Bioinspired Multifunctional Protein Coating for Antifogging, Self

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Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 24504−24511

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Bioinspired Multifunctional Protein Coating for Antifogging, SelfCleaning, and Antimicrobial Properties Haishan Qi,†,‡,∥ Chen Zhang,†,‡,∥ Hongshuang Guo,†,‡ Weiwei Zheng,†,‡ Jing Yang,†,‡ Xiao Zhou,†,‡ and Lei Zhang*,†,‡,§ Department of Biochemical Engineering, School of Chemical Engineering and Technology, and ‡Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering (MOE), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300350, China § Qingdao Institute for Marine Technology of Tianjin University, Qingdao 266235, P.R. China Downloaded via UNIV OF SOUTHERN INDIANA on July 18, 2019 at 06:36:34 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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S Supporting Information *

ABSTRACT: A multifunctional coating with antifogging, self-cleaning, and antimicrobial properties has been prepared based on a mussel-inspired chimeric protein MP-KE, which is the first example that these proteins were successfully applied to fabricate antifogging surfaces. The coating exhibits super hydrophilic properties, as indicated by contact angles less than 5° and high light transmittance similar to bare glass substrates about 90%. The zwitterionic peptides of MP-KE empower water molecules to expand into thin hydrated films rapidly, providing the protein coating with diverse surface functions. Moreover, the coatings have excellent stability and a convenient preparation process because of the mussel adhesive motif of MP-KE which makes the coating anchor onto the surface strongly. As a protein material, this multifunctional coating possesses remarkable biocompatibility and has a potential application prospects in the biomedical and pharmaceutical fields. KEYWORDS: 3,4-dihydroxyphenylalanine, posttranslational modification, protein engineering, surface chemistry, universal, multifunctional protein coating, antifogging, self-cleaning, antimicrobial, biocompatibility

1. INTRODUCTION Zwitterionic materials possess diverse surface functions for drug-delivering, avoiding nonspecific adsorption of proteins, resisting cell attachment, and antifogging, which have attracted increasing attentions in recent years.1−3 Amino acids, as natural zwitterions, have attracted great interests in biomedical applications, such as tissue engineering, synthetic skin applications, and coatings for plastic prostheses.4,5 Jiang and co-workers have constructed a bioinspired zwitterionic peptide which displayed an excellent performance in preventing protein adsorption.6,7 Besides, in our previous work, a mussel-inspired chimeric protein combined with hydrophilic zwitterionic peptide was synthesized and used as a substrateindependent antifouling material.8 Furthermore, its superior hydrophilicity and facile anchoring triggered us to make a thorough inquiry in other applications. Fog on the solid surface is due to the temporary changes of temperature and humidity, which represents a challenge on windshields, camera lenses, eyeglasses, mirrors, and other display devices for blurring the images displayed on or behind them because the tiny water droplets of fogs can strongly scatter visible light.9−14 To alleviate the trouble of fog, superhydrophilic, superhydrophobic, and amphiphilic materials © 2019 American Chemical Society

have been investigated to alleviate fogging hazards. The superhydrophobic polymers can induce a low adherence force to water droplets, resulting in water-repellent abilities.9,10 However, the amphiphilic coatings displayed high water contact angles (CAs) in the beginning and then suddenly fall to low values.11,12 In contrast, on the superhydrophilic surface especially with zwitterionic coating, water droplets can spread quickly to form a hydrated film by forming hydrogen bonds between the coating and surface without affecting light scattering.15 To our knowledge, zwitterionic peptides have not been explored in the antifogging field. Lysine (K) that is positively charged and glutamic acid (E) that is negatively charged are polymerized alternately and can form a superhydrophilic zwitterionic motif, which may endow the surface with outstanding antifogging properties.13,14 In addition, zwitterionic coatings also possess another interesting character, fast removal of dirty oil.15 As a zwitterionic material, KE peptides could strongly bind to water molecules and efficiently eliminate the interaction with oil molecules, which may cause Received: February 25, 2019 Accepted: June 20, 2019 Published: June 20, 2019 24504

DOI: 10.1021/acsami.9b03522 ACS Appl. Mater. Interfaces 2019, 11, 24504−24511

Research Article

ACS Applied Materials & Interfaces the oil molecules to leave the surface with flowing water droplets. Medical optical devices such as colonoscopy and laparoscopic will become blurry by the fog resulted from the temperature difference between the vivo and vitro, and the sight of them will be blocked by lipids in an internal environment.16−18 At present, the antifogging measures of medical equipment still employ relatively conventional methods, for instance, spraying the antifogging reagents (xylitol ester, lauric acid and so on) many a time and heating to balance temperature difference between the vivo and vitro. It not only is a cumbersome operation but also has safety hazards, for example, several patients burn their eyes by using antifogging reagents coated on the goggles.19−21 During the past decades, some antifogging materials had been paid attention to the antimicrobial aspect, whereas the biocompatibility of them was determined infrequently.22,23 As nontoxic and biocompatibility materials, multifunctional chimeric proteins have the obvious advantage property of preventing bacteria adhesion and very low cytotoxicity, so as to avoid biofouling of the medical device and decrease health adventure.8 In addition, adhering of coatings onto chemically distinct substrates with a facile process is still a challenging task. So far, various techniques have been developed to achieve multifunction surfaces such as UV light cross-linking and layer-bylayer assembly, which involves external energy input, fabrication costs, and poor adhesion9,10,24,25 In our previous work, mussel adhesive motif was introduced into the chimeric protein to form a stable connection between the coatings and substrates.8 The excellent adhesion of the protein is due to catechol ligand 3,4-dihydroxyphenylalanine (Dopa), of which two hydroxyl groups can mediate stronger various interactions with surfaces such as double hydrogen bonds, π−π interactions, and covalent cross-links.26−29 In comparison, this “one-step” surface deposition functionalization of various substrates is relatively convenient to be applied to optical devices. In this work, we report the fabrication of multifunction films via deposition on a substrate directly with bioinspired protein MP-KE. The interfacial binders were created by introducing mussel adhesive motif and modifying by tyrosinase in vivo because Dopa is an unnatural amino acid.8 The CA measurements and ultrasonic oscillating on protein-modified surfaces were studied to demonstrate surface superhydrophilic capability and stability. Besides, the transmittance of the protein films under foggy conditions through cold−heat method, heat−cold method, and UV−visible spectrophotometer were explored. Moreover, self-cleaning, icing delay, antimicrobial adhesion, and biocompatibility of the protein coatings were also demonstrated. To our knowledge, the successful fabrication of protein antifogging coatings with selfcleaning ability, icing delay, antimicrobial properties, excellent hemocompatibility, and negligible cytotoxicity has not been reported, which provides a promising direction for fabricating novel multifunctional coatings.

Information. Bradford was used to determine the purity of the protein, and the absorbance at 562 nm was recorded according to BCA Protein Assay Kit method (TIANGEN, China) to determine the protein concentration. 2.2. Coating Preparation and Characterization. The cut standard glass microscope slides (SAIL Brand, China) were washed initially by a KQ-100DV ultrasonic cleaning machine with ethanol and acetone for 30 min, respectively. Then, different MP-KE concentration solutions (0, 1, 2, and 4 mg/mL) dispersed in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.2) were drop/spray-coated onto the clean glass substrates. The coated substrates were dried under room-temperature conditions, leading to the formation of protein films. Finally, all of the samples were washed with deionized water three times and dried again to reach complete formation of protein films. The protein coatings were characterized using scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectra (FTIR), and CA. Four different protein films were fabricated on conductive glass following the above-mentioned preparative protocol. The samples were coated by sputtering with gold and examined by a low-vacuum SEM (S-4800, Hitachi) at an acceleration voltage of 5 kV to observe the morphologies. The roughness and 3D images of different samples were obtained by a CSPM5500A microscope AFM instrument (Benyuan Co. Ltd., China). The chemical composition of different coatings was detected by a K-Aepna XPS instrument using a Probe spectroscope (Thermo Scientific, America) with an Al Kα source gun type. Water CAs of different samples were collected by a JC2000D CA meter (Shanghai Zhongchen Equipment Co. Ltd., China) with deionized water drops. The droplets used were 5 μL from a micro syringe, and the measurements were performed at least three times at random positions on a sample under ambient conditions. FTIR measurements were recorded in reflection mode using an AVATR360 IR spectrometer (Nicolet, America). 2.3. Stability Measurements. The stability of protein coatings was quantified by the following methods: initially, the antifogging performance and dynamic changes of CAs were tested after exposing the coatings to ambient conditions for 40 days; specific test methods are as described above. Besides, the protein-coated glasses were treated with a JY 92-IIN ultrasonic cleaning machine (Scienze, China) for 20 min (5 W), and the variations of CAs were further measured. The surface morphology of the different protein coatings was also observed by SEM and AFM instruments after being stored for 40 days under ambient conditions. 2.4. Antifogging Tests. The microscope glass slides with one-half coated by a protein film were prepared. Then, the samples were placed above a beaker containing hot water for 10 s and in a refrigerator (−20 °C) for 20 min, respectively, and the photographs were taken immediately after the tests. In addition, the light transmission over the 400−800 nm range were monitored with an UV−vis spectrophotometer (UV-1800, Lamda) before and after the samples were stored in a refrigerator (−20 °C). 2.5. Self-Cleaning Determination. The efficiency of selfcleaning was defined as residual volume of contaminations. Dimethyl silicone oil mixed with some carbon powder was added to microscope slides modified by protein coatings, and then, the samples were washed with deionized water three times. Photographs were taken to show residual oil droplets.30 2.6. Anti-Icing Analysis. The anti-icing performance was carried out by detecting the icing delay time with a water CA of a cold stage (Tianjin Jing Yi Industry &Trade Co. Ltd., China) according to previous report.31 Glass slides coated by different concentrations of protein were placed on a cold stage (−15 °C), and 5 μL droplets were added dropwise from a micro syringe. Then, the icing delay times were recorded by taking a photograph of the water droplet shape every 4 s until water droplet is frozen. The measurements were performed at least three times at random positions for every sample. 2.7. Bacterial Adsorption Measurements. The antibacterial property of protein films was disclosed by the numbers of bacteria adhering on the coated surfaces. The round glass disks with a

2. MATERIALS AND METHODS 2.1. Protein Production. The genes encoding the recombinant chimeric protein MP-KE was cloned and expressed as our previous description.8 Besides, the tyrosinase derived from Streptomyces antibioticus was coexpressed in vivo to obtain the Dopa from tyrosine efficiently. High-concentration proteins were obtained by ultrafiltration and freeze-drying, describing in detail in the Supporting 24505

DOI: 10.1021/acsami.9b03522 ACS Appl. Mater. Interfaces 2019, 11, 24504−24511

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Figure 1. SEM images (a) of the protein coatings. Time profile of the water CA values on the different protein coatings within 240 s (b). AFM images over a scope of 5 μm × 5 μm of the different samples (c). diameter φ of 1 cm were modified with protein as described above. The samples were sterilized by UV irradiation before the tests, and then, they were incubated with bacterial suspension which were shaken until OD600 = 0.6 at 60 rpm and washed with 0.9% NaCl solutions. Finally, the bacteria conglutinated on the coatings were shaken by an ultrasonic cleaner (KQ-100DV, China), and 100 μL of vibrated bacteria solution was cultivated with lysogeny broth agar plates. The colony-forming units were counted after 24 h of incubation.32 Three parallel samples of each protein coatings were measured. 2.8. Hemocompatibility Analysis. The hemocompatibility of protein coatings was examined by blood clotting and hemolysis assays. Protein coatings were prepared on round glass dishes (φ 1 cm, 1 mm thick). Whole blood (50 μL) was added to the surface of samples, and then, 5 μL of CaCl2 (0.2 M) was added dropwise into the blood. After incubating for 5 min at 37 °C, 2 mL of ultrapure water was added, and the well plate was shaken at 30 rpm for 10 min. The control group was composed of 50 μL of whole blood with 2 mL of ultrapure water. The absorbance of solutions was measured at 541 nm, and the blood clotting index (BCI) was calculated according to the eq 1. BCI = 100 × (AS/AC)

prepared glass dishes (φ 1 cm, 1 mm thick) containing protein coatings were disinfected by UV light. Then, the samples were cocultured with GLC-82 cell suspensions in 48-well plates, the inoculation concentration of the cells is 1 × 105 cells/mL. After culturing at 37 °C under 5% CO2 for 24 h, the cells were added to MTT solution and incubated for 4 h. Then, the MTT solution was removed and the formazan solution was added to each well, the absorbance was measured at 490 nm. Cell viability was calculated according to the eq 3.

Cell viability (%) = AT/AC × 100%

where AT and AC are the absorbance values of the test and control groups, respectively.

3. RESULTS AND DISCUSSION 3.1. Protein Expression, Coating Fabrication and Characterization. To resolve the coating anchoring problem, a “one-step” dispersion fabrication process was developed to prepare antifogging coatings. Because of the stable bonds between the two hydroxyl groups of Dopa and the substrate, protein MP-KE can anchor directly onto the substrate to form a coating.33,34 Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated the successful expression and purification of MP-KE protein (Figure S1). Based on the preliminary experiment of diversity concentrations protein from low to high, stock protein solutions of 1, 2, and 4 mg/mL were chosen to show the difference. SEM was employed to observe the surface morphology of the protein coatings (Figure 1a). The coatings of 1 and 2 mg/mL displayed more sparse arrangements, whereas the coatings of 4 mg/mL formed a dense membrane. This phenomenon indicated the successful anchoring of proteins onto the substrate. In addition, the morphology of the glass, the protein coatings of 1, 2, and 4 mg/mL were observed by an AFM machine (Figure 1c), and their rootmean-square roughness (Rq) values were 0.03, 0.06, 0.06, and 0.05 nm, respectively, which proved that the protein can form a

(1)

where AS and AC is the absorbance of samples and control, respectively. For the hemolysis assays, fresh blood was diluted with 0.9% NaCl solutions in a ratio of 1:9 and coincubated with different protein coatings for 1 h at 37 °C. Then, the blood solutions were centrifuged at 1000 rpm for 10 min, and the absorbance of supernatants was measured at 541 nm. The blood incubating without protein coating and the blood diluted with water were set as a negative control and a positive control, respectively.32 The hemolysis rate was calculated according to the eq 2.

HR = (AS − AN)/(AP − AN)

(3)

(2)

where HR is the hemolysis rate and AS, AP, and AN are the absorbance of the samples, positive control, and negative control, respectively. 2.9. Cytocompatibility Analysis. The cytocompatibility of protein coatings is explored by the MTT colorimetric method. The 24506

DOI: 10.1021/acsami.9b03522 ACS Appl. Mater. Interfaces 2019, 11, 24504−24511

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Figure 2. Antifogging effect images of the glass slides covered with coatings containing 1, 2, and 4 mg/mL protein, treated with water steam (a), and exposed to laboratory conditions after being frozen at −20 °C for 20 min (b). Light transmission through the protein coatings before (c) and after (d) freezing at −20 °C for 20 min.

the 1 mg/mL protein coating had nonsignificant improvement compared with the uncoated side, and regional letters below can be seen on the glass coated with 2 mg/mL protein (Figure 2b). The excellent antifogging performance is mainly due to the rapid diffusion of water droplets into a hydrated film on the protein-coated surface. The water molecules can generate stable hydrogen bonds with the nitrogen and oxygen atoms of the protein, which promotes the formation of a hydrated film. In addition, the electrostatic attraction of zwitterion peptides in the chimeric protein MP-KE can further make the hydrogenbonded interaction between protein and water molecules stronger.31,36,37 The antifogging properties were further examined by measuring the visible light transmittance of the protein coatings from 400 to 800 nm by an ultraviolet spectrophotometer. The translucency of all coatings has almost no difference between the treatment group and blank glass (86− 91%) before freezing (Figure 2c), which confirms that protein films hardly affect light penetration through the glass.1,38 The light transmittance diversifications of different glasses after freezing are displayed in Figure 2d, and the 4 and 2 mg/mL coatings were about 57% higher than the glass matrix. The transmittance of 4 mg/mL protein coating (about 88−92%) is almost the same as that of the blank one before freezing, which may be attributed to the hydrated film. The aqueous layer acts as the lubricant, and at the same time, it removes the defects on the solid surface which could make the surface much glossy.39 Superhydrophilic protein materials have the ability to quickly absorb small water droplets by hydrogen bonds due to zwitterion peptides, leading to forming of a water film which does not affect the scattering of light.5,40 Aging and function tests were also applied to test proteincoated films. After being placed in the external environment (25 °C, 40% relative humidity) for 40 days, the variations of surface wettability were monitored by measuring CA values. The initial CA of the 4 mg/mL protein film is 23.06° followed by a decrease and less than 5° in 240 s (Figure S4a) similar to the previous trend, revealing that it maintains a superhydrophilic performance. Ultrasonic shock was also carried to further confirm the stability of the coatings. After the coating was ultrasonic shaken for 20 min, the coating is still hydrophilic with the CA shifting from 5° to 10° (Figure S4b). Surface images of protein coatings stored for 40 days were

smooth membrane on the substrate. The protein was successfully immobilized on glass substrates by confirming with the discovery of the feature XPS signals for N1s (BE = 402.0 eV) and Si2p (BE = 102.7 eV) in Figure S2. Besides, two significant amide bond signals at 1616 and 1517 cm−1 of the 4 mg/mL protein coating in FTIR spectra also indicated the presence of protein (Figure S3).10,35 The amide bond signals of the 2 mg/mL protein coating and the 1 mg/mL protein coating were relatively weak, consistent with the SEM results. In summary, 4 mg/mL protein will result in a stronger bond signal leading more dense coatings. Furthermore, dynamic changes of the water CA values were recorded to character the wettability of the protein coatings. As shown in the Figure 1b, the CA of 1 mg/mL coating (44.59°) was lower than the bare glass (69.1°) with slight changes. However, 2 and 4 mg/mL coatings changed greatly, particularly 4 mg/mL coating had a smaller CA value (23.53°) and followed by a fast drop to 10.96° within 90 s and reduced to below 5° within 240 s. This phenomenon is similar to the previous antifogging/anti-icing coatings POSSPDMAEMA-b-PSBMA with a small amount of ethylene glycol dimethacrylate.31 The hydrophilic effects of protein coatings were attributed to the zwitterionic peptide (KE)20 in chimeric protein MP-KE, and the hydrophilicity was positively correlated with the amount of protein. In conclusion, it is reasonable to assume that the 4 mg/mL protein coatings have unique hydrophilic properties. 3.2. Antifogging Performance. To test the antifogging property of the protein coating, cold−heat and heat−cold treatments were adopted.30 As shown in Figure 2a, the 4 mg/ mL protein film exhibited an amazing antifogging effect, and the coated side was visible clearly to observe the alphabets beneath. Evidently, the MP-KE coatings are able to inhibit the fog droplets cluster and ensure the excellent optical transparency. While a large number of small water droplets gathered on the other bare side, leading to smearing the letters (Video S1). In contrast, the 1 mg/mL protein film was as fuzzy as the other half of bare glass. Although the 2 mg/mL protein coating was also able to prevent droplets forming, the clarity was not comparable with that of the 4 mg/mL (Figure 2a). The 4 mg/ mL protein-coated side was so transparent to see beneath letters clearly (Figure 2b) after storing at −20 °C, while the control side was so blurred to see nothing. It is regretful that 24507

DOI: 10.1021/acsami.9b03522 ACS Appl. Mater. Interfaces 2019, 11, 24504−24511

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Figure 3. Optical images of self-cleaning test results of the bare glass (a) and the protein coatings at 1 (b), 2, (c) and 4 mg/mL (d).

collected by AFM and SEM; they did not show a significant difference compared with the ones before being stored (Figures S5 and S6). In addition, the stability of the antifogging performance was demonstrated by the cold−heat and heat−cold experience. The 4 mg/mL coating still inhibits the formation of fog, suggesting their long-lasting utilities (Figure S7). There is no doubt that protein coatings displayed a famous stability, which is important for practical applications. 3.3. Self-Cleaning Property. A large amount of lipid in the body environment influences the light transmission; therefore, the self-cleaning performance is one of the indispensable features provided by medical antifogging coatings.15 The dimethyl silicone mixed with carbon dioxide as an indicator for quantitative estimation of the oil droplets was introduced to contact with different concentration protein coatings. The protein coating with a concentration of 4 mg/ mL displayed almost no residual volume after washing, while the control glass covered a large area of black oil (Figure 3, Videos S2 and S3). The coatings of 1 and 2 mg/mL represent a diminishing amount of oil stain, consistent with the above experimental phenomenon (Figure 3). The main reasons for protein MP-KE coatings with such an eminent self-cleaning performance may be as follows: the first point is that water molecules are more immediately and rapidly absorbed compared with oil molecules by hydrogen bonds with the zwitterionic motif of MP-KE, allowing oil droplets to leave the surface with extra flowing water.41 Besides, the water molecules can squeeze between the oil layer and hydrophilic coatings to form an insulating layer, causing the low adhesion of oil droplets to the surface.15 The difference of the oil droplet residual area of the coatings should be due to hydrophilic ability discrepancy. The more the content of protein, the stronger the bonding force of hydrogen bonds, and the thicker the aqueous lubricating layer formation at the interface. 3.4. Anti-Icing Performance. There have been many reports about superhydrophilic materials used in anti-icing, such as polyelectrolyte brush, the anti-icing coating with an aqueous lubricating layer, and P(SBMA-co-FMA-co-AMA) with different contents of oligoethylene glycol dimethacrylate.39,42,43 Here, MP-KE proteins were also used to determine the ice-resistance, and the experimental results of icing delay are shown in Figure 4. The tip of the water droplet was an important feature of the water droplets becoming ice crystals; thus, the time profile was recorded when water droplets appeared the tip on the bare glass and protein coatings. It is expected to find that the water droplets on the 4 mg/mL protein film became frozen until 52 s compared to the bare glass which just remained for 8 s, and the icing delay times for the 1 and 2 mg/mL protein coatings were 32 and 44 s,

Figure 4. Photographs of the water droplets on the bare glass and the protein-modified samples during the freezing process at −15 °C. TD, the freezing delay time.

respectively. It can be concluded that the protein coating can delay the water from freezing in a certain degree.44 The icing delay effect of protein coatings may be attributed to the following causes: water is diffused into a hydrated film by hydrogen bonding with the hydrophilic zwitterionic segments of the protein MP-KE, allowing for a better performance.43,45 Besides, the free water molecules can be absorbed into protein films, existing in the nonfreezing state. In addition, MP-KE protein may lower the freezing point of water, which is somewhat similar to the reported anti-icing material.31,39 Furthermore, hydrophilic materials are able to organize an aqueous lubricating layer between the coatings and ice, which plays an important role in preventing the formation of ice crystals.43 3.5. Antimicrobial Properties. More and more materials in the medical fields are expected to have excellent antimicrobial capability because bacterial infection can cause severe safety issues, such as pneumonia, meningitis, and sepsis.22,23,46 Here, Escherichia coli and Staphylococcus aureus were selected as representatives of Gram-negative and Grampositive bacteria to determine the antimicrobial ability of different concentrations of protein MP-KE. As illustrated in Figure 5a, the control plates were covered with dense E. coli colonies, of interest is that only a very small number of bacteria adhered to the 4 mg/mL coating. The numbers of colonies in 1 and 2 mg/mL coatings were decreased by 85.4 and 96.8% compared with the control glass disks, whereas that of the 4 mg/mL coating was reduced by 98.1% (Figure 5c). A similar phenomenon about S. aureus can be seen in Figure 5b, the histogram units in Figure 5c further highlighted the significant influence, and about 1% colonies were on the plates of the 4 mg/mL coating. This consequence illustrates that protein coatings possess the ability to resist the adhesion of S. aureus and E. coli, consistent with our previous work.8 Bacterial adhesion to a material surface can be described as a two-phase process including an initial, instantaneous, and 24508

DOI: 10.1021/acsami.9b03522 ACS Appl. Mater. Interfaces 2019, 11, 24504−24511

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Figure 5. Antimicrobial test results of the glass slides with different protein coatings, E. coli (a) and S. aureus (b). Statistic data (c) of E. coli and S. aureus adhesion on different samples.

Figure 6. Hemolysis assays (a) results of the glass slides and the protein-coated samples. Quantitative data of GLC-82 cells vitality, hemolysis ratio (b) and BCI (c) of different samples.

protein coating were more than 96%, comparable with the control statistically (Figure 6b). The above results suggested that the protein coatings have excellent hemocompatibility and negligible cytotoxicity. Moreover, the BCI was also used to quantitatively evaluate the antithrombogenic activity of chimeric protein. Erythrocytes encapsulated in the blood clots were not hemolyzed, which was quantified by a spectrophotometer. In this case, higher BCI values indicate a better compatibility. The BCI of 4 mg/mL protein coatings was 94, 14% higher than that of the control, while the 1 and 2 mg/mL groups maintained intermediate BCI values (Figure 6c). These data indicate that the surface of protein materials would conquer the blood clotting. The size of the clots formed on the surface modified by the protein coating was also decreased substantially as the proportion of protein increases (Figure 6c). The hydrophilic protein coatings may inhibit the adhesion of fibrinogen and platelets in the blood, resulting in a low probability of thrombosis.52 In summary, the excellent biocompatibility of MP-KE protein coatings were demonstrated by hemocompatibility and cytotoxicity. Besides, the artificial prosthesis experiment is also important for practical applications, which is our further plan to evaluate the MP-KE properties in vivo.

reversible physical phase and a time-dependent and irreversible molecular and cellular phase.47 One of the most effective methods to prevent bacterial adhesion is to avoid or reduce the initial, nonspecific, and reversible attachment of bacteria to a surface.48 Zwitterionic MP-KE coatings are extreme hydrophilic because of electrostatically induced hydration, which makes the replacement of surface-bound water molecules by foulants enthalpically unfavorable.49,50 The tightly bound water layer formed by the superhydrophilic MP-KE prevents the interactions between bacteria and the membrane surface and restrains biofilm formation, resulting in a significant antibacterial property.50 It is highly mentioned that this protein coating achieves antimicrobial by resisting bacteria adhesion without chemical reagents, which does not divulge bactericide to harm the environment or cause drug resistance.51 3.6. Biocompatibility Analysis. Hemocompatibility and cytotoxicity of the protein coatings were also carried out to invest the biocompatibility of different amount proteins. As shown in Figure 6a, no visible hemolysis effect can be observed visually in the hemolysis assay. Although the HRs of protein coating were increased slightly as the protein concentration increases, they were both lower than 0.18% (Figure 6b), satisfying the clinical application requirement (HR < 5%).32 As for cytotoxicity test, the MTT solutions were used to detect the proliferation of GLC-82 cells. The absorbances of the cell culture solutions cocultured with different concentrations of 24509

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Research Article

ACS Applied Materials & Interfaces

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4. CONCLUSIONS We developed a multifunctional protein coating MP-KE for antifogging, self-cleaning, antimicrobial, and anti-icing applications with excellent biocompatibility on the basis of musselinspired protein and zwitterionic polypeptide. Superior adhesion of mussel-inspired protein allows coatings to adhere stably to substrate material surfaces with a “one-step” process tenderly. Expectedly, the coatings exhibit a remarkable antifogging performance. Because the superhydrophilic property of the zwitterionic polypeptide make water molecules spread rapidly into pseudo films on the surface, leading to avoiding the accumulation of water molecules and the interference of light transmittance. The superhydrophilicity also imparts protein coatings with a great self-cleaning performance and resistance to bacterial adsorption. Besides, protein coatings also have excellent anti-icing ability to delay the freeze time. Furthermore, this multifunctional coating possesses famous biocompatibility with wonderful hemocompatibility, negligible cytotoxicity, and anticoagulation effect, which offers merits over traditional hydrophilic surface modifications and may find unique applications in medical treatments such as pharmaceutical packaging and medical endoscope.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b03522. Additional SDS-PAGE; SEM and AFM images; XPS and FTIR spectra; stability test data; and videos of protein coating performances test (PDF) Video of protein coatings antifogging performance test (AVI) Video of bare glass slides self-cleaning test (AVI) Video of protein coatings self-cleaning test (AVI)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Lei Zhang: 0000-0003-3638-6219 Author Contributions ∥

H.Q. and C.Z. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the financial support from the National Natural Science Foundation of China (21606165, 21621004 and 21422605); the Natural Science Foundation of Tianjin City (17JCQNJC05600, 18JCYBJC29500); the Qingdao National Laboratory for Marine Science and Technology (QNLM2016ORP0407).

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DOI: 10.1021/acsami.9b03522 ACS Appl. Mater. Interfaces 2019, 11, 24504−24511

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DOI: 10.1021/acsami.9b03522 ACS Appl. Mater. Interfaces 2019, 11, 24504−24511