Enzyme Degradable Polymersomes from Hyaluronic Acid-block-poly(ε

Article pubs.acs.org/Biomac

Enzyme Degradable Polymersomes from Hyaluronic Acid-blockpoly(ε-caprolactone) Copolymers for the Detection of Enzymes of Pathogenic Bacteria Simon Haas,†,‡ Nicole Hain,†,‡ Mohammad Raoufi,†,‡ Stephan Handschuh-Wang,†,‡ Tao Wang,‡,§ Xin Jiang,‡,§ and Holger Schönherr*,†,‡ †

Physical Chemistry I, Department of Chemistry and Biology, University of Siegen, Adolf-Reichwein-Str. 2, 57076 Siegen, Germany Research Center of Micro and Nanochemistry and Engineering (Cμ) and §Institute of Materials Engineering, University of Siegen, Paul-Bonatz-Str. 9-11, 57076 Siegen, Germany

‡

S Supporting Information *

ABSTRACT: We introduce a new hyaluronidase-responsive amphiphilic block copolymer system, based on hyaluronic acid (HYA) and polycaprolactone (PCL), that can be assembled into polymersomes by an inversed solvent shift method. By exploiting the triggered release of encapsulated dye molecules, these HYA-block-PCL polymersomes lend themselves as an autonomous sensing system for the detection of the presence of hyaluronidase, which is produced among others by the pathogenic bacterium Staphylococcus aureus. The synthesis of the enzyme-responsive HYA-block-PCL block copolymers was carried out by copper-catalyzed Huisgen 1,3-dipolar cycloaddition of ω-azide-terminated PCL and ω-alkyne-functionalized HYA. The structure of the HYA-block-PCL assemblies and their enzyme-triggered degradation and concomitant cargo release were investigated by dynamic light scattering, fluorescence spectroscopy, confocal laser-scanning microscopy, scanning and transmission electron, and atomic force microscopy. As shown, a wide range of reporter dye molecules as well as antimicrobials can be encapsulated into the vesicles during formation and are released upon the addition of hyaluronidase.

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INTRODUCTION The application of amphiphilic block copolymer vesicles in drug delivery is a widely discussed topic in the literature.1−4 These nanocapsules can be filled with various hydrophilic as well as hydrophobic compounds, and their composition and properties can be tailored toward various release triggers and dynamics fitting the application of interest. Assemblies, which are formed by self-assembly of the block copolymer,5 will “inherit” very similar physical properties as the single blocks of the amphiphile.6 These properties can be exploited, for example, to develop novel enzyme-responsive block copolymer vesicles, for the detection of pathogenic bacteria such as Staphylococcus aureus in a wound environment.7,8 Because of the impossibility to differentiate between an inflammatory response and a potentially life threatening bacterial infection in bandage-covered burn wounds based on symptoms like elevated temperature or general wound condition, a rapid in situ sensor approach that aids in the detection of infection would be important. A capsule-based approach is depicted in Scheme 1, which can be realized by exploiting a colorimetric response by dye release from a capsule, whose wall is selectively lysed by bacterial enzymes, toxins, and other virulence factors. Compared to lipid vesicle-based approaches,7,8 polymersomes offer enhanced stability and a unique tunability of the response, since the © 2015 American Chemical Society

chemical nature of the vesicle wall material, the wall thickness, and the vesicle size can be varied substantially.1−6 Previous work on block copolymer vesicles of poly(ethylene glycol)block-poly(lactic acid) (PEG-block-PLA) underlined the applicability of the polymersome approach to sense the presence of bacterial enzymes.9 Alternative approaches that are currently under development comprise enzyme-responsive hydrogels10 and nanoporous sensing platforms.11 To target the very relevant bacterium Staphylococcus aureus, a suitable enzyme must be identified. More than 90% of all strains of Staphylococcus aureus are known to secrete hyaluronidase. This enzyme, which is also produced by Clostridium and Streptococcus spp.,12 degrades the polysaccharide hyaluronic acid (HYA) (1a) (Scheme 2).13,14 HYA, which is also produced in the human body, is biocompatible and has been approved by the FDA for medical use.15 It is nowadays widely used in medicine as well as skin and body care products.16 By exploiting HYA-containing nanocarriers, Baier et al. were able to release the antimicrobial polyhexanide in an elegant approach to kill Staphylococcus aureus.17 Received: November 27, 2014 Revised: February 3, 2015 Published: February 5, 2015 832

DOI: 10.1021/bm501729h Biomacromolecules 2015, 16, 832−841

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Scheme 1. Enzymes Produced and Liberated by the Bacterium Degrade the Wall of the Polymersome Capsule. Upon Release from the Capsule Interior, the Dye Is Significantly Diluted and May Fluoresce with High Intensity. Alternatively, the Dye Is Altered Chemically upon Release Resulting in a Marked Light-Up Effect

Scheme 2. Synthesis of Hyaluronic Acid-block-Poly(ε-Caprolactone) Copolymers (1c) by Ring-Opening Polymerization of ε-Caprolactone (3) and Click-Chemistry of the Polymer with a Modified Hyaluronic Acid 1b

Block copolymers of HYA are already known.18,19 One prominent example is a block copolymer with poly(γ-benzyl glutamate),20 which was synthesized from individual blocks by 1,3-Huisgen dipolar cycloaddition. As reported here, we accomplished the synthesis of block copolymers of HYA and PCL as biodegradable synthetic polyester.9 In addition, the applicability of HYAblock-PCL in sensing bacterial enzymes via the enzyme-triggered

The strategy followed here in pursuit of the sensing approach depicted in Scheme 1 is to covalently attach the HYA to poly(ε-caprolactone) (PCL) in order to obtain an amphiphilic block copolymer that can be in parts degraded by hyaluronidase. Likewise, the PCL block is known to be biodegradable and is degraded by certain lipases. Hence, vesicles of this type of block copolymer can be applied in detecting the presence of those enzymes. 833

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were dried in an oven at 120 °C, built-up hot, and cooled in an argon stream prior to the reaction. PCL-N3 (1.00 g; 43.8 μmol) and HYA-Ac (441 mg; 87.5 μmol) were dissolved in DMSO (20 mL) under argon atmosphere. CuBr (12.6 mg; 87.5 μmol) and PMDETA (15.2 mg; 87.5 μmol; 18.3 μL) were added to the solution and stirred for 15 min under argon atmosphere. Afterward, the solution was stirred for 2 d at 60 °C. The solvent was removed in vacuo, which yielded the crude block copolymer (857 mg; 73%). 1H NMR (400 MHz, CDCl3): δ = 1.34− 1.42 (m, 380H, CH2), 1.61−1.69 (m, 796H, 2 × CH2), 2.13−2.16 (t, 2H, 2 × CH), 2.29−2.32 (t, 379H, CH2), 3.63−3.66 (t, 4H, 4 × CH), 3.86−3.89 (t, 2H, 2 × CH), 4.04−4.08 (t, 378H, CH2), 4.22− 4.26 (t, 2H, 2 × CH), 7.53 (s, 0.23 H, triazole). FTIR: 1154 cm−1 (νCOC), 1417 cm−1 (δCH2), 1470 cm−1 (δCH2), 1512 cm−1 (νCN), 1548 cm−1 (νNH), 1661 cm−1 (νCC−N), 1720 cm−1 (νCO), 2780 cm−1 (νCH), 2935 cm−1 (νCH2). Vesicle Preparation. HYA-b-PCL (61.8 mg; 4 w%) was dissolved in chloroform (1 mL). Milli-Q water (3−5 mL) was given into a round-bottom flask. Afterward, the block copolymer solution was added to the water in 10-μL steps over a period of 10 min. Cargo molecules were dissolved in water, except for the hydrophobic dye nile red, which was dissolved in the chloroform phase, before addition of the block copolymer. The mixture was stirred overnight at room temperature. Milli-Q water (5 mL) was added to the solution after 16−18 h. To remove the organic solvent and cargo residues, the vesicle solution was dialyzed (cutoff = 50 000 g/mol) for 1−4 d against Milli-Q water. Vesicle Degradation Experiments. Hyaluronidase (2 mg; 400− 1000 u/mg) was dissolved in acetate buffer (10 mL, 100 mM, pH = 5.6). α-Chymotrypsin (10 mg; 40 u/mg) was dissolved in 10% aqueous sodium chloride solution (5 mL). The vesicle solution (2 mL) was treated with the hyaluronidase solution (1 mL) at 37 °C. If N-succinyl-ala-ala-phe-7-amido-4-methylcoumarin was used as fluorescent dye, a solution of α-chymotrypsin (0.5 mL) was added additionally. Methods. NMR-spectra were measured with a Bruker Advance 400-Spectrometer (1H NMR = 400 MHz; 13C NMR = 100 MHz) at room temperature. The solvent signal was used as internal standard (δH (CHCl3) = 7.25 ppm, δC (CHCl3) = 77.0 ppm). Chemical shifts were specified in ppm. The raw data were analyzed with ACD/NMR Processor Academic Edition 12.01. Gel permeation chromatography (GPC) was performed on a PSS-System consisting of a isocratic pump and an autosampler (1200 Series, Agilent), a 254 nm Lambda 1010 UV-detector (Bischoff, Leonberg), a RI-71 differential refractometer (Shodex), and a column with 103−106 Å pore size (PSS SDV linear 5 μm 8 mm × 300 mm for THF). Tetrahydrofuran (THF) was used as eluent with a flow rate of 1.0 mL per min. The concentration of the polymer solution was 0.1−0.2%, and 20 μL was injected per run. 2,6-Ditert-butyl-4-methylphenol (BHT) was used as internal standard and polystyrene samples for calibration. The average molecular weights were calculated by using the software WinGPC Unity (Polymer Standard Service, Mainz). The solvent was THF/acetic acid (v:v = 99:1) to avoid aggregation. The system was calibrated with a set of PEG standards. Field-emission SEM images and TEM images were measured with a Zeiss Ultra 55cv. For SEM, the samples were dried on a silicon wafer and sputtered with gold afterwards, while TEM samples were dried on a TEM grid and stored under vacuum overnight. The measurements were performed under vacuum (98%) and used without further purification. 1H NMR (400 MHz, CDCl3): δ = 1.35 (s, 2H, CH2), 1.61 (s, 4H, 2 × CH2), 2.26−2.28 (d, 2H, CH2), 4.02−4.03 (d, 2H, CH2). 13 C NMR (100 MHz, CDCl3): δ = 24.5 (CH2), 25.5 (CH2), 28.3 (CH2), 34.0 (CH2), 64.1 (CH2), 173.5 (CO). FTIR: 1193 cm−1 (νCO), 1423 cm−1 (δCH2), 1470 cm−1 (δCH2), 1726 cm−1 (νCO), 2864 cm−1 (νCH), 2941 cm−1 (νCH2). Synthesis of Tosylated Poly(ε-Caprolactone)189 (PCL189OTs).22 Poly(ε-caprolactone) (2.50 g; 115 μmol) and toluene-4sulfonyl chloride (41.6 mg; 218 μmol) were dissolved in dichloromethane (20 mL) under argon atmosphere. Afterward, triethylamine (110 mg; 1.09 mmol; 152 μL) was added dropwise under moderate stirring. The reaction mixture was stirred for 24 h at room temperature. The crude reaction solution was precipitated in cold methanol (200 mL), and the solid compound was filtered off and dried in vacuo. The tosylated PCL was obtained as a white powder (2.53 g; >98%). Synthesis of Azide Functionalized Poly(ε-Caprolactone)189 (PCL189-N3).22 The tosylated PCL (2.00 g; 91.6 μmol) was dissolved in DMS (30 mL) under argon atmosphere. Afterward, sodium azide (113 mg; 1.73 mmol) was added to the solution, and the reaction mixture was stirred for 24 h at room temperature. The solvent was removed in vacuo, and the crude reaction product was dissolved in dichloromethane (50 mL). The solution was washed with an aqueous sodium chloride solution (5%, 4 × 25 mL) and with Milli-Q water (25 mL). The organic phase was dried over magnesium sulfate. The drying agent was filtered off, and the reaction product was precipitated in cold methanol (500 mL). The functionalized PCL was obtained by filtration (1.67 g; 84%). Synthesis of Acetylene Functionalized Hyaluronic Acid13 (HYA13-Ac).20 Hyaluronic acid (2.20 g; 440 μmol) was solubilized in acetate buffer (50 mL; buffer was prepared from sodium acetate (1.80 g; 21.9 mmol) and conc. acetic acid (186 mg; 3.1 mmol), which were mixed with Milli-Q water (250 mL) to yield a buffer of 100 mM, pH = 5.6) under vigorous stirring. Propargylamine (2.42 g; 44.0 mmol; 2.82 mL) and sodium cyanoborohydride (2.77 g; 44.0 mmol) were added to the solution, and the whole mixture was stirred for 5 d at 50 °C. The reaction mixture was precipitated in cold methanol (300 mL), and the solid HYA was collected by filtration. The acetylene functionalized HYA was obtained as glassy solid (1.81 g; 81%). Synthesis of Hyaluronic Acid13-block-Poly(ε-Caprolactone)189 (HYA13-b-PCL189).20 All glass wares used for the reaction 834

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Biomacromolecules AFM measurements were carried out on a MFP-3D Bio (Asylum Research, Santa Barbara, CA). The surface morphologies were scanned in intermittent contact (“tapping”) mode in air using Si probes (type AC160TS) with a nominal spring constant of 40 N m−1 and a resonance frequency of 300 kHz (Olympus, Tokyo, Japan). For imaging in water, V-shaped MLTC cantilevers (Bruker AXS, Camarillo, CA) with a spring constant of 0.5 N m−1 were used. Dynamic light scattering (DLS) experiments were performed with a Zetasizer Nano (Malvern Instruments Limited, Worcestershire, England) at 25 °C, if not stated otherwise. The samples were analyzed employing a refractive index of n = 1.460 for polycaprolactone and water with a viscosity of η25 °C = 0.8872 cP as dispersant. Fourier transform infrared (FTIR) spectra were measured with a Bruker IFS66v equipped with a liquid nitrogen-cooled cryogenic mercury cadmium telluride (MCT) detector. All measurements were performed under vacuum (90% of all strains of Staphylococcus aureus. The degradation of the HYA-bPCL vesicles was done with bovine hyaluronidase with a concentration of 47 u/mL (Figure 9). The sizes of the assemblies before and after treatment with hyaluronidase (for DLS data of neat hyaluronidase solution see

After addition of the enzyme solution, the mean size (maximum of the number distribution) did not change significantly in the first 10 min. Afterward, a fast decrease in size of the assemblies was observed. The degradation was finished after 30 min, when a plateau was reached, and the degradation products did not become smaller anymore (τ1/2 = 18 min). This fulfills the requirement of a fast degradable vesicle system (full degradation within 30 min) for potential applications as signaling device in wound dressings, etc. Enzyme-Triggered Response of HYA-b-PCL Capsules. After the degradation of HYA containing block copolymer vesicles was proven, the release of the cargo was investigated. For this N-succinyl-ala-ala-phe-7-amido-4-methylcoumarin (NSAAPAMC),10 a fluorogenic substrate for the detection of α-chymotrypsin, was encapsulated into the vesicles (Supporting Information, Scheme S-2, Figure S-4). In the protonated form, 7-amino-4-methylcoumarin fluoresces with an emission wavelength of λem = 440 nm upon excitation at a wavelength of λex = 365 nm. However, because of the covalently linked peptide chain, the excitation and emission wavelengths are blue-shifted (λex = 290 nm, λem = 395 nm), and a excitation at 365 nm does

Figure 9. DLS size distributions of HYAlong-b-PCL145 (left) and HYA13-b-PCL189 (right) vesicles treated with hyaluronidase in acetate buffer (pH = 5.6, 100 mM) at 37 °C. The vesicles (blue) are degraded by the enzymes within 30 min (red). 839

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not result in any emission. If chymotrypsin is added to a solution of the fluorogenic substrate, the peptide will be cleaved off of the chromophore and the dye fluoresce in aqueous medium, when excited. In this way, the enzymatic degradation of the fluorogenic substrate can be followed via fluorescence spectroscopy. After thorough dialysis for 5 days, the NSAAPAMCfilled vesicles were treated with hyaluronidase (47 u/mL) and chymotrypsin at 37 °C for 30 min. The fluorescence emission spectra of the vesicle solution were measured before and after the treatment with the enzymes (Figure 11).

Article

ASSOCIATED CONTENT

* Supporting Information S

Assignment of signals in NMR and FTIR spectra, microscopy analyses of polymersomes of HYA13-b-PCL189, TCSPC data of SRhB at high concentrations, DLS data of hyaluronidase, and fluorescence spectra of fluorogenic substrate during enzymatic degradation in solution. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Katrin-Stephanie Tü c king, Manuel Roesener, and Dr. Sergey Druzhinin for stimulating discussions and practical advice. Prof. Ulrich Jonas and Dipl. Lab-Chem. Petra Frank are acknowledged for the GPC analyses. Dr. Thomas Paululat is acknowledged for his help with the NMR experiments and for providing access to the FTIR spectrometer. Prof. Dr. R. Trettin is thanked for access to the DLS device. Finally, the authors gratefully acknowledge financial support from the EU (FP7 project BacterioSafe, Grant No. 245500; ERC project ASMIDIAS, Grant No. 279202), the DFG (Grant No. INST 221/87-1 FUGG), and the University of Siegen.

Figure 11. Fluorescence emission spectra (λex = 365 nm) showing the light-up of 7-amino-4-methylcoumarin upon release of the substrate from HYAlong-b-PCL145 vesicles and subsequent cleavage of the peptide bond by α-chymotrypsin.

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After the release of the fluorogenic substrate and the cleavage of the peptide bond by α-chymotrypsin, a marked increase in fluorescence can be observed. While the substrate showed almost no fluorescence before the treatment, a strong emission was observed after both enzymes were added. Since the capsules can be coated and immobilized on various polymers by deposition from solution, these polymersomes can be utilized as a key component in bacteria-detecting coatings for various applications including self-reporting wound dressings.

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CONCLUSIONS In this paper, we introduced a new block copolymer system based on HYA and poly(ε-caprolactone), which was synthesized following literature known procedures for the single blocks. These blocks were then linked by copper-catalyzed Huisgen 1,3-dipolar cycloaddition, which yielded HYA-b-PCL block copolymers. These copolymers were assembled by an inversed solvent-shift method with chloroform and water, which yielded assemblies in a size range of 50−400 nm. The assemblies, which could be shown by TEM and FLIM to be of vesicular nature, were loaded with a variety of fluorescent dyes and antimicrobials. The degradation of the assemblies was achieved with hyaluronidase, an enzyme that selectively degrades HYA. With DLS, a decrease in diameter was observed with increasing reaction time, which indicates the successful degradation of the assemblies with hyaluronidase. When loaded with a fluorogenic substrate, a spontaneous light-up upon addition of hyaluronidase and α-chymotrypsin was observed by fluorescence spectroscopy. Because of the large fraction of hyaluronidase producing Staphylococcus aureus strains, these HYA-b-PCL block copolymer systems offer a promising indicator system for the detection of these bacteria. 840

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DOI: 10.1021/bm501729h Biomacromolecules 2015, 16, 832−841