Osteotropic peptide-mediated bone targeting for photothermal treatment of bone tumors

Accepted Manuscript Osteotropic peptide-mediated bone targeting for photothermal treatment of bone tumors Yitong Wang, Jian Yang, Hongmei Liu, Xinyu Wang, Zhengjie Zhou, Quan Huang, Dianwen Song, Xiaopan Cai, Lin Li, Kaili Lin, Jianru Xiao, Peifeng Liu, Qiang Zhang, Yiyun Cheng PII:

S0142-9612(16)30617-2

DOI:

10.1016/j.biomaterials.2016.11.010

Reference:

JBMT 17802

To appear in:

Biomaterials

Received Date: 19 August 2016 Revised Date:

30 October 2016

Accepted Date: 8 November 2016

Please cite this article as: Wang Y, Yang J, Liu H, Wang X, Zhou Z, Huang Q, Song D, Cai X, Li L, Lin K, Xiao J, Liu P, Zhang Q, Cheng Y, Osteotropic peptide-mediated bone targeting for photothermal treatment of bone tumors, Biomaterials (2016), doi: 10.1016/j.biomaterials.2016.11.010. 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

Osteotropic Peptide-Mediated Bone Targeting for Photothermal Treatment of Bone Tumors Yitong Wang,a Jian Yang,b Hongmei Liu,d Xinyu Wang,a Zhengjie Zhou,a Quan Huang,b Dianwen Song,b Xiaopan Cai,b Lin Li,b Kaili Lin,c Jianru Xiao,b Peifeng Liu,d,e,* Qiang Zhanga,* and Yiyun Chenga,* a

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Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai, 200241, P.R. China. b Department of Orthopedic Oncology, Changzheng Hospital, the Second Military Medical University, Shanghai, 200003, P.R. China. c School & Hospital of Stomatology, Tongji University, Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Shanghai, 200072, P.R. China. d Central Laboratory, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, P.R. China e State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200032, P.R. China

E-mail: [email protected]; [email protected]; [email protected] Abstract: The treatment of bone tumors is a challenging problem due to the inefficient delivery of therapeutics to bone and the bone microenvironment-associated tumor resistance to chemo-and

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radiotherapy. Here, we developed a bone-targeted nanoparticle, aspartate octapeptide-modified dendritic platinum-copper alloy nanoparticle (Asp-DPCN), for photothermal therapy (PTT) of bone tumors. Asp-DPCN showed much higher affinities toward hydroxyapatite and bone

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fragments than the non-targeted DPCN in vitro. Furthermore, Asp-DPCN accumulated more efficiently around bone tumors in vivo, and resulted in a higher temperature in bone tumors during

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PTT. Finally, Asp-DPCN-mediated PTT not only efficiently depressed the tumor growth but also significantly reduced the osteoclastic bone destruction. Our study developed a promising therapeutic approach for the treatment of bone tumors.

Keywords: photothermal therapy, bone targeting, bone tumor, osteoclastic bone resorption

ACCEPTED MANUSCRIPT 1. Introduction Bone tumors are classified into primary tumors and metastatic ones. Primary bone tumors such as osteosarcoma, Ewing’s sarcoma and fibrosarcoma start in bone or cartilage are frequently diagnosed in children and adolescents.[1,2] Metastatic bone tumors begin elsewhere in the body and

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spread to the bone. Bone metastases occur in 65-80% of patients with advanced breast and prostate cancers, and are frequently found in lung, liver and kidney cancers.[3-6] The extended survival in cancer patients may lead to increased incidence of bone metastases.[7] During bone metastasis, cancer cells secret cytokines to activate the osteoclasts, resulting in increased bone

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resorption and secretion of growth factors from the bone matrix.[8] This causes severe skeletal-related events including pain, pathological fracture, hypercalcemia, bone deformity and

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spinal-cord compression.[9] These complications significantly reduce patients’ quality of life and increase mortality. On the other hand, bone marrow microenvironment provides a fertile soil for cancer cell recruitment, survival and outgrowth. It offers a protective niche for cancer cells to resist clinical treatments including chemotherapy and radiotherapy.[10-12] Excisional surgery may prolong the survival of patients with bone tumors, but the patients are more likely to experience a

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tumor recurrence due to the inadequate surgical margins. Therefore, novel and efficient therapeutic regimens are urgently needed in the clinical treatment of bone tumors. Photothermal therapy (PTT) is an effective strategy for cancer treatment.[13-15] It has been used to successfully ablate diverse tumors and even metastatic tumors.[16-19] However, PTT of bone

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tumors is rarely investigated,[20,21] and targeting nanoparticles to bone is still in its infancy. To date, three major kinds of bone-targeting moieties including bisphosphonate, oligopeptide and aptamer

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are developed for preferentially delivering therapeutic agents to bone.[22-25] Alendronate, a representative bisphosphonate, favorably binding to active bone remodeling sites (both bone resorption and formation surfaces) has been well used to specifically deliver drug-loaded nanoparticles to bone for tumor treatments. [8,26-30] An oligopeptide with six repetitive sequences of aspartate, serine and serine and an octapeptide with eight repeating sequences of aspartate (Asp8) are identified with the ability that preferentially bind to bone-formation and bone resorption surface, respectively.[24,31,32] In a recent study, osteoclast-targeting aptamer modified on lipid nanoparticles efficiently facilitate RNAi-based anabolic therapy. [25] Bone tumors are commonly associated with osteoclastic bone resorption. Hence, we used the

ACCEPTED MANUSCRIPT octapeptide Asp8 as a bone-targeting ligand to deliver photothermal agents to bone tissues for targeted PTT of bone tumors (Scheme 1). Dendritic platinum-copper alloy nanoparticles (DPCN), a near-infrared photothermal agent with high photothermal conversion efficiency, high drug loading capacity, and multimodal imaging modalities,

[33]

were used as the model photothermal

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agent in this study. The bone-targeting oligopeptide Asp8 was modified to the surface of DPCN via a cysteine linker (Asp-DPCN). DPCN modified with a non-targeting octapeptide with eight repeating sequences of glycine (Gly8) (Gly-DPCN) were used as the control material. The in vitro binding affinities of Asp-DPCN and Gly-DPCN to hydroxyapatite and ex vivo bone fragments

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were quantitatively determined. In addition, the in vivo targeted delivery of Asp-DPCN to bone tumors and the efficiency of Asp-DPCN in photothermal ablation of bone tumors were

2. Experimental section 2.1 Materials

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investigated.

Cupric chloride dihydrate (CuCl2·2H2O), hexachloplatinic (IV) acid hexahydrate (H2PtCl6·6H2O),

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potassium iodide (KI), poly (vinylpyrrolidone) (PVP, molecular weight = ~29000), ethylene glycol (EG) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cysteine-Asp8 and cysteine-Gly8 were synthesized by Top-peptide Biotechnology (Shanghai, China).

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2.2 Synthesis of Asp-DPCN and Gly-DPCN.

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DPCN were synthesized according to the previous reported method.[34,35] Briefly, 1 mL H2PtCl6·6H2O (20 mM) and 1 mL CuCl2·2H2O (20 mM) in aqueous solution were added in a Teflon liner under magnetic stirring. 0.025 mL aqueous KI (5 M) was added dropwise in the Teflon liner, and then 160 mg PVP (200 mg/mL) in aqueous solution was added. After that, 10 mL EG was injected in the reaction solution. The liner was sealed in a stainless vessel and heated in an oven at 140 ºC for 90 min. The vessel was then cooled down to the room temperature, and the sample was washed with ethanol/acetone (volume to volume ratio = 1:1) twice and deionized (DI) water twice via centrifugation. Asp-DPCN were prepared by simply mixing DPCN with cysteine-Asp8 at a platinum (Pt)-to-peptide molar ratio of 10:1 in DI water under magnetic stirring. After incubation for 24 h,

ACCEPTED MANUSCRIPT the Asp-DPCN were then isolated via centrifugation and washed three times with DI water. Gly-DPCN were synthesized following the same protocol for the preparation of Asp-DPCN. 2.3 Characterization

The transmission electron microscopy (TEM) images were taken using a transmission

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electron microscope (HT7700, HITACHI, Japan) operated at an accelerating voltage of 100 kV. The scanning electron microscopy (SEM) images were captured using a scanning electronic microscope (S-4800, Hitachi, Japan) operated at 10 kV. The ultraviolet-visible-near infrared (UV-Vis-NIR) spectra were recorded using a Cary 60 UV-Vis spectrophotometer (Agilent

Inductively coupled plasma mass spectrometer (ICP-MS) analysis was

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Technologies, USA).

conducted by using Neptune MC-ICP-MS (Thermo, USA). The hydrodynamic diameters and

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zeta potentials were measured by dynamic light scattering (Zetasizer Nano ZS90, Malvern Instruments, UK). X-ray photoelectron spectroscopy (XPS) measurements were carried out on a RBD upgraded PHI-5000C ESCA system (Perkin Elmer) with with Mg Kα radiation (hν=1253.6 eV) or Al Kα radiation (hν=1486.6 eV).

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2.4 Photothermal effect of Asp-DPCN.

Asp-DPCN (300 µM, 1 mL in DI water) in a cuvette (1 × 1 cm in cross section) was irradiated by an 808-nm NIR laser (MDL-III-808, Changchun New Industries Optoelectronics Technology Co.,

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Ltd, China) at a power density of 5.6 W·cm-2 for 10 min. The temperatures were recorded by using an infrared thermal camera (Magnity Electronics, China). Gly-DPCN were also tested

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following the same protocol, and DI water was used as control. 2.5 The affinity of Asp-DPCN toward hydroxyapatite and bone fragments. The affinity of Asp-DPCN and Gly-DPCN to hydroxyapatite was evaluated in vitro. The highly crystallized hydroxyapatite tablets were kindly gifted by Prof. Kaili Lin. Briefly, the highly crystallized hydroxyapatite tablets (diameter 10 mm, high 2.5mm) were incubated with 100 µM Asp-DPCN or Gly-DPCN for 24 h. The tablets were then rinsed with DI water for 30 min and dried at room temperature. The hydroxyapatite tablets were irradiated by an 808-nm NIR laser at a power density of 5.6 W·cm-2 for 5 min, and the thermographs and the temperature changes of the hydroxyapatite tablets were recorded by using an infrared thermal imaging camera (ThermoX,

ACCEPTED MANUSCRIPT China). After that, the hydroxyapatite tablets were dissolved by aqua regia, and the Pt content was determined by ICP-MS. Furthermore, the hydroxyapatite tablets adhered with Asp-DPCN and Gly-DPCN were also evaluated by SEM. The binding ability of Asp-DPCN and Gly-DPCN to ex vivo bone fragments was also tested.

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The tibias were isolated from sacrificed mice, and then were incubated with 100 µM Asp-DPCN or Gly-DPCN. After incubation for 24 h, the tibias were rinsed with DI water and dried at room temperature. Finally, the tibias were dissolved by aqua regia, and the Pt content was measured by ICP-MS.

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2.6 Cell culture.

MDA-MB-231 cells stably expressing luciferase (MDA-MB-231-luc, a human breast carcinoma

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cell line, ATCC) were cultured in MEM (GIBCO) containing 10% fetal bovine serum (FBS, Wisent), 100 units/mL penicillin and 100 µg/mL streptomycin at 37 ºC under 5% CO2. NIH3T3 cells (a mouse embryo fibroblast cell line, ATCC) were cultured in DMEM (GIBCO), containing 10% FBS, 100 units/mL penicillin and 100 µg/mL streptomycin at 37 ºC under 5% CO2.

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2.7 Cytotoxicity assay.

NIH3T3 cells were seeded in a 96-well plate at a density of 10000 cells per well. After incubation overnight at 37 ºC, the culture media were removed, and Asp-DPCN and Gly-DPCN in the fresh

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culture media were added at different concentrations, respectively. After incubation for another 24 h, the cell viabilities were determined by the standard 3-(4, 5-dimethylthiazol-2-yl)-2,

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5-diphenyltetrazolium bromide (MTT) assay. 2.8 In vitro photothermal killing of cancer cells MDA-MB-231 cells were seeded in 96-well plate at a density of 10000 cells per well and incubated overnight at 37 ºC. The culture media were removed, and then 100 µM Asp-DPCN, Gly-DPCN and DPCN in culture media were added in wells and incubated with MDA-MB-231 cells for 24 h. After that, the culture media were removed again, and the cells were washed twice with MEM to remove the untaken nanoparticles. The MDA-MB-231 cells were then irradiated with an 808-nm NIR laser (3.6 W·cm-2) for 5 min, and then the cell viabilities were determined by a standard MTT assay.

ACCEPTED MANUSCRIPT 2.9 In vivo biodistribution of Asp-DPCN and Gly-DPCN. Male BALB/c nude mice (4 weeks old) with average weight of 20 g were purchased from SLAC 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

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approved by the ethics committee of East China Normal University. MDA-MB-231-Luc cells (2×105 cells in 20 µL phosphate buffer solution (PBS)) were engrafted in the cavum medullare of nude mice tibias. After raising for two weeks, the mice were imaged by using an in vivo imaging system (Lumina- II, Caliper Life Sciences, USA), and the ones with luminescence emission in the

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tumor regions were picked out for use. Mice bearing bone tumors in two groups (three mice in each group) were intravenously injected with Asp-DPCN and Gly-DPCN (0.8 mg/kg, Pt mass), respectively.

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The mice were sacrificed 24 h after injection, and the main organs and tissues including heart, liver, spleen, lung, kidney, tibia (the one bearing tumor) 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 to dissolve the cells and DPCN. The digested samples were then diluted 100 time with 1% aqua regia. Pt contents were measured with ICP-MS (7500A, Thermo, USA).

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Quantification was carried out by external five-point calibration with internal standard correction. The amounts of Asp-DPCN and Gly-DPCN were finally normalized to the injection dose per gram (ID/g).

2.10 In vivo photothermal treatment of bone tumors.

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The animal model was established by injecting MDA-MB-231-Luc cells (2×105 cells in 20 µL PBS) in the cavum medullare of nude mice tibias. After raising for two weeks, the mice were

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imaged by using an in vivo imaging system, and the ones with luminescence emission in the tumor regions were divided into four groups (four mice in each group). Since the luminescence was emitted by the tumor cells, it is suggested that the bone tissue around the tumors was eroded in the mice emitting luminescence. The destroy of bone tissue also allowed the PTT of bone tumors. Each of the mice in three groups were intravenously injected with 100 µL of PBS, Gly-DPCN (0.8 mg/kg, Pt mass) and Asp-DPCN (0.8 mg/kg, Pt mass), respectively. The mice were then treated with NIR irradiation at a power density of 3.6 W·cm-2 for 10 min at a time point of 24 and 48 h post injection. The mice in the fourth group were intravenously administrated with Asp-DPCN (0.8 mg/kg, Pt mass) but were not treated with NIR irradiation. The second and the third round

ACCEPTED MANUSCRIPT injections were performed four and eight days after the first injection, respectively, and the same NIR irradiations were conducted. The luminescence imaging of the tumors was recorded before and after treatment by using an in vivo imaging system. The body weights of mice were recorded every day. The tumor-site temperatures and the thermographic images of mice were obtained

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using the infrared thermal camera. 2.11 In vivo three-dimensional (3D) micro-computed tomography (micro-CT) reconstruction of the tibias.

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The tumor-bearing hind legs were isolated from the sacrificed mice after PTT. The hind legs of mice were placed in a scanning holder, and analyzed using a Siemens Biograph 3D micro-CT device (Skyscan 1076, Antwerp, Belgium). After scanning, the 3D models were reconstructed and

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evaluated using the CTVox program (Bruker micro-CT NV, Antwerp, Belgium).

2.12 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining assay. The tumors in the four groups were harvested and processed with TUNEL staining. The tumor tissues were fixed in 4% formalin solution at room temperature for 48 h, and then were embedded

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in paraffin and sectioned into 4 mm thick slices. The tumor sections were incubated 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 in the

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sections were detected by using a fluorescence microscope.

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3. Results and Discussion

To acquire bone-targeted function, DPCN was modified with Asp8, which has high affinity to bone resorption surfaces,[24,31] and a non-targeting oligopeptide Gly8 was also modified on DPCN to be a negative control. The TEM images indicate that Asp-DPCN and Gly-DPCN had the same morphology with unmodified DPCN (Figure 1a). The elemental compositions of Asp-DPCN, Gly-DPCN and DPCN were analyzed by using XPS, and the result shows that the element sulfur was detected in Asp-DPCN and Gly-DPCN (Figure S1), which indicates the oligopeptides, Asp8 and Gly8, were successfully modified on the surface of DPCN. The dynamic light scattering analysis reveals that Asp-DPCN and Gly-DPCN had the similar hydrodynamic sizes and negative zeta potentials (Figure S2). The two nanoparticles also possessed the excellent biocompatibility in

ACCEPTED MANUSCRIPT a broad concentration range of 0-300 µM (Figure 1b, Pt molar concentration was used here and below for DPCN except statement.). DPCN possessed a continuous absorption in the visible to NIR region (Figure S3), and their mass extinction coefficient (Pt mass used instead of DPCN mass) was 6.267 g-1cm-1 (Figure S4). The photothermal conversion efficiency of DPCN was also

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calculated to be 37.51 % (Figure S5). Our previous investigation suggests that DPCN had an excellent photothermal effect.[33] Asp-DPCN and Gly-DPCN also had the same photothermal conversion efficiency (Figure S6) with DPCN, and could kill cancer cells efficiently (Figure 1c). These similar physicochemical characters of Asp-DPCN and Gly-DPCN were essential for

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comparing their targeting capability and therapeutic effect in vitro and in vivo. The colloid stability of Asp-DPCN and Gly-DPCN in different media including PBS, MEM, and 50% FBS in

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PBS were tested. The results suggest that both of the two nanoparticles were very stable in these media (Figure S7).

The affinities of Asp-DPCN and Gly-DPCN to bone were first assessed in vitro. The mineralized bone tissues are majorly composed of hydroxyapatite.[5] Asp8 had been reported to favorably bind to bone resorption surface,[24] which is characterized by highly crystallized

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hydroxyapatite.[33] Therefore, we first used the highly crystallized hydroxyapatite tablets as a substrate to compare the bone binding affinities of Asp-DPCN and Gly-DPCN. The hydroxyapatite tablet after incubation with Asp-DPCN represented a gray color while the one with

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Gly-DPCN was still white (Figure 1d and e insets), indicating more Asp-DPCN bound to the surface of hydroxyapatite. The SEM images show that there were much more Asp-DPCN than

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Gly-DPCN on the surface of hydroxyapatite tablets (Figure 1d and 1e). Furthermore, the amounts of Asp-DPCN and Gly-DPCN that absorbed on hydroxyapatite tablets were quantitatively determined via analyzing element Pt using ICP-MS. The result reveals that the amount of Asp-DPCN bound to hydroxyapatite was around 14 times over that of Gly-DPCN (Figure 1f). To evaluate the heating capability, Asp-DPCN and Gly-DPCN absorbed hydroxyapatite tablets were further irradiated by 808-nm NIR laser. As shown in Figure 1g, Asp-DPCN absorbed hydroxyapatite tablet was heated to a higher temperature of 76 oC compared with that of 53 oC for Gly-DPCN absorbed one. To practically identify the bone-binding capability of Asp-DPCN and Gly-DPCN in vitro, the bone fragments were isolated from mouse, and then were incubated with Asp-DPCN and Gly-DPCN, respectively. The ICP-MS analysis reveals that the amount of

ACCEPTED MANUSCRIPT Asp-DPCN bound to bone fragments was much larger than that of Gly-DPCN (Figure 1h). All these data suggest that Asp-DPCN had a higher affinity to bone compared with Gly-DPCN. The in vivo bone targeting efficiencies of Asp-DPCN and Gly-DPCN were also evaluated. Mice bearing orthotropic bone tumors were intravenously administrated with Asp-DPCN and

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Gly-DPCN, respectively (Figure 2a), and were then assessed for their biodistribution 24 h post-injection. The data reveal that the accumulation of Asp-DPCN was much higher than Gly-DPCN in the tibia of bone tumors (Figure 2b). However, there was no significant difference between the distributions of Asp-DPCN and Gly-DPCN in the tumor tissue and the main organs

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(Figure 2b). This result suggests that the oligopeptide Asp8 could more efficiently target DPCN to bone tumors.

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The photothermal treatment of bone tumors was further performed in mice orthotropic bone tumors. The mice in four groups were intravenously administrated with PBS, Asp-DPCN (two groups) and Gly-DPCN, respectively. The tumor-site temperature changes upon NIR irradiation at the time points of 24 and 48 h were recorded by using a thermal infrared camera. At the two time points, the mice administrated with Asp-DPCN represented a relative higher temperature on

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tumors compared with the one injected with Gly-DPCN (Figure 2c-e). In view of the similar physicochemical characters (hydrodynamic size and zeta potential) and the same photothermal conversion efficiency processed by Asp-DPCN and Gly-DPCN, the higher tumor-site temperature

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detected in the mice administrated with Asp-DPCN compared with the one injected with Gly-DPCN means that Asp-DPCN more efficiently accumulated in the tumors. The tumor-site temperature for mice administrated with Asp-DPCN finally increased to ~47 oC at 48 h (Figure 2

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e), which is proved to efficiently ablate diverse tumors.[14,36] The whole treatment of bone tumors involved three injections of different agents and two NIR irradiations after each injection (Figure 3a). The luminescence images suggest that mice before PTT had a similar luminescence intensity in tumors (Figure 3b). After PTT, the luminescence intensity of the tumors in mice injected with PBS followed by NIR irradiation (PBS+NIR group) was intensively increased (Figure 3b). The mice injected with Asp-DPCN (without NIR irradiation, Asp-DPCN group) had the similar luminescence intensity in tumor with the mice injected with PBS (Figure 3b and c), indicating Asp-DPCN, the particles themselves, had no influence over the tumor growth. The mice injected with Gly-DPCN followed by NIR irradiation

ACCEPTED MANUSCRIPT (Gly-DPCN+NIR group) represented a slightly weaker luminescence intensity in tumor compared with the mice in PBS+NIR group (Figure 3b and c), suggesting Gly-DPCN-mediated PTT could not effectively depress the tumor growth. However, in the group of mice treated with Asp-DPCN and NIR irradiation (Asp-DPCN+NIR group), the tumor luminescence intensity was significantly

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reduced compared with that of the tumors in the other three groups (Figure 3b and c), and also had no obvious increase compared with that of the tumors before PTT. All of these results suggest that Asp-DPCN-mediated PTT could significantly depress the bone tumor growth. After PTT, the hind leg of mouse in Asp-DPCN+NIR groups had no observable morphology change, while the ones in

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the other three groups swollen differently (Figure S8). All the bone tumors were further insolated and weighed. The excised tumors in Asp-DPCN+NIR group were much smaller than the ones in

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both PBS+NIR group and Asp-DPCN group, and were also smaller than the ones in Gly-DPCN+NIR group (Figure 3d and e). These results confirm that the bone tumor growth was significantly reduced by Asp-DPCN-mediated PTT. During the therapeutic period, the body weights of mice in four groups had no significant changes (Figure 3f), indicating Asp-DCPN and Gly-DPCN had negligible systematic toxicity. The TUNEL assay reveals that the tumor cells from

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Asp-DPCN+NIR groups were majorly apoptotic after PTT, while the ones in PBS+NIR, Asp-DPCN and Gly-DPCN+NIR groups were almost non-apoptotic (Figure 3g). Bone tumors are commonly associated with osteoclastic bone resorption, which causes a series

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of severe complications that threat the patients’ life quality and increase the mortality.[3,4,9] Therefore, inhibiting osteoclastic bone resorption in bone tumors is equally important with the

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depression of tumor growth. In this case, the skeletal morphologies and 3D architecture parameters of tumor-bearing tibias were carefully evaluated after PTT. The in vivo X-ray imaging was first conducted to roughly examine the bone morphologic changes in tumors, and the result indicates that the proximal tibias of mice in PBS+NIR group, Asp-DPCN group and Gly-DPCN+NIR groups were severely damaged, while the ones in the Asp-DPCN+NIR group were less destructed (Figure S9). Furthermore, the 3D micro-CT reconstructions of the tibias were processed. The comminuted fractures in proximal tibia were observed in mice from PBS+NIR, Asp-DPCN, and Gly-DPCN+NIR groups (Figure 4a). However, the tibias in mice from the Asp-DPCN+NIR group kept their shapes well, although some erosive lesions were observed (Figure 4a). These data suggest that Asp-DPCN-mediated PTT could effectively reduce the bone

ACCEPTED MANUSCRIPT resorption. The 3D architecture parameters of the tibias such as bone volume, bone surface, trabecular numbers (Tb. N) and tibia space (Tb. Sp) were also summarized. Compared with the control group (mice did not bear bone tumor), the parameters including bone volume, bone surface and Tb. N in PBS+NIR, Asp-DPCN and Gly-DPCN+NIR groups were significantly decreased,

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while these in the Asp-DPCN+NIR group were slightly reduced (Figure 4b-d). Conversely, Tb. Sp in the Asp-DPCN+NIR group increased a little and had a similar value with that of mouse in the control group, while it in PBS+NIR, Asp-DPCN and Gly-DPCN+NIR groups increased obviously (Figure 4e). These data confirm that Asp-DPCN-mediated PTT could well protect the skeletal

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microstructure of bone from damage in bone tumors. The 3D transverse sections and profiles of the tibias given out more precise details of bone destructions. The tibias in PBS+NIR, Asp-DPCN

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and Gly-DPCN+NIR groups were severely destructed and had large pieces of bone fragments corroded, while the tibias in the Asp-DPCN+NIR group maintained their integrity in shape and only had some erosive holes in bone walls (Figure 4f and g). Overall, the investigation reveals that Asp-DPCN-mediated PTT could not only ablate bone tumors but also well protect the bones from

4. Conclusion

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osteoclastic destruction.

In summary, we developed bone-targeted photothermal nanoparticles for targeted PTT of bone

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tumors. DPCN as a representative photothermal agent, were modified with Asp8 for bone-targeted delivery. The in vitro studies reveal that Asp-DPCN had a higher affinity to hydroxyapatite and ex

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vivo bone fragments compared with the non-targeted Gly-DPCN. The in vivo data demonstrate that Asp-DPCN accumulated in bone tumors more efficiently than Gly-DPCN, resulting in increased temperature for PTT. Finally, the tumor growth and bone resorption were both reduced efficiently by Asp-DPCN-mediated PTT. Our study demonstrates that targeting delivery of photothermal agents to bone tumor is feasible, and thus provides a new and efficient strategy for the treatment of bone tumors.

Acknowledgements This work was supported by the National Key Research and Development Program of China

ACCEPTED MANUSCRIPT (2016YFC0902100), the National Natural Science Foundation of China (Grant No. 81671822), the Basic Research Program of Science and Technology Commission of Shanghai Municipality (14JC1491100), the Fok Ying Tong Education Foundation (No. 151036), and the Shanghai Pujiang Program (Grant No.14PJD016). We would also like to acknowledge the electron

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microscopy center of East China Normal University for sample characterization.

Supplementary Data

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Further data on XPS spectra of DPCN, Asp-DPCN and Gly-DPCN. Hydrodynamic sizes and Zeta-potentials of DPCN, Asp-DPCN and Gly-DPCN. The temperature changes of DPCN, Asp-DPCN and Gly-DPCN. Photographs of the hind legs of tumor-bearing mice after different

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treatments. X-ray images of the mice tibias bearing bone tumors after different treatments.

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Scheme 1. Illustration of bone-targeted PTT of bone tumors. (a) Synthesis of bone-targeted

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Asp-DPCN. (b) Targeting delivery of Asp-DPCN to bone for PTT of bone tumors.

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Figure 1. Particle characterization and in vitro bone targeting evaluation. (a) TEM images of DPCN, Asp-DPCN and Gly-DPCN. (b) Cell viability of NIH3T3 cells after incubation with

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Asp-DPCN and Gly-DPCN at different concentrations for 24 h. (c) Photothermal killing of cancer cells (MDA-MB231) by using DPCN, Asp-DPCN and Gly-DPCN. (d and e) SEM images of Asp-DPCN (d) and Gly-DPCN (e) bound hydroxyapatite tablets. The arrows indicate Asp-DPCN and Gly-DPCN. Insets are photographs of Asp-DPCN and Gly-DPCN absorbed hydroxyapatite

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tablets. (f) Asp-DPCN and Gly-DPCN adhered on hydroxyapatite tablets were quantitatively analyzed by ICP-MS. (g) Temperature changes of Asp-DPCN and Gly-DPCN absorbed

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hydroxyapatite tablets while NIR irradiation. Insets are thermographs of hydroxyapatite tablets taken at the end of NIR irradiation. (h) Asp-DPCN and Gly-DPCN absorbed on bone fragments of mouse’s tibias were quantitatively determined by ICP-MS. No significance difference (N.S.), **p<0.01 and ***p<0.001 analyzed by student’s t-test.

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Figure 2. Temperature evolution in bone tumor while PTT. (a) Schematic represents the mice

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intravenously administrated with Asp-DPCN followed with NIR irradiation. (b) In vivo biodistribution of Asp-DPCN and Gly-DPCN 24 h post-injection. No significance difference (N.S.),

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**p<0.01 analyzed by student’s t-test. (c) Thermographs of mice taken at the end of NIR irradiation

at 48 h post-injection. (d and e) The tumor-site temperature changes recorded while NIR irradiation at 24 (d) and 48 h (e) post-injection. The mice were irradiated by NIR laser at a power density of 3.6 W·cm-2 for 10 min at each time point.

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Figure 3. In vivo bone-targeted PTT of bone tumors. (a) The experimental timeline of PTT. (b) Luminescence imaging of mice before and after PTT. (c) Relative luminescence intensities (Ratio of luminescence intensities after and before PTT) of bone tumors after PTT. (d) Photographs of bone tumors excised from mice. (e) Average weight of the excised tumors. (f) The body weight changes of mice during PTT. (g) Apoptosis (red) of tumor cells after PTT analyzed by a TUNEL assay. The cell nuclei were stained by Hoechst 33342. **p<0.01 and ***p<0.001 analyzed by student’s t-test.

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Figure 4. The evaluation of bone destruction. (a) 3D micro-CT reconstruction of the tibias.

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(b-e) Plots of the architecture parameters of bone (bone volume (b), bone surface (c), and Tb. N (d), and Tb. S (e). (f and g) The 3D transverse sections (f) and profiles (g) of tibia fragments from

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different groups. **p<0.01 and ***p<0.001 analyzed by student’s t-test.