Nanostructured Peptidotoxins as Natural Pro-Oxidants Induced

Research Article Cite This: ACS Appl. Mater. Interfaces 2018, 10, 4569−4581

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Nanostructured Peptidotoxins as Natural Pro-Oxidants Induced Cancer Cell Death via Amplification of Oxidative Stress Hongzhi Qiao,*,†,# Dong Fang,†,# Lei Zhang,† Xiaochen Gu,‡ Yin Lu,†,§ Minjie Sun,∥ Chunmeng Sun,∥ Qineng Ping,∥ Junsong Li,† Zhipeng Chen,† Jun Chen,† Lihong Hu,†,⊥ and Liuqing Di*,† †

State Key Laboratory Cultivation Base for TCM Quality and Efficacy, School of Pharmacy, §Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, and ⊥Jiangsu Key Laboratory for Functional Substance of Chinese Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China ‡ College of Pharmacy, University of Manitoba, 750 McDermot Avenue, Winnipeg, Manitoba R3E 0T5, Canada ∥ State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China Pharmaceutical University, Nanjing 210009, China S Supporting Information *

ABSTRACT: Melittin (Mel), one of the host defense peptides derived from the venom of honeybees, demonstrates substantial anticancer properties, which is attributed to augmenting reactive oxygen species (ROS) generation. However, little has been reported on its pro-oxidation capacity in cancer oxidation therapy. In this study, an ROS amplifying nanodevice was fabricated through direct complexation of two natural pro-oxidants, Mel and condensed epigallocatechin gallate (pEGCG). The obtained nanocomplex (NC) was further covered with phenylboronic acid derivatized hyaluronic acid (pHA) through the ROS-responsive boronate ester coordination bond to produce pHA-NC. Upon undergoing receptor-mediated endocytosis into cancer cells, the inner cores of pHA-NC will be partially uncovered once pHA corona is degraded by hyaluronidase and will then escape from the lysosome by virtue of cytolytic Mel. The elevated ROS level in the tumor cytoplasm can disrupt the boronate ester bond to facilitate drug release. Both Mel and pEGCG could synergistically amplify oxidative stress and prolong ROS retention in cancer cells, leading to enhanced anticancer efficacy. This ROS cascade amplifier based on selective coordination bond and inherent pro-oxidation properties of natural ingredients could detect and elevate intracellular ROS signals, potentiating to move the tumor away from its homeostasis and make the tumor vulnerable. Compared to previously reported chemosynthetic pro-oxidants, the ROS self-sufficient system, fully composed of natural medicine, from this study provides a new insight in developing cancer oxidation therapy. KEYWORDS: peptidotoxins, melittin, polyphenols, drug delivery, oxidative stress, oxidation therapy

1. INTRODUCTION Host defense peptides (HDPs) are pivotal components found in the defense system of many complex life forms. They usually are short, cationic, amphipathic peptides possessing diverse yet selective pharmacological characteristics.1,2 Melittin (Mel), one of the HDPs derived from the venom of European honeybees (Apis mellifera), is a water-soluble peptide with 26 amino acids.3 Mel has been investigated for antitumor activity, and its reported anticancer mechanisms included classical apoptosisinducing cell death and suppression of angiogenesis through the VEGFR pathway.4,5 However, Mel also demonstrated instability and nonspecific cellular lytic activity in vivo.6,7 A series of strategies have been utilized to improve the functionality of Mel.8 Nevertheless, conventional structural modification or preparation molding was not satisfactory. For example, conjugating Mel with an antibody or a targeting protein would not completely shield its original hematoxicity; thus, systemic administration was compromised.9 Encapsulating © 2018 American Chemical Society

Mel with lipid carriers did not work either, because Mel is capable of disrupting the phospholipid bilayer membrane.10 Epigallocatechin gallate (EGCG), one of the active polyphenols isolated from green tea, has shown therapeutic effect against cancer.11 In addition, it is capable of binding with a variety of biological molecules, including proteins and peptides, and creating nanoscaled aggregates.12 The formed nanoaggregates are able to temporarily conceal the toxicity of peptidotoxins through shielding their specific amino acid residues12 and deliver to the tumor site by virtue of enhanced permeability and retention effect (EPR effect).13 Moreover, cumulative data indicated that both Mel and EGCG produced reactive oxygen species (ROS),14,15 which was considered a positive attribute to their anticancer activities because overReceived: December 10, 2017 Accepted: January 16, 2018 Published: January 16, 2018 4569

DOI: 10.1021/acsami.7b18809 ACS Appl. Mater. Interfaces 2018, 10, 4569−4581

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ACS Applied Materials & Interfaces expression of ROS will pose lethal damage to tumor cells.16 Furthermore, the ROS level in abnormal cancer cells was reported to be thousand times higher than that in normal cells, which had been used as a specific signaling molecule to develop an ROS-responsive drug-delivery system (DDS) to target selective drug release into cancer cells.17−19 Previous studies have synthesized some exogenous redox modulators, which could either abrogate antioxidant defense or augment ROS generation to a level, above which tumor cells will not survive.20,21 Nevertheless, little has been reported describing selective natural products as pro-oxidants for efficient cancer oxidation therapy. In this study, an ROS cascade amplification DDS comprising Mel and EGCG was prepared and tested as natural prooxidants to enhance anticancer targeting and efficacy (Scheme 1). EGCG would be concentrated on condensed tannins

ingredients might be of great interest for testing pro-oxidation substances in novel anticancer therapy. Compared to previous ROS-generating systems composed of chemo-synthesized small molecules or polymers, this sophisticated system is fully constituted of natural active components, which have been utilized in traditional medicine for thousands of years, whose functionality and potentials could be further verified and expanded by modern science to offer alternative clinical applications.

2. EXPERIMENTAL SECTION 2.1. Materials. Melittin (Mel, 98%) was synthesized by GL Biochem Ltd (Shanghai, China). EGCG (98%) was purchased from Meilun Biotech Co., Ltd (Dalian, China). HA (10 kDa) was purchased from Haihua Biochem Ltd (Zhenjiang, China). Alizarin red S (ARS, 98%), coomassie blue R 250, 3-aminobenzeneboronic acid (PBA, 98%), nystatin (99%), amiloride (99%), N-hydroxysuccinimide (NHS), and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) were purchased from Adamas (Shanghai, China). Fluorescein isothiocyanate (FITC), rhodamine B isothiocyanate (RBITC), and chlorpromazine hydrochloride (99%) were purchased from TCI (Shanghai, China). Phosphate-buffered saline (PBS), Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium-bromide (MTT), and 0.25% trypsin were purchased from HyClone Laboratories (Logan, UT). Annexin V-FITC/PI double staining apoptosis detection kit, JC-1 detection kit, and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were purchased from KeyGEN BioTECH (Nanjing, China). LysoTracker Red and 4,6-diamidine-2-phenylindole (DAPI) were purchased from Beyotime (Shanghai, China). Cy7 was purchased from Fanbo Biochem Ltd (Beijing, China). Deionized (DI) water with a resistivity of 18.2 MΩ cm−1 was used in all experiments. B16F10 melanoma cells and NIH3T3 fibroblast cells were purchased from American Type Culture Collection. Cells were cultured in DMEM containing 10% FBS and 1% penicillin− streptomycin at 37 °C under an atmosphere of 5% CO2 and 90% relative humidity. 2.2. Synthesis and Characterization of pEGCG. EGCG (1.0 g) was dissolved in a mixture of DI water and dimethylsulfoxide (DMSO, 9:1, v/v, 20% acetic acid). The polymerization reaction was initiated by adding acetaldehyde (7.2 mL) and was maintained at 20 °C (pH 2.0) under a nitrogen atmosphere for 12 h.23 The resulting solution was dialyzed [1 kDa molecular weight cutoff (MWCO)] against DI water and lyophilized to obtain pEGCG. Proton nuclear magnetic resonance (1H NMR) (DSX400, Bruker, Billerica, MA) spectroscopy was used to analyze the structure of pEGCG. Electrospray ionization mass spectrometry (ESI-MS) (LCQ Fleet, Thermo Fisher, Waltham, MA) was used to determine the molecular weight. 2.3. Interaction of Mel and pEGCG. Complexation of Mel and pEGCG was processed under mild conditions to produce Mel/ pEGCG NC. Briefly, aliquots of pEGCG (500 μg/mL) and Mel (1 mg/mL) was mixed in nitrogen for 30 min at room temperature in the dark. Size distribution and morphology of the NC were measured by dynamic light scattering (DLS) (Malvern, Worcestershire, United Kingdom) and transmission electron microscopy (TEM) (H-7650, Hitachi, Japan), respectively. To confirm complexation of pEGCG and Mel, native-polyacrylamide gel electrophoresis (PAGE) technique was used on a gel electrophoresis system (Mini-PROTEAN Tetra, BioRad, Hercules, CA). The sample (20 μL) containing approximately 10 μg of Mel was loaded on the gel, followed by staining with 0.25% coomassie blue R 250 for 30 min. After destaining three times, the gel was scanned under an ImageScanner III (GE Healthcare, Little Chalfont, United Kingdom). The binding affinity between Mel and pEGCG was determined by the microscale thermophoresis (MST) technology on a Monolith NT.115 system (NanoTemper, Germany) using 80% blue lightemitting diode (488 nm) and 1 V infrared laser power.24 Laser on and off times were set at 35 and 5 s, respectively. FITC-labeled Mel was

Scheme 1. Schematic Illustration of a Multipronged Nanocomplex (pHA-NC) Comprising Mel and pEGCG Surrounded by Hyaluronic Acid (HA) through the Coordination Bond for ROS Self-Sufficient Oxidation Therapy of Cancersa

a

I, EPR Effect; II, CD44 Receptor Recognition; III, ReceptorMediated Endocytosis; IV, Endosomal/Lysosome Escape; V, ROSTriggered Disruption of pHA Corona and Drug Release; VI, ROS Generation and Amplification; and VII, ROS Feedback Regulation

(pEGCG) to preferably bind with Mel, and the subsequently formed nanocomplex (NC) would be further covered by phenylboronic acid derivatized hyaluronic acid (pHA) through the boronate ester coordination bond to obtain pHA-NC. Upon delivering to the tumor region by the EPR effect and achieving active binding to the overexpressed receptors in cancer cells, such as CD44, the inner cores of pHA-NC would be partially uncovered once pHA corona is degraded by hyaluronidase (HAase) and then would escape from the lysosome by virtue of cytolytic Mel.22 The elevated ROS level in the tumor cytoplasm would disrupt the boronate ester bond to facilitate drug release while also maintaining a consistent ROS mass above a threshold, in which the tumor cells would not survive. This positive feedback loop constructed by pHANC, as if a powerful pump of ROS, could detect and amplify intracellular ROS signals, potentiating to move the tumor away from its homeostasis and make it more vulnerable. Development of an ROS self-sufficient system made of natural 4570


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ACS Applied Materials & Interfaces synthesized according to the standard operating method. Subsequently, 2 μM FITC-labeled Mel was combined with different concentrations of pEGCG (0.8, 1.6, 3.2, 6.4, 12.5, 25, 50, 200, and 500 μM) to obtain FITC-labeled NC for measurement. The MST curves were recorded, and the dissociation constant (Kd) of Mel and pEGCG was measured using NT analysis software (NT Control v2.1.31). To examine the binding force, a series of chemical reagents, including Tween 20 and Triton X-100 (hydrophobic competitors), urea (hydrogen bond competitor), and NaCl (ionic competitor), were diluted to variable concentrations and added to preformed NC solution (1:1, v/v). After incubation for 30 min at an ambient temperature, size distribution variation of NC was measured using a Zetasizer (Nano ZS, Malvern, United Kingdom). To further confirm the interaction force, the fluorescence resonance energy transfer (FRET) technique was utilized.25 Mel and pEGCG were first labeled with an energy acceptor (RBITC) and an energy donor (FITC), respectively.26 FRET-based NC was obtained by mixing aliquots of FITC-labeled pEGCG (500 μg/mL) and RBITClabeled Mel (1 mg/mL). Next, the FRET-based NC was incubated in 0, 1, 10, and 40 mM Tween 20 for 30 min at room temperature separately. Emission spectra of samples were recorded by a fluorescence spectrophotometer (LS-50B, Perkin-Elmer, Waltham, MA) at 488 nm excitation. Variations in the FRET signal were used to detect dissociation of Mel and pEGCG. The FRET ratio was indicated as IRBITC/(IRBITC + IFITC) × 100%, where IRBITC is the fluorescence intensity of RBITC at 572 nm and IFITC is the fluorescence intensity of FITC at 516 nm. 2.4. Synthesis and Characterization of pHA. HA (0.05 mmol) was dissolved in 50 mL of DI water. Then, 2 mmol EDC/NHS was added to this solution and stirred for 30 min. PBA (2 mmol) was dissolved in 20 mL of 50% ethanol and added dropwise.27 The reaction was carried out for 24 h at room temperature. The resulting solution was dialyzed (3.5 kDa MWCO) against DI water and lyophilized to obtain pHA. The structure of pHA was analyzed by 1H NMR spectra using D2O/DMSO-d6 (1:1, v/v) as the solvent, and the degree of PBA modification in pHA was calculated from the regression line of the integration ratio (7.3−7.7 ppm to 1.8 ppm) to the weight ratio between PBA and HA.27 2.5. Preparation and Characterization of pHA-NC. pHA-NC was prepared via two sequential self-assembly steps in aqueous solution: (1) complexation between Mel and pEGCG to produce the inner core NC; (2) pHA surrounding the preformed NC by the coordination bond to generate hydrophilic corona.28,29 Briefly, the NC was formed by mixing aliquots of pEGCG solution (500 μg/mL) and Mel solution (1 mg/mL) in a nitrogen atmosphere under agitation for 30 min at room temperature. Subsequently, the resultant NC was added to pHA solution (2.5 mg/mL, pH 7) dropwise and stirred for 30 min to obtain pHA-NC. Particle size and zeta potential were measured using a Zetasizer. TEM was utilized to observe the particle morphology. The mechanism of pHA surrounding the NC was studied by a competitive binding assay.30 Briefly, a solution of ARS (1.8 × 10−5 M) and pHA (2.4 × 10−4 M) was mixed with pEGCG or NC at various concentrations. Emission spectra from 480 to 700 nm were recorded using a fluorescence spectrophotometer at 468 nm excitation. Stability of pHA-NC was evaluated in saline and serum. As a control, non-pHA was directly adsorbed onto the NC surface to create physically encapsulated HA/NC. For saline stability, pHA-NC and HA/NC were incubated in 150 mM NaCl at 37 °C, and the particle size and zeta potential were measured at 0, 1, 6, and 12 h. pHA-NC stability in serum was determined by incubating in 10% FBS at 37 °C for 24 h, and the particle size distribution of pHA-NC was monitored using DLS. Dilution stability was studied by diluting pHA-NC in DI water to 1, 10, 100, and 1000-fold, followed by particle size measurement. 2.6. Degradation of pHA Corona in the Presence of HAase. HAase-mediated degradation of pHA was evaluated by monitoring the changes in the surface charge of pHA-NC after treatment with HAase. Briefly, a mixture of pHA-NC and buffer solution (pH 4.5, 6.5, or 7.4, HAase concentration at 0.5 mg/mL) was incubated in a 37 °C water bath under agitation for 4 h. pHA-NC in PBS without HAase was

compared as the negative control. The zeta potential was measured by a Zetasizer. 2.7. In Vitro Release Study. To determine the release kinetics of pHA-NC, 2 mL of pHA-NC containing 0.5 mg of Mel was placed in a dialysis bag (25 kDa MWCO) and immersed in 18 mL of PBS solution at pH 4.5 (with/without 0.5 mg/mL HAase) or pH 7.4 (with/without 1 mM H2O2). At predetermined intervals, 0.2 mL of the sample was withdrawn, followed by replenishing with fresh buffer solution. Concentration of Mel in samples was quantified using the highperformance liquid chromatography (HPLC) method. The chromatographic conditions were the following: mobile phase: acetonitrile/0.2% trifluoroacetic acid water (45:55, v/v); flow rate: 1.0 mL/min; and detection wavelength: 220 nm. HA-NC (HA chemically conjugated NC) was prepared as a control through the standard EDC chemistry procedure and also dialyzed against PBS at pH 7.4 with H2O2. 2.8. H2O2 Production in pH 7.4 Buffer Solution. Mel, pEGCG, NC, pHA-NC or HA-NC (1 mL, 250 μg/mL Mel; 125 μg/mL pEGCG) was added to 9 mL of PBS buffer (pH 7.4). The sample (200 μL) was withdrawn at predetermined intervals and mixed with 800 μL of acetic acid to prevent further oxidation. All samples were stored at −20 °C until HPLC quantification. The acidified sample (1 mL) was mixed with 9 mL of acetonitrile/water (2:1, v/v) containing 0.2 mM triphenylphosphine (TPP), followed by reaction in the dark for 2 h. The H2O2 amount in the samples was proportional to the oxidized TPP, which was measured by HPLC.31 2.9. Intracellular ROS Generation and Retention. B16F10 cells were seeded in 12-well plates (1 × 105 cells/well) and cultured for 24 h. After incubating for different intervals in the dark with Mel, pEGCG, NC, pHA-NC, or HA-NC (Mel at 5 μg/mL; pEGCG at 2.5 μg/mL), B16F10 cells were washed with cold PBS. Fresh culture medium containing DCFH-DA (30 μM) was added to incubate at 37 °C for another 20 min. Intracellular ROS level was analyzed using flow cytometry (FACS Caliber, Becton Dickinson, San Jose, CA). For antioxidation study, the cells were preincubated with 1 mM Nacetylcysteine (NAC) or glutathione (GSH) for 2 h, followed by pHA treatment. To investigate the elimination kinetics of ROS, the ROS concentration that remained in intracellular media was monitored at predetermined intervals after an 8 h pretreatment of B16F10 cells with Mel, pEGCG, NC, pHA-NC, or HA-NC (Mel at 5 μg/mL; pEGCG at 2.5 μg/mL). The fluorescent image of cells at 0 and 6 h after 8 h of pretreatment was captured using a fluorescent microscope (Olympus X51, Tokyo, Japan). Cellular GSH level was also evaluated using Ellman’s reagent.32 GSH level from the untreated cells was used as a baseline value for comparison with that of cells receiving different treatments. 2.10. Cellular Uptake. To investigate the site-specific delivery, B16F10 cells and NIH3T3 cells seeded in 12-well plates (1 × 105 cells/well) were cultured with FITC-labeled pHA-NC or HAasetreated pHA-NC (5 μg/mL Mel) at pH 4.5 for 4 h. Afterward, the cells were washed with cold PBS, trypsinized, resuspended in 0.5 mL of PBS, and analyzed using a flow cytometer. For comparison, the cells were preincubated with free HA (10 mg/mL) for 1 h before pHA-NC treatment. To illustrate the endocytosis pathway of pHA-NC, B16F10 cells were preincubated for 1 h at 37 °C with different specific endocytosis inhibitors: chlorpromazine (10 μg/mL) for clathrin-mediated endocytosis inhibition; nystatin (0.5 μg/mL) for caveolin-mediated endocytosis inhibition; amiloride (150 μg/mL) for macropinocytosis inhibition; and sodium azide (200 μg/mL) for energy inhibition. Subsequently, the cells were treated with FITC-labelled pHA-NC (5 μg/mL Mel) for 4 h and analyzed using a flow cytometer. 2.11. Confocal Microscopy. For intracellular delivery study, B16F10 cells (1.0 × 105 cells/well) were seeded in confocal microscopy dishes. After culture for 24 h, the cells were exposed to FITC-labeled pHA-NC (3 μg/mL Mel) at 37 °C for 1 h and then washed with cold PBS twice, followed by incubation in FBS-free culture medium for another 0.5 or 2 h. Subsequently, the cells were stained with LysoTracker Red (2 μM) for 30 min and DAPI (10 μg/ mL) for 10 min. The samples were then washed with 4 °C PBS twice and immediately observed using confocal laser scanning microscopy 4571


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Figure 1. Preparation and characterization of the NC and pHA-NC. (A) Size distribution and morphology of NC measured by DLS (left) and TEM (right), respectively. (B) Schematic illustration of the MST system and representative MST traces of FITC-labeled Mel (2 μM) with different concentrations of pEGCG (0.8−500 μM). (C) Particle size of the NC incubated with Tween 20, Triton X-100, urea, and NaCl for 30 min (n = 3). (D) Fluorescence emission spectra of FRET-based NC at 488 nm excitation wavelength after incubation with different concentrations of Tween 20. (E) Zeta potential of pHA-NC in the absence and presence of 0.5 mg/mL HAase at pH 4.5, 6.5, and 7.4 (n = 3). (F) In vitro release of pHA-NC in response to HAase (0.5 mg/mL) and H2O2 (1 mM) at pH 4.5 and 7.4, respectively. Non-ROS-sensitive HA-NC incubated in pH 7.4 PBS, with 1 mM H2O2 as the negative control (n = 3). (G) TEM images of pHA-NC (left) and pHA-NC treated with 1 mM H2O2 for 2 h (right). the following equation: CI = D1/Dx1 + D2/Dx2, where D1 and D2 are the concentrations of Mel and pEGCG in combination resulting in the fraction affected (Fa) × 100% growth inhibition and Dx1 and Dx2 are the concentrations of single Mel and pEGCG resulting in Fa × 100% growth inhibition.34 Apoptosis-inducing capability of different preparations was assessed using an Annexin V-FITC/PI apoptosis detection kit. B16F10 cells at a density of 2 × 105 cells/well were seeded and cultured in 6-well plates for 12 h at 37 °C and then incubated with Mel, pEGCG, NC, pHANC, or HA-NC (5 μg/mL Mel and/or 2.5 μg/mL pEGCG) for 12 h. Cellular apoptosis was analyzed using a flow cytometer according to standard kit protocol. MMP was measured with the JC-1 dye using flow cytometry.35 Briefly, B16F10 cells were seeded in 6-well plates at a density of 2 × 105 cells/well and cultured for 12 h at 37 °C. The cells were then incubated with Mel, pEGCG, NC, pHA-NC, or HA-NC (5 μg/mL Mel and/or 2.5 μg/mL pEGCG) for 12 h. After incubation, the cells were collected and incubated in the JC-1 dye for 20 min at 37 °C. Subsequently, the samples were washed twice with cold JC-1 buffer, resuspended in 0.5 mL PBS, and analyzed. The x-axis (FL-1 channel) indicated green fluorescence (JC-1 monomers), whereas the y-axis (FL-2 channel) demonstrated red fluorescence (JC-1 aggregates). Cells with a low MMP were in the lower-right quadrant. 2.13. Animals and Tumor Xenograft Models. Animal studies were conducted in accordance with the Guide for Care and Use of Laboratory Animals, and the protocol was approved by the Animal Care Committee of Nanjing University of Chinese Medicine. C57BL/ 6 mice (male, 18−22 g) were purchased from the College of

(CLSM) (Carl Zeiss LSM 700, Oberkochen, Germany). As a control, Mel was replaced by bovine serum albumin (BSA) labeled with FITC to obtain pHA-NCb (3 μg/mL BSA). To monitor the intracellular dissociation of Mel and pEGCG, B16F10 cells were exposed to FRET-based pHA-NC that was composed of RBITC-labeled Mel (3 μg/mL) and FITC-labeled pEGCG (1.5 μg/mL) for 4 h.33 Afterward, the cells were washed with cold PBS twice, followed by incubation in FBS-free culture medium for additional 0.5, 4, and 8 h. The samples were washed and immediately observed using CLSM. CLSM images were acquired at excitation of 488 nm and emission of 518 and 585 nm. Fluorescence intensity was analyzed by Leica Application Suite Software (Advanced Fluorescence Version: 2.3.0 build 5131). FRET ratio = IRhB/(IRhB + IFITC) × 100%, where IRhB and IFITC are the fluorescence intensities of RBITC-labeled Mel at 585 nm and FITC-labeled pEGCG at 518 nm, respectively. Non-ROS-sensitive FRET-based HA-NC was used as the control. 2.12. In Vitro Antitumor Efficacy. In vitro antitumor efficacy was evaluated for cytotoxicity, cellular apoptosis, and mitochondrial membrane potential (MMP). MTT assay was used to examine the cytotoxicity of different drug-containing preparations in B16F10 cells. Briefly, the cells (5 × 103 cells/well) were seeded and cultured in 96well plates for 24 h at 37 °C. Afterward, the cells were incubated with Mel, pEGCG, NC, HA-NC, pHA-NC, or pHA-NC containing 1 mM NAC with different concentrations for 24 h, followed by addition of 20 μL of MTT (5 mg/mL) to each well and continuing incubation for another 4 h. The medium was then removed, and 150 μL of DMSO was added to each well. Absorbance at 570 nm was measured using a microplate reader. The combination index (CI) was calculated using 4572


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ACS Applied Materials & Interfaces Veterinary Medicine, Yangzhou University. They were subcutaneously injected with 1 × 106 B16F10 melanoma cells in the right flank, and the tumor size was monitored using a vernier caliper. Tumor volume (V) was calculated as V = L × W2/2, where L and W are the length and width of the tumor, respectively. 2.14. ROS Generation in Xenograft B16F10 Tumor-Bearing Mice. The xenograft B16F10 tumor-bearing mice were intratumorally injected with DCFH-DA at a dose of 2.5 mg/kg and then intravenously injected via the tail vein with saline, pHA-NC, or HANC at a dose of 2.5 mg/kg Mel. At predetermined time intervals, the animals were anaesthetized with 2% isoflurane, and images were acquired using an IVIS Lumina imaging system (Caliper Life Sciences Inc., Waltham, MA) at λex/λem = 460 nm/535 nm. At 24 h post administration, the mice were sacrificed, and the main organs (e.g., brain, heart, liver, spleen, lung, and kidney) and tumor tissues were collected for ex vivo imaging. Fluorescence intensity was analyzed using Living Image Software 4.5.2 (PerkinElmer, Waltham, MA). For further examination, frozen sections of the tumor tissue were prepared in 8 μm thickness, stained with DAPI, and observed under a fluorescent microscope. 2.15. In Vivo Biodistribution of pHA-NC. Mel was first labeled with Cy7 for imaging. When the tumor size reached 200−400 mm3, the mice were intravenously injected via the tail vein with Cy7-labeled Mel, NC, or pHA-NC at a Mel dose of 2.5 mg/kg. For HA competition study, the mice were injected with a high dose of free HA (50 mg/kg) 1 h prior to pHA-NC injection. At predetermined time intervals, the animals were anaesthetized with 2% isoflurane and imaged under an IVIS Lumina imaging system at λex/λem = 740 nm/ 790 nm. At 48 h post administration, the tumor-bearing mice were sacrificed, and the main organs (brain, heart, liver, spleen, lung, and kidney) and tumor tissues were collected for ex vivo imaging. Regionof-interests (ROIs) were circled around the organs, and the fluorescence intensity was analyzed using Living Image Software 4.5.2. 2.16. Efficacy and Toxicity Evaluation. Once the tumor size reached 100−200 mm3, the mice were randomly divided into four groups, and they received intravenous injection of saline, pHA-NCb, HA-NC, or pHA-NC at BSA or Mel dose of 2.5 mg/kg every second day for a total of six doses. The tumor size and body weight were regularly monitored. On day 12, one animal from each group was randomly selected and euthanized, and the tumor was harvested, washed with saline thrice, and then fixed in 4% polyformaldehyde for hematoxylin & eosin (H&E) staining and TUNEL apoptosis staining. Furthermore, the healthy mice receiving saline and pHA-NC for 12 days were anesthetized for collecting the blood samples and main organs.36 H&E staining of tissues was subsequently performed. Serum samples were used for biochemistry assay, including blood urea nitrogen, total bilirubin, aspartate aminotransferase, and alanine aminotransferase. Routine blood test included counting of white blood cells, hemoglobin, and platelets. Hemolysis assay was also conducted using fresh mouse red blood cells. 2.17. Statistical Analysis. Data are presented as mean ± standard deviation. Statistical analysis was performed using analysis of variance, followed by Bonferroni post hoc correction, and P < 0.05 was used as the minimal level of statistical significance.

(Figure S1B). ESI-MS detected the presence of the octamer with a cluster of peaks, separated by the regular incremental mass of an EGCG unit linked in the CH3−CH bridge (z = 2, m/z = 228 of EGCG fragment, and m/z = 242 of EGCGCHCH3 fragment) (Figure S1C), further confirming the conjugation of EGCG through the CH3−CH bridge.39 3.2. Preparation and Characterization of Mel/pEGCG NC. The Mel/pEGCG NC was prepared by a mild selfassembled process of adding pEGCG into Mel solution. As shown in Figure 1A, the mean particle size of NC was 127.67 nm, with a polydispersity index (PDI) of 0.24 and zeta potential of +25.40 mV. The obtained NC (pEGCG/Mel = 1:2, w/w) possessed a near-spherical, uniform, and intact morphology. Previous study showed that EGCG could fill the most potential binding sites of peptides such as proline and arginine simultaneously.40 To analyze the evolution process, the native-PAGE technique was applied to distinguish free Mel and the binding peptide packaged by pEGCG (Figure S2). The results indicated that the band of free Mel shallowed when the amount of pEGCG increased; as the ratio of pEGCG to Mel reached 1:2, the band of free Mel almost disappeared, suggesting that Mel was completely bound with pEGCG to form the NC. MST technique was also used to quantify the binding affinity between Mel and pEGCG. As shown in Figure 1B, the MST curve gradually moved down, suggesting that pEGCG was bound with Mel to form the NC. From the data, the equilibrium dissociation constant Kd was calculated as 2.42 ± 0.40 μM. To determine the dominant mode of interaction between Mel and pEGCG, the size change of the NC after adding different competitors was monitored by DLS. As shown in Figure 1C, the NC was effectively dissociated by hydrophobic competitors regardless of Tween 20 and Triton X-100. However, adding urea (aiding in the formation of strong hydrogen bonds) or NaCl (an ionic interaction competitor) was ineffective in dissociating NC. The results are in agreement with previous studies, suggesting that interassociation between Mel and pEGCG was largely driven by hydrophobicity, rather than hydrogen bonding or ionic interaction.39 Furthermore, the formation and dissociation of the NC were studied using FRET technology. FITC-labeled Mel and RBITC-labeled pEGCG were assembled to form the FRET-based NC using the same NC preparation procedures. Changes in the FRET intensity signified the integrity variation of the NC. As exhibited in Figure 1D, the FRET-based NC showed an apparent emission of RBITC at 572 nm after the FITC excitation at 488 nm, demonstrating the integrity of the NC evidenced by the energy transfer from the FITC donor to the RBITC acceptor. Upon introducing Tween 20, however, the FRET ratio decreased with a noticeably increased FITC signal at 516 nm along with a reduced RBITC signal, which corresponds to an increase in the NC dissociation. These findings confirmed that the main mode of binding affinity between Mel and pEGCG was hydrophobic interaction. 3.3. Preparation and Characterization of pHA-NC. Mel, as a cytolytic peptide, would compromise intravenous administration because of its hemolytic potential.7 To further shield the positive charges on the NC surface and to endow site-specific drug release capacity in the tumor, pHA was synthesized for the functional decoration of NC. The reaction was carried out through an EDC/NHS-mediated amidation between the carboxylic acid group of HA and the amine group of phenylboronic acid derivatized PBA. The 1H NMR spectrum

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of pEGCG. Autopolymerization of polyphenols commonly occurs in nature, for instance, during conservation and aging of red wine, which subsequently produces tannins with enhanced anticarcinogenic activity and better stability in vivo.37,38 Inspired by this mechanism, pEGCG was synthesized by polycondensation of EGCG with acetaldehyde under acidic conditions (Figure S1A).23 1H NMR spectrum of pEGCG showed that H6 and H8 in the A ring of EGCG (5.8 ppm) disappeared while a CH3−CH bridge (1.3−1.7 ppm) formed, demonstrating the condensation of EGCG linked through a CH3−CH bridge at C6−C6, C8−C8, and C6−C8 positions 4573


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Figure 2. ROS generation and retention by pHA-NC in vitro and in vivo. (A) H2O2 production from different formulations in pH 7.4 PBS solution (n = 3). (B) Relative ROS levels in B16F10 cells incubated with different formulations corresponding to 5 μg/mL Mel and/or 2.5 μg/mL pEGCG for 1, 4, and 8 h. ROS content was measured by flow cytometry after DCFH-DA staining (n = 4). (C) Remaining ROS amount in B16F10 cells at 1, 2, 4, and 6 h after an 8 h pretreatment with different formulations (n = 4). (D) Fluorescence imaging of ROS generation in B16F10 tumor-bearing mice at 1, 6, 12, and 24 h after intravenous injection of saline (I), pHA-NC (II), and HA-NC (III) at a dose of 2.5 mg/kg Mel. DCFH-DA (2.5 mg/ kg) was intratumorally injected as an indicator of ROS before intravenous injection. (E) Ex vivo fluorescence imaging of ROS in tissues collected from B16F10 tumor-bearing mice 24 h post injection. The tissues are: 1, brain; 2, heart; 3, liver; 4, spleen; 5, lung; 6, kidney; and 7, tumor. (F) ROI analysis of fluorescent signals from tumor and normal tissues (n = 3). **P < 0.01 vs pHA-NC group. (G) ROS generation (green regions represented activated DCF) in tumor tissues from the mice receiving saline, pHA-NC, and HA-NC. Tumor sections were stained with DAPI for nuclei (blue) before observation by a fluorescence microscope (scale bar 100 μm).

of pHA showed the characteristic peaks representing the aromatic protons of PBA (7.3−7.7 ppm) and protons of Nacetyl group of HA (1.8 ppm) (Figure S3), demonstrating the successful conjugation of PBA to HA. The content of PBA in pHA was 3.5%, obtained from the regression line between the weight ratio (PBA/pHA) and the ratio of integration area (7.3−7.7 ppm/1.8 ppm).27 Phenylboronic acid groups of pHA and catechol moieties of pEGCG can form a boronate ester bond under neutral and basic conditions, which was used to achieve pHA surrounding the preformed NC.28,29 pHA-NC was prepared by adding NC solution dropwise to the pHA solution. When the mass ratio of pHA to NC reached 3.3, the hydrodynamic diameter of pHANC measured by DLS was 141.06 nm, with a PDI of 0.13 and zeta potential of −17.53 mV, in comparison to the positive potential of the NC prior to coating. The uniform particle size and negative surface potential of pHA-NC would be desirable for systemic drug delivery using intravenous administration.41

To confirm that the pHA hydrophilic corona coated on the NC surface was anchored through the boronate ester bond, fluorescence quenching technique was used, with ARS as an indicator (Figure S4A). In the presence of pHA, the catechol portion of ARS formed a boronate ester, which was evidenced by a significant fluorescence increase at 562 nm (Figure S4B). As the NC was introduced, the fluorescence intensity decreased proportionally, gradually close to the ARS baseline when the NC concentration reached 188 μg/mL (Figure S4B). Similarly, pEGCG showed significant suppression in the fluorescence intensity produced by ARS and pHA (Figure S4C). This was attributed to the competitive binding of catechol moieties of the NC (or pEGCG) to phenylboronic acid groups of pHA, resulting in more free ARS in the solution and thereby a decrease in the fluorescence. Glynn et al. used NMR spectroscopy to identify the structures and properties of the boron adducts of EGCG and the related polyphenols.42 The observed adducts included both neutral boronate and anionic 4574


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H2O2, with a dissolution profile similar to that at pH 7.4. TEM images also showed no noticeable change of HA-NC after the treatment in H2O2 (Figure S6). The results suggested that the boronate ester coordination bond in pHA-NC responded to the oxidation state and led to accelerated release of drugs. 3.5. ROS Generation and Retention by pHA-NC in Vitro and in Vivo. To determine the oxidizability of pHA-NC, H2O2 production was quantified by HPLC using the TPP method. As shown in Figure 2A, both pEGCG and NC produced H2O2 in pH 7.4 PBS over time, whereas Mel showed no significant H2O2 increase, indicating that the elevated H2O2 amount in the NC mainly originated from pEGCG. It was reported that the oxidation of EGCG by molecular oxygen occurred at the trihydroxyl B ring, which resulted in the formation of the EGCG semiquinone radical and a superoxide radical (•O2−). The •O2− could oxidize another EGCG molecule and generate H2O2 in the process. Autoxidation of EGCG was known to depend on the pH of a medium; a decrease in the pH condition would prevent acid dissociation of the hydroxyl groups of the B ring. Hence, the generation of H2O2 would also be inhibited under acidic conditions.48 By contrast, neither pHA-NC nor HA-NC exhibited efficient H2O2 release, which was possibly attributed to the shield of HA corona that inhibited oxygen permeation and thus H2O2 generation.48,49 Furthermore, ROS generation and retention in B16F10 cells were tested. As illustrated in Figure 2B, Mel and pEGCG, whether single or in combination, led to a significant elevation of ROS. Unexpectedly, although Mel produced little H2O2 in PBS, it produced a comparable amount of ROS to that of pEGCG in cells, presumably attributed to the special electron conductive pathway.14 Previous study reported that the components of the aerobic respiratory chain contributed to the peptide-induced ROS signals. Peptidotoxins possibly triggered a stress response, which led to changes in the transcription/translation profile of the cells.14 Benefited by this mechanism, the highest increase in ROS appeared in cells that were treated with pHA-NC. By contrast, HA-NC showed a slow increase in ROS overall, only a slight upswing after 8 h of incubation, which was probably attributed to the gradual dissociation of chemically coated corona. The intracellular ROS retention after the withdrawal was also investigated. As shown in Figure 2C, pHA-NC and HA-NC demonstrated slower elimination kinetics of ROS. The subsequent fluorescence imaging by a microscope using ROS indicator (DCFH-DA) confirmed more efficient ROS generation and hysteresis effect in ROS elimination of pHA-NC in B16F10 cells (Figure S7). Two factors may have contributed to the persistently elevated ROS by pHA-NC, that is, the synergistic role of generating ROS from Mel and pEGCG and the ROS-sensitive coordinated coating like a “ROS switch” that facilitated continuous drug release. By contrast, the supply of ROS maintained by HA-NC mainly originated from the gradual degradation of HA corona. Accumulating evidence has demonstrated that the tumor cells tolerate oxidative stress by activating antioxidant systems, for instance, through upregulating the GSH.21 Therefore, GSH levels in B16F10 cells after an 8 h-incubation with different formulations were measured. Intracellular GSH levels all reduced to variable degrees afterward (Figure S8), especially in pHA-NC (40% of GSH depletion), implying that cancer cells would consume GSH to offset oxidative damage. A similar conclusion was further supported by in vivo imaging performed on the xenograft B16F10 tumor-bearing mice. In Figure 2D, pHA-NC produced a visible fluorescence

borate derivatives. In the case of EGCG, its ability to make a stable adduct seemed to result from two key factors: (a) the electronic effect that induces the formation of boron adducts at gallate ring D and pyrogallol ring B and (b) the steric effect that facilitates the conversion of anionic cyclic borate species to intramolecular stabilized neutral cyclic borate/EGCG adducts. We next evaluated the colloidal stability of pHA-NC in saline, serum, and under dilution (Figure S5). No significant change in the particle size and zeta potential was observed in 150 mM NaCl solution, suggesting minimal impact of ionic bonds on pHA anchoring. By contrast, physically coated HA/ NC, without the coordination bond, notably increased the particle size and zeta potential in the same medium, indicating instability or dissociation. In serum, pHA-NC still retained its original size with no sign of particle aggregation. The resistance of pHA-NC against the dilution process showed excellent tolerance up to 1000-fold dilution. The coordination bond incorporated in pHA-NC was distinctly more stable than the charge adsorption because of its stronger specificity and affinity, and consequently, pHA-NC was endowed with satisfactory stability and suitability for intravenous administration. 3.4. Dual Response of pHA-NC to HAase and ROS. The pHA corona and the boronate ester in pHA-NC were hypothesized to respond to both HAase and ROS, which are reportedly rich in various tumor matrices and cellular endocytic vesicles (endolysosomes).18,43 We investigated the degradation of pHA by monitoring the changes in the zeta potential of pHA-NC after incubation with HAase under three different pH conditions. As illustrated in Figure 1E, the zeta potential of pHA-NC at pH 7.4 showed no significant change (−16.9 mV vs −17.5 mV) after 4 h of exposure to HAase. As pH decreased, the zeta potential increased to less negative (−7.6 mV) at pH 6.5 and to positive (+6.1 mV) at pH 4.5. In comparison, the zeta potential of pHA-NC in the absence of HAase showed less dramatic changes over pH values (−18.6 mV at pH 7.4 vs −13.6 mV at pH 4.5). Acidic tumor microenvironment (∼pH 6.5) is rich in HAase, which may facilitate unveiling and activation of HAase- and acid-responsive devices.33,44 Of note, compared to the HA-coated device previously reported,43 pHANC seemed to be less sensitive to HAase, evidenced by its maintaining a negative charge. This was partially attributed to the boronate ester coordination bond that increased the stability of HA corona against HAase erosion.45 Therefore, it was speculated that pHA-NC would preferentially enter the cytoplasm intact via the HA receptor-mediated endocytosis pathway,33 which could subsequently minimize the risk of inflammatory response stemmed from pore-forming peptidotoxins.46,47 Release of Mel from pHA-NC was tested using the dialysis method at pH 4.5 and 7.4 in the presence and absence of 0.5 mg/mL HAase or 1 mM H2O2, respectively. As illustrated in Figure 1F, the release of Mel at pH 4.5 was slightly faster than that at pH 7.4, but the cumulative release content was both smaller than 20% over 48 h. By contrast, when HAase or H2O2 was included, the cumulative release significantly increased to 33 and 42%, respectively, indicating that both HAase and H2O2 would facilitate pHA corona stripping and subsequently activate drug release. Furthermore, TEM images confirmed H2O2induced morphological change and drug release from pHA-NC, supported by complete pHA corona disruption after incubating with 1 mM H2O2 for 12 h (Figure 1G). By comparison, the reference HA-NC, prepared by chemically conjugating HA to the NC surface, showed little sign of accelerated release by 4575


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Figure 3. Cellular uptake, subcellular delivery, and cytotoxicity assay of pHA-NC. (A) Cellular uptake of pHA-NC after different treatments in B16F10 and NIH 3T3 cells (n = 3). **P < 0.01 vs pHA-NC. (B) Relative uptake efficiency of pHA-NC in B16F10 cells in the presence of various endocytosis inhibitors. Chlorpromazine, amiloride, nystatin, and sodium azide are inhibitors for clathrin-mediated endocytosis, macropinocytosis, caveolin-mediated endocytosis and energy-dependent endocytosis, respectively (n = 3). **P < 0.01 vs the control. (C) Subcellular distribution of pHA-NC and pHA-NCb in B16F10 cells at different times observed by CLSM. B16F10 cells were incubated with pHA-NC or pHA-NCb at 37 °C for 1 h, followed by incubation with FBS-free culture medium for another 0.5 or 2 h. Mel and BSA were labeled with FITC (green); late endosomes and lysosomes were stained with LysoTracker Red (red); and nuclei were stained with DAPI (blue). (The scale bar is 20 μm). (D) In vitro cytotoxicity of different formulations in B16F10 cells after 24 h of incubation (n = 4). (E) Synergistic analysis of pHA-NC in B16F10 cells. Corresponding CI vs Fa plots of NC and pHA-NC. (F) Apoptosis-inducing capacity of different formulations in B16F10 cells after 12 h of incubation. Viable, early apoptotic, and late apoptotic cell populations (%) are shown in the lower-left, lower-right, and upper-right quadrants, respectively.

signal (represented ROS amount), specifically in the tumor site at 1 h post injection. As time elapsed, the elevated fluorescent signal was captured in pHA-NC groups as compared to saline and HA-NC groups, highlighting the ROS activating capacity of pHA-NC in vivo. After 24 h, the main organs and tumor tissues were harvested for ex vivo imaging. The strongest fluorescence was detected in the tumor of mice receiving pHA-NC treatment, which was 2.8-fold and 1.4-fold higher than that of the saline group and HA-NA group, respectively (Figure 2E,F). The ROS signal observed in the tumor from the HA-NC group was time-lagging and faint, which was attributed to the nonspecific degradation of HA corona. Histopathologic analysis of the excised tumor samples further confirmed that pHA-NC preferentially induced ROS generation and accumulation in the tumor compared to other groups (Figure 2G). All of the above results confirmed that the nanostructured Mel showed potential

as natural pro-oxidants to stimulate and maintain elevated ROS level in vitro and in vivo. ROS regulating the cellular physiology or inducing lethal oxidation entirely depends on the magnitude, duration, and locale of ROS generation.50,51 Evidence generally supports that low ROS levels regulate redox signaling events, whereas high ROS doses are responsible for cell toxicity.21 In view of HA-NC possessing a low overall ROS level, the delayed elimination made little sense. However, pHA-NC sustaining the highest level of intracellular ROS for an extended period of time was highly desirable to potentially compromise tumor cells. 3.6. Cellular Uptake and Intracellular Delivery. To study the cellular transport pathway, the uptake assay of pHANC was performed in both B16F10 cells (CD44 receptor overexpression) and NIH3T3 cells (CD44 receptor deficiency).52 As shown in Figure 3A, the uptake amount of 4576


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results indicated that Mel and pEGCG separated after pHA-NC entered the cytoplasm. In comparison, the non-ROS-sensitive FRET-based HA-NC showed a slower attenuation of the FRET signal. Of note, the release and dissociation of the drug from pHA-NC in PBS was significantly slower than that in cells (Figure 1F), which was probably caused by lack of hydrotropes,56 for example, adenosine triphosphate, which will solubilize proteins and prevent them from aggregation at millimolar concentrations.57 In short, several factors, including endosomal escape assisted by Mel, elimination of hydrophilic corona triggered by ROS, and rapid drug release, collectively build an intracellular network with rapid response and amplification mechanism of ROS. 3.7. Anticancer Effect of pHA-NC in Vitro. To evaluate the effect of the ROS self-sufficient system on cell viability, MTT assay was performed in B16F10 cells. As shown in Figure 3D, the half-maximal inhibitory concentration (IC50) of pHANC was calculated as 1.54 μg/mL, which was 1.6-fold higher in cytotoxicity than the NC (2.49 μg/mL). This might be explained by the specific cellular uptake of pHA-NC in B16F10 cells. Both pHA-NC and NC displayed a significantly stronger growth inhibition on tumor cells than the monotherapy of Mel (IC50 4.55 μg/mL) and pEGCG (IC50 37.07 μg/mL) after 24 h of treatment, indicating that the reversible complexation of Mel and pEGCG exerted no impact on the activity of Mel and instead would provide synergistic potential to increase cytotoxicity. The combined effect of Mel and pEGCG was estimated by the CI value. As shown in Figure 3E, the CI value of both pHA-NC and NC was smaller than 1 over the Fa range of 0.2−0.8, indicating synergism of the drug combination.34 By contrast, HA-NC showed a relatively lower validity (IC50 13.55 μg/mL) in inhibiting the growth of tumor cells, which resulted from the inefficient release of drugs. Furthermore, for the pHANC group, cell viability resumed to some extent after treatment with 1 mM NAC (IC50 6.11 μg/mL), corresponding to decreased ROS levels in the presence of NAC or GSH (Figure S10). It was speculated that the noticed susceptibility of the tumor treated by pHA-NC was possibly caused by an increase in the ROS level above the threshold where cancer cells would not survive. The effect of test preparations to induce cell apoptosis was evaluated using an Annexin V-FITC/PI apoptosis detection kit. The strongest cell apoptosis was found on treatment with pHANC, and its total apoptotic ratio, a sum of the early and late apoptotic ratios, was 67.0% in B16F10 cells (Figure 3F), much higher than that of all other formulations tested. This confirmed that pHA-NC possessed a high ROS-generating efficiency, thus demonstrating a superior apoptosis-inducing capacity. Presence of excessive ROS is known to induce collapse of the MMP, which is considered as an initial and irreversible step in cell apoptosis.58 To further elucidate the mechanism of pHA-NC initiating cell apoptosis, mitochondrial depolarization induced by ROS was tested using the commercial fluorescent dye JC-1.35 As illustrated in Figure S11, when B16F10 cells were treated with pHA-NC, the decrease in red fluorescence (JC-1 aggregates in the cytoplasm) was the lowest at 23.3% among the test samples, indicating increased mitochondrial depolarization. Moreover, the degree of mitochondrial depolarization in different treatment groups was consistent with that of apoptosis-inducing and ROSgenerating effects. Because mitochondrial vulnerability to oxidants was emphasized by their limited ability to interact

pHA-NC was significantly higher in B16F10 cells than in NIH 3T3 cells. For B16F10 cells, the uptake of pHA-NC was obviously inhibited after pretreatment with free HA, a factor to saturate CD44 receptors. On the other hand, after treating pHA-NC with HAase at pH 4.5 for 4 h to destabilize HA corona, cellular uptake was also reduced. These findings confirmed that pHA-NC was preferably transported into B16F10 cells through CD44 receptor-mediated endocytosis. By contrast, for CD44-deficient NIH3T3 cells, uptake of pHANC after pretreatment, regardless of HA saturation or HAase pre-incubation, showed no noticeable changes. Accordingly, pHA-NC surrounded by HA corona endocytosed through the CD44-receptor mediated pathway and enhanced specific targeting to tumor cells. Uptake of pHA-NC in B16F10 cells was further investigated by adding various specific inhibitors to block the corresponding endocytosis pathways. As shown in Figure 3B, the cellular uptake of pHA-NC was significantly suppressed in the presence of sodium azide, indicating that pHA-NC entered the cells via a typical energy-dependent endocytotic pathway.53 In addition, the presence of chlorpromazine or amiloride also significantly inhibited the cellular uptake, suggesting internalization of pHA-NC resulting from the combination of clathrin-dependent and macropinocytosismediated endocytosis.54 Intracellular delivery of pHA-NC was also recorded using CLSM. As pictured in Figure 3C, FITC-labeled pHA-NC was mainly localized in red fluorescent endo−lysosomes, evidenced by the overlaid yellow fluorescence at 0.5 h after withdrawal, indicating that pHA-NC was taken by cells and entrapped in the endosomes.55 Dissociation of red fluorescent endo− lysosomes and green fluorescent pHA-NC was observed as the incubation time extended to 2 h, suggesting that pHA-NC was efficiently separated from endo−lysosomes. Mel was reported to improve endosomal escape through the membrane-disrupting mechanism, which has been used for cytosol delivery of siRNA.22 The endocytosed pHA-NC would be subjected to acid-activated degradation by HAase in endo− lysosomes to expose Mel residues, which subsequently facilitated endosomal disruption and cytoplasm-selective delivery of the drug substance. In Figure 3C, it showed a certain degree of fluorescent separation as well as an increase of punctate green for pHA-NC after 0.5 h of incubation, indicating the rapid endosomal escape of pHA-NC with assistance from Mel. By contrast, pHA-NCb prepared by replacing Mel with BSA showed no significant escape from endo−lysosomes corresponding to yellow fluorescence even after 2 h of incubation. The rapid endosomal escape of pHA-NC to the cytoplasm would be beneficial to protect active therapeutic agents, especially peptides and proteins, from degradation by lysosomal enzyme and acidity.22 At the same time, owing to inefficient ROS-generating capacity of EGCG under low pH conditions,48 the cytoplasm could provide a desirable environment for ROS-triggered drug release and maximizing the ROSgenerating potential of both Mel and pEGCG. These may lead to excellent ROS generation and retention of pHA-NC within cancer cells. To further illustrate the intracellular fate of pHANC, FRET technique was utilized in B16F10 cells.33 CLSM images showed the strong FRET signal (red color) and weak donor emission (green color), resulting in a high FRET ratio (80%) at 0.5 h after withdrawal (Figure S9). This ratio diminished to 40% when the time was extended to 4 h and further suppressed to 10% at 8 h, shown as intense green fluorescence and faint red fluorescence (Figure S9). These 4577


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Figure 4. In vivo and ex vivo fluorescence imaging of pHA-NC. (A) In vivo fluorescence images of B16F10 tumor-bearing mice captured at different intervals post intravenous injection of Cy7-labeled Mel (I), NC (II), and pHA-NC (III), and pHA-NC with preinjection of HA (50 mg/kg) (IV) at a dose of 2.5 mg/kg Mel. (B) Ex vivo fluorescence images of tissues collected from mice injected with different formulations after 48 h. The tissues are 1, brain; 2, heart; 3, liver; 4, spleen; 5, lung; 6, kidney; and 7, tumor. (C) Semiquantitative relative biodistribution of Cy7-labeled Mel delivered by different formulations in various tissue samples, determined by the fluorescence intensities obtained from (B) (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001 vs pHA-NC group.

pHA-NCb possessed efficacy similar to that of saline, which was primarily attributed to Mel being replaced by inactive BSA. Free Mel solution was not tested because of its intense hemolytic activity. Unexpectedly, chemically conjugated HA-NC, which was found to be inefficient in the in vitro release and cytotoxicity study, produced statistically growth suppression to the tumor after administration. It was speculated that the active therapeutic agent was gradually released in vivo as the tumor-targeted HA-NC was degraded for the entire treatment duration. During the efficacy test, no noticeable activity abnormity was observed in any study group and neither was there significant weight loss in the study animals (Figure 5C). The mice receiving pHA-NC had the longest survival among the treatment groups (Figure 5D). Histologic images demonstrated that after receiving pHA-NC, massive cancer cell remission occurred in the tumor tissue (Figure 5E), with no obvious pathological abnormality in other primary organs (Figure S12A). At the same time, images obtained from the in situ TUNEL assay demonstrated the highest level of cell apoptosis in the tumor harvested from mice treated with pHANC (Figure 5E), indicating that the dominant inhibition activity of tumor growth was attributed partially to the increased apoptosis effect induced by pHA-NC. Moreover, no difference in biochemistry parameters (Figure S12B−E) and blood routine (Figure S12F−H) was found among mice receiving the pHA-NC group and saline group, indicating negligible systemic toxicity. Notably, pHA-NC adequately shielded hemolytic toxicity from Mel, demonstrating suitability of pHA-NC for intravenous administration in cancer treatment (Figure S12I). Collectively, these results verified that pHA surrounding the NC through coordination bonds efficiently accumulated at the tumor site, demonstrated effective intracellular transport and ROS-mediated dissociation, and thereby accomplished synergistic antitumor efficacy with minimal side effects.

with ROS, the intrinsic mitochondrial pathway would be plausible to explain the apoptosis mechanism in this study.58 3.8. Biodistribution of pHA-NC in Vivo. To assess the tumor-targeting capability of pHA-NC, the biodistribution of Cy7-labeled Mel-loaded pHA-NC was monitored in mice using a noninvasive near infrared imaging system. As shown in Figure 4A, pHA-NC group exhibited Cy7 signal at the tumor site 1 h post injection. As time elapsed, stronger fluorescent signals were clearly observed in the tumor region in this group than in other groups for 48 h post injection, validating a significant tumor-targeting capacity of pHA-NC. On the other hand, the Mel group and NC group without HA coating did not produce sufficient tumor targeting, with primary accumulation in liver for 48 h. This was mainly attributed to the nonspecific distribution and rapid clearance of the test preparations in vivo.33 To further confirm the role of HA corona in the activetargeting of pHA-NC to the CD44 overexpressing tumor, a high dose of HA solution was injected before the administration of pHA-NC. As projected, a clear attenuation of the Cy7 signal was visualized at the tumor site for all time intervals, suggesting that HA corona not only retained the integrity of pHA-NC in systemic circulation but also endowed pHA-NC with active tumor targetability. After 48 h, the tumor and normal tissues were isolated from the animals for ex vivo imaging. As shown in Figure 4B,C, the strongest Cy7 signal detected in the tumor tissue of the pHA-NC group was 9.1, 2.3, and 1.4-fold higher than that of Mel, NC, and HA pretreated pHA-NC groups, and the fluorescent signal was also much higher than that of normal tissues. 3.9. Efficacy and Toxicity Evaluation. The antitumor efficacy of pHA-NC was evaluated in B16F10 tumor-bearing mice. As shown in Figure 5A,B, the tumor growth was significantly suppressed after successive intravenous injections of Mel-containing formulations including pHA-NC and HANC compared to the negative control saline group. By contrast, 4578


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Figure 5. In vivo antitumor efficacy in B16F10 tumor-bearing mice. (A) Representative images of B16F10 xenograft tumors in mice after different treatments on day 12, tumor size circled. (B) Tumor growth curves of mice after different treatments (n = 5). **P < 0.01 vs pHA-NC group. (C) Body weight changes in B16F10 tumor-bearing mice during treatments (n = 5). (D) Animal survival rates after different treatments. (E) Histological and apoptosis comparison after different treatments. Tumor sections were stained with H&E for histological observation, fluorescein-dUTP (green) for apoptosis, and DAPI for nuclei (blue).

4. CONCLUSIONS In summary, an ROS self-sufficient pHA-NC comprising Mel and EGCG as natural pro-oxidants was successfully constructed for enhanced cancer therapy. pHA coating achieved through coordination bonds potentiated suitability of intravenous drug administration and tumor targeting. This sophisticated design endowed pHA-NC with a series of capabilities, including CD44 receptor-mediated cellular endocytosis, Mel-aided lysosomal escape, and ROS-triggered structural collapse. The released Mel and pEGCG induced constant ROS generation to reach a threshold, above which the tumor cells could not survive. In vitro and in vivo evaluations confirmed that pHA-NC could overcome the defects of peptidotoxins and significantly inhibit tumor progression with minimal side effects. It appeared that DDS based on ROS triggering and cascade amplification maximized the advantages of HDPs and EGCG, improving anticancer therapeutic efficacy with high selectivity against tumor. Compared to previous ROS amplification systems composed of chemo-synthesized small molecules and polymers, pro-oxidants derived from natural products would be more desirable owing to their long usage history in traditional medicine as well as their active defense mechanisms as drug entities. Synergism of the oxidation capacity found in this study

could also offer feasible options for combination cancer therapy in the future.

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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.7b18809. Synthesis route, 1H NMR and ESI-MS spectrum of pEGCG; native-PAGE of Mel and pEGCG; synthesis and 1H NMR spectrum of pHA; illustration of pHA binding pEGCG or NC through the coordination bond; stability of pHA-NC under various conditions; TEM images of HA-NC and HA-NC treated with H2O2; fluorescence images of ROS in B16F10 cells; intracellular GSH level; FRET ratio changes of pHA-NC and HA-NC in B16F10 cells; intracellular ROS level determined by flow cytometry; MMP; and safety evaluation of pHA-NC (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected], [email protected]. Phone: +86 25 85811230. Fax: +86 25 85811072 (H.Q.). 4579


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ACS Applied Materials & Interfaces *E-mail: [email protected] (L.D.).

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ORCID

Hongzhi Qiao: 0000-0003-3154-9584 Author Contributions #

H.Q. and D.F. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the financial support from the National Natural Science Foundation of China (no. 81503259), the Jiangsu Natural Science Foundation of China (BK20151002), the Young Elite Scientists Sponsorship Program by CAST (CACM-2017-QNRC1-01), the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYLX16_1158), the Special Project of Jiangsu Provincial Administration of Traditional Chinese Medicine (ZX2016D1), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the Program for Outstanding Scientific and Technological Innovation Team of Jiangsu Higher Education Institutions.

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