Polymer Nanoreactors with Dual Functionality: Simultaneous

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Polymer Nanoreactors with Dual Functionality: Simultaneous Detoxification of Peroxynitrite and Oxygen Transport Dominik Dobrunz, Adriana C. Toma, Pascal Tanner, Thomas Pfohl, and Cornelia G. Palivan* Chemistry Department, University of Basel, Klingelbergstrasse 80, 4056 Basel, Switzerland S Supporting Information *

ABSTRACT: The design of multifunctional systems is in focus today as a key strategy for coping with complex challenges in various domains that include chemistry, medicine, environmental sciences, and technology. Herein, we introduce protein-containing polymer nanoreactors with dual functionality: peroxynitrite degradation and oxygen transport. Vesicles made of poly-(2methyloxazoline)−poly(dimethylsiloxane)−poly(2-methyloxazoline) successfully encapsulated hemoglobin (Hb), which serves as a model protein because of its dual function in oxygen transport and peroxynitrite degradation. By inserting channel proteins, the polymer membranes of vesicles permitted passage of various compounds that served for the assessment of in situ Hb activity. The requisite conformational changes in the protein structure and the change in oxidation states that took place within the confined space of the vesicle cavity demonstrated that Hb preserved its dual functionality: peroxynitrite degradation and oxygen transport. The functionality of our nanoreactor, combined with its simple procedure of production and extensive stability over several months, supports it as a promising system for further medical applications.

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brains of mice following traumatic injury.10 Combined with its ability to diffuse longer distances than the diameter of a typical cell, peroxynitrite is an extremely effective oxidant that can cause damage even far from its origin. Physiological concentrations of peroxynitrite have been reported on the order of 50 μM;11 however, they reached up to 500 μM in some cases.12 A variety of compounds present in biological systems has been shown to be modified by peroxynitrites and include amino acids, nucleic acids, and membrane lipids.13 Dairou and co-workers reported on an enzyme involved in human breast cancer that was irreversibly inactivated at physiological peroxynitrite concentrations at a rate constant of 104 M−1 s−1.14 In vivo, there are endogenous compounds that exhibit high second-order rate constants with peroxynitrite.15 In this respect, various proteins are capable to detoxify peroxynitrite in natural conditions: peroxiredoxins, Se-containing proteins (glutathion peroxidase), and heme proteins (hemoglobin, cytochrome c oxidase, FeII cytochrome c).16,17,15 Unfortunately, the concentration of antioxidant enzymes is not high enough to overcome the excess peroxynitrite involved in metabolic dysfunctions. A compound active in peroxynitrite decomposition should possess a complex set of properties: (i) It should react quickly with peroxynitrite. (ii) It should be present in large amounts in the desired biological compartment. (iii) It should generate products that are not toxic by themselves. Administration of the above antioxidant proteins is expected to decrease the amount of peroxynitrite involved in

INTRODUCTION New systems and approaches in nanoscience are expected to provide efficient solutions in various domains, such as medicine, environmental and food sciences, and technology. In particular, in medicine, there is a need to improve the patient condition by increasing sensitivity and expanding detection of new biomolecules, decreasing dosages, or providing multifunctionality. In this respect, a challenging scenario involves oxidative stress, mainly based on the effects of reactive oxygen species (ROS) in a variety of pathologic situations, or associated with the toxicity of various inorganic nanoparticles or quantum dots.1−3 When oxidative stress takes hold, efficient biologic strategies for regulation of ROS, including cellular antioxidants (glutathione and ascorbate) and enzymes (glutathione peroxidase, glutathione transferases, catalase, and superoxide dismutase), are overwhelmed. In these situations, ROS, which includes the superoxide radical anion (O2•−), peroxynitrite, the hydroxyl radical, and hydrogen peroxide are constantly produced and are involved in the initiation and progression of chronic inflammation. In this respect, peroxynitrite has been reported to be implicated in the development of neurodegenerative disorders,4,5 cardiovascular diseases,6 and cancer.7 Peroxynitrite is formed by the diffusion-limited reaction (rate constant ∼1 × 1010 M−1 s−1)8 between nitrogen monoxide, which plays a critical role in cell regulation and communication, and superoxide anion, present in every living cell as a result of normal metabolism.9 Large amounts of peroxynitrite are produced when the combination of •NO with O2•− becomes competitive with the dismutation of O2•− by superoxide dismutases, for example, in the mitochondrial respiratory complex. Moreover, peroxynitrite was detected in the © 2012 American Chemical Society

Received: July 6, 2012 Revised: October 9, 2012 Published: October 19, 2012 15889

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molecules.39 The permeability of the polymer membrane to oxygen species will support in situ Hb activity. Based on this type of oxygen-permeable nanovesicle, we have already introduced the concept of antioxidant nanoreactors by the encapsulation of superoxide dismutase/mimics, which served to detoxify in situ superoxide radicals.38 Here, we develop the concept for peroxynitrite degradation. With a proven record of good biocompatibility,40 the membrane of PMOXA-PDMS-PMOXA copolymer possesses high flexibility that serves for a functional insertion of channel proteins, such as OmpF, LamB, or Aquaporin Z.39,41−43 The incorporation of channel proteins within the vesicle membrane was essential to allow substrates/ products to penetrate through and support the nanoreactor functions. In addition, nanoreactors avoid the drawbacks of conventional drug delivery systems, such an uncontrolled release or degradation in biological compartments other than those desired.23 Within this context, we first analyzed the decomposition of peroxynitrite by carboxy- and deoxy-hemoglobin in solution under normal conditions, and under conditions simulating the nanoreactors (concentration, buffers, temperature). Polymeric vesicles were characterized by light scattering (LS) and transmission electron microscopy (TEM), while the protein encapsulation efficiency was established by fluorescence correlation spectroscopy (FCS) combined with UV−vis spectroscopy. Finally, we tested the dual function of our nanoreactor: peroxynitrite decomposition and oxygen transport. The simplicity of production, its robustness, and dual functionality support our nanoreactor for further medical applications.

pathological situations. In this respect, hemoglobin (Hb) is an ideal candidate due to its dual functionality: (i) reduction of peroxynitrites and (ii) oxygen transport. In addition, its reaction with peroxynitrite has a relatively high rate constant (human hemoglobin has a rate constant of 2 × 104 M−1s−1 at pH = 7.4, at 37 °C),18 which supports a medical application. There are significant ongoing efforts to use Hb as an oxygen carrier in the development of artificial blood to avoid transfusion problems related to short shelf life of blood19 and safety risks associated with cross-matching and viral infections.20 A limiting factor in the use of hemoglobin is that it tends to separate into dimers, which are rapidly filtered by the kidneys and excreted.21 The efforts to polymerize hemoglobin in order to reduce dimerization have not been successful because the molecule continues to dissociate.22 The encapsulation of proteins in nanocarriers, such as micelles, vesicles, capsules, or particles represents another way to avoid obstacles such as those encountered by Hb when delivered into biological compartments.23 Liposomes have been extensively developed as hemoglobin carriers,24,25 but their main drawbacks are short lifespan26 and a tendency to aggregate or precipitate.27 To improve their stability, poly(ethylene glycol) (PEG)-modified lipids have been used to encapsulate hemoglobin.28,29 This system already showed promising results reported by enhancement of oxygen transfer and cell proliferation while cell hypoxia decreased.30 In addition, encapsulation of Hb inside PEG modified liposomes attenuates the reaction with nitric oxide and regulates itsdependent vasodilation.31 The use of polymeric vesicles constitutes a better approach because of their increased mechanical stability: the block copolymer membranes are thicker and far more stable than those of liposomes.32 In recent years, various block polymers have been used to encapsulate hemoglobin: poly(ethylene glycol) (PEG) and poly(lactide acid) (PLA) blocks,22,33 poly(butadiene)−poly(ethylene glycol) (PEO/PBD),25 and poly(ethylene glycol)b-(caprolactone) (PEG/PCL).33,34 By measuring the oxygen binding curve of the encapsulated hemoglobin, these studies have also shown that the encapsulated hemoglobin maintains its activity.25 Although encapsulated hemoglobin has, to date, been widely proposed as artificial blood, however, none of these studies has approached the decomposition of peroxynitrite. Moreover, no other proteins involved in the degradation of peroxynitrites have been encapsulated/entrapped in nanocarriers to fight increased amounts of peroxinitrites. To the best of our knowledge, only one study reported the insertion of metalloporphyrins in the membrane of liposomes as a means of peroxynitrite degradation.35 The main aim of our study is to create a protein-containing polymer nanoreactor capable of degrading peroxynitrite. Nanoreactors are formed by encapsulation of active compounds inside polymeric assemblies where they can simultaneously act in situ and are protected from proteolytic attack.36,37 By choosing Hb as model protein for detoxification of peroxynitrites, we implemented a second function in our nanoreactor: the added ability to carry oxygen. There are two possible approaches for designing nanoreactors with dual-functionality: either selecting a combination of enzymes, each with a specific function, or selecting one enzyme with two different functions, which is the case for Hb. We selected poly-(2-methyloxazoline)-block-poly(dimethylsiloxane)block-poly(2-methyloxazoline) PMOXA-PDMS-PMOXA triblock copolymer because this amphiphilic copolymer has been shown to generate vesicles by self-assembly under physiological buffer conditions.38 The vesicle membrane is permeable to oxygen species while it features low permeability to larger

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MATERIALS AND METHODS

Chemicals. Ascorbic acid and methemoglobin were purchased from Sigma (Switzerland). Fluorescent dye Alexa488 carboxylic acid, succinimidyl ester was purchased from Invitrogen (Switzerland), and phosphate buffered saline (PBS) (pH 7.2) was obtained from Fluka (Switzerland). All chemicals were used without further purification. Outer membrane protein F (OmpF) expression was carried out in E. coli (BL21(DE3)omp8)44 according to a previously optimized protocol45 and was stored (700 μg/mL) in 3 wt % N-Octyl-oligo-oxyethylene (OPOE) stock solution at 4 °C. The procedure for the synthesis of peroxynitrite is described in detail elsewhere.46 Hemoglobin Labeling. A solution of 1 mg of Alexa488 in 50 μL dimethyl sulfoxide (DMSO) was slowly added to a solution of 15 mg of methemoglobin in 850 μL of PBS buffer. After 15 min 0.1 mL of a 0.1 M Na2CO3 solution was added. The mixture was stirred for 1 h at room temperature. The free dye was removed by size exclusion chromatography (SEC) on Sephadex G25 (300 mm) equilibrated with PBS. The labeling efficiency was determined by UV spectroscopy, using the Beer−Lambert law. On average, 2.3 Alexa488 dye molecules per Hb were attached. Polymer Vesicle and Nanoreactor Preparation. PMOXAPDMS-PMOXA was synthesized according to the procedure described in ref 45. The copolymer was characterized by NMR and gel permeation chromatography (GPC). Polymer vesicles were prepared by the film rehydration method: 5 mg of triblock copolymer formed a thin film on the walls of a 25 mL round-bottom flask using a rotary evaporator. After the addition of 1 mL of PBS buffer to the polymer film and stirring for 2 h, the solution was extruded through polycarbonate membranes (pore Φ = 400 nm) in order to generate vesicles with a low size distribution. The other method that was used was the cosolvent method. In this method, the polymer is dissolved in a small amount of ethanol and is then slowly added while stirring to a buffer solution. The problem with this method is that it only produced micelles and small vesicles with a radius of around 25 nm. The vesicles were too small to encapsulate Hb. Nanoreactors were prepared by addition of 1 mL of labeled methemoglobin (3 mg/mL in PBS buffer) to the polymer film, stirring 15890

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for 2 h, and extrusion through polycarbonate membranes (pore Φ = 400 nm). Vesicles containing both the protein and the channel protein OmpF were prepared by simultaneous addition to the polymer film of 0.95 mL of metHb (3.16 mg/mL in PBS buffer) and 50 μL of OmpF (8 μM in PBS buffer),45 stirring for 2 h, and extrusion through polycarbonate membranes (pore Φ = 400 nm). Hb initial concentration was selected such to have a similar value as in the case of other Hb-encapsulated vesicles.47 The extrusion process results in a loss of around 50% of the polymer−hemoglobin aggregates, which has been taken into account for the encapsulation efficiency of Hb. In order to purify the unencapsulated methemoglobin, the extruded mixture was dialyzed with 300 kDa molecular weight cut off dialysis bags in PBS at 2−3 °C at a 1:300 v/v dialyzed sample/PBS ratio. In addition, the solution was purified by size exclusion chromatography (SEC) on a Sephadex G25 column (300 mm) equilibrated with PBS. The final solution was analyzed by FCS to determine the fraction of encapsulated hemoglobin. Characterization of Polymer Vesicles and Nanoreactors. Light Scattering (LS). LS experiments were performed with a compact goniometer system ALV/DS/SLS 5000/E correlator from ALV, Langen (Germany), equipped with a helium−neon laser (JDS Uniphase) with λ = 633 nm (T = 20 °C). The diffusion coefficient (D0) was obtained from a second-order fit of dynamic light scattering (DLS) data from dilute suspensions. From D0, the hydrodynamic radius (Rh) was calculated using the Stokes−Einstein relation. Scattering angles were varied between 30° and 150° with each 10 angles measured for 100 s. For static light scattering (SLS) measurements, the data, presented in a Guinier plot extrapolated to zero concentration and zero scattering angle, was used to calculate the radius of gyration.48 Transmission Electron Microscopy (TEM). TEM micrographs were recorded using a CM 100 Philips microscope operating at 120 kV, and equipped with an USC1000-SSCCD 2 k_2 k Gatan camera. The solution of vesicles (empty vesicles and Hb-containing vesicles) was deposited on a carbon-coated copper grid. The excess of solution was removed using absorbent paper, and the sample was allowed to dry at room temperature for 1 min before the images were recorded. The grids were stained with uranyl acetate. Hemoglobin Encapsulation Efficiency. The encapsulation efficiency and the percentage of free Hb were measured by fluorescence correlation spectroscopy (FCS). Solutions of Alexa Fluor 488, Alexa Fluor 488-methemoglobin, and Alexa Fluor 488-metheoglobincontaining vesicles were measured at room temperature in special chambered quartz-glass holders (Lab-Tek; 8-well, NUNC A/S) that provide optimal conditions for the measurement while reducing evaporation of the aqueous solutions. FCS measurements were performed with a Zeiss LSM 510-META/Confocor2 confocal laser scanning microscope (Zeiss AG, Germany) equipped with an argon laser (λ = 488 nm) and a 40× water-immersion objective (Zeiss C/ Apochromat 40×, NA 1.2), with the pinhole adjusted to a diameter of 70 μm. The excitation power of the Ar laser was PL = 40 mW, and the excitation transmission at 488 nm was 3%. Fluorescence intensity fluctuations were analyzed in terms of an autocorrelation function with the LSM 510/Confocor software package (Zeiss, AG). Spectra were recorded over 30 s, and each measurement was repeated 10 times; results are reported as the average of three independent experiments. Adsorption and bleaching effects were reduced by exchanging the sample droplet after 5 min of measurement. To reduce the number of free fitting parameters, the diffusion times of the free dye (Alexa Fluor 488) and of Alexa488-labeled methemoglobin (Alexa488-metHb) were independently determined and fixed in the fitting procedure. Activity Assays for Peroxynitrite Degradation and Oxygen Binding of Hb. The conversion of Hb was similarly established in solution and in situ inside nanoreactors by various reactions: (a) Conversion of metHb was realized by (i) addition of L-ascorbic acid and carbon monoxide and (ii) addition of sodium dithionite. (i) A solution of 2 μmol methemoglobin in 1 mL of PBS was converted by adding 50 μL of L-ascorbic acid solution (1 mol/L in PBS). After reacting for 10 min, the mixture was flushed with carbon monoxide for 10 min.

(ii) Methemoglobin (900 μL of 50 μM metHb solution) was converted by adding 100 μL of sodium dithionite solution (10 mmol/L in PBS) at room temperature. Excess sodium dithionite was removed by SEC. The SEC column consisted of Sephadex G25 (300 mm) equilibrated with PBS. Samples were measured both before and after removal of the sodium dithionite excess. (b) Peroxynitrite degradation was achieved by the addition of 100 μL of 10 mM peroxynitrite solution to 900 μL of a 50 μM oxyHb or deoxyHb solution (pH 7.4, room temperature). At least 8−10 mol ONOO− was required to completely convert 1 mol of oxyHb to metHb. (c) Oxygen binding was tested by conversion of deoxyHb to oxyHb and by conversion of oxyHb to carboxyHb. DeoxyHb obtained after addition of sodium dithionite was passed through a column of Sephadex G25 (100 mm) to remove the excess sodium dithionite, in the presence of oxygen. To obtain carboxyHb, an oxyHb solution was flushed with carbon monoxide for 20 min. The different oxidation states of hemoglobin were measured by UV−vis spectroscopy and Raman spectroscopy. UV−vis measurements were performed at room temperature using a SPECORD 210 spectrophotometer (Analytic Jena, Germany). The spectra were recorded at room temperature, in a range from 390 to 1000 nm. Samples were measured immediately after preparation (within 20 s), to avoid further reaction with nitrate, a ubiquitous contaminant of ONOO− solution. Raman Spectroscopic Characterization. Raman spectra were collected with an alpha300R (WITec GmbH, Ulm, Germany) confocal upright microscope spectrometer (UHTS 300, WITec) equipped with epi-illumination. A water immersion objective lens with glass correction (Olympus UAPON 40XW340 NA1.15WD0.25UV) was used to focus the laser beam on the sample and to collect the Raman backscattered light. Raman scattering was excited by a 532 nm Nd:Yag laser. The laser power at the objective focal point was measured with a power meter (PM100D, Thor Laboratories). For Raman peak intensity optimization, several laser powers and hemoglobin concentrations were tested, and it was concluded that reducing the Hb concentration to around 50 μM allows for longer acquisition times without sample alteration (Supporting Information Figure S1). The laser line was selectively removed from the measured Raman scattering with a notch filter, but allowing good transmission of the Raman scattered light. A multimode optical fiber with a core diameter of 50 μm played the role of a pinhole, and projected the scattered light onto the entrance of the spectrometer. Furthermore, inside the spectrometer, the light is further reflected on a diffraction grating with 600 gratings/mm. The detection of the Raman scattering was accomplished with a charge-coupled device (CCD) camera (DV401A-BV-352, WITec) with 1024 × 128 pixels cooled to −60 °C in order to reduce thermal noise. The spectra were measured with a spectral resolution of 3.5 cm−1. Acquisition and Data Treatment. Each Raman spectrum represented the cumulative signal of 100 measurements of 1 s integration time. Spectra were processed to remove cosmic rays (WITec Project Software) and background corrected by subtraction of the signal acquired with the laser off immediately after each series of 100 consecutive measurements. Sample Preparation for Raman Experiments. Hemoglobin samples were analyzed between two 0.15 mm thick glass cover slides separated by a 50 μm thick double sided tape (Tesa, Switzerland). All measurements were performed with the laser beam focused 20 μm below the top cover slide. The Raman signal could not be collected from encapsulated hemoglobin solutions because the Rayleigh scattering from the 200 nm size vesicles interfered greatly with the spectra acquisition. Moreover, the encapsulated Hb concentration in solution was 1 order of magnitude lower than what would normally be detected using Raman spectroscopy. We thus decided to work at a bulk concentration between 20 and 50 μM, which is relevant to a local protein concentration within one vesicle when considering between 10 and 20 encapsulated protein molecules per polymeric vesicle. 15891

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Stopped Flow Spectroscopy. The kinetics of the reaction of hemoglobin with peroxynitrite was studied by single-wavelength stopped flow spectroscopy under pseudo-first-order conditions (at λ = 430 nm). Peroxynitrite was always present at least at 10-fold excess, to maintain pseudo-first-order conditions. Measurements were performed at room temperature using an SFM-20 instrument equipped with an MOS-200 monochromator. The degradation of peroxynitrite was measured at its absorption maximum λ = 302 nm. The traces correspond to the average of at least 10 single traces, and were analyzed with Biokine32 software. The data were fitted with a second-order exponential fit. In order to acquire a better signal, the nanoreactor solution was concentrated by centrifugation (10 min at 4500 rpm).

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and spherical objects with a hydrodynamic radius of RH = 164 ± 15.4 nm, the latter being the major component (see Supporting Information Figure S3). According to SLS, the spherical objects have a radius of gyration Rg = 194 ± 8.2 nm. The ratio between the hydrodynamic radius and the radius of gyration of RH/Rg = 0.84 indicates that the major population of spherical objects is vesicles. The analysis of the DLS data for empty vesicles and protein-containing vesicles shows that metHb does not influence vesicle formation. TEM micrographs indicate the presence of circular objects that we assume are micelles with a mean radius of around 28 ± 8.5 nm, and of spherical objects with a mean radius of 120 ± 20 nm (Figure 1). The larger value RH obtained by LS experiments is expected, as the RH from DLS experiments represents the sum of the particle radius and the contribution of its surrounding hydration sphere. TEM micrographs of methemoglobin-containing vesicles (Figure 1B) or metHb-containing vesicles with OmpF incorporated in the polymer membrane did not show any significant morphological change as compared to empty vesicles (Figure 1A). Thus, the encapsulation of methemoglobin and insertion of OmpF in the membrane of PMOXA6-PDMS90-PMOXA6 vesicles do not affect the self-assembly process or the morphology of generated nanoobjects, in agreement with other protein−polymer nanoreactors based on this type of amphiphilic copolymer.45 After storage at 4 °C for long periods of time (more than 3 weeks), TEM micrographs revealed no significant changes, suggesting that the nanovesicles from PMOXA6-PDMS90-PMOXA6 are mechanically stable. Protein Encapsulation Efficiency. We used FCS to determine whether metHb was encapsulated in the cavities of vesicles and to analyze how effectively the remaining unencapsulated metHb was removed from the solution surrounding the vesicles. In FCS, the laser-induced fluorescence of the excited fluorescent molecules that pass through the confocal volume is autocorrelated in time and permits obtaining information about their diffusion times. As the diffusion times are proportional to the RH of the fluorescent molecules (Stokes−Einstein equation), they are used to establish the interactions of the fluorescent molecules with larger target molecules, for example, the encapsulation of fluorescently labeled proteins in nanovesicles.38,51 We encapsulated Alexa488-labeled metHb in polymer vesicles and compared its diffusion time to that of the free Alexa488labeled protein.52 Alexa488 had a diffusion time of around 26 μs, while the Alexa488-metHb featured a diffusion time of 95 μs (Figure 2a and b). The hydrodynamic radius of Alexa488metHb, calculated from the diffusion time, is 2.5 nm, in good agreement with the reported value of 3.1 nm.53 The best fit of the FCS data (Figure 2c) indicates the presence of two populations of diffusing objects based on their different molecular weight: a major population with an average diffusion time of around 6930 μs and a minor population with a diffusion time of 95 μs. The mole fraction of the major population containing more than 98% of metHb was attributed to Alexa488-metHb encapsulated in vesicles with a hydrodynamic radius of around 170 ± 15 nm, in good agreement with the LS results. The minor fraction represents free Alexa488-metHb. As the diffusion time of free Alexa488-metHb was not modified by addition of empty vesicles, it clearly indicates that metHb was not attached at the surface of vesicles. To determine the encapsulation efficiency, we first evaluated the average number of Alexa488 molecules connected to one methemoglobin molecule. On average, 2.3 dye molecules were

RESULTS AND DISCUSSION

Formation and Stability of Polymer Vesicles and Nanoreactors. As nanoreactors represent an elegant way to simultaneous protect enzymes from proteolytic attack while allowing them to act in situ, we applied the concept to hemoglobin in order to design nanoreactors exhibiting such a dual-functionality: peroxynitrite degradation and oxygen transport (Scheme 1). MetHb is encapsulated in polymer supramolecular Scheme 1. Conversion of Hb to Different Oxidation States in Solution and in Situ Inside Nanoreactors

assemblies formed by the self-assembly of amphiphilic copolymers. It is then converted in situ to deoxyHb and carbonmonoxyhemoglobin, to serve in the reaction that detoxified peroxynitrites. The conversion of deoxyHb to oxyHb inside nanoreactors supports the function of nanoreactors in transporting oxygen. We chose an amphiphilic copolymer PMOXA-PDMS-PMOXA with six repeating units of PMOXA and 90 repeating units of PDMS, as established by NMR (Supporting Information Figure S2). We used a PMOXA-PDMS-PMOXA copolymer, for reasons of stability and because it has been shown that this type of copolymer, with various hydrophilic to hydrophobic ratios, generates vesicles by self-assembly with sizes in the range of 100 −350 nm.49,50 In order to generate vesicles, we used the film rehydration method (see Materials and Methods) and optimized the protein encapsulation conditions in terms of stirring time. A stirring time of approximately 2 h was optimal, both to significantly decrease the amount of unreacted polymer and to avoid precipitation. To reduce the size distribution of vesicles, and to remove unreacted polymers, the solution was extruded with a filter membrane. Both empty vesicles and protein-containing vesicles prepared under similar conditions were analyzed by light scattering (DLS and SLS) and TEM. DLS data indicate the presence of two populations of nanoobjects: micelles with a hydrodynamic radius of around 45 nm 15892

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Figure 1. TEM images of empty vesicles (A) and vesicles with encapsulated metHb (B). Scale bar = 200 nm.

conversion reactions of free hemoglobin in solution served to optimize the conditions previous to the encapsulation. In order to obtain different oxidation states of hemoglobin, we used two methods: (i) addition of sodium dithionite in excess to obtain deoxyHb, followed by the removal of the remaining sodium dithionite in the presence of oxygen to obtain oxyHb, and (ii) addition of L-ascorbic acid in the presence of carbon monoxide to convert metHb to HbCO (Scheme 1). The inverse conversion reactions of oxyHb, deoxyHb, and HbCO were accomplished by adding freshly prepared peroxynitrite. These reactions represent the activity assays for peroxynitrite detoxification. Hemoglobin Conversion in Solution. Raman spectra of metHb, deoxyHb, and HbCO within the frequency range 1300−1900 cm−1 plotted in Figure 3A (left) agree with the spectra already reported in the literature, and the main peaks have been indexed accordingly.58−60 For clarity, all Raman spectra were shifted in intensity. The laser excited heme chromophores gave rise to vibrational modes that translated into a rich Raman band pattern of between 600 and 1700 cm−1.61 Within this interval, conformational changes associated with ligand (e.g., oxygen, carbonmonoxide, etc.) binding or electron transfer are observed. The most prominent bands found between 1300 and 1400 cm−1 are named oxidation state marker bands, whereas those in the region within 1500 and 1650 cm−1 are named spin state marker bands.59 As a result of a change in hemoglobin’s oxygenation state, unique spectral shifts arise within the above-mentioned spectral intervals, particularly in the peak relative intensities and positions.61 The electronic transition of Hb porphyrins coupled with the 532 nm laser wavelength scatter resonantly. The Raman spectrum of HbCO is highly similar to one of deoxyHb in respect to the position of the major peaks, which occur at the same Raman shifts and with similar intensities.62 Upon conversion of metHb to deoxyHb or HbCO, a change in the Raman peaks intensity occurs in the spin state marker band region. Namely, the intensity of peaks at 1555 and 1610 cm−1 become much more pronounced. Laser power required to follow the conversion reaction in the concentration domain that we used prevented us from demonstrating the final formation of oxyHb (after removal of sodium dithionite excess in the presence of oxygen). We observed only deoxyHb because of the rapid dissociation of oxyHb to deoxHb, as already reported.63 In addition to Raman spectroscopy, complementary structural information was achieved using UV−vis absorption spectroscopy. The Soret absorption band was examined for metHb, HbCO, oxyHb, and deoxyHb (Figure 3A right). The

Figure 2. Autocorrelation function of free Alexa488 (a), Alexa488metHb (b), and Alexa488-metHb-containing vesicles (c).

attached per metHb molecule. The number of Alexa488-metHb molecules per vesicle was estimated via brightness measurements. In this respect, we compared the count rates per molecule (cpm, in kHz) of Alexa488, Alexa488-metHb, and encapsulated Alexa488-metHb. The number of Alexa488-metHb molecules per vesicle, obtained from brightness experiments (for an initial concentration of metHb of 4.7 μM) was then compared to the theoretical number of metHb/vesicle (120 molecules/vesicles), and resulted in an encapsulation efficiency of around 20.6% (see Supporting Information Figure S5). Activity Assays: Peroxynitrite Degradation. We chose to start with metHb because of its stability54,55 and availability, and to convert it in situ, inside the nanoreactors, to deoxyHb and carbonmonoxyhemoglobin, followed by the inverse reaction in the presence of peroxynitrites (Scheme 1). Previous studies showed that peroxynitrite is degraded by the conversion reaction of oxyHb to metHb via the corresponding oxoiron(IV) for of the protein (Scheme 2).56,57 Scheme 2. Peroxynitrite Degradation by Conversion Reaction of oxyHb (Fe(II)O2) to metHb (Fe(III))

We studied the conversion to different oxidation states of hemoglobin, both in solution, and inside nanoreactors. The 15893

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Figure 3. (A) Raman spectra (left) of 50 μM metHb, deoxyHb, and HbCO excited at 532 nm and the corresponding UV−vis absorption spectra (right). (B) Raman spectra and corresponding UV−vis absorbance of 20 μM metHb, HbCO, and HbCO in the presence of 40 μM peroxinitrite, and (C) 50 μM metHb, deoxyHb, and deoxyHb in the presence of 540 μM peroxynitrite.

the main peaks in the Raman spectra shifted to the positions corresponding to the metHb spectrum, in agreement with the shift of the maximum of the Soret absorption band back to 405 nm, as reported by Exner.56 Hemoglobin Conversion Inside Nanoreactors. In order to investigate peroxinitrites detoxification mediated by the nanoreactors, we followed the conversion reactions of hemoglobin similarly to those of the free protein. First, we analyzed the conversion of metHb encapsulated in vesicles to HbCO upon addition of ascorbic acid in the presence of CO. While in the case of nanoreactors without OmpF no shift of the Soret band was observed (Figure 4B), in the case of nanoreactors with the channel proteins inserted in the polymer membrane the shift indicated the

other bands were used only to verify the reactions in the case of protein solution (Supporting Information Figure S6), but not the reaction in nanoreactors because they are not observed due to the low overall protein concentration. The transition from metHb to deoxyHb was observed by a shift in the maximum of the Soret absorption band from 405 to 430 nm (Figure 3A right), while for metHb to HbCO by a shift from 405 to 419 nm. The conversion of deoxyHb to oxyHb after removal of sodium dithionite excess in the presence of oxygen has been demonstrated by a shift of the maximum of the Soret absorption band from 430 to 415 nm.47 Peroxynitrite degradation was monitored by the inverse conversion reactions of hemoglobin with Raman and UV−vis spectroscopy (Figure 3B and C). Upon peroxynitrite addition, 15894

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Figure 4. (A) Normalized UV−vis spectra of metHb-containing nanoreactors with OmpF inserted in the membrane: before (black) and after flushing the solution with carbon monoxide (red). (B) UV−vis spectra of metHb-containing nanoreactors without OmpF inserted in the membrane: before (black) and after flushing with carbon monoxide (red).

conversion of metHb to HbCO (Figure 4A). As expected, only when the polymer membrane was permeable to ascorbic acid via channel protein OmpF insertion did the nanoreactors become fully active. Without OmpF, there is no reduction of the encapsulated metHb inside nanoreactors. This is indirect proof that Hb is completely encapsulated, and only in the presence of channel proteins is it converted in situ. We noticed that, by flushing the solution with CO, a precipitate appeared after 10 min, which induced a slightly decrease of the intensity of the Soret absorption band. The decomposition of peroxynitrites mediated by the nanoreactors was followed via the hemoglobin conversion reactions to metHb under similar conditions as in free protein solution. The shift of the maximum of the Soret absorption band from 408 to 430 nm, observed upon addition of sodium dithionite to the nanoreactor environment, indicated the successful in situ conversion of metHb to deoxyHb (Figure 5). After removing the excess sodium dithionite, encapsulated deoxyHb was converted in the presence of oxygen from the air to oxyHb, according to the shift in the Soret band to 415 nm. After the addition of peroxynitrite, the shift of the maximum of the absorption band from 415 to 408 nm indicates the final conversion to metHb (Figure 6). The slight shift of the maximum of the absorption band of metHb from 405 to 408 nm was already observed when metHb was encapsulated in a silica matrix64 or immobilized on a glass surface.65 Although the local Hb concentration within the vesicles was close to 30 μM, the overall protein concentration in solution (∼100 nM) was far below the Raman detection limit (∼10 μM). Several efforts were made to overcome the concentration limitations, such as vesicle solution concentration. Unfortunately, the Raman signal was impossible to collect from hemoglobin-containing nanoreactors because the Rayleigh scattering from the 200−300 nm size vesicles interfered greatly with the spectra acquisition. Such limitations may be overcome by using surface enhanced Raman scattering (SERS) approaches. The detection of single hemoglobin molecules in the presence of silver nanoparticles, which have the role of enhancing the weak Raman scattering signal of the molecules absorbed on the metal surface, has previously been reported.66 However, the presence of silver nanoparticles (25 nm in size) was shown to perturb the conformation of hemoglobin’s heme pocket. Thus, SERS approach would not be suited to monitor modifications in the heme pocket structure upon changes in the oxygenation state, which constitute the main objective in our study. Kinetics of Peroxynitrite Degradation. There are various kinetics studies on hemoglobin and peroxynitrite to distinguish

Figure 5. UV spectra of metHb-containing nanoreactors: before addition of sodium dithionite (black), after addition of sodium dithionite in excess (red), and after removal of sodium dithionite excess (blue).

Figure 6. UV spectra of oxyHb-containing nanoreactors before (black) and after addition of peroxynitrite (red).

the mechanism of the reaction and the intermediate species involved.56,67,68 We focused here on a different approach to study the activity of hemoglobin-containing nanoreactors, and whether the polymer membrane influences it. To follow the in situ reaction inside nanoreactors, we used a wavelength of 430 nm, 15895

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Figure 7. (A) Normalized trace of the conversion of encapsulated metHb (red) and free metHb (black) with 1 mmol/L sodium dithionite solution. (B) Normalized trace of the conversion of encapsulated deoxyHb (red) and free deoxyHb (black) with 10 mmol/L peroxynitrite solution.

maximum of the absorbance band for peroxynitrite (Supporting Information Figure S7).46 Activity Assay: Oxygen Storage Inside Nanoreactors. As hemoglobin is the active part of our nanoreactors, we also tested them as oxygen carriers. One of the main problems with hemoglobin-based oxygen carriers is that oxyHb is rapidly oxidized to metHb. To avoid this drawback in previous studies, various enzymes such as carbonic anhydrase, catalase, and superoxide dismutase were coencapsulated together with hemoglobin.70 However, limited control of the necessary enzyme amount inside the carrier and increased complexity of the preparation procedure suggest that this approach is still in need of optimization, especially for medical applications. In this respect, our nanoreactors have the advantage of a permeable membrane for molecules up to 600 Da due to the insertion of OmpF into the polymer membrane. OmpF favors the penetration of reducing agents through the polymer membrane: These agents include ascorbic acid or glutathione, which are present in the plasma71 and can enter into the nanoreactor. Once inside the nanoreactor, these reducing agents support the conversion of metHb to deoxyHb. Only in situations in which the amount of reducing agents present in vivo is low can the nanoreactors be modified by coencapsulation of a second biomolecule, for example, NADH-cytochrome b5, known to reduce metHb in red blood cells.72 To establish the oxygen transport ability of Hb in situ, we followed the uptake and release of oxygen inside the nanoreactors and compared that to the free protein solution. When a solution of deoxyHb-containing nanoreactors was passed through a column to remove the excess sodium dithionite in the presence of oxygen, the maximum of the Soret absorption band shifted within the UV−vis spectrum. This indicated the formation of oxyHb and, therefore, proved oxygen binding to the heme pocket inside nanoreactors. The release of oxygen from oxyHb was obtained by flushing the protein/nanoreactors solution with carbon monoxide in an approach slightly modified as compared to that reported by Sakai et al.73 The shift of the maximum Soret absorption band proves that HbCO was formed both under free protein conditions and when encapsulated in the cavities of nanoreactors (Figure 8). Oxygen binding is not affected by the polymer membrane, due to the chemical nature of PMOXA-PDMS-PMOXA copolymers, which do not interact with Hb, and to their intrinsic permeability to oxygen species, which does not limit the penetration of oxygen. We proved the functionality of our nanoreactors separately both in the degradation of peroxynitrites and in the transport of oxygen. These processes run inversely in terms of oxygenated

and not of 586 or 609 nm, due to the low extinction coefficients of these bands. Even if this prevented us from demonstrating the presence of the ferryl intermediate already proposed by Exner and Herold,56 it allowed us to distinguish the conversion to metHb, which represents the activity assay of the nanoreactor. In addition, due to the very small difference between the rate constants for the two steps of the reaction, we are able to obtain a rate constant value that fully characterizes the degradation process of peroxynitrite mediated by the nanoreactor. Sodium dithionite was added to a solution of free metHb and to a solution with nanoreactors containing metHb. Because of light scattering from the nanoreactors, the nanoreactor solution has a higher absorption than the solution containing free Hb. The kon value for the conversion reaction of metHb to deoxyHb in the case of free Hb (4.42 × 106 M−1 s−1) is similar to that of encapsulated Hb in the nanoreactors (kon = 4.19 × 106 M−1 s−1) (Figure 7A). The polymer membrane does not influence the reaction kinetics, as expected, because of both the oxygen permeability of the membrane and the insertion of OmpF into the membrane. After the addition of peroxynitrite to free deoxyHb and to deoxyHb-containing nanoreactors, a decrease in the absorption at 430 nm was observed. This indicates that deoxyHb was converted to a different oxidation state, both in free protein conditions and in Hb-containing nanoreactors. A kon value of 2.75 × 104 M−1 s−1 was obtained for the reaction of peroxynitrite degradation via Hb in the case of free Hb (at 5 μM concentration). When Hb is inside the nanoreactors the rate constant kon has a similar value of 2.28 × 104 M−1 s−1 (Figure 7B). The mild encapsulation procedure, based on the self-assembly process for vesicle formation under conditions that are relevant for hemoglobin (buffer, temperature) does not affect this protein’s activity. The kon values indicate that the activity of the hemoglobin is preserved upon encapsulation, and are in good agreement with the values previously reported for this reaction in free human Hb solution (2.9 × 104 M−1 s−1 for a Hb concentration of 3.0 μM).57 The peroxynitrite degradation inside red blood cells (RBCs), mediated by oxyHb present at a 5 mM concentration, has a rate constant kon of 1.7 × 104 M−1 s−1 at 37 °C and pH 7.4,69 which is on the same order of magnitude as the rate constant that we obtained both for free Hb and for the nanoreactors. To the best of our knowledge, there are no reports of peroxynitrite degradation mediated by Hb-based O2 carriers (HBOCs) or by liposomeencapsulated Hb (LEHs). These systems were proposed for oxygen transport capability only.22,24−26,29−31,33,34 The kinetics of peroxynitrite degradation, in addition, was followed at 302 nm, the 15896

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Figure 8. (A) UV spectra of oxyHb solution before flushing with carbon monoxide (black) and after flushing with carbon monoxide (red). (B) UV spectra in the domain of Soret absorption band of oxyHb containing nanoreactors with OmpF inserted into the polymer membrane before flushing with carbon monoxide (black) and after flushing with carbon monoxide (red).

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Hb species, one inducing their consumption and the other requiring them at a stable amount. Therefore, coencapsulation of a reducing biomolecule in tandem with Hb to favor the oxygen carrier function should be carefully optimized (so as not to obstruct the peroxynitrite degradation function). Depending on the intended medical application, one or the other of the nanoreactors functions is expected to play the major role.

ASSOCIATED CONTENT

S Supporting Information *

Additional experimental details as described in the text. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Notes

CONCLUSION Here, we have introduced a nanoreactor with dual-functionality: detoxification of peroxynitrites, known as reactive oxygen species involved in various pathologic conditions, and a carrier for oxygen to serve as artificial blood. We selected, as a model protein, hemoglobin, because of its natural functions of oxygen transport and peroxinitrites degradation. Nanoreactors were generated by the encapsulation of metHb in vesicles formed by the self-assembly of PMOXA-b-PDMS-b-PMOXA copolymers. Encapsulated hemoglobin preserved its functionality inside the nanoreactor, as established by conversion reactions involving various oxidation states of Hb, such as such as oxyHb, carboxyHb, and deoxyHb. The changes in oxidation states were reversible. The in situ changes in the oxidation state of hemoglobin by reaction with different molecules were allowed by the insertion of channel protein OmpF inside polymer membrane, which served as “gates” for the nanoreactor. We demonstrated that encapsulated oxyHb and deoxyHb react with environmental peroxynitrite, resulting in a degradation of peroxynitrite. Our study is the first to report peroxynitrite degradation mediated by Hb-containing polymer nanovesicles. Kinetic measurements indicate that the activity of the hemoglobin is similar to that of free protein, thus the polymer membrane does not influence the reaction kinetics. In addition, we proved that oxygen can be taken up and released inside nanoreactors, which can then also serve to transport oxygen. For extended periods of time, hemoglobin fulfilled its natural functions of oxygen storage and peroxynitrite degradation inside polymer vesicles. However, as these processes run inversely in relation to oxygenated forms of Hb, further optimization is necessary to provide nanoreactors for specific medical applications. In addition, Hb-containing nanoreactors can be used in tandem with nanoreactors based on the coencapsulation of superoxide dismutase and lactoperoxidase we introduced for superoxide radicals and related H2O2 detoxification.74 Together, they form a nanoplatform for the detoxification of superoxide radicals, peroxynitrites, and H2O2, representing an advanced approach to fighting oxidative stress.

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the Swiss National Science Foundation and by NCCR Nanoscale Science, and this is gratefully acknowledged. The authors thank Prof. W. Meier of the University of Basel for providing the block copolymer and for useful discussions. The authors acknowledge Prof. C. Schönenberger from the University of Basel for access to the Raman spectrometer. A.C.T. thanks the “Forschungsfonds der Universität Basel” for funding and T. Fröhlich for support with Raman measurements. D.D. thanks Dr. Ozana Fischer-Onaca for useful discussions. The authors acknowledge Mark Inglin for reading the manuscript.

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