In Vivo Evaluation of Reduction-Responsive Alendronate-Hyaluronan

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In vivo evaluation of Reduction-Responsive AlendronateHyaluronan-Curcumin Polymer-Drug Conjugates for Targeted Therapy of Bone Metastatic Breast Cancer Kaili Wang, Chunjing Guo, Xue Dong, Yueming Yu, Bingjie Wang, Wanhui Liu, and Daquan Chen Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00266 • Publication Date (Web): 24 May 2018 Downloaded from http://pubs.acs.org on May 24, 2018

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Molecular Pharmaceutics

In vivo evaluation of Reduction-Responsive Alendronate-Hyaluronan-Curcumin Polymer-Drug Conjugates for Targeted Therapy of Bone Metastatic Breast Cancer Kaili Wang, Chunjing Guo, Xue Dong, Yueming Yu, Bingjie Wang, Wanhui Liu, Daquan Chen* Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, School of Pharmacy,Yantai University, Yantai 264005, P. R. China;

KEYWORDS: Reduction-Responsive, CD44 Receptor, Polymer-Drug, Drug Delivery, Bone Tumor Therapy

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ABSTRACT Many cancers such as human breast cancer and lung cancer easily metastasize to bones, leading to the formation of secondary tumors in advanced stage. Based on the CD44targeted effect of oHA and bone-targeted effect of ALN, we prepared a reduction-responsive, CD44

receptor-targeting and bone-targeting nano-micelle, called CUR-loaded ALN-oHA-S-S-CUR micelles. In this study, we aimed to evaluate the antitumor activity and bone-targeting ability of CUR-loaded ALN-oHA-S-S-CUR micelles. The in vivo experiment results showed that a larger number of micelles was gathered in the bone metastatic tumor tissue and reduced the bone destruction. The CUR loaded ALN-oHA-S-S-CUR micelles markedly inhibited the tumor growth. So the CUR-loaded ALN-oHA-S-S-CUR micelles constitute a promising drug delivery system for bone tumor therapy.

Introduction Curcumin (CUR) is an anticancer drug obtained from the Curcuma longa plant. However, despite its reported effectiveness, the low aqueous solubility and poor absorption of CUR considerably limit its application in cancer treatment1, 2. In this regard, polymer-drug conjugates, consisting of water-soluble and biocompatible polymers, can enhance the solubility and stability of hydrophobic drugs3. Polysaccharides are hydrophilic materials characterized by good biocompatibility4. Therefore, using a polysaccharide to form polymer-drug conjugates can be a promising method to enhance the solubility of a hydrophobic drug. Oligosaccharide of hyaluronan (oHA) is a non-toxic, biocompatible hydrophilic polysaccharide5. This kind of oHA


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conjugates possess CD44-targeted effect and can target the CD44-overexpressing tumor cells with high tumor targeting capability6. Given its specific binding to CD44 receptors that are overexpressed in tumor cells, many reports have focused on the capacity of oHA to target rumor cells6. In the present study, we used oHA as a hydrophilic material to be combined with CUR in order to obtain hydrophilic polymer-drug conjugates and enhance CUR solubility in water. The physiological microenvironment of the bone is particularly suitable for cancer cell adhesion and proliferation7, 8. Therefore, many cancers, such as human breast or lung cancer, easily metastasize to bone, leading to the formation of secondary bone tumors9. Alendronate (ALN) is a new bisphosphonate drug that is mainly used in the treatment of osteoporosis and metastatic bone tumors10. This drug can bind calcium and accumulate in the bone at a high concentration11. Therefore, a nanodrug modified with ALN can reasonably be expected to be delivered to the bone, kill tumor cells and enhance bone growth. As an antitumor agent, ALN can also improve the outcome of tumor treatment. Accordingly, numerous studies have sought to enhance the accumulation of antitumor drug in the bone. For instance, a study using ALN as a bone-targeting ligand to modify DOX-hyd-PEG has demonstrated that the DOX-hyd-PEG-ALN micelles enhance the accumulation of the drug in bone12. Furthermore, the PEG-Paclitaxel micelles modified with ALN (PEG-Paclitaxel-ALN) micelles prevent tumor metastasis to the bone13. Based on the findings outlined above, our goal was to design a system that could codeliver ALN and CUR to achieve two effects: enhance the antitumor activity of the drug and prevent tumor metastasis to the bone. A tumor microenvironment-responsive drug delivery system could minimalize undesired toxicity and achieve the maximum desired effect14, 15. The concentration of GSH in tumor cells is higher than in normal cells16. Therefore, to enhance the tumor targeting ability of the drug


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delivery system, we took advantage of the tumor microenvironment and designed a redoxresponsive system with a disulfide bond17. Specifically, the disulfide bond was used as a connection arm that combines the hydrophobic CUR with hydrophilic oHA and ALN to serve as the amphiphilic material capable of self-assembling into micelles in water. Upon entering into tumor cells, the micelles could break down as a result of the disruption of the disulfide bond in the presence of high GSH concentration, thereby causing the drug release. In order to enhance the treatment effect of curcumin (CUR) on bone metastases, the CURloaded ALN-oHA-S-S-CUR micelles were prepared. We hypothesized that ALN-oHA-S-S-CUR material would self-assemble into CUR-loaded micelles (see Fig. 1A) first. Then, due to the ability of ALN to bind to hydroxyapatite, the micelles would accumulate in bone tissue, and deliver the drug to tumor cells by CD44 receptor targeting. Finally, the release of the drug would be caused by the breakage of the disulfide bond under the reducing environment (see Fig. 1B). The drug release, cellular uptake, cytotoxicity and penetration of CUR-loaded ALN-oHA-S-SCUR micelles had been studied as the previously reported18. So, in this study, we aimed to perform in vivo analysis of the antitumor effect, bone-targeting ability and distribution of CURloaded ALN-oHA-S-S-CUR micelles.

2. Material and Methods 2.1 Material Oligosaccharide of hyaluronan (oHA) (molecular weight [MW] < 10kDa) was purchased from Shandong Freda Co. Ltd (Shandong, China). Curcumin (CUR) was obtained from Aladdin Reagent Net (Shanghai, China).

Alendronate (ALN) was purchased from Sigma-Aldrich

(Shanghai, China). 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indotricarbocyanine Iodide (DiR) were


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obtained from Saiersi Biotechnology Co. Ltd (Shangdong, Yantai, China). H＆E were purchased from Bioworld Technology Co. Ltd. (Nanjing, China). All other reagents and solvents were of chemical grade. Deionized (DI) water used was used in all experiments. Human breast cancer cells (MDA-MB-231) were provided by the Experimental Medicine Center, at the Affiliated Hospital of Southwest Medical University (Luzhou, China). Nude mice weighing 14–18 g (3–4 weeks) were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. 2.2 The preparation of ALN-oHA-S-S-CUR materials The ALN-oHA-S-S-CUR materials was synthesized via a series of esterification reaction as previously reported18. And its structure was verified by 1H-NMR spectra. The ALN-oHA-S-SCUR materials were dissolved in D2O:DMSO-D6 (1:1, v/v) for further measurements. 2.3 The preparation of CUR-loaded ALN-oHA-S-S-CUR micelles The amphiphilic materials ALN-oHA-S-S-CUR could successfully self-assemble to form micelles in water. In this study, the CUR-loded ALN-oHA-S-S-CUR micelles was prepared by a dialysis method. The size and morphology of CUR-loaded ALN-oHA-S-S-CUR micelles was measured by Beckman Coulter Particle Analyzer and transmission electron microscope (TEM); the concentration of CUR in micelles was measured by HPLC as previously reported. 2.4 In vivo antitumor activity The CUR-loaded ALN-oHA-S-S-CUR micelles was prepared according to the previous study. The antitumor activity of CUR-loaded ALN-oHA-S-S-CUR micelles in vivo was evaluated in a xenograft model employing nude mice19. The MDA-MB-231 cells (1×108 cells/mice) were injected into the medullar channel of the right tibia of the tumor-bearing mice model by puncture method to induce the bone metastases. After 12 days, the mice were randomized in four groups


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and treated with saline, free CUR (25 mg/kg), CUR-loaded oHA-CUR micelles (25 mg/kg CUR), and CUR-loaded ALN-oHA-S-S-CUR (25 mg/kg CUR) micelles by a tail vein injection. Administration of drugs or saline was performed every other day for 20 days. The tumor volume of nude mice was measured during the administration. The tumor volume was calculated using Eq. (3). Tumor volume = Length × Width2/2

(3)

In order to further evaluate the antitumor activity of CUR-loaded ALN-oHA-S-S-CUR micelles. We also observed the inhibition effect about metastatic bone tumors. The model of metastatic bone tumors was obtained by the tibia injection methods11. 2.5 Histopathological analysis After treatment, the tumors were collected, fixed with formalin, and embedded in paraffin. Sections, 3 µm thick, were obtained and stained with hematoxylin and eosin (H & E)20-22. The specimens were examined using an inverted fluorescence microscope. 2.6 In vivo biodistribution The human breast cancer (MDA-MB-231) cells (1×106 cells/animal) were injected into the right tibia of nude mice to induce metastatic bone tumors21. In order to observe the distribution of the micelles, the tumor-bearing mice were randomly divided into three groups. The free DiR, DiR-loaded oHA-CUR micelles and DiR-loaded ALN-oHA-S-S-CUR micelles (DiR:500 µg/mL) were injected using the tail vein method. The distribution of the micelles was observed using the IVIS Lumina II in vivo imaging system at different time points (1 h, 2 h, 4 h, 8 h, and 12 h). The organs including the heart, liver, spleen, lung, kidney, and tibia were collected at 8 h, washed with PBS, and their fluorescence intensity was measured. Furthermore, the three-dimensional (3D) micro-computed tomography (micro-CT) was used


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to observe the destruction of bone tissue in the leg23. The bone was dissected from tumor-bearing mice, placed into 10% formalin for 48 h, and analyzed using 3D micro-CT technology. 2.7 Statistical analyses All data are reported as a mean ± standard deviation (SD) from at least three independent measurements conducted at three different times. Quantitative data were presented as the mean ± standard deviation (SD). The statistical comparisons between different groups were analysed by the Student's t-test or one-way analysis of variance (ANOVA) at confidence levels of 95 and 99%. The differences were considered to be statistically significant when the P values were less than 0.05 (P < 0.05). Results and Discussion 3.1 The characterization of ALN-oHA-S-S-CUR materials The 1H-NMR spectra of ALN-oHA-S-S-CUR materials was shown in Fig. 2A. The characteristic peak of CUR in ALN-oHA-S-S-CUR was observed in the region between 6.5-8 ppm. The appearance of the signal peak at 7.8 ppm verified the presence of the amide bond (-CONH-) connection between ALN and oHA. In addition, the -CH2- proton peak of 3,3-dithiodipropionic acid was observed at 2.8 ppm. These results indicate that the amphiphilic material, ALN-oHA-SS-CUR, was successfully synthesized. 3.2 The characterization of CUR-loaded ALN-oHA-S-S-CUR micelles As shown in Fig. 2B and C. The particle size of CUR-loaded ALN-oHA-S-S-CUR micelles was 180 nm measured by Beckman Coulter Particle Analyzer at room temperature. But the average size of the CUR-loaded ALN-oHA-S-S-CUR micelles was 80 nm observed by TEM. This difference could have resulted from the dehydration of the micelles and their contraction before being analyzed by TEM. All these results were consistent with previously reported.


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3.3 In vivo antitumor activity of ALN-oHA-S-S-CUR micelles In order to evaluate the antitumor activity of ALN-oHA-S-S-CUR micelles in human breast cancer (MDA-MB-231) xenograft models, we measured the tumor volume of the tumor-bearing mice treated with saline, free CUR, CUR-loaded oHA-CUR micelles, and CUR-loaded ALNoHA-S-S-CUR micelles (25 mg/kg). The results suggest that while the free CUR and CURloaded oHA-CUR micelles do exhibit an antitumor activity, this activity is lower than the one afforded by CUR-loaded ALN-oHA-S-S-CUR micelles (Fig. 3A and B). Specifically, the growth of tumor was severely inhibited after treatment with CUR-loaded ALN-oHA-S-S-CUR micelles, suggesting that their antitumor activity was enhanced. On the other hand, compared to control group, the metastatic bone tumor was markedly inhibited in CUR-loaded ALN-oHA-S-S-CUR micelles treatment group (see Fig. 3C). All these results might have been in virtue of the high CUR concentration in tumor tissue, due to the EPR effect, ability to target CD44 receptors and redox sensitivity of ALN-oHA-S-S-CUR micelles23. 3.4 The results of histopathological analysis The pathological changes in the tumor tissue were observed in tumor sections stained with H ＆E. As shown in Fig. 4, the blue purple parts are nucleus stained by hematoxylin, while the pink parts are the cytoplasm and extracellular matrix dyed by eosin. The results showed that the tumor cells in saline and free CUR groups had obvious cellular morphology and chromatin, and that the cell nuclei were contracted in the CUR-loaded ALN-oHA-S-S-CUR group, indicating active growth of tumor cells in saline and free CUR groups. Therefore, via delivering more CUR to tumor cells, thereby exerting a cytotoxic effect, CUR-loaded ALN-oHA-S-S-CUR micelles have a superior antitumor efficacy. Together, our results demonstrate that CUR-loaded ALN-oHA-SS-CUR micelles act as a promising system to deliver CUR into tumor cells with the lowest


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systemic toxicity and efficient tumor targeting property12. 3.5 In vivo distribution of CUR-loaded ALN-oHA-S-S-CUR micelles To further evaluate the antitumor activity of CUR-loaded ALN-oHA-S-S-CUR micelles, the distribution of the DiR-loaded oHA-CUR micelles, as the control group, and DiR-loaded ALN-oHA-SS-CUR micelles were observed using an in vivo imaging system. As illustrated in Fig. 5 (a), (b) and (c), after the free DiR and the DiR-loaded oHA-CUR micelles injection, the fluorescence of DiR was distributed to the heart, liver, lung and kidney tissue, and the distribution to bone tumor was very low, probably because the free DiR has no tumor targeting ability; also, a reason for increased systemic toxicity. Meanwhile, in the control group DiR-loaded oHA-CUR micelles, the fluorescence of DiR was distributed to the heart, liver, lung and kidney tissue, and the distribution to bone tumor was a little higher than the free DiR group and lower than the DiR-loaded ALN-oHA-S-S-CUR

micelles group, probably due to the tumor targeting capacity of oHA and the bone targeting capacity of ALN. Free DiR and two different micelles can quickly enter the internal circulation system and distribute to tumors and tissues. With the detection at different time points, the distribution of micelles in tumor sites increased significantly. The fluorescence of DiR was mainly distributed in the right leg bone tumor after treated with DiR-loaded ALN-oHA-S-S-CUR micelles due to their EPR effect, redox-sensitivity, as well as the ability to target CD44 receptors and bone13. The high accumulation of DiR in bone tumors can contribute to its antitumor effect. To observe whether CUR-loaded ALN-oHA-S-S-CUR micelles could effectively deliver CUR to the bone tumor and achieve high therapeutic efficacy, the right leg bone density of tumorbearing mice was analyzed post-mortem using the 3D Micro-CT technology. The results showed that bone tumor led to bone destruction (see Fig. 6A). However, after treatment with CURloaded ALN-oHA-S-S-CUR micelles, the destruction of bone was effectively decreased. In comparison with the treatment with free CUR and CUR-loaded oHA-CUR micelles, the bone


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mineral density (BMD) was enhanced after treatment with CUR-loaded ALN-oHA-S-S-CUR micelles (Fig. 6B). This suggests that ALN-modified micelles could be used to create a promising delivery system to prevent tumor expansion in the bone tissue and damage to the bone11. 4. Conclusions In the present study, we used the disulfide bond as a connection arm and successfully prepared ALN-oHA-S-S-CUR polymer-drug conjugates to enhance the DL/EE of CUR and to achieve bone tumor targeting. And the results of in vivo experiments indicated that ALN-oHA-S-S-CUR micelles exhibited a relevant antitumor activity, effectively delivering CUR into tumor cells and thus exerting a cytotoxic effect. Likewise, in vivo distribution assays showed that CUR-loaded ALN-oHA-S-S-CUR micelles were mainly distributed in the target tumors, while the distribution to other normal tissues was considerably lower. Finally, the results of 3D micro-CT imaging suggested that, while in the groups treated with free CUR and CUR-loaded oHA-CUR micelles metastatic bone tumors led to a decrease in bone density, CUR-loaded ALN-oHA-S-S-CUR micelles prevented the formation of metastatic tumors and helped to preserve the structural integrity of the bone. Taken together, results of the present study convincingly demonstrate that CUR-loaded ALN-oHA-S-S-CUR micelles have a great potential for effective delivery of CUR to tumor cells.

Supporting information The Supporting Information is available free of charge on the ACS Publications website at Synthesis and characterization of ALN-oHA-S-S-CUR; Preparation of self-assembly ALN-oHA-


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S-S-CUR micelles; HPLC analysis of concentration of CUR in ALN-oHA-S-S-CUR micelles

Acknowledgements All authors contributed equally to the present work. This research was supported by grants from the National Natural Science Foundation of China (No. 81573614), Taishan Young Scholar Program (No. 20161035). Disclosure statement The authors declare no conflict of interest.

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Fig. 1. (A) Schematic representation of the ALN-oHA-S-S-CUR micelles formation. (B) The micelles enter tumor cells by passive and active targeting, and release the drug by disassembly mediated by a high concentration of GSH.



Fig. 2. (A) The

H-NMR spectra of ALN-oHA-S-S-CUR materials; (B) The size distribution of CUR-loaded ALN-oHA-S-S-CUR micelles; (C) The TEM images of CUR-loaded ALN-oHA-S-S-CUR micelles. 1


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Fig. 3. In vivo antitumor activity of CUR-loaded ALN-oHA-S-S-CUR micelles on tumor-bearing nude mice (10 mg/kg). (A) The tumor volume changes over treatment times. (B) The size of tumor at the end point; (a) the saline group, (b) the free CUR group, (c) the CUR-loaded oHA-CUR micelles group, (d) the CUR-loaded ALN-oHA-S-S-CUR micelles group. (C) The size of metastatic bone tumors after treatment with CUR-loaded ALN-oHA-S-S-CUR micelles. All data are reported as a mean ± SD. n = 6; *indicates p < 0.05.



Fig. 4. H&E tumor staining from tumor-bearing nude mice after treatment with different formulations.

Fig. 5. In vivo imaging of tumor-bearing mice. (A) The distribution after treatment with (a) free DiR (b) DiR-loaded oHA-CUR micelles and (c) DiR-loaded ALN-oHA-S-S-CUR micelles in mice by vein injection. (B) Distribution of micelles in the heart, liver, spleen, lung, kidney, and tibia after treatment with (a) free DiR (b) DiR-loaded oHA-CUR micelles and (c) DiR-loaded ALN-oHA-S-S-CUR micelles.*Indicates p < 0.05.


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Fig. 6. (A) Micro-CT 3D reconstruction image, the 2D transverse sections and the longitudinal sections of tibia. (B) The analysis of bone mineral density (BMD) .*Indicates p < 0.05.




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In Vivo Evaluation of Reduction-Responsive Alendronate-Hyaluronan

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