Targeting nanoplatform synergistic glutathione depletion-enhanced chemodynamic, microwave dynamic, and selective-microwave thermal to treat lung cancer bone metastasis

Abstract

Once bone metastasis occurs in lung cancer, the efficiency of treatment can be greatly reduced. Current mainstream treatments are focused on inhibiting cancer cell growth and preventing bone destruction. Microwave ablation (MWA) has been used to treat bone tumors. However, MWA may damage the surrounding normal tissues. Therefore, it could be beneficial to develop a nanocarrier combined with microwave to treat bone metastasis. Herein, a microwave-responsive nanoplatform (MgFe₂O₄@ZOL) was constructed. MgFe₂O₄@ZOL NPs release the cargos of Fe³⁺, Mg²⁺ and zoledronic acid (ZOL) in the acidic tumor microenvironment (TME). Fe³⁺ can deplete intracellular glutathione (GSH) and catalyze H₂O₂ to generate •OH, resulting in chemodynamic therapy (CDT). In addition, the microwave can significantly enhance the production of reactive oxygen species (ROS), thereby enabling the effective implementation of microwave dynamic therapy (MDT). Moreover, Mg²⁺ and ZOL promote osteoblast differentiation. In addition, MgFe₂O₄@ZOL NPs could target and selectively heat tumor tissue and enhance the effect of microwave thermal therapy (MTT). Both in vitro and in vivo experiments revealed that synergistic targeting, GSH depletion-enhanced CDT, MDT, and selective MTT exhibited significant antitumor efficacy and bone repair. This multimodal combination therapy provides a promising strategy for the treatment of bone metastasis in lung cancer patients.

Graphical abstract

Highlights

• •Magnetic targeting spinel ferrite for enhancing microwave thermal therapy.
• •Nanoparticles loaded with zoledronic acid can target bone metastasis.
• •Synergistic cancer therapy via chemodynamic and microwave dynamic.
• •Depletion of glutathione, zoledronic acid and reactive oxygen species.
• •Zoledronic acid and Mg activate osteogenesis and antitumor.

1. Introduction

Lung cancer is the leading cause of cancer-related death worldwide, and approximately 30 %–40 % of lung cancer patients develop bone metastases [[1], [2], [3]]. Due to the invasion of lung cancer cells into the bone matrix, reactive osteogenesis decreases, resulting in osteolytic destruction [4]. As a consequence, nearly half of patients with bone metastases suffer from skeletal-related events (SREs), such as pathological fractures, spinal cord compression, severe pain, and hypercalcemia, which can greatly decrease quality of life and increase mortality [[5], [6], [7]]. For patients with lung cancer with bone metastases, the main treatment methods are surgery and chemotherapy, and bisphosphonates (BPs) are often used to treat osteolytic destruction [8]. However, due to the lower blood supply relative to that in other tissues, chemotherapeutic drugs do not easily reach tumor tissue, which affects the efficacy of treatment, and the use of higher doses or continuous use of BPs may induce osteonecrosis of the jaw (ONJ) [[9], [10], [11], [12]]. Therefore, it is imperative to develop a novel therapeutic method for the treatment of bone metastases to reduce the incidence of SREs rates and prevent side effects caused by surgery and chemotherapy.

Hyperthermia can directly damage cancer cells and result in enhanced internalization of chemotherapeutic drugs [13]. Compared to other thermotherapies, microwave ablation (MWA) possesses numerous advantages in tumor treatment, including deep penetration, high heating efficiency, minimal side effects, and effective pain relief associated with cancer [[14], [15], [16]]. MWA induces coagulation necrosis by continuously rotating dipoles under the influence of the electromagnetic field, thereby accomplishing microwave thermal therapy (MTT) [17,18]. However, local hyperthermia may induce thermal damage to the surrounding normal tissues and be limited by the ablation zone [19]. With the development and advancement of nanotechnology, a vast array of microwave-responsive nanomaterials have been developed to boost the efficiency of MTT for tumor treatment [20,21]. For example, Tang et al. prepared microcapsules (mPEG-PLGA) based on the MW sensitizers MoS₂ and Fe₃O₄ for enhanced MWA to expand the heated area of the tumor [22]. Therefore, employing MW-sensitive agents in the context of MWA for tumor treatment offers a promising therapeutic strategy.

With the in-depth study of magnetic nanoparticles (NPs), they have been widely used in the biomedical field for applications such as targeted drug delivery, magnetic hyperthermia, and cancer diagnosis [23,24]. For example, Feng et al. used a novel Cu–Zr MOF under microwave irradiation to significantly improve the microwave sensitizing effect [25]. Among these NPs, spinel magnesium ferrite (MgFe₂O₄) NPs can transport drugs to tumor tissue via the enhanced permeability and retention (EPR) effect and an external magnetic field, and can be used as a MW-susceptible agent for tumor-targeted therapy due to their great biocompatibility and electromagnetic wave heat conversion properties [[26], [27], [28]]. The MgFe₂O₄ NPs, serving as promising heterogenous Fenton catalysts, can effectively catalyze H₂O₂ into hydroxyl radicals (·OH) [29]. Moreover, due to the weakly acidic and high concentration of hydrogen peroxide (H₂O₂) and glutathione (GSH) in the tumor microenvironment (TME) [30,31], chemodynamic therapy (CDT) has attracted much attention because of its tumor selectivity and lack of requirement for complicated therapeutic devices [32,33]. However, CDT faces challenges stemming from the limited catalytic efficiency of the conventional Fenton reaction [34,35]. To enhance CDT efficacy, exogenous stimuli are often integrated [36,37]. Consequently, CDT is commonly paired with hyperthermia methods like photothermal therapy to amplify therapeutic outcomes [38,39]. Among thermal treatments, microwaves offer deeper tissue penetration compared to near-infrared light [40,41]. When combined with MgFe₂O₄ NPs, microwaves can expedite the production of cytotoxic reactive oxygen species (ROS) within the tumor microenvironment due to their narrow energy band gap [42]. This therapeutic strategy is termed microwave dynamic therapy (MDT) [43,44]. Hence, it is crucial to develop new approaches that combine MgFe₂O₄ NPs with MWA to enhance the MTT effect and achieve effective MDT.

Moreover, in the treatment of bone metastatic tumors, it is crucial to precisely target and eradicate tumors while simultaneously ensuring bone reconstruction [45,46]. BPs, which have a high affinity and selectivity for bone, are often used as adjuvants to treat or prevent SREs [47]. Zoledronic acid (ZOL), classified as a third-generation BP with powerful anti-bone resorption properties, is commonly recommended as the primary treatment option for bone metastasis [48,49]. For example, Qiao et al. synthesized ZOL-encapsulated silica-covered gadolinium-upconversion nanoparticles (NPs) tethered to plumbagin, which could facilitate bone metastasis treatment [50]. Additionally, ZOL not only stimulates the activity of osteoblasts to decrease the incidence of SREs but also promotes tumor cell apoptosis [51]. Hence, delivering a concentrated dose of ZOL directly to the bones could serve as an effective strategy for treating lung cancer-related bone metastases.

This manuscript reports for the first time the use of a combination of a spinel magnesium ferrite nanocarrier and microwave. First, a hollow mesoporous spinel MgFe₂O₄ nanoparticle-based drug delivery system with microwave sensitivity and precise antitumor activity was constructed. As shown in Fig. 1A, hollow mesoporous MgFe₂O₄ NPs were synthesized via a simple solvothermal method, and the triblock copolymer Pluronic F127 was used as a soft template to shape the mesoporous and spherical nanostructure. Subsequently, ZOL was loaded with the MgFe₂O₄ NPs using N,N′-carbonyldiimidazole (CDI) as an activator. As displayed in Fig. 1B, once the MgFe₂O₄@ZOL NPs were injected, the MgFe₂O₄@ZOL NPs targeted the bone tumor through the influence of their own ZOL and the EPR effect to target bone metastasis. It rapidly decomposed to release the cargos of Fe³⁺, Mg²⁺ and ZOL in the acidic TME and under microwave irradiation. Fe³⁺ could react with intracellular GSH to generate Fe²⁺, which catalyzed H₂O₂ to generate •OH, resulting in GSH depletion-enhanced CDT. When used in combination with MW, the process was further accelerated and enhanced, resulting in MDT. Moreover, the released Mg²⁺ and ZOL could stimulate osteogenesis for bone regeneration and inhibit cancer cells. In addition, when exposed to low-power MW irradiation, MgFe₂O₄ NPs act as an MW sensitizer to selectively heat tumor tissue, enhancing the effect of MTT while minimizing damage to surrounding tissues. Both in vitro and in vivo experiments demonstrated that synergistic targeting, GSH depletion-enhanced CDT, MDT, and selective-MTT via MgFe₂O₄@ZOL combined with MW exhibited excellent efficacy in terms of tumor eradication and osteoblast activation. This approach shows promise as a strategy for treating lung cancer bone metastasis.

Fig. 1.

Schematic illustration of the microwave and MgFe₂O₄@ZOL NPs used in synergistic tumor therapy. (A) The synthesis process of MgFe₂O₄@ZOL NPs. (B) Schematic diagram of synergistic therapy involving targeting, CDT, MDT, and selective-MTT, and bone repair for bone metastasis.

2. Experimental procedure

2.1. Materials

Iron chloride hexahydrate (FeCl₃‧6H₂O), magnesium chloride hexahydrate (MgCl₂‧6H₂O), ethylene glycol (C₂H₆O₂, >99 %) and methylene blue (MB) were bought from Aladdin (Shanghai, China). Pluronic F-127 was obtained from Sigma-Aldrich (USA). Amine-PEG-Amine, acetonitrile, and sodium acetate (NaAc) were purchased from Sinopharm (Shanghai, China). Zoledronic acid (ZOL), N,N′-carbonyldiimidazole (CDI), and triethylamine were purchased from Macklin (Shanghai, China). N,N-Dimethylformamide (DMF) was purchased from Shanghai Boer Chemical Reagent Co., Ltd. Dulbecco's modified Eagle medium (DMEM), penicillin, streptomycin, fetal bovine serum (FBS), and phosphate-buffered saline (PBS) were obtained from Gibco. FITC-xtra was acquired from ATT Bioquest (USA). Cell Counting Kit-8 and the Annexin V-FITC Apoptosis Detection Kit were purchased from Dojindo (Japan). A Live-Dead Cell Staining Kit (propidium iodide (PI) and calcein-AM) was purchased from Bestbio (China). The total glutathione (GSH) assay kit, alkaline phosphatase (ALP) assay kit, and BCIP/NBT alkaline phosphatase color development kit were all purchased from Beyotime Biotechnology Co., Ltd. (China).

2.2. Preparation of MgFe₂O₄ NPs

Pluronic F127 (1.0 g) was added to ethylene glycol (40 mL) and agitated at 60 °C until the mixture turned clear. Then, 0.4 g of MgCl₂‧6H₂O and 1.08 g of FeCl₃·6H₂O were added. The solution was agitated for 30 min to obtain a uniform solution. Next NaAc (1.2 g) was poured into the uniform mixture and mixed vigorously for 60 min. After sonicating for 10 min, the homogeneous suspension obtained was transferred into a 100 mL Teflon-sealed autoclave and put into a vacuum oven at 200 °C for 16 h. The suspension was separated via centrifugation at 10,000 g for 30 min and rinsed three times with distilled water and ethanol to obtain the MgFe₂O₄ nanoparticles.

2.3. Characterization of MgFe₂O₄ NPs

The morphological characteristics of the as-prepared MgFe₂O₄ nanoparticles were observed by transmission electron microscopy and scanning electron microscopy (TEM, FEI Talos-F200S, 200 kV; SEM, TESCAN MIRA LMS). X-ray diffraction (XRD) analysis was performed using graphite monochromatized Cu Kα radiation (PANalytical, the Netherlands) at a scanning rate of 5° min⁻¹ in the 2θ range of 20–80°. Chemical composition analysis was performed utilizing a Fourier transform infrared (FTIR) spectrometer (iS50, Thermo Fisher Scientific, USA). A Thermo Scientific K-Alpha electron energy spectrometer was utilized to record the X-ray photoelectron spectra (XPS) via Al Kα radiation as the X-ray excitation source. The nanoparticles were dispersed in ethanol and subjected to ultrasonic treatment for 10 min. Subsequently, a Malvern Zetasizer Nano ZS90 system was used to determine the particle size range by dynamic light scattering (DLS) and zeta potential. The Brunauer-Emmett-Teller (BET) surface areas were detected by the typical N₂ absorption/desorption method (Micromeritics APSP2460, USA). Thermal analysis of the samples was performed using thermogravimetry (TG; Netzsch, STA 449 C, Germany) under ambient air conditions.

2.4. In vitro MW heating effect of MgFe₂O₄ NPs

The microwave sensitivity of the MgFe₂O₄ NPs was monitored every 30 s using a forward-looking infrared (FLIR) thermal camera. The MgFe₂O₄ NPs were dispersed in phosphate buffer saline (PBS) at different concentrations (0, 1, 2.5, 5, and 10 mg/mL) under MW irradiation (5 W, 2450 MHz) for 5 min in vitro. Additionally, changes in the temperature of the MgFe₂O₄ NPs solution (1 mg/mL) after microwave irradiation at different powers (0, 3, 4, 5, and 6 W) for 5 min were also observed.

To evaluate the MW thermal stability of MgFe₂O₄ NP, a series of MW irradiated cycles were applied. The nanoparticles dispersed in PBS (2.5 mg/mL) was irradiated by MW (5 W) for 5 min. Following this, they were allowed to naturally cool down to room temperature for an additional 10 min for a total of 5 cycles. The temperature change was recorded every 30 s.

2.5. ZOL loading and release assays

ZOL was loaded with MgFe₂O₄ by modified CDI [52]. First, the amine group was functionalized in MgFe₂O₄ NPs by NH₂-PEG-NH₂. Briefly, MgFe₂O₄ and NH₂-PEG-NH₂ were dispersed at a mass ratio of 2:1 in 10 mL of DMSO, followed by constant shaking for 24 h in the dark. Then, ZOL was activated by CDI. Specifically, 50 mg of ZOL was added to DMF (25 mL) with triethylamine (1 mL). Subsequently, CDI (45 mg) was applied under vacuum for 24 h at 60 °C. The suspension was separated via centrifugation at 10,000 g for 30 min, and the precipitate was collected, which was washed thrice using acetonitrile. Then, MgFe₂O₄@ZOL were obtained through an aminolysis reaction between activated ZOL and MgFe₂O₄–NH₂. In detail, MgFe₂O₄–NH₂ (25 mg) and activated ZOL (11.3 mg) were dispersed in 10 mL of DMSO in a vessel containing 1 mL of triethylamine under vacuum for 12 h in the dark. Finally, the MgFe₂O₄@ZOL NPs were dried in a vacuum oven and collected for subsequent use.

To explore the release behavior of ZOL, the effect of pH on the release rate of ZOL from MgFe₂O₄@ZOL was assessed using the dialysis method. Briefly, the MgFe₂O₄@ZOL NP solution (200 μL, 2 mg/mL) was sealed in a dialysis bag with a molecular weight cutoff (MWCO) of 14,000 Da. Subsequently, the bag was placed in a container with 20 mL of PBS (pH = 5.5, 6.5, and 7.4) at room temperature with a shaking rate of 150 rpm min⁻¹. At predefined time points, samples of the external PBS solution containing the released ZOL were collected. The amount of ZOL released was calculated by detecting the absorbance at 210 nm via ultraviolet–visible absorption spectroscopy. The standard curves of ZOL were used to calculate the concentration of ZOL. The drug release rate of ZOL from MgFe₂O₄@ZOL was determined using the following equation (M₁/M₂ × 100 %), where M₁ represents the mass of the released drug in PBS, and M₂ represents the weight of the loaded drug.

2.6. The bone targeting ability of MgFe₂O₄@ZOL NPs

To detect the binding effect of MgFe₂O₄@ZOL NPs on bone tissue in vitro, cow bone slices were co-cultured with the same concentration of MgFe₂O₄@ZOL NPs and MgFe₂O₄ NPs. Cow bone slices were incubated with 100 μg/mL MgFe₂O₄ and MgFe₂O₄@ZOL NPs, and the contents were stirred slowly. The concentration of MgFe₂O₄ NPs was estimated by detecting the absorbance at 563 nm using ultraviolet–visible absorption spectroscopy [53], and then the binding ability was measured by calculating the changes in concentration at various time intervals. The binding rate is equal to the amount of binding to bone divided by the original total.

To further confirm the targeting efficacy of MgFe₂O₄@ZOL NPs in vivo, we conducted the live imaging experiment on mouse models with bone metastasis cancer. Initially, we established a lung cancer bone metastasis model in nude mice by administering intraperitoneal injections of 3 % pentobarbital sodium (30 mg/kg) for anesthesia. Subsequently, A549 cells (10⁶ cells in 20 μL PBS) were directly injected into the left femur using a 26-gauge needle and the nude mice were housed in a specific pathogen-free (SPF) condition. FITC-labeled NPs were then prepared according to the previous literature [54]. 5 μL of FITC-xtra solution (20 mM) was added to 5 mL of MgFe₂O₄ and MgFe₂O₄@ZOL NPs solutions (1 mg/mL). After stirring on ice for 1 h, the mixture was centrifuged at 10,000 rpm for 5 min to remove unbound FITC-xtra. Subsequently, once the tumor is observed on the left femur of the tumor-bearing mouse compared to the contralateral side, the FITC-labeled NPs were administered to the mouse models via the tail vein (2 mg/kg). Finally, the mice were placed in the IVIS spectrum imaging system (PerkinElmer Instruments Co., Ltd, Shanghai) to capture a series of fluorescence images at different time points (3 h, 6 h, and 24 h).

2.7. Detection of singlet oxygen (¹O₂) and hydroxyl radicals (·OH) generation

Firstly, the production of singlet oxygen (¹O₂) was evaluated by RNO-ID (p-nitrosodimethylaniline (RNO)-imidazole (ID)). To elaborate, 0.225 mg RNO and 16.34 mg of ID were added to 30 mL of ultrapure water to form the RNO-ID solution. Then, the RNO-ID solution (500 μL) was added into MgFe₂O₄ solution (500 μL) at various concentrations (0, 200, 500, 1,000, and 2000 μg/mL) and the above groups were/were not exposed to MW. The MgFe₂O₄ solution (200 μg/mL) was irradiated with MW for different durations. After 4 h of reaction at room temperature, the absorption at 440 nm was detected via a microplate reader.

Methylene blue (MB) can be degraded by ·OH, the production of which can be measured by the change in the absorbance of the MB. Different concentrations of MgFe₂O₄ NPs (0, 200, 500, 1,000, and 2000 μg/mL) were added into the MB solution (20 mg/mL) containing 3 % H₂O₂, and the absorption values of the mixtures were measured at 664 nm through a UV–vis absorption spectrometer after the reaction occurred for 30 min. Additionally, the production of ·OH was detected at different pH values (pH = 4.5, 5.5, 6.5, and 7.4) and under microwave irradiation (5 W; 0, 1, 2, 3, 4, and 5 min).

In addition, the production of ·OH was detected by the EPR spectrometer, and 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO) was used as the ·OH trapping reagent in this study. The experimental groups were MgFe₂O₄, MgFe₂O₄ + H₂O₂, and MW + MgFe₂O₄ + H₂O₂. The final concentrations of MgFe₂O₄ and H₂O₂ were 100 μg/mL and 3 %, respectively. Then, BMPO was added into all the experimental groups, and the mixture was allowed to react for 5 min. The characteristic peaks of ·OH were detected by electron paramagnetic resonance (EPR) spectrometry.

2.8. Evaluation of GSH levels and ROS production in cells

A GSH assay kit was used to measure the intracellular GSH level. Human lung adenocarcinoma cells (A549 cells) and solutions containing various concentrations of MgFe₂O₄ and MgFe₂O₄@ZOL NPs (0, 12.5, 25, 50, 100, and 200 μg/mL) were cocultured. Next, the cells were flushed with PBS and centrifuged at 300 g for 5 min to obtain the sediment. The collected sediment was subjected to two rapid freeze-thaw cycles using liquid nitrogen and ultrapure water at 37 °C to lyse the sediment. The resulting mixture was further centrifuged at 10,000 g for 15 min. Finally, the amount of the supernatant was measured with a GSH assay kit to estimate the intracellular GSH concentration.

The 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was utilized to detect ROS production in cancer cells. A549 cells were incubated in medium supplemented with MgFe₂O₄ and MgFe₂O₄@ZOL NPs (100 μg/mL). DCFH-DA reagent (10 μM) was added, and the cells were cocultured for 30 min. Subsequently, the cells were rinsed with PBS and irradiated with MW at 5 W for 5 min. The resulting green fluorescence was detected via fluorescence microscopy.

2.9. In vitro biocompatibility

The CCK-8 assay was used to assess the cytotoxicity of the MgFe₂O₄ NPs. A549 cells, human umbilical vein endothelial cells (HUVECs) and mouse embryo fibroblasts (C3H10) cells, were seeded in 96-well plates (2 × 10³ cells/well) and treated with various concentrations of MgFe₂O₄ NPs (0, 12.5, 25, 50, 100, and 200 μg/mL) for 1, 3, and 5 days. After the respective incubation periods, the cells were flushed twice. Subsequently, a solution consisting of the CCK-8 reagent diluted in DMEM (200 μL, volume ratio = 1:9) was transferred to the well and cocultured for 1 h. The absorption values were measured at 450 nm with a microplate reader to estimate the cell viability.

The biocompatibility of the MgFe₂O₄ NPs was assessed by conducting an in vitro hemolysis test. First, fresh rabbit blood containing EDTA was centrifuged at 300 g for 5 min to separate the red blood cells (RBCs). After rinsing with saline, 200 μL of RBCs were diluted to 10 mL with PBS to obtain a 2 % RBC solution. MgFe₂O₄ NPs (at various concentrations) (0, 12.5, 25, 50, 100, 200, 400, and 800 μg/mL) were used as the experimental group, PBS was used as a negative control, and ultrapure water was used as a positive control. The contents were mixed gently and incubated at 37 °C for 3 h. Next, the mixtures were centrifuged at 300 g for 5 min, and the optical density (OD) at 545 nm of the supernatant was detected with a microplate reader. The hemolysis rate (%) was determined by the following formula: hemolysis rate (%) = (OD_M – OD_NC)/(OD_PC – OD_NC) × 100 %, where OD_M represents the OD of the MgFe₂O₄ NPs solution; OD_PC represents the absorbance of the positive control; and OD_NC represents the absorption of the negative control.

2.10. Cytotoxicity and apoptosis analysis of cancer cells under MW irradiation

To evaluate whether MgFe₂O₄@ZOL can enhance the efficacy of microwave irradiation of tumors in vitro, cytotoxicity was assessed by the CCK-8 assay and live-dead cell staining. Specifically, A549 cells were cocultured with a range of concentrations (0, 12.5, 25, 50, and 100 μg/mL) of MgFe₂O₄ NPs and MgFe₂O₄@ZOL NPs for 24 h at 37 °C. Then, the CCK-8 solution was added to the cells, and the absorbance at 450 nm was detected with a microplate reader. The cell viability was calculated as follows: cell viability (%) = (OD _sample - OD _blank)/(OD _control - OD _blank) × 100.

At the same time, the A549 cells were cocultured with varying concentrations (0, 12.5, 25, 50, and 100 μg/mL) of MgFe₂O₄@ZOL NPs for 24 h. After the nanoparticles were endocytosed, the cells were irradiated by microwave at different powers (0, 4, and 5 W) for 5 min. Finally, the A549 cells were continued to culture for 24 h at 37 °C to calculate the cell survival rate using the CCK-8 assay.

In addition, cell viability was observed using a live-dead cell staining kit. A549 cells were cocultured with 100 μg/mL MgFe₂O₄ and MgFe₂O₄@ZOL NPs for 24 h and then irradiated with MW (5 W, 5 min). Subsequently, the A549 cells were incubated with calcein AM in the dark at 37 °C for 15 min. After two additional flushes, the cells were cocultured with PI for 5 min. Finally, the stained A549 cells were observed by inverted fluorescence microscopy. Subsequently, the number of dead cells and viable cells were counted separately using the ImageJ software.

Flow cytometry was utilized for cell counting to evaluate the apoptosis of cancer cells. First, A549 cells were incubated with MgFe₂O₄ and MgFe₂O₄@ZOL NPs (100 μg/mL) for 24 h. Furthermore, the cells in the MW group were irradiated with MW (5 W for 5 min) and cultured for an additional 6 h. The adherent cells and the medium were collected and then separated via centrifugation at 300 g for 3 min to obtain all the cells. Subsequently, the collected cells were double rinsed and resuspended in binding buffer. The cell apoptosis rate was analyzed by flow cytometry.

2.11. Cellular uptake of MgFe₂O₄@ZOL by TEM

The A549 cells were cultured in a cell culture dish for 24 h to allow attachment. After treatment with 100 μg/mL nanoparticles and exposure to MW, the cells were scraped off and rinsed with PBS. The cells were then centrifuged and harvested. Then, 3 % glutaraldehyde and 1 % osmium tetroxide were used to fix the tumor cells for one day. Subsequently, the cells were dehydrated twice using various concentrations of ethanol (50 % and 70 %) and acetone (80 %, 90 %, and 100 %), after which they were incubated for 10 min at each chemical concentration. The dehydrated cells were embedded in Epon812 and polymerized for 48 h at 70 °C. Ultrathin sections of the samples were obtained via an ultramicrotome and treated with lead citrate and uranyl acetate solution. Finally, the sections were observed using TEM.

2.12. Colony formation of cancer cells

By evaluating the formation of clones, we assessed the population dependence and proliferation of the cells. First, A549 cells were seeded in a 6-well plate (500 cells per well) and treated with the desired concentration of MgFe₂O₄@ZOL NPs and MW as previously described. The cells were then cultured for 14 days, after which the medium was replaced every three days. After the formation of colonies, the proliferating cells were fixed with 4 % paraformaldehyde. Subsequently, crystal violet solution was added to the wells, and the stained cells were rinsed with PBS and photographed.

2.13. Cancer cell migration and invasion

The migration ability of cancer cells was assessed via a wound healing assay. After the confluence of the A549 cells reached approximately 90 %, parallel wounds were created on the cell layers via a sterile tool. The cells were incubated with a mixture of nanoparticles and medium for 12 and 24 h, respectively. After incubation, the process of cell migration in the wound area was visualized and documented using a microscope. The migration ratio was determined by calculating the difference in the migration area between the different time points and dividing it by the original area.

Transwell assay was used to assess cell invasion. A549 cells (1 × 10³ cells) were treated with the abovementioned nanoparticles. The cells were collected and dispersed in serum-free DMEM and then transferred to the upper cavity (pore size: 8 μm). A complete culture solution was poured into the lower plate to support cell growth and to induce cell invasion. After being incubated for 12 h, the cancer cells were fixed using 4 % paraformaldehyde and stained with crystal violet. Images of stained cells were captured through a microscope, and the cells were counted.

2.14. Osteoblast differentiation and mineralization assay of C3H10 cells

The potential for osteoblast differentiation was assessed by evaluating the activity of alkaline phosphatase (ALP). First, C3H10 cells were cocultured with a solution containing MgFe₂O₄ or MgFe₂O₄@ZOL NPs (final concentration: 25 μg/mL). These cells were kept in osteoblast inducer medium (DMEM supplemented with 10 % FBS, 1 % penicillin/streptomycin, 50 μg/mL Vitamin C, 10 mM sodium β-glycerophosphate, and 10 nM dexamethasone, and the prepared induced complete medium was filtered) for 3, 7, and 14 days. Then, the activity of ALP was tested using an ALP color development kit and an ALP assay kit.

Mineralized nodule formation in C3H10 cells was measured by staining the samples with Alizarin Red. After the nanoparticles were endocytosed, the C3H10 cells were cultured in osteoblast inducer medium for 3, 7, or 14 days. Then, the ability to form mineralized nodules was qualitatively analyzed by staining with Alizarin Red and quantitatively evaluated using 10 % hexadecyl pyridinium chloride monohydrate (CPC).

2.15. In vivo animal therapeutic efficacy

The Animal Ethics Committee of the General Hospital of Southern Theatre Command authorized all animal experimental procedures (ethical code SYDW2023007). Male BALB/c nude mice (four weeks old) were selected as model animals for tumor studies and housed under specific pathogen-free (SPF) conditions. Lewis lung carcinoma (LLC) cells (10⁶ cells, 100 μL of DMEM) were administered under the skin near the left scapular region to construct the subcutaneous tumor-bearing mouse model. This injection site was chosen for convenient monitoring and measurement of tumor growth. After the tumor volume reached 50–80 mm³, the mice bearing tumors were randomly allocated into six distinct experimental groups as follows: 1. PBS; 2. MgFe₂O₄ NPs; 3. MgFe₂O₄@ZOL NPs; 4. PBS + MW; 5. MgFe₂O₄ NPs + MW; 6. MgFe₂O₄@ZOL NPs + MW. The mice in different groups received tail-vein administration of either PBS (equal to the volume of the solution containing NPs) or a solution containing nanoparticles (2 mg/kg body weight). After 24 h, the mice in the MW groups received microwave ablation at 5 W for 5 min. The temperature variation induced by the MW was monitored using the FLIR instrument. The weight and tumor dimensions of the experimental animals were recorded every three days. The size of the tumors was determined using the following formula: tumor volume = 0.5 × length × width × width. After 14 days, the mice were humanely euthanized. The organs (i.e., the heart, liver, lungs, spleen, and kidneys) and tumors were collected. The tumor tissues were embedded in paraffin and sliced for different staining procedures, including HE, TUNEL, Ki67, and Prussian blue staining. The major organs of the experimental animals were also sectioned for H&E staining. Finally, all stained slices were observed under a fluorescence microscope.

2.16. Statistical analysis

All results were derived from repeated independent experiments and are expressed as the mean ± standard deviation (SD). The differences among groups were determined by analysis of variance (ANOVA). All differences were considered to be statistically significant at p < 0.05; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

3. Results and discussion

3.1. Synthesis and characterization of MgFe₂O₄ and MgFe₂O₄@ZOL

MgFe₂O₄ NPs were constructed using a straightforward solvothermal method. The XRD pattern (Fig. 2A) of the sample aligned with the spinel ferrite crystal structure (JCPDS card No.01-071-1232), confirming its chemical formula as MgFe₂O₄. The inverse spinel structure of MgFe₂O₄ was determined by the distinctive peaks observed in the crystallographic planes (220), (311), (400), (511), and (440) [55]. The characteristic type-IV isotherms with H1 hysteresis loops validate the hollow and mesoporous structure of the MgFe₂O₄ (Fig. 2B). The BET-specific surface area of the hollow MgFe₂O₄ was determined to be 63.78 m² g⁻¹, and the adsorption average pore diameter was about 5.17 nm, as determined by the pore dimension range plot (Fig. 2B and C). From the SEM and TEM images, the as-obtained MgFe₂O₄ NPs showed a uniform morphology. They were spherical and had a rough surface; the average size of the particles was about 300 nm, indicating their good dispersibility and homogeneity (Fig. 2D and E). Furthermore, TEM images indicate that the prepared MgFe₂O₄ NPs exhibit an inner hollow structure alongside surface mesopores. The presence of a larger surface area and mesoporous structure in MgFe₂O₄ NP enabled it to possess a significantly enhanced loading capacity. The selected area electron diffraction (SAED) pattern, spinel structure, and single-crystal nature of the magnesium ferrite particles are shown in Figure S1A-C [55]. The SAED patterns showed that the crystal structure agreed with the structure obtained via XRD. The EDS elemental mapping (Figure S1D) analysis showed that the Fe, Mg, and O elements were uniformly distributed in the as-synthesized MgFe₂O₄ nanoparticles. Results from the EDS spectra demonstrated that Mg, Fe, and O was present (Figure S2 and Table S1). Based on the XPS analysis, besides Fe, Mg was also detected in the as-synthesized MgFe₂O₄ (Fig. 2H). From the deconvolution, it could be concluded that magnesium existed as Mg²⁺ and iron existed as Fe³⁺ in the MgFe₂O₄ nanoparticles. The average particle size of MgFe₂O₄ measured by DLS was around 330 nm (Fig. 2K). The zeta potential of the nanoparticles was −30.8 mV (Fig. 2L). To summarize, the hollow mesoporous MgFe₂O₄ NPs that could be used for drug loading were synthesized.

Fig. 2.

Synthesis and characterization of MgFe₂O₄ and MgFe₂O₄@ZOL NPs. (A) XRD pattern; (B) N₂ absorption/desorption isotherms; (C) pore size distribution curves; (D) SEM image; (E) TEM image; (F) TEM element mapping; (G) FTIR spectra; (H) XPS survey; (I) high-resolution XPS spectra of N1s; (J) TG analysis curves; (K) DLS; (L) Zeta potential.

As MgFe₂O₄ NPs had a special hollow mesoporous structure, they were suitable for drug delivery [56]. In our study, ZOL was efficiently loaded into the hollow and mesopores of MgFe₂O₄ NPs through physical adsorption. Moreover, a covalent linkage (amide bond) was also established to conjugate ZOL, resulting in the formation of MgFe₂O₄@ZOL NPs. Visual inspection of SEM and TEM images confirmed the presence of a distinct ZOL membrane on the surface of MgFe₂O₄@ZOL NPs (Fig. 2D and E). Moreover, TEM analysis revealed that the internal hollow and surface mesoporous structures of MgFe₂O₄@ZOL NPs were not readily discernible, indicating successful filling of ZOL within the hollow pores and surface mesopores of MgFe₂O₄ NPs. Elemental mapping of MgFe₂O₄@ZOL NPs exhibited characteristic P and N elements associated with ZOL (Fig. 2F). The presence of specific peaks corresponding to ZOL in the FTIR spectra of MgFe₂O₄@ZOL at 1000-1200 cm⁻¹ and 3100-3200 cm⁻¹ further validated the effective loading of ZOL (Fig. 2G). Additionally, a comparative analysis of high-resolution XPS spectra of N1s pre- and post-conjugating of ZOL demonstrated a new deconvoluted peak at 399.3 eV, indicating the formation of amide bonds between ZOL and MgFe₂O₄ (Fig. 2I). Thermal analysis revealed a drug mass fraction of 32.21 % on MgFe₂O₄@ZOL NPs, which exhibited larger size and higher electronegativity compared to MgFe₂O₄ NPs (Fig. 2J-L). Collectively, these findings provide strong evidence supporting the successful loading of ZOL onto MgFe₂O₄ NPs by absorption physically and the formation of amide bonds.

3.2. Microwave heating efficiency in vitro

Magnetic spinel nanoparticles as polar materials can enhance the efficiency of MW heating, and enhance heat through hysteresis losses activated in magnetic fields released from MWA (Fig. 3A) [57,58]. The spinel MgFe₂O₄ NP, which is soft magnetic mental oxide, can absorb microwave due to its narrow energy band gap, great magnetic heating capability, and lower thermal conductivity, resulting in low heat loss during heating [59]. Therefore, MgFe₂O₄ NPs are suitable heating agents. The property of microwave thermal conversion was evaluated using an FLIR imaging instrument. When the concentration of MgFe₂O₄ NPs was 1, 2.5, 5, and 10 mg/mL, as indicated by the infrared images recorded by FLIR (Fig. 3B), the temperature changed to 36.1, 37, 39, and 40 °C, respectively, whereas the temperature of the control group was only 33 °C (Fig. 3C). The above data demonstrated that the temperature of the solution increased in proportion to the concentration of MgFe₂O₄. The temperature exhibited a positive correlation with the microwave power when the same concentration of 1 mg/mL MgFe₂O₄ NPs was irradiated for 5 min (Fig. 3D) (the infrared thermal images are shown in Fig. 3E). In addition, the microwave thermal stability of MgFe₂O₄ NPs (2.5 mg/mL) was analyzed by five irradiation cycles at the power of 5 W. As shown in Fig. 3F, no attenuation in the heating efficiency was demonstrated, illustrating that MgFe₂O₄ NPs possess potential for cyclic MWA in antitumor process. These results shown the excellent microwave heating effect of MgFe₂O₄ NPs in the simulated body fluid and laid the determining the concentration and microwave power in subsequent experiments. It has been reported that temperatures ranging from 43 to 48 °C can induce apoptosis and necrosis in tumor cells [60]. The 5 W and 6 W groups met the temperature requirement for damaging tumor cells, and the power of the 6 W group was not acceptable due to easy damage to the surrounding tissue. MgFe₂O₄ NPs reportedly possess photothermal and magnetic hyperthermia capabilities [59,61], but there have been no reports on the combination of MgFe₂O₄ NPs and MW. Due to the greater penetration of MWs compared to that of near-infrared light, MWs can be applied to deep tissue tumors [62]. Both photothermal heating and alternating magnetic fields (AMF) rely on stimulating the inherent heat generation of MgFe₂O₄, resulting in limited heating efficiency. However, when MgFe₂O₄ NPs are combined with MW, both water and MgFe₂O₄ generate heat, significantly enhancing the local temperature elevation effect. MgFe₂O₄ exhibits superparamagnetism and a narrow hysteresis loop, making it a promising candidate biomaterial for hyperthermia therapy in cancer [63].

Fig. 3.

In vitro microwave heating effect of MgFe₂O₄ and release of MgFe₂O₄@ZOL. (A) The principle of microwave heating. (B) FLIR images of concentration gradients of MgFe₂O₄ NPs when the suspension was exposed to MW irradiation. (C) Temperature variations of MgFe₂O₄ NPs suspension at different concentrations when the solution was exposed to MW at 5 W for 5 min. (D) Changes in the temperature of the MgFe₂O₄ NPs suspension (1 mg/mL) at different microwave power. (E) Infrared thermal images of MgFe₂O₄ NPs irradiated by microwave at different power levels. (F) Microwave thermal stability of MgFe₂O₄ NPs (2.5 mg/mL) under MW irradiation (5 W) for 5 on-off cycles. (G) The UV–vis spectra of ZOL, MgFe₂O₄, NH₂-PEG-NH₂, and MgFe₂O₄@ZOL. (H) The release curves of MgFe₂O₄@ZOL NPs at various acid environments. (I) The in vitro bone affinity of MgFe₂O₄ and MgFe₂O₄@ZOL NPs.

3.3. Drug release in vitro

The loaded capacity and pH-triggered release of MgFe₂O₄ NPs were assessed via the concentration of ZOL. The drug release concentration was determined using the standard concentration curve of ZOL (Figure S41). The successful loading of ZOL was confirmed by detecting an absorption peak at approximately 210 nm following the coupling of MgFe₂O₄ NPs and ZOL (Fig. 3G). The amount of ZOL released from the MgFe₂O₄@ZOL NPs increased as the pH decreased (Fig. 3H). The drug was released in short bursts within the initial 10 h, followed by a sustained slow release of the drug. The release rate and quantity of ZOL were greatly greater at pH 5.5 than at pH 7.4, which indicated that the hydrogen ion (H⁺) concentration of the mixture and the release kinetics of ZOL from the MgFe₂O₄@ZOL NPs were positively correlated. On the one hand, due to the large specific surface area of hollow mesoporous structure of MgFe₂O₄ NP, which allows for the absorption of ZOL onto material. On the other hand, in the acidic environment, protons protonate the amide bonds in MgFe₂O₄@ZOL NP and collapse the mesoporous framework, thereby accelerating drug release [37,64].

3.4. The bone targeting ability of MgFe₂O₄@ZOL NPs

A major hindrance to cancer therapy is nonspecific action, which can lead to serious problems in patients [52]. As shown in Fig. 3I, MgFe₂O₄@ZOL NPs had a stronger affinity for cow bone slices than MgFe₂O₄ NPs in vitro. After 6 h, 67.6 % of the MgFe₂O₄@ZOL NPs were found in bone slices, while 34.3 % of the MgFe₂O₄ NPs were localized in bone slices.

We further assessed the targeting capability of MgFe₂O₄@ZOL towards bone tumors in vivo using the aforementioned tumor-bearing mice. These mice were divided into the MgFe₂O₄-FITC group and the MgFe₂O₄@ZOL-FITC group. Nanoparticles were administered via the tail vein, and fluorescence images were captured using an animal live imaging system after 3, 6, and 24 h, respectively. As illustrated in Figure S3A, the fluorescence intensity was stronger in the 3 h groups compared to the 6 and 24 h groups. Moreover, at the same time points, the fluorescence signals at the bone tumor sites (left femur) were notably more concentrated and intense in the MgFe₂O₄@ZOL group. In vivo imaging conducted 6 h after injection of the MgFe₂O₄ NP group revealed the presence of fluorescence in certain areas of the tumor site. This could be attributed to the EPR effect of MgFe₂O₄ nanoparticles, allowing some nanoparticles to accumulate at the tumor site, while the EPR effect is relatively limited due to the larger particle size of MgFe₂O₄ NPs [5,27]. Additionally, MgFe₂O₄@ZOL not only exploits the EPR effect but also benefits from the strong bone tumor targeting ability of ZOL, resulting in more concentrated fluorescence signals at the bone tumor sites [65]. Mice in the 3 h post-injection group were euthanized, and vital organs (heart, liver, spleen, lungs, kidneys) and lower limbs were collected for fluorescence imaging. Consistent with the live imaging results, the MgFe₂O₄@ZOL group exhibited the most concentrated fluorescence signals at the tumor site (Figure S3B). It was also noted that the nanoparticles were primarily concentrated in the gallbladder and liver, followed by the kidneys. This suggests that the liver and kidneys are the main organs for nanoparticle metabolism and excretion. The aforementioned findings demonstrate that MgFe₂O₄@ZOL NPs possess the ability to effectively target bone tumors.

3.5. Catalytic production of ROS and consumption of GSH

The MgFe₂O₄ NPs reacted with O₂ to produce ¹O₂ under microwave irradiation. The intermediate product of the reaction between ¹O₂ and imidazole oxidizes RNO, resulting in the solution fading, which is the RNO decolorization reaction [36]. As shown in Figure S4B, at the same concentration, the MgFe₂O₄@ZOL group produced more ¹O₂. The OD of RNO decreased as the concentration of MgFe₂O₄ increased and as the MW increased (Fig. 4A). As the duration of exposure to MW increased, a corresponding decrease in the absorption values was recorded after treatment with 200 μg/mL MgFe₂O₄ (Figure S4C). It was found that both MgFe₂O₄, ZOL, and MW can stimulate ROS production as previously reported [66,67].

Fig. 4.

Oxidation-reduction reactions of nanomaterials. (A) Generation of singlet oxygen after treatment with different concentrations of MgFe₂O₄ NPs (0, 200, 500, 1000, 2000 μg/mL) and irradiation of MW. (B) The relative GSH content in cancer cells after treatment with various concentrations of MgFe₂O₄ or MgFe₂O₄@ZOL NPs (0, 12.5, 25, 50, 100, and 200 μg/mL). (C and D) UV–vis absorbance of MB treated with a gradient of concentrations of MgFe₂O₄ NPs or acidic environment (insert: images of MB solutions; the concentrations separately are 0, 200, 500, 1000, and 2000 μg/mL; PH are 4.5, 5.5, 6.5, and 7.4). (E) EPR detection of MgFe₂O₄ under MW irradiation to produce ·OH. (F) The quantity of cellular ROS produced after different treatments. (G) Fluorescence images illustrating ROS in cells.

GSH, a major endogenous antioxidant that can weaken the Fenton/Fenon-like reaction by depleting the generated ·OH, plays a key role in the tumor antioxidant defense system [68,69]. Therefore, improving the scavenging ability of intracellular GSH in cancer cells can augment the effectiveness of CDT [70]. Due to the killing effect of MW, the MW group was not used in this experiment. The consumption of intracellular GSH was evaluated using a GSH assay kit. The depletion of GSH in tumor cells increased with increasing concentrations of MgFe₂O₄ NPs (Fig. 4B), whereas treatment with MgFe₂O₄@ZOL resulted in a greater decrease in intracellular GSH. These findings indicated that MgFe₂O₄ can enhance the effect of CDT by depleting intracellular GSH. Other studies also reported that ZOL can induce perturbations in the biosynthesis of GSH [67,71,72]. The GSH content in cancerous cells is essential for tumor development and drug resistance [73]. MgFe₂O₄@ZOL NPs can deplete GSH by redox reactions to produce Fe²⁺ and GSSH, both of which are toxic to cancer cells, achieving the efficient antitumor effect of GSH depletion-enhanced CDT.

MgFe₂O₄ NPs can be used for real-time detection of high concentrations of MB dye as nanocomposites [74]. However, in this study, MB degradation experiments were performed to assess the catalytic efficiency of MgFe₂O₄ NPs on the production of ·OH. In the present study, H₂O₂ was added to the MB solution to mimic the tumor microenvironment. With increasing concentrations of MgFe₂O₄ NPs and H⁺, the absorption of MB decreased significantly (Fig. 4C and D). Additionally, the blue color faded as the duration of MW irradiation increased (Figure S4D). These results confirmed that MgFe₂O₄ NPs can catalyze the production of ·OH from H₂O₂. The efficiency of MgFe₂O₄ NPs on the production of ·OH increased under acidic conditions and MW irradiation.

The ·OH signal was trapped by BMPO and measured by EPR spectroscopy. The characteristic peaks of ·OH were not detected in the solution containing MgFe₂O₄ NPs (Fig. 4E). However, these peaks were detected when H₂O₂ was added, which suggested that MgFe₂O₄ can catalyze the formation of ·OH by the iron-based Fenton reaction. The intensity of the characteristic peaks of ·OH increased significantly after MW irradiation increased, which indicated that MW irradiation accelerated the Fenton reaction to produce ·OH, thus facilitating MDT. Many studies have reported that MW can accelerate the Fenton reaction [66]. The surface area and temperature of the NPs increased under MW irradiation, and the Fenton reaction rate was also enhanced, thus increasing the efficiency of the MgFe₂O₄ NPs for the formation of ·OH [75].

The ability of MgFe₂O₄@ZOL combined with MW to catalyze ROS production through intracellular Fenton reactions was investigated using the probe DCFH-DA. The A549 cells treated with the nanoparticles exhibited more intense green fluorescence than did the control cells (Fig. 4F and G), which indicated that MgFe₂O₄@ZOL NPs damage cancer cells through CDT [72], and that the overproduction of ROS is generated by ZOL [67,71,72]. The cells irradiated with MW exhibited more intense green fluorescence than those that were not irradiated with MW. These results indicated that ROS production increased considerably after microwave irradiation, and the cells treated with MgFe₂O₄@ZOL and exposed to MW irradiation experienced considerably greater ROS production due to the effects of MDT [66,75]. These findings showed that MgFe₂O₄@ZOL NPs under MW irradiation have an excellent ability to stimulate ROS production.

Traditional treatments for bone metastasis include chemotherapy, radiation, surgery, and medication. However, the limited number of drugs that can successfully reach bone tumor sites often leads to severe side effects. Therefore, a novel cancer treatment strategy known as CDT utilizes the specific properties of the TME to induce the Fenton reaction in vivo, effectively generating ·OH, which induces tumor cell death [76]. Compared to traditional therapy, CDT demonstrates excellent tumor selectivity and regulates the hypoxic and immunosuppressive TME [77]. MgFe₂O₄ NPs, as heterogeneous Fenton catalysts, could increase the rate at which H₂O₂ transforms into ROS (such as ·OH and ¹O₂) [78]. In this paper, the release of Fe³⁺ by MgFe₂O₄, while depleting GSH, can induce the Fenton reaction in lung cancer tissues, generating ROS to induce the apoptosis of tumor cells, thereby effectively reducing the side effects of treatment [79]. MTT increases the sensitivity of tumor cells to heat, resulting in thermal damage to tumor tissues. Additionally, the increase in temperature in the tumor area can promote the Fenton reaction, resulting in enhanced CDT [39]. In addition, microwave energy can induce local resonance coupling to create hot spots in specific areas. These hot spots may cause structural changes within the substance, such as the formation of point defects or weak surface bonds, resulting in the generation of free radicals [62]. Thus, we deduced that MgFe₂O₄@ZOL NPs create hot spots under MW irradiation and local thermal stimulation to dissociate or modify molecules, ultimately accelerating ROS expression via a similar process. As the above experimental results indicate, this combination therapy can synergize effectively with GSH depletion-enhanced CDT/MDT to generate a large amount of ROS, effectively achieving satisfactory tumor therapeutic effects.

3.6. Biocompatibility and toxicity of MgFe₂O₄@ZOL NPs and MW in vitro

To use MgFe₂O₄ NPs as carriers to deliver ZOL for precision therapy, they should have low toxicity, but they may induce toxicity after exposure to MW irradiation. Therefore, first, the biocompatibility of the MgFe₂O₄ NPs was assessed through hemolysis tests and CCK-8 assays. The biocompatibility of the MgFe₂O₄ NPs (at different concentrations) (0, 12.5, 25, 50, 100, and 200 μg/mL) for 1, 3, and 5 days, was evaluated after they were co-cultured with A549 cells (Fig. 5A), HUVECs (Figure S4E), and C3H10 cells (Figure S4F). The minimum cell viability was approximately 85 % after the cells were cocultured with 100 μg/mL MgFe₂O₄ NPs for five days, which showed that the MgFe₂O₄ NPs were highly biocompatible. Hemolysis tests were also conducted on solutions of MgFe₂O₄ NPs (12.5, 25, 50, 100, 200, 400, and 800 μg/mL) using rabbit blood. The blank and standard controls consisted of PBS and deionized water, respectively. At all tested concentrations of MgFe₂O₄ NPs, the hemolysis rates were less than 1 % (Figure S4G), which indicated the strong biocompatibility and negligible effect of these NPs on the erythrocyte membrane. These results were similar to those reported in another study [80], which showed good biocompatibility of the MgFe₂O₄ NPs.

Fig. 5.

The proliferation and viability of the tumor cells. (A) The cell proliferation of A549 cells treated with MgFe₂O₄ NPs for 1, 3, and 5 days. (B) The effects of MgFe₂O₄ NPs and MgFe₂O₄@ZOL NPs on A549 cell proliferation for 24 h. (C) Effects of MgFe₂O₄ NPs combined with MW irradiation on A549 cell proliferation. (D) The live/dead A549 cells staining with Calcein-AM/PI.

The cytotoxicity of the MgFe₂O₄@ZOL NPs and MW treatment were assessed through a CCK-8 assay. The efficacy of the MgFe₂O₄@ZOL NPs showed concentration-dependent changes (Fig. 5B). With increasing nanoparticle concentration, the viability of the A549 cells decreased, and after one day of treatment with 100 μg/mL MgFe₂O₄@ZOL NPs, the cell survival rate decreased to 70 %. There were prominent differences in the viability of cancer cells at different MW power levels. After A549 cells were cocultured with MgFe₂O₄ NPs for the uptake of nanoparticles, they were irradiated at different power levels (i.e., 0, 4, and 5 W) for 5 min to determine the optimum power of the MW to resist cancer cells. There was almost no difference between the A549 cells treated with MW at 4 W and the cells not treated with MW (Fig. 5C). However, after treatment with MW at 5 W, the cell viability decreased to 60 %; specifically, after treatment with 100 μg/mL MgFe₂O₄ NPs, the viability decreased to 30 %. These results can serve as a reference for the subsequent selection of the nanoparticle concentration and microwave power.

The proliferation of the cancer cells treated with the nanoparticles and irradiated with MW was evaluated through a live-dead staining assay. As displayed in Fig. 5D, no red fluorescence in the control group indicated no dead cells. The intensity of the red fluorescence of the cells treated with 100 μg/mL MgFe₂O₄ NPs was slightly stronger than that of the cells in the control group. Although green fluorescence dominated, the intensity of red fluorescence was greater after intervention with MgFe₂O₄@ZOL NPs or MW, because of the antitumor effect of ZOL released from MgFe₂O₄@ZOL and the consequence of MDT/MTT. The intensity of red fluorescence was considerably greater when cancer cells were treated with both MW and MgFe₂O₄ NPs. Almost no live cells were detected after treatment with MW combine with MgFe₂O₄@ZOL NPs due to the effects of the synergistic treatment of ZOL, MDT, and MTT. ImageJ was utilized to statistically analyze the viability of the cells after staining, enabling quantitative observation of the killing effect of MgFe₂O₄@ZOL NPs and MW on cancer cells (Figure S4H, with the numbers representing survival rates). These phenomena reflected the strong anticancer effects of the combination treatment.

3.7. Cell structure alteration

Based on the strong Fenton reaction and the microwave responsiveness of the MgFe₂O₄@ZOL NPs, we further investigated lung cancer cells via TEM to evaluate the degree of intracellular damage. A549 cells were analyzed via TEM after they were incubated with the nanoparticles for 24 h and with MW at 5 W for 5 min. In the absence of treatment, the cells exhibited normal-shaped cytomembrane, nucleus (red arrows), endoplasmic reticulum (blue arrows), and mitochondria (black arrows) without typical apoptotic features (Figs. 6A–1). In this study, 100 μg/mL nanoparticles were used to treat A549 cells. Dark granules (green arrows) were distributed in the cytoplasm after treatment with nanoparticles (Figs. 6A–2), which indicated that they were endocytosed. The diameter of the granules was approximately 340 nm, which matched the results of the DLS analysis. After endocytosis of the MgFe₂O₄ NPs, some mitochondria in the cells appeared slightly swollen (black arrows), indicating that the nanoparticles generated a small amount of ROS, leading to impaired mitochondrial function. In the MgFe₂O₄@ZOL NP group, the mitochondria of the cells were more swollen, autophagosomes (yellow arrows), and lipid droplets (purple arrows) increased, and the chromatin appeared to be slightly condensed compared with MgFe₂O₄ NP group (Figs. 6A–3). After 5 min of 5 W MW irradiation, the A549 cells changed from normal fusiform to irregular round and even lysed. As shown in Figs. 6A–4 and 5, the lung cancer cell lost its normal morphology and exhibited more severe mitochondrial swelling, ridges detachment, vacuolization; expansion of the endoplasmic reticulum, degranulation; chromatin clumping, and edge aggregation; and the appearance of the lipid droplets, apoptotic body, and autophagosomes. The above manifestations occur due to the effects of ROS, ZOL, and hyperthermia, which disrupt oxidative phosphorylation and mitochondrial membrane permeability, leading to cellular damage, degeneration, and apoptosis [81]. As depicted in Fig. 6A–6, the high toxicity of the combination therapy leads to the direct disintegration and necrosis of tumor cells. Li et al. reported that magnetic Fe₃O₄ NPs could activate the IRE1-ASK1-JNK pathway and induce endoplasmic reticulum stress to triggers intrinsic apoptosis to enhance MWA [82]. TEM revealed membrane blebbing and apoptotic bodies, similar to the findings described by Li. The magnetic MgFe₂O₄ NPs and Fe₃O₄ NPs are similar in structure and characteristics. In addition, ZOL can lead to apoptosis through increased oxidative stress and endoplasmic reticulum stress [83]. Consequently, the combination of MgFe₂O₄@ZOL and MW not only enhances the cytotoxic effect of MTT on tumor tissues, but also induces mitochondrial damage and endoplasmic reticulum stress through the production of abundant ROS, hyperthermia, and ZOL, promoting programmed cell death in cancer cells and thus further enhancing antitumor efficacy.

Fig. 6.

In vitro anticancer effects. (A) TEM was used to detect the treatment of A549 cells: (A-1) Blank control; (A-2) MgFe₂O₄ NPs; (A-3) MgFe₂O₄@ZOL NPs; (A-4) MW control; (A-5) MW + MgFe₂O₄, and (A-6) MW + MgFe₂O₄@ZOL. (Red arrow indicates the cell nucleus, blue arrow indicates the endoplasmic reticulum, black arrow indicates the mitochondria, green arrow indicates the nanoparticles, and purple arrow indicates the lipid droplets.) (B) The diagram of cancer cell apoptosis process. (C) Fow cytometry was conducted to show the apoptosis of A549 cells in different treatment groups. (D) The apoptosis rate in different groups.

3.8. Anticancer effects in vitro

For programmed cell death, cancer cell apoptosis is a crucial process that can be triggered by ROS and hyperthermia (Fig. 6B). For a more comprehensive understanding of the health and status of cells treated with the MgFe₂O₄@ZOL NPs, cell viability and apoptosis were detected using flow cytometry. The Annexin V-FITC and PI kits were utilized to assess the integrity of the cell membranes, and the analysis was conducted by fluorescence-activated cell sorting (Fig. 6C and D). The trend described above was reflected in the cancer cell apoptosis rate after the different treatments. The percentage of apoptotic cells significantly increased after MW irradiation. Specifically, in the MW control group, the percentage of apoptotic cells increased from 7.65 % to 41.24 %. In the MW + MgFe₂O₄ NP group, the percentage of apoptotic cells increased from 11.05 % to 68.73 %. The percentage of apoptotic cells in the MW + MgFe₂O₄@ZOL NP group increased from 32.87 % to 90.82 %. These findings indicated that synergistic therapy with MgFe₂O₄@ZOL NPs and MW effectively induced cell apoptosis through the combined effects of ZOL, ROS, and hyperpyrexia.

Since cancer metastasis and poor clinical prognosis are associated with proliferation and invasion of cancer cells, we analyzed the effects of MgFe₂O₄@ZOL and MW on the behavior of cancer cells, including proliferation, migration, and invasion of cells. The capacity for cancer cell proliferation was reflected in colony formation (Fig. 7A and D), and the colony formation rate of the cells in the MW + MgFe₂O₄@ZOL group decreased to 9 %. The invasion capability of the A549 cells was assessed through a transwell assay (Fig. 7B and E). The invasion assay revealed that the number of invasive cells in the control group was approximately 43, whereas, in the MW + MgFe₂O₄@ZOL group, it was approximately 7. Additionally, the migration area of the cells in the control, MgFe₂O₄, and MgFe₂O₄@ZOL groups was evaluated at 12 h and 24 h (Fig. 7C and F). At 12 h, the migration area of the cells in the control group was approximately 27 %, whereas that of the cells in the MgFe₂O₄@ZOL group was approximately 9 %. After 24 h, the migration area of the cells in the control group increased to approximately 60 %, while that of the cells in the MgFe₂O₄@ZOL group increased to 19 %.

Fig. 7.

Biological interactions between MgFe₂O₄@ZOL NPs and A549 cells in vitro. The A549 cells were treated with blank control, MgFe₂O₄, MgFe₂O₄@ZOL, MW + control, MW + MgFe₂O₄, and MW + MgFe₂O₄@ZOL. (A and D) Colony formation and clone formation rate. (B and E) Images of the transwell assay and invasion ability of A549. (C and F) Cell scratch assay and migration capability.

It is well known that the uncontrolled energy of cancer cells for mitochondrial dysfunction induces cancer progression and invasion [84]. Our results demonstrated that the combination of MgFe₂O₄@ZOL NPs and MW inhibited the proliferation, migration and invasion of lung cancer cells. These effects may be attributed to the ability of ROS and hyperthermia to disrupt electron transport chains and destroy oxidative phosphorylation, leading to cell apoptosis. Furthermore, hyperthermia can interfere with the transcription of cancer cell DNA, thereby inhibiting the ability of cancer cells to proliferate and invade other tissues or organs, further enhancing antitumor effects [85,86].

3.9. Osteogenic differentiation

Osteogenic differentiation is an important process in the bone microenvironment. The evaluation was performed after the induction of osteogenic differentiation on days 3, 7, and 14. Alkaline phosphatase (ALP) staining was used to observe osteoblast differentiation and function, to assess the osteogenic differentiation of cells. Although there was no significant difference between the groups on day 3, the differences in ALP activity among the three groups increased substantially as the induction period increased (Fig. 8A–C). Alizarin red staining was utilized to identify mineralized nodule formation, providing a visual assessment of osteogenic induction effectiveness. The formation of mineralized nodules in the MgFe₂O₄ group increased remarkably in the C3H10 cells, whereas the mineralization in the MgFe₂O₄@ZOL group exhibited an even greater increase, with the effect becoming more pronounced over time (Fig. 8D–F). These changes occurred due to the synergistic stimulation of osteogenesis by the combined effects of Mg²⁺ and ZOL [87,88]. Osteolytic bone destruction is common in lung or breast cancer patients with bone metastasis, because cancer cells secrete humoral factors that stimulate osteoclasts and inhibit osteoblasts to cause osteolytic bone destruction and induce SREs [4]. Mg²⁺ mainly promotes the differentiation and adhesion of osteoblasts and enhances the adhesion of human bone-derived cells. Recently, Zhao et al. reported that a hydrogel microsphere (GelMA-BP-Mg) stimulates osteoblasts and endothelial cells while restraining osteoclasts via magnet-inspired Mg²⁺ for the treatment of osteoporotic bone defects [89]. MgFe₂O₄@ZOL released Mg²⁺ and ZOL in bone tumor tissues under MW irradiation to promote osteogenesis and inhibit osteoclasts. Thus, the inhibitory effects of MgFe₂O₄@ZOL NPs on osteoclasts should be further investigated. In other words, MgFe₂O₄@ZOL NPs were suitable for the treatment of osteolytic bone metastasis (such as lung cancer and breast cancer) to restore bone mineral density.

Fig. 8.

Effects of MgFe₂O₄ and MgFe₂O₄@ZOL on the differentiation of osteoblasts in vitro. (A, B, and C) ALP activity of C3H10 cells on days 3, 7, and 14: (A) Macroscopic images; (B) Microscopic images; (C) Quantitative analysis of ALP activity. (D, E, and F) Alizarin red staining of C3H10 cells on days 3, 7, and 14: (D) Macroscopic images; (E) Microscopic images; (F) Quantitative analysis of mineralized nodules.

3.10. Efficacy of anticancer effects in vivo

Based on the excellent therapeutic efficacy and MW sensitivity in vitro, the performance of the combinations of targeting, MTT, CDT, and MDT enhanced by the MgFe₂O₄@ZOL NPs in vivo against tumors in nude mice was further evaluated. The nude mice bearing tumors were randomly assigned to the following groups: 1. PBS; 2. MgFe₂O₄ NPs; 3. MgFe₂O₄@ZOL NPs; 4. PBS + MW; 5. MgFe₂O₄ NPs + MW; 6. MgFe₂O₄@ZOL NPs + MW. The mice were administered solutions intravenously at a dosage of 2 mg/kg through the tail vein. Thermal infrared images (Fig. 9A) and temperature changes were analyzed in the MW groups, and the ablation area of each group was quantitatively analyzed by imageJ software (Figure S4I). The heating area and temperature of the MgFe₂O₄ and MgFe₂O₄@ZOL groups were noticeably greater than those of the PBS group, and the area of MgFe₂O₄@ZOL group was larger than that of MgFe₂O₄ group. The in vivo temperature elevation curve is shown in Fig. 9D, with the MgFe₂O₄@ZOL group displaying the highest peak. These were partly attributed to the tumor-targeting ability, which facilitated the accumulation of the microwave sensitizer in tumors and enhanced the effect of selective MW heating [90,91]. In addition, the in vivo temperatures of all the groups were lower than the in vitro temperatures, probably because of greater heat dissipation caused by blood circulation [92]. These results indicated that the heating efficiency of tumors in the MgFe₂O₄@ZOL group was significantly increased after MW irradiation. The differences in body weight among the groups of mice were not statistically significant (Fig. 9E), which indicated that all the mice were in good condition. Fig. 9F showed that in the groups treated with MgFe₂O₄ NPs, the increase in tumor volume was slightly inhibited compared to that in the PBS group, and the tumors were found to grow rapidly. The group that was treated with MgFe₂O₄@ZOL NPs and MW exhibited significant tumor growth inhibition, probably due to the synergistic effects of the combination of ZOL, MDT, and MTT. The macroscopic images of the tumor and the tumor mass (Fig. 9B and C) in each group on day 14 confirmed these findings. The tumor sections were histologically stained to validate the effectiveness of various treatments in vivo (Fig. 9G–J). Prussian blue staining (Fig. 9G) was performed on tumor slices to evaluate the distribution of Fe³⁺ released by MgFe₂O₄ NPs in the animals. These results demonstrated that there was a higher concentration of Fe³⁺ in the tumor sites of the MgFe₂O₄@ZOL NPs group, suggesting that they possess a certain degree of tumor targeting capability. The histological images of tissues stained with hematoxylin and eosin (H&E) revealed remarkable necrosis in the tumor tissue in the MgFe₂O₄@ZOL + MW group, in stark contrast to the other groups (Fig. 9H). Furthermore, TUNEL staining of cells in the MgFe₂O₄@ZOL + MW group also exhibited a pronounced red fluorescence which represents apoptotic cells, suggestive of the multimodal combination therapy's superior ability to effectively induce cancer cell apoptosis compared to other treatment modalities (Fig. 9I). Additionally, Ki67 staining was carried out to assess the proliferation ability of cancer cells. The finding from Ki67 staining revealed a significant suppression in the synthesis of the nuclear proliferation protein Ki67 within the tumors of the MgFe₂O₄@ZOL + MW group, indicating that the multi-synergistic treatment exhibited promising anticancer efficacy in vivo (Fig. 9J). Histological staining of the major organs was conducted to determine whether adverse effects on the mice occurred throughout the treatment period (Figure S5). The findings revealed that there were no notable pathological alterations detected in the vital organs across all groups, indicating the absence of apparent side effects in the mice throughout the entire course of treatment.

Fig. 9.

In vivo antitumor effects. (A) Images of microwave heating in vivo. (B) Nude mice with tumor at the end of the experiment. (C) Images of tumors excised from mice. (D) Temperature changes caused by microwave heating in vivo. (E) Weight gain in various groups of mice. (F) The increase in tumor volume in mice between groups. (F) The tumor slices were stained with Prussian blue. (G) The tumor slices were stained with Prussian blue (PB) (the arrow points to the dyed Fe³⁺). (H–J) H&E, TUNEL, and KI67 staining of tumor tissue between groups: (H) H&E staining; (I) TUNEL staining; (J) Ki67 staining.

4. Conclusion

To summarize, a novel antitumor nanoplatform, MgFe₂O₄@ZOL, was synthesized based on targeting-tumor and microwave-sensitive nanoparticles to effectively treat lung cancer bone metastasis via CDT, MDT, and selective-MTT. The advanced MgFe₂O₄@ZOL nanosystems could release Fe³⁺, Mg²⁺, and ZOL in the TME and under microwave irradiation. These compounds not only catalyze the decomposition of H₂O₂ to generate abundant cytotoxic ROS and deplete GSH in tumor tissues, inducing cancer cell apoptosis, but also stimulate osteogenesis to improve osteolytic destruction. In addition, the combination of MgFe₂O₄@ZOL and MWA further enhanced the release of ROS and selectively heated the tumor tissue to avoid damage to the surrounding tissues. Thus, this novel strategy is a promising candidate for treating lung cancer bone metastasis.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Ethics approval

The Animal Ethics Committee of the General Hospital of Southern Theatre Command authorized all animal experimental procedures (ethical code SYDW2023007).

CRediT authorship contribution statement

Man Shu: Writing – original draft, Validation, Software, Methodology, Data curation, Conceptualization. Jingguang Wang: Software, Methodology. Ziyang Xu: Visualization, Methodology. Teliang Lu: Methodology, Software. Yue He: Software, Methodology. Renshan Li: Methodology. Guoqing Zhong: Supervision, Methodology. Yunbo Yan: Visualization, Software. Yu Zhang: Supervision, Resources, Project administration. Xiao Chu: Writing – review & editing, Supervision, Resources, Conceptualization. Jin Ke: Resources, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare no conflict of interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article.