Phytic acid-modified manganese dioxide nanoparticles oligomer for magnetic resonance imaging and targeting therapy of osteosarcoma

Abstract

Osteosarcoma is the most common malignant tumor in the skeletal system with high mortality. Phytic acid (PA) is a natural compound extracted from plant seeds, which shows certain antitumor activity and good bone targeting ability. To develop a novel theranostics for magnetic resonance imaging (MRI) and targeting therapy of osteosarcoma, we employed PA to modify manganese dioxide nanoparticles (MnO₂@PA NPs) for osteosarcoma treatment. The MnO₂ NPs oligomer was formed by PA modification with uniformed size distribution and negative zeta potential. Fourier-transform infrared spectroscopy, X-ray diffraction, energy dispersive spectroscopy, X-ray photoelectron spectroscopy, and thermogravimetric analysis demonstrated that PA has been successfully modified on MnO₂ NPs, and the structure of MnO₂@PA NPs is amorphous. In vitro experiments demonstrated that MnO₂@PA NPs oligomer can be efficiently internalized by tumor cell, and the internalized NPs can react with H₂O₂ under acid microenvironment to produce Mn²⁺ and O₂. In vivo experiments demonstrated that MnO₂@PA NPs oligomer can passively accumulate in tumor tissue, and the accumulated NPs can produce Mn²⁺ and O₂ for MRI and targeting therapy of osteosarcoma. In conclusion, we prepared a novel bone-targeting nano theranostics for MRI and therapy of osteosarcoma.

Keywords:

• Phytic acid
• manganese dioxide nanoparticles
• osteosarcoma
• magnetic resonance imaging
• targeting therapy

1. Introduction

Osteosarcoma is the most common malignant tumor in the skeletal system, accounting for 20%–34% of the primary malignant bone tumor with extreme invasiveness and metastasis as well as dismal prognosis (Rathore & Van Tine, 2021). Currently, surgery was the preponderant clinical treatment, but only patients with early-stage osteosarcoma could be cured via surgical resection. In addition, the five-year survival rate after surgery was less than 70% (Siegel et al., 2021). Surgery combined with neoadjuvant chemotherapy can significantly increase the survival rate of patients. Classical anticancer drugs such as cisplatin, doxorubicin, and methotrexate have shown good therapeutic effects to osteosarcoma (Jiang et al., 2022). However, chemical drugs are likely to cause numerous side effects and bone marrow microenvironment-associated drug resistance is inevitable (Yang & Tian et al., 2020).

In recent years, various nanoformulations such as inorganic nanoparticles (NPs) (Li et al., 2020), liposomes (Jing et al., 2022), nanogel (Zhang et al., 2018), lipid NPs (Peira et al., 2022), and polymeric NPs (Heyder et al., 2021) had been widely exploited for targeting treatment of osteosarcoma (Ambrosio et al., 2021; Wu et al., 2022). Nanoformulations can target and deliver therapeutics to osteosarcoma region to improve therapeutic benefits and decrease adverse effects of therapeutics (Jo et al., 2015). Otherwise, NPs can alter tumor microenvironment to decrease tumor cells survival through a serial of chemical, physical, and photogenic reactions. For example, manganese dioxide (MnO₂) NPs synthesized by chemical (Lim et al., 2021) or biological methods (Liu et al., 2021) can catalyze the conversion of endogenous hydrogen peroxide (H₂O₂) in tumor region into O₂ due to its catalase-like activity, thereby alleviating tumor hypoxia. The O₂ generated by MnO₂ can facilitate the cancer cell cycle status to S phase, which is sensitive to chemotherapeutic drugs (Guo et al., 2020). In addition, MnO₂ NPs can enhance radiotherapy efficacy of tumors (Liu et al., 2020; Yang & Ren et al., 2020). MnO₂ NPs also play an important role in osteosarcoma treatment. For example, alendronate/K7M2 cell membranes-coated hollow MnO₂ NPs encapsulated ginsenoside Rh2 was employed for immuno-chemodynamic combination therapy of osteosarcoma (Fu et al., 2022). In addition to chemodynamic therapy, the Mn²⁺ ions produced by MnO₂ NPs can be used as a magnetic resonance imaging (MRI) contrast, which displayed good biodegradation and biocompatibility in vivo (Yang et al., 2018). Consequently, MnO₂ NPs can potentially serve as a theranostics for T1-weighted MRI and therapy of osteosarcoma or other tumor. However, how to improve the targeting capacity of MnO₂ NPs to osteosarcoma need to be further investigated.

Aptamers (Niu et al., 2022), diphosphonate (Wu & Wan, 2012), and aspartic acid-related oligopeptides (Ogawa et al., 2017) exhibited good targeting ability to bones. However, their applications were restricted due to sophisticated synthetic methods or unpredictable biosafety. Phytic acid (PA), an organic phosphoric acid compound extracted from plant seeds, has been widely used as food additives (Zhou et al., 2019), antioxidant (Lux et al., 2022), preserving agent (Zhao et al., 2022), and chelating agent (Chen et al., 2018). PA has good biocompatibility since it was detected in mammalian cells. Interestingly, PA showed certain antitumor activities on colon tumor (Vucenik et al., 2020). PA also displayed special bone-targeting capability due to its strong chelating ability to calcium ion of bone. Consequently, PA-modified NPs was employed for targeting treatment of bone tumors (Zhou & Fan et al., 2019; Wang et al., 2020).

Herein, we hypothesized that PA-modified MnO₂ NPs (MnO₂@PA NPs) could improve the targeting capability and therapy efficacy of MnO₂ for osteosarcoma treatment. To verify this hypothesis, we fabricated MnO₂@PA NPs and investigated the in vivo targeting ability and therapeutic efficacy of NPs for osteosarcoma on a mice model. Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), energy dispersive spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), and thermogravimetric analysis (TGA) indicated that PA was successfully modified on MnO₂ NPs, and the fabricated MnO₂@PA NPs was amorphous. Transmission electron microscopy (TEM) results confirmed that a MnO₂@PA NPs oligomer was formed by PA modification. In vitro experiments demonstrated that MnO₂@PA NPs could be efficiently internalized by various tumor cells and the internalized NPs can release Mn²⁺ under acidic microenvironment. In vivo experiments verified that MnO₂@PA NPs could enhance T1 weighted MRI of tumor and significantly suppress tumor growth in a 143B tumor-bearing mice model. The theranostics exhibits wide prospect clinical applications for MRI and therapy of osteosarcoma.

2. Materials and methods

2.1. Regents and cells

Potassium permanganate (KMnO₄) and manganese sulfate (MnSO₄) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). PA, W = 70% was provided from Sigma-Aldrich (St. Louis, USA). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM) solutions, and Roswell Park Memorial Institute 1640 (RPMI 1640) solution were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Cell Counting Kit-8 (CCK-8, 480 T) and dichlorofluorescein diacetate (DCFA-DA, 2.5 μm) were received from Beyotime Biotechnology Co., Ltd. (Shanghai, China). H₂O₂ colorimetric assay kit (480T) was obtained from Abbkine Biotechnology Co., Ltd. (Shanghai, China). BBcellProbe® P01 probe was purchased from Bestbio Co., Ltd. (Shanghai, China).

All cells including RAW 264.7 (mouse monocyte macrophage), U2OS, 4T1, Ishikawa, MC 38, Panc 02, SW480, and 143B cells were obtained from American type culture collection (ATCC; Manassas, VA, USA). These cells were cultured in DMEM media with 10% FBS, 100 µg/mL streptomycin, and 100 µg/mL penicillin in a 37 °C incubator with fractional concentration of 5% carbon dioxide.

2.2. Synthesis of MnO₂ NPs and MnO₂@PA NPs

A reported method was employed to synthesize MnO₂ NPs (Marin et al., 2020). In brief, 2.455 g of MnSO₄•H₂O and 2.224 g of KMnO₄ were dissolved in 50 mL of ultrapure water and heated to 80 °C, respectively. About 100 mL of distilled water was added to a three-necked flask, then the heated MnSO₄ and KMnO₄ solution were slowly added to the three-necked flask with 1000 rpm stirring. A black suspension was obtained after 2 h reaction at 80 °C. The suspension was cooled to room temperature, ultrasonically dispersed for 10 min, filtered with suction, rinsed three times with distilled water, and dried in a drying oven at 80 °C for 12 h to obtain MnO₂ NPs.

To prepare MnO₂@PA NPs, 0.0512 g of KMnO₄ and 0.25 mL of PA were dissolved in 20 mL deionized water and reacted at 50 °C for 8 h. After finished the reaction, the solution was further stirred at room temperature for 24 h, then centrifuged at 6000 rpm for 5 min and washed three times with deionized water to obtain MnO₂@PA NPs. The reaction conditions were optimized with the same method.

2.3. Characterization of the MnO₂@PA NPs

The size, polydispersity index (PDI), and zeta potential of MnO₂@PA NPs were measured by dynamic light scattering (DLS) and laser Doppler anemometry with a Malvern Zetasizer (Nano ZS, Malvern, UK). To check the stability of the NPs, 0.5 mg/mL of MnO₂@PA NPs was used to determine the size and PDI of NPs at 0, 7, 14, and 28 days.

The structure of MnO₂@PA NPs was characterized by FTIR (FTIR-8400S, Shimadzu, Japan). The morphology of the NPs was observed by TEM (JEM-2100F, JEOL, Japan). The phase structure of the NPs was analyzed by X-ray diffractometer (Max-2550, Rigaku Corporation, Japan). The elements and valence states of manganese and phosphorus in NPs were analyzed by XPS (Escalab 250Xi spectrometer, Thermo Fisher Scientific, USA). The element species and contents of NPs were determined by EDS (Elite T, EDAX Inc, USA). The thermal stability of NPs was tested by thermogravimetric/differential scanning calorimeter (TG209F3, NETZSCH, Germany).

2.4. Cytotoxicity assay

RAW 264.7, U2OS, 4T1, Ishikawa, MC 38, Panc 02, 143B, and SW480 cells were cultured in 96-well plates (10⁴ cells/well) and incubated overnight. To evaluate the cytotoxicity of PA and MnO₂@PA NPs, cells were incubated with different concentrations of PA and MnO₂@PA NPs (0, 12.5, 25, 50, 75, 100, 125, 150 μg/mL) for 4 or 24 h. After incubation, cells were washed with phosphate-buffered saline (PBS) to remove PA and MnO₂@PA NPs, and 100 μL medium containing 10% CCK-8 solution was added to each well with another 30-min incubation, followed by optical density was measured at 450 nm using a Thermo Multiskan Spectrum spectrophotometer (Varioskan Flash, Thermo Scientific Inc., USA).

2.5. Detection of pH value, ROS, and H₂O₂ in cells

Intracellular reactive oxygen species (ROS) levels were detected by DCFH-DA. To evaluate the varieties of ROS in cells with MnO₂@PA NPs treatment, cells were co-cultured with MnO₂@PA NPs in six-well plates (10⁶ cells/well) for 4 h, then the medium was discarded and the cells were washed with PBS to remove MnO₂@PA NPs. After that, trypsinization was added and the cell suspension was transferred into a 1.5-mL Eppendorf tube for centrifugation (1000 rpm, 5 min). The centrifuged cells were re-suspended in 1 mL of serum-free medium, and 1 μL of DCFH-DA reagent was added to each well with 20 min incubation at 37 °C. The cells were then washed with PBS, re-suspended in serum-free medium, and analyzed on a flow cytometer (NovoCyte. ACEA, USA).

To determine the intra- and extracellular H₂O₂ concentration varieties, MnO₂@PA NPs were incubated with cells for 4 h, and the intra- and extracellular H₂O₂ concentrations were determined by H₂O₂ kit according to manufacturer’s instructions.

BBcellProbe® P01 probe was used to detect the pH value in tumor cells. Cells were incubated with PA, MnO₂ NPs, MnO₂@PA NPs for 4 h to determine pH value viability in cells. After incubation, the medium was discarded and cells were washed with Hank’s Balanced Salt Solution (HBSS) to remove PA, MnO₂ NPs, or MnO₂@PA NPs. After washing, trypsin digestion was added and the cell suspension was centrifuged (1000 rpm, 5 min). The centrifuged cells were washed with HBSS again and re-suspended in 1 mL of HBSS. Except for the blank control group, 1 μL of BBcellProbe® P01 probe was added to each well and incubated at 37 °C for 30 min. After incubation, cells were washed with HBSS and re-suspended with 1 mL of HBSS for analysis on a flow cytometer.

2.6. Cell uptake of NPs

To evaluate the cellular uptake behavior of MnO₂@PA NPs, cells were incubated with MnO₂@PA NPs for 24 h. After incubation, cells were digested with trypsin and centrifuged at 1000 rpm for 5 min. The collected cells were washed and centrifuged. The centrifuged cells were re-suspended in 2 mL of DMEM medium and the number of cells were counted by inductively coupled plasma mass spectrometry (ICP-MS) (Agilent ICP-MS 7800, Agilent, USA) to determine the contents of manganese in the cells.

2.7. Preliminary evaluation of biosafety of MnO₂@PA NPs

To evaluate bio-safety of MnO₂@PA NPs in vivo, 20 female Kunming mice were divided into four groups, in which the control group was received 100 μL of saline, and other groups were received 10, 20, and 40 mg/kg of MnO₂@PA NPs, respectively. Drug administration was performed every four days, three times in total. Mice were monitored and weighed every other day. After two weeks, mice were sacrificed and blood was collected for hematological analysis. The main organs of the mice including heart, liver, spleen, lung, and kidney were collected and fixed with 4% paraformaldehyde for hematoxylin–eosin (H&E) staining.

2.8. In vivo antitumor evaluation

Four-week-old female BALB/c nude mice were obtained from the Experimental Animal Center of Army Medical University (Chongqing, China) and kept in a SPF-level sterile animal room. A total of 1 × 10⁶ 143B cells re-suspended in sterile RPMI 1640 medium were subcutaneously implanted into the right back of mice to establish a bone tumor model. The tumor volume was measured and calculated as follows: $$V\left({\text{mm}}^3\right)=L\times W^2/2$$

V is the tumor volume of nude mice; L is the longest diameter of the tumor; and W is the shortest diameter of the tumor. When the tumor volume grew to 100 mm³, mice were randomly divided into four groups (n = 5). Control group received 100 mL of PBS. Treatment groups received 20 mg/kg of PA, MnO₂ NPs, or MnO₂@PA NPs per mice, respectively. Drug administration was performed every four days, three times in total. Tumor volumes and mice weights were measured every two days. After treatment, mice were sacrificed, and the resected tumors were weighted and immersed in 4% paraformaldehyde solution for histological examination and immunohistochemical analysis. The manganese contents in tumor tissues were detected by ICP-MS.

2.9. In vivo T1-weighted MRI

When tumor volume reached 200 mm³, mice received 20 mg/kg of MnO₂@PA NPs via tail vein injection. T1-weighted MRI was performed at 0, 0.5, 2, 4, 8, 24, and 48 h after administration.

2.10. Statistical analysis

Results were expressed as mean ± standard deviation (SD). All measurements included at least three independent experiments. One-way variance (ANOVA) was used for data analysis. Tukey’s multiple comparison test was used for more than three groups, and Student’s t test was used for two groups. Statistical significance was defined as *p < .05, **p < .01, and ***p < .01.

3. Result and discussion

3.1. Preparation and characterization of NPs

Reduction of KMnO₄ is a classical method to prepare MnO₂ NPs (Wang et al., 2017; Gao et al., 2021). Otherwise, MnO₂ NPs can be modified through addition of various ligand (Ma et al., 2021; Zhang et al., 2020) or cell membranes (Huang et al., 2022). MnO₂ NPs or its derivatives were widely used for tumor treatment because MnO₂ can react with H₂O₂ in tumor region to produce O₂ to alleviate tumor hypoxia (Lin and Zhao et al., 2018). PA is a natural extracts from plant seeds and widely used as additives, antioxidant, and chelating agent (Bloot et al., 2021). Interestingly, recent research demonstrated that PA exhibited antitumor activities and special bone targeting ability (Zhou et al., 2019; Wang et al., 2020). Based on these results, we speculated that PA-modified MnO₂ NPs can enhance the antitumor activity of MnO₂ and can serve as targeting therapeutics for osteosarcoma treatment. To verify the hypothesis, the PA-modified MnO₂ NPs was prepared through a simple method (Figure 1). The synthesis method was optimized by adjusting reaction temperature, time, as well as reactant ratio. As shown in Figure 2(A), the size of MnO₂@PA NPs was obviously increased with higher reaction temperature (Figure 2A). Interestingly, with prolonged heating time from 5 h to 6 h, the size of NPs increased obviously. However, when heating time was increased to 8 h, the size of NPs decreased dramatically. Otherwise, the size of NPs increased again when prolonging the heating time continuously (Figure 2(B)). Longer stirring time was also beneficial to decreasing the size of NPs. However, when prolonging the stirring time to 36 h, the size of NPs increased obviously (Figure 2(C)). The size of NPs was also significantly affected by the ratio of KMnO₄ and PA. As displayed in Figure 2(D), when the ratio of KMnO₄ and PA was 1:1, the size of NPs reached the minimum.

Figure 1. Fabrication of MnO₂@PA NPs oligomer for in vivo MRI and therapy of osteosarcoma.

Figure 2. A–D: The size distribution of MnO₂@PA NPs oligomer under various preparation conditions. (A) Reaction temperature, (B) heating time, (C) stirring time, and (D) various molar ratio of KMnO₄ and PA. E: The morphology of MnO₂@PA NPs oligomer observed by TEM, (Scale bar: 100 nm). F and G: The size distribution and zeta potential of NPs. H: The size distribution of MnO₂@ PA NPs oligomer in water under various storage time.

The morphology of NPs was confirmed by TEM. Interestingly, TEM results indicated that MnO₂@PA NPs oligomer was formed (Figure 2(E)). The reason may be that the six phosphate of PA can bond several manganese, which caused the MnO₂@PA NPs oligomer formation. In addition, DLS results demonstrated that the size of MnO₂@PA NPs oligomer was 111.1 ± 1.9 nm (Figure 2(F)) and the PDI was 0.28 ± 0.03. Otherwise, the zeta potential of MnO₂@PA NPs oligomer was –25.9 ± 0.6 mV (Figure 2(G)). Interestingly, the size of MnO₂@PA NPs oligomer was not significantly increased when stored in water for 20 days (Figure 2(H)). These results indicated that the MnO₂@PA NPs oligomer showed good stability in water.

The composition of MnO₂@PA NPs oligomer was confirmed by FTIR and EDS analysis. The FTIR spectra of MnO₂@PA NPs oligomer showed that Mn-O stretching vibration was observed at 514 cm⁻¹ (Figure 3(A)). In addition, the absorption peak of HPO₄^2– and PO₄^3– appeared in the absorption spectrum of MnO₂@PA NPs (Figure 3(A)), indicating that PA was successfully modified on MnO₂@PA. The EDS analysis results showed that C, Mn, O, and P elements was observed in MnO₂@PA NPs (Figure 3(B)), this result further demonstrated that MnO₂@PA NPs was successfully modified by PA.

Figure 3. Characterization of MnO₂@PA NPs oligomer. A: FTIR spectra of PA, MnO₂ NPs, and MnO₂@PA NPs oligomer. B: EDS spectra of MnO₂@PA NPs oligomer. C: XRD spectra of MnO₂ NPs and MnO₂@PA NPs oligomer. D–F: XPS spectra of MnO₂@PA NPs oligomer, Mn and P elements. G: The thermogravimetric curves of MnO₂ NPs and MnO₂@PA NPs oligomer.

The XRD diffractograms of MnO₂ NPs showed many diffraction peaks, which indicated that the MnO₂ NPs had a highly crystalline structure (Figure 3(C)). However, amorphous structure was found in the MnO₂@PA NPs, which implied that the crystal morphology of MnO₂ was destroyed by PA modification. The XPS results showed that the signals of Mn, O, C, and P could be observed in MnO₂@PA NPs, this results further demonstrated that PA was successfully modified on MnO₂ NPs (Figure 3(D)). Otherwise, the spin-orbit coupling level splitting peaks of Mn2p_3/2 and Mn2p_1/2 appeared at 642 eV and 655 eV (Figure 3(E)), and the spectral peak of P2p appeared at 134 eV (Figure 3(F)), respectively. These data further indicated that the MnO₂@PA NPs were successfully fabricated and the structure of MnO₂@PA NPs was amorphous. PA may change the surface free energy of the MnO₂ crystal, which may influence the growth rate of the crystal surface according to the Curie–Wulifu principle. Consequently, the heterogeneous crystal surface may disrupt the crystal morphology of MnO₂. In addition, we used α-Al₂O₃ as a control to analyze the thermal stability of the synthesized NPs under nitrogen atmosphere. As shown in the Figure 3(G), 15% and 2% weight loss was observed in MnO₂@PA NPs and MnO₂ NPs in the low temperature range, respectively. The weight loss may be attributed to the loss of water and the absorbed PA. MnO₂@PA NPs lose 15.6% weight between 150 and 600 °C, while MnO₂ NPs only lost 5.2% weight during the same temperature range (Figure 3(G)). The difference of thermogravimetric loss of the NPs may be contributed to the decomposition of PA in MnO₂@PA NPs. All the results confirmed that MnO₂@ PA NPs oligomer has been successfully fabricated and its structure is amorphous.

3.2. Cytotoxicity assay

The cytotoxicity of PA and MnO₂@PA NPs in RAW 264.7 cells and tumor cells were evaluated by CCK-8. As shown in Figure 4(A), both PA and MnO₂@PA NPs displayed poor cytotoxicity on RAW 264.7 cells with 4-h incubation under various concentration. The cell viability of 4T1, Ishikawa, MC 38, Panc 02, and SW480 cells were decreased with high concentration of PA treatment after 4-h incubation (Figure 4(C–G)), suggesting that PA can inhibit these tumor cells proliferation. In addition, PA displayed poor cytotoxicity on U2OS and 143B cells (Figure 4(B,H)). Interestingly, the cell viability of all the tested tumor cells were decreased with high concentration of MnO₂@PA NPs treatment (Figure 4(B–H)). Importantly, compared to PA, the cell viability of U2OS, 4T1, Ishikawa, SW480, and 143B were significantly decreased with high concentration of MnO₂@PA NPs treatment (Figure 4(B–D,G,H)). These results implied that MnO₂@PA NPs exhibited enhanced in vitro antitumor activity compared to PA.

Figure 4. Cytotoxicity of PA and MnO₂@ PA NPs oligomer on macrophages (A), U2OS cells (B), 4T1 cells (C), Ishikawa cells (D), MC 38 cells (E), Panc 02 cells (F), SW480 cells (G), and 143B cells (H) were measured by CCK-8 assay with 4-h incubation. The data were shown as mean ± SD. *p < .05, **p < .01, ***p < .001, and ****p < .0001 vs. PA group.

After 24-h incubation, the cell viability of RAW 264.7 cells was not significantly decreased with high concentration of PA treatment (Figure S1A, Supporting Information). However, the cell viability of U2OS, 4T1, MC 38, Panc 02, and SW480 cells were obviously decreased with high concentration of PA treatment after 24-h incubation (Figure S1(B,C,E–G), Supporting Information). These results indicate that the antitumor activity of PA was enhanced on U2OS, 4T1, MC 38, Panc 02, and SW480 cells with prolonged incubation time. Interestingly, the cell viability of U2OS, 4T1, Ishikawa, Panc 02, and 143B cells were distinctly decreased by MnO₂@PA NPs treatment compared to that of PA after 24-h incubation (Figure S1(B–D,F,H), Supporting Information). These results further demonstrated that MnO₂@PA NPs displayed increased antitumor activity compared to PA.

3.3. Cellular uptake

To investigate whether MnO₂@PA NPs can be internalized by macrophage or tumor cells, the manganese contents in cells were determined by ICP-MS. As shown in Figure 5(B), only a small amount of manganese were detected in RAW 264.7 cells, suggesting that only few MnO₂@PA NPs was internalized by macrophages. Interestingly, the manganese contents in MC 38, 4T1, and Ishikawa is close to 500 ng per 10⁶ cells, indicating that MnO₂@PA NPs can be efficiently internalized by these cells. Furthermore, the manganese contents in SW480 and 143B cells were further increased, indicating that the cellular uptake of MnO₂@PA NPs was enhanced by these cells. It is worth noting that the highest manganese contents were detected in U2OS cells, implying that U2OS cells had the strongest cellular uptake ability for MnO₂@PA NPs compared to other tumor cells.

Figure 5. A: Detection of intracellular hydrogen peroxide concentrations after MnO₂@PA NPs oligomer treatment. B: The cellular manganese contents when cells treated with MnO₂@PA NPs oligomer. C and D: The fluorescence intensity of cells when cells treated by MnO₂ NPs or MnO₂@PA NPs oligomer (Strong fluorescence indicating to weak acid conditions). E: The intracellular pH values when cells treated by MnO₂ NPs or MnO₂@PA NPs oligomer. The data were shown as mean ± SD. *p < .05, **p < .01, ***p < .001, and****p < .0001 vs. control group.

3.4. Detection of ROS and H₂O₂ concentrations in cells

NPs can significantly influence the ROS concentration in cells (Sun et al., 2022). Otherwise, MnO₂ NPs can react with ROS such as H₂O₂ to produce O₂ to alleviate tumor hypoxia (Guo et al., 2020). Therefore, the cellular ROS concentrations were detected with MnO₂@PA NPs treatment. As shown in Figure 5(A), the fluorescence intensity of tumor cells was obviously increased when cells were treated by MnO₂@PA NPs compared to that of negative control. Semi-quantitative analysis results were consistent with the fluorescence intensity determination (Figure S2, Supporting Information). These results demonstrated that the uptake of MnO₂@PA NPs can elevate the intracellular ROS concentrations, which is consistent with literature report (Hafez et al., 2019). The reason may be that MnO₂ NPs only reacted with H₂O₂ to produce O₂ (Jiang et al., 2020), which didn’t alter other ROS such as •OH, O₂^–, and ¹O₂.

To further investigate the influence of MnO₂@PA NPs on ROS concentration varieties, the intra- and extracellular H₂O₂ concentration in RAW 264.7 and tumor cells were also detected. When cells treated with MnO₂@PA NPs, the extracellular H₂O₂ concentration of all cells was not changed obviously (Figure S3, Supporting Information). In contrast, compared to control group, the intracellular H₂O₂ concentration of U2OS, 4T1, Panc 02, SW480, and 143B was significantly decreased. As previously described, MnO₂@PA NPs can be efficiently internalized by tumor cells, and the internalized NPs can react with H₂O₂ to produce O₂, which may reduce the intracellular H₂O₂ concentration.

As mentioned, MnO₂ NPs can react with H₂O₂ under acid condition to produce O₂. Therefore, the pH value viability in cells was detected. As shown in Figure 5(C–E), the pH value in 143B cells was obviously increased when cells treated with MnO₂ or MnO₂@PA NPs compared to that of untreated group. MnO₂ can exhaust H⁺ to react with H₂O₂ to produce O₂, consequently, the cellular pH values were increased when cells were treated with MnO₂ or MnO₂@PA NPs. In addition, MnO₂@PA NPs treated cells has higher cellular pH value than that of MnO₂ group. The reason could be that MnO₂@PA NPs can be easily internalized by tumor cells than MnO₂ NPs. All the results demonstrated that the internalized MnO₂@PA NPs can react with H₂O₂ under acid conditions to produce O₂ to alleviate tumor hypoxia.

3.5. In vivo imaging on a 143B tumor-bearing mice model

MnO₂ NPs can react with H₂O₂ to produce Mn²⁺, which can serve as T1-weighted MRI contrast for tumor (Lin et al., 2018). Consequently, we checked the T1-weighted MRI ability of MnO₂@PA NPs in tumor-bearing mice. After injection of 20 mg/kg NPs, the T1-weighted MRI at 0.5, 2, 4, 6, 8, 24, and 48 h were recorded. As shown in Figure 6, no enhanced MRI were observed after 0.5 h injection. Interestingly, an enhanced MRI was obtained when mice received MnO₂@PA NPs after 4, 6, and 8 h injection. In addition, the MR signals were attenuated after 24 or 48 h injection. These results indicated that MnO₂ NPs can be passively accumulated in tumor tissues and the accumulated NPs can produce Mn²⁺ under acidic tumor microenvironment. To further investigate if MnO₂@PA NPs can be cleaned from body, the T1-weighted MRI for kidney in tumor-bearing mice was performed. As shown in Figure S4 (Supporting Information), an enhanced T1 signal was observed in the kidney after 4 h injection, and the MR signal was obviously attenuated after 24 h injection, suggesting that the MnO₂@PA NPs can be cleaned from body by kidney.

Figure 6. In vivo T1-weighted MRI of 143B tumor-bearing mice after 0.5, 2, 4, 6, 8, 24, and 48 h injection of MnO₂@PA NPs oligomer.

3.6. In vivo antitumor efficacy

In vitro experiments demonstrated that MnO₂@PA NPs can significantly decrease tumor cells viability, which inspired us to further investigate the in vivo antitumor ability. The in vivo antitumor efficacy of the NPs was evaluated using 143B tumor-bearing mouse model. As shown in Figure S6 (Supporting Information), all of the tumor-bearing mice maintained their weight during the treatment. PA and MnO₂ NPs could obviously inhibit tumor growth compared to saline group during the treatment (Figure 7(B)), because PA and MnO₂ NPs had been confirmed owning antitumor activity in vivo (Masunaga et al., 2019; Liu et al., 2022). Importantly, tumor growth was significantly inhibited when mice treated with MnO₂@PA NPs compared to that of PA and MnO₂ NPs after 16 days of treatment (Figure 7(C)), indicating that MnO₂@PA NPs treatment had better antitumor activity than PA or MnO₂ NPs. The reason may that MnO₂@PA NPs can combine the antitumor activity of PA and MnO₂ NPs (Figure 7(D)), which cause MnO₂@PA NPs show better in vivo antitumor activity than other groups. In addition, we also detected the apoptosis of cancer cells after therapeutics treatment by TDT-mediated dUTP nick end labeling (TUNEL) assay (Figure 7(E)). Compared with saline group, MnO₂@PA NPs could significantly induce 143B cell apoptosis, which was consistent with in vivo antitumor results. In summary, MnO₂@PA NPs showed enhanced in vivo antitumor activity compared to PA or MnO₂ NPs.

Figure 7. In vivo antitumor efficacy evaluation of PA, MnO₂ NPs and MnO₂@PA NPs oligomer in 143B tumor-bearing mice. A: The dosage frequency of therapeutics after tumor inoculation. Mice received 20 mg/kg of PA, MnO₂ NPs or MnO₂@PA NPs. B: The tumor growth curves after i.v. injection of PA, MnO₂ NPs, and MnO₂@PA NPs oligomer. The data were shown as mean ± SD, **p < .01 vs. PA group and MnO₂ NPs group. C: The excised tumor images with PA, MnO₂ NPs, or MnO₂@PA NPs oligomer treatment. D: The contents of manganese in excised tumor. The data were shown as mean ± SD, **p < .01 vs. control group. E: TUNEL detection and immunohistochemistry assay for Ki67 in tumor tissues, (scale bar: 50 μm).

3.7. Preliminary evaluation of the biosafety of MnO₂@ PA NPs

Healthy Kunming mice were selected to evaluate the biosafety of MnO₂@PA NPs. There was no significant difference in body weight and organ weight when mice treated with different concentrations of MnO₂@PA NPs or saline (Figure S8(A,B), Supporting Information). Otherwise, the hematological parameters of mice treated with MnO₂@PA NPs did not show significant difference compared to saline group (Figure S7, Supporting Information). Typical biomarker related to hepatic and renal functions such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and urea also did not show obvious difference compared to saline group. H&E staining showed no changes in heart, liver, spleen, lung, kidney, necrosis, congestion, or vascular morphology (Figure S8(C), Supporting Information). These results demonstrate that MnO₂@PA NPs has good biocompatibility and no obvious adverse effects when administrated intravenously.

4. Conclusion

Patients with osteosarcoma remain high mortality after surgery. Herein, we developed a PA-modified MnO₂ NPs by a simple method for targeting MRI and therapy of osteosarcoma. The physicochemical properties of MnO₂@PA NPs were thoroughly characterized by FTIR, XRD, XPS, EDS, TEM, and TGA. The results demonstrated that PA was successfully modified on MnO₂ NPs, and the structure of MnO₂@PA NPs is amorphous. TEM results verified that a MnO₂ oligomer was formed by PA modification. The MnO₂@PA NPs oligomer has uniform size distribution and negative zeta potential. In vitro experiments confirmed that MnO₂@PA NPs oligomer could be internalized by various tumor cells, and the internalized MnO₂ could react with H₂O₂ to produce Mn²⁺ and O₂ under acid microenvironment. In vivo experiments demonstrated that MnO₂@PA NPs oligomer can accumulate in tumor tissues, and the accumulated NPs can release Mn²⁺ and O₂ for T1-weighted MRI and targeting therapy of osteosarcoma, respectively. Compared to MnO₂ NPs and PA, MnO₂@PA NPs oligomer can significantly inhibit tumor growth without obvious adverse effects. Our results provide novel candidate for targeting MRI and therapy of osteosarcoma.

Author contributions

Qian Ju and Rong Huang contributed equally to this study. Qian Ju and Rong Huang performed the main experiments in this manuscript and co-drafted the manuscript. Ruimin Hu, and Junjie Fan participated in the animal experiments and cell experiments. Dinglin Zhang, Rong Li and Jun Ding designed the project and modified the manuscript. All authors have given approval to the final manuscript.

Ethical approval statement

All animal experiments were performed in accordance with the guidelines for the Care and Use of Laboratory Animals of the Army Medical University (NO. AMUWEC2020121, Chongqing, China). All authors have read the manuscript and consent for publication.

Disclosure statement

The authors declare no conflict of interest.

Additional information

Funding

The authors acknowledge support via the Key Support Object of Army Medical University (no. 410301060191) and the Natural Science Foundation of Chongqing (no. cstc2020jcyj-msxmX0469).