Multifunctional Polymeric Micelles with Amplified Fenton Reaction for

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Multifunctional Polymeric Micelles with Amplified Fenton Reaction for Tumor Ablation Yuheng Wang, Wei Yin, Wendong Ke, Weijian Chen, Chuanxin He, and Zhishen Ge Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01777 • Publication Date (Web): 08 Feb 2018 Downloaded from http://pubs.acs.org on February 8, 2018

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Multifunctional Polymeric Micelles with Amplified Fenton Reaction for Tumor Ablation Yuheng Wang,1 Wei Yin,1,3 Wendong Ke,1 Weijian Chen,1 Chuanxin He,*,2 and Zhishen Ge*,1

1

CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering,

University of Science and Technology of China, Hefei 230026, Anhui, China

2

College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen 518060,

Guangdong, China. 3

Department of Pharmacology, Xinhua University of Anhui, Hefei 230088, Anhui, China

Corresponding Author E-mail: [email protected] (Z. Ge), [email protected] (C. He)

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ABSTRACT

Relative to normal cells, tumor cells lack adequate capability of reactive oxygen scavenging. Thus, tumor cells can be selectively killed by increasing the concentration of reactive oxygen species in tumor tissue. In this report, we construct an integrated multifunctional polymeric nanoparticle which can selectively improve hydrogen peroxide (H2O2) levels in tumor tissue and convert them into more active hydroxyl radicals by Fenton reaction. First, the diblock copolymers containing polyethylene glycol (PEG) and poly(glutamic acid) modified by β-cyclodextrin (β-CD) were synthesized. The block copolymer, ferrocenecarboxylic acid hexadecyl ester (DFc), and ascorbyl palmitate (PA) were co-assembled in aqueous solution to obtain stable core-shell micelles through the inclusion complexation between β-CD moieties in the block copolymer and ferrocene (Fc) groups from DFc. After intravenous injection, the particles achieved significant accumulation in tumor tissue where ascorbic acid at the pharmacological concentration promotes the production of H2O2, and subsequently Fenton reaction was catalyzed by Fc groups to produce hydroxyl radicals to efficiently kill cancer cells and suppress tumor growth. The micellar systems possess great potentials toward cancer therapy through synergistic H2O2 production and conversion into hydroxyl radicals specifically in tumor tissue.

KEYWORDS: reactive oxygen species; Fenton reaction; polymer micelles; cancer treatment; host-guest interaction.


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INTRODUCTION Reactive oxygen species (ROS), including superoxide, H2O2, and hydroxyl radical (•OH) play vital roles in living organisms.1,2 But overproduction of ROS may cause damages to biological molecules, leading to various disorders such as inflammation and cancer.3,4 In tumor tissues, cancer cells are immersed in the oxidative stress with relatively higher H2O2 level compared with normal tissues, which is important for the proliferation of cancer cells and tumor development.5-7 Meanwhile, cancer cells usually possess deficient antioxidant systems with low expression of ROS-depleting enzymes such as catalase, superoxide dismutase, etc.8-10 Cancer cells are welladaptive toward the intrinsically improved tumor oxidative stress while fragile to the exogenous ROS.3,4 Therefore, modulation of oxidative stress in tumor tissues has been proposed as an effective strategy to treat cancers, termed as oxidation therapy.11,12 In general, the primary approaches to perform oxidation therapy are delivery of cytotoxic ROS or ROS generators to the tumor tissue, and inhibition of antioxidant ability of cancer cells.11,12 The former method attracted great attention in recent years due to facile operation as well as versatile materials and reagents for ROS level increase in tumor tissues. For example, a variety of molecules including xanthine oxidase,13 D-amino acid oxidase,14 arsenic trioxide,11,15 glucose oxidase,16-18 cinnamaldehyde,19 and vitamin C (Vc) or Vc-containing molecules,20-24 etc. have been explored to improve the tumor oxidative level to kill cancer cells. Notably, vitamin C (Vc) or Vc-containing derivatives at the pharmacological concentration could suppress tumor growth to some extend or help cancer chemotherapy through improving the hydrogen peroxide (H2O2) which attracted a lively interest due to their high biosafety even at high concentrations.20,25-28 On the other hand, the relatively mild oxidation capability of H2O2 (standard redox potential E(H2O2/H2O)= 1.78 V) is significantly lower than those of hydroxyl radical (•OH) (E(•OH/H2O)= 2.80 V) and singlet oxygen (1O2) (E(1O2/H2O)= 2.17 V).29,30 At a tumor site, the concentration of H2O2 is 3 ACS Paragon Plus Environment


intrinsically higher compared with normal tissues. Based on these two points, one reasonable approach for efficient tumor oxidation therapy is to convert the mild H2O2 into highly active hydroxyl radicals to damage cancer cells more severely and kill them. Fenton reaction as a wellstudied redox reaction generates highly reactive hydroxyl radicals in the presence of H2O2 and a catalyst, which can be used to kill cancer cells more efficiently via severe cellular components damage.30-34 For example, amorphous iron nanoparticles have been used for cancer therapy through a localized Fenton reaction and •OH production in tumor tissue.30 Intriguingly, the combination of improvement of H2O2 level in tumor tissues and conversion of H2O2 into highly active •OH may presumably cause more efficient and continuous cancer cell killing.33-35 For instance, Lee et al.34 prepared an amphiphilic block copolymer consisting of H2O2-producing benzoyloxycinnamaldehyde and ferrocene-containing compounds in the backbone, which can selfassemble into micelles that serve as Fenton reactors to produce highly cytotoxic hydroxyl radicals and kill cancer cells preferentially. Lin and coauthors also demonstrated that the combination of cisplatin and Fe3O4 nanoparticles to enhance anticancer activity of cisplatin through tumor sitespecific intracellular H2O2 generation caused by cisplatin and subsequent Fe2+/Fe3+-catalyzed Fenton reaction by Fe3O4 nanocarriers.36 Nevertheless, more facile nanoparticle preparation and simpler nanocarriers that specifically work in tumor are still urgently desired. In this work, we prepared an integrated micellar nanoparticles via the self-assembly of poly(ethylene glycol)-block-Poly(γ-propargyl-L-glutamat-graft-β-cyclodextrin ) (PEG-b-P(PLG-gCD)), L-ascorbyl palmitate (PA), and ferrocenecarboxylic acid hexadecyl ester (DFc) in aqueous solution (Scheme 1). In the micelles, PA and DFc formed the cores of the micelles and PEG as the shells covered via the host-guest interactions between β-CD and ferrocene (Fc) moieties in the interfaces between PEG coronas and cores. Upon intravenous injection, the nanoparticles can accumulate in the tumor tissues due to the core-shell stable micellar structure which is favorable for the blood circulation and tumor accumulation. PA molecules at the pharmacological 4 ACS Paragon Plus Environment

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concentration act as the prooxidant agents to generate H2O2 in tumor tissues followed by transformation onto highly toxic •OH under the catalysis of Fc via well-known Fenton reaction. Fc moieties not only served for stabilization of the nanoparticles via host-guest interactions but also acted as the catalyst for the Fenton reaction. The combination of PA and Fc moieties was used for cancer therapy via oxidation therapy for the first time, which can work in the tumoral extracellular environment. All the compounds used here showed good biocompatibility including poly(amino acid), β-CD, PA, and DFc, which may possess low systemic toxicity. Moreover, the antitumor results showed that the combination of PA and DFc in one nanoparticles can efficiently kill cancer cells and suppress the tumor growth.

Scheme 1. Schematic illustration for the construction of polymeric micelles and application in cancer therapy. PA/Fc-Micelles were formed via the host-guest interactions between β-CD and Fc moieties. PA molecules generated H2O2 in the tumor environment followed by transformation into highly toxic •OH for cancer killing under the catalysis of Fc via Fenton reaction. 5 ACS Paragon Plus Environment


EXPERIMENTAL SECTION Materials. Dicyclohexylcarbodiimide (DCC, 98%), 4-dimethylaminopyridine (DMAP, 98%), disodium terephthalate (>99%), and ferrocenecarboxylic acid (98%) were purchased from Energy Chemical (Shanghai, China) and used as received. Copper (I) bromide (99%), 2hydroxyterephthalic

acid

(97%),

and

N,N,N′,N′′,N′′-pentamethyldiethylenetriamine

(PMDETA, >99%) were purchased from Sigma-Aldrich. Alexa Fluor 680 (AF680) carboxylic acid succinimidyl ester was purchased from Thermo Fisher Scientific. Reactive Oxygen Species (ROS) Assay Kit (2',7'-dichlorofluorescin diacetate, DCFH-DA) was purchased from Beyotime Institute of Biotechnology (Shanghai, China). 6-Monodeoxy-6-monoazido-β-cyclodextrin (β-CDN3) and alkynyl-functionalized block copolymer poly(ethylene glycol)-b-poly(γ-propargyl-Lglutamate) (PEG113-b-PPLG52) were synthesized according to the previous reports.37-39 All other commercially available solvents and reagents were purchased from Sinopharm Chemical Reagent Co. Ltd. and used as received. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate buffer saline (PBS, 10 mM, pH 7.4), trypsin, 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyltetrazolium bromide (MTT), and hematoxylin and eosin (H&E) staining kit were purchased from Beyotime Institute of Biotechnology (Shanghai, China). The murine hepatic cancer cell line H22, mice breast cancer cells line 4T1, and human breast cancer cell line MCF-7 were purchased from the Shanghai Institute of Cell Biology (Shanghai, China). Female 5-weekold BALB/c mice were purchased from Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). The animal studies were carried out in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (Hefei, revised in June 2013). Characterization. All 1H NMR spectra were recorded on a Bruker AV300 NMR 300 MHz spectrometer using CDCl3, dimethyl sulfoxide-d6 (DMSO-d6), or D2O as the solvent. The molecular weight (MW) and molecular weight distribution (Mw/Mn) were determined by gel 6 ACS Paragon Plus Environment

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permeation chromatography (GPC) equipped with a G1310B Iso. pump, a G1316A PL gel column, and a G1362A differential refractive index detector. The eluent was DMF with 1 g/L LiBr at a flow rate of 1.0 mL/min. A series of low-polydispersity PEG standards were employed for calibration. Particles sizes and particle size distributions were analyzed on a zeta-potential analyzer with dynamic laser light scattering (DLS), equipped a Malvern Zeta sizer Nano ZS90, a He-Ne laser (633 nm), and 173o collecting optics. A Hitachi H-7650 electron microscope at the acceleration voltage of 100 kV was employed to obtain transmission electron microscopy (TEM) images. All data were averaged over three measurements. High performance liquid chromatography (HPLC) analysis was performed on a Shimadzu HPLC system, equipped with a LC-20AP binary pump, a SPD-20A UV-Vis detector, and a Symmetry C18 column. Oxi 7310 meter equipped with dissolved oxygen sensor CellOx 325 were used to measure oxygen. UV-vis spectroscopy and fluorescence spectroscopy were recorded by UV-2401 PC UV-VIS spectrophotometer (Sahimadzu Corporation, Japan) and F-4600 fluorescence spectrophotometer (Hitachi, Japan), respectively. Fluorescence microscopy OLYMPUS X71 was used for fluorescence imaging. Xenogen IVIS spectrum optical imaging device was employed for studying the distribution and accumulation of samples in vivo.

Sample Preparation Synthesis of Block Copolymer PEG-b-P(PLG-g-CD). To a Schlenk tube equipped with a magnetic stirring bar, PEG113-b-PPLG52 (0.1 g, 7.1 µmol), β-CD-N3 (0.88 g, 0.76 mmol), PMDETA (0.17 g, 0.98 mmol), CuBr (0.14 g, 0.98 mmol), and DMF (3 mL) were added. The reaction tube was degassed by three freeze-pump-thaw cycles, sealed under vacuum, and stirred for 24 h at 40 °C. The solution was then dialyzed against distilled water for three days using a



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dialysis bag (cellulose membrane, MW cutoff: 10 kDa). After lyophilization, white powder was obtained (0.29 g, yield: 53.7%. Mn = 33.5 kDa, Mw/Mn = 1.21). For biodistribution observation, AF680-labelled PEG-b-P(PLG-g-CD) polymer was also synthesized. Briefly, amidation reaction was conducted between AF680 carboxylic acid succinimidyl ester and PEG-b-P(PLG-g-CD). AF680 carboxylic acid succinimidyl ester (1.73 mg, 1.5 µmol) and PEG-b-P(PLG-g-CD) (50 mg, 1.5 µmol) were dissolved in DMF (2 mL) and stirred for 24 h. Then, dialysis method (MWCO 7000 Da) was applied to remove unreacted small molecules. The final product was obtained after lyophilization as pale green powder (45 mg, Yield: 87.3%). Synthesis of DFc. Ferrocenecarboxylic acid (2.30 g, 10 mmol), DCC (2.50 g, 12.1 mmol), and DMAP (0.15 g, 1.22 mmol) were dissolved in anhydrous dichloromethane (50 mL) and stirred for 30 min. Hexadecanol (2.67 g, 11 mmol) was then added into the solution. The mixture was stirred for 36 h at room temperature. The resulting solution was filtered by Buchner funnel, and the filtrate was concentrated by a rotary evaporator. The product was purified by silica gel column by using the mixture of ethyl acetate and petroleum ether (v/v, 1:5) as the eluent. After removal of the solvent, orange solid was obtained (2.56 g, yield: 56.3%).1H NMR (300 MHz, CDCl3): δ 4.80 (t, 2H), 4.38 (t, 2H), 4.25-4.14 (m, 7H), 1.71 (dd, 2H), 1.49-1.17 (m, 26H), 0.88 (t, 3H).

13

C NMR

(75 MHz, CDCl3): δ 167.02 (s), 66.80 (s), 66.41 (s), 65.34 (s), 64.95 (s), 59.58 (s), 27.18 (s), 25.10-24.39 (m), 24.18 (s), 21.34 (s), 17.95 (s), 9.38 (s). ESI-MS Calcd. for (C26H40O2Fe + H)+: 455.54; Found: 455.89. Preparation of PA/Fc-Micelles. PEG-b-P(PLG-g-CD) (10 mg), PA (7.5 mg), and DFc (2.5 mg) were dissolved in DMF (1 mL), stirred at room temperature for 2 h. The solution was injected into 5 mL deionized water in one shot under vigorous stirring. The colloidal dispersion was further stirred for 20 min, followed by dialysis (MWCO, 3.5 kDa) against deionized water for 24 h to 8 ACS Paragon Plus Environment

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remove DMF. Free unencapsulated PA and DFc were removed by ultracentrifugation. Fresh water was replaced every 6 h. The final concentration of the nanoparticles was fixed to 2 mg/mL. Particle size and size distribution were monitored by DLS and TEM. For in vivo biodistribution studies, AF680-labelled PA/Fc-Micelles were also prepared using AF680-labelled block polymer. As a control, the empty micellar nanoparticles were also prepared by the complexation between the polymer and DFc without addition of PA molecule. The encapsulation efficiencies (EEs) of DFc or PA loaded in the micelles were determined by UV detection and HPLC, respectively. The standard UV absorbance curve of DFc in methanol and standard HPLC curve of PA with the eluent of methanol were measured, respectively (Figure S1 and S2). The micelle solutions were lyophilized followed by dispersion in methanol solution and analyzed by UV absorbance and HPLC, respectively. EEs was calculated according to the following formula: EE (%) = ((weight of loaded drug)/(weight of initially added drug)) × 100%. Release Profiles Evaluation. In vitro release profiles of PA and DFc from PA/Fc-Micelles were also evaluated via the dialysis method. Briefly, PA/Fc-Micelles (1 mg/mL, 0.5 mL) solution was transferred into a dialysis bag and immersed into PBS (pH 7.4, 10 mL) at 37 oC. At predetermined time intervals, aliquots of samples were withdrawn and an equal volume of PBS was added. After lyophilization, the release amounts of PA and DFc were measured. Evaluation of H2O2 Production. H2O2 production of PA in DMEM medium with 10% FBS was measured by dissolved oxygen (D.O.) meter in the presence of catalase. Briefly, PA at final concentrations of 0.3, 0.5 or 1.0 mM was added to DMEM medium containing 10% FBS (100 mL) and incubated at room temperature. The solution (10 mL) was taken out from the incubation medium at predetermined time intervals. O2 sensor was immersed in the collected medium, followed by addition of 1000 units of catalase solution (200 µL). A prior standard calibration



regarding O2 production and H2O2 concentrations (range: 10-200 µM) was established to determine the H2O2 concentration in the sample. Fenton Reaction Detection. Hydroxyl radicals were detected by using disodium terephthalate as a capture agent, which can react with hydroxyl radicals to generate fluorescent 2hydroxyterephthalic disodium.40-42 Briefly, Fenton reaction was performed by mixing DMEM medium (1.6 mL) with 10% FBS upon addition of disodium terephthalate solution (200 µL, 50.0 mmol/L) and 200 µL PBS, PA (0.66 mg/mL), DFc (0.24 mg/mL), or PA/Fc-Micelles (2 mg/mL) at the PA-equivalent concentration of 0.66 mg/mL and DFc-equivalent concentration of 0.24 mg/mL. The solution was subjected to fluorescence tracing by using a fluorescence spectrometer at the excitation wavelength of 310 nm and emission wavelength of 425 nm. In Vitro Cytotoxicity. 4T1 and MCF-7 cells were used for in vitro evaluation of cytotoxicity via

MTT assay. 4T1 and MCF-7 cells were seeded onto 96-well plates, respectively, at a density of 5 × 103 cells/well in 100 µL DMEM with 10% FBS at 37 °C with 5% CO2 humidified atmosphere and incubated for 24 h. After replacing with 90 µL fresh DMEM containing 10% FBS in each well, PA, DFc, or PA/Fc-Micelles solutions (10 µL) at different final PA concentrations of 3.3, 16.5, 33.0, and 66.0 µg/mL, DFc of 1.2, 6.0, 12.0, and 24.0 µg/mL, or PA/Fc-Micelles of 10, 50, 100, and 200 µg/mL with the same PA-equivalent and DFc-equivalent concentrations were added into each well. After further incubation for 48 h, 100 µL fresh DMEM was replaced for each well and 10 µL MTT solution (5 mg/mL) was added, followed by incubation for another 4 h. The medium was replaced by 200 µL DMSO in each well. Finally, the plate was placed in the dark for 30 min, followed by measurement of the absorbance at 490 nm by a microplate reader. Intracellular ROS Production. Intracellular H2O2 in 4T1 and MCF-7 cells was detected by ROS Assay Kit DCFH-DA. Briefly, 4T1 and MCF-7 cells were seeded onto 12-well plates at density at 5 × 105 cells per well in 1 mL DMEM containing 10% FBS at 37 °C under 5% CO2 and incubated 10 ACS Paragon Plus Environment

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for 24 h. The cells were then treated with PA (66 µg/mL), DFc (24 µg/mL), or PA/Fc-Micelles (200 µg/mL) at the PA-equivalent concentration of 66 µg/mL and DFc-equivalent concentration of 24 µg/mL with PBS solution as control, followed by incubation for another 6 h. The medium was removed. Then, fresh DMEM medium (2 mL) containing DCFH-DA (10 µM) were added followed by further incubation for 20 min. Then, the cells were washed three times with PBS and imaged with fluorescence microscopy. Evaluation of DNA Damage by Comet Assay. DNA damage assay kit was used to evaluate DNA damage after treatment with various formulations.43,44 Briefly, 4T1 and MCF-7 cells were seeded onto 12-well plates at density at 5 × 105 cells per well in 1 mL DMEM containing 10% FBS at 37 °C under 5% CO2 and incubated for 24 h. The cells were then treated with PA (66 µg/mL), DFc (24 µg/mL), or PA/Fc-Micelles (200 µg/mL) at the PA-equivalent concentration of 66 µg/mL and DFc-equivalent concentration of 24 µg/mL with PBS solution as control, followed by incubation for another 6 h. Subsequently the cells were trypsinized, collected and resuspended in PBS (1 mL). The gels were subsequently prepared according to the instructions. Briefly, 100 µL normal melting point agarose PBS solution (0.5%) was coated to a glass slide as the first layer. For the second layer, 20 µL cell solution mixed with 100 µL low melting point agarose PBS solution (0.7%) was anointed to the slides quickly. After the gel was solidified, the slides were immersed into lysis solution (10% DMSO) at 4 °C for 3 h. After that, the slides were washed with PBS solution and immersed into alkaline buffer (1 mM EDTA, 300 mM NaOH) for 30 min to denature, followed by electrophoresis for 20 min (25 V, 200 mA). The samples were neutralized in 0.4 M tris(hydroxymethyl)aminomethane (Tris)-HCl (pH = 7.5) for 10 min and repeated the progress twice. Then the gel was stained with propidium iodide for 10 min in a dark. Fluorescence microscope was engaged to examine the DNA damage. Comet Assay Software Project (CASP) was used to analyze the DNA damage displayed as Tail DNA percent.



In Vivo Distribution and Accumulation of PA/Fc-Micelles. H22 tumor model was first established. Briefly, 5 × 106 H22 cells in 0.1 mL saline were subcutaneously inoculated into the right limb armpits of female BALB/c mice. Tumors were allowed to grow for a week to reach proliferative phase with the size of ~100 mm3. Subsequently, 200 µL of AF680-labelled PA/FcMicelles solution (2 mg/mL) was intravenously injected into the tail vein of H22 tumor-bearing BALB/c mice. A Xenogen IVIS Lumina system was used to monitor the tumor accumulation at determined time points. Antitumor Activity Evaluation. H22 tumors were established and treatment commenced when the average tumor volume reached ~100 mm3. A total of 20 female BALB/c mice (7 weeks old) were assigned to four groups (n = 5) and injected with PBS, PA (6.6 mg/kg), DFc (2.4 mg/kg) controls, or PA/Fc-Micelles (20 mg/kg) at the PA-equivalent dose of 6.6 mg/kg and DFc of 2.4 mg/kg via tail vein at day 0, 2, and 4. Tumor progression and body weights of mice were monitored every other day. Tumor growth was monitored by digital calipers, and the volumes calculated as tumor volume (mm3) = 0.5 × L × W2, where L represents length and W represents width. At day 20, all the mice were sacrificed. The major organs and tumors were dissected, collected, and used for H&E staining after sectioning into thin slices (10 µm).

RESULTS AND DISCUSSION

Fabrication and Characterization of Core-Shell Micelles. The synthesis of β-CDfunctionalized block copolymer PEG-b-P(PLG-g-CD) was performed via the click reaction between PEG113-b-PPLG52 and azide moieties-containing β-CD-N3 at the molar ratio of 1:2 for alkynyl to β-CD-N3 (Scheme 2). Notably, although excessive β-CD-N3 was used, 1H NMR analysis demonstrated that just 30% of the alkynyl groups were reacted with the β-CD-N3 and the 12 ACS Paragon Plus Environment

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final number of β-CD moieties on each block copolymer was 15 by comparing the integration of 2,3-OH in β-CD and PEG peak (h; Figure 1A). This should be attributed to the steric hindrance of β-CD molecules with large molecule volume. The polymer PEG-b-P(PLG-g-CD) was also characterized by GPC measurements with the molecular weight (Mn) of 33.5 kDa and polymer dispersity index(Mw/Mn) of 1.21 compared with the precursor PEG113-b-PPLG52 (Mn = 16.0 kDa, Mw/Mn = 1.12) (Figure 1B). Moreover, we also designed and successfully synthesized the Fccontaining molecules DFc via DCC condensation between ferrocenecarboxylic acid and 1hexadecanol.

Scheme 2. Synthetic routes employed for the preparation of PEG-b-P(PLG-g-CD) and DFc It is well-known that Fc can interact with β-CD via the host-guest interaction which has been used to construct nanoparticles from the Fc and β-CD moieties-containing polymers.45-47 Herein, we prepared the micellar nanoparticles via nanoprecipitation method for the self-assembly of mixed PA, DFc, and PEG-b-P(PLG-g-CD) at the molar ratio of 50:17:1 with Fc/β-CD of 1.1:1, which was denoted as PA/Fc-Micelles. In the nanoparticle, the amphiphilic properties of PA and 13 ACS Paragon Plus Environment


DFc resulted in the formation of small molecules micelles with n-alkyl chains in the cores and ascorbic acid and Fc as the shells, and PEG-b-P(PLG-g-CD) simultaneously covered on the surface via the host-guest interactions between Fc and β-CD moieties. The formation of inclusion complexes between the Fc groups and β-CD groups was investigated by 2D NMR NOESY spectra. Figure 2A shows the NOESY spectrum for the self-assembled micelles of PEG-b-P(PLG-g-CD) and DFc in the mixed solvent of D2O and DMSO-d6. It can be seen that the signals of β-CD correlated with the resonance of the Fc groups at 4.45 and 4.80 ppm, indicating that the Fc groups threaded into the cavity of β-CD to form inclusion complexes.

Figure 1. (A) 1H NMR spectrum recorded for the block polymer PEG-b-P(PLG-g-CD). (B) GPC traces obtained for the polymers amino group-terminated PEG (PEG-NH2, Mn = 5.3 kDa, Mw/Mn = 14 ACS Paragon Plus Environment

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1.06), PEG-b-PPLG (Mn = 16.0 kDa, Mw/Mn = 1.12), and PEG-b-P(PLG-g-CD) (Mn = 33.5 kDa, Mw/Mn = 1.21). After self-assembly into polymeric micelles, we further evaluated EEs of DFc and PA via UV absorbance and HPLC, respectively, according to the standard curves (Figure S1 and S2). They were determined to 80.4% and 86.3%, respectively. DLS results showed that the formed PA/FcMicelles exhibited nanoscaled structure with the average diameter of 83 nm and unimodal size distribution (Figure 2B). TEM characterization also confirmed uniform spherical structures with an average diameter of 69 ± 14 nm. Moreover, to evaluate the stability of PA/Fc-Micelles in the serum-containing medium, we detected their size and polydispersity index for two days after incubation of the micelles in the presence of FBS (10% serum) (Figure 2C). The results showed that the sizes and polydispersity indexes almost maintained constant with slight increase within two days, indicating that the polymer micelles were stable in the serum-containing medium. In sharp contrast, in the absence of the polymer PEG-b-P(PLG-g-CD) during self-assembly, PA and DFc formed the drastically turbid solutions which precipitated after incubation for a while indicating that the formed nanoparticles were not stable likely due to relatively high hydrophobicity of these two molecules, and the hydrophilic head groups cannot stabilize the formed nanoparticles (Figure 2D). The results indicated that the block copolymers can stabilize the formed PA/Fc-Micelles with PEG as the shells, which are favorable for the following in vitro and in vivo applications. Moreover, the release profiles of PA and DFc were also evaluated (Figure S3). Only a small amount of PA and DFc (≤ 10%) was released after incubation for two days in PBS, which also indicated the relatively high stability of the micellar nanoparticles constructed on the basis of hydrophobic and host-guest interactions.



Figure 2. (A) 2D NMR NOESY spectra for PEG-b-P(PLG-g-CD) and DFc in D2O/DMSO-d6. (B) DLS and TEM characterization of PA/Fc-Micelles at the polymer concentration of 1.5 mg/mL. Scale bars represent 500 nm. (C) Time-dependent size and polydispersity index change of PA/FcMicelles after incubation in the 10% serum-containing medium. Mean ± s.d. (n = 3). (D) The pictures of formed solutions of PA (1.32 mg/mL), DFc (0.48 mg/mL), and PA/Fc-Micelles (4 mg/mL) in aqueous solutions. H2O2 and •OH Production. To validate the H2O2 production capability of PA molecules, we first investigated the time-dependent H2O2 concentration increase after incubation with DMEM containing 10% serum (Figure 3A). Apparently, PA could produce H2O2 efficiently in the medium with a PA concentration-dependent manner due to the oxidation reaction between ascorbic acid and certain metal ion-containing proteins.22-24,26,48 The concentration of H2O2 increased rapidly until reached its peak of approximately 100 µM at the PA concentration of 1 mM within 16 ACS Paragon Plus Environment

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approximately 100 min. Subsequently, the H2O2 concentration decreased gradually. The significantly increased H2O2 concentration can provide H2O2 source for the Fenton reaction in the presence of DFc moieties to produce highly active hydroxyl radicals.

Figure 3. (A) Cumulative H2O2 production in DMEM medium (10% serum) in the presence of PA. (B) Fluorescence intensity change of 2-hydroxyterephthalic acid as a function of time by incubation with PA (67 µg/mL), DFc (24 µg/mL), or PA/Fc-Micelles (200 µg/mL) at the PAequivalent concentration of 67 µg/mL and DFc-equivalent concentration of 24 µg/mL in the 10% serum-containing medium. Emission peak at the wavelength of 425 nm was detected. Mean ± s.d. (n = 3). Moreover, to evaluate the production of hydroxyl radicals, PA/Fc-Micelles were incubated with the cell culture medium containing 10% serum. Because it is difficult to detect the production 17 ACS Paragon Plus Environment


of hydroxyl radicals due to their short lifetime, we used disodium terephthalate as a probe to investigate the generation of hydroxyl radicals via the fluorescence emission peak at the wavelength of 425 nm, which is a non-fluorescent molecule and produce fluorescent product (2hydroxyterephthalic disodium) after reaction with the hydroxyl radicals.40-42 As shown in Figure 3B, the fluorescence intensity almost maintained constant for the sole PA and DFc. In sharp contrast, PA/Fc-Micelles showed rapid increase of fluorescence intensity within three hours indicating the efficient production of hydroxyl radicals. Collectively, the results confirmed that PA/Fc-Micelles can produce H2O2 in the serum-containing medium and generate a large amount of hydroxyl radicals under the catalysis of DFc, which can be used to destroy the cancer cells powerfully. Cytotoxicity Evaluation. To evaluate the cytotoxicity of PA/Fc-Micelles, 4T1 and MCF-7 cells were incubated in the presence of the nanoparticles, and the cellular viability was evaluated by using MTT assay (Figure 4A,B). When the cells were treated with free DFc, the viability of the cells had almost no change with Fc concentration increasing for both 4T1 and MCF-7 cells. Although Fc has been previously reported to induce cytotoxicity by catalyzing Fenton reaction of intrinsic H2O2 inside cancer cells,33,34,49 low H2O2 level inside cells and poor water-solubility of DFc may limit their cytotoxicity toward cancer cells. Free PA had some cytotoxicity for both cells which is consistent with the previously published reports.22-24 PA with ascorbic acid head can kill cancer cells through H2O2 generation in cell culture medium which finally induced the cancer cell death by high H2O2 level. Specifically, the half inhibitory concentrations (IC50) of PA with respect to 4T1 and MCF-7 cells were determined to be nearly 39 and 36 µg/mL, respectively. However, PA/Fc-Micelles showed significantly higher cytotoxicity compared with free PA molecules at the same PA-equivalent concentrations. In detail, the IC50 values of PA/Fc-Micelles against 4T1 and MCF-7 cells were both 73 µg/mL with the PA-equivalent concentrations of 24 µg/mL, which were much lower than that of free PA. The results indicated that PA/Fc-Micelles possess the strong 18 ACS Paragon Plus Environment

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capability to kill cancer cells through effective production of H2O2 followed by Fenton reaction to generate •OH which may induce severe damage to the cancer cells. We further used ROS Assay Kit DCFH-DA to observe intracellular ROS (H2O2 and •OH) level as shown in Figure 4C. We can find that the fluorescence intensity of PA/Fc-Micelles group in both 4T1 and MCF-7 cells was significantly higher compared with PA group. Presumably, PA only induced H2O2 production while PA/Fc-Micelles resulted in the production of hydroxyl radicals. Active hydroxyl radicals can react with DCFH-DA more efficiently compared with H2O2 leading to stronger fluorescence intensity.35 ROS (H2O2 and •OH) can kill cancer cells primarily through DNA damage.50 Highly active hydroxyl radicals •OH can damage DNA of cancer cells much more efficiently compared with H2O2.30 To further investigate the mechanism of the cancer cell killing of PA/Fc-Micelles, we performed the comet assay to evaluate DNA damages after treatment by PA and PA/Fc-Micelles, respectively.43,44 The proportions of comet trailing reflect the degree of DNA damage. As shown in Figure 4 D, E, PBS control group and DFc showed very little DNA damage. For free PA group, the comet tailing DNA degrees were determined to be 11% and 16% for 4T1 and MCF-7 cells, respectively. In sharp contrast, for the cells treated with PA/Fc-Micelles, the tails of the comets were significantly higher with the tail DNA percent of 45% for 4T1 cells and 39% for MCF-7 cells. Therefore, the combination of PA and DFc in PA/Fc-Micelles can induce significantly higher cytotoxicity via severe DNA damage due to the production of the highly active hydroxyl radicals.



Figure 4. In vitro cytotoxicity against (A) 4T1 cells and (B) MCF-7 cells based on MTT assay. Mean ± s.d., n = 5. (C) Intracellular ROS level analyzed by DCFH-DA staining after incubation 20 ACS Paragon Plus Environment

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with PBS, DFc, PA or PA/Fc-Micelles for 4T1 and MCF-7 cell. Scale bars represent 50 µm (D) Comet assay for 4T1 cells and MCF-7 cells after treatment with PBS (control), DFc (24 µg/mL), PA (66 µg/mL), or PA/DFc-Micelles (200 µg/mL). (E) DNA damage was expressed by the ratio of comet tail based on the data obtained in comet assay. Scale bars represent 100 µm. Mean ± s.d. (n = 10), **p< 0.01, ***p< 0.001 (t-test). Antitumor Efficacy. To investigate the in vivo performance of PA/Fc-Micelles, we selected murine hepatic carcinoma H22 tumor models in BALB/c mice due to the facile establishment of H22 tumor-bearing mice with uniform tumor sizes. First, the biodistribution and tumor deposition of AF680-labelled PA/Fc-Micelles in tumor tissue were observed by using IVIS Lumina system. As shown in Figure 5A, PA/Fc-Micelles could accumulate effectively in the tumor at 24 h after intravenous injection and reach maximum at 48 h, suggesting that PA/Fc-Micelles can deposit efficiently in tumor tissues for anticancer therapy. We further demonstrated the in vivo anticancer efficacy of PA/Fc-Micelles toward H22 tumor-bearing mice, which were randomly divided into four groups including PBS (control), DFc, PA, and PA/Fc-Micelles. Various formulations were intravenously injected from tail vein at day 0, 2, and 4, respectively, at the PA-equivalent dose of 6.6 mg/kg and DFc of 2.4 mg/kg for each injection. The tumor sizes were monitored. For PBS control, DFc and PA group, the tumor sizes showed a 17-fold, 10-fold and 8-fold increase, respectively, after 20 days treatment (Figure 5B, C). The growth curves revealed that the average tumor size just increased to 3-fold compared with the original size indicting that PA/Fc-Micelles showed significantly better antitumor activity and suppressed the tumor growth.



Figure 5. (A) Biodistribution of AF680-labelled PA/Fc-Micelles in H22 tumor-bearing mice at different time points post intravenous injection. (B) Time-dependent tumor volume change after injection of PBS, DFc, PA, or PA/Fc-Micelles at the Fc-equivalent dose of 2.4 mg/kg, PA of 6.6 mg/kg on day 0, 2, and 4, respectively. Mean ± s.d. (n = 5), ***p < 0.001 (t-test). (C) Tumor tissue images extracted from the mice at the end of treatment by various formulations. (D) H&E staining images of H22 tumor sections harvested from the mice treated with PBS, DFc, PA, or PA/FcMicelles, respectively. Scale bars represent 25 µm. At the end of treatment, the mice were sacrificed and the tumor tissues were sectioned into slices. H&E staining indicated severe coagulative necrosis and tumor tissue damage for PA/FcMicelles-treated group (Figure 5D). However, PBS (control), DFc, and PA groups showed significantly mild tissues necrosis. The results were consistent with the tumor growth profiles. 22 ACS Paragon Plus Environment

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Moreover, the mice body weights were monitored during treatment, and no significant body weight loss was observed indicating no drastic systemic toxicity after treatment by various groups (Figure 6A). In addition, the main organ toxicity was observed in all groups by using H&E staining of organ slices (heart, liver, spleen, lung, and kidney). Figure 6B showed that PA, DFc, and PA/Fc-Micelles did not induce apparent main organ damage. Collectively, the developed PA/Fc-Micelles nanoparticles can significantly inhibit the tumor growth with low systemic toxicity.

Figure 6. (A)Time-dependent body weight change of H22 tumor-bearing mice after treatment by PBS, PA, DFc or PA/Fc-Micelles. (B)H&E staining images of main organs (heart, liver, spleen, 23 ACS Paragon Plus Environment


lung, and kidney) sections harvested from the mice at the end of treatment by PBS, PA, DFc, or PA/Fc-Micelles. Mean ± s.d., n = 5, Scale bars represent 25 µm.

CONCLUSIONS In summary, we developed a novel integrated micellar nanoparticle through self-assembly of βCD-containing block copolymer PEG-b-P(PLG-g-CD), PA, and DFc. PEG shells and host-guest interaction between β-CD and Fc moieties improved the stability of the micelles in serumcontaining medium. PA and DFc cores offered the multifunctional properties of the micelles. The pharmacological concentration of PA promoted the H2O2 generation specifically in tumor tissues, which was followed by Fc-catalyzed Fenton reaction to transform H2O2 into highly active and toxic hydroxyl radicals. Potent cancer cell killing capability can be achieved. Through intravenous injection of PA/Fc-Micelles, effective tumor accumulation resulted in high-efficiency tumor growth suppression with negligible systemic toxicity. Therefore, PA/Fc-Micelles design provides a novel strategy to achieve more efficient oxidation therapy of cancers, which showed great promises to treat cancers with low systemic toxicity.

ASSOCIATED CONTENT Supporting Information. UV absorbance of DFc, HPLC of PA, drug release profiles. This material is available free of charge via the internet at http://pubs.acs.org.


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AUTHOR INFORMATION

Corresponding Author * Email: [email protected] (Z. Ge), [email protected] (C. He)

ORCID Zhishen Ge: 0000-0002-2668-6974

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS

We gratefully acknowledge ﬁnancial support from National Natural Science Foundation of China (NNSFC) (21674104) and the Fundamental Research Funds for the Central Universities (WK3450000002).

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Table of Contents

Self-assembly

PEG -b-P( PLG -g-C D)

D Fc

PA O O

O

Fe

HO HO HO

O

Mn+

H 2O2

OH

O Oxidation


Multifunctional Polymeric Micelles with Amplified Fenton Reaction for

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