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Binary Colloidal Crystal Layers as Platforms for Surface Patterning of Puroindoline-Based Antimicrobial Peptides Andrew Boden, Mrinal Bhave, Peng-Yuan Wang, Snehal R. Jadhav, and Peter Kingshott ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10392 • Publication Date (Web): 28 Dec 2017 Downloaded from http://pubs.acs.org on December 28, 2017

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ACS Applied Materials & Interfaces

Binary Colloidal Crystal Layers as Platforms for Surface Patterning of Puroindoline-Based Antimicrobial Peptides Andrew Boden, Mrinal Bhave, Peng-Yuan Wang, Snehal Jadhav and Peter Kingshott† Department of Chemistry and Biotechnology, School of Science, Faculty of Science, Engineering and Technology, Swinburne University of Technology, Hawthorn, 3122 VIC, Australia. Key words: Binary colloidal crystals; antimicrobial peptides; puroindoline; surface modification; surface patterning; nanoparticles

ABSTRACT: The ability of bacteria to form biofilms and the emergence of antibiotic-resistant strains has prompted the need to develop the next-generation of antibacterial coatings. Antimicrobial peptides (AMPs) are showing promise as molecules that can address these issues, especially if used when immobilized as a surface coating. We present a method that explores how surface patterns together with the selective immobilization of an AMP called PuroA (FPVTWRWWKWWKG-NH2) can be used to both kill bacteria, but also as a tool to study bacterial attachment mechanisms. Surface patterning is achieved using stabilized self-assembled binary colloidal crystal (BCC) layers, allowing selective PuroA immobilization to carboxylated particles using N-(3dimethylaminopropyl)-N’-ethyl carbodiimide (EDC) hydrochloride/N-hydroxysuccinimide (NHS) coupling chemistry. Covalent immobilization of the PuroA was compared with physical adsorption (i.e. without the addition of EDC/NHS). The AMPfunctionalized colloids and BCC layers were characterized by X-ray photoelectron spectroscopy (XPS), zeta potentials, and matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS). Surface antimicrobial activity was assessed by viability assays using Escherichia coli. MALDI-ToF-MS analysis revealed that while not all of the PuroA were successfully covalently immobilized, a relatively low density of PuroA (1.93x1013 molecules/cm2 and 7.14x1012 molecules/cm2 for covalent and physical immobilization, respectively) was found to be sufficient at significantly decreasing the viability of E. coli by 70% when compared to control samples. The findings provide a proof of concept that BCC layers are a suitable platform for the patterned immobilization of AMPs, and the importance of ascertaining the success of small molecule grafting reactions using surfaceMALDI, something that is often assumed to be successful in the field.

INTRODUCTION The ability to prevent the attachment and growth of bacteria by modification of both surface chemistry and topography has the potential to be important role for a range of applications including implantable medical devices,1-4 water purification systems5,6, and food processing equipment.7 Within the biomedical industry, the risk of chronic infection associated with microbial attachment and biofilm formation remains a significant concern with the use of indwelling medical devices such as catheters, artificial bone replacements and stents. Such complications can subsequently lead to patient suffering, prolonged hospitalization, and in many cases patient death.8-10 While the use of proper aseptic techniques and antibiotics has reduced the incidence of such infections, the emergence of antibiotic-resistant bacteria and/or the formation of tenacious biofilms has prompted the need to develop ‘new-generation’ antimicrobial coatings. Recent studies have indicated that two main approaches are used to control bacterial attachment and growth on a surface. These involve; i) prevention of bacterial attachment (i.e. fouling) through unfavorable surface topographies and chemistries, and ii) incorporation of biocidal or biostatic agents to

prevent or inhibit the growth of any attached bacteria.11,12 Several types of antifouling surfaces have been proposed, including the grafting of polymer brushes13-15 and the fabrication of nano/micro-structured topographies inspired from naturally occurring surfaces.16-18 The use of antifouling polymer brushes provides further functionalization possibilities, as a range of antibiotic molecules can be potentially conjugated to terminal chemical groups through various coupling strategies.19 However, long polymerization and processing times6,20, and the presence of brush defects currently limits the use of such coatings in practical settings.

Bacterial attachment to nano/micro-structured surfaces prepared by a range of lithographic techniques have also been extensively studied, and it has been shown that surface topography plays an important role in the attachment processes of bacterial cells.21,22 Previous research has shown that certain nanometer sized topographies are capable of reducing the adhesion of Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa to titanium surfaces compared to controls23, and also that surface topography is the only feature responsible for the bactericidal property of cicada (Psal-

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toda calripennis) wings.18 Recently, a novel approach has been developed for fabricating large area ordered micro- and nanotopographies using binary mixtures of colloidal particles that self-assemble into layers, and are referred to as binary colloidal crystal layers (BCCs).24,25 BCCs enable the fabrication of surfaces with specific topographies and chemistries, which is highly desirable in many applications including photonics26,27, biomedicine28 and have even been applied in the fabrication of surface-enhanced Raman scattering (SERS) substrates.29,30 Recent studies have also shown that highly-ordered mixed protein patterns can be produced using BCCs, which suggests that a range of biomolecules may be immobilized to these layers in a patterned and selective manner.31 Traditionally, incorporation of bactericidal and bacteriostatic agents into biomedical coatings has been limited to antibiotics,32,33 quaternary ammonium compounds34 and metal ions including silver,35 copper and zinc.36 However, these approaches pose some problems such as cytotoxic side-effects and as mentioned earlier, the development of antibioticresistant bacteria. Antimicrobial peptides (AMPs) may be able to overcome such obstacles, as many cationic AMPs possess a range of unique properties that include broad-spectrum activity, high efficacy at low concentrations, and an inherently low propensity to develop bacterial resistance.19,37 AMPs are part of the innate immune system of many organisms and target the negatively charged membranes of bacteria through electrostatic interactions from positively charged amino acids.38 Due to their susceptibility to proteolytic degradation and peptide selfaggregation,39 in a biomedical setting, covalent immobilization of AMPs and their synthetic derivatives is preferred to overcome such problems while also maintaining their antimicrobial properties. In this study, we utilize BCC layers as a suitable platform with inherent nano/microstructures and topographies for the covalent immobilization of AMPs, and present a surface coating method based on AMP-functionalized micro/nano-rough surfaces, along with an examination of their antibacterial activities. For these investigations, a 13-mer highly cationic peptide PuroA (FPVTWRWWKWWKG-NH2); derived from the tryptophan-rich-domain (TRD) of wheat puroindoline proteins was chosen due to its broad-spectrum of antimicrobial activity40, and also its stability at pH 2-12 and temperatures up to 130°C.41 MATERIALS AND METHODS Materials 2 µm carboxylated polystyrene (PSC2) (4% w/v) particles (Invitrogen, Aus.) and 0.110 µm poly(methyl methacrylate) (PMMA011) (4% w/v) particles (Bangs Laboratories, US) were used. The synthetic peptide PuroA (FPVTWRWWKWWKG-NH2) (99% purity; purified in trifluoroacetic acid with HPLC) was obtained from Mimotopes (Clayton, Aus.). Polystyrene (MW 250,000 Da) and the LIVE/DEAD BacLightTM Bacterial Viability Kit (which contains the dyes SYTO9; propidium iodide (PI)) were purchased from ThermoFisher (Scorsby, Aus.). Water used in all experiments was purified using a Millipore system and had a resistivity of 18 MΩ/cm. Toluene, phosphate-buffered saline (PBS) (0.01 M PBS, 0.14 M NaCl; 0.0027 KCl; pH 7.4), 2,5dihydroxybenzoic acid (DHB), 0.1% trifluoroacetic acid

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(TFA), acetonitrile (ACN) (anhydrous, 99.8%), N-(3dimethyl-aminopropyl)-N’-ethyl carbodiimide (EDC) hydrochloride, and N-hydroxysuccinimide (NHS) were purchased from Sigma-Aldrich (Castle Hill, Aus.) and used as received. Bacterial strains, media and culture conditions Escherichia coli (ATCC 25922) was used for all bacterial investigations and obtained from the Swinburne University of Technology microbiology collection. Bacterial stocks were cultivated on nutrient agar (15 g/L agar, 8 g/L nutrient broth) at 37 °C. For analysis, single colonies were inoculated into Mueller Hinton Broth (MHB) and grown at 37 °C until logarithmic growth phase. All media were purchased from ThermoFisher (Scorsby, Aus). Coupling of PuroA to PSC2 particles EDC/NHS zero-length coupling chemistry was used for the covalent coupling of carboxyl groups to free primary amines. The activation of COOH groups with EDC is most efficient at pH 4.5-7.2; however, due to instability of the o-acylisourea active ester intermediate, NHS was also used, as this will form an NHS active ester with o-acylisourea. This active ester is much more stable (half-life 4-5 hr) than EDC-activated COOH groups and is, therefore, expected to increase the coupling efficiency of PuroA with the COOH groups of PSC2 particles. To perform the immobilization, 1 mL of diluted PSC2 particles (0.4 % (w/v)) were centrifuged at 1.5 x 103 rpm for 15 min and the supernatant discarded and replaced with 1 mL of MilliQ (pH 5.4) containing EDC/NHS (125 mM each). Samples were left to react whilst shaking for 15 min at 4 °C to activate COOH groups. The particle suspensions were again centrifuged as above and the supernatant again discarded. For PuroA coupling, the activated PSC2 particles were resuspended in 1 mL diluted PBS (1 mM, pH 7.4), containing 250 µg/mL PuroA and left to react for 20 h at 4 °C whilst shaking. The particles were then washed three times by centrifugation as above with replacing the supernatant with 1 mL of fresh MilliQ water after each wash. Samples were finally suspended in 1 mL of MilliQ water prior to subsequent analysis. Preparation of PuroA-modified BCC monolayers Glass slides were used as the BCC substrate and cut to approximately 1 cm2. Prior to use, the slides were cleaned by sonicating in solutions of Decon (2% v/v) (30 min), MilliQ (10 min) and ethanol (70% v/v) (10 min), then given a final rinse in ethanol (70% v/v), and dried under a flow of compressed N2 gas. Cleaned slides were then coated with polystyrene (PS) by applying 30 µL of PS solution (5% (w/v) in toluene) to the center of the substrate followed by spin-coating at 800 rpm for 100 seconds. All slides were subsequently treated in a UVozone cleaner for 15 min to increase surface hydrophilicity to a level where BCC growth is possible.28 BCC layers were prepared from PuroA modified and unmodified particle solutions (0.4% w/v) by the evaporation induced confined area assembly (EICAA) method.24 Approximate volumes of the large particles (VP in µL) confined in a rubber Oring of diameter (DR in cm) needed to create a single monolayer can be calculated as follows (Equation 1):


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Equation 1. Determination of Particle Volumes for BCC Monolayer Formation

coated with Au (~10 nm) by evaporation using a K975X Turbo Evaporator. Zeta potential analysis

Equation 1. ܸ௉ ൌ

మ ଵ଴ൈగൈఘൈ஽ು ൈ஽ೃ

ଵଶൈ௪

Where ρ is the density (g/cm3), DP is the diameter (µm) of the particle and w is the % solid content of the particle suspension. Large and small particle combinations chosen for this investigation were 2 µm PSC (PSC2) and 0.110 µm PMMA (PMMA011) particles, respectively. These combinations were chosen as PuroA could be selectively tethered to PSC particles, and the BCC topographical features are similar to or smaller than the dimensions of a bacterial cell (≈ 3 µm x 1 µm for E.coli).42 Typically, to produce a PSC2-PMMA011 BCC monolayer covering the area within an O-ring of 1 cm diameter (0.785 cm2), 20.9 µL of PSC (0.4% w/v) and 29.6 µL of PMMA (0.4% w/v) particles were mixed and diluted in MilliQ to a final volume of 100 µL. After being thoroughly vortexed to ensure particles were well suspended, the resulting binary colloidal suspension was then pipetted inside the rubber Oring in an EICAA apparatus and left at room temperature until the solvent had completely evaporated (~12 h). The surfaces were then removed from the EICAA apparatus and the resultant BCC layers were heat-treated for stabilization by placing the prepared surfaces on a hot-plate at 130 °C for 60 s. Scheme 1 summarizes the steps taken to immobilize PuroA to PSC2-PMMA011 BCC monolayers using EDC/NHS as the coupling agent and EICAA as the method for BCC formation.

PSC2

PMMA011

To assess the surface charge of PuroA-modified PSC2 particles, zeta potentials were measured using a Zetasizer Nano ZSP instrument (Malvern Instruments, UK). In brief, 30 µL of the final PuroA-PSC2 coupling suspensions were diluted to 5% v/v using 570 µL of MilliQ water (pH 5.4), and the resulting solution (600 µL) was transferred to a disposable capillary cell. The zeta potential of each colloidal solution was an average of five data points (n =5). X-ray photoelectron spectroscopy (XPS) analysis XPS was used to investigate the elemental composition and chemical states of atoms within the outer 10 nm of the PuroAmodified PSC2 particle surfaces. Initially, PuroA particles were immobilized to PSC2 particles as outlined above, and samples were prepared for XPS analysis by placing concentrated drops of the colloidal particles onto clean glass slides and allowing solvent evaporation (12-16 h). XPS data were obtained using a Kratos Axis Nova spectrometer (Kratos Analytical, UK), equipped with a monochromated aluminum Xray source (Alkα, hν = 1486.6eV) operating at 15 mA and 15 kV (225 W power). Corresponding XPS data was analyzed with CasaXPS software (Casa Software Ltd., UK). Survey spectra were collected from 0 to 1100 eV using a detector pass energy of 160 eV, and high resolution core-level spectra of C 1s, O 1s, N 1s photoelectrons were acquired at 20 eV pass energy. All spectra were calibrated with respect to the C-C/CH peak at 285.0 eV. For curve-fitting of the high resolution spectra, a full width half maximum (FWHM) constraint of 0.21.2 was applied for all components and residual STD of ≤ 1.0 was used to confirm good fits. Samples were prepared in duplicate (n=2) and three spots were analyzed per sample. Control samples were prepared with free PuroA (1 mg/mL in MilliQ pH 5.4), unmodified PSC2 particles, and PuroA physically adsorbed to PSC2 particles (i.e. no EDC/NHS).

EDC/NHS 125 mM

PuroA Evaporation induced confined area assembly (EICAA)

Scheme 1. Overview of PuroA coupling and subsequent BCC formation. PSC2 and PuroA-modified PSC2PMMA011 PMMA011 represent 2 µm carboxylated polystyrene and 0.110 BCC layers µm poly(methyl methacrylate) particles, respectively. Scanning electron microscopy (SEM) analysis

Topographical structures of modified and unmodified PSC2PMMA011 BCC monolayers were observed using field emission SEM (FE-SEM, ZEISS SUPRA 40 VP, Carl Zeiss, Germany) at 3 keV. Prior to SEM imaging, the surfaces were

Matrix Assisted Laser Desorption Ionization – Time of Flight (MALDI-ToF) analysis MALDI-ToF MS was used to confirm the success of the covalent immobilization of PuroA to the PSC2 particles. The socalled surface-MALDI technique can be used to directly analyze proteins at surfaces.43,44 It is known that the soft ionization process of MALDI-ToF analysis does not have enough energy to break covalent bonds, thus successful reaction between the PuroA and PSC2 particles should result in no signals being detected in the resultant spectra.45 PuroA modified PSC2 particles were prepared as described earlier and MALDI-ToF analysis was performed as outlined previously.46 For analysis, each sample was spotted on the MALDI target plate in duplicate (n=2) by adding 1 µL of sample and 1 µL of matrix onto the target plate and mixing by pipetting up and down several times. The matrix used was a 10 mg/mL DHB diluted in a 1:1 water/acetonitrile solution with 0.1% TFA. 2 µL of sample-matrix mixture was set to dry on the sample plate and spectra were acquired in a MALDI-ToFMS Shimadzu Axima Performance (Shimadzu Scientific In-



struments, USA) using a 337 nm nitrogen laser in reflectron mode over a mass range between 100-4000 m/z. Control samples were prepared using PuroA (250 µg/mL in 1 mM PBS pH 7.4), unmodified PSC2 particles, and physically adsorbed PuroA AMPs (i.e. no EDC/NHS). Generated spectra were analyzed using Shimadzu Launchpad Software, and the MALDI-TOF-MS was externally calibrated using standards over a mass range of 190-3658 Da (C10H8NO3 (190.05 Da), Angiotensin 2 (1046.54 Da), Angiotensin 1 (1296.69 Da), Glu-1-fibrinogen (1570.98 Da), N-Acetyl renin (1800.94 Da), ACTH 1-17 (2093.08 Da), ACTH 18-39 (2465.20 Da), ACTH 7-38 (3657.93 Da)). Activity of PuroA in solution The minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial agent that can inhibit the visible growth of a microorganism after an overnight culture.47 The MICs of PuroA (FPVTWRWWKWWKG-NH2) in solution against Escherichia coli (ATCC 25922) removed Pseudomonas aeruginosa (ATCC 9027) suspension cultures were determined using a modified broth dilution method, as described previously40 with final peptide concentrations ranging from 250 µg/mL to 0.5 µg/mL. Control wells were prepared with no bacterial cells, and also cells treated with no PuroA. The microtitre plates were incubated at 37 °C for 24 h. The MIC values of peptides were then determined by measuring absorbance of the samples at 600 nm using a FLUOstar Omega microplate reader. All samples were tested in triplicate (n=3). Indolicidin (unrelated to puroindoline based AMPs) was used as a positive control due to its known activity against a number of microorganisms.40 Antibacterial activity of immobilized PuroA AMPs The viability of E. coli (ATCC 25922) cells attached to the PuroA-modified PSC2-PMMA011 BCC layers was assessed using the BacLightTM Bacterial Viability Kit. Samples were placed into wells of a sterile 12-well flat-bottomed plate and submerged in suspensions of E. coli (1x108 CFU/mL) in MHB. The plates were then incubated at 37 °C for 24 h. After incubation, samples were washed three times with PBS (1 mM, pH 7.4) to remove any non-adherent bacteria and treated with 200 µL of Syto9/PI solution (0.3 % v/v in 1 mM PBS pH 7.4) and left in dark for 15 min. Samples were again washed three times (1 mM PBS pH 7.4) to remove excess staining solution and subsequently imaged using a Nikon Eclipse 50i fluorescence microscope with excitation wavelengths of 485 nm for Syto9 (green) and 561 nm for PI (red). The emission wavelengths of the excited fluorophores were detected through spectral emission filters of 505 nm and 630/22 nm for green and red fluorescence, respectively. All samples were prepared in triplicate (n=3) and three images were taken per sample. Control surfaces were prepared using (i) unmodified BCC monolayers; (ii) physically adsorbed PuroA-NH2, and (iii) flat PS coated glass slides. In brief, E. coli (ATCC 25922) was seeded on to surfaces at 1x108 CFU/mL. After 24 h incubation at 37°C, cell suspensions and substrates were removed and washed three times in PBS (10mM pH 7.4), followed by being fixed in 3.0% glutar-

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aldehyde for 3 h. Surfaces were then dehydrated under an increasing ethanol gradient (60, 70, 80, 90 and 100%) being incubated for 15min at each concentration. Dehydrated surfaces were then coated with Au (~10 nm) by evaporation using a K975X Turbo Evaporator, and then imaged using field emission SEM (FE-SEM, ZEISS SUPRA 40 VP, Carl Zeiss, Germany) at 3 keV. RESULTS AND DISCUSSION Particle modification and characterization Immobilization of PSC2 particles with PuroA was assessed using zeta potential measurements, XPS, and surface-MALDIToF-MS. The zeta potential data at pH 5.4 (Fig. 1) shows a large change in surface charge suggesting that PuroA was successfully immobilized to the surface of the PSC2 particles. Zeta potentials of unmodified PSC2 particles (-25 ± 9 mV) were similar to those of PSC2 particles treated with EDC/NHS but without PuroA (-28 ± 9 mV), suggesting that activation of COOH groups and subsequent hydrolysis of NHS active esters does not significantly affect the surface charge. Results for unmodified PSC2 particles are observed to be somewhat different to previously published data (~-45 mV)28; this is likely due to the lower pH used which may have increased the proportion of protonated COOH groups, or a lower than expected COOH surface concentration. Zeta potentials of samples processed without the addition of coupling reagents (i.e. EDC/NHS) suggested a considerable amount of PuroA was physically adsorbed to the particle surface, which is not surprising considering the highly cationic nature of PuroA and negative zeta potential of unmodified PSC2 particles. There was however, a larger change in zeta potential seen for covalently immobilized PuroA samples (+48 ± 8 mV) compared to physically immobilized ones (+28 ± 5 mV). This could be due to different orientations of the peptide in addition to an increase in the amount of immobilized peptide; as physically adsorbed AMPs will orientate themselves to a position that is electrostatically favorable and minimizes electrostatic repulsive forces.48

PuroA_Phys

PuroA

No PuroA + EDC/NHS

No PuroA

-40

-20

0

20

40

60

Zeta potential (mV)

Figure 1. Zeta potentials of unmodified and PuroA- modified PSC2 Particles. Sample type is indicated on the y-axis and zeta potential in mV is indicated on the x-axis. Values presented are mean (mV) ± standard deviation (n=5). XPS analysis was used to determine the surface chemistry of the PuroA-modified PSC2 particles. The atomic composition results (Table 1) indicate a moderate increase in % nitrogen


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for both covalent and physically adsorbed PuroA samples compared to control samples, which would be expected if PuroA immobilization was successful.31 Low levels of sodium and chlorine (0.1%) were also detected, which is not surprising considering NaCl is a major component of the PBS buffer used. The data for analysis of bulk films of PuroA (Table 1) showed the presence of fluorine, probably due to some residual trifluoroacetic acid present after PuroA purification by HPLC by the supplier. The presence of fluorine is also confirmed in the high-resolution C1s spectra (Fig. 2a) with peaks assigned to CF3 (292.6 eV).49 For calculations of the thickness and surface coverage of PuroA films on PSC2 colloidal monolayers, the contribution of fluorine in these samples was omitted (w/o F) so as to obtain more accurate representations of the % nitrogen in bulk PuroA films. Table 1 also shows the presence of Si for all PuroA-modified PSC2 particles; however, this is attributed to the glass (SiO2) substrate rather than contamination, as the increase in Si was

also accompanied by a considerable increase in % O. Elemental composition of unmodified PSC2 particles showed small traces of nitrogen, most likely attributable to either residual surfactant that is removed by rinsing before PuroA immobilization, or the chemical groups used to generate COOH on the PSC surface. Compared to theoretical O/C ratios for carboxylated PS (~ 0.2), the results showed a lower ratio (~ 0.1), suggesting that the number of COOH groups may be relatively low, which is also supported by the lower than expected zeta potential values. Moreover, for covalently immobilized samples, there was a considerable increase in % N after PuroA incubation (4.1%) when compared to unmodified PSC2 particles (1.6%). This reaffirms the zeta potential data that PuroA immobilization on to the PSC2 surface was successful, albeit below the expected value for complete monolayer coverage (~ 13%) as determined by the % N obtained from bulk films of PuroA.

Table 1. Atomic composition of PuroA-modified and unmodified PSC2 particles Atomic % Sample ID

O

C

N

F

Si

PuroA

14.2 ± 2.7

65.2 ± 4.6

12.4 ± 1.7

8.2 ± 3.5

-

PuroA (w/o F)

15.6 ± 3.6

71.0 ± 2.3

13.4 ± 1.4

-

-

PSC2

9.5 ± 2.9

87.6 ± 3.8

1.6 ± 0.9

-

1.5 ± 1.2

PSC2-PuroA

37.7 ± 1.1

46.8 ± 2.6

4.1 ± 0.5

-

11.4 ± 2.0

PSC2-PuroA Phys

26.0 ± 1.1

63.9 ± 2.2

2.7 ± 0.2

-

8.0 ± 1.2



Equation 2. ‫ ݖ‬ൌ −ߣூெி௉ ൈ ܿ‫ ߠ ݏ݋‬ൈ ݈݊ ቀ1 −

ூ

ூಮ

ቁ

Where I and I∞ are the % N determined from the peptide coated samples and reference peptide respectively. θ is the angle between the sample and the analyzer which is estimated to be 57.3° for randomly rough particle surfaces 51, and λIMFP is the inelastic mean free path of N1s photoelectrons emitted from the peptides (2.5 nm).31 The surface coverage (Θ) of the peptides can subsequently be calculated from z values assuming the density of peptide to be 1.4 g/cm3.50 Table 2 shows calculated thickness and surface coverage values for unmodified and PuroA-modified PSC2 particles. For more accurate estimations of the amount of immobilized PuroA, the calculated PuroA surface coverage from unmodified PSC2 particles was subtracted from other samples before calculating the immobilization density of PuroA (molecules/cm2). Results show that the density of PuroA immobilized to PSC2 colloidal monolayers was 1.93x1013 and 7.14x1012 molecules/cm2 for the covalently and physically immobilized samples, respectively. Using product specifications for PSC2 particles (Lot No. 1071790) (www.thermofisher.com), the total number of COOH groups per cm2 (1.11x1014) can be calculated, and compared to observed PuroA densities for physically and covalently immobilized samples (Table 2) it appears that the efficiency of the immobilization was considerably low (~17% and 6% for covalently and physically immobilized samples, respectively). However, assuming that less than half of the occupied COOH groups will be available for analysis when presented as a colloidal crystal, in reality the immobilization efficiency is expected to be higher than what was observed. Table 2. Thickness and surface coverage of PuroAmodified and unmodified PSC2 particles Sample ID

z (nm)

Θ (mg/m2)

Corrected Θ (mg/m2)

Density (molecules/cm2)

PSC2

0.26

0.36

-

-

PSC2-PuroA

0.72

1.01

0.65

2.11E+13*

PSC2-PuroA Phys

0.45

0.63

0.27

8.88E+12*

*Densities of PuroA calculated using a MW of 1862.0 Da obtained from the supplier’s specifications (Mimotopes; Clayton, VIC).

High-reolution C1s spectra generated from PuroA-modifie PSC2 particle layers (Figure. 2) showed certain peaks common for all samples, including those corresponding to C=C (284.9 eV), C-C/C-H (285.5 eV), and also a weak shake-up satellite peaks from aromatic π-π* transitions (291.6 eV) in the benzene rings of PSC2 particles.31 As the majority of PSC2 particles are comprised of aromatic benzene rings, peak intensities for C=C at approximately 284.9 eV are ob-

served to be greater than the C-C/C-H due to the higher proportion of aromatic carbons. These peaks are also present in the PuroA sample, attributed to its aromatic Trp and Phe residues.52 High-resolution C1s spectra also reveal an increase in the components that can be assigned to (N-C=O) at approximately 288.5 eV,31 both for physically (Figure. 2d) and covalently (Figure. 2c) immobilized samples, again suggesting that the PuroA immobilization was successful. Interestingly, the high-resolution C1s spectra for unmodifiedPSC2 particles (Figure. 2b) shows that no peak could be assigned to O=C-O at approximately 289.5 eV, which would be expected to be present for carboxyl-containing particles.53 Considering the lower than expected zeta potentials, and the considerably low contribution of oxygen obtained for bare PSC2 particles, it is likely that the surface concentration of COOH groups is actually quite low and resulted in the lower than expected surface coverage. Furthermore, films of PuroA (Figure. 2a) show that the contribution of C=C at ~284.8 eV has much less prominence than C-C/C-H, which was unexpected considering 46 of the 97 (47 %) carbons of PuroA possess aromaticity. The result can be explained by a greater contribution from C-C due to the trifluoroacetic acid that is used during the peptide purification process. 4000 3500

Intensity (CPS)

Using atomic % obtained from XPS survey spectra (Table 1), the thickness of the PuroA layers (z) (in a dry state) and surface coverage can be calculated using the overlayer equation (Equation 2).50 This is under the assumption that the particle surface has random roughness with a range of emission angles51:

a)

2500 2000 1500

b)

C-C

π−π∗ shake-up satellite O-C=O C-F3

30000

C-C

C-N

N-C=O

3000

C=C

C-O C=C

20000

π−π∗ shake-up satellite

C-N C-O

10000

1000 500 0

0 294

292

290

288

286

284

282

15000

294

10000

292

290

288

286

284

282

25000

c)

Intensity (CPS)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 12

d)

π−π∗ shake-up satellite O-C=O

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Figure 2. High-resolution C1s spectra of: a) PuroA; b) unmodified PSC2 particles; c) PuroA covalently immobilized to PSC2 particles, and d) PuroA physically adsorbed to PSC2 particles.

Surface-MALDI-ToF-MS was utilized to assess if the PuroA molecules were successfully covalently attached to the PSC particles since no signals should be detected in subsequent MALDI spectra due to the soft ionization process that does not have enough energy to break the amide bond formed between the PuroA and the PSC2 particle.45,54 Corresponding results are shown in Figure 3, and the presence of PuroA can be determined by the peak observed at m/z = 1860.8, which corresponds to the protonated molecular ion [M+H]+ (Figure. 3b), where M corresponds to PuroA (FPVTWRWWKWWKG-NH2). Sodium [M-H+Na]+ and potassium [M-H+K]+ adducts can also be seen at m/z = 1883.4, and m/z = 1899.1 (Fig. 3b), however, the peaks observed at m/z = 1893.0 and m/z = 1908.0 in physically adsorbed samples (Figure. 3d) are yet to be definitively assigned, but could be caused by PuroA complexes formed with the loss of multiple H2O molecules, and the addition of H, K, or Na ions.55


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Figure 3. Surface-MALDI-TOF-MS spectra of PuroAmodified PSC2 particles over an m/z range of 1750-2250 Da. Spectra shown are of: a) unmodified PSC2 particles, b) free PuroA, c) PuroA covalently immobilized to PSC2 particles, and d) PuroA physically adsorbed to PSC2 particles. Peaks are labeled with their detected molecular weights (Da) and signal to noise ratios (sn). For the mass range of 1750-2250 Da, the MALDI-ToF-MS spectra show peaks at m/z = 1860.9 and m/z = 1860.8, confirming the presence of PuroA in covalently and physically immobilized samples, respectively (Fig 3c-d). The considerably low signal-to-noise ratio (sn) for [M+H]+ in covalent immobilized samples (sn = 353), when compared to physically immobilized samples (sn = 2540), indicates that a small proportion of PuroA molecules are physically adsorbed to the PSC particle surfaces. Thus, MALDI-ToF-MS analysis provides a way to qualitatively confirm that the covalent binding of PuroA to PSC2 particles using EDC/NHS chemistry was somewhat successful but there still remains some trace of physically adsorbed PuroA (Fig. 3c). While this finding, to our best knowledge, has yet to be reported for the covalent immobilization of AMPs, comparable results have been observed with DNA based immobilizations54, and small proteins.43 This highlights the importance of a thorough characterization of immobilized AMPs and antimicrobial agents; as potential leaching of physically adsorbed molecules from functionalized surfaces could be cytotoxic in certain applications.37 In addition, given the potential for leaching of non-covalent AMPs the mode of action may be incorrectly interpreted if not all molecules are irreversibly attached to surfaces.

BCC formation and characterization Surface topographies and structures of BCC layers prepared with unmodified 2 µm PSC particles and 0.110 µm PMMA particles (PSC2-PMMA011) were observed using SEM (Fig. 4a). The BCC monolayers were seen to form a hexagonally close-packed arrangement made of the large PSC particles with the smaller PMMA particles assembling within the interstitial spaces between the larger particles. This structure was similar to previous observations made using particle combinations with similar size ratios (γ = 0.055).28 The SEM micrograph (Fig. 4a) also indicates that the prepared particle combination can form a long-range ordered BCC structure,

which facilitates the patterned immobilization of PuroA AMPs and potentially a wide-range of other biomolecules. Similar long-range ordering was observed for PuroAmodified BCC layers (Fig. 4b), however, areas of defects and disordered structures were more prominent within the modified BCC layers (red box). This suggests that the large reversal in surface charge on the modified particles may have perturbed BCC formation, as it is well established that the repulsive electrostatic interactions are important for the production of ordered BCCs.28 Furthermore, the areas between the larger particles occupied by small PMMA particles appears to increase after functionalization with PuroA. This was due to the smaller PMMA particles adsorbing to the PuroA modified PSC2 particles through electrostatic attraction of PMMA to PuroA-modified PSC2 particles caused by the reversal in surface charge after PuroA immobilization.

Figure 4. SEM image of a PSC2-PMMA011 BCC layers showing a) unmodified BCCs, and b) PuroA-modified BCC layers. Size ratio (γ) of the BCC layer is 0.055.

Antibacterial activity investigations In order to assess the activity of unbound PuroA, the MIC was firstly determined against E. coli. PuroA was found to be active at a MIC of 32 µg/mL (data not shown), the value being somewhat higher than its previously reported MIC against E. coli (16 µg/mL).40 The antibacterial activity of PuroA and also a vast majority of other AMPs is attributed to their hydrophobic and cationic properties, allowing them to be attracted to negatively charged bacterial cell walls and insert their hydrophobic motifs into the lipophilic portion of the membrane.37 As this process is highly dependent on AMP concentration and also the number of bacterial cells present, this can make MIC values difficult to compare between studies. In this respect, the control AMP Indolicidin also showed a higher MIC value against E.coli (64 µg/mL) when compared to previous studies (32 µg/mL)40, and the observed differences in activities seen here may be caused by small variations in starting inoculations rather than differences in AMP activity. The antimicrobial properties of PuroA-modified BCCs after 24 h incubation was assessed using propidium iodide (PI) uptake as an indicator of compromised membranes (Fig.5). Similarly to MIC investigations, a 24 h incubation period was chosen for this work as an end-point to determine if the coating can remain active over sustained periods of time which is required for in vivo applications. The results show a substantial increase in the number of PI-positive cells for all PuroA-modified BCCs (Fig. 5c-d) compared to unmodified BCCs (Fig. 5b) and PS coated glass slides (Fig. 5a). The apparent number of adherent E. coli cells per field of view was similar for all samples except PS-coated glass, which



mobilized to PSC2-PMMA011 BCCs, and d) PuroA physically adsorbed to PSC2-PMMA011 BCCs. Images were taken at 1000X magnification under immersion oil, and six images were taken per sample.

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exhibited slightly lower numbers. This observation was somewhat expected, considering E. coli cells may withstand washing procedures when attached to micro- and nanoscale structures found within the BCCs, and additionally, the highly cationic nature of PuroA would present more electrostatic attraction to the negatively charged bacterial membranes.39 The visual observations were also substantiated using ImageJ software, which showed a significant decrease in the viability of E. coli cells for all PuroA-modified BCCs (Fig. 6) when compared to control samples. > 70% and > 50% of adherent E. coli cells were PI-positive for covalently and physically immobilized samples, respectively, compared to approximately 10% for PS-coated glass and 15% for unmodified BCC monolayers. Furthermore, considering the presence of small traces of physically adsorbed PuroA in covalent samples, confirmed using MALDI-ToF (Fig. 4), it is difficult to attribute the activity solely to the covalently immobilized PuroA. However, it is clear that PuroA presents surface-localized activity toward E. coli cells when covalently and/or physically immobilized to PSC2-PMMA011 BCC layers. While the significant decrease seen in the viability of E. coli cells for PuroA physically or covalently immobilized to PSC2-PMMA011 BCCs is promising for production of effective antimicrobial coatings, direct comparisons to other literature are not always appropriate due to different experimental conditions,56 starting inocula,39,48,56 as well as strain or species-specific differences in bacterial responses to surface topography and chemistry.17 Therefore, it is necessary to investigate responses of these surfaces to a number of bacterial species/strains or a better understanding of their spectrum of antimicrobial potential.

PS

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Figure 6. Viability of adherent E.coli cells attached to PuroAmodified PSC2PMMA011 BCC layers. Values presented are percentage Live/Dead ± standard deviation (preliminary y-axis) and the total number of bacteria per field ± standard deviation (secondary y-axis). Sample description is indicated on the xaxis, and statistical analysis was performed using a pairedsamples Students T. Test. Values of p < 0.05 were considered significant and indicated using *.

Figure 6 also indicates the average number of bacteria observed per field after the 24 h incubation suggesting that an increased attachment to the modified surfaces compared to relatively planar PS controls. It is widely known that bacteria will alter their attachment profiles depending on the surface topography presented,57,58 and it is suggested that the inherent nano- and micro-topographies presented here, not only facilitates AMP penetration but also provides attachment sites for bacterial cells (Fig.7).

Figure 7 (left). Visual representation of the proposed mechanism of bacterial attachment and antimicrobial activity. Planktonic cells may adhere to AMP-coated particles causing membrane destabilization and cell death, or may attach within the interstitial spaces of the AMP-modified particles. Figure 5. Representative fluorescent microscopy images after 24 h incubation showing live (green) and membrane compromised (red) E. coli cells adhered to: a) PS-coated glass slides, b) unmodified PSC2PMMA011 BCCs, c) PuroA covalently im-


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Figure 8. Representative SEM micrographs of adherent E. coli cells to a) PS-coated glass slides, b) unmodified PSC2PMMA011 and c-d) PuroA-modified PSC2PMMA011 BCC layers. Scale bar: 1 µm SEM images in Figure 8 show representative micrographs of the necessity for spacers may be reduced by immobilizing adherent E. coli cells to different substrate types. Upon incuAMPs to surfaces with inherent nano/micro-structures, albation with PuroA-modified BCCs, structural differences lowing for greater AMP penetration depth compared to plawere seen where cells appeared to be deflated due to the nar surfaces. leakage of cellular components upon membrane disruption (green ellipse), when compared to cells on unmodified BCC CONCLUSIONS layers.. Deflated cells were only observed on PuroAIn conclusion, PuroA was successfully immobilized to PSC2 modified samples, always being associated with a nearby particles using EDC/NHS zero-length coupling chemistry, large particle, suggesting that the antibacterial activity may and led to a significant decrease in the viability of surfacebe due to the immobilized AMP. Additionally, the images adherent E.coli on the modified surfaces. Thus, presentation shown in Figure 8 confirm our hypothesis that nano- and of PuroA using PSC2-PMMA011 BCC layers shows promismicro-topographies can influence bacterial cell attachment, ing potential for the development of antibacterial coatings, as it was observed that the majority of adherent bacterial which may provide a suitable platform for the site-specific cells were found within in the low-lying sites of the BCC tethering of other AMPs and biomolecules to substrates with layers as opposed to the protruding PSC2 particles. This a range of well-defined surface topographies and chemisresult is quite interesting, as it suggests that specific attachtries. In addition, this investigation also highlighted that dement of bacterial cells can be achieved using patterned BCC claring surface activity solely based on the action of covalayers, which would have potential applications in a number lently immobilized AMPs can be misleading, as the presence of biodiagnostic and biomaterials areas. of physically bound PuroA was confirmed with MALDISurface immobilized AMPs have only been reported a few ToF-MS, which could have contributed to the surfacetimes in the literature, and compared to zero-length immobilocalized antibacterial activity observed. While an antimilization; AMP immobilization using spacer-arms has shown crobial activity was observed here for the PuroA-modified 19,39,48 relatively enhanced activity. For example, zero-length surfaces, future investigations will need to address; the bioimmobilization of chrysophsin-1 (CHY1) resulted in approxcompatibility and lack of cytotoxicity toward mammalian imately 34% of E. coli (ATCC 33694) being killed, whereas cells for applications in a biomedical setting, the use of spacthe addition of a flexible PEG linker resulted in approximateer molecules for reduced steric hindrance and greater pene48 ly 80% killing. The relative amounts of immobilized AMP tration into bacterial membranes, and also the effects of in each case was determined by QCM-D, and calculated to changing particle type and respective sizes of the colloidal 15 14 2 be 1.01x10 and 4.56x10 molecules per cm for zeroparticles to further elucidate how changes in topography length, and PEG-linker immobilization, respectively. This affects bacterial attachment profiles and growth. indicates that higher AMP loading onto a planar surface is not sufficient to counteract the decrease in lateral mobility and penetration depth. Moreover, when immobilized to a AUTHOR INFORMATION planar surface via a flexible PEG linker, the AMP IG-25 13 2 grafted at a density of 1.6x10 molecules per cm could sigCorresponding Author nificantly decrease the viability of P. aeruginosa (GFP†To whom correspondence should be addressed: phone 61-3PA01) cells (> 80%).19 While the relative amount of AMP 9214-5033, e-mail [email protected] covalently immobilized to the surface in this study is compared only to the total amount of AMP used in the reaction, Author Contributions without any subsequent surface analysis it is difficult to deP.K. and M.B. helped to provide the experimental concepts and termine the respective quantities of physically and covalently designs for this investigation. Peng-Yuan Wang performed zeta immobilized AMP, and therefore also difficult to discern the potential experiments. MALDI-ToF experiments were persource of the observed antibacterial activity. Considering the absence of a flexible linker molecule used in our work, yet the relatively high activity for immobilized PuroA, the surface activity noted here is likely to be due to the inherent nano- and micro-patterning of BCCs,24,28 potential AMP leaching, in addition to the high Trp content and potency of PuroA.40 The combination of these properties may have allowed efficient entrance and subsequent destabilization of bacterial membranes, and it is hypothesized that

formed with the help of Snehal Jadhav. All other experiments and data analysis was performed by A.B. The manuscript was written by A.B, with comments made by all authors. All authors have given approval to the final version of the manuscript.

Funding Sources A.B. is supported by a Swinburne University of Technology Australian Postgraduate Award.

ACKNOWLEDGMENTS



A.B. acknowledges James Wang and Rebecca Alfred for technical support as well as their valuable insights and help in instrument training. We also thank De Ming Zhu for analytical support in obtaining XPS data.

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ABBREVIATIONS AMP, antimicrobial peptide; BCC, binary colloidal crystal; Phe, phenylalanine; PMMA, poly(methyl methacrylate); PSC, carboxylated polystyrene; Trp, tryptophan

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