Gadolinium-Doped Hydroxyapatite Nanorods as T1 Contrast Agents

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Gadolinium-Doped Hydroxyapatite Nanorods as T1 Contrast Agents and Drug Carriers for Breast Cancer Therapy Ying Liu, Yuxia Tang, Ying Tian, Jiang Wu, Jing Sun, Zhaogang Teng, Shouju Wang, and Guangming Lu ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b02036 • Publication Date (Web): 24 Jan 2019 Downloaded from http://pubs.acs.org on January 25, 2019

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ACS Applied Nano Materials

Gadolinium-Doped Hydroxyapatite Nanorods as T1 Contrast Agents and Drug Carriers for Breast Cancer Therapy Ying Liu, Yuxia Tang, Ying Tian, Jiang Wu, Jing Sun, Zhaogang Teng*, Shouju Wang* and Guangming Lu* Department of Medical Imaging, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002, P.R. China. *Corresponding authors, E-mail addresses: [email protected] (Z. Teng), [email protected] (S. Wang) and [email protected] (G. Lu) Abstract Hydroxyapatite nanoparticles have drawn much attention in the development of multifunctional nanosystems because of their excellent biocompatibility. In the report, gadolinium-doped hydroxyapatite nanorods (NHA:Gd) were controlled synthesized through adjusting the amount of gadolinium. The NHA:Gd showed a high loading capacity for doxorubicin (118 μg per milligram) and a pH-responsive drug release ability. The r1 value of obtained doxorubicin (DOX)-loaded NHA:Gd (NHA:Gd-DOX) solution reached 0.0472 (μg/mL)−1s−1 and was higher than that of commercial T1 contrast Gd-DTPA (0.0314 (μg/mL)−1s−1). In vivo experiments demonstrated that NHA:Gd-DOX exhibited excellent MR imaging property. The results of therapeutic efficacy demonstrate that the tumor volume treated by NHA:Gd-DOX was significantly reduced compared to that treated by free DOX. In vivo toxicity experiments demonstrated no obvious damage to the major organs in NHA:Gd-DOX treated mice. Considering the excellent biocompatibility, T1WI contrast ability and enhanced therapeutic efficacy, these NHA:Gd-DOX have great potential for applications as MR T1 contrast agents and drug carriers for cancer therapy. Keywords hydroxyapatite nanoparticle, gadolinium, magnetic resonance imaging (MRI), drug carrier, breast 1

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cancer Introduction The development of nanosized carriers to deliver drugs to tumor sites is one of the most active fields of nanomedicine. [1-4] Particularly, the construction of drug carriers with excellent biocompatibility is crucial for the clinical translation of these nanosystems.[5-7] In recent years, hydroxyapatite (HA), as a bone substitute material, has been considered as the building block of nanosized drug carriers for its bioactivity, nontoxicity, and noninflammatory properties.[8-13] Furthermore, various dopants can be easily doped into the lattice of HA, endowing HA nanoparticles desired functionalities for medical applications such as bioimaging.[14-17] Magnetic resonance imaging (MRI) is very popular for high soft tissue contrast, deep penetration depth, non-ionizing radiation, and multiplanar reconstruction ability.[18-20] Nowadays only gadolinium chelates, such as Gd-DTPA, are approved for human use as T1 contrast agents. However, these gadolinium chelates may induce nephrogenic systemic fibrosis (NSF) presenting as cutaneous fibrosing impairment in a few days or months following the injection, which may cause severe renal failure in the future.[21-23] Our previous report has demonstrated that HA exhibits none detectable toxic effects in vivo for two months.[24] Therefore, HA nanoparticles may serve as good T1 contrast agents with high biocompatibility. However, to our knowledge, the full potential of gadolinium-doped HA nanoparticles in MRI and drug delivery was scarcely explored. In our previous report, gadolinium-doped HA was used only for SPECT and MR imaging , but none in vivo tumor therapy applications were carried out.[24] Chen et. al. reported the properties of delivering drugs and MRI of Eu3+/Gd3+ doped HA nanorods ex vivo , but these properties have not been studied in vivo.[25] Herein, the gadolinium-doped HA nanorods (NHA:Gd) are synthesized by liquid–solid–solution (LSS) method and loaded with doxorubicin (DOX). Gadolinium is critical in controlling the morphology and crystallinity of NHA:Gd. The r1 value of the obtained DOX-loaded NHA:Gd (NHA:Gd-DOX) is carefully assessed. Moreover, the 2


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drug release behavior and intracellular distribution of NHA:Gd-DOX is explored. Finally, the therapeutic efficacy and biocompatibility of NHA:Gd-DOX is studied in vitro and in vivo. Experimental Section Materials Gd2O3, sodium oleate and oleic acid were obtained from Sinopharm (Beijing, China). Gd2O3 was dissolved in hydrochloric solution under heating and the water was remomved by evaporating to obtain GdCl3. CaCl2, Na2HPO4·12H2O, cyclohexane, ethanol and dimethyl sulfoxide (DMSO) were purchased from Nanjing Chemical Reagent Co., Ltd. (Nanjing, China), polyethylenimine (PEI) with a molecular weight of 1800 and oleylamine were obtained from Aladdin (Shanghai, China). Injection dimeglumine gadopentetate (Gd-DTPA) were purchased from BeiLu Pharmaceutical Co., Ltd. (Beijing, China). All chemical materials were used directly without further purification. Dulbecco's modified eagle medium (DMEM), phosphate buffered solution (PBS), doxorubicin (DOX) and Methyl thiazolyl tetrazolium (MTT) were obtained from KeyGEN Biotech Co., Ltd. (Nanjing, China). Fetal bovine serum (FBS) was acquired from Gibco/Life Technologies (NY, USA). The human breast cancer cell line (MCF-7 cells) and normal human embryonic kidney cell line (HEK293 cells) were purchased from American Type Culture Collection. Deionized water (Millipore) was used in all experiments. Characterization A JEOL JEM-2100 transmission electron microscope (TEM) was used to capture the images of nanoparticles and energy dispersive X-ray (EDX) was analyzed. X-ray diffraction (XRD) was taken on a Bruker D8 Focus diffractometer (Copper Ka radiation, λ = 0.15406 nm). UV-Vis spectra were acquired with a PerkinElmer Lambda 35 UV-Vis spectrophotometer. Zeta potential and Dynamic Light Scattering (DLS) was measured by using a Brookhaven analyzer. Fourier transform infrared (FT-IR) spectra were performed by a Perkin-Elmer 580B IR spectrophotometer. Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) was used to measure the actual doping concentration of Gd3+ on a PerkinElmer Optima-5300DV spectrometer.

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Synthesis of NHA:Gd and NHA:Gd-DOX Firstly, 10 mL of 1 mmol of CaCl2 and GdCl3 aqueous solution was mixed with solution of 12.5 mmol sodium oleate, 12 mL ethanol, 4 mL oleylamine and 2 mL oleic acid under agitation for 30 min. Subsequently, 10 mL of 0.6 mmol Na2HPO4·12H2O aqueous solution was added drop by drop and the mixture was stirred for 30 min, then transferred to a 50 mL autoclave and heated at 160 oC for 8 h by liquid–solid–solution (LSS) hydrothermal method. The materials were centrifugated, washed with cyclohexane and ethanol, and finally dried in a vacuum. Afterward, 10 mg of the products (NHA:Gd) and 5 µg of PEI were mixed under ultrasonic vibration for 5 min and agitation for 2 h in 10 mL solution of water and ethanol, the PEI coated NHA:Gd (NHA:Gd-PEI) were centrifuged and washed. And the NHA:Gd-PEI was added to 0.5 mg/mL aqueous solution of doxorubicin (DOX). After shaking for 12 h in the dark, the obtained products (denoted as NHA:GdDOX) was centrifuged and washed with water for three times. MRI experiment in vitro The T1 map MRI were performed with a 3.0 T clinical MR scanner (MAGNETOM Trio, SIMENS, Germany). Various concentrations of NHA:Gd-DOX aqueous solutions were placed in 2 mL tubes for T1 map MR images. The parameters were as follows: repetition time (TR) = 15 ms, echo time (TE) = 1.73 ms. Average= 2, slice thickness= 2.0 mm, field of view (FOV) = 113 mm, Flip Angle = 5, 26. After acquiring the T1 map MR images, T1 values were recorded by drawing regions of interest (ROI) for each tube and used to calculate relaxation rates (R1). Cytotoxicity assay MCF-7 Cells were cultured in DMEM supplemented with 10% FBS at 37 oC with 5% CO2. Cytotoxicity of cells was detected using MTT assays. Cells were cultured in 96-well plates for 24 h and then 0.2-0.8 μg/mL DOX equivalent of free DOX, NHA:Gd-PEI (corresponding to concentrations of NHA:Gd-PEI in NHA:Gd-DOX), and NHA:Gd-DOX was added for 24 and 48 h. Then 100 μL of fresh DMEM containing 10 μL of MTT (5 mg/mL) in each well and incubated for 4 h. Then the supernatant was replaced with 150 μL of DMSO. Finally, the absorbance of the plate was 4


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recorded using a BioTek Winooski Vermont microplate reader at 570 and reference 630 nm. Cell viability (%) was calculatedas ((absorbance of test cells - absorbance of reference)/(absorbance of control - absorbance of reference) × 100. For the cytotoxicity experiment of HEK293 cells incubated with NHA:Gd-PEI, the same protocol was used as mentioned above. Cellular Uptake NHA:Gd-DOX or free DOX (1 μg/mL of DOX) was added to MCF-7 cells in 24-well plate for 2, 12, 48 and 72 h. For intracellular distribution observation, cells were washed with PBS for three times and stained with DAPI, and imaged by an Olympus FluoView FV1000 confocal fluorescence microscope. For flow cytometer analysis, the cells were digested and resuspended in PBS. The fluorescent signal of DOX was measured using Beckman Coulter CytoFLEX and analyzed by Flowjo 10. TEM and MRI of MCF-7 cells NHA:Gd-DOX (1 μg/mL DOX equivalent) was added to cells in 6-well palte for 2 h. The cells were washed with PBS. For TEM, cells were fixed with 2.5% glutaraldehyde in PBS and 1% OsO4 in PBS in order. Ultrathin sections were prepared with a LKB Ultracut E ultramicrotome and observed with JEOL JEM-2100 TEM. For MRI experiment, cells were digested with 0.05% trypsin-EDTA and then centrifugated and dispersed in 200 mL of 0.5% (w/v) agarose solution. MRI was performed on a 7.0 T Brucker PharmaScan Micro-MRI instrument. In vivo therapeutic efficacy Animal experiments accorded with the guidelines of the local animal ethics committee. About 5 × 106 of MCF-7 breast cancer cells suspended in PBS (100 μL) were injected into the subcutaneous dorsal of Balb/c mice (female, five-week-old). When the tumor volume was approximately 200 - 250 mm3, saline, NHA:Gd-PEI, free DOX and NHA:Gd-DOX were injected into mice (n = 5) through intratumoral injection at 1 mg /kg DOX equivalent. The tumor volume was calculated via a*b2/2, where a and b is the largest and smallest diameter, respectively. Tumor volume and body weight were recorded every four days for 28 days.

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In vivo MRI The mouse model was the same as the therapeutic experiment. When the tumor volume reached 250 - 300 mm3, NHA:Gd-DOX were injected into tumor. MRI was performed on a 7.0 T Brucker PharmaScan Micro-MRI instrument. Image J software was used to measure signal intensities of tumor area. In vivo toxicity Saline, NHA:Gd-PEI, free DOX and NHA:Gd-DOX were intravenously injected into five-week-old mice (n = 5) at 1 mg /kg DOX equivalent. After 28 days, the heart, liver, spleen, lung, and kidney were harvested and immersed in 4% paraformaldehyde solution. The specimen was embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) and then observed by a IX71 Olympus optical microscope. Results and Discussion NHA:Gd were synthesized using a hydrothermal method with Gd3+/Ca2+ molar ratio of 0.02. TEM images revealed the length of NHA:Gd was 44.3 ± 5.9 nm and the diameter of NHA:Gd was 11.3 ± 1.3 nm (Figure 1a, 1c and 1d). Figure 1b was the HR-TEM image of NHA:Gd and the lattice fringe was 0.34 nm, which was consistent with the (002) lattice planes in the hexagonal HA structure. The results indicated that NHA:Gd preferential grew in the (002) direction and along the c-axis direction. Figure 1e showed that the XRD patterns of NHA:Gd had characteristic diffraction peaks of hexagonal HA according to the standard data (JCPDS No. 09-0432). The EDXA spectrum showed that there was Ca, P, O, and Gd in NHA:Gd (Figure 1f), confirming the Gd ions doped in the lattice of HA. The accurate doping concentration of Gd ions was determined as 1.26 wt% by ICP-AES analysis.

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Figure 1 (a) Low and (b) high magnification of TEM images, distribution of (c) length and (d) diameter, (e) XRD pattern, (f) EDXA spectrum of NHA:Gd.

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Figure 2 TEM images of NHA:Gd synthesized with various molar ratios of Gd3+/Ca2+ in the prepare process: (a) 0, (b) 0.05, (c) 0.10 and (d) 0.20. (e) XRD patterns of NHA:Gd prepared with different molar ratios of Gd3+/Ca2+ from 0 to 0.20.

Then the effect of gadolinium ions on NHA:Gd morphology and crystallinity was investigated with different ranges of molar ratios of Gd3+/Ca2+ from 0 to 0.20. The results revealed that the aspect 8


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ratio of obtained nanorods increased from 4 to 40 and crystallinity decreased with the increasing of molar ratios of Gd3+/Ca2+ (0-0.10) (Figure 2a-c, e). But when the molar ratios of Gd3+/Ca2+ was further increased to 0.20, the particles were amorphous and none characteristic peaks of HA were observed (Figure 2d, e). Furthermore, when the molar ratios of Gd3+/Ca2+ were 0.05 (2.54 wt% by ICP) and 0.1 (4.95 wt% by ICP), the lattice parameter of NHA:Gd were a = b =9.425 Å, c =6.900 Å and a = b =9.424 Å, c =6.953 Å, respectively. Compared with the standard data (a = b = 9.418, c = 6.884Å), these parameters slightly deviated. It might because of the replacement of the Ca2+ by Gd3+ and the structure is not stable, resulting in a change in the ordering of the oxygen groups within columns.[2628] With the increase of the introducing Gd3+, the crystal was preferentially growth along c-axis direction. When the Gd3+/ Ca2+ was further increased to 0.2, the crystallinity structure was difficult to maintain and the obtained materials were amphorous. These results demonstrate that the gadolinium ions are critical in controlling the morphology and crystallinity of NHA:Gd. The preparation for the synthesis of NHA:Gd-DOX was illustrated in Scheme 1. For improve the dispersion of NHA:Gd, a little amount of PEI was modified on surface of NHA:Gd. The DLS of NHA:Gd increased from 56.4 ± 3.2 to 137.4 ± 6.9 nm after coating with PEI (Figure S1). The loading capacity of DOX in NHA:Gd-DOX was measured to be 118 µg per milligram NHA:Gd. The zeta potential of bare NHA:Gd increased from -29.66 ± 0.44 mV to 13.47 ± 0.99 mV after coating with PEI and further increased to 23.82 ± 1.62 mV after DOX loading, which confirmed the successful construction of DOX-loading NHA:Gd (Figure 3a). Due to that NHA:Gd was modified with oleic acid hydrophobic molecules, the DOX can be loaded in NHA:Gd-PEI via the hydrophobichydrophobic interactions between the hydrophobic DOX and the hydrophobic groups modified on the NHA:Gd. The FT-IR spectra of NHA:Gd, NHA:Gd-PEI and NHA:Gd-DOX were displayed in Figure 3b. There was a characteristic peak of OH group at 3590 cm–1 and the specific peaks of PO43– groups at 1090, 1060, 609 and 573 cm–1, demonstrating that HA was successfully synthesized. The specific peak at 1590 cm–1 belongs to N–H groups from primary and secondary amines of oleylamine and PEI (Figure 3b rectangle position). The typical band located at 1700 cm-1 belongs to C=O groups 9



of oleic acid on the NHA:Gd. The characteristic peaks at 1620-1690 cm-1 in the spectra of NHA:GdDOX were attributed to bending vibration of aromatic carboxylic acid of DOX [29], suggesting the successful conjugation of DOX to NHA:Gd. Similarly, there was the same absorbance peak at 498 nm as free DOX in the UV-Vis spectrum of NHA:Gd-DOX, indicating the existence of DOX in NHA:Gd-DOX (Figure S2). The DLS of NHA:Gd-DOX was 119, 121 and 122 nm in H2O, 137, 137 and 142 nm in PBS and 153, 160 and 167 nm in FBS for 0, 24 and 48 h, demonstrating the great stability of NHA:Gd-DOX (Figure S3). Cell uptake of NHA:Gd-DOX prepared at different molar ratios of Gd3+/Ca2+ was performed by flow cytometry analysis. The results demonstrated that NHA:Gd-DOX prepared at Gd3+/Ca2+ ratio of 0.02 had the highest amount of uptake. (Figure S4). Therefore, the Gd3+/Ca2+ ratio of 0.02 was used for further experiments. Scheme 1 Illustration of the process for synthesizing NHA:Gd-DOX.

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Figure 3 (a) zeta potential and (b) FT-IR spectra of NHA:Gd, NHA:Gd-PEI and NHA:Gd-DOX. 10


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MRI is an important clinical imaging modality for its high soft tissue contrast and deep penetration depth. To reveal the T1 contrast potential of NHA:Gd-DOX, the R1 value of NHA:Gd-DOX was accessed and compared to that of commercial T1 contrast Gd-DTPA. As shown in pseudocolor-coded T1 map MR images of NHA:Gd-DOX and Gd-DTPA (Figure 4a inset), with increasing concentration of NHA:Gd-DOX, the T1 values of solutions became shorter. At the same concentration of Gd, the T1 values of NHA: Gd-DOX were slightly shorter than that of Gd-DTPA. Figure 4a showed that the relaxation rate (R1) values of NHA:Gd-DOX and Gd-DTPA solution were linear related to its concentrations (R2=0.99). The calculated T1 relaxivity (r1) value of NHA:Gd-DOX solution were 0.0472 (μg/mL)−1 s−1 and was higher than commercial T1 contrast Gd-DTPA (0.0314 (μg/mL)−1 s−1). Additionally, T1 MRI of MCF-7 cells incubated with NHA:Gd-DOX was tested by a 7.0 T MRI scanner at different concentrations for 2 h and the results show that the MR images turned brighter with the increase of NHA:Gd-DOX concentrations (Figure S3). This result demonstrated that NHA:Gd-DOX have higher T1 contrast than Gd-DTPA, suggesting their further potential applications in MRI.

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Figure 4 (a) Relaxation rate R1 of NHA:Gd-DOX and Gd-DTPA determined by a 3.0 T MRI scanner. Inset: T1 map MRI of different concentrations of NHA:Gd-DOX and Gd-DTPA. Deionized water 11



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was the reference (b) MRI (above) and the color-mapped images (below) of mice post-injection and 15 min after injection of NHA:Gd-DOX at 10 mg/kg. Scale bar, 5 mm.

The MRI of tumor-bearing mice was conducted to determine the contrast enhance effect of NHA:Gd-DOX after intratumoral injection. Figure 4b showed a significant signal increase in the tumor region after injection of NHA:Gd-DOX on original and pseudocolored T1WI. The signal from the signal of tumor increased by 42 ± 2% after NHA:Gd-DOX injection. These results indicated that NHA:Gd-DOX can be used for MRI as T1-weighted contrast agents. To further investigate the drug delivery efficacy of NHA:Gd-DOX, the drug release behavior of NHA:Gd-DOX was tested. As shown in Figure 5b, DOX was released fast in the initial 4 hours after incubated in PBS at pH 5.5 and 7.4. It is worth noting to note that significantly more DOX was released at pH 5.5 than that at pH 7.4, even reached 80% at 10 days (Figure S6), probably due to the slight degradation surface of NHA:Gd at pH 5.5.[11, 30-32] And in the supernatant, no Gd3+ ion was detected by ICP, indicating that no obvious leakage of Gd3+ ion under different conditions. This pHresponsive release behavior is beneficial for drug delivery because of the low pH around tumors and in lysosomes.

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Figure 5 (a) CLSM images of MCF-7 cells treated with NHA:Gd-DOX for 2, 12, 48 and 72 h. Scale bar, 20 µm (b) DOX release profile of NHA:Gd-DOX at pH 7.4 and 5.5. (c) TEM images at different magnifications of MCF-7 cells treated with NHA:Gd-DOX. (d) MCF-7 cells viability treated with DOX, NHA:Gd-PEI, and NHA:Gd-DOX for 24 and 48 h.

The cellular uptake is important for delivering drugs of nanoparticles. To study the cellular uptake of NHA:Gd-DOX, the intracellular distribution of NHA:Gd-DOX was monitored dynamically up to 72 h after co-incubation with MCF-7 cells by CLSM. Figure 5a obviously showed that NHA:GdDOX was gradually accumulated in cell cytoplasm 12 h post incubation. Bright fluorescence signal from DOX and massive nuclear pyknosis could be observed in cell nucleus 48 and 72 h post incubation, indicating the successful translocation of DOX into cellular nuclei and cytotoxicity of NHA:Gd-DOX to MCF-7 cells. Cellular uptake was quantified by flow cytometry. The results 13



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showed the fluorescent intensity of DOX was doubled when the incubation time prolonged from 2 to 72 h (Figure S7). Furthermore, the effective cellular uptake of NHA:Gd-DOX was also observed by TEM (Figure 5c). The TEM images showed engulfed NHA:Gd-DOX distributed in the cytoplasm of MCF-7 cell after 2 h of incubation (arrow, Figure 5c). The therapeutic efficacy of NHA:Gd-DOX on MCF-7 cells for 24 and 48 h were detected by MTT assays. As shown in Figure 5d, the NHA:Gd-DOX showed obvious cytotoxicity at corresponding DOX concentration from 0.2 to 0.8 µg/mL. In contrast, both MCF-7 and normal human embryonic kidney cells (Figure S8) incubated with barely NHA:Gd-PEI exhibited viability higher than 80%, indicating great biocompatibility of NHA:Gd-PEI. It is also noted that at all concentrations, the therapeutic efficacy of NHA:Gd-DOX was significantly better than that of free DOX, which is probably due to the improved intracellular DOX delivery by NHA:Gd-DOX (Figure S9, S10). Next, the therapeutic efficacy of NHA:Gd-DOX in vivo was evaluated by studying the relative volume and H&E stained slices of tumors. Due to the tumor model in this study is breast cancer, which is a superficial tumor. It is easy to reach the tumor site by percutaneous puncture. Therefore, local treatment is very suitable for the treatment of this cancer. Also, intratumoral injection can increase of the accumulation of nano-drugs in the tumor site and alleviate systemic reactions. Therefore, intratumoral injection was chose to manipulate the nano-drugs and the biodistribution and pharmacokinetic of NHA:Gd-DOX was not necessary. Figure 6a indicated that the growth of tumors injected with NHA:Gd-DOX was remarkably inhibited in the following 28 days. The relative tumor volume treated by NHA:Gd-DOX was reduced by 28% than that treated by free DOX (p=0.0005), demonstrating the enhanced therapeutic efficacy of NHA:Gd-DOX in vivo. On the one hand, the improved efficacy might be more DOX was intracellularly delivered by NHA:Gd-DOX than that of the free DOX. Additionally, free DOX would diffused rapidly away from the tumor interstitium, while the NHA:Gd-DOX could retain in the tumor site due to their larger volume. The H&E stained tumor slices also exhibited massive areas of necrosis after treating with both free DOX and NHA:GdDOX, further confirming the cancer-killing effect of NHA:Gd-DOX (Figure 6b). These anti-tumor 14


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results of mice by intratumoral administration very clearly demonstrate the enhanced therapeutic efficacy and MR imaging capability of these nanocarriers. Additionally, the body weights of all mice were slightly increased, indicating that NHA:Gd-DOX has no obvious toxic effects (Figure S11).

Figure 6 (a) The tumor growth of mice intratumorally injected of saline, free DOX and NHA:GdDOX for 28 days. (***P < 0.001). (b) H&E staining of tumor slices of different groups at 28 days. Inset: images of excised tumors. I: saline, II: NHA:Gd-PEI, III: free DOX and IV: NHA:Gd-DOX.

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(c) H&E staining of major organs 28 days after intravenous injection of saline, NHA:Gd-PEI, free DOX and NHA:Gd-DOX. All scale bars, 50 µm.

In vivo biocompatibility is also an important issue for the translation of NHA:Gd-DOX. To address this, the organs of mice after treating of NHA:Gd-DOX were harvested and stained. Figure 6c revealed no obvious damage to the major organs in NHA:Gd-DOX treated mice when compared with those mice treated with free DOX and saline. This result indicated that the NHA:Gd-DOX would not induce apparent damage to major organs at least in four weeks. Conclusion In this report, we synthesize the gadolinium-doped hydroxyapatite nanorods via a hydrothermal method. Also, in this method, gadolinium ions is very important in controlling the aspect ratio of the gadolinium-doped hydroxyapatite nanorods. The obtained NHA:Gd show superior T1 contrast over commercial Gd-DTPA. After loading DOX, the NHA:Gd-DOX exhibit more enhanced therapeutic efficacy both in MCF-7 cells and in vivo than free DOX at the same concentration. Moreover, the NHA:Gd-DOX have minimal systemic toxicity in vivo. Overall, our data indicate the great potential of NHA:Gd-DOX as MR T1 contrast agents and drug carriers for cancer therapy. Acknowledgments We highly appreciate financial support from the National Natural Science Foundation of China (81501537, 81501588, 81601555, 81601556 and 8170071382), the National Science Foundation for Postdoctoral Scientists of China (2014T71012 and 2013M542576), the Jiangsu Planned Projects for Postdoctoral Research Funds (1301023A and 1601090C), and the Natural Science Foundation of Jiangsu Province (BK20160610). ASSOCIATED CONTENT

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Graphical abstract

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Gadolinium-Doped Hydroxyapatite Nanorods as T1 Contrast Agents

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