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A novel nanomedicine for osteosarcoma treatment: triggering ferroptosis through GSH depletion and inhibition for enhanced synergistic PDT/PTT therapy

Journal of Nanobiotechnology volume 23, Article number: 323 (2025) Cite this article

Abstract

Osteosarcoma treatment remains challenging due to the limitations of single-modality therapies. To address this, we designed a carrier-free nanomedicine SRF@CuSO4.5H2O@IR780 (CSIR) for synergistic ferroptosis, photodynamic therapy (PDT), and photothermal therapy (PTT) in osteosarcoma. Interestingly, CSIR could harness the enhanced permeability and retention (EPR) effect to effectively enter tumors. Copper ions (Cu2+) within CSIR could react with the reductive intracellular environment, depleting glutathione (GSH) levels. Near-infrared (NIR) irradiation of CSIR further depleted GSH through reactive oxygen species (ROS) generation. Additionally, CSIR released sorafenib (SRF), which inhibited cystine-glutamate antiporter system xCT (xCT), thereby blocking GSH biosynthesis. RNA sequencing data confirmed ferroptosis induction by CSIR. This synergistic strategy of GSH depletion-induced ferroptosis, enhanced PDT, and photothermal cascade holds promise for improved osteosarcoma treatment and future nanomedicine design.

Introduction

Over the past three decades, despite great progress in the treatment of osteosarcoma (OS) using surgery, neoadjuvant chemotherapy, and radiotherapy, therapeutic results remain unsatisfactory [1,2,3]. The limitations of current osteosarcoma therapies necessitate the exploration of novel treatment strategies. Promising alternatives have emerged in the form of near-infrared (NIR) light-mediated phototherapies like photothermal therapy (PTT) and photodynamic therapy (PDT) [4, 5]. These techniques offer distinct advantages: precise targeting, minimal invasiveness, and ease of use. However, due to tumor heterogeneity, diverse phenotypes, heat-resistant cells, and the activation of anti-apoptotic pathways, single-modality PTT or PDT often fall short of achieving complete tumor eradication [6]. Among synergistic therapies that build upon PTT, PDT has garnered significant interest [7]. The combination demonstrates superior therapeutic efficacy compared to PTT alone. NIR laser irradiation can induce a high local temperature and generate high levels of reactive oxygen species (ROS) within the tumor tissue [8]. ROS considered a potent weapon against cancer cells, plays a crucial role in PDT-mediated cell death [9]. This approach effectively eliminates residual heat-resistant cancer cells that may survive PTT, offering a complementary cell death mechanism via ROS [10].

Ferroptosis typically presents with reduced activity of an enzyme known as glutathione peroxidase 4 (GPX4), enhanced lipid peroxidation, and malfunctioning mitochondria [11,12,13]. A state of ferroptosis occurs when ROS accumulate within the cells beyond the capacity of glutathione (GSH) and the phospholipid hydroperoxidases to maintain redox balance [14, 15]. Furthermore, compared to normal tissues, tumor microenvironment (TME) exhibits mild acidity, hypoxia, and high concentration of GSH, an antioxidant that promotes intracellular ROS scavenging [16]. Indeed, depleting GSH in cells alone does not produce “GSH starvation” given that tumor cells can produce more GSH through metabolic replenishment [17]. Over the years, several studies have shown that GSH, a key antioxidant within cells, is essential for GPX4 function [18, 19]. GSH acts as the fuel for GPX4’s enzymatic reaction that breaks down harmful hydroperoxides [20]. The production of GSH relies on cystine import facilitated by the amino acid transporter SLC7A11 and the subsequent coupling of cysteine with glutamate by the enzyme GCL [21, 22]. Therefore, the cystine-GSH-GPX4 axis is considered the cornerstone of ferroptosis regulation [23]. Research has revealed that nanomaterials can penetrate cancer cell membranes, accumulating in tumors and potentially enhancing treatment efficacy [24]. This suggests an intriguing strategy: developing nanoparticles (NPs) that trigger ferroptosis by depleting and hindering the production of intracellular GSH, potentially boosting the therapeutic effects of PDT or PTT.

Glutathione synthesis requires three essential precursors: glycine, glutamate, and cysteine. Notably, cysteine is the rate-limiting factor in this process [25]. Consequently, the intracellular concentration of GSH can be regulated by controlling the availability of these related amino acids [26]. Cancer cells primarily acquire cysteine through the uptake of extracellular cystine, the oxidized form of cysteine [27]. This uptake is facilitated by the solute carrier family 7 member 11 (SLC7A11), also known as xCT [21]. Sorafenib (SRF) is among the most important approved for HCC treatment due to its ability to induce ferroptosis by inhibiting cystine-glutamate antiporter System Xc [28]. According to studies, sorafenib suppresses cystine-glutamate antiporter-xCT, which leads to ROS accumulation and GSH depletion [29]. Meanwhile, numerous cancers have been demonstrated to be affected by sorafenib-induced ferroptosis and lipid peroxidation [29, 30]. Recent research has demonstrated the efficacy of Cu²⁺-catalyzed Fenton-like reactions in weakly acidic and neutral environments [31]. These reactions exhibit significantly faster rates compared to traditional Fenton and Fenton-like reactions. Upon release from the TME, Cu²⁺ can deplete intracellular GSH and generate Cu⁺ [32]. Notably, the reaction rates of Cu²⁺ (460 M⁻¹ s⁻¹) and Cu⁺ (10,000 M⁻¹ s⁻¹) surpass those of Fe²⁺ by a factor of six and 132 [33], respectively. Furthermore, Cu²⁺ can deplete overexpressed endogenous GSH, subsequently converting to the more potent Cu⁺ [32]. This characteristic highlights its immense potential for amplifying oxidative stress within cancer cells. Additionally, due to its exceptional coordination capability, Cu²⁺ can be readily integrated with various multifunctional materials, such as the photodynamic agent IR780 [34]. Based on these findings, there is a promising avenue for developing GSH-depleting therapeutics that leverage ferroptosis-inducing strategies.

IR780, a water-insoluble dye with peak absorption at 808 nm, demonstrates inherent tumor-targeting properties [35] This characteristic allows for the conversion of light energy into heat and ROS, with a focus on the mitochondria, even without the addition of specific tumor-targeting ligands [36, 37]. Consequently, IR780 holds significant promise for in vivo tumor therapy. During the last few years, carrier-free drug delivery systems (DDSs) for the treatment of cancer have moved forward significantly [38, 39]. Compared with carrier-containing nanoparticles, carrier-free nanoparticles are less likely to cause materials-induced side effects [40]. This study presents the development of a novel nanomedicine, SRF@CuSO4.5H2O@IR780 NPs (CSIR), which embodies a synergistic approach combining ferroptosis, photothermal therapy, and photodynamic therapy. Notably, CSIR functions as a carrier-free nanomedicine, where each component serves a distinct purpose. Inspired by these findings, we designed CSIR to deplete and inhibit GSH synthesis through electrostatic interactions and π-π stacking between Cu2+, Sorafenib, and IR780. Notably, Cu2+ could act as a bridge facilitating strong binding between SRF and IR780. As depicted in Scheme 1, this ingenious nanomedicine was formulated solely from drug molecules, eliminating the need for organic or inorganic carriers. Following accumulation at the tumor site via the enhanced permeability and retention (EPR) effect, CSIR undergoes a redox reaction with GSH, consuming GSH and triggering the decomposition of CSIR, leading to the simultaneous release of IR780, Sorafenib, and Cu2+. A combination of boosted intracellular ROS production and GSH consumption efficiently induced ferroptosis in OS through inactivation of GPX4 and accumulation of lipid peroxides (LPOs). Consequently, this proposed CSIR-based strategy presents a robust approach for achieving synergistic antitumor effects in PDT/PTT therapy. CSIRs effectively deplete intracellular GSH and inhibit its synthesis. This dual action holds significant potential for treating OS. Further research is warranted to explore the mechanisms underlying the synergistic effects observed between CSIRs and PDT/PTT. By elucidating these mechanisms, this approach can potentially become a safe and highly effective strategy for tumor therapy.

Results and discussion

Synthesis and characterization of CSIR

Scheme 1 illustrates the design of a system for depleting intracellular GSH and inhibiting its synthesis to trigger ferroptosis, thereby enhancing the therapeutic efficacy of PDT/PTT. The optimal proportional composition of the system referred to as CSIR, was determined by investigating the mass ratio of sorafenib, Cu2+, and IR780. Particle size and polydispersity index (PDI) were characterized for various feed ratios with different amounts (0.5, 1, 1.5, and 2.0 μg) of IR780 (10 mg mL− 1 in DMSO) dissolved in 1 mL of ultrapure water. As shown in Fig. 1a and b, the CSIR prepared at a feed mass ratio of 1:2:1 exhibited the most favorable nanoparticle size and PDI distribution. This ratio was consequently chosen for further experiments. Figures 1c and d demonstrate that CSIR displayed acceptable changes in hydrodynamic size and PDI after incubation in PBS at different pH values for 9 days, indicating its stability. Further characterization of the CSIR nanomedicine’s composition was achieved using energy-dispersive X-ray spectroscopy (EDS) analysis (Figure S1a, SI) and elemental mapping (Fig. 1e). Additionally, X-ray photoelectron spectroscopy (XPS) (Figure S1b, SI) and Fourier-transform infrared spectroscopy (FTIR) were employed to evaluate the functional groups present within the CSIR nanomedicine. As depicted in Fig. 1f, the absorption peaks observed at 1202 cm⁻¹ and 1155 cm⁻¹ corresponded to the S = O stretching vibration mode of the sulfate group. Similarly, the peaks at 659 cm⁻¹ and 604 cm⁻¹ originated from the S = O bending vibration mode of the same group. The peaks at 1724 cm⁻¹ and 1690 cm⁻¹ were characteristic of the C = O stretching vibration. Notably, the CSIR spectrum primarily resembled that of IR780, although additional peaks were present. The peak near 618 cm⁻¹ indicated the S = O bending vibration of copper sulfate pentahydrate, while the peak near 1700 cm⁻¹ corresponded to the C = O stretching vibration of SRF. These observations suggest the successful incorporation of copper sulfate pentahydrate and SRF into the IR780 matrix. Furthermore, the sample exhibited a noticeable change in the peak shape of the N-H and O-H absorption peaks around 3429 cm⁻¹, potentially indicating the presence of hydrogen bonding interactions between the various components of the CSIR [41]. In addition, the original sharp peaks of each sample disappeared and stretched into broad peak shapes, which was due to the interaction between nanomaterial particles (and quantum size effect), indicating that the synthesized nanomaterials have a large dispersion and small particle size [42]. To gain deeper insight into the formation of CSIR, XPS analysis was conducted (Figure S2, Supporting Information). The C1s spectrum of LICN revealed six distinct peaks corresponding to various bonding configurations: 284.79 eV (C-C/C-H), 285.90 eV (C-O), 287.11 eV (C = O), 288.72 eV (CF), 291.19 eV (CF2), and 292.76 eV (CF3). The F1s spectrum of CSIR displayed a single peak at 688.10 eV (C-F). Additionally, the presence of chlorine (Cl), iodine (I), and copper (Cu) was confirmed by the XPS survey spectrum (Figure S2).

Scheme 1
scheme 1

Schematic illustration of CSIR as a carrier-free nanomedicine induce ferroptosis by depleting intracellular GSH and inhibiting its synthesis to improve the therapeutic effect of PDT /PTT

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Fig. 1
figure 1

Synthesis and characterization of CSIR (a) Dynamic light scattering (DLS) analysis of SRF /Cu2+/IR780 complexes with different feed ratios (n = 3). (b) DLS analysis of hydrodynamic size distribution of CSIR. (c) The hydrodynamic size and polydispersity index (PDI) changes of CSIR in PBS within 9 days. Data were presented as the mean value ± SD, n = 3. (d) The hydrodynamic size changes of CSIR in PBS with different pH within 9 days. (e) TEM image and EDS mapping of CSIR. Scale bars: 100 nm. (f) FTIR analysis of Cu2+, SRF, IR780 and CSIR. (g) Temperature variation curves of PBS, IR780, and CSIR under NIR irradiation (n = 3). (h) Infrared thermal imaging of PBS, IR780, and CSIR for 5 min under NIR 808 nm laser irradiation. (i) Temperature variation curves of CSIR under different laser power densities and (j) various CSIR concentrations (n = 3)

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The UV–Vis absorption spectra indicate that both CSIR and IR780 exhibit a maximum absorption peak at approximately 780 nm, with the absorbance of CSIR being slightly lower than that of IR780 (Figure S4). The molecular structure of sorafenib includes a pyridine ring and ether bonds, where the nitrogen and oxygen atoms possess lone pair electrons that can coordinate with copper ions (Cu²⁺). As a metal center, Cu²⁺ forms coordination bonds with the pyridine ring of sorafenib and with specific groups (such as nitrogen or oxygen atoms) in IR-780 iodide, thereby linking the two compounds together. IR-780 iodide is a lipophilic cationic compound. Its hydrophobic segment, such as the long-chain conjugated structure, can interact with other hydrophobic regions (for example, the hydrophobic domains of sorafenib) via hydrophobic interactions. These interactions promote the close packing of molecules, leading to the formation of the nanoparticle core structure. In particular, the above-mentioned spectral range contains peaks characteristic of the Cu2+ [31]. These findings collectively validate the successful synthesis of CSIR. The content of IR780, SRF, and Cu2+ within CSIR were quantified using UV-visible absorption spectroscopy and inductively coupled plasma mass spectrometry (ICP-MS) for each component, respectively. The encapsulation efficiencies were 82.41% for IR780, 73.60% for SRF, and 78.53% for Cu2+.

Figures 1g and h depict the results of infrared thermal imaging and temperature change curves for PBS, free IR780, and CSIR solutions irradiated with an 808 nm NIR laser at 1.2 W cm− 2 for 5 min. Notably, the PBS group exhibited minimal temperature change under laser exposure. In contrast, the temperature of both the free IR780 and CSIR groups rose significantly, exceeding 80 °C, which was well above the 42 °C threshold required for thermal tumor cell ablation [43]. These findings, corroborated by the similar infrared thermographic images of free IR780 and CSIR (Fig. 1g), demonstrated that CSIR effectively retained the superior photothermal properties of IR780. Furthermore, Fig. 1i and j illustrate that the temperature increase of CSIR accelerated with higher laser power density and concentration. Based on the results presented in Fig. 1g h, we chose to characterize the photothermal conversion efficiency under the conditions of a 40 μg/mL concentration and a laser power density of 1.2 W/cm². Under irradiation with an 808 nm laser at 1.2 W/cm², we recorded the temperature increase of the 40 μg/mL CSIR solution over 600 s. After switching off the laser, the solution was allowed to cool naturally, and the temperature change was similarly recorded over 600 s. Using the method described in reference, we calculated the photothermal conversion efficiency of the CSIR nanomaterials. As shown in Figure S3  illustrates a heating and cooling cycle, and fitting the data to produced a t‑-lnθ plot with a time constant (τs) of 235.6 s. According to the formula provided in the literature, the photothermal conversion efficiency of the CSIR solution was determined to be 68.9%. This observation suggests that the photothermal conversion efficiency of CSIR exhibited a dependence on both laser power and concentration.

Cellular uptake and biodistribution of CSIR

Given the critical role of intracellular uptake in tumor eradication, we employed CLSM to investigate the cellular internalization of CSIR in K7M2 tumor cells. Cellswere incubated with either “free Cu²⁺+SRF + IR780” (a mixture of unbound components) or CSIR. Hoechst 33,342 fluorescent stain (blue) was used to visualize cell nuclei, while IR780 emitted red fluorescence. As depicted in Figure S7a, K7M2 cells treated with CSIR exhibited a significantly stronger red fluorescence intensity than those treated with “free Cu²⁺+SRF + IR780” or PBS alone. This observation suggests a higher degree of cellular uptake for CSIR. These findings were further corroborated by flow cytometry analysis (Figure S7b). Figure 2a further demonstrates a time-dependent increase in red fluorescence intensity, indicating a gradual accumulation of CSIR within the cells over time (0 to 12 h). Notably, the CSIR group displayed a markedly stronger red fluorescence signal than the free drug group (“free Cu²⁺+SRF + IR780”). This observation confirms the effective uptake of CSIR by K7M2 cells. Flow cytometry analysis (Fig. 2b and c) further validated these findings, supporting the feasibility and potential of CSIR for in vivo applications.

Fig. 2
figure 2

Cellular Uptake and Biodistribution of CSIR (a) CLSM, (b) FCM, and (c) MFI analysis of red florescence in K7M2 cells after incubated with “PBS”, “Cu2++SRF + IR780” and “CSIR” for 12 h, respectively. Scale bar: 10 μm. (d) In vivo biodistribution of free “Cu2++SRF + IR780” and “CSIR” in tumor-bearing nude mice within 48 h. (e) The fluorescence intensity of the fluorescently labeled CSIR changes over time. (f) Fluorescence imaging of the major organs and the tumors 48 h after injection of free “Cu2++SRF + IR780” and “CSIR”. (g) A comparison of the fluorescent intensities. (h) Thermal imaging at different times and (i) Temperature variation curves at the tumor sites. Data (c, e, g, i) are presented as the mean value ± SD, n = 3. One-way ANOVA was applied for statistical analysis in “c”, followed by Tukey’s multiple comparisons test, and student’s t-test (two-tailed) were conducted for statistical analysis in “e”, “g”, and “i” (*P < 0.05, **P < 0.01, ***P < 0.001 and****P < 0.0001)

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To evaluate the in vivo tumor tropism of CSIR, we employed a small animal fluorescence imaging system to track the distribution of CSIR formulations within tumor-bearing Balb/c nude mice. The mice were injected with CSIR samples via their tail veins. As shown in Fig. 2d and e, the experimental data demonstrated a higher fluorescence intensity in the tumors of the CSIR group compared to the group receiving “free Cu2++SRF + IR780”. The CSIR group exhibited the most pronounced fluorescence intensity at 36 h post-injection, which remained detectable within the tumor even 48 h later. This significant tumor accumulation of CSIR can be attributed to the EPR effect [44]. In contrast, the group receiving the free drugs (free Cu2++SRF + IR780) displayed weak fluorescence. In conclusion, these findings convincingly demonstrate the tumor targeting and accumulation capabilities of CSIR. Based on the observed intratumoral accumulation profile, the NIR laser irradiation time was set to 24 h post-injection for the subsequent in vivo antitumor efficacy studies.

Following a 48-hour observation period, the ex vivo fluorescence imaging analysis encompassed the tumor and key organs such as the liver, heart, kidneys, lungs, and spleen (Fig. 2f). Notably, most of the fluorescent signal was localized within the tumor region (Fig. 2g), with a secondary but significant accumulation observed in the lung tissue. This pulmonary uptake can be attributed to the non-specific sequestration of IR780 [45]. Overall, these results suggest the promising tumor-targeting potential of CSIR. In tumor therapy, the selective enrichment of therapeutic agents within tumor tissue and cells is a critical determinant of treatment efficacy. Having established the effective accumulation of CSIR, our subsequent investigation focused on elucidating the potential mechanisms of tumor inhibition at both the cellular and in vivo levels.

To further assess the suitability of CSIR for in vivo applications, we evaluated its temperature-modulating capabilities following administration to tumor-bearing mice. Twenty-four hours post-injection, we employed an infrared (NIR) thermal camera to record thermal changes within the tumor region following 5 min of laser irradiation at 808 nm and a power density of 1.20 W cm− 2. This approach facilitated real-time temperature monitoring. Following 5 min of laser irradiation, Balb/c nude mice treated with CSIR developed tumor temperatures higher than those of the “free Cu2++SRF + IR780” group and PBS group (Fig. 2h, i), exhibiting its photo-thermal conversion capabilities in vivo.

In vitro GSH depletion and synergistic therapeutic performance in vitro

Glutathione, the primary cellular antioxidant, safeguards cells against oxidative stress. Elevated GSH levels have been documented in various cancer cells, contributing to their survival. Inhibiting GSH synthesis and depleting GSH stores could diminish the adaptive antioxidant capacity, thereby inducing significant oxidative stress within cancer cells [46]. Furthermore, heightened oxidative stress can render cancer cells more susceptible to GSH depletion. As illustrated in the schematic diagram of Fig. 3a, a subsequent experimental investigation was conducted to elucidate the antitumor effect of CSIR and its mechanism of GSH biosynthesis inhibition and intracellular GSH depletion. Each component of our formulated carrier-free nanomedicine plays a distinct role. SRF, as established, hinders GSH biosynthesis by impeding the Xc- transporter system’s Xc- transport activity [28, 29]. We employed 5,5ʹ-dithiobis-(2-nitrobenzoic acid) (DTNB) to assess GSH depletion. As shown in Fig. 3b, a peak absorption was observed at 407 nm, indicative of a substantial quantity of GSH within the cells. Conversely, a progressive decline in the absorption peak intensity at 407 nm was observed with increasing incubation time, potentially attributable to the reaction between GSH and Cu2+. As depicted in Fig. 3c, a rise in Cu2+ concentration corresponded to a gradual decrease in absorption at 407 nm. These experiments convincingly demonstrate CSIR’s remarkable capacity for GSH depletion.

Fig. 3
figure 3

In vitro GSH depletion and cell damage effect of CSIR a) Schematic illustration of the CSIR inhibits GSH biosynthesis and depletes intracellular GSH. Incubation of GSH with CSIR was performed at different times and different Cu2+ concentrations according to (b) and (c) respectively. d) Microscopic observation and e) MFI of intracellular ROS in K7M2 cells following different treatments. Scale bar: 100 μm. (f and g) K7M2 Cell viability evaluation by a Live/Dead assay. Scale bar: 100 μm

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The generation of reactive oxygen species (ROS) by CSIR in vitro was assessed using a singlet oxygen sensor green fluorescent probe. As shown in Figs. 3d and e, minimal green fluorescence was observed in the PBS control groups. Similarly, negligible fluorescence signals were detected in the “Cu²⁺” and “SRF” groups, indicating insufficient ROS production via the Cu²⁺-mediated Fenton-like reaction and a minimal effect of sorafenib on ROS levels. In contrast, cells treated with samples containing IR780 and exposed to subsequent laser irradiation exhibited a marked increase in green fluorescence, suggesting significant ROS generation by IR780 under laser irradiation. This effect compensates for the limited ROS production from Cu²⁺ and SRF. Notably, the CSIR group displayed the most pronounced fluorescence intensity among all groups (Figure S5c). These results demonstrate the efficient cellular uptake of our carrier-free nanomedicine formulation, which leverages the synergistic effects of its individual components to induce substantial ROS production.

Subsequently, cell viability was assessed by staining the cells with calcein-AM and propidium iodide (Calcein-AM/PI). As shown in Fig. 3g, cells treated with individual drugs (Cu²⁺, SRF, or IR780) primarily displayed live cells (green fluorescence), similar to the PBS control group, indicating that these agents alone do not exhibit significant antitumor effects. Notably, only a few dead cells (red fluorescence) were observed in the SRF + IR780 and Cu²⁺+SRF + IR780 groups, while a substantial number of dead cells were detected in the CSIR group, demonstrating its superior antitumor activity. These observations were further supported by the MTT assay results (Fig. 3f), which indicated a 98% antitumor rate for the CSIR group. As shown in Figure S5a and b, under NIR laser irradiation (1.2 W/cm² for 3 min), cell viability in the PBS + NIR group showed no significant difference compared to the PBS group, suggesting that NIR irradiation alone does not affect cell viability. However, in the absence of NIR irradiation, the survival rate of osteosarcoma cells in the CSIR group was 32.3%, with live/dead fluorescence staining revealing only a small number of dead cells. In contrast, under NIR irradiation, the survival rate of osteosarcoma cells in the CSIR group significantly decreased to 2%. These findings suggest that CSIR, when combined with NIR irradiation, induces a remarkably enhanced antitumor effect via multimodal therapy. Even without the photothermal effect, CSIR exhibits a certain degree of osteosarcoma inhibition by releasing Sorafenib and Cu²⁺, which effectively deplete intracellular GSH and significantly reduce its levels. When exposed to NIR irradiation, the photothermal effect synergizes with other therapeutic modalities, substantially enhancing its antitumor efficacy. We have also included data on the release levels of Sorafenib, Cu²⁺, and IR780 from CSIR in PBS, as shown in Figure S6. After 6 h, the release of Sorafenib from CSIR reached a plateau, with a cumulative release of approximately 30%. Similarly, after 8 h, CSIR released 0.97 mg/L of Cu²⁺ and 35.6% of IR780.

Ability of CSIR to induce ferroptosis

Ferroptosis is a regulated form of cell death characterized by reduced intracellular GSH levels, elevated ROS levels, and accumulation of lipid peroxides. Notably, mitochondrial shrinkage is also a hallmark of ferroptosis. We further investigated whether combining these effects could synergistically induce potent ferroptosis. MMP (mitochondrial membrane potential) serves as an indicator of mitochondrial health, structural integrity, and function. In the “Cu2++SRF + IR780” and “CSIR” treated groups, a significant shift in the cell population from the PE channel to the FITC channel was observed by flow cytometry (FCM) analysis. This shift was most pronounced in the “CSIR” group (Fig. 4a), suggesting severe mitochondrial damage. Furthermore, transmission electron microscopy (TEM) was employed to assess changes in K7M2 cell mitochondrial morphology following treatment with different samples. As shown in Fig. 4b, mitochondria in the mixed drugs treated group exhibited damage, while those in the “Cu2++SRF + IR780” and “CSIR” groups displayed severe disruption, characterized by edema, membrane rupture, and cristae breakdown. These TEM findings corroborate the results obtained by FCM analysis. Collectively, these results indicate that CSIR can amplify mitochondrial damage through ROS accumulation and GSH depletion.

Fig. 4
figure 4

Evaluation of ferroptosis (a) Different treatment groups measuring mitochondrial membrane potential by flow cytometry (FCM). (b) TEM examination of cell mitochondrial integrity. Scale bar: 500 nm. (c) CLSM monitoring the intracellular lipoperoxide accumulation by C11-BODIPY 581/591 probe on the K7M2 after incubation with different samples. Scale bar: 10 μm. (d) Western blot analysis of the expression of xCT and GPX4 in K7M2 cells after different treatments. Gray statistics of (e) xCT and (f) GPX4

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As lipid peroxidation is a hallmark of ferroptosis, we measured the amount of peroxidation in K7M2 cells by staining them with BODIPY-C11 581/591. BODIPY-C11 exhibits a red fluorescence signal that transforms to green upon lipid oxidation. As depicted in Fig. 4c, the PBS and Cu2+ treatment groups displayed the strongest red fluorescence intensity and the weakest green fluorescence intensity. Conversely, a mild decrease in red fluorescence intensity and a moderate increase in green fluorescence intensity were observed following treatment with either SRF or IR780 alone. Notably, these changes were further amplified in the combination treatments of IR780 with Cu2+ or sorafenib. As expected, the combined treatment of IR780 with sorafenib and Cu2+ mostly exhibited green fluorescence, suggesting that the combination of three drugs could significantly promote the accumulation of lipid peroxides. Importantly, a synergistic interaction among the components within our formulated carrier-free nanomedicine effectively mediated a substantial decrease in reduced GSH levels in tumorigenic cells.

Ferroptosis is characterized by the inactivation of GPX4, either through a decrease in GSH levels or direct inhibition during ferroptosis. Additionally, the inhibition of xCT by sorafenib leads to a reduction in GSH synthesis. Western blot analysis of K7M2 cells treated with various interventions revealed alterations in the expression of GPX4 and xCT (Fig. 4d). Notably, the SRF group and groups containing SRF exhibited a reduction in xCT expression compared to the other groups, with the CSIR group showing the lowest expression level (Fig. 4e). Moreover, Fig. 4f demonstrates a significant downregulation of GPX4 expression in the CSIR-treated group upon NIR irradiation. This suggests that CSIR, when exposed to NIR irradiation, can downregulate GPX4 expression via the GSH/GPX4 axis [23], thereby enhancing lipid peroxidation and promoting ferroptosis. Collectively, these results indicate that CSIR induces ferroptosis by depleting GSH and inhibiting GSH biosynthesis.

Potential mechanisms of CSIR mediated ferroptosis

To elucidate the molecular mechanisms underlying the anti-tumor activity of CSIR, a transcriptome sequencing analysis was employed to compare the gene expression profiles of K7M2 tumors treated with CSIR to those of a control group. To elucidate the molecular signaling pathways associated with ferroptosis, we employed DAVID software for differential expression analysis. Principal component analysis (PCA) revealed a substantial divergence in gene expression profiles between the two K7M2 cell groups (Fig. 5a). Figure 5b presents a cluster analysis heatmap of differentially expressed genes (DEGs), where the bottom axis represents samples and the top axis.

Fig. 5
figure 5

Transcriptome sequencing showed that ferroptosis-related genes. (a) PCA and clustering analyses on all gene expression read counts showed a clear distinction between the PBS and CSIR groups. (b) Visualization of the top 100 differentially expressed gene expression in a heat map. c) Volcano plots for differentially expressed genes between the PBS group and the CSIR group. d) GO enrichment analysis, and e) KEGG pathway analysis. f) Ferroptosis signaling pathway ranked at the top

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represents sample clusters. Furthermore, a volcano plot (Fig. 5c) visually depicts the differentially expressed genes between the two groups, with red signifying upregulated DEGs and green signifying downregulated DEGs. Functional enrichment analyses using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were performed on the DEGs identified in CSIR-treated K7M2 tumors (Fig. 5d and e). These analyses revealed a spectrum of functions and pathways associated with the DEGs. GO analysis indicated that the DEGs participate in various biological processes (BP), including small GTPase-mediated signal transduction, chromatin organization, and regulation of cell morphogenesis. Regarding molecular function (MF), the DEGs were enriched for activities such as GTPase regulation, nucleoside-triphosphatase regulation, and GTPase activation. Intriguingly, KEGG pathway enrichment analysis highlighted the intersection between differentially expressed genes, iron death-related genes, and ferroptosis signaling as the most significant pathway (Fig. 5f). Within the enriched ferroptosis.

signaling pathway genes, GPX4 exhibited the most pronounced difference between the CSIR-treated and PBS-treated groups. Furthermore, SLC genes were also ranked among the top 10 most differentially expressed genes (see Figure S8a and b).

In vivo antitumor efficiency of CSIR

To assess the in vivo antitumor efficacy, nude mice harboring K7M2 tumors were randomly assigned to eight groups upon reaching a predetermined tumor volume of approximately 200 mm3. A schematic of the experimental scheme can be found in Fig. 6a. The laser parameters used in the in vivo experiments for CSIR were as follows: laser power of 1.2 W/cm², treatment duration of 5 min, administered once every other day. Tumor volume measurements were obtained every other day, and the resulting data points were used to construct tumor growth curves. Neither the PBS nor the Cu2+ group exhibited a significant effect on tumor growth over the course of 12 days, as shown in Fig. 6b and c. Conversely, the treatment groups receiving SRF and IR780 demonstrated a moderate inhibitory effect on tumor growth. Tumor inhibition was somewhat weaker in the “Cu2++IR780”, in contrast to the “SRF + IR780” group, which had a partial tumor-suppressing effect. The tumor volume in the “Cu2++SRF + IR780” group was considerably reduced after treatment, attributed to Cu2+, sorafenib, and IR780 synergistically inducing ferroptosis to enhance the effect of PDT/PTT. Crucially, nude mice in the CSIR group exhibited the most significant tumor growth inhibition following therapy compared to the combined treatment with Cu2+, SRF, and IR780 mixtures. This finding underscores the remarkable potential and therapeutic value of these self-assembled nanomedicines in enhancing the efficacy of combination therapy. Twelve days after treatment initiation, tumors were excised, photographed, and measured. Notably, ex vivo tumor size and weight in the CSIR group were demonstrably reduced compared to the unassembled drug groups (Fig. 6d and e). In three mice, complete tumor disappearance was observed, highlighting the exceptional tumor ablation efficacy of CSIR. As shown in Supplementary Figure S9, no significant weight loss was observed in any treatment group throughout the study.

As indicated by the tumor growth curves, there was no significant difference in tumor volume between the single free drug groups (Cu2+, SRF, IR780) and the PBS group, which may be attributed to the poor targeting ability of Cu2+ and SRF in vivo, as well as the poor hydrophilicity of IR780, ultimately affecting their anti-tumor efficacy. Although the tumor volume in the Cu2++SRF + IR780 group was reduced after treatment, it remained about 31.1% of that in the PBS group, demonstrating some anti-tumor activity. This is mainly attributed to the synergistic induction of ferroptosis by Cu2+, Sorafenib, and IR780, which enhances the effects of PDT/PTT. Notably, the CSIR nanocomposite drug, self-assembled from Cu2+, Sorafenib, and IR780, exhibited the most impressive anti-tumor effect, with tumor volume significantly different from that of the other seven groups. Furthermore, tumor tissue weight measurements also showed that the CSIR group displayed the best anti-tumor effect. These results indicate that the CSIR nanocomposite drug not only possesses the functions of the individual free drugs but also demonstrates tumor targeting ability, enhancing anti-osteosarcoma therapeutic effects through the induction of ferroptosis combined with PDT/PTT treatment.

Furthermore, analysis of key blood indicators, including uric acid (UA), serum creatinine (CREA), aspartate aminotransferase (AST), alanine transaminase (ALT), blood urea nitrogen (BUN), albumin (ALB), total bile acid (TBA), and direct bilirubin (DBil), revealed values similar to those of the PBS group (Fig. 6f and Figure S12). This suggests the excellent safety profile of CSIR. Additionally, H&E staining of major organs (heart, liver, spleen, lung, and kidney) revealed no discernible pathological changes (Supplementary Figure S10). Moreover, as demonstrated in Supplementary Figure S11, CSIR exhibited good blood compatibility, with hemolysis rates below 8% at various concentrations. Following therapy, tumors were excised for TUNEL, H&E, Ki-67, SLC, and GPX4 staining (Fig. 6g). The results revealed substantial tumor tissue damage and necrosis induction in the CSIR group, whereas the PBS group displayed intact cellular morphology. Furthermore, immunohistochemistry (IHC) analysis was performed to assess the expression of Ki-67 proliferation proteins, along with GPX4 and xCT, which are representative proteins associated with tumor ferroptosis and amino acid transport, respectively. Ki-67, GPX4, and xCT expression levels were significantly lower in the CSIR group compared to other treatment groups. These findings collectively demonstrate the remarkable potential of CSIR nanomedicine for antitumor therapy by triggering ferroptosis in synergy with PDT/PTT therapy.

Fig. 6
figure 6

In vivo antitumor efficiency of CSIR a) Schematic diagram of the strategy for the antitumor of CSIR in subcutaneous K7M2 tumor-bearing mice. b, c) Tumor growth curves in K7M2 tumor-bearing mice. d) Weight of harvested tumors after various treatments. e) Various treatment groups’ tumor tissues. f) Nude mouse blood biochemistry indexes after various treatments. g) TUNEL, H&E, Ki67, xCT, and GPX4 staining of tumors with different treatments. Scale bars: 100 μm. Data are displayed as the mean ± SD (n = 5). One-way ANOVA was applied for statistical analysis in “c”, and “d”, followed by Tukey’s multiple comparisons test (*P < 0.05 and ****P < 0.0001)

Full size image

Conclusion

Our research aimed to develop a novel carrier-free nanomedicine, CSIR, to enhance the efficacy of the combination of PDT and PTT for tumor eradication. CSIR exhibits excellent biocompatibility, effectively disrupts tumor cell antioxidant defenses, possesses high photothermal stability, and demonstrates potent antitumor activity. Experimental findings revealed that Cu2+ depletes intracellular GSH, while SRF inhibits GSH biosynthesis by blocking Xc- transport. Additionally, IR780, under 808 nm laser irradiation, elevates ROS levels within tumor cells, further exacerbating GSH depletion and leading to pronounced tumor ablation in both in vitro and in vivo models. Importantly, the induction of ferroptosis was not only supported by morphological and mechanistic evidence but also definitively validated through subsequent comprehensive RNA-Seq analysis. The ferroptotic capabilities ultimately proved advantageous for tumor eradication, highlighting the value of our efforts in exploring ferroptotic synergy with PDT/PTT for efficient antitumor therapy. This innovative combined antitumor strategy of PDT/PTT/ferroptosis, utilizing a self-assembled carrier-free nanodrug, holds immense potential for OS therapy.

Studies have reported that IR780 exhibits strong absorption in the 780–900 nm range and possesses inherent tumor-targeting properties, making it widely explored in tumor therapy. For instance, Chenxiao Jiang et al. encapsulated hydrophobic IR780 within biodegradable human serum albumin nanoparticles to achieve tumor ablation through PTT and PDT [47]. Similarly, Jyh-Ping Chen et al. loaded IR780 into liposomes to enhance glioblastoma treatment efficacy by overexpressing heat shock proteins via PTT and generating intracellular reactive oxygen species (ROS) through PDT [48]. Although loading IR780 into nanocarriers can improve its hydrophobicity and enhance therapeutic efficiency, the efficiency of single-mode therapy remains limited, and osteosarcoma treatment continues to be challenging. To address this issue, we designed a carrier-free nanodrug (CSIR) for synergistic ferroptosis, PDT, and PTT in osteosarcoma therapy. The combination of intracellular ROS generation and GSH depletion effectively induces ferroptosis in osteosarcoma by inactivating GPX4 and accumulating lipid peroxides (LPO). Therefore, this CSIR-based strategy provides a powerful approach to achieving synergistic antitumor effects in PDT/PTT-based treatments. Nude mice were selected as the tumor treatment model primarily due to their immunodeficient nature, which facilitates the establishment of xenograft tumors and enables a more precise evaluation of tumor progression and therapeutic efficacy. However, to assess the potential immunogenicity of CSIR, further investigations are required using immunocompetent mouse models, such as C57 or Balb/C mice. In these models, post-treatment cytokine levels in the blood will be analyzed to evaluate the extent of the immune response.

Experimental section

Preparation and Characterization of CSIR: CSIR was prepared using a straightforward self-assembly method. First, sorafenib (10 mg) was dissolved in 1 mL of DMSO (10 mg mL− 1), and copper sulfate pentahydrate (CuSO4.5H2O) (39 mg) was dissolved in 10 mL of ultrapure water (10 mg mL− 1). Twenty microliters of the CuSO4.5H2O solution (10 mg mL− 1) and 10 μl of the sorafenib dispersion (10 mg mL− 1) were then slowly mixed in 960 μl of ultrapure water. This mixture was stirred for 1 h, followed by 20 min of sonication. Next, 10 μl of the IR780 solution (10 mg mL− 1) in DMSO were added to the mixture and stirred for an additional 3 h. The resulting dispersion was then sonicated for 20 min to complete the formation of the carrier-free CSIR nanomedicine. Following centrifugation at 20,000 rpm for 30 min, the supernatant was collected to remove any unbound drugs. Particle size and morphology of CSIR were characterized using dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments Ltd., UK) and TEM (JEOL JEM-F200, Japan), respectively. Elemental mapping and chemical composition analysis were performed using a transmission electron microscope (FEI Talos F200S). The coordination structure of CSIR was investigated using FTIR (Thermo Fisher Scientific Nicolet iS20) and XPS (Thermo Scientific K-Alpha). The encapsulation efficiency of Cu2+ in the nanomedicine was determined by inductively coupled plasma optical emission spectrometry (ICP-OES) (Agilent 7850). The encapsulation efficiency of IR780 and SRF was quantified using a UV-vis spectrophotometer (λ = 796 nm, Edinburgh Instruments, DS5). The drug encapsulation efficiency (EE%) was calculated using the formula: EE% = We/Wt × 100% where Wt is the total weight of the drug added, We is the weight of the encapsulated drug.

Cells and culture conditions: Our in n vitro studies utilized the Murine K7M2 OS cell line, which was commercially obtained from WuhaProcell Life Technology Co., Ltd. (Wuhan, China). These cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/milliliter (U/mL) penicillin, and 0.1 milligrams/milliliter (mg mL− 1) streptomycin.

Animals: BALB/c nude mice were obtained from Henan SKobes Biotechnology Co., Ltd. All animal experiments adhered to the established laws and regulations set forth by the Laboratory Animal Center of Anhui Medical University (approval number: LLSC 20200329). To establish tumor models, a K7M2 cell suspension (approximately 5 × 106 cells in 100 μL of PBS was subcutaneously injected into the backs of the BALB/c nude mice.

In vitro photothermal properties: The photothermal performance of CSIR was assessed in vitro. Samples containing 200 μL of free IR780 or CSIR at various IR780 concentrations (0, 10, 20, 40, and 60 μg mL− 1) were prepared. PBS served as a control. Each sample was irradiated with an 808 nm laser at a power density of 1.2 W cm− 2 for 5 min. A thermal imaging camera (Fotric, 225 s L24) was used to record the resulting temperature changes. To further investigate CSIR’s photothermal performance, 200 μL of CSIR with a fixed IR780 concentration of 40 μg mL− 1 was irradiated with an 808 nm laser at different power densities (0.6, 1.2, and 1.8 W cm− 2) for 5 min.

Depletion of GSH: The intracellular glutathione levels were measured using a total glutathione detection kit. Total glutathione (oxidized glutathione, GSSG + reduced glutathione, GSH) predominantly consists of reduced glutathione (GSH). Therefore, by measuring the total glutathione content in cells, we can indirectly reflect the intracellular GSH levels. GSH reacts with the DTNB substrate in the kit, resulting in a yellow product. The amount of total glutathione is proportional to the amount of yellow substrate formed, and thus the total glutathione content can be quantified by measuring the absorbance at 412 nm (A412). Following the kit’s instructions, various stock solutions were prepared, including: (1) a 10 mM GSH stock solution, DTNB stock solution, protein removal reagent solution at a specific concentration, NADPH stock solution (40 mg/ml), and 5x diluted glutathione reductase; (2) the total glutathione assay working solution was prepared by combining 6.6 μL of 5x diluted glutathione reductase, 6.6 μL of DTNB stock solution, and 150 μL of total glutathione assay buffer for each sample. After 24 h of treatment, osteosarcoma cells were collected, washed once with PBS, and centrifuged. The supernatant was then used for total glutathione measurement. Absorbance at 407 nm, measured using a microplate reader (TECAN Spark), was used to calculate the relative GSH content (%) using the following formula: (ODtd − ODtb)/(ODpd − ODpb) × 100%, where ODtd represents the absorbance of treated cells, ODtb represents the background absorbance of treated cells, ODpd represents the absorbance of PBS-treated cells, and ODpb represents the background absorbance of PBS-treated cells.

In vitro ROS generation detection: To assess intracellular ROS production, a DCFH-DA staining assay was employed. K7M2 cells were seeded in 12-well plates (1 × 105 cells/well) and cultured overnight. The following day, the media was replaced with fresh media containing various treatments: PBS (control), individual components (Cu2+, SRF, or IR780), combinations (Cu2++IR780, SRF + IR780, or all three), or CSIR nanomedicine (all at specified concentrations: Cu2+-20 μg mL− 1, SRF and IR780-10 μg mL− 1). After 8 h of incubation, cells in groups receiving IR780 (free or combined) and CSIR were irradiated with an 808 nm laser (1.2 W cm− 2) for 5 minutes. Following an additional 4-hour incubation, cells were washed and then incubated with 10 μM DCFH-DA for 20 min at 37 °C. Fluorescence intensity, which correlates with ROS levels, was visualized using an inverted fluorescence microscope with excitation and emission wavelengths of 488 nm and 525 nm, respectively.

Live/dead cell staining analysis: For the Live/dead assay, K7M2 cells were seeded in 6-well plates at a density of 1 × 105 cells per well. The culture medium was then replaced with fresh medium containing one of the following treatments: PBS (control), free Cu2+, free SRF, free IR780, a combination of free Cu2+ and IR780, a combination of free SRF and IR780, a combination of free Cu2+, SRF, and IR780, or CSIR nanomedicine (all at concentrations of 20 μg mL− 1 for Cu2+, 10 μg mL− 1 for SRF and IR780). After co-incubation at 37 °C for 12 h, laser irradiation (808 nm, 1.20 W cm− 2, 5 minutes) was applied to the groups treated with free IR780, combined free Cu2+ and IR780, combined free SRF and IR780, combined free Cu2+, SRF, and IR780, and CSIR. Following an additional 4-hour incubation, the cells were stained with Calcein-AM and propidium iodide (PI) for 20 min. Fluorescence intensity was measured using a fluorescence microscope (Mshot, MF52-N).

Lipid peroxidation measurements: Lipid peroxidation was assessed using C11-BODIPY (581/591) staining. K7M2 cells seeded on confocal dishes were incubated with the following treatments: PBS, free Cu2+, free sorafenib (SRF), free IR780, free Cu2++IR780, free SRF + IR780, free Cu2+ + SRF + IR780, and CSIR. For groups containing IR780, cells were irradiated with an 808 nm NIR laser at a power density of 1.2 W cm− 2 for 5 min following treatment. Next, a solution of the C11-BODIPY (581/591) probe was prepared by adding 2.5 μL of the probe to 1000 μL of serum-free DMEM medium. Five hundred microliters of this solution were added to each well and incubated for 30 min at 37 °C. Afterward, the cells were washed three times with PBS and replenished with 1 mL of fresh media. Oxidized C11-BODIPY fluorescence, indicative of lipid peroxidation, was visualized at an excitation wavelength of 500 nm and an emission wavelength of 510 nm.

RNA Sequencing Analysis: Following treatment with PBS or CSIR, tumor tissues were collected for RNA isolation. Total RNA was extracted and purified using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA) was used to quantify the amount and purity of RNA in each sample. Illumina Novaseq™6000 sequencing platform (LC-BioTechnology CO., Ltd., Hangzhou, China) was employed for library construction and subsequent sequencing of the RNA samples. Differentially expressed mRNAs were identified using the R package edgeR. Genes with a fold change greater than 2 or less than 0.5 and a statistically significant difference determined by a parametric F-test comparing nested linear models (p-value < 0.05) were considered differentially expressed. DAVID software was then utilized to perform GO and KEGG pathway enrichment analyses on the identified differentially expressed genes.

In Vivo Antitumor Efficiency of CSIR: All animal experiments adhered to the established laws and guidelines set forth by the Laboratory Animal Center of Anhui Medical University (approval number: LLSC20200329). 5 × 106 K7M2 cells were subcutaneously injected into female nude mice. Once tumors reached a volume of approximately 200 mm³, the mice were divided into eight groups (PBS, Cu2+, SRF, IR780, Cu2++IR780, SRF + IR780, Cu2+ +SRF + IR780, CSIR) for treatment. Each group received various drugs intravenously, and all mice were individually marked and housed. The laser parameters used in the in vivo experiments for CSIR were as follows: laser power of 1.2 W/cm², treatment duration of 5 min, administered once every other day. Tumor volumes and body weights were monitored every other day while the mice had ad libitum access to food. Following 12 days of treatment, the mice were euthanized, and blood, major organs, and tumors were collected for further analysis.

Statistical Analysis: This study presents data as the average value ± standard deviation (SD) based on three independent experiments (n = 3). Statistical analysis employed Student’s t-test (two-tailed) to compare two groups and one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test for comparisons across multiple groups. Asterisks indicate statistically significant differences between groups, with * representing p < 0.05, ** representing p < 0.01, *** representing p < 0.001, and **** representing p < 0.0001.

Data availability

No datasets were generated or analysed during the current study.

References

  1. Sole CV, Calvo FA, Alvarez E, Cambeiro M, Cuervo M, San Julian M, Sole S, Martinez-Monge R, Sierrasesumaga L. Adjuvant radiation therapy in resected high-grade localized skeletal osteosarcomas treated with neoadjuvant chemotherapy: long-term outcomes. Radiother Oncol. 2016;119:30–4.

    Article  PubMed  Google Scholar 

  2. Tang F, Tie Y, Lan TX, Yang JY, Hong WQ, Chen SY, Shi HH, Li LQ, Zeng H, Min L, et al. Surgical treatment of osteosarcoma induced distant pre-metastatic niche in lung to facilitate the colonization of circulating tumor cells. Adv Sci (Weinh). 2023;10:e2207518.

    Article  PubMed  Google Scholar 

  3. Zhu W, Zhu L, Bao Y, Zhong X, Chen Y, Wu Q. Clinical evaluation of neoadjuvant chemotherapy for osteosarcoma. J BUON. 2019;24:1181–5.

    PubMed  Google Scholar 

  4. He C, Dong C, Yu L, Chen Y, Hao Y. Ultrathin 2D inorganic ancient pigment decorated 3D-printing scaffold enables photonic hyperthermia of osteosarcoma in NIR-II biowindow and concurrently augments bone regeneration. Adv Sci (Weinh). 2021;8:e2101739.

    Article  PubMed  Google Scholar 

  5. Yu W, Wang Y, Zhu J, Jin L, Liu B, Xia K, Wang J, Gao J, Liang C, Tao H. Autophagy inhibitor enhance ZnPc/BSA nanoparticle induced photodynamic therapy by suppressing PD-L1 expression in osteosarcoma immunotherapy. Biomaterials. 2019;192:128–39.

    Article  CAS  PubMed  Google Scholar 

  6. Wang D, Niu X, Wang Z, Song CL, Huang Z, Chen KN, Duan J, Bai H, Xu J, Zhao J, et al. Multiregion sequencing reveals the genetic heterogeneity and evolutionary history of osteosarcoma and matched pulmonary metastases. Cancer Res. 2019;79:7–20.

    Article  CAS  PubMed  Google Scholar 

  7. He F, Feng L, Yang P, Liu B, Gai S, Yang G, Dai Y, Lin J. Enhanced up/down-conversion luminescence and heat: simultaneously achieving in one single core-shell structure for multimodal imaging guided therapy. Biomaterials. 2016;105:77–88.

    Article  CAS  PubMed  Google Scholar 

  8. Guo R, Peng H, Tian Y, Shen S, Yang W. Mitochondria-targeting magnetic composite nanoparticles for enhanced phototherapy of cancer. Small. 2016;12:4541–52.

    Article  CAS  PubMed  Google Scholar 

  9. Zou H, Zhang J, Wu C, He B, Hu Y, Sung HHY, Kwok RTK, Lam JWY, Zheng L, Tang BZ. Making aggregation-induced emission luminogen more valuable by gold: enhancing anticancer efficacy by suppressing thioredoxin reductase activity. ACS Nano. 2021;15:9176–85.

    Article  CAS  PubMed  Google Scholar 

  10. Lei L, Dai W, Man J, Hu H, Jin Q, Zhang B, Tang Z. Lonidamine liposomes to enhance photodynamic and photothermal therapy of hepatocellular carcinoma by inhibiting glycolysis. J Nanobiotechnol. 2023;21:482.

    Article  CAS  Google Scholar 

  11. Li Y, Wang J, Li Y, Luo J, Liu F, Chen T, Ji Y, Yang H, Wang Z, Zhao Y. Attenuating uncontrolled inflammation by radical trapping chiral polymer micelles. ACSNano. 2023;17:12127–39.

    CAS  Google Scholar 

  12. Liang D, Feng Y, Zandkarimi F, Wang H, Zhang Z, Kim J, Cai Y, Gu W, Stockwell BR, Jiang X. Ferroptosis surveillance independent of GPX4 and differentially regulated by sex hormones. Cell. 2023;186:2748–64. e2722.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Oh SJ, Ikeda M, Ide T, Hur KY, Lee MS. Mitochondrial event as an ultimate step in ferroptosis. Cell Death Discov. 2022;8:414.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Huang L, Feng J, Zhu J, Yang J, Xiong W, Lu X, Chen S, Yang S, Li Y, Xu Y, Shen Z. A strategy of fenton reaction cycloacceleration for high-performance ferroptosis therapy initiated by tumor microenvironment remodeling. Adv Healthc Mater. 2023;12:e2203362.

    Article  PubMed  Google Scholar 

  15. Su F, Descher H, Bui-Hoang M, Stuppner H, Skvortsova I, Rad EB, Ascher C, Weiss A, Rao Z, Hohloch S, et al. Iron(III)-salophene catalyzes redox cycles that induce phospholipid peroxidation and deplete cancer cells of ferroptosis-protecting cofactors. Redox Biol. 2024;75:103257.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Yuan Z, Liu X, Ling J, Huang G, Huang J, Zhu X, He L, Chen T. In situ-transition nanozyme triggered by tumor microenvironment boosts synergistic cancer radio-/chemotherapy through disrupting redox homeostasis. Biomaterials. 2022;287:121620.

    Article  CAS  PubMed  Google Scholar 

  17. Yu J, Xiao H, Yang Z, Qiao C, Zhou B, Jia Q, Wang Z, Wang X, Zhang R, Yang Y, et al. A potent strategy of combinational blow toward enhanced cancer chemo-photodynamic therapy via sustainable GSH elimination. Small. 2022;18:e2106100.

    Article  PubMed  Google Scholar 

  18. Cao W, Zhang X, Feng Y, Li R, Lu A, Li Z, Yu F, Sun L, Wang J, Wang Z, He H. Lipid nanoparticular codelivery system for enhanced antitumor effects by ferroptosis-apoptosis synergistic with programmed cell death-ligand 1 downregulation. ACS Nano. 2024;18:17267–81.

    Article  CAS  PubMed  Google Scholar 

  19. Zhang Y, Swanda RV, Nie L, Liu X, Wang C, Lee H, Lei G, Mao C, Koppula P, Cheng W, et al. mTORC1 couples cyst(e)ine availability with GPX4 protein synthesis and ferroptosis regulation. Nat Commun. 2021;12:1589.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Xia C, Xing X, Zhang W, Wang Y, Jin X, Wang Y, Tian M, Ba X, Hao F. Cysteine and homocysteine can be exploited by GPX4 in ferroptosis inhibition independent of GSH synthesis. Redox Biol. 2024;69:102999.

    Article  CAS  PubMed  Google Scholar 

  21. Chen H, Cao L, Han K, Zhang H, Cui J, Ma X, Zhao S, Zhao C, Yin S, Fan L, Hu H. Patulin disrupts SLC7A11-cystine-cysteine-GSH antioxidant system and promotes renal cell ferroptosis both in vitro and in vivo. Food Chem Toxicol. 2022;166:113255.

    Article  CAS  PubMed  Google Scholar 

  22. Kang YP, Mockabee-Macias A, Jiang C, Falzone A, Prieto-Farigua N, Stone E, Harris IS, DeNicola GM. Non-canonical glutamate-cysteine ligase activity protects against ferroptosis. Cell Metab. 2021;33:174–e189177.

    Article  CAS  PubMed  Google Scholar 

  23. Zhang Q, Qu H, Chen Y, Luo X, Chen C, Xiao B, Ding X, Zhao P, Lu Y, Chen AF, Yu Y. Atorvastatin induces mitochondria-dependent ferroptosis via the modulation of Nrf2-xCT/GPx4 axis. Front Cell Dev Biol. 2022;10:806081.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Lv Y, Liu M, Zhang Y, Wang X, Zhang F, Li F, Bao WE, Wang J, Zhang Y, Wei W, et al. Cancer cell membrane-biomimetic nanoprobes with two-photon excitation and near-infrared emission for intravital tumor fluorescence imaging. ACS Nano. 2018;12:1350–8.

    Article  CAS  PubMed  Google Scholar 

  25. Kanaan MN, Pileggi CA, Karam CY, Kennedy LS, Fong-McMaster C, Cuperlovic-Culf M, Harper ME. Cystine/glutamate antiporter xCT controls skeletal muscle glutathione redox, bioenergetics and differentiation. Redox Biol. 2024;73:103213.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Yan H, Huo F, Yue Y, Chao J, Yin C, Rapid Reaction. Slow dissociation aggregation, and synergetic multicolor emission for imaging the restriction and regulation of biosynthesis of Cys and GSH. J Am Chem Soc. 2021;143:318–25.

    Article  CAS  PubMed  Google Scholar 

  27. Zhu J, Berisa M, Schworer S, Qin W, Cross JR, Thompson CB. Transsulfuration activity can support cell growth upon extracellular cysteine limitation. Cell Metab. 2019;30:865–76. e865.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Sun X, Niu X, Chen R, He W, Chen D, Kang R, Tang D. Metallothionein-1G facilitates sorafenib resistance through inhibition of ferroptosis. Hepatology. 2016;64:488–500.

    Article  CAS  PubMed  Google Scholar 

  29. Tong R, Feng X, Sun J, Ling Z, Wang J, Li S, Yang B, Deng J, He G, Wu J. Co-delivery of siNRF2 and sorafenib by a click dual functioned hyperbranched nanocarrier for synergistically inducing ferroptosis in hepatocellular carcinoma. Small. 2024;20:e2307273.

    Article  PubMed  Google Scholar 

  30. Zhou TJ, Zhang MM, Liu DM, Huang LL, Yu HQ, Wang Y, Xing L, Jiang HL. Glutathione depletion and dihydroorotate dehydrogenase inhibition actuated ferroptosis-augment to surmount triple-negative breast cancer. Biomaterials. 2024;305:122447.

    Article  CAS  PubMed  Google Scholar 

  31. Ma B, Wang S, Liu F, Zhang S, Duan J, Li Z, Kong Y, Sang Y, Liu H, Bu W, Li L. Self-assembled copper-amino acid nanoparticles for in situ glutathione AND H(2)O(2) sequentially triggered chemodynamic therapy. J Am Chem Soc. 2019;141:849–57.

    Article  CAS  PubMed  Google Scholar 

  32. Li S, Ding H, Chang J, Dong S, Shao B, Dong Y, Gai S, He F, Yang P. Bimetallic oxide nanozyme-mediated depletion of glutathione to boost oxidative stress for combined nanocatalytic therapy. J Colloid Interface Sci. 2022;623:787–98.

    Article  CAS  PubMed  Google Scholar 

  33. Hu R, Fang Y, Huo M, Yao H, Wang C, Chen Y, Wu R. Ultrasmall Cu(2-x)S nanodots as photothermal-enhanced fenton nanocatalysts for synergistic tumor therapy at NIR-II biowindow. Biomaterials. 2019;206:101–14.

    Article  CAS  PubMed  Google Scholar 

  34. Li T, Rao B, Xu D, Zhou J, Sun W, Zhi X, Zhang C, Cui D, Xu H. Enzyme-like copper-encapsulating magnetic nanoassemblies for switchable T1-weighted MRI and potentiating chemo-/photo-dynamic therapy. Acta Biomater. 2022;153:431–41.

    Article  CAS  PubMed  Google Scholar 

  35. Zhang A, Pan S, Zhang Y, Chang J, Cheng J, Huang Z, Li T, Zhang C, de la Fuentea JM, Zhang Q, Cui D. Carbon-gold hybrid nanoprobes for real-time imaging, photothermal/photodynamic and nanozyme oxidative therapy. Theranostics. 2019;9:3443–58.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Tan Y, Zhu Y, Wen L, Yang X, Liu X, Meng T, Dai S, Ping Y, Yuan H, Hu F. Mitochondria-responsive drug release along with heat shock mediated by multifunctional glycolipid micelles for precise cancer chemo-phototherapy. Theranostics. 2019;9:691–707.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Yang Z, Wang J, Ai S, Sun J, Mai X, Guan W. Self-generating oxygen enhanced mitochondrion-targeted photodynamic therapy for tumor treatment with hypoxia scavenging. Theranostics. 2019;9:6809–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Zhang W, Wen Y, He DX, Wang YF, Liu XL, Li C, Liang XJ. Near-infrared AIEgens as transformers to enhance tumor treatment efficacy with controllable self-assembled redox-responsive carrier-free nanodrug. Biomaterials. 2019;193:12–21.

    Article  CAS  PubMed  Google Scholar 

  39. Zhang N, Li M, Sun X, Jia H, Liu W. NIR-responsive cancer cytomembrane-cloaked carrier-free nanosystems for highly efficient and self-targeted tumor drug delivery. Biomaterials. 2018;159:25–36.

    Article  CAS  PubMed  Google Scholar 

  40. Fu Z, Wang L, Guo H, Lin S, Huang W, Pang Y. Bacterial flagellum-drug nanoconjugates for carrier-free immunochemotherapy. Small. 2024;20:e2306303.

    Article  PubMed  Google Scholar 

  41. Kabir MP, Orozco-Gonzalez Y, Hastings G, Gozem S. The effect of hydrogen-bonding on Flavin’s infrared absorption spectrum. Spectrochim Acta Mol Biomol Spectrosc. 2021;262:120110.

    Article  CAS  Google Scholar 

  42. Yang YW B, Gao B. L.Zhang,L. Guo. Size-Dependent active site and its catalytic mechanism for CO2 hydrogenation reactivity and selectivity over Re/TiO2. ACS Catal. 2023;13.

  43. Li J, Yang D, Lyu W, Yuan Y, Han X, Yue W, Jiang J, Xiao Y, Fang Z, Lu X et al. A bioinspired photosensitizer performs tumor thermoresistance reversion to optimize the atraumatic mild-hyperthermia photothermal therapy for breast cancer. Adv Mater. 2024;36:e2405890.

  44. Wei G, Wang Y, Huang X, Yang G, Zhao J, Zhou S. Enhancing the accumulation of polymer micelles by selectively dilating tumor blood vessels with NO for highly effective cancer treatment. Adv Healthc Mater. 2018;7:e1801094.

    Article  PubMed  Google Scholar 

  45. Jia Q, Zhang R, Yan H, Feng Y, Sun F, Yang Z, Qiao C, Mou X, Tian J, Wang Z. An activatable near-infrared fluorescent probe for precise detection of the pulmonary metastatic tumors: a traditional molecule having a stunning turn. Angew Chem Int Ed Engl. 2023;62:e202313420.

    Article  CAS  PubMed  Google Scholar 

  46. Han D, Ding B, Zheng P, Yuan M, Bian Y, Chen H, Wang M, Chang M, Kheraif AAA, Ma P, Lin J. NADPH oxidase-like nanozyme for high-efficiency tumor therapy through increasing glutathione consumption and blocking glutathione regeneration. Adv Healthc Mater. 2024;13:e2303309.

    Article  PubMed  Google Scholar 

  47. Jiang C, Cheng H, Yuan A, et al. Hydrophobic IR780 encapsulated in biodegradable human serum albumin nanoparticles for photothermal and photodynamic therapy. Acta Biomater. 2015;14:61–9.

    Article  CAS  PubMed  Google Scholar 

  48. Lu YJ, S A T, Chuang CC, et al. Liposomal IR-780 as a highly stable nanotheranostic agent for improved photothermal/photodynamic therapy of brain tumors by convection-enhanced delivery. Cancers. 2021;13(15):3690.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

T.X, Q.M, and C.Z contributed equally to this work. This work was supported by the Program for Upgrading Basic and Clinical Collaborative Research of Anhui Medical University ( 2020xkjT033 ), the research Fund of Anhui Institute of Translational Medicine ( 2022zhyx-C34 ), the major scientific research projects of the Health Commission of Anhui Province ( AHWJ2023A10008 ), Anhui Provincial Science Research Project Fund for Colleges(2024AH050836), and Universitiesand the mechanism study of miR-23c methylation promoting osteosarcoma metastasis by up-regulating HOXB5 expression ( GDS20240531888744 ).

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Author notes

  1. Tangbing Xu, Qiming Ma and Chi Zhang contributed equally to this work.

Authors and Affiliations

  1. Department of Orthopaedics, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022, China

    Tangbing Xu, Qiming Ma, Chi Zhang, Yunfeng Wu, Kunpeng Qin, Faxue Liao, Ping Zhou, Pengfei Xu, Junjun Yang, Jun Chang & Yong Hu

  2. Anhui Public Health Clinical Center, Heifei, 230012, China

    Tangbing Xu, Chi Zhang, Yunfeng Wu, Kunpeng Qin, Faxue Liao, Ping Zhou, Pengfei Xu, Jialai Yang, Junjun Yang & Jun Chang

  3. School of Life Sciences, Anhui Medical University, Hefei, 230032, China

    Xiaoyan He

  4. Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, 230026, China

    Lei Qiao

  5. Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022, Anhui, China

    Qian Wang

  6. Department of emergency, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022, Anhui, China

    Jialai Yang

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  1. Tangbing Xu
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Contributions

XT, MQ and ZC conceived and designed the experiments, and wrote the manuscript; HX, WQ, WY, QK, LF, ZP, XP, YJ and YJ performed the experiments; CJ, QL and HY coordinated and supervised the work.

Corresponding authors

Correspondence to Jun Chang, Lei Qiao or Yong Hu.

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All animal experiments adhered to the established laws and regulations set forth by the Laboratory Animal Center of Anhui Medical University (approval number: LLSC 20200329).

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Xu, T., Ma, Q., Zhang, C. et al. A novel nanomedicine for osteosarcoma treatment: triggering ferroptosis through GSH depletion and inhibition for enhanced synergistic PDT/PTT therapy. J Nanobiotechnol 23, 323 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12951-025-03380-4

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Journal of Nanobiotechnology

ISSN: 1477-3155

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