Article pubs.acs.org/Langmuir
Effect of Linker Length on Cell Capture by Poly(ethylene glycol)Immobilized Antimicrobial Peptides Lisa C. Shriver-Lake, George P. Anderson, and Chris Rowe Taitt* Center for Biomolecular Science & Engineering, Naval Research Laboratory, 4555 Overlook Avenue, SW, Washington, DC 20375, United States S Supporting Information *
ABSTRACT: Development of antimicrobial peptide (AMP)functionalized materials has renewed interest in using poly(ethylene glycol) (PEG)-mediated linking to minimize unwanted interactions while engendering the peptides with sufficient flexibility and freedom of movement to interact with the targeted cell types. While PEG-based linkers have been used in many AMP-based materials, the role of the tether length has been minimally explored. Here, we assess the impact of varying the length of PEG-based linkers on the binding of bacterial cells by surface-immobilized AMPs. While higher surface densities of immobilized AMPs were observed using shorter PEG linkers, the increased density was insufficient to fully account for the increased binding activity of peptides. Furthermore, effects were specific to both the peptide and cell type tested. These results suggest that simple alterations in linking strategiessuch as changing tether length may result in large differences in the surface properties of the immobilized AMPs that are not easily predictable.
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PEG-based linkers were used,24,26 the effect of the tether length has been minimally explored. Han and colleagues18 assessed the structure/function relationship of self-assembled monolayers (SAMs) decorated with AMPs via two different PEG linkers. While AMP surfaces formed using longer PEG linkers had a lower peptide density and total number of captured cells, these surfaces had a higher cell killing activity, as measured by the dead/live ratio of captured cells. This is in agreement with Bagheri, Dathe, and colleagues, who observed lower antimicrobial activity when AMPs were tethered using short PEG linkers; this lowered activity could not be mitigated even with higher AMP loading on the short PEG linker surfaces.24,28 Here, we assess the ability of four membrane-interacting AMPs to capture two Gram-negative and one Gram-positive bacterial species when immobilized to a microsphere via PEG linkers ranging from 0 to >100 ethylene glycol units in length. Although killing of target cells was not addressed, the trends observed here support those observed previously. However, our results further reinforced the need to empirically test a range of AMP−linker combinations to obtain a surface with the desired bacterial capturing activity.
INTRODUCTION Recently, there has been significant interest in utilizing biomaterials functionalized with antimicrobial peptides (AMPs) for a wide variety of applications including implantable devices,1,2 biofilm prevention,3 drug and gene delivery,4,5 and biosensing.6−13 Key to effective use of any AMP-functionalized material is the ability to maintain AMP activitywhether that be recognition, binding, or a membrane-disruptive activity upon immobilization to a surface. Since small modifications in both the structure and amino acid sequence can alter an AMP’s affinity or specificity for its microbial target in solution (reviewed in refs 14−16), surface immobilization can similarly influence both the conformation and activity of an AMP,17−22 affecting the ultimate utility of the biomaterial or device. For sensing and antimicrobial applications, a critical requirement is the ability of immobilized AMPs to bind the appropriate microbes with broad but semiselective specificity while minimizing interactions with nonrelevant matrix or assay components. For this reason, a number of research groups have turned to covalent immobilization of AMPs using poly(ethylene glycol) (PEG)-based linking strategies.3,18,23−27 PEG-based materials are generally more hydrophilic than other commonly utilized linkers, thus creating a microenvironment where nonspecific binding (often driven by hydrophobic interactions) is decreased. Furthermore, PEG spacers may provide additional mobility to tethered AMPs, which may allow for multivalent interactions. Although a number of studies have shown improved bactericidal activity of AMP-functionalized surfaces when This article not subject to U.S. Copyright. Published XXXX by the American Chemical Society
Received: December 14, 2016 Revised: February 14, 2017 Published: March 2, 2017 A
DOI: 10.1021/acs.langmuir.6b04481 Langmuir XXXX, XXX, XXX−XXX
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Table 1. Peptides and Linkers Used for Immobilization peptide
MW
melittin-C27 histatin-C25 magainin 2-C24 tritrpticin-C14
2951 3140 2570 2005
sequence GIGAVLKVLTTGLPALISWIKRKRQQC DSHAKRHHGYKRKFHEKHHSHRGYC GIGKFLHSAKKFGKAFVGEIMNSC VRRFPWWWPFLRRC MW tether (approx. length of PEG)
linker N-(2-aminoethyl)maleimide (PEG0) maleimide-PEG400-amine (PEG400) maleimide-PEG600-amine (PEG600) maleimide-PEG1000-amine (PEG1000) maleimide-PEG2000-amine (PEG2000) maleimide-PEG3400-amine (PEG3400) maleimide-PEG5000-amine (PEG5K)
26 Da 400 Da (32 Å) 600 Da (50 Å) 1 kDa (82 Å) 2 kDa (160 Å) 3.4 kDa (273 Å) 5 kDa (401 Å)
available amines 4 5 5 1 no. ethylene glycol unitsa 0 9 14 23 45 77 113
a Each preparation is a mixture of different length polymers; the average calculated number of ethylene glycol units is shown. Per NANOCS, the MW of 95% of each preparation is within 10% of the indicated value.
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beads were added for 30 min followed by biotinylated-antibody specific for the target cells. The fluorescence signal after an initial interrogation with streptavidin-phycoerythrin (SA-PE) was amplified using biotinylated antistreptavidin and a second exposure to SA-PE. Signals were read on the Luminex 100. Net fluorescence was calculated as the difference in signals between the measured samples and notarget controls.
EXPERIMENTAL SECTION 18,22
Materials. Four AMPs with C-terminal cysteines were customsynthesized and purified to >90% purity by Biosynthesis, Inc. (Lewisville, TX, USA) (Table 1). Other reagents and chemicals were purchased from the following suppliers: maleimide-PEGn-amine linkers from NANOCS (New York, NY, USA; Table 1); 1-ethyl-3-[3(dimethylamino)propyl]carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (sulfo-NHS), cetyltrimethylammonium bromide (CTAB), and NHS-LC-LC-biotin from ThermoFisher Scientific (Rockford, IL, USA); N-(2-aminoethyl)maleimide trifluoro acetate (AEM), buffers, and detergents from Sigma/Aldrich (St. Louis, MO, USA); heat-inactivated Escherichia coli O157:H7 and anti-E. coli O157:H7 from KPL/SeraCare. (Gaithersburg, MD, USA); goat antiBacillus anthracis, rabbit anti-Yersinia pestis, and irradiated preparations of B. anthracis Sterne and Y. pestis CO-92 from the US Department of Defense Critical Reagents Program; biotinylated goat antistreptavidin from Vector Laboratories (Burlingame, CA, USA); and streptavidin-Rphycoerythrin (SA-PE) from Columbia Biosciences (Frederick, MD, USA). Antibodies were biotinylated by incubation for 30 min with a 5fold molar excess of biotin-LC-LC-NHS ester (in 0.1 M borate, pH 8.5) and separated from unincorporated biotin by Zeba columns (ThermoFisher). Preparation of Luminex Microspheres. Carboxy-functionalized MagPlex beads (25 μL; Luminex Corporation, Austin, TX, USA) were functionalized with antimicrobial peptides using linkers with various length maleimide-PEG-amine linkers as previously described.29 Briefly, carboxyl beads were first treated with EDC and sulfo-NHS (5 mg/mL each) for 30 min at pH 6, rinsed, and then incubated with 1 mg/mL maleimide-PEGn-amine linker (pH 6) for 2 h. After additional rinsing, beads were mixed with peptides (0.25 mg/mL in 0.1 M tricine, pH 7.4, 0.05% CTAB), incubated overnight, and subjected to a final rinse to remove nonimmobilized peptides. To estimate the relative density of attached peptides, peptide-functionalized beads were treated for 60 min with biotin-LC-LC-NHS at 1.9, 7.5, or 30 mg/mL in PBS, pH 7.3. After separation from unincorporated biotin, beads were washed with PBS, exposed sequentially to SA-PE (7.5 μg/mL in PBS), biotinylated goat antistreptavidin (1 μg/mL in PBS), and SA-PE (7.5 μg/mL in PBS, 15 min each), and then counted on the Luminex 100.30 For all wash steps, a magnetic plate (Biotek Instrument Model 96F, Winooski, VT, USA) was used. Previous controls ensured that the three biotin concentrations were sufficient and appropriate to compare the relative surface density of each AMP immobilized via the different PEG linkers (Figures S5 and S6). Immunoassays. Peptide-coated beads were used to capture bacterial cells, which were then detected via a MagPlex fluid array sandwich immunoassay using biotinylated antibodies and SA-PE to provide fluorescent signals.30 The full protocol can be found in the Supporting Information. Briefly, serial dilutions of bacterial cells were prepared in 96-well microtiter plates. To the cells, peptide-coated
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RESULTS AND DISCUSSION PEG-Linker Length Reduces Nonspecific Protein Binding but Does Not Necessarily Improve Assay Sensitivity. The ability to limit or prevent nonspecific adsorption in biosensors and other devices is critical for optimal sensor performance. For this reason, PEG polymer linkers have been used to reduce nonspecific protein binding, presumably through their high hydrophilicity. As expected, the no-target controls showed significantly lower nonspecific binding by the biotinylated antibodies and streptavidin constructs when longer chain PEG linkers were used to link melittin-C27, histatin-C25, and magainin 2-C24 to the microspheres (p < 0.05; Figures S1−S4). Above 1K linker lengths, there appeared to be no further decrease in nonspecific binding. For these three peptides, this trend was observed to be independent of the tracer antibody used and supports the common perception that PEG functionalization prevents nonspecific protein binding. However, it was surprising to find that nonspecific binding to surfaces functionalized with PEG0 linker did not differ from those functionalized with PEG400 and PEG600 linkers (p > 0.05). On the other hand, nonspecific protein adsorption to tethered tritrpticin-C14 was unaffected by linker length in any range (p > 0.05). The high proportion of tryptophan residues in tritrpticin-C14 may drive hydrophobic interactions with solution-phase proteins, leading to higher backgrounds. Figure 1 shows the net signal associated with binding of cells (107 cells/mL) to peptide-decorated microspheres. In the presence of target cells, increasing the PEG chain length was generally associated with reduced binding of target cells. Indeed, signals from Y. pestis (at any concentration) were not above negative control values for any of the peptides immobilized by PEG1000 and longer linkers (p > 0.07). This trend was also evident with E. coli O157:H7 and B. anthracis Sterne, although low (but detectable) signals were observed at high cell concentrations with some peptides (Figure 1; p < 0.045; Supporting Information Figure 1). We observed that B
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increasing densities of immobilized peptides with increasing linker length up to 25−40 ethylene glycol units, followed by a significant decrease in peptide density when immobilized via PEG3400 or PEG5000, independent of peptide (p < 0.03). Figure 3 shows results obtained when peptides were labeled with 7.5 μg/mL biotin; similar results were obtained when biotin-NHS was used at 1.9 and 30 μg/mL (Figures S5 and S6). These results are supported by simulations performed by Han,18 suggesting that peptides immobilized via PEG2000 linkers present lower effective surface concentrations than those immobilized via PEG linkers with n = 4 ethylene glycol units (the “spaghetti” effect). Although this effect may simply be due to a lower effective (versus total) density of the peptide component as linker lengths increase, others have observed that long PEG chains tend to fold up, sometimes burying attached ligands.33,34 While we did not test the PEG linker used by Han (4 ethylene glycol units),18 immobilization of magainin 2-C24, histatin-C25, and melittin-C27 using the shortest PEG linker tested here (PEG400, n = 9 ethylene glycol units) did not result in any significant difference in peptide density over those immobilized via PEG2000 linker (n = 45 ethylene glycol units; p > 0.06), indicating that the PEG2000-linked peptides were as accessible for labeling and SA-PE binding as those on a shorter tether. By contrast with the other peptides (and with the Han simulations), we did observe significantly higher densities of tritrpticin-C14 immobilized via PEG2000 linker than by PEG400 linker (p < 0.01). Interestingly, beads with the highest peptide density did not always correlate with the highest target binding. For example, histatin-C25, magainin 2-C24, and tritrpticin-C14 were immobilized at the highest densities using PEG1000 linker (Figure 3), but the same conditions resulted in low, if any, cell binding activities (Figure 1; Figure S2). To the contrary, a general trend was observed whereby all four peptides immobilized via PEG400 and PEG600 linkers had the highest target capture activity. While these linkers often conferred high peptide densities (Figure 3A−C), the relative difference in densities was not sufficient to account for the almost complete loss of activity when peptides were immobilized via shorter (e.g., N-(2-aminoethyl)maleimide) or longer (PEG2000, PEG3400, PEG5K) linkers (Figure 1). These results imply that linker-associated effects on peptide-target binding are likely due to presentation of the pendant peptides, with density effects playing a smaller role. Linker Length Effects on Binding Specificity Are Peptide- and Target-Dependent. Detailed dose−response curves generated for target binding to PEG400- and PEG600immobilized peptides showed semiselective target binding characteristics (Figure 2). For example, magainin 2-C24 immobilized using both PEG400 and PEG600 linkers was highly effective at capturing all three cell types, whereas high signals with histatin-C25 immobilized with the same linkers were obtained with B. anthracis only. This type of semiselective binding is desired for broad-spectrum detection, enabling broad bacterial categories to be detected and classified on the basis of the pattern of recognition/binding.13,35 In general, higher signals were observed with peptides immobilized using PEG600 linkers, although the relative magnitude of improvement varied depending on both the cell type and peptide used. Signals from E. coli O157:H7 binding to PEG600-linked magainin 2-C24 were twice those of E. coli bound to PEG400-linked magainin 2-C24 (gray curve in Figure 2, top two panels), but the difference for B. anthracis binding to
Figure 1. Net fluorescence obtained from capture of 107 cells/mL bacteria to antimicrobial peptides (abscissa) immobilized via PEGbased linkers with varying molecular weight tethers (indicated, top panel): (A) killed E. coli O157:H7; (B) killed B. anthracis Sterne; (C) killed Y. pestis CO-92. Each symbol represents the mean of n = 3 replicates ± SD.
AMPs immobilized by PEG400 or PEG600 linkers generated both the highest signals and the highest signal-to-noise ratios, suggesting that the optimal linker length was in this range. This was confirmed by more detailed dose−response curves of peptides immobilized via PEG400 and PEG600 linkers (Figure 2), which demonstrated that these higher signals translated into improved detection limits, calculated as the lowest concentration tested resulting in signals >3 standard deviations (SD) above negative control levels (Table 2, Table S1). Peptide Density Is Not Fully Responsible for Improved Detection Sensitivity. To ascertain whether improved detection sensitivity on shorter PEG linkers was due to surface peptide density, we biotin labeled the peptides’ available amines after immobilization; the theoretical number of available amines after immobilization was between 1 (Nterminus; tritrpticin-C14) and 5 (4 lysines + N-terminus; magainin 2-C24 and histatin-C25) (Table 1). The surface density of attached biotins was then estimated by capture of SA-PE. Given the size difference between the attached AMPs (2−3 kDa + PEG-linker) and SA-PE conjugate (>290 kDa), this method cannot precisely quantify the peptides. However, similar detection schemes have been used for qualitative comparisons of surface densities of immobilized peptides.31,32 Using amine-specific biotinylation and detection by SA-PE for qualitative assessment, we observed the overall trend of C
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Figure 2. Dose−response curves with net fluorescence from cell binding to melittin-C27 (blue circles), histatin-C25 (orange diamonds), magainin 2C24 (gray squares), and tritrpticin-C14 (yellow triangles) immobilized via PEG400 (left panels) or PEG600 linkers (right panels). Each symbol represents the mean of n = 3 replicates ± SD; error bars not visible are hidden behind the symbols.
Table 2. Limits of Detection (Colony Forming Units, CFU, per mL) Obtained with Peptides Immobilized via PEG400 and PEG600 Linkers PEG400 linker target AMP melittin-C27 histatin-C25 magainin 2-C24 tritrpticin-C14 a
E. coli O157:H7 9.8 6.3 2.4 2.5
× × × ×
PEG600 linker
B. anthracis Sterne
Y. pestis CO-92
× × × ×
1.0 × 107 nda 6.3 × 105 nd
103 105 103 106
6.3 3.9 3.9 3.9
105 104 104 104
E. coli O157:H7 9.8 2.4 2.4 3.9
× × × ×
104 103 103 104
B. anthracis Sterne
Y. pestis CO-92
× × × ×
6.3 × 105 6.3 × 105 2.5 × 106 nd
2.4 2.4 3.9 2.5
103 103 104 106
No binding above background was detected.
the same peptide was 4-fold (Figure 2, middle panels). Similarly, binding of these same cells to melittin-C27 or tritrpticin-C14 attached via PEG400 was minimally above background but large signals were observed when PEG600 was used (blue and yellow curves, respectively, in Figure 2, top four panels). On the other hand, binding of E. coli and B. anthracis to PEG400- or PEG600-linked histatin-C25 was not greatly affected, with similar signals observed for each species independent of linker (Figure 2, orange curves). Just as important, we were able to tune the specificity of some peptides
based on the linker used. While roughly equivalent signals were obtained with B. anthacis Sterne bound to PEG400immobilized magainin 2-C24 and histatin-C25, a clear preference for magainin 2-C24 was observed when PEG600 was used. Overall, our results suggest that binding of target cells by peptides immobilized by PEG-containing linkers is affected by both peptide density and presentation. The approximately 2fold higher densities of PEG400- and PEG600-linked peptides can potentially account for some differences in cell binding but D
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Figure 3. Relative density of peptides immobilized via PEG-based linkers of various lengths (abscissa), assessed by labeling with biotin-NHS at 7.5 μg/mL. Values on the ordinate represent net fluorescence units. (A) melittin-C27; (B) histatin-C25; (C) magainin 2-C24; (D) tritrpticin-C14. Each bar represents the average of n = 2 replicates ± SD.
movement required for peptide−peptide interactions mediating membrane disruption. This phenomenon may explain the higher binding of bacteria but decreased efficacy of killing, by shorter chain PEG-linked peptides observed by others.18 Others have observed a similar effect with liposome-based delivery vehicles coated with peptide-terminated PEG moieties;34,41 namely, the structure/function relationship of targeting peptides was negatively affected by PEG tethers that were too short or too long, with activity obtained with linkers of 6−18 ethylene glycol units, corresponding to our PEG400 and PEG600 linkers.
not to the magnitudes observed. For example, linking of histatin-C25 and magainin 2-C24 via PEG2000 resulted in approximately equivalent AMP densities as PEG600 but yielded little to no cell binding in comparison. Previous studies with antimicrobial peptides have demonstrated that immobilization with longer linkers typically confers enhanced antimicrobial activity.18−20,24,36 However, the ability of shorter PEG linkers to confer enhanced binding in the current study may be due to the peptides used, the surface to which they were attached, and the nature of the assay (binding versus killing). As one potential factor, long PEG-based linkers may interact directly with some AMPsas well as other peptideseven in the absence of surfaces, affecting membrane permeabilizing activity, if not initial membrane-binding interactions.37,38 These interactions may also cause peptides to be partially “buried” within the PEG layer, hindering accessibility.33 These effects can be peptide-, surface-, and linker-specific.34,39−41 As a second factor, we and others have previously observed that surface immobilization of AMPs with short tether length linkers can have significant effects on their baseline structures.18,42,43 Interactions of the peptides with the surface may constrain the peptides to secondary structures different from those typically assumed in solution, which will vary with the peptides used. While these new secondary structures do not necessarily inhibit their overall ability to interact with target cells or membranes, the mechanism of this interaction may indeed be modified.22 On the other hand, since peptide− peptide interactions are involved in membrane-mediated killing mechanisms postulated for all four of the peptides tested here, it could be that longer tetherswhile less effective at allowing peptide binding to cell surfacesprovide the freedom of
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CONCLUSION
This study provides an initial assessment of the effects of tether length on the ability of AMPs immobilized to microspheres through PEG-based linkers to capture microbial targets in solution. While higher densities of immobilized peptides were obtained with shorter PEG linkers, density alone was not fully responsible for the enhanced binding. Furthermore, while PEG400 and PEG600 linkers typically gave the highest signals and lowest detection limits for the three bacterial species tested, effects were specific to both the peptide and cell type tested. The ability of peptide immobilization via different length PEG linkers to enhance or diminish binding (and potentially killing) of target cells may translate into potentially useful ways to custom-tailor materials and platforms for various applications. Simple alterations in linking strategies, such as those described here, may allow similar surfaces to be adapted for different purposes, depending on the type of peptide activity desired. E
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(9) Kulagina, N. V.; Shaffer, K. M.; Ligler, F. S.; Taitt, C. R. Antimicrobial peptides as new recognition molecules for screening challenging species. Sens. Actuators, B 2007, 121 (1), 150−157. (10) Etayash, H.; Norman, L.; Thundat, T.; Kaur, K. Peptide-bacteria interactions using engineered surface-immobilized peptides from class IIa bacteriocins. Langmuir 2013, 29 (12), 4048−4056. (11) Etayash, H.; Norman, L.; Thundat, T.; Stiles, M.; Kaur, K. Surface-conjugated antimicrobial peptide leucocin A displays high binding to pathogenic Gram-positive bacteria. ACS Appl. Mater. Interfaces 2014, 6 (2), 1131−1138. (12) Silva, R. R.; Avelino, K. Y. P. S.; Ribeiro, K. L.; Franco, O. L.; Oliveira, M. D. L.; Andrade, C. A. S. Optical and dielectric sensors based on antimicrobial peptides for microorganism diagnosis. Front. Microbiol. 2014, 5, 443. (13) Kulagina, N. V.; Taitt, C. R.; Anderson, G. P.; Ligler, F. S. Affinity-based detection of biological targets. USPTO Patent no. 8,658,372 B2, Feb 25, 2014. (14) Fjell, C. D.; Hiss, J. A.; Hancock, R. E. W.; Schneider, G. Designing antimicrobial peptides: form follows function. Nat. Rev. Neurosci. 2012, 11 (1), 37−51. (15) Yeaman, M.; Yount, N. Mechanisms of antimicrobial peptide action and resistance. Pharmacol. Rev. 2003, 55, 27−55. (16) Aoki, W.; Ueda, M. Characterization of antimicrobial peptides toward the development of novel antibiotics. Pharmaceuticals 2013, 6, 1055−1081. (17) Li, Y.; Wei, S.; Wu, J.; Jasensky, J.; Xi, C.; Li, H.; Xu, Y.; Wang, Q.; Marsh, E. N. G.; Brooks, C. L.; Chen, Z. Effects of peptide immobilization sites on the structure and activity of surface-tethered antimicrobial peptides. J. Phys. Chem. C 2015, 119 (13), 7146−7155. (18) Han, X.; Liu, Y.; Wu, F.-G.; Jasensky, J.; Kim, T.; Wang, Z.; Brooks, C. L.; Wu, J.; Xi, C.; Mello, C. M.; Chen, Z. Different interfacial behaviors of peptides chemically immobilized on surfaces with different linker lengths and via different termini. J. Phys. Chem. B 2014, 118 (11), 2904−2912. (19) Costa, F.; Carvalho, I.; Montelaro, R.; Gomes, P.; Martins, M. C. L. Covalent immobilization of antimicrobial peptides (AMPs) onto biomaterial surfaces. Acta Biomater. 2011, 7, 1431−1440. (20) Onaizi, S. A.; Leong, S. S. Tethering antimicrobial peptides: current status and potential challenges. Biotechnol. Adv. 2011, 29 (1), 67−74. (21) Silva, R. R.; Avelino, K. Y.; Ribeiro, K. L.; Franco, O. L.; Oliveira, M. D.; Andrade, C. A. Chemical immobilization of antimicrobial peptides on biomaterial surfaces. Front. Biosci., Scholar Ed. 2016, 8, 129−142. (22) North, S. H.; Taitt, C. R. Application of circular dichroism for structural analysis of surface-immobilized cecropin A interacting with lipoteichoic acid. Langmuir 2015, 31 (39), 10791−10798. (23) Cleophas, R. T. C.; Sjollema, J.; Busscher, H. J.; Kruijtzer, J. A. W.; Liskamp, R. M. J. Characterization and activity of an immobilized antimicrobial peptide containing bactericidal PEG-hydrogel. Biomacromolecules 2014, 15, 3390−3395. (24) Bagheri, M.; Beyermann, M.; Dathe, M. Immobilization reduces the activity of surface-bound cationic antimicrobial peptides with no influence upon the activity spectrum. Antimicrob. Agents Chemother. 2009, 53 (3), 1132−41. (25) Bagheri, M.; Beyermann, M.; Dathe, M. Mode of action of cationic antimicrobial peptides defines the tethering position and the efficacy of biocidal surfaces. Bioconjugate Chem. 2012, 23 (1), 66−74. (26) Gabriel, M.; Nazmi, K.; Veerman, E. C.; Nieuw Amerongen, A. V.; Zentner, A. Preparation of LL-37-grafted titanium surfaces with bactericidal activity. Bioconjugate Chem. 2006, 17 (2), 548−550. (27) Peyre, J.; Humblot, V.; Méthivier, C.; Berjeaud, J.-M.; Pradier, C.-M. Co-grafting of amino−poly(ethylene glycol) and magainin I on a TiO2 surface: tests of antifouling and antibacterial activities. J. Phys. Chem. B 2012, 116 (47), 13839−13847. (28) Bagheri, M. Cationic antimicrobial peptides (AMPs): thermodynamic characterization of peptide−lipid interactions and biological efficacy of surface-tethered peptides. ChemistryOpen 2015, 4 (3), 389−393.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b04481. Table S1, limits of detection for bacteria binding to immobilized AMPs; protocol for MagPlex array sandwich immunoassay; Figure S1, total fluorescence of no-target controls; Figure S2, dose−response curves for E. coli O157 binding (all linkers); Figure S3, dose−response curves for B. anthracis binding (all linkers); Figure S4, dose−response curves for Y. pestis binding (all linkers); Figure S5, relative density of AMPs (biotinylated at 30 μg/mL); Figure S5, relative density of AMPs (biotinylated at 1.9 μg/mL) (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: 202-404-4208. ORCID
Chris Rowe Taitt: 0000-0002-0125-9239 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the Office of Naval Research and NRL internal funding (work unit 6547).
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REFERENCES
(1) Tan, X. W.; Goh, T. W.; Saraswathi, P.; Nyein, C. L.; Setiawan, M.; Riau, A.; Lakshminarayanan, R.; Liu, S.; Tan, D.; Beuerman, R. W.; Mehta, J. S. Effectiveness of antimicrobial peptide immobilization for preventing perioperative cornea implant-associated bacterial infection. Antimicrob. Agents Chemother. 2014, 58 (9), 5229−5238. (2) Gao, G.; Lange, D.; Hilpert, K.; Kindrachuk, J.; Zou, Y.; Cheng, J. T. J.; Kazemzadeh-Narbat, M.; Yu, K.; Wang, R.; Straus, S. K.; Brooks, D. E.; Chew, B. H.; Hancock, R. E. W.; Kizhakkedathu, J. N. The biocompatibility and biofilm resistance of implant coatings based on hydrophilic polymer brushes conjugated with antimicrobial peptides. Biomaterials 2011, 32 (16), 3899−3909. (3) Mishra, B.; Basu, A.; Chua, R. R. Y.; Saravanan, R.; Tambyah, P. A.; Ho, B.; Chang, M. W.; Leong, S. S. J. Site specific immobilization of a potent antimicrobial peptide onto silicone catheters: evaluation against urinary tract infection pathogens. J. Mater. Chem. B 2014, 2, 1706−1716. (4) Ahmad, A.; Ranjan, S.; Zhang, W.; Zou, J.; Pyykkö, I.; Kinnunen, P. K. J. Novel endosomolytic peptides for enhancing gene delivery in nanoparticles. Biochim. Biophys. Acta, Biomembr. 2015, 1848 (2), 544− 553. (5) Keohane, K.; Brennan, D.; Galvin, P.; Griffin, B. T. Silicon microfluidic flow focusing devices for the production of size-controlled PLGA based drug loaded microparticles. Int. J. Pharm. 2014, 467 (1− 2), 60−69. (6) Mannoor, M. S.; Zhang, S.; Link, A. J.; McAlpine, M. C. Electrical detection of pathogenic bacteria via immobilized antimicrobial peptides. Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (45), 19207−19212. (7) Kulagina, N. V.; Lassman, M. E.; Ligler, F. S.; Taitt, C. R. Antimicrobial peptides for detection of bacteria in biosensor assays. Anal. Chem. 2005, 77 (19), 6504−6508. (8) Kulagina, N. V.; Shaffer, K. M.; Anderson, G. P.; Ligler, F. S.; Taitt, C. R. Antimicrobial peptide-based array for Escherichia coli and Salmonella screening. Anal. Chim. Acta 2006, 575 (1), 9−15. F
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(29) Shriver-Lake, L. C.; Anderson, G. P.; Taitt, C. R. Oriented peptide immobilization on microspheres. Methods Mol. Biol. 2016, 1352, 183−197. (30) Anderson, G. P.; Taitt, C. R. Suspension microarray immunoassay signal amplification using multilayer formation. Sens. Lett. 2008, 6 (1), 213−218. (31) Inamori, K.; Kyo, M.; Nishiya, Y.; Inoue, Y.; Sonoda, T.; Kinoshita, E.; Koike, T.; Katayama, Y. Detection and quantification of on-chip phosphorylated peptides by surface plasmon resonance imaging techniques using a phosphate capture molecule. Anal. Chem. 2005, 77, 3979−3985. (32) Krishnamurthy, V. R.; Wilson, J. T.; Cui, W.; Song, X.; Yi, L.; Cummings, R. D.; Chaikof, E. L. Chemoselective immobilization of peptides on abiotic and cell surfaces at controlled densities. Langmuir 2010, 26 (11), 7675−7678. (33) Tirosh, O.; Barenholz, Y.; Katzhendler, J.; Priev, A. Hydration of polyethylene glycol-grafted liposomes. Biophys. J. 1998, 74, 1371− 1379. (34) Stefanick, J. F.; Ashley, J. D.; Kiziltepe, T.; Bilgicer, B. A systematic analysis of peptide linker length and liposomal polyethylene glycol coating on cellular uptake of peptide-targeted liposomes. ACS Nano 2013, 7 (4), 2935−2947. (35) Shriver-Lake, L. C.; North, S. H.; Dean, S. N.; Taitt, C. R. Antimicrobial peptides for detection and diagnostic assays. In Designing Receptors for the Next Generation of Biosensors; Piletsky, S. A., Whitcomb, M. J., Eds.; Springer: Heidelberg, Germany, 2013; pp 85−104. (36) Uzarski, J. R.; Mello, C. M.; Hittle, L. E.; Ernst, R. K. Exploring structure/function relationships of bacterial lipopolysaccharides and lipid A with man-made surfaces presenting antimicrobial peptides. Abstracts of Papers of the American Chemical Society 2014, 248. (37) Imura, Y.; Nishida, J.; Ogawa, Y.; Takakura, Y.; Matsuzaki, K. Action mechanism of tachyplesin I and effects of PEGylation. Biochim. Biophys. Acta, Biomembr. 2007, 1768, 1160−1169. (38) Imura, Y.; Nishida, M.; Matsuzaki, K. Action mechanism of PEGylated magainin 2 analog peptide. Biochim. Biophys. Acta, Biomembr. 2007, 1768, 2578−2585. (39) Morris, C. J.; Beck, K.; Fox, M. A.; Ulaeto, D. O.; Clark, G. C.; Gumbleton, M. PEGylation of antimicrobial peptides maintains the active peptide conformation, model membrane interactions, and antimicrobial activity while improving lung tissue biocompatibility following airway delivery. Antimicrob. Agents Chemother. 2012, 56, 3298−3308. (40) Han, E.; Lee, H. Effects of PEGylation on the binding interaction of magainin 2 and tachyplesin I with lipid bilayer surface. Langmuir 2013, 29 (46), 14214−14221. (41) Stefanick, J. F.; Ashley, J. D.; Bilgicer, B. Enhanced cellular uptake of peptide-targeted nanoparticles through increased peptide hydrophilicity and optimized ethylene glycol peptide-linker length. ACS Nano 2013, 7 (9), 8115−8127. (42) Li, Y.; Wei, S.; Wu, J.; Jasensky, J.; Xi, C.; Li, H.; Xu, Y.; Wang, Q.; Marsh, E. N. G.; Brooks, C. L. Effects of peptide immobilization sites on the structure and activity of surface-tethered antimicrobial peptides. J. Phys. Chem. C 2015, 119, 7146. (43) Gallardo, I. F.; Webb, L. J. Demonstration of α-helical structure of peptides tethered to gold surfaces using surface infrared and circular dichroic spectroscopies. Langmuir 2012, 28 (7), 3510−3515.
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DOI: 10.1021/acs.langmuir.6b04481 Langmuir XXXX, XXX, XXX−XXX
