Surface treatment using immobilized antimicrobial peptide mimetics

Abstract

A method for treating a surface containing a plurality of functional groups is provided, the method comprising reacting the plurality of functional groups with linking groups, thereby creating a surface containing a plurality of linking groups, and attaching a first peptoid to each of the plurality of linking groups, thereby obtaining a first treated surface.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 120,686, filed December 2, 2020, having the same title and having the same inventors, and which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE DISCLOSURE The present disclosure relates generally to antimicrobial surface treatments, and more specifically to antimicrobial surface treatments containing antimicrobial peptoid compositions. [Background technology]

[0003] Bacterial adhesion and colonization of implantable biomedical devices and subsequent infections account for 40–70% of hospital-acquired infections (HAIs) [JW Costerton, PS Stewart, EP Greenberg, Science 1999, 284, 1318–1322; E.M. Hetrick, M.H. Choenfisch, Chem. Soc. Rev. 2006, 35, 780–789; and C. Desrousseaux, V. Sautou, S. Descamps, O. Traore, J. Hosp. Infect. 2013, 85, 87–93]. Water purification systems, food packaging, and offshore operations can also be compromised by microbial contamination. [R. Chmielewski, J. Frank, Compr. Rev. Food Sci. Food Saf. 2003, 2, 22-32; X. Z. Zhao, C. J. He, ACS Appl. Mater. Interfaces 2015, 7, 17947-17953; and D. W. Wang, X. Wu, L. X. Long, X. B. Yuan, Q. H. Zhang, S. Z. Xue, S. M. Wen, C. Y. Yan, J. M. Wang, W. Cong, Biofouling 2017, 33, 970-979]. Despite substantial research, preventing bacterial adhesion and growth on surfaces remains challenging. [C. D. Nadell, K. Drescher, K. R. Foster, Nat. Rev. Microbiol. 2016, 14, 589]. Surface properties such as roughness and topology, chemistry and wettability, and surface molecular configuration are representative of many factors that affect biofouling.[K. Bazaka, R. J. Crawford, E. P. Ivanova, Biotechnol. J. 2011, 6, 1103 - 1114, D. Perera - Costa, J. M. Bruque, M. L. Gonzalez - Martin, A. C. Gomez - Garcia, V. Vadillo - Rodriguez, Langmuir 2014, 30, 4633 - 4641, K. W. Kolewe, J. Zhu, N. R. Mako, S. S. Nonnenmann, J. D. Schiffman, ACS Appl. Mater. Interfaces 2018, 10, 2275 - 2281, and A. Hasan, S. K. Pattanayek, L. M. Pandey, ACS Biomater. Sci. Eng. 2018, 4, 3224 - 3233].

[0004] Proposed strategies to overcome bacterial surface fouling include "antifouling" coatings that inhibit nonspecific protein adsorption and bacterial adhesion, such as by surface grafting of poly(ethylene glycol) (PEG) as polymer brushes. [C. Blaszykowski, S. Sheikh, M. Thompson, Chem. Soc. Rev. 2012, 41, 5599-5612; AD White, A.K. Nowinski, W. Huang, A.J. Keefe, F. Sun, S. Jiang, Chem. Sci. 2012, 3, 3488-3494; and S. Lowe, N.M.O. Brien-Simpson, LA Connal, Polym. Chem. 2015, 6, 198-212]. Other strategies include immobilizing existing antibiotics and antibiotic-releasing coatings. [F. Costa, IF Carvalho, RC Montelaro, P. Gomes, MC L Martins, Acta Biomater. 2011, 7, 1431-1440; A. Andrea, N. Molchanova, H. Jenssen, Biomolecules 2018, 8, 27; and SR Palumbi, Science 2001, 293, 1786-1790]. However, many existing antibacterial agents suffer from a narrow spectrum of activity and the development of resistance to their activity [A. Andrea, N. Molchanova, H. Jenssen, Biomolecules 2018, 8, 27; and SR Palumbi, Science 2001, 293, 1786-1790]. To overcome these problems, antimicrobial peptides (AMPs) have been investigated [see Costa et al. and Andrea et al. above]. However, they are degraded by proteases secreted by both the human host and bacteria.[N.Molchanova,PRHansen,H.Franzyk,Molecules 2017,22,1430, M.Sieprawska-Lupa,P.Mydel,K.Krawczyk,K.Wojcik,M.Puklo,B.Lupa,P.Suder,J.Silberring,M.Reed,J.Pohl,Antimicrob.Agents Chemother.2004, 48, 4673-4679, and M. Xiao, J. Jasensky, J. Gerszberg, J. Chen, J. Tian, ​​T. Lin, T. Lu, J. Lahann, Z. Chen, Langmuir 2018, 34, 12889-12896].

[0005] Poly(N-substituted glycine) "peptoids" represent a promising class of peptide mimetics developed to address the shortcomings of AMPs. They have a non-natural polyglycine backbone with side chains attached to the backbone amide nitrogen atom, providing protease resistance and enhanced lipid membrane permeability. [KH A Lau, Biomater. Sci. 2014, 2, 627-633, and A.S.Knight, E.Y.Zhou, M.B.Francis, R.N.Zuckermann, Adv. Mater. 2015, 27, 5665-5691]. Secondary structure is induced by specific sequences with specific side chains. [See Knight et al., supra, and M.El Yaagoubi, K.M.Tewari, KH A Lau in Self-assembling Biomaterials, Elsevier-Woodhead, Amsterdam, 2018, pp. 95-112]. Several groups have demonstrated highly active peptoid AMP mimetics [see Andrea et al. and Malchanova et al., supra; see also NP Hongsiriwatana, JAPatch, AMCzyzewski, MTD Ohm, A. Ivankin, D. Gidalevitz, R.N. Zuckermann, A.E. Barron, Proc. Natl. Acad. Sci. USA 2008, 105, 2794-2799, and JA Patch, A.E. Barron, J.Am. Chem. Soc. 2003, 125, 12092-12093]. One such peptoid was synthesized as part of a surface-grafted peptoid brush, and high levels of overall bacterial adhesion were observed [A.R. Statz, J.P. Park, NP Hongsiriwatana, A.E. Barron, P.B. Messersmith, Biofouling 2008, 24, 439-448]. Natural AMPs, such as hLf1-11, LL-37, and melamine, have also been immobilized with varying results. [See Costa et al., Andrea et al., and Xiao et al., supra. See also J. He, J. Chen, G. Hu, L. Wang, J. Zheng, J. Zhan, Y. Zhu, C. Zhong, X. Shi, and S. Liu, J. Mater. Chem. B 2018, 6, 68-74.] These studies apply bioconjugation techniques, such as maleimide-thiol, amide, and alkyne-azide "click" coupling, to enable covalent surface immobilization. Alkyne-azide coupling is particularly advantageous because it is orthogonal to reactive groups commonly found on AMPs, but the approach is often limited by the availability of specialized chemical linkers. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] JWCosterton,PSStewart,EPGreenberg,Science 1999,284,1318-1322 [Non-licensed document 2] EMHetrick,MHSchoenfisch,Chem.Soc.Rev.2006,35,780-789 [Non-licensed document 3] C.Desrousseaux,V.Sautou,S.Descamps,O.Traore,J.Hosp.Infect.2013,85,87-93

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[0007] In one aspect, an engineered surface is provided that includes a substrate and a first peptoid attached to the substrate by a linking group.

[0008] In another aspect, a method is provided for treating a surface containing multiple functional groups, the method comprising reacting the multiple functional groups with linking groups, thereby creating a surface containing multiple linking groups, and attaching a peptoid to each of the multiple linking groups. [Brief explanation of the drawings]

[0009] [Figure 1] The chemical structure of the (kss)4 antibacterial sequence and its C and N modifications are shown. B) Chemical structure of the modified sequence for CuAAC "click" coupling. Red circles are used in Figure 2A. [Figure 2] Surface modification schemes for the generation of PEG-tethered (kss)4 (i.e., Scheme A: GOPTS-PEG-N3-(kss)4) and (kss)4 directly immobilized on a surface (i.e., Scheme B: APTMS-N3-(kss)4). B) Water contact angles measured after successive modification steps. [Figure 3]High-resolution C1s (A) and N1s (B) XPS spectra after each surface modification step to obtain GOPTS-PEG-N3-(kss)4. [Figure 4] A series of confocal microscopy images of live (green) and dead / damaged (red) P. aeruginosa on: A) unmodified glass, B) APTMS, C) APTMS-N3-(kss)4, and D) GOPTS-PEG-N3-(kss)4, and E) quantified adhesion data corresponding to confocal measurements. Both actual coverage (q coverage) and coverage normalized by adhesion on unmodified glass (q norm) are shown, with # and ## indicating p<0.005 and p<0.05, respectively (one-way ANOVA). [Figure 5] (A) A series of graphs showing viable bacterial adhesion normalized by levels on unmodified substrates (glass or Ti), and B) the ratio of dead / damaged bacterial adhesion to viable adhesion shown in (A). The inset displays the original dead adhesion data. Open boxes (&) indicate the present study of P. aeruginosa. Other symbols represent literature data for P. aeruginosa (&), E. coli (*), S. aureus (^), L. salivarius (~), and S. sanguinis (*). Adhesion was measured either by imaging stained cells or by reculturing the adherent bacteria. [Figure 6] 1 is a graph of the ratio of dead / injured bacteria to viable adherents as a function of antimicrobial peptoid (AMP) release. [Figure 7] (A) A series of graphs of normalized viable attachment as a function of AMP separation, and (B) normalized dead attachment as a function of AMP separation. [Figure 8] FIG. 1 is a series of graphs of normalized surface coverage of peptoids on different surfaces showing the relative amount of dead / injured bacteria ( P. aeruginosa ) versus viable attachment for (A) 2k PEG and (B) 20k PEG tethers. [Figure 9]FIG. 1 is a series of graphs of normalized surface coverage of peptoids on different surfaces showing the relative amount of dead / injured bacteria (S. aureus) versus viable attachment for (A) 2k PEG and (B) 20k PEG tethers. [Figure 10] 1 is a graph of normalized surface coverage of peptoids on different surfaces, showing the relative amount of dead / injured bacteria (P. aeruginosa) versus viable adhesion. [Figure 11] 1 is a graph of normalized surface coverage of peptoids on different surfaces, showing the relative amount of dead / injured bacteria (S. aureus) versus viable adhesion. [Figure 12] 1 is a series of photomicrographs showing live / dead bacterial counts for unmodified surfaces compared to the same surfaces modified with various linked AMPs of the type disclosed herein. DETAILED DESCRIPTION OF THE INVENTION

[0010] In this study, we use the 12-mer (Nlys-Nspe-Nspe)4 antimicrobial peptoid with an amphiphilic helical structure, first reported by Barron et al.

[11] , as a model AMP mimic to investigate the influence of immobilization design on surface antimicrobial activity. First, we tested the effect of modifying the N- and C-termini of the peptoid with diethylene glycol segments on its minimum inhibitory concentration (MIC) in solution. Next, we demonstrated the conversion of surface-immobilized amines to azides for copper(I)-catalyzed alkyne-azide cyclic coupling (CuAAC) surface coupling of the peptoid, with or without a 2 kDa polyethylene glycol (PEG) tether. We characterized the surface modification step by water contact angle (WCA) analysis and X-ray photoelectron spectroscopy (XPS), and finally assayed the surfaces for protein adsorption and live / dead bacterial adhesion. We hypothesized that sufficient spatial separation between AMPs, and therefore flexibility in molecular configuration such as that allowed by the PEG tether, is necessary to resist bacterial adhesion and retain antibacterial activity on the surface.

[0011] The (Nlys-Nspe-Nspe) parent sequence consists of a repeating "kss" motif, where k and s are the Lys analog N-(4-aminobutyl)glycine (Nlys) and the a-chiral (S)-N-(1-phenylethyl)glycine (Nspe), respectively (Figure 1A). This sequence represents a typical ABB trimer motif, where A is cationic and B is hydrophobic (with Nspe often inducing helicity). Peptoid synthesis was performed using the well-established "submonomer" solid-phase synthesis (SSPS) method [9b], and all sequence modifications were performed on-resin using commercially available building blocks (see ESI). HPLC and LC-MS characterization of the purified sequence are shown in Figures S1 and S2.

[0012] First, we investigated whether the C-terminal amide or the N-terminal amine of (kss)4 was important for its bactericidal effect. 7 CFUmL -1(kss) was incubated in growth medium modified with a diethylene glycol (EG2) linker at either the C- or N-terminus to yield (kss)4-EG2 and EG2-(kss)4, respectively (Figure 1A). The EG2 linker was later used to space (kss)4 from the surface coupling group (see below). Similar MICs were observed with and without terminal modification for the Gram-negative and Gram-positive strains tested (i.e., 16–20 mm against Pseudomonas aeruginosa (PA01), 5–9 mm against Escherichia coli (ATCC25922), and 1–6 mm against Staphylococcus aureus (NCTC4135; see Figure S3 for complete data). A previous report modifying the N-terminus of (kss)4 with a small molecule metal chelator also showed little change in the MIC against E. coli

[14] . Another report of C-terminal modification of a peptoid similar to (kss)4 showed a 2- to 10-fold decrease in activity (i.e., increased MIC) of the nonfunctional 20-residue peptoid, depending on the bacterial strain

[12] . Overall, the data suggest that the N- and C-terminal structures of the peptoid are not essential for activity, but that the steric nature of the modifications may be important. For surface immobilization, we further modified the C-terminus with a residue bearing a pentyne side chain using the regular peptoid SSPS to generate (kss)4-EG2-pentyne (Figure 1B). In parallel, we modified the diamino-PEG-2 pentyne according to established protocols (see ESI). 2k Glass slides were prepared (Figure 2A) that were either silanated with (3-glycidyloxypropyl)trimethoxysilane (GOPTS)

[15] or simply with (3-aminopropyl)trimethoxysilane (APTMS)

[16] . The terminal amines on both surfaces were then converted to azides by overnight incubation with imidazole-1-sulfonyl azide (see ESI).

[17] This allowed for the CuAAC coupling of (kss)4-EG2-pentyne

[18] to PEG. 2kPeptoid-functionalized surfaces with and without tethers were performed, i.e., Scheme A: GOPTS-PEG-N3-(kss)4 and Scheme B: APTMS-N3-(kss)4, respectively, in Figure 2A.

[0013] Figure 2B shows water contact angle (WCA) data, which is consistent with the expected change in surface wettability after each modification step. For Scheme A, the WCA increases with the first GOPTS modification (organosilanes are more hydrophobic than glass), then with diamino-PEG. 2K The WCA decreased after coupling (PEG-amine is hydrophilic). Subsequent conversion of the PEG-terminal amine to a non-cationic azide (N3), followed by CuAAC coupling of (kss)4 bearing multiple hydrophobic Nspe groups, resulted in a continuous increase in WCA. Similarly, for Scheme B, a continuous increase in WCA was observed after silanization of glass with the organosilane APTMS and finally functionalization with (kss)4.

[0014] The surface modification was further confirmed by XPS. In the C1s spectrum (Figure 3A), a peak appeared at 286.5 eV after GOPTS silanization, indicating the expected addition of a CO bond in the epoxide group. PEG attachment was verified both by the further increase of this CO peak, resulting from the presence of an ether bond in the PEG, and by the appearance of an N1s NC peak (401 eV) arising from the terminal amine of the PEG used (Figure 3B). Subsequent azide derivatization was confirmed by the addition of a N-N=N bond at 402.3 eV. - The final peptoid coupling was confirmed by the appearance of peaks due to (kss)4:C1s CC (284.8 eV) and amide (288.3 eV),

[19] and N1s NC=O (399.5 eV) and NH2 (400.8 eV).

[20] By analyzing the decay of the Si2p signal from the SiO2 substrate, the final peptoid surface density was estimated to be 0.3 chains / nm2. 2 (Table S1 and related discussion).

[0015] The PEG tethers of the present invention essentially form polymer brushes, conferring resistance to nonspecific biomolecular adsorption and thus reducing bacterial adhesion. [5a, c] For initial evaluation of this antifouling property, GOPTSPEG-N3-(kss)4 samples were incubated in 10% FBS (2 h, RT). Following established protocols

[21] , ellipsometry measurements showed little change in adlayer thickness before and after incubation (Figure S4: 3.5: 0.6 nm vs. 3.7: 0.5 nm, n = 3), indicating less protein adsorption on the PEG-tethered peptoid surface.

[0016] Next, we focused on evaluating the antibacterial activity of peptoid-functionalized surfaces against P. aeruginosa (PA01) due to its high relevance in HAI and the associated risk of biofilm formation

[22] . Figure 4A-D shows typical images of adherent, live and dead / injured bacteria stained by Syto 9 and propidium iodide (PI), respectively, after a 24-hour adhesion assay (5 V 10 CFU mL at 1,378 °C). Figure 4E shows this data in terms of the actual surface coverage (θ coverage ) and normalized coverage (θ norm ; relative to the unmodified glass control).

[0017] On unmodified glass, relatively high survival of P. aeruginosa θ coverage =10.5%

number

[0018] In the present invention, further control was achieved by using APTMS-modified glass, which yielded an amine-terminated surface (Figure 2A, Scheme B, step [i]), mimicking the positive charge expected on the (kss)4 surface. Figures 4B and E show the θ coverage = 6.5%, consisting mainly of viable bacteria. This adhesion level was significantly higher than that of the control (θ norm = 0.62), a phenomenon occasionally observed on aminosilane surfaces (Figure S5).

[23] However, it was about three times higher than on (kss)4-functionalized surfaces. This suggests the role of antimicrobial sequences in inhibiting adhesion, despite their cationic nature, which may be related to the ability of similarly short surface-grafted peptoids to resist biofouling. [21a, b] A minor fraction of dead / damaged adherent cells (θ norm-dead = 0.06), a role for electrostatic surface adhesion that impairs the fluidity and integrity of the bacterial membrane is possible.

[0019] We also performed adhesion assays with E. coli (ATCC 25922), but little adhesion was observed, and no statistically significant data were obtained. It is possible that some separation occurred under our conditions. Nevertheless, based on the lower MICs measured for our modified peptides against E. coli (and S. aureus) than P. aeruginosa (Figure S3), we expect that GOPTS-PEG-N3-(kss)4 surface modification will be effective against these strains. Overall, our PEG-tethered peptoid results were characterized by low viable bacterial adhesion and a high percentage of dead / damaged cells. An immobilization (lateral) density of 0.3 chains / nm2 (see XPS analysis) implies a maximum "volumetric" separation of approximately 5 nm between immobilized (kss)4 sequences, considering the flexibility of both lateral and vertical movement allowed by the 20 nm contour length of PEG N3-(kss)4 (see ESI for calculations). This corresponds to the average molecular separation found in a 25 mM solution, which is orders of magnitude greater than the MIC of (kss)4. Thus, surface immobilization can generate very high local concentrations of AMPs.

[0020] Indeed, previous studies have focused on increasing the immobilization density of AMPs. [12,23-25] However, AMPs generally possess hydrophobic and cationic groups, both of which promote undesirable bacterial adhesion. Plotting the results alongside previous studies (see ESI) where data for calculating AMP separations was available shows many reports of high viable attachment, particularly with relatively short AMP separations (i.e., high AMP density) (Figure 5A). Because silanization provides a high density of surface coupling groups, direct immobilization on silanized surfaces generally results in the shortest separations. Tethering AMPs at the tips of polymer brushes, including our design, generally increased separation because the polymer chains prevent close packing and allow lateral and vertical movement around the anchor points of the polymer tether. However, some studies attached multiple AMPs along the length of the polymer chain to increase immobilization density [23b,24], which reduced AMP separation. Overall, Figure 5A demonstrates that increasing AMP separation can reduce viable attachment, despite the diverse bacterial species and assay protocols investigated. Furthermore, PEG 2k Our design, coupling a single AMP to the ATP-binding protein, provided the lowest attachment with the greatest AMP separation. This reduced fouling was supported by the observed low FBS adsorption (Figure S6).

[0021] Turning to damaged / dead bacterial adhesion, the inset in Figure 5B shows that AMPs attached at intermediate (3-4 nm) separations on the brush exhibited the highest apparent surface activity (i.e., highest dead adhesion). This is consistent with the hypothesis that polymer tethers can provide flexibility in molecular arrangement and orientation for enhanced membrane interactions. However, attached dead bacteria can still lead to biofilm formation and acute immune responses. Figure 5B plots the same data compared to live adhesion, highlighting cases with low overall adhesion and relatively high activity. This reveals a remarkable correlation between increasing AMP separation and relative activity, despite the diverse experiments compared. Indeed, while the APTMS-N3-(kss)4 design exhibited low relative activity, similar to the other silane surfaces, the GOPTS-PEG-N3-(kss)4 brush design not only exhibited the highest relative activity, but also the highest separation (5 nm). Naturally, one might expect relative activity to decrease at very large AMP separations, implying that AMPs at very low densities are insufficient to disrupt bacterial membranes. Therefore, to demonstrate optimal relative activity, an intermediate AMP separation should be present.

[0022] In conclusion, we have demonstrated that a model antimicrobial peptoid AMP mimic is amenable to modification at both its C- and N-termini and a one-step protocol for introducing azide termini to amino-functionalized surfaces for CuAAC "click" surface coupling. These demonstrations enable the study of AMP immobilization designs, showing that surface activity is strongly enhanced by the polymer (PEG2k) tether, consistent with the importance of spatial flexibility and vertical reach for favorable surface interactions with bacteria. Furthermore, we introduce AMP separation as a new parameter for characterizing the anti-biofouling properties of immobilized AMPs. This parameter highlights the extremely high local AMP concentrations achieved by surface immobilization. Furthermore, compared with literature data, we reveal a strong correlation between increased AMP separation and increased relative surface activity, indicating a higher proportion of dead / injured bacteria exhibiting low levels of adhesion. Indeed, our PEG-coupling design demonstrated the greatest AMP separation and the highest relative activity. Therefore, our results highlight the possibility of optimizing AMP separation, rather than immobilization density, to enable both reduced surface activity and reduced bacterial adhesion.

[0023] In some embodiments of the products and methodologies disclosed herein, the treated peptoid-containing surface can be derived from a surface containing suitable functional groups. Such functional groups can contain oxygen, nitrogen, sulfur, phosphorus, boron, or metals. Such metals can include, for example, Mg, Li, Cu, and Al. Examples of such functional groups include, but are not limited to, hydroxyl groups, carbonyl groups, aldehydes, haloformyls, carbonate esters, carboxyl, carboalkoxyl, methoxy, hydroperoxy, peroxy, ethers, hemiacetals, hemiketals, acetals, orthoesters, metylenedioxy, orthocarbonate esters, and carboxylic acid anhydride groups; carboxamides, primary amines, secondary amines, tertiary amines, ammonium, primary ketimines, secondary ketimines, primary aldimines, secondary aldimines, imides, azides, azos, cyanates, isocyanates, nitrates, nitriles, isonitriles, nitrosoxy, nitro, nitroso, oximes, pyridyls, and caprylic / caprylic acid groups. Included may be carbamate groups; sulfhydryl, sulfide, disulfide, sulfinyl, sulfonyl, sulfino, sulfo, thiocyanate, isothiocyanate, carbonothioyl, carbothioic S-acid, carbothioic O-acid, thiol ester, thionoester, carbodithioic, and carbodithio groups; phosphino, phosphono, and phosphate groups; borono, O-[bis(alkoxy)alkylboronyl], hydroxyborino, and O-[alkoxydialkylboronyl] groups; alkyllithium, alkylmagnesium halide, alkylaluminum, and silyl ether groups; alkenes and alkynes; and groups containing one or more radicals, such as, for example, carboxylic acid acyl radicals.

[0024] In some embodiments of the products and methods disclosed herein, the engineered peptoid-containing surface may comprise two or more peptoids, which may be different. For example, such a surface may be derived by creating a surface containing a first tethered peptoid and applying a second peptoid to the surface that binds to the first peptoid via covalent, ionic, hydrogen, or van der Waals forces.

[0025] The above description of the present invention is illustrative and not intended to be limiting. It should be understood, therefore, that various additions, substitutions, and modifications can be made to the above-described embodiments without departing from the scope of the present invention. Accordingly, the scope of the present invention should be interpreted with reference to the appended claims. For convenience, some features of the claimed invention may be set forth separately in certain dependent or independent claims. However, it should be understood that these features can be combined in various combinations and subcombinations without departing from the scope of the present disclosure. By way of example, and not limitation, the limitations of two or more dependent claims may be combined with each other without departing from the scope of the present disclosure.

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Claims

1. A treated surface, comprising: A substrate; A polymer brush; a plurality of poly(N-substituted glycines) having antibacterial activity; wherein the poly(N-substituted glycine) is tethered to the substrate by the polymer brush, and the poly(N-substituted glycine) tethered to the substrate is attached with a separation of 1-20 nm.

2. The treated surface of claim 1, wherein the polymer brush comprises a polyethylene glycol (PEG) polymer.

3. The treated surface of claim 2, wherein the polyethylene glycol polymer is 2k PEG or 20k PEG.

4. The treated surface of any of the preceding claims, wherein the substrate is a glass substrate.

5. The treated surface of any of the preceding claims, wherein each of the poly(N-substituted glycines) tethered to the substrate is attached with a separation of at least about 2 nm.

6. The treated surface of claim 1, wherein the poly(N-substituted glycine) tethered to the substrate is attached with a separation of 3-4 nm.

7. The treated surface of claim 1, wherein the poly(N-substituted glycine) tethered to the substrate is attached with a separation of less than about 5 nm.

8. The treated surface of claim 1, wherein the poly(N-substituted glycine) tethered to the substrate is attached with a separation of less than about 10 nm.

9. The treated surface of claim 1, wherein the poly(N-substituted glycine) tethered to the substrate is attached with a separation of about 20 nm.

10. The treated surface of any preceding claim, wherein the antimicrobial activity comprises activity against one or more of P. aeruginosa, E. coli, or S. aureus.

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