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. Author manuscript; available in PMC: 2021 Oct 14.
Published in final edited form as: ACS Appl Mater Interfaces. 2020 Sep 30;12(41):46991–47001. doi: 10.1021/acsami.0c13492

Antimicrobial Polymer–Peptide Conjugates Based on Maximin H5 and PEG to Prevent Biofouling of E. coli and P. aeruginosa

Valerie Ortiz-Gómez 1, Victor D Rodríguez-Ramos 2, Rafael Maldonado-Hernández 3, José A González-Feliciano 4, Eduardo Nicolau 5
PMCID: PMC8177746  NIHMSID: NIHMS1704095  PMID: 32937073

Abstract

Many pathogens, such as Pseudomonas aeruginosa and Escherichia coli bacteria can easily attach to surfaces and form stable biofilms. The formation of such biofilms in surfaces presents a problem in environmental, biomedical, and industrial processes, among many others. Aiming to provide a plausible solution to this issue, the anionic and hydrophobic peptide Maximin H5 C-terminally deaminated isoform (MH5C) has been modified with a cysteine in the C-terminal (MH5C-Cys) and coupled to polyethylene glycol (PEG) polymers of varying sizes (i.e., 2 kDa and 5 kDa) to serve as a surface protective coating. Briefly, the MH5C-Cys was bioconjugated to PEG and purified by size exclusion chromatography while the reaction was confirmed via SDS-PAGE and MALDI ToF. Moreover, the preventive antimicrobial activity of the MH5C-Cys-PEG conjugates was performed via the growth curves method, showing inhibition of bacterial growth after 24 h. The efficacy of these peptide–polymer conjugates was extensively characterized via scanning electron microscopy (SEM), minimum inhibition concentration (MIC), minimum biofilm inhibition concentration (MBIC), and minimum biofilm eradication concentration (MBEC) assays to evaluate their ability to eradicate and prevent the biofilms. Interestingly, this work demonstrated a critical PEG polymer weight of 5 kDa as ideal when coupled to the peptide to achieve inhibition and eradication of the biofilm formation in both bacteria strains. According to the MICs (40 μM) and MBICs (300 μM), we can conclude that this conjugate (MH5C-Cys-5 kDa) has an action that prevents/inhibits the formation of biofilms and the eradication of biofilms (MBEC 500 μM). In contrast, the MH5C-Cys peptide with PEG polymer of 2 kDa did not show inhibition or eradication of the biofilms.

Keywords: Maximin H5, polymer–peptide conjugates, antimicrobial peptides, biofouling

Graphical Abstract

graphic file with name nihms-1704095-f0001.jpg

2. INTRODUCTION

Of all the pathogenic bacteria species present in nature, Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli) represent two of the most deleterious. E. coli, a facultative anaerobic coliform is implicated in urinary tract infections, biliary sepsis, pneumonia, gastroenteritis, and many other illnesses. This bacteria is an indicator of fecal contamination in water and has been shown to remain in surfaces as a biofilm.1 P. aeruginosa is an opportunistic waterborne pathogen with the ability to thrive in inhospitable environments.2 It is a frequent nosocomial infectious agent that can affect patients with cystic fibrosis and burn wounds that can lead, or infections that can cause, septicemia, meningitis, and pneumonia.3 One of the reasons P. aeruginosa represents a challenge, similar to E. coli, is because of its ability to develop bacterial biofilms or colonies of microorganisms that can grow on any surface.4 This microenvironment has become more tolerant to natural defenses and antibiotics, making it a problem;5 for instance, surface modifications with polydopamine (PDA) and those that are polyphenol-based have become quite common. The main mechanisms by which these modifications lead to biofilm inhibition is due to the formation of a hydration shell and steric hindrance effects.6 Polyphenols are organic compounds with significant biocompatibility, and their essential function is preventing microbial infections in biomedical applications. One typical example of surface modifications with PDA and polyphenol-based ones is a class of PDA particles that have been designed to improve the physiological stability and properties of potential antimicrobial and therapeutic agents. Other studies suggest the modification of surfaces with hydrophilic metal oxides, antimicrobial polymers, and, more recently, antimicrobial peptides.7,8

Recent investigations have proven that in comparison to other materials antimicrobial peptides (AMPs) exhibit high bactericidal and bacteriostatic activity.9–11 AMPs are small biomolecules made of 5–100 amino acids that are found in living organisms and whose antimicrobial activity has proven to be significant.12 Amphibians are classic examples of living organisms that have evolved to secrete AMPs. In fact, they are the first group of organisms to form a bond between terrestrial and aqueous ecosystems and have been able to adapt in habitats with large communities of pathogenic microorganisms from both ecosystems.13 As a survival mechanism, amphibians secrete AMPs that specifically target bacteria which frequent in their environment, possibly including pathogens of interest. For example, the hydrophobic antimicrobial peptide Maximin H5 (MH5) that can be abundantly found in the skin and brain of the Chinese frog Bombina maxima is an AMP with interesting features. This peptide is 20 amino acids in length with a molecular weight of 2 kDa; it contains three aspartate residues with no basic amino acid residues and possesses an α-helical host defense. The primary native structure of this peptide is ILGPVLGLVSDTLDDVLGIL-NH2 (MH5N) and is the first type of anionic AMPs reported in amphibians.14

In a previous study, Lai et al. found for the first time the antimicrobial activity of MH5 against Gram-positive bacteria (i.e., Staphylococcus aureus) with MIC of 80 μM.14 Thereafter, Dennison et al. described the role of C-terminal deamidation in the antimicrobial activity of MH5. The study showed that the peptide with the structure ILGPVLGLVSDTLDDVLGIL-COOH (MH5C) had antimicrobial activity against Gram-negative bacteria but lower activity in Gram-positive bacteria. According to this study, the C-terminal deamination plays a critical role in the activity of Gram-negative bacteria. This behavior is ascribed to the hydrogen-bonding interactions that occur between the C-terminal amide group of MH5C and the bacteria’s membrane surface that helps to increase the antimicrobial activity.15

Even though this peptide has been extensively characterized, there are no studies where the peptide is conjugated to other functional molecules such as polymers. In this study, the principal aim is the characterization of the MH5 C-terminally deaminated isoform (MH5C) modified with cysteine in the C-terminal (MH5C-Cys) and coupled to PEG of different sizes (i.e., 2 kDa and 5 kDa). Specifically, we evaluate the preventive antimicrobial potential of the polymer-peptide conjugates with an emphasis on the physical and biological characterization. PEG is widely known for the benefits it provides when coupled to peptides such as improvements in solubility, stability, and its ability to form micelles for particular uses like drug delivery and other biomedical applications.16,17 Also, studies have shown that coupling antimicrobial peptides with polymers protect the peptides from proteolysis.18 When coupled to a polymer, these peptides could then be transferred as coatings to serve as protective barriers in antibiofouling surface applications. The MH5C-Cys is a hydrophobic peptide with antimicrobial properties that could be susceptible to adsorption from other hydrophobic foulants. However, MH5C-Cys has been modified with the hydrophilic PEG polymer, and this can protect the peptide from other organic matter.

3. EXPERIMENTAL SECTION

3.1. Materials and Reagents.

The MH5C-terminally deaminated isoform (MH5C) (ILGPVLGLVSDTLDDVLGIL-COOH, 2022.39 Da, 95% purity), MH5 with cysteine (MH5C-Cys) ILGPVLGLVSDTLDDVLGILC–COOH, 2125.54 Da, 95% purity), and Tet-20 (KRWRIRVRVIRKC–COOH, 1769.23 Da, 95% purity) AMPs were purchased from GenScript Co. Dimethyl sulfoxide (DMSO) molecular biology 99% purity, tris(2-carboxyethyl) phosphine hydrochloride (TCEP), and Dulbecco’s Phosphate Buffered Saline (DPBS) were purchased from Sigma-Aldrich and used without further purification. For the reaction, the PEG polymers were used with the following specifications: mPEG-Maleimide monofunctional 2 kDa (cat. number PSB-235) and mPEG-Maleimide 5 kDa (cat. number PLS-234) were obtained from Creative PEG Works Co. Nanopure water (np) was used at all times (Milli-Q Direct 16, Millipore, Burlington, MA).

To determine the antimicrobial activity of the peptides against the pathogenic bacteria P. aeruginosa (ATCC 27853)19 and E. coli (ATCC 25922),20 the following materials were used: Costar 96-well plates Flat Bottom (Sterile Low Evaporation Lid, Corning, U.S.A.), Calgary Biofilm Device (Innovotech Inc.), and glycerol 100% purity. The nutrient agar and broth used were BBL Trypticase Soy Agar II (TSA) and BBL Trypticase Soy Broth (TSB) and were purchased from Becton, Dickinson and Company (BD).

3.2. Physical Characterization.

3.2.1. Dynamic Light Scattering (DLS).

AMPs were dissolved in DMSO and nanopure water (DMSO/np water mixture; 20% v/v) with the final concentration at 1.0 mg/mL. First, the samples were ultracentrifuged (29 100 rpm, 1 h at 20 °C),21 and later the size average of these biomolecules in solution were studied using a Malvern Zetasizer Nano instrument (4 mW 632.8 nm laser). The individual peak method was used to determine the polydispersity index (PdI), as published elsewhere.22

3.2.2. Circular Dichroism (CD) Spectroscopy.

CD spectra was recorded on a Jasco J-1500 CD Spectrometer (Jasco, Inc., Easton, MD). Both peptides (MH5C, MH5C-Cys) were dissolved according to the protocol published by Dennison et al. with 2,2,2-trifluoroethanol (TFE) and np water (TFE/np mixture; 50% v/v) as cosolvent for this peptide.23 The final concentration was set to 90 μM and the samples were ultracentrifuged (29 100 rpm) for 1 h at 20 °C. Subsequently, 400 μL of the supernatant of each peptide was added to a rectangular quartz cell 1 mm path length to evaluate secondary structures. The wavelength range used was from 250 to 190 nm and scanning speed was 50 nm/min at 20 °C. The data obtained was performed in triplicate and then averaged for each sample. In addition, the thermodynamics and unfolding of the peptides were studied with this equipment. This technique was used to observe the secondary structure at wavelength complete spectra as a function of temperature.24 Temperatures ranging from 20 to 90 °C were used.

3.3. Microbiological Assays.

For these assays, bacteria of interest were thawed and serial-cultured to reach optimal metabolic activity of bacteria after frozen. Optical density (O.D.), using Genesys 10S UV–vis Spectrophotometer (O.D.600 nm), was utilized as a measure of bacterial concentration, 1.0 × 108 CFU/mL, as standardized and approved by the Clinical and Laboratory Standards Institute (CLSI);25 the final concentration of the solvent (vehicle control) was DMSO 10%.

3.3.1. Bacterial Growth Curve Test.

To create growth curves of the bacteria with and without peptides and to determine the lowest concentration in which these antimicrobial peptides presented inhibition of bacterial growth, MIC was carried out. After thawing, incubating at 37 °C for 24 h, and achieving target O.D., the bacteria was placed on the 96-well plate with a lid together with the peptides in various concentrations. Finally, the 96-well plate was taken to the Synergy H1 Hybrid Multi-Mode Microplate Reader to study the antibacterial effect of the peptides during different time points (0.5, 5, 12, 18, and 24 h) at 37 °C. This process was repeated for the evaluation of growth using the MH5C-Cys and Tet-20 as a control peptide for comparison.

3.3.2. Scanning Electron Microscopy (SEM): Study of Prevention in Biofilms Formation.

SEM was employed to evaluate the formation of biofilms and its interaction with these peptides. For this examination, a JEOL 6480LV under high vacuum mode operating at a range of 10–20 kV was used. In brief, 100 μL of 1.0 × 108 CFU/mL were grown in tubes at 37 °C in continuous shaking for 2 h (direct contact between bacteria and antimicrobial peptides, (Figure S1). The control group is bacteria and nutrient broth, while the experimental group includes bacteria with antimicrobial peptides (90 μM). After this procedure, samples were placed on a glass coverslip that were previously deposited with poly-L-lysine (0.1% w/v Sigma-Aldrich) to aid adhesion. Subsequently, the samples were placed in 24-well plates and incubated for 72 h at 37 °C. Sample fixation and dehydration were performed according to Bello et al., as well as HMDS drying protocol as published elsewhere.26,27 Finally, the samples were mounted on aluminum mount holders (12.2 mm) and sputtered with a thin gold film (about 5 nm thick). This process was repeated in the evaluation of biofilms using the MH5C-Cys.

3.3.3. Determination of Minimum Biofilm Inhibition Concentration (MBIC).

MBICs were determined by the adherence assay using the Calgary Biofilm Device (Innovotech Inc.). The device consisted of a plate containing the inoculated test medium and a PEG lid with 96 identical wells in which the microbial biofilm formed under shaker incubation. The assay was conducted according to the protocol as supplied by the manufacturer. The inoculum of each bacteria was prepared in TSB to a final density of 1.0 × 108. In this assay, the bacteria and compounds (MH5C-Cys and peptide–polymer conjugates) were placed together in the PEG wells and the assay plate lid for 72 h to allow growth. Afterward, colony forming unit (CFU) and optical density measurements for each well were recorded at 650 nm, and clear wells and growth inhibition curves were taken as evidence of biofilm inhibition.

3.3.4. Determination of Minimum Biofilm Eradication Concentration (MBEC).

In order to assess the MBEC of these compounds, biofilms of the P. aeruginosa and E. coli were grown in a Calgary Biofilm Device (Innovotech Inc.) as explained in Section 3.3.3. Into each well of the 96-well plates, 150 μL of the inoculated media was transferred. Assay plates were placed in an incubator for 72 h to allow growth; a biofilm comparison was also performed for each bacteria strain. After 72 h, the PEG lid of the MBEC assay was transferred to a “challenge plate”. Essentially, serial dilutions of each conjugate (total weights of 4 kDa and 7 kDa), antimicrobial peptides, and PEG polymers were prepared in TSB to a final volume in the well plates of 200 μL (control and conjugates at 90, 300, and 500 μM). After exposure of the biofilm to the antimicrobial challenge for 24 h, the PEG lid was removed from the challenge plate, and O.D. measurements for each plate were recorded at 650 nm. Clear wells were taken as evidence of biofilm eradication and MBEC values were assigned as the lowest concentration at which no growth was observed after 24 h of incubation.28,29 This process was repeated in the evaluation of growth using the Tet-20 as a control peptide for comparison.

3.4. Peptide Coupling to PEG Polymers.

3.4.1. Michael Addition Reaction.

A maleimide-modified PEG was utilized for reaction with the antimicrobial peptides. First, the samples of modified antimicrobial peptide (MH5C-Cys) and PEG polymer (2 kDa and 5 kDa) were dissolved in DPBS 1×, pH 7 (4 and 60 mg/mL). Prior to the reaction, oxygen was removed from each PEG polymer solution using nitrogen gas. MH5C-Cys was linked through disulfide bonds formed by the addition of a cysteine group in the amino terminal of the peptide. To break these disulfide bonds, 10 mM TCEP at pH 7.0 was added to the peptide sample at a ratio of 1:1 (MH5C-Cys/10 mM TCEP; thus, the TCEP final concentration was 5 mM).30,31 Then, the modified PEG was added to the MH5C-Cys mixture and allowed to react for 12 h at 4 °C in absence of oxygen. This biomolecule is MH5C-Cys conjugated with PEG (MH5C-Cys-PEG 2 kDa-5 kDa).

3.4.2. Size Exclusion Chromatography (SEC).

The isocratic method was performed for the MH5C-Cys conjugated using a ÄKTA Explorer 100 protein purification system (GE Healthcare). The sample of peptide conjugated was loaded onto the Superdex peptide 10/300 gel column at 0.25 mL/min monitoring at 220 nm of absorbance. The DPBS 1×, pH 7 solvent was used for this experiment. The elution fractions were collected using an automatic fraction collector.

3.4.3. SDS-PAGE.

This method was used to validate the conjugation of MH5C-Cys peptide and PEG polymer through differences in molecular weights. Briefly, 1 mg/mL (MH5-C) and 1.5 mg/mL (MH5-C conjugates) were then loaded into a precast 16.5% Criterion Tris-Tricine Gel. A ladder Precision Plus Protein Dual Xtra Prestained Protein Standard was used for this process. Subsequently, electrophoresis was performed at room temperature for 1 h and 20 min at 125 V with running buffer solution (1× Tris/Tricine/SDS Running Buffer). Once the SDS-PAGE run was completed, gels were rinsed in a fixing solution (40% methanol, 10% acetic acid, and 50% np water) for 30 min. Thereafter, gels were stained using Bio-Safe Coomassie G-250 Stain for 1 h. Finally, gels were washed for 30 min three times and recorded with ChemiDoc XRS System.

3.4.4. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI ToF).

Mass spectrometry analysis was used to determine the specific molecular weight of the PEG-peptide conjugates. For purification and concentration of PEG-peptide conjugates, a reversed phase chromatography (Pierce C18 Spin Columns, catalog number: 89870) was used. After purification, samples containing 2 mg/mL of the peptide (MH5-C), 2 mg/mL of peptide–polymer conjugates, and 30 mg/mL of PEG polymer were mixed (1:6, sample/matrix solution) with the MALDI matrix solution (sinapinic acid in 70% acetonitrile and 0.07% of trifluoroacetic acid). Then samples were spotted on to MALDI target plate and allowed to dry at room temperature. MALDI MS data was acquired in positive ion linear mode on a 4800 Plus MALDI TOF/TOF Analyzer (Sciex) which was calibrated externally using the Opti ToF TIS Calibration Insert (Sciex) (027943, Sciex).

3.4.5. Statistical Analysis.

A statistical analysis was performed using the GraphPad Prism 6 software. All data was expressed as means ± standard errors of the means (S.E.M.). A comparison of the means for the treatments was also made at the 5% significance level. The data was analyzed with two-way ANOVA with Dunnett’s Test for multiple comparisons and the differences were statistically indicated with **** or p < 0.0001.

4. RESULTS AND DISCUSSION

4.1. Biophysical and Microbiological Characterization of MH5C and MH5C-Cys Peptides.

The antimicrobial peptide MH5C is a hydrophobic anionic molecule with 20 amino acids. In this research, the peptide MH5C (ILGPV-LGLVSDTLDDVLGIL-COOH) was coupled to polymers (PEG) of varying molecular weights. In order to promote the Michael addition reaction of the peptide with the polymer, both molecules were modified with terminal reactive groups. A modification to the C-terminal of the peptide was introduced in the form of a terminal cysteine (MH5C-Cys; ILGPV-LGLVSDTLDDVLGILC-COOH), whereas the polymer contains a terminal maleimide.

Before coupling the peptide to the polymer, it was of interest to assess the structural and antimicrobial properties of the peptide’s C-terminal modification. As such, we first conducted physical and biological characterization of the MH5C and MH5C-Cys peptides. First, the stability of the peptide in solution was determined by means of DLS for both peptides (MH5C and MH5C-Cys). The average apparent hydrodynamic diameter results showed that MH5C peptide has a hydrodynamic diameter of 0.72 ± 0.11 nm (PDI = 0.03), whereas MH5C-Cys, displayed a small decrease resulting in 0.52 ± 0.44 nm (PDI = 0.01).32 These results suggest that the peptides do not tend to aggregate and are stable under experimental conditions after having performed the modification.21 Likewise, zeta potential was conducted in order to determine the overall charge of the modified and unmodified peptide. This is an important condition for the stability of the formulation, as no precipitation will occur under the conditions of the experiment.33 MH5C resulted in having positive zeta potential values (46.6 ± 0.9) compared to the MH5C-Cys (−94 ± 4). In any case, a formulation with zeta potential values above and below ±30 is considered to be in a stable form.34 Therefore, adding the cysteine to the peptide for further conjugation does not affect the peptides’ stability as per zeta potential measurements.

Another measure to certify that the peptide’s modification does not lead to structural changes is by verifying the peptide’s secondary structure via CD spectroscopy. The secondary structure is fundamental for the stability and efficiency of AMPs in terms of their biological activity.35 Thus, changes in secondary structures may imply a decrease in the stability and antimicrobial function of the peptides. Analysis of the CD spectra showed MH5C and MH5C-Cys had a 95% α helix structure and a 0% β strand structure (Figure S2). Spectra in these peptides presented negative bands in 208 and 220 nm which is consistent to previous studies.36 The secondary structure analysis was also performed with temperature variations from 20 to 90 °C in order to assess the thermal stability (Figure S3).

The microbiological characterization of the peptide molecules (MH5C and MH5C-Cys) was conducted, and MIC values were determined. The MIC values were determined for E. coli and P. aeruginosa resulting in a value of 90 μM (Figure S4) for both peptides and bacterial strains. Moreover, the growth curves for the two different bacteria strains were conducted in the presence of the antimicrobial peptides, Figure 1A,C. From these results, it can be observed that at 24 h, the MH5C and MH5C-Cys did not allow bacterial growth. In fact, the peptides demonstrate bactericidal effect in both bacteria after 12 h of growth.37 It is also noted that after 0.5 h the peptides caused the death of both of the bacteria that were studied. According to the literature, a bactericidal antimicrobial agent in contact with a microorganism under treatment produces a total and irreversible death. In contrast, a bacteriostatic agent inhibits and halts the growth until the reproduction of the microorganism is not allowed, keeping them in a stationary phase.38 These results are demonstrated in Figure 1B,D with significant statistical differences (p < 0.0001). In addition, it was found that the solvent (vehicle control) does not have any effect in the behavior of the bacteria strains.

Figure 1.

Figure 1.

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Bacterial growth curve test used in MH5C and MH5C-Cys peptides. Control (bacteria and nutrient broth), vehicle control (DMSO 10%), and MH5C, MH5C-Cys AMPs samples (90 μM). (A,C) Growth curves of bacteria, (B,D) indicate statistically differences. The concentration of these bacteria is 1.0 × 108 CFU/mL. The statistically significant differences between control and AMPs are demonstrated in asterisks (P < 0.0001; n = 3 in each time point).

The antibacterial activity of MH5C peptides is comparable to that of other anionic AMPs such as GH-1l L and GH-1l D from the frog Bombina orientalis.39 These results confirm that the peptides MH5C and MH5C-Cys exhibit activity against these microorganisms when in the planktonic phase. The bactericidal antimicrobial activity in the peptides under study is likely due a combination of hydrophobic effects and the α-helical structure. The most accepted mechanism of action for the peptide MH5 is the carpet model. Interestingly, the high content of hydrophobic residues in this peptide provides for a tilted conformation that allows interaction of the α-helical AMP with the lipids in the membrane in an angle of 30° to 60° that contributes to their ability to penetrate membranes. It is worth indicating that the net negative charge of MH5C is the result from an internal cluster of D residues (aspartic acid) and appears to play no direct role in the membrane interactions.15,40 The most accepted mechanism of this model relies in the formation of a carpet-like complex on the surface of the bacterial membrane. This event occurs when the peptides have a specific critical concentration interacting with the bacteria.41 The interaction occurs via the parallel orientation of the peptide with the lipid bilayer while forming an expanded carpet. In the carpet model, the peptide diminishes the function of the bacterial membrane causing a collapse. According to previous research, the secondary structure is what provides for the exceptional antimicrobial activity in AMPs. In general, all AMPs have a three-dimensional arrangement with amphiphilic properties, where the hydrophobic portions interact with the lipid bilayer of the bacterial membrane.42 Various studies have determined that the secondary structure is a necessary element for the stability and efficiency of AMPs.35,43–45 Overall, the α-helical peptides have higher antimicrobial activity in Gram-positive and Gram-negative bacteria when compared to ß-sheet peptides; this type of structure is the most present in nature.46 Moreover, the α-helical structure can provide severe disturbances in the bacterial membrane, and it is postulated that amino acids such as lysine (K), leucine (L), and phenylalanine (F) play an essential role in this action.47–49 The antimicrobial activity of MH5C was tested in this study and it displayed activity against Gram-negative bacteria P. aeruginosa and E. coli, which is consistent to previous reports.15

To further confirm the ability of the peptides to diminish the proliferation of microorganisms on a surface, contact studies were conducted via the SEM technique. As can be qualitatively appreciated in Figures S5 and S6, the antimicrobial peptides exhibited a prevention of the biofilm formation for P. aeruginosa and E. coli. The effect of preventing biofilm formation is solely ascribed to the peptides since PEG controls did not show any effect (Figures S7–S9).

For the purposes of comparison, Tet-20 was utilized as a control peptide and MIC are 40 and 15 μM in P. aeruginosa and E. coli, respectively (Figure S10). Tet-20 antimicrobial peptide is a traditional control with antifouling activity. The primary structure of this peptide is KRWRIRVRVIRKC and has a broad antimicrobial activity. In addition, the value of the MBEC assay is 300 μM in both bacteria. In Figures S11 and S12, a bactericidal effect in both bacteria was observed. This effect is attributed to the hydrophilic and cationic properties of this peptide.50

4.2. Synthesis and Characterization of the Polymer–Peptide Conjugate: MH5C-Cys-PEG.

Once the modified peptide with a C-terminal cysteine was thoroughly characterized, the reaction between the PEG and modified MH5C-Cys was performed. In this reaction, the thiol group performs a nucleophilic attack to the carbon of the double bond located in the maleimide linked to PEG. This reaction occurs due to maleimide specific reactivity toward thiols as stated in previous articles.16,51,52

After the reaction, the conjugates were purified via SEC in order to remove any unreacted molecules from the conjugation process by their hydrodynamic volume.53 Thereafter, the fractions of interest were collected and analyzed by SDS-PAGE and MALDI-ToF (Figure 2). As can be observed in this figure, SDS-PAGE and MALDI-ToF results show clear bands and molecular weight migration corresponding to the synthesized conjugates. The analytical SEC results present three peaks corresponding to the elution fraction profile of the conjugates (Figure 2A). In SDS-PAGE, for the conjugate with the PEG 2 kDa there are two main bands at 2 kDa and 4 kDa. Similarly, in the conjugate with the PEG 5 kDa two main bands at 2 kDa and 7 kDa are observed with the first bands corresponding to the MH5C-Cys and the subsequent ones to the latter to the MH5C-Cys conjugated with PEG (Figure 2B).

Figure 2.

Figure 2.

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Characterization of the peptide–polymers conjugate. (A) Analytical SEC at 220 nm. Presented in red is the conjugate with PEG 5 kDa, in blue is the conjugate with PEG 2 kDa, and finally in black is the MH5C-Cys. (B) SDS PAGE in peptide and polymer conjugate. Lane 1, Dual Xtra Standards (ladder); lane 2, MH5C-Cys; and lane 3, MH5C-Cys conjugated with PEG. (C) MALDI-ToF spectra; black and gray lines denote the PEG and the PEG-MH5C-Cys conjugated, respectively. These techniques were employed with the objective of characterizing and analyzing the Michael addition reaction through molecular weight. The assay includes 2 mg/mL of the MH5-C, 2 mg/mL of the conjugates and 30 mg/mL of PEG. These samples were mixed with sinapinic acid (1:6) in 70% acetonitrile and 0.07% of trifluoroacetic acid.

Additionally, the MALDI-ToF MS was used to determine the specific molecular weights of these conjugates. Since the peptide utilized in these experiments (i.e., MH5C-Cys) has a molecular weight of 2126 Da and the PEG used has a molecular weight of 2 and 5 kDa, an increase in the molecular weight is expected for the peptides-conjugates (e.g., 2126 (MH5C-Cys) + 2000 Da= 4126 Da). Indeed, results in Figure 2C show a displacement to the right for the peptides-conjugates, which correspond to an increase in the molecular weights.54

After the synthesis of the polymer–peptide conjugates, MIC values were determined at 40 μM for both conjugates (Figures S13 and S14). Futhermore, to investigate whether the conjugates of MH5C-Cys and PEG polymer were able to retain the preventive antimicrobial efficacy, bacterial growth curves and SEM images with both bacteria strains were performed. As can be observed in Figure 3, the synthesized conjugates presented antimicrobial activity after the conjugation with the polymer (i.e., PEG 2–5 kDa). In Figure 3A,B, the growth curves for bacteria P. aeruginosa and E. coli are presented, respectively. Undeniably, both of the synthesized conjugates presented a bactericidal effect and did not allow bacterial growth. Additionally, the PEG polymer does not present any effect in preventing the growth of the bacteria, which suggests that the conjugated peptide remains active after conjugation. Figure S15 shows the statistically significant differences between the control and MH5C-Cys conjugates (P < 0.0001; n = 3 in each time point). Consistently, SEM images were recorded, and a prevention of the bacteria biofilm is observed. This suggests that the conjugates do not permit the quorum sensing of bacteria in the biofilm process; see Figure 3D,E (P. aeruginosa) and Figure 3G,H (E. coli).55 This is an important finding as it proves that the peptide’s function is retained after the covalent attachment into a conjugate.

Figure 3.

Figure 3.

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Bacterial growth curve test and SEM using MH5C-Cys conjugated. (A,B) Growth curves of bacteria. (C,F) Control in biofilms formation. (D,E) Prevention of biofilms in P. aeruginosa, MH5C-Cys-PEG 2 kDa and MH5C-Cys-PEG 5 kDa, respectively, and (G,H) in E. coli, MH5C-Cys-PEG 2 kDa and MH5C-Cys-PEG 5 kDa, respectively. In both assays (growth curve test and SEM), the concentration of the conjugates is 90 μM. The initial concentration in both bacteria is 1.0 × 108 CFU/mL.

To extend the understanding of the peptide-polymer conjugates efficacy to inhibit or eradicate the biofilm, the MBIC and MBEC assays were performed (Figures 4–6). The purpose of the MBIC analysis is to observe the ability of these antimicrobial agents to inhibit biofilms. As presented in Figure 4, we only found an effect on the viability of bacteria when the MH5C-Cys-PEG 5 kDa conjugate was utilized. In both bacteria under treatment with this conjugate, a reduction in their growth is observed when compared to the control. As shown in the image, it has a confluent growth and is identified as Too Many To Counts (TMTC).56 Treatments with MH5C-Cys and MH5C-Cys-PEG 2 kDa also showed as having a confluent growth and no inhibitory effect as they could continue with the formation of biofilms (Figure S16). On the basis of these results, we can identify that the MH5C-Cys-PEG 5 kDa conjugate can inhibit biofilms at 300 μM in both bacteria. It can be observed at a conjugate concentration of 180 μM that more isolated colonies of P. aeruginosa are present when compared to the MH5C-Cys. Nevertheless, at 300 μM the conjugate does not allow the growth of these bacteria. In turn, for E. coli the MBIC value resulted in 300 μM, although more isolated colonies were observed compared to the MH5C-Cys. Distinct from other assays like MICs and growth curves, this conjugate shows antimicrobial activity at 90 μM. Thus, it is likely that a higher concentration is needed to inhibit the biofilm community, in contrast to when the bacteria are in the planktonic phase or free state. Hence, when dealing with biofilm inhibition, it is understandable that a higher concentration may be required to obtain the desired effect.

Figure 4.

Figure 4.

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MBIC results using a CFU via spread plate method. The initial concentration in both bacteria is 1.0 × 108 CFU/mL. The samples treated with MH5C-Cys-PEG 5 kDa showed an effect on the viability of the bacteria. The isolated colonies were observed in E. coli at 300 μM. Meanwhile, in P. aeruginosa the isolated colonies were observed at 180 μM and at 300 μM in the absence of bacterial growth.

Figure 6.

Figure 6.

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Bioactivity MBEC results after 24 h in E. coli. The initial concentration of bacteria is 1.0 × 108 CFU/mL. (A) MH5C-Cys peptide, (B) MH5C-Cys-PEG 2 kDa, and (C) MH5C-Cys-PEG 5 kDa. The samples were loaded at different concentrations (90, 300, and 500 μM).

Previously, we had clarified that biofilms are complex communities with a high degree of resistance to commonly used drugs and antibiotics.57 In this study, we were able to identify a type of bactericidal antimicrobial agent that may be used in the future aid in biomedical and environmental applications. Now, in order to assess the biofilm eradication activity, the MBEC assay was performed and the results showed that the MH5C-Cys and conjugates are able to eradicate the biofilm at a concentration of 500 μM. For each bacteria, this effect is observed during the first growth phase. Indeed, the bacteria is not able to start the logarithmic growth phase until after 10 h when compared to the MH5C-Cys. In Figures 5A,B and 6A,B, we can observe that they initially have an effect for approximately 8–10 h, where they did not allow bacterial growth. After this time, slow bacterial growth is observed compared to control samples (control, nutrient broth and bacteria; PEG 2 kDa, PEG 5 kDa, nutrient broth, bacteria and PEG polymer), see also Figure S17. Interestingly, this effect is more noticeable for the MH5C-Cys-PEG 5 kDa conjugate. These findings lead to suggest that the chemical and physical interactions of the conjugate with the bacteria are enhanced when a larger polymeric hydrophilic tail is utilized. Thus, it is likely that PEG 5 kDa protects the conjugate from proteases in the biofilm formation.

Figure 5.

Figure 5.

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Bioactivity MBEC results after 24 h in P. aeruginosa. The initial concentration of bacteria is 1.0 × 108 CFU/mL. (A) MH5C-Cys peptide, (B) MH5C-Cys-PEG 2 kDa, and (C) MH5C-Cys-PEG 5 kDa. The samples were loaded at different concentrations (90, 300, and 500 μM).

In summary (Table 1), the MICs and MBICs demonstrate that both Gram-negative bacteria remain in the planktonic phase after direct contact with the proposed conjugated compounds. In general, the peptide MH5C-Cys with PEG polymer 5 kDa exhibited prevention and inhibition of biofilm formation for P. aeruginosa and E. coli. In addition, this conjugate presented biofilm eradication activity. According to the MICs (90 μM, MH5C-Cys and 40 μM, MH5C-Cys-PEG conjugated) and MBICs (300 μM), we can conclude that this conjugate has an action that prevents the formation of biofilms and eradicates biofilms (MBEC 500 μM). In contrast, the MH5C-Cys peptide with PEG polymer 2 kDa did not show inhibition (Figure 4) nor eradication (Figure 5 and Figure 6) of the biofilm. Thus, this study also demonstrates the dependency of the polymer’s weight to the antimicrobial activity of the conjugates when employing MH5C-Cys.

Table 1.

Summary of Antimicrobial Activity Assays Values in P. aeruginosa (ATCC 27853) and E. coli (ATCC 25922)a

antimicrobial assays MH5C-Cys MH5C-Cys-PEG 2 kDa MH5C-Cys-PEG 5 kDa
MIC 90 40 40
MBIC NAD NAD 300
MBEC NAD NAD 500
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a

All concentrations are reported as μM; values were obtained from three to six independent experiments and were determined using a spectrophotometer (96-well plates) and Colony Forming Units (CFU/mL). These values are the same in both bacteria. NAD: no activity detected.

The results presented in this study may suggest that the molecular weight of the PEG polymer has an important role in inhibiting biofilms. Here we have identified a type of synergism between the hydrophilic PEG polymer and the MH5C-Cys antimicrobial peptide with hydrophobic properties. From these results, it is deduced that MH5C-Cys in the presence of PEG may have a better effect on bacteria, specifically against the biofilm community. Numerous scientific investigations have shown that PEG helps improve the biological activity of various antimicrobial agents such as peptides.18,58,59 The exact mechanism for the observed synergism between both molecules (PEG and MH5C-Cys) is not known. However, it is suggested that PEG may be contributing its excellent properties to repel proteins such as proteases and other enzymes. As previously noted, its properties related to repelling proteins are due to the formation of an amphiphilic brushes.60,61 Bacteria can use proteases to protect themselves from antimicrobial biomolecules, which is a known form of resistance in biofilms. An important example is in biomaterials that are used for biomedical implants where bacteria depend on iron from the host’s body to regulate adhesins that are essential to adhere and form biofilms. In the case of the host’s immune system, it works with lactoferrin that binds iron and reduces the amount of iron that is supplied to protect bacteria. At the same time, bacteria use proteases against lactoferrin to maintain and be able to continue biofilm formation.62

5. CONCLUSIONS

This work demonstrates the synthesis and extensive characterization of a new class of polymer–peptide conjugate based on the antimicrobial peptide MH5C. The effects of varying the polymer weight were assessed after purification. The antimicrobial potential of the polymer-peptide conjugates was determined via bacterial growth test. Also, this manuscript presents a biophysical characterization of the MH5C peptide and a modified version with a Cysteine (MH5C-Cys). It was shown that the MH5C-Cys peptide retains its biophysical and antimicrobial characteristics. The synthesized polymer-peptide conjugates were able to eradicate biofilm formation. However, we found an interesting dependency on the polymer’s weight attached to the peptide and the eradication efficacy. Interestingly, the peptide coupled to the PEG 5 kDa showed both eradication and inhibition of the biofilm. The exact mechanism by which this dependency exists for this particular system remains unclear. In future studies, such dependency will be further explored to provide sound answers. Also, the incorporation of the conjugates in relevant surfaces to aid in biomedical and environmental applications is foreseen.

Supplementary Material

Supplementary material

ACKNOWLEDGMENTS

This work was possible thanks to the funding from NASA MIRO-Puerto Rico Space Partnership for Research, Education, and Training (PR-SPRInT) under Grant 80NSSC19M0236, NASA Experimental Program to Stimulate Competitive Research (EPSCoR) under Grant NNX14AN18A, Maximizing Access to Research Careers of undergraduate students (MARC), Program Grant 5T34GM007821-38, Research Initiative for Scientific Enhancement (RISE Program), Program Grant 5R25GM061151, and the PR NASA Space Grant NNX15AI11H. The authors acknowledge the UPR Materials Characterization Center (MCC) for providing support during the attainment of this work. Special thanks to Dr. Edwin Ortiz-Quiles and Claudia S. Pérez-Draper for their helpful discussion and comments on this work. Also, special thanks to Markus Harzdford for providing us with an actual image of a juvenile toad Bombina maxima from his professional herpetology gallery.

Footnotes

The authors declare no competing financial interest.

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsami.0c13492.

SEM results for the MH5C peptides and PEG polymers, MIC results for the MH5C peptides, MH5C-Cys conjugates and Tet-20, MBEC control results, and secondary thermal stability CD spectra (PDF)

Contributor Information

Valerie Ortiz-Gómez, Department of Biology, University of Puerto Rico, Ŕio Piedras Campus, San Juan, Puerto Rico 00925-2537; Molecular Sciences Research Center, University of Puerto Rico, San Juan, Puerto Rico 00931-3346.

Victor D. Rodríguez-Ramos, Department of Biology, University of Puerto Rico, Ŕio Piedras Campus, San Juan, Puerto Rico 00925-2537; Molecular Sciences Research Center, University of Puerto Rico, San Juan, Puerto Rico 00931-3346

Rafael Maldonado-Hernández, Department of Biology, University of Puerto Rico, Ŕio Piedras Campus, San Juan, Puerto Rico 00925-2537; Molecular Sciences Research Center, University of Puerto Rico, San Juan, Puerto Rico 00931-3346.

José A. González-Feliciano, Molecular Sciences Research Center, University of Puerto Rico, San Juan, Puerto Rico 00931-3346

Eduardo Nicolau, Department of Chemistry, University of Puerto Rico, Ŕio Piedras Campus, San Juan, Puerto Rico 00925-2537; Molecular Sciences Research Center, University of Puerto Rico, San Juan, Puerto Rico 00931-3346.

REFERENCES

  • (1).Russo TA; Johnson JR Medical and economic impact of extraintestinal infections due to Escherichia coli: focus on an increasingly important endemic problem. Microbes Infect. 2003, 5 (5), 449–456. [DOI] [PubMed] [Google Scholar]
  • (2).Loveday HP; Wilson JA; Kerr K; Pitchers R; Walker JT; Browne J Association between healthcare water systems and Pseudomonas aeruginosa infections: a rapid systematic review. Journal of Hospital Infection 2014, 86 (1), 7–15. [DOI] [PubMed] [Google Scholar]
  • (3).Driscoll JA; Brody SL; Kollef MH The epidemiology, pathogenesis and treatment of Pseudomonas aeruginosa infections. Drugs 2007, 67 (3), 351–368. [DOI] [PubMed] [Google Scholar]
  • (4).Luca V; Stringaro A; Colone M; Pini A; Mangoni LM Esculentin (1–21),an amphibian skin membrane-active peptide with potent activity on both planktonic and biofilm cells of the bacterial pathogen Pseudomonas aeruginosa. Cell. Mol. Life Sci. 2013, 70 (15), 2773–2786. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (5).Roux A; Beloin C; Ghigo JM Combined inactivation and expression strategy to study gene function under physiological conditions: application to identification of new Escherichia coli adhesins. J. Bacteriol. 2005, 187 (3), 1001–1013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (6).Wang Z; Yang H-C; He F; Peng S; Li Y; Shao L; Darling SB Mussel-inspired surface engineering for water-remediation materials. Matter 2019, 1 (1), 115–155. [Google Scholar]
  • (7).Qian Y; Qi F; Chen Q; Zhang Q; Qiao Z; Zhang S; Wei T; Yu Q; Yu S; Mao Z; Gao C; Ding Y; Cheng Y; Jin C; Xie H; Liu R Surface modified with a host defense peptide-mimicking β-peptide polymer kills bacteria on contact with high efficacy. ACS Appl. Mater. Interfaces 2018, 10 (18), 15395–15400. [DOI] [PubMed] [Google Scholar]
  • (8).Yang H-C; Xie Y; Chan H; Narayanan B; Chen L; Waldman RZ; Sankaranarayanan SK; Elam JW; Darling SB Crude-oil-repellent membranes by atomic layer deposition: oxide interface engineering. ACS Nano 2018, 12 (8), 8678–8685. [DOI] [PubMed] [Google Scholar]
  • (9).Xu L; Shao C; Li G; Shan A; Chou S; Wang J; Ma Q; Dong N Conversion of broad-spectrum antimicrobial peptides into species-specific antimicrobials capable of precisely targeting pathogenic bacteria. Sci. Rep. 2020, 10 (1), 1–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (10).Chung C-R; Kuo T-R; Wu L-C; Lee T-Y; Horng J-T Characterization and identification of antimicrobial peptides with different functional activities. Briefings Bioinf. 2020, 21 (3), 1098–1114. [DOI] [PubMed] [Google Scholar]
  • (11).Rončević T; Vukičević D; Krce L; Benincasa M; Aviani I; Maravić A; Tossi A Selection and redesign for high selectivity of membrane-active antimicrobial peptides from a dedicated sequence/function database. Biochim. Biophys. Acta, Biomembr. 2019, 1861 (4), 827–834. [DOI] [PubMed] [Google Scholar]
  • (12).Teixeira MC; Carbone C; Sousa MC; Espina M; Garcia ML; Sanchez-Lopez E; Souto EB Nanomedicines for the Delivery of Antimicrobial Peptides (AMPs). Nanomaterials 2020, 10 (3), 560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (13).Lai R; Zheng YT; Shen JH; Liu GJ; Liu H; Lee WH; Tang SZ; Zhang Y Antimicrobial peptides from skin secretions of Chinese red belly toad Bombina maxima. Peptides 2002, 23 (3), 427–435. [DOI] [PubMed] [Google Scholar]
  • (14).Lai R; Liu H; Hui Lee W; Zhang Y An anionic antimicrobial peptide from toad Bombina maxima. Biochem. Biophys. Res. Commun. 2002, 295, 796–799. [DOI] [PubMed] [Google Scholar]
  • (15).Dennison SR; Mura M; Harris F; Morton LH; Zvelindovsky A; Phoenix DA The role of C-terminal amidation in the membrane interactions of the anionic antimicrobial peptide, maximin H5. Biochim. Biophys. Acta, Biomembr. 2015, 1848 (5), 1111–1118. [DOI] [PubMed] [Google Scholar]
  • (16).Hamley IW PEG–peptide conjugates. Biomacromolecules 2014, 15 (5), 1543–1559. [DOI] [PubMed] [Google Scholar]
  • (17).Du J; Bandara H; Du P; Huang H; Hoang K; Nguyen D; Mogarala SV; Smyth HD Improved biofilm antimicrobial activity of polyethylene glycol conjugated tobramycin compared to tobramycin in pseudomonas aeruginosa biofilms. Mol. Pharmaceutics 2015, 12 (5), 1544–1553. [DOI] [PubMed] [Google Scholar]
  • (18).Kumar P; Takayesu A; Abbasi U; Kalathottukaren MT; Abbina S; Kizhakkedathu JN; Straus SK Antimicrobial peptide–polymer conjugates with high activity: Influence of polymer molecular weight and peptide sequence on antimicrobial activity, proteolysis, and biocompatibility. ACS Appl. Mater. Interfaces 2017, 9 (43), 37575–37586. [DOI] [PubMed] [Google Scholar]
  • (19).Mena KD; Gerba CP Risk assessment of Pseudomonas aeruginosa in water. Rev. Environ. Contam. Toxicol. 2009, 201, 71–115. [DOI] [PubMed] [Google Scholar]
  • (20).Edberg S; Rice E; Karlin R; Allen M Escherichia coli: the best biological drinking water indicator for public health protection. J. Appl. Microbiol. 2000, 88, 106S–116S. [DOI] [PubMed] [Google Scholar]
  • (21).Lorber B; Fischer F; Bailly M; Roy H; Kern D Protein analysis by dynamic light scattering: Methods and techniques for students. Biochem. Mol. Biol. Educ. 2012, 40, 372–382. [DOI] [PubMed] [Google Scholar]
  • (22).Karow AR; Götzl J; Garidel P Resolving power of dynamic light scattering for protein and polystyrene nanoparticles. Pharm. Dev. Technol. 2015, 20 (1), 84–89. [DOI] [PubMed] [Google Scholar]
  • (23).Dennison SR; Mura M; Harris F; Morton LH; Zvelindovsky A; Phoenix DA The role of C-terminal amidation in the membrane interactions of the anionic antimicrobial peptide, maximin H5. Biochim. Biophys. Acta, Biomembr. 2015, 1848 (5), 1111–1118. [DOI] [PubMed] [Google Scholar]
  • (24).Greenfield NJ Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions. Nat. Protoc. 2006, 1 (6), 2527–2535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (25).Jorgensen JH; Turnidge JD Susceptibility test methods: dilution and disk diffusion methods. Manual of Clinical Microbiology, Eleventh Edition 2015, 1253–1273. [Google Scholar]
  • (26).Bello SA; de Jesús Maldonado I; Rosim Fachini E; Sundaram PA; Diffoot Carlo N In vitro evaluation of human osteoblast adhesion to a thermally oxidized γ-TiAl intermetallic alloy of composition Ti–48Al–2Cr–2Nb (at.%). J. Mater. Sci.: Mater. Med. 2010, 21 (5), 1739–1750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (27).Santillán J; Dwomoh EA; Rodríguez Avilés YG; Bello SA; Nicolau E Fabrication and Evaluation of Polycaprolactone Beads-on-String Membranes for Applications in Bone Tissue Regeneration. ACS Applied Bio Materials 2019, 2 (3), 1031–1040. [DOI] [PubMed] [Google Scholar]
  • (28).Carson L; Chau PKW; Earle MJ; Gilea MA; Gilmore BF; Gorman SP; McCann MT; Seddon KR Antibiofilm activities of 1-alkyl-3-methylimidazolium chloride ionic liquids. Green Chem. 2009, 11 (4), 492–497. [Google Scholar]
  • (29).Lemire J; Demeter M; Turner RJ Protocols for Harvesting a Microbial Community Directly as a Biofilm for the Remediation of Oil Sands Process Water. Hydrocarbon and Lipid Microbiology Protocols 2015, 131–152. [Google Scholar]
  • (30).Burns JA; Butler JC; Moran J; Whitesides GM Selective reduction of disulfides by tris (2-carboxyethyl) phosphine. J. Org. Chem. 1991, 56 (8), 2648–2650. [Google Scholar]
  • (31).Van Leeuwen LA; Hinchy EC; Murphy MP; Robb EL; Cochemé HM Click-PEGylation–a mobility shift approach to assess the redox state of cysteines in candidate proteins. Free Radical Biol. Med. 2017, 108, 374–382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (32).Bhattacharjee S DLS and zeta potential–what they are and what they are not? J. Controlled Release 2016, 235, 337–351. [DOI] [PubMed] [Google Scholar]
  • (33).Gerzhova A; Mondor M; Benali M; Aider M Study of total dry matter and protein extraction from canola meal as affected by the pH, salt addition and use of zeta-potential/turbidimetry analysis to optimize the extraction conditions. Food Chem. 2016, 201, 243–252. [DOI] [PubMed] [Google Scholar]
  • (34).Jarujamrus P; Amatatongchai M; Thima A; Khongrangdee T; Mongkontong C Selective colorimetric sensors based on the monitoring of an unmodified silver nanoparticles (AgNPs) reduction for a simple and rapid determination of mercury. Spectrochim. Acta, Part A 2015, 142, 86–93. [DOI] [PubMed] [Google Scholar]
  • (35).Mai X. t.; Huang J; Tan J; Huang Y; Chen Y Effects and mechanisms of the secondary structure on the antimicrobial activity and specificity of antimicrobial peptides. J. Pept. Sci. 2015, 21 (7), 561–568. [DOI] [PubMed] [Google Scholar]
  • (36).Gopal R; Park JS; Seo CH; Park Y Applications of circular dichroism for structural analysis of gelatin and antimicrobial peptides. Int. J. Mol. Sci. 2012, 13 (3), 3229–3244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (37).Cavaleri J; Rankin D; Harbeck J; Sautter LR; McCarter SY; Sharp SE; Spiegel AC Manual of antimicrobial susceptibility testing. American Society for Microbiology 2005, 12, 53–42. [Google Scholar]
  • (38).Bernatova S; Samek O; Pilat Z; Sery M; Jezek J; Jakl P; Siler M; Krzyzanek V; Zemanek P; Hola V; Dvorackova M; Ruzicka F Following the mechanisms of bacteriostatic versus bactericidal action using Raman spectroscopy. Molecules 2013, 18 (11), 13188–13199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (39).Mangoni ML; Grovale N; Giorgi A; Mignogna G; Simmaco M; Barra D Structure-function relationships in bombinins H, antimicrobial peptides from Bombina skin secretions☆. Peptides 2000, 21 (11), 1673–1679. [DOI] [PubMed] [Google Scholar]
  • (40).Dennison SR; Morton LH; Harris F; Phoenix DA Low pH enhances the action of maximin H5 against Staphylococcus aureus and helps mediate lysylated phosphatidylglycerol-induced resistance. Biochemistry 2016, 55 (27), 3735–3751. [DOI] [PubMed] [Google Scholar]
  • (41).Lee TH; N Hall K; Aguilar MI Antimicrobial peptide structure and mechanism of action: a focus on the role of membrane structure. Curr. Top. Med. Chem. 2015, 16 (1), 25–39. [DOI] [PubMed] [Google Scholar]
  • (42).Izadpanah A; Gallo RL Antimicrobial peptides. J. Am. Acad. Dermatol. 2005, 52 (3), 381–390. [DOI] [PubMed] [Google Scholar]
  • (43).Gopal R; Park JS; Seo CH; Park Y Applications of circular dichroism for structural analysis of gelatin and antimicrobial peptides. Int. J. Mol. Sci. 2012, 13 (3), 3229–3244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (44).Louis-Jeune C; Andrade-Navarro MA; Perez-Iratxeta C Prediction of protein secondary structure from circular dichroism using theoretically derived spectra. Proteins: Struct., Funct., Genet. 2012, 80 (2), 374–381. [DOI] [PubMed] [Google Scholar]
  • (45).Louis JC; Andrade NM; Perez IC Prediction of protein secondary structure from circular dichroism using theoretically derived spectra. Proteins: Struct., Funct., Genet. 2012, 80 (2), 374–381. [DOI] [PubMed] [Google Scholar]
  • (46).Yeaman MR; Yount NY Mechanisms of antimicrobial peptide action and resistance. Pharmacol. Rev. 2003, 55 (1), 27–55. [DOI] [PubMed] [Google Scholar]
  • (47).Rao AG Conformation and antimicrobial activity of linear derivatives of tachyplesin lacking disulfide bonds. Arch. Biochem. Biophys. 1999, 361 (1), 127–134. [DOI] [PubMed] [Google Scholar]
  • (48).Zasloff M; Martin B; Chen HC Antimicrobial activity of synthetic magainin peptides and several analogues. Proc. Natl. Acad. Sci. U. S. A. 1988, 85 (3), 910–913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (49).Brogden KA Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol. 2005, 3 (3), 238–250. [DOI] [PubMed] [Google Scholar]
  • (50).Gao G; Lange D; Hilpert K; Kindrachuk J; Zou Y; Cheng JTJ; Kazemzadeh-Narbat M; Yu K; Wang R; Straus SK; Brooks DE; Chew BH; Hancock REW; Kizhakkedathu JN The biocompatibility and biofilm resistance of implant coatings based on hydrophilic polymer brushes conjugated with antimicrobial peptides. Biomaterials 2011, 32 (16), 3899–3909. [DOI] [PubMed] [Google Scholar]
  • (51).Nair DP; Podgorski M; Chatani S; Gong T; Xi W; Fenoli CR; Bowman CN The thiol-Michael addition click reaction: a powerful and widely used tool in materials chemistry. Chem. Mater. 2014, 26 (1), 724–744. [Google Scholar]
  • (52).Lowe AB Thiol-ene “click” reactions and recent applications in polymer and materials synthesis. Polym. Chem. 2010, 1 (1), 17–36. [Google Scholar]
  • (53).Braun AC; Gutmann M; Mueller TD; Lühmann T; Meinel L Bioresponsive release of insulin-like growth factor-I from its PEGylated conjugate. J. Controlled Release 2018, 279, 17–28. [DOI] [PubMed] [Google Scholar]
  • (54).Evgrafova Z; Voigt B; Baumann M; Stephani M; Binder WH; Balbach J Probing Polymer Chain Conformation and Fibril Formation of Peptide Conjugates. ChemPhysChem 2019, 20 (2), 236–240. [DOI] [PubMed] [Google Scholar]
  • (55).Packiavathy IAS; Maruthamuthu S; Gnanaselvan G; Manoharan S; Paul JBJ; Annapoorani A; Kannappan A; Ravi AV The control of microbially induced corrosion by methyl eugenol–A dietary phytochemical with quorum sensing inhibitory potential. Bioelectrochemistry 2019, 128, 186–192. [DOI] [PubMed] [Google Scholar]
  • (56).Asri LA; Crismaru M; Roest S; Chen Y; Ivashenko O; Rudolf P; Tiller JC; van der Mei HC; Loontjens TJ; Busscher HJ A shape-adaptive, antibacterial-coating of immobilized quaternary-ammonium compounds tethered on hyperbranched polyurea and its mechanism of action. Adv. Funct. Mater. 2014, 24 (3), 346–355. [Google Scholar]
  • (57).Cepas V; López Y; Muñoz E; Rolo D; Ardanuy C; Martí S; Xercavins M; Horcajada JP; Bosch J; Soto SM Relationship between biofilm formation and antimicrobial resistance in gram-negative bacteria. Microb. Drug Resist. 2019, 25 (1), 72–79. [DOI] [PubMed] [Google Scholar]
  • (58).Wu X; Wei PH; Zhu X; Wirth MJ; Bhunia A; Narsimhan G Effect of immobilization on the antimicrobial activity of a cysteine-terminated antimicrobial Peptide Cecropin P1 tethered to silica nanoparticle against E. coli O157: H7 EDL933. Colloids Surf., B 2017, 156, 305–312. [DOI] [PubMed] [Google Scholar]
  • (59).Chen Q; Yu S; Zhang D; Zhang W; Zhang H; Zou J; Mao Z; Yuan Y; Gao C; Liu R Impact of antifouling PEG layer on the performance of functional peptides in regulating cell behaviors. J. Am. Chem. Soc. 2019, 141 (42), 16772–16780. [DOI] [PubMed] [Google Scholar]
  • (60).Zalipsky S Functionalized poly (ethylene glycols) for preparation of biologically relevant conjugates. Bioconjugate Chem. 1995, 6 (2), 150–165. [DOI] [PubMed] [Google Scholar]
  • (61).Veronese FM; Pasut G PEGylation, successful approach to drug delivery. Drug Discovery Today 2005, 10 (21), 1451–1458. [DOI] [PubMed] [Google Scholar]
  • (62).Schmidtchen A; Frick IM; Andersson E; Tapper H; Björck L Proteinases of common pathogenic bacteria degrade and inactivate the antibacterial peptide LL-37. Mol. Microbiol. 2002, 46 (1), 157–168. [DOI] [PubMed] [Google Scholar]

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Supplementary Materials

Supplementary material
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