An Environmentally Benign Antimicrobial Coating ... - ACS Publications

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An Environmentally Benign Antimicrobial Coating Based on a Protein Supramolecular Assembly Jin Gu,†,⊥ Yajuan Su,‡,⊥ Peng Liu,*,§ Peng Li,*,‡,∥ and Peng Yang*,† †

Key Laboratory of Applied Surface and Colloids Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710119, China ‡ Center for Biomedical Engineering and Regenerative Medicine, Frontier Institute of Science and Technology, Xi’an Jiaotong University, Xi’an 710049, China § College of Biomedical Engineering, Chongqing University, Chongqing 400044, China ∥ Key Laboratory of Flexible Electronics (KLOFE) and Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), Nanjing 210009, China S Supporting Information *

ABSTRACT: The use of antimicrobial materials, for example, silver nanoparticles, has been a cause for concern because they often exert an adverse effect on environmental and safety during their preparation and use. In this study, we report a class of green antimicrobial coating based on a supramolecular assembly of a protein extracted from daily food, without the addition of any other hazardous agents. It is found that a self-assembled nanofilm by mere hen egg white lysozyme has durable in vitro and in vivo broad-spectrum antimicrobial efficacy against Gram-positive/negative and fungi. Such enhanced antimicrobial capability over native lysozyme is attributed to a synergistic combination of positive charge and hydrophobic amino acid residues enriched on polymeric aggregates in the lysozyme nanofilm. Accompanied with high antimicrobial activity, this protein-based PTL material simultaneously exhibits the integration of multiple functions including antifouling, antibiofilm, blood compatibility, and low cytotoxicity due to the existence of surface hydration effect. Moreover, the bioinspired adhesion mediated by the amyloid structure contained in the nanofilm induces robust transfer and self-adhesion of the material onto virtually arbitrary substrates by a simple one-step aqueous coating or solvent-free printing in 1 min, thereby allowing an ultrafast route into practical implications for surfacefunctionalized commodity and biomedical devices. Our results demonstrate that the application of pure proteinaceous substance may afford a cost-effective green biomaterial that has high antimicrobial activity and low environmental impact. KEYWORDS: surface coating, antimicrobial, biocompatibility, protein phase transition, lysozyme

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ecosystems.23 For instance, silver nanoparticles as one of most classical and important materials have been recognized as a potential environmental hazard, and their use is starting to be regulated in many countries.23 Recent attention on novel antimicrobial materials was paid to simultaneously combine multiple functions into one material toward the integrated solution of the above concern, although up to date such a combination is highly critical4−17 and often turns to bring new issues such as complex chemical processing/synthesis, poor coating stability and reuseability, as well as strict demand on surface architecture and geometry.10 Such situations thereby noticeably restrict the sustainable development of green

INTRODUCTION Bacterial adhesion and growth on substrate surfaces is a widespread problem in medicine and industry. It causes infections and mortality in our daily life and medical surgery,1,2 and also leads to speed reduction, corrosion, and increased energy and fuel costs in the utilization of aquatic vessels.3 As a result, endowing a surface of medical implants, daily commodities, or equipment with antimicrobial property is of great importance in a variety of areas including health care,4−17 packaging,18 textile industry,19 water purification,20 and marine antibiofouling.21 For this aim, various antimicrobial materials have been developed such as metals/metal oxides, quaternary ammonium salts, photosensitizers, peptides, synthetic and natural polymers, as well as structured black silicon.4−23 Nevertheless, it is known that a majority of the above materials are associated with a big concern about adverse effects on © XXXX American Chemical Society

Received: October 24, 2016 Accepted: December 16, 2016 Published: December 16, 2016 A

DOI: 10.1021/acsami.6b13552 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

To address the above-mentioned hurdles, we herein present a new class of supramolecular assembled proteinaceous material to confer broad-spectrum antimicrobial properties with excellent biocompatibility toward blood and mammalian cells (Scheme 1). Lysozyme, a class of important functional enzyme in natural defense system, 25 widely distributes among eukaryotes and prokaryotes to catalyze the hydrolysis of microbial cell wall components.26,27 It is commercially available at low cost, and classified as GRAS grade (generally recognized as safe) by the Food and Drug Administration (FDA, US) and as a food additive by the European Union (E 1105). Unlike the risky synthetic or metal-based biocides, the lysozyme-derived antimicrobials could be normally served in an edible application.28 Accordingly, the development of lysozymebased antimicrobial material is significantly important toward an environmentally benign antimicrobial field. However, the fully exploitation of lysozyme in antimicrobial application is hampered because the antimicrobial performance of lysozyme is heavily dependent on its origin, and different types of lysozyme often present differentiated antimicrobial activity.29−35 In general, most commonly used wild-type lysozymes, for example, hen egg white lysozyme (HEWL), have many disadvantages such as poor activity toward Gram-negative bacteria and fungi, instability under ambient conditions, and difficulties to be robustly incorporated into daily commodities without losing their antimicrobial activity.29−35 In addition, the

Scheme 1. Strategy To Develop Phase-Transited Lysozyme (PTL) Nanofilm toward a Broad-Spectrum Antimicrobial Coating on Virtually Arbitrary Materials

antimicrobial materials from laboratory research to practical implication.24

Figure 1. Characterization on the surface hydrophobicity, charge state, morphology, and resultant antimicrobial performance toward three kinds of microbes. (A) The Raman spectra of the PTL nanofilm surface and native lysozyme in which the characteristic peaks for amino acid residues were used to calculate the propensity; (B) the propensity diagram for typical amino acid residues that existed on the PTL nanofilm surface and in the native lysozyme; (C) the typical surface morphology of the PTL nanofilm revealed by AFM; and (D) the killing efficiency of the PTL nanofilm toward E. coli, S. aureus, and C. albicans. B


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Figure 2. Antimicrobial test by FESEM images on the PTL nanofilm surface. (A,C,E) Typical images of the control (without the PTL coating) and (B,D,F) the treated (PTL nanofilm-coated) surfaces after contacting with three kinds of microbes. As indicated by the white arrows in (B,D,F), the cell wall of microbes was deformed or destroyed on the PTL nanofilm, indicating a possible cell membrane perturbation by the PTL.

Figure 3. Optical (A,C,E,G,I,K) and fluorescence (B,D,F,H,J,L) images of SYTOX Green assay for control (without the PTL nanofilm treatment) and the PTL nanofilm-treated bacteria cells. (A,B), (E,F), and (I,J) were for control sample; (C,D), (G,H), and (K,L) were for treated sample.

controllable conformation transition of HEWL extracted from egg to form phase-transited lysozyme (PTL) nanofilm is capable of affording a new class of antimicrobial material with a broad-spectrum contact-killing action toward both Grampositive/negative bacteria and fungi (Scheme 1). Such pure

integration of multiple functions into a general type of lysozyme besides antimicrobial activity is typically challenging and usually relies on complex chemical or physical modifications.5,10,12,29−35 We herein propose a strategy that without complex chemical and physical treatments, the C


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charge that could represent mammalian cells did not interact with the PTL nanofilm noticeably (Figure S3).40 The result from negatively charged GUV reflected that the electrostatic force exerted on microbial membrane with a net negative charge by the PTL nanofilm might be lethal to microbes. In contrast, it could be expected that the PTL nanofilm would not disturb mammalian cells with overall neutral charge, because the results from the GUV with net zero charge implied the absence of a strong electrostatic interaction with the PTL nanofilm surface. It has been proved that hydrophobicity-induced aggregation occurred during the phase transition, in which lyzosome chains rotate tryptophan (Trp) residues in their hydrophobic domain outward the molecular surfaces.37−39 Accordingly, the surface of the PTL product contained a certain amount of hydrophobic blocks, which could enhance the perturbation of lysozyme on the cell walls of microbes.41 The existence of hydrophobic domains on the PTL nanofilm surface was reflected by XPS and Raman spectra. The C1s signal assigned to aliphatic and aromatic hydrocarbon groups in XPS weighted over 40% contribution among all kinds of carbon-derived structures on the PTL nanofilm surface, and the coexistence of the hydrophobic and polar groups resulted in a water contact angle on the nanofilm surface as 75° (Figure S2). By the Raman spectral deconvolution analysis (Figure S1), it was further shown that the propensity of hydrophobic Trp and phenylalanine (Phe) residues on the PTL nanofilm surface was 5-fold higher for Trp and 3-fold higher for Phe, respectively, than those from native lysozyme (Figure 1A,B). The hydrophobic perturbation on cell walls of microbes to improve microbial killing performance could be further assisted by polymeric form of proteins,42−44 which was found in the amyloid-like structure45 contained in the PTL nanofilm. Such a polymeric form developed into an aggregation of protein nanoparticles (Figure 1C),39 which was driven by the hydrophobicity-induced aggregation and subsequent disulfide formation in aggregates to maintain a stable assembled structure.46 During this aggregation process, translocation of hydrophobic Trp residues to expose outward37−39 induced the enrichment of Trp and its adjacent Arg residues on the PTL nanofilm surface, as shown in Figure 1A,B. Taken together, the effective integration of positive charge and hydrophobic amino acid residues enriched by polymeric aggregation into the PTL nanofilm surface facilitated the perturbation toward the microbial cell walls, and finally led to the death of microbes.17,26,29,31,41,47 To prove the above-mentioned antimicrobial expectations, we tested the killing capability of the PTL nanofilm toward typical Gram-positive bacteria S. aureus (Staphyloccus aureus, ATCC 6538), Gram-negative bacteria E. coli (Escherichia coli, ATCC 8739), and fungus C. albicans (Candida albicans, ATCC 10231). The PTL nanofilm coated on glass slides presented the killing ratios of 95%, 92%, and 94% toward E. coli, S. aureus, and C. albicans, respectively (Figure 1D). Such killing ratios were repeatably obtained through sufficient contact between the PTL nanofilm surface and microbes. For HEWL used in the present work, a good killing ability toward Gram-positive bacteria S. aureus could be rationally expected. However, high killing ratios of 95% and 94% toward Gram-negative bacteria E. coli and fungus C. albicans were distinctive from conventional recognitions that HEWL as a general wild-type lysozyme should have poor antimicrobial activity to Gram-negative and fungi. These results implied that the PTL nanofilm presented a novel antimicrobial activity different from conventional

Figure 4. Loading and leakage of the membrane potential sensitive dye DiSC3(5) from E. coli challenged with the PTL nanofilm.

proteinaceous supramolecular nanofilm has multiple capabilities including in vitro and in vivo broad-spectrum antimicrobial activity, antibiofilm, biocompatibility, as well as transferrable robust adhesion to virtually arbitrary materials. These conspicuous advantages of this PTL nanofilm make it possible to behave as a reliable and cost-effective biocompatible antimicrobial coating in a one-step water-borne ultrafast (1 min) process onto a variety of daily commodities and biomedical implants/devices (e.g., catheter, guide wire, surgical blade, and titanium screw) with variable shapes, geometry, and substrate types. The PTL nanofilm was made by a unique conformation change of native lysozyme under quasi-physiological condition, in which lysozyme dissolved in a neutral buffer could transform to insoluble suprastructures when mixing it with tris(2carboxyethyl)phosphine (TCEP) buffer at neutral pH.36−40 After the disulfide bonds in the lysozyme chain were reduced by TCEP, the resultant partially unfolded monomer aggregated to form amyloid-like protein nanospheres that further hierarchically assembled into 2D nanofilm at the solution surface.39 The design of antimicrobial capacity on such phase transition structure required understanding the interactions of the assembly with the intended microbes. As discussed below, the PTL nanofilm surface presented an enrichment of net positive charge and hydrophobic amino acid residues, which synergistically contributed to the antimicrobial capability. For lysozyme, the positive charge may be attributed to arginine (Arg) and lysine (Lys) residues in its sequence, and the Raman spectral deconvolution analysis (Figure S1) clearly reflected that the propensity of Arg domains on the PTL nanofilm surface was 5-fold higher than that of the native lysozyme (Figure 1A,B). The total propensity for Arg and Lys to provide cationic site was significantly higher than the combined content of aspartic acid (Asp) and glutamic acid (Glu) to offer anionic site, which further implied a net positive charge on the PTL nanofilm surface. The X-ray photoelectron spectroscopy (XPS) reflected a content ratio of protonated amino to carboxyl groups on the nanofilm surface as 1.25:1 (Figure S2), which also supported the fact of a net positive charge on the PTL nanofilm surface. The zeta potential measurement on the PTL nanofilm surface reflected a +6 mV at pH 7.4. Previously, the surface positive charge of PTL nanofilm was fully utilized to bind anions and negatively charged particles.37−39 By exploiting lipid giant unilamellar vesicles (GUV) as a cell model, it was found in this work that the PTL nanofilm surface induced a membrane rupture on the negatively charged GUV that could represent anionic bacteria cells, while the GUV with net zero D


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Figure 5. Implantation of the PTL nanofilm on a variety of engineering materials and commodity articles with different geometry through the contact-printing. (A) Schematic cartoon to describe the lift-up technique in which the PTL nanofilm was first formed and floating at the solution surface, and then transferred onto an agarose gel stamp (the star-shaped object in the cartoon) through soaking the stamp into the solution and then passing through the solution surface; (B) schematic cartoon to describe the contact-printing technique in which the star-shaped agarose gel stamp coated with the PTL nanofilm through method A was first stained with Rhodamine and then stamped onto a material surface to transfer the nanofilm onto the material surface. The nanofilm with the same shape as the stamp (as visualized as a red star) was then formed on the surfaces of a variety of engineering materials and commodity articles after peeling the stamp from the material surfaces.

microscopy (FESEM) images revealed that the cell wall of microbes was deformed or destroyed by the contact with the nanofilm (as indicated by the white arrow), reflecting a possible cell membrane perturbation by the PTL (Figure 2). The membrane perturbation was then confirmed by SYTOX Green

lysozyme. Upon the contact of microbes with the substrate, an initial killing ratio of 55% was obtained after the contact time between the PTL nanofilm surface and the microbes reached 30 min, and such a killing ratio then elevated to 95% at 2 h and then 98% at 6 h (Figure S4). Field-emission scanning electron E


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to an expanded broad-spectrum antimicrobial capability toward Gram-positive/negative bacteria and fungi. This result was in accordance with our expectations, and attributed to the synergistic combination of surface positive charge and hydrophobic amino acid residues enriched by polymeric aggregation in the PTL nanofilm. In contrast to most antimicrobial substances that need certain complex chemical or physical methods to incorporate them into bulk materials, it was shown that three simple onestep transfer techniques could be flexibly adopted by the antimicrobial PTL nanofilm, resulting in an easy and robust coating onto virtually arbitrary materials/devices with various compositions and shapes (Figure 5).39 In the first method, after simply soaking a material into the phase transition buffer, the phase transition instantly transferred the assembled lysozyme onto an immersed material surface to form conformal PTL nanofilm coating. For the second method, the PTL nanofilm could be directly in situ formed at the solution surface of the phase transition buffer, and with a substrate being lifting up from the bottom of the phase transition solution to pass through the solution surface, the antimicrobial PTL nanofilm was spontaneously transferred onto the substrate surface to form a conformal adhered film coating (Figure 5A). In addition to the above two wet coating techniques, the third solvent-free coating could be achieved by a contact-printing method. In this case, the PTL nanofilm being preformed onto an agarose gel surface via the lift-up technique could be transferred within 1 min onto a material surface through stamping/peeling the gel on a target surface (Figure 5B). All of the methods afforded the coatings with consistent surface morphology (Figure S5) and antimicrobial capability (Figure S6). Particularly, the contactprinting ensured a special “ready-to-use” utility in which any target device or material could be rapidly switched to antimicrobial state within 1 min. The water-free feature in the contact-printing method was also exclusively favorable to those water-labile articles being unstable or damaged in aqueous environment, for example, display screens or paintings. This fast process thus offered great potential to perform instant disinfection on daily used articles through a convenient customer-controlled mode, which is important to human life for tackling off the microbes during everyday contacts with these materials. To test the generality of this contact-printing method, the colorless PTL nanofilm was first stained with Rhodamine for visualization and molded into a star shape via the use of a star-shaped gel slab. As shown in Figure 5, through the contact-printing, the red star-shaped PTL nanofilm was stably transferred onto a wide range of substrates covering widely used biomedical materials such as Si, Ag, Au, Pt, Ni, Ti, ZrO2, polydimethylsiloxane (PDMS), polytetrafluoroethylene (PTFE), poly(methyl methacrylate) (PMMA), polystyrene (PS), and cloth, as well as daily contacted materials/devices such as mica, glass, quartz, indium tin oxide (ITO), paper, Cu, Al, polyethylene terephthalate (PET), biaxially oriented polypropylene (BOPP), polycarbonate (PC), polyimidie (PI), wood, painting, cell phone, and steel ruler. Accordingly, great potential for serving the PTL nanofilm in a wide range of antimicrobial applications could be expected in the fields such as medical catheter (e.g., Si, PDMS),50 dental materials (e.g., Ti, PS, PMMA, ZrO2),51,52 orthopedic materials (e.g., stainless steel, Ni, Ti, or their alloy),53,54 wound dressing (e.g., cloth),55 packaging (e.g., paper, PET, BOPP, Al, PC),56 and electronic devices (e.g., PI, ITO, cell phone).57 It goes without saying that the instant antimicrobial functionalization in the above-

Figure 6. Cell viability of osteoblasts cultured onto the control and PTL nanofilm-coated substrates after 1, 4, and 7 days incubation. Error bars represent means ± SD for n = 6, *p < 0.05, **p < 0.01.

uptake assay (Figure 3). SYTOX is a nucleic acid-sensitive dye that is only able to penetrate cells when the integrity of the intact plasma membrane of live cells is effectively compromised.48 By this assay, the microbes as E. coli, S. aureus, and C. albicans cells seeded on the PTL nanofilm surface showed bright green fluorescence, while control cells without the contact with the nanofilm did not show obvious fluorescence. The membrane disruption was further verified by adding the membrane potential sensitive dye DiSC3(5), which is adsorbed into intact membrane bilayers and released upon subsequent membrane disruption.49 In Figure 4, the first peak reflected the addition of DiSC3(5) and gradual incorporation into E. coli membrane. Subsequently, the contact of E. coli with the PTL nanofilm resulted in an immediate fluorescence increase that indicated release of the dye due to cell membrane disruption. Taken together, the studies of the FESEM, SYTOX, and DiSC3(5) assays provided strong evidence that the PTL nanofilm induced the disruption and/or permeabilization of bacterial membrane. The effective killing efficiency of the PTL nanofilm toward both Gram-positive/negative bacteria and fungi is an interesting result, because in conventional recognition, lysozyme usually is poorly effective toward Gram-negative bacteria26 and fungi.27 This is because that lysozyme could enzymatically hydrolyze β1,4-glycosidic linkages in peptidoglycans that constitute a Gram-positive bacteria cell wall. However, a Gram-negative bacteria cell wall is different in constitution by surrounding its peptidoglycan layer with an outer membrane containing lipopolysaccharides, proteins, and lipids, so the contact of lysozyme with peptidoglycans in Gram-negative cell wall is obscured to raise lysis resistance.29 For fungi, general wild-type lysozymes, for example, HEWL and human lysozyme, are usually inactive to kill them due to the absence of peptidoglycans in the fungal cell wall.27 A variety of chemical and physical modifications on general lysozyme have been attempted to expand its antimicrobial spectrum, such as sequence mutation, conjugation with hydrophobic peptides or synthetic molecules, thermochemical treatment, etc.29−35 It has also been found that the increase of the surface hydrophobicity of lysozyme molecules could induce an enhanced perturbation on the cell lipid membrane, so as to promote the killing efficacy on microbes.29−35 Without the previously reported chemical and physical modifications, the above-mentioned results indicated that our PTL nanofilm generated by a simple phase transition of pure and single wild-type lysozyme (HEWL) led F


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Figure 7. Evaluation of antifouling ability toward the nonspecific adsorption of proteins and blood platelets on the PTL nanofilm surface. (A) QCM measurements to check the nonspecific adsorption of BSA (left) and fibrinogen (right) on the PTL nanofilm surface, with the top row being the resonance frequency F (Hz) and the bottom row being the energy dissipation factor D (Hz); (B,C) the SEM images of the blank (B) and the PTL nanofilm-coated (C) surfaces after the incubation in the suspension of the blood platelet.

reagents.60,61 We investigated the leaching behavior of the PTL nanofilm when it was attached onto a flat glass substrate (Figure S7). After the PTL-coated substrate was flushed in PBS buffer or Tween-20 solution, the UV−vis adsorption of the rinsing buffer was monitored to record the leaching curve of lysozyme into the solution. The results indicated that the PTL coating exhibited robust bonding with the underlying substrate, as there was no detectable leaching of PTL found in the rinsing buffer with or without surfactants added. This result also implied the antimicrobial activity of the PTL nanofilm was achieved through a contact killing mode. The biological stability of the coating was further evaluated in an undiluted serum. As revealed by AFM, the thickness of the dried PTL nanofilm did not change significantly after incubating the coating in the bovine serum for different time intervals (Figure S8). This

mentioned applications is of great importance, because frequent skin or body contact with the microbial world in these applications highly requires a rapid and strict hygienic demand. On the basis of the foldability of the agarose gel stamp, it was further proved that the PTL nanofilm could be stably implanted onto a curved surface as represented by a ceramic spoon or plastic tube of a medical injector through the seamless contactprinting from a bend gel stamp (Figure 5). The mechanism for the robust adhesion between the PTL nanofilm and underlying substrate is attributed to a bioinspired adhesion from the amyloid structures58,59 majorly contained in the PTL nanofilm.36−39 High adhesion stability is helpful to maintain a low leaching ability for the coating, which is desirable in the nonleachable antimicrobial material application toward a stable and long-term performance of the active G


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guaranteed the high-quality stealthy coating on real devices (Figure S9). For use as a coating on a biomedical device/ implant, the coating biocompatibility is also highly required. In this respect, unlike bacteria and fungi that usually possess negatively charged outer surface to initiate a contact-killing on the PTL coating, the mammalian cytoplasmic membranes usually present as nearly neutral in net charge.63 As mentioned above, the PTL nanofilm surface did not exert an obvious interaction with neutral GUV (Figure S3); it was thus expected that the PTL coating may exhibit a low cytotoxicity toward the mammalian cells,63 which could be supported by the following biocompatibility tests. The hemocompatibility of the PTL coating was assessed by the hemolysis assay that examined the hemoglobin release due to the rupture of red blood cells upon the contact with the coating. As shown in Figure S10, the lysozyme-based PTL coating afforded a hemocompatible interface without significant hemolysis. The in vitro cytotoxicity of the PTL coating was further investigated with the CCK-8 cell viability assay of murine osteoblast cells (SD mouse) (Figure 6). The viability of osteoblasts contacting with the PTL coating was statistically higher than those of osteoblasts onto the control bare glass after 1 day culture (p < 0.01). After culture for 4 days, although the average cell viability of osteoblasts adhered to the PTL coating was higher than that of the glass, no statistically significant difference was observed (p > 0.05).

Figure 8. Evaluation of antibiofilm performance on the PTL nanofilm coated on glass. The fluorescent images shown here were for the control (A) and the PTL nanofilm-coated (B) surfaces after the incubation in the biological media containing bacteria for the formation of biofilm. In this case, representative microscopic images of fluorescently stained Pseudomonas aeruginosa attached to suspension for 5 days. Living bacteria were stained with Syto9 (green), and dead bacteria were stained with propidium iodide (red).

result implied a potential for a long-term use in biological microenvironment. The hemostability of the PTL nanofilm was also ascribed to the amyloid structure contained in the PTL that afforded an endurable chemical stability.59 The durable adhesion and hemostability facilitate the use of the PTL material in nonmigrating antimicrobial applications.60,61 For a high-quality implication in practical devices, a critical issue is to combine multiple functions with antimicrobial efficacy.62 The good optical transparency of the PTL nanofilm

Figure 9. Antibiofilm evaluation on the PTL nanofilm-coated medical devices after seeding and incubating with bacterial colony. Top, control (A, without the PTL coating) and PTL-coated medical catheter (B); middle, control (C, without the PTL coating) and PTL-coated guide wire (D); bottom, control (E, without the PTL coating) and PTL-coated titanium screw (F). The PTL nanofilm coating was simply achieved by soaking the medical devices into the phase transition buffer for a given time. H


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Figure 10. Rat experimental biomaterial-associated infection evaluation for the PTL coating. (A) The control (without the PTL coating) and treated (with the PTL coating) medical catheters after challenge with E. coli that were implanted under skin of the rats; (B) the wounds shown in (A) after sewing up and being cultured for 4 days; (C) the sutured wound in (B) with the control catheter implanted was reopened after the culture for 4 days; (D) the sutured wound in (B) with the treated catheter implanted was reopened after the culture for 4 days; (E) the microbe retrieved from the control catheter explanted from the rats wound after culturing on agarose gel media; (F) the microbe retrieved from the treated catheter explanted from the rats wound after culturing on agarose gel media; and (G) total number of CFU (log) detected on control (without the PTL coating) and treated (with the PTL coating) medical catheters explanted from the wounds in rats shown in (C) and (D). Statistical analysis was assessed by Mann−Whitney tests, and the horizontal line represents the median value per group.

nanofilm as demonstrated in the present work further supported a mild hydrated biointerface toward good biocompatibility (vide infra). The antifouling capability of an antimicrobial material to suppress the nonspecific adsorption of proteins and platelets is critically significant for biocompatibility and reuseability.62 Typically, amphiphilicity is a common feature for proteins, and in Figure 1A, except for hydrophobic amino acid residues, we also recognized noticeable Raman signals assigned to typical hydrophilic amino acids such as Asp, Glu, Arg, Lys, and Cysteine (Cys) on the PTL nanofilm surface. These hydrophilic amino acid residues with significant content on the nanofilm surface (Figure 1B) initiated an obvious hydration effect after the material was incubated in a biological media or buffer for mammalian cells or microbes culture. In comparison with the dried PTL nanofilm, the ATR-FTIR spectrum of the PTL nanofilm after the aqueous incubation for 2 h showed new peaks around 3400 cm−1, which symbolized the OH stretch mode of asymmetrically bonded water on the surface (Figure S11A,B).64 This change implied a detectable surface hydration after incubating the nanofilm in the aqueous solution. It should be noted that in comparison with a surface hydration time (typically 2 h), the WCA measuring time was very short (tens of seconds). This contrast meant that during the WCA measurement, a surface hydration due to instant contact of tiny water droplet on the dried surface was ignorable. Accordingly, the surface wettability reflected by WCA (75°) was contributed from both hydrophilic and hydrophobic blocks on the PTL film (Figure 1, Figures S1 and S2) without an obvious influence from surface hydration. By AFM, it was further found that after the surface hydration, the diameter of particles contained in the nanofilm and surface roughness increased, which reflected a hydration-induced swelling process during the incubation

Figure 11. Microscopic observations on the tissue slice by the histological section. The samples were collected from the wounds after subcutaneous implantation of the control (blank) or treated (PTLcoated) catheter in the back of rats and culture for 3 days. In the image for control sample (left), the inflammatory exudates and necrosisor granulation tissue, accompanied by acute and chronic inflammatory cell infiltration, were found. In the image for treated sample (right), the inflammatory exudation and inflammatory cell infiltration were largely reduced, and there were a lot of new blood capillaries and mesenchymal cells formed. For cell staining, the subcutaneous tissue cells removed from rat were fixed by Formalin to ensure the original morphological structure, and then stained with Hematoxylin-Eosin.

Furthermore, after culture for 7 days, osteoblasts grown on the PTL coating displayed statistically higher cell viability than those grown on glass (p < 0.05). Overall, by choosing the red blood cell and osteoblasts as the models, it was observed that a negligible cytotoxicity from the PTL coating was exerted on mammalian cells. Certain cell viability enhancement on the PTL nanofilm surface might be due to the change from rigid glass substrate in the control sample to more biocompatible soft protein interface of the PTL nanofilm. As studied by thermogravimetric analysis in previous work,39 there was ∼20 wt % internal water bonded in the PTL nanofilm, which could impart certain biocompatibility to the nanofilm. Moreover, as shown below, the effective surface hydration on the PTL I


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specific adsorption of proteins on the nanofilm (Figure 7A) could decrease the mask effect of a large amount of proteins existing in the blood on the antimicrobial coating surface, and further introduce potential antibiofilm performance. In contrast to control glass surface, under the condition for the formation of biofilm of Pseudomonas aeruginosa, a much lower number of bacteria was found on the PTL-coated surface (Figure 8). In this way, various invasive biomedical devices/implants that are under threat of microbe contamination could be easily coated with the PTL nanofilm to prevent the formation of biofilm. As shown by FESEM observation, after challenging with the bacteria (E. coli) on the surfaces of medical catheter, guide wire, and titanium screw, the PTL-coated surfaces showed few bacteria colonies, while the control surfaces without the PTL coating were remarkably dominated by a large amount of bacteria biofilm (Figure 9). This result underlines a promising use of PTL coating to prevent transmitting diseases and nosocomial infections during the wide utilization of biomedical devices/implants. Encouraged by the above advantages, animal tests toward a potential clinic application on the PTL-coated medical catheter were further performed. The PTL-coated catheter and blank catheter without the PTL coating were simultaneously seeded with E. coli at the same condition and then implanted under the skin of rats (Figure 10A). After stitching up the wounds and culturing the rats for a determined time (Figure 10B), the wounds were reopened to compare the infection state. It was clearly shown that the wound implanted with the PTL-coated catheter presented a normal tissue without the inflammation, while in sharp contrast, the control wound with the blank catheter embedded was festering with serious inflammation and suppuration (Figure 10C,D). Such inflammation contrast in the wound was further reflected by the histological section study (Figure 11). As shown in the microscopic observation on the tissue slice, the inflammatory exudates, necrosis, and granulation tissue, accompanied by acute and chronic inflammatory cell infiltration, were found in the control sample. In contrast, the inflammatory exudation and inflammatory cell infiltration were largely reduced in the treated wound, and there were a lot of new blood capillaries and mesenchymal cells formed. The bacteria grown on the catheters taken out from the rats were then collected and incubated on the agar plates to count the numbers. We observed an average 4−5-Log reduction of bacteria number on the PTL-coated surface in comparison with the control surface (Figure 10E−G). This result verified that the inhibition on the microbe growth was successfully achieved in vivo on the PTL-coated catheters.

(Figure S11C−G). We thus rationalized that such hydration potency was able to afford an effective hydration microenvironment nearby the surface during the incubation in a biological media or buffer, and thereby inhibit the nonspecific adsorption of proteins.65 The quartz crystal microbalance (QCM) measurements supported such a hypothesis, as the PTL coating profoundly led to a nondetectable adsorption level of albumin from bovine serum (BSA) and fibrinogen when the protein buffer was flowing through the coating surface (Figure 7A). The QCM results were further supported by MicroBCA assay that characterized a low amount of nonspecific adsorbed proteins on the PTL film surface (Figure S12). As it is generally believed that platelet adhesion on a surface is related to adsorbed proteins especially fibrinogen,66 the excellent resistance to nonspecific adsorption of proteins on the PTL nanofilm surface further implied a good antiadhesion of platelets in bloods. In contrast to massive distribution of platelets on the unmodified control surface (Figure 7B), the treated surface coated with the PTL nanofilm repelled noticeably the adhesion of blood platelets (Figure 7C), indicating the great potential to be not thrombogenic.5 The collaborative results from Figure 6 and Figure 7B,C reflected that the PTL nanofilm surface exhibited a biomimetic property to simultaneously support the growth of osteoblasts (to some extent) and inhibit nonspecific adhesion of blood platelets. Unlike platelets usually suspending in a medium, osteoblasts as natural adherent cells have stronger adhesion tendency onto a surface. A good platform to support the growth of osteoblasts was the surface bioactivity especially the binding activity toward calcium ions (to be helpful for the formation of active hydroxyl apatite) as well as the facilitation on the secretion of extracellular matrix (ECM). In our opinion, the PTL nanofilm surface possessed these features due to several reasons. First, the enriched carboxyl and hydroxyl groups on the film as characterized by Raman and XPS (Figure 1, Figures S1 and S2) had great potential to bind effectively with calcium ions67 from the medium for osteoblasts culture. Second, it is previously found that lysozyme could promote the secretion of collagen in dermal fibroblasts and induce the synthesis of ECM proteins in the dermis of mice,68 and both of these events are important for the growth of osteoblasts. Intact antifouling behavior was helpful to produce repeatable antimicrobial activity. For instance, after a repeat use of the PTL nanofilm 10 times, the killing ratio toward Gram-positive/negative bacteria and fungi still maintained around 90% (Figure S13). Succeeding in this process implied that the dead bacteria and fungi could be washed away in the rinsing step to regenerate the protein nanofilm surface with few dead microbial occupied. During this process, the existence of the above-mentioned surface hydration effect between the microbial and underlying positively charged/ hydrophobic residues was important to maintaining a delicate force balance, in which both effective microbial-killing due to electrostatic and hydrophobic perturbation on the microbial membrane and effective removal of dead microbial by external dynamic flow peeling could be achieved. The antibiofilm potential was further evaluated for the PTL coating. A biofilm is a group of microorganisms in which cells stick to each other and adhere to a living or nonliving surface.69 Typically, antimicrobial agents fail to penetrate the full depth of a biofilm, and thus biofilm-forming microbes become resistant to antimicrobial agents,7,16 leading to notorious health problems and biofouling of biomedical implants/devices.70,71 In the present work, the antifouling property toward non-

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CONCLUSION We have reported a class of antimicrobial PTL nanofilms with the use of one-step water-borne self-assembly of pure HEWL. This kind of proteinaceous material exhibits broad-spectrum antimicrobial action toward Gram-positive/negative bacteria and fungi as well as antifouling and antibiofilm activity, while using mere lysozyme without the loading of any other environmental hazards. Such performance was attributed to an intrinsical triple-combination of positive charged and hydrophobic residues as well as surface hydration effect in the protein nanofilm. The amyloid-like structure in the PTL material boosts the reliable bonding with virtually arbitrary materials/devices in minutes and ambient conditions via simple aqueous soaking or contact printing to afford a robust coating. By the contact printing, a surface of virtually arbitrary material J


Research Article


Development (Grant no. 2016YFC1100300) and the Natural Science Foundation of China (51303218).

could be transformed into an antimicrobial state in 1 min, representing one of the fastest ways for antimicrobial surface functionalization. In vitro cytotoxicity assays indicate that the proteinaceous coating favors good hemocompatibility and cytocompatibility toward mammalian cells. An in vivo antimicrobial test on invasive implants indicates that this proteinaceous coating on an implanted catheter presents an excellent infection inhibition effect in a rat model. In comparison with other antimicrobials such as silver nanoparticles, synthetic polymers, or peptides, HEWL as a nutrient extracted from daily food could offer lower environmental impact and be directly supplied at large-scale from low-cost renewable feedstock, for example, egg, without the use of physical doping or chemical synthesis. The antimicrobial results shown herein illustrate how protein selfassembly could be manipulated to provide a new class of antimicrobial nanomaterial with increased activity. We further expect that a new avenue on the biomolecular assembly engineering may be opened toward green and sustainable antimicrobial biomaterials.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b13552. Full details of experimental details and characterization methods, and additional supporting evidence (Figures S1−S13) (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Peng Li: 0000-0002-5876-2177 Peng Yang: 0000-0002-0463-1024 Author Contributions ⊥

J.G. and Y.S. contributed equally to this work.

Notes

The authors declare the following competing financial interest(s): P.Y. and J.G. declare that a Chinese patent regarding to this work is on the application. (Application No. 201510822799.7).

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ACKNOWLEDGMENTS P.Y. is thankful for the funding from the National Natural Science Foundation of China (nos. 21374057, 51303100, and 51673112), the Fundamental Research Funds for the Central Universities (nos. GK201502001, GK201301006), the 111 Project (no. B14041) and Program for Changjiang Scholars and Innovative Research Team in University (no. IRT_14R33), the Natural Science Basic Research Plan in Shaanxi Province of China (no. 2015JM2048), as well as the Open Project of State Key Laboratory of Supramolecular Structure and Materials (no. sklssm201626). P.Li and Y.S. are thankful for the funding from the National Natural Science Foundation of China (no. 51403173), the Natural Science Basic Research Plan in Shaanxi Province of China (no. 2015JQ5139), and the Fundamental Research Funds for the Central Universities. P.Liu is thankful for the funding from the State Key Project of Research and K


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