Multifunctional bone cement for synergistic magnetic hyperthermia ablation and chemotherapy of osteosarcoma

Journal Pre-proof Multifunctional bone cement for synergistic magnetic hyperthermia ablation and chemotherapy of osteosarcoma

Bing Liang, Deyu Zuo, Kexiao Yu, Xiaojun Cai, Bin Qiao, Rui Deng, Junsong Yang, Lei Chu, Zhongliang Deng, Yuanyi Zheng, Guoqing Zuo PII:

S0928-4931(19)32761-4

DOI:

https://doi.org/10.1016/j.msec.2019.110460

Reference:

MSC 110460

To appear in:

Materials Science & Engineering C

Received date:

28 July 2019

Revised date:

8 November 2019

Accepted date:

17 November 2019

Please cite this article as: B. Liang, D. Zuo, K. Yu, et al., Multifunctional bone cement for synergistic magnetic hyperthermia ablation and chemotherapy of osteosarcoma, Materials Science & Engineering C (2019), https://doi.org/10.1016/j.msec.2019.110460

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© 2019 Published by Elsevier.

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Multifunctional bone cement for synergistic magnetic hyperthermia ablation and chemotherapy of osteosarcoma Bing Liang a,f,1, Deyu Zuoa,1, Kexiao Yub,1, Xiaojun Caic*, Bin Qiaoa, Rui Dengd, Junsong Yange, Lei Chud, Zhongliang Dengd, Yuanyi Zhengc* and Guoqing Zuoa,b* a

Institute of Ultrasound Imaging of Chongqing Medical University; The Second Affiliated

Hospital of Chongqing Medical University, 76 Linjiang Road, Yuzhong Distinct, Chongqing,

Chongqing Hospital of Traditional Chinese Medicine, 6 Panxi Road, Jiangbei Distinct,

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b

f

400010, P. R. China.

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Chongqing, 400021, P. R. China.

c Shanghai Institute of Ultrasound in Medicine, Shanghai Jiao Tong University Affiliated Sixth

Department of Orthopaedics, The Second Affiliated Hospital of Chongqing Medical University,

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d

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People’s Hospital, 600 Yishan Road, Xuhui Distinct, Shanghai, 200233, P. R. China.

76 Linjiang Road, Yuzhong Distinct, Chongqing, 400010, P. R. China.

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China.

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e Department of Spine Surgery, Honghui Hospital, Xi’an Jiaotong University, Xi’an, 710000, P. R.

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f Department of gastroenterology and hepatology, The Second Affiliated Hospital of Chongqing Medical University, 76 Linjiang Road, Yuzhong Distinct, Chongqing, 400010, P. R. China. 1 Bing Liang, Deyu Zuo and Kexiao Yu are co-first authors who contributed equally to this study. * Corresponding authors: Dr. Xiaojun Cai, Shanghai Institute of Ultrasound in Medicine, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, 600 Yishan Road, Xuhui Distinct, Shanghai, 200233, P. R. China. E-mail: [email protected]; Prof.Yuanyi Zheng, Shanghai Institute of Ultrasound in Medicine, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, 600 Yishan Road, Xuhui Distinct, Shanghai, 200233, P. R. China. E-mail: [email protected]; Prof. Guoqing Zuo, Institute of Ultrasound Imaging, The Second Affiliated Hospital of Chongqing Medical University, 76 Linjiang Road, Yuzhong Distinct, Chongqing, 400010, P. R. China. E-mail: [email protected]. 1

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Abstract: Myelosuppression, gastrointestinal toxicity and hypersensitivities always accompany chemotherapy of osteosarcoma (OS). In addition, the intricate karyotype of OS, the lack of targeted antitumor drugs and the bone microenvironment that provides a protective alcove for tumor cells reduce the therapeutic efficacy of chemotherapy.

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Here, we developed a multifunctional bone cement loaded with Fe3O4 nanoparticles

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and the antitumor drug doxorubicin (DOX/Fe 3O4@PMMA) for synergistic MH

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ablation and chemotherapy of OS. The localized intratumorally administered

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DOX/Fe3O4@PMMA can change from liquid into solid at the tumor site via a

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polyreaction. The designed multifunctional bone cement was constructed with Fe3O4 nanoparticles, PMMA, and an antitumor drug approved by the U.S. Food and Drug

drug

release

profile,

and

synergistic

therapeutic

effect

of

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controlled

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administration (FDA). The injectability, magnetic hyperthermia (MH) performance,

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DOX/Fe3O4@PMMA in vitro were investigated in detail. Furthermore, the designed DOX/Fe3O4@PMMA controlled the release of DOX, enhanced the apoptosis of OS tissue, and inhibited the proliferation of tumor cells, demonstrating synergistic MH ablation and chemotherapy of OS in vivo. The biosafety of DOX/ Fe3O4@PMMA was also evaluated in detail. This strategy significantly reduced surgical time, avoided operative wounds and prevented patient pain, showing a great clinical translational potential for OS treatment. Keywords: osteosarcoma, magnetic hyperthermia ablation, chemotherapy, PMMA bone cement 2

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1 Introduction Osteosarcoma (OS), which is the most common malignant bone tumor, causes decreased range of motion, intense and painful swelling, and pathological fracture in patients1. Traditional therapeutic modalities, including surgery and chemotherapy2, play important roles in OS therapy. Surgery can intraoperatively remove tumors and

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provide internal fixation to repair bone defects caused by OS invasion3. However, the

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typical approach for removing OS and preserving bone structure with internal fixation

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has some pitfalls, such as large incisions and serious surgical damage 4. Moreover, OS

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patients exhibit cachexia and anemia5, making surgery an extremely risky strategy. Chemotherapy, which is intended as an alternative to or synergistic therapy with

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surgery6 to decrease operative damage, is another efficient therapeutic strategy for OS.

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Unfortunately, myelosuppression, gastrointestinal toxicity and hypersensitivities

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always accompany chemotherapy7. In particular, the intricate karyotype of OS8, the

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lack of targeted antitumor drugs and the bone microenvironment that provides a protective alcove for tumor cells reduce the therapeutic efficacy of chemotherapy9, 10. Therefore, it is greatly necessary to develop a noninvasive and efficient treatment of OS while simultaneously reducing its side effects. In the past decade, magnetic hyperthermia (MH), as a potential noninvasive treatment for OS, has been employed to convert electromagnetic energy into heat under an external alternating magnetic field (AMF), selectively increasing the temperature to ablate the tumor11. The heating magnetizable particle used, including magnetofluid12, ferromagnetic thermoseeds13, magnetic seeds14, and promising 3

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magnetic mesoporous bioactive glasses15, is a key factor in MH. Because of the lack of efficient targeting strategies, intratumor injection is the best route of administration of the magnetofluid to achieve MH for OS16. Without the limitations of effective depth and biological barriers17, 18, MH has shown significant outcomes in some clinical trials and many basic studies19-21. For example, a pilot clinical study reported the transperineal injection of magnetic nanoparticles into prostate cancer patients for

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achieving minimally invasive treatment22. Another investigation used magnetite

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cationic liposomes and an AMF in a mouse OS model to inhibit local tumor and lung

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metastases23. The histology of tumor tissues showing necrosis demonstrated the good

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curative effect of this treatment. However, the magnetofluid may escape from the tumor into normal tissue, resulting in the risk of particle accumulation in sensitive

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organs (e.g., the brain, lung and heart) and thus damaging healthy tissue24. To avoid

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the diffuse leakage of the magnetofluid, an efficient strategy, “injectable smart

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phase-transformation implants with magnetofluid inside,” was proposed to treat tumors25. The injection of this liquid-solid phase-change hybrid composite material solidified with magnetic powders, into tumors can quickly increase the local tumor temperature, causing tumor ablation. Nonetheless, the efficacy of this treatment is still limited by tumor regrowth and residual tumor. Because of the incomplete ablation issue, some researchers have proposed the synergistic use of hyperthermia ablation and chemotherapy for tumors26. Accordingly, there has been recent progress on a combination of MH ablation and chemotherapy. Compared with monotherapy, this combined treatment with poly(lactic-co-glycolic acid) (PLGA)/Fe/doxorubicin (DOX) 4

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plus MH can result in higher mean levels of cell apoptosis and lower mean levels of cell proliferation26. Because hyperthermia can kill tumor cells or sensitize them to chemotherapy, it can effectively reduce the tumor recurrence rate 27. However, PLGA is limited by its own weak mechanical properties and might lack of the mechanical support of the skeleton. Poly(methyl methacrylate) (PMMA) bone cement, a U.S. Food and Drug

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Administration (FDA)-approved injectable bone repair material29, has been used as a

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grouting agent with appropriate mechanical strength in total joint replacement surgery 30, 31

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. PMMA provides mechanical support to weakened bone and consequently

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relieves pain from fractures caused by OS24. Moreover, PMMA can carry nanoparticles and drugs and prevent their escape into the surrounding tissues and

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circulatory system. Fe3O4 nanoparticles, as a mediator for magnetic-thermal energy

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conversion32, and DOX, as an FDA-approved classic anticancer drug, are widely used

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in clinical treatment34. The setting time of PMMA bone cement is approximately 5 minutes, which is much less than the 30-60 minutes required using calcium phosphate cement. Additionally, PMMA bone cement, with its fast liquid-solid phase transition property, it is able to be injected into tumors in a minimally invasive manner. Here, we aimed to develop a smart material to mediate a minimally invasive modification to remove malignant OS and reduce the damage caused by surgery while simultaneously overcoming the low chemotherapeutic efficacy arising from the bone microenvironment. Furthermore, this material can provide inter-mechanical support for relieving pain from OS fractures. Therefore, in this study, we developed a 5

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multifunctional bone cement (DOX/Fe3O4@PMMA) for synergistic MH ablation and chemotherapy of OS. DOX/Fe3O4@PMMA was directly injected into OS in a minimally invasive manner. This strategy significantly reduced surgical time, avoided operative wounds and prevented patient pain. According to a previous study33, PMMA could provide imbedded internal fixation to repair bone defects by OS. Under an AMF,

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DOX/Fe3O4@PMMA induced hyperthermia ablation of OS. In addition, DOX was

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released from this multifunctional bone cement in response to pH and MH and was

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maintained at a sufficiently high concentration in local OS, avoiding the limitation of

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the intricate OS karyotype. This constructed multifunctional bone cement with good

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biocompatibility completely eradicated OS without recurrence via synergistic MH ablation and chemotherapy; thus, it may improve therapeutic efficacy against OS

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while reducing the side effects, thereby promoting the clinical translation of this

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therapy.

2 Materials and Methods 2.1 Materials

Fe3O4 nanoparticles were obtained from Chengdu Aike Reagent (China). Doxorubicin was obtained from Xian Chonghua Reagent and Biomaterials Company (China). PMMA powder and methyl methacrylate (MMA) monomer were obtained from an FDA-approved clinical bone repair material manufacturer (OSTEOPAL, Germany).

6

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2.2 Synthesis of DOX@PMMA, Fe3O4@PMMA and DOX/Fe3O4@PMMA The composites of Fe3O4 nanoparticles with PMMA in different proportions were prepared by varying the mass ratio of Fe 3O4 nanoparticles in PMMA bone cement. The PMMA bone cement was compounded with PMMA powder (weight) and MMA monomer (volume) at a ratio of 2.6 w/v according to the operation instructions.

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Following the requirements of the study protocol, DOX and Fe 3O4 nanoparticles were

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interfused (separately or in combination) into PMMA powder by a mechanical

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vibration method. Then, the mixed powders were added into the liquid MMA

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monomer after a 1.5min response time to obtain DOX@PMMA, Fe 3O4@PMMA and

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DOX/Fe3O4@PMMA. For this study, the ultimate concentrations of Fe3O4 were 2%,

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4%, and 8%, and the DOX concentration was 1%.

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2.3 Morphological and structural analysis of DOX/Fe 3O4@PMMA

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The morphological features of the Fe 3O4 nanoparticles, DOX, PMMA powder, liquid monomer and prepared DOX/Fe3O4@PMMA were observed by digital imaging. Harvested DOX/Fe3O4@PMMA samples were dried for 6 h and placed in SEM single-mount storage tubes to obtain advanced surface conductivities. Using a JSM-7800F field emission scanning electron microscope (JEOL, Japan), the microstructures of Fe3O4 nanoparticles, DOX, and PMMA powder were characterized by SEM imaging. The DOX/Fe3O4@PMMA with and without exposure to an external AMF was evaluated by SEM imaging and the corresponding energy-dispersive X-ray spectrometry (EDS) results. The magnetic properties of DOX/Fe 3O4@PMMA were 7

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tested using a physical property measurement system (PPMS-9) at room temperature. The pore characteristics of DOX/Fe3O4@PMMA with and without exposure to an external AMF were evaluated using a mercury porosimeter (Micromeritics autopore 9500, USA). Inductively coupled plasma optical emission spectrometer (ICP-OES) quantitative measurements were performed to calculate the escape of Fe3O4 NPs of

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DOX/Fe3O4@PMMA.

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2.4 In vitro and ex vivo hyperthermia effect of DOX/Fe3O4@PMMA by MH

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2.4.1 In vitro MH effect of DOX/ Fe3O4@PMMA

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To evaluate the magnetic thermal performance of DOX/Fe 3O4@PMMA, varying mass ratios of Fe3O4 nanoparticles (2%, 4% and 8%) and different volumes of

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DOX/Fe3O4@PMMA (50 μL, 75 μL, or 100 μL, refers to DOX/Fe3O4@PMMA after

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polymerization) were placed into saline solution in Eppendorf tubes. A far-infrared

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thermometer (FOTRIC225, ZXF Laboratories, US) was used to record the magnetic thermal performance of DOX/Fe3O4@PMMA by analyzing the temperature changes from thermal images (AnalyzIR 7.1 software) acquired during exposure to the homemade MH analyzer (frequency: 626 kHz, output current: 28.6 A, turns of coil: 2, coil length: 1 cm, coil diameter: 3 cm, field strength: 5.72 K/Am). After placing different types of DOX/Fe3O4@PMMA into Eppendorf tubes with saline and exposing these tubes to an AMF, the peak temperatures of the saline solution were taken every 20 s for 200 s. With the same approach, the tube containing saline without DOX/Fe3O4@PMMA was used as the control group. All of the above experiments for 8

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each group were performed three times.

2.4.2 Ex vivo hyperthermia effect of DOX/Fe3O4@PMMA Based on the above experimental results, 75 μL of DOX/4%Fe 3O4@PMMA was chosen for further study. For this work, freshly excised ex vivo bovine liver (2 cm × 2

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cm × 2 cm) was used to build a mimic in vivo model. Then, 75 μL of

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DOX/4%Fe3O4@PMMA was implanted into the center of the ex vivo bovine liver and

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exposed to the heating coil of the abovementioned MH analyzer for predetermined

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time durations (t=40, 80, 120, 160 and 200 s). The temperature variations in the model were detected every 20 s. Then, the volumes of coagulative necrotic liver tissues were

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calculated by the following formula: V (mm3) =π/6 × length × width × depth47.

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Additionally, ultrasound (US) images of the model before and after ablation for

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various time durations were recorded to evaluate the synergistic MH ablation effect of

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DOX/Fe3O4@PMMA during the procedure by observing grayscale changes. With the same approach, ex vivo bovine liver injected with saline was used as the control group. All of the above experiments for each group were performed three times.

2.5 In vitro DOX release 2.5.1 Evaluation of DOX thermochemical properties To ensure the stabilization of the DOX pharmaceutical effect, the thermochemical properties of DOX were assessed. DOX was dissolved in a neutral solution (phosphate buffer solution, pH:7.4) to obtain a standard DOX solution (0.075 mg/mL). The 9

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absorbency of the DOX standard solution was measured using ultraviolet-visible (UV-Vis) spectrophotometry (UV-2550 Shimadzu, Japan) and a high performance liquid chromatography (HPLC) system (Agilent 1290HPLC, USA) before and after exposure to laser irradiation (808 nm, for 60 s, achieving 80 ℃) and a water bath

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(achieving 80 ℃ ), and the absorption spectrum was determined.

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2.5.2 In vitro DOX release behavior at various pH values

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To evaluate the DOX release behavior, DOX/Fe 3O4@PMMA was initially

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prepared at various pH levels. First, 75 µL of DOX/Fe3O4@PMMA was injected into a

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dialysis bag (8 to 12 kDa molecular weight cutoff [MWCO]) with 1 mL of solution at various pH values (7.4, 6, 5.5) and then placed into a 50 mL centrifuge tube with 29

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mL of same solution as before, followed by shaking at 100 rpm at 37°C. At

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predetermined time points (0 to 12 h at 30 min intervals and 24, 36, 48 and 72 h), 2

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mL samples were harvested and replaced by an equal volume of the corresponding solution. The DOX concentration in each sample was determined using UV-Vis spectrophotometry. The efficiency of DOX release was calculated by the following equation: Release efficiency (%) = weight of DOX in each sample/weight of DOX in DOX/Fe3O4@PMMA ×100%. The weight of DOX in each sample was detected from the DOX standard curve. All of the above experiments for each group were performed three times.

2.5.3 In vitro DOX release behavior triggered/enhanced by MH at different pH values 10

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The DOX release behavior with or without MH triggering at various pH values was explored. As previously described, 75 μL of DOX/Fe 3O4@PMMA and DOX@PMMA were first injected into dialysis bags at a pH of 7.4 with and without exposure to an AMF for 30 s, 60 s, 120 s and 180 s, following by shaking at 100 rpm at 37°C. In addition, 75 μL of DOX/Fe3O4@PMMA was injected into dialysis bags to

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add the material to different pH value solutions (pH 7.4, 6, and 5.5), and tubes

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containing the dialysis bags were exposed to an AMF for 60 s. At predetermined time

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points (0 to 12 h at 30 min intervals and 24, 36, 48 and 72 h), 2 mL samples were

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harvested from every tube and were replaced by an equal volume of the corresponding

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solution. The DOX concentration in each sample was detected using UV-Vis spectrophotometry, and the efficiency of DOX release was calculated by using the

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performed three times.

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abovementioned equation. All of the above experiments for each group were

2.6 In vitro cytotoxicity assay and cell culture Human 143B OS cells were obtained from Chongqing Medical University (Chongqing, China) and were used for in vitro cell and in vivo experiments. These cells were cultured in high-glucose Dulbecco's modified Eagle medium (DMEM) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS) in an incubator with an atmosphere containing 5% CO 2 at 37°C. The cytotoxicity of DOX/Fe3O4@PMMA was evaluated by the classic cell-counting kit 8 (CCK-8) viability (Sihai Bio-Tech, Shanghai, China) assay. Culture 11

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medium containing varying concentrations of the DOX/Fe 3O4@PMMA suspension (0, 25, 50, 100, 200, and 400 μL/mL) was coincubated with 143B cells pre-seeded into 96-well plates (5×10 cells/well) for 12, 24, or 48 h. After 24 h of incubation, the medium was removed and replaced with 10 μL of CCK-8 solution, followed by another 3 h of coincubation. Finally, the absorbance of each well at 490 nm was

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determined by an enzymatic reader (BIO-TEK EL×800, USA). The wells with 143B

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cells without DOX/Fe3O4@PMMA and the wells without cells in each plate were used

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as controls and blanks, respectively. The cell viability rate (%) was calculated by the

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following equation: cell viability (%) = (ODw-ODb)/(ODc-ODb) ×100%, where ODw,

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ODc and ODb represent the optical densities (ODs) of the DOX/Fe3O4@PMMA,

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control and blank groups, respectively.

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2.7 In vitro chemo/MH synergistic therapy effect of DOX/Fe 3O4@PMMA

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To assess the synergistic therapeutic effect between MH and DOX, cell viability was measured after different treatments. As in the above experiment, medium containing DOX/Fe3O4@PMMA suspension was exposed to an AMF for various periods (0, 30, 60, 120, and 180 s). After coincubation with 143B cells for 24 h, cell viability was calculated using the previously mentioned formula. Human 143B OS cells were seeded into 3 cm culture dishes and cultured for 24 h for adherence. The dishes were treated with different regimens, including the control, Fe 3O4@PMMA, Fe3O4@PMMA+MH, DOX@PMMA, DOX@PMMA+MH, DOX/Fe 3O4@PMMA, and DOX/Fe3O4@PMMA+MH groups. The culture dishes with the MH groups were 12

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exposed to an AMF for 60 s. After coincubation with 143B cells for 24 h, cell viability was evaluated and calculated using the above equation.

2.8 In vivo biosafety All animal studies were performed following protocols approved by the Animal

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Ethics Committee of Chongqing Medical University. Healthy 6- to 8-week-old

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BALB/c mice were randomly divided into three groups (n=6 for each group): the first

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was injected with 75 μL of DOX/Fe3O4@PMMA subcutaneously in vivo following

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exposure to an AMF for 60 s, the second was injected without exposure to an AMF,

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and the third did not receive any treatment. At predetermined time points (pre-injection and days 1, 7, and 14 post-injection), serum and blood were collected

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from all mice for biochemical assays and hemanalysis using an automated biochemical

China).

The

serum

biochemical

indexes

included

aspartate

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BC-2800vet,

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analyzer (Hitachi 7600-110, Japan) and an automated hematology analyzer (Mindray,

aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), lactate dehydrogenase (LDH-L), creatine kinase isoenzyme (CK) and creatine kinase muscle/brain isoenzyme (CK-MB). The hemanalysis indexes included white blood cells (WBCs), red blood cells (RBCs), hemoglobin (HGB), mean corpuscular volume (MCV) and platelets (PLTs). As described above, healthy 6- to 8-week-old BALB/c mice were randomly divided into seven groups (n=6 for each group) and treated with the

following

DOX@PMMA,

methods:

control,

DOX@PMMA+MH,

Fe3O4@PMMA, DOX/Fe

Fe3O4@PMMA+MH, Fe3O4@PMMA,

or 13

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DOX/Fe3O4@PMMA+MH. The mice in the MH groups were exposed to an AMF for 60 s. After 21 days, all mice were sacrificed to obtain and weigh the main organs, including the heart, liver, spleen, lung and kidney. The organ sections were subjected to physiological analysis using hematoxylin-eosin (H&E) staining after fixation with 4%

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paraformaldehyde for 24 h.

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2.9 Establishment of the nude mouse 143B OS xenograft model

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Briefly, 4-week-old SPF BALB/c-nude mice (nude mice) (weights of 10-12 g)

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were purchased from and fed at the Animal Experiment Center of Chongqing Medical University. This study was performed after the nude mice were allowed to acclimate

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for one week. Human 143B OS cells (1×106 cells) were suspended in 100 μL of

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phosphate-buffered saline (PBS), and the cells were then subcutaneously injected into

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the left back regions of the mice to establish animal tumor xenografts. The tumor

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volumes were measured every 5 days. After the tumors grew to nearly 200 mm3, the mice were used for further treatment and therapeutic evaluation.

2.10 Synergistic effect of DOX/Fe3O4@PMMA for magnetic ablation and chemotherapy The OS tumor-bearing nude mice were randomly divided into the following seven groups (n=5): control group (treated with 75 μL saline), Fe 3O4@PMMA (treated with 75

μL Fe3O4@PMMA only),

Fe3O4@PMMA+MH

(injected

with

75

μL

Fe3O4@PMMA followed by MH), DOX@PMMA (treated with 75 μL DOX@PMMA 14

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only), DOX@PMMA+MH (injected with 75 μL DOX@PMMA followed by MH), DOX/Fe3O4@PMMA (treated with 75 μL DOX/Fe3O4@PMMA only), and DOX/ Fe3O4@PMMA+MH (injected with 75 μL DOX/Fe3O4@PMMA followed by MH). After a day to allow full solidification of the material, the mice were transferred to the center of an electromagnetic induction heating coil for magnetically induced

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hyperthermia and magnetically controlled DOX release. During the treatment, the

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temperatures of the tumors were evaluated using thermal images recorded every 20 s.

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After 180 s, the ablated mice were maintained for further observation.

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On the first day following treatment, 3 mice were randomly selected from each

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group to obtain fresh tumor tissue, followed by excision for histopathological and immunohistochemical assays. To observe cell structure and necrosis, the tumor tissues

fragmentation

during

cell

apoptosis

and

growth

fraction,

terminal

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DNA

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were sliced for H&E after fixation with 4% paraformaldehyde for 24 h. To detect

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deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and proliferating cell nuclear antigen (PCNA) assays were used. All of the remaining mice were fed for further observation. Body weights and tumor volumes were measured every three days. In addition, mice with tumors larger than 1000 mm3 were euthanized according to the standard animal protocol.

2.11 Statistical analysis All data are shown as the means ± standard deviations (SDs). An independent-samples t-test and one-way ANOVA were used for intergroup 15

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comparisons. A paired t-test was used to compare the data with the SPSS program package. (*p<0.05, **p<0.01, ***p<0.001), assuming unequal variances between the two data sets. Probability levels of <0.05, <0.01 and < 0.001 were considered to be the thresholds for significance.

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3 Results and Discussions

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3.1 Design, synthesis and characterization of DOX/Fe 3O4@PMMA

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To overcome chemotherapy resistance resulting from the protective alcove of bone

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and tumor invasion and pathological fracture caused by OS, we developed an

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injectable multifunctional bone cement. As shown in Table 1, cements with 2, 4 and 8% Fe3O4 and 1% DOX were designed to test the heating efficacy and DOX release

Composition of DOX/Fe3O4@PMMA.

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Table 1.

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properties with different iron contents.

Powder (g) Fe3O4 DOX PMMA DOX/2%Fe3O4@PMMA 0.076 0.037 2.6 DOX/4%Fe3O4@PMMA 0.152 0.038 2.6 DOX8%Fe3O4@PMMA 0.315 0.039 2.6

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Bone Cement

As shown by the

MMA monomer (mL) 1 1 1

graphical overview of the preparation process of

DOX/Fe3O4@PMMA in Figure 1A, DOX and Fe3O4 nanoparticles were interfused separately or in combination into PMMA powders by a mechanical vibration method. PMMA has been clinically utilized in orthopedic surgery35, particularly percutaneous vertebroplasty (PVP) and percutaneous kyphoplasty (PKP)36,

37

, because of its

relatively high compressive strength38. Some studies have reported that PMMA bone cement could be a carrier to load therapeutic drugs, such as antibiotics, in the form of 16

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small antibiotic-impregnated PMMA beads strung together in long chains 29, 31, which inspired us to integrate DOX into PMMA to act as a chemotherapeutic implant for tumor treatment. After heating up DOX with IR or a water bath (Figure S1), the UV absorbance spectra and peak area (by HPLC) of DOX were not changed, illustrating that DOX is thermally stable (Figures S2 and S3). SEM images show the internal

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features of solid DOX/Fe3O4@PMMA before (Figure 1B) and after (Figure 1C)

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exposure to an AMF. After exposure to an AMF, the internal morphology of

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DOX/Fe3O4@PMMA appeared to be a porous structure, which indicated that it was a

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good drug carrier for releasing DOX, possibly through the its tiny and relatively dense pores. In addition, pore diameter of DOX/Fe3O4@PMMA was 18.40±2.58 nm before

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exposure to AMF and 82.73 ± 4.94 nm after that (p<0.001). The porosity of

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DOX/Fe3O4@PMMA was 8.4±1.41% without applying AMF, while it increased into

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17.7±1.85% after exposure to AMF, showing that MH could trigger pores increase.

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The performance of these materials could enable this multifunctional implant to provide the best possible results. To ensure that DOX/Fe3O4@PMMA was suitable for minimally invasive injection into the tumor, its injectability was evaluated. Fe 3O4 particles, DOX and prepolymer powder could be evenly mixed with a liquid monomer, and polymerization of PMMA and the monomer could be triggered (Figure S4). In addition, the material could be loaded into a standard syringe (1 mL) (Figure 1D), thereby proving the excellent syringeability and fluidity of DOX/Fe3O4@PMMA in its as-prepared liquid state (Figure

S4).

After

the

liquid

form

transformed

into

a

stringy

state, 17

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DOX/Fe3O4@PMMA was suitable for injection, which took 1.5-2 min; this time was much shorter than the reported setting time of calcium phosphate bone cement (CPC)39. It was found that DOX/Fe3O4@PMMA could easily pass through the syringe because of the low viscosity of the stringy state; subsequently, the stringy form of DOX/Fe3O4@PMMA transformed into a strong solid form (Figure S4). Based on the

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obvious US contrast enhancement (Figure S5), Fe3O4 nanoparticles could firmly

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combine with PMMA inside the tumor, preventing the nontherapeutic materials from

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leaking into the blood vessels and surrounding normal tissue and facilitating

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subsequent MH. Furthermore, the culture supernatants co-incubated with PBS and the

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Fe3O4@PMMA sample were pure and lacked a black impurity (Figure S6). Before and after co-incubation, with the application of ICP-OES quantitative measurement,

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the iron concentrations of solid Fe3O4@PMMA were 37.53±0.88 mg/g and 38.03±

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1.55mg/g, with no significant difference between each other (p＞0.05). These results

Jo u

indicated that the Fe3O4@PMMA matrix was immobile and that no Fe3O4 escaped. The presence of Fe3O4 NPs and DOX was observed by SEM images of DOX/Fe3O4@PMMA powders (Figure S7). The size of Fe3O4 NPs was 128.4±43.25 nm (Figure S9A). The elemental energy spectrum of solid DOX/Fe3O4@PMMA were showed in Figure 1E, indicating the homogeneous dispersity of Fe3O4, DOX and PMMA powder within the matrix of the solid composite implant after the polymerization reaction. The corresponding atomic proportion (Figures 1F and 1G) coincided with the above distribution of elements. As shown in Figure 1H and Figure S8 for DOX/Fe3O4@PMMA with a Fe3O4 mass fraction of 2%, 4% and 8%, the shape 18

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of the hysteresis curve was long and narrow (DOX/4%Fe3O4@PMMA saturation magnetization: 7.54 emu/g, coercive force: 63.23 Oe), and similar to that of pure Fe3O4 NPs (Figure S9B, saturation magnetization: 88.57 emu/g, coercive force: 104.14 Oe), indicating that DOX/Fe3O4@PMMA had a low coercive force and residual magnetization value with soft magnetic performance. As a soft magnetic

f

material with relatively low hysteresis loss properties and coercive force,

oo

DOX/Fe3O4@PMMA could be easily heated by an AMF because the coercive force of

e-

pr

the material was inferior to that of the applied magnetic field 40.

The

magnetic

thermal

Pr

3.2 In vitro MH performance of DOX/Fe3O4@PMMA properties

and

appropriate

formulation

of

al

DOX/Fe3O4@PMMA were evaluated. MH consists of heating magnetic materials

rn

through the application of an external AMF41 by converting electromagnetic energy

Jo u

into heat without loss, permitting its utilization in superficially deep to deeply located tumors. The SAR value for DOX/Fe3O4@PMMA with a Fe3O4 mass fraction of 2%, 4% and 8% were 147.7±9.7W/g, 255.1±2.7 W/g and 495.3±5.3 W/g, respectively. Thermal images of the MH process for tubes containing saline and different proportions of DOX/Fe3O4@PMMA are shown in Figure 2A. These images demonstrate that the presence of DOX/Fe 3O4@PMMA could effectively increase its own temperature and that of the surrounding saline solution because the electromagnetic energy was converted into thermal energy by the Fe 3O4 nanoparticles. Comparatively, the pure saline solution without a magnetic constituent showed no 19

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temperature variations. With increasing Fe3O4 nanoparticle concentrations, 50 μL of DOX/Fe3O4@PMMA with an Fe3O4 mass fraction of 8% only required 28.2 s to reach a temperature over 55 ℃, whereas 50 μL of DOX/Fe3O4@PMMA with an Fe3O4 mass fraction of 2% took the same time to reach 29.5 ℃ (Figure 2B). Moreover, at the end of the exposure to an AMF, the temperature of DOX/Fe3O4@PMMA with an Fe3O4

f

mass fraction of 8% reached 98.4±2.1 ℃, which is extremely high and difficult to

oo

control and might lead to damage to and necrosis of the surrounding normal tissue. In

pr

contrast, for DOX/Fe3O4@PMMA with an Fe3O4 mass fraction of 2%, the temperature

e-

curve was mildly increased and only reached 52.9 ± 0.6 °C at 200 s. It is difficult to

Pr

achieve the treatment temperature range, and the action time 21 would cause recurrence of residual tumors and might unfortunately lead to increased permeability of the tumor

al

vessel, subsequently allowing making tumor cells to metastasize easily. It was found

rn

that the MH efficiency proportionately increased with the Fe 3O4 quantity, the

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DOX/Fe3O4@PMMA volume, and the AMF exposure time. A volume of 75 μL of DOX/Fe3O4@PMMA was determined to reach a temperature of 48 ± 1.1°C in 40 s (Figure 2C). For 100 μL of DOX/Fe3O4@PMMA, the temperature increase rate was too fast to control; thus, it is not suitable for in vivo treatment. In contrast, the thermal behavior of 50 μL of DOX/Fe3O4@PMMA was too mild to achieve a therapeutic effect. Considering the above results, we chose DOX/Fe 3O4@PMMA with an Fe3O4 mass fraction of 4% for further experiments in this study. Based on the thermal images and the corresponding time-temperature curve, 75 μL of DOX/Fe3O4@PMMA could exert a thermotherapy effect at a moderate temperature, 20

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ranging from 42-47 °C 42, and could gradually transmit thermal energy into the whole tumor tissue within the next 160 s. According to the above results, 75 μL of DOX/Fe3O4@PMMA was rationally chosen for the following experiment. To further prove the magnetic hyperthermal ablation efficiency of DOX/Fe3O4@PMMA, ex vivo bovine liver was initially used for evaluation. The thermal image (Figure 2D) and corresponding temperature curve (Figure 2E) of the bovine liver with/without the

oo

f

implantation of 75 μL of DOX/Fe3O4@PMMA demonstrated that only the tissue with

pr

the magnetic materials could be increased in temperature when exposed to an AMF.

e-

The temperature of the center of the bovine liver with the implantation of 75 μL of

Pr

DOX/Fe3O4@PMMA persistently and mildly increased due to significant tissue ablation. After MH ablation, the ablation volumes and morphology of the bovine livers

al

were measured, showing obvious coagulative necrosis with pale tissue after treatment

rn

(Figure 2D). As expected, the ablation efficiency depended on the exposure duration

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of DOX/Fe3O4@PMMA (Figure 2F). It is suggested that the exposure duration is selected based on the tumor volume, which potentially offers an individual treatment scheme to patients with various sizes of OS tumors by varying the exposure duration and implanted content of DOX/Fe3O4@PMMA. After MH ablation, the US signal intensity of the livers was highly echogenic (Figure 2D) and the echo intensity was increased (Figure S10), consistent with the ablation volume results and the expectation that DOX/Fe3O4@PMMA would be capable of inducing tumor hyperthermia.

21

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3.3 In vitro magnetic-enhanced and pH-responsive DOX release. Controlled drug release plays a vital role in accelerating the delivery of chemotherapeutic agents to targeted sites while ensuring that only minor drug leakage occurs in healthy tissues under various pH, US, or temperature conditions 13,43,44. Encouraged by the pore structure of DOX/Fe 3O4@PMMA after exposure to an AMF

f

(Figure 1C) and the corresponding porosity of the magnetic materials, we wondered

oo

whether this DOX/Fe3O4@PMMA bone cement was not only a carrier that can load

pr

therapeutic drugs but also a material capable of magnetically triggered synergistic

e-

drug release. The experimental procedure for analyzing the drug release behaviors of

Pr

DOX/Fe3O4@PMMA at different pH values with or without exposure to an AMF for various application periods is illustrated in Figure S11. To evaluate the effects of MH

al

and pH on DOX release from DOX/Fe3O4@PMMA, the dose of DOX released from

rn

DOX/Fe3O4@PMMA was verified by UV-Vis spectroscopy. As shown in Figure 3A,

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the cumulative release percentage of DOX from DOX/Fe 3O4@PMMA and DOX@PMMA without enhancement by MH was not significantly different. The percentage of DOX released at 72 h increased to 40.33±1.93% with a single treatment of MH. The release percentage of DOX is related to the exposure time of MH treatment. When the MH treatment time was rapidly increased to 180 s, 67.76± 2.34% of DOX was released after 72 h (Figure 3B). In addition, the color of the supernatants deepened, indicating that drug release can be controlled by effective magnetic thermal application and duration. According to previous studies, the intracellular pH value is approximately 5.0, which is lower than that of the tumor 22

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microenvironment (pH: 6.0-6.8) and especially that of normal tissues (pH: 7.35-7.45)45, 46

. This variation makes it possible to increase acid-sensitive DOX release in tumor

cells while minimizing DOX release in normal cells and tissues 47. The DOX release percentage increased with decreasing pH (Figure 3C), presumably because of the higher solubility of DOX at lower pH values48. Moreover, upon MH action, the release

Compared with the DOX release percentage at pH 7.4 without

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1.99%, respectively.

f

percentages of DOX at pH 6 and pH 5.5 after 72 h were 68.17±2.13% and 78.11±

pr

exposure to an AMF (7.01±0.15%), the release percentage at pH 5.5 with MH

Pr

e-

exposure was approximately 10-fold higher Figure 3D.

3.4 Biosafety of DOX/Fe3O4@PMMA

al

As DOX/Fe3O4@PMMA is a new therapeutic agent, it is imperative to evaluate its

rn

in vitro toxicity before its application to in vivo therapy. The cytotoxicity of

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DOX/Fe3O4@PMMA was measured by the typical CCK-8 method. As shown in Figure S12, DOX/Fe3O4@PMMA showed no cytotoxicity even when its concentration reached 400 ppm for various incubation durations, which indicated that DOX/Fe3O4@PMMA has good biosafety without exposure to an AMF and that the use of an AMF was necessary to achieve drug release. Although DOX/Fe3O4@PMMA had no significant toxic effects in cell experiments without magnetic induction as mentioned above, in vivo biosafety has been a restriction that has greatly impacted the materials and drugs used in clinical treatment. To assess the biosafety and biocompatibility of DOX/Fe 3O4@PMMA, the drug-free 23

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magnetic

material

(Fe3O4@PMMA),

drug-loaded

PMMA without

magnetic

nanoparticles (DOX@PMMA) and all materials exposed to an AMF (Figure S13), the in vivo toxicity in healthy BALB/c mice was analyzed. BALB/c mice and BALB/c-nude mice are congenic strains, and with the latter being BALB/c mice with a deteriorated or absent thymus, thus ensuring the consistency of subsequent in vivo

f

experiments. In the bio-functional analysis, the functions of the liver (AST and ALT)

from those

in the

other

groups

(untreated

group,

pr

significantly different

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and kidney (BUN) in the treated mice (DOX/Fe 3O4@PMMA+MH) were not

e-

DOX/Fe3O4@PMMA without MH group and control group) at days 1, 7, and 14 after the injection of DOX/Fe3O4@PMMA (Figure 4A, B, C, D, E, F and G). Despite the

Pr

cardiac toxicity of DOX49, the normal levels of CK and CK-MB and the reasonable

al

CK-MB/CK ratio showed that DOX/Fe3O4@PMMA delivered a therapeutic dose of

rn

DOX without cardiac toxicity. The WBC counts (Figure 4H) indicated that

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DOX/Fe3O4@PMMA caused no acute inflammatory reaction. In addition, the RBC, HGB, MCV and PLT counts showed that DOX/Fe 3O4@PMMA induced no noticeable side effects or abnormal changes in the hematopoietic system (Figure: 4I, J, K and L). Furthermore, the results of the histopathological analysis of the treated groups (Figure 5A) showed no difference from those of histopathological analysis of the control group. The general form (Figure S14) of the organs, the lack of hemorrhagic spots or injuries and the weights (Figure 5B) of the major organs (i.e., heart, liver, spleen, lung and kidney) were not significantly different between groups, demonstrating that there was no obvious damage or material deposition in the tissues of the treated and 24

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untreated groups. Additionally, DOX/Fe3O4@PMMA did not cause a marked change in the body weight of the BALB/c mice and had no impact on body growth and development (Figure S15); thus, this material demonstrates within good biosafety. From these findings, we concluded that DOX/Fe 3O4@PMMA has high histocompatibility and no significant toxicity to mice at the therapeutic dose. The

f

major reason for the safety of DOX/Fe3O4@PMMA might be that its components are

oo

all safely utilized for tumor therapy. DOX, as a typical anticancer drug, has been

pr

extensively applied in the clinic47. Fe3O4 nanoparticles have been authorized by the

e-

FDA for use in clinical practice50. PMMA bone cement, as a bone defect filler and repair material, has been used as grouting agent in joint replacement surgery for more

Pr

than 50 years51. Thus, DOX/Fe3O4@PMMA, as a drug-loaded MH and bone repair

al

agent, is highly biocompatible and clinically acceptable, which is a benefit to OS

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rn

patients and might accelerate its clinical translation.

3.5 Synergistic magnetically induced ablation and chemotherapy of OS A standard CCK-8 assay was used to investigate the in vitro cytotoxicity of various proportions of materials with or without exposure to an AMF for different durations. With exposure to an AMF at different times, the viability of cancer cells incubated with DOX/Fe3O4@PMMA solution for 24 h was decreased in a stepwise fashion (Figure 6A). Furthermore, the relative cancer cell viability rate dropped sharply to 30.6% after incubation with DOX/Fe3O4@PMMA solution and exposure to an AMF, indicating that the cell-killing effect of DOX/Fe3O4@PMMA was MH, duration and 25

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drug

dose

dependent.

In

contrast,

the

control,

Fe3O4@PMMA,

and

Fe3O4@PMMA+MH groups showed no significant cell damage or decrease in cell viability.

For

the

DOX@PMMA,

DOX@PMMA+MH,

and

DOX/Fe3O4@PMMA-only groups, the relative cancer cell viability rates were approximately 83% with minor cell damage (Figure 6B). The development of

f

DOX/Fe3O4@PMMA meets the need of reducing the side effects of chemotherapy due

oo

to its smart release system, which can be controlled via magnetic thermal application

pr

and duration.

e-

Next, we evaluated the in vivo antitumor efficacy of DOX/Fe3O4@PMMA using

Pr

the established 143B OS xenograft model. The evaluation process was implemented according to the protocol shown in Figure 7A. Three weeks after 143B OS

al

implantation, thirty-five nude mice bearing 143B OS were randomly divided into

rn

seven groups and were assessed to observe the therapeutic efficacies of different

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treatments. According to the pre-experimental targeting temperature and duration, mice were injected with various formulations at a dose of 75 μL, followed by exposure to an AMF for 180 s. As shown in Figure 7B, the MH processes were recorded by thermal imaging every 20 s, which obviously indicated that the efficient injection of DOX/Fe3O4@PMMA into the tumor could quickly increase the tumor temperature upon exposure to an AMF and that the results were similar to those obtained in vivo. The temperature of the tumor increased from 23.8 ℃ to 44.6 ℃ after exposure to an AFM, and the temperature increased steadily and slowly for 130 s (Figure 7C). At the end of the procedure, the temperature reached 65.5°C, which demonstrated that 26

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DOX/Fe3O4@PMMA could effectively yield a sufficient target temperature for an adequately long exposure time to ablate the tumor tissue. In contrast, the tumor temperature in the MH-only group increased by 1.5 ℃. Tumor volume measurements (Figure 7D) and imaging were used to monitor tumor growth every three days (until day 22), and photos of representative tumor-bearing mice in each group are presented

f

in Figure 7E. Both MH and chemotherapy, as assisted by various proportions of

oo

materials, only mildly restricted tumor growth (Figure 7D and 7E); unfortunately,

pr

distinct residual tumor and tumor recurrence appeared. At 13 days after ablation, all of

e-

the tumors were increased in size to varying extents except for those in the

Pr

DOX/Fe3O4@PMMA+MH group, and at 22nd day, the tumors were completely obliterated, leaving only a tiny scar, demonstrating the high synergistic efficacy of

al

combined MH and chemotherapy for removing OS (Figure 7E). In particular, the

rn

tumor inhibition rate results suggest that the 91.8% inhibition rate in the

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DOX/Fe3O4@PMMA+MH group, which was significantly higher than those in the other groups, was the same as the morbidity-free survival results (Figure 7F). The survival rate in the DOX/Fe3O4@PMMA+MH group was also highest among the seven groups over the monitoring duration of 45 days (Figure 7G). Throughout the entire duration, the body weights were not different among the seven groups (Figure 7H), indicating that the DOX/Fe3O4@PMMA systems were efficacious and safe for therapeutic applications. As shown in Figure 8A, only the DOX@PMMA+MH and Fe3O4@PMMA +MH groups showed slight changes in cell status, while the other groups showed no obvious 27

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changes in tumor cytomorphology. Comparatively, the microscopic structure of the tumors in the DOX/Fe3O4@PMMA+MH group showed homogeneous eosin-stained cellular debris and either coagulated, darkly stained nuclei or no nuclei upon H&E staining. Moreover, the edge of the ablated tumor tissue could be easily observed under a microscope. The immunofluorescence histochemical double-staining results

f

(Figure 8B and 8C) showed that the number of apoptotic tumor cells (green staining

oo

in TUNEL images) in the DOX/Fe3O4@PMMA+MH group was significantly higher

pr

than those in the other groups and that the number of proliferating cells (red staining in

e-

PCNA images) in the DOX/Fe3O4@PMMA+MH group was significantly lower than

Pr

that in the other groups, demonstrating that DOX/Fe 3O4@PMMA in combination with MH could have certain cytostatic and proapoptotic properties. However, the detailed

al

mechanism of the involvement of DOX/Fe 3O4@PMMA in tumor cell death still needs

rn

to be further studied. Furthermore, the merged images in Figure 8D were almost

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consistent with the H&E staining results, confirming the synergistic effects induced by the combination of MH and chemotherapy assisted by DOX/Fe 3O4@PMMA as a chemo-hyperthermal agent. Combined with the high anticancer efficiency, the high level

of

in

vivo

MH

ablation

and

the

chemotherapy

performance

of

DOX/Fe3O4@PMMA endow it with a promising clinical translation potential for combating OS.

4 Conclusion In this study, a multifunctional bone cement (DOX/Fe3O4@PMMA) based on the 28

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FDA-approved polymer PMMA was constructed for synergistic MH ablation and chemotherapy of OS. DOX/Fe3O4@PMMA can be injected into OS in a minimally invasive manner to reduce surgical trauma and to release DOX locally and effectively by magnetic enhancement. The heat generated upon MH not only caused significant tumor regression but also enhanced the tumor chemotherapeutic efficacy by triggering

f

fast release of the encapsulated DOX from the PMMA matrix. OS could be erased

oo

without recurrence using DOX/Fe3O4@PMMA with MH. Importantly, the Fe3O4

pr

nanoparticles do not escape into the surrounding tissues. Therefore, potential side

e-

effects and local complications are significantly mitigated. The multifunctional bone

Pr

cement is prepared with a convenient handling and administrating procedure. With one injection, magnetically enhanced ablation, gradual release of a therapeutic drug and

al

potential cavity support are achieved along with tumor coagulative necrosis, enhanced

rn

apoptosis of OS tissue, and restrained proliferation of tumor cells. Thus, this novel

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agent possesses a promising clinical translation potential for MH ablation and chemotherapy of OS with high therapeutic efficacy.

Supporting information Electronic supplementary information (ESI) is available: Figure S1-15.

Ethical statement All animal procedures were performed in accordance with the Guidelines of the Ministry of Science and Technology of Health Guide for Care and Use of Laboratory 29

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Animals, China, and approved by the institutional ethical committee (IEC) of Second Affiliated Hospital of Chongqing Medical University.

Author Contribution and Conflicts of Interest All authors have approved to the final version of the manuscript. There are no

f

conflicts to declare. Bing Liang, Deyu Zuo and Kexiao Yu are co-first authors who

pr

oo

contributed equally to this study.

e-

Acknowledgements

Pr

We acknowledge the financial supports from NSF for Distinguished Young Scholars (Grant No. 81425014),

National Key R&D Program of China

al

(2018YFC0115200), NSFC Key Projects of International Cooperation and Exchanges

rn

(81720108023) and Shanghai S&T Major project (2018SHZDZX05). We thank

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associate Prof. Li Chunhong of Chongqing University Of Science & Technology, who provided magnetic hysteresis loop of DOX/Fe3O4@PMMA magnetic materials in this work.

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Figure 1: Composition and structural characterization of the DOX/Fe 3O4@PMMA magnetic materials. A). Scheme of preparing the DOX/Fe3O4@PMMA magnetic materials, B). Low (left image; the scale bar represents 50 μm) and high (right image; the scale bar represents 20 μm) magnification SEM images of DOX/Fe 3O4@PMMA. 36

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C). Low (left image; the scale bar represents 50 μm) and high (right image, the scale bar represents 20 μm) magnification SEM images of DOX/Fe 3O4@PMMA after exposure to an AMF for 180 s. D).Digital photographs showing the general appearance of the PMMA powder (left upper image), DOX powder (left middle image), MMA monomer (left lower image), Fe 3O4 nanoparticles (right upper image) and injectable DOX/Fe3O4@PMMA (right lower image). E). Elemental energy

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spectrum of DOX/Fe3O4@PMMA (the scale bar represents 50 μm). F). Weight

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percentage and atomic percentage of each element in the sample. G). Corresponding

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elemental energy spectrum. H). Magnetic hysteresis loop of the DOX/Fe3O4@PMMA

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magnetic materials.

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Figure 2: In vitro MH performance. A). Thermal images of the DOX/Fe 3O4@PMMA magnetic materials with different volumes and mass fractions of Fe 3O4 after exposure to

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alternating

current

(AC)

magnetic

field

(with

saline

without

DOX/Fe3O4@PMMA as a control group). B) and C) The corresponding time-temperature curve of DOX/Fe3O4@PMMA at different Fe3O4 (B. 50 μl Fe3O4) concentrations and (DOX/4%Fe3O4@PMMA) adopted volumes. D). Ablative efficiency in an ex vivo bovine liver model evaluated by thermal images, US images 38

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and digital photos of macroscopic necrosis. E). The corresponding time-temperature curve of the ex vivo bovine liver (the control group was injected with saline). F). The corresponding necrotic volumes of the ex vivo bovine livers after different ablation

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times. (The data are expressed as the means ± sd. **p<0.01, ***p<0.001)

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Figure 3: In vitro magnetic-enhanced and pH-responsive DOX release. A). The DOX

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release-time curves with (AMF for 30 s) and without exposure to AMF (inset: digital

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photos of the differently exposed DOX supernatants at 72 h). B). The DOX release-time curves with exposure to AMF for different exposure times in PBS solution (inset: digital photos of DOX supernatant with different exposure times at 72 h). C). The DOX release-time curves at different pH levels without exposure to AMF (inset: digital photos of DOX supernatants in different pH solutions at 72 h). D). The DOX release-time curves at different pH levels after exposure to AMF for 30 s (inset: digital photos of DOX supernatants in different pH solutions after exposure to AMF for 60 s at 72 h). (The data are expressed as the means ± sd. *p<0.05, **p<0.01, ***p<0.001) 40

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Figure 4: In vivo biochemical analysis. Histograms depicting variations in the serum biochemical parameters for (A) AST, (B) ALT, (C) BUN, (D) LDH-L (E) CK, (F) CK-MB and (G) CK/CK-MB at different time points during the 14 days with different treatments. Histograms depicting variations in the blood parameters for (H) WBCs, (I) RBCs, (J) HGB, (K) MCV, (L) MCV and (M) PLTs at different time points during the

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14 days with different treatments.

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Figure 5: In vivo histocompatibility. A). H&E staining images of the major organs of Balb/c mice after subcutaneous injection of 75 μL of different materials with diverse treatments for 14 days of feeding (100x magnification) and B). the corresponding organs weight.

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Figure 6: In vitro synergistic MH-chemo therapeutic efficacy of DOX/Fe3O4@PMMA magnetic materials. A). Relative viabilities of 143B cells after coincubation with

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DOX/Fe3O4@PMMA solution medium at exposure to AMF for 0, 30, 60, 120, 180 s

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for 24 h. B). Relative viabilities of 143B cells after different treatments. (The data are

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expressed as the means ± sd. **p<0.01, ***p<0.001)

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Figure 7: In vivo synergistic MH-chemo therapeutic efficacy of DOX/Fe 3O4@PMMA magnetic materials. A). Treatment and follow-up regimen. B). The thermal images of nude mice bearing 143B OS after the intratumoral injection of 75 μL DOX/Fe3O4@PMMA with MH for the 180 s C). The corresponding time-temperature curve for the tumors of 143B OS-bearing mice with varied treatments. D). The time-tumor volume curves of 143B OS-bearing mice in the different treatment groups. E). Digital photos of 143B OS-bearing mice and their tumor regions after the various 45

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treatments on the 13th day (Inset yellow arrow: 143B OS-bearing mice after the injection of 75 μL of DOX/Fe3O4@PMMA under an AMF and its tumor region on the 22th day). F). Tumor-inhibition rate of 143B OS-bearing mice with the various treatments. G). Survival curves of 143B OS-bearing mice with the various treatments. H). Time-body weight curves of 143B OS-bearing mice in the different treatment

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groups. (The data are expressed as the means ± sd. ***p<0.001).

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Figure 8: Immunohistochemical staining results of the tumors. A). H&E staining in the tumor region of each group (100x magnification: the red dotted line marks: the edge of the necrotic area). B). PCNA assay, C). TUNEL immunohistochemistry staining and D) merged images in the tumor region of each group (100x magnification).

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Author Contribution and Conflicts of Interest All authors have approved to the final version of the manuscript. There are no conflicts to declare. Bing Liang, Deyu Zuo and Kexiao Yu are co-first authors who

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contributed equally to this study.

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Journal Pre-proof Highlights

1. Developed a multifunctional bone cement (DOX/Fe3O4@PMMA) 2. Determined the drug release properties of DOX/Fe 3O4@PMMA 3. Provided a minimally invasive modality for removal of osteosarcoma 4.Explored the synergistic magnetically induced ablation and the chemotherapy of

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osteosarcoma

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