEM per well) for 24 h, and then 1 × PBS (pH = 7.4), HTPS or HTPS_Niclo solutions of different concentrations were added to the culture medium. After incubation for 12 h, the cells were irradiated under 808 nm laser for 5 min (0.2 W/cm²). After incubation for another 24 h, the culture medium was removed, and cells were gently washed by fresh PBS. Fresh supplemented DMEM (100 μL per well) mixed with MTS (0.1 mg/mL, 20 μL per well) was then added to cells. After incubation for 3 h, the absorbance of the culture medium at 490 nm was recorded by SpectraMax M5 microplate/cuvette reader. Cell viability was calculated as the ratio of absorbance in different groups to that of control cells. Cells in the hypoxic group were cultured in hypoxic environments for 6 h and then treated with PDT treatment as introduced above.

Intracellular ROS detection

4T1 cancer cells were seeded in confocal cell culture dishes at a density of 1 × 10⁵ cells/dish and incubated with HTPS or HTPS_Niclo ([BTP-OD] = 5 μg/mL) for 24 h after adherence. H₂DCFDA probe or DHR123 was then added to the cells and co-cultured for 30 min. The cells were irradiated with NIR irradiation for 5 min (808 nm, 0.2 W/cm²). Then the treated cells were washed with PBS for confocal imaging. Cells in the hypoxic group were cultured in hypoxic environments for 6 h and then treated with PDT treatment as introduced above.

In vitro DC maturation and M2 macrophages downregulation

BMDCs and BMDMs were isolated from the bone marrow of BALB/c mice according to established protocols. 4T1 cancer cells were first seeded in confocal cell culture dishes at a density of 5 × 10⁴ cells/dish and then cultured overnight for cell adherence. Then the cells were incubated with HTPS or HTPS_Niclo ([BTP-OD] = 5 μg/mL) for 24 h. Afterward, the cells were treated with NIR irradiation (808 nm, 0.2 W/cm) for 5 min. After incubation for another 24 h, the cell supernatants after different treatments were collected and added to the BMDCs or BMDMs culture dishes. After incubation for another 24 h, BMDCs or BMDMs was blocked with CD16/32 and stained with anti-CD11c, anti-CD80, and anti-CD86 antibodies or anti-F4/80 and anti-iNOS, for 30 min. After washing three times, the cells were analyzed by Fortessa X20 (BD Biosciences).

Western blotting

BMDCs were incubated with supernatants from 4T1 tumor cells for different groups, followed by gentle washing with cold PBS three times. The cells were then lysed using a cell lysis buffer, and the resulting supernatant was collected by centrifugation at 12,000 × g for 10 min at 4 °C. The protein in the supernatant was quantified using an enhanced BCA protein quantification kit. Equal volumes of the supernatant and loading buffer (1:1) were mixed and incubated in a 95 °C water bath for 30 min. The samples were then subjected to SDS-PAGE gel electrophoresis. After two hours, proteins were transferred from the gel to a PVDF membrane. After that, the membrane was incubated with 5% (w/v) non-fat milk in TBST for 2 h. The membrane was washed three times with TBST in a shaker after each step. Primary antibodies against p-STAT3, STAT3, or actin were added and incubated overnight at 4 °C. The membrane was subsequently incubated with horseradish peroxidase-conjugated secondary antibodies at 37 °C for 2 h. After three washes with TBST, western blotting images were captured using an ECL detection system. Uncropped and unprocessed scans of the western blot data are provided in the supporting information and source data.

Animal ethnics statement

Animal experiments were conducted in accordance with the guidelines for the care and use of laboratory animals set by the Nanyang Technological University-Institutional Animal Care and Use Committee (NTU-IACUC) and were approved by the Institutional Animal Care and Use Committee (IACUC) of Singapore, under protocol number A21078. Mice were housed in groups in ventilated, transparent plastic cages, maintained at a controlled ambient temperature (~22 °C) and humidity (50%), with a standard 12-h light/dark cycle. Sex was not a factor in the study design.

Establishment of mouse tumor model

Five-week-old female BALB/c mice were purchased from InVivos (Singapore). For tumor inoculation, 4T1 cancer cell suspension in PBS were subcutaneously inoculated at the flank region of right hind legs of each mouse (1 × 10⁶ cells). The mice were used for imaging and therapy when tumor size reached about 100 mm³ unless otherwise stated. The experimental endpoints were defined when the tumor size exceeded 1500 mm³.

In vivo fluorescence imaging of 4T1 tumor-bearing mice

Real-time in vivo fluorescence imaging was conducted in 4T1 tumor-bearing mice at t = 0, 1, 4, 12, 24, 36, 48, 72, 96 h after intravenous administration of HTPS or HTPS_Niclo (200 µL, [BTP-OD] = 200 μg/mL). Fluorescence images of the mice were conducted on an IVIS spectrum CT system with excitation at 570 nm and emission at 780 nm. At 96 h post-injection time point, the mice for in vivo fluorescence imaging were euthanized and the resected organs were imaged for ex vivo biodistribution analysis. Fluorescence intensity of the tumor and the resected organs from the mice were quantified by the ROI analysis using Living Image 4.3 software.

In vivo evaluation for HTPS_Niclo-mediated photo-immunotherapy

4T1 cancer cell suspension in PBS were subcutaneously inoculated at flank region on -7 d (1 × 10⁶ cells, primary tumor) and -1 d (2 × 10⁵ cells, distant tumor). The mice were systematically administrated with 200 µL PBS, HTPS or HTPS_Niclo (200 µL, [BTP-OD] = 200 μg/mL). At 1 d, 3 d, and 5 d post-injection, primary tumors in HTPS or HTPS_Niclo with NIR irradiation group were irradiated for 5 min (808 nm, 0.2 W/cm²). The body weights and tumor sizes were monitored every 2 days for 20 days. The equation for tumor size calculation is volume = a × b²/2, where a represents tumor length and b represents tumor width. After 20 days, the mice in each group were monitored for 40 days to record survival rates.

In vivo generation of O₂ ^•-

Mice with established tumors (~100 mm³) were intravenously administered with 200 µL PBS, HTPS or HTPS_Niclo (200 µL, [BTP-OD] = 200 μg/mL). At 24 h post-injection, 30 µL DHR123 (50 µM) was intratumorally injected into the tumors of the mice in each group. After 30 min, the tumors of the mice were irradiated with NIR for 5 min (808 nm, 0.8 W/cm²). The mice were then euthanized, and the tumors were collected, fixed in 4% paraformaldehyde (PFA), and cryo-sectioned. The tumor sections were stained with Hoechst 33342 and imaged using LSM800 confocal laser scanning microscope. The fluorescent intensity of DHR123 was quantified to evaluate in vivo O₂^•- generation in each group using the image processing software ImageJ.

DC maturation and cytokine production

Three days after different treatments, the mice in each group were euthanized and their blood and tumor draining lymph nodes (TDLNs) were collected. Single cell suspension was obtained by directly grounding and straining TDLNs using a 70-µm cell strainer. Cells were then incubated with anti-CD16/32 antibody and stained with anti-CD11c, anti-CD80, and anti-CD86 antibodies for flow cytometric analysis. The blood serum was collected from the supernatant of the blood samples for cytokine detection (IL-12, IFN-γ, TNF-α, and IFN-β) by ELISA according to manufacturer’s protocols.

Flow cytometric analysis of immune infiltrate phenotyping

Seven days after different treatments, the mice in each group were euthanized, and tumors and spleens were collected. Single cell suspensions from tumor and spleens were first blocked with anti-CD16/32 antibody, followed by stained with anti-CD45, anti-CD3, anti-CD4, and anti-CD8a antibodies. For flow cytometric analysis of Tregs, the cells were stained with anti-CD45, anti-CD3, anti-CD4, followed by fixation in 0.5 mL/tube Fixation and Perm Buffer and incubated with PE anti-mouse FOXP3. After washing three times, the cells were analyzed by Fortessa X20 (BD Biosciences).

Immunofluorescence staining of tumor sections

According to the standard protocol, tumors were fixed in 4% PFA and cryo-sectioned with a thickness of 10 µm. The tumor sections were incubated with anti-caspase 3, HIF-1 and CD31 at 4 °C followed by staining the nuclei with Hoechst 33342. The fluorescent images were captured using LSM800 confocal laser scanning microscope. The quantification of the fluorescent intensities was performed using software ImageJ.

Histological studies and metastasis analysis

The resected organs from the 4T1 tumor-bearing mice in each group were fixed with 4% PFA, dehydrated with 30% sucrose and sectioned into thin slices (10 μm).

Rechallenged tumor model

Mice bearing 4T1 tumors were systematically administrated with HTPS or HTPS_Niclo (200 µL, [BTP-OD] = 200 μg/mL). At 1 d, 3 d, and 5 d post-injection, tumors in HTPS or HTPS_Niclo + NIR group were irradiated with NIR for 5 min (808 nm, 0.8 W/cm²). On day 33, mice were re-inoculated with 1 × 10⁶ 4T1 cells in PBS on the left flank without any further treatment. Tumor size was monitored every two days for two weeks. Spleen from 4T1 tumor-bearing mice at 57 d were collected for population analysis of memory T cells using flow cytometry. TDLNs were directly ground and strained with a 70-µm cell strainer to obtain single cell suspension. Then, the cells were blocked with anti-CD16/32 antibody for 10 min, followed by stained with FITC anti-CD3, PE anti-CD8, APC anti-CD62L, and BV605 anti-CD44 antibodies. After washed thrice with cell staining buffer, the cells were analyzed by Fortessa X20 (BD Biosciences).

Statistical analysis

Technical replicates were used in all experiments, and data were calculated and expressed as mean ± standard deviation (S.D.) unless stated otherwise. Investigators were blinded to group allocation during experiments. The specific statistical methods are indicated in the figure legends. Statistical differences between two groups were tested with a two-tailed student’s t-test and more than three groups were determined by one-way analysis of variance followed by Tukey’s post hoc test. The p values of survival curves were analyzed using the Kaplan–Meier method via the log-rank test. For all tests, P values less than 0.05 were considered statistically significant. *p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. All statistical calculations were performed using GraphPad Prism 8.0, including assumptions of tests used.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data needed to evaluate the conclusions in the paper are presented in the paper and/or the Supplementary Materials. Source data are provided with this paper. All other data are available from the corresponding authors upon request. Source data are provided with this paper.

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Acknowledgements

Y.Z. thanks National Natural Science Foundation of China (22322406) for financial support. K.P. thanks Singapore National Research Foundation (NRF) (NRF-NRFI07-2021-0005) and the Singapore Ministry of Education Academic Research Fund Tier 2 (MOE-T2EP30220-0010, MOE-T2EP30221-0004) for financial support. This article was also supported by china postdoctoral science foundation under grant number GZC20240525 (J.Y.).

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Authors and Affiliations

National Engineering Research Centre for Nanomedicine, College of Life Science and Technology, Hubei Key Laboratory of Bioinorganic Chemistry and Materia Medical, Huazhong University of Science and Technology, Wuhan, PR China
Jie Yu & Yan Zhang
School of Chemistry, Chemical Engineering and Biotechnology, Nanyang Technological University, Singapore, Singapore
Jiayan Wu, Jingsheng Huang, Cheng Xu, Mengke Xu, Clarence Zhi Han Koh & Kanyi Pu
Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore
Kanyi Pu

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Jie Yu
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Contributions

K.P. and J.Y. conceived the concept and designed the research. J.Y. performed material preparation and in vivo experiments. K.P., Y.Z., and J.Y. contributed to the interpretation of the results. K.P., Y.Z., J.Y., J.W., J.H., C.X., M.X., and C.K. contributed to the writing of this manuscript.

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Correspondence to Kanyi Pu or Yan Zhang.

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Yu, J., Wu, J., Huang, J. et al. Hypoxia-tolerant polymeric photosensitizer prodrug for cancer photo-immunotherapy. Nat Commun 16, 153 (2025). https://doi.org/10.1038/s41467-024-55529-8

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Received: 07 August 2024
Accepted: 16 December 2024
Published: 02 January 2025
DOI: https://doi.org/10.1038/s41467-024-55529-8

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