Article www.acsabm.org
Cite This: ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Pseudomonas Infection Responsive Liquid Crystals for Glycoside Hydrolase and Antibiotic Combination Chelsea R. Thorn,†,‡ Clive A. Prestidge,† Ben J. Boyd,§ and Nicky Thomas*,†,‡
Downloaded via UNIV OF SYDNEY on July 14, 2018 at 22:47:56 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
†
School of Pharmacy and Medical Science, and ARC Centre for Excellence in Bio-Nano Science and Technology, University of South Australia Cancer Research Institute, North Tce, Adelaide, South Australia 5000, Australia ‡ Biofilm Test Facility, Sansom Institute, University of South Australia, City East Campus, Frome Road, Adelaide, South Australia 5001, Australia § Drug Delivery Disposition and Dynamics, and ARC Centre for Excellence in Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052 Australia S Supporting Information *
ABSTRACT: Bacterial biofilms account for up to 80% of all communityacquired infections for which bacterial eradication is currently not achievable using conventional antimicrobial treatments. The protective matrix that engulfs biofilm-associated bacteria frequently renders antibiotics ineffective. Glycoside hydrolases are a class of enzymes that break down the biofilm matrix, thereby increasing the effectiveness of antibiotics. Herein, nanostructured liquid crystals composed of glyceryl monooleate (GMO) were investigated as an infection responsive delivery system for alginate lyase (glycoside hydrolase) and gentamicin (antibiotic) to treat Pseudomonas biofilms. The presence of Pseudomonas lipase triggered the release of alginate lyase and gentamicin from the GMO liquid crystals. Treatment with the liquid crystals containing alginate lyase and gentamicin resulted in a greater than 2-log reduction in mucoid Pseudomonas aeruginosa (clinical isolate) biofilm. The anti-biofilm activity of alginate lyase and gentamicin from the liquid crystals was sustained for 2 days and equivalent to the respective unformulated solution treatments. Accordingly, GMO based liquid crystals are a promising responsive delivery system for alginate lyase and gentamicin to combat topical Pseudomonas infections. KEYWORDS: alginate lyase, gentamicin, biofilm, liquid crystals, glyceryl monooleate
■
INTRODUCTION
antibiotic gentamicin, which is otherwise electrostatically immobilized by alginate.8,9 The therapeutic use of alginate lyase is however limited due to instabilities (e.g., susceptibility to proteolysis) in addition to inadequate delivery and release of the enzyme at the infection site.10 Moreover, combined therapy with antibiotics is required, given that alginate lyase does not have any antimicrobial activity itself. Thus, a delivery system for alginate lyase and gentamicin that protects and delivers both compounds to a biofilm infection site would be beneficial. Various delivery systems have been investigated for the oral delivery of alginate lyase including hyaluronan−cholesterol hydrogels,11 cross-linking enzyme aggregated pectins,12 alginate, and high methoxylated pectin microspheres and hydrogels.13,14 However, of the delivery systems explored, none show any advantage of effective delivery of alginate lyase to the infection site. While oral delivery is patient friendly, biofilm infections typically occur at a localized site (i.e., in the
The global threat of antimicrobial resistant bacteria is more prevalent than ever, with the mortality rate expected to rise to 10 million deaths per year by 2050.1 It is paramount that novel antimicrobials are developed in response to life-threatening bacterial infections. Of particular concern are bacterial biofilms: communities of bacteria surrounded by a thick matrix composed of extracellular polymeric substances (EPS).2 Clinically, biofilms are associated with approximately 65−80% of all chronic infections, for instance in chronic rhinosinusitis and nonhealing wounds.3,4 Currently, no effective antimicrobial therapy is available against biofilm infections, where bacteria are up to 1000-fold more tolerant to antimicrobials compared to nonattached, planktonic bacteria.5 The EPS is a key barrier of biofilms, preventing antimicrobial entry.6 New developments have shown glycoside hydrolases to be promising tools to disrupt the polysaccharides within EPS, thereby allowing entry of antimicrobials and increasing their effectiveness.7 The glycoside hydrolase, alginate lyase, has been shown to degrade matrices rich in alginate formed by Pseudomonas aeruginosa and to enhance the action of the © XXXX American Chemical Society
Received: May 1, 2018 Accepted: June 29, 2018
A
DOI: 10.1021/acsabm.8b00062 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Article
ACS Applied Bio Materials
keV. The sample to detector distance was 1529 mm, giving a q range 0.010−0.67 Å−1. The scattering was acquired for 1.0 s on a Pilatus 1 M detector. The computer program ScatterBrain was used to acquire and convert 2D patterns to 1D intensity versus q profiles. Bragg peaks were indexed to corresponding phase structure and were calculated in addition to the lattice parameters using known relationships as described by Hyde.21 In Vitro Release Studies. In Phosphate Buffered Saline (PBS). The liquid crystal gels (0.20 g) were loaded into the welled pillars of a custom designed aluminum multisample plate holder (see Supporting Information, Figure S1), fabricated at the South Australian Node of the Australian National Fabrication Facility based on a design from Bisset, Boyd, and Dong.22 The aluminum multisample plate holder was inverted into a 24-well plate containing PBS at pH 7.40. At specific time points, 200 μL samples were taken and replaced with fresh media. Samples containing AL were quantified for enzyme content through the Pierce BCA Protein Assay Kit (ThermoFisher Scientific, MA, USA). Samples containing gentamicin were analyzed for content through a microbiological technique, as previously described.23 Briefly, Staphylococcus aureus ATCC strain 29213 from a freshly streaked agar plate was diluted in sterile 0.90% saline to a 7.0 McFarland Standard and then diluted 1:1250 in Mueller-Hinton agar (at 50 °C). After the inoculated agar was poured and dried, 6.0 mm diameter holes were punched and filled with 25.0 μL samples and incubated for 24 h at 37 °C. Inhibition zones of the samples from the release study where compared to known concentrations of gentamicin using a standard curve. In Presence of Porcine Pancreatic and Pseudomonas Lipases. The release of AL and gentamicin from the GMO liquid crystals was investigated in the presence of lipase from porcine pancreas (20 mg/ mL) and Pseudomonas species (1.0 mg/mL). Briefly, lipases were dissolved in digestion media containing 50 mM Tris maleate, 0.15 M NaCl, 5 mM CaCl2·2H2O, and Milli-Q water. The pH was adjusted to 7.55 using NaOH. For porcine pancreatic lipase, 5 mM bile salts and 1.3 mM phosphatidylcholine were also dispersed throughout the media.24 The liquid crystal gels containing AL and gentamicin were loaded into the welled pillars of the aluminum multisample plate holder and inverted into the media containing either porcine pancreatic lipase or Pseudomonas lipase. At specific time points, 200 μL samples were withdrawn and replaced with fresh digestion media. The samples were then centrifuged at 21 130 × g for 90 min at 4.0 °C. The supernatant was then analyzed for AL content through the BCA assay and gentamicin through the microbiological assay, as previously described. Alginate Lyase Stability. AL degrades alginate by β-elimination at glycosidic 1 → 4 O-linkage, forming a C-4 and C-5 carbon−carbon π bond at the new nonreducing termini, which strongly absorbs light at 235 nm.25 The reaction between alginate and AL was therefore quantified through mapping the product over time using ultraviolet− visible spectroscopy. Briefly, 190 μL of alginate (0.15 mg/mL) was added to the wells of a UV-transparent 96-well plate (Greiner BioOne, Kremsmünster, Austria). To begin the reaction, 10.0 μL of a freshly prepared AL solution (60 μg/mL) and a solution treated the same as the fabrication of the liquid crystals (60 μg/mL) were added to wells containing alginate. In addition, AL (60 μg/mL) stored for 48 h at 4.0 °C, and 37 °C in PBS, pH 7.40, were also reacted with alginate (0.15 mg/mL). Upon mixing, the plate was transferred into a plate spectrometer (Inspire Multimode Plate reader, PerkinElmer, Waltham, MA), and the formation product was read at an absorption of 235 nm every 5 min for 2 h. In Vitro Biofilms Studies. Liquid Crystals Compared to Unformulated Solutions . Mucoid P. aeruginosa (clinical isolate) from a freshly streaked agar plate was suspended in 0.90% saline and adjusted to 0.5 ± 0.1 McFarland standard. The suspension was further diluted 1 to 100 in LB media and added to the wells of a 24-well plate, leaving the final column for sterile LB media to act as the negative control. The plate was wrapped in aluminum foil and incubated for 24 h at 37 °C to grow biofilms.
sinuses or wounds); thus, delivery systems that promote topical administration require further exploration. Nanostructured lipid liquid crystals are multidimensional constructs of lipids intercalated within water channels to form mesophases whose structure is dependent on molecular shape, concentration of amphiphile, and environment conditions.15 Lipid liquid crystals are gaining interest as drug delivery systems for a range of different molecular weight compounds.15 Release of compounds can be modulated by different phase structures of the liquid crystals including the reverse hexagonal (H2), inverse bicontinuous cubic (Im3m, Pn3m, Ia3d), and discontinuous micellar cubic phases (Fd3m).15,16 Glyceryl monooleate (GMO) is a lipid commonly used to fabricate liquid crystals of the inverse bicontinuous cubic phase, in which the bulk material is a highly viscous gel that affords topical delivery.17 GMO is digested into glycerol and oleic acid by lipasemediated hydrolysis and is thus considered to be biodegradable and biocompatible.17 In oral drug delivery, the digestion of GMO by pancreatic lipases is unfavorable as drug is released prematurely, limiting drug absorption.18 Pseudomonas also produces lipases as a virulence factor to invade and disrupt macrophages and platelets in the human immune response.19 The digestibility of GMO in the presence of bacterial lipases is unknown, hence it is hypothesized that Pseudomonas lipases can degrade GMO, prompting responsive release of alginate lyase and gentamicin at the local Pseudomonas infection site. The aim of this study was to investigate the feasibility of GMO liquid crystals to protect and deliver alginate lyase together with gentamicin as a novel delivery system to control P. aeruginosa biofilms.
■
METHODS
Materials. Alginate lyase (AL) (≥10 000 units/g solid), gentamicin USP, phosphate buffered saline (PBS), Trizma maleate, sodium chloride (NaCl), sodium hydroxide (NaOH) pellets, calcium chloride dihydrate (CaCl2·2H2O), lipase from Pseudomonas bacteria, pancreatin from porcine pancreas, piperazine-1,4-bis (2-ethanesulfonic acid) (PIPES) buffer, hexamethyldisilazane (HDMS), Luria− Bertani (LB) media, Mueller-Hinton media, and agar were purchased from Sigma-Aldrich (St. Louis, MO, USA). Glyceryl monooleate (Myverol 18−99K) was kindly donated by Kerry Ingredients and Flavours (Egham, Surrey, UK). Myverol 18−99K was composed of >90% unsaturated monoglycerides and >60% glycerol monooleate. Staphylococcus aureus strain ATCC 29213 (American Type Culture Collection, Manassas, VA) and mucoid Pseudomonas aeruginosa clinical isolate (alginate-producing) were obtained from the institutional culture collection (UniSA, Adelaide). Preparation of the Liquid Crystals. Bulk liquid crystals were prepared by weighing 0.70 g of GMO and 0.30 g of PBS (pH 7.4) into glass vials. The lipid and aqueous phases were combined through three, alternate (60 s) cycles of heating to 60 °C and vortex mixing. Subsequently, the mixture was then centrifuged for 10 min at 21 130 × g (ambient) and then left to equilibrate at 37 °C for 5 days. The blank liquid crystals were prepared as described, whereas the loaded liquid crystals contained either (1) AL (1.0% w/w), (2) gentamicin (0.0050% w/w), or (3) combined AL (1.0% w/w) and gentamicin (0.0050% w/w) in the 0.3 g aqueous phase. Structural Determination of the Liquid Crystals. The structure of the liquid crystals was determined by small-angle X-ray scattering (SAXS). Equilibrated liquid crystal gels were loaded into the bottom of a 96-well plate at ambient temperatures and sealed with Kapton tape immediately. Measurements were performed at the Australian Synchrotron on the SAXS/WAXS beamline.20 At least five measurements were recorded on each gel at different locations within the well. The wavelength of the X-rays was 0.95 Å at an energy of 13 B
DOI: 10.1021/acsabm.8b00062 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Article
ACS Applied Bio Materials The biofilms in the wells of the microtiter plate were then washed twice with 0.90% saline to remove nonadherent bacterial cells. For solution treatments, AL (0.10 mg/mL), gentamicin (6.0 μg/mL), and a combination of the two in LB media were added to the biofilm. For AL and gentamicin in the GMO liquid crystals, 0.20 g (equivalent concentrations of AL and gentamicin to the solutions) was loaded into the pillars of the aluminum multisample plate holder and inserted into the wells of the microtiter plate (containing fresh media). Crystal Violet Assay. To quantify biofilm biomass, biofilms were fixed onto the wells by the addition of methanol (200 μL). Following methanol removal, the wells were exposed to 0.10% (w/v) crystal violet for 15 min, and excess dye was washed away with deionized water. The remaining dye that stained the biofilms was solubilized by the addition of 30% (w/v) acetic acid. The biofilm biomass was quantified by absorption at 595 nm by spectrometry (Inspire Multimode Plate reader, PerkinElmer, Waltham, MA).26 Bacteria Enumeration. To quantify the total number of bacterial cells within the biofilm, the biofilms were extracted with 0.90% saline from the wells (two alternating cycles of sonication followed by vortexing), as previously described.27 Then 500 μL of the bacterial suspensions were serial diluted in LB agar, and the colony forming units (CFU) were counted after 18 h incubation at 37 °C. Time Dependent Anti-biofilm Activity . To ascertain the effect of the release of AL and gentamicin from the GMO liquid crystals, release samples at 0.50 h, one-, two, three-, and five-days were taken as described above and sterilized through a syringe filtration unit (Filtropur S 0.45, Sarstedt, Technology Park, SA, Australia). Using MBEC microtiter (Innovotech, Alberta, Canada) plates, biofilms were grown from mucoid P. aeruginosa (clinical isolate) for 24 h as described above. Biofilms grown on the pegs were washed twice with sterile 0.90% saline before placing the lid into another base plate containing the release samples from the liquid crystals and freshly prepared AL (0.1 mg/mL) and gentamicin (6.0 μg/mL) solution treatments. The MBEC plate was then incubated for a further 24 h with the treatments. Thereafter, each individual peg was removed from the plate with sterile pliers, inserted into 1.00 mL of sterile 0.90% saline, and briefly sonicated to disperse the biofilm bacteria. Following 500 μL of bacterial suspensions were serial diluted into 900 μL of sterile 0.90% saline and was used to plate (LB agar) and enumerate bacteria after 18 h incubation at 37 °C.27 Scanning Electron Microscopy of Biofilms Following Treatment. The morphologies of P. aeruginosa biofilms formed on the MBEC device before and after treatment were further visualized through scanning electron microscopy (SEM). To prepare the samples, P. aeruginosa biofilms were formed and treated on the MBEC device, as previously described. Following removal of treatments, the bacteria were fixed on to the pegs by immersion in 2.5% (v/v) glutaraldehyde in PIPES buffer (0.20 M, pH 7.40) for 30 min, followed by 1.0% (w/ v) osmium tetraoxide for 60 min. To dehydrate the samples, the pegs were subjected to an ethanol series (25%, 50%, 75%, 90%, and 100% (v/v)) for 15 min each. Finally, the pegs were dried following immersion in hexamethyldisilazane (HMDS) before being mounted onto carbon tape and sputter coated with 8.0 nm of gold. The pegs were imaged by a Zeiss Gemini 2 scanning electron microscope (Zeiss, Oberkochen, Germany) at an accelerating voltage of 2.0 kV. Statistical Analysis. Data are reported as mean ± standard deviation. Student t tests were used to compare the in vitro release samples, liquid crystals to solution treatments, and activity of AL following different storage conditions. One-way analysis of variance (ANOVA) assessed the difference between in vitro biofilm treatments, and a two-way ANOVA compared the difference in the timedependent biofilm treatments. Statistical significance was evaluated at the 95% confidence interval. All tests were performed using GraphPad Prism (version 7.00 for Windows; GraphPad Software, La Jolla, CA).
Figure 1. Small angle X-ray scattering (SAXS) profiles of the GMObased liquid crystals containing no compounds (blank), gentamicin, alginate lyase (AL), and AL + gentamicin combination. Downward facing arrows represent the additional hexagonal phase shoulders (√1: √3: √4). Mean data are represented, n = 5.
Bragg peak ratios of, √2: √3: √4: √6: √8: √9, for all GMO liquid crystals. The lattice parameter was 8.51 ± 0.15 nm and was consistent for all samples. For the neat GMO liquid crystals and when gentamicin was incorporated within the material, additional peaks at spacing ratios √1: √3: √4 were apparent (downward facing arrows in Figure 1), indicating the hexagonal phase (H2) structure coexisting with the cubic Pn3m phase. The H2 phase was absent from samples containing AL in the GMO liquid crystals. In Vitro Release Studies. AL was released slowly from the liquid crystals in PBS, with 18% of total AL released after five days (Figure 2a). This release profile did not change when gentamicin was added in combination with AL in the liquid crystals (data not shown). However, gentamicin showed a considerably faster rate of release (77% after 48 h) (Figure 2b). The release from the liquid crystals was controlled by diffusion and correlated strongly with the Higuchi diffusion kinetic model.28 From the diffusion coefficients stated in Table 1, gentamicin was released at a 14-fold faster rate than AL, yet both were considerably slower than diffusion in water.29,30 When lipases from porcine pancreas and Pseudomonas bacteria were added in the release media, the percentage of AL and gentamicin released increased significantly (P = 0.001 and 0.025, respectively), (Figure 2). Within 8 h, 40% of AL was released in the presence of Pseudomonas lipase compared to 16% released without lipases. After 5 days, 60% of AL was released in the presence of Pseudomonas lipases, as opposed to 18% of AL released without lipase. While at 3 h, the release of AL was significantly greater from the inclusion of porcine pancreatic lipase compared to Pseudomonas lipase (P = 0.010), after 48 h, the percent of AL released did not differ (P = 0.50). For gentamicin, the addition of lipase resulted in greater than 85% release within 2 h and complete release within 1 day. Similar to the release of AL, there was no significant difference between Pseudomonas lipase and porcine pancreatic lipase (P = 0.47). Alginate Lyase Stability. As illustrated in the Supporting Information (Figure S2), the activity of AL was maintained after it had been exposed to three 60 s cycles of 60 °C, during the preparation of the liquid crystals. However, the exposure of the enzyme to 37 °C for 48 h significantly (P = 0.014) reduced the activity of AL, compared to storage at 4 °C.
■
RESULTS Structural Determination of the Liquid Crystals. The SAXS profiles shown in Figure 1 demonstrate the formation of the inverse bicontinuous cubic (Pn3m) phase structures, with C
DOI: 10.1021/acsabm.8b00062 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Article
ACS Applied Bio Materials
Figure 3. (A) Biomass reduction quantified by crystal violet staining and (B) bacterial cell count reduction (log10) of mucoid P. aeruginosa (clinical isolate) biofilm following treatment with either; saline control (NaCl), solutions of gentamicin (GEN) (6.0 μg/mL), AL (0.10 mg/mL), and combined AL + gentamicin, or from the liquid crystals (LC) (AL LC and AL+ GEN LC) for 48 h. Data represented as mean ± standard deviation, n = 4, ANOVA ∗∗∗∗P < 0.0001.
To correlate the biofilm biomass data with bacteria load, bacteria were extracted from biofilms grown in the microtiter plate and enumerated. Similar to the crystal violet data, the liquid crystals maintained the anti-biofilm activity of AL and gentamicin (Figure 3b). Co-treatment with AL and gentamicin was significantly greater (P < 0.00010) than AL treatment alone, with a greater than 2-log reduction in bacterial cells (compared to 1-log reduction from AL). However, the AL and gentamicin combination treatment was not significantly greater (P = 0.11) than single gentamicin treatment alone, which produced a 1.8-log reduction in bacterial cells. Time Dependent Anti-biofilm Activity. To investigate the anti-biofilm effect in relation to the release of AL and gentamicin from the liquid crystals, P. aeruginosa biofilms grown on the MBEC device were treated with sterilized samples taken during a release study. From Figure 4, it was evident that five-day sustained release did not result in an enhanced reduction of biofilm bacteria. In fact, the greatest anti-biofilm activity (greater than 2-log reduction from combined AL and gentamicin) was seen only up to 2 days post release from the liquid crystals (P = 0.0001). The earlier
Figure 2. Total percentage of (A) alginate lyase (AL) and (B) gentamicin released from the GMO liquid crystals, all using PBS (pH 7.40) (green squares) and pancreatic lipase in stimulated fasted digestion media (pH 7.55) (red triangles) compared to Pseudomonas lipase in digestion media (blue circles). For some points, the error bar is smaller than the height of the respective symbol. Data represented as mean ± standard deviation, n = 4.
In Vitro Biofilm Studies. Liquid Crystals Compared to Unformulated Solutions. Compared to the unformulated simple solutions, the liquid crystals containing AL and gentamicin demonstrated similar anti-biofilm effects, with no statistical difference between each group (P > 0.99) (Figure 3). On the basis of the crystal violet assay in Figure 3a, indicating biomass reduction, treatment with AL and gentamicin showed a significantly greater reduction in biofilm biomass of 34% (P < 0.00010), than treatment with gentamicin or AL separately. In addition, AL treatment produced a similar reduction (P = 0.45) in biofilm biomass to gentamicin treatment.
Table 1. Kinetic Model Data for AL and Gentamicin Released from GMO Liquid Crystalsa Liquid Crystals
Higuchi Goodness of Fit (r2)
Diffusion Constant (× 108 cm2/s)
Diffusion in Water (× 108 cm2/s)
AL Gentamicin
0.921 0.913
0.850 ± 0.0590 12.3 ± 1.55
10429 46030
Data represented as mean ± standard deviation, n = 4.
a
D
DOI: 10.1021/acsabm.8b00062 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Article
ACS Applied Bio Materials
Figure 4. Log10 reduction in bacterial cells of P. aeruginosa biofilms grown using the MBEC model, following treatment with samples from an in vitro release study (denoted as days of release), compared to solution treatment controls equal to the concentration released at 1 day (0.10 mg/mL AL, 6.0 μg/mL gentamicin (GEN)). For some points, the error bar is smaller than the respective symbol. Data represented as mean ± standard deviation, n = 2, two-way ANOVA ∗∗∗∗P < 0.0001.
compounds into GMO has the ability to change the mesophase.31 The hydrophilic portions of AL are suspected to interact with the hydrophilic fatty acid headgroup of GMO, decreasing the critical packing parameter of the lipid and the negative curvature of the mesophase, resulting in the neat cubic phase.32 Moreover, it is not unusual for commercial mixed lipid systems, such as the different Myverol grades, to exhibit mixed cubic and hexagonal phases at ambient temperatures due to suppression of the temperature dependent phase boundary between the cubic and hexagonal phase. The presence of the hexagonal phase in the neat and gentamicin containing liquid crystals is not an equilibration issue; in such cases, the less hydrated Ia3d cubic phase, lamellar phase, or even an inverse micellar L2 phase is observed. The Pn3m cubic phase is preferred for sustained delivery of large molecular weight compounds, unlike the hexagonal phase, where the release of large compounds is restricted.16 Bisset, Boyd, and Dong22 showed that higher molecular weight compounds are released to a lower extent than low molecular weight compounds, where approximately 10% release is expected for compounds with molecular weights of 40 000 Da. For GMO based Pn3m cubic liquid crystals, with an average lattice parameters of 10 nm (consistent with the present study), large molecular weight molecules (>20 000 Da) are released at rates below 1.0 × 108 cm2/s. In comparison, smaller molecules (∼500 Da) are released at rates that are at least an order of magnitude higher and continually increase with decreasing molecular size.33 AL has a molecular weight of approximately 36 000 Da.34 Consequently, it is released from the GMO liquid crystals to a limited extent (18% total released, Figure 2a) and slow rate (0.85 × 108 cm2/ s, Table 1). In comparison, the release of gentamicin was fourfold greater than the extent of AL, due to its lower molecular weight of 477.6 Da.35 Also corresponding to similar sized molecules, the rate of gentamicin released was 14-fold greater than AL (12.3 × 108 cm2/s, Table 1). GMO is hydrolyzed into glycerol and oleic acid by lipases, degrading the liquid crystal cubic phase structure and thereby increasing the release of compounds.18 Lipases are well distributed physiologically for a variety of purposes including aiding metabolism of dietary triglycerides, cell signaling, and
time points (up to two-days) were also similar to the solution treatment (P = 0.659). Following two-days, the release samples had comparable effect to the negative control (saline treatment). Scanning Electron Micrographs of Biofilms Following Treatment. The biofilms grown on the MBEC device were imaged by SEM to elucidate the morphology of the P. aeruginosa biofilms before and after exposure to AL and gentamicin. The micrographs in Figure 5 showed a dense P. aeruginosa biofilm formed. In concordance with the results reported above, the exposure to AL, gentamicin, and a combination resulted in a consecutive decrease in visible biofilm bacteria.
■
DISCUSSION Glyceryl monooleate (GMO) nanostructured liquid crystals were investigated as a topical delivery system for glycoside hydrolase, alginate lyase (AL), and the antibiotic, gentamicin to treat Pseudomonas aeruginosa biofilms. Constructed with lipids intercalated with water channels, liquid crystals can encase a broad range of different molecular weight compounds.21 The GMO bulk cubic phase forms a viscous gel, which could be applied to a local site such as the skin or sinuses, for efficient delivery. While increased drug release from GMO liquid crystals has been described because of enzymatic digestion by pancreatic lipases,18 it was hypothesized that the release of AL could also be triggered by exposure to lipases produced by Pseudomonas bacteria, thereby creating an ondemand responsive delivery system for AL and gentamicin at the site of infection. The feasibility of a Pseudomonas infection responsive delivery was explored along with the anti-biofilm performance of the liquid crystals containing AL and gentamicin compared to solution treatments against P. aeruginosa (clinical isolate). The GMO liquid crystals could effectively encapsulate AL and gentamicin, forming the inverse bicontinuous (Pn3m) cubic phase with 70% (w/w) GMO, in concordance with previous literature.16 While the neat GMO liquid crystals and the samples containing gentamicin alone also had SAXS peaks resembling the hexagonal phase, the addition of AL reduced this phase structure. Combining hydrophobic or hydrophilic E
DOI: 10.1021/acsabm.8b00062 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Article
ACS Applied Bio Materials
Figure 5. Representative scanning electron micrographs of (A) P. aeruginosa biofilm grown on MBEC device for 24 h before treatment, and following treatment with (B) AL (0.10 mg/mL), (C) gentamicin (6.0 μg/mL), and (D) a combination of AL and gentamicin.
inflammation.36 Consequentially, GMO liquid crystals are not suitable for oral delivery purposes as the exposure to pancreatic lipases results in premature release of compounds from the carrier.37 In the present study, the release of AL and gentamicin was increased by the addition of lipases in the release media (Figure 2). However, the susceptibility of GMO to lipase was not only specific to (porcine) pancreatic lipases, but also to lipases produced by Pseudomonas bacteria. As a virulence factor, Pseudomonas bacterium produce lipases to disengage the human immune response through restricting the action of macrophages and platelets.19 Porcine pancreatic lipase and Pseudomonas lipase have similar structures, constituting an α/β hydrolase fold, Ser-His-Asp catalytic triad and an oxyanion hole, permitting comparable activity.38 While porcine pancreatic lipases enhanced the release of AL by three-fold within 8 h, the lipase from Pseudomonas bacteria enhanced the release of AL by two-fold within the same time. However, by five-days there was an equivalent, three-fold increase in release from both the porcine pancreatic lipase and Pseudomonas lipase. Moreover, porcine pancreatic and Pseudomonas lipases both stimulated full release of gentamicin from the liquid crystals. Thus, the disadvantage of GMO liquid crystal degradation in oral delivery can be exploited in topical delivery to Pseudomonas infection sites, creating a novel bioresponsive delivery system. As AL is a protein sensitive to chemical and environmental changes, including degradation by proteases, pH, and temperature,29 the overall aim for a delivery carrier is to effectively protect the stability of AL. The fabrication of the GMO liquid crystals, involving heating cycles, did not disrupt the activity of
AL (Figure S2). In addition, the activity of AL from the GMO liquid crystals was maintained against an in vitro mucoid P. aeruginosa biofilm compared to an unformulated solution (Figure 3), confirming effective delivery from the carrier. While AL has been investigated in various polymeric delivery systems intended for oral administration, to maintain stability, the effect of the carrier in respect to bacterial biofilms has not been studied. The polymeric systems explored often have other advantages to improve its functionality. As such, the hyaluronan− cholesterol nanogels were established to have fabrication techniques that could be up-scaled to an industrial level.11 Alginate and pectin based microspheres had benefits of selfdegradation in response to the lower pH of the intestines14 and also the ability to protect AL from toxic environmental conditions (i.e., low pH and high bodily temperatures within the gastrointestinal tract).13 However, the advantages for each system are based on oral delivery, with no specificity to bacterial biofilm infections. Topical therapy offers greater advantages for biofilm related infections to gain direct exposure of the agent at the site of infection.4 Thus, in the present study, the enhanced release of AL and gentamicin in the presence of lipases produced by Pseudomonas creates specific advantages for topical delivery to bacterial biofilm infection sites in addition to maintaining the stability of AL. The effectiveness of AL to reduce biofilm growth in combination with gentamicin is consistent with previous literature.7,39 AL is a glycoside hydrolase that degrades alginate, a polysaccharide present in the extracellular polysaccharide substance matrix (EPS) of P. aeruginosa biofilms.8 AL has F
DOI: 10.1021/acsabm.8b00062 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Article
ACS Applied Bio Materials minimal antimicrobial properties, as demonstrated in the scanning electron micrographs (Figure 5), and is widely suggested to affect the biofilm EPS or, indirectly, the biofilm’s biomass.7,40 However, there are varying reports on the activity of AL in the literature based on the mechanism of action of AL,39 including the contrary view that AL is a food source for the bacteria, thereby increasing their metabolic state and, consequently, their susceptibility to antibiotics.9 Exploring the mechanistic action of AL was beyond the scope of the current study. Yet, analogous with the literature, AL combined with gentamicin was shown to be superior to gentamicin in reducing biofilm biomass (Figure 3a), although not the bacterial load of the biofilm (Figure 3b). While other groups have investigated nanoparticulate delivery systems that display sustained release of AL,11,13,14 there is no understanding of the sustained release effect on the anti-biofilm performance of AL and gentamicin. Sustained release from the liquid crystals is favorable to reduce administration times for patients and improve the efficacy of compounds by maintaining a constant concentration over time.41 On the basis of the anti-biofilm performance as a function of release time (Figure 4), it was evident in the present study that the anti-biofilm effect of AL and gentamicin peaked within the first 2 days. Following 2 days, the antibiofilm activity reduced to become equivalent with the control. The decreasing anti-biofilm activity over time is also the same for both single treatments of AL and gentamicin. As gentamicin is a concentration-dependent antibiotic, the antimicrobial effect is independent of the time of exposure.42 Hence, increasing the time of exposure, that is, sustained released, is not beneficial, resulting in the decreasing antibiofilm activity. For AL, the exposure to 37 °C for longer than 48 h during the in vitro release study was demonstrated in the Supporting Information (Figure S2) to reduce the activity of AL. Thus, while the 48-h treatment of AL from the liquid crystals maintains the anti-biofilm activity of AL (Figure 3), a longer period of release results in sustained exposure to high temperatures of 37 °C and AL loses stability.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Clive A. Prestidge: 0000-0001-5401-7535 Ben J. Boyd: 0000-0001-5434-590X Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This research is supported by the Australian Government and the Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology. N.T. is supported by a grant from the National Health and Medical Research Council (GNT1090898). This work was performed in part at the South Australian node of the Australian National Fabrication Facility under the National Collaborative Research Infrastructure Strategy to provide nano- and microfabrication facilities for Australia’s researchers. The SAXS studies were conducted on the SAXS/WAXS beamline at the Australian Synchrotron. Andrew Clulow is acknowledged for assistance in completing the SAXS studies.
■
ABBREVIATIONS AL, alginate lyase; EPS, extracellular polymeric substances; GMO, glyceryl monooleate; GEN, gentamicin; LB, Luria− Bertani; PBS, phosphate buffered saline; SAXS, small-angle Xray scattering; SEM, scanning electron microscopy
■
REFERENCES
(1) O’Neill, J. Antimicrobial Resistance: Tackling a Crisis for the Health and Wealth of Nations, 2014; pp 1−16. (2) Costerton, J. W.; Lewandowski, Z.; Caldwell, D. E.; Korber, D. R.; Lappin-Scott, H. M. Microbial Biofilms. Annu. Rev. Microbiol. 1995, 49, 711−745. (3) Hoiby, N.; Bjarnsholt, T.; Moser, C.; Bassi, G. L.; Coenye, T.; Donelli, G.; Hall-Stoodley, L.; Hola, V.; Imbert, C.; Kirketerp-M?ller, K.; Lebeaux, D.; Oliver, A.; Ullmann, A. J.; Williams, C. ESCMID Guideline for the Diagnosis and Treatment of Biofilm Infections 2014. Clin. Microbiol. Infect. 2015, 21 (Suppl 1), S1−25. (4) Ciofu, O.; Rojo-Molinero, E.; Macià, M. D.; Oliver, A. Antibiotic Treatment of Biofilm Infections. APMIS 2017, 125 (4), 304−319. (5) Lewis, K. Persister Cells and the Riddle of Biofilm Survival. Biochemistry (Moscow) 2005, 70 (2), 267−274. (6) Colvin, K. M.; Irie, Y.; Tart, C. S.; Urbano, R.; Whitney, J. C.; Ryder, C.; Howell, P. L.; Wozniak, D. J.; Parsek, M. R. The Pel and Psl Polysaccharides Provide Pseudomonas Aeruginosa Structural Redundancy within the Biofilm Matrix. Environ. Microbiol. 2012, 14 (8), 1913−1928. (7) Kaplan, J. B. Therapeutic Potential of Biofilm-Dispersing Enzymes. Int. J. Artif. Organs 2009, 32 (9), 545−554. (8) Christensen, B. E.; Ertesvag, H.; Beyenal, H.; Lewandowski, Z. Resistance of Biofilms Containing Alginate-Producing Bacteria to Disintegration by an Alginate Degrading Enzyme (Algl). Biofouling 2001, 17 (3), 203−210. (9) Lamppa, J. W.; Griswold, K. E. Alginate Lyase Exhibits CatalysisIndependent Biofilm Dispersion and Antibiotic Synergy. Antimicrob. Agents Chemother. 2013, 57 (1), 137−145. (10) Lalani, R.; Misra, A.; Amrutiya, J.; Patel, H.; Bhatt, P.; Patel, V. Challenges in Dermal Delivery of Therapeutic Antimicrobial Protein and Peptides. Curr. Drug Metab. 2017, 18 (5), 426−436.
■
CONCLUSION GMO nanostructured liquid crystals can be fabricated to encase AL alone and in combination with gentamicin. The slow release of AL from the cubic phase liquid crystals was limited in comparison to gentamicin. However, lipases from Pseudomonas bacteria could increase the release of AL and gentamicin, signifying a Pseudomonas infection responsive delivery system. Furthermore, the in vitro anti-biofilm activity of AL and gentamicin in the GMO liquid crystals was comparable to unformulated solution treatments against mucoid Pseudomonas aeruginosa (clinical isolate), with the greatest anti-biofilm activity within the first 2 days of the sustained release profile. The development of Pseudomonas infection responsive liquid crystals for glycoside hydrolase and antibiotic combination has promising potential as a novel topical therapy for difficult to treat biofilm related infections in the sinuses or wounds.
■
Image of aluminum multisample plate holder and stability data of alginate lyase (PDF)
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.8b00062. G
DOI: 10.1021/acsabm.8b00062 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
Article
ACS Applied Bio Materials
Gentamicin from Collagen Matrices. Pharm. Res. 1995, 12 (8), 1205− 10. (31) Milak, S.; Zimmer, A. Glycerol Monooleate Liquid Crystalline Phases Used in Drug Delivery Systems. Int. J. Pharm. 2015, 478 (2), 569−587. (32) Yaghmur, A.; Laggner, P.; Zhang, S.; Rappolt, M. Tuning Curvature and Stability of Monoolein Bilayers by Designer Lipid-Like Peptide Surfactants. PLoS One 2007, 2 (5), 479−484. (33) Lee, K. W. Y.; Nguyen, T.-H.; Hanley, T.; Boyd, B. J. Nanostructure of liquid crystalline matrix determines in vitro sustained release and in vivo oral absorption kinetics for hydrophilic model drugs. Int. J. Pharm. 2009, 365 (1−2), 190−199. (34) Davidson, I. W.; Lawson, C. J.; Sutherland, I. W. An Alginate Lysate from Azotobacter Vinelandii Phage. J. Gen. Microbiol. 1977, 98 (1), 223−229. (35) Marcus, Y.; Sasson, K.; Fridkin, M.; Shechter, Y. Turning LowMolecular-Weight Drugs into Prolonged Acting Prodrugs by Reversible Pegylation: A Study with Gentamicin. J. Med. Chem. 2008, 51 (14), 4300−4305. (36) Lowe, M. E. The Catalytic Site Residues and Interfacial Binding of Human Pancreatic Lipase. J. Biol. Chem. 1992, 267 (24), 17069− 73. (37) Han, N.; Zhao, Q.; Wan, L.; Wang, Y.; Gao, Y.; Wang, P.; Wang, Z.; Zhang, J.; Jiang, T.; Wang, S. Hybrid Lipid-Capped Mesoporous Silica for Stimuli-Responsive Drug Release and Overcoming Multidrug Resistance. ACS Appl. Mater. Interfaces 2015, 7 (5), 3342−3351. (38) Kim, K. K.; Song, H. K.; Shin, D. H.; Hwang, K. Y.; Suh, S. W. The Crystal Structure of a Triacylglycerol Lipase from Pseudomonas cepacia Reveals a Highly Open Conformation in the Absence of a Bound Inhibitor. Structure 1997, 5 (2), 173−185. (39) Germoni, L. A.; Bremer, P. J.; Lamont, I. L. The Effect of Alginate Lyase on the Gentamicin Resistance of Pseudomonas Aeruginosa in Mucoid Biofilms. J. Appl. Microbiol. 2016, 121 (1), 126−135. (40) Zhu, B.; Yin, H. Alginate lyase: Review of major sources and classification, properties, structure-function analysis and applications. Bioengineered 2015, 6 (3), 125−131. (41) Langer, R. New Methods of Drug Delivery. Science 1990, 249 (4976), 1527−1533. (42) Tam, V. H.; Kabbara, S.; Vo, G.; Schilling, A. N.; Coyle, E. A. Comparative Pharmacodynamics of Gentamicin against Staphylococcus aureus and Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 2006, 50 (8), 2626−2631.
(11) Montanari, E.; Di Meo, C.; Sennato, S.; Francioso, A.; Marinelli, A. L.; Ranzo, F.; Schippa, S.; Coviello, T.; Bordi, F.; Matricardi, P. Hyaluronan-Cholesterol Nanohydrogels: Characterisation and Effectiveness in Carrying Alginate Lyase. New Biotechnol. 2017, 37 (Part A), 80−89. (12) Islan, G. A.; Martinez, Y. N.; Illanes, A.; Castro, G. R. Development of Novel Alginate Lyase Cross-Linked Aggregates for the Oral Treatment of Cystic Fibrosis. RSC Adv. 2014, 4 (23), 11758−11765. (13) Islan, G. A.; Bosio, V. E.; Castro, G. R. Alginate Lyase and Ciprofloxacin Co-Immobilization on Biopolymeric Microspheres for Cystic Fibrosis Treatment. Macromol. Biosci. 2013, 13 (9), 1238− 1248. (14) Islan, G. A.; Dini, C.; Bartel, L. C.; Bolzán, A. D.; Castro, G. R. Characterization of Smart Auto-Degradative Hydrogel Matrix Containing Alginate Lyase to Enhance Levofloxacin Delivery Against Bacterial Biofilms. Int. J. Pharm. 2015, 496 (2), 953−964. (15) Rizwan, S. B.; Boyd, B. J.; Rades, T.; Hook, S. Bicontinuous Cubic Liquid Crystals as Sustained Delivery Systems for Peptides and Proteins. Expert Opin. Drug Delivery 2010, 7 (10), 1133−1144. (16) Phan, S.; Fong, W.-K.; Kirby, N.; Hanley, T.; Boyd, B. J. Evaluating the Link Between Self-Assembled Mesophase Structure and Drug Release. Int. J. Pharm. 2011, 421 (1), 176−182. (17) Kulkarni, C. V.; Wachter, W.; Iglesias-Salto, G.; Engelskirchen, S.; Ahualli, S. Monoolein: A Magic Lipid? Phys. Chem. Chem. Phys. 2011, 13 (8), 3004−3021. (18) Boyd, B. J.; Khoo, S.-M.; Whittaker, D. V.; Davey, G.; Porter, C. J. H. A Lipid-Based Liquid Crystalline Matrix that Provides Sustained Release and Enhanced Oral Bioavailability for a Model Poorly Water Soluble Drug in Rats. Int. J. Pharm. 2007, 340 (1−2), 52−60. (19) Jaeger, K.-E.; Ransac, S.; Dijkstra, B. W.; Colson, C.; van Heuvel, M.; Misset, O. Bacterial Lipases. FEMS Microbiol. Rev. 1994, 15 (1), 29−63. (20) Kirby, N. M.; Mudie, S. T.; Hawley, A. M.; Cookson, D. J.; Mertens, H. D. T.; Cowieson, N.; Samardzic-Boban, V. A LowBackground-Intensity Focusing Small-Angle X-ray Scattering Undulator Beamline. J. Appl. Crystallogr. 2013, 46 (6), 1670−1680. (21) Hyde, S. T. Identification of Lyotropic Liquid Crystalline Mesophases 2001, 2, 299−332. (22) Bisset, N. B.; Boyd, B. J.; Dong, Y. D. Tailoring Liquid Crystalline Lipid Nanomaterials for Controlled Release of Macromolecules. Int. J. Pharm. 2015, 495 (1), 241−248. (23) Mugabe, C.; Azghani, A. O.; Omri, A. Preparation and Characterization of Dehydration−Rehydration Vesicles Loaded with Aminoglycoside and Macrolide Antibiotics. Int. J. Pharm. 2006, 307 (2), 244−250. (24) Thomas, N.; Holm, R.; Rades, T.; Müllertz, A. Characterising Lipid Lipolysis and Its Implication in Lipid-Based Formulation Development. AAPS J. 2012, 14 (4), 860−871. (25) Wong, T. Y.; Preston, L. A.; Schiller, N. L. Alginate Lyase: Review of Major Sources and Enzyme Characteristics, StructureFunction Analysis, Biological Roles, and Applications. Annu. Rev. Microbiol. 2000, 54 (1), 289−340. (26) Thomas, N.; Dong, D.; Richter, K.; Ramezanpour, M.; Vreugde, S.; Thierry, B.; Wormald, P.-J.; Prestidge, C. A. Quatsomes for the Treatment of Staphylococcus aureus Biofilm. J. Mater. Chem. B 2015, 3 (14), 2770−2777. (27) Richter, K.; Thomas, N.; Claeys, J.; McGuane, J.; Prestidge, C. A.; Coenye, T.; Wormald, P. J.; Vreugde, S. A Topical Hydrogel with Deferiprone and Gallium-Protoporphyrin Targets Bacterial Iron Metabolism and Has Antibiofilm Activity. Antimicrob. Agents Chemother. 2017, 61 (6), e00481−17. (28) Higuchi, W. I. Diffusional Models Eseful in Biopharmaceutics. Drug Release Rate Processes. J. Pharm. Sci. 1967, 56 (3), 315−324. (29) Swift, S. M.; Hudgens, J. W.; Heselpoth, R. D.; Bales, P. M.; Nelson, D. C. Characterization of AlgMsp, an Alginate Lyase from Microbulbifer sp. 6532A. PLoS One 2014, 9 (11), 1129−1139. (30) Singh, M. P.; Stefko, J.; Lumpkin, J. A.; Rosenblatt, J. The Effect of Electrostatic Charge Interactions on Release Rates of H
DOI: 10.1021/acsabm.8b00062 ACS Appl. Bio Mater. XXXX, XXX, XXX−XXX
