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Scavenging ROS: Superoxide Dismutase/Catalase Mimetics by the Use of an Oxidation-Sensitive Nanocarrier/Enzyme Conjugate Ping Hu† and Nicola Tirelli*,‡,§ †

School of Pharmacy and Pharmaceutical Sciences, ‡School of Materials, §School of Biomedicine, University of Manchester, Oxford Road, Manchester, M13 9PT, United Kingdom S Supporting Information *

ABSTRACT: Reactive Oxygen Species (ROS) are quintessential inflammatory compounds with oxidizing behavior. We have successfully developed a micellar system with responsiveness at the same time to two of the most important ROS: superoxide and hydrogen peroxide. This allows for an effective and selective capture of the two compounds and, in perspective, for inflammation-responsive drug release. The system is composed of superoxide dismutase (SOD) conjugated to oxidation-sensitive amphiphilic polysulfide/PEG block copolymers; the conjugate combines the SOD reactivity toward superoxide with that of hydrophobic thioethers toward hydrogen peroxide. Specifically, here we have demonstrated how this hybrid system can efficiently convert superoxide into hydrogen peroxide, which is then “mopped-up” by the polysulfides: this modus operandi is functionally analogous to the SOD/catalase combination, with the advantages of (a) being based on a single and more stable system, and (b) a higher overall efficiency due the physical proximity of the two ROS-reactive centers (SOD and polysulfides).

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INTRODUCTION Reactive Oxygen Species (ROS), such as superoxide anion, hydroxyl radical, or hydrogen peroxide, are ubiquitous byproducts of cell metabolism with a double role of hazardous agents capable of extensive cellular damage but also signaling molecules;1 ROS can stimulate a variety of phenomena, from mitosis to angiogenesis, and also tumorigenesis, but in particular they are closely associated with virtually all main inflammatory pathologies.2−5 A central target of our group is the development of oxidationresponsive (nano)materials, that can be used to develop advanced anti-inflammatory therapies. We have specifically focused on thioether-containing organic polymers (polysulfides) in the forms of micelles,6 vesicles,7 and nanoparticles,8 which have shown promising results as in vivo carrier structures.9 Their responsiveness is based on the morphological changes associated to the oxidation of hydrophobic thioethers to more hydrophilic sulfoxides and sulfones; this may lead to swelling or solubilization and, e.g., release of an encapsulated drug to an extent proportional to the concentration of oxidants. The synthetic strategies and the oxidation response of polysulfides are discussed in a recent review.10 In a previous study, we have demonstrated how polysulfide nanoparticles can also respond to oxidants that are generated enzymatically in situ:11 by decorating the nanoparticle surface with glucose oxidase, hydrogen peroxide can be produced in close proximity to the polysulfide material in the presence of glucose; the resulting oxidation and swelling of the matrix permitted the release an encapsulated drug (doxorubicin). © 2012 American Chemical Society

However, the responsiveness of polysulfides may depend on the nature of the oxidant: for example, the low solubility of very polar or ionic molecules may determine a slow or negligible response. Superoxide is a ubiquitous ROS, most commonly produced by NAPDH oxidase in activated leukocytes,2 although alternative sources exist, e.g., xanthine oxidase.12 It is also one of the most dangerous ROS; besides its direct toxic action, it can produce a number of cytotoxic compounds, such as hydroxy radicals, peroxynitrite, or hydrogen peroxide. It has long been shown that superoxide can be scavenged by the combined action of superoxide dismutase (SOD) and catalase,13−15 where the first enzyme converts it to hydrogen peroxide, which is then removed by the second one; however, this scavenging activity is rather short-lasting, due to the rather limited lifetime of the enzymes. Superoxide is anionic, thus poorly soluble in a hydrophobic matrix; it is also highly reactive and unselective, so scarce solubility in polysulfides is likely to result in negligible advantageous effects. In the present research, we have focused on polysulfidecontaining micelles (block copolymers of poly(propylene sulfide) (PPS) and PEG), studying their possible superoxidescavenging activity and tackling the issue of improving it by conjugation with the most common naturally occurring, selective superoxide scavenger, i.e., superoxide dismutase. Received: August 16, 2011 Revised: January 4, 2012 Published: January 31, 2012 438

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Scheme 1. Sequence of Preparative Steps Leading to PPSPEG Reactive Micelles That Allow the Incorporation of SOD through Michael-Type Addition of SOD Nucleophilic Groups onto Vinyl Sulfone Polymeric Termini

A third, more proven way has seen the use of SOD conjugates. Poly(ethylene glycol) (PEG) is a “gold standard” to improve protein stability in vivo, reduce enzymatic degradability and immunogenicity, improve solubility, and reduce renal clearance. PEG has been conjugated to SOD using a variety of coupling reagents, e.g., cyanuric trichloride,30 phenyl chloroformiate,31 or carbonyl diimidazole.32 There is ample evidence that PEGylation per se does not substantially alter SOD activity, as demonstrated in a number of in vitro and in vivo33 models and also in clinical trials.34 Other polymers have also been conjugated to SOD for the same purposes, e.g., PEG amphiphilic block copolymers, such as Pluronics,35 or poly(N-vinyl pirrolidone).36 PEG has also been used as a spacer for the conjugation of SOD to a payload, such as hemoglobin,37 to protect the latter from the action of free radicals. In this study, we have prepared PPSPEG block copolymers through sequential ring-opening anionic polymerization of propylene sulfide initiated by benzyl mercaptane and endcapped by monovinyl sulfone terminated PEG (Scheme 1); the synthesis of these copolymers is described in a recent publication.6 We have specifically focused on a low PPS length (PPS10-PEG44) to maximize the surface-to-volume ratio of the micelles and therefore maximize their response rate to waterborne oxidants. A vinyl sulfone-terminated block copolymer (PPS10-PEG44-VS) was prepared with an identical procedure and used to prepare mixed micelles that were then functionalized with SOD through Michael-type addition. We have then investigated the superoxide-scavenging ability of PPSPEG micelles with or without SOD.

SOD is a general name for a class composed of four families of enzymes, i.e., Cu,Zn-, Fe-, Mn-, and Ni-SODs; as a common element, they all accelerate the transformation of the superoxide radical anion into H2O2 and molecular oxygen. This activity renders SOD one of the most ubiquitous enzymes for the defense of both prokaryotic and eukaryotic organisms from oxidative damage. A recent review of Bafana et al.16 summarizes the state of the art about physiological and pathophysiological roles of SOD. SODs, and more specifically Cu,Zn-SOD, has been long researched as a possible means to treat a number of pathologies of inflammatory or tumoral nature.17 In particular, a clear link has been established between forms of amyotropic lateral sclerosis and inactivation of SOD due to mutation,18 but also other inflammatory pathologies with oxidative character, such as inflammatory bowel diseases,19 have shown beneficial effects of SOD administration.20 Although SOD has shown efficacy in several inflammatory pathologies, it has not found widespread clinical application: its rapid renal clearance associated with a slow extravasation in peripheral tissues minimizes its beneficial effects on peripheral, inflamed tissues.21 A number of strategies have been attempted to overcome the poor pharmacokinetic behavior of SOD. A first approach focuses on the in situ sustained production of the enzyme through the use of genetically engineered cells22 or bacteria;23 although promising, this avenue is complicated by the generally low acceptance of therapies based on engineered cells. Another approach has tackled the development of SOD-mimetic compounds,24,25 generally based on low molecular weight26 or polymeric27 Mn porphyrin but including also cerium oxide28 or platinum29 nanoparticles; although significant results have been reached, the rapid clearance of low molecular weight compounds and the regulatory approval of new active principles are significant hurdles.

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EXPERIMENTAL PROCEDURES Materials. Acetic acid, poly(ethylene glycol) monomethyl ether (Mn = 2000 and 5000 g/mol), sodium hydride, divinyl

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Reaction with Reduced SOD. 2.5 mg of SOD were dissolved in 2 mL of 10 mM sodium phosphate buffer, pH 8.5; 47 μL of a 0.01 M TCEP (4.7 × 10−4 mmol) solution were then added and the solution was allowed to react for 2 h at room temperature. In control experiments, the solution was transferred into a dialysis tube (MWCO = 3000 g/mol), and dialyzed against 1 L 10 mM PBS buffer, pH 8.5, for 24 h to remove the reducing agent. The solution of reduced SOD with or without dialysis was then reacted with PEG-VS as previously described. Conjugation of SOD to PPS10-PEG44-VS Micelles. 0.5 mL of a PPS10-PEG44-VS micelles dispersion (4.8 mg of a 9:1 PPS10-PEG44/PPS10-PEG44-VS molar ratio for a total 1.6 × 10−3 mmol of polymer chains) was employed in stead of a PEG-VS solution. Purification through Size Exclusion Chromatography (SEC). The reaction mixtures (volume ∼2.5 mL) were loaded on the top of a prewetted Sephadex G100 column (1.5 × 500 mm) and the system was eluted with the help of a pump (LDC Analytical, constaMetric 3200) operating at 1 mL/min, using 10 mM PBS buffer. The eluted profile of the sample was monitored using absorbance at 260 nm, collecting different fractions that were further characterized by SDS-PAGE. Analytical Assays on PEGylated SOD. SDS-PAGE. SDSPAGE analysis was performed on 4−12% polyacrylamide gels. First, the protein concentration of all samples was determined by the BCA assay using SOD as standard (see later). Then each sample was freeze−dried and redissolved in water to achieve the same concentration of protein (1 mg/mL). Each solution was mixed with the SDS-PAGE buffer (250 mM Tris pH 6.8, 25% glycerol, 2% SDS, 5% β-mercaptoethanol and 0.01% bromophenol blue) in a 1:1 volume ratio and heated at 95 °C for 5 min. The slab of gel was composed of 2 cm of stacking gel (4% acrylamide, 62.5 mM Tris pH6.8) and 6 cm of resolving gel (12% acrylamide, 375 mM Tris pH 8.8) with a thickness of 1.5 mm. The electrophoresis was performed at 120 V for 10 min, then increased to 200 V for the rest of the run. The gel was then incubated in a staining solution (0.025% Coomassie brilliant blue R-250 in 40% acetic acid, 20% methanol) for 1 h with gentle shaking, and washed with a destaining solution (40% acetic acid, 20% methanol) to remove excess stain. Barium Iodide Assay. The concentration of PEG was determined by a modified barium iodide method:38 25 μL of barium chloride (5%, in 1 M HCl) was added to 100 μL of sample solution in a 96 well plate. The solution was mixed for 5 min at room temperature before 25 μL of iodine solution (0.1 M) was added to the well. The plate was allowed to stand for 15 min at room temperature to develop the color which was read at 535 nm by a plate reader (BioTek Synergy 2). The concentrations of PEG in PEG-SOD and PPSPEG-SOD samples were measured and calculated by comparing the readings with a PEG5k/PEG2k standard calibration curve. The influence of SOD to this method was investigated by measuring the absorbance of PEG standard solutions added with different amounts of (noncovalently bound) SOD. BCA (Bicinchonic Acid) Assay. The concentration of total proteins was determined with a standard BCA assay: in a 96 well plate 100 μL of BCA assay reagent (Sigma-Aldrich) were added to 100 μL of SOD-containing solution/dispersion (e.g., PPSPEG-SOD micelles) previously purified through SEC. The plate was kept at room temperature for 16 h before the absorbance at 562 nm was read using a plate reader. The

sulfone, superoxide dismutase (Cu,Zn-SOD from bovine erythrocytes, lyophilized powder, 3780 U/mg protein, MW: 32.5 kDa), tris(2-carboxyethyl)phosphine hydrochloride solution (0.5 M), Sephadex G-100, glycerol, sodium dodecyl sulfate, β-mercaptoethanol, bromophenol blue, Coomassie brilliant blue R-250, barium chloride, iodine solution (0.05 M in water), 2,4,6-trinitrobenzenesulfonic acid solution (5% in water), BCA assay kit, SOD assay kit, Triton X-100, sodium hydroxide, Nile Red, hydrogen peroxide (30 wt % solution), xanthine, xanthine oxidase (from bovine milk, ammonium sulfate suspension, 1.0 U/mg protein, MW: 270 kDa), Catalase (from bovine liver, lyophilized powder, 4342 U/mg protein, MW: 250 kDa), and Celltiter 96 Aqueous Cell Proliferation assay kit (containing MTS and PES) were used as received from Sigma-Aldrich (Gillingham, United Kingdom). Macrophages J774.2 were obtained from the European Collection of Cell Cultures (Porton Down, UK). Phosphate buffered saline (PBS) was prepared from phosphate buffered saline (Dulbecco A) tablets (Oxoid limited, Basingstoke, UK). Water was purified through a Milli-Q system (Millipore, UK). Physicochemical Characterization. Molecular Characterization. 1H NMR spectra were recorded on 1 wt % polymer solutions in deuterated chloroform using a 300 MHz Bruker spectrometer. FT-IR spectra were recorded in ATR mode (Golden gate) on a Tensor 27 Bruker spectrometer. GPC was performed in THF on a Polymer Laboratories GPC 50 equipped with refractive index and viscosity detectors, using universal calibration with poly(styrene) standards. UV−vis and fluorescence readings were obtained on a BioTek Synergy 2 multiwell plate reader. Dynamic Light Scattering (DLS). Size distributions were measured with the help of a Zetasizer Nano ZS Instrument (model ZEN2500, Malvern Instruments Ltd., UK). All the samples were analyzed at an angle of 114° and at a temperature of 25 °C. Preparative Procedures. Polymer Synthesis. PEG2k-VS (Mn = 2100 g/mol, yield: 78 wt %), PEG5k-VS (5200 g/mol, yield: 81 wt %) were prepared via reaction of the corresponding monomethoxy PEG derivatives with an excess divinyl sulfone after deprotonation with catalytic amounts of NaH, as described in a previous publication.6 PPS10-PEG44 and PPS10PEG44-VS were prepared through propylene sulfide anionic polymerization initiated by benzyl mercaptane and end-capped with PEG2k mono- or bis(vinyl sulfone) (Mn ∼ 3000 g/mol, polymerization yield: 62 wt %, end-capping conversion: 94 mol % end-capping conversion 94 mol % calculated from the ratio of the 1H NMR resonance of PEG terminal CH3 at 3.4 ppm and that of the aromatic group of the initiator at 7.4 ppm6). Most SOD PEGylation experiments were conducted with PEG5k-VS, in order to have a hydrodynamic volume comparable to PPS10-PEG44-VS; PEG2k-VS was used only as a control for SDS-PAGE experiments. Conjugation Experiments. Reaction with Native SOD. 2.5 mg of SOD were dissolved in 2 mL of 10 mM sodium phosphate buffer at pH 8.5. 8.8 mg of PEG5k-VS (1.6 × 10−3 mmol) were dissolved in 0.5 mL of the same buffer (corresponding to a 20:1 PEG-VS/SOD molar ratio) and were added to the SOD solution. The reaction mixture was allowed to react for 72 h at room temperature. A drop of NaOH (0.01 M) solution was then added to deactivate any unreacted vinyl sulfone groups, followed by a few drops of HCl solution (0.01 M) to restore neutrality. 440

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the absorbance of the test sample and [A]control is the absorbance of a control sample (well containing cell culture medium without PPS10-PEG44 micelles). Nile Red Loading and Encapsulation Efficiency. 0.1 mL of dichloromethane solutions of Nile Red with variable concentrations (0.0625 to 2 mg/mL) were mixed with 1 mL of 1.0 mg/mL of pure PPS10-PEG44 or of PPS10-PEG44/PPS10PEG44-SOD 9:1 micellar dispersions, evaporating the organic solvent in a rotary evaporator (400 mbar for 5 min, followed by 80 mbar for 30 min, T = 25 °C). Any nonencapsulated payload was removed by centrifugation at 4500 rpm. To measure the amount of loaded Nile Red, 0.1 mL of N,N-dimethylformamide was added to 0.1 mL of drug-loaded micellar dispersion in order to solubilize the encapsulated compounds. The concentration of hydrophobe was then measured via its emission intensity at 620 nm (excitation in the window 540 ± 25 nm, emission in the window 620 ± 40 nm). The concentrations of samples were calculated by comparing the readings to a standard curve of Nile Red in 50% DMF. Drug loading (DL) and encapsulation efficiency (EE) were calculated as follows: DL (w/w) = (amount of loaded drug)/(amount of polymer), EE (wt %) = (actual amount of loaded drug)/ (theoretical amount of loaded drug). Oxidation Responsiveness of PPS10-PEG44(-SOD) Micelles. Responsiveness to Hydrogen Peroxide. DLS. One mL of a 1 mg/mL dispersion of PPS10-PEG44 micelles prepared as previously described was added to 1 mL of a solution of hydrogen peroxide solution with variable concentration to yield a final H2O2 concentration of 0, 2, 5, and 10 wt %. The kinetics of oxidation was followed by recording the size distribution and the overall scattering intensity at room temperature as a function of time. Nile Red Method. The experiments were prepared as above, diluting a 1 mg/mL micellar dispersion with a 15 μg/mL concentration of Nile Red with 1 mL of a solution of hydrogen peroxide solution. The fluorescence of Nile Red was measured at defined times (0, 0.1 h, 1 h, 2 h, 4 h, 5.5 h, 8 h, 12 h) using a plate reader with excitation filter at 540 ± 25 nm and emission filter at 620 ± 40 nm. Responsiveness to Superoxide. Nile Red Assay. 0.5 mL of a 40 mM solution of xanthine and 0.1 mL of a 4 × 10−2 mM solution of xanthine oxidase were added to 2 mL of a Nile Redloaded PPSPEG (3.3 mM of sulfides,15 μg/mL of Nile Red) or PPSPEG-SOD (3.3 mM of sulfides, 7.7 × 10−4 mM of conjugated SOD, 18 μg/mL of Nile Red) micellar dispersion. In different experiments 0.2 mL of a 6.1 × 10−3 mM solution of SOD and/or 0.2 mL of a 8 × 10−4 mM solution of catalase were added too. The molar concentrations of the enzymes are calculated by dividing their concentration in weight/volume by the molecular weight of the enzymes; this does not take into account the different purity of the preparations, and indeed these molar concentrations are just indicative values; it is important to note that the apparent molarity of SOD and catalase is much lower than that of XO, but the activity of their solutions is comparable. Finally, variable amounts of PBS buffer (pH 7.4, 10 mM) was added to the dispersion which resulted in 4 mL of final mixture containing 1.7 mM of sulfides, 5 mM of xanthine, and 1 × 10−3 mM of xanthine oxidase without SOD (3.1 × 10 −4 mM) and catalase (4 × 10−5 mM). The fluorescence intensity of the sample was then measured via its emission intensity at 620 nm (excitation in the window 540 ± 25 nm, emission in the window 620 ± 40 nm) at fixed times (0, 0.5, 1, 2, 3, 4, and 6 h).

concentration of SOD in the sample was calculated by comparing the reading with a standard SOD calibration curve. The degree of PEGylation was calculated as the ratio molar concentration of PEG/molar concentration of SOD. TNBS Assay. The number of PEG residues attached to SOD lysines was determined by measuring the primary amine content of SOD before and after conjugation through the colorimetric reaction with TNBS. Briefly, solutions at different concentrations of the native SOD and of PEGylated SOD were prepared in borate buffer (pH 9.3, 0.1 M). 50 μL of TNBS reagent (0.01 M) and 50 μL of 10% SDS were added to 50 μL of protein solution with 100 μL of borate buffer in a 96 well plate. The solution was allowed to react at 25 °C for 2 h. The absorbance at 420 nm of the sample was then measured and plotted versus the protein concentration: the number n of the reacted lysine residues on SOD is n = 20 × (1 − (slopePEG‑SOD)/ (slopeSOD)) where 20 represents the number of the lysine residues present in SOD. Evaluation of the Superoxide-Scavenging Activity. The superoxide-scavenging activity of SOD, PPS10-PEG44, and PPS10-PEG44-SOD was determined by the Sigma Aldrich (Dojindo) SOD assay kit, which is based on xanthine oxidase (XO) as the generator of superoxide and WST-1 as the chromophore’s parent compound. Briefly, 20 μL of a solution of SOD, or of a dispersion of PPS10-PEG44-SOD at different concentrations and 20 μL of a solution of XO at variable concentration were added to 200 μL of a WST-1 standard solution in wells of a 96 well plate. The plate was incubated at 37 °C for 20 min and the absorbance was read at 450 nm using a plate reader. The activity of the superoxide scavenger was expressed as the inhibition percentage in relation to the absorbance of a blank sample. Preparation and Characterization of PPS10-PEG44(-SOD) Micelles. PPS10-PEG44 micellar dispersions were prepared through direct dispersion of the block polymers in PBS buffer (pH 7.4) at a concentration of 1 mg/mL and then possibly further diluted. PPS10-PEG44-SOD micelle dispersions in the same buffer were prepared and purified by size exclusion chromatography (SEC) on Sephadex G-100. Cytotoxicity of PPS10-PEG44 Micelles. Cytotoxicity of PPS10PEG44 micelles was assessed on a line of murine macrophages (J774.2) using a CellTiter 96 Aqueous Cell Proliferation Assay. Cells were seeded in a clear 96-well flat bottomed plate at a concentration of 2.5 × 104 cells per well and kept in 37 °C, 5% CO2 incubator for 24 h to allow for cell adherence. On the next day, the cells were washed once with PBS, then PPS10-PEG44 micelles in cell culture medium at concentrations of 1.9, 2.5, 3.8, 5, 7.5, 10, 15, and 20 mg/mL were added into each well and plain medium was used as a control. The plate was then incubated for 48 h at 37 °C, 5% CO2 environment. After 48 h of incubation, the supernatant medium was removed, and the wells were rinsed twice with PBS (pH 7.4, 10 mM). Then 100 μL of culture medium without FBS and phenol red supplemented with 20 μL of MTS reagent was added into each well. After 3 h the absorbance of each well was read on a microplate reader (μQuant, Biotek) at 490 nm. Finally, in each well the cells were washed with PBS and were then exposed to a lytic solution of 0.5 wt % Triton X-100 in 0.2 N sodium hydroxide for 10 min at 37 °C, which was then employed to analyze the protein content through the QuantiPro BCA Assay Kit according to the manufacturer’s recommendations. The relative cell viability (%) was calculated by ([A]test/[protein content]test)/([A]control/[protein content]control), where [A]test is 441

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solubilization, which occurred at a time increasingly longer for lower H2O2 concentrations (Figure 2A). On the other hand, their scattering gradually decreased in intensity, more rapidly with increasing H2O2 concentration (Figure 2B). Since the scattering intensity is proportional to the refractive index difference between micellar core and water, this result would indicate a gradual increase in the water content of PPS domains. This was confirmed by monitoring the fluorescence of Nile Red, whose fluorescence is quenched by the presence of water; this peculiarity is often used in studies of micellar aggregates.39,40 Nile Red is encapsulated with high efficiency in PPS10-PEG44 micelles (see later, Figure 8), its fluorescence decreased with time and H2O2 concentration (Figure 2C), with a kinetics very similar to that of the scattering intensity. The only noticeable difference was a certain retardation of Nile Red quenching (see Supporting Information, Figure 1SI); this lag is probably due to the need of a defined water concentration to achieve an appreciable quenching of NR emission in an apolar solvent.6 The PPS10-PEG44 micelles appear therefore to undergo a gradual hydophobic-to-hydrophilic transition when exposed to a nonionic model oxidant, such as H2O2. However, the ionic nature of superoxide may limit its solubility in a polysulfide matrix, thus decreasing the possible response of PPS. Due to its instability, superoxide was generated in situ through the xanthine oxidase (XO)-mediated conversion of xanthine to uric acid; this method provides equimolar amounts of superoxide and H2O2 (Scheme 2, left), so we have examined the effects of XO on PPS10-PEG44 micelles also in the presence of catalase, which selectively decomposes H2O2, and SOD, in order to separate the effects of the two oxidants. The kinetics of oxidation was followed using Nile Red fluorescence (Figure 3); XO produced a significant and timedependent hydrophilization, but no effect was recorded when XO was used in combination of catalase, with or without SOD; furthermore, the combination of XO and SOD, which increases the concentration of H2O2 and decreases that of superoxide, slightly accelerated the process of PPS hydrophilization (=lower NR fluorescence at all time points). These results indicated that XO could oxidize the PPS micellar core through the production of H2O2, while the presence of superoxide appeared to have no effect. We have confirmed that the negligible effects of superoxide of PPS translated in a negligible superoxide-scavenging activity of PPS micelles, by monitoring their effect on the kinetics of a superoxide-mediated reaction: the oxidative conversion of MTS to MTS formazan (Scheme 2, right). SOD was used as a positive control and, as expected, it showed excellent superoxide scavenging activity (Figure 4, top): with [XO] and [SOD] = 2 × 10−6 − 2 × 10−4 mM, dose-dependent SOD effects were recorded for all combinations except when XO was present in large excess (XO 100 times more concentrated than SOD). On the other hand, the presence of PPS 10-PEG 44 micelles did not produce any significant variation or delay in formazan production, even with concentrations up to 8.3 mM of sulfide groups (2.5 mg/mL) (Figure 4, bottom). It could therefore be concluded that PPS10-PEG44 micelles had a poor superoxide-scavenging capacity, due to the negligible reaction of the sulfide groups with superoxide. Conjugation (PEGylation) of SOD. The SOD−PPSPEG conjugation chemistry should minimize the disruption to the protein secondary and tertiary structure, while also avoiding the

The fluorescence intensity was presented as the percentage of the fluorescence intensity at 0 h. Formazan (MTS) Assay. Superoxide was generated in situ through a xanthine−xanthine oxidase system and its presence was monitored via the reduction of MTS added to the system. Typical experiments were conducted in 96 well plates; in each well, 50 μL of PPS10-PEG44 micellar dispersions (0, 0.033, 0.33, 3.3 mM of polymer) or 20 μL of SOD solution (0, 2 × 10−5, 2 × 10−4, 2 × 10−3 mM of enzyme) were added to 50 μL of 2 mM xanthine, 20 μL of xanthine oxidase (2 × 10−5, 2 × 10−4, 2 × 10−3 mM), 50 μL of 0.2 mM MTS, and variable amounts of PBS buffer (pH 7.4, 10 mM) to yield 200 μL of final solution which contains PPS10-PEG44 micelles (0, 8.3 × 10−2, 8.3 × 10−1, 8.3 mM sulfides) or SOD (0, 2 × 10−6, 2 × 10−5, 2 × 10−4 mM), xanthine (0.5 mM), xanthine oxidase (2 × 10−6, 2 × 10−5, 2 × 10−4 mM) and MTS (0.05 mM). The enzymatic kinetics was monitored through the absorbance of MTS at 490 nm. A purified PPS10-PEG44-SOD micelle dispersion (containing 0.33 mM polymer and 7.7 × 10 −4 mM conjugated SOD) was also reacted with the superoxide-generating system. A final solution containing 8.3 × 10−1 mM sulfides and 1.9 × 10−4 mM conjugated SOD, 0.5 mM xanthine, 2 × 10−5 mM xanthine oxidase, and 0.05 mM MTS was employed for identical kinetics studies.

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RESULTS AND DISCUSSIONS Low Toxicity, Oxidation Responsiveness, but Poor Superoxide Scavenging Ability of PPS10-PEG44. PPSbased PEGylated materials (block copolymers in vesicular or micellar morphology, nanoparticles, etc.) are generally characterized by negligible cytotoxic effects on cells. For example, the exposure of a rather sensitive cell line, J774.2 murine macrophages, to PPS10-PEG44 micelles caused no significant reduction in cell viability for PPSPEG concentrations up to 4 mg/mL; an IC50 was recorded in the region of 15 mg/mL (1.5 wt %).

Figure 1. Viability of J774.2 macrophages as a function of the concentration of PPSPEG micelles after a 48 h exposure. The mitochondrial reductase activity (MTS assay) was normalized in relation to the protein content and reported as % of the control. The dashed line allows the identification of the IC50.

The reaction of PPS with oxidants produces polar species, such as sulfoxides. As a result, when treated with a nonionic oxidant such as hydrogen peroxide, PPS10-PEG44 micelles eventually solubilize in water (Figure 2). The micelles maintained their size substantially unchanged until complete 442

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Figure 2. Influence of the concentration of hydrogen peroxide on volume-average micellar size (panel A, top left), on the scattering intensity of the suspension (panel B, top right), and on the fluorescence of encapsulated Nile Red (panel C, bottom left). PPS10-PEG44 concentration: 0.5 mg/mL; Nile Red concentration: 7.5 μg/mL.

Scheme 2. Superoxide Generated in Situ through the Enzymatic Conversion of Xanthine to Uric Acid Can Be Detected Colorimetrically Using the Oxidation of Tetrazolium to Purple-Coloured Formazan47 a

a

Two water-soluble tetrazolium derivatives were used: MTS for most measurements and WST-1 for the assessment of SOD activity, which employed a commercially available, WST-1 based kit (Dojindo) The presence of other oxidizable compounds, such as polysulfides, or of superoxide dismutase (SOD) can reduce the conversion of tetrazolium at any given time point.

other Michael-type acceptors such as α,β-unsaturated esters and amides, their reaction products do not bear hydrolyzable groups and resist to an oxidizing environment. On the other hand, vinyl sulfones also react with water/hydroxyl anions (Scheme 3); this limits the efficiency of the bioconjugation

binding of multiple micelles to the same molecule of SOD that would lead to large-scale aggregation. We have employed PEG or PPSPEG vinyl sulfones, whose preparation is described in a recent publication.6 Vinyl sulfones are highly reactive Michael-type acceptors; differently from 443

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Figure 3. The fluorescence of Nile Red loaded in PPS10-PEG44 micelles (15 μg NR per mg of PPSPEG) was examined as a function of time in the presence of XO as an oxidizing enzyme. In water buffers, XO is in a colloidal, rather opaque state, and this makes DLS measurements unreliable; on the other hand, Nile Red fluorescence was previously shown (Figure 2) to follow rather closely the phenomenon of PPS oxidation. Concentrations: PPS10-PEG44, 0.5 mg/mL (1.7 mM of sulfides); Nile Red, 15 μg/mL, 5 mM of xanthine, 1 × 10−3 mM of xanthine oxidase with/out SOD (3.1 × 10−4 mM) and catalase (4 × 10−5 mM)). The symbols on the right (√ or ⊗), respectively, indicate the presence or absence of the ROS, showing a response only when hydrogen peroxide was present.

Figure 4. Formazan assay for assessing superoxide scavenging activity: the absorbance of oxidized MTS (at 490 nm) was recorded as a function of time and of the concentration of xanthine oxidase and of superoxide dismutase (SOD, upper half of the figure) or of PPSPEG (lower part of the figure). The presence of the latter did not influence the kinetics of the MTS conversion at any combination of XO and micelle concentrations. On the other hand, SOD showed a distinct dose-dependent retardation effect, which can be ascribed to the removal of superoxide.

SOD to be derivatized (see Supporting Information, Figure S2). Similar results were obtained on micelles containing 10 mol % of functional polymers (PPS10-PEG44-VS). The reaction products can be easily separated from unreacted SOD via SEC on Sephadex G100 (Figure 5). The purified products showed that roughly 2.5 mol % of vinyl sulfones reacted with SOD, corresponding to the presence one SOD every 420

reaction, which also allows minimizatio of the extent of multiple binding and therefore also the likelihood of micelle aggregation. Due to the competitive reaction with water, a high yield of SOD PEGylation was obtained only by employing a large stoichiometric excess of vinyl sulfones, e.g., a 20-fold molar excess sulfone-terminated PEG (2 or 5 kDa) for a majority of 444

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Scheme 3. Possible Reactions of Vinyl Sulfone-Terminated PEG Constructs with Nucleophiles in a Protein-Containing Water Solution

Table 1. PEGylation Data for SOD (PEG5k-VS 20:1 Vinyl Sulfone/SOD Molar Ratio) after SEC Purification Average number of PEG chains per SODa Lysines modified by PEGb

Native SOD

Reduced SOD

3.74 4.15

3.59 3.93

a

Calculated dividing the weight fractions of PEG (barium iodide assay) and SOD (BCA assay) by their respective molecular weights. b Difference in the number of free amines (TNBS assay) before and after the conjugation reaction.

SDS-PAGE showed no trace of di-, tri-, or penta-PEGylated derivatives. SOD has been shown to aggregate under mild conditions even in a mature and metalated form:46 it is likely that, while aggregated SOD would be poorly reactive, the introduction of even only one PEG chain would solubilize it, thus allowing reaction of the vinyl sulfones with all readily available nucleophiles, i.e., four lysine residues per protein. The introduction of SOD on micelles was not accompanied by any sound increase in size (Figure 6), thus confirming the

polymer chains (from the weight ratio of PEG and protein measured through barium iodide and BCA assays, respectively). The aggregation numbers of PEG-based amphiphilic block copolymers with comparable molecular weight and similar micellar dimensions are often reported to be in the region of 50−100: 70 for high propylene oxide-content Pluronic P123,41 70−100 for hydrophobically modified PEGs,42 40− 50 for PEG-PHB-PEG triblock copolymers.43 We therefore assume to have about one conjugated SOD roughly every 5− 10 micelles. In terms of the regiochemistry, bovine Cu,Zn SOD is a 32.5 kDa dimeric structure, where each monomer presents 3 cysteine residues (C6 free, C55 and C146 involved in an intramolecular disulfide44) and 10 lysine ones, therefore allowing in principle for both N- and S-alkylation. The analysis of the purified PEG-SOD products showed a substantial identity between number of PEG chains per SOD and number of reacted lysine residues, even after reduction of the intramolecular disulfide with TCEP (Table 1; see also the SDS page analysis in Supporting Information, Figure S3), which demonstrates a conjugation through N-alkylation. It is worth pointing out that this may not apply to human Cu,Zn SOD, which bears a different number of cysteine residues (four, with C57 and C146 involved in the intramolecular disulfide45). It is also noteworthy that the PEG vinyl sulfone/SOD molar ratio affected the ratio between tetra-PEGylated and native SOD, but not the number of PEG chains introduced: independently on the vinyl sulfone/SOD stoichiometric ratio,

Figure 6. Size distribution of PPS10-PEG44 micelles before and after functionalization with SOD. In the micelles, nonfunctional (methoxyterminated) and functional (vinyl sulfone- or SOD-terminated) block copolymers were present in a 9:1 molar ratio.

Figure 5. Left: Typical SEC elution curves of SOD before conjugation (top), after conjugation with PEG5k-VS (middle), and after conjugation with PPS10-PEG44-VS (bottom). Using appropriate SEC conditions (1 mL/min on Sephadex G100), the samples can be separated into three fractions, which are illustrated in the example of PEG-SOD. It is noteworthy that both the micelles and PEG5k-SOD are close to or beyond the exclusion limit of the column (hence their sharp peak), while the breadth of the SOD peak derives from the colloidal nature of the protein in water buffers (see also caption to Figure 3). Right: SDS-PAGE results for SOD (control), for the bioconjugation reaction product and the three different fractions obtained through SEC purification. Nonconjugated SOD is absent from fraction 1. 445

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Figure 7. Loading (left) and encapsulation efficiency (right) of Nile Red in micelles of PPS10-PEG44 or PPS10-PEG44-SOD was measured by adding DMF to a 1 mg/mL dispersions of loaded micelles and recording the emission of the resulting water/DMF 1:1 solutions. For weight ratios above 50 μg/mg polymer the micelles appear to reach saturation; however, the encapsulation efficiency was strongly reduced well before that point, which suggests the loading process not to be totally governed by a simple partition.

Despite the significant drop in SOD activity, the enzymecontaining micelles were still able to significantly inhibit the superoxide-induced WST-1 oxidation at concentrations orders of magnitude lower than those affecting cell viability: for comparison, 0.03 mg/mL PPSPEG-SOD caused a 50% reduction in the WST-1 reaction rate, while a 50% reduction in L929 viability is obtained at a 500× larger concentration (15 mg/mL). Additionally, the conjugation of SOD to PPSPEG appeared to provide remarkable stability to the enzyme. For example, PPSPEG-SOD showed a negligible IC50 increase after storage in PBS buffer (pH 7.4, 10 mM) for up to 3 months, whereas under the same conditions the activity of free SOD decreased by almost 3 orders of magnitude (see the increase in IC50 in Figure 9, right; compare black symbols to white ones). The encapsulation of organic hydrophobes in PPSPEG micelles makes use of an organic solvent (dichloromethane); neither this nor the presence of appreciable amounts of Nile Red (up to 1.5 wt % in relation to the polymers) appeared to affect the SOD activity (crossed symbols in Figure 10, right). Due to the negligible effect of the Nile Red encapsulation, we have used the Nile Red assay to highlight any effect of the proximity of SOD on the H2O2 scavenging activity of PPSPEG. As previously discussed, xanthine oxidase generates also hydrogen peroxide, causing PPS oxidation also without SOD (see Figure 3). However, the activity of SOD increases the H2O2 concentration, supposedly accelerating the oxidation kinetics; a further acceleration may derive from the proximity between PPS and SOD, which would mean a higher local H2O2 concentration and therefore a higher efficiency in its uptake. The experiments showed that free SOD already caused a slightly quicker PPS oxidation (= lower Nile Red fluorescence at any time point; compare white circles to black squares in Figure 10, left); however, the effect was much more noticeable for PPSPEG-SOD. This can be better seen in a plot of relative quenching efficiency (Figure 10, right), which more clearly depicts the higher efficiency of hydrogen peroxide capture following the chemical conjugation of SOD to PPSPEG chains. Therefore, the lower superoxide scavenging activity of the conjugates may be more than overcome by the higher efficiency in hydrogen peroxide scavenging, due to the physical proximity of PPS and SOD.

absence of multiple binding to SOD. Further, the presence of the enzyme had insignificant effects on the solubilization capacity of PPS10-PEG44 micelles: using Nile Red as a model hydrophobic compound, nonfunctional and SOD-containing micelles exhibited a very similar maximum encapsulation capacity, i.e., 15 μg of dye per mg of block copolymer = 1.5 wt % (Figure 7, left), with an identical decrease of the encapsulation efficiency with increasing Nile Red concentration (Figure 7, right). Superoxide- and Peroxide-Scavenging Behavior of PPSPEG-SOD. Differently from PPS-PEG, PPSPEG-SOD showed a clear capacity to inhibit the oxidative conversion of MTS, thus demonstrating a superoxide scavenging ability (Figure 8). In comparison to free SOD, the molar activity of the

Figure 8. Results of the formazan assay (absorbance of MTS formazan as a result of superoxide production by XO and xanthine) for PPSPEG-SOD in comparison to SOD and PPSPEG alone. Concentration of XO: 2 × 10−5 mM.

conjugated enzyme significantly dropped, possibly because of increased steric hindrance and/or partial denaturation during conjugation. We have quantified this effect by measuring the decrease in the rate of WST-1 formazan production as a function of the molar concentration of free or conjugated SOD; expressing these results in terms of a percentage of inhibition (Figure 9, left), free SOD showed a half-maximal inhibitory concentration (IC50) of 5.6 × 10−9 mM, which was reduced of about 1 order of magnitude by its conjugation either to PEG5k or to PPSPEG micelles. 446

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Figure 9. Inhibitory effect of free or conjugated SOD on the oxidation of WST-1 by superoxide. The percentage of inhibition was calculated from the slope of the absorbance at 450 nm vs time (see also Figure 4) considering the slope of the control sample (no SOD, no PPSPEG) to be the 0% inhibition. The IC50 is the SOD concentration necessary to achieve a 50% reduction in the slope. Left: comparison between free and conjugated (PEG5k or PPSPEG-SOD-containing micelles) SODs. Right: comparison between free SOD and PPSPEG-SOD after SEC purification and after a 3 month storage at 4 °C. The conjugated SOD showed no significant change in activity upon storage, and also upon loading with Nile Red.

Figure 10. Left: Fluorescence of Nile Red as a function of time in the presence of XO, without SOD or with SOD free or conjugated to PPSPEG. Right: The data of the left graph are reported in a relative quenching scale, which is obtained dividing the fluorescence intensity of NR in PPSPEG micelles without SOD from that in the presence of free or conjugated enzyme. Concentrations: PPS10-PEG44, 0.5 mg/mL (1.5 mM of sulfides); Nile Red, 15 μg/mL, 5 mM of xanthine, 1 × 10−3 mM of xanthine oxidase without SOD (3.1 × 10−4 mM for both free and conjugated enzyme).

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CONCLUSIONS We have demonstrated that PPSPEG block copolymers are sensitive to hydrogen peroxide but not to superoxide; however, in combination with SOD they are capable of removing both ROS from a given environment. When the two components are covalently conjugated, the SOD-mediated removal of superoxide is considerably more efficient and the enzyme appears to be remarkably more stable. This result is clearly limited to in vitro measurements, and should be confirmed by studies in vivo or at least in plasma, where the presence of proteic components may affect both activity and stability of the enzymatic system. The PPSPEG-SOD micellar conjugate shows therefore a functional similarity to the combination of SOD and catalase. A couple of points should be taken into account. (A) Safety: the final products of the scavenging cascade are sulfoxidecontaining, water-soluble polymers. Their low molecular weight (max ∼3000 g/mol) should ensure a relatively rapid renal clearance when in a nonaggregated (fully oxidized) form; on the other hand, their short-term toxicity is probably negligible: an ongoing study in our group (subject of a forthcoming publication) has shown very low cytotoxicity (IC50 > 20 mg/mL in L929 fibroblasts) independent of molecular weight. (B) Stability: PEGylation is known to reduce protein immunogenicity and proteolytic degradation, and we have shown that it has significantly increased the SOD stability against denaturation.

We therefore expect PPSPEG-SOD to have a significantly more sustained superoxide scavenging effect than SOD/ catalase.

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ASSOCIATED CONTENT

S Supporting Information *

Comparison of DLS and Nile Red quenching data for nonconjugate PPSPEG micelles, SDS pages for SOD PEGylation reaction products (different ratios SOD/PEG, reducing and nonreducing conditions). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +44 161 275 24 80. Fax: +44 161 275 23 96. E-mail: [email protected].

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ACKNOWLEDGMENTS P.H. thanks the University of Manchester for the award of an Overseas Research Studentship. The help of Ms. Jureerat Laliturai in cell culture is also gratefully acknowledged.

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ABBREVIATIONS DVS, Divinyl sulfone; PPS, Poly(propylene sulfide); PEG, Poly(ethylene glycol); SOD, Superoxide dismutase; TCEP, Tris(2-carboxyethyl)phosphine; SDS, Sodium dodecyl sulfate; 447

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BCA, Bicinchoninic Acid; WST, Water-soluble tetrazolium; MTS, 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium; PES, Phenazine ethosulfate

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