Osteosarcoma (OS) is the most prevalent form of primary bone malignancy among children and young adults (Kansara et al., 2014; Isakoff et al., 2015; Roessner et al., 2021). These tumors often develop in the long diaphysis, with tumors of the proximal tibia and distal femur being particularly common (Bielack et al., 2002; Whelan et al., 2012; Kollar et al., 2019). OS is associated with a highly aggressive and malignant disease course characterized by high rates of pulmonary metastasis, with ∼80% of metastatic nodules ultimately developing in the lungs (Bielack et al., 2002; PosthumaDeBoer et al., 2011). The early diagnosis of osteosarcoma is difficult due to the limitations of available imaging technologies and the atypical symptoms associated with early-stage disease. Even with a combination of surgery and adjuvant chemotherapy, only 65–70% of OS patients achieve curative outcomes (PosthumaDeBoer et al., 2011; Whelan and Davis, 2018), and 5-year overall survival rates for metastatic OS patients are just 20% (Meyers et al., 2011; Doyle, 2014; Setsu, 2015). These low survival rates are primarily attributable to a combination of high rates of pulmonary metastasis and the frequent emergence of chemoresistance in treated patients (Snyder et al., 1990; Gadd et al., 1993; Kempf-Bielack et al., 2005; Simon et al., 2005). Over the past four decades, little progress has been made in improving OS patient survival rates (Gill and Gorlick, 2021), underscoring the need for the development of novel treatment approaches for affected patients.

Photothermal therapy (PTT) is a noninvasive therapeutic modality in which the energy-absorbing properties of particular agents, known as photosensitizers, are leveraged such that when they are exposed to near-infrared (NIR) light, they convert that NIR energy into heat to selectively ablate tumor cells (Wang et al., 2015; Wang et al., 2016; Zhang J. et al., 2019; Shramova et al., 2020; Bu et al., 2021; Gao et al., 2021; Zhang et al., 2021). Owing to its promise, NIR laser-induced PTT has emerged as a prominent form of noninvasive tumor treatment (Hou et al., 2018; Liu et al., 2019). When photosensitizers convert laser energy into heat, local tissue temperatures can rise to 45°C or higher, resulting in localized necrotic cell death (Hildebrandt et al., 2002). To effectively mediate PTT, nano-scale materials that absorb light across a wide range of the NIR spectrum and exhibit high photothermal conversion efficiency are critical. Suitable nanomaterials developed to date have included gold nanoparticles (NPs) (Zhang Y. et al., 2019; Alvi et al., 2021), carbon-based nanomaterials (Shen et al., 2020; Yu et al., 2020), and semiconductor nanostructures (Guo et al., 2017; Wang et al., 2019; Han et al., 2021). Fe₃O₄ NPs have previously been used selectively as contrast agents in the context of T2 magnetic resonance (MR) imaging, shortening the transverse relaxation time to improve negative contrast in T2-weighted images (Cheng et al., 2011; Cheng et al., 2012). These Fe₃O₄ NPs are highly stable, exhibit good photothermal conversion efficiency, and are both non-toxic and biocompatible under physiological conditions (Shen et al., 2015; Ren et al., 2016; Xiang et al., 2019; Lu et al., 2021). Polydopamine (PDA) is a biopolymer that exhibits good photothermal conversion efficiency and can be employed as a multi-functional coating agent (Beik et al., 2016), with PDA-coated nanomaterials having been employed for photothermal research and to diagnose and treat a variety of tumors (Xi et al., 2017; Schille et al., 2020).

In the present report, Fe₃O₄@PDA NPs were successfully synthesized and evaluated to establish their in vitro and in vivo utility as both contrast agents for T2 MR imaging and as therapeutic tools. Overall, our results clearly demonstrate that these Fe₃O₄@PDA NPs were able to effectively inhibit OS tumor growth and pulmonary metastasis, underscoring the value of leveraging these and similar nanomaterials for the diagnosis and treatment of OS.

Initially, Fe₃O₄ NPs were synthesized using ferric trichloride as precursor via a hydrothermal approach, with a PDA coating then being applied to yield Fe₃O₄@PDA nanocomposites (Figure 1A). When these NPs were evaluated via transmission electron microscopy (TEM) and scanning electron microscopy (SEM) (Figures 1B–D), they were found to be monodispersed spheres that were 3–9 nm in diameter. Following PDA coating, the size of these nanocomposites rose to 200–300 nm. To explore the structural characteristics of these Fe₃O₄@PDA nanocomposites, they were analyzed via high-resolution TEM, revealing small Fe₃O₄ NPs within the overall nanocomposite, consistent with successful Fe₃O₄ NP encapsulation within PDA polymers.

Next, the Fourier transform infrared (FTIR) spectra for PDA, Fe₃O_4, and Fe₃O₄@PDA nanocomposites were generated (Figure 1E). PDA exhibited characteristic peaks at 3,210 cm⁻¹ (ν_N–H), 2,930 cm⁻¹ (ν_C–H) (Fang et al., 2010), 1,635 cm⁻¹ (ν_arC-C), 1,400 cm⁻¹ (ν_N–C), and 1,113 cm⁻¹ (ν_arC-O) (Liao et al., 2020) corresponding to N-H bond, C-H bond, aromatic ring, N-C bond, and C-O bond stretching vibrations, respectively. The Fe₃O₄ spectrum exhibited a characteristic peak at 584 cm⁻¹(ν_Fe-O) corresponding to the Fe-O bond. Fe₃O₄@PDA nanocomposites exhibited all characteristic peaks associated with both Fe₃O₄ and PDA polymers.

The crystalline structures of Fe₃O₄@PDA nanocomposites were assessed via X-ray diffraction (XRD) (Figure 1F). Peaks at (220), (311), (400), (422), (511), and (440) were clearly evident for both Fe₃O₄ and Fe₃O₄@PDA samples, consistent with the PDA polymer coating processing having not damaged the inverse spinel Fe₃O₄ (JCPDS NO. 19-0629)_.

The robust absorption of the prepared Fe₃O₄@PDA nanocomposites in the FTIR region (Figures 2A,B) led us to explore their photothermal efficacy. Upon NIR laser irradiation (808 nm, 1 W/cm², 15 min), the temperature for a Fe₃O₄/PDA nanocomposite solution rose significantly up to 42°C in a dose-dependent manner as compared to pure water (17°C), underscoring the potential utility of these Fe₃O₄@PDA as photothermal agents. The photothermal conversion efficiency of Fe₃O₄@PDA nanocomposites was also calculated to be 31.9% (Supplementary Figure S1), which was slightly lower than the pure PDA nanomaterials.

T₂-weighted MR images of prepared Fe₃O₄@PDA solutions were next generated using a 9.4 T MRI magnet, revealing that these nanocomposites mediated a clear dose-dependent contrast effect in the resultant images (Figure 3A), with a calculated T₂ relaxivity (r₂) of 45.0 mM⁻¹ s⁻¹ (Figure 3B).

An MTT assay was then performed to gauge the biocompatibility and toxicity of prepared NP solutions when applied to 143B cells and LO2 cells (Supplementary Figure S2). Overall, these Fe₃O₄@PDA nanocomposites exhibited low cytotoxicity, with 85.10% of cells remaining viable even at a nanocomposite concentration of 200 μg/ml. In order to test the stability of the Fe₃O₄@PDA nanpcomposites in cell culture medium, 200 ppm of the nanocomposites were dispersed in cell culture medium for 2 h no precipitation was observed, indicating that the Fe₃O₄@PDA nanpcomposites are very stable in culture (Supplementary Figure S3). Further MTT assay-based analyses of the PTT treatment efficacy of these nanocomposites were then performed, revealing a dose-dependent increase in cytotoxicity such that at a 50 μg/ml Fe₃O₄@PDA dose, 90.06% cell death was achieved following irradiation (808 nm, 2 W/cm², 5 min), consistent with satisfactory in vitro PTT efficacy. When these nanocomposite concentrations were increased to 100 μg/ml, the increase in overall cell death was relatively limited (4.05%), and a dose of 50 μg/ml was thus selected for further experimentation.

To more fully explore the effects of PTT treatment when using Fe₃O₄@PDA nanocomposites in vitro, Calcein-AM and PI were used to stain 143B cells in different treatment groups as a means of visualizing cell viability. While negligible cell death was evident in the first three treatment groups, near-total cell death was observed in the Fe₃O₄@PDA+NIR group (Figure 4C), confirming the ability of these nanocomposites to efficiently kill tumor cells upon laser-mediated excitation.

Following PTT, rates of apoptotic cell death in the control, saline+NIR, Fe₃O₄@PDA, and Fe₃O₄@PDA+NIR groups were 13.09, 16.17, 15.15, and 63.7%, respectively (Figure 4D), thus reaffirming the ability of these nanocomposites to mediate PTT.

To expand on the above results and explore the in vivo utility of our prepared nanocomposites, mice were intravenously injected with Fe₃O₄@PDA solutions via the tail vein (5 mg/kg of a 3.0 mg/ml solution in saline), after which T2-weighted MR images were captured with a 3.0 T instrument at baseline and at 1, 2, 4, and 6 h post-injection (Figure 5A). Prior to injection, the signal-to-noise ratio (SNR) for these orthotopic tumors was 4.92 ± 1.61, but it had risen to 3.23 ± 1.39 at 6 h post-injection, with the SNR for the tumor area being 34.34 ± 2.78% lower at this time point relative to baseline (Figure 5B). The contrast of these T2-weighted images gradually improved over time as evidenced by the darkening of the tumor area, thus improving overall MR imaging quality of these OS tumors in a manner that should be conducive to their early detection and treatment. This effect is likely primarily attributable to the enhanced permeability and retention (EPR) effect characteristic of the tumor-associated vasculature, which can enable iron oxide-based nanomaterials to remain in the tumor area for extended periods of time in a manner amenable to improved PTT treatment utilization.

Next, orthotopic tumor-bearing nude mice were intratumorally injected with 50 μl of a 2 mg/ml Fe₃O₄@PDA solution or an equivalent volume of normal saline. Laser irradiation was then performed, with the temperature being monitored via infrared thermal imaging, revealing clear differences in temperature values between the saline+NIR and Fe₃O₄@PDA+NIR groups under laser irradiation (808 nm, 2 W/cm², 5 min) (Figures 6A,B). Tumor temperatures rose to over 50°C within 4 min in the Fe₃O₄@PDA+NIR group, with local temperatures as high as 53.4 ± 0.3°C after 8 min.

These temperatures would be sufficient to induce thermal damage to the tumor, resulting in extensive necrotic cell death and consequent tumor ablation. In contrast, temperatures in the saline treatment group only rose to 40.0 ± 0.1°C.

In mice in the Fe₃O₄@PDA+NIR PTT treatment group, tumor growth was effectively controlled (Figures 6C,D). While tumor volumes in saline-treated control animals rose to 1,722.0 ± 112.6 mm³, they decreased to 146.0 ± 8.0 mm³ in animals that underwent Fe₃O₄@PDA+NIR treatment (Figure 6E). Tumor volumes for mice in the two other treatment groups were largely the same as those in control mice (p > 0.05). No significant differences in murine body weight were observed among groups over time (Figure 6F). In summary, these data indicated that Fe₃O₄@PDA+NIR treatment was sufficient to mediate the effective PTT-based ablation of orthotopic OS tumors in mice.

Tumor tissue sections from mice in the different treatment groups were subjected to H&E staining, revealing no evidence of necrotic cell death in the control, saline+NIR, or Fe₃O₄@PDA groups, with cell morphology remaining intact (Figure 7A). In contrast, extensive necrotic tumor cell death and a loss of cellular morphology were evident in the Fe₃O₄@PDA+NIR group (Figure 7A). Ki-67 and CD31 immunohistochemical staining in the Fe₃O₄@PDA+NIR groups was reduced relative to that in the three other groups (Figures 7B,C).

Biosafety concerns are one of the primary barriers to the more widespread application of PTT. To that end, the histology of major organs collected from mice in the different treatment groups was assessed, revealing no evidence of necrotic cell death or morphological abnormalities following treatment in the brain, heart, spleen, kidneys, or liver (Figure 7D). Metastatic nodules were evident in the lungs of mice in all treatment groups other than the Fe₃O₄@PDA+NIR group (Figures 7D–F), while no metastases were observed in other organs. Metastatic tumor nodules in the lungs exhibited hyperstaining with heteromorphic changes and clear boundaries relative to the normal surrounding pulmonary tissue (Figures 7D,E). In contrast, lungs from mice in the three other treatment groups exhibited multiple solid metastatic nodules that were 1–3 mm in diameter with a fine texture (Figure 7F), with metastases being observed in 40–60% of mice in the first three treatment groups despite being evident in 0% of mice in the Fe₃O₄@PDA+NIR group.

Blood ALP levels were significantly lower for mice in the Fe₃O₄@PDA+NIR group, consistent with the ability of these nanocomposites to effectively inhibit OS tumor growth following NIR laser irradiation without inducing off-target toxicity in other major organs. Consistently, analyses of lung tissue samples from these mice indicated that Fe₃O₄@PDA+NIR treatment reduced both primary tumor size and the incidence of pulmonary metastasis, which has the potential to significantly improve OS patient prognostic outcomes (Lindsey et al., 2017; Gill and Gorlick, 2021). These results thus further underscore the promising utility of NP-based platforms for tumor-targeted PTT. However, additional pharmacokinetic and pharmacodynamic studies will be critical to the future clinical application of these materials.

In conclusion, we herein developed Fe₃O₄@PDA nanocomposites that exhibit excellent photothermal properties and are well-suited to use in both MR imaging and PTT treatment applications. When intravenously administered to mice, these particles increased the tumor relaxation (R2) value significantly, thereby enhancing T2 imaging contrast and thus increasing the odds of successful early-stage OS tumor diagnosis. Further in vitro and in vivo analyses revealed that these Fe₃O₄@PDA nanocomposites were biocompatible and largely non-toxic. When excited via NIR laser irradiation, these Fe₃O₄@PDA nanocomposites mediated robust antitumor activity and prevented OS tumor pulmonary metastasis, underscoring the broad potential of these nanomaterials for use in the treatment of this deadly form of cancer.