Hyaluronan

Article pubs.acs.org/cm

Triggered Release of Bacteriophage K from Agarose/Hyaluronan Hydrogel Matrixes by Staphylococcus aureus Virulence Factors Jessica E. Bean,† Diana R. Alves,† Maisem Laabei,† Patricia P. Esteban,‡ Naing Tun Thet,† Mark C. Enright,† and A. Toby A. Jenkins*,† †

Department of Chemistry, University of Bath, Bath, U.K., BA2 7AY Department of Chemical Engineering, University of Bath, Bath, U.K., BA2 7AY

‡

S Supporting Information *

ABSTRACT: The use of hydrogels as safe, biocompatible materials for wound healing has been widely utilized in recent years. Here, we investigated the use of a composite hydrogel to impart a “trigger” mechanism into an antimicrobial hydrogel system. The system was comprised of a bilayer hydrogel architecture: a lower agarose layer containing the antimicrobial virus Bacteriophage K (ΦK) and an upper layer formed of photo-cross-linkable hyaluronic acid methacrylate (HAMA) which creates the hydrogel trigger. This trigger is sensitive to the enzyme hyaluronidase, an enzyme known to be secreted by the majority of Staphylococcus aureus strains. In the presence of hyaluronidase, HAMA is degraded, releasing ΦK into the surrounding environment which consequently go on to kill surrounding bacteria. Our results show that on incubation with hyaluronidase (purified or from S. aureus), large pores form in HAMA as degradation goes on, which facilitates ΦK release. rated into nanoparticles and nanoemulsions8 as well as being used to functionalize a variety of surfaces such as polymers and wound dressings.9 None of these formulations however have contained mechanisms whereby the secretion of bacterial virulence factors directly trigger bacteriophage release. Hydrogels have been used for decades in medical applications. In wounds concerning dermal tissue, the highly aqueous, nontoxic environment provides optimum conditions to reduce pain, promote cell movement, and retain tissue hydration and structure.10 Hyaluronic acid (HA) was chosen for the hydrogel trigger in this study as the molecule is a nonimmunogenic biopolymer. The molecule can be functionalized easily through conjugation with its carboxylic acid, Nacetyl, and alcohol groups, as well as through oxidation of saccharide rings, leading to highly versatile chemistries.11 Modified and unmodified HA have also been used extensively in a wide range of applications including tissue engineering,12 cosmetics,13 and drug delivery.14 Hyaluronic acid is known to have a beneficial effect on wound healing, especially on skin and burn wounds.15 HA encourages wound healing by promoting cell proliferation and migration, with the specific mechanism still under debate.16 The high number of repeating units in HA also aids cell-to-cell interactions, with one molecule interacting with more than one cell at once. Endothelial cells possess a number of receptors for

1. INTRODUCTION The increasing prevalence of bacterial resistance to conventional antibiotics is a critical problem facing modern medicine today. The drive to find alternatives has resulted in renewed interest in natural antimicrobials to target bacterial infection; these include bacteriocins, phage lysins, antimicrobial enzymes, and heavy metals.1 In this article we will focus on Staphylococcus aureus infection and the natural viral predators of bacteria, bacteriophage (specifically the lytic anti-Staphylococcal Bacteriophage K (ΦK)).2 The resistance of S. aureus to previously effective antibiotics, notably the emergence of methicillinresistant S. aureus MRSA but also recently the advent of vancomycin resistant strains, is a major problem. S. aureus is the most common Gram-positive bacterium found in skin wound infections.3 As well as mild skin infections, the bacterium is responsible for a variety of severe infections in humans such as pneumonia, meningitis, sepsis, and Toxic Shock Syndrome. Many bacteriophage have been isolated that are active against S. aureus and which pose a promising substitute for current therapeutics.4 ΦK is a lytic bacteriophage that shows a broad host range over both MRSA and methicillin sensitive S. aureus strains; for this reason, it was selected and characterized for the present studies.5 The delivery of bacteriophage is as important as the bacteriophage itself in the bid to provide efficient treatment of infection.6 Local skin delivery of bacteriophage to an infection site has been investigated in a range of studies, with the bacteriophage successfully administered either in buffer, hydrogels, or creams.7 Bacteriophage have also been incorpo© 2014 American Chemical Society

Received: October 28, 2014 Revised: November 24, 2014 Published: December 8, 2014 7201

dx.doi.org/10.1021/cm503974g | Chem. Mater. 2014, 26, 7201−7208

Chemistry of Materials

Article

Figure 1. Schematic of bilayered hydrogel design. Lower layer contains ΦK particles (green hexagons) embedded in 0.7% w/v agarose. Upper layer comprises cross-linked HA based gel that is degraded by secreted bacterial HAase. (a) S. aureus infects host tissue, excreting virulence factors into the surrounding environment. (b) Secreted hyaluronidase diffuses into the porous network and degrades the cross-linked HAMA layer. (c) Bacteriophage K embedded in a bottom agarose layer are then able to freely diffuse and infect S. aureus, causing bacterial death and bacteriophage proliferation.

HA, notably CD44 and RHAMM. A hydrogel dressing that contains this biopolymer utilizes these beneficial properties to aid healing. Hyaluronidase (HAase) is an important virulence factor known to be secreted by certain pathogenic bacteria involved in skin infections, these include Staphylococcus, Clostridium, and Streptococcus spp.17 HAase is expressed by the extracellular hysA gene in S. aureus; the expression has been shown to be regulated by the agr gene and sarA genes, with significantly higher HAase concentrations seen in sarA and sarAagr mutants.18 The enzyme cleaves HA at the β-1,4 position, resulting in unsaturated disaccharide unit products. The mechanism of HA degradation by bacterial HAase (in this case Streptococcus pneumoniae) has been described by Jedrzejas et al. (Figure 2).19 The mechanism of bacterial HAase is different to that of mammalian HAase, in that breakdown is due to β-elimination. It is thought to be expressed by S. aureus as a spreading factor to aid the bacterial invasion of tissues;20 it may also have a role in the utilization of HA as a carbon source. A number of investigations have been carried out into the prevalence of HAase expression in Staphylococci, with at least 90% of the strains exhibiting activity. Of late, a number of triggered release systems have been investigated, most commonly with the utilization of responsive polymers in hydrogels or nanoparticles.21 Enzymatic triggers offer an elegant way for external stimuli to elicit an effect on a polymeric system.22 In most cases they are highly specific to one substrate, giving a more sensitive response than temperature or pH cues. In the case of this system the HA/HAase system was chosen because, as previously described, the enzyme is secreted in the majority of S. aureus strains, with little to no excretion in Gram-negative strains. A triggered system was chosen, as opposed to a passive system, in order to selectively release therapeutic (ΦK) only in the presence of HAase producing S. aureus. In this way, the risk of resistance occurring is eased; organisms are not continuously in contact with bacteriophage, delaying the selection pressure usually seen with continuous use. First, we proposed that agarose could be used as an inert, robust hydrogel to act as a bacteriophage reservoir. Bacteriophage would be stable in the hydrogel and could move fluidly through the matrix on infection with S. aureus. To provide a triggered release mechanism for the hydrogel, a photo-cross-linkable HA derivative was then employed (see Figure 1) which would be sensitive to secreted bacterial HAase.

Figure 2. Schematic of cleavage of HA by bacterial HAase. Amino acids Asn349, Tyr408, and His399 coordinate the gluocaronate sugar subunit, catalyzing scission of the ethoxy linkage.

2. MATERIALS AND METHODS Materials. All reagents were purchased from Sigma-Aldrich and were used as received unless stated otherwise. S. aureus strains used were grown overnight at 37 °C in tryptic soy broth (TSB) with constant shaking. Aliquots were then stored at −80 °C in 15% v/v glycerol until needed. Synthesis and Characterization. ΦK Propagation. ΦK was propagated on the bacterial host S. aureus H560 unless otherwise stated. Volumes of 100 μL of ΦK lysate and 100 μL of S. aureus H560 overnight culture were mixed and added to 3 mL of soft agar (TSB supplemented with 0.65% w/v bacteriological agar). The mixture was then poured onto a tryptic soy agar (TSA) plate and incubated overnight at 37 °C to achieve confluent lysis (widespread absence of bacterial growth). To harvest the bacteriophage, 3 mL of SM buffer (100 mM NaCl, 8.5 mM MgSO4, 50 mM Tris-HCl [pH 7.5], 0.01% gelatin in distilled water) was added to each plate, and the plates were incubated with mild shaking at 37 °C for 4 h. Phage lysate solution was then removed and 2% v/v chloroform added before centrifugation (4 000g, 15 min) to aid the removal of bacterial debris. Lysate was then filter sterilized (0.22 μm) and kept at 4 °C until needed. Live ΦK titer was calculated using standard protocols.23 Formation of ΦK Containing Agarose. Agarose (280 mg) was first dissolved in 36 mL of SM buffer with incubation at 95 °C until fully solvated. The molten agarose was then cooled to 50 °C and kept at this temperature until needed. ΦK lysate was mixed with the cooled molten agarose at a ratio of 1:9 ΦK/agarose, giving a final ΦK concentration of 108 pfu/mL and final agarose concentration of 0.7% 7202



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w/v. The mix was then cast in the required well plate or mold and allowed to set at room temperature for 10 min or until firm. Synthesis of HAMA. Photo-cross-linkable HA methacrylate (HAMA) was prepared using the method described by Leach et al.24 In brief, 1 g of HA (Mw = 1.8 × 106 g/mol) was dissolved in water overnight to form a 1% w/v solution. Volumes of 2.2 mL of triethylamine and 4.4 mL of glycidyl methacrylate and 2.2 g of tetrabutyl ammonium bromide were then added sequentially, with each reagent fully dissolved before the addition of the next. The solution was stirred at room temperature for 24 h, followed by a 1 h incubation at 60 °C. HAMA was then recovered by precipitation with acetone (20× volumes) and redissolved in distilled water to remove excess reactants. The HAMA was lyophilized and stored at 4 °C until needed. 1H NMR was used to determine the percentage methacrylation. HAMA samples were dissolved in D2O, and spectra were recorded with a Bruker 450 MHz NMR. Photo-Cross-Linking of HAMA. Solutions containing varying concentrations of HAMA (0.5−2% in H2O) and PEG diacrylate (0−10%) were mixed overnight at room temperature. The 1% Irgacure 2959 was then added and the hydrogel was mixed and incubated at 50 °C for 5 min to aid solvation. The mixture was then kept in the dark at room temperature for up to 2 weeks until needed. To photo-cross-link the HAMA mix, 400 μL of the mix was added per well to a 12-well plate with the plates then incubated under UV light (Dymax 5000 Flood curing system, 400 W) for 60 s. HA Degradation Assay. The activity of HAase enzymes in bacterial supernatant was quantified using a modified version of the Carbazole assay described by Makris et al.18 Bacterial overnight cultures (∼109 cfu/mL) in TSB broth were centrifuged at 4 000 rpm for 15 min and the supernatant filter sterilized with 0.22 μm filters. A volume of 125 μL of supernatant was then added to 250 μL of prewarmed HA solution (0.6% w/v HA, 1% w/v NaN3 and 200 mM NaCl). The vials were mixed and incubated at 37 °C for 2 h with mild shaking (100 rpm). After this time, 125 μL was removed and added to vials containing 125 μL of water and 25 μL of 0.8 M sodium tetraborate. The vials were then boiled at 95 °C for 3 min to stop further enzyme activity. Samples were cooled and stored at 4 °C until needed. A standard curve was simultaneously carried out using 125 μL of Nacetyl-glucosamine (0.5−0.05 mM) mixed with 125 μL water and 25 μL sodium tetraborate (0.8 M). Vials were boiled at 95 °C for 3 min and again allowed to cool. A color change was seen on the addition of 750 μL of 0.1× DMAB reagent (10% w/v p-dimethylaminobenzaldehyde, 12.5% v/v 10 M HCl, 87.5% v/v glacial acetic acid) and subsequent incubation at 37 °C for 20 min. The absorbance at 544 nm was measured on a BMG SPECRAstar spectrometer. Degradation Analysis of Cross-Linked HAMA. HAase solution of between 0.01 and 1 mg/mL (7−700 U/mL) was made up in SM buffer. In bacterial supernatant assays, bacteria were grown for 18 h at 37 °C. Supernatant was then harvested by centrifugation at 4 000 rpm for 10 min and consequent filter sterilization through 0.22 μm filters. A volume of 1 mL per well of HAMA cross-linking mix was cross-linked per well in a 12-well plate. A volume of 1 mL per well of either HAase or bacterial supernatant was then added and incubated at 37 °C with shaking. The degradation of cross-linked HAMA hydrogels by HAase and bacterial supernatant was followed using the Carbazole assay. Samples of 125 μL of solution were removed at varying time points and the concentration of breakdown products analyzed as described previously. Bacteriophage Release from Hydrogels. A volume of 1 mL of molten ΦK containing agarose solution (108 pfu/mL) was cast into a 12-well plate and allowed to set. The samples of 0.2%, 0.4%, and 0.7% w/v final agarose concentration were analyzed to show release with soft, medium, and hard set agarose. SM buffer (1 mL) was then added to each well and the ΦK titer in SM samples measured for each time point. Preparation of Bilayered HAMA/Agarose Hydrogels Containing Bacteriophage. ΦK containing agarose (108 pfu/mL) was formed with 1 mL per well in a 12-well plate. Once set, 400 μL of HAMA solution (2% w/v HAMA, 1% v/v PEG diacrylate, 1% w/v Irgacure

2959 in water) was placed in each well and allowed to completely cover the set agarose. The HAMA gel was then photo-cross-linked by curing in UV light for 60 s as previously described. ΦK Release from Bilayered HAMA/Agarose Hydrogels. Bilayered HAMA/agarose hydrogels containing ΦK were formed, and 1 mL of test solution (HAase and supernatant) was added per well. Plates were then incubated for the desired time at 37 °C with shaking and the ΦK titer determined. Hydrogel Characterization. Swelling studies were carried out by immersing 10 mm gel discs in PBS buffer. Discs were incubated in PBS overnight to achieve total swelling. After 18 h, discs were removed, blotted, and weighed (Ws). They were then dried at 60 °C overnight until a constant weight was seen (Wd). The swelling ratio QM was calculated as Ws/Wd. SEM analysis of the degradation of HAMA gels was measured after 2 h incubation with HAase and supernatant. Hydrogels samples were freeze-dried overnight and sputter coated with a thin layer of gold (Edwards S150B). Images were gained using a JEOL SEM6480LV SEM.

3. RESULTS AND DISCUSSION FTIR analysis verified methacrylation with peaks at 1455 cm−1 corresponding to CC stretching. The structure of HAMA was also confirmed by 1H NMR. Resonances at 5.6 and 6.2 ppm verified the presence of methylene protons coupled to the grafted methacrylate. The integration ratio of methacrylate methylene protons and the N-acetyl glucosamine methyl proton peak allowed the approximate % methacrylation to be calculated as 7%. Characterization of HAMA-PEG Diacrylate Hydrogels. HAMA (2% w/v) was co-cross-linked with a range of concentrations of PEG diacrylate, along with the photoinitiator Irgacure 2959 through free radical polymerization. The hydrogels could be cross-linked for up to 1 min without visible signs of degradation (e.g., structure breakdown or fluid exudate). The swelling ratio changed inversely with the concentration of PEG diacrylate in the hydrogel. For 0, 1, 5, and 10% w/v PEG diacrylate, the swelling ratio was determined as 68.4 ± 3.9, 42.5 ± 0.7, 15.6 ± 0.1, and 8.7 ± 0.3, respectively (n = 3). All values showed statistical significance as determined by the ptest. This indicated increasing levels of cross-linking and a decreasing mesh size, which correlated well with SEM images. A better understanding of hydrogel networks can be found using Florey−Rehner calculations; however, in this case the heterogeneity of the system means a too simplistic approximate is given. For 0% PEGDA HA hydrogels (a one component system well explained by Florey−Rehner), the mesh size was found to be approximately 6.6 μm, with a cross-link density of 1.65 × 10−8 mol cm−3 (Supporting Information). On the addition of PEGDA, however, Florey−Rehner cannot now be reliably used. An indication of mesh size and cross-link density can be gained from swelling data by utilizing the Florey parameter and specific dry polymer volume of HA throughout. By doing this, a mesh size of 3.1, 0.6, and 0.2 μm, respectively, was seen for 1%, 5%, and 10% PEGDA hydrogels; however, this is not completely illustrative of the real system. HAase Degradation of HAMA-co-PEG Gel. HAMA-coPEG hydrogels were incubated in a range of concentrations of the HA hydrolyzing enzyme, HAase (Supporting Information). Pure HAase was obtained from the naturally HAase secreting Streptomyces hyalurolyticus; although this could not be a direct comparison to S. aureus HAase, the commercial HAase was still derived from a Gram-positive bacterium. In this respect the enzyme was used as a model HAase with a similar derivation. 7203



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Figure 3. Top view SEM images after 2 h incubation of 1% PEG diacrylate + 2% HAMA hydrogels with (a) PBS, (b) 0.01 mg/mL HAase, and (c) 1 mg/mL HAase.

The enzyme has a calculated size of approximately 52 nm × 44 nm × 39 nm,25 although large for an enzyme, this is still small enough to diffuse into the HAMA network based on theoretical mesh size calculations. The concentration of the HA breakdown product N-acetyl glucosamine (NAG) was quantified using the Carbazole assay (see the Supporting Information). In brief, the assay involves the reaction of HA NAG at the reducing end with borate to form a monoanhydro sugar. On subsequent addition of acidified p-dimethylaminobenzaldehyde (DMAB), the DMAB is able to react with the sugar to form a red/purple complex with maximum absorbance at 544 nm. On incubation with HAase, an increase in the concentration of NAG was seen compared to hydrogels incubated with PBS buffer solution, showing that although modified with methacrylate, HAMA was still susceptible to enzymatic degradation. Hydrogels were also seen to disappear by 6 h in all HAase concentrations, with degradation continuing past this time as longer polymer chains were made smaller. The degradation process was analyzed through SEM after 2 h incubation with HAase at 37 °C (Figure 3). On incubation with PBS, no damage to hydrogel morphology was seen; gel homogeneity and a general absence of cavities implied the gel does not undergo significant hydrolysis with buffer. Hydrogels incubated with 0.01 mg/mL HAase showed small pores of approximately 5 μm in diameter, which were consistent through the gel. An increased concentration of 1 mg/mL gave considerably larger pores of 15−20 μm, with evidence of collapse between the layers also being apparent. HAMA hydrogels containing a range of PEG diacrylate concentrations were incubated with HAase, and the extent of breakdown of the cross-linked polymer measured after 24 h by the measurement of the formation of NAG breakdown product using the Carbazole assay (Figure 4). It can be seen that an increase in HAase concentration increases breakdown of cross-linked HAMA; an increase in the relative proportion of PEG-diacrylate however decreases the susceptibility of the cross-linked polymer to HAase. The breakdown of 10% PEGDA cogels is roughly a third of that seen for non-cross-linked HAMA gel. It is hypothesized that in high copolymer hydrogels, the viscosity of the polymer gel greatly increases slowing diffusion of HAase into the matrix. Any further work on the HAMA hydrogel was then carried out on the following optimal formulation: 2% w/v HAMA, 1% v/v PEG diacrylate, 1% w/v Irgacure 2959 (HAMA-co-PEG). This gave optimal physical properties while retaining enzymatic sensitivity.

Figure 4. Degradation of HAMA hydrogels containing 0%, 1%, 5%, and 10% PEGDA co-cross-linker. A positive correlation with gel breakdown and HAase concentration and a negative correlation with cross-link density is observed.

HAase Expression in S. aureus. A screen of 116 clinical strains was carried out to assess HAase in a wide range of bacterial strains and species (see the Supporting Information), which gave an 82.7% overall activity and 86% activity in S. aureus. These included hospital and community acquired MSSA and MRSAs, coagulase positive and negative Staphylococci, as well as other medically important species such as Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus epidermidis. The majority of S. aureus strains analyzed showed moderate HAase expression, with approximately 10% strains showing high activity and 10% no measurable activity. Little to no expression was seen in Gram-negative and coagulase negative strains; however, S. epidermidis strains did exhibit moderate HAase expression. It is known that some Gram-negative organisms secrete HAase periplasmically; consequently, because of this reduced diffusion, concentrations are markedly lower and the enzyme is not thought to play a role in pathogenesis.26 The secretion of HAase was also then monitored over the growth cycle of both HAase positive (H560) and negative (Mμ2) S. aureus strains (Figure 5). In S. aureus H560, HAase expression was predominantly seen in the early exponential phase of bacterial growth, with a peak after 4 h growth, followed by a gradual decrease in expression once the stationary phase is reached (Figure 5a). This was consistent with the indication that HAase is a spreading factor involved in early 7204



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The Carbazole assay was again used to quantify the concentration of NAG breakdown products on incubation with supernatant (Figure 6). First, no significant breakdown

Figure 6. Degradation of HAMA hydrogels by HAase positive (green) and HAase negative (red) S. aureus supernatant.

was seen in HAase negative strains (Mμ2 and hys-) compared to the negative control (TSB). With strains known to produce HAase, however, the concentration of NAG breakdown products increases as the polymer is broken down. The rate at which this is done is different between strains. High HAase producers RN6390B, H560 with RN6390B, and lac exhibited relatively fast breakdown with a plateau seen after 6 h. Visually, hydrogels were completely dissolved after incubation with HAase positive S. aureus supernatant. ΦK Propagation and Sensitivity. High titer (>109 pfu/ mL) ΦK was successfully propagated using S. aureus H560 as a host strain. The standard protocol for determination of bacteriophage titer is the plaque assay, where bacteriophage solution is serially diluted and plated onto a lawn of host bacterium. The titer calculated is the number of live infectious bacteriophage per mL. The assay does not measure “dead” or damaged virions. ΦK was able to be stored at 4 °C for a number of months without a significant loss in titer being seen (data not shown). ΦK is a well characterized bacteriophage with the genome already sequenced by O’Flaherty et al.; recently Gill et al. have also published additions to this sequence.28 TEM imaging of the bacteriophage has been described in previous publications;7 ΦK is in the class Myoviridae with an icosahedral head and 200 nm contractile tail.29 In a previous study by the authors, approximately 64% of a range of hospital and community acquired S. aureus strains (including MRSAs) exhibited susceptibility to ΦK, with a further 34% having intermediate susceptibility.30 ΦK Efficacy and Diffusion from Agarose. ΦK were mixed with molten agarose in SM buffer to form a stable, aqueous environment in which bacteriophage were immobilized but still able to diffuse and infect. The diffusion of bacteriophage into buffer solution was assessed in 0.2%, 0.4%, and 0.7% w/v agarose gels (Figure 7). A fast release of bacteriophage was seen in all agarose concentrations with a burst release by 30 min incubation. After 6 h incubation, the highest ΦK titer of 1.6 × 106 ± 2.2 × 105 pfu/mL was seen in the most aqueous gel, 0.2% agarose; 0.7% agarose gels showed the least bacteriophage diffusion with 6 × 104 ± 5.4 × 103 pfu/

Figure 5. HAase expression (blue, 103 mmol NAG released/mL/ min−1OD600) with bacterial growth (black, OD600) in HAase positive, H560, (a) and negative, Mμ2, (b) S. aureus strains.

bacterial invasion of hosts.20 In HAase negative S. aureus Mμ2, no expression of HAase was detected. Bacterial Supernatant Degradation of HAMA-co-PEG Gel. HAMA-co-PEG hydrogels were incubated with overnight bacterial supernatant for 2 h. Top view SEM images of the hydrogels after incubation showed significant differences in hydrogel morphology (see the Supporting Information). HAMA hydrogels incubated with strains known to secrete HAase (images a, b, c) all exhibited clear visual signs of enzymatic degradation. Large areas of hydrogel loss were seen that permeated through the entire matrix. This enzymatic hydrolysis leads to increased permeability and the eventual dissolution of the gel. Hydrogels were also incubated with S. aureus strains that showed no HAase activity in the initial strain screen. S. aureus hys-,27 a genetic mutant containing a transposon in the hysA gene (image d) showed no changes compared to the negative control (TSB growth medium, image f). On incubation with TSB growth medium (negative control), despite the apparent surface roughness of the gel, no pores were seen in the matrix. The hydrogel top layer remained confluent with no pores formed. On incubation with Mμ2 (image e), a phenotypic mutant where despite hysA being present, no activity was seen in the strain screen, small circular pores were seen in some parts of the hydrogel. This brings into focus the limitations of the Carbazole assay, as a loss of sensitivity is seen below 0.05 mM NAG. In the case of Mμ2, HAase could still be produced in very low concentrations but is not detected. 7205



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Figure 8. ΦK release from 0.7% agarose (blue) hydrogels and gels after 2 h, 4 h, and 6 h incubation with SM buffer (red) and 1 mg/mL HAase (green). ***p < 0.001.

Figure 7. ΦK release from 0.2% (black), 0.4% (red), and 0.7% (blue) w/v agarose (initial titer 108 pfu/mL).

release is then seen in the presence of purified HAase compared to buffer; a 103 pfu/mL ΦK difference after 2 h, 104 pfu/mL after 4 h, and 102 pfu/mL after 6 h. In the presence of no HAase (SM buffer negative control) “background release” was observed, as some bacteriophage were able to diffuse through larger pores in the porous HAMA matrix. Notwithstanding, a statistically significant increase in ΦK titer was still seen on incubation with HAase. Triggered Release of ΦK by Bacterial Supernatant. In order to establish if triggered ΦK release was seen from the dual agarose-HAMA system, we incubated the gels with bacterial supernatant of both HAase positive (RN6390B, Not380, H050960412, D98, Cuba4005, CDC201078USA700, C3, CAN6820-0616) and negative (963Small, D470, D473, HT2001-634, hys-, HT2002-0635, MRSA378, C154) S. aureus strains. The strains were chosen to cover a broad range of S. aureus types, with both MRSAs and MSSAs of varying virulence being assessed. After 4 h incubation, the concentration of bacteriophage released was calculated (Figure 9).

mL. The difference in this titer was thought to be due to the partial entanglement of bacteriophage at increased concentrations of agarose polymer. The diffusion of bacteriophage from a range of agarose concentrations was also visualized by incubating hydrogel discs on an S. aureus H560 bacterial lawn (see the Supporting Information). Larger zones of inhibition were seen with decreasing agarose concentration; 0.2% agarose allowed facile diffusion up to 21 ± 0.7 mm whereas in 0.7% agarose only 10 ± 0.3 mm was seen. Although allowing greater bacteriophage diffusion, 0.2% and 0.4% agarose hydrogels were too soft to be practical. The 0.7% agarose enabled bacteriophage to diffuse into the surrounding environment while also being the most robust. The ΦK-agarose hydrogel was incubated with live bacterial culture to ensure bacteriophage were still able to prevent bacterial growth in agarose. Normal bacterial growth was seen when incubated with pure agarose gel. The growth of bacteria in ΦK-agarose however was completely prevented with no increase in OD600 seen. Preparation of ΦK-Agarose/HAMA-co-PEG Bilayer Hydrogels. Bilayer hydrogels were formed by cross-linking HAMA solution on top of set 0.7% agarose which contained 108 pfu/mL ΦK. It has long been known that UV irradiation has an inactivating effect on bacteriophage, with a decrease in bacteriophage titer being seen.31 The 1 min UV irradiation needed for HAMA cross-linking was not found to cause a significant loss in bacteriophage titer released from agarose. On the contrary, previous experiments have been carried out with poly vinyl (alcohol) (PVA) as the immobilization hydrogel which showed near complete loss of bacteriophage titer (data not shown); this was attributed to highly damaging radical species being formed in PVA which effect bacteriophage, which do not occur in agarose. Triggered Release of ΦK by HAase. ΦK-agarose/ HAMA-co-PEG hydrogels were first incubated with pure HAase to assess if triggered release of bacteriophage could be seen on addition of purified enzyme (Figure 8). On the addition of the HAMA layer, first we saw no detectable ΦK diffusion through the gel matrix compared to ΦK-agarose. Although the HAMA hydrogel is porous, it is not as porous as agarose and is still capable of preventing a significant amount of ΦK diffusion into the surrounding environment. A significant

Figure 9. Triggered release of ΦK after 4 h incubation with HAase positive (green) and negative (red) S. aureus supernatant. Less than 102 pfu/mL was below the detection limit for titer calculation. Each data point is a result of three independent experiments. 7206



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Potter and John Mitchels, MAS Suite, University of Bath, for their assistance with imaging.

In HAase positive strains, the HAMA layer was seen to be completely removed. A significant increase in the amount of ΦK released was also seen with hydrogels incubated with these strains. The HAase present in the S. aureus supernatant was able to degrade the HAMA layer, allowing ΦK diffusion. These bacteriophage can then go on to infect S. aureus present in the surrounding environment. The average concentration of ΦK seen was approximately 105 pfu/mL; a concentration that is sufficient to cause a significant, if not total, loss of bacteria. Conversely, little to no release was seen on incubation with HAase negative strain supernatant. The HAMA layer still remained after incubation, implying no loss of gel integrity. Less than 5 × 102 pfu/mL ΦK were seen in solution after 4 h incubation. This concentration is not enough to cause significant bacterial killing.

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ABBREVIATIONS HAMA, hyaluronic acid methacrylate; HA, hyaluronic acid; TSB, tryptic soy broth; HAase, hyaluronidase; ΦK, Bacteriophage K; OD600, optical density 600 nm; NAG, N-acetyl glucosamine

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4. CONCLUSIONS This system described here could be used for the triggered release of a large range of therapeutics aimed at S. aureus eradication. The novel release mechanism allows therapeutic to only be delivered in the presence of HAase secreting strains (namely, S. aureus), thus slowing the formation of resistant strains occurring. The matrix would allow a “smart” treatment method which makes treatment automated, avoiding the extra time and costs associated with a healthcare professional. The use of bacteriophage instead of antibiotics in this system also utilizes the elegant bacterial control system seen in nature. In many ways the use of ΦK here is a “model” system; the use of other bacteriophage and bacteriophage cocktails can be used to tune sensitivity. It is generally believed that in nature, bacteria do not exist as planktonic organisms but as complex biofilms, where bacteria are protected from the environment by a polysaccharide matrix. It is known that HAase expression is regulated by sarA, a key gene that is also involved in biofilm formation. The expression of HAase in biofilms has been investigated in Streptococcus intermedius strains by Pecharki et al.; HAase expression was seen from biofilms (in a similar way described in Figure 4a), with a significant upregulation on the addition of HA to the growth media.32 Our release system must also be sensitive to biofilm HAase in order to be viable in a clinical environment.

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ASSOCIATED CONTENT

S Supporting Information *

FTIR and 1H NMR spectra of HAMA molecules, swelling measurements, HAase breakdown of HAMA, incubation of S. aureus with agarose-ΦK, and 116 strain screen of bacterial HAase expression. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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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 support of the BBSRC Grant No. APG20594 and EPSRC Healthcare Partnership Grant (No. EP/027602/1) and support from The Healing Foundation funded Children's Burns Research Centre are gratefully acknowledged. We thank Ursula 7207



Article

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