Delivery of Doxorubicin from Hyaluronic Acid-Modified Glutathione

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Delivery of doxorubicin from hyaluronic acid modified glutathioneresponsive ferrocene micelles for combination cancer therapy Hong-Lin Mao, Feng Qian, Shun Li, Jia-Wei Shen, Cheng-Kun Ye, Lei Hua, Long-Zhen Zhang, Dong-Mei Wu, Jun Lu, Ru-Tong Yu, and Hong-Mei Liu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b00862 • Publication Date (Web): 09 Jan 2019 Downloaded from http://pubs.acs.org on January 10, 2019

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Molecular Pharmaceutics

Delivery of doxorubicin from hyaluronic acid modified glutathione-responsive ferrocene micelles for combination cancer therapy Hong-Lin Mao‡, Feng Qian‡, Shun Li‡, Jia-Wei Shen, Cheng-Kun Ye, Lei Hua, Long-Zhen Zhang, Dong-Mei Wu, Jun Lu, Ru-Tong Yu*, Hong-Mei Liu* ‡Honglin Mao, Feng Qian and Shun Li contributed equally to this manuscript.

H.-L. Mao, F. Qian, S. Li, J.-W. Shen, Dr. C.-K. Ye, Dr. L. Hua, Prof. R.-T. Yu, A/Prof. H.-M. Liu Institute of Nervous System Diseases Xuzhou Medical University Xuzhou, 221002, P. R. China. Email: [email protected] (A/Prof. H.-M.Liu), [email protected] (Prof. R.-T.Yu), H.-L. Mao, F. Qian, S. Li, J.-W. Shen, Dr. C.-K. Ye, Dr. L. Hua, Prof. R.-T. Yu, A/Prof. H.-M. Liu Brain Hospital 1 ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Affiliated Hospital of Xuzhou Medical University Xuzhou, 221002, P. R. China

Prof. L.-Z. Zhang Department of Radiation Oncology Affiliated Hospital of Xuzhou Medical University Xuzhou, 221002, P. R.China.

Prof. L.-Z. Zhang Cancer Institute of Xuzhou Medical University Xuzhou, 221002, P. R.China.

A/Prof. D.-M. Wu, Prof. J. Lu Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province School of Life Science Jiangsu Normal University 2 ACS Paragon Plus Environment

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Xuzhou, 221116, P. R. China.

KEYWORDS：combination cancer therapy, GSH-responsive, prodrug micelle, DOX delivery

ABSTRACT ： A combination of different chemotherapy approaches can obtain the best response for many cancers. However, the greatest challenge is the development of a nanoparticle formulation that can encapsulate different chemotherapeutic agents to achieve the proper synergetic chemotherapy for the tumor. Here, amphiphilic ferrocenium-tetradecyl (Fe-C14) was constructed to form cationic micelles in an aqueous solution via self-assembly. Then, it was coated by Hyaluronic acid (HA) through electrostatic interactions to generate HA-Fe-C14 micelles. The HA-Fe-C14 micelles were used to deliver doxorubicin (DOX), and it showed that the DOX could be released rapidly under a high-GSH tumor environment. The HA-Fe-C14/DOX micelles were able to accumulate efficiently in tumor, and showed significant anticancer effect both in vitro and in vivo. These results suggest that HA-Fe-C14/DOX micelles are a useful drug delivery system that enhances synergic antitumor treatment effects.

1. Introduction

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Cancer remains an unconquered fatal diseases worldwide. Therefore, the fight against cancer is still challenging for modern medicine and basic medical science. The combination of two drugs can improve the therapeutic efficiency of cancer.1, 2 Despite the advantages of combination treatment, the cocktail administration does not necessarily lead to improvement of therapeutic efficacy.3 Plethora of drug delivery systems in nano-size have been designed for combination therapy in cancer and have made great progress in therapeutic applications. The codelivery systems should have the ability to lower negative reactions, such as the adverse effects of chemotherapeutics, and the ability of being stimuli-responsive in the tumor microenvironment.4 Meanwhile, stimuli-responsive nanoparticles are stable in the blood circulation and under the normal physiological environment but is activated by acidic pH,5, 6 enzymatic upregulation,7-9 or hypoxia10, 11 after they extravasate into the tumor microenvironment. Therefore, designing a successful nanoparticle for the precise and controlled delivery of therapeutic agents is still challenging in cancer combination treatment application. DOX, a topoisomerase II inhibitor, can induce DNA damage for the treatment of various types of cancer.12, 13 Despite extensive clinical utilization, cardiotoxicity is a major adverse side effect of DOX.14 Thus, developing a carrier to deliver DOX into the tumor can reduce side effects and improve the curative efficacy against tumors.


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Ferrocene derivatives have shown antineoplastic activities against cancer and are of great interest because of their good biocompatibility.15-17 Meanwhile, the ferrocene derivatives have received a lot of attentions in designing supramolecular assemblies and drug delivery research due to its unique sandwich structure, hydrophobicity and redox character.18, 19 Hydrophobic ferrocene can be transformed into hydrophilic ferrocenium in presence of mild oxidants, and the procedure is reversible under certain conditions.20-22 Therefore, the hydrophilic ferrocenium cation can be reduced to hydrophobic ferrocene by GSH.23 Due to these characteristics, ferrocenes usually were used to assemble ferrocene-based redox polymers.24, 25 And the drug or target with negative charge can be loaded into positively charged ferrocenium through electrostatic adsorption via intermolecular interaction similar to GSH-responsive bonds, 26 The polyanionic HA could bind to receptors of cell surface and serve as an active tumor-targeting ligand with good biocompatibility and biodegradability, such as CD44.27-30 Moreover, the HA is over expressed in many tumors. HA-modified nanoparticles accumulate effectively in the tumor through receptor-mediated endocytosis.31, 32 Therefore, their targeting characteristics make HA nanoparticles ideal materials for embedding anticancer drugs to cure cancer.



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To overcome these limitations, we synthesized a Fe-C14 prodrug, prepared HA-modified

reduction-responsive

HA-Fe-C14

micelles

based

on

the

properties of ferrocene in the tumor microenvironment and encapsulated DOX to generate HA-Fe-C14/DOX micelles for combination cancer therapy. The HA-Fe-C14 micelle mainly consisted of two distinctive functional constituents: 1) HA, the primary CD44-binding molecule, which enhanced the drug encapsulation efficiency and lowered down the toxicity of positively charged ferrocenium; 2) hydrophilic cationic ferrocene, which was reduced to hydrophobic ferrocene by GSH, disintegrated, provoked the release of DOX and improved the curative effect by the combination of DOX and ferrocene derivatives. The formation of the HA-Fe-C14 micelles and the mechanism of action after intravenous administration are illustrated in Scheme 1. In this paper, clear evidence was provided that the HA-Fe-C14 micelles effectively combined with ferrocene and DOX to inhibit the growth of tumor both in vitro and in vivo.


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Scheme 1. Illustration of the drug release process of GSH-responsive HA-Fe-C14/DOX micelles. A) Mechanism of GSH-triggered dissociation based on the charge change of Fe-C14 under the GSH microenvironment; B) schematic diagram illustrating the assembly of HA-Fe-C14/DOX micelles and the mechanism of GSH-triggered release of DOX from HA-Fe-C14/DOX; C) GSH-responsive delivery of DOX by HA-Fe-C14 prodrug micelles for combination treatment of tumor. I, accumulation of HA-Fe-C14/DOX at the tumor through passive and active targeting; II, receptor-mediated endocytosis; III, GSH-triggered DOX release into the cytosol; IV, DOX entering the nucleus to kill tumor cells. 2. Materials and methods



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2.1. Materials Unless otherwise specified, all the solvents, including dichloromethane (DCM), petroleum ether, ethyl acetate, dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), and they were used as received. Ferrocenylacetic acid and tetradecyl alcohol were from Tokyo Chemical Industry Co., Ltd. (Shanghai, China). Hyaluronic acid (HA, 100 kDa), doxorubicin hydrochloride (DOX.HCl) were obtained from Dalian Meilun Biochem Co., Ltd. (Dalian, China). 4′,6-Diamidino-2-Phenylindole dihydrochloride (DAPI) were purchased from Sigmaalrich. 2.2. Synthesis of Fe-C14 and Preparation of Micelles According to our previous study, Fe-C14 were synthesized33. 1H NMR (300 MHz, CDCl3): δ 4.21 (s, 2H), δ 4.17 – 4.00 (m, 9H), 3.34 (s, 2H), δ 1.96 - 1.16 (m, 24H), δ 0.93 - 0.84 (m, 3H). 13C NMR (75 MHz, CDCl3): = 14.1, 22.7, 24.7, 25.5, 25.9, 28.6, 29.2, 29.4, 29.5, 29.6, 29.7, 29.7, 29.7, 31.9, 34.9, 35.7, 55.7, 64.8, 67.8, 68.7, 68.8, 80.5, 139.8, 171.2. MS m/z (ESI): calculated for C26H40O2FeNa+ 463.42, Found 463.22. The preparation of HA-Fe-C14 and HA-Fe-C14/DOX micelles followed our previous report.24 The critical micelle concentration (CMC) values was tested through the pyrene assay, ranged from 2.0×10-5 to 0.2 mg mL-1 for the Fe-C14 micelles. 8 ACS Paragon Plus Environment

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The fluorescent emissions were obtained at 372 and 383 nm by a given 330 nm excitation wavelength. 2.3. DOX Loading and Characterization of Micelles The loading content and loading efficiency of DOX in the HA-Fe-C14/DOX were determined by UV-vis spectrophotometer (Bio Tek Synergy 2) at 480 nm (The maximum absorbance of Dox) and calculated using the following formulas:34 Loading efficiency (%) = (weight of loaded drug/weight of drug in feed) ×100% Loading content (%) = (weight of loaded drug/weight of HA-Fe-C14/DOX) ×100% TEM was used to observe the morphology of the HA-Fe-C14/DOX micelles under normal conditions, or HAase (0.5 mg mL-1), or HAase (0.5 mg mL-1) and GSH (10 mM). The HA-Fe-C14/DOX micelles were dyed using 2% phosphotungstic acid. DLS measurements of the HA-Fe-C14/DOX micelles (under normal conditions, or HAase (0.5 mg mL-1) or HAase (0.5 mg mL-1) and GSH (10 mM) conditions) were measured by Malvern Zetasizer Nano ZS. 2.4. In Vitro DOX Release The release of DOX in vitro from free DOX and HA-Fe-C14/DOX micelles was carried out on a shaking table at 37 ℃. HA-Fe-C14/DOX micelles were carried out in 9 ACS Paragon Plus Environment


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PBS (pH 7.4), and PBS (pH 7.4) with 2, 5 and 10 mM GSH concentrations. 2 mL of micelles containing DOX was loaded into a dialysis bag and placed in 20 mL of the corresponding media. At the scheduled timepoint, 1 mL of release medium was removed, and the equivalent volume of fresh medium was added. The fluorescence of DOX was then determined by a F-4600 type fluorescence spectrophotometer to determine the solubility of DOX in the release medium at different time points. Each release experiment was done three times in parallel. 2.5. Cell Culture Prostatic cancer (PC3) cells were cultured in RPMI with FBS (10%, v: v), penicillin (100 U mL-1) and streptomycin (100 μg mL-1) in an incubator (Thermo Scientific) at 37 °C under 5% CO2 and 90% relative humidity. 2.6. MTT Assay The MTT assay was used to test the cell viability of free DOX, HA-Fe-C14 and HA-Fe-C14/DOX micelles (doses: 0.75, 1.5, 3, 6, 12, 24 and 48 μg mL-1 DOX; 0.01, 0.02, 0.04, 0.08, 0.16, 0.32 and 0.64 mmol mL-1 Fe-C14). PC3 cells were seeded into 96-well microplates at the density of 1 × 104 cells per well. After incubation for 24 h,

freshly

prepared

different

concentrations

of

the

HA-Fe-C14

and

HA-Fe-C14/DOX micelles with 10% FBS-medium were replaced by the medium and incubated for another 72 h. 20 μL of MTT solution was added to each well and incubation continued for 4 h, the MTT-formazan generated by 10 ACS Paragon Plus Environment

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live cells and were dissolved in 100 μL DMSO. The optical density (OD) was measured at 570 nm by a microplate reader. Cell survival fraction was calculated by dividing OD 570 test by OD 570 control. Three independent experiments were performed. 2.7. Animals and Tumor Models BALB/c nude mice (male, 16-20 g) were brought from Beijing HFK Bioscience Co., Ltd. (Beijing, China). The PC3 cell-bearing nude mice were established. Measure tumor lengh and width and calculate the tumor volume using the following equation: V = L × W2/2.35, 36

2.8. In Vivo Distribution of HA-Fe-C14/DOX Micelles

Mice were intravenously injected with HA-Fe-C14/DOX (doses: 2.5 mg kg-1 DOX and 15.6 mg kg-1 Fe-C14) and 2.5 mg kg-1 DOX (free DOX). The mice were sacrificed at post-injection 6 h and 12 h . The DOX fluorescence of main organs (tumor, heart, liver, spleen, lung and kidney) were visualized via in vivo real-time fluorescence imaging system. 2.9. In Vivo Antitumor Efficacy When the tumors reached to around 50 mm3, PBS, free DOX, HA-Fe-C14 micelles and HA-Fe-C14/DOX micelles were injected intravenously into the PC3-bearing mice

every two day and three times in all. The tumor volumes



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were measured with a Vernier caliper, and weighed every other day. The tumors were collected, weighed, and imaged after 16 days of treatment. For the H＆E staining, the normal organs, including the heart, liver, spleen, kidney and lung, of the mice were embedded in paraffin blocks and visualized by optical microscopy. 2.10. Statistical analysis

The results (Mean ± SD) were subjected to statistical analysis by one-way ANOVA and shown in the figures (*p < 0.05, **p < 0.01). 3. Discussion and Results 3.1. Characterization of Fe-C14 and HA-Fe-C14 Micelles Fe-C14 was synthesized (Figure 1). The chemical structure of Fe-C14 were confirmed by 1H NMR and mass spectrometry (MS) . 1H NMR spectroscopy revealed that Fe-C14 was successfully synthesized (Figure 1B), the molecular formula and weight of Fe-C14 were calculated as C26H40FeO2Na: 463.42, the it was found to be MS (m/z): 463.22 (+ Na+) (Figure 1C). These results indicated that Fe-C14 was obtained.


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Figure 1. (A) Synthetic route of Fe-C14. (B) 1H NMR spectra of Fe-C14. (CDCl3, 300 MHz) (C) HRMS (Found m/z 463.22 (+ Na+). Calcd for C26H40FeO2Na, 463.42) spectra of Fe-C14. They were solubilized in CDCl3 for HRMS analysis. The oxidation of the ferrocenyl groups on Fe-C14 by FeCl3 provided stoichiometry, resulting in polarity reversal from the hydrophobic Fe-C14 to the amphipathic Fe-C14+, which could undergo self-assembly in water to Fe-C14 micelles. The result of CMC revealed that HA-Fe-C14/DOX had a low CMC of 4.1 mg L-1 in 10 mM PB at pH 7.4 (Figure S1). The Fe-C14 micelles had highly positive charged surface due to the hydrophilic ferrocenium cations, which were coated with HA via the electrostatic interaction. The anionic HA is overexpressed by many tumors, behave as an active tumor-targeting ligand with good biocompatibility and 13 ACS Paragon Plus Environment


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biodegradability and target the cell surface receptors, such as CD44.27-30 As shown in Figure 2A and B, the average size of Fe-C14 micelles and HA-modified Fe-C14 micelles (HA-Fe-C14) were 87.6 ± 1.12 nm and 117.6 ± 0.77 nm. After coating HA with Fe-C14, the zeta potential was changed from +51.8 mV to -25.7 mV. As shown in Figure 2C, the particle size of the HA-Fe-C14/DOX micelles was 99.6 ± 1.05 nm. The TEM image of HA-Fe-C14/DOX micelles showed a micellar structure (Figure 2D). According to the drug loading equation, DOX was loaded in the HA-Fe-C14 micelles at 5.97%.

Figure 2. (A) The average size of Fe-C14 micelles and HA-Fe-C14 micelles. (B) The zeta potentials of Fe-C14 micelles and HA-Fe-C14 micelles. (C) The average size of HA-Fe-C14/DOX micelles. (D) TEM image of HA-Fe-C14/DOX micelles.


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Hyaluronidase (HAase) is highly expressed in tumor microenvironment, which can promote the degradation of HA shell of Fe-C14 micelles.31 To demonstrate HA degradation by HAase, zeta potentials of the HA-Fe-C14 micelles were compared between before and after incubation with HAase. The zeta potential of the HA-Fe-C14 micelles varied from -25.7 mV to -12.3 mV after exposure to HAase for 15 min in PBS at pH 7.4 (Figure 3A). The structure of HA-Fe-C14 micelles remained intact after incubation with HAase. The structural integrity was supported by the average size and TEM image after the HAase treatment (Figure 3B and C). And then, HA-Fe-C14 micelles were entered into tumor cells.

Figure 3. (A) The zeta potential of HA-Fe-C14 micelles after incubation with HAase. (B) Particle size of HA-Fe-C14 micelles after incubation with HAase. (C) TEM image of HA-Fe-C14/DOX micelles after incubation with HAase. GSH-responsive disassembly of the HA-Fe-C14 micelles was tested. Treated by HAase and reductant GSH.37, Yellow precipitates formed gradually, The average diameter of the HA-Fe-C14 micelles became broader and shifted to a larger size (Figure 4A), and the TEM imaging showed no micelles (Figure 15 ACS Paragon Plus Environment


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4B) under the HAase and GSH conditions. These results demonstrated the micelles were disintegrated through the reduction of the ferrocenium by GSH. The HA-Fe-C14 micelles accelerated DOX release by GSH in the tumor microenvironment. The in vitro DOX release profiles of the HA-Fe-C14/DOX micelles were assessed by incubating the micelles in PBS containing 2, 5 and 10 mM GSH and in PBS without GSH. As shown in Figure 4C, the burst release of DOX happened initially in the presence of 2, 5 and 10 mM GSH. This release profile was attributed to the transformation of the hydrophilic ferrocenium cations into the hydrophobic ferrocenyl groups by GSH.

Figure 4. Characterization of HA-Fe-C14 micelles. (A) DLS testing HA-Fe-C14 micelles and HA-Fe-C14 micelles under HAase and GSH conditions. (B) TEM imaging of HA-Fe-C14 micelles under HAase and GSH conditions. Bar = 200 nm. (C) The in vitro cumulative DOX released from the HA-Fe-C14/DOX 16 ACS Paragon Plus Environment

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micelles in PBS (pH = 7.4) without GSH and under 2, 5 and 10 mM GSH at 37 °C. 3.2. Internalization and Intracellular DOX Release from Micelles We next investigated the intracellular uptake of HA-Fe-C14 micelles to test their abilities to target CD44-overexpressing cancer cells. PC3 cell lines expressing high levels of CD44 and a DU145 cell line which had extremely little expression of CD44 were utilized to assess the CD44-targeting capability of HA-Fe-C14/DOX micelles38. As shown in Figure 5, HA-Fe-C14/DOX entered CD44-overexpressing PC3 cells more efficiently than CD44-negative DU145 cells. The results indicated that HA-Fe-C14/DOX were efficiently accumulated into the cancer cells through specific HA-CD44 interactions. The above experimental results demonstrated that HA-Fe-C14/DOX micelles had good GSH response. The cell lines with different GSH levels were used to test the GSH-activated fluorescence recovery of DOX in cell level. It was reported that there is more GSH existing in tumor cells than in normal cells38. In this study, we used PC3 tumor cell lines with high levels of GSH and 293T normal cell lines to evaluate the GSH-responsive of HA-Fe-C14/DOX39. Experimental results in Figure S2 indicated that PC3 cells had stronger DOX fluorescence than those of 293T cells. These results suggested that HA-Fe-C14/DOX rapidly released DOX in a high-GSH tumor environment.



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Figure 5. Enhanced delivery of DOX to CD44-overexpressed cancer cells. (A) Fluorescence images of PC3 and DU145 cells treated by HA-Fe-C14/DOX micelles for 4 h. Scale bar: 100 μm; (B) Flow cytometry analysis of PC3 and DU145 treated by HA-Fe-C14/DOX micelles for 4 h. The in vitro cytotoxicity assays of HA-Fe-C14 micelles with or without DOX were carried out in PC3 cells for 72 h. As shown in Figure 6, all the groups exhibited cytotoxicity in a dose-dependent manner. Comparing with HA-Fe-C14 group and free DOX group, HA-Fe-C14/DOX micelles group were highly toxic to PC3 cells. This result showed that HA-Fe-C14/DOX combine ferrocene and DOX effectively to inhibit tumor growth in vitro.


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Figure 6. Cell viability of PC3 cells in presence of micelles and free DOX with different DOX concentrations and Fe-C14 concentrations for 72 h. 3.3. In Vivo Antiglioma Efficacy of HA-Fe-DOX Micelles.

The in vivo targeting ability of HA-Fe-C14/DOX for tumor was tested using in vivo bioluminescence imaging. As shown in Figure 7A, B, HA-Fe-C14/DOX groups had the strongest DOX fluorescence, suggesting that the HA-Fe-C14/DOX micelles were effectively delivered into the tumor by the enhanced permeability and retention (EPR) effect and active effect. Nevertheless, rare DOX fluorescence was observed since the body could clear the free DOX rapidly. The heart, liver, spleen, lung and kidneys were collected and imaged after euthanized mice. As shown in Figure 7C, D, the distribution of HA-Fe-C14/DOX micelles was mainly in liver and kidneys after 6 h and 12 h tail vein injection in mice. These results suggested that free DOX and HA-Fe-C14/DOX micelles distributed rapidly and widespreadly in liver and kidneys.



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Figure 7. (A) Fluorescence images and quantitative analysis of free DOX and HA-Fe-C14/DOX micelles in PC3 tumor-bearing nude mice. Error bars indicate s.d. (n = 3). (B) Fluorescence microscopy images showing the distribution of DOX in PC3 tumor-bearing nude mice after intravenous injection of free DOX or HA-Fe-C14/DOX micelles. DOX fluoresced red; cell nuclei were stained with DAPI (blue). Scale bar: 200 µm. (C) Ex-vivo organ distribution of the HA-Fe-C14/DOX micelles. (D) HA-Fe-C14/DOX micelles distribution in the major organs determined by fluorescence intensity of DOX after (a) 6 h and (b) 12 h intravenous injection. 20 ACS Paragon Plus Environment

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Next, the combination therapy of the HA-Fe-C14/DOX micelles were tested in vivo. PC3 tumor-bearing nude mice were divided randomly into four groups (n = 5): PBS group, free DOX group, HA-Fe-C14 group and HA-Fe-C14/DOX group (doses: 2.5 mg kg-1 DOX and 15.6 mg kg-1 Fe-C14). After administering the different treatments, the tumor volumes were measured with calipers every 2 days. The tumor treatments with HA-Fe-C14 (7.49 ± 2.38) and free DOX (6.15 ± 1.08) were shown a delay in the growth of the tumor, compared that with PBS (10.23 ± 2.78) alone. This result suggested that the ferrocene groups showed antitumor bioactivity, which is consistent with previous report. As depicted in Figure 8A, B, HA-Fe-C14/DOX (4.03 ± 0.89) inhibited the growth of tumor cells more effectively than free DOX and HA-Fe-C14 did. The mice were sacrificed, and the tumors were dissected and weighed after 16 days of treatment. The tumor weights significantly decreased on the mice treated by the HA-Fe-C14/DOX (Figure 8C). The animal body weight did not undergo a significant change during the treatment with the HA-Fe-C14/DOX micelles (Figure 8D). Moreover, the HA-Fe-C14/DOX group did not exert significant damage against the normal organs, including the heart, liver, spleen, lung, and kidneys, of the mice (Figure S3), suggesting that HA-Fe-C14/DOX has s negligible adverse effects on normal tissues. Collectively, these results



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demonstrate that HA-Fe-C14/DOX has a predominantly synergic therapeutic efficacy against tumors.

Figure 8. In vivo evaluation of the antitumor efficacy of HA-Fe-C14/DOX micelles. (A) Images of the tumors on the 16th day after treatments. (B) Tumor growth curves

of mice by different treatments. Error bars indicate s.d.

(n = 5). *P < 0.05, **P < 0.01 (two-tailed Student’s t-test). (C) Average tumor weight on the 16th day for each group. (D) The body weights of mice by different treatments. *P < 0.05, **P < 0.01 (two-tailed Student’s t-test). 4. Conclusion In summary, a novel hyaluronic acid-modified and GSH-responsive HA-Fe-C14 micelles to deliver a hardly soluble anticancer drug and attain the synergic treatment of tumors. The excellent GSH-responsive ability of the HA-Fe-C14 22 ACS Paragon Plus Environment

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micelles had been demonstrated in this study. Remarkably, the in vivo results showed that the treatment with HA-Fe-C14/DOX significantly inhibited tumor growth in our subcutaneous tumor models of BALA/c nude mice compared with the other treatment groups. Therefore, our HA-Fe-C14 micelles can provide a new and efficient strategy for the combination treatment of tumor. Supporting Information Supporting Information is available from the Wiley Online Library or from the author. Corresponding Author A/Prof. H.-M. Liu, Prof. R.-T. Yu Institute of Nervous System Diseases Xuzhou Medical University Xuzhou, 221002, P. R.

China.

Email: [email protected] (A/Prof. H.-M.Liu), [email protected] (Prof. R.-T.Yu), Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.



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‡These authors contributed equally. Acknowledgements This work was financially supported by National Natural Science Foundation of China (NO. 81772665, 81502153, 81472345), Natural Science Foundation of Jiangsu Province (NO. BK20150221), China Postdoctoral Science Foundation funded project ( NO. 2016M591926; 2017T100409), Jiangsu Province, Key Research & Development Plan of Jiangsu Province (NO. BE2016646), and Jiangsu provincial Commission of Health and Family Planning (NO. Q201608), Six Talents Peak Foundation of Jiangsu Province (NO. 2018-WSW-071), Postgraduate Research & Practice Innovation Program of Jiangsu Province (Grants NO. KYCX17_1733). Honglin Mao, Feng Qian and Shun Li contributed equally on this manuscript. The authors declare no conflicts of interest in this work. REFERENCES 1.

Mignani, S.; Bryszewska, M.; Klajnert-Maculewicz, B.; Zablocka, M.; Majoral,

J.-P. Advances in combination therapies based on nanoparticles for efficacious cancer treatment: An analytical report. Biomacromolecules 2015, 16, (1), 1-27. 2.

Parhi, P.; Mohanty, C.; Sahoo, S. K. Nanotechnology-based combinational drug

delivery: an emerging approach for cancer therapy. Drug Discov Today 2012, 17, (17), 1044-1052. 3.

Hu, C.-M. J.; Zhang, L. Nanoparticle-based combination therapy toward

overcoming drug resistance in cancer. Biochem Pharmacol 2012, 83, (8), 1104-1111. 4.

He, Q.; Shi, J. MSN anti‐cancer nanomedicines: chemotherapy enhancement, 24 ACS Paragon Plus Environment

Page 25 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


overcoming of drug resistance, and metastasis inhibition. Adv Mater 2014, 26, (3), 391-411. 5.

Guan, X.; Li, Y.; Jiao, Z.; Chen, J.; Guo, Z.; Tian, H.; Chen, X. A pH-sensitive

charge-conversion system for doxorubicin delivery. Acta Biomater 2013, 9, (8), 7672-7678. 6.

Shi, J.; Liu, Y.; Wang, L.; Gao, J.; Zhang, J.; Yu, X.; Ma, R.; Liu, R.; Zhang, Z.

A tumoral acidic pH-responsive drug delivery system based on a novel photosensitizer (fullerene) for in vitro and in vivo chemo-photodynamic therapy. Acta Biomater 2014, 10, (3), 1280-1291. 7.

Bernardos, A.; Aznar, E.; Marcos, M. D.; Martínez‐Máñez, R.; Sancenón, F.;

Soto, J.; Barat, J. M.; Amorós, P. Enzyme‐responsive controlled release using mesoporous silica supports capped with lactose. Angew Chem Int Ed Engl 2009, 48, (32), 5884-5887. 8.

de la Rica, R.; Aili, D.; Stevens, M. M. Enzyme-responsive nanoparticles for drug

release and diagnostics. Adv Drug Delivery Rev 2012, 64, (11), 967-978. 9.

Gao, L.; Zheng, B.; Chen, W.; Schalley, C. A. Enzyme-responsive

pillar[5]arene-based polymer-substituted amphiphiles: synthesis, self-assembly in water, and application in controlled drug release. Chem Commun 2015, 51, (80), 14901-14904. 10. Alimoradi, H.; Matikonda, S. S.; Gamble, A. B.; Giles, G. I.; Greish, K. Hypoxia responsive drug delivery systems in tumor therapy. Curr Pharm Design 2016, 22, (19), 2808-20. 11. Liu, H.; Zhang, Y.; Xie, Y.; Cai, Y.; Li, B.; Li, w.; Zeng, L.; Li, Y. L.; Yu, R. Hypoxia-responsive ionizable liposome delivery siRNA for glioma therapy. Int J Nanomed 2017, 12, 1065-1083. 12. Du, J.-b.; Cheng, Y.; Teng, Z.-h.; Huan, M.-l.; Liu, M.; Cui, H.; Zhang, B.-l.; Zhou, S.-y. pH-triggered surface charge reversed nanoparticle with active targeting to enhance the antitumor activity of doxorubicin. Mol Pharmaceut 2016, 13, (5), 1711-1722. 13. Rao, W.; Wang, H.; Han, J.; Zhao, S.; Dumbleton, J.; Agarwal, P.; Zhang, W.; Zhao, G.; Yu, J.; Zynger, D. L.; Lu, X.; He, X. Chitosan-decorated doxorubicin-encapsulated nanoparticle targets and eliminates tumor reinitiating cancer stem-like cells. ACS Nano 2015, 9, (6), 5725-5740. 25 ACS Paragon Plus Environment


Page 26 of 29

14. Michaela, F.; Jaromir, G.; Tibor, S.; Martina, R.; Anna, S.; Marketa, V.; Vojtech, A.; Petr, B.; Marie, N.; Michal, M. Reduction of doxorubicin-induced cardiotoxicity using nanocarriers: A review. Curr Drug Metab 2017, 18, (3), 237-263. 15. Hillard, E. A.; Vessières, A.; Jaouen, G., Ferrocene functionalized endocrine modulators as anticancer agents. Medicinal Organometallic Chemistry 2010, 81-117. 16. Nguyen, A.; Vessières, A.; A. Hillard, E.; Top, S.; Pigeon, P.; Jaouen, G., Ferrocifens and ferrocifenols as new potential weapons against breast cancer. Chimia 2007, 9, (61), 716-724. 17. W. Neuse, E., Macromolecular ferrocene compounds as cancer drug models. J Inorg Organomet P 2005, 15, 3-31. 18. Ma, Y.; Dong, W. F.; Hempenius, M. A.; Mohwald, H.; Vancso, G. J. Redox-controlled molecular permeability of composite-wall microcapsules. Nat Mater 2006, 5, (9), 724-729. 19. Peng, F.; Li, G.; Liu, X.; Wu, S.; Tong, Z. Redox-responsive gel-sol/sol-gel transition in poly(acrylic acid) aqueous solution containing Fe(III) ions switched by light. J Am Chem Soc 2008, 130, (48), 16166-16167. 20. Long, B.; Liang, S.; Xin, D.; Yang, Y.; Xiang, J. Synthesis, characterization and in vitro antiproliferative activities of new 13-cis-retinoyl ferrocene derivatives. Eur J Med Chem 2009, 44, (6), 2572-2576. 21. Manosroi, J.; Rueanto, K.; Boonpisuttinant, K.; Manosroi, W.; Biot, C.; Akazawa, H.; Akihisa, T.; Issarangporn, W.; Manosroi, A. Novel ferrocenic steroidal drug derivatives and their bioactivities. J Med Chem 2010, 53, (10), 3937-3943. 22. Nakahata, M.; Takashima, Y.; Yamaguchi, H.; Harada, A. Redox-responsive self-healing materials formed from host–guest polymers. Nat Commun 2011, 2, 511. 23. Beasley, C. A.; Murray, R. W. Voltammetry and redox charge storage capacity of ferrocene-functionalized silica nanoparticles. Langmuir 2009, 25, (17), 10370-10375. 24. Chang, Y.; Yang, K.; Wei, P.; Huang, S.; Pei, Y.; Zhao, W.; Pei, Z. Cationic vesicles based on amphiphilic pillar[5]arene capped with ferrocenium: A redox‐responsive system for drug/siRNA co‐delivery. Angew Chem Int Ed Engl 2014, 53, (48), 13126-13130. 25. van Staveren, D. R.; Metzler-Nolte, N. Bioorganometallic chemistry of ferrocene. Chem Rev 2004, 104, (12), 5931-5986. 26. Jewell, C. M.; Hays, M. E.; Kondo, Y.; Abbott, N. L.; Lynn, D. M. Chemical 26 ACS Paragon Plus Environment

Page 27 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


activation of lipoplexes formed from DNA and a redox-active, ferrocene-containing cationic lipid. Bioconjugate Chem 2008, 19, (11), 2120-2128. 27. Hu, K.; Zhou, H.; Liu, Y.; Liu, Z.; Liu, J.; Tang, J.; Li, J.; Zhang, J.; Sheng, W.; Zhao, Y.; Wu, Y.; Chen, C.

Hyaluronic acid functional amphipathic and

redox-responsive polymer particles for the co-delivery of doxorubicin and cyclopamine to eradicate breast cancer cells and cancer stem cells. Nanoscale 2015, 7, (18), 8607-8618. 28. Knüpfer, M. M.; Poppenborg, H.; Hotfilder, M.; Kühnel, K.; Wolff, J. E. A.; Domula, M. CD44 expression and hyaluronic acid binding of malignant glioma cells. Clin Exp Metastas 1999, 17, 71-76. 29. Liu, L.; Cao, F.; Liu, X.; Wang, H.; Zhang, C.; Sun, H.; Wang, C.; Leng, X.; Song, C.; Kong, D.; Ma, G. Hyaluronic acid-modified cationic lipid–PLGA hybrid nanoparticles as a nanovaccine induce robust humoral and cellular immune responses. ACS Appl Mater Inter 2016, 8, (19), 11969-11979. 30. Zhao, Q.; Geng, H.; Wang, Y.; Gao, Y.; Huang, J.; Wang, Y.; Zhang, J.; Wang, S.

Hyaluronic acid oligosaccharide modified redox-responsive mesoporous silica

nanoparticles for targeted drug delivery. ACS Appl Mater Inter 2014, 6, (22), 20290-20299. 31. Bae, K. H.; Tan, S.; Yamashita, A.; Ang, W. X.; Gao, S. J.; Wang, S.; Chung, J. E.; Kurisawa, M. Hyaluronic acid-green tea catechin micellar nanocomplexes: Fail-safe cisplatin nanomedicine for the treatment of ovarian cancer without off-target toxicity. Biomaterials 2017, 148, 41-53. 32. Sun, H.; V Benjaminsen, R.; Almdal, K.; Andresen, T., Hyaluronic acid immobilized polyacrylamide nanoparticle sensors for CD44 receptor targeting and pH measurement in cells. Bioconjugate Chem 2012, 23, (11), 2247-2255. 33. Sun, Q.; Kang, Z.; Xue, L.; Shang, Y.; Su, Z.; Sun, H.; Ping, Q.; Mo, R.; Zhang, C. A collaborative assembly strategy for tumor-targeted siRNA delivery. J Am Chem Soc 2015, 137, (18), 6000-6010. 34. Liu, H.; Xie, Y.; Zhang, Y.; Cai, Y.; Li, B.; Mao, H.; Liu, Y.; Lu, J.; Zhang, L.; Yu, R. Development of a hypoxia-triggered and hypoxic radiosensitized liposome as a doxorubicin carrier to promote synergetic chemo-/radio-therapy for glioma. Biomaterials 2017, 121, 130-143. 35. Mo, R.; Jiang, T.; Disanto, R.; Tai, W.; Gu, Z., ATP-triggered anticancer drug 27 ACS Paragon Plus Environment


Page 28 of 29

delivery. Nat Commun 2014, 5, 3364. 36. Yao, H.; Qiu, H.; Shao, Z.; Wang, G.; Wang, J.; Yao, Y.; Xin, Y.; Zhou, M.; Wang, A. Z.; Zhang, L. Nanoparticle formulation of small DNA molecules, Dbait, improves the sensitivity of hormone-independent prostate cancer to radiotherapy. Nanomed-Nanotechnol 2016, 12, (8), 2261-2271. 37. Hong, R.; Han, G.; Fernandez, J. M.; Kim, B. J.; Forbes, N. S.; Rotello, V. M. Glutathione-mediated delivery and release using monolayer protected nanoparticle carriers. J Am Chem Soc 2006, 128, (4), 1078-9. 38. Draffin, J. E.; McFarlane, S.; Hill, A.; Johnston, P. G.; Waugh, D. J. CD44 potentiates the adherence of metastatic prostate and breast cancer cells to bone marrow endothelial cells. Cancer Res 2004, 64, (16), 5702-11. 39. Fan, H.; Yan, G.; Zhao, Z.; Hu, X.; Zhang, W.; Liu, H.; Fu, X.; Fu, T.; Zhang, X. B.; Tan, W. A smart photosensitizer-manganese dioxide nanosystem for enhanced photodynamic therapy by reducing glutathione levels in cancer cells. Angew Chem Int Ed Engl 2016, 55, (18), 5477-82.


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82x44mm (300 x 300 DPI)

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Delivery of Doxorubicin from Hyaluronic Acid-Modified Glutathione

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