Peptoid-loaded microgel-modified substrates for resistance to bacterial colonization

Abstract

A method is provided for treating a surface of a biomedical device. The method comprises depositing a polyanionic microgel on a surface of the biomedical device and loading the deposited polyanionic gel with a peptoid. In one aspect, a method is provided for treating a surface of a biomedical device. The method comprises depositing a polyanionic microgel on a surface of the biomedical device and loading the deposited polyanionic microgel with a peptoid. In another aspect, a biomedical device is provided that includes a surface selected from the group consisting of a metal surface, a ceramic surface, and a polymeric surface. A polyanionic microgel is disposed on the surface and a peptoid is disposed within the polyanionic microgel.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 214,782 (Libera et al.), entitled "SUBSTRATES MODIFIED WITH PEPTOID-LOADED MICROGELS FOR RESISTANCE TO BACTERIAL COLONIZATION," filed June 25, 2021, and incorporated herein by reference in its entirety.

[0002] Government Rights This invention was made with government support under Grant #W911NF2010277 awarded by the Army Research Office, Grant #DMR-1608406 awarded by the National Science Foundation, Grant #1DP1 OD029517-01 awarded by the US Public Health Services, and Grant No. DE-AC02-05CH11231 awarded by the US Department of Energy.

[0003] The present disclosure relates generally to surface treatments, and more specifically to methods of rendering substrates resistant to bacterial colonization through treatment with peptoid-loaded microgels. [Background technology]

[0004] Although surgical operating rooms (ORs) are commonly referred to as being sterile or aseptic, in reality, the OR atmosphere contains microorganisms from many different sources. Such sources include ventilation systems, shedding from clothing, sneezing or coughing by OR personnel, and pedestrian traffic [1]. These airborne bacteria can precipitate directly onto the surfaces of implantable biomedical devices when the devices are removed from their sterilized packaging. Early measurements [2] have shown that the number of microorganisms present is approximately 3 × 106 CFU / m 2 With improved sanitation practices, the settling rate steadily decreased to about 10 3 ~10 4 CFU / m 2 The sedimentation rates were reduced to 10-20 h [3]. However, these sedimentation rates suggest that even under these improved conditions, devices are often contaminated with hundreds or thousands of bacteria after implantation. Once implanted and exposed to favorable growth conditions in the body, a subset of these bacteria can develop into a biofilm and cause chronic device-associated infections.

[0005] A variety of implantable devices, such as hip / knee prostheses [4], heart valves [5], pacemakers [6], cochlear implants [7], shunts [8], surgical meshes [9], sutures

[10] , and tissue engineered constructs

[11] , are susceptible to device-associated infection. It is well established that the incidence of surgical site infection increases linearly with time in the OR

[12] , and there is strong consensus that intraoperative contamination is responsible for at least some device-associated infections

[13] . Summary of the Invention [Means for solving the problem]

[0006] In one aspect, a method for treating a surface of a biomedical device is provided, the method comprising depositing a polyanionic microgel onto the surface of the biomedical device and loading the deposited polyanionic microgel with a peptoid.

[0007] In another aspect, a biomedical device is provided that includes a surface selected from the group consisting of a metallic surface, a ceramic surface, and a polymeric surface, a polyanionic microgel disposed on the surface, and a peptoid disposed within the polyanionic microgel.

[0008] In a further aspect, a self-protective surface is provided that includes a substrate, a polyanionic microgel disposed on the substrate, and a peptoid disposed within the polyanionic microgel.

[0009] In another aspect, a biomedical implant is provided, the biomedical implant comprising a substrate and a release surface disposed on the substrate, the release surface releasing a peptoid in response to the presence of a pathogen, the release surface comprising a 3D crosslinked colloidal structure.

[0010] In a further aspect, a method is provided for treating a surface of a biomedical implant to render it resistant to bacterial culture, the method comprising: (a) priming the surface of the implant with poly(allylamine hydrochloride), thereby obtaining a primed surface, (b) applying a submonolayer of poly(acrylic acid) (PAA) microgel to the primed surface, thereby obtaining a 3D crosslinked colloidal structure, and (c) loading the colloidal structure with a peptoid by forming a complex between the PAA microgel and the peptoid. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows the molecular structure of TM1, a 12-meric antibacterial peptoid with 5 positive charges at physiological pH (circles). [Diagram 2] Figure 2A: Graph showing time resolved change in microgel diameter indicating TM1 loading (1 mg / mL TM1 in 0.01 M phosphate buffer) by complexation within PAA microgels. Inset optical micrographs show the same set of hydrated microgels before / after loading. Figure 2B: Graph showing lack of change in microgel diameter indicating that complexed TM1 remains sequestered within PAA microgels upon exposure to PBS containing no TM1. Sequestration is maintained for 4 weeks (inset) with daily replacement of PBS. Each data point / error bar represents the mean / standard deviation of n=5 measurements. [Diagram 3]Figure 1 illustrates the fouling resistance of Ti rods subjected to each of four different surface treatments. The top of the figure is a series of schematic diagrams of the four surface treatments applied to the Ti rods. The bottom of the figure is a series of SEM images of the surfaces of rods modified with unfilled PAA microgels (bottom left) and TM1-filled PAA microgels (bottom right). [Figure 4] Results of the colony formation resistance assay are shown. The top left image shows a TSA plate assayed for culturable colonies on Ti-A (unmodified) rods. The white arrow indicates the rod after 4 rotations on the agar surface. SEM images of Ti-A rods (bottom left) after incubation show substantial MSSA colony formation. The corresponding images of Ti-D rods (TM1 loaded microgel modified) show no culturable colonies. The Ti-D SEM images show that the loaded microgels remain intact on the rod surface, but no MSSA is present. Images of both MSSA and S. epidermidis contamination for all four (A-D) surface modifications are presented as Figures 10-11. The right panels quantify the total number of loosely bound and tightly attached MSSA and S. epidermidis CGUs for each of the four conditions. Means are shown and error bars represent standard deviation of n=3 samples. * indicates p<0.05, ** indicates p<0.005. [Diagram 5] Results of a cytocompatibility assay using human fetal osteoblasts are shown. Metabolic activity of hFOB cells, averaged per surface treatment, normalized to its day 1 value. Asterisks indicate statistically significant differences (t-test at p<0.05) between fully modified Ti-D and Ti-A unmodified controls. (Bottom) Confocal fluorescence images (DAPI / Phalloidin staining) of hFOB cells grown on Ti rods with each of the four surface treatments (scale bar is 100 μm). [Figure 6]We show that many of the imaging complications associated with the curved and rough surfaces of Ti rods can be avoided by the use of glass substrates. A series of confocal images shows hFOB cells cultured on unmodified glass (top left) and TM1-loaded microgel-modified glass (top right) after 7 days of culture. The white arrow (top right) indicates autofluorescence from the TM1-loaded microgel. The bottom SEM image shows that hFOB cells are able to spread on the TM1-loaded microgel. [Figure 7] FIG. 11 is a size distribution histogram of PAA microgel diameters when hydrated in 0.01 M phosphate buffer (pH 7.4, [Na+]=0.016 M). The solid line represents a Gaussian distribution fitted to the data with mean and standard deviation of 6.0 μm and 2.0 μm, respectively. [Figure 8] Graph and series of images showing no change in microgel diameter, where TM1 remains sequestered within the PAA microgels when exposed to serum-containing DMEM or equine synovial fluid. (Top) Stable diameter indicates that complexation-loaded TM1 remains sequestered in DMEM+10% FBS and equine synovial fluid (ESF). (Bottom) Images show TM1-loaded microgel-modified glass surface after exposure to DMEM+10% FBS for 20 hours (left) and ESF for 15 hours (right). Scale bar corresponds to 10 μm. TM1 remains sequestered within the PAA microgels when exposed to DMEM or equine synovial fluid, as indicated by no change in microgel diameter. [Figure 9] A series of images of a TSA plate (24 h incubation) after rolling and vortexing an MSSA-contaminated Ti rod (white arrow). (Bottom) SEM image of the contaminated rod after rolling and incubation. These images show the results of the MSSA colony formation assay for all four (A-D) surface modifications. [Figure 10]A series of images of a TSA plate (24 h incubation) after rolling and vortexing a Ti rod contaminated with S. epidermidis (white arrow). (Bottom) SEM image of the contaminated rod after rolling and incubation. These images show the results of the S. epidermidis contamination assay for all four (A-D) surface modifications. [Figure 11] MTS assay of hFOB metabolic activity on the four modified Ti surfaces. The assay characterizes the metabolic activity of hFOB cells on the four (A-D) surface modifications. [Figure 12] Figure 12. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Self-protective surfaces

[14] represent a new strategy that may be able to inhibit bacterial colonization due to OR contamination. The term "self-protective" was introduced by Boulmedais et al.

[15] for the case of bacteria-induced antimicrobial release. The mechanism at work in self-protective surfaces is fundamentally different from conventional methods of inhibiting bacterial colonization by elution-based drug delivery. In elution-based techniques, antimicrobial compositions are continuously released from the surface, whether they are needed or not. In contrast, self-protective mechanisms release very small amounts of antimicrobial compositions locally, only when and where they are needed. Importantly, in the absence of a microbial challenge, self-protective surfaces do not release any antimicrobial compositions at all.

[0013] Self-protective surfaces have been developed by Sukhishvili et al.

[16] , who designed a polyelectrolyte thin-film coating that releases specific antimicrobial compositions in response to local pH changes. Cado, Boulmedais et al. [15a] designed a coating that harnesses microbial enzyme secretion and incorporates substrates for those specific enzymes such that local coating degradation releases antimicrobial compositions. Both approaches rely on bacterial metabolism to become active. However, prior to implantation, contaminated device surfaces lack a nutrient source and therefore the occurrence of significant bacterial metabolism is unlikely. Thus, metabolism-dependent self-protective approaches may not be appropriate for addressing OR contamination.

[0014] An alternative self-protective mechanism, termed "contact transfer" [14c], has been developed and may be better suited to the problem of OR contamination. Contact transfer has been shown to respond to the presence of challenging bacteria even in the absence of bacterial metabolism

[17] . In doing so, contact transfer can drive the responsive release of specific antimicrobial compositions from the polyelectrolyte microgel coating and transfer them to the challenging bacteria. This transfer is due to the presence of a high concentration of negative charges on the bacterial envelope and the hydrophobicity of that envelope, such that the complexation strength between the bacteria and the antimicrobial composition is greater than that between the microgel and the antimicrobial composition. The proximity of the bacteria to the loaded microgel then creates a steep chemical potential gradient, promoting the decomplexation of the antimicrobial agent from the microgel and its transfer to the bacteria.

[0015] It has now been discovered that antimicrobial peptoids can be utilized on certain contact transfer surfaces to create effective self-protecting synthetic surfaces. Such surfaces can be fabricated, for example, by electrostatically depositing microgels of poly(acrylic acid) (PAA) to form sub-monolayer coatings, for example, on glass coverslips or titanium rods. In a subsequent self-assembly step, the sub-monolayer coatings can be loaded with suitable cationic antimicrobial peptoids. These self-protecting surfaces can significantly inhibit bacterial colonization, even in the absence of nutrients for metabolism. As described herein, the effectiveness of these surfaces has been verified by testing in an in vitro model of OR contamination.

[0016] Although the use of a peptoid called TM1 (also known as peptoid 1) is preferred, a variety of peptoids may be utilized in the self-protective surfaces disclosed herein.

[18] Peptoids are analogs of peptides in which the side groups are located on the amide nitrogen rather than on the α-carbon. In general, they are not subject to proteolysis,

[19] improving their biostability and reducing their immunogenicity.[19a] TM1 is a helical and amphiphilic dodecamer peptoid consisting of four repeated trimers (NLys-Nspe-Nspe) terminated with secondary amine groups (see Figure 1). Each of the four NLys moieties contains a primary amine group. All five of these amine groups are protonated under physiological conditions, and thus TM1 has a net electrostatic charge of +5. In addition, each Nspe contains an aromatic moiety that can contribute to complexation interactions.

[20]

[0017] In situ optical microscopy can be utilized to follow the peptoid loading process by imaging the deswelling of the microgels during complexation with the peptoids. Such imaging can also assess the ability of the peptoids to remain sequestered within the PAA microgels upon exposure to physiologically relevant media. A digitally controlled aerosolization system can be utilized to spray well-defined amounts of bacteria such as staphylococci (either S. aureus or S. epidermidis) onto the Ti rods. Subsequent in vitro assays of bacterial viability and osteoblast response show that the peptoid-loaded microgel-modified surfaces highly effectively inhibit bacterial colonization while still maintaining cytocompatibility comparable to unmodified controls.

[0018] Materials and Methods Microgel synthesis, loading, and sequestration Poly(acrylic acid) (PAA) microgels were synthesized by thermally initiated membrane emulsification. The precursor solution was prepared by mixing 1.0 ml of acrylic acid (Sigma), 0.47 g of sodium hydroxide (NaOH, Sigma), 4 ml of deionized (DI) water (Millipore type 1), 100 mg of ammonium phosphate sulfate (APS, Sigma), and 100 μl of poly(ethylene glycol) diacrylate (PEGDA, M n =575 Da). This aqueous solution was forced via N2 pressure (40 kPa) through a ceramic membrane (1.5 μm pore size (Shirasu Porous Glass (SPG)) into a stirred (400 rpm) oil phase consisting of 2.56 g Span® 80 and 160 ml paraffin oil. The resulting emulsion was then deoxygenated by N2 bubbling for 30 min, followed by heating to 70° C. and holding under continuous stirring (500 rpm) for 4 h. After cooling to room temperature, the paraffin oil was removed by centrifugation and resuspension first in cyclohexane twice, then in ethanol 10 times, and finally in DI water 10 times. The resulting PAA microgels were suspended in sterile DI water and stored at 4° C.

[0019] For in situ studies of microgel / antimicrobial interactions, 12 individual reaction chambers were defined on top of a glass microscope slide using polydimethylsiloxane (PDMS) gaskets. Holes of 6 mm diameter were drilled from a cast sheet of cured PDMS (approximately 4 mm thick). The drilled gasket was then pressed onto a pre-cleaned (oxygen plasma for 3 min) glass slide and annealed at 70 °C for 20 min. The volume of each glass-bottom chamber was approximately 100 μl. The glass surface in each chamber was primed with positively charged poly(allylamine hydrochloride) (PAH, Sigma, Mw = 17.5 kDa) using an aqueous solution of 0.2 mg of PAH / ml for 1 h, followed by gentle rinsing using DI water and then drying by flowing N2 gas. A submonolayer of PAA microgels was then electrostatically deposited on the primed glass surface by filling each chamber with a colloidal aqueous microgel suspension at room temperature and allowed to soak for 30 min. The chambers were then rinsed with DI water and filled with 0.01 M phosphate buffer (pH 7.4, 4 mM NaH2PO4 and 6 mM Na2HPO4), [Na+]=0.016 M) before subsequent loading with the antimicrobial agent.

[0020] TM1(M w = 1819 Da) were synthesized as previously described.

[21] The filling was achieved by complexation within the microgels by replacing the buffer in a particular reaction chamber with a solution of 1 mg TM1 / ml in 0.01 M phosphate buffer. The filling process was followed by in situ time-resolved optical microscopy using an inverted microscope (Nikon, Eclipse Ti-E) equipped with a 14-bit CCD camera (pco.pixelfly) and a CFI S Fluor ELWD 20× objective lens (NA = 0.45, WD = 8.2-6.9 mm). Images were collected at 1-min intervals during the first 10 min of filling, and then at 5-min intervals thereafter. After data acquisition, the time-resolved diameters of at least five different microgels were measured using ImageJ.

[22] Each diameter was normalized using the initial diameter measured in 0.01 M phosphate buffer without TM1.

[0021] Following sequestration of TM1 complexed within the microgels, in situ imaging was again performed. After filling, the TM1 solution was removed by gentle washing (three times) using DI water. Each reaction chamber was then filled with either autoclaved 0.01 M phosphate buffer or autoclaved PBS (phosphate buffered saline, pH 7.4, ionic strength = 0.138 M). The buffer was replaced with fresh buffer every day for 28 days. Optical micrographs of the same set of microgels were collected daily and digital image analysis was used to measure the microgel diameter as a function of immersion time from at least five different microgels.

[0022] The amount of TM1 loaded into the microgels was quantified by UV absorption. Two aliquots of 10 μl of aqueous PAA microgel suspension were prepared. One was completely dehydrated to measure the dry microgel weight. The other was added to 1 ml of 0.01 M phosphate buffer containing 0.25 mg of TM1. After 60 min, the loaded microgels were pelleted by centrifugation and the UV absorption (230 nm) of the supernatant was measured using a Synergy HT BioTek Spectrometer. The amount of TM1 loaded was then quantified by comparison to a calibration curve made using solutions of known TM1 concentrations in 0.01 M phosphate buffer. The zeta potential of the microgels was measured before and after TM1 loading using a Malvern ZETA SIZER Nano series from aliquots of microgels suspended in pure 0.01 M phosphate buffer.

[0023] OR pollution model Commercially pure, surgical grade 1 titanium rods (1 cm long) were cut from wire (Temco RW0469) with an average diameter of 1.29 mm. The rods were rinsed twice with 70% ethanol, sonicated in ethanol (15 min), washed, sonicated in autoclaved DI water, and finally dried using a stream of N2 gas. The dried rods were then exposed to oxygen plasma for 10 min. Further modifications were made to create four surfaces: (Ti-A) plasma-treated but otherwise unmodified Ti, (Ti-B) PAH-primed Ti, (Ti-C) microgel-modified (unloaded) Ti, and (Ti-D) TM1-loaded microgel-modified Ti. PAH priming, electrostatic PAA microgel deposition, and TM1 loading all followed the procedures described above for glass substrates. Surfaces of each condition were imaged by scanning electron microscope (SEM; Zeiss Auriga). TM1 filling in condition D was confirmed by exposing the modified pins to an aqueous solution of fluorescein isothiocyanate (FITC, Sigma) in sterile DI water for 30 min, followed by repeated washing with DI water. FITC-stained Ti-D rods were then imaged while hydrated using a Nikon Eclipse Ti-E inverted microscope with a C3 confocal attachment.

[0024] A digitally controlled aerosolization system was used to contaminate the rod surface with well-controlled amounts of bacteria in each of the four surface conditions. Details of the system configuration, operation, and characteristics have been described elsewhere.

[23] The system is capable of reproducibly spraying well-defined bursts of aerosolized bacteria suspended in a nutrient-free buffer onto the test surface. Depending on the spray conditions, the density of the sprayed bacteria could be as high as 10 2 ~10 3 / cm 2of the order of magnitude and thus can mimic the contamination conditions of an operating room. The experiments here used Methicillin-susceptible Staphylococcus aureus (MSSA, ATCC 29213) and Staphylococcus epidermidis (ATCC 35984). For each set of experiments, a single bacterial colony was inoculated into 10 ml of tryptic soy broth (TSB) and grown to stationary phase at 37°C under gentle shaking for 18 hours. The bacteria were pelleted by centrifugation and resuspended twice in sterile PBS to remove the TSB. After the final pelleting, the bacteria were suspended in sterile PBS to an optical density (OD600) of 0.0005A for MSSA and 0.0004A for S. epidermidis. These densities were approximately 10 2 CFU / cm 2

[23] The solution was kept at room temperature and used within 3 hours of preparation. Under these preparation conditions, it has been shown that over 90% of bacteria are culturable.

[23]

[0025] Two sets of three Ti rods from each of the four surface modification conditions were sprayed with a 100 ms bacterial aerosol burst. One set was sprayed with MSSA and the other with S. epidermidis. The three rods of each set were sprayed simultaneously. After spraying, the samples were allowed to dry in air for 30 min in a laminar flow biosafety hood. The degree of contamination was then quantified in terms of the number of (i) loosely attached cultivable bacteria, and (ii) tightly attached cultivable bacteria. Loosely attached bacteria were recovered by immersing each rod in 1.25 ml of sterile PBS followed by gentle vortexing. Three 250 μl aliquots of buffer were then spread on separate tryptic soy agar (TSA) plates. Tightly attached bacteria were assayed by gently washing each vortexed Ti rod using sterile PBS and then rolling each rod using flame-sterilized forceps over the surface of a TSA plate. Both sets of TSA plates were then incubated at 37° C. for 18 h. The number of colony forming units (CFU) was counted manually. The statistical significance of the differences in bacterial colony formation of the different surface modification treatments was determined using a two-tailed Student's t-test assuming unequal variances for treated and control rods.

[0026] cytocompatibility Human fetal osteoblasts (hFOB, ATCC, VA) were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium containing 10% fetal bovine serum (FBS, Atlantic Biologicals) and 2.5 mM L-glutamine (without phenol red). Prior to use, cultures were maintained at 34 °C, 5% CO2 and 95% humidity until 70-80% confluence. To achieve equal and uniform cell distribution on individual substrates (Ti rods or glass coverslips) and among multiple substrates, hFOB cells were seeded and cultured according to a specific protocol. Briefly, Ti rods and coverslips sampling each of the four surface modification conditions (A-D) were sterilized using 70% ethanol and then washed with sterile DI water (three times) for 2 min. All samples were sterilized again under UV irradiation for 20 min before cell seeding. Each rod was first seeded with 100 μl of hFOB cell suspension, and then the cell-seeded rods were incubated for 15 min (34° C., 5% CO2, and 95% humidity) to promote cell attachment. The seeded rods were then turned over and exposed again to 100 μl of the hFOB cell suspension, resulting in 2×10 4 cells / cm 2 A final seeding density of 10 4 cells / cm 2 Each well was seeded with 200 μl of hFOB cell suspension to a seeding density of 100 μl. After 30 min of total incubation, all cell seeded samples were transferred to a new 6-well plate for further culture. Culture medium was refreshed every 2 days.

[0027] The adhesion (1 day) and proliferation (4 and 7 days) of hFOB cells on the rods were evaluated using the MTS assay. Briefly, after 1, 4, and 7 days, cultured rods (n=3 for each condition) were harvested, placed in 12-well plates, and gently rinsed twice with cold PBS. Then, 1.4 mL of a mixture containing 400 μL of MTS reagent (assay kit purchased from Promega, Madison, WI) and 1 mL of culture medium was added to each well. The plates were incubated at 37°C with 5% CO2 for 2 hours. 200 μL of supernatant from each sample was transferred to a 96-well plate. The absorbance at 490 nm was recorded using a BioTek Synergy microplate reader (BioTek Instruments, Inc., Vermont, USA). Statistically significant differences were assessed using one-way t-tests when the Ti-A surface was used as a control and ANOVA tests with Tukey's mean comparisons (p<0.05) when the Ti-A, B, and C surfaces were used as controls.

[0028] After 1, 4, and 7 days of culture, cell morphology on Ti wires and coverslips was characterized. Specimens were fixed with 4% (w / v) paraformaldehyde for 15 min and washed with PBS for 2 min (3 times). Samples were then rinsed 3 times with 0.5% Triton® X-100 and blocked with 3% (w / v) BSA to permeabilize cell membranes. Cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, Sigma, USA) and cytoskeletal filament actin (F-actin) was stained with Alexa Fluor 488-conjugated phalloidin. After staining, samples were immersed in PBS and imaged while hydrated using a Nikon Eclipse 80i epifluorescence microscope (Japan) and a Zeiss LSM880 confocal laser scanning microscope (Germany).

[0029] Results and Discussion When hydrated in 0.01 M phosphate buffer at pH 7.4 and [Na+] = 0.016 M, the as-synthesized PAA microgels have an average diameter of 6 ± 2 μm (Figure S1). It has been previously shown that the carboxyl groups in PAA are fully deprotonated under these conditions

[20] . Thus, they can complex with oppositely charged macroions, such as the TM1 peptoid in this study. The fact that TM1 can be loaded into PAA microgels by complexation is shown in Figure 2A. The plot follows the average microgel diameter during exposure to 0.01 M phosphate buffer with 1 mg / ml TM1. The data are normalized to the unloaded diameter (6.0 μm) at time t = 0 measured in 0.01 M phosphate buffer without TM1. After 10 min, the normalized diameter decreases to about 55%. In a separate experiment, measuring the decrease in TM1 concentration from the loading solution by UV absorption, shows that 39.6 μg of dried microgels load 206 μg of TM1. These values ​​indicate a ratio of 4.8 acrylic acid groups for each molecule of TM1, which is very close to the stoichiometry of 5 -1 acid groups for each +5 TM1 molecule. The zeta potential of the microgels further increased from -33.5 ± 4.5 mV (n = 6) in the unloaded state (t = 0) to a value of 3.6 ± 1.2 mV (n = 6) in the loaded state (both measurements were performed in 0.01 M phosphate buffer), again indicating charge neutralization due to TM1 / PAA complexation.

[0030] Since colonization of the device is possible not only before implantation when the device is dry, but also before surgical site closure, an important question centers on whether TM1 remains stably complexed within the PAA microgel when the device is in contact with physiological fluids. At a constant pH of 7.4, when the ionic strength increases from [Na+] = 0.016 M in 0.01 M phosphate loading buffer to [Na+] = 0.14 M typical of physiological conditions, the additional salt can shield the electrostatic pairing between the amine groups on TM1 and the acid groups within the PAA microgel. Such shielding could lead, for example, to a burst release of colistin from the PAA microgel. [17, 20] The fact that Figure 2B shows no change in microgel diameter when the loaded microgel is exposed to PBS without TM1 indicates stable TM1 / PAA complexation. Similar experiments using running buffers with increasing concentrations of added NaCl indicate that a threshold [Na+] of approximately 0.35 M at pH 7.4 is required to allow rapid release of complexed TM1. This enhanced resistance to salting out of TM1 / PAA compared to colistin / PAA is consistent with our recent observation that aromaticity enhances complexation strength.

[20] Exposure of TM1-loaded PAA microgels to serum-containing DMEM or equine synovial fluid did not induce any change in the diameter of the loaded microgels, again indicating stable complexation in these more heterogeneous media (Figure S2). The inset in Figure 2B further shows stable TM1 / PAA complexation in PBS over a 4-week period.

[0031] Having developed a means to create TM1-loaded PAA-microgel modified surfaces by directed self-assembly, the fouling resistance of Ti rods subjected to each of four different surface treatments was investigated. These are shown diagrammatically in the top panel of Figure 3. The SEM images in the bottom panel of Figure 3 show the surfaces of rods modified with unloaded microgels (Ti-C) and TM1-loaded microgels (Ti-D). The microgels form a submonolayer coating on the rough Ti surface, with spacing between the microgels on the order of a few microns. The PAH-primed Ti surface is exposed between the microgels. Hydrated (unloaded) microgels contain approximately 97% water

[20] . Thus, when dried for SEM imaging, they flatten and show little topography. In contrast, the TM1-loaded microgels have substantially more mass and less hydration, and show a very characteristic topography when dried.

[0032] To mimic intraoperative contamination with airborne bacteria, three Ti rods were sprayed and each of the four surface conditions was sampled with an aerosol of 0.0067 M phosphate buffer (1x PBS with 0.0067 M PO4, pH 7.0-7.2) containing MSSA or S. epidermidis. After spraying, the rods were allowed to dry and then assayed to determine the number of culturable bacteria that were loosely bound or tightly attached to the Ti surface. These two cases were mechanically distinguished by vortexing each rod in 0.0067 M phosphate buffer. Loosely bound bacteria were collected in the buffer, and tightly attached bacteria were collected after rolling and vortexing the contaminated rod on an agar plate.

[0033] Figure 4 summarizes the results of these experiments. The images compare the TM1 loaded microgel modified Ti-D with the unmodified control Ti-A in the case of MSSA contamination. The results are strikingly different. MSSA colonies (white dots) are evident in each of the four rolling paths of the Ti-A, while none can be seen for the fully modified Ti-D. Recognizing that not all culturable bacteria are necessarily transferred from the rod (white arrows in the top image) to the agar, the rod surface was imaged by SEM. The bottom left image in Figure 4 shows extensive colonization of the unmodified Ti-A. However, no bacteria were found by SEM imaging of the Ti-D surface, although the microgels clearly remained present. The fact that the microgels remain loaded is reflected by their topography. The graph in Figure 4 quantifies the amount of loosely bound (solid blue line) and tightly attached (solid orange line) MSSA for each of the four surface treatments. The cross-hatched data show the results of a similar experiment using S. epidermidis. The trends are very similar. The PAH-primed rods (Ti-B) show the highest degree of contamination, consistent with the fact that at pH 7.4 PAHs are positively charged

[24] and the surface should attract bacteria such as staphylococci, whose surface is negatively charged. Compared to unmodified Ti-A, the (unloaded) microgel-modified Ti-C shows slightly fewer viable bacteria. This finding may be due to the fact that the microgels are less susceptible to colonization than the PAH-primed Ti surfaces, and that the microgels block part of the Ti surface.

[0034] Importantly, the number of culturable bacteria found on TM1-loaded microgel-modified Ti-D samples was very low. In three tests, an average of only 2 CFU of MSSA and 1 CFU of S. epidermidis were found. All of these low numbers of CFU were recovered from the buffer after vortexing the contaminated rods and therefore corresponded to loosely attached colonies. No CFU were found from bacteria that were tightly attached to the Ti-D rods.

[0035] Since all three control surfaces are significantly colonized, it is assumed that the Ti-D rods are similarly exposed to culturable bacteria as a result of the aerosolization process. The fact that only very few colonies are subsequently found on the Ti-D rods suggests that the majority of these bacteria are killed by interaction with the surface. Since neither the surface nor the medium (0.067 M phosphate buffer) in which the bacteria are suspended during the aerosolization process provide nutrients, it is expected that the bacteria undergo little or no metabolism at the surface. Therefore, it is unlikely that a local pH drop

[25] could cause the release of TM1 from the loaded microgels. Instead, these findings support the concept of contact transport, where the local chemical potential of TM1 is lower within the bacterial envelope than within the microgel, [14c, 17] and, as a result, some TM1 decomplexes from the microgel and recomplexes within the staphylococcal cell envelope when the bacteria are in close proximity. Such complexation interactions with bacteria have been extensively studied for several host defense peptides and are attributed to the abundance and high concentration of anionic, hydrophobic, and aromatic moieties in the bacterial outer membrane.

[26]

[0036] The fact that the microgels remain hydrophilic even when loaded promotes the opportunity for microgel-bacteria contact. When an aerosolized droplet impacts a drying microgel-modified surface, water from the droplet locally hydrates one or more microgels. As the entire droplet evaporates, the microgel becomes the most hydrophilic element of the surface and finally dries completely. Without wishing to be bound by theory, it is believed that surface forces associated with the edge of the drying droplet attract any bacteria it may contain to the microgel, concentrating those bacteria on or near the microgel once evaporation of the droplet is complete.

[0037] Additionally, in vitro models of OR contamination use concentrated inocula (e.g., 10 μg / mL) to induce infection in control animals such as rats and mice. 6 ~10 8It is also noteworthy that the TM1-depleted or partially depleted ToD rods (CFU / mL) do not blanket the surface with bacteria as is the case for in vivo infection models, which are typically required. The OR contamination model sprays a small amount of bacteria onto the rod surface. The probability that any particular microgel will interact with bacteria is low. For that (much larger) subset of microgels that are not challenged, the antimicrobial agent remains sequestered within these microgels (see SEM image of a ToD rod in Figure 4) and can face a subsequent bacterial challenge if it occurs. That much smaller subset of microgels that interact with bacteria presents a high local concentration of the antimicrobial agent, which the assay in Figure 4 shows is sufficient to prevent surface colonization. The overall low number of bacteria involved in the contamination model again creates a situation where the probability of a second droplet landing on a TM1-depleted or partially depleted microgel is very low. Thus, refilling of the microgels, although possible in principle, is not necessary.

[0038] Cell morphology (imaging) and metabolic activity (MTS) were used to assess the short-term cytocompatibility of the various surfaces. In the case of microgel-modified surfaces, only a portion of the surface is covered by the microgel. The underlying PAH-primed Ti surface is exposed between adjacent microgels, and because PAHs are cationic, upon exposure to serum-containing medium, this exposed surface is expected to be covered by negatively charged serum proteins (e.g., albumin, fibronectin, etc.). Such microgel-modified surfaces have previously been shown to remain highly compatible with both osteoblasts and macrophages. [17,27] It is noted that unmodified Ti surfaces have an inherent roughness over lateral scales on the order of 2–10 μm, and this roughness is further perturbed by the addition of TM1-loaded microgels with characteristic dimensions on the order of a few microns. Such surface topographies have long been known to affect, and in many cases promote, cell adhesion, spreading, and proliferation.

[28]

[0039] Figure 5 shows the results of our cytocompatibility assay using human fetal osteoblasts. Cell metabolic activity was analyzed with an MTS assay (Figure 5 top). To better assess the proliferation rate independently of the initial cell attachment, the absorbance of each sample set is normalized to the day 1 value. Raw MTS absorbance data are represented in Figure S5. Importantly, the metabolic activity measured from the fully modified Ti-D sample is higher than that of the unmodified control sample (Ti-A). This indicates that the modified surface promotes the proliferation of hFOB cells. The imaging data in the bottom of Figure 5, although qualitative, show that hFOB cells attach, spread, and proliferate on the various rods in a similar manner. These results indicate that TM1-loaded microgels are not only effective in inhibiting bacterial colonization but also do not cause significant cytocompatibility, at least as observed in these short-term in vitro assays.

[0040] A glass substrate was utilized to avoid many of the imaging complications associated with the curved, rough surface of Ti rods. The top image in Figure 6 shows that the morphology of hFOB cells cultured for 7 days on unmodified glass and on TM1-loaded microgel-modified glass is comparable. The top right image also shows a striking green contrast (see white arrows) distributed across the surface. This fluorescence is only observed from the TM1-loaded microgels when the samples are dried after loading and rehydrated for the subsequent culture experiments. This contrast with TM1 self-organization within the microgels can be attributed to hydrophobic interactions, hydrogen bonding, and π-π stacking, which have been recently documented when TM1 self-organizes into helix bundles.

[29] Importantly, modified Ti rods (Ti-D) were treated identically (loaded, dried, and then rehydrated), indicating that antibacterial properties are retained even when TM1 forms such aggregated bundles. The autofluorescence not only indicates the location of the microgels, but further indicates that TM1 remains loaded within the microgels despite the presence of hFOB cells. This finding is consistent with previous results showing that culturing hFOB cells on PAA microgels loaded with the Sub5 antimicrobial peptide does not result in AMP release.

[17] SEM imaging (Figure 6, bottom) shows that hFOB cells can grow directly on top of TM1-loaded microgels.

[0041] conclusion Exposure to the OR atmosphere between the time the medical device is removed from its sterile packaging and the time the wound site is fully closed can lead to contamination of the device by bacterial settling from the atmosphere. The process has been modeled using an aerosolization system that can spray small amounts of bacteria onto titanium rods. Despite the relatively low number of bacteria contaminating each rod, upon exposure to culture medium, the bacteria sprayed onto unmodified Ti rods develop into growing colonies. The experiments described herein show that Ti surfaces modified with polyanionic microgels loaded by complexation with a cationic antimicrobial peptoid (preferably TM1) can almost completely inhibit bacterial colonization. Nutrients are unavailable to enable bacterial metabolism during the contamination process, indicating that the bacteria trigger local TM1 release by contact transport and not by a local change in pH. Due to the low number of contaminating bacteria, most of the loaded microgels are not challenged during contamination, so their TM1 payload remains sequestered. However, neither the loaded peptoids nor the additional topography introduced by the microgels reduces the cytocompatibility of the modified surfaces as assayed by in vitro experiments with human fetal osteoblasts. Thus, this surface modification strategy suggests a promising approach to inhibit intraoperative bacterial colonization of exposed biomedical devices resulting from contamination in the operating room.

[0042] Although TM1 (H-(NLys-Nspe-Nspe)4-NH2) is frequently referred to herein, a variety of peptoids can be utilized in the devices, methodologies, and compositions disclosed herein. Suitable peptoids include, for example, those described in US 8,445,632 (Barron et al.), entitled "Selective Poly-N-Substituted Glycine Antibiotics," which is incorporated herein by reference in its entirety, and those having the names and structures set forth in Tables 1-2 below. [Table 1]

[0043] Various cyclic peptoids disclosed herein can also be utilized in the devices, methodologies, and compositions, including, but not limited to, US 9,938,321 (Kirshenbaum et al.), US 9,315,548 (Kirshenbaum et al.), and US 8,828,413 (Kirshenbaum et al.), all of which are incorporated herein by reference in their entireties.

[0044] In some embodiments, the peptoids disclosed herein have the formula [ka] (In the formula, A is a terminal N-alkyl substituted glycine residue, n is an integer, B is selected from the group consisting of NH2, one and two N-substituted glycine residues, said one and two N-substituted glycine residues having N-substituents independently selected from natural alpha amino acid side chain moieties, isomers and carbon homologs thereof; X, Y, and Z are independently selected from the group consisting of N-substituted glycine residues, wherein the N-substituents are independently selected from the group consisting of natural alpha amino acid side chain moieties, isomers and carbon homologs thereof, and proline residues. In some embodiments, these peptoids are poly N-substituted glycine compounds of the formula: [ka] (In the formula, C is the formula [ka] R is an alkyl group, m is an integer, D is a terminal N-alkyl substituted glycine residue selected from the group consisting of NH, one and two N-substituted glycine residues, said one and two N-substituted glycine residues having N-substituents independently selected from natural alpha amino acid side chain moieties, isomers and carbon homologs thereof; J, K, and L are independently selected from the group consisting of N-substituted glycine residues, wherein the N-substituents are independently selected from the group consisting of natural alpha amino acid side chain moieties, isomers and carbon homologs thereof, and proline residues, and may be utilized in combination with at least one other peptoid that is a poly N-substituted glycine compound.

[0045] The peptoids in the compositions described herein may be alkylated, preferably with terminal alkylation, where alkylation (and especially terminal alkylation) with a C10 or C13 tail is particularly preferred. It has been found that such terminal alkylation can enhance the effectiveness of peptoids in certain applications and in some cases can cause peptoids with low antibacterial activity to have significant antibacterial activity. Thus, in the above formula, R can be selected from linear, branched, and cyclic alkyl moieties from about C4 to about C20. R preferably contains at least 8 carbon atoms, more preferably contains at least 10 carbon atoms, and most preferably contains at least 12 carbon atoms. Particularly preferred are embodiments in which R is a decyl or tridecyl moiety.

[0046] It will be further understood that various salts or precursors of the aforementioned peptoids can be utilized in the compositions and methodologies disclosed herein. As an example of the latter, these compositions and methodologies can include one or more precursors of the general formula A-P, where P is one of the aforementioned peptoids (or a salt thereof) and A is a moiety that functions as a leaving group to generate P when the precursor undergoes an in vivo reaction (such as, for example, proteolytic degradation).

[0047] Various halogenated peptoids and halogenated oligomers of N-substituted glycine (and salts thereof) may also be utilized in the compositions and methodologies disclosed herein. These include, but are not limited to, various halogenated analogs of the aforementioned peptoids and oligomers of N-substituted glycine. Examples of such halogenated peptoids are described, for example, in WO2020 / 223581A1 (Molchanova et al.), entitled "Halogenated Antimicrobial Peptoids," which is incorporated herein by reference in its entirety. These halogenated compositions may be halogenated in a variety of ways. For example, these compounds may contain any number of halogen substitutions with the same or different halogens. In particular, these compounds may contain one or more fluoro, chloro, bromo, or iodo substitutions, and may contain substitutions with two or more different halogens. However, the use of one or two bromo or chloro substitutions is preferred in many applications. Additionally, the peptoids described herein can be halogenated at a variety of positions; para-halogenation on peptoids containing aryl rings is particularly preferred for many applications, although ortho- and meta-substitutions, or even per-halogenation, may be useful in some applications.

[0048] Any of the peptoids disclosed in [Nam, HY, Choi, J., Kumar, SD, Nielsen, JE, Kyeong, M., Wang, S., Kang, D., Lee, Y., Lee, J., Yoon, M.-H., Hong, S., Lund, R., Jenssen, H., Shin, SY, Seo, J., 2020 Helicity Modulation Improves the Selectivity of Antimicrobial Peptoids. ACS Infectious Diseases 6, 2732-2744. doi:10.1021 / acsinfecdis.0c00356], which are incorporated herein by reference in their entirety, may also be utilized in the compositions and methodologies disclosed herein. These specifically include the peptoids shown in Table 2 below. [Table 2]

[0049] A variety of biomedical devices may be treated in accordance with the teachings herein. These may include, for example, hip / knee prostheses, heart valves, pacemakers, cochlear implants, shunts, surgical meshes, sutures, and tissue engineered constructs. These biomedical devices may be of the type designed to be implanted within the body of a subject, which may be a human or animal subject.

[0050] A preferred embodiment of the methodology disclosed herein involves treating the surface of the biomedical device by depositing a polyanionic microgel (or in some embodiments, a nanogel) onto the surface of the biomedical device and loading the deposited polyanionic microgel with a peptoid. In some embodiments, the surface of the biomedical device may be primed with a polycation layer prior to depositing the polyanionic microgel. The polyanionic microgel may be synthesized by a process including film emulsification and UV photopolymerization, and is preferably deposited as a quasi-monolayer coating on the surface of the biomedical device. Preferably, the polyanionic microgel comprises polyacrylic acid.

[0051] The surfaces of biomedical devices treated according to the methodology disclosed herein may include a variety of materials, including but not limited to materials selected from the group consisting of metals, metal oxides, and metal alloys, such as, for example, alumina, zirconia, and zirconia-reinforced alumina (ZTA); refractory metals, such as Zr, Ta, V, Nb, W, Mo, and alloys thereof; metal alloys, such as, for example, Ti-6Al-4V alloy, Ti-5Al-2.5Fe alloy, Ti-6Al-7Nb alloy, and β-Ti alloys (including Ti-12Mo-6Zr-2Fe (TMZF)); titanium alloys, such as, for example, Co-Cr-Mo alloys, stainless steel alloys, and refractory metal alloys; zirconium oxide; alumina, zirconia, and zirconia-reinforced alumina (ZTA); magnesia-partially stabilized zirconia (MgPSZ); and yttria-stabilized oxide (Y-TZP). The surfaces of biomedical devices treated according to the methodologies disclosed herein may also include various organic polymers such as, for example, polyethylene (including ultra-high molecular weight polyethylene (UHMWPE) and highly cross-linked polyethylene (HXLPE)), polytetrafluoroethylene (PTFE), or polyetheretherketone (PEEK).

[0052] The self-protective surfaces disclosed herein may be adapted to release one or more peptoids in the presence of various pathogens, including various bacteria, fungi, and viruses. Specific examples of such pathogens may include Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella, Proteus, Enterobacter, Clostridium difficile, and Salmonela, Streptococci; Candida albicans, Aspergillus spp., Nocardia, Pneumocystis carinii, Cryptococcus neoformans, and Cryptosporidium; respiratory syncytial virus, cytomegalovirus, human immunodeficiency virus (HIV), Ebola, rotavirus, enterovirus, influenza A (including subtypes H2N2 and H3N3), hepatitis, and herpes viruses.

[0053] A variety of peptoid release surfaces may be made according to the teachings herein. These release surfaces preferably comprise microgels, but in some embodiments may also comprise nanogels. The release surfaces preferably comprise zwitterionic polymers such as polybetaines. Such polymers may contain cationic groups, such as, for example, quaternary ammonium groups, and anionic groups, such as, for example, carboxylate, sulfonate, phosphate, phosphinate, and phosphonate groups.

[0054] In some embodiments, a method is provided for treating a surface of a biomedical implant to render it resistant to bacterial culture. In such embodiments, the method may include (a) priming the surface of the implant with poly(allylamine hydrochloride), thereby obtaining a primed surface; (b) applying a submonolayer of poly(acrylic acid) (PAA) microgel to the primed surface, thereby obtaining a 3D crosslinked colloidal structure; and (c) loading the colloidal structure with the peptoid by forming a complex between the PAA microgel and the peptoid. The poly(allylamine hydrochloride) is preferably positively charged, and treating the surface of the implant with poly(allylamine hydrochloride) may include (a) applying an aqueous solution of poly(allylamine hydrochloride) to the surface, thereby forming a treated surface; (b) rinsing the treated surface, thereby obtaining a rinsed surface; and (c) drying the rinsed surface. Applying a sub-monolayer of PAA microgel to the primed surface may include electrostatically depositing PAA onto the primed surface, which may include exposing the primed surface to a colloidal aqueous microgel suspension. Similarly, loading the PAA microgel with a peptoid may include exposing the PAA microgel to a buffered solution of the peptoid, with the use of a phosphate buffer being particularly preferred. The loading process preferably includes forming a complex between the PAA microgel and the peptoid.

[0055] PAA microgels of various dimensions can be utilized in the constructs and methodologies described herein. Preferably, the PAA ls have an average diameter in the range of 6±2 μm prior to loading the PAA microgels with peptoids. Once the PAA microgels are loaded with peptoids, the ratio of acrylic acid groups to peptoids, R aa / pep will be in the range of 1 to 50, more preferably 2 to 20, even more preferably 3 to 10, and most preferably 4 to 5. The zeta potential of the microgel preferably increases from -33.5±4.5 mV in the unloaded state to a value of 3.6±1.2 mV in the loaded state.

[0056] The above description of the present invention is illustrative and not intended to be limiting. It should be understood that various additions, substitutions, and modifications can be made to the above embodiments without departing from the scope of the present invention. The scope of the present invention should therefore be interpreted with reference to the appended claims.

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Claims

1. A method of manufacturing a substrate, comprising: a surface selected from the group consisting of a metal surface, a ceramic surface, and a polymer surface; a polyanionic microgel disposed on the surface; a peptoid disposed within the polyanionic microgel; 1. A biomedical device comprising: The biomedical device is a hip prosthesis, a knee prosthesis, a heart valve, a pacemaker, a cochlear implant, a shunt, a surgical mesh, a suture, or a tissue engineered construct.

2. The surface is At least one material selected from the group consisting of metals, metal oxides, metal alloys, and polymeric materials; or At least one material selected from the group consisting of alumina, zirconia, and zirconia-toughened alumina (ZTA); or at least one refractory metal; or at least one titanium alloy; or stainless steel 10. The biomedical device of claim 1, comprising:

3. The surface comprising a polymeric material, the polymeric material comprising: At least one material selected from the group consisting of polyethylene, polytetrafluoroethylene (PTFE), and polyetheretherketone (PEEK); or At least one material selected from the group consisting of ultra-high molecular weight polyethylene (UHMWPE) and highly cross-linked polyethylene (HXLPE).

10. The biomedical device of claim 1, comprising:

4. The biomedical device of claim 1, wherein the surface comprises at least one metal alloy selected from the group consisting of Co-Cr-Mo alloys, stainless steel alloys.

5. The biomedical device of claim 1, wherein the surface comprises a refractory metal selected from the group consisting of Zr, Ta, V, Nb, W, Mo and alloys thereof.

6. The surface is an alloy selected from the group consisting of Ti-6Al-4V alloy, Ti-5Al-2.5Fe alloy, Ti-6Al-7Nb alloy, and β-Ti alloy; or Zirconium oxide (oxinium) 6. The biomedical device of claim 5, comprising:

7. The biomedical device of claim 6, wherein the surface comprises Ti-12Mo-6Zr-2Fe (TMZF).

8. The surface is the group consisting of alumina, zirconia, and zirconia-toughened alumina; or A group consisting of magnesia partially stabilized zirconia (MgPSZ) and yttria stabilized zirconia (Y-TZP) 10. The biomedical device of claim 1, comprising a material selected from:

9. The biomedical device of claim 1, wherein the peptoid is H-(NLys-Nspe-Nspe) 4 -NH 2 .

10. The peptoid of claim 1, H-(NLys-Nspe-Nspe) 4 -NH 2 , H-(NLys-Nspe-Nspe(p-Br)) 2 -NH 2 , H-NLys-Nspe-Nspe-NLys-Nspe-Nspe(p-Br)-NH 2 , H-((NLys-Nspe(p-Br)-Nspe(p-Br)) 2 -NH 2 , H-Ntridec-NLys-Nspe-Nspe-NLys-NH 2 , H-(NLys-Nspe-Nspe) 3 -NLys-Nspe-NH 2 , H-(NLys-Nspe-Nspe) 2 -NH 2 , H-Ndec-(NLys-Nspe-Nspe) 2 -NH 2 , H-Ndec-(NLys-Nspe-Nspe(p-Br)) 2 -NH 2 , H-Ntridec-(NLys-Nspe-Nspe(p-Br)) 2 -NH 2 , H-(NLys-Nspe-Nspe) 4 -NLys-NH 2 , H-(NLys-Nspe-Nspe(p-Br)) 2 -NLys-NH 2 , H-NLys-Nspe-Nspe-NLys-Nspe-Nspe(p-Br)-NLys-NH 2 , H-(NLys-Nspe(p-Br)-Nspe(p-Br)) 2 -NLys-NH 2 , H-Ntridec-NLys-Nspe-Nspe-NLys-NLys-NH 2 , H-(NLys-Nspe-Nspe) 3 -NLys-Nspe-NLys-NH 2 , H-(NLys-Nspe-Nspe) 2 -NLys-NH 2 , H-Ndec-(NLys-Nspe-Nspe) 2 -NLys-NH 2 , H-Ndec-(NLys-Nspe-Nspe(p-Br)) 2 -NLys-NH 2 , H-Ntridec-(NLys-Nspe-Nspe(p-Br)) 2 -NLys-NH 2 , and H-(NLys-Nssb-Nssb) 4 -NH 2 ; or H-(NLys-Nspe-Nspe(p-Br)) 2 -NH 2 , H-NLys-Nspe-Nspe-NLys-Nspe-Nspe(p-Br)-NH 2 , H-((NLys-Nspe(p-Br)-Nspe(p-Br)) 2 -NH 2 , H-Ndec-(NLys-Nspe-Nspe(p-Br)) 2 -NH 2 , H-Ntridec-(NLys-Nspe-Nspe(p-Br)) 2 -NH 2 , H-(NLys-Nspe-Nspe(p-Br)) 2 -NLys-NH2 , H-NLys-Nspe-Nspe-NLys-Nspe-Nspe(p-Br)-NLys-NH 2 , H-(NLys-Nspe(p-Br)-Nspe(p-Br)) 2 -NLys-NH 2 , H-Ndec-(NLys-Nspe-Nspe(p-Br)) 2 -NLys-NH 2 , and H-Ntridec-(NLys-Nspe-Nspe(p-Br)) 2 -NLys-NH 2 ; or H-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NH 2 、H-NLys-Npm-Npm-NLys-Npm-Npm-NLys-Npm-Npm-NLys-Npm-Npm-NH 2 、H-NLys-Nspe-Npm-NLys-Npm-Npm-NLys-Npm-Npm-NLys-Npm-Npm-NH 2 、H-NLys-Npm-Nspe-NLys-Npm-Npm-NLys-Npm-Npm-NLys-Npm-Npm-NH 2 、H-NLys-Npm-Npm-NLys-Nspe-Npm-NLys-Npm-Npm-NLys-Npm-Npm-NH 2 、H-NLys-Npm-Npm-NLys-Npm-Nspe-NLys-Npm-Npm-NLys-Npm-Npm-NH 2 、H-NLys-Npm-Npm-NLys-Npm-Npm-NLys-Nspe-Npm-NLys-Npm-Npm-NH 2 、H-NLys-Npm-Npm-NLys-Npm-Npm-NLys-Npm-Nspe-NLys-Npm-Npm-NH 2 、H-NLys-Npm-Npm-NLys-Npm-Npm-NLys-Npm-Npm-NLys-spe-Npm-NH 2 、H-NLys-Npm-Npm-NLys-Npm-Npm-NLys-Npm-Npm-NLys-Npm-Nspe-NH 2 、H-NLys-Nspe-Npm-NLys-Npm-Npm-NLys-Npm-Npm-NLys-Npm-Nspe-NH 2 、H-NLys-Nspe-Nspe-NLys-Npm-Npm-NLys-Npm-Npm-NLys-Npm-Npm-NH 2 、H-NLys-Nspe-Nspe-NLys-Npm-Npm-NLys-Npm-Npm-NLys-Npm-Nspe-NH 2 、H-NLys-Nspe-Npm-NLys-Npm-Npm-NLys-Npm-Npm-NLys-Nspe-Nspe-NH 2 、H-NLys-Npm-Npm-NLys-Npm-Nspe-NLys-Nspe-Nspe-NLys-Npm-Npm-NH 2 、H-NLys-Nspe-Nspe-NLys-Npm-Npm-NLys-Npm-Npm-NLys-Nspe-Nspe-NH 2and the group consisting of H-NLys-Npm-Npm-NLys-Nspe-Nspe-NLys-Nspe-Nspe-NLys-Npm-Npm-NH 2 .

10. The biomedical device of claim 1, wherein the biomedical device is selected from:

11. The peptoid of claim 1, wherein the peptoid has the formula 【Chemistry 1】 (In the formula, A is a terminal N-alkyl substituted glycine residue; n is an integer, B is selected from the group consisting of NH 2 , one and two N-substituted glycine residues, said one and two N-substituted glycine residues having N-substituents independently selected from natural alpha amino acid side chain moieties, isomers and carbon homologs thereof; X, Y, and Z are independently N-substituted glycine residues, wherein the N-substituents are independently selected from the group consisting of natural alpha amino acid side chain moieties, isomers, and carbon homologs thereof, and proline residues.

2. The biomedical device of claim 1, wherein the poly N-substituted glycine compound is

12. The biomedical device of claim 1, wherein the peptoid is a cyclic peptoid.

13. The biomedical device of claim 1, wherein the polyanionic microgel releases the peptoid in the presence of a pathogen selected from the group consisting of bacteria, fungi, and viruses.

14. The polyanionic microgel The group consisting of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella, Proteus, Enterobacter, Clostridium difficile, and Salmonella, Streptoccoci; or the group consisting of Candida albicans, Aspergillus spp., Nocardia, Pneumocystis carinii, Cryptococcus neoformans, and Cryptosporidium; or The group consisting of respiratory syncytial virus, cytomegalovirus, human immunodeficiency virus (HIV), Ebola, rotavirus, enterovirus, influenza A (including subtypes H2N2 and H3N3), hepatitis, and herpes viruses 10. The biomedical device of claim 1, wherein the peptoid is released in the presence of a pathogen selected from the group consisting of:

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