Tumor Targeting and Lipid Rafts Disrupting Hyaluronic Acid

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Biological and Medical Applications of Materials and Interfaces

Tumor Targeting and Lipid Rafts Disrupting Hyaluronic AcidCyclodextrin-Based Nanoassembled Structure for Cancer Therapy Song Yi Lee, Seung-Hak Ko, Jae-Seong Shim, Dae-Duk Kim, and Hyun-Jong Cho ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b08243 • Publication Date (Web): 09 Oct 2018 Downloaded from http://pubs.acs.org on October 9, 2018

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ACS Applied Materials & Interfaces

Tumor Targeting and Lipid Rafts Disrupting Hyaluronic Acid-Cyclodextrin-Based Nanoassembled Structure for Cancer Therapy Song Yi Lee,† Seung-Hak Ko,‡ Jae-Seong Shim,‡,§ Dae-Duk Kim,ㅗ Hyun-Jong Cho†,*

†College

of Pharmacy, Kangwon National University, Chuncheon, Gangwon 24341, Republic of Korea. ‡Biogenics §Skin

ㅗCollege

Inc., Daejeon 34027, Republic of Korea.

& Tech Inc., Cheongju 28116, Republic of Korea.

of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 08826, Republic of Korea.

KEYWORDS: cancer therapy, cholesterol depletion, cyclodextrin, CD44 receptor,

hyaluronic acid, nanoassembly

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ABSTRACT: CD44 receptor targeting and lipid rafts destroying nanoassembly (NA) was developed for breast cancer therapy. Methyl-β-cyclodextrin (MbCD), as a cholesterol depletion moiety, was conjugated to hyaluronic acid-ceramide (HACE) structure via an ester linkage. HACE-MbCD NA with 198 nm hydrodynamic size, unimodal size distribution, and spherical shape was fabricated by self-assembly property. By filipin III staining, it was identified that HACE-MbCD NA extracted cholesterol from the cellular membrane of MDA-MB-231 (human breast adenocarcinoma) cells more efficiently rather than MbCD and HACE NA. Efficient lipid rafts disruption of HACE-MbCD NA, compared with MbCD and HACE NA groups, seems to lead to the increment in apoptosis and antiproliferation efficiencies in MDA-MB-231 cells. Improvement in tumor targeting efficiency of HACE-MbCD NA, compared with HACE NA, in MDA-MB-231 tumor-bearing mice can be explained by the extraction process of cellular cholesterol by MbCD. Following intravenous injection in MDA-MB-231 tumor-bearing mice, the most efficient suppression of tumor growth and highest apoptotic region were observed in HACEMbCD NA group rather than MbCD group. All these findings suggest that CD44 receptortargetable HACE-MbCD NA, retaining cholesterol depletion activity from cancer cells, may be one of remarkable nanosystems for breast cancer therapy.


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■ INTRODUCTION A lot of strategies regarding cancer diagnosis, tumor-selective drug delivery, and cancer therapy have been suggested.1-5 Precise and elaborated delivery of anticancer agents to tumor tissue can maximize anticancer activities and minimize unwanted toxicities to normal organs and tissues. Solid tumors have defective vascular structure and immature lymphatic system, thus macromolecules (> 40 kDa) can be diffused into the tumor tissue.6 For the efficient extravasation from the leaky vasculature, the particle size of nanocarrier should not exceed 400 nm.7 Increment in the vascular permeability and tumor accumulation has been called as an enhanced permeability and retention (EPR) effect, regarded as a main mechanism for passive tumor targeting.8,9 However, EPR effect-guided passive tumor targeting also has several intrinsic limitations such as tumor vascularization-dependent targeting efficiency and elevated interstitial fluid pressure in solid malignant tumors. To overcome these drawbacks, various types of targeting ligands (e.g., antibodies, nucleic acids, peptides, and small chemical entities) for cancers have been introduced to the surface of nanocarriers.7,10 The interactions between ligand and receptor can improve tumor targeting efficiency and that strategy was known as an active tumor targeting.7 Among various kinds of tumor targeting ligands, hyaluronic acid (HA) has been widely used as one of targeting ligands for CD44 receptor, presented in several kinds of solid tumors.11 HAbased nanocarriers have been widely investigated for cancer diagnosis and therapy.12,13 Particularly, self-assembled HA oligomer-based nanocarriers have been tested for anticancer drug delivery to CD44 receptor-positive cancer and its imaging in our reports.14-23 Amphiphilic hyaluronic acid-ceramide (HACE) conjugate was prepared and its self-assembly and nano-size properties were demonstrated. Moreover, several functional moieties (e.g., dopamine, phenylboronic acid, and poly(ethylene glycol)) to improve tumor directing and infiltrating 3 ACS Paragon Plus Environment


potentials were attached to the HACE structure.16,17,21 Hydrophobic anticancer agents can be loaded in the internal space of nanoparticles composed of HACE or its derivatives. Nano-size related characteristics (for passive targeting) and HA-CD44 receptor interaction (for active targeting) may provide accurate drug delivery efficiency to malignant tumor tissues with less offtargeting effects in normal tissues and organs. With the solubilization capacity of cyclodextrin (CD), it also has been reported as a lipid rafts disrupting molecule and its anticancer activities have been identified.24,25 CD can extract cholesterol from the cell membrane and it can affect downstream signal pathways of apoptosis.24,26 Among CD derivatives, methyl-β-CD (MbCD) exhibited a specific cholesterol-binding affinity without binding or inserting into the plasma membrane.27 CD concentration, exposure period, and cell type may influence on the extraction efficiency of cholesterol.28 However, MbCD can also interact with hydrophobic amino acids and protein domains.28 The interactions between CD and protein may induce hemolysis in the blood stream. Therefore, it is necessary to develop tumor targetable CD for reducing the administration dose, which can prevent hemolysis, as well as increasing therapeutic efficacies against cancers.29 Herein, MbCD was linked to the amphiphilic HACE conjugate and self-assembled MbCDmodified HACE nanoassembly (NA) was prepared. Nano-size properties, interactions between HA and CD44 receptor, and installation of cellular cholesterol capturable MbCD were combined to make single HACE-MbCD NA for improving anticancer activities via inducing an apoptosis with minimal toxicities.

■ EXPERIMENTAL METHODS Materials. Oligo HA (4‒8 kDa of molecular weight (MW) range) was supplied from SK 4 ACS Paragon Plus Environment

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Bioland Co., Ltd. (Cheonan, Republic of Korea). DS-Y30 ceramide (CE) was obtained from Doosan Biotech Co., Ltd. (Yongin, Republic of Korea). Doxorubicin hydrochloride (DOX) was acquired from Boryung Pharmaceutical Co., Ltd. (Seoul, Korea). Dimethyl sulfoxide-d6 (DMSOd6), MbCD, N-hydroxysuccinimide (NHS), N, N´-dicyclohexylcarbodiimide (DCC), N, Ndimethylpyridin-4-amine

(DMAP),

N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide

hydrochloride (EDC), tetra-n-butylammonium hydroxide (TBA), water-d2 (D2O), and 4(chloro)methylbenzoyl chloride were purchased from Sigma–Aldrich (Saint Louis, MO, USA). Cy5.5-NH2 was supplied by Lumiprobe Corp. (Hunt Valley, MD, USA). Phosphate buffered saline (PBS), DMEM, RPMI 1640, fetal bovine serum (FBS), and penicillin/streptomycin manufactured by Gibco Life Technologies, Inc. (Grand Island, NY, USA) were used. Synthesis and Physicochemical Characterization Tests of HACE-MbCD. HACE was synthesized from HA oligomer and CE according to the reported method.14 MbCD was covalently bonded to HACE via an ester linkage in this study. Hydroxyl (‒OH) group of MbCD was linked to the carboxylic acid (‒COOH) group of HA in HACE with the aid of DCC and DMAP. MbCD (262.0 mg), DCC (41.5 mg), and DMAP (6.4 mg) were solubilized in dimethyl sulfoxide (DMSO, 20 mL). HACE (116 mg) was also solubilized in DMSO (20 mL). Those two solution groups were mixed and stirred for 12 h. Then, it was transferred to the dialysis membrane (molecular weight cut-off (MWCO): 6‒8 kDa) and stirred in methanol and distilled water (DW) for 12 h and 48 h, respectively. The dialyzed resultant was freeze-dried and stored at room temperature prior to its use. The conjugation of HACE-MbCD was confirmed by proton nuclear magnetic resonance (1HNMR; Varian FT-500 MHz, Varian Inc., Palo Alto, CA, USA) analysis.21 For calculating the content of MbCD in HACE-MbCD, samples with different weight ratios of MbCD to HACE 5 ACS Paragon Plus Environment


dissolved in the blend of DMSO-d6 and D2O (50:50, v/v). With the results of 1H-NMR assay, the regression line of ratios of integration areas (5.0 ppm/1.8 ppm) on the weight ratios (MbCD/HACE) was plotted. Using the established standard curve, the content of MbCD in the synthesized HACEMbCD was determined. For the preparation of NAs, HACE or HACE-MbCD (at 5 mg/mL concentration) was dispersed in DW. Then, it was passed through individual syringe filter (pore size: 0.45 μm) and further freeze-dried. Each sample (at 5 mg/mL concentration) was then dispersed in DW and the mean diameter and polydispersity index (dynamic light scattering (DLS) method) and zeta potential (laser Doppler method) were determined (ELS-Z1000; Otsuka Electronics, Tokyo, Japan). The particle shape of HACE-MbCD NA was investigated by transmission electron microscopy (TEM). That dispersion of HACE-MbCD NA in DW was loaded onto the copper grids with films, stained with 2% (w/v) phosphotungstic acid, and destained with DW. The specimen was dried and then its images were taken with TEM (JEM 1010; JEOL, Tokyo, Japan).20 Incubation time-dependent in vitro particle stability of HACE-MbCD NA was evaluated in PBS and FBS (50%, v/v). Lyophilized HACE-MbCD NA (at 5 mg/mL concentration) was dispersed in PBS or FBS solution and stored at 37°C for 24 h. Their hydrodynamic size values were determined after incubating for 1, 3, 6, and 24 h by described DLS method. Cellular Internalization Assay. Cellular internalization and distribution of HACE-MbCD NA were tested in MDA-MB-231 cells. Cells were acquired from Korean Cell Line Bank (Seoul, Korea). RPMI 1640 medium containing FBS (10%, v/v), penicillin (100 U/mL), and streptomycin (100 µg/mL) were used for the culture of MDA-MB-231 cells in this study. In vitro cellular location of HACE-MbCD NA was observed by confocal laser scanning microscopy (CLSM). As 6 ACS Paragon Plus Environment

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a fluorescence labeling agent, Cy5.5 was covalently bonded to HACE-MbCD in this study. Amide bond between the amine group of Cy5.5-NH2 and the carboxylic acid group of HA (in HACEMbCD) was formed via an EDC/NHS-coupled reaction. HACE (48 mg) or HACE-MbCD (48 mg) was solubilized in DMSO (20 mL) or the mixture of DMSO and DW (75:25 (v/v), 20 mL), respectively. EDC (28.8 mg) and NHS (17.3 mg) were added to the above HACE or HACE-MbCD solution and they were agitated for 15 min in the cause of activation of the carboxylic acid group of HA. Cy5.5-NH2 (4.7 mg) was solubilized in that mixture and incubated for 1 day. It was then moved to the dialysis membrane (MWCO: 3.5 kDa) and stirred with DW for 2 day. The dialyzed resultant was further purified by using a PD-10 desalting column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The resultant was freeze-dried for further uses. The contents of Cy5.5 were determined by monitoring its absorbance value at 675 nm with a SpectraMax i3 multi-mode detection platform (Molecular Devices, Sunnyvale, CA, USA). MDA-MB-231 cells (1.0 × 105 cells per well) were put into the chambered cell culture slides (4-wells; BD Falcon, Bedford, MA, USA) and those cells were incubated at 37C for 1 day. Cy5.5 tagged HACE NA or Cy5.5 tagged HACE-MbCD NA (at 10 μg/mL concentration of Cy5.5) was applied to the cells and they were incubated for 4 h at 37°C. PBS (pH 7.4) was used for washing cells to remove remained NAs and cells were immersed in 4% formaldehyde solution for the fixation of specimen. After fixation of specimen, VECTASHIELD antifade mounting medium (H1200; Vector Laboratories, Inc., Burlingame, CA, USA) with 4',6-diamidino-2-phenylindole (DAPI) was treated to the cells for counterstaining nuclei and inhibiting photobleaching. Intracellular fluorescent intensities were monitored by CLSM imaging analysis (LSM 880, CarlZeiss, Thornwood, NY, USA). Cholesterol Capture Study. Extraction of cellular cholesterol by MbCD was assessed with 7 ACS Paragon Plus Environment


filipin III staining in MDA-MB-231 cells. Cells (5.0 × 104 cells per well) were put onto the glass bottom dishes. MbCD, HACE, or HACE-MbCD (at 200 μg/mL of MbCD concentration) was applied to the cells and incubated for 1 day. Cellular cholesterol was quantitatively analyzed with cholesterol assay kit (Abcam, Cambridge, MA, USA). Intracellular fluorescence intensities of filipin III were detected by CLSM (LSM 880, Carl-Zeiss). Antiproliferation Efficacy Test. Cytotoxicities of MbCD and HACE-MbCD were tested in NIH3T3, HUVEC, and MDA-MB-231 cells using the colorimetric cell proliferation assay. NIH3T3 cells were supplied by Korean Cell Line Bank (Seoul, Korea) and DMEM containing FBS (10%, v/v), penicillin (100 U/mL), and streptomycin (100 µg/mL) was used for the culture of NIH3T3 cells. Human umbilical vein endothelial cell (HUVEC) was purchased from Thermo Fisher Scientific (Waltham, MA, USA) and those cells were cultured with endothelial cell growth medium 2 (PromoCell GmbH, Heidelberg, Germany). MDA-MB-231 cells were grown up under the above described conditions. NIH3T3, HUVEC, or MDA-MB-231 cells, at a seeding density of 5.0 × 103 cells per well, were loaded onto the 96-well plate and cells were incubated for 1 day. MbCD (at 1, 2.5, 5, 10, 25, 50, 100, 250, 500, and 1000 μg/mL MbCD concentrations) and HACEMbCD (at 10, 50, 100, 250, and 500 μg/mL corresponding MbCD concentrations), dispersed in the serum-free cell culture media (for NIH3T3 and MDA-MB-231 cells) or complete media (for HUVEC),30 were treated to the cells and they were incubated for 3 days. After eliminating each sample, CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) kit (Promega Corp., Fitchburg, WI, USA) was applied to the cells by suggested protocols. Absorbance value (at 490 nm wavelength) in each sample was scanned by a SpectraMax i3 multi-mode detection platform (Molecular Devices). Cell viability (%) was estimated by dividing the absorbance values of tested sample into those of control (no treatment). 8 ACS Paragon Plus Environment

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Apoptosis Assay. Apoptotic efficacy of fabricated HACE-MbCD NA in MDA-MB-231 cells was tested by Annexin V-FITC/propidium iodide (PI)-based assay. Cells were loaded onto to the 6-well cell culture plate at 1.0 × 105 cells per well and cells were incubated for 1 day. MbCD, HACE, or HACE-MbCD (at 100 μg/mL MbCD concentration) was applied to the cells and those were treated for 24 and 48 h. Cells were then rinsed with PBS (pH 7.4) to remove remained each sample. Cells were detached from the bottom and harvested by centrifugation. Then, those cells were added to the reaction buffer of FITC Annexin V Apoptosis Detection Kit (BD Pharmingen, BD Biosciences, San Jose, CA, USA) for co-staining with Annexin V-FITC and PI. FITC and PIbased cellular fluorescence intensities were quantitatively measured by FACSCalibur fluorescence-activated cell sorter (FACSTM) system (Becton Dickinson Biosciences, San Jose, CA, USA). Animal Experiments. Mice were raised at 55 ± 5% relative humidity and 22 ± 2°C. All animal protocols were approved by the Animal Care and Use Committee of the Kangwon National University.31 Animal studies were followed by the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978). Real-Time Near-Infrared Fluorescence (NIRF) Imaging Tests. In vivo fate of injected NAs was observed by NIRF imaging in MDA-MB-231 tumor-bearing mice. For the detection of NIRF signals, HACE and HACE-MbCD was modified with Cy5.5. The contents of Cy5.5 in Cy5.5tagged HACE and Cy5.5-tagged HACE-MbCD were determined by detecting the absorbance value (at 675 nm wavelength) with a SpectraMax i3 multi-mode detection platform (Molecular Devices). Female BALB/c nude mice (5-weeks-old, Charles River, Wilmington, MA, USA) were used to make MDA-MB-231 tumor-bearing mice. Cell suspension (2.0 × 106 cells in 0.1 mL cell culture 9 ACS Paragon Plus Environment


media) was subcutaneously injected into the dorsal side of the mouse. The tumor volume (V, mm3) was determined with the following formula: V = 0.5 × longest diameter × (shortest diameter)2.17 Mice with similar tumor volume (150-200 mm3) were used for NIRF imaging test.17 Aliquot (0.1 mL) of dispersion of Cy5.5-tagged HACE NA or Cy5.5-tagged HACE-MbCD NA (Cy5.5 dose: 200 µg/kg) was intravenously injected to the mouse model. Mouse was anesthetized using isoflurane (2.5%) via an inhalation route prior to NIRF imaging tests. In vivo NIRF signals in mouse models were detected at 0, 2, 6, and 24 h postinjection by fluorescence imaging system (FOBI; NeoScience Co., Ltd., Suwon, Korea) installed with a red laser.31 At 24 h postinjection, liver, heart, lung, spleen, kidney, and tumor were obtained from the mouse for ex vivo imaging (FOBI; NeoScience Co., Ltd.) with a red laser.31 In Vivo Anticancer Activity Tests. In vivo anticancer activities of cholesterol capturable NA were evaluated in the mouse model xenografted with MDA-MB-231 tumor after intravenous administration. BALB/c nude mice (female, 5-weeks-old, Charles River, Wilmington, MA, USA) were utilized for making mouse model xenografted with MDA-MB-231 tumor. Cell suspension (2.0 × 106 cells in 0.1 mL cell culture media) was innoculated into the dorsal side of mouse. Tumor volume (V, mm3) was determined with the following formula: V = 0.5 × longest diameter × (shortest diameter)2.17 Mice with approximately 150 mm3 tumor volume were allocated to each group. The tumor volume and body weight were measured during test period. HACE (at 5 mg/kg MbCD dose), MbCD (at 5 mg/kg MbCD dose), DOX (at 5 mg/kg DOX dose), or HACE-MbCD (at 5 mg/kg MbCD dose) was injected intravenously to the tumor-bearing mouse on day 6, 10, 12, and 14. Tumor tissues were dissected on day 17 and they were immersed in formaldehyde (4%, v/v) solution for fixation prior to histological stainings. Slices of tumor tissue (6-μm thickness) were acquired for deparaffinization and hydration with ethanol.31 Then, they were stained with 10 ACS Paragon Plus Environment

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hematoxylin and eosin (H&E) reagent. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was conducted to evaluate apoptosis in tumor tissues.31 In TUNEL assay of tumor tissues, 3,3ʹ-diaminobenzidine (DAB) was used for reaction with the horseradish peroxidase (HRP)-labeled tissues to develop brown color at the site of deoxyribonucleic acid (DNA) fragmentation which may be produced by the apoptotic signals.31 DAB-developed portion was quantitatively determined by calculating optical density (OD) values with “Fiji” version of Image J.32 In Vivo Toxicity Tests. In vivo toxicity of NAs was investigated by blood tests and histological stainings in ICR mouse (male, approximately 20 g of body weight; Orient Bio, Sungnam, Korea). MbCD, HACE NA, and HACE-MbCD NA (5 mg/kg dose of MbCD) were injected via an intravenous route. Aliqouts of blood were obtained by cardiac puncture at 24 h postinjection. The levels of blood urea nitrogen (BUN), aspartate transaminase (AST), alanine transaminase (ALT), albumin, and total cholesterol (TC) were quantitatively determined by UREAL (Roche Diagnostics, Manheim, Germany), Aspartate Aminotransferase acc. to IFCC (Roche Diagnostics), Alanine Aminotransferase acc. to IFCC (Roche Diagnostics), ALB2 (Roche Diagnostics), and CHOL2 (Roche Diagnostics) kits, respectively, using Cobas 8000 C702 chemical autoanalyzer (Roche Diagnostics). Heart, kidney, liver, lung, and spleen were also excised at 24 h postinjection. They were fixed in formaldehyde (4%, v/v) solution for H&E staining. Slices of tumor tissue (6-μm thickness) were acquired for deparaffinization and hydration with ethanol.31 Then, they were stained with H&E reagent. Data Analysis. Statistical analysis was performed with Student’s t-test and analysis of variance (ANOVA). Each value is shown as the mean ± standard deviation (SD).



■ RESULTS AND DISCUSSION Synthesis and Physicochemical Properties of HACE-MbCD. In our previous reports, several kinds of nanovehicles composed of HACE derivatives were prepared for the precise delivery of anticancer agents to tumor region.14-23 HA can be one of ligands for CD44 receptor expressed in various types of cancers. Therefore, HA-based nanocarriers can possess passive and active tumor targeting effects (EPR effect and interactions between HA and CD44 receptor) after their intravenous administration.12,13,17 HACE-based particles exhibited nano-size distribution in the aqueous environment due to the self-assemble property of HACE. To elevate tumor targeting capability after intravenous administration, several moieties (i.e., dopamine, folic acid, and polyethylene glycol) were introduced to the HACE structure.16,17,21 In this study, MbCD was covalently bonded to the HACE structure (Figure 1). It is expected that MbCD can extract cholesterol from the cell membrane and it can be lead to apoptosis in malignant tumor cells.24,26 Interestingly, MbCD can efficiently extract cholesterol from cell membranes, however it may hardly interact with phospholipids.29 The internal cavity and external surface of CD have hydrophobic and hydrophilic property, respectively. Thus, lipid-soluble molecules (i.e., cholesterol) may get into the internal cavity of CD and that CD complex may behave as a hydrophilic molecule in the aqueous environment (Figure 1). HACE-MbCD was synthesized via an ester linkage between ‒COOH group of HA (in HACE) and ‒OH group of MbCD (Figure 2A). DCC/DMAP-mediated Steglich esterification strategy was introduced to synthesize HACE-MbCD in this study.15,33 Synthesis of HACE and its verification were already reported in our previous study.14 In short, linker-CE was conjugated with HA-TBA and the chemical structure of HACE was verified by 1H-NMR assay.14,21 Corresponding chemical shifts of protons in methyl group of CE and N-acetyl group of HA were presented at 0.8–0.9 ppm 12 ACS Paragon Plus Environment

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and 1.8 ppm, respectively.14,17 The degree of substitution of CE to HA was reported to be 2.38%.14 To confirm the existence of MbCD in HACE-MbCD, the chemical shift at 5.0 ppm (MbCD) was used with that at 1.8 ppm (HA) in this study (Figure 2B). The linear regression line of integration ratio (5.0/1.8 ppm) on the weight ratio (MbCD/HACE) was drawn with mixtures of HACE and MbCD (Figure S1). By the result of 1H-NMR assay, the percentage portion of MbCD included in HACE-MbCD was 13.3 ± 1.6%. Based on the feeding ratio of MbCD to HACE in this synthetic condition, HACE-MbCD is likely to be produced rather than the other by-products. Considering the substitution degree of CE in HACE and the content of MbCD in HACE-MbCD conjugate, most portion of carboxyl groups in HA seems to be remained as a free (unreacted) form. It implies that those carboxyl groups can participate in CD44 receptor targeting after intravenous administration. To fabricate nano-sized assembly of HACE-MbCD, its dispersion was filtered and freezedried. In this study, self-assembly property of HACE was used to prepare HACE-MbCD NA in the aqueous environment. The combination of hydrophilic backbone (HA) and hydrophobic residue (CE) can make an amphiphilic conjugate and produce nano-sized self-assembly in aqueous media. It was reported that the critical aggregation concentration of HACE was 42 µg/mL.14 Hydrophilic MbCD might be located on the exterior face of HACE-MbCD NA and it can easily access to the cell surface and extract cellular cholesterol. In this investigation, the tumor targetability and anticancer activities of HACE-MbCD NA were demonstrated without loading anticancer agents. Averaged hydrodynamic size values of HACE NA and HACE-MbCD NA were 176 and 198 nm, respectively (Table 1). As shown in Figure 3A and Table 1, HACE-MbCD NA exhibited approximately 0.2 polydispersity index and unimodal size distribution. According to the TEM 13 ACS Paragon Plus Environment


image, HACE-MbCD NA has a spherical shape (Figure 3A). Judged from zeta potential values (Table 1), the modification of HACE with MbCD makes it less negative than unmodified HACE (p < 0.05). It suggested that carboxylic acid group of HA was participated in the synthesis of ester linkage between HACE and MbCD. Significant change (shift to zero value) in zeta potential value of HACE-MbCD NA, compared with HACE NA, may mean the attachment of MbCD molecules to HACE structure. Observed particle size and size distribution may be suitable for EPR effectrelated passive tumor targeting strategy.9,34 With the interactions between HA and CD44 receptor, particle characteristics-related EPR effect may contribute to the selective arrival of developed HACE-MbCD NA in the tumor tissue after intravenous administration. Particle stability of fabricated HACE-MbCD NA was tested in artificial biological fluids (Figure 3B). Incubation time-dependent hydrodynamic size of HACE-MbCD NA in PBS and FBS solution was measured. Mean diameters of HACE-MbCD NA were 219‒264 nm in PBS and 141‒167 nm in FBS solution, respectively, during 24 h of incubation period. Considering the hydrodynamic size (198 ± 13 nm) of HACE-MbCD NA in DW (Table 1), the difference of particle size between DW and FBS solution was lower than the gap between PBS and FBS solution. Pharmaceutical salts included in PBS seem to increase the hydrodynamic size of HACE-MbCD NA in this study. On the other hand, slight decrease of particle size in FBS solution group compared to DW group might be due to the interactions between serum proteins of FBS and HA exposed in the outer surface of HACE-MbCD NA. Dramatic change was absent in both PBS and FBS solution groups during the tested incubation period and observed particle size ranges may be sufficient for exerting EPR effect, easily connected to the efficient delivery of nanocarriers to tumor region. Upon intravenous injection, developed HACE-MbCD NA may show unperverted circulation and less side effects (e.g., embolization) in the blood stream. 14 ACS Paragon Plus Environment

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Cellular Internalization and Location of NAs. Cellular distribution patterns of developed HACE-MbCD NA in MDA-MB-231 cells were tested (Figure 4). For tracing cellular movement of developed NAs, Cy5.5-tagged HACE and Cy5.5-tagged HACE-MbCD were synthesized via an amide bond formation between Cy5.5-NH2 and HA. The intracellular fluorescence signal of Cy5.5 was detected by CLSM imaging and it indicates the cellular location of NAs. As shown in Figure 4, red fluorescence signal, indicating Cy5.5, in HACE-CD NA group was much stronger than that in HACE NA group. HACE-MbCD-treated group exhibited 1.84-fold higher mean intensity of red fluorescence rather than HACE NA-treated group. It means that higher amounts of HACE-MbCD NA seems to be accumulated in the cells rather than HACE NA in tested experimental conditions. Cellular internalization of HACE-based nanosystems in cancer cells via HA-CD44 receptor binding was verified in previous works.14-23 Considering higher uptake efficiency of HACEMbCD NA rather than HACE NA in MDA-MB-231 cells, additional internalization mechanisms into the cells seem to be existed in addition to HA-CD44 receptor interaction. Cholesterol capturing capability of MbCD in HACE-MbCD NA may be one of those cellular association mechanisms. Further cellular cholesterol seizable ability of HACE-MbCD NA was assessed in following sections. Cellular Cholesterol Capturing Ability of HACE-MbCD NA. MbCD was chemically conjugated to HACE for extracting cholesterol from lipid rafts of malignant tumor cells. For determining cholesterol levels of cellular membrane, filipin III staining method was used.35,36 Filipin III (as one of filipin isomers) can selectively bind to free (i.e., unesterified) cholesterol in the biological membrane.37 Therefore, the relationship between the fluorescence intensity of filipin III and cholesterol level in cell membrane can be used to estimate cholesterol efflux from cell membrane. As shown in Figure 5, cholesterol in the cell membrane was stained with filipin III 15 ACS Paragon Plus Environment


after applying MbCD, HACE NA, and HACE-MbCD NA. HACE-MbCD group exhibited the lowest intensity of blue color (filipin III) among all experimental groups. It means that cholesterol in the cell membrane was efficiently extracted by HACE-MbCD NA rather than MbCD and HACE. CD44 receptor-mediated endocytosis of HACE-MbCD NA made a substantial contribution to its improved cholesterol efflux activity rather than the other groups in MDA-MB-231 cells. Superior cholesterol capturing efficiency of HACE-MbCD NA may lead to following signaling cascades for cancer cell death. In Vitro Anticancer Potentials. Anticancer potentials of HACE-MbCD NA in MDA-MB231 cells were examined (Figure 6). Antiproliferation and apoptotic efficacies of HACE-MbCD NA were tested by MTS test and Annexin V-PI assay, respectively. Antiproliferation potentials of MbCD and HACE-MbCD were examined in NIH3T3, HUVEC, and MDA-MB-231 cells. NIH3T3 (normal fibroblast) cell was selected as a control cell line (non-malignant tumor cell) in this test. HUVEC was chosen for evaluating the toxicity of HACE-MbCD against vascular endothelial cells.30 HACE did not show severe cytotoxicities in MDA-MB-231 and NIH3T3 cells as confirmed in previous studies,16,22 therefore those data were excluded in this study. As shown in Figure 6A, both MbCD and HACE-MbCD exhibited higher cell viability profiles in NIH3T3 cells and HUVEC rather than MDA-MB-231 cells. In NIH3T3 cells and HUVEC, the IC50 data of MbCD and HACE-MbCD were over 3 mg/mL, implying feeble cytotoxicity on non-cancer cells. It is assumed that injected HACE-MbCD may not induce severe toxicities to vascular endothelium compared to other chemotherapeutic agents.38 On the other hand, both MbCD and HACE-MbCD may have tumor selectivity in terms of antiproliferation efficacies. Notably, in MDA-MB-231 cells, IC50 value of HACE-MbCD (416 ± 25 μg/mL) was obviously lower than MbCD (903 ± 77 μg/mL) in tested conditions (p < 0.05). Considering the negligible cytotoxicity of HACE, improved 16 ACS Paragon Plus Environment

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antiproliferation efficacy of HACE-MbCD, rather than MbCD, can be supported by the internalization of HACE-MbCD NA through the interaction with CD44 receptor in MDA-MB231 cells. Enhanced antiproliferation potentials of HACE-MbCD, compared with MbCD, may contribute to the increment in anticancer activities after the delivery of HACE-MbCD NA to the neighboring region of malignant tumors. Cholesterol depletion-induced cell death by MbCD has been explained by apoptosis in cancer cells.29 It is already reported that cholesterol synthesis inhibitor (e.g., simvastatin), except for CD derivatives, can reduce raft cholesterol content and induce apoptosis in cancer cells.39 It is assumed that the modulation of cholesterol content in lipid raft of cancer cell membrane can affect the anticancer potentials. In this study, by using Annexin V-PI assay, apoptotic efficacies of MbCD, HACE, and HACE-MbCD were assessed in MDA-MB-231 cells (Figure 6B). In Annexin V-PI assay, lower right (LR) panel and upper right (UR) panel present initial and terminal status of apoptosis, respectively. Thus, the percentage of (LR + UR) parts has been used to mean the apoptosis rates. At 24 and 48 h incubation groups, the apoptotic percentage presented in (LR + UR) panels of HACE-MbCD NA group was the highest among all tested groups (p < 0.05). In particular, those apoptotic percentages of HACE-MbCD NA group at 24 and 48 h were 1.9 and 2.8-fold higher than those of MbCD group (p < 0.05). Considering inappreciable contribution of HACE to apoptosis in MDA-MB-231 cells, enhanced apoptosis in HACE-MbCD-treated group, compared with MbCD-treated group, could be supported by the interactions of HA and CD44 receptor. In particular, MDA-MB-231 cells include more lipid rafts and show higher sensitivity rather than normal counterpart.24 Therefore, the installation of MbCD in HACE nanostructure may induce efficient and selective death of MDA-MB-231 cells via apoptotic events.



CD44 Receptor-Expressed Tumor Targeting. Biodistribution pattern of intravenously injected HACE NA and HACE-MbCD NA was monitored by an in vivo optical imaging (Figure 7). NIRF molecule (Cy5.5) was covalently bonded to HACE structure via an amide bond in this optical imaging test. HACE NA or HACE-MbCD NA modified with Cy5.5 was injected into the MDA-MB-231 tumor-innoculated mice and the scanned image of body was acquired at 2, 6, and 24 h postinjection (Figure 7A). At 2 h postinjection, both HACE NA and HACE-MbCD NA were distributed in normal tissues and organs as well as tumor tissue. However, 24 h later, the fluorescence signal of tumor region in HACE-MbCD NA was higher than that in HACE NA. Integrated density, considering mean fluorescence intensity and surface area of tumor region, of HACE-MbCD NA group was 74% higher than that of HACE NA group as shown in Figure 7B (p < 0.05). In the images of dissected organs and tissues (Figure S2), the fluorescence signal of tumor in HACE-MbCD NA group was stronger than HACE NA group. Although the fluorescence intensity of kidney was higher than tumor in HACE-MbCD NA group, accumulation in kidney seems to be normal excretion process of injected HACE-based nanocarriers as reported.16 Improved tumor targetability of HACE-MbCD NA, compared with HACE NA, can be explained by cholesterol detectable characteristics of MbCD in HACE-MbCD NA. With HA-CD44 receptor interaction, cholesterol capturable property of MbCD seems to contribute to the enhancement of tumor targetability. In Vivo Antitumor Potentials. In vivo anticancer activities of prepared HACE-MbCD NA were examined in MDA-MB-231 tumor implanted mice (Figure 8). Tumor growth, body weight, and histological stainings of tumor region were evaluated after multiple intravenous injection of HACE NA, MbCD, DOX, and HACE-MbCD NA. Calculated tumor volume of HACE-MbCD NA-injected group was smaller than those of control, HACE NA-injected, and MbCD-injected 18 ACS Paragon Plus Environment

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groups on day 10, 12, 14, and 17 as shown in Figure 8A (p < 0.05). DOX was added as one of commercial chemotherapeutic agents to evaluate the anticancer activities of developed HACEMbCD NA in this study. Although there was no significant difference between HACE-MbCD NA group and DOX group in tumor volume, SD value of tumor volume was higher in DOX group rather than HACE-MbCD NA group. It indicates that the inter-individual anticancer responses can be shown in DOX-injected group while HACE-MbCD NA group can provide relatively constant anticancer activities. Also, the body weight of DOX-injected group was lower than HACE-MbCD NA-injected group at day 17 (p < 0.05) (Figure 8B). It implies the presence of systemic toxicities of DOX rather than HACE-MbCD NA after their intravenous injection. In the images of TUNEL assay (Figure 8C and Figure S3), higher apoptotic events, which was shown as a brown color, occurred in HACE-MbCD NA group rather than the other groups (p < 0.05). Efficient tumor growth suppression of HACE-MbCD NA seems to be based on the increment in apoptosis of tumor after its multiple dosing. Improved anticancer activities of HACE-MbCD NA can be supported by the more accurate tumor targeting capability (Figures 7 and 8). Moreover, higher cholesterol extractable capacity of HACE-CD, which can lead to the elevation in apoptosis and antiproliferation, rather than MbCD and HACE in MDA-MB-231 cells may contribute to improve anticancer activities in tumor-xenografted mouse models. In Vivo Toxicity. In vivo toxicity of developed HACE-MbCD NA was evaluated by blood assays and H&E staining in mice (Table 2 and Figure 9). HACE and its derivatives have been utilized for the fabrication of nanovehicles and cancer imaging in our previous studies,14,16-23 and no severe toxicities were observed during pharmacokinetic and antitumor efficacy studies in rodent models after intravenous administration. The levels of BUN, AST, ALT, albumin, and TC of control, MbCD, HACE NA, and HACE-MbCD NA-injected groups were determined (Table 2). 19 ACS Paragon Plus Environment


The functions of kidney (BUN and albumin) and liver (AST, ALT, and albumin) can be evaluated by this blood chemistry test. Generally, compared with control group, HACE-CD NA group did not show significant differences in BUN, AST, and ALT levels. It implies that the intravenous administration of HACE-MbCD NA does not induce severe damages to the kidney and liver at tested administration dose. TC level in plasma was also quantitatively analyzed to estimate the interactions between MbCD (in HACE-MbCD NA) and blood cholesterol. Compared with the TC level in the control group, no significant difference was presented in HACE-MbCD NA group. The intravenous injection of HACE-MbCD NA did not affect the TC level in the blood. Observed results of blood tests support the safe application of developed HACE-MbCD NA for cancer therapy. Along with blood assay data, H&E stained images of heart, kidney, liver, lung, and spleen were also obtained (Figure 9). There was no significant difference among all experimental groups (control, MbCD, HACE NA, and HACE-MbCD NA) in H&E staining images of heart, kidney, liver, lung, and spleen. Those findings also support the safe application of developed HACEMbCD NA in cancer therapy.

■ CONCLUSIONS Lipid rafts disrupting nanostructure composed of HACE and MbCD was developed for breast cancer therapy. MbCD was conjugated to HACE via an ester linkage and it may extract cholesterol on the membrane of cancer cells. Nano-size (approximately 200 nm mean diameter) of HACEMbCD NA and HA-CD44 receptor interaction are able to be employed for passive and active targeting of CD44 receptor-positive cancers, respectively. Unimodal and narrow size distribution, spherical morphology, and the maintenance of particle stability in the serum media were also observed in HACE-MbCD NA group. Compared with MbCD and HACE NA groups, more 20 ACS Paragon Plus Environment

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efficient cholesterol depletion from cancer cell membrane was shown in HACE-MbCD NA group. Disruption of lipid rafts by the application of HACE-MbCD NA was lead to the enhancement in apoptosis and antiproliferation. Precise cancer targeting of HACE-MbCD NA, compared with HACE NA, in MDA-MB-231 tumor-implanted mice can be explained by the extraction process of cholesterol by MbCD. HACE-MbCD NA provided more efficient suppression of cancer growth and apoptotic events in tumor tissues. Developed HACE-MbCD NA may be used as CD44 receptor targeting and lipid rafts disrupting nanosystem for breast cancer therapy.

■ ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: (1) Regression line of weight ratio (MbCD/HACE)-dependent ratio of integration area (5.0/1.8 ppm) (Figure S1). (2) Ex vivo NIRF images of dissected liver, heart, lung, spleen, kidney, and tumor at 24 h postinjection (Figure S2).

■ AUTHOR INFORMATION Corresponding Author *(H.-J.Cho)

Tel.: +82-33-250-6916. Fax: +82-33-259-5631. E-mail: [email protected].

Notes The authors declare no competing financial interest.

■ ACKNOWLEDGEMENTS



Page 22 of 42

This research was supported by the National Research Foundation of Korea (NRF), funded by the Korean

government

(MSIP)

(No.

NRF-2015R1A1A1A05027671

and

NRF-

2017R1E1A1A01074584).

■ REFERENCES (1) Amoozgar, Z.; Goldberg, M.S. Targeting Myeloid Cells Using Nanoparticles to Improve Cancer Immunotherapy. Adv. Drug Deliv. Rev. 2015, 91, 38‒51. (2) Choi, J.H.; Lee, Y.J.; Kim, D. Image-Guided Nanomedicine for Cancer. J. Pharm. Investig. 2017, 47, 51‒64. (3) Kim, H.S.; Lee, D.Y. Photothermal Therapy with Gold Nanoparticles as an Anticancer Medication. J. Pharm. Investig. 2017, 47, 19‒26. (4) Liu, Y.; Xu, C.F.; Iqbal, S.; Yang, X.Z.; Wang, J. Responsive Nanocarriers as an Emerging Platform for Cascaded Delivery of Nucleic Acids to Cancer. Adv. Drug Deliv. Rev. 2017, 115, 98‒114. (5) Mitra, A.K.; Agrahari, V.; Mandal, A.; Cholkar, K.; Natarajan, C.; Shah, S.; Joseph, M.; Trinh, H.M.; Vaishya, R.; Yang, X.; Hao, Y.; Khurana, V.; Pal, D. Novel Delivery Approaches for Cancer Therapeutics. J. Control. Release 2015, 219, 248‒268. (6) Fang, J.; Nakamura, H.; Maeda, H. The EPR effect: Unique Features of Tumor Blood Vessels for Drug Delivery, Factors Involved, and Limitations and Augmentation of the Effect. Adv. Drug Deliv. Rev. 2011, 63, 136‒151. (7) Danhier, F.; Feron, O.; Préat, V. To Exploit the Tumor Microenvironment: Passive and Active Tumor Targeting of Nanocarriers for Anti-Cancer Drug Delivery. J. Control. Release 2010, 148, 135–146. 22 ACS Paragon Plus Environment

Page 23 of 42 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


(8) Iyer, A.K.; Khaled, G.; Fang, J.; Maeda, H. Exploiting the Enhanced Permeability and Retention Effect for Tumor Targeting. Drug Discov. Today 2006, 11, 812‒818. (9) Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. Tumor Vascular Permeability and the EPR Effect in Macromolecular Therapeutics: a Review. J. Control. Release 2000, 65, 271– 284. (10) Kim, C.H.; Lee, S.G.; Kang, M.J.; Lee, S.; Choi, Y.W. Surface Modification of LipidBased Nanocarriers for Cancer Cell-Specific Drug Targeting. J. Pharm. Investig. 2017, 47, 203‒227. (11) Mattheolabakis, G.; Milane, L.; Singh, A.; Amiji, M.M. Hyaluronic Acid Targeting of CD44 for Cancer Therapy: from Receptor Biology to Nanomedicine. J. Drug Target. 2015, 23, 605‒618. (12) Liang, X.; Fang, L.; Li, X.; Zhang, X.; Wang, F. Activatable Near Infrared Dye Conjugated Hyaluronic Acid Based Nanoparticles as a Targeted Theranostic Agent for Enhanced Fluorescence/CT/Photoacoustic Imaging Guided Photothermal Therapy. Biomaterials 2017, 132, 72‒84. (13) Yoon, H.Y.; Kim, H.R.; Saravanakumar, G.; Heo, R.; Chae, S.Y.; Um, W.; Kim, K.; Kwon, I.C.; Lee, J.Y.; Lee, D.S.; Park, J.C.; Park, J.H. Bioreducible Hyaluronic Acid Conjugates as siRNA Carrier for Tumor Targeting. J. Control. Release 2013, 172, 653‒661. (14) Cho, H.J.; Yoon, H.Y.; Koo, H.; Ko, S.H.; Shim, J.S.; Lee, J.H.; Kim, K.; Kwon, I.C.; Kim, D.D. Self-Assembled Nanoparticles Based on Hyaluronic Acid-Ceramide (HA-CE) and Pluronic® for Tumor-Targeted Delivery of Docetaxel. Biomaterials 2011, 32, 7181–7190.



(15) Cho, H.J.; Yoon, H.Y.; Koo, H.; Ko, S.H.; Shim, J.S.; Cho, J.H.; Park, J.H.; Kim, K.; Kwon, I.C.; Kim, D.D. Hyaluronic Acid-Ceramide-Based Optical/MR Dual Imaging Nanoprobe for Cancer Diagnosis. J. Control. Release 2012, 162, 111‒118. (16) Cho, H.J.; Yoon, I.S.; Yoon, H.Y.; Koo, H.; Jin, Y.J.; Ko, S.H.; Shim, J.S.; Kim, K.; Kwon, I.C.; Kim, D.D. Polyethylene Glycol-Conjugated Hyaluronic Acid-Ceramide SelfAssembled Nanoparticles for Targeted Delivery of Doxorubicin. Biomaterials 2012, 33, 1190– 1200. (17) Jeong, J.Y.; Hong, E.H.; Lee, S.Y.; Lee, J.Y.; Song, J.H.; Ko, S.H.; Shim, J.S.; Choe, S.; Kim, D.D.; Ko, H.J.; Cho, H.J. Boronic Acid-Tethered Amphiphilic Hyaluronic Acid DerivativeBased Nanoassemblies for Tumor Targeting and Penetration. Acta. Biomater. 2017, 53, 414‒426. (18) Jin, Y.J.; Termsarasab, U.; Ko, S.H.; Shim, J.S.; Chong, S.; Chung, S.J.; Shim, C.K.; Cho, H.J.; Kim, D.D. Hyaluronic Acid Derivative-Based Self-Assembled Nanoparticles for the Treatment of Melanoma. Pharm. Res. 2012, 29, 3443–3454. (19) Lee, J.Y.; Yang, H.; Yoon, I.S.; Kim, S.B.; Ko, S.H.; Shim, J.S.; Sung, S.H.; Cho, H.J.; Kim, D.D. Nanocomplexes Based on Amphiphilic Hyaluronic Acid Derivative and Polyethylene Glycol–Lipid for Ginsenoside Rg3 Delivery. J. Pharm. Sci. 2014, 103, 3254–3262. (20) Lee, J.Y.; Termsarasab, U.; Park, J.H.; Lee, S.Y.; Ko, S.H.; Shim, J.S.; Chung, S.J.; Cho, H.J.; Kim, D.D.,. Dual CD44 and Folate Receptor-Targeted Nanoparticles for Cancer Diagnosis and Anticancer Drug Delivery. J. Control. Release 2016, 236, 38–46. (21) Lee, S.Y.; Park, J.H.; Ko, S.H.; Shim, J.S.; Kim, D.D.; Cho, H.J. Mussel-Inspired Hyaluronic Acid Derivative Nanostructures for Improved Tumor Targeting and Penetration. ACS Appl. Mater. Interfaces 2017, 9, 22308‒22320.


Page 24 of 42

Page 25 of 42 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


(22) Park, J.H.; Cho, H.J.; Termsarasab, U.; Lee, J.Y.; Ko, S.H.; Shim, J.S.; Yoon, I.S.; Kim, D.D. Interconnected Hyaluronic Acid Derivative-Based Nanoparticles for Anticancer Drug Delivery. Colloids Surf. B Biointerfaces 2014, 121, 380–387. (23) Park, J.H.; Cho, H.J.; Yoon, H.Y.; Yoon, I.S.; Ko, S.H.; Shim, J.S.; Cho, J.H.; Park, J.H.; Kim, K.; Kwon, I.C.; Kim, D.D. Hyaluronic Acid Derivative-Coated Nanohybrid Liposomes for Cancer Imaging and Drug Delivery. J. Control. Release 2014, 174, 98–108. (24) Li, Y.C.; Park, M.J.; Ye, S.K.; Kim, C.W.; Kim, Y.N. Elevated Levels of CholesterolRich Lipid Rafts in Cancer Cells Are Correlated with Apoptosis Sensitivity Induced by Cholesterol-Depleting Agents. Am. J. Pathol. 2006, 168, 1107‒1118. (25) Llaverias, G.; Danilo, C.; Mercier, I.; Daumer, K.; Capozza, F.; Williams, T.M.; Sotgia, F.; Lisanti, M.P.; Frank, P.G. Role of Cholesterol in the Development and Progression of Breast Cancer. Am. J. Pathol. 2011, 178, 402‒412. (26) Badana, A.K.; Chintala, M.; Gavara, M.M.; Naik, S.; Kumari, S.; Kappala, V.R.; Iska, B.R.; Malla, R.R. Lipid Rafts Disruption Induces Apoptosis by Attenuating Expression of LRP6 and Survivin in Triple Negative Breast Cancer. Biomed. Pharmacother. 2018, 97, 359‒368. (27) Ilangumaran, S.; Hoessli, D.C. Effects of Cholesterol Depletion by Cyclodextrin on the Sphingolipid Microdomains of the Plasma Membrane. Biochem. J. 1998, 335, 433‒440. (28) Zidovetzki, R.; Levitan, I. Use of Cyclodextrins to Manipulate Plasma Membrane Cholesterol Content: Evidence, Misconceptions and Control Strategies. Biochim. Biophys. Acta. 2007, 1768, 1311‒1324. (29) Onodera, R.; Motoyama, K.; Okamatsu, A.; Higashi, T.; Kariya, R.; Okada, S.; Arima, H. Involvement of Cholesterol Depletion from Lipid Rafts in Apoptosis Induced by Methyl-βCyclodextrin. Int. J. Pharm. 2013, 452, 116‒123. 25 ACS Paragon Plus Environment


(30)

Qu,

J.;

Wang,

Q.Y.;

Chen,

K.L.;

Luo,

Page 26 of 42

J.B.;

Zhou,

Q.H.;

Lin,

J.

Reduction/Temperature/pH Multi-stimuli Responsive Core Cross-linked Polypeptide Hybrid Micelles for Triggered and Intracellular Drug Release. Colloids Surf. B Biointerfaces 2018, 170, 373‒381. (31) Lee, S.Y.; Cho, H.J. An α-Tocopheryl Succinate Enzyme-Based Nanoassembly for Cancer Imaging and Therapy. Drug Deliv. 2018, 25, 738‒749. (32) Schindelin, J.; Arganda-Carreras, I.; Frise, E; Kaynig, V.; Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.; Schmid, B.; Tinevez, J.Y.; White, D.J.; Hartenstein, V.; Eliceiri, K.; Tomancak, P.; Cardona, A. Fiji: An Open-Source Platform for Biological-Image Analysis. Nat. Methods 2012, 9, 676‒682. (33) Neises, B.; Steglich, W. Simple Method for the Esterification of Carboxylic Acids. Angew. Chem. Int. Ed. 1978, 17, 522‒524. (34) Ernsting, M.J.; Murakami, M.; Roy, A.; Li, S.D. Factors Controlling the Pharmacokinetics, Biodistribution and Intratumoral Penetration of Nanoparticles. J. Control. Release 2013, 172, 782–794. (35) Rosenbaum, A.I.; Zhang, G.; Warren, J.D.; Maxfield, F.R. Endocytosis of BetaCyclodextrins Is Responsible for Cholesterol Reduction in Niemann-Pick Type C Mutant Cells. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 5477‒5482. (36) Yokoo, M.; Kubota, Y.; Motoyama, K.; Higashi, T.; Taniyoshi, M.; Tokumaru, H.; Nishiyama, R.; Tabe, Y.; Mochinaga, S.; Sato, A.; Sueoka-Aragane, N.; Sueoka, E.; Arima, H.; Irie, T.; Kimura, S. 2-Hydroxypropyl-β-Cyclodextrin Acts as a Novel Anticancer Agent. PLoS One 2015, 10, e0141946.


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(37) Maxfield, F.R.; Wüstner, D. Analysis of Cholesterol Trafficking with Fluorescent Probes. Methods Cell Biol. 2012, 108, 367‒393. (38) Soultati, A.; Mountzios, G.; Avgerinou, C.; Papaxoinis, G.; Pectasides, D.; Dimopoulos, M.A.; Papadimitriou, C. Endothelial Vascular Toxicity from Chemotherapeutic Agents: Preclinical Evidence and Clinical Implications. Cancer Treat Rev. 2012, 38, 473‒483. (39) Zhuang, L.; Kim, J.; Adam, R.M.; Solomon, K.R.; Freeman, M.R. Cholesterol Targeting Alters Lipid Raft Composition and Cell Survival in Prostate Cancer Cells and Xenografts. J. Clin. Invest. 2005, 115, 959‒968.



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Table 1. Particle Characterizations of NAs. Formulation

Mean diameter (nm)

Polydispersity index

(mV)

HACE NA

176 ± 19

0.13 ± 0.01

-27.2 ± 0.8

HACE-MbCD NA

198 ± 13

0.22 ± 0.01

-19.4 ± 4.0#

HACE or HACE-MbCD was dispersed in DW at 5 mg/mL. Data are presented as the mean ± standard deviation (SD) (n ≥ 3). #p

Zeta potential

< 0.05, compared with HACE NA group.


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Table 2. Blood Chemistry Test of Developed NAs after Intravenous Administration in Mice. Parameter

Control

MbCD

HACE NA

HACE-MbCD NA

BUN (mg/dL)

16.5 ± 1.7

15.6 ± 1.1

13.8 ± 1.0*,&

16.4 ± 2.3

AST (U/L)

104 ± 17

98 ± 24

100 ± 19

64 ± 26

ALT (U/L)

24 ± 9

20 ± 5

23 ± 7

18 ± 6

Albumin (g/dL)

3.7 ± 0.1

3.3 ± 0.1*

3.5 ± 0.1*

3.4 ± 0.2*

TC (mg/dL)

84 ± 16

77 ± 14

85 ± 14

89 ± 10

Note: BUN: blood urea nitrogen; AST: aspartate transaminase; ALT: alanine transaminase; TC: total cholesterol. Data are shown as the mean ± SD (n = 4‒5). *p

< 0.05, compared with control group.

&p

< 0.05, compared with MbCD group.



Figure legends

Figure 1. Schematic illustration regarding tumor targeting and therapeutic strategy of HACEMbCD NA.

Figure 2. Synthesis and identification of HACE-MbCD. (A) Synthetic scheme of HACE-MbCD. (B) 1H-NMR spectrum of HACE-MbCD. The chemical shifts ‘1’ (1.8 ppm) and ‘2’ (5.0 ppm) indicate HA and MbCD group, respectively. For 1H-NMR (500 MHz) analysis, HACE-MbCD was solubilized in the mixture of DMSO-d6 and D2O mixture (1:1, v/v).

Figure 3. Particle characterization of HACE-MbCD NA. (A) Particle size distribution and particle shape (observed by TEM image) of HACE-MbCD NA. HACE-MbCD NA was dispersed in DW at 5 mg/mL concentration prior to its analysis. The length of the scale bar in the TEM image is 1 μm. (B) Particle stability of HACE-MbCD NA after incubation in PBS (pH 7.4) and FBS (50%, v/v). Hydrodynamic size of HACE-MbCD NA according to the incubation time (~24 h) is presented. Each point represents the mean ± SD (n ≥ 3).

Figure 4. Cellular accumulation assay of HACE-MbCD NA in MDA-MB-231 cells. Cy5.5-HACE NA and Cy5.5-HACE-MbCD NA were applied to the cells and the fluorescence signal of Cy5.5 was monitored by CLSM imaging. Blue and red colors mean DAPI and Cy5.5, respectively. The length of the scale bar in the CLSM image is 20 μm.

Figure 5. Cholesterol capture assay of HACE-MbCD NA in MDA-MB-231 cells. Blue color 30 ACS Paragon Plus Environment

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indicates filipin III staining in the CLSM image. The length of the scale bar in the CLSM image is 20 μm.

Figure 6. In vitro anticancer activity tests of HACE-MbCD NA. (A) Antiproliferation assay of MbCD and HACE-MbCD in NIH3T3, HUVEC, and MDA-MB-231 cells. Cell viability values according to the concentrations of MbCD and HACE-MbCD are plotted. Each point represents the mean ± SD (n ≥ 3). (B) Apoptotic efficacies, measured by Annexin V-FITC and PI-based assay, of HACE-MbCD NA in MDA-MB-231 cells. Population percentages of each quadrant group after 24 h and 48 h incubation are shown. Population percentages of (UR+LR) panels are also presented. UL, UR, LL, and LR indicate upper left, upper right, lower left, and lower right, respectively. Each point represents the mean ± SD (n = 3). *p < 0.05, compared with control group. &p < 0.05, compared with MbCD group. #p < 0.05, compared with HACE NA group.

Figure 7. NIRF imaging data in MDA-MB-231 tumor-xenografted mouse model. Cy5.5conjugated HACE NA and HACE-MbCD NA were injected to the tail vein of the mouse model. (A) Real-time images taken at 0 (pre), 2, 6, and 24 h. Scanned images of whole body are presented. Yellow dashed circle indicates the tumor mass. (B) Integrated density (area × mean of intensity) values of fluorescence signals in tumor region. Each point represents the mean ± SD (n = 3). #p< 0.05, compared to HACE NA group.

Figure 8. In vivo anticancer activity tests in MDA-MB-231 tumor-bearing mouse models. (A) Tumor growth (presented by tumor volume) profiles of control, HACE NA, MbCD, DOX, and HACE-MbCD NA groups. Each sample was injected intravenously on day 5, 8, and 12. Each point 31 ACS Paragon Plus Environment


indicates the mean ± SD (n = 6‒8). *p < 0.05, compared with control group. #p < 0.05, compared with HACE NA group. &p < 0.05, compared with MbCD group. (B) Body weight profiles of control, HACE NA, MbCD, DOX, and HACE-MbCD NA groups. Each point indicates the mean ± SD (n = 6‒8). +p < 0.05, compared with DOX group. (C) Microscopic images of dissected tumors after H&E and TUNEL stainings. The microscopic images of H&E (left panel) and TUNEL (right panel) stainings are presented. The length of scale bar in the microscopic images is 100 μm.

Figure 9. In vivo toxicity tests after intravenous injection of developed nanosystems in mouse. H&E staining images of heart, kidney, liver, lung, and spleen of control, MbCD, HACE NA, and HACE-MbCD NA groups are shown.


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Figure 1 254x190mm (300 x 300 DPI)

ACS Paragon Plus Environment


Fig. 2 254x190mm (96 x 96 DPI)


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Fig. 3 190x254mm (96 x 96 DPI)



Figure 4 254x190mm (300 x 300 DPI)


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Figure 5 142x254mm (96 x 96 DPI)



Fig. 6 (revised) 190x254mm (96 x 96 DPI)


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Figure 7 180x180mm (300 x 300 DPI)



Fig. 8 190x338mm (96 x 96 DPI)


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Fig. 9 239x220mm (96 x 96 DPI)



graphical abstract 210x110mm (96 x 96 DPI)


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Tumor Targeting and Lipid Rafts Disrupting Hyaluronic Acid

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