Mussel-Inspired Hyaluronic Acid Derivative Nanostructures for

Jun 16, 2017 - College of Pharmacy, Kangwon National University, Chuncheon, Gangwon 24341, Republic of Korea. ‡College of Pharmacy and Research Inst...
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Mussel-Inspired Hyaluronic Acid Derivative Nanostructures for Improved Tumor Targeting and Penetration Song Yi Lee,† Ju-Hwan Park,‡ Seung-Hak Ko,§ Jae-Seong Shim,§,⊥ Dae-Duk Kim,‡ and Hyun-Jong Cho*,† †

College of Pharmacy, Kangwon National University, Chuncheon, Gangwon 24341, Republic of Korea College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 08826, Republic of Korea § Biogenics Inc., Daejeon 34027, Republic of Korea ⊥ Skin & Tech Inc., Seongnam, Gyeonggi 13135, Republic of Korea ‡

S Supporting Information *

ABSTRACT: An amphiphilic hyaluronic acid-ceramide-dopamine (HACE-d) conjugate was prepared, and HACE-d-based nanoparticles (NPs) including phloretin (as an inhibitor of glucose transporter (GLUT1)) were fabricated. Mussel-inspired property of d was introduced to HACE NPs, and it may improve tumor targetability and penetration in addition to passive (based on enhanced permeability and retention effect) and active (interaction between HA and CD44 receptor) tumor targeting effects. HACE-d/phloretin NPs with 279 nm mean diameter, ∼0.2 polydispersity index, and −18 mV zeta potential were successfully fabricated, and a sustained drug release pattern was observed. HACE-d/phloretin NPs exhibited enhanced cellular accumulation efficiency and antiproliferation property, compared with HACE/phloretin NPs, in MDA-MB-231 cells (GLUT1 and CD44 receptor-expressed human breast adenocarcinoma cells). In a MDA-MB-231 spheroid model, HACE-d NPs group showed better tumor penetration efficiency and spheroid growth inhibitory effect rather than HACE NPs group. According to the optical imaging test in MDA-MB-231 tumor-xenografted mouse, HACE-d NPs group exhibited more selective distribution in tumor region and deeper infiltration into the inner part of tumor compared with HACE NPs group. After intravenous injection, HACEd/phloretin NPs group also exhibited improved antitumor efficacies rather than the other experimental groups in MDA-MB-231 tumor-xenografted mouse. All these findings suggested that HACE-d/phloretin NP may be a promising tumor targetable and penetrable nanosystem for the therapy and imaging of GLUT1 and CD44 receptor-expressed cancers. KEYWORDS: dopamine, hyaluronic acid-ceramide, nanoparticle, phloretin, tumor targeting, tumor penetration

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INTRODUCTION Various tumor targeting strategies to deliver anticancer agents selectively to tumor region have been developed.1−3 After the entry of nanocarriers into the bloodstream, avoidance of clearance from renal filtration (5 nm cutoff) and mononuclear phagocyte system (MPS; principally resided in bone marrow, liver, and spleen) is necessary for the successful arrival of nanocarriers to the surroundings of tumor tissue.4 An enhanced permeability and retention (EPR) effect has been used as a passive targeting for tumors. Solid tumors have leaky vasculatures; thus, macromolecules with over 40 kDa can be transported into the tumor tissues.5 Therefore, nanocarriers with certain physicochemical characteristics (i.e., particle size) can move to tumor region via this EPR effect.6 The extravasation and penetration of nanocarriers can be enhanced by reducing particle size and modulating tumor vasculatures.4 Physiological characteristics of tumor tissue, such as irregular and heterogeneous tumor vasculature, increased interstitial fluid © XXXX American Chemical Society

pressure (IFP), dense stroma, and the existence of tumor associated macrophage (TAM), should be modulated for the efficient tumor penetration.4,7 It is also known that the physicochemical characteristics of nanocarriers, such as size, surface charge, and the installation of ligands, influence their tumor penetration.4 Cellular internalization of nanocarriers can be influenced by several factors such as size, shape, charge of surface, surface modification, and ligands.4 In particular, ligand−receptor interaction can be adopted as an active targeting strategy for tumors to complement the drawbacks (i.e., lack of specific tumor selectivity) of passive targeting strategy.8 Recently, several approaches for tumor targeting and penetration have been developed.9−11 In this investigation, Received: May 10, 2017 Accepted: June 16, 2017 Published: June 16, 2017 A

DOI: 10.1021/acsami.7b06582 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

(119 mg) was solubilized in dimethyl sulfoxide (DMSO, 20 mL), and its pH value was reduced to 4 by 1 N HCl. EDC (57.5 mg) and NHS (34.5 mg) were added to the above solution, and its pH value was elevated to 7 by 1 N NaOH. After d (37.9 mg) was dissolved in DMSO (4 mL), it was put into HACE/EDC/NHS mixture and stirred for 1 day at room temperature. Then the resultant was moved to dialysis bag (molecular weight cutoff [MWCO]: 3.5 kDa) and dialyzed in distilled water (DW) for 2 days. Final material was acquired by lyophilization. HACE-d was dissolved in D2O/DMSO-d6 mixture (1:1, v/v) for proton nuclear magnetic resonance (1H NMR; Varian FT-500 MHz, Varian Inc., Palo Alto, CA, USA) analyses. To calculate the content of d in HACE-d, regression line of integration ratios of peaks (6.5−6.7 ppm/1.8 ppm) according to the weight ratios (d/HACE) was plotted. Fluorescence spectra of HACE (50 μg/mL), d (50, 100, 250, 500, and 1000 ng/mL), and HACE-d (50 μg/mL), dissolved in 50% (v/v) MeOH, were obtained at 290−500 nm emission wavelength with 279 nm excitation wavelength (FP-6500, Jasco Co., Tokyo, Japan). The content (w/w) of d in HACE-d was calculated using the regression line with standard samples of d. Fabrication and Particle Characterization of PhloretinIncluded NPs. Phloretin was incorporated to the NPs as one of hydrophobic anticancer agents. For preparing phloretin-loaded NPs, HACE or HACE-d (8 mg) and phloretin (1 mg) were dissolved in 50% (v/v) dimethylformamide (DMF, 1 mL). The solvents were removed by heating at 70 °C for 2 h with the flow of N2 gas. The dispersion of NPs was prepared by vortex-mixer and probe sonicator (VC-750; Sonics and Materials, Inc., Newtown, CT, USA) after DW (1 mL) was added. The mean diameter and polydispersity index of NPs were measured by dynamic light scattering (DLS) method (ELS-Z1000; Otsuka Electronics, Tokyo, Japan), and their zeta potential values were detected by laser Doppler method (ELS-Z1000; Otsuka Electronics), respectively, according to the manufacturer’s protocols. To measure drug encapsulation efficiency, the dispersion of phloretin-loaded NPs was mixed with five-times the volume of DMSO and further diluted for high-performance liquid chromatography (HPLC) analysis. The content of phloretin in NPs was quantitatively analyzed by an HPLC system equipped with a pump (PU-2089 Plus; Jasco, Tokyo, Japan), an UV−vis detector (UV-1575), and an automatic injector (AS-2050 Plus). A guard column (SecurityGuard Guard Cartridge kit, Phenomenex, Torrance, CA, USA)-connected reverse phase C18 column (Gemini, 250 mm × 4.6 mm, 5 μm; Phenomenex, Torrance, CA, USA) was utilized for the drug analysis. The mobile phase was composed of acetonitrile, DW, and phosphoric acid (50:50:0.08, v/v/ v), and the flow rate was 1 mL/min. The injection volume was 20 μL, and the absorbance was measured at 288 nm by UV−vis detector. The analytical method of phloretin was validated. The morphological shape of phloretin-loaded NPs was monitored by transmission electron microscopy (TEM). Phosphotungstic acid (2%, w/v) was used to stain the dispersion of NPs. The aliquot of sample was loaded onto the copper grids, and the liquid content was eliminated by drying for 10 min under the gentle air stream prior to the observation by TEM (JEM 1010; JEOL, Tokyo, Japan). Stability Test of NPs. Stability of drug-loaded NPs was assessed by measuring the mean diameter of NPs in PBS (pH 7.4) for 24 h. The hydrodynamic size of HACE/phloretin NPs and HACE-d/ phloretin NPs was determined by a DLS method (ELS-Z1000; Otsuka Electronics) according to the manufacturer’s manual. Drug Release Test. Aliquots of NPs dispersion (approximately 0.15 mL), containing 200 μg of phloretin, were put into the dialysis tube (Mini-GeBAflex tubes, MWCO 14 kDa; Gene Bio-Application Ltd., Kfar Hanagide, Israel). They were transferred to PBS (pH 7.4; 10 mL) containing Tween 80 (0.3%, w/w) and agitated in a water-bath with 50 rpm at 37 °C. NPs-loaded tubes were moved to fresh release media at 2, 4, 6, 24, 48, 72, 96, 120, and 168 h, and 0.2 mL of release media was gathered for HPLC analysis. The concentration of phloretin in each sample was quantitatively analyzed by the HPLC system. Cellular Uptake Studies. The cellular internalization efficiency of developed NPs was determined by flow cytometry. MDA-MB-231

dopamine (d) was introduced to hyaluronic acid (HA)-based nanoparticles (NPs) for improving tumor penetration and cellular internalization. The mussel-inspired property of d included in marine mussel adhesive proteins had been reported, and d with catecholamine groups had been used for enhancing cellular adhesion.12,13 Polydopamine, which can be formed by self-polymerization of d, coating has been used as facile and versatile surface modification method to improve adhesion properties.14 Instead of polydopamine coating onto the surface of NPs, chemical conjugation of d to hydrophilic polymers (i.e., HA and chitosan) was tried, and biomedical applications were reported.15,16 In this study, amphiphilic hyaluronic acidceramide-dopamine (HACE-d) conjugate was synthesized, and phloretin-loaded HACE-d NPs were fabricated. Phloretin is one of dihydrochalcones, and it is abundant in apples.17 Phloretin can inhibit the cellular uptake of glucose via glucose transporter (GLUT), and it has antioxidant properties.18−21 Therefore, it has been used as an anticancer agent for various kinds of cancer cells.19,21,22 In particular, GLUTs were expressed in breast cancer cell lines (e.g., MDA-MB-231 cells), and phloretin exhibited in vitro antitumor efficacies in those cancer cells.21,23 As CD44 receptor and GLUT were expressed in MDA-MB-231 cell line, it can be used for evaluating tumor targeting efficiency based on the interaction between HA and CD44 receptor and glycolysis inhibition based on the blockade of glucose transport via GLUT. Herein, HACE-d/phloretin NPs were developed for cancer targeting and penetration based on the HA-CD44 receptor binding and cellular adhesion property of d. The antitumor efficacies of phloretin were assessed in two and three-dimensional cell culture models, and the in vivo biodistribution of HACE-d NPs was monitored by optical imaging techniques.

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EXPERIMENTAL METHODS

Materials. 3-Hydroxytyramine hydrochloride (dopamine; d), phloretin, and Tween 80 were obtained from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). DS-Y30 (ceramide 3B; mainly Noleoylphytosphingosine) and HA oligomer (average molecular weight (MW): 4.7 kDa) were provided by Doosan Biotech Co., Ltd. (Yongin, Republic of Korea) and Bioland Co., Ltd. (Cheonan, Republic of Korea), respectively. Adipic acid dihydrazide, chloromethylbenzoyl chloride, coumarin 6 (C6), hexadeuterodimethyl sulfoxide (DMSOd6), N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), tetra-n-butylammonium hydroxide (TBA), and 1-hydroxybenzotriazole (HOBt) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Aminefunctionalized Cy5.5 (FCR-675 amine) was acquired from BioActs (Incheon, Korea). RPMI 1640, heat-inactivated fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Gibco Life Technologies, Inc. (Grand Island, NY, USA). All used reagents were analytical grade and used without further purification. Synthesis and Identification of HACE-d. HACE was synthesized according to the reported method.24 In short, HA (12.21 mmol) and TBA (9.77 mmol) were dispersed in double-distilled water (DDW, 60 mL) by stirring for 30 min. HA-TBA was obtained by freeze-drying. DS-Y30 ceramide (8.59 mmol) and triethylamine (9.45 mmol) were dissolved in tetrahydrofuran (THF, 25 mL), and 4chloromethylbenzoyl chloride (8.59 mmol) was solubilized in THF (10 mL), respectively. They were mixed and stirred for 6 h at 60 °C. The DS-Y30-included linker was acquired by enrichment and recrystallization. DS-Y30 linker (0.41 mmol) and HA-TBA (8.10 mmol) were dissolved in the solvent (THF/acetonitrile = 4:1, v/v), which was then stirred at 40 °C for 5 h. By eliminating impurities and organic solvents, HACE was purified. HACE-d was prepared via the formation of amide bond between −NH2 group (from d) and −COOH group (from HACE). HACE B

DOI: 10.1021/acsami.7b06582 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces cells were seeded in six-well plates (6.0 × 105 cells per well) and cultured for 24 h at 37 °C. As a fluorescent dye, C6 was loaded to NPs instead of drug. HACE or HACE-d (40 mg) and C6 (0.5 mg) were dissolved in 75% (v/v) DMSO (5 mL). That mixture was added in the dialysis bag (MWCO: 3.5 kDa), and it was stirred in DW for 6 h. C6loaded NPs were acquired by freeze-drying. For flow cytometry analysis, C6-loaded HACE NPs and C6-loaded HACE-d NPs, at 2 μg/ mL C6 concentration, were added to MDA-MB-231 cells, and they were incubated for 1 and 4 h. To elucidate the roles of d residue during the uptake process of HACE-d NPs, the excess amount of d (1 mg/mL) was treated to MDA-MB-231 cells, and they were preincubated for 2 h in case of (HACE-d/C6 NPs + d) group. Then cells were treated with HACE-d/C6 NPs for 2 h. After cells were washed with PBS (pH 7.4), they were gathered by centrifugation. FBS solution (2%, v/v) was used to resuspend cells. The cellular accumulation capability, represented as a counted cell number according to the fluorescence intensity, was evaluated by a FACSCalibur Fluorescence-activated Cell Sorter (FACS) equipped with CELLQuest software (Becton Dickinson Biosciences, San Jose, CA, USA).25 Intracellular location of prepared NPs was assessed by confocal laser scanning microscopy (CLSM).26 MDA-MB-231 cells were seeded on cell culture slides (BD Falcon, Bedford, MA, USA) at a density of 1.0 × 105 cells per well (1.7 cm2 surface area per well) and incubated for overnight at 37 °C.26 HACE NPs or HACE-d NPs, including C6 (2 μg/mL) as a fluorescent dye, were incubated for 1 and 4 h at 37 °C. Those cells were then washed with PBS (pH 7.4) at least thrice. Cells were put into formaldehyde solution (4%), and they were taken out after 10 min of fixation. After the liquid contents were removed, VECTASHIELD mounting medium including 4′,6-diamidino-2phenylindole (DAPI) (H-1200; Vector Laboratories, Inc., Burlingame, CA, USA) was treated for staining nuclei and inhibiting fluorescence photobleaching. They were then observed by CLSM (LSM 780, CarlZeiss, Thornwood, NY, USA).27 Cellular uptaken amounts of drug were evaluated by described HPLC method. MDA-MB-231 cells were seeded at 6.0 × 105 cells per well in six-well plates and cultured for 1 day at 37 °C. Then phloretin solution, HACE/phloretin NPs, and HACE-d/phloretin NPs (at 10 μg/mL drug concentration) were treated to the cells, and they were cultured for 6 h at 37 °C. PBS (pH 7.4) was added to rinse the cells at least thrice, and DW (0.5 mL) was put into the well. Then those cells were collected by scraping and disrupted by probe sonication for 30 s (VC-750; Sonics and Materials, Inc.). Obtained cell lysates (0.4 mL) and acetonitrile (0.6 mL) were blended by vortex-mixing and were centrifuged. Afterward, the aliquot of supernatant (0.9 mL) was moved to another microcentrifuge tube and heated at 80 °C for 3 h. Remained contents were mixed with acetonitrile (0.12 mL), and they were centrifuged. Aliquots (20 μL) of supernatants were used for determining drug concentrations by the HPLC system. Drug in cell lysates was measured by described HPLC method. Cytotoxicity Tests. Cytotoxicity of synthesized HACE-d was tested in MDA-MB-231 cells. Cells (at 5.0 × 103 cells per well) were seeded onto a 96-well plate and grown for 1 day at 37 °C. HACE-d (∼500 μg/mL) was treated to the cells and cultured for 72 h at 37 °C. Then, MTS-based CellTiter 96 AQueous One Solution Cell Proliferation Assay Reagent (Promega Corp., Fitchburg, WI, USA) was added to the cells, and they were incubated at 37 °C according to the manufacturer’s protocols. By using an EMax Precision Microplate Reader (Molecular Devices, Sunnyvale, CA, USA), the absorbance value of each sample was detected at 490 nm. In vitro antiproliferation efficiency of drug-loaded NPs was tested in MDA-MB-231 cells using the colorimetric assay. Cells (at 5.0 × 103 cells per well) were seeded onto 96-well plate and cultured for 1 day at 37 °C. Phloretin solution, HACE/phloretin NPs, and HACE-d/ phloretin NPs, at 0, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, and 50 μg/mL phloretin concentration, were added to the cells, and they were incubated for 72 h at 37 °C. Then MTS-based CellTiter 96 AQueous One Solution Cell Proliferation Assay Reagent (Promega Corp.) was added to the cells and they were incubated at 37 °C in compliance with the procedures provided by the manufacturer. By using an EMax

Precision Microplate Reader (Molecular Devices), the absorbance at 490 nm was determined, and the cell viability was calculated. Tumor Penetration Studies. For preparing MDA-MB-231 spheroids, cells were seeded (at 500 cells per well) onto roundbottomed cell culture plates (96-well), covered with agarose (2% [w/ v] in Hank’s balanced salt solution).9 They were incubated with mild shaking at 37 °C.9 Penetration efficiency of developed NPs in MDAMB-231 spheroids was tested by CLSM. When MDA-MB-231 spheroids reached 200−300 μm mean diameter, they were incubated with HACE NPs and HACE-d NPs containing C6 (2 μg/mL) for 24 h at 37 °C. Then spheroids were rinsed with PBS (pH 7.4) thrice and fixed in formaldehyde solution (4%, v/v) for 10 min.9 Fixed spheroids were loaded onto a coverglass bottom dish, and the fluorescent signals were detected using CLSM (LSM 780, Carl-Zeiss).9 Spheroid growth inhibition efficiency of phloretin-loaded NPs was assessed in MDA-MB-231 spheroids. MDA-MB-231 cells were seeded (at 500 cells per well) onto round-bottomed cell culture plates (96well), covered with agarose (2% [w/v] in Hank’s balanced salt solution).9 They were incubated with mild shaking at 37 °C.9 After MDA-MB-231 spheroids reached 200−300 μm mean diameter, phloretin solution, HACE/phloretin NPs, HACE-d/phloretin NPs, HACE NPs, and HACE-d NPs (10 μg/mL phloretin) were treated, and they were incubated for 1 day. The drug solution or NPs were removed, and fresh cell culture medium was added. Morphology of MDA-MB-231 spheroid was observed using an inverted microscopy (Eclipse TS100-F, Nikon, Tokyo, Japan) over 2 days. The volume (V, mm3) of spheroid was determined according to the following formula: V = 0.5 × (longest diameter) × (shortest diameter)2.9 Optical Imaging. Biodistribution of developed NPs in the tumorxenografted mouse was evaluated by real-time NIRF imaging. For NIRF imaging study, Cy5.5-included HACE NPs and HACE-d NPs were prepared. HACE or HACE-d (8 mg) and Cy5.5 (0.1 mg) were dissolved in 50% (v/v) DMF (1 mL). Solvent was eliminated by heating that solution for 2 h at 70 °C with a mild N2 gas flow. Cy5.5loaded NPs were acquired by freeze-drying. By detecting the absorbance at 680 nm, the content of Cy5.5 included in NPs was calculated.25 To prepare the MDA-MB-231 tumor-xenografted mouse, BALB/c nude mice (∼20 g body weight, female, 5 weeks old; Charles River, Wilmington, MA, USA) were used.25 Mice were reared in a lightcontrolled room at 22 ± 2 °C and 55 ± 5% relative humidity (Animal Center for Pharmaceutical Research, College of Pharmacy, Seoul National University, Seoul, Korea).25 All animal experiments were approved by the Animal Care and Use Committee of the College of Pharmacy, Seoul National University.25 Cells were cultured as mentioned above, and the cell suspension (2 × 106 cells in 0.1 mL) was inoculated into the back of mice. Tumor volume (V, mm3) was determined by the following formula: V = 0.5 × (longest diameter) × (shortest diameter)2.9 After they reached 150−200 mm3, HACE NPs and HACE-d NPs containing Cy5.5 (at 100 μg/kg dose) were injected into the tail vein of the mice. Fluorescence signals in the mice were measured by Optix MX3 (ART Advanced Research Technologies Inc., Saint-Laurent, QC, Canada).27 A laser diode (670 nm) was introduced for the excitation of Cy5.5. Images were scanned at 1, 3, 6, and 24 h after injection. At 24 h, heart, kidneys, liver, lungs, spleen, and tumor were separated from the mouse, and their fluorescent intensity values were quantitatively analyzed. Penetration capability of developed HACE-d NPs into the tumor tissue was assessed by three-dimension (3D) optical imaging technique. By using OptiView 3D Reconstruction module (version 3.2; ART Advanced Research Technologies Inc.), fluorescence intensity in 3D space of tumor tissue was produced from time-resolved fluorescence, and volumetric data were acquired.9 The fluorescence intensity of the region of interest (ROI) (i.e., the tumor tissue) was reconstructed, and the penetration efficiency of NPs was evaluated by comparing the sliced planes of the ROI.9 In Vivo Anticancer Activity Tests. MDA-MB-231 tumorxenografted mouse was prepared with BALB/c nude mice (female, 5-weeks-old, Charles River, Wilmington, MA, USA) to evaluate in vivo antitumor efficacies of phloretin-loaded NPs. As described in above section, mice were reared in similar conditions. The protocol of animal C

DOI: 10.1021/acsami.7b06582 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 1. Schemes regarding tumor targeting and penetration strategy of HACE-d/phloretin NPs. Phloretin (as a hydrophobic drug) was loaded to self-assembled HACE-d NPs. The physicochemical properties (i.e., particle size) of NPs and HA-CD44 receptor interaction may provide passive and active tumor targetability after intravenous injection. In addition, dopamine-cell adhesion may enhance the tumor penetration efficiency of HACE-d NPs. Phloretin may inhibit glucose uptake via GLUT1 thereby induce anticancer activities. study was approved by the Animal Care and Use Committee of Kangwon National University. Cells were cultured as described above, and the aliquot (2 × 106 cells in 0.1 mL) of cell suspension was inoculated into the back of mice. Tumor volume (V, mm3) was determined by the following formula: V = 0.5 × (longest diameter) × (shortest diameter)2.9 After it reached approximately 100 mm3 tumor volume, it was monitored. Phloretin solution, HACE/phloretin NPs, and HACE-d/phloretin NPs (2.5 mg/kg phloretin dose) were intravenously injected to the mouse (day 12, 14, and 17). Body weight of all experimental groups was measured with the tumor volume. After monitoring of tumor volumes and body weights was finished, tumor was dissected from the mouse and fixed in formaldehyde (4%, v/v) solution. Tumor tissues with 6-μm thickness were acquired for deparaffinization and hydration with ethanol. According to the standard protocols, the tumors were stained by hematoxylin and eosin (HE) and used for a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.9 For the TUNEL assay, the chromogen diaminobenzene (DAB) was used, and the tissue samples were incubated for color development to detect deoxyribonucleic acid (DNA) fragmentation resulting from apoptotic signaling cascades.9 Statistical Analysis. Statistical analysis was conducted with analysis of variance (ANOVA) and a two-tailed t-test. All experiments were repeated at least thrice. Data are shown as the mean ± standard deviation (SD).

glycol and folic acid) were covalently bonded to HACE, and biofunctional materials (i.e., lipid and poloxamer) were physically mixed with HACE for improving tumor targeting efficiency. Polydopamine coating onto NPs has been used to provide mussel-inspired characteristics and improve cellular adhesion.12,14,33 Instead of polydopamine coating onto NPs, mussel-inspired functional residue (d) was conjugated to amphiphilic HACE via amide bond formation in this study (Figure 1). The synthesis and confirmation of HACE was already reported.24 Linker-ceramide (CE) was covalently bonded to HA-TBA, and it was demonstrated by 1H NMR analysis.24 Chemical shifts for the protons of the methyl group of CE (0.9−1.0 ppm), N-acetyl group of HA (1.8 ppm), and aromatic ring of the linker (7.5−8.0 ppm) in HACE conjugate were confirmed in our previous study.24 To synthesize HACEd, amine group of d was linked to carboxylic acid group of HA backbone via amide bond (Figure 2A). After TBA salt was removed from HACE by making acidic environment (around pH 4), activated −COOH group of HA was reacted with −NH2 group of d. The proposed interaction between the exofacial thiol groups of cell membrane and d of HACE-d conjugate is shown (Figure 2B). As reported,15 amine or thiol group can be conjugated to dopamine molecule. It could explain the cellular adhesion of HACE-d NPs. Synthesized HACE-d was identified by 1H NMR assay (Figure 2C). Chemical shifts of protons in N-acetyl group (a, 1.8 ppm) and phenyl ring group (b−d, 6.5−6.7 ppm) indicate HA oligomer and d, respectively. The regression line of integration ratios (6.5−6.7/1.8 ppm) to weight ratios (d/HACE) was constructed with the physical mixtures of HACE and d (Figure S2). The ratio of integration area obtained from the NMR spectra of synthesized HACE-d can be put into the equation of linear regression line (Figure S2) and corresponding weight ratio (d/ HACE) can be calculated. The content of d in HACE-d was 1.3

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RESULTS AND DISCUSSION Synthesis and Verification of HACE-d. HACE-based selfassembled NPs for tumor-targeted drug delivery and cancer imaging have been reported.24,28 Hydrophobic CE was conjugated to hydrophilic HA oligomer for preparing an amphiphilic HACE.24 Linker (chloromethylbenzoyl chloride)CE conjugate was attached to HA-TBA via an ether bond (Figure S1).24 It was reported that the substitution degree of CE to HA, estimated from the NMR spectra, was 2.38%.24 In previous studies,24,28−32 functional moieties (i.e., polyethylene D

DOI: 10.1021/acsami.7b06582 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 2. Synthesis and characterization of HACE-d. (A) Scheme of the synthesis of HACE-d is shown. (B) Proposed interaction between exofacial thiol group of cellular membrane and HACE-d is presented. (C) 1H NMR spectrum of HACE-d is presented. The shifts “a” (1.8 ppm) and “b−d” (6.5−6.7 ppm) indicate HA and d group, respectively. HACE-d was dissolved in the mixture of DMSO-d6 and D2O mixture (1:1, v/v) for 1H NMR (500 MHz) analysis. (D) Fluorescence intensity profiles of HACE (50 μg/mL), d (50−1000 ng/mL), and HACE-d (50 μg/mL) are shown. Emission spectra (290−500 nm) of HACE, d, and HACE-d were scanned at 279 nm excitation wavelength.

Table 1. Characterization of Phloretin-Loaded NPsa composition

mean diameter (nm)

polydispersity index

zeta potential (mV)

encapsulation efficiency (%)b

loading content (%)c

HACE/phloretin NPs HACE-d/phloretin NPs

276 ± 11 279 ± 14

0.22 ± 0.01 0.22 ± 0.01

−30 ± 1 −18 ± 1d

86.0 ± 6.2 90.5 ± 2.2

10.7 ± 0.8 11.3 ± 0.3

a

Data are presented as mean ± standard deviation (SD) (n = 3).

c

mass of drug in NPs after purification total mass of drug and HACE (or HACE ‐ d)

Loading content (%) =

b

Εncapsulation efficiency (%) =

actual amount of drug in NPs input amount of drug in NPs

× 100

× 100 dp < 0.05, compared with HACE/phloretin NPs group.

± 0.3% (w/w). The synthesis of HACE-d was also verified by the measurement of fluorescence intensity (Figure 2D). The content of d in HACE-d was also quantitatively determined by

measuring fluorescence intensity. According to the results of fluorescence spectra, the average content of d in HACE-d was approximately 1% (w/w). The results of 1H NMR assay and E

DOI: 10.1021/acsami.7b06582 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 3. Characterization of phloretin-loaded NPs. (A) TEM images of HACE/phloretin NPs and HACE-d/phloretin NPs groups are shown. The length of the scale bar is 200 nm. (B) Size distribution, plotted as differential intensity profiles according to the mean diameter, of prepared NPs is presented.

fluorescence intensity measurement indicate the successful synthesis of HACE-d. Fabrication and Evaluationof Phloretin-Included NPs. HACE-d/phloretin NPs were prepared for tumor-targeted delivery and cancer imaging in this study. As a model drug with poorly water-solubility, phloretin was incorporated in the internal region of HACE-d NPs. The other poorly watersoluble drug (i.e., docetaxel, doxorubicin, and resveratrol) was successfully encapsulated into the HACE-based NPs in our previous studies.24,27−29,31 Hydrophobic CE residue was chemically conjugated to hydrophilic HA oligomer and the self-assembled structure of HACE in the aqueous phase (critical aggregation concentration: 42 μg/mL) was already identified in our previous report.24 As d is a hydrophilic material, it is expected that d may be existed in the exterior of HACE-d NPs in the aqueous phase. Exposed d in the outer surface of HACEd NPs may participate in cellular adhesion. As reported,24,27−29,31 HA shell in NPs can interact with CD44 receptor existed in many kinds of malignant tumors and it may lead to the receptor-mediated endocytosis.

Figure 4. In vitro release profiles of phloretin from NPs. The released amounts of phloretin (%) from NPs, at pH 7.4, according to the incubation time are presented. Each point represents the mean ± SD (n = 3).

F

DOI: 10.1021/acsami.7b06582 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Figure 5. Cellular uptake study in MDA-MB-231 cells. (A) Cellular accumulation amounts of C6 were quantitatively analyzed by flow cytometry. C6 (2 μg/mL)-loaded HACE NPs and HACE-d NPs were incubated for 1 and 4 h for their analysis. (B) Cellular distribution of NPs was observed by CLSM. C6 (2 μg/mL)-loaded HACE NPs and HACE-d NPs were incubated for 1 and 4 h and the fluorescence signals were detected by CLSM. Blue and green colors indicate DAPI and C6, respectively. The length of the scale bar in the image is 20 μm. (C) Accumulated amounts of phloretin, after 6 h incubation, in MDA-MB-231 cells were quantitatively by HPLC. Each point represents the mean ± SD (n = 3). #, p < 0.05 compared with phloretin solution group. ∗, p < 0.05 compared with HACE/phloretin NPs group.

diameter of NPs in the aqueous buffer cannot provide complete information about their actual size in the blood flow. Particle size in serum media (50% FBS) may simulate the real particle size of nanocarriers in the biological fluids. As shown in Figure S4, HACE/phloretin NPs exhibited small portion of aggregates in both 0 and 24 h incubation groups. In contrast, HACE-d/ phloretin NPs group did not show the formation of aggregates even after 24 h incubation in serum. The initial mean diameter of HACE-d/phloretin NPs was maintained during 24 h incubation period. Improved stability of HACE-d/phloretin NPs, compared with HACE/phloretin NPs, may be explained by the location of d in the outer surface in the aqueous environment. Hydrophilic d residue of HACE-d NPs seems to contribute to the maintenance of stability and reduce the formation of aggregates. Observed stability of HACE-d/ phloretin NPs may guarantee the efficient tumor targetability of NPs after their intravenous injection. Drug Release Study. The in vitro release profiles of phloretin from HACE/phloretin NPs and HACE-d/phloretin NPs were assessed at pH 7.4 (Figure 4). Sustained release pattern of drug may be one of requisites for the development of injection dosage forms. It can provide the longer circulation of cargo in the bloodstream. Additionally, it can decrease the dosing frequency and improve patient compliance. As shown in Figure 4, no significant difference was shown in the released amounts of drug between HACE/phloretin NPs and HACE-d/ phloretin NPs groups at pH 7.4 buffer including 0.3% Tween 80. Because of the higher degradation rate of phloretin at normal physiological pH (pH 7.4), rather than acidic pH conditions,36 NPs-loaded dialysis tube was moved to fresh

Phloretin was loaded to HACE-d NPs by a reported solvent evaporation method.28,29,31 The hydrodynamic diameters of HACE/phloretin NPs and HACE-d/phloretin NPs were 276 and 279 nm, respectively, and the polydispersity index values of both NPs were approximately 0.2, implying narrow size distribution (Table 1 and Figure 3). In TEM images (Figure 3A and Figure S3), similar particle size (as determined by DLS method) and spherical morphology of phloretin-loaded NPs were observed. Magnified TEM images of NPs, indicating the round shape, are presented in Figure S3. HACE-d/phloretin NPs group (−18 ± 1 mV) displayed less negative zeta potential value than that of HACE/phloretin NPs group (−30 ± 1 mV) (p < 0.05). The number of negatively charged free carboxylic group of HACE-d seems to be reduced, compared with HACE, via amide bond formation with d residue. It implies the successful conjugation of d to HACE and the existence of d group in the exterior of HACE-d NPs. The mean encapsulation efficiency values of phloretin in HACE NPs and HACE-d NPs were 86% and 91%, respectively. Several physicochemical characteristics (i.e., mean diameter, size distribution, and zeta potential) of NPs may determine the passive tumor targeting.4,34 Observed hydrodynamic size of phloretin-loaded NPs may be appropriate for passive targeting (related to an EPR effect) for tumors.4,9,35 It is hypothesized that EPR effect, HA-CD44 receptor binding, and the musselinspired property of d may totally explain the tumor-specific drug delivery in this investigation. In Vitro Stability Test. The stability of phloretin-loaded NPs in FBS (50%) was evaluated by monitoring the hydrodynamic size of NPs (Figure S4). Hydrodynamic G

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fluorescent dye for flow cytometry and CLSM studies (Figure 5A,B), and it was incorporated into the NPs instead of phloretin. The different physicochemical properties (i.e., solubility and lipophilicity) of C6, compared to phloretin, can be considered to explain this result. As shown in CLSM images (Figure 5B), intracellular fluorescence signals in HACE-d NPs group were also stronger than those of HACE NPs group after 1 and 4 h incubation. To investigate the influence of d residue in HACE-d NPs on the cellular accumulation efficiency, HACE-d with low feeding ratio of d to HACE (HACE-dlow) was synthesized, and NPs based on HACE-dlow containing C6 were prepared. The mean content of d in HACE-dlow was 0.3% according to the 1H NMR analysis, and it was lower than that of HACE-d (1.3%) (Figure S2). As shown in Table S1 and Figure 5A, the fluorescence intensity of HACE-dlow NPs was similar to that of HACE NPs and significantly lower than that of HACE-d NPs (p < 0.05). It implies that exposed d in the exterior of HACE-d NPs can contribute to the cellular uptake of HACE-d NPs. To elucidate the cell adhesion mechanisms of HACE-d NPs, free d was used as an inhibitor for the cellular uptake study of HACE-d NPs (Figure S5). The relative fluorescence intensity of (HACE-d/C6 NPs + d) group was 54% of that of HACE-d/C6 NPs group after 2 h incubation. The conjugation of free d molecule with the thiol group of cellular membrane may hamper the cellular adhesion of HACEd NPs (Figure 2B). In addition, it was reported that dopamine receptors are highly present in MDA-MB-231 cells.37 Therefore, the preincubation of free d may inhibit the dopamine receptor-mediated cellular entry of HACE-d NPs. Both mechanisms can explain the enhanced cellular internalization efficiency of HACE-d NPs, rather than that of HACE NPs. The intracellular accumulation property of HACE-d NPs was further evaluated with the measurement of the uptaken amounts of phloretin (Figure 5C). After 6 h incubation, a significant difference between phloretin solution group and HACE/phloretin NPs group was not presented. However, HACE-d/phloretin NPs group exhibited 72% and 71% higher cellular accumulated amounts of drug, compared with phloretin solution group and HACE/phloretin NPs group (p < 0.05). Differences in physicochemical and membrane permeability properties between C6 and phloretin and incubation period seem to affect the improvement of cellular accumulation efficiency of NPs. Although the cellular internalization amount of phloretin in longer incubation period (e.g., > 24 h) was not determined due to its cytotoxicity, the result of antiproliferation assay (Figure 6) can contribute to identify the improved cellular internalization efficiency of HACE-d NPs rather than HACE NPs. HACE-d NPs accomplished an additional elevation in the cellular accumulation capability, rather than HACE NPs. It can make a contribution to the improvement of tumor targetability. Cytotoxicity Test. Cytotoxicity of synthesized HACE-d and in vitro antitumor efficacy of phloretin-loaded NPs were evaluated in MDA-MB-231 cells (Figure 6). That cell line was adopted as a CD44 receptor-expresssed cancer cell in previous studies.27,32,38 HACE did not show serious cytotoxicity to cancer cells, including MDA-MB-231 cells, in our previous reports.24,27−29,31,38 After incubating for 72 h, cell viability according to HACE-d concentration was measured in MDAMB-231 cells (Figure 6A). In tested concentration range (∼500 μg/mL), the cell viability was over 80%. It implies that induced antitumor efficacy of phloretin-loaded NPs may be based on the pharmacological actions of phloretin and the biofunctions of fabricated NPs, not by the cytotoxicity of synthesized

Figure 6. Cytotoxicity test in MDA-MB-231 cells. (A) Cell viability profiles according to the concentrations of HACE-d are shown. HACE-d, at various concentrations (∼500 μg/mL), was incubated for 72 h and the cell viability was measured. Each point represents the mean ± SD (n = 3). (B) Cell viability values according to the concentration of drug are presented. Phloretin solution, HACE/ phloretin NPs, and HACE-d/phloretin NPs were incubated for 72 h, and the cell viability was measured by colorimetric method. Each point represents the mean ± SD (n = 4).

release media at each sampling point in this study. In both groups, the released amounts reached the equilibrium state after 48 h incubation. The released amounts of phloretin from HACE/phloretin NPs and HACE-d/phloretin NPs were 85.7 ± 2.6% and 83.8 ± 1.6%, respectively, after 168 h incubation. Although pH-dependent drug release was not observed in this study due to the replacement of release media (data not shown), lower degradation of phloretin in more acidic pH, compared to normal physiological pH, implies the efficient cancer therapy based on the inhibition of glucose transport into the cells.36 Cellular Uptake Study. Cellular uptake efficiency and intracellular location of NPs were tested in MDA-MB-231 cells (Figure 5). It was hypothesized that hydrophilic d existed in the outer layer of HACE-d NPs can improve the cellular adhesion. In our previous studies,24,27−29,31 HA-CD44 receptor interaction-mediated endocytosis was already demonstrated; thus, the elucidation of its detailed mechanism was not included in this study. For monitoring in vitro movement of developed NPs in MDA-MB-231 cells, NPs containing a fluorescent dye (i.e., C6) were prepared. As shown in Figure 5A, HACE-d NPs exhibited higher cellular uptake efficiency rather than HACE NPs at both incubation time groups (1 and 4 h). The mean fluorescence intensity values of HACE-d NPs after 1 and 4 h incubation were 41% and 14% higher than each value of HACE NPs, respectively (p < 0.05). In early incubation period (i.e., 1 h), the uptake rate of HACE-d NPs seems to be higher than that of HACE NPs. The difference between two groups seemed to decrease at 4 h incubation group rather than 1 h group. However, the fluorescence intensity of HACE-d NPs group was still higher than HACE NPs group. C6 was selected as a H

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Figure 7. Tumor penetration studies in MDA-MB-231 spheroid model. (A) Tumor distribution of C6-loaded NPs in MDA-MB-231 spheroids was observed by CLSM after incubating for 24 h. The length of scale bar in the image is 50 μm. (B) Spheroid growth inhibition profiles after incubating for 24 h are shown. The volume of spheroids was observed for 48 h. Each point represents the mean ± SD (n ≥ 3). &, p < 0.05 compared with control group. #, p < 0.05 compared with phloretin solution group. ∗, p < 0.05 compared with HACE/phloretin NPs group. +, p < 0.05 compared with HACE-d NPs group. (C) Optical images of MDA-MB-231 spheroids after 48 h incubation are presented. The length of scale bar in the image is 100 μm.

to improve tumor penetration after reaching the surrounding region of tumor.4,9 HACE-d NPs were designed and fabricated for the elevation of cellular adhesion and subsequent improved tumor penetration in this study. The distribution of developed NPs including C6 in spheroid model was observed by CLSM, and the inhibitory effect of phloretin-loaded NPs for spheroid growth was assessed (Figure 7). The fluorescence signal of HACE-d NPs group was markedly stronger than HACE NPs group in MDA-MB-231 spheroid model (Figure 7A). Notably, the fluorescence signal in core region of spheroid was also stronger in HACE-d NPs group rather than HACE NPs group. Improved cellular accumulation efficiency of NPs and uptaken amounts of phloretin in two-dimensionally cultured cell model can explain this result (Figure 5). Exposed d in the surface of HACE-d NPs may play a role regarding cellular adhesion and their entry into the tumor spheroid. Spheroid growth inhibitory effect of phloretin-loaded NPs was also evaluated after 48 h incubation (Figure 7B and 7C). After 48 h incubation, the spheroid volume of HACE-d/phloretin NPs group was obviously smaller than control (no treatment), phloretin solution, HACE/phloretin NPs, and HACE-d NPs groups (p < 0.05). Enhanced infiltration efficiency of HACE-d/phloretin NPs seems to provide better spheroid growth inhibitory effect rather than phloretin solution and HACE/phloretin NPs group. HACE NPs and HACE-d NPs groups were used as control groups (without phloretin loading). They were used to identify that the inhibitory effects of phloretin-loaded NPs on spheroid growth were based on the pharmacological efficacies of phloretin, not by the toxicity of blank HACE or HACE-d

HACE-d. Phloretin can inhibit the uptake of glucose via GLUT; thus, it can be used as an anticancer agent.18−21 It was known that GLUT was expressed in MDA-MB-231 cells; therefore, it can inhibit glycolysis and contribute to the antitumor efficacies.23,39 As shown in Figure 6B and Table S2, IC50 values of phloretin solution, HACE/phloretin NPs, and HACE-d/phloretin NPs groups were calculated after 72 h incubation. The IC50 values were arranged as follows: phloretin solution > HACE/phloretin NPs > HACE-d/phloretin NPs (p < 0.05). The improvement in cellular internalization capability of HACE-d NPs, compared with HACE NPs, may be related to the elevated antitumor efficacy of HACE-d NPs (Figures 5 and 6). In Figure 5C, no significant difference existed in the cellular uptaken amounts of phloretin between drug solution and HACE NPs groups (p > 0.05). However, the IC50 value of HACE/phloretin NPs group was obviously lower than phloretin solution group (p < 0.05). It can be explained by the difference in detailed experimental methods (i.e., incubation time and cell seeding density). Enhanced in vitro antitumor efficacy of HACE-d/phloretin NPs, compared with HACE/ phloretin NPs, may guarantee the improved in vivo antitumor efficacy after NPs’ approach in tumor tissue followed by intravenous administration. Tumor Penetration Studies. Infiltration of nanocarriers toward the internal region of solid tumor is very critical for producing efficient antitumor efficacies. Because of the higher IFP and firmness of extracellular matrix, the homogeneous distribution of anticancer agent in whole tumor mass cannot be easily accomplished.40 Various approaches have been adopted I

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Figure 8. NIRF imaging study in MDA-MB-231 tumor-xenografted mouse models. Cy5.5-loaded HACE NPs and HACE-d NPs were intravenously injected in the mouse model. Real-time images were acquired at 0 (pre), 1, 3, 6, and 24 h. (A) Scanned images of whole body are shown. Red dashed circle means the tumor region. (B) Fluorescence intensity (average NC) profiles, according to the time, in tumor region are presented. Each point represents the mean ± SD (n = 3). ∗, p < 0.05 compared with HACE NPs group. (C) Fluorescence intensity (average NC) values in each organ or tissue are presented. Each point represents the mean ± SD (n = 3). ∗, p < 0.05 compared with HACE NPs group. (D) Tumor penetration efficiency in MDA-MB-231 tumor-xenografted mouse model was evaluated by three-dimensional NIRF imaging technique. Z-stack NIRF images of tumor at 24 h postinjection are shown.

NPs. Therefore, the significant difference between HACE NPs and HACE-d NPs is not expected result in this study. In this study, there was no significant difference in spheroid volumes between HACE/phloretin NPs and HACE-d/phloretin NPs groups after 24 h incubation in spite of smaller spheroid volume of HACE-d/phloretin NPs group rather than HACE/phloretin NPs group (Figure 7B). However, the significant difference in spheroid volume between HACE/phloretin NPs and HACE-d/ phloretin NPs groups was observed at 48 h incubation group (p < 0.05) (Figure 7B). The smaller projected size of spheroid in HACE-d/phloretin NPs group rather than HACE/phloretin NPs group at 48 h (Figure 7C) can support the difference in spheroid volume data (Figure 7B). It is expected that spheroid volume data (considering three-dimensional structure) may be more accurate than spheroid image (two-dimensional image) to

predict the virtual size of spheroids. The combination of all data in Figure 7 (CLSM images, spheroid volume, and projected image of spheroid) can be used to identify the improved spheroid growth inhibitory effects of HACE-d/phloretin NPs, rather than HACE/phloretin NPs. Optical Imaging Test. Tumor targetability and penetration of developed NPs were assessed in the tumor-xenografted mouse using a real-time NIRF imaging (Figure 8 and Figure S6). Cy5.5 (NIRF dye) was conjugated to the nanomaterials or encapsulated in NPs to monitor the in vivo biodistribution of NPs in our previous study.9,24,29,31,35 Hydrophobic Cy5.5 was encapsulated in HACE NPs and HACE-d NPs, and they were intravenously administered to MDA-MB-231 tumor bearing mouse in this study. The fluorescence signal of HACE-d NPs group was stronger than HACE NPs group at 24 h (Figure 8A). J

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Figure 9. In vivo anticancer activity studies in MDA-MB-231 tumor-xenografted mouse models. (A) Tumor growth profiles of control, phloretin solution, HACE/phloretin NPs, and HACE-d/phloretin NPs groups are shown. Tumor volumes were measured for 20 days and phloretin solution and phloretin-loaded NPs were injected via tail vein on day 12, 14, and 17. Each point indicates the mean ± SD (n ≥ 3). &, p < 0.05 compared with the control group. #, p < 0.05 compared with phloretin solution group. ∗, p < 0.05 compared with HACE/phloretin NPs group. (B) Profiles of body weight of control, phloretin solution, HACE/phloretin NPs, and HACE-d/phloretin NPs-injected groups are presented. It was monitored for 20 days. Each point indicates the mean ± SD (n ≥ 3). (C) Staining results of dissected tumor tissues after treatment of drug solution and drug-loaded NPs are presented. The images of HE staining (left) and TUNEL assay (right) are presented. The length of scale bar in the image is 10 μm.

can be distributed in tumor region rather than HACE NPs. The fluorescence intensity in cross-section of tumor mass (coronal plane) was further measured using a three-dimensional analysis technique (Figure 8D).9 The fluorescence intensity of HACE-d NPs group was 37% higher, relative to HACE NPs group (p < 0.05). It indicates that HACE-d NPs can penetrate into the deep core region of tumor rather than HACE NPs. All of these NIRF image data suggest that HACE-d NPs have improved tumor targetability and penetration capability compared with HACE NPs. Surface modification of NPs with d residue may provide another tumor targeting and penetration strategy for cargo delivery and cancer imaging. In Vivo Anticancer Activity Tests. Anticancer activities of developed NPs were tested in tumor bearing mouse (Figure 9). Tumor size and body weight were measured after multiple injection of phloretin solution and phloretin-loaded NPs via intravenous route. As shown in Figure 9A, relative tumor

According to the quantitative analysis (Figure 8B), the fluorescence intensity of HACE-d NPs group was 47−60% stronger than HACE NPs group for 24 h. At 24 h, several organs and tissues, such as liver, spleen, lungs, kidneys, heart, and tumor, were separated from the mouse, and their fluorescence signals were also quantitatively analyzed (Figure 8C). Although the fluorescence intensity of HACE-d NPs group in spleen and liver was slightly higher compared to HACE NPs group, no obvious difference between two groups was presented (p > 0.05). Because of the existence of MPS in spleen and liver, they may be responsible for the clearance of nanocarriers from the bloodstream.4,41 Interestingly, the fluorescence level of HACE-d NPs group in tumor was 74% higher than HACE NPs group (p < 0.05). Also, the averaged fluorescence intensity ratios of tumor to (liver + spleen) in HACE NPs group and HACE-d NPs group were 0.46 and 0.69, respectively. It implies that the higher amount of HACE-d NPs K

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volume ratios (%) of phloretin solution, HACE/phloretin NPs, and HACE-d/phloretin NPs groups, in comparison with control group, were 56%, 40%, and 26%, respectively, on day 20 (p < 0.05). Both phloretin-loaded NPs groups exhibited efficient suppression of tumor growth than that of phloretin solution group. Notably, the tumor volume of HACE-d/ phloretin NPs group was 64% of that of HACE/phloretin NPs group (p < 0.05). No significant difference was presented in body weight of mouse models among experimental groups (Figure 9B). It means that developed NPs have no serious systemic toxicity after intravenous administration. The result of TUNEL assay implies the higher portion of apoptosis in HACE-d/phloretin NPs group rather than the other groups (Figure 9C). All these improved anticancer activities can be explained by enhanced antiproliferation activity, tumor penetration capability, and in vivo tumor targetability of HACE-d NPs.

AUTHOR INFORMATION

Corresponding Author

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

Hyun-Jong Cho: 0000-0002-5070-9371 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Research Foundation of Korea (NRF), funded by the Korean government (MSIP) (No. NRF-2015R1A1A1A05027671).

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REFERENCES

(1) Biswas, S.; Torchilin, V. P. Nanopreparations for OrganelleSpecific Delivery in Cancer. Adv. Drug Delivery Rev. 2014, 66, 26−41. (2) Danhier, F.; Feron, O.; Préat, V. To Exploit the Tumor Microenvironment: Passive and Active Tumor Targeting of Nanocarriers for Anti-Cancer Drug Delivery. J. Controlled Release 2010, 148, 135−146. (3) Lammers, T.; Kiessling, F.; Hennink, W. E.; Storm, G. Drug Targeting to Tumors: Principles, Pitfalls and (Pre-) Clinical Progress. J. Controlled Release 2012, 161, 175−187. (4) Ernsting, M. J.; Murakami, M.; Roy, A.; Li, S. D. Factors Controlling the Pharmacokinetics, Biodistribution and Intratumoral Penetration of Nanoparticles. J. Controlled Release 2013, 172, 782− 794. (5) Matsumura, Y.; Maeda, H. A New Concept for Macromolecular Therapeutics in Cancer Chemotherapy: Mechanism of Tumoritropic Accumulation of Proteins and the Antitumor Agent SMANCS. Cancer Res. 1986, 46, 6387−6392. (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 Delivery Rev. 2011, 63, 136−151. (7) Minchinton, A. I.; Tannock, I. F. Drug Penetration in Solid Tumours. Nat. Rev. Cancer 2006, 6, 583−592. (8) Sun, T.; Zhang, Y. S.; Pang, B.; Hyun, D. C.; Yang, M.; Xia, Y. Engineered Nanoparticles for Drug Delivery in Cancer Therapy. Angew. Chem., Int. Ed. 2014, 53, 2−47. (9) Lee, J. Y.; Chung, S. J.; Cho, H. J.; Kim, D. D. Phenylboronic Acid-Decorated Chondroitin Sulfate A-Based Theranostic Nanoparticles for Enhanced Tumor Targeting and Penetration. Adv. Funct. Mater. 2015, 25, 3705−3717. (10) Li, H. J.; Du, J. Z.; Liu, J.; Du, X. J.; Shen, S.; Zhu, Y. H.; Wang, X.; Ye, X.; Nie, S.; Wang, J. Smart Superstructures with Ultrahigh pHSensitivity for Targeting Acidic Tumor Microenvironment: Instantaneous Size Switching and Improved Tumor Penetration. ACS Nano 2016, 10, 6753−6761. (11) Teesalu, T.; Sugahara, K. N.; Ruoslahti, E. Tumor-Penetrating Peptides. Front. Oncol. 2013, 3, 216. (12) Lee, H.; Dellatore, S. M.; Miller, W. M.; Messersmith, P. B. Mussel-Inspired Surface Chemistry for Multifunctional Coatings. Science 2007, 318, 426−430. (13) Lee, H.; Lee, Y.; Statz, A. R.; Rho, J.; Park, T. G.; Messersmith, P. B. Substrate-Independent Layer-by-Layer Assembly by Using Mussel-Adhesive-Inspired Polymers. Adv. Mater. 2008, 20, 1619− 1623. (14) Park, J.; Brust, T. F.; Lee, H. J.; Lee, S. C.; Watts, V. J.; Yeo, Y. Polydopamine-Based Simple and Versatile Surface Modification of Polymeric Nano Drug Carriers. ACS Nano 2014, 8, 3347−3356. (15) Kim, K.; Kim, K.; Ryu, J. H.; Lee, H. Chitosan-Catechol: A Polymer with Long-Lasting Mucoadhesive Properties. Biomaterials 2015, 52, 161−170.

CONCLUSIONS An amphiphilic HACE-d conjugate was synthesized and the mussel-inspired properties of d were introduced into the NPs for tumor-specific delivery of phloretin and in vivo visualization of cancer. HACE-d/phloretin NPs with 279 nm mean diameter, 0.2 polydispersity index, and −18 mV zeta potential were successfully fabricated. Along with the HA-CD44 receptor binding-intermediated endocytosis of HACE-d NPs, d group exposed in the outer layer of nanocarriers seems to improve cellular accumulation efficiency and subsequent in vitro antitumor efficacy in MDA-MB-231 cells (CD44 receptor and GLUT1-expressed breast adenocarcinoma). Enhanced infiltration and superior spheroid growth inhibitory effect of HACE-d NPs, in comparison with HACE NPs, were observed in MDAMB-231 spheroid model. According to the results of real-time optical imaging studies in the tumor bearing mouse, improved tumor targeting and penetration can be achieved with HACE-d NPs rather than HACE NPs. HACE-d/phloretin NPs group also exhibited the improved anticancer activities, compared with the other experimental groups, in the tumor bearing mouse. The combination of passive targeting (an EPR effect), active targeting (HA-CD44 receptor interaction), improved cellular adhesion and tumor penetration of d residue, and glycolysis inhibition of phloretin may be an unprecedented tumor targeting and penetration approach, and it can be successfully applied for the therapy and imaging of CD44 receptor and GLUT1-expressed cancers.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b06582. Synthetic scheme of HACE; relationship between weight ratio and integration ratio with the physical mixture of HACE and d; magnified TEM image of developed NPs; in vitro stability of phloretin-loaded NPs in serum media; cellular accumulation inhibition study of HACE-d NPs in MDA-MB-231 cells; ex vivo NIRF image of dissected liver, spleen, lungs, kidneys, heart, and tumor; content of d in HACE-dlow and cellular accumulation efficiency of HACE-dlow NPs; IC50 values of phloretin solution and phloretin-loaded NPs in MDA-MB-231 cells (PDF) L

DOI: 10.1021/acsami.7b06582 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

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DOI: 10.1021/acsami.7b06582 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX