Rational design of amphiphilic peptides and its effect on antifouling

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Rational design of amphiphilic peptides and its effect on antifouling performance Sheng Long Gaw, Gowripriya Sakala, Sivan Nir, Abhijit Saha, Zhichuan J. Xu, Pooi-See Lee, and Meital Reches Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00587 • Publication Date (Web): 03 Aug 2018 Downloaded from http://pubs.acs.org on August 6, 2018

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Rational design of amphiphilic peptides and its effect on antifouling performance Sheng Long Gawa, Gowripriya Sakalab, Sivan Nirb, Abhijit Sahab, Zhichuan J. Xua, Pooi See Leea, and Meital Rechesb a. School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore b. Institute of Chemistry, The Hebrew University of Jerusalem, Edmond J. Safra Campus, Givat Ram, Jerusalem 9190401 KEYWORDS: Antifouling, Biofouling, Amphiphilic Peptide, Peptide, Coating

Abstract Biofouling, the unwanted adhesion of organisms to surfaces, has a negative impact on energy, food, water, and health resources. One possible strategy to fight biofouling is to modify the surface using a peptide-based coating that will change the surface properties. We reveal the importance of rational design and positioning of individual amino acids in an amphiphilic peptide sequence. By just manipulating the position of the amino acids within the peptide chain having the same chemical composition, we improved the antifouling performance of an amphiphilic peptide-based coating - Phe(4-F)-Lys-DOPA - by 30%. We have judiciously tailored the peptide configurations to achieve the best antifouling performance by i) positioning the amino acid lysine adjacent to the DOPA moiety in the linear peptide chain for better adhesion, ii) having a linear fluorinated N-terminal to improve the packing density of the film by straightening the peptide chain, and iii) placing DOPA at the C-terminal. We have also compared the antifouling performances of amphiphilic, hydrophobic, hydrophilic, and alternately arranged peptides. Our results show a reduction of ~ 80% in bacterial adhesion for an amphiphilic peptidecoated surface when compared to a bare titanium surface. This work provides important strategic design guidelines for future peptide-related materials that have effective antifouling properties.

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Introduction Biofouling is the undesirable accumulation of organisms on surfaces. The interactions between microorganisms and the substrate play a critical role in this process.1 To prepare materials that resist biofouling, termed antifouling, the surface properties must be changed by either chemistry or topography or both.2-5 Environmentally friendly antifouling solutions have been extensively studied due to stringent regulations.6-11 Amphiphilic polymer coatings result from combining hydrophobic and hydrophilic monomers, which can serve as an effective antifouling coating.2, 5, 12-15

Most of the proposed amphiphilic polymers consist of long repeating monomer units with

random, block, or alternate sequencing of two different properties that increase the complexity and unpredictable properties of the polymer. This often leads to uncertainty regarding the polymer’s mechanism of action. 2, 5, 12-15

Peptides are prepared by combining several amino acids into a molecular chain by amide bonds.16,17 It is an environmentally friendly and biocompatible material and can easily combine different properties of individual amino acids to attain its desired functionality.16-19 To date, several peptides have been designed specifically for antifouling applications.6,

20-23

The

arrangements of the amino acids reported are usually in an alternate or random arrangement.20,23 Therefore, it is of a great interest to determine the effect of positioning individual amino acids within the peptide chain to strategically manipulate and enhance the antifouling performance.

Here, we used several elements for designing the peptide: 3, 4-dihydroxy-L-phenylalanine (DOPA) is an amino acid with adhesive properties. It is the main constituent of mussel adhesive proteins (MAPs) that bind to different materials.24,

25

DOPA binds onto different types of

materials since it can form various bonds with the surface.25-27 Incorporating DOPA into a peptide sequence can attach the peptide to the surface easily.28, 29 Our group has anchored two 4fluoro-phenylalanine residues (Phe(4-F)) to the surface using DOPA to change the surface hydrophobicity, which consequently produces an antifouling coating.6 We have also used positively charged Lysine (Lys) of a hydrophilic nature. It has also been well reported for its antibacterial effect.30,

31

Since the sequence of amino acids within a peptide chain can be

manipulated during the synthesizing process, we hypothesized that the position of the amino acids might affect the antifouling performance of the peptides. 2 ACS Paragon Plus Environment

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Here, we have investigated an amphiphilic peptide that consists of three amino acids: i) DOPA, which serves as a binding unit, ii) the hydrophobic Phe(4-F) moiety, and iii) a hydrophilic, positively charged lysine (Lys) residue, providing self-assembly capability; these amino acids are of particular interest for antifouling applications.

This work shed light on the importance of the rationalizing and positioning of individual amino acids in the peptide chain. We have shown that the antifouling performance differs by 30% by strategic positioning of the amino acids within the peptide without altering the overall chemical composition. Having the established sequences, we also compared the antifouling performances of amphiphilic, hydrophobic, and hydrophilic peptides. Our results clearly showed a reduction of 80% in the number of bacteria on the amphiphilic peptide-coated surface when compared with a titanium-coated silicon surface. To further emphasize the importance of the positioning of amino acids within a peptide chain, we compared our established amphiphilic peptide to an alternative configuration peptide; both peptides had the same chemical composition. The results showed that our strategically amino acid-positioned peptide performed the best. Stability tests have shown a consistent antifouling performance of ~80% bacteria reduction after 7 days of incubation in E.coli inoculum when compared with the initial result.

Experimental Section Peptide Synthesis and Characterization: Peptides were manually synthesized using manual solid-phase peptide chemistry synthesis. Fmoc-DOPA(acetonide)-OH (GL Biochem (Shanghai)), L-4-fluoro phenylalanine-OH (GL Biochem (Shanghai)), Fmoc-L-Lys(Boc)-OH (Matrix Innovation (Canada)), HATU (Matrix Innovation (Canada)), and Fmoc-Rink Amide resin (Matrix Innovation (Canada)) were purchased and used. Coupling conditions using Amino Acids/HATU/ N,N-Diisopropylethylamine (DIPEA) (Bio-lab (Israel)) were employed to obtain the desired peptides. The Fmoc group was removed using 20% Piperidine in Dimethylformamide (DMF) (Bio-lab (Israel)) for 15 min three times, followed by rinsing thrice with DMF to remove any residuals. Amino acids were coupled in an excess of 5-fold in all reactions. The coupling reaction lasted 2 hours. The peptide was cleaved off from the resin support by using Trifluoroacetic acid (Bio-lab (Israel)), Triisoproplysilane (Tokyo Chemical Industry), Deionized 3 ACS Paragon Plus Environment


water at a ratio of 95/2.5/2.5 for 3 hours, and precipitation in cold diethyl ether (Bio-lab (Israel)). The precipitate was centrifuged down, re-dissolved in a mixture (50% acetonitrile and 50% water), and lyophilized to obtain the peptide. The purity of the peptide was confirmed by using analytical high-performance liquid chromatography (HPLC) (Merck Hitachi). The peptide was then characterized using high-resolution liquid chromatography mass spectrometry (LCMS) (Agilent) and nuclear magnetic resonance spectroscopy (NMR) (Bruker).

Bacterial growth: E. coli bacteria starter (ATCC#25922, which forms biofilms) was prepared by incubating a colony of E. coli at 37 °C for 6 hours in 15ml LB Broth (Merck Millpores) and then shaking it at 120 rpm. The E. coli starter was then diluted with LB Broth to a UV-Vis absorbance reading of 0.3 and at a wavelength of 600 nm to attain a bacterial concentration of 5x107 cells/mL. The absorption was measured by a 1650PC UV-vis spectrophotometer (Shimadzu, Kyoto, Japan). To access the antifouling properties on the surface, E. coli biofilms were scrapped off the substrate using a sterilized cotton swab. Serial dilutions were performed before performing the Plate Count Methods (PCM).

Crystal Violet Staining: The substrates were immersed in 0.2% crystal violet (Merck, Darmstadt, Germany) for 15 minutes and then rinsed three times with ultra-pure distilled water. The samples were dried at room temperature and placed under an optical microscope (Carl Zeiss, Axio Vision) for imaging.

Live/Dead® Assay Kit: A QIA76 Live/Dead Double Staining assay kit (Merck Millpores) was used. Substrates were stained in accordance with the manufacturer’s protocols. Fluorescent microscopy images were obtained by fluorescence microscopy (Carl Zeiss, Axio Vision).

X-ray Photoelectron Spectroscopy (XPS): The measurements were performed using a Kratos Axis Ultra X-ray photoelectron spectrometer (Karatos Analytical, Ltd., Manchester, UK) using an Al Kα monochromatic radiation source (1,486.7 eV) with a 90° takeoff angle (normal to 4 ACS Paragon Plus Environment

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analyzer). The high-resolution XPS spectra were collected with a pass energy of 20 eV and a 0.1 eV step. The binding energies were calibrated relative to the C 1s peak energy position as 285.0 eV. Data analyses were performed using Casa XPS (Casa Software, Ltd.).

Quartz Crystal Microbalance with dissipation monitoring (QCM-D): The measurements were performed using a quartz crystal microbalance (Q-Sense AB, Biolin Scientific, Gothenburg, Sweden) in a flow module system. A titanium sensor was purchased and used. Prior to the experiments, the sensor was rinsed with ethanol and DI water, followed by nitrogen air drying, and 3 minutes of oxygen plasma (Harrick Plasma Cleaner, NY). Acquisition of the data starts when the system stabilizes. Next, 0.5mg/ml of a peptide-methanol mixture was introduced into the system for 45 minutes. It was then switched to pure methanol for 10 minutes to determine the binding of peptide onto the surface.

Results and Discussion

Figure 1. Molecular structures of the designed peptides.



To complete the full combination of the tripeptide sequencing, we synthesized a total of six different peptides (Figure 1) using solid phase synthesis method (experimental section). The synthesized peptides were characterized by analytical HPLC (Figure S1 (a-f)), 1H NMR (Figure S3-S8), 13C NMR (Figure S9-S14), and LCMS (Table S1), respectively.

Each peptide was dissolved in methanol at a concentration of 0.5 mg/mL. Titanium-coated silicon substrates were used as the surface for the study. The substrate was immersed in the peptide solution for 8 hours at 23°C to form the coating. The coated substrate was rinsed twice with methanol and dried with nitrogen. The contact angle was measured 3 times for each sample. Table S2 shows the mean contact angles measured on the different samples. The control sample that was immersed in methanol displayed a contact angle of 22°±1, whereas peptide-coated samples displayed a higher contact angle for all the samples. The increase in the hydrophobicity is due to the fluorinated side chain present in the peptides.


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Figure 2. XPS analysis (a) Nitrogen XPS spectra of all samples. (b) Fluorine XPS spectra of all samples. (c) Titanium XPS spectra of all samples. (d) Oxygen XPS spectra of all samples. 7 ACS Paragon Plus Environment


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We used a quartz crystal microbalance with dissipation monitoring (QCM-D) and X-ray photoelectron spectroscopy (XPS) measurements to ascertain the binding of the peptides on the titanium-coated silicon surface. Figure S15 shows the QCM-D results from all peptides. From the results, a change in resonance frequency is observed. This indicates the binding of peptides on the titanium surface despite washing with methanol at the end of the experiment. Figure 2 shows the results obtained by XPS measurements obtained from the coatings of the 6 different peptides and the control sample (bare Ti on silicon). We performed Lorentzian-Gaussian peak fitting on all the spectra obtained. In the nitrogen spectra (Figure 2a), two peaks can be fitted. The 400 eV peak corresponds to C-N bonding, which is present in all the samples due to the organic molecules adsorbed on the surface.32-34 The second peak, which is at ~401.9eV, is attributed to the N-H bonding that exists in the chemical structure of peptides.32-34 This peak could only be detected in those samples coated with the peptides. In the fluorine spectra (Figure 2b), two peaks can also be fitted. The ~684.5eV peak is consistently found in all samples, including the control sample, and was identified as Ti-F bonding.35 The presence of the peak is attributed to the contamination of the titanium-coated silicon substrates during its preparation or ions implanted into the silicon substrates. Another peak at ~687.4eV is attributed to the C-F bonding.36 The presence of the C-F bonding is due to the fluorine-terminated group in Phe(4-F) amino acid. Therefore, this peak was present in all the spectra and exists in all samples coated with peptides.

DOPA has been extensively reported before for its adhesive properties.28, 29, 37 The proposed adhesive mechanism between DOPA and a titanium surface works through catechol binding.25 The hydroxide terminal of DOPA will bind to the surface, forming Ti-O linkage between the peptide and the titanium surface. This will lead to a change in the Ti-O binding energy between a coated and a non-coated peptide titanium surface. Therefore, the Ti and O XPS analyses were systematically studied in this work.

There are 8 peaks that can be fitted in the titanium spectra (Figure 2c).38,

39

There are no

significant peak shifts to the doublet peaks assigned to Ti, Ti3+, and Ti4+. In contrast, there is a consistent peak shift observed in the doublet peaks of Ti2+ on the peptide-coated samples when 8 ACS Paragon Plus Environment

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compared to the control.38,

39

The Ti2+ peak for the control sample has a binding energy of

~455.04eV, whereas there is a consistent positive shift of ~0.2-0.3 eV in the Ti2+ peak positions for all six peptide-coated samples. This indicates a change in the chemical bonding of the Ti-O bonding on the surface of the control and the peptide-coated substrate.

In the oxygen XPS spectra (Figure 2d), the TiO2, Ti-O, and C=O peaks can be fitted.38, 40 There were no significant shifts observed in the TiO2 and C=O peaks among the samples. However, we were able to detect a significant peak shift for the Ti-O binding peak. The Ti-O binding peak for the control sample has a binding energy of 531.07eV, whereas there is a consistent ~0.1-0.2eV positive shift of binding energy for all the peptide-coated samples. A change in the chemical bonding of the elements will cause the binding energy of a core electron of elements to deviate. Owing to the change in the chemical bonding of the bare titanium surface and the strong binding to the self-assembled peptide surface, the binding energy becomes altered when the peptide is chemically anchored onto the surface. Indeed, both oxygen and titanium XPS results display a consistent positive shift in the binding energy, which supports the hypothesis of DOPA and titanium catechol binding through the formation of Ti-O bonding.

Figure 3. Schematic drawing of the proposed chemical arrangement of the peptides on the surface. Based on the XPS results, we proposed a different chemical arrangement for each peptide, which differ in the position of the DOPA moiety (Figure 3). Peptide A has DOPA at the C-terminal of the peptide with adjacent Phe(4-F) and Lys at the N-terminal, whereas peptide B has the opposite arrangement. Based on the molecular arrangement of peptides A and B, we expect that both 9 ACS Paragon Plus Environment


peptides would form a monolayer of linear chains where Lys would be exposed to the surroundings in either the C-terminal (Peptide A) or the N-terminal (Peptide B) (see Figure 3). Peptides C and D both contain DOPA in the middle of the peptide between Phe(4-F) and Lys. Peptide C has Phe(4-F) at the C-terminal and Lys at the N-terminal, but peptide D has the opposite structure. We therefore expected the formation of a “Y”-branched peptide layer for these two peptides. In peptides E and F, Lys is adjacent to the DOPA moiety. We anticipated the formation of a monolayer having a linear chain where its Phe(4-F) moiety at the N-terminal (Peptide E) or the C-terminal (Peptide F) would be exposed to the surroundings.

Figure 4. Normalized bacteria growth (E. coli) (%) obtained from Plate Count Methods (PCM) on Ti-coated silicon substrates (n=9; p=Table S3).

To test the antifouling performance of the peptide-coated surfaces, the substrates were immersed in a medium containing E. coli at 37°C for 16 hours for substantial biofilm formation.41 We then performed the Plate Count Methods (PCM) for each surface.42 Figure 4 summarized the results from the three triplicate experiments. Peptide A reduced the number of bacteria by an average of 30%, whereas peptide B reduced it by an average of 25% when compared to a bare substrate. Peptides C and D reduced the number of adherent bacteria by 43%, on average. Peptides E and F reduced the number of bacteria by 56% and 51%, respectively. From these results, we can summarize the following findings: First, peptides C, D, E, and F performed better than peptides


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A and B. This might be due to the enhanced adhesive properties of the peptides to the surface due to DOPA and the lower surface energy due to the “Teflon-like” properties resulting from exposing the fluorinated terminal of phe(4-F).6, 43 Second, the linear chain tripeptide (peptides E and F) has a better antifouling performance than does the “Y”-branched (peptides C and D) (Figure 3). It was noted that peptides E and F have the best antifouling properties. This is probably due to the exposed fluorinated side chain located at the end of the chemical chain, which improves the ability to drag the chain toward the surface, enabling the formation of film with better packing density (Figure 3).9 Lastly, we have also observed a consistent trend between peptide A/B and peptide E/F, where there is a ~5% mean improvement in the antifouling performance when DOPA is on the C-terminal than on the N-terminal. However, due to variations between the repeat experiments, we have noted insignificant results obtained from the t-test (Table S3).

Based on these results, we found that the following peptide configurations achieve the best antifouling performance: (1) a linear chain peptide monolayer with Lys adjacent to the DOPA for better adhesion, (2) a linear fluorinated N-terminal to improve the packing density of the film by straightening the peptide chain, and (3) DOPA positioned at the C-terminal.



Figure 5. Molecular structures of the studied peptides.

To evaluate the effect of amphiphilicity on the antifouling performance, we designed and synthesized three additional peptides [G, H, and I] via solid-phase synthesis (Figure 5) based on a configuration established earlier. We characterized the peptides by analytical HPLC (Figure S2a-S2c) and LCMS (Table S1). Peptide G is an amphiphilic peptide consisting of three Phe(4-F) [hydrophobic], three Lys residues [hydrophilic], and one DOPA moiety. Peptide H is a hydrophobic peptide consisting of three Phe(4-F) [hydrophobic], one Lys [hydrophilic], and one DOPA, whereas Peptide I is a hydrophilic peptide consisting of one Phe(4-F) [hydrophobic], three Lys [hydrophilic], and one DOPA. We repeated the experiments under the same conditions reported earlier with peptides G, H, and I.


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Figure 6. Normalized bacteria growth (E. coli) (%) obtained from Plate Count Methods (PCM) on Ti-coated silicon substrates. (n=9; p=Table S3).

Figure 6 shows the antifouling performance of peptides G, H, and I. Peptide G reduced the number of bacteria by 80%, on average, whereas peptide H and I reduced it by 50% when compared with the uncoated surface. Indeed, from the above results, better antifouling performance can be obtained from the amphiphilic peptide when compared with the hydrophobic or hydrophilic peptide.

To further emphasize the importance of the positioning of amino acids within a peptide chain, using solid-phase synthesis, we synthesized peptide J (Figure 5), which has the same chemical composition as peptide G but the positions of the amino acids are arranged in an alternate manner. The synthesized peptide was characterized by analytical HPLC (Figure S2d) and LCMS (Table S1). Antifouling performance results (Figure 6) obtained from PCM show an average of 40% reduction of the bacteria when compared with the bare titanium-coated Si substrate. This result provided evidence indicating the importance of positioning the amino acids within a peptide chain.



Figure 7. Optical microscopy images (50x) of crystal violet-stained substrates (a1) Titaniumcoated silicon substrate. (b1) Phe(4-F)- Phe(4-F)- Phe(4-F)- Lys- Lys-Lys-DOPA [G]-coated titanium substrate. Fluorescence microscopy image (50x) of Live/Dead assay-stained substrate. (a2) Bare titanium substrate. (b2) Phe(4-F)- Phe(4-F)- Phe(4-F)- Lys- Lys-Lys-DOPA [G]coated titanium substrate. Scanning electron microscopy images of substrates. (a3) Titaniumcoated silicon substrate. (b3) Phe(4-F)- Phe(4-F)- Phe(4-F)- Lys- Lys-Lys-DOPA [G]-coated titanium substrate. (Fluorescence Microscopy Image: Red indicates dead E. coli; green indicates live E. coli)

To better understand the antifouling mechanism, we stained the bacteria with crystal violet and used a live/dead kit. The tests were conducted on the bare titanium-coated silicon substrate and the peptide G-coated substrate. Figure 7 (a1 and b1) shows the optical microscopic images of the crystal violet stained surface. From the images, it can be observed that the number of bacteria that adhered to the surfaces indeed is consistently reduced on the peptide G-coated samples, compared with the control. Figure 7 (b1) shows the lowest number of bacteria on the substrate. This result correlates with the bacterial count obtained from the PCM (Figure 6). Figure 7 (a2 and b2) shows the fluorescent microscope images of the live/dead kit-stained surface. These 14 ACS Paragon Plus Environment

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results are consistent with the optical microscope images as well as the results obtained from the bacterial counts. These results have also indicated that the number of dead bacteria on all the peptide-coated surfaces is not substantially high. Therefore, based on these optical microscope images, we can conclude that the reduction in the bacterial count is due to the antifouling properties and not because of the antibacterial properties. From the high-resolution scanning electron microscopy images (Figure 7 (a3) and (b3), when comparing the two substrates, we can see that the bacteria did not undergo any morphological changes.

Figure 8. Normalized bacteria growth (E. coli) (%) obtained from Plate Count Methods (PCM) on Ti-coated silicon substrates. (n=9; p=

Rational design of amphiphilic peptides and its effect on antifouling

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