A photoactivatable nano-liposome containing tripartite therapeutics for photothermal-triggered chemotherapy

Abstract

Chemotherapy represents a conventional method for cancer treatment, but it inevitably has the issues of low clinical efficacy, therapy resistance and severe side effects. In view of the unique characteristics of nanosystems that can deliver drugs in an effective and safe manner, we report photoactivatable nano-liposomes containing a hypoxia-responsive prodrug tirapazamine (TPZ), glucose oxidase (GOx) and indocyanine green (ICG) for photothermal-triggered chemotherapy of subcutaneous metastatic breast cancer. The nano-liposomes (termed IGT@NPs) are fabricated using a thermo-responsive liposome component to enable photoactivatable drug delivery via the photothermal effect. IGT@NPs mediate the local temperature increase under near-infrared (NIR) laser irradiation, not only allowing for photothermal therapy (PTT), but also achieving on-demand TPZ and GOx release. In the tumor microenvironment, GOx catalyzes the consumption of glucose and oxygen, resulting in aggravated hypoxia levels. As a consequence, TPZ is activated through the aggravated hypoxic microenvironment to trigger the chemotherapeutic action. Therefore, photothermal-triggered chemotherapy is achieved by IGT@NPs, which leads to the effective inhibition of primary tumor growth and metastatic tumor occurrence in subcutaneous 4T1 tumors. This current study thus provides a photoactivatable nanosystem containing tripartite therapeutics for cancer treatment with controllable and combined functions.

---

Introduction

Chemotherapy is one of the main means of cancer treatment involving the use of drugs to kill cancer cells or inhibit their growth.^1–3 Unlike local effects of surgery or radiotherapy, chemotherapy drugs can reach the whole body through the blood circulation, thus showing the ability to kill cancer cells in both primary and metastatic sites and reduce the risk of recurrence.^4–7 However, due to the low selectivity of drugs, chemotherapy can also damage normal cells causing serious side effects.^8–10 In addition, some types of cancer cells will develop resistance to chemotherapy drugs, which results in treatment failure or recurrence.^11–15 In clinical practices, chemotherapy is often used in combination with other therapeutic strategies such as targeted therapy and immunotherapy to improve the efficacy, but the potential off-target effects and therapy resistance still limit the further applications.^16–18

Photothermal therapy (PTT) is a minimally invasive cancer treatment technology based on the photothermal conversion effect.^19–22 PTT uses near-infrared (NIR) light to irradiate photothermal agents (such as gold nanorods, carbon nanomaterials and semiconducting polymers) generating localized high temperatures at the tumor sites, to selectively kill cancer cells while reducing the damage to normal tissues.^23–25 Compared with traditional tumor treatment methods, PTT has the advantages of minimal invasiveness, high spatiotemporal selectivity and low systemic toxicity.^26–28 Because of the limited tissue penetration capability of light, PTT is particularly suitable for superficial tumors or deep-seated tumors via light delivering technology.²⁹ However, excessive temperature will also damage the nearby normal tissues, but mild heat leads to unsatisfactory antitumor effects due to the heat shock protein expression of tumor cells.^30–34 Thus, PTT-combined chemotherapy has emerged as a promising strategy for cancer treatment.^35–37 Such a combination can improve the therapeutic outcomes via overcoming therapy resistance, and also obviously relieve the side effects by reducing the drug dose.^38–40

Nanoparticle-based drug delivery systems have provided a promising tool for combining PTT with chemotherapy as both drugs and photothermal agents can be integrated.⁴¹ Nevertheless, most of these systems only achieve the combined action of PTT and chemotherapy, but fail to precisely deliver drugs into the location of the lesion, and thus their therapeutic efficacy and selectivity still need to be improved.^42–45 In contrast, intelligent nanosystems that can be activated by tumor microenvironment characteristics (such as enzymes, hypoxia, reactive oxygen species and acidic pH) to achieve drug release have been reported.^46–48 Because PTT involves light irradiation, photoactivatable nanosystems that show well-controlled drug delivery only after the PTT effect can offer a more precise and effective treating strategy compared to tumor microenvironment-responsive nanosystems.^49–51 In view of the complicated tumor microenvironment that limits therapeutic effects, photoactivatable nanosystems with well-designed components and functions should be expected for effective cancer therapy.

In this study, a photoactivatable nano-liposome containing tripartite therapeutics is fabricated for treating subcutaneous metastatic breast cancer through photothermal-triggered chemotherapy. Via co-loading of a photothermal agent indocyanine green (ICG), glucose oxidase (GOx) and a hypoxia responsive prodrug tirapazamine (TPZ) in thermal-responsive liposomes, IGT@NPs are constructed (Fig. 1a). As DSPE-PEG could form a hydrophobic barrier on the liposome surface, enhancing particle stability and prolonging the circulation time, DPPC, as the main component, ensured the biocompatibility and membrane fluidity of the carrier, which was favorable for drug encapsulation and release regulation. The resulting IGT@NPs have good a photothermal effect and stability, and the release of GOx and TPZ can be controlled under NIR laser irradiation. Via an effective enrichment into tumors after systemic injection and local irradiation of tumors by NIR light, a temperature increase is triggered for PTT and on-demand releases of TPZ and GOx by destroying the thermal-responsive liposome shell. Besides PTT, the released GOx consumes oxygen to further aggravate tumor hypoxia, which leads to the activation of TPZ for chemotherapy (Fig. 1b). In the subcutaneous 4T1 tumor mice and 4T1 tumor-derived bone metastasis models, this photothermal-triggered chemotherapy by IGT@NPs is demonstrated to have a much higher efficacy in inhibiting tumor growth and metastases compared to PTT alone.

Fig. 1 The design of photoactivatable IGT@NPs for photothermal-triggered chemotherapy. (a) The schematic showing the construction method of IGT@NPs. (b) The schematic showing antitumor and anti-metastasis mechanisms of IGT@NPs via photothermal-triggered chemotherapy.

Results and discussion

Photothermal effect and drug release analysis

A liposomal synthesis procedure was used to fabricate IGT@NPs containing ICG, GOx and TPZ.^52,53 I@NPs containing only ICG were also fabricated as the control counterparts. TEM analysis showed that IGT@NPs and I@NPs had similar spherical shapes (Fig. 2a). The hydrodynamic diameters were also similar, which were 27.8 and 32.3 nm for IGT@NPs and I@NPs, respectively (Fig. 2b). The zeta potentials were measured as −40.3 and −44.3 mV for IGT@NPs and I@NPs, respectively (Fig. 2c). Moreover, in order to assess the sample's stability, its particle sizes remained virtually constant over a period of fourteen days (Fig. S1, SI). The IGT@NPs and I@NPs similarly exhibited an obvious absorbance peak at around 790 nm, which should be due to the loading of ICG in both IGT@NPs and I@NPs (Fig. 2d). In addition, IGT@NPs and I@NPs were found to exhibit obvious fluorescence signals in the wavelength from 750 to 850 nm, which is pivotal for cellular uptake and tumor accumulation analysis (Fig. 2e).

Fig. 2 Photothermal effect and drug release analysis. (a) TEM analysis of I@NPs and IGT@NPs. (b) Hydrodynamic diameter curves of IGT@NPs and I@NPs in aqueous solution. (c) Zeta potentials of IGT@NPs and I@NPs in aqueous solution (n = 3). (d) Absorbance property analysis of IGT@NPs and I@NPs. (e) Fluorescence property analysis of IGT@NPs and I@NPs. (f) Temperature changes of solutions containing IGT@NPs at the concentration of 12.5, 25, 50 and 100 µg mL⁻¹ under 808 nm laser irradiation. (g) Temperature changes of solutions containing I@NPs at the concentration of 12.5, 25, 50 and 100 µg mL⁻¹ with 808 nm laser irradiation. (h) Cumulative GOx release efficacy for IGT@NPs with 808 nm laser irradiation. (i) TPZ release analysis for IGT@NPs with 808 nm laser irradiation.

Photothermal property analysis was conducted by irradiating the solutions containing IGT@NPs and I@NPs using an 808 nm laser. With laser irradiation, the temperature of solutions containing IGT@NPs gradually increased, and a higher concentration of IGT@NPs enabled a more remarkable temperature increment (Fig. 2f). After 300 s of laser irradiation, the temperature could reach 36.2, 42.2, 47.9 and 53.2 °C for IGT@NPs at the concentrations of 12.5, 25, 50 and 100 µg mL⁻¹. Compared to PBS, the solution of I@NPs also exhibited obvious temperature increment with laser irradiation (Fig. 2g). The maximum temperature reached 35.9, 42.0, 47.5 and 53.7 °C under laser irradiation if the concentration of I@NPs was 12.5, 25, 50 and 100 µg mL⁻¹. IGT@NPs and I@NPs were demonstrated to have good stability of photothermal properties for at least 5 cycles of irradiation (Fig. S2, SI). These data indicated that both IGT@NPs and I@NPs exhibited good photothermal properties due to the loading of the photothermal agent ICG.

IGT@NPs and I@NPs were designed to contain a thermo-responsive material DPPC, whose photothermal property-induced temperature increase could trigger the phase transition of DPPC, leading to disruption of the liposomal shell and controlled drug release. The release profile of GOx and TPZ from IGT@NPs with 808 nm laser irradiation was determined. The GOx release efficacy was negligible for IGT@NPs without laser irradiation, which, however, reached 36.6%, 67.9% and 71.6% after 100, 200 and 300 s of 808 nm laser irradiation (Fig. 2h). To further quantify GOx release, BCA protein analysis was used for accurate quantification of GOx release (Fig. S3, SI). Similarly, an obvious TPZ release property was only observed for IGT@NPs with 808 nm laser irradiation (Fig. 2i). These data confirmed that IGT@NPs showed a photoactivatable property to achieve on-demand release of GOx and TPZ only with laser irradiation.

In vitro cellular uptake and therapeutic efficacy analysis

After 24 h incubation of 4T1 cancer cells with IGT@NPs and I@NPs (3–50 µg mL⁻¹), the viability of 4T1 cells was still similar to that in the PBS control group (Fig. 3a). This indicated that IGT@NPs and I@NPs showed good cytocompatibility. The cellular uptake efficacy of IGT@NPs and I@NPs by 4T1 cells was confirmed via a flow cytometer as these cells after IGT@NPs and I@NPs treatment had exhibited remarkable fluorescence signals (Fig. 3b). The fluorescence changes for IGT@NPs and I@NPs treatment reached a similar 9.2-fold compared to that of PBS control group (Fig. 3c), which verified the consistent cellular uptake efficacy for IGT@NPs and I@NPs.

Fig. 3 In vitro cellular uptake and therapeutic efficacy analysis. (a) Analysis of 4T1 cell viability after IGT@NPs and I@NPs incubation for 24 h (n = 3). (b) Cellular uptake analysis of IGT@NPs and I@NPs by 4T1 cells via flow cytometry. (c) Fluorescence fold changes for 4T1 cells after IGT@NPs and I@NPs treatment (n = 3). (d) Analysis of 4T1 cell viability after IGT@NPs and I@NPs incubation and 808 nm laser irradiation (n = 3). (e) Analysis of 4T1 cell viability after IGT@NPs and I@NPs incubation and 808 nm laser irradiation in hypoxic conditions (n = 3).

The in vitro therapeutic efficacy was then analyzed after incubation of 4T1 cells with IGT@NPs and I@NPs, followed by laser irradiation of the treated cells (Fig. 3d). Note that the cell viability in PBS, PBS + laser, IGT@NPs and I@NPs was similarly approximately 100%, which, however, was only 46.9% and 40.5% for I@NPs + laser and IGT@NPs + laser groups, respectively. The remarkable cell viability decline should be attributed to the PTT effect of IGT@NPs and I@NPs under laser irradiation. In addition, to further verify the generation of reactive oxygen species (ROS) during photothermal therapy, we used DCFH staining to fluorescently image the intracellular ROS levels and quantitatively analyze the amount of H₂O₂ generated (Fig. S4). The results showed that the PBS and I@NPs + laser groups exhibited almost no obvious fluorescence signals, whereas the IGT@NPs + laser group produced strong green fluorescence signals corresponding to a significantly higher concentration of H₂O₂, suggesting that it could induce a stronger oxidative stress response under laser irradiation. The therapeutic effect in hypoxic conditions was also demonstrated. After cell incubation under hypoxic conditions and laser irradiation, the cell viability in IGT@NPs + laser group was only 31.9%, much lower relative to that in I@NPs + laser group (48.2%) (Fig. 3e). This should be because TPZ in IGT@NPs was activated in hypoxic conditions to kill cancer cells. PTT-combined chemotherapy was achieved for the IGT@NPs + laser group, which showed an amplified therapeutic efficacy compared to the I@NPs + laser group.

Tumor accumulation and bio-distribution analysis

Subcutaneous 4T1 tumor mouse models were injected with IGT@NPs or I@NPs via the tail vein to analyze the in vivo tumor accumulation and bio-distribution (Fig. 4a). After injection of IGT@NPs and I@NPs, the obvious fluorescence signals could be detected in tumor regions, which gradually brightened and reached a peak at 24 h post-injection point (Fig. 4b). These data suggested that both IGT@NPs and I@NPs showed effective tumor accumulation effect. The fluorescence intensity for tumor sites of mice after IGT@NPs and I@NPs injection could increase to the maximum value (1.02 × 10⁹ and 1.05 × 10⁹ p s⁻¹ cm⁻² sr⁻¹) after 24 h of injection, which indicated their similar tumor accumulation efficacy (Fig. 4c).

Fig. 4 Tumor accumulation and bio-distribution analysis. (a) Schedule of the establishment of subcutaneous 4T1 cancer models. (b) In vivo tumor accumulation analysis of IGT@NPs and I@NPs after injection via the tail vein. (c) Fluorescence intensity analysis of tumor sites for mice after IGT@NPs and I@NPs injection. (d) Fluorescence images of major organs and tumors for mice after IGT@NPs and I@NPs injection. (e) Fluorescence intensity analysis of the major organs and tumors from mice after IGT@NPs and I@NPs injection.

The in vivo bio-distribution of IGT@NPs and I@NPs after injection was then evaluated. Obvious fluorescence signals of IGT@NPs and I@NPs were only detected in liver and tumors of mice (Fig. 4d). These findings suggested that IGT@NPs and I@NPs mainly accumulated in the tumors and livers, but rarely accumulated in kidneys, lungs, heart and spleen. The maximal fluorescence intensity was observed in liver for both IGT@NPs and I@NPs injection (Fig. 4e). In addition, the fluorescence intensity of tumors for IGT@NPs and I@NPs injection was similar. Such a favorable accumulation efficacy in tumor tissues for IGT@NPs and I@NPs is expected to contribute to enhanced antitumor efficacy antitumor efficacy.

In vivo tumor growth inhibition evaluation

Subcutaneous 4T1 tumor mouse models were utilized to analyze in vivo tumor growth inhibition efficacy after injection of IGT@NPs and I@NPs and laser irradiation of tumors. The growth of tumors in IGT@NPs + laser and I@NPs + laser groups was remarkably suppressed compared to that in PBS, PBS + laser, IGT@NPs and I@NPs groups (Fig. 5a). After 14 days of treatment, the tumor volume in the IGT@NPs + laser group was only 13.1 mm³, whereas it was 170.1 mm³ in the I@NPs + laser group and more than 840.8 mm³ in the other 4 groups. As shown in the tumor photographs, the tumors in IGT@NPs + laser group exhibited the smallest sizes with complete elimination observed in 2 tumors out of 5 mice (Fig. 5b). Besides the IGT@NPs + laser group, the tumors in the I@NPs + laser group also exhibited smaller sizes compared to those in other groups. Tumor weight was measured to be 1.1, 1.0, 1.0, 0.9, 0.2 and 0.03 g for PBS, PBS + laser, IGT@NPs, I@NPs, I@NPs + laser and IGT@NPs + laser groups, respectively (Fig. 5c). Thus, the tumors in the IGT@NPs + laser group showed the lowest weight after treatment, which should be due to the PTT-amplified chemotherapy for tumor cell killing. The tumor inhibition efficacy in I@NPs + laser groups was caused by the sole PTT effect.

Fig. 5 In vivo tumor growth inhibition evaluation. (a) Tumor growth curves of mice in the treated six groups (n = 5). (b) Tumor photograph of mice in the treated six groups after 14 days of treatments (n = 5). (c) Analysis of the tumor weights of mice in the treated six groups (n = 5). (d) H&E staining images of tumors in the treated six groups. (e) Fluorescence staining images of tumors showing the levels of hypoxia after various treatments.

H&E staining analysis showed that tumor cell apoptosis was only detected in I@NPs + laser and IGT@NPs + laser groups due to the therapeutic effects, while the tumors in other 4 groups did not show any apoptotic regions (Fig. 5d). Moreover, the apoptosis level in the IGT@NPs + laser group was much more severe than that in the I@NPs + laser group, further verifying their different antitumor effects. To verify the aggravated hypoxia level for activating TPZ chemotherapy, the hypoxia condition of tumors after different treatments was evaluated. A significantly elevated hypoxia level was observed exclusively in tumors from the IGT@NPs + laser group (Fig. 5e). The intensity of hypoxia fluorescence staining signals in the IGT@NPs + laser group was increased by at least 2.4-fold compared to that in the other groups (Fig. S5, SI). To further validate tumor hypoxia, the oxygen indicator was then used. Compared with all control groups, the IGT@NPs + laser group exhibited a 9-fold augmentation in hypoxia-specific fluorescence intensity (Fig. S6, SI). This was because the PTT effect in IGT@NPs + laser group caused the temperature increase to enable GOx release for consuming oxygen via catalyzing glucose consumption.

The body weights of all these treated mice in the six groups were stable for 14 days (Fig. S7, SI). H&E staining analysis of heart, spleen and kidney verified that the treatment did not cause any damage to these organs (Fig. S8, SI). All these results confirm the in vivo biosafety of this therapeutic strategy.

In vivo tumor metastasis inhibition analysis

To verify the better antitumor efficacy of the IGT@NPs + laser group compared to the I@NPs + laser group, tumor metastasis inhibition effects were also evaluated. As shown in the H&E staining images of lungs, the metastatic tumor nodules were detected in PBS, PBS + laser, IGT@NPs, I@NPs and I@NPs + laser groups, but not in the IGT@NPs + laser group (Fig. 6a). The tumor metastasis in the I@NPs + laser group was not as serious as compared to that in PBS, PBS + laser, IGT@NPs and I@NPs groups. The number of metastatic tumor nodules in PBS, PBS + laser, IGT@NPs, I@NPs and I@NPs + laser groups was 14.6, 13.8, 15.2, 17.8, and 3.8, respectively, which, however, was only 1.2 in IGT@NPs + laser group (Fig. 6b). The obvious metastatic tumor nodules were found in the H&E staining images of PBS, PBS + laser, IGT@NPs, I@NPs and I@NPs + laser groups, while almost no tumor metastasis was detected in the livers of IGT@NPs + laser group (Fig. 6c). The mean number of metastatic tumor nodules in the IGT@NPs + laser group was only 2.6, whereas it reached 51.4, 56, 58.2, 56.6, and 10.2 for PBS, PBS + laser, IGT@NPs, I@NPs and I@NPs + laser groups, respectively (Fig. 6d). These findings indicated that the IGT@NPs + laser group showed a much higher efficacy in inhibiting tumor metastasis compared to the I@NPs + laser group.

Fig. 6 In vivo tumor metastasis inhibition analysis. (a) H&E staining images of metastatic tumor nodules in lungs in the treated six groups. (b) Analysis of metastatic tumor nodule number of lungs in the six treated groups (n = 5). (c) H&E staining images of metastatic tumor nodules in livers in the treated six groups. (d) Analysis of metastatic tumor nodule numbers of livers in the treated six groups (n = 5).

Conclusions

Based on a thermo-responsive liposome, a photoactivatable nano-liposome containing ICG, GOx and TPZ was fabricated for metastatic breast cancer therapy. The formed IGT@NPs not only exhibited good photothermal property under 808 nm laser irradiation due to the loading of photothermal agent ICG, but also displayed light-triggered release of GOx and TPZ via destroying the thermal-responsive liposome shell by the generated heat. In such a therapeutic nanoplatform, PTT and TPZ chemotherapy were integrated to achieve synergetic action. Besides PTT, the generated heat under 808 nm irradiation could mediate on-demand release of GOx and TPZ in tumor sites, which aggravated tumor hypoxia to activate the TPZ prodrugs for PTT-triggered chemotherapy. This therapeutic strategy was found to obviously inhibit both subcutaneous and bone metastasis tumor growth and tumor metastases in lungs and livers in a more effective manner compared to PTT alone. Such a good antitumor efficacy should also be attributed to the effective accumulation of IGT@NPs into tumor tissues, even without surface conjugation of tumor targeting ligands. This current study offers a photoactivatable therapeutic nanoplatform with the integration of tripartite therapeutic actions for cancer treatment, and their good properties may enable their application in various cancer types. Moreover, the fabrication procedures of such nanosystems are simple, which provides more opportunities for further application.

In summary, this study showed good efficacy in mouse 4T1 subcutaneous and metastatic breast cancer models, but these models still exhibit limitations in reflecting the complex heterogeneity of human tumors and the clinical microenvironment, even though they are widely used for pre-treatment evaluation of tumors. Follow-up work will further validate the efficacy and biosafety of this nanoplatform in more clinically relevant xenograft models or spontaneous tumor models to facilitate its clinical application.

Experimental section

Materials sources

TPZ was purchased from MedChemExpress (USA). ICG and GOx were purchased from Sigma-Aldrich (USA). 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG) were purchased from AmyJet Scientific Inc. (China).

Characterization

A Tecnai G2 transmission electron microscope (TEM) was used to obtain TEM images. A Malvern Zetasizer Nano ZS was used to measure the hydrodynamic diameters and zeta potentials. A TU-1810 Persee spectrophotometer was used to obtain UV-vis absorbance spectra. SHIMADZU fluorescence spectrophotometer was used to collect fluorescence spectra. High performance liquid chromatography (HPLC) was used to determine TPZ releases. The temperature was measured using a thermal imager.

Synthesis of IGT@NPs

The chloroform containing DPPC (20.0 mg) and DSPE-PEG (20.0 mg) were evaporated to form a thin film and then the mixed solution of ICG (0.5 mg), TPZ (0.5 mg) and GOx (0.5 mg) was added into the thin film. The solution was heated to 55 °C for hydration under stirring and then sonicated in an ice-bath. After washing with PBS via ultrafiltration, the product IGT@NPs were synthesized. In a similar method, the control nanoparticles (I@NPs) were synthesized via addition of ICG (0.5 mg) into the thin film, followed by hydration, sonication and ultrafiltration.

Evaluation of the photothermal effect

PBS and the solutions containing IGT@NPs or I@NPs (12.5, 25, 50 and 100 µg mL⁻¹) were used to evaluate the photothermal properties under 808 nm laser irradiation (1.0 W cm⁻², total 300 s). The temperature changes of the irradiated solutions were recorded.

Analysis of GOx and TPZ release properties

The solutions of IGT@NPs (20 µg mL⁻¹) were irradiated via an 808 nm laser (1.0 W cm⁻²) for 100, 200 and 300 s, respectively and then filtered to collect the liquid supernatants. The concentrations of GOx and TPZ were analyzed via BCA assay kit and HPLC to evaluate their release properties.

Assessment of ROS generation in vitro

GOx was evaluated for catalytic stability in vitro. After incubation with PBS, I@NPs and IGT@NPs (25 µg mL⁻¹), 4T1 cells were treated with an 808 nm laser in a medium containing H2DCFDA (10 µM). The images of 4T1 cells were obtained using fluorescence microscopy (Leica DMi8, Germany) to analyze intracellular ROS levels.

In vitro cytotoxicity analysis

After incubation of 4T1 cancer cells with IGT@NPs or I@NPs (3, 6, 12, 25 and 50 µg mL⁻¹) in 96-well culture dishes for 24 h, the cells were then treated with CCK-8 solution and their cell viabilities were determined.

In vitro cellular uptake analysis

After incubation of 4T1 cancer cells with PBS, IGT@NPs or I@NPs (25 µg mL⁻¹) for 24 h, the cells were washed 3 times with PBS. The cells were analyzed via flow cytometry by measuring the fluorescence intensity for cellular uptake evaluation.

In vitro therapeutic effect evaluation

After incubation of 4T1 cancer cells with PBS, IGT@NPs or I@NPs (25 µg mL⁻¹) for 24 h, the cells were treated by 808 nm laser irradiation (1.0 W cm⁻², 5 min). After further 6 h of culture, the cell viability of 4T1 cells was determined via CCK-8 analysis. In hypoxic conditions, the IGT@NPs- or I@NPs-treated cells were also irradiated by 808 nm laser (1.0 W cm⁻², 5 min) and the cell viability was determined to evaluate the in vitro therapeutic effect.

Tumor mouse models

Animal experiments in this study were performed under the approval of the Institutional Animal Care and Treatment Committee of Donghua University. Female Balb/c mice (4 to 6 weeks old, Shanghai JieSiJie Laboratory Animal Co., Ltd) were utilized to establish subcutaneous 4T1 tumor models via injecting 4T1 cancer cells into mouse right flank.

Tumor accumulation and bio-distribution analysis

IVIS imaging system was used to analyze the tumor accumulation and bio-distribution after the injection of IGT@NPs or I@NPs (250 µg mL⁻¹, 0.2 mL) into subcutaneous 4T1 tumor mouse models via the tail vein. At various post-injection points, the fluorescence images of injected mice were obtained and the tumor fluorescence signals were analyzed. After 24 of post-injection, the injected mice were euthanized and the major organs and tumors were separated to obtain fluorescence images and their fluorescence signals were determined.

In vivo tumor growth inhibition analysis

Subcutaneous 4T1 tumor models were used to analysis in vivo tumor growth inhibition efficacy, which included six groups: PBS, IGT@NPs, I@NPs, PBS + laser, IGT@NPs + laser, and I@NPs + laser. The mice were injected with PBS, IGT@NPs or I@NPs (250 µg mL⁻¹, 0.2 mL) via the tail vein, and then the tumors were irradiated using an 808 nm laser (1.0 W cm⁻², 10 min) for PBS + laser, IGT@NPs + laser, and I@NPs + laser groups. After treatment, the tumor sizes of the mice in each group were measured for 14 days to calculate tumor volumes. On day 14, the tumors in each group were separated from mice and photographed, and then tumor weights were recorded. In addition, the body weights of mice during treatments were recorded for 14 days.

Histological staining analysis

After treatments, the major organs and tumors were separated from mice in each group and used for H&E staining and the obtained staining images were analyzed for evaluating antitumor effects and biosafety. Moreover, the tumor tissues were used for HIF-α fluorescence staining to determine the hypoxia levels in tumor tissues.

In vivo tumor metastasis inhibition analysis

After treatment, lungs and livers were separated from mice in each group and utilized for H&E staining to visualize tumor metastases. For quantitative analysis of the anti-metastasis effect, the numbers of tumor metastatic nodules in lungs and livers were recorded.

Tumor inhibition evaluation in bone metastasis models

4T1 tumor-derived bone metastasis models were established by injecting 4T1 cancer cells into the right posterior tibia of mice. These mice were injected with PBS, IGT@NPs or I@NPs (250 µg mL⁻¹, 0.2 mL) via the tail vein. After injection, the bone metastasis sites of mice in PBS + laser, IGT@NPs + laser, and I@NPs + laser groups were irradiated using an 808 nm laser (1.0 W cm⁻², 10 min). To evaluate the tumor inhibition efficacy, the tumor sizes were measured to calculate tumor volumes. After 14 days, the tumors were detached for photographing, weighing and H&E staining analysis.

Statistical analysis

Values are presented in mean ± SD. Sample size (n) was explicitly specified for statistical analysis *(p < 0.05), **(p < 0.01), and ***(p < 0.001). The two-tailed unpaired t-test was employed to evaluate the significant differences in experimental data between the two groups. Statistical analysis was performed using GraphPad Prism version 8.0 software.

Humanitarian endpoint indicators

Tumor volume exceeded 1500 mm³, body weight loss exceeded 20%, severe activity limitation, or the onset of significant pain. Once any of the endpoint criteria was met, euthanasia was performed immediately to alleviate the animal's suffering.

Ethics statement

All experimental protocols involving animals were performed in accordance with the institutional ethical standards reviewed and approved by Donghua University's Institutional Animal Care and Use Committee (Protocol No. DHUEC-NSFC-2022-16).

Author contributions

N. Zhu, Y. Men, and J. Li conceived the idea, designed the experiments, supervised the research and revised the manuscript. Y. Li, Y. Zhan, Y. Liu and J. Su performed the experiments, analyzed the data and wrote the draft.

Conflicts of interest

The authors declare no competing financial interest.

Data availability

All data supporting the findings of this study are available in the paper and its SI. See DOI: https://doi.org/10.1039/d5tb01368b

Acknowledgements

This work was supported by grants from the Donghua University 2024 Cultivation 798 Project of Discipline Innovation, DHU Distinguished Young Professor Program (LZB2025003).

Notes and references

1. A. Letai and H. de The, Conventional chemotherapy: millions of cures, unresolved therapeutic index, Nat. Rev. Cancer, 2024, 209–218.
2. Y. Guo, Y. Fan, Z. Wang, G. Li, M. Zhan, J. Gong, J. P. Majoral, X. Shi and M. Shen, Chemotherapy mediated by biomimetic polymeric nanoparticles potentiates enhanced tumor immunotherapy via amplification of endoplasmic reticulum stress and mitochondrial dysfunction, Adv. Mater., 2022, 34(47), 2206861.
3. D. Hao, Q. Meng, B. Jiang, S. Lu, X. Xiang, Q. Pei, H. Yu, X. Jing and Z. Xie, Hypoxia-activated PEGylated paclitaxel prodrug nanoparticles for potentiated chemotherapy, ACS Nano, 2022, 16(9), 14693–14702.
4. J. Choi, M. K. Shim, S. Yang, H. S. Hwang, H. Cho, J. Kim, W. S. Yun, Y. Moon, J. Kim and H. Y. Yoon, Visible-light-triggered prodrug nanoparticles combine chemotherapy and photodynamic therapy to potentiate checkpoint blockade cancer immunotherapy, ACS Nano, 2021, 15(7), 12086–12098.
5. B. Zheng, Z. Liu, H. Wang, L. Sun, W.-F. Lai, H. Zhang, J. Wang, Y. Liu, X. Qin and X. Qi, R11 modified tumor cell membrane nanovesicle-camouflaged nanoparticles with enhanced targeting and mucus-penetrating efficiency for intravesical chemotherapy for bladder cancer, J. Controlled Release, 2022, 351, 834–846.
6. F. Jin, J. Qi, D. Liu, Y. You, G. Shu, Y. Du, J. Wang, X. Xu, X. Ying and J. Ji, Cancer-cell-biomimetic Upconversion nanoparticles combining chemo-photodynamic therapy and CD73 blockade for metastatic triple-negative breast cancer, J. Controlled Release, 2021, 337, 90–104.
7. L. Cao, H. Tian, M. Fang, Z. Xu, D. Tang, J. Chen, J. Yin, H. Xiao, K. Shang and H. Han, Activating cGAS-STING pathway with ROS-responsive nanoparticles delivering a hybrid prodrug for enhanced chemo-immunotherapy, Biomaterials, 2022, 290, 121856.
8. N. M. Kuderer, A. Desai, M. B. Lustberg and G. H. Lyman, Mitigating acute chemotherapy-associated adverse events in patients with cancer, Nat. Rev. Clin. Oncol., 2022, 19(11), 681–697.
9. Q. Weng, H. Sun, C. Fang, F. Xia, H. Liao, J. Lee, J. Wang, A. Xie, J. Ren and X. Guo, Catalytic activity tunable ceria nanoparticles prevent chemotherapy-induced acute kidney injury without interference with chemotherapeutics, Nat. Commun., 2021, 12(1), 1436.
10. H. Hu, D. Xu, Q. Xu, Y. Tang, J. Hong, Y. Hu, J. Wang and X. Ni, Reduction-responsive worm-like nanoparticles for synergistic cancer chemo-photodynamic therapy, Mater. Today Bio., 2023, 18, 100542.
11. S. Shen, X. Xu, S. Lin, Y. Zhang, H. Liu, C. Zhang and R. Mo, A nanotherapeutic strategy to overcome chemotherapeutic resistance of cancer stem-like cells, Nat. Nanotechnol., 2021, 16(1), 104–113.
12. Y. Guo, M. Wang, Y. Zou, L. Jin, Z. Zhao, Q. Liu, S. Wang and J. Li, Mechanisms of chemotherapeutic resistance and the application of targeted nanoparticles for enhanced chemotherapy in colorectal cancer, J. Nanobiotechnol., 2022, 20(1), 371.
13. S. Siemer, T. A. Bauer, P. Scholz, C. Breder, F. Fenaroli, G. Harms, D. Dietrich, J. Dietrich, C. Rosenauer and M. Barz, Targeting cancer chemotherapy resistance by precision medicine-driven nanoparticle-formulated cisplatin, ACS Nano, 2021, 15(11), 18541–18556.
14. X. Dong, Y. Sun, Y. Li, X. Ma, S. Zhang, Y. Yuan, J. Kohn, C. Liu and J. Qian, Synergistic combination of bioactive hydroxyapatite nanoparticles and the chemotherapeutic doxorubicin to overcome tumor multidrug resistance, Small, 2021, 17(18), 2007672.
15. P. Xie, Y. Wang, D. Wei, L. Zhang, B. Zhang, H. Xiao, H. Song and X. Mao, Nanoparticle-based drug delivery systems with platinum drugs for overcoming cancer drug resistance, J. Mater. Chem. B, 2021, 9(26), 5173–5194.
16. Y. Hou, J. Jin, H. Duan, C. Liu, L. Chen, W. Huang, Z. Gao and M. Jin, Targeted therapeutic effects of oral inulin-modified double-layered nanoparticles containing chemotherapeutics on orthotopic colon cancer, Biomaterials, 2022, 283, 121440.
17. X. Gao, G. Lei, B. Wang, Z. Deng, J. Karges, H. Xiao and D. Tan, Encapsulation of platinum prodrugs into PC7A polymeric nanoparticles combined with immune checkpoint inhibitors for therapeutically enhanced multimodal chemotherapy and immunotherapy by activation of the STING pathway, Adv. Sci., 2023, 10(4), 2205241.
18. M. Ding, Y. Zhang, N. Yu, J. Zhou, L. Zhu, X. Wang and J. Li, Augmenting Immunogenic Cell Death and Alleviating Myeloid-Derived Suppressor Cells by Sono-Activatable Semiconducting Polymer Nanopartners for Immunotherapy, Adv. Mater., 2023, 35(33), 2302508.
19. K. Yang, S. Zhao, B. Li, B. Wang, M. Lan and X. Song, Low temperature photothermal therapy: Advances and perspectives, Coord. Chem. Rev., 2022, 454, 214330.
20. M. Overchuk, R. A. Weersink, B. C. Wilson and G. Zheng, Photodynamic and photothermal therapies: synergy opportunities for nanomedicine, ACS Nano, 2023, 17(9), 7979–8003.
21. S. G. Alamdari, M. Amini, N. Jalilzadeh, B. Baradaran, R. Mohammadzadeh, A. Mokhtarzadeh and F. Oroojalian, Recent advances in nanoparticle-based photothermal therapy for breast cancer, J. Controlled Release, 2022, 349, 269–303.
22. J. Ma, N. Li, J. Wang, Z. Liu, Y. Han and Y. Zeng, In vivo synergistic tumor therapies based on copper sulfide photothermal therapeutic nanoplatforms, Exploration, Wiley Online Library, 2023, 20220161.
23. J. Lv, B. Li, T. Luo, C. Nie, W. Pan, X. Ge, J. Zheng, Y. Rui and L. Zheng, Selective Photothermal Therapy Based on Lipopolysaccharide Aptamer Functionalized MoS2 Nanosheet-Coated Gold Nanorods for Multidrug-Resistant Pseudomonas aeruginosa Infection, Adv. Healthcare Mater., 2023, 12(15), 2202794.
24. S. I. Amaral, R. Costa-Almeida, I. C. Goncalves, F. D. Magalhaes and A. M. Pinto, Carbon nanomaterials for phototherapy of cancer and microbial infections, Carbon, 2022, 190, 194–244.
25. Y. Liu, F. Li, Y. Lyu, F. Wang, L. T. O. Lee, S. He, Z. Guo and J. Li, A Semiconducting Polymer NanoCRISPR for Near-Infrared Photoactivatable Gene Editing and Cancer Gene Therapy, Nano Lett., 2025, 25(11), 4518–4525.
26. J. Li, X. Zhen, Y. Lyu, Y. Jiang, J. Huang and K. Pu, Cell membrane coated semiconducting polymer nanoparticles for enhanced multimodal cancer phototheranostics, ACS Nano, 2018, 12(8), 8520–8530.
27. L. Zhao, X. Zhang, X. Wang, X. Guan, W. Zhang and J. Ma, Recent advances in selective photothermal therapy of tumor, J. Nanobiotechnol., 2021, 19, 1–15.
28. X. Deng, Z. Shao and Y. Zhao, Solutions to the drawbacks of photothermal and photodynamic cancer therapy, Adv. Sci., 2021, 8(3), 2002504.
29. Y. Liu, R. Lu, M. Li, D. Cheng, F. Wang, X. Ouyang, Y. Zhang, Q. Zhang, J. Li and S. Peng, Dual-enzyme decorated semiconducting polymer nanoagents for second near-infrared photoactivatable ferroptosis-immunotherapy, Mater. Horiz., 2024, 11(10), 2406–2419.
30. G. Gao, X. Sun and G. Liang, Nanoagent-promoted mild-temperature photothermal therapy for cancer treatment, Adv. Funct. Mater., 2021, 31(25), 2100738.
31. X. Zhang, J. Du, Z. Guo, J. Yu, Q. Gao, W. Yin, S. Zhu, Z. Gu and Y. Zhao, Efficient near infrared light triggered nitric oxide release nanocomposites for sensitizing mild photothermal therapy, Adv. Sci., 2019, 6(3), 1801122.
32. G. Gao, Y. W. Jiang, W. Sun, Y. Guo, H. R. Jia, X. W. Yu, G. Y. Pan and F. G. Wu, Molecular targeting-mediated mild-temperature photothermal therapy with a smart albumin-based nanodrug, Small, 2019, 15(33), 1900501.
33. M. R. Ali, H. R. Ali, C. R. Rankin and M. A. El-Sayed, Targeting heat shock protein 70 using gold nanorods enhances cancer cell apoptosis in low dose plasmonic photothermal therapy, Biomaterials, 2016, 102, 1–8.
34. S. Wang, Y. Tian, W. Tian, J. Sun, S. Zhao, Y. Liu, C. Wang, Y. Tang, X. Ma and Z. Teng, Selectively sensitizing malignant cells to photothermal therapy using a CD44-targeting heat shock protein 72 depletion nanosystem, ACS Nano, 2016, 10(9), 8578–8590.
35. X.-Q. Pu, X.-J. Ju, L. Zhang, Q.-W. Cai, Y.-Q. Liu, H.-Y. Peng, R. Xie, W. Wang, Z. Liu and L.-Y. Chu, Novel multifunctional stimuli-responsive nanoparticles for synergetic chemo–photothermal therapy of tumors, ACS Appl. Mater. Interfaces, 2021, 13(24), 28802–28817.
36. R. Fan, C. Chen, H. Hou, D. Chuan, M. Mu, Z. Liu, R. Liang, G. Guo and J. Xu, Tumor acidity and near-infrared light responsive dual drug delivery polydopamine-based nanoparticles for chemo-photothermal therapy, Adv. Funct. Mater., 2021, 31(18), 2009733.
37. W. Zhang, Z. Guo, D. Huang, Z. Liu, X. Guo and H. Zhong, Synergistic effect of chemo-photothermal therapy using PEGylated graphene oxide, Biomaterials, 2011, 32(33), 8555–8561.
38. 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.
39. W. Li, J. Peng, L. Tan, J. Wu, K. Shi, Y. Qu, X. Wei and Z. Qian, Mild photothermal therapy/photodynamic therapy/chemotherapy of breast cancer by Lyp-1 modified Docetaxel/IR820 Co-loaded micelles, Biomaterials, 2016, 106, 119–133.
40. G. Zhang, X. Chen, X. Chen, K. Du, K. Ding, D. He, D. Ding, R. Hu, A. Qin and B. Z. Tang, Click-reaction-mediated chemotherapy and photothermal therapy synergistically inhibit breast cancer in mice, ACS Nano, 2023, 17(15), 14800–14813.
41. Y. Wang, G. Wei, X. Zhang, X. Huang, J. Zhao, X. Guo and S. Zhou, Multistage targeting strategy using magnetic composite nanoparticles for synergism of photothermal therapy and chemotherapy, Small, 2018, 14(12), 1702994.
42. A. Granja, R. Lima-Sousa, C. G. Alves, D. de Melo-Diogo, C. Nunes, C. T. Sousa, I. J. Correia and S. Reis, Multifunctional targeted solid lipid nanoparticles for combined photothermal therapy and chemotherapy of breast cancer, Biomater. Adv., 2023, 151, 213443.
43. M. Zheng, C. Yue, Y. Ma, P. Gong, P. Zhao, C. Zheng, Z. Sheng, P. Zhang, Z. Wang and L. Cai, Single-step assembly of DOX/ICG loaded lipid–polymer nanoparticles for highly effective chemo-photothermal combination therapy, ACS Nano, 2013, 7(3), 2056–2067.
44. B. Liu, W. Wang, J. Fan, Y. Long, F. Xiao, M. Daniyal, C. Tong, Q. Xie, Y. Jian and B. Li, RBC membrane camouflaged prussian blue nanoparticles for gamabutolin loading and combined chemo/photothermal therapy of breast cancer, Biomaterials, 2019, 217, 119301.
45. A. M. Itoo, M. Paul, N. Jain, V. Are, A. Singh, B. Ghosh and S. Biswas, Biotinylated platinum (IV)-conjugated graphene oxide nanoparticles for targeted chemo-photothermal combination therapy in breast cancer, Biomater. Adv., 2025, 168, 214121.
46. S. Bai, Y. Zhang, D. Li, X. Shi, G. Lin and G. Liu, Gain an advantage from both sides: Smart size-shrinkable drug delivery nanosystems for high accumulation and deep penetration, Nano Today, 2021, 36, 101038.
47. M. Chen, Z. Huang, M. Xia, Y. Ding, T. Shan, Z. Guan, X. Dai, X. Xu, Y. Huang and M. Huang, Glutathione-responsive copper-disulfiram nanoparticles for enhanced tumor chemotherapy, J. Controlled Release, 2022, 341, 351–363.
48. Y. Hao, Y. Chen, X. He, R. Han, C. Yang, T. Liu, Y. Yang, Q. Liu and Z. Qian, RGD peptide modified platinum nanozyme Co-loaded glutathione-responsive prodrug nanoparticles for enhanced chemo-photodynamic bladder cancer therapy, Biomaterials, 2023, 293, 121975.
49. Q. Lei, W. X. Qiu, J. J. Hu, P. X. Cao, C. H. Zhu, H. Cheng and X. Z. Zhang, Multifunctional mesoporous silica nanoparticles with thermal-responsive gatekeeper for NIR light-triggered chemo/photothermal-therapy, Small, 2016, 12(31), 4286–4298.
50. F. Liu, Z. Cheng and H. Yi, NIR light-activatable dissolving microneedle system for melanoma ablation enabled by a combination of ROS-responsive chemotherapy and phototherapy, J. Nanobiotechnol., 2023, 21(1), 61.
51. X. Wang, Z. Xuan, X. Zhu, H. Sun, J. Li and Z. Xie, Near-infrared photoresponsive drug delivery nanosystems for cancer photo-chemotherapy, J. Nanobiotechnol., 2020, 18, 108.
52. T. Shibata, Y. Shibamoto, K. Sasai, N. Oya, R. Murata, T. Takagi, M. Hiraoka, M. Takahashi and M. Abe, Tirapazamine: hypoxic cytotoxicity and interaction with radiation as assessed by the micronucleus assay, J. Cancer Suppl., 1996, 27, S61–64.
53. S. B. Bankar, M. V. Bule, R. S. Singhal and L. Ananthanarayan, Glucose oxidasean overview, Biotechnol. Adv., 2009, 27, 489–501.

---

Footnote

† Ynna Li, Yiduo Zhan and Yifang Liu contributed equally to this work.

---