Polymeric quaternary ammonium salts (PQASs), quaternary phosphonium salts (PQPSs), polymeric guanidine (PGSs) and biguanidine salts (PBGSs) are classes of cationic polymer materials with high potential as antimicrobial agents, due to the high and permanent cationic character of their quaternary groups^{[39][40][41][42][43][44][45]}.

Designed just to mimic CAMPs, polynorboranes (PNBs)-based antimicrobial polymers possess an amphiphilic structure characterized by having the cationic hydrophilic fragment segregated onto one region (or face) of the macromolecule and the hydrophobic portion, usually constituted by hydrocarbon chains, distinctly onto the opposite face.

In this regard, such polymers are called “facially amphiphilic” (FA) and were synthetized by polymerizing FA norbornene-based monomers, with different protonated groups, such as primary amine, guanidine or pyridine, located on a side alkyl chain, pending from the nitrogen atom of the bicyclic norbornane structure.

The mechanism of action of these polymers, involves as usually, an initial interaction with OM, the creation of pores, the insertion of the biocide into the bacteria cell wall, a second electrostatic interaction with CM, the impairment of its integrity, the progressive increase of its permeability up to its disruption, loss of cytoplasmic material and bacteria death. The type of counterions, the length of alkyl side chains and also the molecular charge density strongly influence the activity and the selectivity of PNBs polymers.

Thanks to their amphiphilic structure, antimicrobial PNBs possess particular ability in inserting and disrupting the CM of bacteria.

Alkyl hydrophobic norbornene-type polymers and the analogous oxanorbornene-based hydrophilic macromolecules, containing primary alkyl ammonium groups as cationic moieties were prepared by Ilker et al. (2004)^[70]. The first ones, although very active versus representatives of Gram-negative bacteria, proved to be not selective for pathogens, thus resulting considerably toxic on mammalian cells, as established by the vesicle-dye leakage assays. On the contrary, the latter were less cytotoxic, but practically inactive^[70].

By random copolymerization of two types of alkyl hydrophobic norbornene monomers, it was possible to tune the overall hydrophobicity of the polymer achieving CAPs with high selectivity (>100) and considerable activity against E. coli (MIC [µg/mL, (µΜ)] = 40, 2.6–3.3).

Similar results were obtained later by Gabriel et al. (2009), for slightly modified oxanorbornene-based hydrophilic polymers that proved to be endowed with low cytotoxicity and good selectivity, but were practically inactive^[71].

Later a good solution, in terms of preparing compounds with high antimicrobial activity on bacteria and low hemolytic toxicity on human cells, was to replace the primary ammonium group onto the side alkyl chain with the guanidinium one^[72].

In this regard, a polyguanidinium oxanorbornene (PGON) compound was synthesized from norbornene monomers via ring-opening metathesis polymerization (ROMP), which in time killing studies proved to be lethal for bacteria and not only bacteriostatic^[72].

A broad library of highly active antimicrobial FA oxanorbornene monomers were prepared and after ROMP and deprotection, provided several series of polynorbornene-derived polymers with tunable activity and selectivity^[73]. Polyamine oxanorbornene-based antimicrobial random copolymers, with high hydrophobicity were prepared by performing two different approaches. One strategy involved the copolymerization of two hydrophobic FA monomers with cationic primary ammonium groups on side alkyl chains, while the other consisted in copolymerizing one cationic primary ammonium oxanorbornene monomer and a hydrophobic alkyl amine oxanorbornene comonomer.

By following the second strategy, a series of copolymers endowed with significant antibacterial activity and tunable selectivity were prepared^[71].

Amphiphilic polyoxanorbornene-based polymers having different quaternary alkyl pyridinium side chains were synthesized by Eren et al. (2008), but with questionable success^[74].

Compounds with a C₄ side chain or shorter proved low antimicrobial activity and low hemolytic toxicity on human red blood cells, while compounds with a side chain longer than C₆ proved high antimicrobial effect, but low selectivity for bacterial over mammalian cells^[74].

For years, it was thought that fixing permanent cationic charges on polymers by quaternization of amine or phosphorus groups could be the best way to achieve polymers with enhanced antimicrobial effects. To disprove this belief, polymer systems encompassing not quaternary protonated amine groups were synthetized and their antimicrobial activity was evaluated and compared to that of N-quaternized analogous derivatives.

In this regard, polystyrene-based polymers, containing tertiary amine groups susceptible of reversible protonation, exerted bactericidal activity similar to that of the peptide toxin melittin and somewhat lower activity than that of a potent derivative of the host defense peptide known as magainin II^[75].

For clarity, host defense peptide is another broader term to call CAMPs, which takes into account, that small cationic amphipathic peptides have strong potential not only as antimicrobials, but also as antibiofilm agents, immune modulators and anti-inflammatories^[76].

The not quaternary compounds, compared to the permanently cationic corresponding N-quaternized macromolecules, showed far higher antimicrobial activity, suggesting that reversible N-protonation leads to greater biocidal activity than irreversible N-quaternization^[75].

Unfortunately, protonable amine polymers, not exerting their antibacterial activity by a detergent like membrane disruption mechanism, lacked the selectivity of magainin II and showed high hemolytic toxicity, mimicking the not selective melittin behavior^[75].

Amphiphilic methacrylamide random copolymers, bearing reversibly protonated primary or tertiary amine groups and encompassing a hydrocarbon hydrophobic side chains, were prepared and their antimicrobial and hemolytic properties were compared with those of similar macromolecules, containing quaternary ammonium groups^[77].

The not quaternized copolymers owing the primary amine groups proved considerable antimicrobial activity on E. coli by a membrane-disrupting action^[77] and were tunable in order to achieve CAPs with considerable antimicrobial activity and low hemolytic toxicity. Concerning this, Palermo et al. (2009) demonstrated that antimicrobial activities and biocompatibility depend in a different manner on the mole fraction of the alkyl side chains, on the length of alkyl groups and on ionic charge density^[78].

As examples, dense cationic charge leads to cytotoxicity, whereas excessive hydrophobicity leads to hemolysis associated to higher antimicrobial activity and a careful balance of structural features is necessary for achieving a well-performant antimicrobial device with low level of toxicity.

Analogs macromolecules containing tertiary amine groups proved minor antimicrobial activity by 100 times and less selectivity, while the quaternized co-polymers, in order to exert acceptable antimicrobial activity required a greater amount of hydrophobic comonomer and therefore showed poor selectivity and high hemolytic toxicity^[77].

Water-soluble poly(diallylamines) (PDAAs) with cationic charges, thanks to the presence of pyrrolidine links with secondary or tertiary amine groups, protonated with trifluoroacetic acid, revealed potent antimicrobial activity against a representative set of bacteria and versus Candida albicans^[51].

In particular, the less active poly(diallylammonium trifluoroacetate) (PDAATFA) derivative with MW = 24 kDa was bactericidal at 125 μg/mL and bacteriostatic at 62 μg/mL concentrations versus E. coli at all the conditions adopted for the experiments^[51][79].

The analogs tertiary poly(diallylmethylammonium trifluoroacetate) (PDAMATFA) proved to be bactericidal versus E. coli even at the lower concentration of 62 μg/mL. PDAATFA and PDAMATFA derivatives with higher MW (62 kDa and 55 kDa, respectively) proved to be bactericidal also against P. Aeruginosa, P. Mirabilis and K. Pneumoniae ^[79][80].

According to what reported in 2009, without however presenting numeric data as proof, the quaternary hydrophobic polymers of this series and in particular poly(diallyldimethylammonium chloride) (namely PDADMAC in the cited work) would own week antimicrobial activity^[51].

On the contrary, more recent studies showed that PDADMAC, differently named PDDA (poly (diallyldimethyl) ammonium chloride), displayed the capability of reducing of CFU counting to one of P. Aeruginosa MDR and K. Pneumoniae KPC+ at minimal concentrations of 1.5 and 0.9 µg/mL, respectively^[80] and excellent microbicidal action against E. coli ATCC 25,922 (5 µg/mL) and P. Aeruginosa (2 µg/mL) at dosage where hemolysis was 0% ^{[79][81][82][83][84]}.

The antimicrobial activity against Gram-negative bacteria of PDAA series increases with MW and with the hydrophobic-hydrophilic balance of the cationic polymers^[79].

In a study by Yang et al. (2014), it was examined whether by converting the hydrophobic moiety of a synthetic antimicrobial peptide (SAMP) into a hydrophilic one could provide hydrophilic cationic polymer compounds with maintained antimicrobial activity, but enhanced biocompatibility and selectivity for bacteria cells^[85].

In this regard, not quaternary primary ammonium trifluoroacetate copolymers (SAMPs) were prepared from N-(tert-butoxycarbonyl)aminoethyl methacrylate and butyl methacrylate. Then, by replacing butyl methacrylate with 2-hydroxyethyl methacrylate (HEMA), hydrophilic cationic mutants of previously prepared SAMPs were obtained.

The reactions were performed via AIBN-initiated free radical copolymerization or via RAFT copolymerization. The so obtained BOC-protected copolymers, after deprotection with trifluoroacetic acid provided the copolymer products. Antibacterial assays showed that long hydrophilic-and-cationic mutants of SAMPs were membrane active against bacteria but showed strikingly reduced hemolytic toxicity and dr^[85]astically enhanced selectivity^[68][85].

Polymers, encompassing both tertiary amines groups protonable in a reversible way and permanent protonated azetidinium moieties, were prepared by a simple two steps procedure^[86]. Briefly, waterborne multifunctional poly(vinylamine)s were first, prepared modifying commercial poly(vinyl amine), through a reaction with functional cationic couplers, in order to improve its hydrophobicity. Second, the modified poly(vinyl amine)s were furtherly functionalized by reaction with a bifunctional coupler, thus inserting azetidinium groups and alkyl chains.

A library of cationic polymer compounds was achieved, whose structure-activity relations, antimicrobial activities against Gram-positive and Gram-negative bacteria and hemolytic toxicity were determined.

Finally, the best polymer was used to prepare antimicrobial cotton surfaces, which were tested on E. coli establishing a 99.9% bacterial growth inhibition^[86].

Cationic polymers bearing sulfonium groups are similar to quaternary ammonium materials in terms of charge, but few studies were performed for evaluating their antibacterial and/or hemolytic activity.

In this regard, a study performed in 1990s reports the synthesis of poly(p-vinylbenzyl tetramethylene sulfonium tetrafluoroborate salts with different MW values and the assessment of their biocidal activity against S. aureus and E. coli in comparison to those of the corresponding monomer^[87].

The low MW monomer showed no activity against both the Gram-positive and Gram-negative bacteria, while even if practically ineffective against E. coli, the polymer macromolecules, exhibited acceptable antimicrobial activity, increasing with the increase of the MW, versus S. aureus.

In particular, the best performant polymer (MW = 46,800) was able to kill all the bacterial cells within 30 min at the concentrations of 100 and 10 µg/mL and was capable of destroying more than 99.9% of S. aureus cells at the lowest concentration of 1 µg/mL within 120 min of contact^[87].

The sequence of events in the mode of action of sulfonium tetrafluoroborate polymers matched the common mode of cationic biocides, which involves a phase of adsorption onto the bacterial OM, followed by impairments of membrane integrity and diffusion through the cell wall.

A second phase of binding to the CM, followed by its disruption and release of cytoplasmic constituents such as K⁺ ions, DNA and RNA up to cell death, follows.

As far as our knowledge allows, only another study dealing with sulfonium compounds with antimicrobial activity was reported. Although it does not deal with polymeric materials but concerns a library of 14 not polymeric low MW sulfonium salts, it has however reported. The prepared sulfonium salts were evaluated both for their antimicrobial activity and biocompatibility and the results showed that the major part of sulfonium salts proved higher biocompatibility and lower toxicity than those of the less toxic compound among the ammonium and phosphonium salts, taken as references. Concerning antimicrobial activity, the well performant compound was more active against S. aureus and B. subtilis by 3–4 times if compared to the best ammonium and/or phosphonium salts, but less active versus E. coli and P. aeruginosa by 5–8 times. Although sulfonium salts could be advisable as highly biocompatible devices to counteract infections by Gram-positive bacteria, their clinical applications are limited by their low thermal stability^[88].

In this regard, the author suggest that the conversion of the best representatives of the reported library in polymeric compounds could be an idea for both improving antibacterial effects and enhancing stability of the small molecules.

A series of synthetic biodegradable polycarbonates containing propyl and hexyl halogenated side chains were prepared via ring-opening polymerization (ROP) performed under an inert atmosphere in a nitrogen-filled glovebox. Subsequently, they were quaternized under ambient laboratory conditions with different heterocycles, such as methyl, ethyl and butyl imidazoles and pyridines. Alkyl imidazole, as well as pyridine and dimethylamine pyridine (DMAP) polymer derivatives were achieved and were investigated concerning their antimicrobial activity (MIC) against S. aureus, E. coli, P. aeruginosa and C. albicans (fungus)^[89].

In addition, hemolytic cytotoxicity (HC₅₀) were assessed and the results were compared with those from analogous polymer scaffolds quaternized with trimethylamine (TMA). All compounds, TMA derivatives included, showed very low hemolytic toxicity, but concerning antimicrobial activity, heterocyclic compounds proved higher activity than TMA macromolecules, especially against S. aureus.

However, remaining within the interests of this review, heterocyclic polymers proved higher activity against E. coli and lower on P. Aeruginosa and hexyl derivatives both containing pyridine, DMAP or alkyl-imidazole groups were the best performants and the MIC observed against E. coli were < 4 µg/mL and in the range 31–63 and 8–31 µg/mL, respectively^[89].

Studies concerning the mechanisms of action performed on E. coli confirmed the usual a-specific disruptive action on bacterial cell membranes that assure a minor tendency to develop drugs resistance.

A copolymer of 4-vinylpyridine (4VP), styrene (St) and divinylbenzene (DVB), namely P(4VP-St-DVB), was prepared by suspension polymerization and subsequently was quaternized with excesses of halohydrocarbons (RX) such as benzyl bromide (BzBr), C₄H₉Cl, C₄H₉Br and C₄H₉I to prepare series of insoluble pyridinium-type polymers, namely Q-P(4VP-St-DVB)-RX^[14].

Living and death cells of E. coli, suspended in sterilized and distilled water, were the selected candidates on which the capability of the prepared pyridinium CAPs of interacting and adhering to bacteria cells wall was investigated, while living cells were used to evaluate their antimicrobial properties by a colony count method^[14].

The results showed that, except for the compound containing iodine, insoluble pyridinium-type polymers, were capable to imprison both living and death bacterial cells by a partially irreversible adsorption or adhesion process, without killing the living cells. From these results, the cationic polymers developed could be advisable for the treatment of waste waters^[14].

A number of polymers, such as high-density polyethylene (HDPE), low-density poly ethylene (LDPE), polypropylene (PP), nylon 6/6 and poly(ethylene terephthalate) (PET) were functionalized with poly(vinyl-N-hexyl pyridinium bromide) (hexyl-PVP) obtaining quaternary pyridinium antimicrobial surfaces whose antibacterial effects were essayed on S. aureus and E. coli.

Suspensions of bacteria in distilled water were sprayed on the hexyl-PVP-modified polymer slides, to simulate airborne bacteria. The slides were incubated overnight and the results from the counting of survived colonies showed that all polymers provided high bactericidal activity on contact, managing to kill up to 99% of bacteria^[15].

The proposed methodology is eligible to render numerous products bactericidal, with limited costs, being the surfaces renewable by periodic washings.

Previously, the same authors was reported the same idea, by functionalizing glass slides with the same hexyl-PVP and performing the same procedure. Antimicrobial quaternized pyridinium glass surfaces were obtained, that proved to be able to kill on contact the 94%, > 99%, > 99.8% and > 99% of S. aureus (ATCC, strain 33,807), S. epidermidis (wild type), P. aeruginosa (wild type) and E. coli (ZK 605), respectively^[16].

Unfortunately, their extensive applications are strongly limited by their poor water solubility, low biocompatibility and high risk of skin irritation. In order to address these issues, it was reported the preparation of more hydrophilic methacrylate-based copolymers with bactericidal activity and biocompatibility higher than those of quaternized poly(vinylpyridine)^[90].

As comonomers were used either biocompatible HEMA and poly(ethylene glycol) methyl ether methacrylate (PEGMA). First, copolymers with different content of comonomers were prepared starting from vinylpyridine, by radical polymerization.

Second, the copolymers were quaternized with hexylbromide.

Pathogenic E. coli (O157:H7) was treated with the copolymers, in order to assess their antimicrobial effects and the results showed that several of the copolymers possessed antibacterial activity ∼20 times greater than that of pure quaternized poly(vinylpyridine) homopolymer. Even if the evidences of the study supported the hypothesis of good biocompatibility, the authors made no claim as to the biocompatibility of these materials^[90].

In the same year, Allison et al. (2007) studied the biocompatibility of analogous pyridinium co-polymers by interaction with human red blood cells, to analyze hemolysis. The results showed that blood compatibility does not depend on the length of PEG chain in copolymers containing PEGMA. A critical weight ratio PEGMA/VP was determined which divide copolymers with no-hemolysis activity from those with 100% hemolysis^[91].

Later, the hemolytic cytotoxicity expressed as HC₅₀, the minimum bactericidal concentrations (MBC) determined by the ability of the antimicrobial materials to kill 10⁶ colonies of E. coli O157 and the selectivity for some of the quaternized poly(vinylpyridine) and poly(ethylene glycol) methyl ether methacrylate copolymers, at different content of vinylpyridine (VP) [P(VP-co-PEGMA 1100)-HB] were investigated^[92].

In addition, biocompatibility was evaluated by cell viability assays performed on human intestinal epithelial cells cultivated in vitro, that offer specific advantages over red blood cells (RBC) hemolysis assays, as a measure of biocompatibility of these copolymers.

The results confirmed acceptable MBC values (70 µg/mL), associated to very low HC₅₀ (10,000 µg/mL) and high cells viability (1000 µg/mL) and therefore both low hemolytic activity and good selectivity and cells viability for P(VP-co-PEGMA 1100)-HB containing 50% VP.

Copolymers containing < 50% VP were endowed with high biocompatibility and very low HC_50, but were ineffective as antimicrobials, while copolymers containing 75% and 90% VP were more effective but endowed with very low biocompatibility and high EC_50^[92].

The VP monomer was employed also to prepare poly(4-vinyl pyridine/poly(vinylidene fluoride) (P4VP/PVDF) polymeric microbeads, by the phase inversion technique^[93].

PVDF was used as filler to achieve beads with proper mechanical strength. N-alkylation of the P4VP moieties was developed by using alkyl chains of different lengths, because known to be able to affect the antibacterial efficacy of the pyridinium-type polymers based on their number of carbon atoms^[15][16].

Several P4VP/PVDF were achieved and were investigated for their antimicrobial efficacy against both bacterial and fungal spores. E. coli and Aspergillus niger were the representative pathogens of the two categories, respectively^[93].

The pyridinium groups quaternized with C₄–C₁₀ alkyl chains proved the highest antimicrobial activity by a membrane disruptive action and in particular an number of beads of 0.8 wt % killed almost the 100% of pathogens within 20 min of an E. coli suspension of 105 CFU/mL.

A larger number of beads was necessary to kill A. niger spores at the same time, because of the more resistant nature of the fungal wall. The developed antimicrobial beads are highly stable and allows repeated applications, maintaining effective micro biocidal properties^[93].

Polyethylenimine (PEIs) or polyaziridine are polymers in which the monomeric unit of the N,N-diethylamine is repeated, forming compounds encompassing amine groups and C₂ aliphatic spacers.

PEIs can be linear, branched or dendrimeric, the first ones containing all secondary amines, while the others primary, secondary and tertiary amino groups.

They are produced on industrial scale, are commercially available and find applications in many fields, due to their positively charged character^[94].

Among the several applications, PEIs are reported to be an effective permeabilizer of the OM of Gram-negative bacteria^[19].

In this regard, the effect of commercial b-PEI (50 kDa) on the OM of representatives of Gram-negative bacteria as E. coli, P. aeruginosa and S. typhimurium was investigated by evaluating the bacterial uptake of l-N-phenylnaphthylamine, which is a hydrophobic probe indicating increased hydrophobic permeation of the OM. The uptake was prominent at the low concentration of 20 µg/mL. In addition, PEIs were able to sensitize the bacteria under study to the hydrophobic antibiotics as clindamycin, erythromycin, fucidin, novobiocin and rifampicin, to the lytic action of the detergent SDS and concerning P. aeruginosa, also to the non-ionic detergent Triton X-100. From the results, it was reported that PEIs showed to be a potent permeabilizer of the OM of Gram-negative bacteria, even if it does not inhibit the growth of bacteria to any significant extent^[19].

On the contrary, alkylated quaternized PEIs attached to flat macroscopic surfaces and to nanoparticles proved high bactericidal effects toward both Gram-positive and Gram-negative pathogenic bacteria.

Concerning this, the findings regarding the antimicrobial properties of surfaces observed by immobilizing quaternized alkyl polyvinylpyridinium salts onto coating glass and plastic slides were extended to b-PEIs, used in place of PVP.

b-PEI (≥ 25 kDa) were first, attached to NH₂-glass slides and to magnetic Fe₃O₄ nanoparticles containing NH₂ groups, then were either alkylated only with an alkyl bromide derivative or furtherly methylated by reaction with iodomethane^[20].

After a screening performed on S. aureus to detect the candidate suitable for further investigation, the hexyl-PEI glass slides and NPs were tested against other airborne S. epidermidis, P. aeruginosa and E. coli with promising results. The bactericidal efficiency versus Gram-negative bacteria of our interest was in the range 67%–74% for the devices containing only the C₁–C₁₈ alkyl chain and 95%–97% for the furtherly methylated ones^[20].

The year later, following an analogous synthetic pathway, the same authors immobilized 750 kDa b-PEIs onto cotton, wool, nylon or polyester cloths and furtherly alkylated the PEIs-modified materials with ethyl and methyl chains achieving permanently cationic PEIs-based textiles. Their micro biocidal efficiencies were assessed towards airborne bacteria and fungi^[21].

The promising results showed that micro biocidal efficiency against E. coli was in the range 96%–99%, whereas versus P. aeruginosa in the range 97%–98%.

Heine and co-workers reported the synthesis of two kinds of amphiphilic compounds (series B-I and B-II) and then the preparation of three kinds of amphiphilic poly(ethylene imine)s (series PEI-I, PEI-II and PEI-III) randomly linked to cationic and hydrophobic groups (PEI-I), to compounds of series B-I (PEI-II) and to compounds of series B-II (PEI-III).

In particular, compounds B-I encompass alkyl chains directly attached to the cationic group, while compounds B-II have the cationic group and the alkyl chains connected by a spacer^[95].

All compounds B-I and B-II and modified PEI polymers were tested to evaluate their antibacterial properties against Bacillus subtilis, S. aureus and E. coli, while the more active were selected to investigate the hemolytic toxicity. Concerning amphiphilic compounds, the highest activity against E. coli of our interest, was showed by two compounds of B-I type having C₁₄–C₁₈ chains (MIC = 8 and 10 µg/mL), but while the most active was hemolytic at concentration far higher than MIC (EC₅₀ = 22 µg/mL), the other showed an EC₅₀ lower than MIC (4 µg/mL).

The most active among compounds of B-II type similarly possessed C₁₄–C₁₈ chains and showed a higher MIC of 20 µg/mL and an EC₅₀ = 28 µg/mL.

Polymer materials were less active than amphiphilic compounds and showed higher hemolytic toxicity. On E. coli, PEI-II polymers were the most active (MIC of 60–100 µg/mL) but were also the most toxic for red blood cells (EC₅₀ << 1 µg/mL)^[95].