Nanostructured peptidotoxins as natural pro-oxidants induced cancer

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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 ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18809 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 16, 2018

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ACS Applied Materials & Interfaces

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,ǁ ⊥

Chunmeng Sun,ǁ Qineng Ping,ǁ Junsong Li,† Zhipeng Chen,† Jun Chen,† Lihong Hu,†, and Liuqing Di*,† †

State Key Laboratory Cultivation Base for TCM Quality and Efficacy, School of

Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China ‡

College of Pharmacy, University of Manitoba, 750 McDermot Avenue, Winnipeg, MB,

R3E 0T5, Canada §

Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia

Medica, Nanjing University of Chinese Medicine, Nanjing 210023, China ǁ

State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China

Pharmaceutical University, Nanjing 210009, China ⊥

Jiangsu Key Laboratory for Functional Substance of Chinese Medicine, Nanjing

University of Chinese Medicine, Nanjing 210023, China #

These authors contributed equally to this work.

*

Address for Correspondence: Hongzhi Qiao, Associate Professor & Liuqing Di, Professor School of Pharmacy Nanjing University of Chinese Medicine 138 Xianlin Avenue Nanjing, China 210023 Phone: +86 25 85811230 Fax: +86 25 85811072 Email: [email protected], [email protected]; [email protected]

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ABSTRACT: Melittin (Mel), one of the host defense peptides (HDPs) derived from venom of honeybees, demonstrates substantial anticancer properties, which is considered 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, a 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 ROSresponsive 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 (HAase), and then escape from lysosome by virtue of cytolytic Mel. The elevated ROS level in 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 tumor away from its homeostasis and make tumor vulnerable. Compared to previously reported chemosynthetic pro-oxidants, ROS self-sufficient system fully composed of natural medicine from this study provide a new insight in developing cancer oxidation therapy.

KEYWORDS: peptidotoxins, melittin, polyphenols, drug delivery, oxidative stress, oxidation therapy


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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 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 apoptosis-inducing cell death and suppression of angiogenesis through VEGFR pathway.4-5 However, Mel also demonstrated instability and nonspecific cellular lytic activity in vivo.6-7 A series of strategies has 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 Mel with lipid carriers did not work either, because Mel is capable of disrupting 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 was capable of binding with a variety of biological molecules including proteins and peptides and creating nanoscaled aggregates.12 The formed nano-aggregates are able to temporarily conceal the toxicity of peptidotoxins through shielding their specific amino acid residues12 and deliver to tumor site by virtue of enhanced permeability and retention effect (EPR effect).13 Moreover, cumulative data indicated both Mel and EGCG produced reactive oxygen species (ROS),14-15 which was considered a positive attribute to their anticancer activities,



since overexpression of ROS will pose lethal damage to tumor cells.16 Furthermore, 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 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 the 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, a ROS cascade amplification DDS comprising Mel and EGCG was prepared and tested as natural pro-oxidants to enhance anticancer targeting and efficacy (Scheme 1). EGCG would be concentrated on condensed tannins (pEGCG) to preferably bind with Mel, the subsequently formed nanocomplex (NC) would be further covered by phenylboronic acid derivatized hyaluronic acid (pHA) through boronate ester coordination bond to obtain pHA-NC. Upon delivering to tumor region by EPR effect and achieving active binding to the overexpressed receptors in cancer cells, such as CD44, inner cores of pHA-NC would be partially uncovered once pHA corona is to be degraded by hyaluronidase (HAase), and then escape from lysosome by virtue of cytolytic Mel.22 The elevated ROS level in tumor cytoplasm would disrupt the boronate ester bond to facilitate drug release, while also maintaining a consistent ROS mass above the threshold in which tumor cells would not survive. This positive feedback loop constructed by pHA-NC, as if a powerful pump of ROS, could detect and amplify intracellular ROS signals, potentiating to move tumor away from its homeostasis and make it more vulnerable. Development of a ROS self-sufficient system made of natural ingredients might be of great interest for testing


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

Scheme 1. Schematic illustration of a multipronged nanocomplex (pHA-NC) comprising Mel and pEGCG surrounded by hyaluronic acid through coordination bond for ROS selfsufficient oxidation therapy of cancers. I, EPR effect; II, CD44 receptor recognition; III, receptor-mediated endocytosis; IV, endosomal/lysosome escape; V, ROS-triggered disruption of pHA corona and drug release; VI, ROS generation and amplification; VII,



ROS feedback regulation.

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). Hyaluronic Acid (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 obtained from HyClone Laboratories (Logan, UT). Annexin VFITC/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 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 the experiments. B16F10 melanoma cells and NIH3T3 fibroblast cells were purchased from American Type Culture Collection (ATCC). Cells were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin at 37 °C under an atmosphere of 5% CO2 and 90% relative 6 ACS Paragon Plus Environment

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humidity. 2.2. Synthesis and Characterization of pEGCG 1.0 g of EGCG was dissolved in a mixture of DI water and DMSO (9:1, v/v, 20% acetic acid). The polymerization reaction was initiated by adding acetaldehyde (7.2 mL) and maintained at 20 °C (pH 2.0) under a nitrogen atmosphere for 12 h.23 The resulting solution was dialyzed (1 kDa MWCO) against DI water and lyophilized to obtain pEGCG. Proton nuclear magnetic resonance (1H-NMR) (DSX400, Bruker, Billerica, MA) was used to analyze the structure of pEGCG. Electrospray ionisation 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 in mild condition to produce Mel/pEGCG NC. Briefly, an aliquot of pEGCG (500 µg/mL) and Mel (1 mg/mL) was mixed in nitrogen for 30 min at room temperature in dark. Size distribution and morphology of NC were measured by dynamic light scattering (DLS) (Malvern, Worcestershire, United Kingdom) and transmission electron microscope (TEM) (H-7650, Hitachi, Japan), respectively. To confirm complexation of pEGCG and Mel, native-PAGE technique was used on a gel electrophoresis system (Mini-PROTEAN Tetra, Bio-Rad, Hercules, CA). 20 µL of sample containing approximately 10 µg Mel was loaded on the gel, followed by staining with 0.25% Coomassie Blue R 250 for 30 min. After destaining for three times, the gel was scanned under ImageScanner III (GE Healthcare, Little Chalfont, United Kingdom). The binding affinity between Mel and pEGCG was determined by microscale thermophoresis (MST) technology on a Monolith NT.115 (NanoTemper, Germany) using 7 ACS Paragon Plus Environment


80% blue LED (488 nm) and 1 V IR-laser power.24 Laser on and off times were set at 35 s and 5 s, respectively. FITC-labeled Mel was synthesized according to standard operating method. Subsequently, 2 µM of 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 FITClabeled NC for measurement. The MST curves were recorded, and 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 respectively diluted to variable concentrations and added to preformed NC solution (1:1, v/v). After incubation for 30 min at ambient temperature, size distribution variation of NC was measured using a Zetasizer (Nano ZS, Malvern, United Kingdom). To further confirm the interaction force, a 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 aliquot of FITC-labeled pEGCG (500 µg/mL) and RBITC-labeled Mel (1 mg/mL). Next, FRET-based NC was incubated in 0, 1, 10 and 40 mM Tween 20 for 30 min at room temperature, respectively. Emission spectra of samples were recorded by a fluorescence spectrophotometer (LS-50B, Perkin-Elmer, Waltham, MA) at 488 nm excitation. Variations in FRET signal were used to detect dissociation of Mel and pEGCG. The FRET ratio was indicated as IRBITC/(IRBITC +IFITC) × 100%, where IRBITC, fluorescence intensity of RBITC at 572 nm and IFITC, fluorescence intensity of FITC at 516 nm, respectively.


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2.4. Synthesis and Characterization of pHA 0.05 mmol HA was dissolved in 50 mL DI water. Then 2 mmol EDC/NHS were added to this solution and stirred for 30 min. 2 mmol PBA was dissolved in 20 mL 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 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 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 pre-formed NC by 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 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 particle morphology. Mechanism of pHA surrounding NC was studied by a competitive binding assay.30 Briefly, 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, non9 ACS Paragon Plus Environment


phenylboronic acid derivatized HA was directly adsorbed onto 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 particle size and zeta potential were measured at 0, 1, 6 and 12 h, respectively. pHA-NC stability in serum was determined by incubating in 10% FBS at 37 °C for 24 h, and 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 Presence of HAase HAase-mediated degradation of pHA was evaluated by monitoring changes in 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 37 °C water bath under agitation for 4 h. pHA-NC in PBS without HAase was compared as negative control. The zeta potential was measured by a Zetasizer. 2.7. In Vitro Release Study To determine release kinetics of pHA-NC, 2 mL of pHA-NC containing 0.5 mg 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 sample was withdrawn followed by replenishing with fresh buffer solution. Concentration of Mel in samples was quantified using an HPLC method. The chromatographic conditions were, mobile phase: acetonitrile/0.2% trifluoroacetic acid water (45:55, v/v), flow rate: 1.0 mL/min, detection wavelength: 220 nm. HA-NC (HA chemically conjugated NC) was prepared as a control through standard EDC chemistry procedure and also dialyzed against PBS at pH 7.4 with H2O2. 10 ACS Paragon Plus Environment

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2.8. H2O2 Production in pH 7.4 Buffer Solution 1 mL of Mel, pEGCG, NC, pHA-NC or HA-NC (250 µg/mL Mel; 125 µg/mL pEGCG) was added to 9 mL PBS buffer (pH 7.4). 200 µL sample was withdrawn at predetermined intervals, mixed with 800 µL acetic acid to prevent further oxidation. All samples were stored at -20 °C until HPLC quantification. 1 mL of acidified sample was mixed with 9 mL acetonitrile/water (2:1, v/v) containing 0.2 mM triphenylphosphine (TPP) followed by reaction in dark for 2 h. H2O2 amount in samples was proportional to 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 incubated for different intervals in the dark with Mel, pEGCG, NC, pHA-NC, or HANC (Mel at 5 µg/mL; pEGCG at 2.5 µg/mL), B16F10 cells were washed with cold PBS. Fresh culture media 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 anti-oxidation study, cells were pre-incubated with 1 mM N-acetylcysteine (NAC) or glutathione (GSH) for 2 h, followed by pHA treatment. To investigate elimination kinetics of ROS, ROS concentration 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). Fluorescent image of cells at 0 h and 6 h after 8 h 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 untreated cells was used as a baseline value for comparison with that of cells receiving different treatments. 11 ACS Paragon Plus Environment


2.10. Celluar Uptake To investigate the site-specific delivery, B16F10 cells and NIH3T3 cells seeded in 12well plates (1 × 105 cells/well) were cultured with FITC-labeled pHA-NC or HAase-treated pHA-NC (5 µg/mL Mel) at pH 4.5 for 4 h. Afterwards, cells were washed with cold PBS, trypsinized, resuspended in 0.5 mL PBS, and analyzed using a flow cytometer. For comparison, cells were pre-incubated with free HA (10 mg/mL) for 1 h before pHA-NC treatment. To illustrate endocytosis pathway of pHA-NC, B16F10 cells were pre-incubated 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; sodium azide (200 µg/mL) for energy inhibition. Subsequently, cells were treated with FITClabelled 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, 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 h or 2 h. Subsequently cells were stained with LyosTracker Red (2 µM) for 30 min and DAPI (10 µg/mL) for 10 min. Samples were then washed with 4 °C PBS twice, and immediately observed using confocal laser scanning microscopy (CLSM) (Carl Zeiss LSM 700, Oberkochen, Germany). As a control, Mel was replaced by BSA labeled with FITC to obtain pHA-NCb (3 µg/mL BSA). In order to monitor intracellular dissociation of Mel and pEGCG, B16F10 cells were 12 ACS Paragon Plus Environment

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exposed to FRET-based pHA-NC that composed of RBITC-labeled Mel (3 µg/mL) and FITC-labeled pEGCG (1.5 µg/mL) for 4 h.33 Afterwards, cells were washed with cold PBS twice, followed by incubation in FBS-free culture medium for additional 0.5, 4 and 8 h. Samples were washed and immediately observed using CLSM. CLSM images were acquired at excitation of 488 nm, and emission of 518 nm 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 fluorescence intensity 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 a control. 2.12. In Vitro Antitumor Efficacy In vitro antitumor efficacy was evaluated in cytotoxicity, cellular apoptosis and mitochondrial membrane potential. MTT assay was used to examine cytotoxicity of different drug-containing preparations in B16F10 cells. Briefly, cells (5×103 cells/well) were seeded and cultured in 96-well plates for 24 h at 37 °C. Afterwards, 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 adding 20 µL MTT (5 mg/mL) to each well and continuing incubation for another 4 h. Medium was then removed and 150 µL DMSO was added to each well. Absorbance at 570 nm was measured using a microplate reader. The combination index (CI) was calculated using the following equation: CI＝ D1/Dx1＋D2/Dx2, where D1 and D2 are concentration of Mel and pEGCG in combination resulting in the fraction affected (Fa) × 100% growth inhibition, and Dx1 and Dx2 are concentration 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, pHA-NC 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. Mitochondrial membrane potential was measured with 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. 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, cells were collected and incubated in JC-1 dye for 20 min at 37°C. Subsequently, 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), while the y-axis (FL2 channel) demonstrated red fluorescence (JC-1 aggregates). Cells with low mitochondrial membrane potential were in the lower right quadrant. 2.13. Animals and Tumor Xenograft Models Animal studies were conducted in accordance with Guide for Care and Use of Laboratory Animals, protocol approved by Animal Care Committee of Nanjing University of Chinese Medicine. C57BL/6 mice (male, 18-22g) were purchased from College of Veterinary Medicine, Yangzhou University. They were subcutaneously injected with 1×106 B16F10 melanoma cells in the right flank, 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 14 ACS Paragon Plus Environment

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The xenograft B16F10 tumor-bearing mice were intratumorally injected with DCFHDA at a dose of 2.5 mg/kg, and then intravenously injected via tail vein with saline, pHANC or HA-NC at a dose of 2.5 mg/kg Mel. At predetermined time intervals, animals were anaesthetized with 2% isoflurane, and images 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, mice were sacrificed, main organs (e.g., brain, heart, liver, spleen, lung, kidney) and tumor tissue were collected for ex vivo imaging. Fluorescence intensity was analyzed using Living Image Software® 4.5.2 (Perkin-Elmer, Waltham, MA). For further examination, frozen sections of 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 tumor size reached 200-400 mm3, mice were intravenously injected via tail vein with Cy7-labeled Mel, NC or pHA-NC at a Mel dose of 2.5 mg/kg. For HA competition study, mice were injected with a high dose of free HA (50 mg/kg) 1 h prior to pHA-NC injection. At predetermined time intervals, 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, tumor-bearing mice were sacrificed, main organs (brain, heart, liver, spleen, lung, kidney) and tumor tissues collected for ex vivo imaging. Region-of-interests was circled around the organs, and fluorescence intensity analyzed using Living Image Software® 4.5.2. 2.16. Efficacy and Toxicity Evaluation Once tumor size reached 100-200 mm3, mice were randomly divided into 4 groups and received intravenous injection of saline, pHA-NCb, HA-NC or pHA-NC at BSA or Mel 15 ACS Paragon Plus Environment


dose of 2.5 mg/kg, every second day for a total of 6 doses. Tumor size and body weight were regularly monitored. On Day 12, one animal from each group was randomly selected and euthanized, and tumor harvested, washed with saline thrice, and then fixed in 4% polyformaldehyde (PFA) for HE staining and TUNEL apoptosis staining. Furthermore, healthy mice receiving saline and pHA-NC for 12 days were anesthetized for collecting blood samples and main organs.36 HE staining of tissues was subsequently performed. Serum samples were used for biochemistry assay, including blood urea nitrogen (BUN), total bilirubin (T-Bil), aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Routine blood test included counting of white blood cells (WBC), hemoglobin (Hb), and platelets (PLT). Hemolysis assay was also conducted using fresh mouse red blood cells (RBC). 2.17. Statistical Analysis Data are presented as Mean ± standard deviation (S.D.). Statistical analysis was performed using ANOVA followed by Bonferroni post hoc correction, and P < 0.05 used as the minimal level of statistical significance.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of pEGCG Auto-polymerization of polyphenols commonly occurs in nature, for instance during conservation and aging of red wine, which subsequently produces tannins with enhanced anti-carcinogenic activity and a better stability in vivo.37-38 Inspired by this mechanism, pEGCG was synthesized by polycondensation of EGCG with acetaldehyde under acidic


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condition (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, C8C8 and C6-C8 positions (Figure S1B). ESI-MS detected the presence of octamer with a cluster of peaks, separated by the regular incremental mass of an EGCG unit linked in CH3CH bridge (z=2, m/z = 228 of EGCG fragment and m/z = 242 of EGCG-CHCH3 fragment) (Figure S1C), further confirming the conjugation of EGCG through a CH3-CH bridge.39 3.2. Preparation and Characterization of Mel/pEGCG NC The Mel/pEGCG NC was prepared by a mild self-assembled process of adding pEGCG into Mel solution. As shown in Figure 1A, the mean particle size of NC was 127.67 nm, with a PDI of 0.24 and zeta potential of +25.40 mV. The obtained NC (pEGCG:Mel=1:2, w/w) possessed near-spherical, uniform and intact morphology. Previous study showed that EGCG could fill most potential binding sites of peptides such as proline and arginine simultaneously.40 To analyze the evolution process, native-PAGE technique was applied to distinguish free Mel and 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 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 bound with Mel to form NC. From the data, the equilibrium dissociation constant Kd was calculated as 2.42 ± 0.40 µM.



Figure 1. Preparation and characterization of NC and pHA-NC. (A) Size distribution and morphology of NC measured by DLS (left) and TEM (right), respectively. (B) Schematic illustration of 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 NC incubated with Tween 20, Triton X-100, urea and NaCl for 30 min, respectively (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 negative control


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(n=3). (G) TEM images of pHA-NC (left) and pHA-NC treated with 1 mM H2O2 for 2 h (right).

In order to determine dominant mode of interaction between Mel and pEGCG, the size change of NC after adding different competitors was monitored by DLS. As shown in Figure 1C, 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 were in agreement with previous studies, suggesting that inter-association between Mel and pEGCG was largely driven by hydrophobicity, rather than hydrogen bonding or ionic interaction.39 Furthermore, the formation and dissociation of NC was studied using FRET technology. FITC-labeled Mel and RBITC-labeled pEGCG were assembled to form the FRET-based NC using the same NC preparing procedures. Changes in FRET intensity signified the integrity variation of NC. As exhibited in Figure 1D, FRET-based NC showed an apparent emission of RBITC at 572 nm after the FITC excitation at 488 nm, demonstrating the integrity of NC evidenced by 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 was corresponding to increase in 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 due to its



hemolytic potential.7 To further shield the positive charges on NC surface and to endow site-specific drug release capacity in tumor, phenylboronic acid derivatized hyaluronic acid (pHA) was synthesized for 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). 1H-NMR spectrum of pHA showed the characteristic peaks representing aromatic protons of PBA (7.3-7.7 ppm) and protons of N-acetyl group of HA (1.8 ppm) (Figure S3), demonstrating 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 (7.37.7 ppm/1.8 ppm).27 Phenylboronic acid groups of pHA and catechol moieties of pEGCG can form boronate ester bond under neutral and basic conditions, which was used to achieve pHA surrounding the pre-formed NC.28-29 pHA-NC was prepared by adding NC solution dropwise to pHA solution. When mass ratio of pHA to NC reached 3.3, the hydrodynamic diameter of pHA-NC measured by DLS was 141.06 nm, with a PDI of 0.13 and zeta potential of -17.53 mV, in comparison to positive potential of NC prior to coating. 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 NC surface was anchored through 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 NC was introduced, fluorescence intensity decreased proportionally, gradually close to ARS baseline when NC


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concentration reached 188 µg/mL (Figure S4B). Similarly, pEGCG showed significant suppression in fluorescence intensity produced by ARS and pHA (Figure S4C). This was attributed to competitive binding of catechol moieties of NC (or pEGCG) to phenylboronic acid groups of pHA, resulting in more free ARS in the solution and thereby, decrease in fluorescence. Glynn et al used NMR spectroscopy to identify the structures and properties of the boron adducts of EGCG and related polyphenols.42 The observed adducts included both neutral boronate and anionic borate derivatives. In the case of EGCG, its ability to make a stable adduct seemed to result from two key factors: (a) electronic effect that induces the formation of boron adducts at the gallate ring D and the pyrogallol ring B, and (b) steric effect that facilitates the conversion of anionic cyclic borate species to intramolecular stabilized neutral cyclic borate/EGCG adducts. We next evaluated colloidal stability of pHA-NC in saline, serum and under dilution (Figure S5). No significant change in particle size and zeta potential was observed in 150 mM NaCl solution, suggesting minimal impact of ionic bonds on pHA anchoring. In contrast, physically coated HA/NC, without coordination bond, notably increased 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 dilution process showed excellent tolerance up to 1000-fold dilution. Coordination bond incorporated in pHA-NC was distinctly more stable than charge adsorption because of its stronger specificity and affinity, 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 changes in zeta potential of pHA-NC after incubation with HAase under three different pH conditions. As illustrated in Figure 1E, 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, zeta potential of pHA-NC in 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, comparing to HA-coated device previously reported,43 pHA-NC seemed to be less sensitive to HAase evidenced by its maintaining negative charge. This was partially attributed to the boronate ester coordination bond that increased the stability of HA corona against HAase erosion.45 Thereby, it was speculated that pHA-NC would preferentially enter the cytoplasm intact via 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 a dialysis method at pH 4.5 and 7.4, in presence and absence of 0.5 mg/mL HAase or 1 mM H2O2 respectively. As illustrated in Figure 1F, release of Mel at pH 4.5 was slightly faster than that at pH 7.4, but cumulative release content was both smaller than 20% over 48 h. In contrast, when HAase or H2O2 was included, 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 H2O2-induced


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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 through chemically conjugating HA to NC surface, showed little sign of accelerated release by H2O2, with a similar dissolution profile 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 boronate ester coordination bond in pHA-NC responded to 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 TPP method. As shown in Figure 2A, both pEGCG and NC produced H2O2 in pH 7.4 PBS over the time, while Mel showed no significant H2O2 increase, indicating that elevated H2O2 amount in NC was mainly originated from pEGCG. It was reported that oxidation of EGCG by molecular oxygen occurred at the trihydroxyl B ring, which resulted in the formation of 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 pH condition would prevent acid dissociation of the hydroxyl groups of B ring. Hence the generation of H2O2 would also be inhibited under acidic condition.48 In 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 was tested. As illustrated in Figure 2B, Mel and pEGCG, whether single or in combination, led to significant elevation of ROS. Unexpectedly, while Mel produced little H2O2 in PBS, it produced comparable amount of



ROS to that of pEGCG in cells, presumably attributed to the special electron conductive pathway.14 Previous study reported that components of aerobic respiratory chain contributed to the peptide-induced ROS signals. Peptidotoxins possibly triggered a stress response, which led to changes in 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. In contrast, HA-NC showed slow increase in ROS overall, only a slight upswing after 8 h of incubation, which was probably attributed to 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 microscope using ROS indicator (DCFHDA) 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, i.e., 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 was mainly originated from gradual degradation of HA corona. Accumulating evidence has demonstrated that 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 afterwards (Figure S8), especially in pHA-NC (40% of GSH depletion), implying that cancer cells would consume GSH to offset oxidative damage. Similar conclusion was further supported by in vivo imaging performed on the xenograft B16F10 tumor-bearing mice. In Figure 2D, pHA-NC produced visible


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fluorescence signal (represented ROS amount) specifically in tumor site at 1 h post injection. As time elapsed, elevated fluorescent signal was captured in pHA-NC groups as compared to saline and HA-NC groups, highlighting ROS activating capacity of pHA-NC in vivo. After 24 h, main organs and tumor tissue 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 saline group and HA-NA group, respectively (Figure 2E and F). The ROS signal observed in tumor from HA-NC group was time-lagging and faint, which was attributed to nonspecific degradation of HA corona. Histopathologic analysis of the excised tumor samples further confirmed that pHA-NC preferentially induced ROS generation and accumulation in tumor compared to other groups (Figure 2G). All above results confirmed that nanostructured Mel showed potential as natural pro-oxidants to stimulate and maintain elevated ROS level in vitro and in vivo.



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


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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. DCFHDA (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; 7 tumor. (F) Region-of-interest (ROI) analysis of fluorescent signals from tumor and normal tissues (n=3). **P

Nanostructured peptidotoxins as natural pro-oxidants induced cancer

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