Anti-Trop2 antibody-conjugated bioreducible nanoparticles for targeted triple negative breast cancer therapy

Accepted Manuscript Title: Trop2 antibody-conjugated bioreducible nanoparticles for targeted triple negative breast cancer therapy Authors: Soyoung Son, Sol Shin, Vijayakameswara Rao, Wooram Um, Jueun Jeon, Hyewon Ko, V.G. Deepagan, Seunglee Kwon, Jun Young Lee, Jae Hyung Park PII: DOI: Reference:

S0141-8130(17)32831-3 https://doi.org/10.1016/j.ijbiomac.2017.10.113 BIOMAC 8408

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

31-7-2017 30-9-2017 17-10-2017

Please cite this article as: Soyoung Son, Sol Shin, Vijayakameswara Rao, Wooram Um, Jueun Jeon, Hyewon Ko, V.G.Deepagan, Seunglee Kwon, Jun Young Lee, Jae Hyung Park, Trop2 antibody-conjugated bioreducible nanoparticles for targeted triple negative breast cancer therapy, International Journal of Biological Macromolecules https://doi.org/10.1016/j.ijbiomac.2017.10.113 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

(Manuscript for International Journal of Biological Macromolecules) Invited Research Article for a Special Issue: BMDIT-2017

Trop2 antibody-conjugated bioreducible nanoparticles for targeted triple negative breast cancer therapy Soyoung Sona,1, Sol Shina,1, Vijayakameswara Rao b, Wooram Uma, Jueun Jeonb, Hyewon Koa, V. G. Deepaganb, Seunglee Kwonb, Jun Young Leeb, Jae Hyung Parka,b,* a

Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University,

Suwon 14619, Republic of Korea b

School of Chemical Engineering, College of Engineering, Sungkyunkwan University,

Suwon 14619, Republic of Korea 1

These authors contributed equally to this work

*Corresponding author: Jae Hyung Park, Ph.D. School of Chemical Engineering College of Engineering Sungkyunkwan University, Suwon 14619, Republic of Korea Tel: +82-31-290-7288; fax: +82-31-299-6857; e-mail: [email protected]

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Abstract Trop2, a transmembrane glycoprotein, has emerged as a biomarker for targeted cancer therapy since it is overexpressed in 80% of triple negative breast cancer (TNBC) patients. For the site-specific delivery of the anticancer drug into TNBC, Trop2 antibody-conjugated nanoparticles (ST-NPs) were prepared as the potential nanocarrier, composed of carboxymethyl dextran (CMD) derivatives with bioreducible disulfide bond. Owing to its amphiphilicity, the CMD derivatives were self-assembled into nano-sized particles in an aqueous condition. Doxorubicin (DOX), chosen as a model anticancer drug, was effectively encapsulated into the nanoparticles. DOX-loaded ST-NPs (DOX-ST-NPs) rapidly released DOX in the presence of 10 mM glutathione (GSH), whereas the DOX release is significantly retarded in the physiological condition (PBS, pH 7.4). Confocal microscopic images and flow cytometry analysis demonstrated that DOX-ST-NPs were selectively taken up by MDA-MB-231 as the representative Trop2-expressing TNBC cells. Consequently, DOX-ST-NPs exhibited higher toxicity to Trop2-positive MDA-MB-231 cancer cells, compared to DOX-loaded control nanoparticles without the disulfide bond or Trop2 antibody. Overall, ST-NPs might be a promising carrier of DOX for targeted TNBC therapy. Keywords: Triple negative breast cancer; Trop2 antibody; bioreducible nanoparticle

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1. Introduction Breast cancer has become the most common cancer as well as the second leading cause of death in women worldwide [1]. Among the subtypes of the breast cancer, triple negative breast cancer (TNBC) is the most aggressive one, characterized by lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) [2-6]. Therefore, TNBC is not effectively treated by the conventional chemotherapy, primarily ascribed to lack of the appropriate target. Although limited success was demonstrated by combination of multiple agents [7] and epidermal growth factor receptor-targeted antibody [8], there is an urgent need to develop the carrier which can effectively deliver the drug into the site of action in TNBC. Over the past two decades, numerous nanocarriers have been developed for targeted drug delivery for cancer therapy in forms of liposomes, dendrimers, micelles, and polymer-drug conjugates [9-12]. Owing to the enhanced permeability and retention effect by the leaky vasculature and lack of lymphatic drainage, nanocarriers have exhibited preferential accumulation into the tumor tissue after their systemic administration, implying their potential for targeted delivery of the drug [13,14]. To facilitate the site-specific release of anticancer drugs into tumor tissues, several stimuli-responsive nanocarriers have been extensively investigated [15-19]. They are designed to exhibit physicochemical alterations by responding to internal or external stimuli such as pH, hypoxia, glutathione, and temperature [20-23]. In particular, glutathione (GSH), a thiol-containing tripeptide capable of reducing disulfide bonds, has received increasing attention to develop the nanocarriers for the intracellular drug delivery since it is abundant in the cytoplasm of the cell (1-10 mM) [24,25]. Moreover, the GSH concentrations in cancer cells of pathological sites have been demonstrated to be at least 4-fold higher than that of 3

normal tissues [26]. This has encouraged the development of bioreducible nanoparticles for the GSH-triggered drug release [27]. For the effective therapy of TNBC, it is obviously required to develop the nanocarriers which specifically recognize the tumor cells. Trop2, a 46-kD transmembrane glycoprotein involving numerous intracellular signaling pathways, has emerged as a biomarker for targeted cancer therapy since it is overexpressed in several cancers compared to normal tissue [28-30]. Of note, Trop2 is expressed in 80% of TNBC patients, indicating that Trop2 is attractive target for TNBC therapy. In this study, we developed Trop2 antibody-conjugated bioreducible nanoparticles (ST-NPs) for active targeting TNBC, allowing for rapid drug release at the intracellular compartment via the reduction of disulfide bond (Fig. 1). As controls, the GSHsensitive nanoparticles without Trop2 antibody (SS-NPs), GSH-insensitive nanoparticles without Trop2 antibody (CA-NPs), and GSH-insensitive nanoparticles with Trop2 antibody (CT-NPs) were also prepared to investigate the potential of Trop2 antibody and bioreducible bond for TNBC-targeted therapy. The physicochemical properties of ST-NPs were characterized using various instruments including 1H NMR, transmission electron microscopy (TEM), and dynamic light scattering (DLS). The anticancer drug, doxorubicin (DOX), was physically encapsulated into nanoparticles by the emulsion method, and its GSH-dependent release behavior was assessed as a function of time. Also, Trop2-mediated endocytosis by TNBC cells was evaluated using flow cytometry analysis. 2. Materials and methods 2.1. Materials Carboxymethyl dextran sodium salt (CMD, Mn=10-20 kDa, the carboxyl content = 1.1-1.5 mmol g-1), 5β-cholanic acid (CA), 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC), 4

dicyclohexyl

carbodiimide

(DCC),

N-hydroxysuccinimide

(NHS),

ethylenediamine,

doxorubicin·hydrochloride (DOX·HCl), and trimethylamine were purchased from SigmaAldrich Co. (St. Louis, MO, USA). Cystamine·dihydrochloride was obtained from Tokyo Chemical Industries (Tokyo, Japan). N-hydroxysulfosuccinimide (sulfo-NHS) was purchased from Thermo Scientific (Rockford, IL, USA). Fetal bovine serum (FBS), RPMI 1640, Dulbecco's phosphate buffered saline (DPBS), antibiotic-antimycotic (AA) solution, and trypsin-EDTA were purchased from WelGENE (Seoul, Korea). Monoclonal anti-Trop2 antibody was obtained from BD Biosciences Pharmingen (San Diego, CA, USA). The water used in the experiments was prepared by an AquaMax-Ultra water purification system (Younglin Co., Anyang, Korea). All other chemicals and reagents were of analytical grade and used without further purification. 2.2. Characterization The chemical structures of the conjugates were characterized using 1H NMR (Palo Alto, CA, USA) operating at 500 MHz. D2O and CD3OD were used as the solvents. The hydrodynamic sizes, size distribution, and zeta potentials of nanoparticles were measured using a Zetasizer Nano ZS90 (Malvern Instruments, Worcestershire, UK) with a He-Ne laser (633 nm) at 90 collecting optics. TEM (JEOL-2100F, Tokyo, Japan) and field emission scanning electron microscopy (FESEM, JEOL-7600F, Tokyo, Japan) images were obtained to observe the morphology of nanoparticles. For TEM analysis, nanoparticles were dropped on a 400-mesh copper grid and operated at an accelerating voltage of 200 kV. For FE-SEM analysis, dried samples were directly mounted onto double-sided carbon tape and operated at 15 kV. 2.3. Synthesis of CMD-SS-CA conjugate Bioreducible nanoparticles were synthesized in a two-step process (Fig. 2). In the first step, CA derivatives were synthesized as reported previously [25]. CA (1 g, 2.8 mmol) was dissolved 5

in tetrahydrofuran (25 ml), followed by the addition of DCC (0.74 g, 3.6 mmol) and NHS (0.41 g, 3.6 mmol) at room temperature. The reaction mixture was stirred for 12 h under a nitrogen atmosphere, and then the precipitated dicyclohexylurea was removed by filtration. The filtrate was precipitated in hexane, filtered, and dried at room temperature under vacuum to obtain NHSactivated CA (CA-NHS). The disulfide-containing CA derivative (CA-SS-NH2) was synthesized by reacting CA with cystaminedihydrochloride. In brief, CA-NHS (1 g, 2.1 mmol) was dissolved in 5 ml of DMF, and the solution was mixed with cystaminedihydrochloride (4.7 g, 21 mmol) in triethylamine. The reaction was continued for 6 h at room temperature, and the product was precipitated in deionized water, filtered, and dried under vacuum. CA-NH2, chosen as the control without the disulfide bond, was prepared by reacting CA-NHS (1 g, 2.1 mmol) with ethylenediamine (12.6 g, 210 mmol) in an identical method. In the second step, the CA derivatives were conjugated to the CMD backbone through amide formation. In brief, EDC (0.128 g, 0.66 mmol) and NHS (0.09 g, 0.66 mmol) were dissolved in the CMD (0.2 g, 0.84 mmol) solution in a mixture of formamide and dimethyl formamide. To prepare the CMD-SS-CA conjugates, CA-SS-NH2 (0.026 g, 0.05 mmol) in DMF was added to the reaction mixture and allowed to stir for one day at room temperature. The resulting solution was dialyzed (MWCO = 6-8 kDa) against an excess amount of distilled water/methanol (1/3-1/1 v/v) for 1 day and against distilled water for 2 days, followed by lyophilization. As a control sample, the CMD-g-CA conjugate without the disulfide bond was prepared by the chemical conjugation of CA-NH2 to CMD in an identical manner. 2.4. Conjugation of Trop2 antibody to nanoparticle Antibody-conjugated nanoparticles were prepared through the EDC chemistry. The carboxyl group of the conjugate in 1 ml PBS (pH 6.5, 10 mg/ml) was activated with EDC (10 μl, 6

200 mM) and sulfo-NHS (100 μl, 200 mM) for 30 min at room temperature. Monoclonal Trop2 antibody (12 μl, 0.5 mg/ml) was added to the solution and incubated for 2 h under gentle stirring at 4 °C. Purification was carried out by using Zeba spin desalting columns (7K MWCO, Thermo Scientific, Rockford, IL, USA) in PBS (pH 7.4). 2.5. Preparation of DOX-loaded nanoparticles DOX-loaded nanoparticles were prepared by the emulsion method. In brief, DOXHCl (3 mg) was dissolved in chloroform with 3.0 equimolar amount of triethylamine. The resulting solution was added to the aqueous solution of SS-NPs (30 mg), leading to the formation of an oil-in-water emulsion. This emulsion was kept in the dark condition overnight under stirring to allow evaporation of chloroform, and the solution was dialyzed (MWCO = 6-8 kDa) for 12 h against distilled water. The resulting solution was then filtered through a 0.8 μm syringe filter to remove unloaded DOX, followed by lyophilization to obtain DOX-loaded bioreducible nanoparticles (DOX-SS-NPs). The same method was applied to prepare the DOX-loaded control sample without the disulfide bond (DOX-CA-NPs). For the preparation of DOX-loaded targeted nanoparticles (DOX-CT-NPs and DOX-ST-NPs), Trop2 antibody was conjugated with the carboxyl group of DOX-loaded nanoparticles through the EDC/sulfo-NHS coupling reaction. The content and loading efficiency of DOX in the nanoparticles were determined by measuring the absorbance at 480 nm using a UV-Vis spectrophotometer (Optizen 33320, Mecasys Inc., Korea). For this experiment, a calibration curve was obtained using DOX solutions at different concentrations in DMSO/deionized water (3v/1v). The loading efficiency (LE) and loading content (LC) were calculated according to the following formulae: LE (%) = (weight of the loaded drug/ weight of drug in feed) × 100 LC (%) = (weight of the loaded drug/ weight of the polymer) × 100 7

2.6. In vitro drug release The in vitro drug release behaviors of DOX-CA-NPs, DOX-SS-NPs, DOX-CT-NPs, and DOX-ST-NPs were investigated in the absence and presence of 10 mM GSH in PBS (pH 7.4). Each sample was gently shaken at 37 °C and 100 rpm. Briefly, DOX-loaded nanoparticles (1 mg/ml) were placed in a dialysis membrane bag (MWCO = 1 kDa). Next, the membrane bag was immersed in PBS (pH 7.4) with or without 10 mM GSH. At predetermined time intervals, the medium was refreshed by transferring the tube to fresh release medium, and the DOX concentration was measured using a UV-Vis spectrophotometer at 480 nm. Release experiments were conducted in triplicate. 2.7. In vitro cellular uptake MDA-MB-231 cells, Trop2-expressing TNBC cell line, were obtained from the American Type Culture Collection (Rockville, MD, USA). HCC-1395 cells, defined as Trop2-negative TNBC line, were obtained from the Seoul National University Cell Bank (Seoul, Korea). MDAMB-231 cells and HCC-1395 cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% antibiotics. The TNBC cells were maintained at 37 °C in a humidified atmosphere with 5% CO2. To observe the cellular uptake and intracellular drug release behavior, TNBC cells were seeded onto cover slips in 6-well plates (1  105 cells per well) and incubated for 24 h in 2 ml medium with 10% FBS and 1% AA. Thereafter, the cells were treated with DOX-loaded nanoparticles (DOX dose = 5 μg/ml) and incubated for 30 min at 37 °C in a humidified 5% CO 2containing atmosphere. The cells were then washed twice with DPBS and fixed with 4% paraformaldehyde solution, followed by staining the cell nucleus using 4,6-diamidno-2phenylindole (DAPI). Fixed cells were monitored using a confocal microscope (LSM 510 META 8

NLO, Heidelberg, Germany).

The cellular uptake of DOX-loaded nanoparticles was also

quantified through flow cytometric analysis. Harvested cells were suspended in DPBS and centrifuged at 1500 rpm for 3 min. Cell pellets were washed twice with DPBS and resuspended in 300 μl DPBS, followed by analysis using flow cytometry (guava easyCyte, EMD Millipore, USA) with laser excitation at 488 nm. Untreated cells were used as a control. 2.8. In vitro cytotoxicity The cytotoxicity of bare nanoparticles and DOX-loaded nanoparticles was evaluated using the MTT assay. MDA-MB-231 cells (1  104 cells per well) and HCC-1395 cells (2  104 cells per well) were seeded in 96-well plates and incubated for 24 h. Subsequently, bare nanoparticles were incubated with MDA-MB-231 cells for 24 h and HCC-1395 cells for 72 h. For DOX-loaded nanoparticles, MDA-MB-231 and HCC-1395 cells were treated at different concentrations of DOX for 15h and 70h, respectively. Then, the medium in each plate was replaced with medium containing 10% MTT solution (5 mg/ml in PBS), followed by incubation for 2 h. After removal of the medium, the MTT crystals were dissolved in 200 μl of DMSO. Cell viability was determined by measuring the absorbance at 570 nm using a microplate reader (BioTek, Seoul, Korea). 3. Results and discussion 3.1. Synthesis and characterization of conjugates Amphiphilic CMD-based conjugates were prepared by the chemical conjugation of hydrophobic CA derivatives to the hydrophilic CMD backbone. First, the carboxyl group of CA was converted to amine group in the presence of cystamine or ethylenediamine, resulting in the formation of CA-SS-NH2 or CA-NH2, respectively. The amine-functionalized CA derivatives were chemically conjugated to CMD in the presence of EDC and NHS to prepare CMD-SS-CA 9

and CMD-g-CA conjugates. The chemical structures of the conjugates were confirmed using 1H NMR (Fig. 3). The characteristic peaks of CA appeared at 0.6-1.8 ppm and the CMD peaks were observed at 3.41-4.29 ppm, indicating the formation of amphiphilic conjugates. The degree of substitutions (DSs) of CA, defined as the number of CA per 100 CMD repeating units, were 3.46 and 4.12 for CMD-g-CA and CMD-SS-CA, respectively (Table 1). DS was determined by the integration ratio of the proton peak from the methyl group of CA (0.70 ppm) and that from the carboxymethylene proton of CMD (1.02 ppm). Owing to their amphiphilic properties, CMD-g-CA and CMD-SS-CA could be selfassembled into GSH-insensitive nanoparticles (CA-NPs) and GSH-sensitive bioreducible nanoparticles (SS-NPs), respectively. FE-SEM images revealed that both nanoparticles were spherical with unimodal size distributions (Fig. 4(a)). The mean diameters of CA-NPs and SSNPs, determined using DLS, were 169 nm and 187 nm, respectively. As shown in Fig. 4(b), the stability of CA-NPs and SS-NPs were examined by measuring the size of nanoparticles in PBS (pH 7.4) as a function of time. No significant changes in sizes for both nanoparticles were observed for 5 days. When Trop2 antibody was conjugated onto the nanoparticles, their sizes slightly increased without affecting their stability (Figs. 4(c) and (d)). The zeta potentials of all the CMD-based nanoparticles were negative, indicating the presence of the carboxyl groups of CMD on the nanoparticular surface (Table 1). It was also found that the zeta potential values of nanoparticles decreased after conjugation of negatively charged Trop2 antibody. 3.2 GSH-triggered drug release behavior DOX, a model anticancer drug for TNBC, was encapsulated into the nanoparticles by an emulsion method. The physicochemical characteristics of DOX-loaded nanoparticles were summarized in Table 2. For both of DOX-CA-NPs and DOX-SS-NPs, the loading efficiencies of 10

DOX were higher than 75% and their particle sizes were slightly larger than bare nanoparticles without DOX. To evaluate the effect of GSH on drug release, the release behaviors of DOX from nanoparticles were monitored for 2 days. As shown in Fig. 5, all the nanoparticles released DOX in a sustained manner under the physiological condition (0 mM GSH). On the other hand, DOX was rapidly released from the bioreducible nanoparticles (DOX-SS-NPs and DOX-ST-NPs) in PBS containing 10 mM GSH, mimicking reductive environment of the intracellular level. Specifically, 96% and 92% of DOX were released from DOX-SS-NPs and DOX-ST-NPs within 48 h, respectively. These results suggest that bioreducible nanoparticles containing the disulfide bond can rapidly release the drug, responding to the GSH level.

3.3. In vitro cellular uptake and intracellular release of DOX To investigate the effect of Trop2 antibody as a TNBC-targeting ligand, cellular uptake of DOX-loaded nanoparticles was examined using confocal microscopy after an incubation with Trop2-positive TNBC cell line (MDA-MB-231) and Trop2-negative TNBC cell line (HCC1395). As demonstrated in Fig. 6(a), for Trop2 antibody-conjugated nanoparticles (DOX-CTNPs and DOX-ST-NPs), strong fluorescences were observed in MDA-MB-231 cells. On the other hand, Trop2-negative HCC-1395 cells, treated with targeted and non-targeted nanoparticles, did not exhibit a noticeable difference in DOX fluorescence (Fig. 6(b)). These results suggest that Trop2 antibody can effectively target Trop2-expressing TNBC. Furthermore, bioreducible nanoparticles (DOX-SS-NPs and DOX-ST-NPs) exhibited higher fluorescence in the nucleus for both MDA-MB-231 and HCC-1395 cells than the reduction-insensitive nanoparticles (DOX-CA-NPs and DOX-CT-NPs). These results imply that DOX-SS-NPs and

11

DOX-ST-NPs are responsive to GSH at the intracellular level, leading to effective release of DOX. The cellular uptake of DOX was also quantitatively investigated using flow cytometry, in which cells without any DOX treatment were used as the negative control. For Trop2-positive MDA-MB-231 cells, the highest fluorescence intensity was found for DOX-ST-NPs (Fig. 7(a)). In particular, compared to DOX-SS-NPs, DOX-ST-NPs exhibited 3.1-fold higher fluorescence intensity, indicating that Trop2 antibody in DOX-ST-NPs plays an important role in cellular uptake. Unlike Trop2-expressing cells, HCC-1395 cells showed negligible difference in fluorescence intensity among the samples tested (Fig. 7(b)). These results indicated that Trop2 antibody can be an effective target for Trop2-positive TNBC. 3.4. In vitro cytotoxicity assay Cell viability of bare nanoparticles, free DOX and DOX-loaded nanoparticles was analyzed by the MTT assay. Owing to their biocompatibility, bare nanoparticles exhibited no significant cytotoxicity to both MDA-MB-231 and HCC-1395 cells. On the other hand, all the DOX-loaded nanoparticles showed dose-dependent cytotoxicity to both TNBC cells (Fig. 8). Interestingly, the cytotoxicity of GSH-sensitive nanoparticles was significantly greater than that of GSHinsensitive nanoparticles, which might be due to the rapid release of DOX by the cleavage of the disulfide bond at the intracellular environment. It should be emphasized that DOX-ST-NPs elicited the strongest cytotoxic effects for MDA-MB-231 cells, compared to DOX-SS-NPs, DOX-CT-NPs, and DOX-CA-NPs. On the other hand, for HCC-1395 cells, cytotoxicity of DOXST-NPs was not significantly different from that of DOX-SS-NPs. Overall, it was evident that enhanced cytotoxic effect of DOX-ST-NP was due to the specific binding to Trop2-positive TNBC cells, followed by rapid DOX release triggered at the intracellular reducing environment. 12

4. Conclusion In this study, we investigated the potential of Trop2 antibody-conjugated bioreducible nanoparticles as the carrier of DOX. The bioreducible nanoparticles, based on the disulfidebearing CMD conjugate, exhibited GSH-dependent release of DOX. It was demonstrated that DOX-ST-NPs could specifically target Trop2-expressing TNBC cells and rapidly release DOX via the reduction of disulfide bond in an intracellular environment. Therefore, ST-NPs might be a promising nanocarrier for targeted TNBC therapy.

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Acknowledgments This work was financially supported by the Global Research Laboratory Program (NRF2016K1A1A2A02942563) and the Basic Science Research Programs (20100027955 and 2015R1A2A2A05001390) of NRF. Declaration of interest 'Conflicts of interest: none'. References [1] F. André, C.C. Zielinski, Optimal strategies for the treatment of metastatic triple-negative breast cancer with currently approved agents, Ann. Oncol. 23 (2012) 46-51. [2] C.L. Griffiths, J.L. Olin, Triple Negative Breast Cancer: A Brief Review of its Characteristics and Treatment Options, J. Pharm. Pract. 25 (2012) 319-323. [3] W.J. Irvin, L.A. Carey, What is triple-negative breast cancer?, Eur. J. Cancer 44 (2008) 27992805. [4] D.G. Song, Q. Ye, M. Poussin, J.A. Chacon, M. Figini, D.J. Powell, Effective adoptive immunotherapy of triple-negative breast cancer by folate receptor-alpha redirected CAR T cells is influenced by surface antigen expression level, J. Hematol Oncol. 9 (2016) 56. [5] S.K. Pal, B.H. Childs, M. Pegram, Triple negative breast cancer: unmet medical needs, Breast Cancer Res. Treat. 125 (2011) 627-636. [6] R.V. Kutty, S.-S. Feng, Cetuximab conjugated vitamin E TPGS micelles for targeted delivery of docetaxel for treatment of triple negative breast cancers, Biomaterials 34 (2013) 10160-10171. [7] J.M. Miller-Kleinhenz, E.N. Bozeman, L. Yang, Targeted Nanoparticles for Image-guided Treatment of Triple Negative Breast Cancer: Clinical Significance and Technological Advances, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 7(6) (2015) 797-816. 14

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[17] S. Aleksanian, B. Khorsand, R. Schmidt, J.K. Oh, Rapidly thiol-responsive degradable block copolymer nanocarriers with facile bioconjugation, Polym. Chem. 3 (2012) 2138-2147. [18] T. Thambi, H.Y. Yoon, K. Kim, I.C. Kwon, C.K. Yoo, J.H. Park, Bioreducible Block Copolymers Based on Poly(Ethylene Glycol) and Poly(γ-Benzyl l-Glutamate) for Intracellular Delivery of Camptothecin, Bioconjugate Chem. 22 (2011) 1924-1931. [19] H.S. Han, K.Y. Choi, H. Lee, M. Lee, J.Y. An, S. Shin, S. Kwon, D.S. Lee, J.H. Park, GoldNanoclustered Hyaluronan Nano-Assemblies for Photothermally Maneuvered Photodynamic Tumor Ablation, ACS Nano 10 (2016) 10858-10868. [20] V. Rao N, S. Mane, A. Kishore, J. Das Sarma, R. Shunmugam, Norbornene Derived Doxorubicin Copolymers as Drug Carriers with pH Responsive Hydrazone Linker, Biomacromolecules 13 (2012) 221-230. [21] V. Rao N, M.N. Ganivada, S. Sarkar, H. Dinda, K. Chatterjee, T. Dalui, J. Das Sarma, R. Shunmugam, Magnetic Norbornene Polymer as Multiresponsive Nanocarrier for Site Specific Cancer Therapy, Bioconjugate Chem. 25 (2014) 276-285. [22] T. Thambi, S. Son, D.S. Lee, J.H. Park, Poly(ethylene glycol)-b-poly(lysine) copolymer bearing nitroaromatics for hypoxia-sensitive drug delivery, Acta Biomater. 29 (2016) 261-270. [23] S. Qin, Y. Geng, D.E. Discher, S. Yang, Temperature-Controlled Assembly and Release from Polymer Vesicles of Poly(ethylene oxide)-block- poly(N-isopropylacrylamide), Adv. Mater. 18(21) (2006) 2905-2909. [24] A.N. Koo, H.J. Lee, S.E. Kim, J.H. Chang, C. Park, C. Kim, J.H. Park, S.C. Lee, Disulfidecross-linked PEG-poly(amino acid)s copolymer micelles for glutathione-mediated intracellular drug delivery, Chem. Commun. 0(48) (2008) 6570-6572.

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[25] R. Hong, G. Han, J.M. Fernández, B.-j. Kim, N.S. Forbes, V.M. Rotello, GlutathioneMediated Delivery and Release Using Monolayer Protected Nanoparticle Carriers, J. Am. Chem. Soc. 128(4) (2006) 1078-1079. [26] K. Miura, T.W. Clarkson, Reduced Methylmercury Accumulation in a MethylmercuryResistant Rat Pheochromocytoma PC12 Cell Line, Toxicol. Appl. Pharmacol. 118(1) (1993) 3945. [27] T. Thambi, D.G. You, H.S. Han, V.G. Deepagan, S.M. Jeon, Y.D. Suh, K.Y. Choi, K. Kim, I.C. Kwon, G.-R. Yi, J.Y. Lee, D.S. Lee, J.H. Park, Bioreducible Carboxymethyl Dextran Nanoparticles for Tumor-Targeted Drug Delivery, J. Control. Release 3 (2014) 1829-1838. [28] D.M. Goldenberg, T.M. Cardillo, S.V. Govindan, E.A. Rossi, R.M. Sharkey, Trop-2 is a novel target for solid cancer therapy with sacituzumab govitecan (IMMU-132), an antibody-drug conjugate (ADC), Oncotarget 6 (2015) 22496-22512. [29] A. Shvartsur, B. Bonavida, Trop2 and its overexpression in cancers: regulation and clinical/therapeutic implications, Genes Cancer 6 (2015) 84-105. [30] T.M. Cardillo, S.V. Govindan, R.M. Sharkey, P. Trisal, R. Arrojo, D. Liu, E.A. Rossi, C-H. Chang, D.M. Goldenberg, Sacituzumab Govitecan (IMMU-132), an Anti-Trop-2/SN-38 Antibody-Drug Conjugate: Characterization and Efficacy in Pancreatic, Gastric, and Other Cancers, Bioconjugate Chem. 26(5) (2015) 919-931. Table 1. Physicochemical characteristics of nanoparticles Sample

Size (nm)a)

PDIb)

Zeta potential [mV]

Feed ratio [%]c)

DS [%]d)

CA-NP

169.2±6.62

0.18

-10.7±0.70

0.05

3.46

SS-NP

187.9±1.27

0.22

-21.1±0.07

0.05

4.12

17

CT-NP

195.1±2.19

0.16

-21.7±1.15

-

-

ST-NP

232.9±6.23

0.20

-25.9±0.92

-

-

a)

Mean diameters were measured using a particle analyzer

b)

Polydispersity index is abbreviated as PDI

c)

Molar feed ratio of the CA to sugar residues of CMD backbone.

d)

Degree of substitution was estimated using 1H NMR

18

Table 2. Characteristics of DOX-loaded nanoparticles DOX

Loading

Loading Size (nm)b)

Sample feed ratio (%)

efficiency (%)

a)

content (%)

a)

DOX-CA-NP

10

78.1

7.8

195.1±2.19

DOX-SS-NP

10

75.2

7.5

202.6±2.33

a)

Determined using UV-Vis spectrophotometer

b)

Determined using the particle analyzer.

19

Figure Caption

Fig.1. Schematic illustration of (a) the preparation of DOX loaded bioreducible nanoparticles conjugated with Trop2 antibody (DOX-ST-NPs) and (b) receptor-mediated endocytosis of DOXST-NP and GSH-responsive drug release.

Fig. 2. Synthetic scheme of CMD-SS-CA and CMD-g-CA conjugates Fig. 3. 1H NMR spectra of (a) CA-NH2 and (b) CA-SS-NH2 in CD3OD. 1H NMR spectra of (c) CMD-SS-CA and (d) CMD-g-CA conjugates in D2O/CD3OD (1v/1v).

Fig. 4. (a) Size distribution and FE-SEM images of CA-NPs and SS-NPs. (b) Stability of CANPs and SS-NPs as a function of time in a PBS (pH 7.4). (c) Size distribution and TEM images of CT-NPs and ST-NPs. (d) Stability of CT-NPs and ST-NPs as a function of time in a PBS (pH 7.4). The error bars in the graph represent standard deviations (n=3)

Fig. 5. In vitro drug release profile of DOX-loaded nanoparticles in the absence and presence of GSH: (a) DOX-CA-NPs and DOX-SS-NPs (b) DOX-CT-NPs and DOX-ST-NPs. The error bars in the graph represent standard deviations (n=3)

Fig. 6. Confocal microscopic images of (a) MDA-MB-231 and (b) HCC-1395 cells incubated with DOX-loaded nanoparticles for 30 min. Nuclei were stained with DAPI (blue).

Fig. 7. Flow cytometry analysis and mean fluorescence intensity (MFI) of DOX in TNBC cells treated with non-targeted nanoparticles (DOX-CA-NPs and DOX-SS-NPs), or targeted nanoparticles (DOX-CT-NPs and DOX-ST-NPs), or medium alone (control) for 30 min at 37 °C (DOX concentration: 5 μg/ml). Representative histogram plots showing the intracellular DOX content and MFI of DOX in (a) MDA-MB-231 and (b) HCC-1395 cells.

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Fig. 8. In vitro cytotoxicity of bare nanoparticles and DOX-loaded nanoparticles. The error bars represent standard deviations (n=3). Asterisks (*) denote statistically significant differences (p<0.05).

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Figure 1. Son et al 22

Figure 2. Son et al

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Figure 3. Son et al 24

Figure 4. Son et al

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Figure 5. Son et al

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Figure 6. Son et al

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Figure 7. Son et al 28

Figure 8. Son et al

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