Permeability Effects on the Efficiency of Antioxidant Nanoreactors

Enzyme-loaded polymeric vesicles, or polymersomes, can be regarded as nanoreactors, which, for example, can be applied as artificial organelles...
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Permeability Effects on the Efficiency of Antioxidant Nanoreactors Iria Louzao† and Jan C. M. van Hest* Radboud University Nijmegen, Institute for Molecules and Materials, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands S Supporting Information *

ABSTRACT: Enzyme-loaded polymeric vesicles, or polymersomes, can be regarded as nanoreactors, which, for example, can be applied as artificial organelles. We implemented a naturally occurring enzymatic cascade reaction in two types of polymersomes, which are known to possess different permeability properties. The selected cascade reaction involved the antioxidant enzymes superoxide dismutase (SOD1) and catalase (CAT) for combating oxidative stress. The activities of both enzymes were investigated by spectrophotometric and electrochemical assays. Whereas the SOD1 substrate (the radical anion superoxide, O2•−) was able to penetrate both membranes equally well, the CAT substrate (H2O2) showed different rates of diffusion. When O2•− was generated inside polymersomes filled with both SOD1 and CAT, the activities of the two systems were comparable again.

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INTRODUCTION Polymeric vesicles, also known as polymersomes, have found broad applications in the nano- and biomedical sciences.1−8 The term polymersome originates from the structural analogy with liposomes, as both types of vesicles are composed of amphiphilic moieties forming a bilayer structure. The main difference between these two vesicular assemblies is that polymersomes are composed of amphiphilic block copolymers, whereas the liposomal bilayer consists of phospholipids. The higher molecular weight of the block copolymer with respect to the phospholipid leads to a larger membrane thickness, which translates to a higher robustness but also a lower permeability.9,10 Membrane permeability is an important factor that determines the potential applications of polymersomes.11,12 For instance, to be used as biocatalytic nanoreactor, the nanocontainer needs to be on the one hand permeable enough for substrates and products because they must be able to diffuse across the bilayer and on the other hand able to retain the enzymes inside. Methods for tuning permeability include the use of block copolymer blends and the inclusion of nanoparticles or channel proteins in the membrane.9,10,11−13 Intrinsically permeable polymersome nanoreactors were developed when the polymer shell was composed of polyelectrolytes or the block copolymer polystyrene40-b-poly(L-isocyanoalanine(2-thiophen-3-ylethyl)amide)50 (PS40-bPIAT50).14,15 More recently, polymersome-based nanoreactors were successfully internalized in living cells,16−18 retaining enzymatic activity of, for example, the model enzyme horseradish peroxidase. The decoration of the polymersome surface with cell penetrating peptides allowed the spontaneous uptake of these nanoreactors by different cell lines. The application of © 2013 American Chemical Society

polymersomes as artificial organelles, that is, introducing functional enzymes encapsulated in vesicles in the cellular environment,16−18 is a promising strategy to increase intracellular enzyme activity or to replace malfunctioning proteins. For this application to be of interest, therapeutically relevant enzymes are demanded to carry out their tasks inside polymersomes. Reactive oxygen species (ROS) are formed upon partial reduction of molecular oxygen (O2). These species are naturally formed during mitochondrial metabolism, especially in the complex I of the electron transport chain and during the tricarboxylic acid (TCA) cycle.19−21 Superoxide radical anion (O2•−) is known as the “primary” ROS, which can be subsequently and spontaneously converted into other ROS including hydrogen peroxide (H2O2). Under normal circumstances, the cell self-regulates the levels of these reactive species by means of antioxidant enzymes, for example, superoxide dismutase (SOD)22 and catalase (CAT), which neutralize O2•− and H2O2, respectively. When ROS production exceeds the capacity of the cellular defenses, these highly reactive species produce severe damage to cell structures, including lipids and membranes, proteins, and DNA.23 This deleterious process is known as oxidative stress, and it is related to several pathologies and diseases, such as mitochondrial dysfunction, cardiovascular disease, cancer,24 neurological disorders (including Parkinson25 and Alzheimer’s diseases), diabetes, ischemia/reperfusion, and the normal process of aging.23,26 A recent attempt to combat ROS consisted of an antioxidant cascade reaction implemented in poly(2-methyloxazoline)-bReceived: April 8, 2013 Revised: May 29, 2013 Published: May 30, 2013 2364

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space and in peroxisomes.21,32 Catalase is a homotetramer of 250 kDa33 situated in the mitochondria, cytosol, and peroxisomes.21,32 As nanocontainers, the intrinsically permeable PS-PIAT (1, Figure 1B) and the more traditional polystyrene160-b-poly(ethylene glycol)24 (PS160-b-PEG24, 2) polymersomes were chosen (Figure 1B) because of the previously observed large differences in enzymatic activity when enzymes were enclosed in the lumen of these two different types of polymersomes (Figure 1C).15,34 The catalytic activities of the coencapsulated enzymes were analyzed either by adding H2O2 or by generating O2•− in situ by means of the reaction of β-nicotine adenine dinucleotide in reduced form (NADH) with the electron carrier phenazine methosulfate (PMS).35 Spectrophotometric and electrochemical methods were used to study the enzymatic activities. Although the substrates for the two enzymes are small in size, differences in activity were observed and related to the different permeability properties of the polymersomes used.

poly(dimethylsiloxane)-b-poly(2-methyloxazoline) (PMOXAPDMS-PMOXA) polymersomes as well as an enzyme mimic.18,27 The enzymatic cascade involved the action of superoxide dismutase (SOD) and a lactoperoxidase (LPO) for the detoxification of ROS.18 SOD dismutates O2•− into water and H2O228 (Figure 1A). Then, LPO oxidized a reporter

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EXPERIMENTAL SECTION

Materials. PS-b-PEG 1 was prepared as previously described.36 CuBr (Aldrich, 99.999%), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA) (Sigma-Aldrich, 99%), tert-butyl-2-bromo-isobutyrate (Fluka, 99%), Anisole (Acros, 99%), deuterated chloroform (CDCl3) (Aldrich, 99.8%), PEG22-OMe (Fluka), InCl3 (Aldrich), bis(2,2′-bipyridine)-(5-isothiocyanato-phenanthroline)ruthenium bis(hexafluorophosphate) (Sigma-Aldrich), nitro blue tetrazolium (Aldrich), Purpald (Sigma-Aldrich), H2O2, nicotine adenine dinucleotide in reduced form (NADH), superoxide dismutase (SOD1, Sigma) from bovine erythrocytes, catalase from bovine liver (CAT, Aldrich), and bovine serum albumin (BSA) fraction V (Sigma) were used as received. PS40-b-PIAT50 was purchased from Encapson. Styrene (Aldrich) was distilled before use. Transmission Electron Microscopy (TEM). TEM was performed on a JEOL JEM 1010 microscope with an acceleration voltage of 60 kV equipped with a charge-coupled device (CCD) camera. Sample specimens were prepared by placing a drop (10 μL) of a diluted aqueous vesicle solution on an EM science carbon-coated copper grid (200 mesh) for 15 min. The grid was washed with Milli-Q water. The grid was finally air-dried for at least 3 h and analyzed without further treatment. Scanning Electron Microscopy (SEM). SEM was performed on a JEOL 6330 cryo field emission scanning electron microscope (FESEM). A fraction of 10 μL of the sample to be visualized was placed on a silicon wafer substrate. The excess of the sample was removed from the substrate after 15 min and gently washed with MilliQ. The substrate was allowed to dry overnight. Samples were coated in 1.5 nm Pt/Au by using a BALZERS sputter machine. The samples were visualized by the scanning electron microscope. Enzymatic Activities. Enzymatic activities were analyzed with a UV−visible Cary spectrometer (Varian), in either kinetic or scan mode (23 °C). Oxygen Measurements. Oxygen measurements were performed with a Clark-type oxygen sensor (Hansatech Instruments). The electrode possesses a platinum cathode and a silver anode. KCl (17.5 g/100 mL Milli-Q water) was used as electrolyte. The platinum cathode was separated from the sample chamber by a paper spacer and a PTFE membrane. All experiments were performed at 25 °C. Preparation of PS-PIAT and PS-PEG Polymersomes. A solution of PS-PIAT or PS-PEG in THF (1.0 or 1.5 mg/mL, respectively) was prepared. 0.5 mL of this solution was injected into 2.5 mL of phosphate buffer (20 mM, pH 7.4) at a rate of 1 mL h−1 and at a stirring rate of 350 rpm. The buffered solution contained either 1150 μg of catalase, 150 μg SOD1, or both. The resulting suspension was allowed to self-assemble for 30 min. The unencapsulated enzymes and the organic solvent were removed by dialysis (1000 kDa MWCO, Spectra/Por Biotech) against phosphate buffer. The polymersome

Figure 1. Biocatalytic reactions involving SOD and CAT (A). Chemical structures of the block copolymers, 1: PS-PIAT, 2: PSPEG (B). Schematic representation of the enzymatic cascade reaction in SOD- and CAT-loaded polymersomes (C).

molecule, amplex red, in the presence of H2O2 to obtain a fluorescent product, resorufin. The low permeability of PMOXA-PDMS-PMOXA polymersomes needed to be increased for the LPO substrate and product by incorporating the outer membrane protein F (OmpF). Because primary ROS are small molecules, it could be expected that they have the ability to cross (almost) every polymersome membrane, even though it was already described that some polymersome membranes have a very low permeability even for water.11,29 The present report aims to investigate the catalytic activity of enzymes involved in ROS decomposition when enclosed in polymersomes with different levels of permeability. For this study, two enzymes were encapsulated to perform a natural cascade reaction to regulate ROS levels: SOD, more specifically copper−zinc superoxide dismutase (Cu/Zn SOD or SOD1), and catalase (CAT). CAT decomposes H2O2 in dismutase and peroxidase reactions.30 Whereas in the case of the dismutase reaction H2O2 is the sole substrate for CAT (Figure 1a), peroxidase activity requires other molecules such as alcohols or aldehydes to be oxidized, while H2O2 is reduced. SOD1 is a homodimeric protein of 32.5 kDa located mostly in the cytosol31 but also in the mitochondrial intermembrane 2365

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Figure 2. TEM (A,C) and SEM (B,D) micrographs of SOD1- and CAT-loaded PS-PIAT (A,B) and PS-PEG (C,D) polymersomes. Scale bars: 1 μm. suspension was dialyzed against phosphate buffer for 36 h, changing the buffer solution up to four times. After dialysis, the presence of free enzymes was checked by using an Amicon Ultra Free-MC centrifugal filter with a cut off of 0.1 μm. The suspension was centrifuged at 3000 rpm at 4 °C for 10 min and the filtrate was analyzed for enzymatic activity. Ru-Labeling of Enzymes. The enzymes SOD1 and CAT were dissolved in carbonate buffer (0.1 M, pH 9.0) at 1 mg/mL. Bis(2,2′bipyridine)-(5-isothiocyanato-phenanthroline) ruthenium bis(hexafluorophosphate) (10 and 5 equiv for SOD and CAT, respectively) dissolved in ultrapure water was added to the enzyme solution (1 mg/mL) in carbonate buffer (0.1 M, pH 9). The brightyellow solution was shaken at 4 °C. The unbound ruthenium complex was removed by size exclusion chromatography using Sephadex G-50. Fractions containing the labeled protein were concentrated using an Amicon Ultra centrifugal filter with either 10 (SOD1, 32 kDa) or 100 kDa (CAT, 250 kDa) cutoff membranes. The enzymatic solution was concentrated by centrifugation (5 min, 10.000 rpm) and refilled several times with phosphate buffer to remove any remaining unbound complex. The polymersome preparation with one of the labeled enzymes dissolved in Milli-Q or phosphate buffer was performed by using the same concentrations and procedure used for the unlabeled enzymes, with one exception: for the polymersomes prepared in phosphate buffer, the outer buffered solution was gradually decreased in ionic strength to reach Milli-Q water during dialysis to avoid any interference during ICP-MS measurements. Inductively Coupled Plasma−Mass Spectrometry (ICP-MS). The polymersome suspensions, containing Ru-labeled enzymes, and a solution of the Ru-labeled enzyme of known concentration were lyophilized and subsequently digested in concentrated nitric acid (65%, 0.50 mL) for 3 h at 80 °C. After cooling the samples to room temperature, an internal standard of InCl3 (1.50 mL of 0.49 mg/mL) was added. The total volume of each sample was adjusted to 5.00 mL with Milli-Q. ICP-MS measurements were performed on a Thermo Fisher Scientific Xseries I quadrupole machine. The measured values of Ru in ppb were translated into encapsulated amount of protein by

comparing the results to samples containing a known amount of Rulabeled enzyme. SOD1 Assay.37 Stock solutions of NADH (0.59 mg/mL in phosphate buffer 20 mM pH 7.4), NBT (0.49 mg/mL in phosphate buffer 20 mM pH 7.4), and PMS (88 μg/mL in Milli-Q) were prepared. A sample of polymersomes (typically 150 μL) was diluted with phosphate buffer to 975 μL, and subsequently NADH (100 μL) and NBT (100 μL) solutions were added. The UV−vis measurement at 560 nm started as soon as PMS (25 μL) was added to the solution (total volume: 1.2 mL). Data were taken during the first 5 min of the experiments. Oxygen Electrode. Catalase Assay. The oxygen electrode was calibrated just before use. Calibration was performed in a temperaturecontrolled (25 °C) cylindrical chamber (borosilicate glass) equipped with magnetic stirring (30 rpm). Oxygraph software (Hansatech Instruments) calibrated the electrode by setting the temperature and pressure (101.32 kPa was used) and by measuring the oxygen content of air-saturated deionized water, followed by zero oxygen water. The first solution was prepared by vigorous shaking fresh deionized water, and the latter one was prepared by dissolving sodium dithionite in deionized water. The chamber was carefully washed and rinsed with water and buffer before starting the measurements to remove any sodium dithionite that could affect the measurements. Measurements were taken every 0.5 s. A known volume of polymersome sample representing ∼10% of a described batch (typically 300 μL of 3.0 mL) and additional buffer up to 1.1 mL (800 μL) was placed in the chamber. Once the oxygen measurement was stable (plateau signal), 30 μL of a H2O2 solution (44 mM in phosphate buffer) was added. The increase in the oxygen concentration (in nmol/mL) was recorded. The slopes were compared with a Catalase activity calibration curve. Oxygen Electrode. Cascade Reaction Assay. The oxygen electrode was calibrated just before use. Calibration was performed in a temperature-controlled (25 °C) cylindrical chamber (borosilicate glass) equipped with magnetic stirring (30 rpm). Oxygraph software (Hansatech Instruments) calibrated the electrode by setting the temperature and pressure (101.32 kPa was used) and by measuring the oxygen content of air-saturated deionized water, followed by zero oxygen water. The first solution was prepared by vigorously shaking 2366

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Figure 3. Representation of the covalent labeling of lysine residues of SOD1 (PDB ID 1SXS) with bis(2,2′-bipyridine)-(5-isothiocyanatophenanthroline) ruthenium bis(hexafluorophosphate) 3 (A). Schematic description of the different combinations of (un)labeled enzymes and polymersomes for the estimation of encapsulation efficiencies (B).

Table 1. Encapsulation Efficiencies of Encapsulated Enzymes in Both PS-PIAT and PS-PEG Polymersomes

a

labeled enzyme

block copolymer

enzymatic system

SOD SOD CAT CAT SOD SOD CAT CAT

PS-PIAT PS-PEG PS-PIAT PS-PEG PS-PIAT PS-PEG PS-PIAT PS-PEG

SOD,CAT SOD,CAT SOD,CAT SOD,CAT SOD SOD CAT CAT

absolute amount encapsulated (nmol)a 0.13−0.15 0.11−0.13 1.38−1.67 2.18−2.24 0.18−0.22 0.07−0.11 2.34−2.93 2.28−2.84

(0.58b) (0.63b) (0.35b) (0.24b) (0.99b) (0.76b)

efficiency (%) 3.0 ± 0.3 2.6 ± 0.3 33.2 ± 3.1 (12.7b) 48.0 ± 0.7 (13.6b) 4.4 ± 0.4 (7.8b) 2.0 ± 0.4 (5.3b) 57.3 ± 6.5 (21.5b) 55.7 ± 6.1 (16.5b)

Initial amounts 1:1 molar ratio (4.60 nmol). bEncapsulation performed in ultrapure water instead of phosphate buffer (20 mM, pH 7.4).

fresh deionized water, and the latter one was prepared by dissolving sodium dithionite in deionized water. The chamber was carefully washed and rinsed with water and buffer before starting the measurements to remove any sodium dithionite that could affect the measurements. Measurements were taken every 0.5 s. Stock solutions of NADH (0.59 mg/mL in phosphate buffer 20 mM pH 7.4), NBT (0.49 mg/mL in phosphate buffer 20 mM pH 7.4), and PMS (88 μg/ mL in Milli-Q) were prepared. A known volume of polymersome sample representing ∼10% of a described batch (typically 300 μL of 3.0 mL), 100 μL of the NADH stock solution, and additional buffer up to 1.1 mL (700 μL) was placed in the chamber. Once a stable level of oxygen was reached, 20 μL of the PMS stock solution was added. The oxygen levels were recorded (in nmol/mL). Free enzyme cascade: Absolute amounts of 0.56 μg of SOD1 and 45 μg of CAT, which are in the range of the amounts encapsulated in polymersomes, were used for the assays. Data treatment: The pressure was set as 760 mmHg

(101.32 kPa) and was not corrected. Therefore, the variations in the oxygen levels were transformed into Δ[O2] (nmol/mL) to eliminate the variability of the starting oxygen levels.

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RESULTS AND DISCUSSION Coencapsulation of the Enzymes in Polymersomes. Because the product of SOD1 is H2O2, another ROS, both SOD and CAT can work in tandem to neutralize ROS (Figure 1A,C). This can be most efficiently done by coencapsulating both enzymes in the polymersomes. The enzyme-filled polymersomes were prepared by the nanoprecipitation method, following procedures previously described.16 An organic solution of the block copolymers in THF (1 mg/mL, 0.5 mL) was injected into a buffered solution containing both enzymes in a 1:1 molar ratio (1.9 μM, 2.5 mL). After 30 min of 2367

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Figure 4. Reduction of NBT to diformazan by superoxide, and inhibition of this reaction by SOD1 (A). Example of a kinetic reduction (duplo) in the presence of empty PS-PIAT polymersomes (control, blue) and SOD1,CAT-loaded PS-PIAT polymersomes experiment (green) (B). TEM micrograph of SOD1,CAT-loaded PS-PEG polymersomes after the kinetic experiment. (C) Scale bar: 200 nm.

polymersomes, respectively. The corresponding number of coencapsulated CAT molecules was 623 ± 186 and 1270 ± 200 for PS-PIAT and PS-PEG polymersomes, respectively (see Supporting Information for details, Table S1 and Figure S2). These encapsulation numbers for both enzymes are significantly higher than expected based on statistical encapsulation, which demonstrates that polymer−protein interactions take place upon polymersome formation. Although of course a distribution in number of enzymes per polymersome exists, the average values are sufficiently high to diminish the probability of a significant number of empty or single-enzyme encapsulating polymersomes. Interestingly, we found different values for CAT loading depending on the encapsulation conditions. When the enzymes were dissolved in ultrapure water, the encapsulation efficiencies were much lower than by using phosphate buffer (20 mM, pH 7.4). These higher encapsulation efficiencies in buffer furthermore substantiate the hypothesis that interactions take place between polymer and protein during the polymersome formation, where the presence of hydrophobic domains may have an influence on the encapsulation process.18 Enzymatic Activities. SOD Assays. SOD1 activity was measured by a colorimetric assay. The redox indicator, nitro blue tetrazolium chloride (NBT), is transformed into diformazan upon reduction by O2•−.35,37 This reduction induces a change in color from yellow to blue; therefore, the product shows a maximum in absorbance at 560 nm (Figure 4A). The formation of diformazan can be monitored when O2•− is generated in situ.35 When SOD1 is present, the disproportionation of O2•− into H2O2 and O2 is catalyzed, so the reduction of NBT is inhibited to a certain extent, which depends on the SOD1 concentration (Figure 4B). The level of

equilibration, the organic solvent and the unencapsulated enzymes were removed by dialysis. The resulting suspension was visualized by transmission and scanning electron microscopy techniques (TEM and SEM, respectively). Micrographs showed spherical shapes for the obtained structures (Figure 2). Encapsulation Efficiencies. The encapsulated amount of each enzyme determines of course the maximum expected efficiency of the enzymes when compared with the free enzyme activities. To estimate the encapsulation efficiencies of each enzyme in the coencapsulated system, we covalently labeled both proteins by means of reaction of some of the lysine residues with a commercially available ruthenium complex containing an isothiocyanate functional group (3, Figure 3A).38 These labeled enzymes (Ru-SOD and Ru-CAT) were separately encapsulated together with the corresponding unlabeled enzyme (Figure 3B), in both PS-PIAT and PS-PEG polymersomes. TEM analysis did not show significant differences in polymersome morphology when compared with the unlabeled enzyme-loaded polymersomes (Figure S1, Supporting Information). The ruthenium content of each polymersome batch was measured by inductively coupled plasma-mass spectrometry (ICP-MS) and compared with the ruthenium content of a known amount of labeled protein. The results are shown in Table 1. ICP-MS results showed much lower encapsulation efficiencies for SOD1 than for CAT. Similar differences regarding the encapsulation efficiencies of several proteins were already reported,18 even for similar proteins such as hemoglobin and myoglobin.39 According to these results, the average number of SOD1 molecules in a polymersome with a diameter of 120 nm was 60 ± 10 and 58 ± 8 in PS-PIAT and PS-PEG 2368

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Table 2. Observed SOD1 Activity Expressed as Percentage of Inhibition of NBT Reduction When Compared with the Activity of the Initial Amount of Free SOD1 Used for Encapsulationa

a

enzymatic system

block copolymer

SOD/CAT SOD/CAT SOD SOD

PS-PIAT PS-PEG PS-PIAT PS-PEG

observed activity (%)b 3.05 2.89 6.98 2.77

± ± ± ±

encapsulation efficiency (%)

activity/encapsulation efficiency ratio

± ± ± ±

0.8−1.3 0.9−1.3 1.0−2.2 0.9−2.1

0.38 0.14 1.92 0.69

3.0 2.6 4.4 2.0

0.3 0.3 0.4 0.4

SOD1 encapsulation efficiencies are used to determine the activity/encapsulation efficiency ratio.40 bCalculated in the first 5 min of the experiment.

Table 3. Activity of CAT in SOD1,CAT-Loaded PS-PIAT and PS-PEG Polymersomes, Relative to Free CAT CAT,SOD PS-PIAT polymersomes time (s) 5 10 15 20 time (s) 5 10 15 20

observed activity (%) 1.32 1.00 1.03 1.12

± ± ± ±

V (10−7 M s−1)

a

0.49 0.41 0.30 0.24

5.37 ± 2.00 4.06 ± 1.65 4.20 ± 1.21 4.55 ± 0.9 CAT,SOD PS-PEG polymersomes V (10−7 M s−1)

observed activity (%)a 0.14 0.16 0.18 0.18

± ± ± ±

apparent kcat/KM (M−1 s−1)b

0.02 0.04 0.04 0.03

1.77 2.15 2.35 2.36

± ± ± ±

0.12 0.22 0.23 0.21

(3.1 (2.4 (2.4 (2.6

± ± ± ±

1.2) 1.0) 0.7) 0.6)

× × × ×

103 103 103 103

apparent kcat/KM (M−1 s−1)b (7.6 (9.2 (10.1 (10.2

± ± ± ±

0.5) 0.9) 1.0) 0.9)

× × × ×

102 102 102 102

a Activity is given in % of the activity of the amount of free CAT (358 and 544 μg for PS-PIAT and PS-PEG polymersomes. respectively). b[E] = 1.17 × 10−7 M for PS-PIAT and 1.58 × 10−7 M for PS-PEG; [S] = 1.47 mM.

interesting dismutase, or catalytic activity (Figure 1A) of CAT. To investigate whether CAT was active inside PS-PEG polymersomes, a sample was added to a solution containing H2O2. An immediate increase in the oxygen content of the solution was detected. This fact allowed us to conclude that CAT was active inside PS-PEG polymersomes and that the membrane was permeable for H2O2. A further quantification of the CAT activity was also carried out. For this purpose, a calibration was prepared by using different concentrations of free CAT (Figure S5, Supporting Information). The slope of the O2 formation just after adding H2O2 was used for correlating concentration and activity, and a linear behavior was obtained. The O2 production profile of CAT-loaded polymersomes was different for the two types of capsules (Figure S6, Supporting Information), showing that CAT activity was higher in PS-PIAT capsules (Table 3). For PS-PIAT polymersomes, the reaction rate was, as expected, maximal at the start of the experiment. In the case of filled PS-PEG polymersomes (Table 3), the maximum reaction rate was reached 15 s after adding the substrate, which indicates that a diffusion process precedes the actual enzyme reaction. These kinetic trends were similar for polymersomes filled with both SOD1 and CAT (Table 3) or with CAT only (Table S2, Supporting Information). Because of the allosteric character of CAT, KM is a function of the concentration of substrate. KM values can be found in the literature, for instance, 372 mM (93 mM per subunit)41 and 1.1 M.42 CAT activity was also described to follow a Michaelis− Menten-like behavior at concentrations of H2O2 below 200 mM. In this case, and assuming that the initial concentration of H2O2 in the experiments (1.47 mM) is negligible in comparison with the Michaelis−Menten constant, KM, the relationship between velocity (v), kcat, KM, enzyme ([E]0), and

inhibition can be compared with a calibration curve based on the activity of known concentrations of SOD1 (Figure S3, Supporting Information). The activities, expressed as percentage of inhibition, were determined for SOD1 encapsulated both with and without CAT and in both PS-PIAT and PS-PEG polymersomes (Table 2). The activities were normalized to the encapsulation efficiencies measured by ICP-MS. The corresponding activity ratio between free and encapsulated enzyme was in all cases close to 1 (Table 2), which suggests that there is not a significant effect of the encapsulation on SOD1 activity, that is, the diffusion of O2•− across the polymersome membrane. The integrity of the polymersomes after being in contact with superoxide was furthermore confirmed by TEM (Figure 4C). Enzymatic Activities. CAT Assays. When a spectrophotometric assay was used for determining the peroxidatic activity of catalase, CAT activity was observed when the enzyme was encapsulated in PS-PIAT polymersomes, and an absence of activity was found in the case of PS-PEG polymersomes. The experiment was based on the ability of CAT to oxidize methanol to formaldehyde in the presence of H2O2. A dye for the quantification of the outgoing formaldehyde was used (Figure S4, Supporting Information). This lack of activity can be attributed to a very low permeability of the PS-PEG polymersome membrane for at least one of the reactants in this assay, not necessarily being H2O2. To ensure that only the permeability for H2O2 was taken into account, the dismutase activity of CAT was studied instead. Consequently, a Clark-type oxygen electrode was utilized. The oxygen sensor consists of an electrochemical cell equipped with two electrodes, a silver anode and a platinum cathode, immersed in an electrolyte solution (KCl). By the reduction of the dissolved O2 in the cathode, the O2 concentration in the sample is measured. The advantage of this method resides in the simplicity of the required reagents, measuring the more 2369

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substrate ([S]) concentration represented in eq 1 can be simplified, as is shown in eq 2. v=

kcat[E][S] KM + [S]

(1)

v=

kcat [E][S] KM

(2)

Therefore, the catalytic efficiency, kcat/KM, is directly obtained from the reaction rates as a parameter to evaluate the effect of both vesicles on the CAT performance. According to these data, PS-PIAT-based nanoreactors are 4.1 and 2.5 times more efficient than PS-PEG polymersomes at a reaction time of 5 and 20 s, respectively, for the coencapsulated system. Cascade Reaction. The variation of oxygen levels during the process of generation and consumption of ROS makes the oxygen sensor also suitable for enzymatic cascade activity tests. After O2•− is produced, it will dismutate into H2O2 and O2 by the action of SOD1 mainly inside polymersomes. As a result, H2O2 is produced in the presence of CAT that should catalyze its conversion into H2O and O2, without the necessity of the diffusion of the substrate. The released O2 is then measured. O2•− was generated in situ as previously described, by means of NADH and PMS. To generate O2•−, O2 dissolved in an airequilibrated solution was consumed, which was detected by the initial decrease in O2 levels. First, an experiment was performed with SOD1 and CAT separately encapsulated in polymersomes. PMS was added into an air-equilibrated buffered solution containing NADH (Figure 5), thereby lowering the O2 concentration. Upon adding SOD1-loaded polymersomes, the O2 content increased, according to the reaction scheme shown in Figure 5. The regenerated oxygen can enter again in the catalytic cycle for O2•− formation until all NADH is consumed. Next, CATloaded polymersomes were added, increasing again the level of O2 in the solution, which indicates that the H2O2 formed during the SOD1-catalyzed process was being consumed (Figure 5). Next, the activity of both types of polymersomes in which SOD1 and CAT were coencapsulated was measured using the oxygen electrode method. For this purpose, PMS was added to an air-equilibrated buffered solution containing NADH and the enzyme-filled polymersomes. The profile of the oxygen levels during the experiment was V-shaped in all cases, showing that at the beginning of the reaction O2 levels decreased during the formation of O2•− and subsequently increased again due to SOD1 and CAT activity. The oxygen production of both polymersomes was three times less than a cascade reaction performed with the free enzymes, which was used as positive control. BSA-loaded polymersomes were used as a negative control, and these values were subtracted from the O2 level profiles of the corresponding loaded polymersomes to obtain the data shown in Figure 6.43,44 The complete kinetic behavior of the different systems can be found in the Supporting Information (Figure S7). The activity of the PS-PIAT and PS-PEG polymersome nanoreactors is similar, and the previously observed differences in reaction rate between CAT-filled polymersomes are thus not observed for these cascade reactions. This can be explained by the fact that H2O2 production takes place inside the polymersome nanoreactors, (due to SOD1 activity) and

Figure 5. Oxygen level profile during a cascade reaction catalyzed by SOD1 and CAT encapsulated separately in PS-PIAT (A) and PS-PEG (B) polymersomes. Superoxide is generated in situ upon addition of PMS to a buffered solution containing NADH (1). SOD1-loadedpolymersomes catalyze the formation of O2 and H2O2 (2). Subsequently, CAT-loaded polymersomes catalyze the dismutation of H2O2 into H2O and O2. Non-normalized graphics.

Figure 6. Oxygen levels registered in the presence of SOD,CAT PSPEG and SOD,CAT-PS-PIAT polymersomes as well as for the free enzymes in solution (in duplo, from left to right) upon in situ generation of superoxide. Normalized with respect to experiments performed with BSA-filled polymersomes (data taken at 400 s of experiment).

diffusion of H2O2 across a barrier is not necessary anymore to reach CAT. The difference in activity between the encapsulated and free enzymes can be explained by a confinement effect. Although the overall concentrations of SOD1 and CAT are similar in all experiments (9−16 and 105−161 nM of SOD1 and CAT, respectively), the effective enzyme concentration inside the polymersomes is much higher ([SOD1] is 879 μM and [CAT] 2370

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Biomacromolecules is 9.1 mM for 120 nm diameter PS-PIAT polymersomes; [SOD1] is 270 μM and [CAT] is 4.8 mM for 120 nm diameter PS-PEG polymersomes). The confinement therefore provokes an inhomogeneity regarding the concentration of enzymes in the reaction vessel with respect to the free enzymes. Because the substrate concentration is homogeneous, enzyme usage will be less efficient because many enzymes will not have a chance to encounter substrates.45 This is especially the case for CAT. Because of the encapsulation ratio of SOD and CAT (1:10 and 1:18 respectively for PS-PIAT and PS-PEG polymersomes), CAT is certainly not saturated.

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REFERENCES

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CONCLUSIONS We have presented a comparison of the performance of a naturally occurring enzymatic cascade reaction inside two types of polymersomes, composed of PS-PIAT and PS-PEG. The cascade reaction focused on counteracting oxidative stress, that is, in regulating the levels of ROS. For this purpose, SOD1 (to neutralize O2•−) and CAT (to neutralize H2O2) were encapsulated. When single-enzyme reactions were studied, clear differences were observed between different types of polymersomes and types of ROS. The activity of SOD1 in PSPEG or PS-PIAT polymersomes was comparable, showing that the diffusion of O2•− across either membrane does not seem to significantly impair SOD1 activity. The differences in CAT kinetics and activity were noticeable, which can be explained by a different rate of diffusion of H2O2 across the polymersome membranes, with the PS-PIAT polymersome being the more permeable. When both enzymes were encapsulated, H2O2 was generated inside the vesicles, and the cascade reaction showed similar activity in both nanoreactors. The activity of the cascade nanoreactors was lower than that for the free enzymes. This can be explained by the confinement of enzymes in a small polymersome volume, which lowers the efficient use of especially the CAT enzyme. Our enzyme-filled polymersomes are able to counteract the effects of ROS, but the effectiveness depends on the type of ROS and the permeability of the vesicle. Whereas both vesicles possess a similar permeability for O2•−, the difference in permeability regarding H2O2 is enough to modify the kinetic behavior of CAT. ASSOCIATED CONTENT

S Supporting Information *

Calculation of the encapsulated enzymes, particle size distributions measured by nanoparticle tracking analysis (NTA), synthesis and characterization of PS-PEG block copolymer, calibration details, additional assays, and EM pictures. This material is available free of charge via the Internet at http://pubs.acs.org.

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ACKNOWLEDGMENTS

We thank the Dutch Science Foundation (NWO) for funding. I.L. thanks the “Barrie de la Maza” Foundation and the Spanish Ministry of Economy and Competitiveness for financial support. Jan van Hest acknowledges funding from the Ministry of Education, Culture and Science (Gravity program 024.001.035). We also thank Dr. Sanne Schoffelen for helpful discussions regarding enzyme kinetics.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +31 24 36 53204. Fax: (+31) 24-36-53393. E-mail: J. [email protected]. Present Address †

School of Pharmacy, The University of Nottingham, University Park, Nottingham NG7 2RD, UK.

Notes

The authors declare no competing financial interest. 2371

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