2,2′-azobisisobutyronitrile (AIBN), Triton X-100 and the bee venom toxin melittin (purity > 85%) were purchased from Sigma-Aldrich and used without further purification. Methylmethacrylate (MMA), butylmethacrylate (BMA), methyl 3-mercaptopropionate (MMP), ethanolamine, di-tert-butyldicarbonate, methacryloyl chloride, and MgCl₂ were purchased from Acros and used without further purification. Reagent grade solvents, NaCl, and CaCl₂ were purchased from Fisher and used without further purification. The antimicrobial peptide magainin −2 (purity > 90%) was purchased from AnaSpec Inc. Antibiotics norfloxacin (NOR; purity ∼99.7%) and ciprofloxacin (CIP; purity ∼98%) were obtained from MP Biomedicals, and LKT Laboratories, respectively. Mueller-Hinton broth (MHB), brucella broth and agar were purchased from Difco Laboratories. Human red blood cells (Red Blood Cells Leukocytes Reduced Adenine Saline Added) were obtained from the American Red Cross Blood Services Southeastern Michigan Region.

Free radical polymerization of N-(tert-butoxycarbonyl)aminoethyl methacrylate (Boc-AEMA) with an alkyl methacrylate was carried out as previously described [40] with some modifications. Briefly, Boc-AEMA and alkyl methacrylates (various ratios, 0.5 mmol total), MMP (16.7 μL, 0.15 mmol) and AIBN (0.82 mg, 0.005 mmol) dissolved in acetonitrile (0.5 mL) in a sealed borosilicate glass test tube were deoxygenated with N₂ bubbling for 2 min and then stirred at 60–70 °C in a mineral oil bath for 20 h. Solvent was evaporated and the crude polymer was purified by size exclusion chromatography (Sephadex LH-20 gel, methanol) monitored by thin layer chromatography (ethyl acetate:hexane 1:1). Fractions containing unreacted monomers and MMP were discarded. The remaining fractions were concentrated, dissolved in 1.25 M HCl in methanol (5–10 mL), and stirred at room temperature for 2 h to cleave the protecting groups. Excess acid was removed by N₂ flushing and the polymers were twice precipitated from methanol into diethylether. The precipitates were collected by centrifugation and lyophilized to afford the random copolymers bearing primary amine groups in the form of ammonium chloride salts. The polymers were characterized by ¹H NMR to determine the mole percentage of alkyl groups (MP_alkyl) and degree of polymerization (DP) as previously described in detail [30,39,40].

Antibacterial activity of polymers was determined in a standard microbroth dilution assay according to the Clinical and Laboratory Standards Institute guidelines (CLSI [41]) with suggested modifications by R.E.W. Hancock Laboratory (University of British Columbia, Vancouver, British Columbia, Canada [42]) and Giacometti et al. [43] for testing cationic agents. Each polymer was dissolved in 0.01% acetic acid and acetic acid was used as a solvent control. The bacterial strains Escherichia coli ATCC^® 25922™, Staphylococcus aureus ATCC^® 25923™, Pseudomonas aeruginosa ATCC^® 27853™, Salmonella enterica subsp. enterica serovar Typhimurium ATCC^® 14028™, and Bacillus subtilis ATCC^® 6633™ were aerobically cultured in MHB. MHB was prepared according to manufacturer's instructions and contained approximately 130 mM Na⁺, 110 mM Cl⁻, 0.4 mM Ca²⁺ and 0.15 mM Mg²⁺ (pH 7.3 [44]). Because of slow growth of Enterococcus faecalis ATCC^® 29212™ in MHB, tryptic soy broth was used for cultivating of this strain. An overnight culture of bacterial strains was regrown to exponential phase (OD₆₀₀ of 0.5–0.6) and diluted to give the final concentration of bacteria on the microplate approximately 5 × 10⁵ CFU/mL. After addition of the test compounds at a 1/10 volume into a 96-well sterile assay plate (Corning #3359), the assay plate was incubated at 37 °C for 18 h. Bacterial growth was detected at OD₆₀₀ using Varioskan Flash microplate reader (Thermo Fisher). Where indicated, fixed concentrations of NaCl, MgCl₂, and CaCl₂ were added into MHB. Heat-inactivated serum was used to examine the effect of serum on antibacterial activity of polymers in the absence potential problems with complement-mediated cell lysis. Anaerobic strain Propionibacterium acnes ATCC^® 6919™ was grown in brucella broth supplemented with hemin (5 μg/mL) and vitamin K₁ (10 μg/mL) in the anaerobic chamber with an atmosphere of 10% CO₂, 10% H₂, and 80% N₂. Activity of polymers against P. acnes was determined by the broth dilution method according to CLSI M11-A6 guidelines [45]. The inoculum was 10⁶ CFU/mL. The inoculated microplates (Falcon #3077) were incubated for 72 h at 37 °C in the anaerobic chamber. Each MIC experiment was independently repeated at least three times in triplicate on different days. The minimum inhibitory concentration (MIC) was defined as the lowest polymer concentration to completely inhibit bacterial growth. Because biological activity of compounds often depends on the assay conditions and the selected model organisms, it would be impractical to compare biological activity for different compounds reported in the literature. Therefore, we selected the natural host-defense peptide magainin-2 and the bee venom toxin melittin as reference standards in all biological assays.

Time-kill studies were performed for the Gram-negative E. coli ATCC 25922 and the Gram-positive S. aureus ATCC 25923 to measure the time dependence of bactericidal activity by PMAs. Cells in exponential or stationary phase were diluted in MHB to give the final concentration of bacteria approximately 5 × 10⁵ CFU/mL and treated with 2 × MIC of PMAs. Aliquots from the assay were removed at certain time intervals and immediately diluted with 0.9% saline to remove the effects of the polymer. Viability was enumerated by serial dilution plating. After 24-h incubation, no regrowth of E. coli or S. aureus cultures containing PMAs was observed. Experiments were carried out two times and produced similar results.

The first MIC determination of polymers and two antibiotics CIP and NOR against E. coli ATCC 25922 was performed as described above. Bacteria samples from duplicate wells at the concentration of one-half MIC were removed and used to prepare the bacterial inoculation (5 × 10⁵ CFU/mL) for the next experiment. This bacteria solution was then replaced on new 96-well plates with fresh dilutions of compounds. After incubation for 18 h at 37 °C, the change of MIC values was determined. This experiment was repeated each day for 21 successive passages.

Human red blood cells (RBCs; 1 mL) were suspended in 9 mL of TBS buffer (10 mM Tris buffer, 150 mM NaCl, pH 7.3) and rinsed three times. The cell suspension (1 mL) was diluted by 29 mL of TBS to give 3.3% RBC (v/v). Cells were counted using a hematocytometer and ∼2 × 10⁷ cells were seeded per well. Polymer solutions (10 μL) and the RBCs (90 μL) were mixed on a 96-well assay plate (Corning #3359), and incubated with orbital shaking at 37 °C. After 1 h, the plate was centrifuged at 780 × g for 5 min, and an aliquot of supernatant (8 μL) from each well was diluted within TBS buffer (92 μL), and the absorbance of the released hemoglobin at 415 nm was measured. Hemolysis was determined relative to the positive lysis control TRITON-X (0.1 % v/v in water) and negative control buffer. HC₅₀ was defined as the polymer concentration causing 50% hemolysis, which was estimated with 95% confidence intervals by a curve fitting with the following equation: H = 100/(1 + (HC₅₀/[polymer])ⁿ) where H is the percentage of hemolysis measured, and [polymer] is the total concentration of polymer. The fitting parameters were n and HC₅₀. Final HC₅₀ is reported as the average value from at least three experiments independently performed in triplicate on different days.

A colony of Escherichia coli D31 was inoculated in a solution of Mueller-Hinton Broth (Difco) and Isopropyl β-D-1-thiogalactopyranoside (IPTG) (2 mM). The culture was incubated at 37° for 18 h. The overnight culture was then diluted 1:100 in fresh media and incubated at 37° until the OD₆₀0 was 0.200–0.500. Using a 96-well polystyrene plate, PMAs and CTAB (cetyl trimethyl ammonium bromide) were serially diluted from stocks. Next Z-buffer (56.25 μL) was added to each well followed the E. coli culture (18.75 μL). Just prior to reading, ortho-Nitrophenyl-β-galactoside (ONPG) dissolved in Z-buffer (15 μL, 4 mg/mL) was added to the wells. The absorbance at 420 nm was then monitored using a ThermoSkan plate reader for 90 min with shaking between individual readings to prevent cell settling. All assays were performed at least in triplicate.

A colony of Escherichia coli D31 was inoculated in a solution of Mueller-Hinton Broth (Difco) and ampicillin (100 μg/mL). The culture was incubated at 37° for 18 h and then diluted 1:100 in fresh media and incubated at 37° until the OD₆₀₀ was ≈0.200. The culture was then centrifuged at 2,500 rpm for 15 min, and the pellet resuspended in an equal volume of PBS buffer (10 mM phosphate, 200 mM NaCl, pH 7.0). The resuspended bacterial cells were added to the wells of a 96-well plate containing serially diluted PMAs or polymyxin B as a control. Nitrocefin was added to the solution to a concentration of 50 μg/mL. The plate was then monitored on a plate reader in the same format as the IM permeability procedure, except the sample absorbance was measured at 486 nm. All assays were performed at least in triplicate.

Amphiphilic polymethacrylate derivatives (PMAs) containing cationic and hydrophobic alkyl side chains (methyl or butyl; Figure 1) in random sequence (Table 1) were prepared by free radical polymerization in the presence of a chain transfer agent, according to our previous report [40]. The polymers have low molecular weights of 2.2–2.8 kDa, which are comparable to those of the natural peptides magainin-2 and melittin. The hydrophobicity of polymers was varied by altering mole percentage of alkyl groups relative to the total number of monomeric units (MP_alkyl) and by the length of the alkyl chains (methyl or butyl groups). The polydispersities of these polymers were unable to be determined due to the low solubility to a standard GPC solvent (THF). In our previous study, the boc-protected precursors of selected polymers prepared by the same polymerization method displayed a polydispersity index (PDI) of <1.5. We speculate that the polymers studied in this report also have a similar PDI. The PMA polymers are denoted as PM_x or PB_x (PM: copolymers with methyl groups, PB: copolymer with butyl groups, subscript x: mole percentage of alkyl side chains relative to the total number of monomeric units in a polymer chain, MP_alkyl); for example, the PMA containing 47% methyl groups is referred to as PM₄₇.

The PMAs were tested against a panel of clinically relevant bacterial pathogens (Table 2). We determined the minimum inhibitory concentration (MIC) by a turbidity-based broth microdilution assay as a first assessment of antibacterial activity of polymers. In general, the activity of PMAs critically depends on the hydrophobicity of the PMAs and the species of bacteria. In general, the MIC values of PM polymers (copolymers with methyl groups) for Gram-negative bacteria tested here decreased as the MP_alkyl was increased (Figure 2). The homopolymer P₀ (no hydrophobic groups) displayed a high MIC of ≥500 μg/mL, and the MIC of PM copolymers started to decrease by an order of magnitude around MP = 30% and reached low MIC (16–31 μg/mL) at the higher MP_alkyl contents. This indicates that the polymer hydrophobicity enhances the antimicrobial activity, which is in agreement with the previous reports on PMAs [39,40]. As the MP_alkyl is increased, the number of cationic amine groups decrease, which should reduce the electrostatic binding of polymers to negatively charged bacterial surfaces, potentially resulting in lower activity. However, the activity was enhanced with increasing MP_alkyl, which indicates that the effect of hydrophobicity appears to be more influential factor in the polymers' antimicrobial activity. Although PMAs with MP_alkyl higher than 63% were not examined, the activity could potentially be increased; however, the PMAs with high MP_alkyl often precipitate in MH broth due to non-specific binding to the components in the broth [30]. The PMAs also likely form solution aggregates due to association of alkyl groups [39] which may result in no activity enhancement or even a decrease in activity. The MIC values at high MPs are lower or compatible to those of magainin-2 and melittin, indicating that the antibacterial activities of PMAs are comparable to or even higher than those of the natural peptides. The MIC for Gram-positive Enterococcus faecalis and Bacillus subtilis also decreased with increasing the MP in parallel with the results for Gram-negative bacteria. However, increasing MP_methyl from 10% to 63% had no or little effect on the MIC against S. aureus, suggesting that the activity of PMAs against S. aureus is not strongly sensitive to changes in polymer hydrophobicity or the number of cationic groups in contrast to other bacteria. Notably, all polymers tested were active against the methicillin-resistant S. aureus (MRSA) with the same MICs as against the susceptible strain, although the control peptide magainin-2 was not active against MRSA (MIC > 500 μg/mL). The P₀ and other PMAs with low MP_methyl tend to be more selective to S. aureus and B. subtilis over other bacteria tested. Lienkamp et al. previously reported that cationic polynorbornenes showed significantly higher activity against S. aureus compared to E. coli [47,48]. The authors demonstrated that the double membrane structure of E. coil cell wall likely limits the access of the polymers to the cytoplasmic membrane [48]. Epand et al. reported that an acyl-Lys oligomer is bacteriostatic, and it does not compromise the cell membrane of S. aureus [49]. The authors speculate that the cell wall binding of acyl-Lys oligomer limits the diffusion of nutrients from extracellular environment and causes starvation. The same molecular mechanisms may explain the activity of PMAs in part; however, the basis of this effect by PMAs remains unclear. In addition, the selected PMAs were also highly active (MIC ≤ 2 μg/mL) against anaerobic bacteria P. acnes which is a major etiological agent of acne vulgaris; increasing resistance of this bacteria to standard antibiotic therapies reduces the efficacy of therapeutic agents and often leads to failure of dermatological treatment [50,51].

As a first assessment of polymer toxicity, we evaluated the hemolytic activity of PMAs against human red blood cells (RBCs). The hemolytic activity was determined by monitoring release of hemoglobin into solution from RBCs, which reflects membrane permeabilization and cell lysis induced by the polymers [46]. The homopolymer and PM polymers did not exhibit adverse hemolytic activities (HC₅₀ > 2,000 μg/mL), except for PM₆₃ which showed an HC₅₀ value of 114 μg/mL (Table 2) [46]. To quantify the selectivity of polymers against bacteria over RBCs, selectivity index, defined as HC₅₀/MIC, for Gram-negative E. coli and Gram-positive S. aureus as representatives, was summarized in Table 3. Examining the selectivity indexes, the polymers PM₄₇ and PM₆₃ are relatively selective against bacteria over RBCs (Table 3).

The PMA with longer alkyl chains (butyl), PB₂₇, exhibited MIC at low μg/mL against all tested bacteria. PB₂₇ displayed an order of magnitude lower MIC value (MIC = 16 μg/mL against E. coli) than PM₂₈ (MIC = 500 μg/mL) while they have the similar mole percentages of hydrophobic groups (27–28 mol %) in a polymer chain and polymer length (DP = 15–16). This result indicates that polymers with more hydrophobic side chains are more active. Considering the activity enhancement with increasing MP_alkyl for the PM polymers, these results support the notion that the overall hydrophobicity of PMAs increases the activity. PB₂₇ also caused significant hemolysis with an HC₅₀ value of 13 μg/mL. The hemolytic activity of PB₂₇ likely reflects the hydrophobicity of butyl side chains; increasing the hydrophobicity beyond a threshold results in the loss of selectivity because the hydrophobic nature of polymers causes non-specific binding of polymers to RBCs and disruption of the cell membrane. This result is in agreement with our previous studies [39,40].

It has been previously reported that synthetic cationic polymers including random nylon copolymers [27,52], cationic polynorbones [53], poly(2-(dimethylaminoethyl)methacrylate derivatives [54], and oligo(oxazoline)s [36] are broad spectrum antimicrobials. In addition, a recent report from Wynne and coworkers indicated that random copolyoxetanes with PEG-like groups and C₁₂-modified quaternary ammonium groups in the side chains showed MICs of low μg/mL against selected Gram-positive and negative bacteria [38]. These studies also support the notion that the polymer design using amphiphilic polycationic structures would have potential for new development of antimicrobials. It should be noted that the MIC and HC₅₀ values strongly depend on the assay conditions including bacterial strains, broth, assay plates, incubation time, etc. [55]. Therefore, the quantitative comparison of MIC and HC₅₀ values may be ambiguous. Accordingly, we limited the discussion in the qualitative comparison of activity profiles between other reports and the results in this study.

We next determined bactericidal kinetics for selected PMAs, PM₆₃ and PB₂₇, against Gram-negative E. coli and Gram-positive S. aureus (Figure 3). As shown in Table 3, PM₆₃ is a relatively selective antimicrobial agent against E. coli over RBCs, and PB₂₇ exhibits non-selective biocidal characteristics which make these two polymers interesting models to examine with respect to their mechanism and characteristics. In order to examine the effect of bacterial growth phase on the activity of the polymers, the bacteria were subcultured from either the exponential or stationary growth phases. At a concentration of two times MIC, the polymers reduced the number of viable cells by 4-log or 99.99% within 60 min for E. coli and 60–120 min for S. aureus in both cultures. This implies that the killing kinetics of the PMAs is independent of the growth phase of the bacteria (Figure 3). It should be noted that PB₂₇ showed somewhat faster killing of E. coli in the exponential phase relative to the stationary phase. It is, however, not clear at this point why PB₂₇ displayed the different behavior. These results indicate that the PMAs are bactericidal against bacteria in both exponential and stationary phases, which suggests that the mechanism may not rely on any growth phase-specific metabolic activity or cellular physiology of the bacterial cells.

One of the hallmarks of natural antimicrobial peptides is low level of resistance development in bacteria. In order to examine the likelihood of development of PMA-resistant bacteria, E. coli were serially subcultured in MHB containing sub-lethal concentrations (one-half MIC) of selected polymers of the selective PM₆₃ and biocidal PB₂₇ for up to 21 passages. The MIC values of PM₆₃ and PB₂₇ did not increase by more than a single two-fold dilution throughout the experiment (Figure 4). In contrast, the MIC values of the antibiotic fluoroquinolones norfloxacin (NOR) and ciprofloxacin (CIP), which inhibit DNA synthesis [56,57], started to increase after as few as four passages and finally increased 512-fold and 256-fold, respectively. After the 21st passage, the obtained NOR- and CIP-resistant strains of E. coli were subcultured in antibiotic-free medium. After 15 antibiotic-free passages, the acquired resistance was persistent, indicating that the antibiotic resistance was not simply a physiological adaption. These CIP- and NOR-resistant strains were both still fully susceptible to PMAs, with the same MIC values as the CIP- and NOR-susceptible strains. Also, the E. coli incubated with PM₆₃ and PB₂₇ were still susceptible to CIP and NOR, with no increase in their MIC values after 21 passages. These results indicate that no cross-resistance developed between these fluoroquinolone antibiotics and the selected antibacterial PMAs tested here. It has been also reported that membrane-active cationic oligomers displayed no development of resistance in S. aureus [58,59]. These results highlight the potential of amphiphilic PMAs as an attractive class of new antimicrobials.

It has been reported that the activity of antimicrobial peptides was significantly reduced in high ionic strength solutions [60-62]. It has been postulated that the increased salt concentration screens the electrostatic attraction between the cationic peptides and anionic binding sites on the bacterial cell envelopes where potential polymer binding sites such as anionic phospholipids, lipopolysaccharides (LPS) in Gram-negative bacteria, and teichoic acids in Gram-positive bacteria are occupied by the salt cations [63,64]. This masking effect reduces the affinity of antimicrobial peptides for bacterial surface, resulting in reduction of antibacterial activity. Similarly, the physiological salts could impair the electrostatic binding of the cationic PMAs to bacteria. In addition, since the salt ions also curtain the cationic charges of polymer side chains, the polymers may have more compact conformations or undergo aggregation under these conditions, which could reduce the effective concentration of active polymer chains and result in lower activity. The antibacterial activity of PB₂₇ was shown to be salt-resistant, indicating that the strong hydrophobicity of PB₂₇ likely outweighs the contribution of electrostatic affinity to binding with anionic targets on bacteria cell membranes.

In order to investigate these possibilities, the effects of additional physiological salts in MH broth on the antibacterial activity of the selected PMAs were determined. The activity of these PMAs against E. coli and S. aureus was inhibited by the addition of physiological salts as evidenced by up to an eight-fold increase in the MIC values (Figure 5). The MICs of PM₄₇ and PM₆₃ against E. coli increased two- to four-fold by addition of CaCl₂ and MgCl₂ to the growth medium. These salt conditions had no effect on the activity of PB₂₇, while the activity of melittin decreased four- to eight-fold. In contrast to CaCl₂ and MgCl₂, the addition of NaCl did not significantly affect the activity of PMAs against E. coli, even at a substantially higher NaCl concentration (150 mM) than those of CaCl₂ (2 mM) or MgCl₂ (1 mM). The MIC values of PMAs and melittin against S. aureus in the presence of CaCl₂ and MgCl₂ were equal to or two-fold greater than those in MH broth without salt additives. In addition, the MICs of PM₄₇ and PM₆₃ for S. aureus in the presence of NaCl increased four-fold.

In addition to potential effects on PMA binding to bacterial surfaces, the divalent cations Mg²⁺ and Ca²⁺ can neutralize and bridge the phosphate groups of LPS molecules on the outer membrane of Gram-negative strains, which is essential to maintaining the membrane integrity [65,66]. Hence, the inhibitory effect of Mg²⁺ and Ca²⁺ on the activity of PMAs against E. coli may indicate that the antibacterial mechanism involves polymer binding to LPS and subsequent disruption of E. coli outer membrane.

In order to probe the antimicrobial mechanism of PMAs, we determined the effect of PMAs on the permeability of the outer (OM) and inner membranes (IM) of E. coli. The assays measure the ability of a chromogenic substrate to cross either the OM or the OM and IM to access a periplasmic or cytoplasmic enzyme, respectively. The OM permeability assay uses the periplasmic enzyme β-lactamase and the chromogenic substrate nitrocefin while the IM assay employs the cytoplasmic enzyme β-galactosidase and the chromogenic substrate ortho-nitrophenyl-β-galactoside (ONPG). These substrates show very low permeability across the membrane under control conditions, but the permeability is enhanced if a polymer, peptide, or other membrane disrupting agent is present [67]. As the substrates cross the membrane(s), the appropriate enzymes produce the chromogenic product which can be monitored by changes in the absorbance in the samples [68,69].

The PMAs did not cause any permeabilization of the IM up to their MIC concentrations (Figure S1 in Supplementary Data) although they disrupt the OM (Figure 6(A)) (discussed below) and model membranes (Figure S2 in Supplementary Data) at polymer concentrations lower than MICs. This indicates that the IM permeabilization may not be the primary factor for bacterial growth inhibition or killing by the PMAs, and other mechanisms may exert the antimicrobial effect against E. coli. On the other hand, the PMAs induced significant permeabilization of the OM at low μg/mL concentrations (Figure 6(A)) compared to the positive control of polymyxin B. Cationic antimicrobial peptides are known to permeabilize the E. coli OM, which promotes uptake of the peptides into the peptidoglycan layer (self-promoted uptake) [70]. The OM permeabilization by PMAs could also enhance their access to the peptidoglycan layers. The inhibition of PMA activity by CaCl₂ and MgCl₂ (Figure 5), which stabilizes the OM structure, may support the notion that the PMA antimicrobial mechanism involves OM permeabilization as uptake process to cell surfaces.

The homopolymer P₀ and copolymers PM₁₂ and PM₂₈ displayed the same OM permeabilization effect at 16 μg/mL and relatively high production rates of products although these polymers are much less active against E. coli (MIC = 500 μg/mL) than other more active polymers. The MIC value and absorbance (product formation) at 30 min after incubation (Figure 6(C)) are constant up to 30% MP_methyl and decreased as the MP_methyl was further increased. This suggests that PMAs with more cationic groups are more advantageous in OM permeabilization, but they are less active against E. coli. This reverse relationship between the activity and OM permeabilization suggests that the ability of PMAs to permeabilize the E. coli OM does not directly reflect their antibacterial activity. This observed phenomenon may be a function of kinetically “trapping” the most cationic PMAs in the negatively charged lipopolysaccharide layer of the OM and peptidoglycan layer resulting in altered OM permeability but an inability to transit to the IM. In addition, since the PMAs could permeabilize model membranes at low μg/mL concentrations (Supplementary Data), the concentration of polymers which transient to the IM would be significantly lower than in bulk solution or the total bound to the bacterial cell. This could support the notion that the cationic PMAs are trapped in the cell wall structures before reaching to the IM.

It has been reported that some peptides and synthetic mimics exert their antimicrobial effect by mechanisms other than permeabilization of inner or cytoplasmic membranes. For example, the antimicrobial peptide buforin II penetrates through bacterial cell membranes and interacts with DNA/RNA leading to lethal action [71], while the peptide mersacidin has been shown to inhibit cell wall synthesis by inhibiting lipid II synthesis [2,72]. A synthetic polynorbornene modified with guanidine groups also showed antimicrobial activity without disrupting bacterial membranes [53]. Pleurocin-derived peptides inhibit macromolecular synthesis in E. coli at sub-lethal concentrations but did not induce IM permeabilization over the same concentration range [73]. Similarly, PMAs could diffuse into the peptidoglycan layers after the OM permeabilization and perturb peptidoglycan integrity and functions, which may result in inhibition of the cell wall synthesis or cellular uptake of metabolites, which has been postulated as a potential component of the mechanism of action of some peptides [74]. The PMAs might also penetrate the inner membranes without significant disruption and interact with intracellular targets. Although the molecular mechanisms and cellular targets remain unclear at this point, PMAs could have multiple targets, and the underlying individual mechanisms may not be mutually exclusive, but rather complementary or synergistic. Further investigation will provide insight into the mode and mechanism of antimicrobial action of these polymers.