Tumor ablation using novel photothermal Na_xWO₃ nanoparticles against breast cancer osteolytic bone metastasis

Introduction

Breast cancer is the second leading cause of cancer-related deaths among women worldwide,¹ and is also the most common cancer among Chinese women.² Osteolytic bone metastasis accounts for approximately 80% of patients with advanced-stage breast cancer,³ which inevitably results in skeletal morbidity, including pathological fractures, hypercalcemia and neurological compression.⁴ Currently, conventional treatment for bone metastasis of breast cancer such as chemotherapy is limited by its high toxicity and drug resistance.⁵ Removal of tumors by surgery is not suitable for small, poorly defined foci. Based on the generally osteolytic nature of the lesion induced by breast cancer, bisphosphonates and denosumab have become classic medications, however, bisphosphonates still have serious adverse effects such as osteonecrosis of the jaw and subtrochanteric fractures. Denosumab was reported to increase the risk of pancreatitis and endocarditis, erysipelas and infectious arthritis.^6,7 Newly reported drugs such as Cathepsin K inhibitor odanacatib have been withdrawn from the clinical trials for safety reasons.⁸ Other treatments such as c-Src inhibitor are still in clinical trials to date.⁸ Hence, novel safe and efficient therapies in the treatment of breast cancer bone metastasis are needed.

Nowadays, approaches of nanomedicine have gained increasingly number of attention^9,10 in the treatment of intractable cancers, especially breast cancer metastasis. Profiting from the discovery of enhanced permeability and retention (EPR) effect and the development of nanotechnology, photothermal therapy (PTT) has been widely developed by researchers. PTT based on near-infrared (NIR) radiation is a promising alternative or supplement to traditional cancer therapy.^11,12 For instance, gold nanoparticles,^13–21 transition-metal chalcogenides,^22–24 and carbon-based nanostructures^25,26 have been explored as photothermal agents because of their effective photothermal conversion ability. Recently, oxygen vacancy-rich transition-metal oxides are particularly appealing for PTT due to their metallic features and strong surface plasmon resonance (SPR) effect.^27–29 Also, non-stoichiometric MoO_3-x nanosheets showed efficient tumor homing capabilities and effective tumor ablation ability due to their strong localized-SPR effects in the NIR region.³⁰

In this study, for the first time, we developed a novel oxygen vacancy-rich tungsten bronze nanoparticles (Na_xWO₃) through a simple pyrogenic decomposition process for PTT. Deep blue Na_xWO₃ nanoparticles show strong absorbance in NIR region (980 nm), which enable the deeper penetration into bone tissue than common 808 nm laser that is significant for the treatment against cancer bone metastasis to eradicate tumor cells. Under the irradiation of NIR laser upon Na_xWO₃, obvious temperature increase was observed derived from excellent photothermal conversion ability of Na_xWO₃ nanoparticles. Therefore, our efficient PTT strategy of Na_xWO₃ could provide brand-new insight into future cancer bone metastasis treatment clinically, which also expand the research horizons of PTT nanomaterials.

Materials and methods

Materials

Dimethyl sulfoxide and 1-octadecene (90%) were purchased from Sinopharm Group (Sinopharm, Hongkong, China). Oleic acid and cyclohexane (C₆H₁₂) were obtained from Adamas-beta Company (Adamas-beta, Shanghai, China). Sodium tungstate dehydrate (Na₂WO₄·2H₂O) was purchased from Sigma-Aldrich (St Louis, MO, USA). All chemical reagents were used as bought without any purification. All aqueous solution used in the experiment was deionized water (18.2 MΩ.cm) obtained from Milli-Q water purification system.

Synthesis of Na_xWO₃ nanoparticles

In a typical synthesis of Na_xWO₃ nanorods, 10 mL of oleic acid and 30 mL of 1-octadecene and placed into a 100 mL flask, after that, 5 mL of ammonia solution with 2 mmol of Na₂WO₄·2H₂O was added into the flask drop wisely with magnetic stirring. After sufficient stir, the mixed solution was heated to 80°C under argon environment so as to evaporate ammonia. After the evaporation, the solution was heated to 280°C and maintained for 1 hr and then cooled to room temperature. Finally, the deep blue solution was centrifuged (13,000 rpm, 10 mins), the blue precipitate was re-dispersed in 10 mL of cyclohexane and 5 mL of ethanol-mixed solution and was centrifuged (13,000 rpm, 10 mins) to scour off the surface oleic acid. The final product was re-dispersed in 30 mL of deionized water for further use.

Materials characterization

Size and morphology of Na_xWO₃ nanoparticles were investigated using transmission electron microscopy (TEM) (JEOL 200CX, Tokyo, Japan). X-ray diffraction (XRD) was measured on a Rigaku D/MAX-2250 V at Cu Kα (λ=0.154056 nm) with a scanning rate of 4°min⁻¹ in the 2θ range of 10°–80°. Photothermal performance of Na_xWO₃ nanoparticles was monitored by a photothermal camera (FLIRTM A325SC camera, Wilsonville, Oregon, USA).

Photothermal evaluations of Na_xWO₃ nanoparticles

Photothermal performance was measured and analyzed by illuminating the cuvette containing different concentrations of Na_xWO₃ nanoparticles using a 980-nm wavelength laser with several different power intensity. One milliliter of Na_xWO₃ nanoparticles at different concentrations (0, 0.25, 0.5, 1 mg/mL) were illuminated by a 980-nm laser for 5 mins. One milliliter of Na_xWO₃ nanoparticles (1 mg/mL) solution was irradiated at various of power density (0.5, 1, 1.5, 2 W/cm²) for 5 mins. In order to evaluate the stability of the photothermal performance of Na_xWO₃ nanoparticles, four cycles of heating (980 nm laser, 1 W/cm², 5 mins) and cooling periods (without laser irradiation) were performed. The temperature changes during all experiments were monitored by a photothermal camera (FLIRTM A325SC camera).

Cell ablation experiment

293T cells, murine breast cancer 4T1 cells and RAW264.7 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured under standard conditions (37°C, 5% CO₂). To test the cytotoxicity of Na_xWO₃ nanoparticles, 293T cells in 96-well plates were incubated with various concentrations of Na_xWO₃ nanoparticles (0, 12.5, 25, 50, 100, 200 mg/mL) for 24 or 48 hrs, after that, the MTT assay was carried out to determine the relative cell viabilities.

To test the photothermal cell ablation effectivity of Na_xWO₃ nanoparticles, 4T1 cells in 96-well plates were incubated with various concentrations of Na_xWO₃ nanoparticles (0, 12.5, 25, 50, 100, 200 mg/mL). After 4 hrs of incubation, the cells were irradiated by 980-nm laser at a power density of 1.5 W/cm² for 5 mins. After another 24 hrs of incubation, the MTT assay was carried out to determine the relative cell viabilities compared with the untreated group.

Subcutaneous 4T1 tumor ablation experiment

To develop the tumor model, 1×10⁶ 4T1 cells suspended in 100 μL PBS were subcutaneously injected into the back of each mouse. After 1 week, the average tumor sizes would reach to about 60 mm³. For in vivo PTT, 4T1 tumor-bearing mice were randomly divided into four treatment groups: the first group was intravenously injected with saline solution (100 μL); the second group was exposed to 980-nm laser with a power density of 1.5 W/cm² for 8 hrs after injection with saline solution; the third group was intravenously injected with Na_xWO₃ nanoparticles (20 mg/kg) without NIR laser irradiation; and the fourth group was exposed to 980-nm laser with an output power density of 1.5 W/cm² for 5 mins at 8 hrs after intravenous injection of Na_xWO₃ nanoparticles (20 mg/kg). Tumor temperature and thermal images were visualized and recorded using the FLIRTM E50 camera. Hematoxylin-eosin (H&E) staining was performed after treatments to compare the therapeutic efficacy of different treatment groups. The tumor volumes and body weights after treatments were recorded every 3 days for 2 weeks using caliper measurements and analytical balance. (The tumor volume was measured using the following equation (V = L×W²/2, in which L means the length of tumor and W means the width of tumor).)

Biodistribution test

Female nude mice were purchased from Nanjing Peng Sheng Biological Technology Co, Ltd (Peng Sheng Biological Technology Co, Ltd, Nanjing, China). Animal experiments were carried out following animal ethics. For biodistribution test, 100 μL of Na_xWO₃ nanoparticles (20 mg/kg) in PBS was intravenously injected into subcutaneous 4T1 tumor-bearing nude mice (n=9). Three main organs and tumor were obtained and dissolved by aqua regia at various time points (4, 8, 12 hrs) post-injection. To calculate the percentage of injected dose per gram of tissue (%ID/g) values in mouse organs and tumors, concentration of W was measured by inductively coupled plasma.

4T1 bone metastasis tumor ablation experiment

The Animal Care Committee of Central South University reviewed and approved all the animal care protocols and experimental procedures in this study. All animal experiments were conducted in accordance with the guiding principles of the Animal Care Committee of Central South University. A total of 20 BALB/c-nu/nu mice (4 weeks old, female) were purchased Shanghai Laboratory Animal Company, CAS (SLACCAS, Shanghai, China) and nurtured in specific pathogen-free (SPF) plastic-isolator cages and maintained in SPF laboratory animal facilities of Shanghai Lab, Animal Research Center. After acclimating to the facility for 7 days, all mice were anesthetized with 1% pentobarbital sodium. Subsequently, mice were subjected to an injection of 100 μL resuspended 4T1 cells at a density of 10⁷ cells/mL in the tibiae plateau using a percutaneous approach to establish the breast cancer bone metastasis model. Two weeks later, after establishment of noticeable 4T1 breast cancer tissues in tibiae, the mice were randomly divided into four group; control group (0.9% NaCl), PTT group, Na_xWO₃ group (10 mg/kg nanoparticles), and Na_xWO₃+ PTT group (10 mg/kg nanoparticles). The second injection of nanoparticles and PTT were performed at 4th week. Body weight and tumor volume were recorded every 2 weeks. The tumor volume was measured using the following equation ((V=0.2618×L×W ×(L + W)), where W: the average distance in the proximal tibia at the level of the knee joint in the anterior-posterior and medial-lateral planes; and L: the distance from the edge of the proximal of the tumor to the distal extent of tumor^4,31).

μCT analysis

Six weeks after 4T1 injection, all mice were sacrificed for μCT scanning to evaluate cancer-associated osteolysis after treatments. The harvested tibiae were fixed in 4% paraformaldehyde. A three-dimensional reconstruction of breast cancer-bearing knee joint and quantitative bone volume fractions (BV/TV) was acquired as previously described.^32–35

Western blot

Murine breast cancer 4T1 cells were administered with different treatments, respectively. Afterward, total proteins of cells were harvested under radioimmunoprecipitation assay (RIPA) lysis buffer containing phenylmethylsulfonyl fluoride. Western blot experiments for Bax, p-Akt, Sclerostin, RANKL and Sclerostin were performed based on previous reports with antibodies purchased from Abcam/Cell signaling Technology (Abcam, Shanghai, China and CST, Danvers, MA, USA).

TRAP staining after CM treatments

After treatments against 4T1 cells by PTT, Na_xWO₃ and Na_xWO₃+ PTT, conditioned medium (CM) from breast cancer cells were collected. Next, relative CM was used to administer osteoclast precursor RAW 264.7 cells.

RAW 264.7 cells seeded in 96-well plates were administered with different CM treatments and 50 ng/mL RANKL, respectively, for 7 days. After the maturation of RAW 264.7 cells, 4% paraformaldehyde (PFA) was used to fix the cells followed by the complete rinse of PBS. Next, the tartrate-resistant acid phosphatase (TRAP) staining was used to label the matured multi-nucleated osteoclasts, among which the stained osteoclast with at least three nuclei was classified as TRAP-positive osteoclasts.³⁶

Statistical analysis

One-way ANOVA was used to determine the significance among groups. All values are expressed as mean ± SD. P-value ≤0.05 was considered statistically significant.

Result and discussion

The size and morphology of Na_xWO₃ nanoparticles were investigated using TEM, as shown in Figure 1A and B, the nanocrystals were cube-like and about 150–200 nm in length. In addition, as the high-resolution TEM (Figure 1C) shown, clear lattice fringe indicating the good crystallinity of Na_xWO₃ nanoparticles. The XRD was also performed, as demonstrated in Figure 1D, all peaks were well matched with Na_xWO₃ phase indicating the successful preparation of Na_xWO₃ nanoparticles.

Figure 1 (A) and (B) TEM images of NaxWO3 nanoparticles. (C) High-resolution TEM image of a NaxWO3 nanoparticle. (D) XRD pattern of NaxWO3 nanoparticles.Abbreviations: TEM, transmission electron microscopy; XRD, X-ray diffraction.

Next, we evaluated the photothermal performance of Na_xWO₃ nanoparticles in vitro. As the UV-vis-NIR absorbance of Na_xWO₃ nanoparticles solution (Figure 2A) indicated, the absorption of Na_xWO₃ nanoparticles solution increasing obviously from 650 to 1000 nm. After that, the photothermal experiments were carried out. Different concentrations of Na_xWO₃ nanoparticles solution was irradiated under 980-nm laser for 5 mins at power intensity of 1 W/cm², the temperature of higher concentration (1 mg/mL) of Na_xWO₃ nanoparticles solution exhibited sharply increased from 25.8°C to 41.8°C in 5 mins. However, temperature of pure water sample was exhibited limited increase (Figure 2B and C). In addition, Na_xWO₃ nanoparticles solution (1 mg/mL) was irradiated under 980-nm laser for 5 mins at different power intensity (0.5, 1, 1.5, 2 W/cm²), as shown in Figure 2D and E, the photothermal performance of Na_xWO₃ nanoparticles was heightened along with the increasing of laser power intensity. Beyond that, the photothermal stability of Na_xWO₃ nanoparticles was also monitored. As temperature profile showed in Figure 2F, no distinct recession could be found during the four cycles, which indicates the good photothermal stability of Na_xWO₃ nanoparticles.

Figure 2 (A) UV-Vis-NIR absorbance of NaxWO3 nanoparticles solution (200 μg/mL), photograph of NaxWO3 nanoparticles solution was shown in the insert. (B) Temperature increase curves of a NaxWO3 nanoparticles solution under an 980 nm-wavelength laser irradiation at various concentrations (0, 0.25, 0.5, 1 mg/mL) at a power intensity of 1 W/cm2 for 5 mins. (C) Representative thermal images of water or NaxWO3 nanoparticles solution (1 mg/mL) under 980 nm-wavelength laser irradiation at a power intensity of 1 W/cm2. (D) Temperature increase curves of NaxWO3 nanoparticles solution (1 mg/mL) under various power intensity (0.5, 1, 1.5, 2 W/cm2) of 980 nm-wavelength laser for 5 mins. (E) Representative thermal images of NaxWO3 nanoparticles solution (1 mg/mL) under 980 nm-wavelength laser irradiation at a power intensity of 0.5 W/cm2 or of 2 W/cm2. (F) Four heating and cooling cycles of NaxWO3 nanoparticles solution (1 mg/mL) (980 nm laser, 1 W/cm2).Abbreviation: NIR, near-infrared.

Encouraged by the good photothermal ability of Na_xWO₃ nanoparticles, we tested its cytotoxicity and ablation efficient further. As shown in Figure 3A, after 24 or 48 hrs co-incubated with various concentrations (0, 12.5, 25, 50, 100, 200 mg/mL) Na_xWO₃ nanoparticles, negligible 293T cells death were observed, which indicated tiny cytotoxicity of Na_xWO₃ nanoparticles. Next, we test photothermal ablation efficient to 4T1 cells. As shown in Figure 3B, 4T1 cells were then treated with 980-nm laser (1.5 W/cm², 5 mins) of various concentrations of Na_xWO₃ nanoparticles (0, 12.5, 25, 50, 100, 200 mg/mL). It was found that the relative viabilities of the 4T1 cells decreased remarkably at elevated Na_xWO₃ concentration. In contrast, negligible cell killing was found for the groups without any treatment or treatment with NIR laser only.

Figure 3 (A) Relative viabilities of 293T cells after they were incubated for 24 and 48 hrs with NaxWO3 nanoparticles of various concentrations (0, 12.5, 25, 50, 100, 200 mg/mL) (n=5, mean ± s.d.). (B) Inhibition of the growth of 4T1 cells incubated with NaxWO3 nanoparticles of various concentrations (0, 12.5, 25, 50, 100, 200 mg/mL) and then irradiated with 980 nm-wavelength lasers (1.5 W cm/2) for 5 mins (n=5, mean ± s.d.).Abbreviation: PTT, photothermal therapy.

The good tumor cells ablation result encouraged us for applying it to in vivo tumor therapy. Before tumor therapy, it is necessary to know the bio-distribution of Na_xWO₃ nanoparticles concentration in mice so as to confirm suitable time point for PTT. As shown in Figure 4A, an obvious gathering of Na_xWO₃ was detected after 4 hrs intravenous injection owing to enhanced permeability effect (EPR), and reached to a peak level in 8 hrs. Therefore, 8 hrs tail vein post-injection was selected for PTT experiments.

Figure 4 (A) Biodistribution profile of NaxWO3 nanoparticles in 4T1 tumor-bearing mice at various time intervals (4, 8, and 12 hrs) after the i.v. injection of NaxWO3 nanoparticles, as determined by measuring W in homogenized tissue solutions (n=3, mean ± s.d.). (B) Representative thermal images of bilateral 4T1 tumor-bearing mice that were irradiated with a 980 nm-wavelength laser (1.5 W cm/2) 8 hrs after injection with saline or NaxWO3 nanoparticles (20 mg/kg). (C) Time-dependent change of relative tumor volume after various treatments (n=5, mean ± s.d.). (D) Time-dependent change of mice body weight after various treatments (n=5, mean ± s.d.). (E) Representative images of H&E-stained tumor sections of various groups (control, control + 980 nm-wavelength laser, NaxWO3, NaxWO3 980 nm-wavelength laser).Abbreviation: PTT, photothermal therapy.

After that, in vivo 4T1 tumor PTT was carried out. The tumor temperature and thermal images were visualized with a thermal camera. As shown in Figure 4B, the temperature of the tumor rapidly increased to above 49.6°C in 5 mins under 980-nm laser irradiation (1.5 W/cm²), which was sufficient to thermally ablate the tumor, while the control group (without Na_xWO₃ nanoparticles injection) showed only limited temperature increase. For the treated group, 4T1 tumor growth was eliminated (Figure 4C), whereas the control groups demonstrated rapid tumor growth. Hematoxylin and eosin (H&E) staining of tumors from different groups further confirmed the above results, where the treatment group shows the most tumor damage (Figure 4E). No abnormal behavior or significant weight loss was observed in any group (Figure 4D), indicating minimal side effects of Na_xWO₃ nanoparticles.

On the basis of excellent PTT performance and previous in vitro inhibitory effect and in vivo percutaneous 4T1 breast cancer model of Na_xWO₃ nanoparticles, we hypothesized that Na_xWO₃ nanoparticles could also prevent breast cancer bone establishment in vivo. Herein, we constructed a breast cancer bone metastasis model by the injection of 4T1 breast cancer cells intratibially. In the control group and PTT group, as shown in Figure 5A, severe osteolytic bone lesions and destruction of cortices were observed. In contrast, there were fewer osteolytic lesions in the Na_xWO₃ group, and the cortices remained intact. Such bone protective effect was more obvious in Na_xWO₃+PTT group. Quantitative analysis of the bone parameters confirmed these results, as evidenced by a significant increase in BV/TV (Figure 5B). In addition, despite that there was no significant difference in the body weight of four groups during observation (Figure 5C), mice treated with Na_xWO₃+ PTT resulted in a significant decrease of tumor volume (Figure 5D), indicating the significant biosafety as well as tumor ablation abilities of Na_xWO₃ nanoparticles simultaneously.

Figure 5 (A) Representative images of μCT of the tibia trabecular bone medial compartment treated with 0.9% sodium chloride (control group), PTT (PTT group), NaxWO3 (NaxWO3 group), and NaxWO3+PTT (NaxWO3+PTT group). Scale bar, 100 μM. (B) Bone volume/tissue volume (BV/TV) was measured by quantitative analysis of μCT. The significance was determined as indicated in methods (**P<0.01 versus control). (C) Evaluation of cachexia by body weight that was recorded every 2 weeks until 6th week. (D) Quantitation of tumor volume of mice in each group.Abbreviation: PTT, photothermal therapy.

More interestingly, we also found that both the Bax (pro-apoptotic protein) and p-Akt levels in 4T1 breast cancer cells were increased after Na_xWO₃+PTT treatment, regardless of the similar tendency in PTT and Na_xWO₃ group compared with control group. These results indicated that Na_xWO₃+PTT treatments facilitated the breast cancer cells apoptosis by augmenting the Bax and phosphorylated Akt expressions. Importantly, both osteoclastic stimulus RANKL and Sclerostin expressions were significantly decreased after Na_xWO₃+PTT treatments compared with individual treatment and control group, signifying that Na_xWO₃+PTT treatments could attenuate the release of osteoclastic factors from breast cancer 4T1 cells (Figure 6A). Hence, we collected the CM from 4T1 cells to further administer osteoclastic RAW 264.7 cells. Figure 6B shows that in control-CM group, large and red-stained matured osteoclasts were formed together with PTT-CM and Na_xWO₃-CM groups. Nonetheless, in Na_xWO₃+PTT group, less and smaller osteoclasts were stained by TRAP solution, indicating the significant inhibition by Na_xWO₃+PTT treatments against 4T1 cells to induce subsequent osteoclast formation. Based on the above WB results, this could be ascribed from the reduced RANKL and Sclerostin secretion from 4T1 cells after Na_xWO₃+PTT treatments. Collectively, our results indicated that Na_xWO₃+PTT could prevent bone destruction of breast cancer and inhibit tumor growth significantly both in vitro and in vivo.

Figure 6 (A) Proteins expressions for Bax, p-Akt, Sclerostin and RANKL in 4T1 cells. (B) Osteoclastogenesis of RAW 264.7 cells at 7th day after stimulations by CM from 4T1 cells treated with control, PTT, NaxWO3, and NaxWO3+PTT. Scale bar, 250 μM.Abbreviations: PTT, photothermal therapy; CM, conditioned medium.

Conclusion

Herein, for the first time, we developed a novel oxygen vacancy-rich tungsten bronze nanoparticles (Na_xWO₃) through a simple pyrogenic decomposition process for PTT. As the results indicated, Na_xWO₃ nanoparticles showed excellent photothermal conversion ability and photothermal stability in vitro. In addition, percutaneous 4T1 tumor was eliminated without recurrence during 2 weeks observation after Na_xWO₃+PTT administration. More importantly, intratibial breast cancer model confirmed the desirable PTT effects of novel Na_xWO₃ nanoparticles against 4T1 breast cancer cells, showing decreased bone destruction as well as tumor volume in vivo. Mechanistic studies showed that Na_xWO₃+PTT treatments increased apoptotic Bax and p-Akt expressions. More importantly, Na_xWO₃+PTT treatments inhibited the osteoclastic RANKL and Sclerostin expressions from 4T1 cells, therefore further attenuated downstream osteoclastogenesis. Therefore, our novel PTT strategy of Na_xWO₃ could provide brand-new insight into future cancer bone metastasis treatment clinically, which also expand the research horizons of PTT nanomaterials.