Poly(dimethylsiloxane) - American Chemical Society

Mar 20, 2014 - Institute of Chemistry and Bioanalytics, School of Life Sciences, University of Applied Sciences and Arts Northwestern Switzerland,...
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Poly(N‑vinylpyrrolidone)-Poly(dimethylsiloxane)-Based Polymersome Nanoreactors for Laccase-Catalyzed Biotransformations Mariana Spulber,† Patric Baumann,† Sina S. Saxer,‡ Uwe Pieles,‡ Wolfgang Meier,† and Nico Bruns*,†,§ †

Department of Chemistry, University of Basel, Klingelbergstrasse 80, 4056 Basel, Switzerland Institute of Chemistry and Bioanalytics, School of Life Sciences, University of Applied Sciences and Arts Northwestern Switzerland, Gründenstrasse 40, 4132 Muttenz, Switzerland § Adolphe Merkle Institute, University of Fribourg, Rte de l’Ancienne Papeterie, P.O. Box 209, 1723 Marly 1, Switzerland ‡

S Supporting Information *

ABSTRACT: Laccases (Lac) are oxidizing enzymes with a broad range of applications, for example, in soil remediation, as bleaching agent in the textile industry, and for cosmetics. Protecting the enzyme against degradation and inhibition is of great importance for many of these applications. Polymer vesicles (polymersomes) from poly(N-vinylpyrrolidone)-block-poly(dimethylsiloxane)-block-poly(N-vinylpyrrolidone) (PNVP-b-PDMS-b-PNVP) triblock copolymers were prepared and investigated as intrinsically semipermeable nanoreactors for Lac. The block copolymers allow oxygen to enter and reactive oxygen species (ROS) to leave the polymersomes. EPR spectroscopy proved that Lac can generate ROS. They could diffuse out of the polymersome and oxidize an aromatic substrate outside the vesicles. Michaelis−Menten constants Km between 60 and 143 μM and turn over numbers kcat of 0.11 to 0.18 s−1 were determined for Lac in the nanoreactors. The molecular weight and the PDMS-to-PNVP ratio of the block copolymers influenced these apparent Michaelis−Menten parameters. Encapsulation of Lac in the polymersomes significantly protected the enzyme against enzymatic degradation and against small inhibitors: proteinase K caused 90% less degradation and the inhibitor sodium azide did not affect the enzyme’s activity. Therefore, these polymer nanoreactors are an effective means to stabilize laccase.

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electroimmunoassays.26 Recent papers indicated their antiproliferative activity against cancer cells27 and their ability to degrade endocrine disruptors known to induce abnormalities in hormone release, immune system, and cell proliferation.28 Laccases couple one-electron oxidation of substrates such as phenols and aromatic amines to the four electron reduction of O2 to water.1−3 Whether or not laccases are also able to generate reactive oxygen species (ROS) is an ongoing scientific debate and the published results are controversial.29−32 In view of the laccases’ wide range of applications, the optimization of their stability and activity is of high interest. This can be achieved, for example, by means of protein engineering2 or by immobilization.2,33−35 An alternative is the stabilization of laccase within micro- and nanocapsules. Laccase has been encapsulated into poly(ethyleneimine) (PEI) microcapsules. However, PEI interacts with the enzyme’s substrate, preventing it from leaving the capsules.36 Liposomes have been used to stabilize laccases.37,38 Such lipid vesicles can protect their guests from digestion by proteases. However, liposomes can be unstable over time and leaky.39 Polymer vesicles, also

INTRODUCTION Laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) are enzymes with a broad range of industrial, chemical, and biotechnological applications as oxidizing agents.1−3 They have been used extensively in the textile industry for bleaching and dyeing,2 for the synthesis of dyes,4 or for modification of the surface of fibers.5 Moreover, they are able to oxidize lignin6 and have therefore been used in paper manufacturing. Their applications in soil remediation are related to their ability to degrade persistent environmental pollutants,2,7,8 for example, polycyclic aromatic hydrocarbons.9 Moreover, they have been used to immobilize degradation products of trinitrotoluene onto soil organic matter.10 When encapsulated in a conductive polymer matrix such as polypyrrole, laccases can be used in biofuel cells.11,12 In chemical synthesis, laccases are efficient catalysts for the preparation of anticancer drugs,13−15 and for the oxidation of polyaromatic hydrocarbons16 and of fullerene.17 The enzymes can furthermore catalyze polymerization reactions18−22 and can be added as environmentally friendly curing agent to coatings.23 In cosmetics, they can be used as oxidizing agents, milder than hydrogen peroxide.2 Their applications expand into the biomedical field due to the ability of laccases to function as biosensors for phenolic agents,2 azides,2 morphine and codeine,24 chatecolamines,25 or in © 2014 American Chemical Society

Received: January 17, 2014 Revised: March 18, 2014 Published: March 20, 2014 1469

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doubly distilled H2O, adjusting the pH with HCl and completing the volume to 1 L. Synthesis of PNVP-b-PDMS-b-PNVP. The synthesis of amphiphilic PDMS-PNVP triblock copolymers was performed in analogy to a protocol described by Simionescu and co-workers.62 In brief, α,ωbis(aminopropyl)poly(dimethylsiloxanes) (H2N-PDMS-NH2) were synthesized by equilibrium polymerization of D4 and APTES in the presence of the catalyst tetramethylamonium siloxanolate. The catalyst was synthesized from D4 and tetramethylammonium hydroxide (80 °C, 24 h in dry toluene). Two polymers were prepared: H2N-PDMSNH21 [Mn = 1400 g mol−1 (determined by 1H NMR), Mn = 1480 g mol−1, PDI = 1.2 (determined by GPC in chloroform)] and H2NPDMS-NH22 [Mn = 2900 g mol−1 (1H NMR), Mn = 3020 g mol−1, PDI = 1.2 (GPC in chloroform)]. The condensation of H2N-PDMSNH2 with 4,4-azobiscyanovaleryl chloride in equimolar amounts yielded polydimethylsiloxanes with azo end groups. They were used as macroinitiators for radical polymerizations of N-vinylpyrrolidone, yielding PDMS-PNVP triblock copolymers (Scheme 1 and Table 1).

termed polymersomes, are substantially more stable and robust than lipid vesicles.40,41 Therefore, they are better suited for technical applications. In order to use polymersomes as nanoscale reaction vessels, two main requirements have to be fulfilled.40 First, catalytically active guests should be encapsulated within their inner compartment to confine the reaction into the polymersomes, and second the polymer membrane that separates the inner aqueous compartment from the outer bulk solution should allow the exchange of substrates between the outside and the inside of polymersomes.40−43 The encapsulation of enzymes into polymersomes is a straightforward process and has been demonstrated, for example, with peroxidases,44−46 penicillin acylase,47 superoxide dismutase,48−51 catalase,50,51 myoglobin,52−54 lysozyme,54 lipase,55,56 phenylacetone monooxygenase,56 alcalase,56 alcohol dehydrogenase,56,57 and alkaline phosphatase.58 To the best of our knowledge, laccase-containing polymersomes have not been reported. The permeabilization of polymersome membranes is more difficult to achieve than the encapsulation of enzymes. Successful permeabilization strategies include the reconstitution of membrane proteins,47,59 the use of block copolymers that form porous membranes,44 or the reaction of photoactive reagents with block copolymer membranes.46,52 Here we report the encapsulation of Lac in polymersomes that were specifically designed to function as nanoreactors for laccase. In order to reduce the complexity of the nanoreactor preparation and therefore to increase the chance of its technological application, we chose to assemble polymersomes from block copolymers that are highly permeable to oxygen and to reactive oxygen species. Poly(N-vinylpyrrolidone) and poly(dimethylsiloxane) are polymers known for their exceptional high permeability to these substances.48,49,60,61 Moreover, they are of opposing hydrophilicity, so that block copolymers thereof are amphiphilic. Therefore, poly(N-vinylpyrrolidone)block-poly(dimethylsiloxane)-block-poly(N-vinylpyrrolidone) triblock copolymers (PNVP-b-PDMS-b-PNVP; for simplicity, abbreviated here as PNVP-PDMS) with the ability to form vesicles were synthesized. Laccase was encapsulated into these polymersomes and was shown to be stabilized, while still being accessible for its key substrate oxygen. ROS produced by the enzyme was able to leave the vesicle and react with other substrates in a nonenzymatic radical reaction outside the polymersomes. Such PDMS-PNVP polymersomes proved to be simple, yet effective nanoreactor for laccase reactions.

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Scheme 1. PDMS-PNVP Triblock Copolymer

1 H NMR (400.1 MHz, CDCl3) δ/ppm = 3.7−3.85 (CH2−CH-N), 3.0−3.2 (CH2−CH2−N−CO), 2.7−3.0 (CH2−CH2−CONH) 2.3− 2.4 (CH2−CH2−CH2−CO), 1.5−2.0 (CH2−CH−N−CO, CH2− CH2−CH2−CO, CH2−CH2−CONH, CH2-CH2−C, CH2−CH2−C, CH2−CH2−C(CH3)−CN), 1.45−1.50 (CH2−CH2−CH2), 0.54 (−SiCH2), 0.1 (Si−(CH3)2). Preparation of Polymersomes. Polymerosomes were formed using a film rehydration method.63 One milliliter of a 5 mg mL−1 solution of PDMS-PNVP copolymers in chloroform was slowly evaporated in a rotary vacuum evaporator at reduced pressure until a film formed on the flask wall. In a second step, the polymer film was rehydrated under magnetic stirring for 12 h with either 1 mL of a solution of PS to form empty vesicles, or 1 mL of a 2 mg mL−1 solution of Lac or HRP in PS to form enzyme-loaded vesicles. The solutions were extruded through 200 nm track edge polycarbonate filters (Whatman). The polymersomes containing Lac or HRP were separated from free enzyme by size exclusion chromatography (SEC) through a Sepharose 2B column (mobile phase PS, 20 cm3 stationary phase). Polymersomes eluted as one fraction before the free enzyme. The enzyme content of each vesicle batch was determined by UV−vis spectroscopy. Activity Assays of Enzymes. Laccase activity was measured by determining the Michaelis−Menten parameters and turn over numbers with ABTS.64 The assay was carried out at room temperature in 1 cm quartz cuvettes by mixing 0.1 mL of a sample solution (0.5 mg mL−1 free Lac in PS, or polymersome-encapsulated Lac in PS (0.42 mg mL−1 in PDMS-PNVP1, 0.36 mg mL−1 in PDMS-PNVP2, 0.25 mg mL−1 in PDMS-PNVP3, 0.21 mg mL−1 in PDMS-PNVP4 and 0.15 mg mL−1 in PDMS-PNVP5)) with 0.5 mL ABTS solution in PS and 0.4 mL PS. The final concentration of the reagent ranged from 5 μM to 800 μM. Absorbance at 414 nm was recorded every 1 min. Apparent Michaelis−Menten parameters Km and Vmax were obtained by fitting the monosubstrate Michaelis−Menten equation to plots of the initial reaction rate versus ABTS concentration using the software Origin 8 pro in analogy to the literature.65 kcat was calculated from Vmax by dividing it with the Lac concentration. HRP activity was measured with the ABTS assay as reported previously.46 Digestion of Lac with Proteinase K. A 0.5 mL sample of a 200 μg mL−1 proteinase K solution in PS was added to 0.5 mL sample solution (0.55 mg mL−1 Lac-filled PDMS-PNVP1 vesicles, or 2 mg mL−1 Lac solution). The reaction mixtures were incubated at 37 °C for 24 h. Then, the residual Lac activity was evaluated by the ABTS assay

MATERIALS AND METHODS

Materials. 4,4′-azobis(4-cyanovaleryl) chloride was synthesized from 4,4′-azobiscyanopentanoic acid (Sigma-Aldrich; dried by azeotropic distillation from toluene) according to a method reported by Simionescu and co-workers.60 1-Vinyl-2-pyrrolidinone (SigmaAldrich) was purified before usage by vacuum distillation and kept under nitrogen. Chloroform (Sigma-Aldrich) was dried over P2O5 and purified by distillation. Toluene (Sigma-Aldrich) was distilled over Na wire. Octamethylcyclotetrasiloxane (D4; Sigma-Aldrich) and 1,3-bis(3aminopropyl)-1,1,3,3-tetramethyldisiloxane (APTES; Alfa-Aesar) were used as received. Laccase (Lac) from Trametes versicolor (≥10 U/mg), horseradish peroxidases (HRP; highly stabilized, essentially salt-free, lyophilized powder, 200−300 units mg−1 solid), 2,2′-azino-bis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS), Sepharose 2B, sodium azide (NaN3), hydrogen peroxide (H2O2), and 5,5-dimethyl-1pyrroline N-oxide (DMPO) were purchased from Sigma-Aldrich and used as received. Proteinase K (recombinant PCR grade) was obtained from Roche. Ten millimolar phosphate saline solution (PS) (pH 4.25, 136 mM NaCl, 2.6 mM KCl) was prepared by dissolving 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4 in 800 mL 1470

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Table 1. Characteristics of PNVP-b-PDMS-b-PNVP Triblock Copolymers

a

polymer

DP siloxane block (1H NMR)

DP vinyl block (1H NMR)

vinyl/siloxane molar ratio

Fa

Mn (GPC)

Mw/Mn (GPC)

PDMS-PNVP1 PDMS-PNVP2 PDMS-PNVP3 PDMS-PNVP4 PDMS-PNVP5

17 17 17 37 37

11 13 17 19 30

0.65 0.75 1 0.5 0.8

0.87 1.03 1.34 0.72 1.14

2360 2860 4200 5400 6160

1.49 1.45 1.52 1.59 1.54

F = Mnvinyl/Mnsiloxane

using 0.15 mL of sample solution, 0.5 mL of ABTS solution and 0.35 mL of PS at a final ABTS concentration of 5 μM. Inhibition of Lac with NaN3. Inhibition conditions were adapted from Johannes and Majcherczyk.66 A 0.5 mL sample of a 1 mM NaN3 solution in PS was added to 0.5 mL of sample solution (0.61 mg mL−1 Lac-filled PDMS-PNVP1, or 2 mg mL−1 Lac solution). The reaction mixtures were incubated at room temperature for 2 h. Then, the residual Lac activity was evaluated by the ABTS assay using 0.12 mL of vesicle solution, 0.5 mL of ABTS solution, and 0.38 mL of PS at a final ABTS concentration of 5 μM. Additionally, the activity of the same batch of Lac-filled polymersomes was measured as reference. Sample Preparation for Electron Paramagnetic Resonance (EPR). The DMPO spin adducts were obtained by mixing 0.1 mL of 1 mM DMPO solution in PS with 0.1 mL of 5 mg mL−1 Lac solution in PS. Methods. The size of the extruded vesicles was determined by dynamic and static light scattering (DLS, SLS). The measurements were performed on an ALV goniometer (Langen, Germany), equipped with an ALV He−Ne laser (λ = 632.8 nm) using serial dilutions to produce polymer concentrations ranging from 0.03 to 0.5 mg mL−1. Light scattering was measured in 10 mm cylindrical quartz cells at angles between 30 and 150° at 293 K. The photon intensity auto correlation function g2(t) was determined with an ALV-5000E correlator. The obtained data were processed using ALV static and dynamic fit and plot software (version 4.31 10/01). SLS data were processed according to the Guinier-model, and DLS data by using a Williams−Watts function. The morphology as well the size of the formed polymersomes was characterized by transmission electron microscopy (TEM) on a Philips EM400 electron microscope that was operated at 100 kV. Polymersome dispersions were deposited on a carbon-coated copper grid and negatively stained with 2% uranyl acetate solution. UV−vis spectroscopy was measured on a Specord 210 plus spectrometer (Analytik Jena, Germany) with a slit width of 4 nm in 1 cm quartz cuvettes (Hellma). In order to determine Lac concentration in samples of enzyme-loaded polymersomes, the absorption at 330 nm, characteristic for the T3 copper center, was measured.67 An extinction coefficient of 4500 M−1 cm−1 (determined experimentally by a dilution series in PS) was used. The concentration of HRP was calculated from the absorbance at 403 nm, using an extinction coefficient of of 0.9 × 105 M−1 cm−1.46 The polymers’ number average molecular weight (Mn) and polydispersity index (PDI) was determined by gel permeation chromatography (GPC) on a Viscotek GPC max system equipped with four Agilent PLgel columns (10 μm guard; mixed C; 10 μm, 100 Å; 5 μm, 103 Å), using chloroform as eluent at a flow rate of 1 mL min−1 at 40 °C. Signals were recorded with a refractive-index detector and calibrated against polystyrene standards (Agilent). 1H NMR spectra were recorded on a Bruker DPX-400 spectrometer operated at 400.140 MHz in CDCl3 and processed with MestReNova software. Chemical shifts are reported in parts per million relative to tetramethylsilane. The degree of polymerization of siloxane blocks (DP siloxane) was determined from 1H NMR spectra of H2N-PDMS-NH2 using the integrals of the peaks of CH2−NH2 at 2.7−2.85 ppm and of Si−(CH3)2 at 0.1 ppm. The degree of polymerization of the vinyl blocks was calculated from the block copolymers’ 1H NMR spectra using the relation DP vinyl = DP siloxane*vinyl/siloxane molar ratio from the ratio in between the integrals of peaks at 3.7−3.85 ppm corresponding to CH2−CH−N and Si−(CH3)2 at 0.1 ppm. EPR measurements were performed on a Bruker CW EPR Elexsys-500 spectrometer equipped with a variable

temperature unit. The spectra were recorded at 298 K with the following parameters: microwave power 2 mW, conversion time 61.12 ms, number of scans up to 20, resolution 2048 points, modulation amplitude 1 G, sweep width 100 G. EPR spectra were simulated using the WINSIM (NIEHS/NIH) simulation package.

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RESULTS AND DISCUSSIONS

Synthesis of Triblock Copolymers. PDMS-PNVP copolymers were synthesized according to a method described by Simionescu and co-workers, starting from α,ω-bis(aminopropyl) poly(dimethylsiloxane) (H 2 N-PDMSNH2).60,68,69 These precursor polymers were obtained by equilibration of octamethylcyclotetrasiloxane in presence of APTES. They were converted into azo-containing macroinitiators by condensation with 4,4-azobiscyanovaleryl chloride in equimolar amounts. In a final step, polymerization of Nvinylpyrrolidon was initiated by thermal decomposition of the azo groups, yielding PDMS-PNVP triblock copolymers (Scheme 1). The PDMS-PNVP copolymers described in literature had a high ratio of vinyl to siloxane repeating units, ranging from 20 to 37. They were quite hydrophilic and self-assembled into micelles.60,68,69 Considering that for our application the formation of polymersomes was mandatory, we have chosen to synthesize block copolymers with smaller hydrophilic blocks and a molar ratio of vinyl to siloxane units between 0.5 and 1. This corresponds to a hydrophilic to hydrophobic molecular weight ratio F of 0.72 to 1.34. In order to screen for polymers that readily self-assemble into vesicles, five PDMS-PNVP triblock copolymers were synthesized from two PDMS macroinitiators. The latter differed in their degree of polymerization. The molecular characteristics of the block copolymers are summarized in Table 1. Preparation and Characterization of PDMS-PNVP Polymersomes. First, the copolymers were tested for their ability to form vesicles in the absence of enzyme, using the film rehydration method.63 To this end, PDMS-PNVP block copolymers were self-assembled in phosphate saline solution (PS) at a pH of 4.25, which is the pH-optimum of Lac.66 TEM micrographs of all five polymers show spherical structures with radii of 80−110 nm, which most likely are polymersomes (Figure 1A and Supporting Information Figure S1A−D). Moreover, micelles with a diameter below 20 nm are also present. The radius of gyration (Rg) and the hydrodynamic radius (Rh) of the nano-objects were measured by LS (Table 2). The ratio Rg/Rh (ρ-parameter) indicates the morphology of the structures. The Rg of the self-assembled structures is between 70 and 120 nm and the ρ-parameter between 0.87 and 1.01. The latter is between the theoretical ρ-parameter of hollow spheres (1.0) and of solid spheres (0.77), indicating the presence of vesicles.70 Lac-filled vesicles were prepared by film rehydration in a solution of Lac in PS at pH 4.25. The driving force for Lac 1471

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Figure 1. TEM micrographs of PDMS-PNVP1 nanostructures formed by self-assembly of the polymer in phosphate saline solution at pH 4.25. (A) Empty polymersomes; (B) polymersomes encapsulating Lac.

Table 2. Light Scattering Data of Polymersomes system

Rg/nm

Rh/nm

ρ = Rg/Rh

PDMS-PNVP1a PDMS-PNVP2a PDMS-PNVP3a PDMS-PNVP4a PDMS-PNVP5a PDMS-PNVP1-Lacb PDMS-PNVP2-Lacb PDMS-PNVP3-Lacb PDMS-PNVP4-Lacb PDMS-PNVP5-Lacb

120 86 72 70 118 97 82 74 68 82

118 98 79 78 129 104 89 82 80 91

1.01 0.87 0.91 0.89 0.91 0.93 0.92 0.90 0.85 0.90

Figure 2. ABTS assay to test for enzymatic activity of Lac. Comparison of free Lac and Lac encapsulated in PDMS-PNVP polymersomes.

also shows that oxygen could reach the encapsulated enzyme and that ABTS was transformed into its colored product, albeit at a smaller rate than in a reaction catalyzed by nonencapsulated Lac. ABTS is unlikely to cross the polymer membrane, as the reagent is hydrophilic and does not permeate block copolymer membranes.46 EPR spectroscopy showed that Lac produces ROS under the given reaction conditions (vide infra). Therefore, the most probable explanation for the observed conversion of ABTS is that Lac generated ROS in the polymersomes, which diffused through the polymersome membrane and oxidized ABTS outside of the vesicles. This is in line with previous reports that showed PDMS-based blockcopolymer membranes to be permeable for superoxide radicals.48,49 As Lac resides inside of the polymersomes and converts oxygen into ROS, the PDMS-PNVP polymersomes can be defined as nanoreactors for Lac. Kinetic assays were carried out with ABTS concentrations ranging from 2.5 to 400 μM. Apparent Michaelis−Menten parameters Km and kcat were calculated from this set of reactions by fitting the Michaelis−Menten equation to plots of the initial reaction rate versus ABTS concentration.65 The Lac used for this study exhibited a Km of 33 μM when not encapsulated into polymersomes (Table 3). This is slightly

a

Self-assembled in the absence of enzyme. bSelf-assembled in the presence of Lac.

encapsulation is most likely random entrapment. Nonencapsulated Lac was removed from the enzyme-containing polymersomes by size exclusion chromatography. The concentration of Lac after this purification step was determined by UV−vis spectroscopy. The results for each vesicle preparation are summarized in the experimental section. Encapsulation efficiencies of 10−20% with respect to the amount of enzyme that was used for film rehydration were achieved. PDMSPNVP1 was the most efficient copolymer for Lac entrapment. TEM revealed that the copolymers formed mostly spherical structures with radii around 90−100 nm, next to some smaller micelles (Figure 1B and Supporting Information Figure S1E to H). Rg was determined by LS to be between 68 and 97 nm, and the ρ-parameters close to 1 (Table 2). These results conclude that the five copolymers self-assembled into polymersomes in the presence of Lac. Lac-loaded PDMS-PNVP polymersomes are stable in time, as indicated in Supporting Information Figure S3. No change in the morphology of PDMS-PNVP1 encapsulating Lac was observed even after three months of storage at room temperature. Activity of Lac. The enzyme-containing polymersomes were tested for enzymatic activity in order to assess if the enzyme is functional after encapsulation. Polymersomes are known to be impermeable to many small molecules including water.40,46 Because PDMS-PNVP polymersomes were used as produced and not further treated with a permeabilization reagent, the activity tests should also indicate if PNVP-PDMS polymersomes allow laccase’s substrates to access the encapsulated enzyme. Lac activity and kinetics were evaluated by an ABTS assay.64 The enzyme oxidizes ABTS to a metastable radical cation with blue-green color. The observed increase in absorbance at 414 nm (Figure 2) therefore shows that Lac remained active during the encapsulation procedure. It

Table 3. Apparent Michaelis−Menten Parameters of Free and Encapsulated Lac, Using ABTS as Substrate Km/μM

system Lac PDMS-PNVP1-Lac PDMS-PNVP2-Lac PDMS-PNVP3-Lac PDMS-PNVP4-Lac PDMS-PNVP5-Lac

33 60 64 73 128 143

± ± ± ± ± ±

3 3 5 2 4 4

Vmax/μM min−1 239 75 73 71 66 42

± ± ± ± ± ±

7 7 4 5 2 5

kcat/s−1 0.29 0.11 0.12 0.17 0.18 0.17

± ± ± ± ± ±

0.07 0.07 0.04 0.05 0.02 0.05

lower than a Km of 38 μM reported in the literature,64 indicating a good affinity of the enzyme for the substrate. When encapsulated into polymersomes, the Km of the enzyme increased by a factor of 2 for the polymers with small PDMS block and approximately 4-fold for polymers with the larger PDMS block (Table 3). The kcat of free Lac is 0.29 s−1 in accordance with the value reported in the literature (0.25 s−1).71 Encapsulated Lac displayed lower kcat between 0.11 and 0.18 s−1. The conversion of the substrate ABTS is slower when the Lac is encapsulated in polymersomes and the reaction rates 1472

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solution of Lac-loaded PDMS-PNVP1 polymersomes, as well as to a solution of free Lac and incubated at 37 °C for 24 h. After the digestion, activity assays with ABTS were carried out. As indicated in Figure 4A, Lac in the PDMS-PNVP1 nanoreactors

appear to depend on the molecular weight of the block copolymers, that is, the membrane thickness. It is unlikely that changes of the kinetic parameters upon encapsulation in the polymersomes are due to a change of the enzyme’s conformation or structure, as in that case the molecular weight of the PDMS block should not have an influence on Km. Instead, the changes of Km and kcat are of apparent nature and could be caused by several effects. The polymer membrane is a diffusion barrier for oxygen and ROS and might slow down the reaction. Furthermore, a portion of ROS could decompose before reaching the substrate. Finally, the mechanism of ABTS conversion should be different. ROS act as redox shuttles between the encapsulated enzyme and the colorimetric substrate, while the free enzyme is able to directly bind and convert ABTS. Studying the Ability of Lac To Form ROS. The ability of Lac to oxidize substrates by ROS intermediates is a controversial discussion in the literature. Lüdemann and coworkers29 as well as Duan and co-workers30,31 suggested that ROS are involved in laccase-induced oxidation of lignin, while Skibsted and co-workers32 argued that ROS are not released by laccase. Therefore, we used EPR spectroscopy with 5,5dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap to investigate whether ROS were formed by Lac in the absence of any organic substrate. These conditions mimic the microenvironment of the Lac in the polymersomes, where it is accessible for oxygen but not for ABTS. A spin adduct formed when DMPO was incubated for 1 min with Lac in solution (Figure 3). The signal was attributed to a DMPO/OH adduct

Figure 4. (A) Protection of Lac in PDMS-PNVP1 polymersomes against an externally added protease. Samples of free Lac and of encapsulated Lac were incubated for 24 h at 37 °C in a solution of proteinase K. Then, the activity of Lac was probed with the ABTS assay. For comparison, a kinetic run of encapsulated Lac without proteinase K digestion is also shown. (B) Protection of Lac in PDMSPNVP1 polymersomes against externally added inhibitor NaN3. The activities of free Lac and of encapsulated Lac were probed with the ABTS assay after incubating samples for 2 h at room temperature in a solution of NaN3. For comparison, a kinetic run of encapsulated Lac that was not exposed to NaN3 is also shown.

retained at least 90% of its activity, while free Lac was completely deactivated. The protease therefore digested Lac that was accessible from the external aqueous phase, while Lac in the polymersomes was protected against proteolytic attack. If the polymer membrane of PDMS-PNVP polymersomes is only permeable for gases and ROS, it should protect the encapsulated enzyme against small molecules. Free Lac that was exposed for 2 h to NaN3, a common inhibitor for laccase,74 lost its activity. On the other hand, encapsulated Lac was not inhibited by NaN3 (Figure 4B). The polymersomes are therefore efficient nanoreactors that shield the enzyme from inhibitors. Control Experiment To Assess if PDMS-PNVP Polymersomes Are Intrinsically Porous. Polymersomes that are based on PDMS as the hydrophobic block have been found to be impermeable to ABTS and H2O2. This manifests itself in the absence of color formation when poly(2-methyl-2-oxazoline)block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline)

Figure 3. Spin trapping of ROS species formed in a 5 mg mL−1 laccase solution in phosphate saline solution at pH 4.25.

with hyperfine splittings of aN = 14.9 G and aH = 14.9 G, which were similar to the values reported in the literature.72 These experiments prove that Lac released hydroxyl radicals. It is also possible that the enzyme generated other ROS such as superoxide radicals. However, they could not be trapped due to their short lifetime, because they can transform into hydroxyl radicals, and because DMPO has a much higher affinity toward hydroxyl radicals than toward superoxide radicals.72,73 Protection of Encapsulated Enzyme against a Degrading Agent and an Inhibitor. In order to prove that encapsulation in polymersomes protects the Lac against macromolecular degrading agents, digestion experiments were carried out with proteinase K, an enzyme that cleaves peptide bonds with a broad specificity. The protease was added to a 1473

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ACKNOWLEDGMENTS We gratefully acknowledge the KTI/CTI, the Holcim Stiftung Wissen, the Swiss National Science Foundation, the Swiss Nanoscience Institute, the Marie-Curie-Actions of the European Commission and the University of Basel for financial support. We thank the Zentrum für Mikroskopie Basel (ZMB) for the TEM images and Ruth Pfalzberger for helping to prepare the graphics.

polymersomes that encapsulated horseradish peroxidase (HRP) were incubated with ABTS and H2O2.46 Gases and ROS can, however, penetrate the block copolymer membrane of such polymersomes.48,49,61 In the light of these previous reports, the experiments reported here suggest that oxygen can enter the PDMS-PNVP polymersomes and ROS can diffuse out and subsequently react with ABTS outside of the polymersomes. However PDMS-PNVP polymersomes might be intrinsically porous and therefore permeable for ABTS. To evaluate this possibility, HRP was encapsulated into the polymersomes. In contrast to Lac, HRP oxidizes ABTS under consumption of hydrogen peroxide.75,76 HRP-loaded PDMS-PNVP3 polymersomes were prepared by a film rehydration method. The concentration of encapsulated HRP was determined to be 2.21 μM by UV−vis spectroscopy at 403 nm, the Soret band of HRP. The morphology and size of HRP-containing selfassembled structures were characterized by TEM and DLS. Electron microscopy images revealed spherical structures with a diameter of approximately 200 nm (Supporting Information Figure S2), while DLS resulted in an Rh of the self-assembled objects of 86 nm. An ABTS assay performed in the presence of H2O2 indicated no color reaction (Supporting Information Figure S3). In comparison, free HRP readily catalyzed the reaction.46 These findings conclude that ABTS and H2O2 did not diffuse into the polymersomes, that is, that the membrane of PDMS-PNVP polymersomes is not porous. This conclusion is further supported by the observation that the polymersomes shield encapsulated enzyme from inhibitors (vide supra).

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CONCLUSIONS PDMS-PNVP triblock copolymers self-assembled into polymersomes in acidic conditions and allowed the encapsulation of laccase under retention of the enzyme’s activity. The polymersomes act as nanoreactors for the biocatalyst, as they are permeable for oxygen. Lac was found to produce ROS, which diffused out of the polymersomes and then oxidized an aromatic compound. The polymersomes shield Lac from proteolytic enzymes as well as from small inhibitors. At the same time, they allow efficient biocatalysis without the need for complex or costly permeabilization methods of their membrane. Therefore, the nanoreactors are simple enough for industrial or technical applications. Nanoreactors that stabilize and protect Lac are of interest for many of laccase’s applications, for example, in the cosmetics field, in environmentally friendly oxidation and bleaching technologies, and in biosensing. Moreover, polymersomes could be used to modulate the activity of laccases by varying the thickness and composition of the vesicle membrane. ASSOCIATED CONTENT

* Supporting Information S

TEM micrographs of empty and Lac-filled polymersomes, TEM micrograph of HRP-filled polymersome, ABTS activity assay of encapsulated HRP. This material is available free of charge via the Internet at http://pubs.acs.org.

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