Multifunctional melanin-like nanoparticles for bone-targeted chemo-photothermal therapy of malignant bone tumors and osteolysis

Accepted Manuscript Multifunctional melanin-like nanoparticles for bone-targeted chemo-photothermal therapy of malignant bone tumors and osteolysis Yitong Wang, Quan Huang, Xiao He, Hui Chen, Yuan Zou, Yiwen Li, Kaili Lin, Xiaopan Cai, Jianru Xiao, Qiang Zhang, Yiyun Cheng PII:

S0142-9612(18)30587-8

DOI:

10.1016/j.biomaterials.2018.08.033

Reference:

JBMT 18839

To appear in:

Biomaterials

Received Date: 2 August 2018 Revised Date:

17 August 2018

Accepted Date: 17 August 2018

Please cite this article as: Wang Y, Huang Q, He X, Chen H, Zou Y, Li Y, Lin K, Cai X, Xiao J, Zhang Q, Cheng Y, Multifunctional melanin-like nanoparticles for bone-targeted chemo-photothermal therapy of malignant bone tumors and osteolysis, Biomaterials (2018), doi: 10.1016/j.biomaterials.2018.08.033. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Multifunctional Melanin-like Nanoparticles for Bone-targeted Chemo-photothermal Therapy of Malignant Bone Tumors and Osteolysis Yitong Wang1,4†, Quan Huang2†, Xiao He1, Hui Chen1, Yuan Zou3, Yiwen Li3, Kaili Lin4, Xiaopan Cai2, Jianru Xiao2, Qiang Zhang1* and Yiyun Cheng1* Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal

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1

University, Shanghai, 200241, P.R. China. 2

Department of Orthopedic Oncology, Changzheng Hospital, the Second Military Medical

University, Shanghai, 200003, P.R. China.

College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials

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3

Engineering, Sichuan University, Chengdu 610065, P.R. China.

Department of Oral & Cranio-Maxillofacial Surgery, Shanghai Ninth People’s Hospital, College

of

Stomatology,

Shanghai

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4

Jiao

Tong

University

School

of

Medicine;

National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology, Shanghai 200011, China. E-mail: [email protected]; [email protected] The authors contributed equally on this manuscript.

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†

Abstract: Malignant bone tumors associated with aggressive osteolysis are currently hard to be

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cured by the clinical strategies. Nevertheless, nanomedicine might provide a promising therapeutic opportunity. Here, we developed a multifunctional melanin-like nanoparticle for bone-targeted

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chemo-photothermal treatment of malignant bone tumors. The particles was originally fabricated from alendronate-conjugated polydopamine nanoparticles (PDA-ALN) that exhibited excellent photothermal effect and high affinity to hydroxyapatite. PDA/Fe-ALN significantly enhanced the magnetic resonance contrast of the bone tumors in vivo, suggesting that more PDA-ALN accumulated at the osteolytic bone lesions in the tumors compared with the non-targeting PDA. Chemodrug SN38 was efficiently loaded on PDA-ALN, and the drug release could be triggered by near-infrared irradiation and acidic stimulus. Finally, the combined chemo-photothermal therapy efficiently suppressed the growth of bone tumors and reduced the osteolytic damage of bones at a mild temperature around 43 oC. This study provides an efficient and robust nanotherapeutics for

ACCEPTED MANUSCRIPT the treatment of malignant bone tumors.

Keywords: malignant bone tumor, melanin-like nanoparticles, bone-targeted delivery, magnetic

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resonance imaging, chemo-photothermal therapy

1. Introduction

The treatment of malignant bone tumors including primary and metastatic ones is still a clinical challenge.

[1-3]

Osteosarcoma, accounts for ~60% primary malignant bone tumors, causes severe

focus.

[4]

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focal bone destruction, and the five-year survival rate is only 15-30% in patients with metastatic Bone metastases frequently occur in 65-80% of patients with metastatic breast and

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prostate cancers, [5, 6] and its incidence in other cancers is also high, [7] owing to the fertile soil of bone microenvironment and the ‘vicious cycle’ between tumor cells and osteoclast.

[5, 8-10]

Malignant bone tumors are also associated with severe skeletal events, manifesting as unbearable bone pain, hypercalcemia and pathological fracture, which contribute extensively to morbidity, mortality and cost in cancer patients. [11] The major clinical treatments of bone tumors including

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surgery, radiotherapy and chemotherapy usually fail in suppressing bone tumor progression. [12, 13] Additionally, the engagement with bone stromal cells induces tumor cell dormancy that promotes resistance to chemotherapeutic attack. [14] Other drugs like bisphosphonates and receptor activator

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of NF-κB ligand antibodies that effect on osteoclastogenesis significantly improve the therapeutic outcomes in the reduction of skeletal events, and have been a gold standard of care for patients with bone metastases. [15, 16] However, 30-50% of patients receiving the anti-osteolysis treatments

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still develop new bone metastases and malignant skeletal complications. [17] Nanomedicine designed with multiple functions including targeted delivery, contrast imaging and combination therapies significantly improves the therapeutic outcomes and reduces side effects, [18-20] which lights up hopes for the clinical treatment of malignant bone tumors. In light of the clinical features, the inhibition of both tumor and osteolysis progression is equally crucial to alleviate the pain in patients with bone tumors. As such, diverse bone-targeted nano-delivery systems were developed to deliver chemical drugs, anti-osteolysis agents and photothermal agents to bone tumors.

[21-28]

The improved therapeutic outcomes emphasize that the importance of

developing new therapies for optimizing the therapeutic efficacy of malignant bone tumors.

ACCEPTED MANUSCRIPT Chemo-photothermal therapy (CPT) has proven to be an optimal choice to improve the therapeutic efficacy of various tumors,

[29-33]

and possesses the incidental advantages that allow minimal

administration of photothermal agents and chemical drugs to reduce dose-related side effects, and meanwhile eradicates tumors at a mild temperature to minimize hyperthermia-induced tissue

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damage and inflammation. [29, 34-36] Herein, we reported a multifunctional melanin-like nanoparticle for bone-targeted chemo-photothermal treatment of malignant bone tumors (Scheme 1). The nanoparticle was an alendronate (ALN)-anchored polydopamine nanoparticle (PDA-ALN), which represented much

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higher affinity to hydroxyapatite and significantly enhanced accumulation at the osteolytic bone lesions compared with PDA. The ferric ion (Fe)-doped PDA-ALN (PDA/Fe-ALN) highly

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enhanced the T1-weighted magnetic resonance imaging (MRI) contrast in bone tumors, indicating a high efficient accumulation of the nanoparticles in bone tumors. The combined CPT associated by anticancer drug 7-ethyl-10-hydroxycamptothecin (SN38)-loaded PDA-ALN (PDA-ALN/SN38) was conducted at a mild temperature around 43 oC, and resulted in higher therapeutic efficiency than individual photothermal therapy (PTT) or chemotherapy. The in vivo results revealed that

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bone tumor and osteolysis were efficiently regressed by the combined therapy.

2. Materials and Methods

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2.1 Materials

Dopamine hydrochloride was purchased from Sigma-Aldrich (St. Louis, USA). ALN was obtained

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from Macklin Biochemical Co., Ltd. (Shanghai, China). Iron (III) chloride hexahydrate was obtained

from

Aladdin Reagent Co., Ltd. (Shanghai, China).

Tris

(2-amino-2-hydroxymethylpropane-1, 3-diol) was purchased from Maclin Biochemical Co., Ltd. (Shanghai, China). SN38 was bought from Aladdin Reagent Co., Ltd. (Shanghai, China). Dimethylsulfoxide (DMSO) was obtained from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). 2.2 Synthesis of PDA-ALN PDA was synthesized according to a previously reported method. [37] Typically, 8 mL ethanol and 18 mL deionized (DI) water were mixed in a water bath at 30 oC under mildly magnetic stirring.

ACCEPTED MANUSCRIPT 0.6 mL ammonia aqueous solution (28-30%) was added in the reaction solution, and then 2 mL dopamine hydrochloride (50 mg/mL) in DI water was added. 12 h later, the product was collected by centrifugation (15000 rpm, 15 min) and washed three times with DI water. The concentration of PDA was determined by weigh after lyophilization.

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PDA-ALN was prepared by simply mixing PDA with ALN at a mass ratio of 1:1 in alkaline buffer solution (pH = 8.5) under magnetic stirring for 24 h. The product was purified by centrifugation and washed three times with DI water. 2.3 Synthesis of Fe-doped PDA (PDA/Fe) and PDA /Fe-ALN [38]

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PDA/Fe was prepared according to a previously reported method.

Briefly, 45 mg of dopamine

hydrochloride and 6.2 mg iron (III) chloride hexahydrate were fully dissolved in 130 mL of DI under

magnetic

stirring

at

room

temperature

for

1

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water

h.

10

mL

of

Tris

(2-amino-2-hydroxymethylpropane-1, 3-diol) aqueous solution (45 mg/mL) was then quickly injected into the above solution. The reaction was allowed to proceed for 2 h. After that, the product was separated via centrifugation and washed three times with DI water. The Fe content of PDA/Fe was 5.3% detected by inductively coupled plasma mass spectrometry (ICP-MS).

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PDA/Fe-ALN was prepared by simply mixing PDA/Fe with ALN at a mass ratio of 1:1 in alkaline buffer solution (pH=8.5) under magnetic stirring for 24 h. The product was purified via centrifugation and washed three times with DI water.

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2.4 Characterization

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Transmission electron microscopy (TEM) images were taken using a Hitachi microscope (HT7700, Hitachi, Japan) operating at an acceleration voltage of 100 kV. The hydrodynamic diameters and zeta potentials were measured recorded with Zetasizer Nano ZS90 (Malvern, UK). The high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) images and the elemental mapping images were obtained using a JEOL electron microscope (JEM-2100, JEOL, Japan) operated at an accelerating voltage of 200 kV. Scanning electron microscopy (SEM) images were collected via a scanning electron microscope (S4800, Hitachi, Japan) operated at 10 kV. The ultraviolet-visible (UV-Vis) spectra were recorded by using a UV-Vis spectrometer (Cary60, Agilent Technologies, USA). The quantitative analysis of Fe in PDA and in tissues were conducted by ICP-MS (7500A, Thermo, USA).

ACCEPTED MANUSCRIPT 2.5 Photothermal effect In a typical assay, 1 mL PDA or PDA-ALN in aqueous solution at different concentrations (0, 50, 100, 150, 200 µg/mL) were held in a cuvette (1 × 1 cm in cross section), and then were irradiated by an 808-nm near-infrared (NIR) laser (MDL-III-808, Changchun New Industries

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Optoelectronics Technology Co., Ltd, China) at a power density of 3.6 W cm-2 for 10 min. The temperatures were recorded by using an infrared thermal camera (Magnity Electronics, China). 2.6 In vitro hydroxyapatite binding evaluation

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The binding affinity of PDA and PDA-ALN to hydroxyapatite was evaluated. The highly-crystallized hydroxyapatite tablets (diameter 10 mm, high 2.5 mm) were kindly gifted by

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Prof. Kaili Lin. The hydroxyapatite tablets were incubated with PDA or PDA-ALN (100 µg/mL, 150 µL, at equivalent PDA concentration) in DI water for 24 h. After that, the tables were collected, and then were washed with DI water for 30 min. After dried at room temperature, the tablets were characterized by SEM or irradiated by NIR laser (5.6 W cm-2, 5 min). To quantitatively analyze the bone-binding capability, PDA/Fe, PDA/Fe-ALN and SN38-loaded

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PDA/Fe-ALN (PDA/Fe-ALN/SN38) (100 µg/mL, 150 µL, at equivalent PDA/Fe concentration) incubated with hydroxyapatite tablets and bone fragments of mouse tibias in DI water or phosphate buffering saline (PBS) containing 10% fetal bovine serum (FBS) for 12 h. After that, the tablets were collected, and then were washed with DI water for 30 min. After dried at room

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temperature, the tablets were irradiated by an 808-nm NIR laser (5.6 W·cm-2, 5 min) and their thermographs and the temperature changes were recorded by using an infrared thermal imaging

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camera. After that, the tablets were dissolved by aqua regia, and the Fe content was determined by ICP-MS.

2.7 Drug loading of PDA-ALN PDA-ALN was suspended in a mix of water and DMSO (10:1, v/v). 5 mg SN38 was added in the PDA-ALN suspension (3 mg/mL, 3 mL), and then the solution was stirred at room temperature for 12 h. The un-dissolved SN38 was removed by centrifugation at a low speed (2000 rpm) for 5 min, and then the supernatant was collected and centrifuged at a high speed of 15000 rpm for 10 min to precipitate PDA-ALN/SN38. The un-dissolved SN38 and the SN38 in the supernatant were

ACCEPTED MANUSCRIPT collected in the drug loading assay, and the concentration of unloaded SN38 were determined by their typical absorption at 380 nm in the UV-Vis spectrum. The as-obtained PDA-ALN/SN38 was then washed three times with DI water. After that, the UV-Vis spectra of PDA-ALN/SN38 was recorded. The typical absorption at 380 nm for SN38 was used to determine the drug loading

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capacity of PDA-ALN (Figure S5). The absorbance of SN38 on PDA-ALN was acquired by deducting PDA-ALN absorbance from PDA-ALN/SN38. The drug loading ratios were calculated according to the formula of drug loading ratio = drug mass × 100% / (drug mass + PDA mass). The calculated drug loading ratios were 8.53% (by measuring unloaded SN38 in supernatant) and

methods were reasonable to calculate the drug loading ratio.

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2.8 Stability evaluation of PDA-ALN/SN38

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8.45% (by measuring SN38 on PDA-ALN/SN38), respectively. The result suggested that the two

The stability of PDA-ALN/SN38 (8.45% drug loading ratio) was assessed via incubating the particles with different media including PBS, cell culture medium, and PBS containing 50% FBS for 72 h. Further, the drug-retaining capability of PDA-ALN/SN38 was evaluated. At different time points, 0.1 mL of the above solution was collected for test. The solution was centrifuged at

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15000 rpm for 10 min to remove PDA-ALN/SN38, and then the supernatant was collected for SN38-releasing analysis by using high performance liquid chromatography (HPLC). The retained SN38 on PDA-ALN was calculated by subtracting the released SN38 from the original

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determination.

2.9 Stimuli-responsive drug release

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NIR light-triggered drug release was first evaluated. PDA-ALN/SN38 (80 µg/mL, 1 mL, 8.45% drug loading ratio) in DI water was held in a quartz cuvette, and was irradiated by an 808-nm NIR laser at a power density of 3.6 W·cm-2 for different times. After that, the supernatant was collected via centrifugation (15000 rpm, 10 min) and then analyzed by HPLC to determine the amount of released SN38. The temperature-dependent drug release from PDA-ALN/SN38 was also conducted. Typically, PDA-ALN/SN38 suspension (1 mL) in 1.5 mL centrifuge tubes (three groups, three tubes per group) were incubated with DI water at different temperatures (20, 37 and 43 ºC), and the released SN38 was measured at different time points by HPLC. To assess drug release of PDA-ALN/SN38 upon acidic pH stimulation, 1 mL PDA-ALN/SN38

ACCEPTED MANUSCRIPT was packaged in a dialysis bag (molecular weight cut-off = 3.5 kDa) and then immersed in 49 mL PBS. The pH value of PBS was adjusted to 7.4 and 5.0 for the pH-responsive release. 0.1 mL of the PBS solution was collected at different time points, and then was analyzed by HPLC to measure the concentration of released drugs. For each measurement, 0.1 mL fresh PBS was added

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into the PBS solution to keep the volume in constant. 2.10 Cell culture

NIH 3T3 cells (mouse embryonic fibroblast cell line, ATCC) were cultured in DMEM (GIBCO)

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containing 10% FBS, 100 units per mL penicillin and 100 µg/mL streptomycin at 37 ºC under 5% CO2. Mouse bone marrow mesenchymal stem cells (MSCs) were cultured in α-MEM (GIBCO) containing 10% FBS, 100 units per mL penicillin and 100 µg/mL streptomycin at 37 ºC under 5%

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CO2. MDA-MB-231 cells stably expressing luciferase (MDA-MB-231-Luc, a human breast carcinoma cell line, ATCC) were cultured in MEM (GIBCO) containing 10% FBS, 100 units per mL penicillin and 100 µg/mL streptomycin at 37 ºC under 5% CO2. PC-9 cells (a non-small-cell lung cancer cell line, ATCC) were cultured in RPMI 1640 medium (GIBCO), containing 10% FBS, 100 units per mL penicillin and 100 µg/mL streptomycin at 37 ºC under 5% CO2.

osteogenic genes

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2.11 Real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis of

The expression of alkaline phosphatase (ALP) and osterix (OSX) mRNA in the bone marrow was

analyzed

by

RT-PCR

using

ALP

specific

primers

(ALP-forward:

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MSCs

5’-AACCCAGACACAAGCATTCC-3’; ALP-reverse: 5’- GAGAGCGAAGGGTCAGTCAG-3’)

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and OSX specific primers (OSX-forward: 5’- ACTGGCTAGGTGGTGGTCAG-3’; OSX-reverse: 5’-GGTAGGGAGCTGGGTTAAGG-3’). Bone marrow MSCs (8000 cells per well) seeded in 96-well plate were incubated with PDA and PDA-ALN (60 µg/mL) for 7 days. Total RNA was isolated from the MSCs and reverse-transcribed into cDNA using a cDNA Synthesis Kit (TaKaRa, Dalian, China). The cDNA was subjected to RT-PCR analysis targeting ALP, OSX and 18S ribosomal RNA (18S rRNA) using a SYBR Green Real time PCR Master Mix (TaKaRa, Dalian, China). The data were normalized to 18S as the endogenous reference (18S-forward: 5’-GGACACGGACAGGATTGACA-3’; 18S-reverse: 5’-GACATCTAAGGGCATCACAG-3’), and relative to that of untreated cells.

ACCEPTED MANUSCRIPT 2.12 In vitro killing of cancer cells MDA-MB-231-Luc cells or PC-9 cells were seeded in 96-well plate with a density of 10000 cells per well and incubated overnight at 37 ºC. The photothermal-killing effect of PDA-ALN was first evaluated. PDA-ALN was added into wells at various concentrations (20, 40, 60, 80, 100 µg/mL),

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and then the cells were irradiated by an 808-nm NIR laser at 3.6 W cm-2 for 5 min. After an additional incubation for 24 h, the relative cell viabilities were determined by a standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

To evaluate the synergetic effect of CPT, the cells were incubated with SN38, PDA, PDA-ALN

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and PDA-ALN/SN38 (equivalent concentration for PDA and PDA-ALN = 60 µg/mL, equivalent SN38 concentration = 5.54 µg/mL), respectively. The cells were immediately irradiated by an

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808-nm NIR laser at 3.6 W cm-2 for 5 min. 2 h later, the culture media were replaced by fresh ones. After incubation for another 24 h, the cell viabilities were analyzed by a standard MTT assay. 2.13 MRI of bone tumor-bearing mice injected with PDA/Fe and PDA/Fe-ALN BALB/c nude mice (4 weeks old) with an average weight of 20 g were purchased from SLAC

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Laboratory Animal Co. Ltd. (Shanghai, China). The animal experiments were carried out according to the National Institutes of Health guidelines for care and use of laboratory animals and approved by the ethics committee of East China Normal University. The orthotopic bone-tumor model was established by injecting MDA-MB-231-Luc cells (2×105 in 20 µL PBS) in the cavum

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medullare of BALB/c nude mice tibias. The MR images were acquired on a Bruker 7.0 T magnet with Avance II hardware equipped with a 72 mm quadrature transmit/receive coil. The parameters

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for 7 T MRI are TR = 750.0 ms, TE = 12.6 ms, echo =1/1, FOV = 6.91/3.12 cm, slice thickness = 2 mm, nex = 2 mm, matrix= 256 × 116. Two mice with orthotopic bone tumor were intravenously injected with 200 µL PDA/Fe and PDA/Fe-ALN (30 mg/kg, both concentrations calculated by the mass of PDA/Fe, Fe content in PDA/Fe was 5.3%), respectively. The MR images were collected before and 24 h after injection. 2.14 Biodistribution of PDA/Fe and PDA/Fe-ALN The orthotopic bone-tumor model was established as above. After raising for two weeks, the mice were imaged by using an in vivo imaging system (Lumina- II, Caliper Life Sciences, USA), and

ACCEPTED MANUSCRIPT the ones with luminescence emission in the tumor regions were picked out for use. Mice bearing bone tumors in two groups (three mice in each group) were intravenously injected with PDA/Fe (30.00 mg/kg, the Fe content of PDA/Fe is 5.3%) and PDA/Fe-ALN (30.00 mg/kg of PDA/Fe concentration), respectively. The mice were sacrificed 24 h after injection, and the main organs

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and tissues including heart, liver, spleen, lungs, kidneys, tibias (tumor-bearing one and the healthy one) and tumor were collected. For ICP-MS analysis, the organs and tissues harvested from mice were weighted and milled. After that, the organs or tissues were digested by 2 mL of aqua regia. The digested samples were then diluted 100 times with 1% aqua regia. Fe contents were measured

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with ICP-MS. Quantification was carried out by external five-point calibration with internal standard correction. The amounts of PDA/Fe and PDA/Fe-ALN were finally normalized to the

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injection dose per gram (ID/g). 2.15 In vivo anticancer efficacy evaluation

The orthotopic bone-tumor model was established as above. After 2 weeks for tumor progression, the mice were divided into four groups with five mice in each group, and then were intravenously injected with 100 µL of PBS, PDA (30.00 mg/kg) and PDA-ALN (30.00 mg/kg of PDA

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concentration, two groups), respectively. The groups injected with PBS and PDA and one of the groups injected with PDA-ALN were treated with NIR irradiation (3.6 W cm-2, 5 min) at a time point of 24 and 48 h post-injection. The same administration and NIR irradiation were performed

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twice with an interval of four days. The tumor growth in the animals was monitored by luminescence imaging. The body weights of each mouse were recorded every day. The tumor-site

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temperature and the thermographs were recorded by an infrared thermal camera. For bone-targeted CPT, the tumor-bearing mice were divided into four groups with five mice in each group, and then were intravenously injected with 100 µL SN38 (2.77 mg/kg in a mix of Tween-80 and PBS (v/v = 1

49)), PDA-ALN (30.00 mg/kg) and PDA-ALN/SN38 (PDA-ALN:

30.00 mg/kg of PDA concentration; SN38: 2.77 mg/kg, 8.45% drug loading ratio, two groups), respectively. The group treated with PDA-ALN and one of the groups treated with PDA-ALN/SN38 were irradiated by an 808-nm NIR laser as above. The other two injections and NIR irradiations were also conducted every four days, and the detection and analysis of the mice were performed as described above.

ACCEPTED MANUSCRIPT 2.16 Ex vivo three-dimensional micro-computed tomography (3D micro-CT) reconstruction of the tumor-bearing tibia The tumor-bearing legs were amputated from the sacrificed mice and analyzed using a Siemens Biograph micro-CT device (Skyscan 1077, Antwerp, Belgium). After scanning, the 3D models

Belgium). 2.17 The

terminal

deoxyribonucleotidyl

transferse

(TdT)-mediated

biotin-16-dUTP

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nick-end labelling (TUNEL) staining assay

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were reconstructed and evaluated using the CTVox program (Bruker micro-CT NV, Antwerp,

Tumor tissues were fixed in 4% formalin solution at room temperature for 48 h, and then were embedded in paraffin and sectioned into 4 mm thick slices. The tumor sections were incubated

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with proteinase K, TUNEL reaction solution and Hoechest 33342 according to the standard protocol of the in situ apoptosis detection kit (Roche, Mannheim, Germany).The apoptotic cells were imaged by a fluorescence microscope.

3

Results and Discussion

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The bone-targeted nanomedicine was constructed by anchoring ALN on the surface of PDA followed with the loading of anticancer drug SN38 (Figure 1a). ALN was modified on the surface of PDA via Michael addition/Schiff base reaction, [39] and SN38 was loaded on PDA via π-π

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stacking. [40] The as-prepared PDA-ALN had a spherical shape and an average dimeter size of 105±16 nm revealed by TEM image (Figure 1b). In comparison with fresh PDA, no morphology

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change for PDA-ALN was detected (Figure 1b and S1). To verify the successful decoration of ALN on the surface of PDA, a HAADF-STEM image was taken for a single particle of PDA-ALN (Figure 1c), and the corresponding energy-dispersive X ray (EDX) mapping revealed that the element of oxygen (O, major from PDA) and the element of phosphorus (P, represents ALN) homogenously distributed over entire nanoparticle (Figure 1c). The hydrodynamic size of PDA was increased from 119 nm to 124 nm after the modification of ALN (Figure S2), and the zeta potential of PDA became more negative due to the additional phosphate groups (Figure S3). All these data demonstrated that ALN was successfully modified on the surface of PDA. The in vitro water-heating assay revealed that the photothermal conversion efficacy of PDA was not changed

ACCEPTED MANUSCRIPT after the modification of ALN (Figure S4). The drug loading capability of PDA-ALN was further evaluated. After the oxidative polymerization, there were abundant of phenyl groups on the surface of PDA, which could be utilized to load hydrophobic drug SN38 via π-π stacking. As shown in Figure 1d, SN38 had a typical absorption peak at 380 nm. A linear calibration curve of

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absorbance versus concentration was determined for SN38 (Figure S5), which was further used for the determination of SN38 loading on PDA-ALN. After incubation with SN38, the typical absorbance peak of SN38 emerged in the spectrum PDA-ALN (Figure 1d), which suggested that SN38 was loaded on PDA-ALN. The average diameter size of PDA-ALN/SN38 was measured to

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be 107±17 (Figure S6), which is slightly larger than PDA-ALN (Figure 1b). The hydrodynamic diameter of PDA-ALN/SN38 was also increased from 124 to 128 nm (Figure S2), and the zeta

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potential of PDA-ALN/SN38 was enhanced from -47 to -25 mV (Figure S3). The variation of hydrodynamic size and zeta potential of PDA-ALN should be attributed to the loading of SN38 on the particle surface. To determine whether PDA-ALN/SN38 was stable in the physiological condition, we incubated the particles with PBS, cell culture media, and PBS containing 50% FBS, respectively. No aggregates were observed in the suspension after 72 h incubation, and negligible

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drug release was detected during the incubation (Figure S7), all which suggested that PDA-ALN/SN38 was very stable at physiological condition. Furthermore, we demonstrated that the loaded SN38 could be released in response to the stimuli of NIR light and mild acidic pH.

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Upon NIR irradiation at 3.6 W·cm-2, the release of SN38 was time dependent, and after 30 min NIR irradiation, there was over 50% SN38 released from PDA-ALN (Figure 1e). The [41]

which led to the

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hyperthermia could disturb the π-π interaction between SN38 and PDA,

dissociation of SN38 from the nanoparticle. This mechanism was demonstrated by incubating PDA-ALN/SN38 at different temperatures, in which assay a higher temperature of 43 ºC more efficiently triggered the release of SN38 from PDA-ALN/SN38 (Figure S8). Moreover, a faster release of SN38 was observed in the case of incubating PDA-ALN/SN38 in PBS with a pH value of 5.4 (Figure S9). The cytotoxicity of PDA-ALN was tested on NIH3T3 cells and bone marrow MSCs. As showed in Figure 1f and S10, there was no detectable cytotoxicity observed in a large concentration range of 0-100 µg/mL PDA-ALN, indicating PDA-ALN was highly biocompatible. We also evaluated the mRNA expression level of ALP and OSX in bone marrow MSCs, which were essential for osteoblast differentiation and bone formation. [42,43] The data revealed that both

ACCEPTED MANUSCRIPT ALP and OSX had no obvious change in PDA-treated cells compared with the control cells, while in the PDA-ALN-treated cells, the expression of ALP and OSX was obviously higher than that in the control cells and PDA-ALN-treated ones (Figure S11). That should be attributed to functions [19]

of ALN on PDA-ALN.

Furthermore, we demonstrated that PDA-ALN associated PTT

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efficiently killed the MDA-MB-231-Luc and PC-9 cancer cells, while minimal cytotoxicity was detected for PDA-ALN without NIR irradiation (Figure S12a and b). After loading with SN38, it was observed that the combined CPT more efficiently killed the cancer cells in comparison with the individual therapies (Figure 1g and S12c).

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To characterize the potential bone-binding affinity of PDA-ALN, the highly-crystalized hydroxyapatite tablets were employed for the in vitro bone-targeting assay. Hydroxyapatite was [44]

The erosive bone surface was composed of

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the major inorganic components of bone.

highly-crystalized hydroxyapatite in contrast with the new forming bone interface that was major composed of amorphous hydroxyapatite. [45] Therefore the high crystalized hydroxyapatite tablet well mimics the erosive skeletal surface around bone tumors. The hydroxyapatite tablets were incubated with PDA and PDA-ALN for 24 h, respectively, and then were characterized by SEM.

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As shown in Figure 2b, PDA-ALN represented dramatically higher affinity to hydroxyapatite than PDA. Furthermore, the hydroxyapatite tablets were irradiated by a NIR laser. The result showed that the tables adsorbed with PDA-ALN were faster heated to a higher temperature than the one

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with PDA (Figure S13), which confirmed that there were much more PDA-ALN adsorbed on the surface of hydroxyapatite tablet. To quantitatively assess the bone binding capability, PDA/Fe, PDA/Fe-ALN and PDA/Fe-ALN/SN38 were further prepared according the previous reported

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method (Figure 2a). [38] The Fe content in PDA was 5.3%. The three nanoparticles were incubated with hydroxyapatite tablets and bone fragments of mouse tibias in DI water or PBS containing 10% FBS for 12 h, and the adsorbed nanoparticles were quantified via the analysis of Fe content by ICP-MS. As shown in Figure 2d and e, the amounts of PDA/Fe-ALN adsorbed on hydroxyapatite were ~11 times higher than these of PDA/Fe in both DI water and PBS containing 10% FBS, and the amounts of PDA/Fe-ALN adsorbed on bone fragments were ~9 times higher than these of PDA/Fe. The data suggested that PDA/Fe-ALN had a stronger bone-binding affinity than PDA/Fe, and the physiological solution had no observable influence over the bone-binding capability of PDA/Fe-ALN. In additionally, PDA/Fe-ALN/SN38 represented a similar bone-binding affinity

ACCEPTED MANUSCRIPT with PDA/Fe-ALN (Figure 2d and e), indicating that the loaded drug had minimal influence over the bone-binding affinity of the nanoparticles. Finally, the particle-adsorbed hydroxyapatite tablets were irradiated by NIR laser. The result showed that the temperature of PDA/Fe-ALN- and PDA/Fe-ALN/SN38-adsorbed hydroxyapatite tablets fast increased to 75 oC, while that of

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PDA/Fe-adsorbed ones just increased to 53 oC (Figure S14), which confirmed that PDA/Fe-ALN had a higher binding affinity to bone.

In order to evaluate the bone-targeting efficacy in vivo, PDA/Fe and PDA/Fe-ALN were used for MRI. To evaluate the MR contrast enhancement, the T1 relaxivity of PDA/Fe in aqueous

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solution at a 7.0 T MR system was determined. With the increased particle concentrations, we observed increased signals in T1-weighted MR images (Figure 3a), indicating that PDA/Fe acted

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as a positive contrast agent. The longitudinal relaxivity value (r1) was 5.74 mM-1s-1 (Figure S15). We then conducted the T1 modal MRI for PDA/Fe and PDA/Fe-ALN in bone tumor-bearing mice with a 7.0 T MR scanner. The T1-weighted MR images before and 24 h after intravenous injection of PDA/Fe and PDA/Fe-ALN were acquired. As shown in Figure 3b, an obviously brighter T1-weighted signal was observed in the tumor injected with PDA/Fe-ALN, while the T1-weighted

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signal in the tumor of mice injected with PDA/Fe were barely increased. The analysis of T1-weighted signal changes in tumor-associated bones suggested that there was an obvious intensity increase of 34% in the osteolytic bone lesions before and after injection of PDA/Fe-ALN

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(Figure 3c). The result suggested that PDA/Fe-ALN was able to act as a MRI contrast agent for bone tumors, and the significantly enhanced T1-weighted MRI signal in mice injected with

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PDA/Fe-ALN demonstrated that ALN significantly enhanced the bone-tumor-targeting efficacy of the nanoparticles. The quantitative analysis of Fe content in different organs and tissues was further conducted by using ICP-MS. The data revealed that PDA/Fe-ALN exhibited a much higher concentration than PDA/Fe in the tumor-bearing tibias due to the high affinity of ALN to bone tissue (Figure 3d).

[19]

The two nanoparticles had similar low concentrations in the tumor tissues

(Figure 3d), which because that they had no tumor cell-targeting moiety. The two nanoparticles also had similar low concentrations in the healthy tibias (Figure 3d), as ALN favorably recognized the high turnover sites in bone but not the moderate health bone tissue.

[7]

Taken together,

PDA/Fe-ALN represented a higher bone-tumor-targeting efficiency and was able to act as a MRI contrast agent.

ACCEPTED MANUSCRIPT We primarily evaluated the effect of PDA-ALN mediated PTT in an orthotopic bone tumor model. PDA and PDA-ALN were intravenously injected to bone tumor-bearing mice and irradiated by an 808-nm NIR laser (Figure 4a). The tumor-site temperature while NIR irradiation (3.6 W cm-2, 5 min) was recorded by an infrared camera. As shown in Figure 4b and c, the

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tumor-site temperature in the group injected with PDA-ALN increased to a final temperature of 43 C, while the one in the group injected with PDA only increased to 39 oC. The enhanced

tumor-site temperature indicated that PDA-ALN was more efficiently accumulated around the bone tumors in comparison with PDA. In most cases, a much higher therapeutic temperature was

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adopted for PTT of tumors (typically >50 oC), [37, 46, 47] under which the tumors were efficiently regressed but the hyperthermia also induced serious damage to health tissues and inflammations.

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Here, we precisely controlled the tumor-site temperature at a mild temperature around 43 oC. Such a temperature did not induce serious damage to the health tissues around the tumors.

[48, 49]

Meanwhile, at such a temperature, the tumors in PDA-ALN+NIR irradiation group were efficiently suppressed (Figure 4d and S16a). In contrast, the tumor growth in the PDA group was minimally reduced (Figure 4d and S16a). The quantitative analysis of tumor luminescence and

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tumor weight confirmed that tumors in the PDA-ALN group were more significantly suppressed compared with the ones in PDA group after PTT (Figure 4e, 4f and S16b). TUNEL staining assay revealed that much more tumor cells in the PDA-ALN plus NIR irradiation group were apoptotic

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than others (Figure S16c). The loss of body weight in the PBS, PDA-ALN only, and PDA plus NIR irradiation groups should be due to malignant tumor progression (Figure S16d). Furthermore,

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osteolysis in the bone tumor bearing mice was evaluated by 3D micro-CT. As shown in Figure 5a, comminuted fractures were observed in the proximal tibias of mice in all the control groups, while the erosion of tibias in the PDA-ALN group was efficiently inhibited after PTT. The 3D architecture parameters of the tibias including bone volume, bone surface, trabecular numbers (Tb. N.) and tibia space (Tb. Sp.) suggested that the microstructure of bone was well protected in the bone-targeted PTT group in comparison with the control groups (Figure 5b-e). Based on the above results, PDA-ALN mediated PTT could efficiently retard the growth of bone tumors. However, the luminescence images revealed that the luminescence intensity of bone-residing tumors in the PDA-ALN group was still increased five folds after PTT. This data suggested that PDA-ALN-mediated PTT could not completely suppress the tumor growth at a

ACCEPTED MANUSCRIPT mild temperature of 43 oC. If we enhance the therapeutic temperature, hyperthermia-induced side effects would emerge. Therefore a paradox between efficient tumor regression and minimal side effect is in front of us. To address this problem, we treated bone tumors by targeted CPT under the same temperature

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of 43 oC. PDA-ALN/SN38 was used to carry out the combined CPT, and PDA-ALN conducted PTT was tested as a control (Figure 6a). The final tumor-site temperature for NIR irradiated mice was controlled at 43 oC (Figure 6b and c), which was consistent with the temperature monitored for PDA-ALN treated mice in Figure 4c. The luminescence images of mice were recorded before

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and after the combined therapy. As shown in Figure 6d and 7a, the mice treated with SN38 or PDA-ALN/SN38 represented an obviously malignant progression associated with serious erosion

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of the tumor-site tibias. In the NIR irradiation groups, the tumors of mice treated with PDA-ALN were efficiently suppressed but still had a 6.6±1.7 fold increase in the luminescence intensity (Figure 6d and e), which was consistent with the data shown in Figure 4e. However, the combined CPT mediated by PDA-ALN/SN38 displayed a much more efficient regression of bone tumors. The relative luminescence intensity was only 1.5±0.8 fold in contrast with the ones before

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treatment (Figure 6d and e). A lung metastasis occurred in the SN38 and PDA-ALN/SN38 groups (Figure 6d), which indicated chemotherapy alone could not efficiently suppress the bone tumors at such a dose. The average tumor weight in bone-targeted CPT group was extremely smaller than

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that in the SN38 and PDA-ALN/SN38 groups, and was also obviously smaller than that in the PDA-ALN plus NIR irradiation group (Figure 6f and S17). Meanwhile, the 3D micro-CT images

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revealed that the tibias in bone-targeted CPT group well preserved the integrity of bone morphology in comparison with the ones in control groups (Figure 7a). The 3D architecture parameters such as bone volume, bone surface, Tb. N. and Tb. Sp. conformed that there was a significantly improved protection of bone microstructures in the bone-targeted CPT group (Figure 7b-e). The mice treated with bone-targeted CPT had minimal change in body weight, while the ones in control groups showed decreased body weight due to malignant tumor progression (Figure 6g). The TUNEL staining assay revealed that tumor cells in the PDA-ALN/SN38 plus NIR irradiation group were more apoptotic than others (Figure 6h). 4

Conclusion

In summary, we reported a bone-targeted chemo-photothermal treatment of malignant bone

ACCEPTED MANUSCRIPT tumors. The therapy was conducted by using multifunctional melanin-like nanoparticles. ALN significantly enhanced the affinity of PDA to hydroxyapatite and the accumulation of PDA at osteolytic bone lesions. PDA/Fe obviously enhanced the magnetic resonance contrast in bone tumors. The targeted CPT-mediated by PDA-ALN/SN38 efficiently suppressed the growth of bone

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tumors as well as tumor-induced osteolysis under a therapeutic temperature around 43 oC. This study suggested that bone-targeted CPT is a promising strategy for the treatment of malignant bone tumors. Ackknowledgements

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This work is supported by the National Natural Science Foundation of China (21725402 and 81671822), the National Key Research and Development Program of China (2016YFC0902100)

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the Fok Ying Tong Education Foundation (151036), and the Science and Technology Commission of Shanghai Municipality (17XD1401600).

Supplementary Data

References

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Supplementary data related to this article can be found at http://dx.doi.org

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Scheme 1. Illustration depicts PDA-ALN/SN38-associated bone-targeted chemo-photothermal treatment of malignant bone tumor.

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Figure 1. Characterizations of PDA-ALN and PDA-ALN/SN38. (a) Schematic depicts the modification of ALN on PDA and the loading of SN38 on PDA-ALN. (b) TEM image of PDA-ALN. (c) HAADF-STEM image of a single PDA-ALN and the corresponding elemental

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mapping of O (green) and P (red). (d) UV-Vis spectra revealed the loading of SN38 on PDA-ALN. (e) NIR irradiation triggered the release of SN38 from PDA-ALN/SN38. (f) The cytotoxicity of

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PDA-ALN on NIH3T3 cells. (g) Chemo-photothermal killing of MDA-MB-231-Luc cancer cells by using PDA-ALN/SN38. No significance (N.S.) and ***p < 0.001 analyzed by student’s t-test.

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Figure 2. In vitro bone-binding capability. (a) Preparation of PDA/Fe and PDA/Fe-ALN. (b and c) SEM images of PDA (b) and PDA-ALN (c) adsorbed hydroxyapatite tablets. The arrows

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indicate PDA and PDA-ALN. Insets are photographs of PDA and PDA-ALN adsorbed hydroxyapatite tablets. (d and e) The quantitatively analysis of PDA/Fe, PDA/Fe-ALN and PDA/Fe-ALN/SN38 adsorbed on the hydroxyapatite tablets (d) and bone fragments (e). The nanoparticles were incubated with hydroxyapatite tablets and bone fragments in DI water or PBS containing 10% FBS for 12 h.

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Figure 3. MR images and biodistribution of PDA/Fe and PDA/Fe-ALN. (a) T1-weighted MR images PDA/Fe at different concentrations in water. (b and c) T1-weighted in vivo MR images of bone tumor-bearing mice (b) and bone tumors (c) before and 24 h after intravenous injection of PDA/Fe and PDA/Fe-ALN. The tumors were circled by red dash lines in (b), and the

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tumor-associated bones by green dash lines in (c). (d) Biodistribution of PDA/Fe and PDA/Fe-ALN analyzed by ICP-MS 24 h after injection. N.S., **p < 0.01 and ***p < 0.001

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analyzed by student’s t-test.

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Figure 4. Bone-targeted photothermal treatment of bone tumors at a mild temperature. (a) Scheme illustrates the protocol for PDA-ALN administration and NIR irradiation. (b and c) Thermographs of mice (b, taken at the end of NIR irradiation) and the tumor-site temperature changes (c) recorded during NIR irradiation at 24 h post-injection. (d) Luminescence imaging of mice before and after PTT. (e) Relative luminescence intensities of bone tumors after PTT. (f)

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Average weights of the excised tumors. ***p < 0.001 analyzed by student’s t-test.

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Figure 5. Evaluation of bone tumor induced-osteolysis after PTT. (a) 3D micro-CT reconstruction

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of the tumor-bearing tibias after treatment. (b-e) Plots of the architecture parameters of bone including bone volume (b), bone surface (c), and Tb. N. (d), and Tb. S. (e). *p < 0.05, **p < 0.01

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and ***p < 0.001 analyzed by student’s t-test.

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Figure 6. Bone-targeted chemo-photothermal treatment of bone tumors at a mild temperature. (a) Schematic illustrates the protocol for PDA-ALN/SN38 administration and NIR irradiation. (b and c) Thermographs of mice (b, taken at the end of NIR irradiation) and the

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tumor-site temperature changes (c) recorded while NIR irradiation at 24 h post-injection. (d) Luminescence imaging of mice before and after CPT. (e) Relative luminescence intensities of

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bone tumors after CPT. (f) Average weight of the excised tumors. (g) Body weight changes during the therapeutic period. (h) Apoptosis (red) of tumor cells analyzed by a TUNEL assay. The cell nuclei were stained by Hoechst 33342. *p < 0.05 and ***p < 0.001 analyzed by student’s t-test.

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Figure 7. Evaluation of bone tumor induced-osteolysis after CPT. (a) 3D micro-CT

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reconstruction of the tumor-bearing tibias after treatment. (b-e) Plots of the architecture parameters of bone including bone volume (b), bone surface (c), and Tb. N. (d), and Tb. S. (e).

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N.S., *p < 0.05, **p < 0.01 and ***p < 0.001 analyzed by student’s t-test.