Article pubs.acs.org/Biomac
Characterization and Activity of an Immobilized Antimicrobial Peptide Containing Bactericidal PEG-Hydrogel Rik T. C. Cleophas,† Jelmer Sjollema,‡ Henk J. Busscher,‡ John A. W. Kruijtzer,† and Rob M. J. Liskamp*,† †
Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands ‡ Department of Biomedical Engineering, University of Groningen and University Medical Center Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands ABSTRACT: A single step immobilization-polymerization strategy of a highly active antimicrobial peptide into a soft hydrogel network on a poly(ethylene terephthalate) surface using thiol−ene chemistry is described. The bactericidal hydrogel was molecularly characterized via Coomassie and Lowry assay protein staining agents as well as by X-ray photoelectron spectroscopy. The bactericidal activity was established against Staphylococcus aureus and Staphylococcus epidermidis, two bacterial strains commonly associated with biomaterial infections. To gain further insight into the biological stability, the hydrogels were incubated with human serum prior to activity testing without loss of activity. These studies revealed a promising bactericidal hydrogel with good stability under physiological conditions.
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INTRODUCTION
diminished antimicrobial activity, albeit without affecting the broad spectrum antibacterial activity.25,26 The majority of these studies involved the immobilization of AMPs (e.g., polymyxin B and nisin) on a hard surface, such as titanium or glass.27,28 A multistep procedure was needed to introduce a spacer between the bioactive peptide and the surface to ensure a certain degree of freedom for the AMP for orientation toward the pathogens. Alternatively, modification of for example commercially available ocular lenses with melimine gave rise to a soft antimicrobial surface.29 The combination of a stabilized AMP with a polyethylene glycol (PEG) network was earlier developed in our group.30 PEG hydrogels are intrinsically resistant to protein adsorption and cell adhesion and thus, serve as an excellent starting point in designing antimicrobial surfaces of biomaterials.31,32 The versatility and tunability of PEG hydrogels permits their use in various biomedical applications.33 A one-step synthesis of such hydrogels was recently achieved by applying thiol−ene chemistry.34 Simple surface modifications further allowed introduction of highly functionalized molecular systems, using robust, simple and high yielding chemistry. Thus, we utilized this method in combination with the previously tested stabilized antimicrobial peptide, inversoCysHHC10 (H-ckrwwkwirw-NH2, all D-enantiomer) to establish one of the first highly bactericidal hydrogel surface coatings in a single step procedure (Scheme 1). Membrane disruption
Implantation of medical devices is growing widely and is essential for maintaining quality of life, especially in view of the general increased life expectancy. However, this growth of implantation of permanent or temporary devices is also accompanied by an increasing incidence of biomaterialassociated infections leading to severe patient discomfort, hospitalization, and high costs.1,2 The spreading of (multi)resistant bacterial strains further complicates this issue.3,4 Efforts to prevent bacterial colonization of medical implants have been mainly directed toward the release of antibiotics such as gentamicin5,6 and ciprofloxacin,7,8 and antimicrobial peptides9−12 for local delivery. However, successful, long-term use of these systems is discouraged by its dose gradient around the device thereby inducing resistance of bacteria retracted in tissue niches.13,14 In addition, the use of silver in vivo proved to be ineffective in reducing the incidence of urinary tract infections and further investigation is needed to firmly establish tissue toxicity.15,16 The use of antimicrobial peptides (AMPs) is highly advantageous over conventional antibiotics due to its broadspectrum activity, selectivity, and minimal bacterial resistance observed so far.17,18 An increase of metabolic stability by using D-amino acids makes covalently attached antimicrobial peptides in principle suitable for long-term use while avoiding the risk of inducing resistance.19 So far, several combinations of surfaces modified with AMPs as the active antibiotic have been reported.20−24 Other studies have clearly demonstrated that immobilization of AMPs led to a © 2014 American Chemical Society
Received: June 19, 2014 Revised: August 7, 2014 Published: August 9, 2014 3390
dx.doi.org/10.1021/bm500899r | Biomacromolecules 2014, 15, 3390−3395
Biomacromolecules
Article
Scheme 1. Synthesis of Hydrogel Coating Containing Immobilized AMPs Using Thiol−Ene Click Chemistry and the Reaction Mechanism30
with a 365 nm wavelength lamp (UV-fusion Systems, D-bulb) for 5 consecutive runs (each ca. 15 s) on a conveyor (UV-Fusion Systems, DRS10/12 Conveyor Systems) to give a clear hydrogel film. Bactericidal Activity Coatings. To evaluate the bactericidal activity of the hydrogel coatings (20 × 20 mm2) against Staphylococcus aureus (ATCC 49230), a slightly modified Japanese Industrial Standard JIS Z 2801:200036 apparatus was used. All samples were washed to remove any nonbound peptide by shaking in 15 mL of water for at least 16 h at 150 rpm at room temperature and were subsequently sterilized in 70% ethanol and dried in a sterile environment for at least 30 min prior to bacterial inoculation. An overnight culture was diluted 100-fold with tryptic soy broth (TSB) and incubated for another 3 h to yield a logarithmically growing test bacteria (S. aureus ATCC 49230), which were used to prepare a suspension with a of concentration of 2 × 105 colony-forming units (CFU) per milliliter in 10 mM phosphate buffer pH 7.0 containing 1% (v/v) TSB (PT). Samples of hydrogel coated PET were inoculated with 32 μL of bacterial suspension and diluted with 32 μL of PT, and sterilized parafilm (18 × 18 mm2) (i.e., slightly smaller than that of the coated surface) was placed on top of the inoculated hydrogel coatings. As a positive control, 32 μL of 1.2 mM Ac-HHC10 in PT was added instead of the PT. All hydrogel coatings with bacteria and parafilm were placed individually in wells of a 6-well plate (Corning Inc., New York) and incubated at 37 °C for 24 h. After incubation, 1.6 mL of 0.1% Tween80 in phosphate buffered saline (PBS) was added to each well followed by sonication of the plate for 30 s and gently shaking for 2 min. This procedure does not affect bacterial viability. Seven 10-fold serial dilutions in PT were made in a 96-well plate. Subsequently, duplicate 10 μL aliquots of the undiluted suspension and of the seven dilutions were pipetted onto blood agar plates (Oxoid, Basingstoke, U.K.). The blood agar plates were incubated overnight at 37 °C, and the numbers of colonies were counted the following day. A similar procedure was used for testing against Staphylococcus epidermidis ATCC 35984.
by the net positive charge of the immobilized peptides gives rise to bacterial cell death. The incorporation of the antimicrobial peptide in the entire hydrogel and not only on its surface permits (small) unavoidable damages during for example surgical handling, thereby preserving its membrane disruptive bactericidal activity. The characterization of covalently bound antimicrobial peptides in such a hydrogel network is less well established. Previously described antimicrobial assays proved to be reliable, but are laborious and time-consuming.35 We therefore wish to report here additional simple, reliable, and rapid tests to quantify the peptide concentration in a hydrogel network. Using these tests, it was also possible to determine the minimal AMP peptide concentration needed to prevent biomaterial associated infections in vitro.
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EXPERIMENTAL SECTION
Peptide Synthesis. Peptides were synthesized by solid phase peptide synthesis on Rink Amide resin (0.24 mmol/g) (Rapp Polymere GmbH, Tübingen, Germany) on a 0.25 mmol scale. The peptide was assembled using an automatic ABI 433A peptide synthesizer, equipped with a UV-monitoring system as described before.30 Coating Synthesis. Pentaerythritol tetrakis(3-mercaptopropionate) (PTMP) (1.56 mL, 4.1 mmol), poly(ethylene glycol) diacrylate (PEGDA) (Mn 700, 6.25 mL, 10 mmol), and
