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A Feasibility Study Exploring the Potential of Novel Battacin Lipopeptides As Antimicrobial Coatings Gayan Heruka DeZoysa, and Vijayalekshmi Sarojini ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b15859 • Publication Date (Web): 19 Dec 2016 Downloaded from http://pubs.acs.org on December 29, 2016
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A Feasibility Study Exploring the Potential of Novel Battacin Lipopeptides As Antimicrobial Coatings Gayan Heruka De Zoysa and Vijayalekshmi Sarojini* School of Chemical Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand *Author for correspondence Vijayalekshmi Sarojini Phone: + 64 9 9233387 E-mail:
[email protected]
Key Words: Lipopeptide, Surface Immobilization, Biofilm, Scanning Electron Microscopy, Membrane Lysis, X-ray Photoelectron Spectroscopy, Contact Angle, Haemolysis Abstract Colonization of medical implant surfaces by pathogenic microorganisms causes implant failure and undermines their clinical applicability. Alarming increase in multidrug resistant bacteria poses serious concerns with the use of medical implants. Antimicrobial peptides (AMPs) that form part of the innate immune system in all forms of life are attractive alternatives to conventional antibiotics to treat multidrug resistant bacterial biofilms. The aim of this study was to assess the in vitro antibacterial potency of our recently discovered lipopeptides from the Battacin family upon immobilization to various surfaces. To achieve this glass, silicon and titanium surfaces were functionalized through silanization followed by addition of the heterobifunctional crosslinker, succinimidyl-[N-maleimidopropionamido]-
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polyethylene glycol ester to generate maleimide functionalized surfaces. The lipopeptide, GZ3.27, with an added N-terminal cysteine was covalently coupled to the surfaces via a thioether bond through a Michael-type addition between the cysteine sulfhydryl group and the maleimide moiety. Success of surface immobilization and antimicrobial activity of the coated surfaces was assessed using water contact angle measurements, X-ray photoelectron spectroscopy, ellipsometry, scanning electron microscopy, CFU assays and biofilm analysis. The lipopeptide coated surfaces caused significant damage to the cellular envelop of Pseudomonas aeruginosa and Escherichia coli upon contact and prevented surface colonization by P. aeruginosa and E. coli biofilms. The lipopeptides investigated in this study were not haemolytic to mouse blood cells in solution. Findings from this study indicate that these lipopeptides have the potential to be developed as promising antimicrobial coatings on medical implants. Introduction Medical implants ranging from contact lenses to artificial organs are increasingly being used in humans. The major challenge with the use of medical implants is the colonization of the implant surfaces by drug resistant microbes, necessitating their surgical replacement which is economically nonviable and increases patient stress and mortality. Drug resistance is often associated with microbial biofilms that grow on biotic and abiotic surfaces.1 The extracellular polymer matrix of biofilms acts as a barrier against conventional antibiotics resulting in poor antibiotic penetration. Additionally, antibiotics do not have efficacy against the dormant cells within biofilms making antibiotic therapy against biofilms ineffective.1-4 Rendering antimicrobial property to materials used in medical implants is an active area of research in biomedical and materials chemistry. Approaches used to minimize surface colonization by microbes include the use of quorum-sensing inhibitors as well as coating the surfaces with antimicrobial polymers, quaternary ammonium salts or metal ions either as slow release or
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covalent coatings.5-8 Potential toxicity to the host from the released coatings, and loss of efficacy over long periods of time are issues associated with slow release coatings. Passive coatings, on the other hand, do not release the antimicrobial agent, but inhibit the adhesion of bacteria to the surface and, in many cases, kill bacteria on contact.9-11 The use of antimicrobial peptides (AMPs) to generate passive coatings of medical implant surfaces is attractive for reasons listed below. Antimicrobial peptides are part of the innate immune systems in almost all forms of life, have broad spectrum antimicrobial activity, including anti-endotoxin activity, high potency and less chance of resistance development.12-13 Lipopeptides are non-ribosomally synthesized, structurally diverse and carry a range of fatty acids on their N-termini.14 Daptomycin and polymyxin B, produced by soil bacteria, are two clinically relevant lipopeptide antibiotics. Daptomycin is active only against Gram positive bacteria and is used in the treatment of methicillin resistant Staphylococcus aureus (MRSA) infections. Polymyxin is active only against Gram negative infections and, is used as the last line of defence, against Gram negative bacterial infections, untreatable by other antibiotics.1518
While some progress has been made on the immobilization of AMPs to various surfaces
resulting in reduced surface colonization by microbes19-26 literature is scarce on surface immobilization studies using antimicrobial lipopeptides. An earlier study reported the antimicrobial activity against Escherichia coli of polymyxin B conjugated to an agarose bead.27 A more recent study reported the surface antimicrobial activity of glass slides with covalently immobilized polymyxin B using E. coli as an example.28 However, the chemistry followed in this study, did not provide selectivity to the anchoring point on the peptide which had multiple free amines, any of which could have reacted with the epoxide counterpart on the glass surface. To the best of our knowledge, there are no other literature reports on the immobilization of antimicrobial lipopeptides to medical implant surfaces.
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Battacin is a close analogue of the lipopeptide antibiotic polymyxin B, the clinical application of which is compromised because of its nephrotoxicity. We have reported on several potent, linear analogues of Battacin with promising antibacterial and antibiofilm activities.29 The most potent peptides from our battacin library were not haemolytic to mouse red blood cells at 1 mM, making these potential lipopeptide antibiotics and ideal candidates to generate passive coatings for medical implants. Given the importance of antibacterial coated surfaces in modern society and the limited number of studies using surface tethered antimicrobial lipopeptides, we undertook a feasibility study to explore the potential of battacin based lipopeptides to be used as antibacterial surface coatings using titanium and silicon which are commonly used in the medical implant industry. Glass was also included in our study as a model surface for standardization of reaction conditions. The surfaces were covalently immobilized with the N-terminal cysteinylated derivative of the linear battacin analogue. The lipopeptide coated surfaces were characterized using X-ray photoelectron spectroscopy, ellipsometry and water contact angle measurements. The antimicrobial potency of the lipopeptide immobilized surfaces against P. aeruginosa and E. coli was evaluated using a colony-forming unit (CFU) assay and the antibiofilm potency against P. aeruginosa and E. coli using Live/Dead staining microscopy respectively. Morphology of P. aeruginosa and E. coli cells treated with the lipopeptide immobilized surfaces was studied using scanning electron microscopy. Results from these investigations, presented in this paper, indicate the feasibility of using linear battacin peptides as antibacterial surface coatings to prevent bacterial colonization and biofilm formation.
Results and Discussion Cysteinylated Battacin Analogues and their Antimicrobial Activity in Solution
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Three cysteinylated versions of the linear battacin lipopeptide GZ3.27 (Table 1) with a cysteine residue added to the N-terminus, the C-terminus and the side chain of Dab2 were chemically synthesized. Addition of cysteine residue to the parent peptide was essential in order to achieve controlled immobilization through chemoselective reaction with the maleimide groups on the PEG coated surfaces. Our previous alanine scanning experiments had shown that all Dab residues except Dab2 are essential for the antimicrobial activity of the parent peptide. Hence Dab2 was chosen for side chain modification.29 The cysteine added lipopeptides were synthesized following standard Fmoc solid phase peptide synthesis protocols, as described in detail under the experimental section. Table 1: Amino Acid Sequences of the Cysteinylated Lipopeptides Peptide
Sequence
GZ3.27
R1-D-Dab-Dab-Dab-Leu-D-Phe-Dab-Dab-Leu-NH2
GZ3.163
R1-Cys-D-Dab-Dab-Dab-Leu-D-Phe-Dab-Dab-Leu-NH2
GZ3.160
R1-D-Dab-Dab-Dab-Leu-D-Phe-Dab-Dab-Leu-Cys-NH2
GZ3.155
R1-D-Dab-Dab(Cys)-Dab-Leu-D-Phe-Dab-Dab-Leu-NH2
R1 = 4-methylhexanoyl The minimal inhibitory concentrations (MICs) of the cysteine modified lipopeptides against the Gram negative pathogens E. coli and P. aeruginosa and the Gram positive pathogen Staphylococcus aureus (Table 2) and their percentage of haemolysis of mouse blood cells were determined using standard protocols, as described in detail in the experimental section. The N and C-terminally cysteine conjugated lipopeptides retained similar potency as the parent peptide against P. aeruginosa while two to three-fold decrease in activity was observed against S. aureus and E. coli. Conjugation of cysteine to the side chain of Dab2 was
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comparatively less tolerated resulting in a 10 fold decrease in activity against E. coli and about 5 fold decrease in activity against S. aureus. However, this agrees well with the MIC reported for our Ala2 analogue previously.29 Moore et al. have reported that polymyxin is capable of binding to multiple binding sites of the P. aeruginosa lipopolysaccharide, because of the higher number of negative charges on the outer membrane of this pathogen.30 This possibility of greater electrostatic interaction with the lipopolysaccharide of P. aeruginosa seems to be relevant to the lipopeptides reported in this study as well, all of which showed consistently higher potency against P. aeruginosa than the other pathogens investigated. The percentage of haemolysis of mouse blood cells by the peptides was negligible and ranged from 1.8 to 4.3 %. These results indicate that the addition of cysteine does not change the antibacterial and haemolytic activities of the parent peptide significantly. The antimicrobial potency (low micromolar) and negligible haemolysis exhibited by cysteinylated lipopeptide analogues of battacin reported in this paper encouraged us to explore their potential for use as antibacterial coatings for medical implant surfaces. The N-terminal cysteinylated peptide was used for surface immobilization studies. Table 2: Antibacterial Activity and Percentage of Haemolysis of Mouse Blood Cells by the Cysteinylated Lipopeptides in Solution MIC (µM) Before Surface Attachment % Haemolysis at 100 µM* Peptide
E. coli
P. aeruginosa
S. aureus
GZ3.27
2.5-5
1-2.5
1-2.5
1.8 ± 0.45
GZ3.163
10-15
1-2.5
2.5-5
4.3 ± 0.42
GZ3.160
10-15
1-2.5
2.5-5
2.8 ± 0.30
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GZ3.155
25-50
1-2.5
5-10
2.2 ± 0.93
* Average of three independent experiments performed in triplicates Lipopeptide Immobilization The N-terminally cysteinylated lipopeptide (GZ3.163) was covalently immobilized on glass, titanium and silicon following the reaction shown in scheme 1 using 3-aminopropyl triethoxysilane (APTES) as the linker and polyethylene glycol (PEG) as the spacer. The experimental section has the detailed protocol followed in this reaction scheme.
Scheme 1: Reaction chemistry used for the selective immobilization of lipopeptide GZ3.163 onto solid surfaces. Covalent immobilization strategy is desirable for surface modifications, since this strategy prevents peptide leaching and enhances the long-term stability of the immobilized state. In order to increase the number of surface available hydroxyl groups essential for optimal silanization, the surfaces were thoroughly cleaned with an oxidising mixture consisting of
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H2O2 and H2SO4. Surface oxidised titanium slides appeared more rough in comparison to the untreated ones and showed significant porosity under the scanning electron microscope (Figure S6: supporting info) indicative of increased surface area, as expected. Silanization is widely used for immobilizing biomolecules on inorganic surfaces. Organic silanes such as 3aminopropyl triethoxysilane (APTES) act as intermediate spacers between organic and inorganic materials and promote the adhesion process.31-34 Functionalized polyethylene glycol (PEG) molecules, ranging in molecular weight from 3000 to 5400, as spacers facilitate peptide lateral diffusion on the immobilized surface and are widely recommended in the surface immobilization of peptides. PEG has the additional advantage of being anti-adhesive that helps to minimize non-specific peptide attachment to surfaces.35-38 The NHS ester of the PEG cross linker facilitated coupling to the free amino groups of APTES on the surfaces, whereas the maleimide groups on the other end of the cross linker allowed the formation of a stable thio-ether linkage with the cysteine SH groups on the lipopeptide. Characterization of Lipopeptide Immobilized Surfaces Contact Angle Analysis Surface wettability is an important parameter that influences the attachment of bacterial cells. Changes in surface wettability are strong indicators of surface hydrophobicity and can be determined through static water contact angle measurements. Changes in water contact angles observed following each step of surface modification with the lipopeptide, confirmed the success of the process. The observed water contact angles for the three surfaces are summarised in Table 3. Increase in surface hydrophilicity following surface oxidation because of treatment with the basic piranha solution resulted in significant reduction in water contact angles in comparison to untreated glass (32.19 ± 6.3 to 20.37± 3.0), silicon (55.07 ± 2.9 to 46.7 ± 2.3) and titanium (62.5 ± 6.3 to 36.4 ± 6.1) (Table 3). Silanization increases
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surface hydrophobicity due to the presence of the propyl chain in APTES and, consequently, the water contact angles increased to 95.2 ± 6.3, 79.3 ± 4.3 and 64.7 ± 5.2 for glass, silicon and titanium respectively. Further conjugation to the amphipathic NHS-PEG-MAL led to decrease in water contact angles by 18.3, 12.7 and 8.8 on the three surfaces. Upon lipopeptide immobilization, water contact angles decreased by 17.1, 6.8 and 12.6 respectively on glass, silicon and titanium. Table 3: Water Contact Angles for Various Stages of Lipopeptide Immobilization on the Surfaces Surface Layer
Water Contact Angle (°) Glass
Silicon
Titanium
SS
32.19 ± 6.3
55.07 ± 2.9
62.5 ± 6.3
SS1
20.37± 3.0
46.7 ± 2.3
36.4 ± 6.1
SS1—APTES
95.2 ± 6.3
79.3 ± 4.3
64.7 ± 5.2
SS1--APTES--PEG
76.9 ± 7.0
66.6 ± 8.0
55.9 ± 5.2
SS1--APTES--PEG--GZ3.163
59.8 ± 4.5
59.8± 1.2
43.3 ± 5.2
SS = untreated solid surface, SS1 = piranha treated solid surface, APTES = 3-aminopropyl triethoxysilane, PEG = bi-functional polyethylene glycol spacer. Chemical Composition by X-ray Photoelectron Spectroscopy and Surface Thickness using Sulfo-SDTB Spectroscopy Assay and Ellipsometry Surface modification of silicon was studied using X-ray photoelectron spectroscopy (XPS)3940
and ellipsometry, while that of titanium using XPS (Figure 1 and Table 4). For the silicon
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surfaces, successful silanization with APTES is indicated by an increase (Table 4) in carbon content from 9.02 to 58.03 atomic % due to the presence of the propyl chain as well as decrease in percentages of elemental oxygen (24.68 to 21.04 atomic % and silicon 48.98 to 14.0 atomic %). As expected, PEGylation resulted in higher percentages of elemental oxygen (21.04 to 24.21%) and nitrogen (3.43 to 5.67%). A further increase in percentage of nitrogen to a total of 7.25 atomic % was observed after lipopeptide immobilization due to the presence of side chain amines and peptide bonds. XPS survey spectra (supporting information Figures S4 and S5) for each step of the surface modification on silicon and titanium revealed characteristic peaks for carbon, oxygen, nitrogen, silicon and titanium. Small traces of fluorine, sulphur and phosphorous were also observed. These can be attributed to the sulphuric acid in the piranha treatment and phosphate buffer washings. Higher resolution scans were carried out on carbon, oxygen, nitrogen and silicon to further analyze the chemical environments surrounding each element. Particularly informative for assessing successful peptide immobilization is the nitrogen region shown in Figure 1. Characteristic peaks in the carbon and oxygen regions (Figure 1) also confirm success of each step of surface conjugation.
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O 1s
N 1s
C 1s
Figure 1: High resolution XPS spectra of the O 1s (top left) N 1s (top right) and C 1s (bottom) regions for the step by step chemo-selective immobilization of lipopeptide GZ3.163
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onto silicon surface. The black line in each spectrum is raw data while the coloured peaks correspond to the fitted Gaussian-Lorentzian peaks (30% Lorentzian). The nitrogen environments in the APTES functionalized surface can be attributed to protonated and non-protonated amines in APTES. The intense peak at 400 eV upon PEGylation can be attributed to amide bond formation between the silanated surface and the PEG molecule. After lipopeptide immobilization, the N 1s spectrum shows three peaks at 399.4 eV, 400.0 eV and 401.6 eV which can be assigned to nitrogen in the side chain amines of Dab, amide bonds of the peptide and protonated Dab side chains respectively. The immobilized lipopeptide is a 11-mer amide with only five Dab residues (sources of free amines) that constitute only 45% of the total peptide composition. Thus, as expected, the nitrogen signal from the amide bonds, which are more abundant than the amines, is more intense. The protonated amine peak at 401.6 eV is slightly less intense (Figure 1) than the non-protonated peak at 399.4 eV suggesting that not all of the Dab molecules have been protonated since the peptide was coupled to the PEGylated silicon at pH 7.4. Similar observations were made for the modification of the titanium surface as can be seen in the titanium XPS spectra provided in the supporting information. The high resolution carbon spectrum of the silicon surface identified three carbon environments after APTES conjugation at 285 eV, 286.2 eV and 287.8 eV. These can be attributed to C-C and C-H, C-O and C-N, C=O respectively. The main peak observed at 285 eV is due to the presence of the propyl chain. The less intense peak at 286.2 eV is due to the presence of the ether bonds of the tri-ethoxy functional groups and the C-N bond of the terminal amine group. The carbonyl peak at 287.8 eV could not be assigned and is likely to be from a contamination. In the presence of the bi-functional PEG linker, more intense peaks were observed at 286.4 eV (C-O) and 288.3 eV (C=O) that can be attributed to repeating ethylene oxide units of PEG and the carbonyl moiety of the amide bond between APTES and
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the PEG linker. The C 1s spectrum after lipopeptide immobilization, as expected, is not significantly different and showed the C-O and C=O peaks at 286.4 eV and 288.2 eV respectively. Extensive characterization of the titanium surface modification was also carried out using XPS analysis. Relevant spectra are provided in the supporting information. The last column in table 4 represents the thickness of the different layers of coatings on silicon wafers determined by ellipsometry. APTES coated silicon wafer produced a uniform ~10 nm (107 Å) layer which agrees with the reported literature value for 24 hour APTES treatment.41 PEGylation of the APTES coated silicon wafer produced a layer 4.1 nm (41 Å) thick. Mishra et.al reported a similar thickness (34 Å) when a 24 carbon chain long heterofunctional PEG layer was coated to the APTES functionalized silicon wafer.24 Lipopeptide coating resulted in a layer of 26 Å thickness consistent with a peptide monolayer coating on the PEGylated surface. Film thickness obtained from ellipsometry measurements were used to calculate the surface concentration of the lipopeptide using the modified Lorentz-Lorenz equation (experimental). A surface lipopeptide concentration of 190 ± 51 ng cm2 consistent with peptide monolayer coating was obtained from these calculations ruling out peptide aggregation on the surface. The immobilized peptide concentration of the three surfaces were determined by Sulfo-SDTB assay.42 Under basic conditions, Sulfo-SDTB reacts with any free amines presented on the immobilized surfaces in stoichiometric amount. Treatment with perchloric acid will release the surface bound 4,4’-dimethoxytrityl cation (DMTr ion) which has a strong absorbance at 498 nm. The amount of DMTr ions present in the perchloric acid solution directly correlates to the amount of free amines present on the immobilized surfaces hence the amount of peptide covalently attached to the surfaces. The amount of immobilized peptide attached to glass, silicon and titanium surfaces determined using Sulfo-SDTB assay are 200 ± 22 ng cm2, 111 ± 8 ng cm2 and 140 ± 18 ng cm2 respectively. The sulfo-SDTB assay highlights similar
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extent of peptide coverage across the three surfaces. Additionally, the sulfo-SDTB assay and ellipsometry measurement of the silicon immobilized lipopeptide showed similar immobilized peptide concentration. Table 4: Chemical Composition (XPS) and Dry-Thickness (ellipsometry) of Si at Different Stages of Surface Modification Percentage atom composition Layer
Thickness (Å) C
O
N
Si
Piranha treated Si
9.02
24.68
0.00
48.98
17 ± 4
Si--APTES
58.03
21.04
3.43
14.08
107 ± 3a
Si--APTES---PEG
52.91
24.21
5.67
14.09
41 ± 6b
Si--APTES--PEG--GZ3.163 47.49
23.65
7.25
17.06
26 ± 7c
a
Thickness of the APTES layer
b
Thickness of the PEG layer
c
Thickness of the lipopeptide layer
Antibacterial and Anti-Biofilm Performance of the Lipopeptide Immobilized Surfaces Antibacterial Performance The antibacterial activity of the GZ3.163-immobilized surfaces against P. aeruginosa and E.
coli was assessed, in comparison to controls, using a colony forming unit assay of the surface adhered bacteria extracted into solution through sonication. Results shown in Figure 2 (a) to (c) clearly show that significant reduction in bacterial (P. aeruginosa) adhesion was observed upon coating with the lipopeptide onto each of these surfaces in comparison to the respective controls. Similar reduction in adhesion between lipoppeptide coated and uncoated surfaces was observed for E. coli (Fig. 2 d to f).Data in Table 5 indicate that the PEG coated surfaces
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moderately (21-44%) inhibit adhesion of P. aeruginosa and E. coli onto the three surfaces. The inhibitory effect of the PEGylated surfaces can be attributed to the anti-adhesive property of PEG because of its high mobility and steric hindrance. The lipopeptide coated surfaces were significantly better and showed 98.6-99.9% inhibition of P. aeruginosa and E. coli adhesion onto the surfaces. These observations indicate that the cystenine added lipopeptide (GZ3.163) remains antibacterial against P. aeruginosa and E. coli when immobilized to various surfaces.
Figure 2: Antibacterial activity determination by CFU counting of P. aeruginosa (top panel) and E. coli (bottom panel) adhered on glass, silicon and titanium surfaces with no coating (uncoated positive with bacterial treatment serving as the control) PEG coating and lipopeptide coating. Error bars represent standard deviation from colony counts from five independent experiments each done in triplicate plates. Uncoated negative indicates uncoated surfaces with no bacterial treatment to serve as the negative control. * Indicates P < 0.05.
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Table 5: Percentage of Bacterial Growth Inhibition of Coated and Uncoated Surfaces. % of Growth Inhibition* P. aeruginosa
PEG
GZ3.163
GZ3.163 (after soft cleaning)
E. coli
Glass
34.4 ± 12.1
99.9 ± 0.1
99.3 ± 0.1
Silicon
20.7.3 ± 17.6
98.6 ± 1.0
97.8 ± 2.0
Titanium
27.6 ± 5.9
99.6 ± 0.1
99.3 ± 0.1
Glass
33.3 ± 14.5
99.3 ± 0.1
98.9 ± 0.4
Silicon
29.2 ± 14.5
99.9 ± 0.1
97.8 ± 3.4
Titanium
44.0 ± 7.0
99.0 ± 0.1
97.5 ± 2.1
*calculated using the formula: % of inhibition = (1-(CFU sample/CFU uncoated surface)*100) Stability Assessment of the Lipopeptide-Immobilized Surfaces by Soft Cleaning The surfaces were immersed in MHB medium for 24 hours (soft cleaning), and then transferred to 1 mL of fresh MHB containing 106 CFU per mL of P. aeruginosa and E. coli which were then allowed to grow overnight. The surfaces were then rinsed, sonicated and a CFU determination of the detached bacteria carried out. Results from this soft-cleaning assay done to test potential peptide leaching are shown in Figure 3.
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Figure 3: Stability assessment of the antibacterial lipopeptide coatings. Antibacterial activity determination by CFU counting of P. aeruginosa (top panel) and E. coli (bottom panel) adhered to surfaces with no coating (uncoated)
lipopeptide (GZ3.163) coating
after
immersion in MHB medium for 24 hours (soft cleaning to test if the coatings leach out). Glass (a and d), silicon (b and e) and titanium (c and f). Error bars represent standard deviation from five independent experiments done in triplicate plates each time. Uncoated negative indicates that these uncoated surfaces were immersed in broth alone with no bacterial cells present to serve as the negative control. * Indicates P < 0.05. As can be seen from Figure 3 and Table 5, all the three lipopeptide-immobilized surfaces exhibited significant level of antibacterial activity (97-99% reduction in bacterial growth) after soft cleaning. These results confirmed that antibacterial activity of the immobilized peptide is retained at least up to 24 hours under soft cleaning conditions. The ability of the immobilized lipopeptide to prevent biofilm formation of P. aeruginosa and E. coli was studied using a Live/Dead staining assay. At 24 and 48 hours, the uncoated glass, silicon and titanium surfaces showed a high fluorescence confirming that biofilms readily
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colonize the uncoated surfaces. However, in the presence of the immobilized lipopeptide, fewer biofilm cells could be observed on the surfaces, indicative of the lipopeptide’s ability to protect the surfaces from biofilm attachment (Figure 4). These observations prove that the immobilized cysteinylated lipopeptide retains the antibiofilm activity exhibited by its parent analogue 29 in solution.
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Figure 4: Representative fluorescence images of P. aeruginosa (top) and E. coli (bottom) biofilm formed for 24 (left) and 48 (right) hours in the absence (control) and presence of the immobilized lipopeptide (GZ3.163) on glass, silicon and titanium. Individual images were viewed separately using FITC 2 and Texas Red filters and merged using the in-built ACT-2U software. Bacterial Cell Morphology Scanning electron microscopy images of P. aeruginosa and E. coli in the absence and presence of immobilized GZ3.163 and GZ3.160 are shown in Figure 5. The rod-shaped P. aeruginosa and E. coli cells maintained a smooth cell surface morphology on all of the uncoated surfaces. Similar cell morphology was also maintained on PEGylated surfaces indicating that the linker alone does not cause damage to the bacterial cells. However, bacterial cells on the lipopeptide immobilized surfaces showed clear signs of damage to the cellular membrane. These included blister formation, protruding bubbles as well as completely burst cells. Positively charged AMPs are known to displace the Mg2+ ions present in the LPS layer of Gram negative outer membranes allowing the AMPs to penetrate in and cause localised disruption to the inner membrane. This can be manifested as blisters and bubbles without complete disruption of the outer membrane. The bubble formation was particularly evident with P. aeruginosa on glass and silicon surfaces with GZ3.163, whereas on titanium surfaces the cells showed hole formation, wrinkling and in some cases complete rupture and cell lesions. E. coli cells did not show bubbles, but more holes and ruptured nature was evident. Overall, both bacterial cells on lipopeptide coated surfaces looked significantly damaged in comparison to the uncoated and PEG coated surfaces, even though the nature of observed damages can be considered as heterogeneous, as described above. SEM data alone is not sufficient to conclude on the actual state of the cellular membrane which will require detailed analyses using Transmission Electron Microscopy. Nevertheless,
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our current SEM data is enough to conclude that the immobilized lipopeptides cause significant damage to the bacterial cell envelope, similar to that exhibited by the parent lipopeptide in solution. Morphological changes in P. aeruginosa cells on glass and silicon coated with GZ3.163 closely resemble those reported by Hilpert et al
19
in the presence of a
series of immobilized cationic AMPs from the cathelicidin family.
Figure 5: Representative SEM images of P. aeruginosa (top) and E. coli (bottom) in the absence (control) and presence of PEGylated, GZ3.163 and GZ3.160 immobilized on glass, silicon and titanium surfaces.
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Conclusions A cysteinylated battacin lipopeptide was covalently immobilized on glass, silicon and titanium using the heterobifunctional PEG cross-inker NHS-PEG24-MAL following a chemoselective reaction. Extensive characterization of the various steps of the immobilization confirmed the success of each step of covalent immobilization. Sulfo-SDTB spectroscopic assay of glass, silicon and titanium coated surfaces and ellipsometry measurements of silicon coated peptide surface identified that a thin monolayer of the lipopeptide (110-200 ng cm2) was sufficient to inhibit bacterial growth on the surfaces. The antibacterial activity of the lipopeptide-immobilized surfaces was validated by SEM imaging of P. aeruginosa and E. coli immobilized on the surfaces and CFU assay. A soft-cleaning procedure carried out proved the stability of immobilized state which proved to be antibacterial after the soft-cleaning procedure. Immobilization of the lipopeptide on glass, silicon and titanium surfaces successfully prevented E. coli and P. aeruginosa biofilms colonising the surfaces, as determined by Live/Dead biofilm assay. This study forms the first report analysing the potential of battacin based lipopeptides as antibacterial surface coatings. Results from these investigations have implications in the development of surface coatings for a wide range of applications including medical implants, food processing plants and antifouling coatings for marine vessels. Experimental Chemicals and Reagents All solvents were of analytical grade and were used without further purification. Fmocprotected amino acids were purchased from GL Biochem (Shanghai, China). The NHSPEG24-MAL linker was purchased from Fisher Scientific. 3-aminopropyl triethoxysilane (APTES) was purchased from Sigma Aldrich. Glass slides (25 mm x 75 mm, 1 mm thick)
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were purchased from Global Science. Silicon wafers (76 mm diameter, 0.4 mm thickness) were purchased from EL-CAT Inc. (New Jersey, USA). Titanium (99.2%) foil (100 mm x 100 mm, 2 mm thick) was purchased from Alfa Aesar (Heysham, England). Titanium foil was polished with silicon carbide and washed thoroughly with Milli-Q water. The foil was further polished using a diamond-polishing disk. The polished foil was then cut into 20 mm x 20 mm square pieces, using a diamond-cutting saw, for the immobilisation studies. Synthesis of the Lipopeptides29 The lipopeptides investigated in this paper have been synthesized following standard solid phase peptide synthesis protocols using Fmoc chemistry. Experimental details of this synthesis and purification have been reported in our previous publication on this family of peptides.29 The experimental protocols for lipopeptide synthesis previously reported by us are described here again. Briefly, the peptides were synthesized on Tentagel-S-NH2 resin (substitution level of 0.29 mmol/g) as C-terminal amides using rink amide linker, following Fmoc/tBu strategy on a 0.1 mmol scale. All couplings were carried out in DMF using TBTU (3.9 eq.) as the coupling reagent and HOBt (3.9 eq.) as the additive and DIPEA (10 eq.) as the base. With the exception of the side chain amino group of Dab2 for GZ3.155, which was protected using the acid labile protecting group Mtt, all other Dab side chains were protected using Boc. Removal of the Mtt protecting group was achieved through repeated washings with 1.8% TFA in DCM for 3 minutes.43 Excess TFA was removed by washing the resin with DCM and DMF before coupling Fmoc-Cys(Trt)-OH to Dab2 side chain. A TFA cocktail mixture (TFA-TIS-H2O - 95:2.5:2.5 v/v) 10 mL per gram of the resin was used to cleave the peptides from the resin. This was followed by a standard procedure for TFA removal and precipitation of the crude peptides using excess cold diethyl ether and lyophilization to generate the crude peptides as white fluffy soilds. The crude peptides were purified to homogeneity using reversed-phase high performance liquid chromatography (RP-HPLC) on a
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GE Pharmacia ÄKTA purifier 10 system on a Phenomenex Luna 5 micron C18 100Å (250 x 10 mm) column using 0.1 % TFA in water as solvent A and 0.1 %TFA-and 0.9 % water in 99 % acetonitrile as solvent B at a flow rate of 4 mL per minute. The peptides were eluted using a linear gradient of 10-55 % of solvent B over 25 to 30 minutes with UV detection of 214 nm. Homogeneity (> 95% purity) of the purified peptides was determined by analytical RPHPLC. Analytical reversed phase HPLC was performed using a Phenomenex Luna 5 micron C18 100Å (250 x 4.6 mm) column using the same solvent system as above at flow rate of 1 mL per minute. The identity of the peptides was established using electrospray ionisation mass spectrometry (ESIMS) recorded on a Bruker micrOTOF-Q mass spectrometer. Peptide Immobilization Piranha treatment of the Surfaces All the surfaces were thoroughly washed with Milli-Q water prior to piranha treatment. The surfaces were placed in piranha solution (150 mL) consisting of 30% hydrogen peroxide and 70% concentrated sulphuric acid for 30 minutes. The temperature of the piranha solution was maintained between 80-130°C during the cleaning. After piranha treatment, the substrates were soaked in Milli-Q water, thoroughly rinsed and dried under nitrogen.44 Silanization with APTES Piranha-treated glass slides and silicon wafers were silanized in 1% APTES in dry toluene (20 mL), for 16 hours. The APTES treatment of titanium slides was carried out under a glove box, to ensure a dry environment to facilitate the silanization. The silanized samples were washed thoroughly with acetone and ethanol, soaked in toluene (20 mL) and sonicated for 10 minutes. The soaked surfaces were thoroughly washed with ethanol to remove any traces of toluene, and dried at 121 °C for one hour to stabilize the APTES monolayer on the surface.4445
The silanized surfaces were dried under nitrogen.
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PEGylation using NHS-PEG24-MAL23 The
APTES
coated
surfaces
were
functionalized
with
succinimidyl-[N-
maleimidopropionamido]-polyethylene glycol ester (NHS-PEG24-MAL). A solution of the NHS-PEG24-MAL ester (0.2 mg/mL; 1 mL) in dry DMF was reacted with the silanized surface for 4 hours at room temperature. The surfaces were then thoroughly washed with DMF, PBS buffer and Milli-Q water and dried under nitrogen. Conjugation of GZ3.163 on to the PEGylated Surfaces 23, 46 The cysteine-modified lipopeptide GZ3.163 was coupled to the PEG functionalized surfaces using selective sulfhydryl chemistry. The PEGylated surfaces were immersed in the lipopeptide solution in PBS buffer at pH 7.4 (2 mg/mL; 1 mL) and left for 16 hours at room temperature. The coated surfaces were thoroughly washed with PBS buffer and Milli-Q water and dried under nitrogen. The lipopeptide-coated surfaces were stored at -20 °C for further use. Surface Characterization Water Contact Angle Measurements Contact angle measurements were done using a KSV Cam 100 Goniometer (Biolin Scientific) at the School of Chemical Sciences, University of Auckland with Attension Theta analysis software (Biolin Scientific, 2014) for contact angle calculations. A water droplet with uniform size (0.5 µL) was deposited on to the surface using a Hamilton syringe. Droplets were allowed to stand for 10 seconds before measurements were taken using ainbuilt planar CCD camera. The water contact angles were calculated using the in-built CAM 100 software and the measurements were repeated in three different regions, to arrive at a representative value. The contact angle values are reported as average ± standard deviation.
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Ellipsometry The thickness of the immobilized lipopeptide coated on the silicon wafer was measured using a Beaglehole Ellipsometer (Beaglehole Instruments, 2009) at variable angles (80°-60°) at 4o increments, at 632 nm. The film thickness was determined using the Thin Film Companion (SemiconSoft Inc., USA) software. A refractive index of 1.457 was used for the uncoated SiO2 layer. The refractive index for APTES was set up at 1.422 while that of the PEG and peptide layer was set at 1.460. The modified Lorentz-Lorenz equation used for calculating the surface concentration of the immobilized lipopeptide is shown below.47 σ = d ρ° = 0.01dMw n2 – 1
A
n2 +2
where, σ = Mass/ unit area (µg cm-2) ρ°= Density (mass per unit volume) d= Thickness of adhered layer (Å) Mw = Molecular weight of the peptide (kDa) A = Molar refractivity of absorbed surface, set at 0.414 n = Refractive index of peptide, set at 1.460 Immobilized
peptide
concentration
determination
by
Sulfo-succinimidyl-4-0-(4,4’-
dimethoxytrityl)-butyrate (Sulfo-SDTB) spectroscopic assay42 The amount of immobilized lipopeptide on glass, silicon and titanium were determined by Sulfo-SDTB assay. Sulfo-SDTB (3 mg, Santa Cruz Biotechnology) was first dissolved in DMF (1 mL) to which was added 10 mM sodium bicarbonate (pH 8.5, to a total volume of 50 mL). The peptide immobilised surfaces were immersed in 1.5 mL of the above Sulfo-SDTB solution and an additional 1.5 mL of sodium bicarbonate and incubated for 1 hour at 25 °C. The surfaces were thoroughly washed with milli-Q water and immersed in 35% perchloric
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acid (3 mL) for 30 minutes to release the bound dye. The absorbance of the perchloric acid solution was measured at 498 nm using UV Pharma Spec 1700 UV-Visible spectrometer. The concentration of the released dye, and therefore the amount of free amines on immobilized peptide was calculated using Beer-Lambert law with excitation coefficient of 70,000 M-1 cm1. The experiment was carried out in triplicates. X-ray Photoelectron Spectroscopy (XPS) A Kratos Axis UltraDLD equipped with a hemispherical electron energy analyser was used to collect XPS data. XPS analyses were conducted using monochromatic Al Kα X-rays (1486.69 eV) with the X-ray source working at 10 mA and 15 kV (analysis area 300 × 700 µm spot). All measurements were collected in normal emission geometry at chamber pressures of approximately 10-9 Torr. For the survey scans, an energy step of 0.1 eV and pass energy of 160 eV was used, whereas for core level scans, a pass energy of 20 eV was used. The C 1s signal from saturated hydrocarbons (285.0 eV) was used as an internal standard, to correct the binding energy scale for charging specimen and the neutraliser shift. Data was analysed using CasaXPS (www.casaxps.com) with Shirley backgrounds and relative sensitivity factors provided by the instrument. In some cases, where required, XPS peaks were fitted with Gaussian-Lorentzian peaks (30% Lorentzian). Bacterial Strains and Growth Media E. coli DH5α, P. aeruginosa ATCC 27853 and S. aureus were obtained from the microbial culture collection of School of Biological Sciences or the Faculty of Medical and Health Sciences, at the University of Auckland. The strains were stored in 50% glycerol at -20°C and -80°C. For routine use, bacteria were plated on Muller Hinton Broth agar plates. Single colonies were re-plated every two weeks. All media for bacterial growth and glassware were autoclaved at 120°C for 1 hour using a Tomy SX 500E high-pressure steam steriliser.
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Determination of Minimal Inhibitory concentration (MIC) the Lipopeptides in Solution MIC determination was conducted following the protocol described in our previous publication.29 Briefly, bacteria were grown in Luria broth (1.25%) prepared in sterilised Milli-Q water which was autoclaved at 120 °C for 1 hour. A single colony of the bacterial strain was transferred to 20 mL of the Luria broth and grown at 37°C overnight. The optical density (OD) of the overnight culture was measured and adjusted to be 0.6 (OD600: 0.6). Stock solutions of the peptides and the antibiotic controls were prepared in Luria broth, and a diluted as necessary. For the assay 50 µL each of the peptide solutions at various concentrations and the diluted bacterial culture were incubated together in 96-well microtiter plates, with three replicates of each peptide concentration tested against each bacterial strain. MIC was determined as the minimum concentration where no growth was observed using optical density (OD600) measurements which were conducted using an EnSpire Multimode plate reader at the Faculty of Medical and Health Sciences, University of Auckland. The Haemolytic Assay29,48 Protocol used for the haemolytic assay is the same as we have reported before.29 Freshly collected mouse blood cells were centrifuged at 1000g for 5 minutes to remove the buffy coat. The blood cells were washed thrice in Tris buffer (10 mM Tris, 150 mM NaCl, pH 7.4), and re-suspended in 2% (v/v) Tris buffer. The peptides were dissolved in Tris buffer at concentrations ranging from 1 mM to 1 µM. The peptide solution (100 µL) was added to the re-suspended blood cells (100 µL) in 96 well plates, and the plates incubated for 1 hour, at 37°C without agitation.. The plates were centrifuged at 3500 g for 10 min. The supernatant from each sample (100 µL) was transferred to new 96 well plates, and the absorbance at 540 nm measured. The tris buffer solution and 0.1% Triton X-100 were used as the negative and positive controls respectively. All of the samples and controls were tested in triplicate
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Percentage haemolysis at each peptide concentration was calculated using the following equation, %haemolysis = (Aexp ─ ATris) / (A100% ─ ATris) x 100 where, Aexp is the experimental absorbance at 540 nm measurement, ATris is the absorbance of the negative control, where only Tris buffer was added to mouse blood cells and A100%, is the absorbance of the positive control, where 0.1% Triton X-100 was used to cause lysis of 100% mouse blood cells present. Antibacterial Activity of the Immobilized Lipopeptide Colony Forming Unit (CFU) assay The procedure reported in the literature was slightly modified to test the antibacterial activity of the immobilized lipopeptide GZ3.163.22,49 A single colony, from a fresh plate, of either P. aeruginosa or E. coli was transferred to 20 mL MHB and grown at 37°C overnight. The overnight culture was then diluted to 105 colony-forming units (CFU). The uncoated, PEG coated and GZ3.163 coated glass, silicon and titanium surfaces were immersed in the diluted bacterial culture (1 mL) onto and incubated at 37°C overnight at 70 rpm. After overnight incubation, the samples were washed five times with sterile saline (0.9% NaCl) solution to remove any non-adhered bacteria. The washed surfaces were transferred to 1 mL of sterile saline solution and were sonicated for 5 minutes at 30 W to retrieve bacteria adhered to the surfaces. This bacterial solution was centrifuged at 10,000 rpm for 5 minutes. The retrieved E. coli and P. aeruginosa solutions were diluted 1000 and 10000 and 10000 and 100,000 respectively with sterile saline, 100 µL of the diluted cultures were plated on triplicate MHB agar plates and the plates were incubated at 37°C overnight. After overnight incubation, the number of colonies on each plate was counted to assess antibacterial activity. The CFU assay
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was repeated five times. Growth curves of initial bacterial suspensions were followed up to 25 hours and the results are presented in Fig. S8 of the supporting information. Stability of the Lipopeptide-Immobilized Surfaces The stability of the lipopeptide-immobilized surfaces was determined following a soft cleaning procedure where one lipopeptide coated and two blank surfaces (controls) were initially submerged in 1 mL of MHB medium for 24 hours. The submerged surfaces were then transferred to fresh MHB (900 µL) into which 100 µL of overnight bacterial cultures (P. aeruginosa and E. coli) diluted to 105 CFU/mL was added followed by overnight incubation at 37°C and 70 rpm. The antibacterial activity of the surfaces was determined following the same CFU count procedure after 5 min. sonication at 30 W, as described above. Inhibition of Bacterial Biofilm formation using Live/Dead Staining29 Overnight grown P. aeruginosa and E. coli cultures were diluted to 105 CFU/mL. The control and lipopeptide immobilized glass and silicon surfaces were cut into 10 mm x 10 mm squares with a diamond cutter, and inserted into the wells of the 12 well plates whereas the titanium surfaces (20 mm x 20 mm) (control and coated) were placed in a large petri dish. The surfaces were covered with 1 mL of Luria broth, and 50 µL of the diluted bacterial suspension added and incubated for 24 and 48 hours, without shaking, at 37°C. These were then carefully washed with Milli-Q water to remove any planktonic bacteria, and dried. The adhered bacteria on the surfaces were stained with, Live/Dead BacLight (Invitrogen) solution following our previously reported protocol.29.. The stained biofilms were visualized at 1000 X magnification using a Nikon Eclipse E600 microscope. The stained biofilms were viewed separately using FITC 2 and Tx Red filters and the images were captured using an in-built digital sight DS-U1 camera. The Live/Dead staining assay was done in triplicates.
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Scanning Electron Microscopy29 An overnight culture of P. aeruginosa and E. coli was diluted to OD600: 0.3 and 1 mL of the diluted culture was incubated with the control and lipopeptide immobilized surfaces for 4 hours, at 37°C, without shaking. Excess bacteria were removed by washing with 10 mM phosphate buffer (pH 7.4) and fixed with 4% glutaraldehyde solution for 1 hour. The slides were washed with 10 mM sodium phosphate buffer and dehydrated with graded ethanol series (25-100 %). The dried slides were sputter- coated with platinum, for 2 minutes, at 20 mA, before viewing under high vacuum, using an FEI Quanta 200 F ESEM microscope, at 10 kV at the Faculty of Engineering, University of Auckland.
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References (1) Costerton, J. W.; Stewart, P. S.; Greenberg, E. P., Bacterial Biofilms: a Common Cause of Persistent Infections. Science 1999, 284 (5418), 1318-22. (2) Wentland, E. J.; Stewart, P. S.; Huang, C. T.; McFeters, G. A., Spatial Variations in Growth Rate within Klebsiella pneumoniae Colonies and Biofilm. Biotechnol. Prog 1996, 12 (3), 316-21. (3) Davies, D., Understanding Biofilm Resistance to Antibacterial Agents. Nat. Rev. Drug Discov 2003, 2 (2), 114-22. (4) Hall-Stoodley, L.; Costerton, J. W.; Stoodley, P., Bacterial Biofilms: From the Natural Environment to Infectious Diseases. Nat. Rev. Microbiol. 2004, 2 (2), 95-108. (5) Siedenbiedel, F.; Tiller, J. C., Antimicrobial Polymers in Solution and on Surfaces: Overview and Functional Principles. Polymers-Basel 2012, 4 (1), 46-71. (6) Monteiro, D. R.; Gorup, L. F.; Takamiya, A. S.; Ruvollo, A. C.; Camargo, E. R.; Barbosa, D. B., The Growing Importance of Materials That Prevent Microbial Adhesion: Antimicrobial Effect of Medical Devices Containing Silver. Int. J. Antimicrob Ag. 2009, 34 (2), 103-110. (7) Hetrick, E. M.; Schoenfisch, M. H., Reducing Implant-Related Infections: Active Release Strategies. Chem. Soc. Rev. 2006, 35 (9), 780-789. (8) Ravikumar, T.; Murata, H.; Koepsel, R. R.; Russell, A. J., Surface-Active Antifungal Polyquaternary Amine. Biomacromolecules 2006, 7 (10), 2762-2769. (9) Nagel, J. A.; Dickinson, R. B.; Cooper, S. L., Bacterial Adhesion to Polyurethane Surfaces in the Presence of Pre-adsorbed High Molecular Weight Kininogen. J. Biomater. Sci. Polym. Ed. 1996, 7 (9), 769-780. (10) Kaper, H. J.; Busscher, H. J.; Norde, W., Characterization of Poly(Ethylene Oxide) Brushes on Glass Surfaces and Adhesion of Staphylococcus epidermidis. J. Biomater. Sci. Polym. Ed. 2003, 14 (4), 313-324. (11) Kingshott, P.; Wei, J.; Bagge-Ravn, D.; Gadegaard, N.; Gram, L., Covalent Attachment of Poly(Ethylene Glycol) to Surfaces, Critical for Reducing Bacterial Adhesion. Langmuir 2003, 19 (17), 6912-6921.
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(23) Lim, K.; Chua, R. R.; Saravanan, R.; Basu, A.; Mishra, B.; Tambyah, P. A.; Ho, B.; Leong, S. S., Immobilization Studies of an Engineered Arginine-Tryptophan-Rich Peptide on a Silicone Surface with Antimicrobial and Antibiofilm Activity. ACS Appl. Mater. Interfaces 2013, 5 (13), 6412-22. (24) Mishra, B. B., M., Saravanan, R., Li Xiang, L., Lim Kai Yang, L.K.; and Leong, S. S. J., Lasioglossin-III: Antimicrobial Characterization and Feasibility Study for Immobilization Applications. Rsc Adv. 2013, 3, 9534–9543. (25) Godoy-Gallardo, M.; Mas-Moruno, C.; Fernandez-Calderon, M. C.; Perez-Giraldo, C.; Manero, J. M.; Albericio, F.; Gil, F. J.; Rodriguez, D., Covalent Immobilization of hLf111 Peptide on a Titanium Surface Reduces Bacterial Adhesion and Biofilm Formation. Acta Biomater. 2014, 10 (8), 3522-3534. (26) Costa, F. M.; Maia, S. R.; Gomes, P. A.; Martins, M. C., Dhvar5 Antimicrobial Peptide (AMP) Chemoselective Covalent Immobilization Results on Higher Antiadherence Effect than Simple Physical Adsorption. Biomaterials 2015, 52, 531-8. (27) LaPorte, D. C.; Rosenthal, K. S.; Storm, D. R., Inhibition of Escherichia coli Growth and Respiration by Polymyxin B Covalently Attached to Agarose Beads. Biochemistry 1977, 16 (8), 1642-8. (28) Mohorcic, M.; Jerman, I.; Zorko, M.; Butinar, L.; Orel, B.; Jerala, R.; Friedrich, J., Surface with Antimicrobial Activity Obtained Through Silane Coating with Covalently Bound Polymyxin B. J. Mater. Sci. Mater. Med. 2010, 21 (10), 2775-82. (29) De Zoysa, G. H.; Cameron, A. J.; Hegde, V. V.; Raghothama, S.; Sarojini, V., Antimicrobial Peptides with Potential for Biofilm Eradication: Synthesis and Structure Activity Relationship Studies of Battacin Peptides. J. Med. Chem. 2015, 58 (2), 625-39. (30) Moore, R. A.; Bates, N. C.; Hancock, R. E., Interaction of Polycationic Antibiotics with Pseudomonas aeruginosa Lipopolysaccharide and Lipid A Studied by using DansylPolymyxin. Antimicrob. Agents Chemother. 1986, 29 (3), 496-500. (31) Nanci, A.; Wuest, J. D.; Peru, L.; Brunet, P.; Sharma, V.; Zalzal, S.; McKee, M. D., Chemical Modification of Titanium Surfaces for Covalent Attachment of Biological Molecules. J. Biomed. Mater. Res. 1998, 40 (2), 324-335. (32) Nakagawa, T.; Tanaka, T.; Niwa, D.; Osaka, T.; Takeyama, H.; Matsunaga, T., Fabrication of Amino Silane-coated Microchip for DNA Extraction from Whole Blood. J. biotechnol. 2005, 116 (2), 105-111.
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(44) Martin, H. J.; Schulz, K. H.; Bumgardner, J. D.; Walters, K. B., XPS Study on the Use of 3-Aminopropyltriethoxysilane to Bond Chitosan to a Titanium Surface. Langmuir 2007, 23 (12), 6645-6651. (45) Xiao, S.-J.; Textor, M.; Spencer, N. D.; Sigrist, H., Covalent Attachment of CellAdhesive,(Arg-Gly-Asp)-containing Peptides to Titanium Surfaces. Langmuir 1998, 14 (19), 5507-5516. (46) Gabriel, M.; Nazmi, K.; Veerman, E. C.; Amerongen, A. V. N.; Zentner, A., Preparation of LL-37-grafted Titanium Surfaces with Bactericidal Activity. Bioconjugate Chem. 2006, 17 (2), 548-550. (47) Cuypers, P. A.; Corsel, J. W.; Janssen, M. P.; Kop, J. M. M.; Hermens, W. T.; Hemker, H. C., The Adsorption of Prothrombin to Phosphatidylserine Multilayers Quantitated by Ellipsometry. J. Biol. Chem. 1983, 258 (4), 2426-2431. (48) Stark, M.; Liu, L. P.; Deber, C. M., Cationic Hydrophobic Peptides with Antimicrobial Activity. Antimicrob. Agents Chemother. 2002, 46 (11), 3585-90. (49) Humblot, V.; Yala, J. F.; Thebault, P.; Boukerma, K.; Hequet, A.; Berjeaud, J. M.; Pradier, C. M., The Antibacterial Activity of Magainin I Iimmobilized onto Mixed Thiols Self-Assembled Monolayers. Biomaterials 2009, 30 (21), 3503-12.
Supporting Information Analytical HPLC traces, ESI-MS and 1H NMR spectra of all peptides, XPS core and survey spectra, titanium plates (photographs and SEM images) and SEM images showing P. aeruginosa and E. coli coverage on control and peptide coated surfaces.
Acknowledgements We thank Dr. Colin Doyle from the Research Centre for Surface and Materials Science, Faculty of Engineering, University of Auckland for XPS data collection.
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