Article pubs.acs.org/Biomac
Development of Elastin-Like Recombinamer Films with Antimicrobial Activity André da Costa,†,‡ Raul Machado,*,†,‡ Artur Ribeiro,† Tony Collins,† Viruthachalam Thiagarajan,§,∥ Maria Teresa Neves-Petersen,∥,⊥ José Carlos Rodríguez-Cabello,#,○ Andreia C. Gomes,† and Margarida Casal*,† †
CBMA (Centre of Molecular and Environmental Biology), Department of Biology, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal § School of Chemistry, Bharathidasan University, Tiruchirappalli − 620 024, India ∥ BioPhotonics Group, Nanomedicine Department, International Iberian Nanotechnology Laboratory (INL), P-4715-310 Braga, Portugal ⊥ Faculty of Medicine, Aalborg University, DK-9220 Aalborg, Denmark # Bioforge (Group for Advanced Materials and Nanobiotechnology), Centro I+D, Universidad de Valladolid, Valladolid, Spain ○ Networking Research Centre on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), E-47011 Valladolid, Spain S Supporting Information *
ABSTRACT: In the present work we explored the ABP-CM4 peptide properties from Bombyx mori for the creation of biopolymers with broad antimicrobial activity. An antimicrobial recombinant protein-based polymer (rPBP) was designed by cloning the DNA sequence coding for ABP-CM4 in frame with the N-terminus of the elastin-like recombinamer consisting of 200 repetitions of the pentamer VPAVG, here named A200. The new rPBP, named CM4-A200, was purified via a simplified nonchromatographic method, making use of the thermoresponsive behavior of the A200 polymer. ABP-CM4 peptide was also purified through the incorporation of a formic acid cleavage site between the peptide and the A200 sequence. In soluble state the antimicrobial activity of both CM4-A200 polymer and ABP-CM4 peptide was poorly effective. However, when the CM4-A200 polymer was processed into free-standing films high antimicrobial activity against Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi was observed. The antimicrobial activity of CM4-A200 was dependent on the physical contact of cells with the film surface. Furthermore, CM4A200 films did not reveal a cytotoxic effect against both normal human skin fibroblasts and human keratinocytes. Finally, we have developed an optimized ex vivo assay with pig skin demonstrating the antimicrobial properties of the CM4-A200 cast films for skin applications.
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Synthetic polymeric materials offer flexibility for combination and functionalization with antimicrobial molecules and display intrinsic properties, which makes them suitable for medical devices.7,8 Such use of synthetic materials is, however, limited by many factors, including a poor control of their composition and biocompatibility.9 On the other hand, advances in protein engineering and recombinant DNA technology now allow for the design and synthesis of biocompatible, complex, and defined recombinant protein-based polymers (rPBPs) with exquisite control of the composition. In fact, the ability to build DNA duplexes encoding virtually any desired amino acid sequence creates an unprecedented opportunity for the de novo synthesis of advanced protein-based materials. Indeed, it
INTRODUCTION
Antimicrobial resistance, resultant of an extensive use of antibiotics and a lack of novel antimicrobials, is a major world threat due to an increasing number of resistant bacterial and fungal pathogens.1−3 Indeed, healthcare-associated infections related to resistant microorganisms already result in 37000 and 99000 deaths every year in Europe and U.S.A., respectively, and place an enormous economic burden on healthcare systems worldwide.4 The human skin microbiome displays a precarious balance between harmful and pathogenic populations that can lead to infections, namely, in acute and chronic wounds or in immune deficiency situations.5,6 In addition, medical devices, both noninvasive and invasive (e.g., catheters), are major sources of such infections, and new materials with antimicrobial activity for incorporation in such devices are therefore a priority. © 2015 American Chemical Society
Received: November 18, 2014 Revised: January 7, 2015 Published: January 12, 2015 625
DOI: 10.1021/bm5016706 Biomacromolecules 2015, 16, 625−635
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Figure 1. Designing and purification of the recombinant proteins. (a) Schematic representation of the CM4-A200 polymer genetic construction. (b) SDS-PAGE (copper chloride reverse staining) of E. coli BL21(DE3) cell crude extract after 22 h of growth in autoinduction medium. (c) Schematic representation of the purification protocol using hot and cold incubation and centrifugation steps. (d) SDS-PAGE analysis of the purified CM4-A200 (approximately 89 kDa) stained with copper chloride (image on the left) and SDS-PAGE of purified ABP-CM4 peptide (approximately 3.8 kDa) stained with Coomassie blue (image on the right).
(A200) was found to fold at 33 °C, but did not unfold until the temperature was reduced to 12 °C.23,24 Therefore, once selfassembled, the ELR A200 is stable over a wide range of temperatures and hence suitable for biomedical applications. In this work, we report the synthesis of a functional rPBP with the antimicrobial peptide ABP-CM4 from the Chinese silkworm25 linked to ELR A200.23 ABP-CM4 is a 35 amino acid peptide with a linear α-helical structure and belongs to the cecropin peptide family,26 which displays antibacterial,25 antifungal,27 and antitumor28 activities. Therefore, through the genetic fusion of ABP-CM4 with the ELR A200 (CM4A200) coding sequences, we generated a new class of recombinant biopolymers with broad antimicrobial efficacy, and this was explored as a base material for skin applications.
enables the creation of functional rPBPs with novel properties not found in their natural counterparts.10 Furthermore, and more specifically, it is possible to combine rPBPs with antimicrobial peptides (AMPs) and thereby produce functional chimeric polymers for potential use as novel antimicrobial biopolymers.11 Typically, AMPs are small molecular weight peptides with a positive net charge, amphipathic structure, and exhibit broad antimicrobial activity.12,13 They are mainly found in multicellular organisms but are also produced by unicellular organisms for competitive advantage over other microorganisms.13,14 Elastin-like recombinamers (ELRs) are repetitive artificial rPBPs derived from amino acid sequences found in the hydrophobic domain of tropoelastin. ELRs most frequently consist of repeats of the pentamer (VPGXG)n, where X, the guest residue, is any amino acid except proline and n represents the number of repeats.15 A feature commonly shared by ELRs is their reversible temperature-dependent phase-transitional behavior,15,16 which has been explored both for biomedical and biotechnological applications, such as in drug delivery systems,17 enzymatic processing of textile fibers,18 and as protein purification tags.19−21 Apart from the aforementioned applications, ELRs have been thus far poorly explored as base materials in the production of functional advanced materials, as their thermoresponsive behavior affects the materials structural stability at reduced temperatures. Below the transition temperature, ELRs are highly soluble in aqueous solutions, as the polymer chains are extended in a disordered state,15 and therefore, the application of non-cross-linked ELR films is hampered by the loss of structural integrity. A potential means of overcoming such a limitation is by periodically including lysine residues in the ELR sequence and thereby providing free amine groups to react with chemical cross-linkers, such as glutaraldehyde, and hence improve material stability.22 However, AMP sequences are also enriched in positively charged amino acids, such as lysine, and therefore, cross-linking reactions would also affect the peptide’s antimicrobial activity. Another approach for improving polymer stability is the substitution of the most commonly used elastin pentamer sequence VPGVG by VPAVG, which causes the temperatureinduced reversible phase transition to display an acute hysteresis.23,24 The ELR based on 200 repetitions of VPAVG
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EXPERIMENTAL SECTION
Gene Construction and Protein Production. The CM4-A200 biopolymer was constructed by standard genetic engineering techniques, fusing the ABP-CM4 amino acid sequence in-frame with the N-terminus of the ELR polymer. The ELR A200 gene was previously cloned in a modified pET25b(+) (Novagen) expression plasmid,24 and the chemically synthesized (Genscript) ABP-CM4 sequence was ligated at the 5′ end of this gene, with inclusion of a formic acid cleavage site aspartate−proline between the two sequences (Figure 1a). This new plasmid was transformed into E. coli BL21(DE3) and used for production in autoinduction medium with Terrific Broth (yeast extract 24 g, tryptone 12 g, glycerol 5.04 g, K2HPO4 12.54 g, KH2PO4 2.31 g per liter) supplemented with 2 g/L lactose and 50 mg/L kanamycin, followed by incubation for 22 h at 37 °C, 200 rpm.29 Cells were collected by centrifugation, resuspended in TE buffer (50 mM Tris-HCl, 1 mM EDTA, pH 8.0), and disrupted by sonication (Sonics VCX 750). The pH was adjusted to 3.5 with 1.6 M HCl, and the precipitated contaminants were removed by centrifugation. Polymer purification was achieved with three cycles of hot (37 °C) and cold (4 °C) incubation (60 min at each temperature) and centrifugation steps (Figure 1b). The purified polymer fraction was freeze-dried and stored at room temperature prior to use. Purification of ABP-CM4 Peptide. The ABP-CM4 peptide was separated from the ELR domain by chemical cleavage with formic acid 70% (v/v) solution, for 48 h at 37 °C using 1.0 mg of protein/mL. Ultrapure water was added to the solution with three times the volume of the initial reaction; samples were frozen and lyophilized. For the purification of the peptide, hot and cold cycles already described for the purification were used. The samples were resuspended in water, 626
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Biomacromolecules heated at 37 °C for 2 h, and centrifuged at 9500 rpm at 37 °C for 20 min. The supernatant, containing the pure peptide, was transferred to a new tube, and the pellet resuspended in cold water. This cycle was repeated three times, and the supernatant was lyophilized in order to concentrate the ABP-CM4 peptide. The lyophilized sample was resuspended in sterile phosphate buffered saline (PBS) 1× (NaCl 8 g, KCl 0.2 g, Na2HPO4 1.44 g, KH2PO4 0.24 g, per liter, at pH 7.4) and quantified by the Lowry method using the Total Protein Kit (SigmaAldrich). Purity of the ABP-CM4 peptide was assessed by SDS-PAGE (Figure 1d) using coomassie blue-stained 16% Tricine-SDS gels.30 Characterization of the Phase Transition Behavior of CM4A200. The thermal events associated with the phase transition of CM4-A200 were assessed by differential scanning calorimetry (DSC) in a Mettler Toledo DSC 822e with liquid nitrogen as cooler. Calibration of enthalpy and temperature was made with a standard indium and zinc sample. An empty aluminum pan was used for baseline correction. The analyses were carried out with 25 mg/mL of CM4-A200 in PBS and the DSC run was performed with 20 μL of polymer solution placed in a hermetically sealed aluminum pan and studied under a four-stage thermal program: (i) isothermic stage, 5 min at 0 °C; (ii) heating stage, heating from 0 to 50 °C at a +5 °C/ min rate; (iii) isothermic stage, 5 min at 50 °C; and (iv) cooling stage, cooling from 50 to 0 °C at a −5 °C/min rate. The onset values were calculated with the provided STARe software. The size of the self-assembled particles was determined by dynamic light scattering (DLS) using filtered (0.22 μm) samples of CM4-A200 and A200 (used as control) with a concentration of 0.001 mg/mL. Measurements were performed at 37 °C with a 5 min equilibration time in a Zetasizer Nano ZS (Malvern) using disposable cells with four clear sides and a 10 mm pathway length. Particle size was determined as the average of 10 readings, with an automatically defined number of runs determined by the software. The hydrodynamic radii of the A200 and CM4-A200 particles was calculated based in the analysis of the scattered light intensity, defined at 90° scattering angle. Data analyses were performed with the Zetasizer Software (Malvern). For morphological analysis, lyophilized self-assembled particles of CM4A200 were coated with a thin gold layer using a sputter coater (Polaron model SC502) and analyzed by scanning electron microscopy (SEM, Leica Cambridge) with an accelerating voltage of 20 kV. Mean particle size was determined as the average value of 100 measurements using ImageJ image processing software.31 For circular dichroism (CD) analysis, samples were prepared in citrate buffer with a 10 μM final concentration. To avoid NaCl interference in the UV-region of the CD spectrum citrate buffer was chosen over PBS. Quartz cells in a Jasco J-815 Circular Dichroism Spectropolarimeter with a 150 W Xe lamp and a Peltier element were used. The spectra were obtained as the average of three measurements in the wavelength range 185−450 nm with a resolution of 1 nm at 10 or 30 °C. Secondary structure analysis and quantification were performed with the SOMCD algorithm.32 Antibacterial Activity of Soluble Protein. Antibacterial assays were performed in sterile 96-well plates with cultures of Escherichia coli HB101, Pseudomonas aeruginosa ATCC10145, Staphylococcus aureus ATCC6538, and Bacillus subtilis 48886 grown in LB (yeast extract 5 g, sodium chloride 5 g, Tryptone 10 g per liter). Aliquots of 300 μM stock solutions of protein were prepared in sterile PBS. A total of 50 μL of protein solution and 50 μL of bacterial suspensions (1 × 107 CFUs/mL) were mixed. Growth was determined by optical density measurements at 600 nm after an 18 h incubation at 37 °C. For agar diffusion method overnight cultures of P. aeruginosa and S. aureus were diluted in LB medium, agar (0.8% w/v), to a final cell density of 1 × 106 cells/mL and layered on LB medium, agar (1.5% w/ v) plates. Cellulose diffusion discs (Oxoid) infused with 20 μL of CM4-A200 (75, 150, 300 μM) were then placed in contact with the plate top-layer surface and incubated overnight at 37 °C. Blank disks embedded with PBS and Kanamycin disks (30 μg, BD Biosciences) were used as negative and positive controls, respectively. After overnight incubation at 37 °C, the diameters of growth inhibitory zones were evaluated.
Preparation of Solvent-Cast Films. Solvent-cast films were prepared with 10% w/v of protein, using as solvents ddH2O, hexafluoro-2-propanol (HFIP), formic acid, or acetic acid (50% ddH2O). Polytetrafluoroethylene (PTFE) molds of 10 mm diameter were used and solvent was evaporated for 48 h at room temperature under extraction. The resulting free-standing films were sterilized by UV exposure and used in subsequent experiments. Effect of Short-Chain Fatty Acids in the Sol−Gel Transition of CM4A-200. Lyophilized CM4-A200 was dissolved in four different short-chain fatty acid solutions of increasing hydrocarbon chain length, formic acid (1 C), acetic acid (2 C), propionic acid (3 C), and butyric acid (4 C), to a final concentration of 10% w/v in microtubes on ice. After 15 min, the tubes were placed upside down to observe the gelation status and digitally recorded. In Vitro Evaluation of Antimicrobial Activity of Films. Antimicrobial activity of cast films (disks of 10 mm diameter) was assessed via a protocol described previously.33 Briefly, 50 μL of a cell suspension (1 × 106 CFUs/mL of bacterial cells or 1 × 105 CFUs/mL of yeast cells) were placed in contact with the disks and incubated over different times as indicated in each experiment (at 30 °C for yeast cells or 37 °C for bacterial cells) in 24-well plates. After incubation, 1 mL of sterile PBS was added, followed by agitation and plating on LBagar or YPD (yeast extract 5 g, peptone 5 g, glucose 10 g, per liter) for bacteria or yeast CFUs enumeration, respectively. The bacterial species used were Escherichia coli HB101, Pseudomonas aeruginosa ATCC10145, Staphylococcus aureus ATCC6538, Bacillus subtilis 48886, and Staphylococcus epidermidis isolate. The yeasts species tested were Saccharomyces cerevisiae PYCC 4072, Candida albicans PYCC 3436, and Candida glabrata CBS 138. The 10 mm polystyrene PS disks were used as a negative control. Results were expressed as % kill by the formula
%kill =
(control CFUs − sample CFUs) × 100 control CFUs
In vitro assays were also extended to the filamentous fungus Aspergillus nidulans. Spores (conidia) were collected with the help of a sterile toothpick and plated in complete medium (CM, salt solution 20 mL, vitamin solution 10 mL, casamino acids 1 g, yeast extract 1 g, peptone 2 g, glucose 10 g, per liter at pH 6.8). Disks were placed near the spore inoculation sites on the agar plates and incubated at 37 °C. Digital images were recorded with a Chemidoc XRS system (BioRad) for registration of cell growth at 48 and 72 h. Microscopy Analysis. A total of 50 μL of a cell suspension of 1 × 107 CFUs/mL of B. subtilis were incubated over film disks for 2 h at 37 °C. Cells were collected by filtration with black Nucleopore polycarbonate membranes (GE Healthcare) followed by LIVE/ DEAD BacLight bacterial viability kit (Life Technologies) assay, according to manufacturer’s instructions. Micrographs were recorded using a Leica DM5000B fluorescence microscope. For SEM analysis, 50 μL of a cell suspension of 1 × 107 CFUs/mL of Escherichia coli HB101, Pseudomonas aeruginosa ATCC10145, Staphylococcus aureus ATCC6538, Bacillus subtilis 48886, Staphylococcus epidermidis isolate, Saccharomyces cerevisiae PYCC 4072, Candida albicans PYCC 3436, and Candida glabrata CBS 138 were placed in contact with CM4-A200 films for 2 h at 37 or 30 °C, for bacteria and yeasts, respectively. Similarly, in a spore germination assay, inoculum containing spores of A. nidulans were placed in contact with CM4-A200 films and PS disks (control) at 37 °C for 18 h. Sample fixation was performed by soaking the films in a fixation solution (1 mL of 2.5% v/v glutaraldehyde in PBS) for 1 h at room temperature, rinsed with distilled water (1 mL), and dehydrated through immersion for 30 min in a series of successive ethanol−water solutions (0.5 mL of 55.0, 70.0, 80.0, 90.0, 95.0, and 100.0% v/v of ethanol). The samples were then dried at room temperature and coated with a thin Au/Pd layer using a sputter coater prior to scanning electron microscopy (SEM, NanoSEM - FEI Nova 200) analysis with a 5.0 kV voltage and a through-lens detector (TLD). Antimicrobial Activity of Films through Ex Vivo Tests. Fresh pig skin, generously supplied by Matadouro Central de Entre Douro e Minho (Portugal) was cleaned of its fat, cut into 2 × 2 cm pieces and 627
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Figure 2. Thermal and structural characterization of CM4-A200. (a) Differential scanning calorimetry analysis of A200, CM4-A200, prosubtilisinA200, BMP2-A200; arrows point the transition temperatures corresponding to the beginning of the self-assembly in the heating stage (1) and resolubilization in the cooling stage (2). (b) DLS measurement of A200 and CM4-A200 self-assembled particles. (c) SEM micrographs of CM4A200 protein below (1) and above (2) its transition temperature. (d) Circular dichroism spectra (190−230 nm) of A200, CM4-A200, and soluble ABP-CM4, collected at 10 and 30 °C showing the transition temperature effect in the secondary structures of the polymers and peptide. (e) Secondary structure analysis of CM4-A200, A200, and ABP-CM4 peptide using SOMCD algorithm (α: α-helical structures; β: β-structures; turn: turn structures; random: random structures). preserved in a vacuum at −20 °C until used. The skin pieces were thawed and sterilized by washing with 70% (v/v) ethanol followed by sterile water washing. The skin was then placed between two stainless steel metal plates with a 6 mm rubber O-ring to delimit the infection region and adjusted using the upper metal plate coupled with wing nuts, as illustrated in Figure 7a,b. The skin was infected with 50 μL of 1 × 106 CFUs/mL or 1 × 105 CFUs/mL of bacterial or yeast cell suspensions, respectively. After 2 h of infection at 32 °C, the O-ring was removed and the films to be tested were placed in contact with the infected skin by adjusting the upper metal plate. After 3 h at 32 °C the skin samples were washed with 1 mL of sterile NaCl solution (0.87% w/v) with the aid of sterile cotton swabs and the suspension plated on LB-agar or YPD-agar for counting of bacterial or yeast CFUs, respectively. The % kill was calculated using CFUs from washed
infected skin samples prior contacting with films. All experiments were performed in triplicate with at least three independent assays. Film Characterization. Infrared (FTIR) spectra were acquired after 24 scans with a resolution of 4 cm−1 at room temperature with a Bruker Alpha spectrometer from Bruker Optics in attenuated total reflection mode (ATR) from 4000 to 600 cm−1. Spectra analysis and secondary structure determination was performed in OriginPro 8.1 (OriginLab, Northampton, MA). Solvent cast samples were coated with a thin gold layer using a sputter coater (Polaron model SC502) and analyzed by SEM with an accelerating voltage of 20 kV. Surface roughness was estimated by atomic force microscopy (AFM) with a scanning area of 5 μm × 5 μm using a Multimode scanning probe microscope and NanoScope IIIa controller (Digital Instruments, Veeco) in tapping mode at a scan rate of 1.0 Hz. NCHV 628
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Biomacromolecules silicon probes (Veeco) were used. The mean surface roughness was calculated with the SPIP v6 software (Scanning Probe Image Processor). Before quantification, images were flattened with a first order line-wise correction. In vitro degradation of CM4-A200 films was evaluated in PBS 1X solution. Round samples of 78.5 mm2 were immersed in 15 mL of PBS and incubated at 37 °C for 15 days. The PBS solution was renewed every 72 h. Samples were removed after 1, 5, 15, and 30 days, washed with ultrapure water, and air-dried at room temperature until constant mass was reached. Samples were weighed before and after incubation in an analytical microbalance (Mettler Toledo, error ± 0.1 mg). All measurements were performed in triplicate. The extent of hydrolytic degradation was calculated according to the following equation:
temperature (Tt) for the CM4-A200 polymer, by differential scanning calorimetry analysis (DSC), was 31.6 °C. The particle hydrodynamic radii estimated by dynamic light scattering (DLS) was 150.4 and 173.6 nm, for A200 and CM4A200 samples, respectively, with a polydispersity index of 0.3 in both cases (Figure 2b). The spherical shape of the selfassembled particles is observed by SEM (Figure 2c). The structural conformational changes attributed to the phase transition behavior of A200 and CM4-A200 associated with the self-assembly process are well represented in the CD spectra; however, no remarkable differences were found between both proteins (Figure 2c,d). Below the transition temperature (10 °C) random coils account for 96% of the overall secondary structure content, for both proteins. On the other hand, above 30 °C the structural content was characterized by a strong decrease of random coil structure followed by an increase of the other secondary structure components as α helix (A200, 46 ± 5.7% and CM4-A200 37.4 ± 2.7%), turns (A200, 13.1 ± 2.3% and CM4-A200 11.7 ± 1.7%), and β sheets (A200, 9.5 ± 14.2% and CM4-A200 22.4 ± 5.3%). As for the free peptide ABP-CM4 (Figure 2d), the secondary structure revealed to be mostly composed of random coil structures (95.3 ± 17.1%). In Vitro Antibacterial Activity of Soluble CM4-A200 and ABP-CM4. CM4-A200 and ABP-CM4 peptide (300 μM, PBS 1×) were incubated with Gram negative (Pseudomonas aeruginosa and Escherichia coli) and Gram positive (Staphylococcus aureus and Bacillus subtilis) bacteria for 18 h at 37 °C (Figure 3a,b). The % kill for P. aeruginosa was of 17.3 ± 1.9 and 29.3 ± 6.4% for CM4-A200 and ABP-CM4, respectively. The antimicrobial effect against the remaining bacteria species is relatively low, with the highest value observed for E. coli, where ABP-CM4 showed a % of kill of 35.7 ± 12.1%. In addition, no significant inhibition halos were found for soluble CM4-A200 and ABP-CM4 in contact with P. aeruginosa and S. aureus (Figure 3c). In Vitro Antimicrobial Activity of Cast Films. The purified and lyophilized CM4-A200 polymer was processed into free-standing films by solvent casting using formic acid as solvent. The cast films were shown to be optically clear and easily detached from the casting mold. Processing of ABP-CM4 into stable films was not feasible. Topographical characterization of the CM4-A200 polymer cast films by AFM revealed a smooth surface with a mean roughness (Ra) of 1.8 ± 2.3 nm and with a periodic pore distribution over its surface. The antibacterial activity of the CM4-A200 films was evaluated using P. aeruginosa, E. coli, B. subtilis, S. aureus, and Staphylococcus epidermidis bacteria, for 30 and 120 min incubation times. Disks of polystyrene (PS), a well-established polymer used for medical devices,9 were used as control. As shown in Figure 4a, upon an incubation of 30 min the CM4A200 films promoted a % kill of 53.0 ± 3.7, 97.4 ± 1.6, and 99.9 ± 0.1% in E. coli, P. aeruginosa, and B. subtilis, respectively. The % kill was not so significant for S. aureus or S. epidermidis (4.7 ± 0.3 and 28.3 ± 0.1%, respectively); however, after 120 min, it increased to 86.5 ± 0.7% for E. coli, 69.7 ± 4.5% for S. aureus, and 100.0 ± 0.0% for S. epidermidis, P. aeruginosa, and B. subtilis. SEM analysis performed for all bacteria placed in contact with CM4-A200 polymer films clearly show the loss of cell shape integrity, cell wall disruption, and cytoplasmic content being released to outside the cell, a behavior not found on PS disks (Figure 4b). It was also possible to note the ability of S.
⎛ M ⎞ %ML = ⎜1 − f ⎟ × 100 Mi ⎠ ⎝ where ML is mass loss, Mf is the sample mass after incubation time, and Mi is the initial sample mass. Cytotoxicity Evaluation of Films. Cytotoxicity was assessed with BJ-5ta (telomerase-immortalized normal human skin fibroblasts) and NCTC 2544 (human keratinocytes) cell lines. UV sterilized films were incubated with 750 μL of cell culture medium for 24 h at 37 °C, 5% CO2 in a humidified environment. In parallel, 100 μL of cell suspension (6.6 × 104 cells/mL) were seeded and cultured in surface treated 96-well plates (Nunclon polystyrene 96-well MicroWell, Thermo Scientific). After 24 h, cell culture medium was replaced with medium conditioned by contact with the films. Cell viability was then evaluated after 48 and 72 h, using the MTS assay (CellTiter 96 Aqueous One Solution Cell Proliferation, Promega) according to manufacturer’s instructions. Results were expressed as percentage of proliferation as compared to the positive control (cells grown in standard culture medium set as 100% viability). Statistics and Data Analysis. One-way analysis of variance (ANOVA) with Bonferroni’s post-test was carried out with GraphPad Prism 5 software to compare the means of different data sets within each experiment. A value of p < 0.05 was considered to be statistically significant. All experiments were performed in triplicate.
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RESULTS Cloning, Production, and Purification of the Recombinant Protein-Based Polymer. Chemically synthesized ABPCM4 coding sequence was ligated in frame to the 5′ end of the A200 elastin-like recombinamer (ERL) gene previously cloned and expressed into the Escherichia coli pET vector through appropriated restriction sites (Figure 1a).34 A formic acid cleavage site (D−P) was placed between ABP-CM4 and A200 polymer, and this new construct was named CM4-A200. After transformation, the recombinant polymers A200 and CM4-A200 were successfully produced in E. coli BL21(DE3) (Figure 1b) and purified by exploring the thermoresponsive behavior of the A200 sequence (Figure 1c,d). Aliquots of 10 mg/L and 120 mg/L of pure lyophilized A200 and CM4-A200 were obtained in batch cultures, respectively. Using the formic acid cleavage site and the thermoresponsive properties of the ELR was possible to obtain the soluble recombinant peptide ABP-CM4 free from the A200 domain of the construct (Figure 1d). Characterization of the Self-Assembly Process. As for A200,17,24 CM4-A200 also presents a thermal hysteresis behavior; however, a shift in the transition temperatures was depicted (Figure 2a). Indeed, the same type of thermoresponsive shift also exists in the other A200 constructs fused with prosubtilisin18 or with Bone Morphogenetic Protein-2 (Casal, M. personal communication). The calculated transition 629
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the CM4-A200 films affected the germination and morphology of A. nidulans spores. Indeed, spores placed on top of the CM4A200 films presented inhibition of germination and a collapsed form when compared to the control, as depicted by SEM micrographs (Figure 5d). Structural Features of CM4-A200 Cast Films. Structural analysis by FTIR of CM4-A200 and A200 films amide bands revealed a shoulder in the CM4-A200 film amide I band at 1642−1652 cm−1 and a small deviation in the amide II band (Figure S1). Amide I band qualitative and quantitative analysis by second derivative spectra and curve fitting methods has shown no significant differences in the secondary structure assignments between the A200 and CM4-A200 films. Processing of CM4-A200 into free-standing films was performed by the use of different solvents in the casting process, such as water, hexafluoro-2-propanol (HFIP), formic acid, and a 50/50 mixture of water/acetic acid. The use of short-chain carboxylic acids (formic and acetic acid) was effective in promoting gelation of the mixture, and this property was shown to be proportional to the size of the acid hydrocarbon chain used (Figure S2). Structural analysis by FTIR revealed similar protein amide bands for all the different CM4-A200 films (Figure 6a), regardless the solvent used. The only exception was for HFIP polymer films that have additional peaks in the 1285−1090 cm−1 range, which is associated with the solvent used.35 For the remaining spectra, no characteristic solvent peaks were found. Secondary structure qualitative and quantitative analysis of the Amide I band showed no significant differences between the films (Figure S3). The films produced from formic acid and water/acetic acid were found to be extremely effective, with 100.0 ± 0.0% P. aeruginosa cell death after 120 min of incubation; on the other hand, films produced from HFIP and water displayed 84.8 ± 2.2 and 53.0 ± 3.9% cell death, respectively (Figure 6b). PS disks were treated with the aforementioned solvents and tested against P. aeruginosa. No antimicrobial activity was observed for all the samples, and inclusively, an unexpected proliferative effect was found for PS disks treated with HFIP, with a % kill of −82.2 ± 16.2% (Figure S4). Ex Vivo Antimicrobial Activity Assays. An optimized methodology to assess antimicrobial activity of films was developed in the scope of this work. Pig skin samples were supported by holed metal pieces and with rubber o-rings to delimit an infection zone and screws with wingnuts were placed at the extremities of the metal places enables adaption to the skin piece thickness (Figure 7a,b). Through this method the CM4-A200 cast films proved to be very effective against P. aeruginosa and C. albicans (Figure 7c,d) with a % kill of 76.9 ± 7.7 and 91.9 ± 5.7%, respectively. The CM4-A200 antifungal activity against C. albicans was very similar to that obtained with itraconazol (discs impregnated with 25 mg/mL) that revealed a % kill of 96.2 ± 1.5%. CM4A200 activity against P. aeruginosa was as effective as kanamycin (50 mg/mL) impregnated disks, with a % kill of 99.5 ± 0.4%. For polylactic acid (PLA) films, here used as controls, no killing effect was found (15.5 ± 21.9 and 2.7 ± 14.3% against P. aeruginosa and C. albicans, respectively). CM4-A200 Film Cytotoxicity and Hydrolytic Degradation. Cell viability in response to CM4-A200 films was assessed by indirect contact with a normal human skin fibroblast and NCTC 2544 cell lines (Figure 8a). Interestingly, after a 48 h incubation, a proliferative effect was found for the
Figure 3. Antimicrobial activity of CM4-A200 and ABP-CM4 resuspended in PBS: (a) CM4-A200 and (b) ABP-CM4 (300 μM) were incubated with the indicated bacterial species for 18 h, 37 °C; bars represent means ± SD (ns, non significant, *p ≤ 0.05, **p ≤ 0.01). (c) CM4-A200 (75, 150, 300 μM) and ABP-CM4 (300 μM) were tested against P. aeruginosa and S. aureus, in a layer of agar, 0.8% (w/v), for 18 h, 37 °C.
aureus cells to form aggregates, reducing the direct contact with the film surface. Furthermore, the Live/Dead bacterial fluorescence assay revealed the membrane lytic activity of CM4-A200 polymer films (Figure 4c). Like PS disks, CM4-A200 did not reveal cellular growth inhibition halo formation against P. aeruginosa (Figure 4d), contrarily to what was found for kanamycin disks (30 μg). Studies evaluating the antimicrobial properties of CM4-A200 cast films were further extended to yeast species with clinical relevance, with 31.4 ± 2.3, 12.4 ± 3.7, and 28.3 ± 3.9% of % kill after 30 min incubation for Candida albicans, Candida glabrata, and Saccharomyces cerevisiae, respectively (Figure 5a). Increasing incubation time to 120 min resulted in a killing efficiency of 98.4 ± 1.1, 99.8 ± 0.1, and 98.9 ± 0.9%, for C. albicans, C. glabrata, and S. cerevisiae, respectively. As depicted by SEM micrographs, yeast cells in contact with CM4-A200 films presented loss of cell integrity, with holes in the cell wall as well as release of cytoplasmic content (Figure 5b). Studies performed against the filamentous fungi Aspergillus nidulans demonstrated the effective inhibition of the mycelium growth by CM4-A200 films when compared to PS disk. Positive controls of itraconazol (25 mg/mL) impregnated disks were also used (Figure 5c). Besides affecting vegetative growth, 630
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Figure 4. Antimicrobial activity of CM4-A200 films. (a) In vitro assay against bacterial species for 30 and 120 min at 37 °C; sterile PS disks were used as reference for 100% survival; bars represent means ± SD (ns, nonsignificant, ****p ≤ 0.0001). (b) SEM micrographs of cells in contact with PS (left) and CM4-A200 (right) surfaces for 120 min, 37 °C; scale bar: 1 μm. (c) Micrographs of fluorescent Live/Dead assay with B. subtilis cells in contact with PS (left) and CM4-A200 (right) disks for 120 min, 37 °C; probe SYTO 9 labels metabolically active cells with green fluorescence; propidium iodide labels with red fluorescence cells with a loss of membrane integrity; scale bar: 50 μm. (d) Diffusion assay of disks on top of LB medium - agar (0.8% w/v) layer with 1 × 106 cells/mL of P. aeruginosa for 18 h, 37 °C.
normal human skin fibroblasts (160.0 ± 7.3%), and after 72 h incubation, no statistically significant cytotoxicity was observed, with 82.0 ± 9.4% of proliferation. Regarding the keratinocytes cell line, no statistically significant cytotoxicity was found, with 96.9 ± 2.9 and 102.4 ± 7.9% of proliferation for 48 and 72 h, respectively. Hydrolytic degradation assays revealed that CM4-A200 films remained stable over 30 days at 37 °C, in PBS solution, without any significant hydrolytic degradation, with a final % mass loss of 1.8 ± 1.6%, at the 30th day (Figure 8b).
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effective separation of the cleaved ABP-CM4 by the use of hot and cold incubation and centrifugation steps. These thermoresponsive properties were observed by SEM. The micrographs of the self-assembling process (Figure 2c) revealed the conformational change above and below the polymer Tt, with the formation of spherical particles above the Tt, a characteristic already exploited for the encapsulation of molecules.17 When compared to A200, the DLS analysis revealed an increased particle size most probably due to the slightly larger sequence size and the side chains of the new amino acids introduced in the CM4-A200 sequence (Figure 2a). The DSC analysis enabled Tt calculation of several ELRbased recombinant proteins (Figure 2a). While the actual Tt is different in each protein, the same thermal hysteresis behavior is maintained and proved to be a characteristic of all elastin-like recombinamers based in the pentamer VPAVG. Circular dichroism was also used to study conformational changes in the recombinant proteins. Studies were performed using solutions of A200, CM4-A200, and the purified ABPCM4 in citrate buffer (Figure 2d,e), as chloride ions of PBS interfered in the UV-region spectra (not shown). Analysis of the secondary structure content of these samples revealed no significant difference between the A200 and CM4-A200. This can be related with the reduced content on α-helical structure of the purified ABP-CM4, which can be affecting the CM4A200 antimicrobial activity in solution.26 But comparing the structural content of A200 and CM4-A200 samples below and above the Tt, the change in structure of the recombinant
DISCUSSION
By the use of standard molecular genetics techniques, we were able to functionalize an ELR with an antimicrobial peptide. The fusion of the ABP-CM4 coding sequence at the 5′ end of the A200 gene promoted a dramatic increase in the volumetric productivity as it had been already reported for other constructions.18,24 The reasons underlying these differences are not completely understood as depending on the host and ELR sequence utilized, production values can range from a few milligrams to grams.24,36,37 The addition of a chemical cleavage site (D−P) in the construct enabled the purification of the soluble recombinant ABP-CM4 peptide. This cleavage process was dependent on the temperature (37 °C) and formic acid concentration (70% v/v) during the reaction, as lower acid concentration (e.g., 50% v/v) or higher incubation temperatures (e.g., 50 °C) were not as effective.38 The thermoresponsive properties of the A200 played an important role in the 631
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Figure 6. Characterization of CM4-A200 films produced with different solvents. (a) ATR-FTIR spectra of lyophilized CM4-A200 (1) and cast films using (2) H2O, (3) HFIP, (4) acetic acid/H2O, and (5) formic acid as solvents; arrows indicate peaks of amide I (1600−1700 cm−1), associated with CO stretch vibrations and amide II (1510−1580 cm−1) associated with N−H bending and C−N stretching vibrations. (b) Antimicrobial assays against P. aeruginosa for 120 min, 37 °C; PS disks were used as reference for 100% survival; bars represent means ± SD (ns, nonsignificant, **p ≤ 0.01, ****p ≤ 0.0001).
Figure 5. Antifungal activity of CM4-A200 films. (a) In vitro assays against different yeast species, for 30 and 120 min, 30 °C; sterile PS disks were used as reference for 100% survival; bars represent means ± SD (ns, nonsignificant, **p ≤ 0.01, ****p ≤ 0.0001). (b) SEM micrographs of yeast cells in contact with PS and CM4-A200 disks for 120 min, 30 °C; scale bar: 1 μm. (c) A. nidulans grown in contact with (1) itraconazol impregnated disks, (2) PS disk, (3) no sample, and (4) CM4-A200 disk; scale bar: 11.6 mm. (d) SEM micrographs of germinating spores of A. nidulans in contact with PS and CM4-A200 disks for 18 h, 37 °C; scale bar: 5 μm.
the bacteria tested, it differs from specie to specie, with the lowest antimicrobial activity registered against S. aureus. Most probably this behavior can be due to the known intrinsic ability of this specie to degrade cationic antimicrobial peptides at the extracellular facet of the outer membrane41 and to the cell’s ability to form aggregates, reducing the direct contact with the film, as suggested by the SEM micrographs (Figure 4b). As the occurrence of fungal infections in human skin and mucosa is growing and since there is a strong demand for new antifungal strategies,42 our next step was to test the activity of CM4-A200, both against yeasts and a model filamentous fungi, Aspergillus nidulans. The CM4-A200 films promoted holes in the yeast cell wall, reduced germ tube formation on Aspergillus nidulans, and collapse of the exosporium structure (Figure 5). Our results further indicate that the activity of the CM4A200 films is mediated by direct contact and not related with diffusion of its content and that these films could be directly active against the microbial cell wall, promoting its disruption and subsequent cell death. This hypothesis is clearly visible with diffusion assays against P. aeruginosa incubated in LB agar plates, where the CM4-A200 films showed no cell growth
polymers when they are above their Tt is observable, while below this temperature they maintain a majority of random structures. This process is expected and associated with a type III β-turn formation at temperatures above the Tt, where βsheet-like structures and hydrogen bonding also appear but were not related with the turn formation.39,40 CM4-A200 and purified ABP-CM4 in solution displayed antibacterial activity against Gram-negative bacteria.25 However, this activity was unexpectedly not evident in Grampositive bacteria (Figure 3). It remains to be clarified how soluble CM4-A200 and ABP-CM4 are only effective against Gram-negative bacteria. Additionally, diffusion assays revealed no antimicrobial activity for the CM4-A200 and ABP-CM4 (Figure 3c). The processing of CM4-A200 polymer into films induced a dramatic change in their antimicrobial activity, with 100% kill against P. aeruginosa, B. subtilis, and S. epidermidis (Figure 4a). Our results demonstrate that this activity is time-dependent and not Gram-dependent. While showing generalized activity for 632
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Figure 7. Ex vivo assessment of antimicrobial activity of polymers in fresh pig skin. (a) Schematic representation of the device developed. (b) Exploded view of the device. (c, d) Antimicrobial ex vivo assay against (c) P. aeruginosa and (d) C. albicans after 5 h, 32 °C; kanamycin (50 mg/mL) and itraconazol (25 mg/mL) disks were used as antibacterial and antifungal controls; PLA film is used as negative control; bars represent means ± SD (ns, nonsignificant, ***p ≤ 0.001, ****p ≤ 0.0001).
related to secondary structures attribution. Due to its small molecular weight (approximately 4 kDa), the AMP component in CM4-A200 promotes undetectable modification in the protein film secondary structure of A200 polymer (with approximately 85 kDa). A factor influencing CM4-A200 films activity was the solvent used for the casting process. As depicted in Figure 6b, the carboxylic acids (formic acid or 50/50 mixture of water/acetic acid) improved the antimicrobial activity of films in comparison to HFIP and ddH 2 O. No direct involvement in the antimicrobial activity of PS disks treated with the aforementioned solvents was found (Figure S4). An increased effect of carboxylic acids in the AMPs antimicrobial activity had been already described in literature.45 FTIR analysis of the films revealed similar spectra for all the samples, with no peaks found for the solvents as an indication of complete solvent evaporation, except for HFIP (Figure 6a). The antimicrobial properties observed for CM4-A200 films motivated its exploitation for skin tissue applications. With that in mind we optimized a system to perform ex vivo studies using pig skin. The system differs from other methods by the lack of glued components (with normal or chirurgical glues) that can affect the infection itself, either by killing the microorganisms or by keeping them in the glued surface. The results obtained against P. aeruginosa and C. albicans, important human pathogens, revealed levels of antimicrobial activity similar to kanamycin and itraconazol. The ex vivo skin assay reinforces the broad antimicrobial activity suggesting the potential use of CM4-A200 film for skin applications. Film degradability in wet conditions is a very important parameter in skin applications, humidity is key factor.
inhibition halo (Figure 4d). Results point to the lack of ABPCM4 peptide release from the film despite the presence of a DP formic acid cleavage site in the construction (Figure 1a). Indeed, this can be explained by the conditions used for film processing, which are very different from the required for optimal DP cleavage reaction as follows: (i) chemical conditions for DP site cleavage with formic acid were: protein concentration 0.1% w/v, formic acid 70% w/v, 37 °C, 48 h; (ii) conditions for film processing were protein concentration 10% w/v, formic acid 100% w/v, 20 °C, 48 h. Although we used 48 h for processing conditions, solid state films are already observed within 3 h, reducing significantly the reaction time. Analysis of different A200-based films were essential to understand the influence of the AMP sequence in the antimicrobial activity of CM4-A200 films. The low level of A200 production hampered its antimicrobial activity assessment. In substitution, formic acid BMP2-A200 and prosubtilisin-A200 films were used as controls (Figure S5). In conditions where CM4-A200 cast films caused 97.4 ± 1.6% kill to P. aeruginosa, upon 30 min of contact the BMP2-A200 films, showed a 19.8 ± 4.7% kill and the prosubtilisin-A200 promoted cell proliferation with an increase of 14.0 ± 2.5% in CFUs. These results point for the antimicrobial activity found in CM4-A200 to be attributed to the AMP component of the polymer. By studying amide bands I and II by FTIR, it was possible to observe differences between A200 and CM4-A200 films (Figure S1), most probably related with the presence of lysines and arginines in the AMP sequence.43,44 However, no significant differences were found in the amide I band curve fitting analysis between A200 and CM4-A200 formic acid films, a process 633
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The CM4-A200 solvent cast films showed a smooth surface (1.8 ± 2.3 nm) but with a periodic hollow distribution. These topographic features, which have been previously described for ELRs deposited on hydrophobic surfaces,47 correlate with the casting surfaces used in this work (PTFE molds) and influence cell adhesion and cytotoxicity.48
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CONCLUSIONS In this study we describe for the first time the fabrication of stable ELR films displaying antimicrobial activity while avoiding the use of cross-linking agents. The fact that CM4-A200 polymer maintains the same thermal hysteresis behavior as A200, improves the stability of the polymer films over a range of temperatures. CM4-A200 cast films exhibited antimicrobial properties against a wide range of microorganisms, including Grampositive and Gram-negative bacteria and fungi, promoting microbial cell disruption as well as inhibiting the growth of filamentous fungi and spore germination. Additionally, we have developed an optimized ex vivo methodology for testing the antimicrobial activity of film-type materials directed for skin applications. This methodology can potentially be used to test any type of material, while avoiding external interference with the use of glued systems. CM4-A200 films revealed no cytotoxic effect in in vitro cultures of normal human skin fibroblasts and human keratinocytes. This work represents a major breakthrough in the development and application of protein-based materials and demonstrates the high versatility of CM4-A200 films for skin applications.
Figure 8. Cytotoxicity and hydrolytic degradation profile of CM4A200 films. (a) Indirect contact viability assay on normal human skin fibroblasts (BJ-5ta cell line) and human keratinocytes (NCTC 2544 cell line) using the MTS assay, represented as % proliferation related to the untreated control; bars represent means ± SD (ns, nonsignificant, ***p ≤ 0.001). (b) Hydrolytic degradation profile in PBS for 30 days at 37 °C represented in terms of % of mass loss compared to the initial weight; dots represent means ± SD.
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ASSOCIATED CONTENT
S Supporting Information *
FTIR analysis of CM4-A200 film compared to A200 film, gelation properties of CM4-A200 using different carboxylic acids, solvents contribution in the antimicrobial activity, and FTIR curve fitting analysis of CM4-A200 films made with different solvents. This material is available free of charge via the Internet at http://pubs.acs.org.
Regarding this parameter, CM4-A200 showed no significant hydrolytic degradation in PBS after 30 days at 37 °C. This is a remarkable property as no cross-linking agents were used for film production. In fact, from our experience, the CM4-A200 films remain stable in solution over a wide range of temperature, solubilizing only when a strong undercooling is reached; this correlates with the thermal hysteresis of CM4A200 (Figure 2a). Cytotoxicity is an important parameter to be investigated when an interface with the human body is expected, as in the development of topical treatments. CM4-A200 leachables displayed no significant in vitro toxicity against BJ-5ta and NCTC 2544 cell lines after 72 h, and a proliferative effect after just 48 h incubation for the BJ-5ta cell line. This effect has been previously reported for a murine myoblast cell line (C2C12 cells) when in contact with very low concentrations of A200.17 However, in the present study, cells were exposed to leachables and, although no significant hydrolytic degradation was observed, the leachables presented a proliferative effect. Furthermore, different solvents were used in the two assays: hydrolytic degradation assays were performed in PBS, while the cytotoxicity tests were performed with culture medium supplemented with fetal bovine serum, known to contain several proteases that can induce degradation of biobased polymers and content release to the leachables.46
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Author Contributions ‡
These authors contributed equally (A.d.C. and R.M.).
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by FEDER through POFC − COMPETE, by Portuguese funds from FCT through Project PEst-OE/BIA/UI4050/2014, and by the Spanish Minister of Economy and Competitiveness (MAT2012-38043-C02-01) and Junta de Castilla y León-JCyL (VA152A12-2 and VA155A12-2), Spain. The authors also thank Matadouro Central Carnes de Entre Douro e Minho, Lda for their availability to provide pig skin. A.C. and R.M. acknowledge FCT for SFRH/BD/75882/2011 and SFRH-BPD/86470/2012 Grants, respectively. T.C. is thankful to the FCT for its support through Programa Ciência 2008. 634
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