Iridium nanoparticles with anti-inflammatory activity for improved tumor photothermal therapy

Abstract

Due to its high efficiency and minimally invasive nature, PTT has received widespread attention. However, traditional PTT leads to the generation of excessive reactive oxygen species (ROS) and inflammatory responses, which exacerbates tumor metastasis and limits its therapeutic efficacy. In this study, we synthesized a polyethylene glycol modified iridium nanoparticle (IrNpP) with high photothermal conversion capacity and ROS scavenging activities. The IrNpP effectively inhibits the tumor cell growth and suppresses the tumor tissue growth. More importantly, the IrNpP extensively eliminates ROS, which significantly mitigates the inflammatory response and effectively inhibits tumor metastasis. Besides, the IrNpP exhibits negligible side effects, suggesting its high potential for biomedical applications. This strategy effectively achieves ablation of tumor cells while minimizing the side effects of photothermal therapy, overcoming the shortcomings of PTT in tumor treatment and providing a new avenue for its application.

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1. Introduction

As a non-invasive tumor treatment, photothermal therapy (PTT) is activated by near-infrared (NIR) irradiation in the range of 700–1350 nm.^1–3 It utilizes photothermal agents (PTAs) including metal nanoparticles, small molecular organic dyes and inorganic nanomaterials to increase the temperature (over 45.0 °C) in tumor tissue,^4,5 leading to thermal damage and necrosis of tumor cells.^6–8 Moreover, PTT is temporally and spatially controllable,^9–11 allowing precise control of the location and depth of the light-exposed area for accurate treatment of the targeted tumor tissue.¹² Although various PTAs with strong NIR light absorption and high photothermal conversion efficiency (PCE) have been developed, their clinical application in PTT remains challenging due to latent biosafety dangers, difficulties in large-scale preparation, and low photothermal stability.¹³ Besides, during PTT treatment, the structure of the cell membrane was damaged due to the heat accumulation by the photothermal effect.^14,15 The extravasation of intracellular substances produces a large number of reactive oxygen species (ROS), which triggers inflammation responses through the recruitment and activation of immune cells.^16–18 Inflammation is a common defense response of tissues to external injury.^19,20 This undesirable inflammation stimulates tumor regeneration and induces tumor cells to release tumor necrosis factor (TNF-α) and interleukins (IL-6 and IL-1β),²¹ promoting tumorigenesis, progression and metastasis.^22–24 Given the current limitations of inflammation in PTT, inhibiting PTT-induced oxidative stress to reduce the inflammatory response could limit tumor metastasis and significantly improve the efficacy of PTT.^25,26

Based on this mechanism, researchers have begun to develop nanoplatforms that can specifically scavenge ROS. Achieving anti-inflammatory effects by eliminating ROS to inhibit tumor metastasis has become a highly promising therapeutic strategy.^27,28 Currently, the latest advances in nanotechnology demonstrate the development of various nanoparticles with high antioxidant activity (such as carbon-based, platinum-based and manganese-based nanoparticles).^29–31 Moreover, researchers have discovered that certain PTAs not only possess photothermal conversion capabilities but also exhibit enzyme-like activities.^32,33 These PTAs can effectively scavenge ROS and reduce inflammation responses during PTT treatment. The dual functionality of these PTAs ablates tumor cells and restrains tumor metastasis, which is beneficial to improve the efficacy of PTT.³⁴ In recent years, Ir as a transition metal has attracted widespread research interest in various fields due to its unique optical properties.³⁵ According to existing research reports, iridium-based nanoparticles have great potential for tumor phototherapy. Iridium-based nanoparticles exhibit high photothermal conversion capability and low cytotoxicity.³⁶ More importantly, iridium-based nanoparticles are recognized as outstanding candidates due to their high activity, stability and stereoselectivity.^37,38 Compared with other traditional PTAs, iridium-based nanoparticles can efficiently decompose ROS to alleviate inflammation.^39,40 These characteristics are significant for exploring iridium-based nanoparticles for tumor therapy.

Herein, we present an IrNpP with high photothermal conversion capacity and ROS scavenging activity for improved PTT (Scheme 1). Under NIR light irradiation, the IrNpP is capable of absorbing NIR light and effectively converting light into heat to increase the temperature of the tumor site, resulting in effective tumor cell ablation and tumor growth suppression. Moreover, a series of in vitro experiments demonstrated that the IrNpP exhibits ROS clearance activity that enables it to eliminate various free radicals, including ˙OH, H₂O₂, ¹O₂, O₂˙⁻, ABTS and DPPH, thereby showing highly efficient antioxidant properties. In the LPS induced macrophage inflammation models, the IrNpP displayed significant potential in scavenging ROS, enabling it to reduce PTT triggered ROS levels and alleviate the associated inflammation response. Since inflammation promotes distant tumor metastasis, the ROS clearance ability of the IrNpP indirectly inhibits tumor metastasis. This effect offers greater efficacy in suppressing tumor metastasis after photothermal therapy. This work proposes an anti-inflammatory strategy following photothermal therapy for tumors, lays a foundation for subsequent anti-metastatic treatment and addresses the limitations of PTT in anti-tumor treatment.

Scheme 1 Schematic illustration of the synthesis of IrNpP and their mechanism of killing tumor cells through photothermal effects, scavenging ROS to inhibit inflammation and achieving anti-tumor metastasis.

2. Results and discussion

2.1. Synthesis and characterization of the IrNpP (IrNp)

IrNp was synthesized via thermal decomposition, as shown in Scheme 1. To verify the successful synthesis of IrNp, the size distribution and morphology were characterized by transmission electron microscopy (TEM). As shown in Fig. 1a, the TEM image indicates that IrNp was uniform spherical nanoparticles with a diameter of 3.0 ± 0.8 nm. The crystal structure of IrNp was analyzed using X-ray powder diffraction (XRD) (Fig. 1b). It appears that the position of the characteristic diffraction peaks of the prepared product matches well with iridium (JCPDS no. 06-0598). Additionally, the X-ray photoelectron spectroscopy (XPS) spectra of IrNp further confirms the successful preparation of IrNp (Fig. 1c and d). We observed a typical peak at about 60.0 eV, which could be assigned to Ir 4f (Fig. 1c). Furthermore, the XPS spectrum of IrNp exhibits two peaks at 60.9 eV and 64.0 eV, indicating that Ir exists in the form of Ir⁰ in IrNp (Fig. 1d). Due to the attachment of oleylamine and oleic acid on the surface of IrNp, we further transferred the prepared IrNp to an aqueous medium by surface modification of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (DSPE-PEG₂₀₀₀ amine). As shown in Fig. S1, the TEM image revealed that the IrNpP was well dispersed in the aqueous medium without aggregation and any morphological changes, confirming the successful modification of DSPE-PEG₂₀₀₀ amine on IrNp. Fourier transform infrared (FT-IR) spectra showed that the IrNpP had characteristic peaks corresponding to DSPE-PEG₂₀₀₀ amine, which further confirmed the successful modification of IrNp with DSPE-PEG₂₀₀₀ amine (Fig. S2). The hydrodynamic diameter and zeta potential of the IrNpP were measured using dynamic light scattering (DLS). The hydrodynamic diameter of the IrNpP was 28.5 ± 3.9 nm and the zeta potential was 16.7 ± 0.5 mV (Fig. 1e and f).

Fig. 1 Characterization of the IrNpP (IrNp). (a) TEM image and size distribution histogram of IrNp. The scale bar is 20 nm. (b) XRD image of the IrNp. (c) XPS survey spectrum of the IrNp. (d) Ir 4f XPS spectra of the IrNp. (e) Hydrodynamic size distribution of the IrNpP. (f) Zeta potentials of the IrNpP in H₂O.

2.2. Photothermal properties and ROS scavenging ability of the IrNpP

The photothermal properties of photothermal agents (PTAs) are crucially important to their efficacy; thus, we evaluated the photothermal effect of the IrNpP. As shown in Fig. S3, the absorption spectra indicated that the IrNpP showed broad absorption from the visible to the NIR region. Besides, the absorbance of the IrNpP increased with increasing IrNpP concentration. These results indicated the potential of the IrNpP for a PTA in PTT. As shown in Fig. 2a, the temperature increase of solutions with IrNpP (0–75 µg mL⁻¹) at a fixed laser power density (808 nm, 1.0 W cm⁻², 8 min) was observed by thermal imaging. After 8 min of laser irradiation, the increased temperature of IrNpP solutions at concentrations of 25, 50 and 75 µg mL⁻¹ was 19.1, 28.6 and 35.0 °C, respectively (Fig. 2b). Subsequently, we investigated the influence of laser power density on the photothermal conversion capacity of IrNpP. As shown in Fig. 2c, the temperature of IrNpP solutions increased with enhancing the laser power density. The increased temperature of IrNpP solutions was 13.8, 20.6 and 28.8 °C when irradiated at laser power densities of 0.5, 0.8 and 1.0 W cm⁻² (808 nm, 8 min), respectively (Fig. 2d). These results indicated that the photothermal conversion capacity of IrNpP was dependent on the concentration and laser power density. To obtain the photothermal conversion efficiency (η), the heating and cooling processes of IrNpP and water were recorded (Fig. 2e and Fig. S4). The η and heat transfer time constant (τ_s) were calculated to be 21.0% and 119.7 s (Fig. 2f), which was significantly higher than that of the commercial photothermal agent ICG.⁴¹ Additionally, the photothermal performance of IrNpP showed no apparent change after 5 repeated laser on/off cycles (Fig. 2g). Consistent with the photothermal performance analyses, no noticeable changes in the size and morphology of the IrNpP were observed by TEM (Fig. S5). These results demonstrated the high stability of the IrNpP during laser irradiation.

Fig. 2 Photothermal conversion and ROS scavenging activity of the IrNpP. (a) Thermal images and (b) photothermal curves of IrNpP solution at different concentrations with varying irradiation times. (c) Thermal images and (d) photothermal curves of IrNpP solution (50 µg mL⁻¹) under different laser power densities with varying irradiation times. (e) Photothermal effect of IrNpP aqueous solution (50 µg mL⁻¹) and the laser was turned off after irradiation for 8 min. (f) Negative natural logarithm of the temperature driving force versus cooling time. (g) Photothermal stability of the IrNpP at 50 µg mL⁻¹ after 5 irradiation cycles. (h) UV-Vis absorption curves and images (inset) of MB solution treated with different concentrations of IrNpP, mediated by the Fe²⁺/H₂O₂ Fenton reaction. (i) TEMP was used as the spin trap to detect the ¹O₂ generation in the reaction systems under treatment conditions with different IrNpP concentrations (0–15 µg mL⁻¹). (j) O₂˙⁻, (k) ABTS and (l) DPPH scavenging ability and images (inset) of the IrNpP with different concentrations. Irradiation: 808 nm, 1.0 W cm⁻², and 8 min. Data are expressed as mean ± SD (n = 3).

To verify whether the IrNpP could eliminate the ROS, we investigated the ROS scavenging ability of the IrNpP. Methylene blue (MB) was used to assess the clearance of ˙OH (Fig. 2h). It appears that the existence of the IrNpP significantly increases the characteristic absorption peak of MB at 665 nm. In addition, the absorption peak of MB increased with the increase of IrNpP concentration. These results indicated that the IrNpP could effectively eliminate ˙OH in a physiological environment. Moreover, the hydrogen peroxide (H₂O₂) scavenging ability of IrNpP was assessed. After decomposition of H₂O₂, its absorbance at 240 nm decreases. As shown in Fig. S6, the absorbance at 240 nm decreased with increasing IrNpP concentration. When the IrNpP concentration was 114 µg mL⁻¹, the scavenging efficiency reached 93.7%. This confirmed that the IrNpP is capable of scavenging H₂O₂. Electron paramagnetic resonance (EPR) spectra were utilized to evaluate the ¹O₂ scavenging capacity of IrNpP (Fig. 2i). The EPR spectra indicated that the signal intensity of ¹O₂ is obviously reduced after introducing IrNpP. Subsequently, the O₂˙⁻ scavenging ability of IrNpP was evaluated. Since the mixture of riboflavin and L-methionine generates O₂˙⁻ under 365 nm UV light irradiation, the O₂˙⁻ specifically increases the absorbance of nitrotetrazolium blue chloride (NBT) at 560 nm.⁴² The absorbance of NBT at 560 nm decreased with the increase in IrNpP concentration (Fig. 2j). When the IrNpP concentration is 114 µg mL⁻¹, the scavenging efficiency reaches 98.7%. We assessed the O₂˙⁻ scavenging capacity using the WST-1 method. At 60 µg mL⁻¹ of IrNpP, the O₂˙⁻ scavenging rate reached 98.5% (Fig. S7). These results clearly indicated the O₂˙⁻ scavenging of IrNpP with high efficiency. In addition, we found that the IrNpP could also efficiently scavenge ABTS and DPPH radicals. At 114 µg mL⁻¹ of IrNpP, the scavenging efficiency of ABTS and DPPH radicals reached 87.0% and 76.6% (Fig. 2k and l). These unique features endow IrNpP with various free radical scavenging abilities to reduce the inflammatory response during PTT and tumor metastasis.

2.3. In vitro antitumor activity of the IrNpP

The biosafety of the IrNpP was assessed by determining the viability of several types of cells, including cancer cells (4T1) and normal cells (Hs578bst and HUVEC), using a cell counting kit-8 (CCK-8). As shown in Fig. 3a and b, the viabilities of all cells exceeded 82.0% after treatment with 75 µg mL⁻¹ IrNpP, suggesting high biocompatibility of IrNpP. Subsequently, we evaluated the therapeutic effect of IrNpP on 4T1 cells under 808 nm laser irradiation (1.0 W cm⁻²). After 8 min of laser exposure, the viability of 4T1 cells decreased with the increase of IrNpP concentration (Fig. 3b). When the IrNpP concentration reached 50 µg mL⁻¹, the cell viability decreased to 21.6%. Consistent with the CCK-8 assessment, the calcein-AM and PI double staining images showed that the 4T1 cells treated with PBS, NIR and IrNpP displayed evident green fluorescence and negligible red fluorescence. In contrast, a large number of dead cells stained with red fluorescence were observed after the 4T1 cells were treated with the IrNpP (50 µg mL⁻¹) and 808 nm laser irradiation (1.0 W cm⁻², 8 min, Fig. 3c). These results demonstrated that the IrNpP could effectively kill tumor cells under laser irradiation.

Fig. 3 Photothermal cancer cell-killing and anti-inflammatory effects of IrNpP in vitro. (a) Cell viability of Hs578bst and HUVEC treated with different concentrations of IrNpP. (b) Cell viability of 4T1 cells at various concentrations of IrNpP with/without 808 nm NIR irradiation. (c) Live/dead staining of 4T1 cells after various treatments. Dead cells were stained red with propidium (PI), and live cells were stained green with calcein-AM. The scale bar is 100 µm. (d) ROS levels of 4T1 cells treated by PBS, Rosup, IrNpP, IrNpP + Rosup, NIR and IrNpP + NIR. The scale bar is 100 µm. (e) Evaluation of fluorescence intensity in 4T1 cells in PBS, Rosup, IrNpP, IrNpP + Rosup, NIR and IrNpP + NIR groups using flow cytometry. (f) Fluorescence images were used to analyze mitochondrial membrane potential using JC-1 probes. The scale bar is 100 µm. (g) TNF-α, (h) IL-6 and (i) IL-1β levels of macrophage treated with media of 4T1 cells with PBS, LPS, IrNpP, IrNpP + LPS, NIR and IrNpP + NIR. (j) Representative images of the transwell assay of 4T1 cells treated with various concentrations of IrNpP under 808 nm NIR irradiation. The scale bar is 100 µm. (k) Quantitative analysis of the migration inhibition rate of 4T1 cells in the transwell assay. Irradiation: 808 nm, 1.0 W cm⁻², and 8 min. *p < 0.05, ***p < 0.001 and ns indicates no statistical significance. Data are expressed as mean ± SD (n = 3).

2.4. In vitro anti-inflammatory and migration inhibition effects of the IrNpP

Excessive ROS promotes the development of inflammation-related diseases.⁴³ Lowering the ROS levels in tumors serves as an effective method for controlling and regulating inflammation.^44,45 Given that IrNpP showed free radical scavenging activity, we investigated the role of IrNpP in the treatment of inflammation. We assessed the ROS levels in 4T1 cells by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and Hoechst 33342 staining (Fig. 3d). Compared to the control group, the NIR, IrNpP and IrNpP + NIR groups showed a decreased intensity of green fluorescence, suggesting that IrNpP + NIR treatments had no particular effects on ROS levels in 4T1 cells. After Rosup treatment, the intensity of green fluorescence was significantly increased, indicating that Rosup effectively increased the ROS levels in 4T1 cells. However, ROS levels were markedly reduced in the cells treated with Rosup after introducing IrNpP. We used flow cytometry to measure intracellular ROS levels (Fig. 3e). Consistent with the DCFH-DA/Hoechst 33342 staining analyses, the flow cytometry assessment indicated that the ROS levels in the IrNpP + NIR group were comparable to those in the control group while lower than that of the Rosup group. Besides, the ROS levels in Rosup treated cells could be obviously reduced by the IrNpP. These results demonstrated that IrNpP effectively scavenged ROS and maintained ROS within the normal range during PTT. The intracellular ROS content increased, leading to mitochondrial damage and mitochondrial membrane potential (MMP) changes. JC-1 monomers produced red fluorescence in healthy mitochondria with high MMP and green fluorescence in damaged mitochondria with low MMP. Therefore, the changes of MMP were further monitored by JC-1 (Fig. 3f). The strongest green fluorescence was observed in the Rosup group, indicating that mitochondria were significantly damaged after Rosup treatment. In addition, strong red fluorescence was observed in the control, IrNpP and IrNpP + Rosup groups, proved that no significant disruption of the MMP. These results suggested that the IrNpP could efficiently scavenge ROS and protect cells from oxidative stress-induced damage.

RAW 264.7 macrophages are often used as cell models to evaluate the anti-inflammatory effects of bioactive compounds.⁴⁶ We used lipopolysaccharide (LPS) to induce the differentiation of macrophages into pro-inflammatory macrophages and analyzed the anti-inflammatory effects of IrNpP. After different treatments, the levels of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) were tested using ELISA kits (Fig. 3g–i). The IrNpP + NIR group showed comparable TNF-α, IL-6 and IL-1β levels to the control group. At the same time, the levels of TNF-α, IL-6 and IL-1β in the LPS group tended to increase, indicating that the in vitro inflammation model was successfully established. However, the levels of pro-inflammatory cytokines in cells treated with LPS were significantly reduced by IrNpP, indicating the role of IrNpP in attenuating the inflammatory response triggered by PTT. These results proved that the IrNpP could effectively scavenge ROS, thereby reducing the levels of pro-inflammatory cytokines during PTT. In addition, we conducted the transwell assay to evaluate the effects of IrNpP on the proliferation and migration of 4T1 cells by PTT. Representative images in Fig. 3j demonstrated a significant decrease in the number of migrating cells with increasing IrNpP concentrations. Quantitative analysis showed that the migration of 4T1 cells treated with IrNpP (50 µg mL⁻¹) was inhibited by nearly 83.3% (Fig. 3k). These results indicated that IrNpP can effectively kill tumor cells after irradiation with an 808 nm NIR laser, thereby inhibiting tumor cell proliferation and migration.

2.5. The biodistribution/metabolism of IrNpDP

We first labeled the IrNpP with the fluorescent dye DiD (IrNpDP) and evaluated IrNpDP aggregation at the tumor site using an in vivo imaging system (IVIS) of a mouse. As shown in Fig. S8a, there was a significant fluorescence signal at the tumor site in the mouse 48 h after tail vein injection. The major organs (heart, liver, spleen, lung, kidney and tumor) of the mouse were removed and each organ was imaged ex vivo (Fig. S8b). These results showed significant fluorescence at the tumor site, confirming the accumulation of IrNpDP. Inductively coupled plasma optical emission spectrometry (ICP-OES) was used to determine the biodistribution and metabolism of IrNpDP. As shown in Fig. S9 and S10, the concentration of Ir in the circulation decreased with time and the circulating half-life of Ir was calculated to be 0.6 ± 0.2 h. At 24 h post-injection, the content of Ir in the major organs (heart, liver, spleen, lung, kidney and tumor) of mice was measured by ICP-OES. These results showed that the IrNpDP was delivered to tumor tissue via a passive targeting manner. In addition, we detected the changes in fluorescence intensity of IrNpDP in vivo. As shown in Fig. S11, the fluorescence intensity of IrNpDP was strongest at 1 h after injection and decreased over time, further confirming the good biosafety of the IrNpP and its potential for use in subsequent tumor PTT.

2.6. In vivo anti-tumor effect of the IrNpP

To explore the in vivo tumor therapeutic effect of the IrNpP, we established 4T1 tumor-bearing mouse models by subcutaneously injecting 4T1 cells into mice (Fig. 4a). When the tumor volume of mice reached approximately 60 mm³, all tumor-bearing mice were randomly divided into four treatment groups: PBS, NIR, IrNpP and IrNpP + NIR. One hour after intravenous injection of either PBS or IrNpP, the tumor areas were irradiated with 808 nm laser (1.0 W cm⁻²) for 8 min. At the same time, we recorded temperature curves to monitor the localized temperature variations in the tumors during PTT using a thermal camera (Fig. 4b). As shown in Fig. 4c, the temperature in the tumor area of mice in the IrNpP + NIR group increased from the body temperature (32.5 °C) to 47.5 °C after laser irradiation, which is significantly higher than that in the NIR group. These results suggested that the IrNpP could effectively convert light energy into heat energy in vivo and raise the temperature of tumors, thus achieving an efficient PTT effect.

Fig. 4 In vivo antitumor effects of IrNpP based photothermal therapy. (a) Schematic illustration of the photothermal treatment procedure on the 4T1 tumor-bearing mice model. (b) The thermal images of tumor tissues with intravenous injection of PBS and IrNpP (8 mg kg⁻¹) under laser exposure. (c) The photothermal curves of tumor tissues after intravenous injection of IrNpP (8 mg kg⁻¹ in 100 µL PBS) and an equal volume of PBS, followed by 808 nm laser irradiation 1 h later. (d)–(g) Tumor growth curves of mice with various treatments. (h) Relative tumor volume of 4T1 tumor-bearing mice in different treatments. (i) The photograph of tumors extracted from mice after different treatments. (j) In vitro dissected tumor weights of different groups. (k) H&E, TUNEL and Ki67 stained images of samples from various treatment groups. The scale bar is 100 µm. Irradiation: 808 nm, 1.0 W cm⁻², and 8 min. ***p < 0.001. Data are expressed as mean ± SD (n = 5).

After 14 days of treatment, there was no significant difference in the weight changes of mice in different treatment groups (Fig. S12), indicating that these treatments had no significant effect on the weight of mice. Subsequently, the growth of tumors was evaluated (Fig. 4d–h). Compared to the PBS group, the mice treated with NIR and IrNpP showed a less effect on tumor growth. Due to the photothermal therapeutic effect of IrNpP, the tumor volume growth of the IrNpP + NIR group was significantly lower than that of other groups. The images of tumor tissues showed that the 808 nm laser irradiation of IrNpP treatment had a significant tumor suppression effect (Fig. 4i). Consistent with the tumor growth analyses, the tumor weight of the IrNpP + NIR group was the lightest among all groups (Fig. 4j). Subsequently, we collected tumor tissues and conducted hematoxylin–eosin (H&E) and immunostaining to assess the PTT effect of IrNpP (Fig. 4k). The H&E staining images indicated that the PBS, NIR and IrNpP groups showed negligible histological damage. However, the tumor tissue in the IrNpP + NIR group showed extensive necrosis and severe damage. These results were further verified by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The TUNEL staining images revealed that the immunofluorescence of the IrNpP + NIR group was stronger than that of other groups, suggesting that IrNpP + NIR treatment resulted in significant apoptosis of tumor cells. Besides, the Ki67 staining images indicated that the proliferation activity of tumor cells in the IrNpP + NIR group was the lowest among all groups. These results implied that IrNpP has the capacity to delay tumor proliferation through the photothermal effect.

2.7. Biocompatibility of the IrNpP

The hemolysis rate of red blood cells is less than 3.0% at 100 µg mL⁻¹ IrNpP concentration (Fig. 5a), demonstrating the good biocompatibility of IrNpP. Moreover, the systemic toxicity of IrNpP was assessed by H&E staining of vital organs of mice (heart, liver, spleen, lungs and kidney) and blood biochemical analyses. As shown in Fig. 5b, no significant tissue damage or necrosis was observed in all groups. In addition, blood biochemical analyses indicated that the levels of kidney (urea: UREA; blood urea nitrogen: BUN; creatinine: CREA) and liver (alanine aminotransferase: ALT; aspartate aminotransferase: AST; alkaline phosphatase: ALP) injury markers were within the normal range without any pathological changes in all groups (Fig. 5c–e). These results demonstrated the good biosafety of IrNpP in vivo and its potential for biomedical applications in clinic.

Fig. 5 Safety evaluation of IrNpP in vivo. (a) Hemolysis assay with different concentrations of IrNpP on red blood cells. (b) H&E stained images of major organs from various groups. The scale bar is 100 µm. (c)–(e) Blood biochemical analysis in the serum of mice with PBS and IrNpP treatments. Irradiation: 808 nm, 1.0 W cm⁻², and 8 min. Data are expressed as mean ± SD (n = 3).

2.8. In vivo anti-inflammatory and metastatic inhibitory effects of the IrNpP

Our above findings demonstrated the satisfactory tumor PTT effects of the IrNpP. Subsequently, we performed ROS staining on the tumor tissues of each group. As shown in Fig. 6a, the IrNpP successfully reduces the expression of ROS during PTT, indicating the inhibitory effect on inflammatory processes of IrNpP. Besides, we further tested the levels of inflammatory cytokines during PTT to investigate the anti-inflammatory effects of IrNpP. As shown in Fig. 6b–d, no significant difference was observed in the expression levels of inflammation-related cytokines (TNF-α, IL-6 and IL-1β) between the IrNpP + NIR group and the other treatment groups, indicating the good anti-inflammatory capacity of IrNpP during PTT.

Fig. 6 In vivo anti-inflammation effects of IrNpP. (a) ROS and DAPI stained images of mice tumor tissues in different groups. The scale bar is 100 µm. (b)–(d) Cytokine (TNF-α, IL-6 and IL-1β) levels in the serum of mice from indicated groups. Irradiation: 808 nm, 1.0 W cm⁻², and 8 min. ns indicates no statistical significance. Data are expressed as mean ± SD (n = 3).

Inflammation has the potential to induce tumor metastasis and recurrence, while tumor metastasis is the most important factor leading to death.⁴⁷ We therefore investigated the inhibitory effect of the IrNpP on tumor metastasis. The picture shows the experimental process of lung metastasis inhibition by IrNpP in vivo (Fig. 7a). After 37 days of tumor treatment, the lung tissues of mice in different treatment groups were collected for photography and H&E staining to analyze the inhibition of tumor metastasis (Fig. S13 and Fig. 7b). Significantly fewer metastatic nodules on the surface of the lungs were observed in the IrNpP + NIR group than in the PBS, NIR and IrNpP groups. These similar results were further confirmed by H&E staining analysis of lung tissue sections in each group. The IrNpP + NIR group exhibited the lowest number of lung metastatic nodules among all groups (Fig. 7c). These results indicated that the IrNpP eliminates the ROS generated during photothermal therapy, thus alleviating the inflammatory response and effectively inhibiting the distant metastasis of tumors.

Fig. 7 In vivo anti-metastasis effects of IrNpP. (a) Schematic illustration of the lung metastasis inhibition process in 4T1 tumor-bearing mice. (b) The representative images and H&E stained images of lung tissues from mice after various treatments. Yellow arrows and yellow circles marked the tumors. The scale bars are 2 mm and 200 µm. (c) The number of lung metastasis nodules in different experimental groups. Irradiation: 808 nm, 1.0 W cm⁻², and 8 min. *p < 0.05; ns indicates no statistical significance. Data are expressed as mean ± SD (n = 3).

3. Conclusions

In this study, we successfully prepared an IrNpP with high photothermal conversion and ROS clearance activity. The IrNpP exhibits good photothermal stability, photothermal conversion efficiency and biocompatibility. Under 808 nm near-infrared (NIR) irradiation, the IrNpP is capable of effectively increasing the temperature of the tumor and inhibiting tumor growth. In addition, the IrNpP exhibits extensive reactive oxygen species (ROS) scavenging activity. In vivo and in vitro experimental results show that the IrNpP is able to scavenge excessive ROS caused by PTT, thereby alleviating the inflammatory response and inhibiting tumor metastasis without systemic toxicity. In conclusion, our study opens new avenues to address the question of subsequent inflammatory treatment in PTT.

Ethics approval

All animal experiments were performed in accordance with the guidelines evaluated and approved by the ethics committee of Chongqing Medical University (20230106).

Author contributions

Xianghua Yang: methodology, investigation, data curation, software, formal analysis, writing – review and editing, and writing – original draft. Siwen Yi: methodology, investigation, formal analysis, and data curation. Meiling Liu: methodology, investigation, formal analysis, and software. Linlin Huo: methodology and formal analysis. Mingya Tan: investigation, methodology, formal analysis, and software. Jiayi Zhao: methodology. Taotao Chu: methodology. Zhenghuan Zhao: conceptualization, validation, supervision, methodology, investigation, formal analysis, data curation, funding acquisition, writing – review and editing, and writing – original draft.

Conflicts of interest

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

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information: additional experimental materials, methods, TEM analyses, FT-IR spectra analyses, absorption spectra analyses, heating and cooling curves of aqueous solutions analyses, ROS scavenging, IVIS, blood circulation, biodistribution, body weights of mice, lung metastasis. See DOI: https://doi.org/10.1039/d5qm00711a.

Acknowledgements

The scheme used in this study was created with BioRender.com. This work was supported by the Natural Science Foundation of Chongqing (CSTB2023NSCQ-LZX0033) and the Program for Youth Innovation in Future Medicine, Chongqing Medical University (W0105).

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