Biomimetic semiconducting polymer dots for dual targeted NIR-II phototheranostic and multimodal coordinated immunostimulatory therapy

Abstract

Breast cancer is a global health challenge necessitating more precise and effective treatment strategies. In this study, we developed a novel drug-loaded therapeutic nanoplatform, OCPdots@CTe, which integrated near-infrared-II (NIR-II) window phototheranostic for targeted treatment of orthotopic breast tumors. The outer membrane vesicles (OMVs) can stimulate more immune responses based on precise targeting, while chelerythrine (CTe) can induce apoptosis by generating reactive oxygen species (ROS), thereby enhancing the therapeutic effect. Owing to the excellent optical properties of polymer dots (Pdots), this nanoplatform can also monitor the in vivo distribution of drugs with dual-module imaging. Moreover, the biomimetics significantly improved the biocompatibility of Pdots@CTe and provided precise delivery. Our results revealed that OCPdots@CTe significantly improved the outcome of breast tumor treatment with minimal side effects. Notably, we found that this combined therapy with multi-platform immune stimulation enhanced the anticancer effect. Together, this multifunctional nanoplatform offers a powerful versatile strategy for breast cancer treatment.

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1. Introduction

Breast cancer is the most common and deadliest malignancy for women worldwide.^1–3 Despite the substantial progress made by traditional treatments such as surgery, chemotherapy, and radiotherapy in improving patient survival rates,⁴ these methods still face limitations such as incomplete tumor removal, high recurrence rates, and severe side effects.^5,6 There is an urgent need for innovative treatment strategies with enhanced efficacy and reduced toxicity.^2,7,8 Recent advancements in nanotechnology have opened up new avenues for the development of multifunctional nanoplatforms that can simultaneously detect and treat malignancies, which are ideally suited for addressing the limitations associated with conventional cancer theranostics.^9–12 Among them, the integration of NIR light-responsive materials in the second window into such platforms especially holds promise for deep tissue penetration and minimized side effects on healthy tissues.^13,14 Semiconducting polymer dots (Pdots), in particular, have emerged as promising candidates for biomedical imaging, drug carriers and cancer phototherapy, owing to their unique optical properties and high biocompatibility.^15–18 Phototheranostics, on the other hand, include both photothermal therapy (PTT) and photodynamic therapy (PDT). However, PTT can cause damage to the surrounding tissues at high temperatures, while photodynamic PDT is limited by its oxygen dependence and insufficient delivery of photosensitizers.^19–21 Overall, designing Pdots that serve as efficient NIR-II photothermal and photodynamic therapeutic agents for deep tumor penetration, enhance therapeutic efficacy, and reduce thermal toxicity entailed by laser irradiation remains largely underexplored.^18,22

Chelerythrine (CTe), a natural benzophenanthridine alkaloid, exhibits significant anti-cancer properties.^23–25 Studies have shown that CTe can inhibit tumor growth by inducing mitochondrial dysfunctions, triggering apoptosis pathways, and generating ROS.^23,26,27 These properties make the compound a promising candidate for amplifying the efficacy of PDT through ROS generation.^28,29 However, the clinical application of CTe is limited by its poor water solubility and low bioavailability, which may reduce its tumor-targeting capability. In contrast, loading CTe into functionalized nanocarriers like Pdots (Pdots@CTe) can resolve these limitations, achieving highly efficient drug delivery and enhanced efficacy of tumor treatment.^30,31

Furthermore, bacterial OMVs are natural secretory immune stimulators which can activate dendritic cells (DCs) and T lymphocytes, improving the anti-tumor immune response. Despite their immunological benefits, OMVs exhibit a lack of tumor targeting capability.^32–34 Conversely, encapsulation of cancer cell membrane fragments on material surfaces has been shown to significantly enhance their targeting specificity, albeit with limited immunostimulation. A compromised approach involves the co-coating of nanomaterials with components derived from natural 4T1 cancer cell membranes and OMVs, which endows the nanoplatform with high targeting ability and immune stimulation, rendering an ideal vector for drug delivery and combined photoimmunotherapy of breast cancer.^35,36 This innovative surface modification offers several advantages. First, the combination of 4T1 cell membranes and OMVs can improve the recognition and targeting of nanoparticles to tumor cells.³⁷ This dual-component strategy not only improves the specificity of tumor targeting but also amplifies the immune response, which is critical for the long-term suppression of tumors and the establishment of immune memory. Additionally, the immunomodulatory effects of OMVs are pivotal in activating key immune cells, such as cytotoxic T lymphocytes (CTLs) and CD4+ helper T cells, which are essential for the sustained tumor control and the development of an immunological memory response.^38,39 Moreover, this dual-membrane structure on the nanoplatform effectively evades clearance by the host immune system, thereby prolonging the nanoparticle circulation within the body and enhancing their therapeutic efficacy. This extended circulation time also allows for a more sustained release of therapeutic agents at the tumor site, which can lead to improved treatment outcomes and reduced side effects associated with traditional cancer therapies.⁴⁰

In this study, we have developed a novel therapeutic nanoplatform, OCPdots@CTe, which integrated multiple functionalities for enhanced cancer therapy. Initially, we encapsulated CTe within Pdots, augmenting the PDT efficacy through PTT at a relatively safe temperature. Subsequently, to ensure the targeting specificity of Pdots@CTe through coating with 4T1 cancer cell membranes, we constructed a genetically modified msbB mutant E. coli strain (MG1655)⁴¹ and utilized its OMVs to further enhance both targeting and immune modulation (Fig. 1a). Consequently, the synthesized OCPdots@CTe not only exhibits remarkable optical properties for NIR-II fluorescence (FL) and photoacoustic (PA) imaging, facilitating tumor detection and enabling real-time monitoring of the therapeutic process, but also achieves a potent synergistic anticancer effect through multimodal immune stimulation. The development of this multifunctional nanoplatform represents a significant step forward in advancing cancer therapy and improving patient outcomes. By exploiting the unique properties of CTe and OMVs, OCPdots@CTe offers targeted drug delivery, enhanced immune response, and precise imaging capabilities (Fig. 1b). This study introduces a novel dual-biointerface design that integrates bacterial outer membrane vesicles (OMVs) with 4T1 cancer cell membranes, effectively resolving the limitations of single-membrane systems in achieving simultaneous immune activation and homologous targeting. This innovation has the potential to advance cancer management by providing a more effective and personalized treatment option for patients.

Fig. 1 Schematic illustration of the synthesis and therapeutic mechanism of OCPdots@CTe. (a) Chemical structural formula and schematic of OCPdots@CTe. (b) Schematic of multimodal coordinated immunostimulatory therapy of OCPdots@CTe for the targeted treatment of breast cancer in situ.

2. Results and discussion

2.1. Synthesis and characterization of OCPdots@CTe

In this section, we detail the preparation and characterization of Pdots and Pdots@CTe, synthesized via a reprecipitation method by using functional polymers such as polystyrene-graft-polyethylene glycol (PS-PEG-COOH), chelerythrine (CTe), and a second NIR-II emitting polymer (PBTQ4F).^42,43 To confirm the successful incorporation of CTe into Pdots, we analyzed the absorption and emission spectra of CTe, Pdots, and Pdots@CTe (Fig. 2b–d). Pdots@CTe exhibited characteristic peaks of both CTe and Pdots, indicating successful construction of the assembly. The introduction of CTe into Pdots@CTe caused an increase in size and zeta potential, as shown in Fig. 2e and g. The successful coating of the two cell membrane proteins on the surface of Pdots@CTe was confirmed by dynamic light scattering (DLS) measurements and transmission electron microscopy (TEM) imaging (Fig. 2a). The protein coating did not affect the absorption peak of Pdots at 950 nm (Fig. 2c), but increased the size of the nanoparticles, as shown in Fig. 2f. The stability of OCPdots@CTe over time was also inspected, showing no significant changes in size (Fig. S1, ESI†). The zeta potential of OCPdots@CTe was changed due to the potential difference between membrane proteins and Pdots@CTe, as shown in Fig. 2g.

Fig. 2 Characterization of OCPdots@CTe. (a) Transmission electron microscopy (TEM) imaging of the samples (scale bar = 100 nm). (b) UV-vis absorption spectra of Pdots, CTe and Pdots@CTe. (c) UV-vis absorption spectra of Pdots, OPdots@CTe and OCPdots@CTe. (d) FL spectra of Pdots, CTe and Pdots@CTe. (e) Representative DLS results of Pdots and Pdots@CTe. (f) Representative DLS results of OPdots@CTe and OCPdots@CTe. (g) Zeta potential values of Pdots, Pdots@CTe, OPdots@CTe, OCPdots@CTe, OMVs of E. coli bacteria, and the 4T1 cancer cell membrane (CCM).

Under 808 nm laser irradiation, the PA signal of Pdots increased linearly with the concentration of Pdots from 6.25 to 100 μg mL⁻¹ (Fig. 3d). Likewise, the FL signal of Pdots also gradually increased with increased concentration from 3.125 to 100 μg mL⁻¹ (Fig. 3d). All the nanoparticles (Pdots, Pdots@CTe, OPdots@CTe, and OCPdots@CTe) exhibited bright FL in the NIR-II window. The hydrodynamic diameter of 120 nm was optimized for enhanced permeability and retention (EPR)-mediated tumor accumulation. The near-neutral zeta potential minimized the clearance using a reticuloendothelial system (RES), thereby extending the circulation time. In addition, SDS–PAGE analysis (Fig. 3f) confirmed the presence of the key membrane OMV marker and the 4T1 membrane protein to ensure the fidelity of targeting. These results collectively demonstrated successful biological membrane modification and drug incorporation onto the surface of OCPdots@CTe without compromising their optical properties. Additionally, ROS production was detected upon laser irradiation, as shown in Fig. S2 and S3 (ESI†).

Fig. 3 Photothermal properties of OCPdots@CTe. (a) Photothermal heating curves of OCPdots@CTe solutions at different concentrations (0, 6.25, 12.5, 25, 50, and 100 μg mL⁻¹) under 808 nm laser irradiation at 0.5 W cm⁻² for 6 min. (b) Photothermal heating curves of Pdots and OCPdots@CTe dispersions (25 μg mL⁻¹) irradiated using an 808 nm laser at varied power densities (0.1, 0.25, 0.5, and 0.75 W cm⁻²). (c) Photothermal performance of OCPdots@CTe and dWater under 808 nm laser irradiation for heating and cooling. (d) PA intensities at 808 nm as a function of concentrations of OCPdots@CTe. Insets are the PA images of OCPdots@CTe at different concentrations (μg mL⁻¹). (e) FL intensity of OCPdots@CTe. (f) SDS–PAGE protein analysis of protein markers, Pdots, Pdots@CTe, the cancer cell membrane (CCM), CPdots@CTe, OMVs, OPdots@CTe, and OCPdots@CTe, respectively.

2.2. Photothermal properties of OCPdots@CTe

Given the significant and broad absorption characteristics of OCPdots@CTe in the NIR-II window, they can serve as contrast agents for NIR-II PA imaging and photothermal agents for NIR-II PTT. To assess their photothermal properties, we examined temperature changes in OCPdots@CTe solutions in vitro under 808 nm laser irradiation. The temperatures at different concentrations (12.5, 25, 50, and 100 μg mL⁻¹) are shown in Fig. 3a. Even at relatively low concentrations, the temperature of the solution rapidly increased, reaching approximately 60 °C within 5 min of laser irradiation at 100 μg mL⁻¹. We also evaluated the photothermal properties at different laser power densities (0.1, 0.25, 0.5, and 0.75 W cm⁻²), and Fig. 3b shows that OCPdots@CTe exhibited strong laser power-dependent photothermal effects under continuous irradiation. To further assess the photothermal stability of OCPdots@CTe, we performed cyclic temperature change tests. The results, as shown in Fig. S4 (ESI†), indicate no significant changes in the maximum temperature over five laser on/off cycles, demonstrating the excellent photostability of OCPdots@CTe.

To determine the photothermal conversion efficiency, OCPdots@CTe solutions were irradiated with an 808 nm laser for 6 min until a stable temperature was reached. The laser was then turned off, and the solutions were allowed to cool to room temperature, with pure water serving as a negative control. The photothermal conversion efficiency of OCPdots@CTe was calculated to be approximately 52%, based on previously described methods (Fig. 3c and eqn (S1)–(S9), ESI†). This efficiency is significantly higher compared to that of other studies, as shown in Table S1 (ESI†). Therefore, the high extinction coefficient, excellent photothermal stability, high NIR-II photothermal conversion efficiency, and superior biocompatibility of OCPdots@CTe make them exceptional candidates for dual-mode PA/FL imaging-guided PTT.

2.3. In vitro uptake and therapeutic effects of OCPdots@CTe

To evaluate the biocompatibility and cellular uptake of OCPdots@CTe leading to enhanced therapeutic effects, we first examined cellular uptake of biomimetic OCPdots@CTe using confocal fluorescence imaging (Fig. 4a and Fig. S6, ESI†). Due to the lack of a NIR-II confocal imaging system, we incorporated a NIR-II emitting polymer (PBTQ4F) and an orange-emitting polymer (CN-PPV) to produce nanoparticles with FL in the visible light region. The absorption and excitation spectra of these nanoparticles are provided in Fig. S7 and S8 (ESI†). Staining of the cell membranes and nuclei revealed that OCPdots@CTe was predominantly internalized by 4T1 cells, a result further corroborated by flow cytometry analysis (Fig. 4b and Fig. S9, ESI†). Additionally, we used the 5-(2,4-dinitrophenyl)-3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazolium salt (WST-8) cell counting kit (CCK-8) to assess the biocompatibility and cytotoxicity of OCPdots@CTe. The results showed that CTe exhibited significant cytotoxicity against normal 293T cells, but the toxicity was greatly reduced when OCPdots@CTe was encapsulated by membrane proteins. After co-incubation with 293T cells for 24 h, OCPdots@CTe showed no significant toxicity even at a high concentration of 100 μg mL⁻¹ (Fig. 4c).

Fig. 4 In vitro therapeutic effects of OCPdots@CTe. (a) Confocal FL images of 4T1 cancer cells after incubation with (i) PBS, (ii) Pdots, and (iii) and (iv) OCPdots@CTe (25 μg mL⁻¹) for 12 h. The yellow FL indicated nanoparticles, cell nuclei (blue) were stained with 4′,6-diamidino-2-phenylindole (DAPI), and the (iii) cell membrane was stained with Dil. (b) Flow cytometry analysis of 4T1 cancer cells after incubation with PBS and nanoparticles (25 μg mL⁻¹) for 12 h. (c) CCK8 assay of 293T normal cells treated with nanoparticles at various concentrations for 24 h to assess the cell viability. (d) Cell viability assessment of HeLa and 4T1 cells after incubation with 25 μg mL⁻¹ nanoparticles for 12 h: cells treated with irradiation. (e) Cell viability assessment of 4T1 cells after incubation with 25 μg mL⁻¹ nanoparticles for 12 h: cells treated with or without irradiation (0.5 W cm⁻² 808 nm NIR-II laser for 10 min). (f) FL images of live/dead HeLa cells incubated with nanoparticles for 12 h first and then irradiated. (g) Flow cytometry analysis of 4T1 cancer cells done by DCFH-DA after incubation with nanoparticles for 12 h first and then irradiated. (h) Flow cytometry analysis of 4T1 cancer cells done using an ANNEXIN V-FITC/PI dye after incubation with nanoparticles for 12 h first and then irradiated (all the irradiation were done using an 808 nm laser at 0.5 W cm⁻² for 10 min, and all error bars indicated standard deviation (n = 3)).

To further evaluate the PTT effect on live cells, we conducted photothermal ablation experiments on 4T1 and HeLa cancer cells using the CCK-8 kit and FL imaging. Different concentrations of the nanoparticles were co-incubated with cancer cells under 808 nm laser irradiation. The CCK-8 results showed that the cell viability was significantly decreased with increasing cellular uptake of the materials (Fig. 4c). From Fig. 4e, it is evident that the specific targeting of 4T1 membrane proteins resulted in a weaker uptake of OCPdots@CTe by HeLa cells compared to that of OPdots@CTe. Furthermore, FL microscopy imaging of calcein/propidium iodide (PI) co-stained HeLa cells demonstrated that OCPdots@CTe exhibited higher PTT ablation efficiency at very low laser power densities than unmodified nanoparticles (Fig. 4f and Fig. S10, ESI†), indicating their potential as efficient photothermal agents for enhanced PTT. Due to the drug mechanism of CTe and the PDT effect of OCPdots@CTe, in vitro treatment also induced cell apoptosis. As shown in Fig. 4g, different bio-functionalized nanoparticles produced varying levels of ROS signals at the same concentration, and the addition of CTe, which has strong absorption at 488 nm, significantly enhanced the ROS signal (Fig. S11, ESI†). Notably, OCPdots@CTe resulted in optimal ROS generation. Additionally, flow cytometric analysis of ANNEXIN V-FITC/PI-stained cells after treatment revealed that up to 50% of the cells entered the apoptosis phase, and over 70% of the cancer cells died (Fig. 4h and Fig. S12, ESI†).

2.4. Dual-mode PA and FL imaging of OCPdots@CTe in the NIR-II window

In vivo NIR-II PA imaging and FL imaging were conducted to visualize the increased accumulation and homing targeting of OCPdots@CTe at the tumor site. A 4T1 tumor-bearing nude mouse model was established, and the mice were injected with the nanoparticles via the tail vein. For in vivo NIR-II FL imaging, a detectable FL signal was observed at the tumor site within 1 h of injection, and the signal intensity gradually increased, reaching a peak at 24 h (Fig. 5a and b). Importantly, OCPdots@CTe exhibited stronger FL signals at each time point compared to Pdots@CTe and OPdots@CTe (Fig. 5b). Even seven days after injection, the strongest signal was still detected in the tumor region of mice injected with OCPdots@CTe (Fig. 5c and Fig. S13, ESI†).

Fig. 5 In vivo dual-mode PA and FL imaging of OCPdots@CTe. (a) In vivo FL imaging of nude mice after intravenous (i.v.) injection of nanoparticles (at a dose of 2.0 mg kg⁻¹) at different time intervals. (b) FL signal intensity in the tumor region at different time points after drug injection. (c) FL intensity of various organs dissected from mice after 7 days of the i.v. injection of nanoparticles. (d) PA intensity in the tumor region at different time points after drug injection. (e) In vivo PA imaging of nude mice after the intravenous (i.v.) injection of nanoparticles (at a dose of 2.0 mg kg⁻¹) at different time intervals.

We then monitored the PA signal intensity at the tumor site using our in-house-built NIR-II PA imaging system at different time points (Fig. S14, ESI†). Fig. 5d shows the in vivo PA images and corresponding PA signal intensities at the tumor site after injection of the nanoparticles and 808 nm laser irradiation. The enhancement efficiency and effect of the PA signals were similar to that of FL imaging, indicating that the accumulation of nanoparticles at the tumor site reached a peak at 24 h due to the enhanced permeability and retention (EPR) effect and the homing targeting ability. Since Pdots@CTe, OPdots@CTe, and OCPdots@CTe exhibited different absorption and FL intensities at the same concentration, OCPdots@CTe demonstrated shorter tumor accumulation times and enhanced NIR-II PAI and FL capabilities compared to Pdots@CTe and OPdots@CTe.

2.5. In vivo therapeutic effects of OCPdots@CTe in the NIR-II window

To further investigate the PTT, PDT and combine immune therapy effects of OCPdots@CTe, we used the same mouse model as described in Section 2.4, with all procedures conforming to the guidelines of the Animal Care and Use Committee at the University of Macau. Nude mice with approximately 100 mm³ tumors were randomly divided into six groups (n = 5), and each group was intravenously injected with Pdots and OCPdots@CTe at a dose of 2.0 mg kg⁻¹ or an equal volume of PBS. The anti-tumor effects of different treatments were observed. The body weight and tumor volume of the mice were recorded every 2 days. After 14 days, the mice were sacrificed to collect tissue samples (Fig. 6a).

Fig. 6 In vivo phototheranostic therapeutic effects of OCPdots@CTe. (a) Frequency and duration of OCPdots@CTe for NIR-II PTT. (b) Body weight changes of mice after various treatments over 14 days. Each data point represents the mean ± SD from n = 5 animals. (c) Relative tumor volume curves. Each data point denotes the mean ± SD from n = 5 animals. (d) H&E and TUNEL analysis results for tumor tissues collected from the mice of different groups at the end of treatment. (e) H&E analysis results for organ tissues collected from mice of different groups. (+: 808 nm laser, 0.3 W cm⁻², and 5 min).

The relative tumor volume plot showed that the OCPdots@CTe + laser group exhibited superior tumor inhibition (Fig. 6c). Additionally, the size and weight of the tumor tissues in vivo were statistically consistent with those observed in vitro (Fig. S15 and S16, ESI†). After treatment, the tumor tissues were collected and analyzed using hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The results showed that there was a large amount of necrotic tissue caused by apoptosis in the OCPdots@CTe + laser group (Fig. 6d and Fig. S17, ESI†). However, no significant fluctuations in body weight were observed in any of the experimental groups (Fig. 6b). Nevertheless, to avoid thermal damage due to high temperatures, we used a lower laser power for treatment, which limited the local temperature increase to below 42 °C (Fig. S18 and S19, ESI†). Consequently, complete tumor inhibition could not be achieved solely through PTT and drug effects. Furthermore, H&E staining of important organs in vitro confirmed that OCPdots@CTe did not produce toxic side effects (Fig. 6e and Fig. S20, ESI†).

Recent studies have indicated that the combined use of PTT/PDT and immunotherapy constitutes an effective synergistic strategy for treating solid tumors. Therefore, we explored the potential of combining Pdots with PTT, PDT, CTe, and OMVs for enhanced immune responses. A bilateral in situ breast cancer tumor-bearing mouse model was constructed, and the mice were divided into six groups (n = 5). When the primary tumor volume reached approximately 100 mm³ and the distal tumor volume reached about 50 mm³, the experimental groups were intravenously injected with Pdots@CTe, OCPdots@CTe, and PBS. After 24 h, one group was treated with an 808 nm laser, while the other group was not. On the 6^th day of the treatment cycle, a second laser irradiation was performed, and the body weight and tumor volume were recorded every 2 days (Fig. 7a). The body weights of the tumor-bearing mice showed no significant differences across groups (Fig. 7b). All tumor-bearing mice were sacrificed on day 14 to collect tissue samples. The relative volume change curve of the primary tumor (Fig. 7c) shows that the OCPdots@CTe + laser group exhibited almost complete tumor ablation. The relative volume change curve of the distal tumor (Fig. 7d) indicates that only the OCPdots@CTe + laser group showed a significant tumor growth inhibition effect. The sizes and weights of the isolated tumor tissues were consistent with the in vivo measurements on day 14 (Fig. 7h, i and Fig. S21, ESI†).

Fig. 7 In vivo multimodal coordinated immunostimulatory effects of OCPdots@CTe. (a) Frequency and duration of OCPdots@CTe for NIR-II phototheranostics and chemoimmunotherapy. (b) Body weight changes of mice after various treatments over 14 days. Each data point represents the mean ± SD from n = 5 animals. (c) Relative tumor volume curves for the primary tumor. Each data point denotes the mean ± SD from n = 5 animals. (d) Relative tumor volume curves for distal tumor. Each data point denotes mean ± SD from n = 5 animals. (e)–(g) Flow cytometry analysis of (e) CD8a, CD4, (f) CD11c, CD86, and (g) CD11b and CD49b infiltrating primary and distal tumor tissues. (h) Weights of primary tumor tissues at the end of the treatment cycle, ± SD from n = 5 animals. (i) Weights of distal tumor tissues at the end of the treatment cycle, ± SD from n = 5 animals. (+: 808 nm laser, 0.3 W cm⁻², 5 min).

Notably, following phototherapy and biomimetic modification, OCPdots@CTe demonstrated superior targeting and immunological effects, particularly in suppressing the growth of primary tumors. Flow cytometry analysis of isolated tumor tissues revealed that the expression levels of multiple immune-related molecules were significantly upregulated in both primary and metastatic tumors in the OCPdots@CTe plus laser treatment group, indicating that these cells were more activated and involved in antitumor immunotherapy during the treatment process. Specifically, the signals for CD4 and CD8a in the laser treatment group exceeded 50% (Fig. 7e and Fig. S22, ESI†). Furthermore, we examined the expression of other immune signaling molecules, including CD11c, CD86, CD11b, and CD49b. The results showed that these immune-related molecules also exhibited significant changes in expression levels in the OCPdots@CTe plus laser treatment group, with CD11c and CD86 eliciting over 40% expression in metastatic tumors (Fig. 7f and Fig. S23, ESI†). The enhanced expression of CD11b also suggested an increased inflammatory response and recruitment of immune cells in the tumor tissue, while the increased expression of CD49b highlighted the enhanced antitumor activity of NK cells (Fig. 7g and Fig. S24, ESI†). These findings further confirmed the crucial role of OCPdots@CTe in combination with phototherapy and biomimetic modification in modulating immune cell functions within the tumor microenvironment and enhancing antitumor immune responses.

3. Conclusions and discussion

In conclusion, we have devised a novel biomimetic nanocomposite, OCPdots@CTe, which combined the advantages of NIR-II Pdots with the therapeutic potential of chelerythrine (CTe). The dual encapsulation with 4T1 cancer cell-derived membrane proteins and OMVs endowed the nanoplatform with remarkable tumor targeting specificity and enhanced the immunotherapeutic response. In combination with CTe-amplified mitochondrial ROS generation, this strategy also overcomes the oxygen dependence inherent in conventional photodynamic therapy (PDT), resulting in over 70% regression of distal tumors (Fig. 7d) and demonstrating robust systemic antitumor immunity. In the treated tumor tissues, there was a significant upregulation of immune-related molecules. This immune activation was multi-faceted: PTT stimulated tumor cells to release heat-shock proteins and damage-associated molecular patterns (DAMPs), which promoted the recruitment and activation of immune cells, especially T cells, leading to the upregulation of CD4 and CD8a signals. In PDT, the reactive ROS generated by CTe caused cellular stress and death, resulting in the release of tumor antigens. These antigens were then taken up by DCs. After maturation, DCs presented the antigens to T cells, further amplifying the adaptive immune response and leading to the enhancement of CD11c and CD86 signals. Meanwhile, OMVs activated monocytes and macrophages, leading to the enhancement of signals of their markers CD11b and CD49b. Our findings not only demonstrated the OCPdots@CTe's ability to modulate the tumor microenvironment and augment antitumor immune responses but also established its usage in inhibiting primary tumor growth and activating immune cells due to the synergistic effects. Thus, OCPdots@CTe is anticipated to act as a promising candidate for a multimodal immunotherapeutic platform.

Looking ahead, we plan to investigate the long-term effects of OCPdots@CTe on immune memory and its potential to prevent tumor recurrence. Additionally, exploring the scalability of OCPdots@CTe production and its application across various cancer types is crucial for translating this research into clinical practice. It is equally important to address the persistent challenge of Pdot clearance in vivo. Although our study did not identify any significant adverse effects, ongoing monitoring and assessment are imperative as research progresses. In summary, OCPdots@CTe represents a significant advancement in the field of nanomedicine, offering a multifunctional platform for combined phototherapy and immunodrug therapy. Its unique properties and the positive outcomes observed in our study warrant further exploration and development to harness its full potential for cancer treatment.

Author contributions

Jintong Guo: methodology, formal analysis, investigation, visualization, and writing – original draft. Xianyuan Wei: methodology and data curation. Ye Liu: methodology. Yun Li: methodology. Pu Chun Ke: writing – review and editing. Zhen Yuan: supervision, project administration, writing – review and editing, and funding acquisition.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

All data generated or analyzed during this study are included in this published article and its ESI† files.

Acknowledgements

This work was supported by the University of Macau (MYRG2022-00054-FHS, MYRG-GRG2023-00038-FHS-UMDF, and MYRG-GRG2024-00259-FHS) and the Macao Science and Technology Development Fund (FDCT 0014/2024/RIB1).

References

1. F. Bray, et al., Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, Ca-Cancer J. Clin., 2024, 74(3), 229–263.
2. R. L. Siegel, A. N. Giaquinto and A. Jemal, Cancer statistics, Ca-Cancer J. Clin., 2024, 74(1), 12–49.
3. N. Ssenyonga, et al., Worldwide trends in population-based survival for children, adolescents, and young adults diagnosed with leukaemia, by subtype, during 2000–14 (CONCORD-3): analysis of individual data from 258 cancer registries in 61 countries, Lancet Child Adolesc. Health, 2022, 6(6), 409–431.
4. V. T. DeVita, et al., Clinical cancer research: The past, present and the future, Nat. Rev. Clin. Oncol., 2014, 11(11), 663–669.
5. F. Bray, et al., Global cancer transitions according to the Human Development Index (2008–2030): a population-based study, Lancet Oncol., 2012, 13(8), 790–801.
6. K. D. Miller, et al., Cancer treatment and survivorship statistics, Ca-Cancer J. Clin., 2022, 72(5), 409–436.
7. C. Xia, et al., Cancer statistics in China and United States, 2022: profiles, trends, and determinants, Chin. Med. J., 2022, 135(05), 584–590.
8. C. E. DeSantis, et al., Breast cancer statistics, 2015: Convergence of incidence rates between black and white women, Ca-Cancer J. Clin., 2016, 66(1), 31–42.
9. D. Fan, et al., Nanomedicine in cancer therapy, Signal Transduction Targeted Ther., 2023, 8(1), 293.
10. J. Guo, et al., A review of biomodified or biomimetic polymer dots for targeted fluorescent imaging and disease treatments, iRadiology, 2023, 1(3), 209–224.
11. K. Deng, et al., Recent progress in near infrared light triggered photodynamic therapy, Small, 2017, 13(44), 1702299.
12. M. P. Melancon, M. Zhou and C. Li, Cancer theranostics with near-infrared light-activatable multimodal nanoparticles, Acc. Chem. Res., 2011, 44(10), 947–956.
13. M.-C. Chen, Z.-W. Lin and M.-H. Ling, Near-infrared light-activatable microneedle system for treating superficial tumors by combination of chemotherapy and photothermal therapy, ACS Nano, 2016, 10(1), 93–101.
14. T. Sun, et al., Second near-infrared conjugated polymer nanoparticles for photoacoustic imaging and photothermal therapy, ACS Appl. Mater. Interfaces, 2018, 10(9), 7919–7926.
15. G. Jeon and Y. T. Ko, Enhanced photodyamic therapy via photosensitizer-loaded nanoparticles for cancer treatment, J. Pharm. Invest., 2019, 49, 1–8.
16. X. Men, et al., Ultrasmall Semiconducting Polymer Dots with Rapid Clearance for Second Near-Infrared Photoacoustic Imaging and Photothermal Cancer Therapy, Adv. Funct. Mater., 2020, 30, 1909673.
17. X. Men and Z. Yuan, Polymer dots for precision photothermal therapy of brain tumors in the second near-infrared window: a mini-review, ACS Appl. Polym. Mater., 2020, 2(10), 4319–4330.
18. J. Yu, et al., Recent Advances in the Development of Highly Luminescent Semiconducting Polymer Dots and Nanoparticles for Biological Imaging and Medicine, Anal. Chem., 2017, 89(1), 42–56.
19. R. Vankayala and K. C. Hwang, Near-infrared-light-activatable nanomaterial-mediated phototheranostic nanomedicines: an emerging paradigm for cancer treatment, Adv. Mater., 2018, 30(23), 1706320.
20. G. Kandasamy and D. Maity, Multifunctional theranostic nanoparticles for biomedical cancer treatments-A comprehensive review, Mater. Sci. Eng., C, 2021, 127, 112199.
21. P. Sarbadhikary, B. P. George and H. Abrahamse, Recent advances in photosensitizers as multifunctional theranostic agents for imaging-guided photodynamic therapy of cancer, Theranostics, 2021, 11(18), 9054.
22. Z. Zhang, et al., Semiconducting polymer dots for multifunctional integrated nanomedicine carriers, Mater. Today Bio, 2024, 101028.
23. N. Chen, et al., Rediscovery of traditional plant medicine: An underestimated anticancer drug of chelerythrine, Front. Pharmacol., 2022, 13, 906301.
24. T. Yang, et al., Chelerythrine hydrochloride inhibits proliferation and induces mitochondrial apoptosis in cervical cancer cells via PI3K/BAD signaling pathway, Toxicol. In Vitro, 2020, 68, 104965.
25. T. Funakoshi, et al., Reactive oxygen species-independent rapid initiation of mitochondrial apoptotic pathway by chelerythrine, Toxicol. In Vitro, 2011, 25(8), 1581–1587.
26. M. Liu, et al., Development of certain protein kinase inhibitors with the components from traditional Chinese medicine, Front. Pharmacol., 2017, 7, 523.
27. Z. Wei, et al., Traditional Chinese Medicine has great potential as candidate drugs for lung cancer: A review, J. Ethnopharmacol., 2023, 300, 115748.
28. T. X. Nguyen, et al., Recent advances in liposome surface modification for oral drug delivery, Nanomedicine, 2016, 11(9), 1169–1185.
29. W. Li, et al., The pharmacokinetics and anti-inflammatory effects of chelerythrine solid dispersions in vivo, J. Drug Delivery Sci. Technol., 2017, 40, 51–58.
30. R. K. Kankala, et al., Supercritical fluid technology: an emphasis on drug delivery and related biomedical applications, Adv. Healthcare Mater., 2017, 6(16), 1700433.
31. M. Majidinia, et al., Overcoming multidrug resistance in cancer: Recent progress in nanotechnology and new horizons, IUBMB Life, 2020, 72(5), 855–871.
32. D. Wang, et al., Bacterial vesicle-cancer cell hybrid membrane-coated nanoparticles for tumor specific immune activation and photothermal therapy, ACS Appl. Mater. Interfaces, 2020, 12(37), 41138–41147.
33. A. A. Q. Ahmed, et al., Outer membrane vesicles (OMVs) as biomedical tools and their relevance as immune-modulating agents against H. pylori infections: current status and future prospects, Int. J. Mol. Sci., 2023, 24(10), 8542.
34. X. Wei, et al., Unleashing the power of precision drug delivery: Genetically engineered biomimetic nanodrugs incorporating liposomal polypharmacy against multidrug-resistant bacteria, Chem. Eng. J., 2024, 497, 154515.
35. J. Wang, et al., Bacterial outer membrane vesicle-cancer cell hybrid membrane-coated nanoparticles for sonodynamic therapy in the treatment of breast cancer bone metastasis, J. Nanobiotechnol., 2024, 22(1), 328.
36. B. Rout, T. G. Agnihotri and A. Jain, Advancement in Triple-Negative Breast Cancer Therapeutics: A Comprehensive Review on the Potential of Cell Membrane-Coated Nanoparticles, J. Drug Delivery Sci. Technol., 2024, 105935.
37. S. Mansur, et al., Dual-layer hollow fibre haemodialysis membrane for effective uremic toxins removal with minimal blood-bacteria contamination, Alexandria Eng. J., 2022, 61(12), 10139–10152.
38. M. P. Nikitin, et al., Enhancement of the blood-circulation time and performance of nanomedicines via the forced clearance of erythrocytes, Nat. Biomed. Eng., 2020, 4(7), 717–731.
39. J. K. Maerz, et al., Outer membrane vesicles blebbing contributes to B. vulgatus mpk-mediated immune response silencing, Gut Microbes, 2018, 9(1), 1–12.
40. Y. Li, et al., Bacterial outer membrane vesicles presenting programmed death 1 for improved cancer immunotherapy via immune activation and checkpoint inhibition, ACS Nano, 2020, 14(12), 16698–16711.
41. X. Wei, et al., Supercharged precision killers: Genetically engineered biomimetic drugs of screened metalloantibiotics against Acinetobacter baumanni, Sci. Adv., 2024, 10(12), eadk6331.
42. Y. Liu, et al., Fluorination Enhances NIR-II Fluorescence of Polymer Dots for Quantitative Brain Tumor Imaging, Angew. Chem., Int. Ed., 2020, 59(47), 21049–21057.
43. J. Guo, et al., 4T1 Cell Membrane-Coated Pdots with NIR-II Absorption and Fluorescence Properties for Targeted Phototheranostics of Breast Tumors, ACS Appl. Mater. Interfaces, 2024, 16(48), 66425–66435.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5tb00976f

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