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Cite This: Chem. Mater. 2018, 30, 344−357

Manganoporphyrin-Polyphenol Multilayer Capsules as Radical and Reactive Oxygen Species (ROS) Scavengers Aaron Alford,†,∥ Veronika Kozlovskaya,†,∥ Bing Xue,† Nirzari Gupta,† William Higgins,† Dana Pham-Hua,‡ Lilin He,§ Volker S. Urban,§ Hubert M. Tse,*,‡,∥ and Eugenia Kharlampieva*,†,∥ †

Department of Chemistry, University of Alabama at Birmingham, Birmingham, Alabama 35294, United States Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294, United States § Neutron Scattering Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States ‡

S Supporting Information *

ABSTRACT: Local modulation of oxidative stress is crucial for a variety of biochemical events including cellular differentiation, apoptosis, and defense against pathogens. Currently employed natural and synthetic antioxidants exhibit a lack of biocompatibility, bioavailability, and chemical stability, resulting in limited capability to scavenge reactive oxygen species (ROS). To mediate these drawbacks, we have developed a synergistic manganoporphyrin-polyphenol polymeric nanothin coating and hollow microcapsules with efficient antioxidant activity and controllable ROS modulation. These materials are produced by multilayer assembly of a natural polyphenolic antioxidant, tannic acid (TA), with a synthesized copolymer of polyvinylpyrrolidone containing a manganoporphyrin modality (MnP-PVPON) which mimics the enzymatic antioxidant superoxide dismutase. The redox activity of the copolymer is demonstrated to dramatically increase the antioxidant response of MnP-PVPON/TA capsules versus unmodified PVPON/TA capsules through reduction of a radical cationic dye and to significantly suppress the proliferation of superoxide via cytochrome C competition. Inclusion of MnP-PVPON as an outer layer enhances radical-scavenging activity as compared to localization of the layer in the middle or inner part of the capsule shell. In addition, we demonstrate that TA is crucial for the synergistic radical-scavenging activity of the MnP-PVPON/TA system which exhibits a combined superoxide dismutase-like ability and catalase-like activity in response to the free radical superoxide challenge. The MnP-PVPON/TA capsules exhibit a negligible, 8% loss of shell thickness upon free radical treatment, while PVPON/TA capsules lose 39% of their shell thickness due to the noncatalytic free-radical-scavenging of TA, as demonstrated by small angle neutron scattering (SANS). Finally, we have found the manganoporphyrin-polyphenol capsules to be nontoxic to splenocytes from NOD mice after 48 h incubation. Our study illustrates the strong potential of combining catalytic activity of manganoporphyrins with natural polyphenolic antioxidants to design efficient free-radical-scavenging materials that may eventually be used in antioxidant therapies and as free radical dissipating protective carriers of biomolecules for biomedical or industrial applications.

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INTRODUCTION

Exogenous metalloporphyrins have demonstrated a strong potential to mimic the catalytic redox activity of CAT and SOD and diminish oxidative stress by antioxidant and immunomodulatory actions.6−11 The most effective Mn(III) porphyrins can scavenge a broad range of oxidants such as superoxide, hydrogen peroxide, peroxynitrite, and lipid peroxyl radicals.12,13 The utility of catalytic metalloporphyrin antioxidants to ameliorate inflammatory-mediated processes has been demonstrated in an adoptive transfer model of Type 1 diabetes (T1D),14 endotoxic shock,15 protection of neuronal

Reactive oxygen species (ROS) are important for cellular signaling and defense against microbial pathogens, but can directly cause damage to DNA, lipids, and proteins through free-radical-mediated oxidative stress. Oxidative stress can facilitate tissue damage and cell death in a variety of human diseases including diabetes, atherosclerosis, Alzheimer’s disease, kidney disease, and cancer.1−3 Imbalanced ROS in the body are managed by endogenous enzymatic antioxidants, including superoxide dismutases (SODs), which can dismutate the superoxide radical (O2•−) into hydrogen peroxide (H2O2), and catalase (CAT), which dissipates hydrogen peroxide into water and oxygen.4,5 © 2017 American Chemical Society

Received: August 18, 2017 Revised: November 24, 2017 Published: December 18, 2017 344

DOI: 10.1021/acs.chemmater.7b03502 Chem. Mater. 2018, 30, 344−357

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Chemistry of Materials cells from apoptosis,16 inhibition of lipid peroxidation,13 and blocking of hydrogen peroxide-induced mitochondrial DNA damage.17 Local modulation of the oxidative stress is advantageous over global ROS suppression, as ROS are crucial in many biochemically important events including cell-to-cell communication, cellular differentiation, apoptosis, and defense against pathogens. Local suppression of pro-inflammatory ROS has been shown through conjugation of SOD to nanocarriers,18 or embedding in/copolymerization of SOD mimetics into polymer matrices.19,20 However, protein molecules may suffer from limited in vivo stability and be potentially immunogenic, while polymerizable SOD mimetics may involve harsh reactants and reaction conditions.21 Conversely, noncomplexed natural or synthetic small molecule antioxidants may suffer from low bioavailability,22 elution from the parent matrices,23 and various degrees of cytotoxicity10 when diffusing into neighboring biological locales. Nanoengineered coatings with noncovalent inclusion of ROS scavengers have demonstrated success for localized ROSscavenging. For instance, 5 μm microcapsules produced via multilayer assembly of ionically paired poly(styrenesulfonate) and poly(allylamine hydrochloride) (PSS/PAH) with embedded 4 nm iron oxide nanoparticles or CAT were shown to be moderately effective in reducing oxidation of encapsulated bovine serum albumin by hydrogen peroxide.24 Similarly, 21 nm polyelectrolyte complexes of CAT and cationic block polyethylenimine-poly(ethylene glycol) increased the catalytic degradation of H2O2.25 Among natural antioxidants, tannic acid (TA) has been widely exploited for the design of nanoengineered coatings and biomedical applications due to its ability to participate in ionic pairing,26 hydrogen bonding,27,28 and metal coordination29−31 because of its 25 phenolic groups on digalloyl ester branches connected to a glucose core.32−39 We previously demonstrated that hydrogen-bonded multilayers of TA and nonionic poly(Nvinylpyrrolidone) (PVPON) were nontoxic for conformal coating of pancreatic islets and helped them maintain in vitro and in vivo function.32,40 We showed that, by disproportionating ROS, a (PVPON/TA) multilayer could affect the activation of redox-dependent signaling pathways that contribute to the synthesis of pro-inflammatory cytokines and chemokines, which resulted in attenuated adaptive immune T cell effector responses involved in autoimmune activity and islet graft rejection.32,41 Moreover, disproportionation of free radicals with (PVPON/TA) coatings also suppressed the activation of pro-inflammatory macrophages which are key for destruction of insulin-producing pancreatic β-cells in T1D.40 Despite the antioxidant potential of the (PVPON/TA) coating system, the TA can lose its ROS-scavenging capability due to formation of quinones upon oxidation by radicals. Moreover, because of the quinone transformation, the coating integrity might be lost with time due to competing interactions of the TA phenol groups with free radicals versus H-bonds with PVPON. To mediate these drawbacks, we have designed a synergistic polyphenol-manganoporphyrin polymeric coating using covalent coupling of the SOD-mimetic manganoporphyrin modality to a PVPON copolymer which can be assembled with TA into an efficient ROS-scavenging (TAmanganoporphyrin-PVPON) multilayer coating. In contrast to a traditional approach, where metalloporphyrins have been simply added to solution or noncovalently embedded into

polymer matrices, covalent inclusion of the manganoporphyrin as a pendant group should allow for highly controllable ROS modulation. We explore the stability of manganoporphyrin-PVPON (MnP-PVPON) in aqueous solutions and the effect of pH conditions on its assembly with TA using in situ ellipsometry and surface wettability measurements. The possible cytotoxicity of manganoporphyrin-PVPON/TA multilayer capsules (due to cationic pyridinium groups and manganese cations in the porphyrin molecule) was tested by treatment of splenocytes from nonobese diabetic (NOD) mice with various capsule concentrations for 48 h. We also study the effect of inclusion of the MnP-PVPON instead of PVPON, the effect of the localization of MnP-PVPON layers within the capsule shell, and the overall number of functional MnP-copolymer layers present in the capsule shell on the radical-scavenging properties of the manganoporphyrin-polyphenolic hollow capsules compared to polyphenolic (PVPON/TA) capsules. We analyze the shell thickness of pure polyphenolic (PVPON/ TA) and the manganoporphyrin-polyphenolic capsules using small angle neutron scattering (SANS) measurements of the capsules in solution before and after free radical treatment. Our work illustrates the strong potential of combining catalytic activity from manganoporphyrins with natural polyphenolic antioxidants to design highly efficient and stable free-radicalscavenging systems. These systems have strong potential for antioxidant therapies and for free-radical-scavenging protective carriers that could eventually see use with sensitive biomolecules for biomedical and industrial applications.

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EXPERIMENTAL SECTION

Materials. N-Vinylpyrrolidone (VPON), poly(N-vinylpyrrolidone) (PVPON, Mw = 1 300 000 Da), mono- and dibasic sodium phosphate, ethyl-1-bromopentanoate, tetrahydrofuran (THF), tannic acid (TA, MW = 1700 Da), petroleum ether, diethyl ether, methanol, isopropanol, dioxane, trolox, 2,2-azino-bis(3-ethylbenzothiazoline-6sulfonic acid) diammonium salt (ABTS), potassium persulfate, cytochrome C, xanthine, xanthine oxidase, ethylene diamine tetraacetic acid (EDTA), and catalase (CAT) were purchased from Fisher Scientific and used without further purification except where otherwise mentioned. N-(t-BOC-aminopropyl)methacrylamide (tBOC) was purchased from Polysciences, Inc. Solid silica cores were purchased from Cospheric. 3-(3-(Dimethylamino)propyl)-1ethyl-carbodiimide hydrochloride (EDC) was purchased from ChemImpex International. Ultrapure deionized water with a resistivity of 18.2 Ω cm was used in all experiments (Evoqua Water Technologies). Meso-tetra(4-pyridyl)porphine was purchased from Frontier Scientific and used without further purification. NMR were recorded on Bruker 300 MHz NMR, and FTIR on a Bruker alpha ATR-FTIR with the bare ATR crystal in open air as the background. Synthesis of the Manganese(III) Meso-tetrakis(1-methylpyridinium-4-yl)porphyrin Pentaacetic Acid (MnP). The carboxyl monofunctionalized tetrapyridyl manganese(III) porphyrin was synthesized according to a modified procedure.42 Briefly, 1 g of meso-tetra(4-pyridyl)porphine (1) was refluxed with 4.78 g of ethyl bromopentanoate (∼14 equiv) in 300 mL of 25% (v/v) ethanol/ CHCl3 for 7 days. The solvent was evaporated, and the resulting 5-(1(4-(ethoxycarbonyl)butyl)pyridinium-4-yl)-10,15,20-tripyridylporphyrin bromide (2) was dried under vacuum at 50 °C for 24 h. Then, 1.06 g of unpurified 2 was added to 50 mL of DMSO and brought to 42 °C with 1.25 mL of methyl iodide (CH3I). After 2 h, another 1.25 mL of CH3I was added. After another 3 h, the solvent was evaporated under vacuum, and the alkylated product, tetracationic porphyrin (3), was dried in a vacuum oven at 50 °C for 24 h. To hydrolyze the ester function, 750 mg of 3 was added to 70 mL of 1 M HCl and refluxed for 3 h. The mixture was cooled to room temperature and filtered 345


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Chemistry of Materials through a sintered glass funnel. The filtrate was evaporated and washed with deionized (DI) water three times. For metalation of the obtained meso-tetrakis(1-methylpyridinium-4-yl)porphyrin monopentaacetic acid (4) with manganese(II) chloride, a 54 mg portion of 4 was added to 5 mL of deionized water along with 30 mg of manganese(II) chloride tetrahydrate and refluxed for 80 min. The metalation to the corresponding manganese(III) meso-tetrakis(1methylpyridinium-4-yl)porphyrin pentaacetic acid (5) was confirmed by UV−vis spectroscopy (Varian Cary Bio50). The mixture was cooled to room temperature, precipitated in 50% (v/v) ethyl ether/ isopropanol, and centrifuged to collect the precipitated carboxyl monofunctionalized tetrapyridyl manganese(III) porphyrin (MnP). Synthesis of MnP-Functionalized Poly(N-vinylpyrrolidone) (MnP-PVPON). To obtain MnP-PVPON, the PVPON-NH2-20% copolymer (20% represents the molar percentage of amino-groupcontaining polymer units) was synthesized using gradual feeding copolymerization of VPON and tBOC as we reported previously.32 Briefly, 6.3 g of freshly distilled VPON in dioxane (25 mL) was degassed with three freeze−pump−thaw cycles. N-(t-BOCaminopropyl)methacrylamide (tBOC, 2.404 g) was dissolved in dioxane (12 mL), filtered through a 0.2 μm syringe filter, and diluted to 25 mL. AIBN (30 mg) in 1 mL of dioxane was added to the VPON solution, and the solution was heated to 70 °C under nitrogen and constant stirring for 3.5 h during which the tBOC solution was gradually added using a syringe pump. After that, the mixture was cooled to room temperature, and the PVPON-tBOC copolymer was precipitated in petroleum ether, collected, and purified by dissolving and precipitating in dichloromethane and petroleum ether, respectively, two times. The tBOC groups were hydrolyzed from PVPON-tBOCc (210 mg) in methanol (17 mL) containing hydrochloric acid (3 mL) for 3 days, followed by neutralizing the acid with 1 M NaOH. The PVPON-NH2 copolymer was dialyzed against deionized water using a Spectra/Por Float-A-Lyzer with a MWCO of 50 000 Da and freeze-dried (Labconco). The composition of the PVPON-NH2 random copolymer after the hydrolysis was determined with 1H NMR in D2O. The molecular weight of the copolymer (Mw = 198 000 g mol−1, Đ = 1.2) was determined with GPC (Waters) in dimethylformamide using linear polystyrene standards. The PVPON-NH2-20 copolymer (135 mg) dissolved in 10 mL of 0.01 M phosphate buffer (pH = 6) was added to a MnP solution which was prepared by dissolving 123 mg of MnP and 41 mg of EDC in 6 mL of 0.01 M phosphate buffer (pH = 6) and stirring for 30 min. The polymer solution was stirred overnight in the dark, followed by exhaustive dialysis against 0.01 M phosphate buffer (pH = 8, 0.1 M NaCl) and DI water (MWCO 20 000 Da) to remove free porphyrin until no trace of porphyrin color was observed in the external solution. The MnP-polymer solution was freeze-dried and stored in the dark. Synthesis of (TA/PVPON) and (TA/PVPON/TA/MnP-PVPON) and (PVPON)6 Multilayer Capsules. Hollow hydrogen-bonded multilayer spherical capsules were prepared by coating 4 μm solid silica particles with (PVPON/TA)n multilayers via alternating exposure of the particles to 0.5 mg mL−1 PVPON, TA, and MnPPVPON polymer solutions (0.01 M phosphate buffer, pH = 5) followed by particle dissolution, where “n” denotes the number of (PVPON/TA) bilayers. Briefly, 40 mg of silica cores suspended in 1 mL of phosphate buffer (0.01 M, pH = 5) was pelleted in a 1.5 mL Eppendorf centrifuge tube and resuspended in PVPON solution. The polymer was allowed to deposit on the surface for 5 min followed by rinsing with the phosphate buffer (0.01 M, pH = 5) followed by adsorption of TA for 5 min. Three rinses were applied after each layer’s deposition. Alternating multilayer deposition was performed until the desired number of polymer layers was achieved. The following multilayer architectures were created: (PVPON/TA)5; (PVPON/TA) 5.5 ; and (MnP-PVPON/TA) 2 (PVPON/TA) 3.5 , (PVPON/TA) 2 (MnP-PVPON/TA) 2 (PVPON/TA) 1 . 5 , and (PVPON/TA)4(MnP-PVPON/TA)1.5. The porphyrin-containing capsules, referred to as S1, S2, and S3, respectively, with the number denoting the location of the MnP-containing bilayer in the inner, middle, or outer part of the (PVPON/TA) multilayer shell, were

covered or kept in the dark. Silica cores were dissolved in hydrofluoric acid (8 wt %) to yield hollow polymeric capsules. The capsules were dialyzed against 0.01 M phosphate buffer (0.1 M NaCl, pH = 8) for 2 days, followed by DI water dialysis for 2 days using 1 mL Float-ALyzers (MWCO 20 000 Da, Spectrum Laboratories). The TA-free (PVPON)6 capsules were prepared as described in our previous work.41 Hydrogen-bonded multilayers of poly(methacrylic acid) (PMAA) with amino-containing poly(N-vinylpyrrolidone) (PVPON-NH2-7, Mw = 143 884 g mol−1, Đ = 1.55) copolymer with a 7% molar percentage of amino-group-containing polymer units were deposited on the silica particles. Assembly of the hydrogenbonded layers was performed at pH = 3, starting from PVPON-NH2 followed by PMAA. Each deposition cycle was followed by rinsing three times with a buffer solution at pH = 3 to remove excess polymer, followed by centrifugation of suspensions at 2000 rpm for 2 min to remove supernatant. After six bilayers of (PVPON-NH2/ PMAA) were deposited, chemical cross-linking of PVPON-NH2 was performed. For that, the core−shell particles were exposed to glutaric aldehyde solution (25 wt %) at pH = 5 for 12 h. After that, core dissolution was performed as described above; resultant (PVPON)6 capsules were purified by dialysis in deionized water for 3 days. For accurate capsule concentrations in all experiments, capsule suspensions were counted (capsules mL−1) using a hemocytometer and an optical microscope with a 40× objective. Nuclear Magnetic Resonance (NMR). 1H NMR spectra were recorded on a Bruker 300 MHz NMR spectrometer. The porphyrin solutions (1 mg mL−1) in (CD3)2SO (Sigma-Aldrich) and PVPONNH2 copolymer in D2O were measured at 25 °C. Scanning Electron Microscopy (SEM). SEM was performed using an FEI Quanta FEG microscope at 10 keV. Samples were prepared by depositing a drop of a capsule suspension on a silicon wafer and allowing it to dry at room temperature. Before imaging, dried specimens were sputter-coated with 5 nm silver film using a Denton sputter-coater. Wettability Measurements. Thin film wettability was assessed by contact angle measurements with a Theta Lite 101 tensiometer (Biolin Scientific) using a sessile drop experiment. A 5 mL drop of deionized water was placed on the film surface at room temperature, and the contact angle was acquired with a CCD camera and analyzed using Attension software. The measurements were done in five different spots, and the average contact angle value and its standard deviation were calculated. Ellipsometry. Film thickness was measured using a M2000U spectroscopic ellipsometer (Woollam). Single-side polished silicon wafers 500-μm-thick (University Wafer) of 20 mm × 50 mm were cleaned in a mixture of concentrated sulfuric acid and Nocromix (Godax Laboratories) for 24 h, washed with DI water, dried with a stream of nitrogen (Airgas), and used immediately thereafter. First, a precursor layer of poly(ethylenimine) (PEI, Sigma-Aldrich) was adsorbed onto the wafer surface from 1 mg mL−1 PEI aqueous solution for 10 min. After that, the deposition of TA, PVPON, and MnP-PVPON on Si wafers coated with a PEI layer was performed using a 5 mL liquid flow-through cell (Woollam) at pH = 5 and pH = 7.2 (0.01 M phosphate buffer). The pH values of 0.01 M phosphate buffers were adjusted using 0.1 M HCl and NaOH aqueous solutions. Dry thickness measurements were performed between 400 and 1000 nm at 65°, 70°, and 75° angles of incidence, while wet thickness studies were monitored at 75° incidence angle. For data interpretation, the ellipsometric angles Ψ and Δ were fitted using a multilayer model composed of silicon, silicon oxide, and the multilayer film to obtain the thickness of films. The thickness of silicon oxide was determined using known optical constants. The thickness of the dry multilayer film was obtained by fitting data with the Cauchy approximation with the refractive index as B C n(λ) = A n + 2n + 4n , with An = 1.5, Bn = 0.01, and Cn = 1.3 × λ λ 10−5. The thickness of the in situ grown film was obtained by fitting data with the Cauchy approximation with permitted fitting of An, Bn, and Cn. The mean squared error for data fitting was less than 30. 346


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Chemistry of Materials Free-Radical-Scavenging Activity of (TA/PVPON) and (TA/ MnP-PVPON) Capsules Using 2,2-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) Radical Cation (ABTS•+) Assay. ABTS•+ was prepared according to a previously published procedure.43 ABTS was dissolved in DI water at a concentration of 0.14 M and treated with 100 μL of a 0.12 M solution of potassium persulfate in DI water. The mixture was left in the dark at room temperature overnight before being stored at 3 °C. Before use, the solution was diluted to reach an absorbance of 1.2 at 734 nm. A 100 μL portion of a 1 × 108 capsule mL−1 suspension (0.01 M phosphate buffer, pH = 7.4) was added to a UV−vis quartz cuvette containing the dye, and mixed while recording the absorbance at 734 nm every 30 s for 20 min. Superoxide Dismutation by Cytochrome C Assay. A steady stream of superoxide radicals was produced in situ over 15 min through a xanthine/xanthine oxidase reaction and monitored via the reduction of cytochrome C (detected at 550 nm).20 The rate of superoxide dismutation was followed in 0.05 M phosphate buffer with 0.1 mM EDTA at pH = 7.8 in a 1 cm UV−vis quartz cuvette in the presence of (PVPON/TA)5.5 or (PVPON/TA)4(MnP-PVPON)1.5 capsules and in the presence and absence of 10 μg/mL catalase. Absorbance at 550 nm from 10 μM cytochrome C was used as the radical indicator in the presence or absence of 10 μg mL−1 catalase. The xanthine stock was oxygenated before each use, and the experiments were carried out at 23 °C. A 100 μL portion of a 1 × 108 capsule mL−1 suspension (0.01 M phosphate buffer, pH = 7.4) was added to a UV−vis quartz cuvette containing the radical, and mixed while recording the absorbance at 550 nm every 15 s for 15 min. Small Angle Neutron Scattering (SANS). SANS measurements were carried out at the CG3 Bio-SANS beamline at the High Flux Isotope Reactor (HFIR) at Oak Ridge National Laboratory. A constant neutron wavelength of 6 Å with a relative wavelength spread of 15% was used in all measurements. One single instrument configuration was able to cover the q range of 0.003 to 0.7 Å−1 by utilizing a combination of a main and a wing detector. The scattering intensity profiles I(q) versus q were obtained by azimuthally averaging the processed 2D images, which were normalized using water as a secondary standard, and corrected for detector dark current, pixel sensitivity, and scattering from backgrounds (D2O and quartz cell). The capsules were suspended in D2O before being placed in titanium tumbler cells with quartz windows. The tumbler cells were rotated constantly during the scans to prevent the capsules from settling. ABTS was activated with potassium persulfate in D2O overnight in accordance with the procedure used in the UV−vis experiments. The (PVPON/TA)5.5 and (PVPON/TA)4(MnP-PVPON-TA)1.5 capsules were scanned in the Bio-SANS instrument before being added to the ABTS•+ solution and again after the reaction had gone to completion to measure shell thickness change. For data analysis, scattering curves were fitted using the LamellarFF model,44 where the scattering intensity is returned; scale*I(q) + incoherent bkg and I(q) is given by I(q) =

standard optical microscopy. The starting values of the shell thicknesses were taken from ellipsometry measurements and adjusted to fit the scattering curves. Fit quality was determined by minimizing goodness-of-fit parameter (χ2), and parameters were constrained to physically reasonable values of thickness and consistency with other measurements, such as ellipsometry and atomic force microscopy. Mice. NOD/ShiLtJ mice were bred and housed under pathogenfree conditions at the Research Support Building animal facility at the University of Alabama at Birmingham. Female mice between 8 and 16 weeks of age were used. Mice received standard chow, and all animal studies were performed in accordance with the UAB Institutional Animal Use and Care Committee in compliance with the laws of the United States of America. Cell Viability. An MTT cell viability assay was performed according to the manufacturer’s protocol (Sigma-Aldrich). Splenocytes from NOD mice were purified and cultured in 96-well plates at 5.0 × 106 cells μL−1. Each plate contained quadruplicates of control and capsule-treated (2.5 × 106 or 5.0 × 106 counts μL−1) splenocytes incubated for 48 h. Absorbance readings were analyzed on a Synergy 2 microplate reader (BioTek) using Gen5 software. Percentage cell viability was determined by subtracting the absorbance reading from the blank with all samples and then dividing the test sample with the control and multiplying by 100. Statistical Analysis. Data were analyzed using GraphPad Prism Version 5.0 statistical software. Determination of the difference between mean values for each experimental group was assessed using the 2-tailed Student’s t test, with p < 0.05 considered significant. All experiments were performed at least three separate times with data obtained in quadruplicate wells for each experiment.

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RESULTS AND DISCUSSION Synthesis of the Mn(III) Porphyrin-PVPON Copolymer. The carboxyl monofunctionalized tetrapyridyl Mnporphyrin (MnP) was synthesized via monoquaternization of meso-tetra(4-pyridyl)porphine with bromopentanoate, followed by methylation of the remaining pyridyl groups with methyl iodide and hydrolysis of the ester function to obtain meso-tetrakis(1-methylpyridinium-4-yl)porphyrin monopentaacetic acid (Figure 1; compounds 1−4). The 1H NMR analysis of 2−4 confirmed the presence of the oxoethyl proton signals at 4.27 ppm (2H, OCH2CH3) and 0.6−1 ppm (3H, OCH2CH3) before and after methylation and the appearance of the proton signals at 4.8 ppm [from methyl groups after

2 2 4π Δρ2 [1 − cos(qδ)e−q σ /2] 4 δq

where q = (4π/λ)sin(θ/2), λ is the neutron wavelength, θ is the scattering angle, δ is the shell thickness, σ is the variation in shell thickness, and Δρ2 is the contrast factor (the difference between the scattering length densities (SLD) of the solvent and shell material). The data were analyzed with Igor Pro using a macro provided by National Institute of Standards and Technology.45,46 The fitting parameters are presented in Tables S1−S4 of the Supporting Information. The Lamellar model (instead of the core−shell model) was applied due to the following considerations: using the equation for Rg of a solid sphere Rg2 = 3R2/5, the minimum q needed to resolve size information on 4 μm spheres would be q ∼ 0.0001 (Guinier scattering at qRg < 1.3), which is beyond the lower limit of the instrument. However, at higher q, Porod surface scattering of a locally flat sheet can be modeled with a lamellar bilayer. Since the capsule wall effectively scatters as a flat sheet at the q range covered in our experiment, the lamellar model can be used to extract the shell thickness and ignore the size parameter which is easily resolved by

Figure 1. Synthesis of carboxyl monofunctionalized tetrapyridyl Mnporphyrin (MnP). 347


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Chemistry of Materials methyl iodide reaction (Figures S1 and S2)].47 The disappearance of the oxoethyl proton signals also was confirmed during the complete hydrolysis of the ester group (Figure S3). The metalation of manganese(III) meso-tetrakis(1-methylpyridinium-4-yl)porphyrin pentaacetic acid (Figure 1; 5) was performed by reflux with manganese(II) chloride in deionized water for 80 min. The UV−vis spectroscopy analysis in Figure 2 shows that the Soret band of the porphyrin shifted from 420

MnP by extensive dialysis for 96 h. FTIR analysis revealed the disappearance of the carboxylic acid of the porphyrin observed at 1715 cm−1 due to the amide bond formation (Figure 3b). The amide band is combined with the PVPON amide at 1640 cm−1 in the obtained MnP-PVPON copolymer. The new peaks in the MnP-PVPON copolymer around 1060 and 978 cm−1 are due to the characteristic porphyrin peaks48 that shifted from 1174 and 1004 cm−1 to lower frequencies due to steric quenching49 from the surrounding polymer chain (Figure 3b). To additionally confirm the covalent modification of PVPON-NH2 with MnP versus noncovalent hydrophobic or polar interactions between the copolymer and MnP molecules, UV−vis spectrophotometric analysis of a solution of PVPONNH2 mixed MnP in the absence of EDC followed by 96 h dialysis in water was conducted. Figure 4a shows that, in

Figure 2. UV−vis spectra of carboxyl monofunctionalized tetrapyridyl porphyrin before (dotted) and after (solid) metalation with Mn(II). The spectra were taken from diluted aliquots of the aqueous metalation reaction (∼0.2 mg mL−1).

to 462 nm after introduction of manganese ions into the porphyrin core.47 To obtain the SOD mimicking PVPON, the Mn-porphyrin was covalently conjugated to the PVPON-NH2 copolymer via carbodiimide-assisted reaction of the free amine groups on the methyl aminopropylacrylamide units of the copolymer and the pentanoic acid function of the porphyrin (Figure 3a). The 20% MnP-PVPON was selected as it has the highest NH2-containing molar percentage we have synthesized that still results in stable (TA/MnP-PVPON) multilayer growth. The MnP-polymer was purified from the unreacted Figure 4. (a) UV−vis spectra of the PVPON-NH2 copolymer (blue ◇), MnP-PVPON (green ○), and a mixture of free MnP and PVPON-NH2 after 96 h dialysis (red □) in 0.01 M phosphate buffer at pH = 7.4. (b) UV−vis spectrum of the MnP-PVPON solution at pH = 7.4 (0.01 M) after being dialyzed for 1, 2, 5, 24, 48, and 120 h.

contrast to the characteristic Soret band of the Mn(III) porphyrin in the spectrum of MnP-PVPON at 464 nm, the spectra of PVPON-NH2 and extensively dialyzed PVPONNH2 mixture with MnP (Figure 4a, MnP-PVPON (mixed)) presented no major absorbance bands in the visible spectrum. We further explored the stability of the porphyrin attachment to the PVPON copolymer and the manganese entrapment in the porphyrin core using UV−vis spectroscopy of aqueous solutions of (1 mg mL−1) MnP-PVPON at pH = 5 and pH = 7.4. The overlaid UV−vis spectra in Figure 4b demonstrate that no change in the Soret band absorbance occurs after dialysis of the MnP-PVPON solution at pH = 7.4 for 120 h. Since the Soret band significantly shifts toward lower energies during metalation of the porphyrin (Figure 2), any loss of manganese ions would result in the appearance of a Soret band at the original wavelength of 420 nm which is not observed during the dialysis (Figure 4b). Similarly, no decrease of the band absorbance after MnP-PVPON dialysis confirms retention of the porphyrin pendant functionalities during extended time in

Figure 3. (a) Synthesis of tetrapyridyl Mn-porphyrin functionalized copolymer of poly(N-vinylpyrrolidone) (MnP-PVPON). (b) FTIR spectra of the PVPON copolymer before (PVPON-NH2, blue) and after (MnP-PVPON, green) coupling to porphyrin (MnP, red). 348


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Chemistry of Materials

Figure 5. (a) Schematics for multilayer assembly of tannic acid (TA), poly(N-vinylpyrrolidone (PVPON), and Mn-porphyrin functionalized PVPON (MnP-PVPON) on spherical sacrificial silica particles to obtain antioxidant porphyrin-polyphenolic hollow capsules. (b) The multilayer architecture of the PVPON/TA hollow capsules with the porphyrin copolymer bilayer of MnP-PVPON/TA localized in the inner (S1, (MnPPVPON/TA)2(PVPON/TA)3.5), middle (S2, (PVPON/TA)2(MnP-PVPON/TA)2(PVPON/TA)1.5), or outer (S3, (PVPON/TA)4(MnPPVPON/TA)1.5) part of the multilayer shell.

total bilayers of (PVPON/TA) after core dissolution and dialysis revealed that the MnP-PVPON-containing capsules are much thinner than their (PVPON/TA) counterparts, which is in agreement with the ellipsometry results at the same deposition pH = 5 (Figure 6e). As seen, in contrast to (PVPON/TA)5 capsules which collapse upon drying leaving protruding thick folds and rigid creases, the porphyrinpolyphenolic capsules are fully collapsed demonstrating minimal wrinkling, a typical feature of soft multilayer capsules (Figure 6a−d).52 Similarly, the increased shell thickness of (PVPON/TA) capsules was demonstrated to result in increased rigidity of the capsule shell which could support their three-dimensional spherical structure upon drying.33,53 Since SEM morphology analysis of dry polymer multilayers can be ambiguous due to possible artifacts, e.g., increased roughness, loss of material, or drying, the SEM results were rationalized by studying the TA/MnP-PVPON multilayer growth at slightly acidic and neutral pH values using in situ ellipsometry. Figure 6e shows in situ thicknesses of the polymer layers adsorbed from polymer solution at pH = 5 followed by that at pH = 7.2 (0.01 M phosphate buffer) on Si wafer surfaces as measured inside a 5 mL liquid cell without intermediate drying, i.e., wet thickness. We found that while MnP-PVPON can be successfully adsorbed on top of the TA layer in both cases, the bilayer thickness of (TA/MnPPVPON) assembled at pH = 5 is smaller than that at pH = 7.4. The (TA/MnP-PVPON) bilayer at pH = 7.4 resulted in 1.8 ± 0.1 nm, a 2-fold larger growth than that at pH = 5, which gave

aqueous solution. These results clearly suggest that the PVPON-NH2 copolymer does not produce noncovalent assemblies with the porphyrin in aqueous solutions and all free porphyrin molecules can be removed via water dialysis. Multilayer Assemblies of the MnP-PVPON with TA and their Cytocompatibility. We studied the capability of MnP-PVPON to assemble with TA at pH = 5 and pH = 7.4 into multilayer coatings on silica surfaces. Figure 5a schematically shows that multilayer assembly between TA phenolic groups and MnP-PVPON carbonyls can occur through hydrogen bonding.50 However, there is a possibility that cationic MnP moieties on PVPON chains can inhibit multilayer assembly of TA and MnP-PVPON due to an increase of the ratio of positive to negative charges within the multilayer and the resulting mutual repulsions between pyridinium positive charges on MnP.51 To test the capability of MnP-PVPON to assemble with TA, we synthesized three types of hollow multilayer capsules 4 μm in diameter with the shell architecture denoted as S1, S2, and S3, where the porphyrin copolymer bilayer of MnP-PVPON/ TA was localized in the inner (MnP-PVPON/TA)2(PVPON/ TA) 3 . 5 , middle (PVPON/TA) 2 (MnP-PVPON/TA) 2 (PVPON/TA)1.5, or outer (PVPON/TA)4(MnP-PVPON/ TA)1.5 part of the multilayer shell, respectively (Figure 5b), using multilayer deposition of the polymers from 0.01 M phosphate buffer solutions at pH = 5. The SEM analysis of the (PVPON/TA)5 (Figure 6a,b) and (PVPON/TA)2(MnPPVPON/TA)2(PVPON/TA) capsules (Figure 6c,d) with five 349


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only 0.9 ± 0.1 nm (TA/MnP-PVPON) bilayer thickness. In contrast, the opposite thickness trend was observed for porphyrin-free TA/PVPON growth, where increasing the deposition pH value from pH = 5 to pH = 7.4 led to a 2fold decrease in capsule thickness with 4.2 and 1.3 nm per a bilayer for pH = 5 (0.01 M) and pH = 7.4 (0.01 M), respectively.50 The pH-dependent difference in the (TA/ PVPON) bilayer thickness was explained by unshielded ionization of TA at high deposition pH which resulted in mutual repulsion of the neighboring layers of TA and slightly thinner coatings at pH = 7.4.50 This effect was also reported for other multilayer systems: for instance, glutaraldehyde-assisted growth of cationic PAH or poly(L-lysine) occurred only at high salt conditions (0.1 M) which allowed the screening of similar positive charges on adjacent polyelectrolyte chains.54,55 Our findings suggest that, at low 0.01 M salt conditions, the MnPPVPON interactions with TA at pH > 5 can involve both hydrogen bonding and ionic pairing due to increased TA ionization which should also allow 38% thicker PVPON/TA coatings when MnP-PVPON is used compared to that of (PVPON/TA) when MnP-free PVPON is employed during the multilayer deposition. We additionally characterized the stability of the MnP-PVPON/TA multilayers dried from pH = 2.5, since ionization of TA is minimized at this pH (below the pKa of TA) which could result in destabilization of the film. We found that the MnP-PVPON/TA bilayer did not show any significant thickness changes after exposure to pH = 2.5 as compared to the film stability at pH = 5 (Table S5). Moreover, the presence of cationic pendant MnP moieties on the PVPON backbone resulted in a decreased hydrophobicity of the coating as observed from sessile drop contact angle measurements using water as a contact liquid. The contact angle of a water drop decreased from 61 ± 0.1° to 49 ± 0.2° when the last layers in (PVPON/TA)5.5 were changed to (MnP-PVPON/TA) 1.5 (Figure 6f,g). The observed increased wettability of the (MnP-PVPON) 1.5 surface compared to (PVPON/TA)1.5 as a top stack is due to the

Figure 6. SEM images of (a, b) (PVPON/TA)5 and (c, d) (PVPON/ TA)2(MnP-PVPON/TA)2(PVPON/TA) multilayer capsules assembled at pH = 5 (0.01 M phosphate buffer) and dried on Si wafers from DI water solutions. (e) Growth of TA/PVPON/MnPPVPON multilayers at pH = 5 and at pH = 7.2 (0.01 M phosphate buffer) on Si wafer surfaces as measured by in situ ellipsometry inside a 5 mL liquid cell. (f, g) Optical images of a water drop placed on (f) a (PVPON/TA)5.5 and (g) a (PVPON/TA)4(MnP-PVPON/TA)1.5 multilayer.

Figure 7. Viability (%) of splenocytes from NOD mice after 48 h incubation with (a) 2.5 × 106 and (b) 5 × 106 capsules of (PVPON/TA)5, (PVPON/TA)5.5, (PVPON/TA)2(MnP-PVPON/TA)2(PVPON/TA), and (PVPON/TA)2(MnP-PVPON/TA)2(PVPON/TA)1.5 (labeled as (MnP-PVPON)* and (MnP-PVPON)**, respectively), as obtained from the MTT assay (absorbance was measured at 540 nm). 350


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Figure 8. (a) Schematics for ABTS oxidation to produce ABTS•+ radical cation (left) and a catalytic cycle of metalloporphyrin reduction/oxidation. (b) UV−vis spectra of ABTS•+ decolorization by (PVPON/TA)5.5 capsules. (c) ABTS•+ discoloration by MnP-PVPON copolymer, (PVPON)6 hydrogels, (PVPON/TA)5.5, (MnP-PVPON/TA)2(PVPON/TA)3.5 (S1), (PVPON/TA)2(MnP-PVPON/TA)2(PVPON/TA)1.5 (S2), and (PVPON/TA)4(MnP-PVPON/TA)1.5 (S3) capsules. (d) Images of ABTS•+ solutions before (left) and after 20 min treatment with (PVPON/ TA)5.5 and S3 capsules.

nontreated control and both negative cytotoxicity controls. The 2-fold increased capsule concentration of 5 × 106 μL−1 did not induce cell cytotoxicity from MnP-PVPON-containing capsules (Figure 7b) further demonstrating the stability of the porphyrin-polyphenolic system in vitro. Antioxidant and Free-Radical-Scavenging Properties of Metalloporphyrin-Polyphenolic Multilayers. We studied how the presence of MnP functionalities within (PVPON/ TA) capsules can change their radical-scavenging ability by measuring the kinetics of their interaction with 2,2′-azinobis(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS•+) radical cation. When ABTS is oxidized to the stable radical (Figure 8a), an absorbance at 734 nm from a brilliant blue-green solution of the ABTS•+ can be used to monitor its decolorization upon reduction by an electron donor (Figure 8b). We found that inclusion of the MnP-PVPON copolymer dramatically enhanced the antioxidant ability of the materials. Figure 8c shows that, in the first 1−2 min, quick ABTS•+ decolorization occurred in the presence of (PVPON/TA)5.5 capsules followed by a slower decomposition of ABTS•+ for the remaining 18 min, which is in agreement with our previous findings on free-radical-scavenging activity of the (PVPON/ TA) capsules as measured by luminol oxidation assay.41 The ability of TA to preserve its antioxidant properties being ionically paired into a multilayer with various polycations has also been demonstrated previously.43

high hydrophilicty of the porphyrin moiety which implies positive implications for use of the system in a biomedically relevant environment, as hydrophilic polymers are reported to prevent negative immune response and quick body clearance.56−59 Despite the hydrophilic nature of TA/MnP-PVPON, there is a reasonable concern about its cytotoxicity due to cationic pyridinium groups60 and manganese cations61,62 of the MnP pendant groups which can be cytotoxic if released from PVPON. To examine the potential cytotoxic effects of TA/ MnP-PVPON, we performed an MTT assay to assess the viability of splenocytes from NOD mice, a spontaneous mouse model of Type 1 diabetes,63 following a 48 h incubation with (PVPON/TA) 2 (MnP-PVPON/TA) 2 (PVPON/TA), and (PVPON/TA)2(MnP-PVPON/TA)2(PVPON/TA)1.5 capsules, with (PVPON/TA)5 and (PVPON/TA)5.5 as negative cytotoxicity controls.40 Future studies will utilize TA/MnPPVPON encapsulation of pancreatic islets for transplantation studies into NOD mice to reverse diabetes and delay immunemediated rejection. Therefore, examining the potential cytotoxic effects of TA/MnP-PVPON on immune cells is essential for defining the feasibility of this material for future in vivo studies. Figure 7a displays noncytotoxic MnP-PVPONcontaining capsules at the concentration of 2.5 × 106 μL−1 and percent viability from formazan, a product of intracellular enzyme cleavage of the initial MTT, that are similar to a 351


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Chemistry of Materials Remarkably, a drastically enhanced response to the ABTS•+ radical cation challenge was observed when two PVPON layers were exchanged for MnP-PVPON in the outer (PVPON/ TA)1.5 stack. The optical density of the ABTS•+ solution decreased in the presence of (PVPON/TA)4(MnP-PVPON/ TA)1.5 capsules by 4-fold more than that by (PVPON/TA)5.5 capsules in 20 min indicating a reduction of 4-fold more radical cation species during the same time. The images of ABTS•+ solutions before and after 20 min treatment with (PVPON/ TA)5.5 and (PVPON/TA)4(MnP-PVPON/TA)1.5 (labeled as S3) capsules demonstrate that a complete loss of ABTS•+ color is observed in the presence of S3 capsules while a significant but incomplete decolorization of ABTS•+ solution occurred in the case of (PVPON/TA)5.5 capsules (Figure 8d). We also changed the localization of the MnP-PVPON layers within the 5.5-bilayer capsule shell and found that positioning the porphyrin stack away from the outer surface slows down the ABTS•+ decolorization and decreases the radicalscavenging ability of the system (the number of the radical cation species being reduced per unit of time) (Figure 8c). Thus, for instance, the initial ABTS•+ optical density decreased 8.5-fold by S3 (outer) capsules, and 6- and 3.2-fold by S2 (middle) and S1 (inner) capsules, respectively, while it was reduced only by 2.1-fold when MnP-PVPON-free capsules of (PVPON/TA)5.5 were used (Figure 8c). These results can be explained by the fact that ABTS•+ radical cations diffuse through a different environment and are being reduced by different species in the case of (PVPON/TA)5.5 and S1, S2, and S3 capsules. In the case of (PVPON/TA)5.5 and S1 and S2, the outer PVPON partially shields TA from the radical cations due to hydrogen bonds between the phenolic groups in TA and the carbonyls in PVPON. These bonds need to be broken to reduce the radical which slows the rate of the radical cation reduction.43 In contrast, in S3 capsules the outer stack contains active MnP groups and is able to immediately reduce the incoming ABTS•+ radical cations. Importantly, the control (PVPON)6 hydrogel capsules and MnP-PVPON polymer did not demonstrate any significant change to the ABTS•+ concentration during the test time of 20 min (Figure 8c). This result additionally confirms the absence of PVPON antioxidant activity as we demonstrated previously using the luminol oxidation assay41 and provides some insight into the synergistic relationship between TA and the MnPPVPON for the radical-scavenging activity of (TA/MnPPVPON) capsules. As reported, manganese porphyrins have manganese ions in the 3+ oxidation state.64 A single oneelectron MnP catalytic loop reduces the manganese to the 2+ oxidation state by accepting an electron followed by an oxidation to the 3+ oxidation state again (Figure 8a).64 However, decolorization of the ABTS•+ radical occurs through the reduction of the radical to ABTS in the ground state (Figure 8a).65 The half-cell reduction potential of the opposite step, i.e., further oxidation of ABTS•+ to ABTS2+, is 1.1 V, which is higher than the oxidation-limited catalysis of Mn(II)Cl265,66 and did not appear to occur during our assay (characterized by an increase in absorbance near 565 nm). Since we did not observe any absorbance shift over time in the presence of the free MnP-PVPON polymer, we suggest that MnP-PVPON on its own does not assist redox reactions where the first step is oxidation of the material, and therefore, TA is required to activate the catalytic MnP loop. In this case the close proximity of TA phenoxy radicals formed through initial oxidation by ABTS•+ may allow the porphyrin to participate in

catalytic cycles where ABTS•+ is reduced alongside formation and disproportionation of peroxides as manganoporphyrins have been shown to exhibit some catalase-like activity.67,68 It is clear from the shape of the curves in the ABTS reaction that the reduction mechanism is complex, with the initial sharp decrease followed by a slow, steady decline indicative of multiple steps. While characterizing the instantaneous rate of the reaction was beyond the scope of this study the overall reduction in ABTS•+ can clearly be seen. Importantly, since the thickness of the multilayer was shown to be decreased by inclusion of the MnP-PVPON at the assembly pH = 5, the increased antioxidant activity per capsule can be even more heavily attributed to inclusion of the manganoporphyrin within the capsule shell. We also found that increasing the number of MnP-PVPON bilayers instead of PVPON layers was not necessary to further increase the rate of ABTS•+ reduction and that incorporation of the MnP-PVPON polymer as the outer two layers of the capsule shell was sufficient to greatly enhance the radicalscavenging ability of the (PVPON/TA) capsules. Thus, for instance, substitution of two additional MnP-PVPON layers into (PVPON/TA)6(MnP-PVPON/TA)1.5 capsules to obtain (PVPON/TA)4(MnP-PVPON/TA)3.5 resulted in similar radical-scavenging abilities of the MnP-containing systems. In the higher layer capsules, the discoloration occurred 2 times more quickly with (PVPON/TA)4(MnP-PVPON/TA)3.5 capsules than when (PVPON/TA)7.5 was used with both showing a plateau of effectiveness after 10 min in the ABTS•+ assay (Figure S4). Superoxide Dismutation by Marganoporphyrin-Polyphenolic Capsules. Since superoxide is among the main radical species that trigger many inflammatory agonists and can result in cascades of inflammatory responses and related autoimmunity, we studied the ability of the MnP-PVPON/TA system to dismute superoxide radicals by using a cytochrome C competition assay. The xanthine/xanthine oxidase reaction was used as a source of superoxide radicals which, upon reduction of cytochrome C in solution, can be traced by a steadily increasing absorbance at 550 nm (Figure 9a). The rate of superoxide dismutation by MnP-PVPON/TA capsules was evaluated in comparison with (PVPON)6 hydrogel capsules and MnP-free (PVPON/TA)5.5 capsules in the presence of catalase to prevent reoxidation of cytochrome C by hydrogen peroxide, which is a major product of O2•− dismutation.69 Figure 9b shows that no reduction in the observed rate of superoxide radical production from the hydrogel capsules was observed, which agrees with our results on the absence of activity from (PVPON)6 capsules in scavenging ABTS•+ radical cations (Figure 8c). In contrast, a significant lowering of the rate of cytochrome C reduction is observed in the presence of (PVPON/TA)5.5 capsules (Figure 9b) due to the antioxidant properties of TA. Using the average literature value of Δε (reduced−oxidized) of cytochrome C at 550 nm70,71 = 20.3 mM−1 cm−1, and the slope of the linear fit to absorbance change in Figure 9c, the rate of superoxide production in the control experiment (no capsules were added) was found to be 189 nM min−1. The MnP-free capsules were found to scavenge the superoxide radical at a rate of 47 nM min−1 (189 nM min−1 minus the observed rate of 142 nM min−1 in the presence of 1 × 107 (PVPON/TA)5.5 capsules). Remarkably, the same concentration of porphyrin-containing (PVPON/TA)4(MnPPVPON/TA)1.5 capsules increased the superoxide-scavenging rate to 77 nM min−1 which represents a 64% increase in the 352


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shell in solution before and after ABTS•+ radical cation treatment. The SANS data was fitted to a lamellar model (see Experimental Section) (Figure 10), and the shell thickness was extracted from model parameters as has been studied previously in SANS studies of polyelectrolyte capsules in solution using the core−shell model.73,74 We found that the 5.5-bilayer (PVPON/TA)4(MnP-PVPON/TA)1.5 capsules were thinner than their MnP-free (PVPON/TA)5.5 counterparts with the capsule shell thicknesses of 15.3 and 18 nm, respectively which is in agreement with our SEM and in situ ellipsometry results (Figure 6a−d and Figure 6e, respectively) and with the previously reported thickness of (PVPON/TA) capsules assembled at pH = 5.28,75 These (PVPON/TA) thickness results suggest that the MnP-PVPON/TA bilayer thickness fits to 2.8 nm (in contrast to 3.3 nm per PVPON/TA bilayer) which is in agreement with the thinner MnP-PVPON/ TA bilayers as measured by in situ ellipsometry (Figure 8e). The moderate expansion of thickness may be attributed to the reduced ability of D2O to screen charges and the reduced hydrogen-bond-driven solvation in D2O due to the higher energy required to disrupt the O−D bond network.76 The importance of the thinner MnP-PVPON/TA shell is the implication that the antioxidant activity of the MnP-PVPON capsules can be attributed more to the porphyrin moiety, since TA represents a reduced compositional percentage in (PVPON/TA)4(MnP-PVPON/TA)1.5 capsules compared to the (PVPON/TA) capsules. Remarkably, despite the limited TA inclusion MnP-containing capsules still exhibit drastically enhanced ROS-scavenging activity (Figures 8c and 9c). We also found that, along with the synergistic relationship between TA and MnP in oxidative environments, inclusion of the manganoporphyrin polymer had an impressive protective effect on the integrity of the capsule shell. Thus, for example, when the same capsules from the initial SANS scattering experiments were challenged with the ABTS•+ radical, the thickness of the (PVPON/TA)4(MnP-PVPON/TA)1.5 capsule shell decreased by only 8% of the initial value with the aftertreatment thickness of 14 nm (Figure 10a,b). In contrast, the MnP-free shell of (PVPON/TA)5.5 capsules lost 39% of the initial thickness resulting in a thickness of 11 nm after the radical challenge (Figure 10c,d). Apparently, the ability of the manganoporphyrin to disproportionate radicals catalytically drastically increased the stability of the PVPON/TA capsule shell by minimizing material loss due to degradation of TA in the oxidative environment. In the case of the MnP-free shell, the radicals are likely absorbed through conversion of phenol hydrogens to stabilized phenoxy radicals77 upon conversion of the phenols in TA into quinones which would result in slow partial disruption of hydrogen bonds between polyphenol molecules and the carbonyls of PVPON. The observed loss of the (PVPON/TA) shell thickness can therefore be attributed to this H-bond disruption.

Figure 9. (a) UV−vis spectra of the reduction of cytochrome C by superoxide. (b) Cytochrome C assay with (PVPON)6 hydrogel capsules. (c) Cytochrome C assay for (PVPON/TA)5.5 and (PVPON/TA)4(MnP-PVPON/TA)1.5 capsules in the presence of catalase.

radical-scavenging efficiency per capsule for the MnPcontaining capsules over their porphyrin-free predecessors and can be seen as a marked decrease in the fitted slope (Figure 9c). Our results on superoxide dismutation by MnP-PVPON/TA capsules are in excellent agreement with the SOD-like activity reported for various free porphyrins. It is worth noting that, without catalase to scavenge H2O2 produced during O2•− dismutation, the rate of superoxide reduction by MnPPVPON-containing capsules was still linear unlike the (PVPON/TA)5.5 capsules which produced kinetic curves with uneven gradation of the slope (Figure S5). Importantly, this observed difference can indicate the combined ability of the MnP-PVPON/TA system to convert both superoxide to H2O2 (superoxide dismutase-like ability) and H2O2 to water (catalase-like activity). Manganic porphyrins have been previously reported to catalytically consume H2O2 and protect endothelial cells against the damaging effects of hydrogen peroxide based on the mechanism similar to catalase. The ring conjugation was speculated to be essential in delocalizing charge for the redox active metal and providing the stability of the manganic porphyrins in oxidative environments (Figure S6).6 In contrast to that, TA scavenges H2O2 noncatalytically and may be slowly polymerized during the process.72 To probe the effect of the free radical challenges on the shell material of MnP-free and MnP-containing (PVPON/TA) multilayer capsules, we employed small angle neutron scattering (SANS) and evaluated the thickness of the capsule

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CONCLUSIONS We report on the synthesis of manganoporphyrin-polyphenolic hollow capsules made via multilayer assembly of TA and a novel PVPON copolymer with managanoporphyrin pendant functionalities (20% monomer unit ratio) synthesized by coupling a PVPON-NH2 copolymer with a carboxyl monofunctionalized tetrapyridyl Mn-porphyrin via carbodiimide chemistry. We show the MnP-PVPON copolymer to be stable in aqueous solutions at physiological pH with no change in its manganese coordination. We also demonstrate that MnP353


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Figure 10. SANS data for (a, b) (PVPON/TA)5.5 and (c, d) (PVPON/TA)4(MnP-PVPON/TA)1.5 capsules before (a, c) and after (b, d) ABTS•+ challenge. The y-axis unit is (cm−1). Solid lines represent model fits to SANS data.

phyrins with natural polyphenolic antioxidants to design highly efficient and stable free-radical-scavenging systems which could eventually be useful for antioxidant therapies as well as for freeradical-scavenging protective carriers of sensitive materials for biomedical or industrial applications.

PVPON can be successfully assembled with TA with the bilayer thickness of (MnP-PVPON/TA) assembled at pH = 5 being smaller than that at pH = 7.4 (0.9 ± 0.1 nm versus 1.8 ± 0.1 nm, respectively), which was the counter-trend to MnPfree (PVPON/TA) assembly. The MnP-PVPON/TA multilayer capsules exhibit a highly hydrophilic nature and are found to be nontoxic to NOD splenocytes after 48 h incubation. The inclusion of the MnP-PVPON in the multilayer capsule shell dramatically enhances the radical-scavenging properties of (MnP-PVPON/TA) capsules compared to (PVPON/TA) capsules. We have found MnP-PVPON included as an outer layer to be more potent as compared to its localization in the middle or inner part of the capsule shell. In addition, we demonstrate that TA is crucial for the synergistic radicalscavenging activity of the (MnP-PVPON/TA) system which exhibits a combined superoxide dismutase-like ability and catalase-like activity in response to free radical superoxide challenge. In contrast to pure polyphenolic (PVPON/TA), the polyphenolic-manganoporphyrin (MnP-PVPON/TA) capsules exhibit a negligible loss of the shell thickness upon free radical treatment, while the former show 39% thickness loss due to noncatalytic free-radical-scavenging of TA as demonstrated by SANS measurements. The current study illustrates the strong potential of combining the catalytic activity of manganopor-

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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/acs.chemmater.7b03502. 1 H NMR spectra for porphyrin compounds; ABTS assay for 7.5-bilayer (PVPON/TA)6(MNP-PVPON)1.5 and (PVPON/TA)4(MnP-PVPON)3.5 capsules in comparison to (PVPON/TA)7.5 capsules; catalase-free cytochrome C assay for (PVPON/TA)5.5 and (MnPPVPON/TA)1.5(PVPON/TA)4, (PVPON/TA)2(MnPPVPON/TA)1.5(PVPON/TA)2, and (PVPON/TA)4(MnP-PVPON/TA)1.5 capsules; SANS model parameters for (PVPON/TA)5.5 and (PVPON/TA)4(MnPPVPON/TA)1.5 capsules before and after ABTS treatment; and stability of the (TA/MnP-PVPON) film at pH = 2.5 (PDF) 354


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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Volker S. Urban: 0000-0002-7962-3408 Eugenia Kharlampieva: 0000-0003-0227-0920 Author Contributions ∥

A.A. and V.K. contributed equally to this work. The corresponding authors (H.M.T. and E.K.) share equal seniority.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by NSF-DMR 1608728, and by NIH/NIDDK R01 award (DK099550) (H.M.T.), American Diabetes Association Career Development Award (7-12-CD11) (H.M.T.), and a Juvenile Diabetes Research Foundation Award (1-SRA-2015-42-A-N) (H.M.T.). The UAB High Resolution Imaging facility (scanning electron microscopy) is also acknowledged. Neutron scattering research conducted at the Bio-SANS instrument, a DOE Office of Science, Office of Biological and Environmental Research resource, used resources at the High Flux Isotope Reactor, a DOE Office of Science, Scientific User Facility operated by the Oak Ridge National Laboratory. Dr. Boualem Hammouda (NIST) is also warmly thanked for model fitting suggestions and SANS instruction.

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