Title: Calcium phosphate-reinforced reduction-sensitive hyaluronic acid micelles for

Title: Calcium phosphate-reinforced reduction-sensitive hyaluronic acid micelles for delivering paclitaxel in cancer therapy Bing Deng a,1, Mengxin Xia a,1, Jin Qian a, Rui Li a, Lujia Li a,b, Jianliang Shen c, Guowen Li b, Yan Xie a,* a Research Center for Health and Nutrition, Shanghai University of Traditional Chinese Medicine, Shanghai 0, China b Pharmacy Department, Shanghai TCM-integrated Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 000, China c Department of Nanomedicine, Houston Methodist Research Institute, Houston 00, USA 1 1 1 1 1 1 1 1 0 1 1 These authors contributed equally to this work. * Corresponding author: Yan Xie, Ph.D. Professor Research Center for Health and Nutrition Shanghai University of Traditional Chinese Medicine 0 Cailun Road Shanghai, China 0 E-mail: rosexie_1@hotmail.com; yxie@shutcm.edu.cn Phone: +(1) Fax: +(1) 1

1 1 1 1 1 1 1 1 0 1 1. Materials Hyaluronic acid (HA, Mw = kda) was purchased from Bloomage Freda Biopharm Co., Ltd. (Shandong, China). 1-Ethyl- (-dimethylaminopropyl) carbodiimide (EDC), N- Hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide (Sulfo-NHS), deoxycholic acid (DOCA), dicyclohexylcarbodiimide (DCC), and glutathione (GSH) were purchased from Aladdin Reagent Inc. (Shanghai, China). Cystamine was purchased from TCI Development Co., Ltd. (Shanghai, China). Pyrene was purchased from Sigma-Aldrich (Shanghai, China).,-Diamidino--phenylindole (DAPI) was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). Dulbecco s Modified Eagle s Medium (DMEM) and Minimum Essential Medium (MEM) were purchased from Thermo-Fisher Biochemical Product (Waltham, MA, USA). Fetal bovine serum (FBS) was obtained from Gibco Laboratory (Grand Island, NY, USA). Monoclonal rabbit anti-cd antibody (Alexa Fluor ) was perchased from Abcam (Cambridge, United Kingdom). The human breast cancer (MDA-MB- 1) cell line and human hepatoma (HepG) cell line were purchased from Institute of Biochemistry and Cell Biology, SIBS, CAS (Shanghai, China). All other chemicals were of analytical grade.. Synthesis of HA-SS-DOCA conjugates.1. Synthesis of cystamine modified HA (HA-CYS) The synthesis of cystamine modified HA (HA-CYS) was carried out as previously described with some modifications. 1 First, 0.0 mm (moles of the carboxyl group) of HA was dissolved in 0 ml PBS solution (0.01 M, ph.). EDC (.0 mm) and sulfo-nhs (0.0 mm) were added to the HA solution and stirred for 0 min to activate the carboxylic group of HA. Then,.0 mm of cystamine was added and the reaction was allowed to proceed under stirring for h at room temperature. The resulting solution was dialyzed (MWCO 00) exhaustively against 0.1 M NaCl and then distilled water to remove the unreacted CYS, EDC, and sulfo- NHS coupling agents. Finally, the solution was freeze-dried to obtain HA-CYS powder and

1 1 1 1 1 1 1 1 0 1 stored in a desiccator until further use... Synthesis of NHS ester of deoxycholic acid (DOCA-NHSE) The carboxylic group of deoxycholic acid was activated as reported previously., Briefly, DOCA ( mm) was mixed with DCC (1 mm) and NHS (1 mm) in 0 ml of tetrahydrofuran (THF). The mixture was reacted for h at room temperature, and then the precipitated dicyclohexylurea was removed by filtration. The filtrate was poured into cold n- hexane and the DOCA-NHSE precipitate was filtered off and washed thoroughly with cold n- hexane, followed by vacuum drying at room temperature. The DOCA-NHSE was stored at - 0 C before use... Synthesis and characterization of amphiphilic HA-SS-DOCA conjugates The amphiphilic HA-SS-DOCA conjugates were prepared by chemical grafting DOCA- NHSE to HA-CYS through amide formation as previously reported. 1, In brief, HA-CYS (0. mm) was dissolved in ml formamide, and DOCA-NHSE (0.0 mm) dissolved in ml N, N-dimethylformamide (DMF) were added drop wise to the solution. The reaction was performed for h at room temperature with gentle stir. The resulting solution was dialyzed against the excess amount of water/ethanol (1:1, v/v) for 1 day and distilled water for days. After being freeze dried, the final products were obtained as a white flocculence powder and stored at C until further use. 1 H NMR spectra of HA-SS-DOCA conjugates were acquired on a Bruker-00 1 H NMR spectrometer. HA-CYS, DOCA-NHSE, and HA-SS-DOCA conjugates were dissolved in D O, DMSO-d, and D O/CD OD (1:1, v/v), respectively. The DS (defined as the number of DOCA per 0 sugar residues of HA) values for HA-SS-DOCA, was obtained from the ratio of integration between the characteristic peaks of the 1 st methyl group in DOCA (0. ppm, [H]) and the N-acetyl group in HA (.01 ppm, [H]). FT-IR was taken on a Nicolet is spectrophotometer (Thermo-Fisher Scientific Inc., Waltham, MA, USA) over the region from 000 to 00 cm -1. The critical micelle concentration (CMC) of amphiphilic HA-SS-DOCA

1 1 1 1 1 1 1 1 0 1 conjugates was evaluated by fluorescence spectroscopy using pyrene as the probe.. Preparation and characterization of micelles.1. Preparation of HA-SS-DOCA micelles The HA-SS-DOCA micelles were prepared by sonication. Briefly, mg of HA-SS-DOCA conjugates were dissolved in ml of distilled water followed by sonicating at 0 W for min in an ice bath by a probe-type ultrasonicator (JY-D, Ningbo Scentz Biotechnology Co., Ltd., Ningbo, China). The resulting micellar solution was filtrated through a 0. μm pore-sized membrane... Reduction triggered destabilization of HA-SS-DOCA micelle The disassembly of HA-SS-DOCA micelles in response to GSH was monitored by Dynamic light scattering (DLS) measurement (Malvern Instruments Ltd., Worcestershire, UK). Briefly, 0 mm GSH was added in the solution of HA-SS-DOCA micelles, and HA-SS-DOCA micelles without addition of GSH were used as a control. To record the disassembly of micelles, the size distribution was monitored at different time points by the above measurement.. CD expression analysis CD expression levels were determined by immunocytostaining. 0. MDA-MB-1 cells and HepG cells were seperately seeded in glass bottom culture dishes and cultured in DMEM and MEM respectively with % FBS overnight. Then cells were washed with cold phosphate buffer saline (PBS) for three times, and fixed with % formaldehyde ( min) away from light. After washed off with PBS, cells were treated with 1% Triton-0 for min and washed with PBS again for three times. After that, cells were incubated with anti-cd antibody ( μg/ml) overnight, washed off with PBS and stained by DAPI ( μm, min). Next, all reagents were removed and cells were examined under an inverted confocal laser scanning microscope (CLSM, Leica, Wetzler, Germany).. Results and discussion

1 1 1 1 1 1.1. Synthesis of HA-SS-DOCA conjugates In the present study, amphiphilic reduction-sensitive HA-SS-DOCA conjugates were successfully synthesized as previously reported with some modification. 1 In this case, the hydrophobic DOCA groups were conjugated to an HA backbone using cystamine containing a disulfide bond as the reduction-sensitive connecting bridge. The chemical structures of HA-SS-DOCA conjugates were confirmed by 1 H NMR and FT- IR spectra. As shown in Fig. S1A, the structure of HA-CYS (b) was confirmed by the characteristic peaks at.0 ppm of HA and the cystamine was found at the range of.-. ppm. The presence of DOCA moieties in the HA-SS-DOCA conjugates (c) was evaluated by the characteristic peaks appearing at 0.-. ppm. As expected, the characteristic peaks ( cm -1 and cm -1 ) of DOCA that corresponds to C-H vibrations were also appeared in the FT-IR spectra of HA-SS-DOCA (Fig. S1B (c)), indicating that DOCA was introduced to HA. Meanwhile, the appearance of peaks at 1 cm -1 (amide I band), 1 cm -1 (amide Ⅱ band), and 1 cm -1 (C=O) in the IR spectrum of HA-SS-DOCA confirms the formation of an amide linkage between amino groups of CYS and carboxyl groups of HA as well as DOCA. These results indicated that the HA-SS-DOCA conjugates had been successfully synthesized. The critical micelle concentration (CMC) is a principal parameter used to evaluate the 1 stability of the aggregates in the blood circulation system post administration. 1 The 1 0 1 fluorescence intensity ratio (I/I) was plotted according to the concentrations of HA- SS-DOCA conjugates (Fig. S), and the CMC value of HA-SS-DOCA conjugates was 0.001 mg/ml. Such a low CMC value suggested that the HA-SS-DOCA conjugates could easily form self-assembled micelles, and guarantee the micelles to retain their original morphology under highly diluted conditions before reaching the targeting site... Characterization of HA-SS-DOCA micelles Reduction-sensitive HA-SS-DOCA micelles could be easily self-assembled in aqueous condition owing to the amphiphilic property of HA-SS-DOCA conjugates. DLS measurement

1 1 1 1 1 1 1 1 0 1 (Table S1) confirmed that the micelles possessed narrow size distribution with a broad range particle size from. to 1. nm. Interestingly, the micelles particle size decreased as the DS of DOCA increased, which was likely due to the increasing hydrophobic interaction between DOCA groups offered a tight micellar inner core. 1, The zeta potential of the micelles in distilled water is negative owing to the carboxylic group of HA, from -. to -. mv, which provided a repelling force between the particles and increased the stability of the micelles. The TEM image of HA-SS-DOCA micelles (Fig. SA) showed that the micelles were almost spherical in shape. Nevertheless, the size obtained from TEM was smaller than that from DLS, which was likely ascribed to that the samples for TEM undergo a shrinkage caused by water evaporation under air drying. To investigate the disassembly of HA-SS-DOCA micelles and evaluate the reduction responsive behaviors in response to GSH, the change of size distribution of HA-SS-DOCA micelles in 0 mm and 0 mm GSH, was monitored for h using DLS measurement. As shown in Fig. SB and SC, the particle size of HA-SS-DOCA micelles showed negligible size variation incubated with 0 mm GSH for h. On the contrary, aggregation of micelles was observed when incubated with 0 mm GSH, i.e., the diameter of HA-SS-DOCA micelles increased from. nm to 0. nm within h, indicating the rapid disassembly of micelles under reducing environment. The particle size change in 0 mm GSH confirmed that the HA-SS-DOCA micelles are sensitive to reducing condition... CD expression As shown in Fig. S, obvious green fluorescence of anti-cd antibody was observed in MDA-MB-1 cells, while this fluorescence was hardly observed in HepG cells, indicating that CD was high-expressed on MDA-MB-1 cells but was low-expressed on HepG cells. Considering hyaluronic acid can specifically bind with CD receptor, MDA-MB-1 cell was used as CD high expressive cell line and HepG cell as CD low expressive cell line (negative control) to investigate the targeted delivery of mineralized micelles in the following

1 1 1 1 1 1 1 1 study. References (1) Li, J.; Huo, M.; Wang, J.; Zhou, J.; Mohammad, J. M.; Zhang, Y.; Zhu, Q.; Waddad, A. Y.; Zhang, Q. Redox-sensitive micelles self-assembled from amphiphilic hyaluronic acid-deoxycholic acid conjugates for targeted intracellular delivery of paclitaxel. Biomaterials 01,, -0. () Hamidi, M.; Azadi, A.; Rafiei, P. Hydrogel nanoparticles in drug delivery. Adv. Drug Deliv. Rev. 00, 0, 1-1. () Kim, S. K.; Kim, K.; Lee, S.; Park, K.; Park, J. H.; Kwon, I. C.; Choi, K.; Kim, C.-Y.; Byun, Y. Evaluation of absorption of heparin-doca conjugates on the intestinal wall using a surface plasmon resonance. J. Pharm. Biomed. Anal. 00,, 1-0. () Han, S.-Y.; Han, H. S.; Lee, S. C.; Kang, Y. M.; Kim, I.-S.; Park, J. H. Mineralized hyaluronic acid nanoparticles as a robust drug carrier. J. Mater. Chem. 0, 1, -001. () Yu, C.; Gao, C.; Lü, S.; Chen, C.; Huang, Y.; Liu, M. Redox-responsive shell-sheddable micelles selfassembled from amphiphilic chondroitin sulfate-cholesterol conjugates for triggered intracellular drug release. Chem. Eng. J. 01,, 0-. () Huang, J. B.; Zhang, H.; Yu, Y.; Chen, Y.; Wang, D.; Zhang, G. Q.; Zhou, G. C.; Liu, J. J.; Sun, Z. G.; Sun, D. X.; Lu, Y.; Zhong, Y. Q. Biodegradable self-assembled nanoparticles of poly (D,L-lactide-coglycolide)/hyaluronic acid block copolymers for target delivery of docetaxel to breast cancer. Biomaterials 01,, 0-. 0

1 1 1 1 1 1 1 1 0 1 Supplementary Figure Captions Fig. S1. (A) 1 H NMR spectra of HA (a), HA-CYS conjugate (b), and HA-SS-DOCA conjugate (c). (B) FT-IR spectrum of HA (a), DOCA (b), and HA-SS-DOCA (c). Fig. S. CMC determination of HA-SS-DOCA conjugates. Fluorescence intensity ratio (I/I) versus HA-SS-DOCA concentration was plotted and CMC was estimated from the threshold concentration of self-assembled micelles. Fig. S. (A) TEM images of blank HA-SS-DOCA micelles. (B) Time dependent changes of the mean diameter of HA-SS-DOCA micelles with or without GSH addition. (C) Size distribution of HA-SS-DOCA micelles after incubated with 0 mm (a) or 0 mm (b) GSH for h, respectively. Fig. S. Release behavior of PTX from HA-SS-DOCA micelles in PBS with 0 mm or 0 mm GSH. Data expressed as the mean ± SD (n = ). Fig. S. CLSM images of (A) MDA-MB-1 cells and (B) HepG cells after incubation of anti-cd antibody overnight. Images from left to right show the green fluorescence of anti- CD antibody, the blue fluorescence of DAPI, and the merged fluorescence of anti-cd antibody and DAPI. Fig. S. Confocal laser scanning microscopy (CLSM) images of MDA-MB-1 cancer cells after h incubation with (A) C, (B) C loaded micelles, and (C) C loaded mineralized micelles, respectively. Scale bars correspond to μm in all the images. Fig. S. Optical microscopy images of H&E staining major organs and tumor after treated with different PTX formulations and % glucose as control. Magnification: 0.

Table S1 Characteristics of HA-SS-DOCA conjugates (n = ). Samples a Feed ratio b DS a Size (nm) c PDI d Zeta (mv) HA-SS-DOCA. 0.1. 1. ± 0. 0.1 ± 0.0 -. ± 1. HA-SS-DOCA.0 0..0 1.0 ± 1.1 0.1 ± 0.01 -. ± 0.1 HA-SS-DOCA. 0.. ± 1.0 0.0 ± 0.01 -. ± 0. a Degree of substitution of deoxycholic acid. b Molar feed ratio of DOCA-NHSE to sugar residuals of HA polymer. c Mean diameters of micelles. d Polydispersity index of micelles size.

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