Photoacoustic-imaging nanomotors enhance tumor penetration and alleviate hypoxia for photodynamic therapy of breast cancer

Abstract

Breast cancer is the most prevalent malignancy worldwide, yet conventional therapies are invasive and prone to resistance, recurrence, and metastasis. Photodynamic therapy (PDT) is a promising noninvasive modality, but its efficacy is limited by tumor hypoxia and poor photosensitizer delivery. Here, we report a photoacoustic-imaging nanomotor, PPIC, which addresses these challenges through integrated functions of oxygen production, deep tissue penetration and photoacoustic imaging. The PPIC nanomotor is constructed by covalently anchoring indocyanine green (ICG) and chlorin e6 (Ce6) on Janus mesoporous Pt-organosilica (JMPO) nanoparticles. The Pt of PPIC catalyzes the decomposition of tumor hydrogen peroxide (H₂O₂) to generate oxygen, which replenishes oxygen for PDT and propels the nanomotor to penetrate deeper into the tumor. PPIC produces reactive oxygen species (ROS) to induce tumor cell apoptosis under 660 nm laser irradiation. This integrated design overcomes tumor hypoxia and enhances photosensitizer delivery, enabling effective PDT. In a 4T1 breast cancer model, PA imaging showed the distribution of PPIC in tumors, accompanied by a significant increase in hemoglobin oxygenation signals, indicating effective in situ oxygen generation. PPIC-mediated PDT reduced the hypoxic marker hypoxia-inducible factor-1 (HIF-1α) expression and achieved complete tumor regression in 4 out of 6 (66.7%) treated mice. In vitro and in vivo safety evaluations demonstrated negligible systemic toxicity. These results indicated that PPIC-mediated PDT can overcome tumor hypoxia and significantly improve therapeutic outcomes, providing a promising strategy for effective cancer phototherapy.

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1. Introduction

According to the World Health Organization's International Agency for Research on Cancer (IARC), breast cancer has surpassed lung cancer as the most frequently diagnosed malignancy worldwide, representing 11.6% of all new cancer cases in 2022.¹ Clinically, breast cancer is primarily treated with surgery and chemotherapy. However, surgery is inherently invasive and may lead to complications, cosmetic deformities, and limited effectiveness in advanced or metastatic cases. Moreover, chemotherapy is often associated with significant side effects and the development of drug resistance, leaving limited therapeutic options in cases of recurrence, progression, or metastasis.² Therefore, the development of novel and precise diagnostic and therapeutic approaches is critical for improving outcomes for breast cancer patients.

Photodynamic therapy (PDT) is an increasingly attractive treatment option for breast cancer because of its non-invasiveness, minimal resistance, and spatiotemporal control.^3,4 However, the therapeutic efficacy of PDT is fundamentally constrained by two intrinsic features of solid tumors. The hypoxic microenvironment restricts the oxygen-dependent generation of reactive oxygen species (ROS), while the dense extracellular matrix hinders the deep penetration of photosensitizers.^5–8 To overcome these challenges, researchers have devoted themselves to enhancing both photosensitizer transport and oxygen supply. Nanomotors have made significant strides for biomedical applications^9–11 such as targeted drug delivery and non-invasive microsurgery by enabling the conversion of chemical fuels, electromagnetic waves, light, or ultrasound into self-propelling mechanical motion.^12–16 For example, Wang et al. utilized Chlamydomonas reinhardtii as a natural O₂-producing motor to alleviate tumor hypoxia through photosynthesis, enhancing their tumor accumulation and PDT efficacy.¹⁷ Similarly, He et al. developed an acoustically driven and magnetically guided micro-motor capable of actively transporting O₂ and photosensitizers to enhance PDT.¹⁸ Overall, previous efforts have focused on enhancing tumor penetration and relieving hypoxia.^19–21 However, the imbalance of oxygen generation and photosensitizer distribution in the tumor environment affects the therapeutic efficacy of PDT. It is very important to monitor the generation of O₂ and the distribution of photosensitizers in the tumor microenvironment simultaneously, which is helpful for obtaining better treatment timing and therapeutic benefits.^22–24

Photoacoustic imaging (PAI), a hybrid modality that combines optical contrast with ultrasound resolution,²⁵ provides an ideal platform for simultaneously tracking photosensitizer distribution and O₂ dynamics within the tumor microenvironment.^26,27 Here, we report a PAI nanomotor, denoted as PPIC, constructed by modifying the PAI agent ICG and the photosensitizer Ce6 on Janus mesoporous Pt-organosilica (JMPO) nanoparticles. PPIC effectively generates O₂ through catalyzing the endogenous hydrogen peroxide (H₂O₂), thereby driving the nanomotors to penetrate deep into the tumor microenvironment. Meanwhile, the endogenous H₂O₂ decomposition continuously replenishes O₂ to sustain PDT efficacy. PPIC converts O₂ to singlet oxygen (¹O₂) upon 660 nm laser irradiation, inducing oxidative stress and triggering apoptosis of breast cancer cells. Importantly, PAI is employed to confirm the intratumoral penetration of photosensitizers and to monitor that the nanomotor effectively alleviates tumor hypoxia. The PPIC nanoplatform integrates PA imaging, autonomous motion and sustained oxygen supply, thereby providing an effective theranostic strategy for enhancing PDT performance.

2. Results and discussion

Janus mesoporous Pt-organosilica (JMPO) nanoparticles were first synthesized using a previously reported method by our group.²⁸ In brief, mesoporous Pt nanoparticles were synthesized using F127 as the structure-directing agent and H₂PtCl₆ as the precursor. Mesoporous organosilica was then asymmetrically grown on one side of the mesoporous Pt nanoparticles using cetyltrimethylammonium bromide (CTAB) as the structure-directing agent (Scheme 1). Pt-organosilica nanoparticles were modified with ICG-Mal via a thiol–maleimide click reaction and Ce6 via an amide reaction, respectively. The final product was denoted as PPIC. Transmission electron microscopy (TEM) images of mesoporous Pt nanoparticles showed a uniform size of approximately 80 nm and good dispersity (Fig. 1a). Low- and high-magnification TEM images of JMPO displayed the asymmetric structure and excellent uniformity (Fig. 1b and c). Moreover, the organosilica was coated on one side of the mesoporous Pt nanoparticle, confirming the successful preparation of the heterogeneous structure. Elemental mapping images showed the presence of C, O, Si, and Pt elements in the Janus particles, indicating the composition of mesoporous Pt nanoparticles with mesoporous organosilica (Fig. 1d). High-angle annular dark-field scanning TEM (HAADF-STEM) imaging showed that Pt elements were partially exposed and partially embedded within the mesoporous organosilica, further confirming the heterogeneous structure. In addition, the nitrogen adsorption–desorption isotherm of JMPO showed a typical type IV curve with a hysteresis loop in the relative pressure range of 0.4 to 1.0, indicating the presence of a mesoporous structure (Fig. 1e and f). The pore size distribution curve showed a pore size of approximately 2.8 nm, providing an ideal mesoporous channel for effective modification of ICG and Ce6. The hydrodynamic diameters of JMPO and PPIC were found to be approximately 285 nm and 291 nm, respectively (Fig. 1g). The zeta potentials of JMPO and PPIC were −28.6 mV and −26.05 mV, respectively, indicating that modification with ICG and Ce6 did not significantly alter the surface charge of the nanomotor (Fig. 1h).

Scheme 1 Illustration of the fabrication of the photoacoustic-imaging nanomotor (PPIC) and PA imaging-directed PDT for breast cancer.

Fig. 1 (a) TEM image of mesoporous Pt nanoparticles. (b and c) TEM images of JMPO. The inset shows the cartoon of JMPO. (d) HAADF-STEM image and elemental mapping of JMPO. (e) Nitrogen adsorption–desorption isotherm, (f) pore size distribution curve, (g) dynamic light scattering (DLS), and (h) zeta potentials of JMPO and PPIC.

The UV-visible spectra of Ce6, ICG, JMPO, and PPIC were measured to assess the chemical modification of Ce6 and ICG on JMPO (Fig. 2b). PPIC exhibited an absorption peak at 660 nm, corresponding to the absorption peak of Ce6, confirming the successful modification of Ce6. The loading amount of Ce6 was 80 μg per milligram of JMPO using a standard curve generated from UV absorbance measurements at 660 nm (Fig. 2c and Fig. S1a).²⁹ In addition, PPIC displayed an absorption peak at 815 nm, indicating the successful modification of ICG. The calculation showed that 12.8 μg of ICG was chemically modified per milligram of JMPO (Fig. 2d and Fig. S1b). To investigate the efficiency of ROS generation for PPIC, we used 1,3-diphenylisobenzofuran (DPBF) as an indicator to detect the production of ¹O₂ (Fig. 2a).³⁰ The UV-visible absorption spectra showed a decrease in the DPBF characteristic peak after laser irradiation, showing that PPIC generates ¹O₂ (Fig. 2e and f).

Fig. 2 (a) Schematic illustration of the detection of ROS generated from PPIC. Ultraviolet-visible absorption spectra of (b) ICG, Ce6, PPIC and JMPO, (c and d) different concentrations of Ce6 and ICG, and (e and f) PPIC (e) and PBS (f) containing DPBF, respectively. (g) Optical photos of PPIC and mesoporous Pt nanoparticles (MPN) after the addition of H₂O₂ at 1 min (left) and 5 min (right). (h) The motion trajectories, (i) MSD, (j) hydrodynamic size distribution, (k) diffusion coefficient, and (l) velocity of PPIC, which is treated with H₂O₂ (20 mM) or not.

Next, we conducted in vitro studies to evaluate the O₂ generation and movement of PPIC. Optical images showed that PPIC produced numerous bubbles upon the addition of 0.5 mL of 100 mM H₂O₂, whereas no bubbles appeared in the H₂O₂ solution group (Fig. 2g). This was attributed to the reaction between Pt of PPIC and H₂O₂, resulting in the production of O₂.^31,32 The Pt particle group produced many bubbles within 1 min after adding H₂O₂, while PPIC generated bubbles more gently and persistently. The PPIC group produced bubbles continuously for 5 min with a noticeable increase in the liquid level, indicating significant bubble production.

PPIC exhibited a wider range of motion and more directional movement in the presence of H₂O₂, indicating that H₂O₂ enhances the motility of PPIC (Fig. 2h). To quantify this behavior, we extracted the mean squared displacement (MSD) scatter plot from the recorded movement trajectories of PPIC using nanoparticle tracking analysis (NTA) (Fig. 2i). The MSD of PPIC increased markedly upon H₂O₂ addition, indicating enhanced self-propulsion. This observation was further supported by a leftward shift in the hydrodynamic size distribution, with the apparent hydrodynamic diameter decreasing from 276.5 nm to 221.3 nm after adding 20 mM H₂O₂ (Fig. 2j). The shift of hydrodynamic diameter in DLS analysis is ascribed to increased translational diffusivity rather than a reduction in physical size. Specifically, the diffusion coefficient of PPIC increased from 0.98 μm² s⁻¹ without H₂O₂ to 2.54 μm² s⁻¹ following H₂O₂ addition (Fig. 2k). Similarly, the velocity of PPIC increased from 1.36 μm s⁻¹ to 3.32 μm s⁻¹ under the same conditions (Fig. 2l). Overall, the results showed that H₂O₂ alters the motion behavior of PPIC and significantly improves its propulsion efficiency by increasing both the diffusion coefficient and velocity.

Building on the O₂ generation and motion characteristics of the PPIC nanomotor, we further evaluated its biocompatibility, cellular uptake, and therapeutic efficacy. PPIC exhibited minimal cytotoxicity across multiple cell lines, including 4T1, MCF-7, and 3T3 cells, with cell viability remaining above 80% after 24 h of incubation at various concentrations (Fig. 3a). Notably, PPIC showed superior biocompatibility compared to free Ce6, particularly at higher doses (Fig. 3b). To further evaluate hemocompatibility, the whole blood was incubated with PPIC at concentrations ranging from 12.5 to 100 μg mL⁻¹. The hemolysis rate remained at 6.2–7.6%, confirming the blood compatibility of PPIC (Fig. S2).

Fig. 3 (a and b) Cytotoxicity of PPIC and Ce6 via incubation with 4T1, MCF-7, and 3T3 cells for 24 h (n = 6). (c) Schematic illustration of the penetration of PPIC against multicellular spheroids incubated. (d) CLSM Z-stack scanning images at 40 μm and surface plot images of 4T1-multicellular spheroids cultured with PPIC. (e) Relative fluorescence intensity of MCS sections cultured with PPIC at different depths. (f) CLSM images of 4T1 cells co-incubated with PPIC. (g) CLSM images of 4T1 cells stained with DAPI (blue) and RDPP (green) after different treatments for 4 h under hypoxic conditions. (h) Fluorescence images of 4T1 cells stained with the ROS fluorescent probe after various treatments. (i) Cell survival of 4T1 co-incubated with different materials and then irradiated with a laser for 3 min (n = 4). (j and k) Live/dead staining and flow cytometry assay of 4T1 cells after undergoing various treatments. The laser irradiation parameters were set at 660 nm and 1 W cm⁻². All data are shown as mean ± SD. p values were calculated by one-way ANOVA (*P < 0.05 and ****P < 0.0001, and “ns” indicates not significant).

To evaluate the in vitro penetration capability of PPIC, we constructed a 3D multicellular spheroid (MCS) model (Fig. 3c). MCSs were incubated with PPIC and subsequently stimulated with 2 mM H₂O₂ for 5 min. Under stimulation with H₂O₂, the MCSs displayed significantly higher fluorescence signals at a scanning depth of 40 μm, suggesting enhanced penetration of PPIC into deeper regions of the MCSs (Fig. 3d). Quantitative analysis showed that the average fluorescence intensities in the H₂O₂-treated group were 82.3, 74.8, and 65.7 at depths of 40, 50, and 60 μm, respectively, which were higher than those without adding H₂O₂ (Fig. 3e). These results confirmed that H₂O₂ enhances PPIC penetration into MCSs, suggesting that PPIC can effectively infiltrate tissue and deliver photosensitizers to deeper regions in tumors with elevated H₂O₂ levels.

Next, we evaluated the cellular uptake of PPIC in 4T1 breast cancer cells (Fig. 3f). Fluorescence microscopy showed a time-dependent increase in Ce6 signal, indicating efficient internalization of PPIC by 4T1 cells. Based on the time-dependent observations, a 12 h incubation period was selected for the subsequent assays. Ru(dpp)₃Cl₂ (RDPP) and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were used as fluorescent probes to investigate intracellular oxygen generation and ROS production. Confocal laser scanning microscopy (CLSM) images revealed a significant decrease in the RDPP fluorescence intensity in the PPIC + H₂O₂ group relative to untreated controls and cells treated with PPIC alone (Fig. 3g), confirming that PPIC catalyzes H₂O₂ decomposition to produce oxygen and relieve hypoxia. In addition, PPIC-treated cells exhibited markedly increased green fluorescence of DCFH-DA following 660 nm laser irradiation (Fig. 3h), showing efficient production of ROS toward killing cancer cells.

Cell viability after in vitro PDT was evaluated by a CCK-8 assay (Fig. 3i). The control groups, including PBS, PBS + laser and JMPO + laser, exhibited negligible cytotoxicity. In contrast, PPIC co-incubated with 4T1 cells and exposed to 660 nm laser irradiation reduced cell viability to approximately 18.3%, showing potent photodynamic-mediated cytotoxicity. Time-dependent studies revealed that as few as 3 min of laser exposure were sufficient to lower cell viability in the PPIC group, while laser treatment alone had negligible effects (Fig. S3), suggesting that the cytotoxicity was attributable to ROS generation mediated by PPIC under laser activation. CLSM and the apoptosis assay further confirmed that PPIC exhibited excellent cytotoxicity and superior PDT efficacy under laser irradiation (Fig. 3j and k).

PAI enables noninvasive spatiotemporal tracking of photosensitizer distribution, offering a valuable imaging strategy for directing PDT. Therefore, a time-resolved PAI analysis in 4T1 tumor-bearing nude mice was conducted. Tumors treated with PPIC showed obvious photoacoustic signals over 24 h (Fig. 4a). PPIC-treated tumors exhibited a broader photoacoustic signal distribution and approximately 4-fold higher signal intensity compared to the periodic mesoporous organosilica nanoparticle-modified ICG and Ce6 (PIC) groups at 24 h post-injection (Fig. 4b). This highlights the superior tumor penetration and imaging capabilities of PPIC, underscoring its potential as an efficient agent for image-guided PDT. For oxygenation monitoring, hemoglobin oxygen saturation (HbO₂) signals were used as an indirect indicator of tumor oxygen levels.³³ PA imaging revealed a gradual increase in HbO₂ levels following the administration of the PPIC nanoparticle (Fig. 4c). Notably, HbO₂ levels peaked at 3 h post-injection, reaching a maximum intensity of 62.3% (Fig. 4d). This indicates that PPIC effectively catalyzes the decomposition of endogenous H₂O₂ to O₂, thereby alleviating the hypoxic tumor microenvironment.

Fig. 4 (a) In vivo PA/US images of nude mice after the injection of PPIC and PIC nanoparticle solution (10 mg kg⁻¹) at different time points. (b) Quantitative PA signal of the tumor at 24 h. (c) PA/US images of HbO₂ of tumor tissues after the injection of PPIC at different time points. (d) HbO₂ signal intensity of the tumor corresponding to (c). All data are shown as mean ± SD. p values were calculated by t-unpaired two-tailed Welch’ s t-test (****P < 0.0001, n = 3).

Encouraged by the promising in vitro results, the in vivo synergistic anticancer effects of the PPIC nanomotor were investigated on a 4T1 tumor-bearing mouse model. BALB/c mice bearing subcutaneous breast tumors were assigned to the following groups: PBS, PPIC, Ce6 + laser and PPIC + laser (Fig. 5a). Continuous 660 nm laser irradiation (1 W cm⁻², 5 min) was applied at 6 h following tail-vein administration of different therapeutic agents. The tumors exhibited rapid growth in the PBS group, owing to the high malignancy of the model (Fig. 5b and c). Moderate tumor growth inhibition was observed initially in both the PPIC and Ce6 + laser groups. However, tumor volumes began to increase again after day 8 for the groups, suggesting limited and transient efficacy. In contrast, the PPIC + laser group exhibited a continuous tumor volume reduction throughout the treatment period, with complete tumor regression achieved in four mice. These results indicated that the combination of PPIC and laser irradiation significantly enhances PDT efficacy and enables effective tumor eradication in vivo. At the end of the 14-day treatment period, tumors were excised and weighed. The PPIC + laser group showed the lowest tumor mass among all the groups (Fig. 5e and f), further confirming the enhanced therapeutic effect of the combined strategy. In addition, body weight monitoring revealed no significant weight loss during the treatment in any group (Fig. 5d), indicating good biocompatibility and low systemic toxicity of the PPIC formulation.

Fig. 5 In vivo antitumor efficacy and mechanistic analysis of the photoacoustic-imaging nanomotor, PPIC. (a) Schematic illustration of the 4T1 tumor model establishment and therapeutic regimen. (b and c) Tumor volume curves during the 14-day treatment period. (d) Dynamic changes in the body weights of mice across the groups. (e) Final tumor weights and (f) representative photographs of the excised tumors. (g) H&E staining of tumor tissues, showing the tumor cell morphology. (h) HIF-1α staining (red) and (i) immunofluorescence analysis of Ki67 expression (green) in the tumor sections. (+) represents the treatments with a laser. All data are shown as mean ± SD. p values were calculated by one-way ANOVA (*P < 0.05, ***P < 0.001 and ****P < 0.0001, n = 6).

At the end of the treatment, blood samples and major organs were collected for the subsequent analysis. Hematoxylin and eosin (H&E) staining of the major organs in all the groups revealed normal histological characteristics, indicating minimal systemic toxicity (Fig. 5g). Tumor tissues were subsequently subjected to histological analysis. Notably, tumor sections from the PPIC + laser group displayed an almost complete absence of spindle-shaped cancer cell nuclei, whereas the other groups exhibited a higher density of cancer cell nuclei (Fig. 5g). This indicated that PPIC under laser irradiation effectively inhibits cancer cell proliferation, thereby exhibiting superior PDT efficacy. HIF-1α expression related to tumor hypoxia was analyzed by immunofluorescence (Fig. 5h). The PBS and Ce6 groups exhibited a high expression of HIF-1α, which was almost undetectable in the PPIC + laser group. Ki67 immunofluorescence staining showed diminished green fluorescence in the PPIC + laser group, suggesting a significant reduction in tumor cell proliferation (Fig. 5i). These results suggested that the PPIC nanomotor can significantly suppress tumor growth in vivo. Collectively, these findings showed that PPIC-mediated PDT exhibits potent anti-tumor activity, highlighting its potential as an effective and safe photodynamic therapeutic platform.

To rigorously assess the biosafety of PPIC for potential clinical translation, in vivo toxicity was further investigated via hematological profiling and histopathological examination. Blood parameters, including white blood cell count, hemoglobin levels, and platelet counts, all fell within normal physiological ranges in PPIC-treated mice (Fig. 6a). No significant differences in the organizational structure were observed between the PPIC group and the PBS control group (Fig. 6b). These results showed that PPIC exhibits negligible systemic toxicity and outstanding biocompatibility, fulfilling the essential criteria for further clinical development.

Fig. 6 Preliminary toxicity analysis: blood chemistry analysis and histological analysis of the major organs. (a) Blood samples were collected after 14 days for further analysis. The following parameters were measured: hematocrit (HCT), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), erythrocyte mean corpuscular volume (MCV), red cell distribution width (RDW), white blood cells (WBCs), mean platelet volume (MPV), platelet distribution width (PDW) and red blood cells (RBCs) (n = 3, mean ± SD). (b) H&E staining of the major organs, including the heart, liver, spleen, lungs, and kidneys.

3. Conclusion

In this study, we developed a photoacoustic-imaging nanomotor for photodynamic therapy. The PPIC nanomotor possesses a uniform diameter of approximately 291 nm and a unique asymmetric structure and exhibits H₂O₂-driven self-propulsion, which collectively enable enhanced tissue penetration and active delivery of photosensitizers. In vitro assays showed that the PPIC-mediated delivery of Ce6 increased tumor cell killing by 81.7%, and cytotoxicity and hemolysis tests confirmed excellent biocompatibility. PA imaging showed the distribution of PPIC in tumors, accompanied by a significant increase in hemoglobin oxygenation signals, indicating effective in situ oxygen generation. The PPIC nanomotor induced tumor regression and achieved complete responses in four of six mice under 660 nm laser irradiation. Hematological analyses, organ histology analysis, and body weight monitoring further verified the biosafety of the PPIC platform, validating the PAI-guided PDT platform as a safe and highly effective approach for the treatment of breast cancer. In the future, the long-term biosafety of the nanomotor requires systematic validation before clinical translation. Future efforts can also be devoted to using clinically approved materials and multimodal imaging strategies for tracking the penetration of nanomotors in the tumor environment.

4. Experimental section

4.1 Materials

Cetyltrimethylammonium bromide (CTAB, ≥99%), concentrated ammonia aqueous solution (NH₃·H₂O, 25 wt%), hydrochloric acid (HCl, 37%), anhydrous ethanol, potassium bromide (KBr, ≥99%), hydrogen peroxide (H₂O₂, 30% w/w, GR) and chloroplatinic acid hexahydrate (H₂PtCl₆·6H₂O) were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). L-Ascorbic acid (L-AA, ≥99%), Pluronic F127 (PEO₁₀₆PPO₇₀PEO₁₀₆, M_w = 12 600), 3-mercaptopropyltriethoxy silane (MPTES, 97%), 1,2-bis(triethoxysilyl)-ethane (BTSE, 96%), (3-aminopropyl) triethoxysilane (APTES, 98%), Ru(dpp)₃Cl₂ (RDPP), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, ≥98%), N-hydroxysulfosuccinimide (NHS, ≥98%), and chlorine e6 (Ce6) were purchased from Sigma-Aldrich Co., Ltd (Shanghai, China). Indocyanine green maleimide (ICG-Mal) was purchased from J&K Scientific Ltd (Beijing, China). 4,6-Diamino-2-phenylindole (DAPI), phosphate buffered saline (PBS, pH 7.4), Dulbecco's modified Eagle's medium (DMEM, high glucose), and cell counting kit-8 (CCK-8) were purchased from Nanjing Keygen Biotech Co., Ltd (Nanjing, China). Fetal bovine serum (FBS) was purchased from BioChannel Biological Technology Co., Ltd (Nanjing, China). 1,3-Diphenylisobenzofuran (DPBF) and 20,70-dichlorofluorescein diacetate (DCFH-DA) were obtained from Beyotime Biotechnology (Shanghai, China). Deionized water (≥18M Ω cm) was obtained from a Millipore water purification system. All animal experimental procedures were reviewed and approved by the Animal Protection Committee of Jinling Hospital, which is affiliated with Nanjing University. The animal experiments were conducted in strict accordance with the guidelines provided by the Animal Protection Committee.

4.2 Characterization studies

Transmission electron microscopy (TEM) images were conducted at 100 kV using a Hitachi HT7700 (Japan). High-angle annular dark-field scanning TEM (HAADF-STEM) and energy dispersive X-ray (EDX) analyses were conducted on a FEI Talos F200X electron microscope at 200 kV, equipped with an EDX detector system. UV-Vis spectra were recorded using a Shimadzu UV-3600 (Japan). Zeta potentials and hydrodynamic sizes were measured using a Brookhaven ZetaPALS analyzer (USA). Nitrogen sorption isotherms were obtained using a Micromeritics ASAP 2020 analyzer at −196 °C. Fluorescence imaging was performed using a Leica inverted fluorescence microscope (Germany). Cell apoptosis analysis was conducted using a MoFlo XDP flow cytometer (Beckman Coulter, USA). Photoacoustic (PA) imaging was performed using the Vevo® LAZR multi-mode, high-resolution photoacoustic/ultrasonic imaging system (Canada).

4.3 Preparation

First, 1.8 g of F127 and 4 g of KBr were dissolved in 60 mL of 0.1 M L-AA aqueous solution using ultrasonication. After adding 1 mL of 0.2 M H₂PtCl₆ aqueous solution, the mixture was incubated at 70 °C in a water bath for 24 h. The resulting product was collected via centrifugation (10 000 rpm, 5 min) and washed three times with deionized water.

Next, 0.12 g of CTAB was dissolved in a mixture of 60 mL of deionized water and 4 mL of anhydrous ethanol, followed by the dispersion of 4.8 mg of mesoporous platinum nanoparticles under stirring. The solution was maintained in a 35 °C water bath and stirred at 500 rpm for 15 min. After adding 2.16 mL of ammonia, the solution was thoroughly mixed and ultrasonicated for 10 min. Then, 40 μL of BTSE was added, and the mixture was stirred at 1000 rpm for 4 h at 35 °C. The final product was dispersed in 200 mL of ethanol containing 400 μL of concentrated hydrochloric acid. To remove the CTAB template, the solution underwent three extraction cycles at 60 °C. The resulting material was then washed three times with ethanol, yielding Janus mesoporous platinum-organosilica (JMPO) nanocomposites.

Subsequently, 300 μL of MPTES was added to 5 mL of JMPO ethanol solution (1 mg mL⁻¹) and allowed to react for 12 h. After centrifugation (10 000 rpm, 10 min) and washing with ethanol 3 times, the product was redispersed in 5 mL of ethanol. Mal-ICG was conjugated on the JMPO surface via a thiol–maleimide click reaction. 100 μL of ICG-Mal ethanol solution (1 mg mL⁻¹) was added to the mixture and incubated for another 12 h. After three ethanol washes, the JMPO-ICG product was obtained and stored in 5 mL of ethanol. Surface modification of JMPO-ICG was achieved using the organosilica source precursor APTES. Subsequently, Ce6 was conjugated to the nanoplatform via an amide reaction between the amine groups and the carboxyl groups of the photosensitizer. 500 μL of APTES was added to the above-prepared 5 mL JMPO-ICG ethanol solution and reacted for 12 h. Following centrifugation (10 000 rpm, 10 min) and washing with ethanol 3 times, the product was redispersed in 5 mL of ethanol. Separately, 2 mg of Ce6, 2 mg of EDC and 2 mg of NHS (each dissolved in 20 mg mL⁻¹ DMF) were mixed and allowed to react in the dark at room temperature for 3 h on a shaker. This activated Ce6 solution was then added to the functionalized JMPO-ICG and further reacted in the dark for 12 h. After three ethanol washes, the final product, PPIC, was obtained and stored in 5 mL of ethanol.

4.4 Detection of ROS and O₂ production in vitro

The generation of ROS was assessed by DPBF assays. Briefly, 3 mL of PPIC (20 μg mL⁻¹) in PBS was mixed with 10 μL of DPBF (20 mM in DMSO). For comparison, PBS alone was exposed to 660 nm laser irradiation at a fixed interval (1 W cm⁻²). The absorbance intensity of DPBF was recorded using UV-Vis spectra. To assess O₂ production, 3 mL of PPIC and Pt nanoparticle aqueous solution with different concentrations were mixed with 0.5 ml of 20 mM H₂O₂, and the formation of O₂ bubbles was observed.

4.5 Cytotoxicity and hemolysis

3T3, MCF-7 and 4T1 cells were purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (Beijing, China). Cells were cultured in DMEM supplemented with 10% (v/v) FBS at 37 °C under 5% CO₂. Cytotoxicity was assessed via cell viability analysis. Briefly, 4T1, MCF-7 and 3T3 cells were seeded in 96-well plates (100 μL per well) and incubated with PPIC at various concentrations (10, 25, 50, 75, and 100 μg mL⁻¹) for 24 h. Following incubation, cells were washed three times with PBS and further incubated in fresh DMEM containing 10 μL of CCK-8 solution for 1 h. Cell viability was quantified by measuring absorbance at 450 nm using a microplate reader.

For hemolysis analysis, 700 μL of blood was mixed with 300 μL of PPIC with different concentrations (10, 25, 50, 75, and 100 μg mL⁻¹) and incubated at 37 °C for 4 h. H₂O and PBS served as the positive and negative controls, respectively. The supernatants were collected, and the absorption values were measured at 416 nm. The hemolysis rate was calculated by using the following formula:

4.6 Cell uptake

4T1 breast cancer cells were seeded at a density of 1 × 10⁴ cells per well and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin under 95% relative humidity and 5% CO₂ at 37 °C. After 12 h of cell adhesion, cells were incubated with 100 μL of PPIC suspension in complete DMEM for varying durations. Cells were then washed three times with PBS, resuspended in 100 μL of PBS, and imaged using a CLSM. Fluorescence intensity and localization were analyzed quantitatively using ImageJ software.

4.7 Tumor penetration assessment in three-dimensional multicellular spheroids (MCSs)

An in vitro MCS model was established using 4T1 cells. First, 96-well plates were pre-coated with 50 μL of hot agarose solution (1.5% w/v) and then cooled to room temperature. Then, 4T1 cells (10 000 cells per well) were seeded in the pre-coated wells and incubated at 37 °C for 4 days to allow spheroid formation. MCSs were incubated with PPIC (50 μg mL⁻¹) for 1 h, followed by the addition of an equal volume of saline or H₂O₂ (2 mM) for 5 min. Spheroids were then harvested, washed with PBS, and imaged using CLSM. Fluorescence intensity and penetration depth were analyzed using ImageJ.

4.8 In vitro antitumor activity

In vitro O₂ measurement using RDPP. To evaluate the O₂ production capacity of PPIC, 4T1 cells (1 × 10⁵ cells per dish) were seeded in confocal dishes and incubated at 37 °C for 24 h. The cells were then cultured under hypoxic conditions (2% O₂) overnight. Following this, the cells were incubated with a fresh medium containing RDPP (5 μM) at 37 °C for 30 min. After different treatments under hypoxia (2% O₂) for 4 h, fluorescence images were captured using a CLSM with excitation at 485 nm and emission at 545 nm.

Intracellular ROS production assay. Intracellular ROS production was evaluated using DCFH-DA staining. 4T1 cells were seeded in CLSM-compatible culture dishes and incubated at 37 °C for 24 h, followed by co-incubation with PPIC (75 μg mL⁻¹) for an additional 12 h. After removing the medium and washing with PBS three times, cells were incubated with DCFH-DA in DMEM for 40 min. Subsequently, cells were exposed or not exposed to 660 nm laser irradiation (1.0 W cm⁻², 3 min). Intracellular ROS levels were then quantified by measuring the fluorescence intensity. Fluorescence images were then acquired using a CLSM with an excitation wavelength of 488 nm and emission collected at 525 nm.

Cell viability assay. 4T1 cells (1 × 10⁴ cells per well) were seeded into 96-well plates and incubated for 24 h. Cells were then treated with 100 μL of PPIC, JMPO and Ce6 for 12 h, respectively. The culture medium was aspirated and the cells were rinsed three times with PBS. 100 μL of fresh complete medium was added, and laser irradiation was performed for 3 min (660 nm laser, power: 1 W cm⁻²). The culture medium in each well was aspirated and replaced with 100 μL of complete medium containing 10 μL of CCK-8. After 1 h, the absorbance of each well at 490 nm was detected using an enzyme labeling instrument. Each experimental group had 4 replicate wells.

Flow cytometry analysis. 4T1 cells were cultured in 6-well plates at a cell density of 1 × 10⁵ cells per well for 24 h. The culture medium was replaced by fresh DMEM containing PPIC (75 μg mL⁻¹). After incubation for 12 h, 4T1 cells were dealt with/without laser irradiation (660 nm laser, 1 W cm⁻², 3 min). After an additional 12 h of co-culture, all treated cells were harvested and incubated with Annexin V-FITC (10 μg mL⁻¹) and PI (5 μg mL⁻¹) for 30 min. The fluorescence of FITC/PI was detected by using a flow cytometer.

4.9 Photoacoustic imaging

We performed time-resolved PAI in BALB/c nude mice bearing subcutaneous 4T1 tumors, established by injecting 1 × 10⁶ 4T1 cells into the right flank. The photoacoustic signal intensity of ICG was monitored over time, serving as an indicator of PPIC distribution and enrichment. The O₂ generation capacity of the nanoparticles in tumors was evaluated by monitoring the HbO₂ signal intensity from the PA images. PA imaging was performed using a multimodal, high-resolution small animal photoacoustic/ultrasound imaging system (Vevo® LAZR). Baseline PA images of tumors in nude mice (n = 3) were acquired prior to nanoparticle injection. After injecting the nanoparticle solution (10 mg kg⁻¹) into the tumor, images of the tumor were taken at 0, 1, 3, 5, 7 and 24 h after injection, respectively.

4.10 Tumor photodynamic therapy

BALB/c mice (14–16 g) were purchased from GemPharmatech Co., Ltd. 5 × 10⁵ 4T1 cells were inoculated into the armpit of the mice to establish a 4T1 breast tumor-bearing mouse model. In vivo antitumor experiments were conducted when the sizes of tumors reached 80–120 mm³. Female BALB/c mice bearing 4T1 tumors were randomly assigned into 4 groups (n = 6 each) for different treatments: control group (untreated), PPIC group, Ce6 + laser and PPIC + laser group (injected with PPIC nanoparticle solution through the tail vein). The nanoparticle dose was 100 μL at a concentration of 1 mg mL⁻¹. For the laser treatment groups, 660 nm laser irradiation was applied at a power density of 1 W cm⁻² for 5 min. Mouse body weight and tumor volume were measured every 2 days.

4.11 Blood chemistry analysis

At the end of the treatment period, BALB/c mice were anesthetized with isoflurane, and approximately 0.8 mL of blood was collected via retro-orbital sinus puncture using a heparin-free capillary tube. Samples were allowed to clot at room temperature for 30 min, followed by centrifugation at 3000 rpm for 10 min to obtain the serum. Serum samples were immediately transported on ice to Nanjing Keygen Biotech Co., Ltd (Nanjing, China) for analysis. Hematocrit (HCT), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), erythrocyte mean corpuscular volume (MCV), red cell distribution width (RDW), white blood cells (WBCs), mean platelet volume (MPV), platelet distribution width (PDW) and red blood cell (RBC) levels were determined using an automatic biochemical analyzer (Dimension EXL 200, Siemens Healthcare Diagnostics Inc., Newark, DE, USA) according to the company's standard protocols.

4.12 Pathological analysis

Fourteen days after the mice received photodynamic therapy, all mice were sacrificed, and the tumors and major organs were dissected and fixed in 4% formaldehyde for 24 h. They were then embedded in paraffin and stained with hematoxylin and eosin (H&E) or immunostained for Ki67 or HIF-1α. The stained slides were then examined using the TEKSQRAY Slide Scan System SQS1000 (Shenzhen Shengqiang Technology Co. Ltd, China).

4.13 Statistical analysis

All quantitative data are presented as mean ± standard deviation (SD) unless otherwise stated. Statistical analyses were performed using GraphPad Prism 9.0 software (GraphPad Software, San Diego, CA, USA). Between-group comparisons were performed using unpaired two-tailed Welch's t-test. Multiple group comparisons were performed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. A p-value of less than 0.05 was considered statistically significant.

Author contributions

T. Y. S.: conceptualization, data curation, writing – original draft, and writing – review & editing. Y. W., Z. W. Z. and J. Y. X.: data curation, methodology and project administration. X. Z. S. and M. D.: data curation and investigation. J. L.: validation and resources. L. Z.: data curation, visualization and writing – review. X. L. M. and K. M.: resources and writing – review. Y. X. T.: methodology, resources, supervision and writing – review. Z. G. T.: conceptualization, writing – review & editing, supervision, project administration 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

The data supporting this article have been included as part of the SI. SI contains calibration curves of Ce6 and ICG, hemolysis assay results, and cell viability data under different laser irradiation times. See DOI: https://doi.org/10.1039/d5bm01112d.

Acknowledgements

We are thankful for financial support from the National Natural Science Foundation of China (No. 22275099), the Project of State Key Laboratory of Organic Electronics and Information Displays from the Nanjing University of Posts & Telecommunications (No. GDX2022010014), a key project grant by the Medical Science and Technology Development Foundation, the Nanjing Department of Health (ZKX21023), and the Postgraduate Research & Practice Innovation Program of Jiangsu Province (SJCX23_1404).

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