Molecular engineering of antimicrobial peptide (AMP)-polymer conjugates

Abstract

As antimicrobial resistance becomes an increasing threat, bringing significant economic and health burdens, innovative antimicrobial treatments are urgently needed. While antimicrobial peptides (AMPs) are promising therapeutics, exhibiting high activity against resistant bacterial strains, limited stability and toxicity to mammalian cells has hindered clinical development. Attaching AMPs to polymers provides opportunities to present AMPs in a way that maximizes bacterial killing while enhancing compatibility with mammalian cells, stability, and solubility. Conjugation of an AMP to a linear hydrophilic polymer yields the desired improvements in stability, mammalian cell compatibility, and solubility, yet often markedly reduces bactericidal effects. Non-linear polymer architectures and supramolecular assemblies that accommodate multiple AMPs per polymer chain afford AMP-polymer conjugates that strike a superior balance of antimicrobial activity, mammalian cell compatibility, stability, and solubility. Therefore, we review the design criteria, building blocks, and synthetic strategies for engineering AMP-polymer conjugates, emphasizing the connection between molecular architecture and antimicrobial performance to inspire and enable further innovation to advance this emerging class of biomaterials.

Figure 1.

Molecular engineering of AMP-polymer conjugates to modulate structure, properties, and performance.

Figure 2.

Examples of conjugation strategies and possible architectures: (a) grafting-to; (b) grafting-from, illustrated here as a peptide growing from a polymer backbone (polymer can also be grown from a peptide functionalized with an initiator or chain transfer agent); and (c) grafting-through methods. The left column shows conjugation reactions for generating comb/brush polymers, and the right column illustrates other possible architectures that can be realized by these methods.

Figure 3.

AMP-polymer conjugation via grafting-to approaches: a) thiol-ene reaction and example conjugation of a thiol-containing AMP to maleimide-functionalized chitosan, adapted with permission from Pranantyo et al., copyright © 2018 American Chemical Society; b) thiol-yne reaction and example conjugation of a thiol-containing AMP to an alkyne-functionalized polyphosphoester, adapted with permission from Pranantyo et al., copyright © 2016 American Chemical Society; c) iodoacetamide-cysteine reaction and example conjugation of a thiol-containing AMP to an iodoacetamide-functionalized hyperbranched polyglycerol (HPG), adapted with permission from Kumar et al, copyright © 2015 American Chemical Society; d) disulfide formation and example conjugation of a thiol-terminated AMP to thiolated chitosan via disulfide linkages, adapted with permission from Costa Petrin et al., copyright © American Chemical Society 2019; e) azide-alkyne reaction and example conjugation of alkyne-terminated AMPs to azide-functionalized chitosan, adapted with permission from Barbosa et al., copyright ® 2017 Elsevier; and f) amidation reactions of amines with carboxylic acids, activated esters, and anhydrides. As an example, we show the conjugation reaction of an amine-terminated AMP to poly(maleic anhydride), followed by capping of acid groups with methyl esters using trimethylsilyldiazomethane, adapted with permission from Liu et al., copyright © 2006 American Chemical Society. In all schemes, R and R’ can represent either polymer or AMP.

Figure 4.

Grafting-from AMP-polymer conjugation: a) NCA ROP scheme and an example ROP of lysine (lys) and valine (val) NCA monomers from PAMAM dendrimers, adapted with permission from Lam et al., copyright © 2016 American Chemical Society; b) RAFT polymerization scheme and an example of grafting poly(2-hydroxyethylmethacrylamide) from an AMP-functionalized chain transfer agent (CTA), adapted with permission from Luppi et al., copyright © 2019 The Royal Society of Chemistry; c) ATRP and d) NMP schemes and sequential polymerization of tert-butyl acrylate and styrene from a resin-bound AMP. Subsequent removal of the conjugates from the resin and removal of tert-butyl groups yielded AMP-poly(acrylic acid)-block-polystyrene conjugates. Adapted with permission from Becker et al., copyright © 2005 American Chemical Society.

Figure 5.

Additional approaches to prepare AMP-polymer conjugates: a) Grafting-through synthesis of comb conjugates by conventional radical polymerization of vinyl-terminated lysine-based AMPs. b) Cross-linking linear conjugates prepared by RAFT polymerization into stars by chain-extension: example preparation of star conjugates by chain extension of polylysine- and glucosamine-containing linear polymer arms with bisacrylamide. Adapted with permission from Wong et al., copyright © 2016 American Chemical Society.

Figure 6.

AMP-polymer conjugates designed in a) linear, b) comb/brush, c) star, and d) hyperbranched architectures.

Figure 7.

AMP-polymer conjugate assemblies in aqueous solution: a) AMP-functionalized poly(acrylic acid)-block-polystyrene assembles into micelles with polystyrene cores, polyacrylic acid shells, and the AMP on the outermost surface, adapted with permission from Becker et al., copyright © 2005 American Chemical Society; b) comb polymer with pendant AMPs assembles into nanoparticles postulated to kill bacteria by membrane disruption, adapted with permission from Zhen et al., copyright © 2019 Royal Society of Chemistry; and c) hyperbranched AMP-polymer conjugates form nanosheets that disrupt bacterial membranes, adapted with permission from Gao et al., copyright © 2016 American Chemical Society.

Figure 8.

Schematic illustrations of stimuli-responsive AMP-polymer conjugate systems. a) Redox-responsive poly(2-ethyl-2-oxazoline) (PEtOx)-cyclic peptide nanotube (CPNT) conjugates are activated by intracellular glutathione-triggered disulfide reduction, cleaving the PEtOx polymer to reveal bactericidal CPNTs. b) Conjugates of the AMP LK₁₃ and PEGylated chitosan form nanospheres in aqueous environments. The neutral surface charge of the nanospheres allows for diffusion into the negatively charged extracellular matrix surrounding P. aeruginosa. Interaction with the bacterial membrane causes disassembly of the nanospheres and conversion of LK₁₃ from random coil to a bactericidal α-helical conformation. c) Emulsion-templated fabrication of nanoparticles from pH-responsive linear triblock copolymers composed of poly(d,l-lactic-co-glycolic acid) (PLGA), poly(ethylene glycol) (PEG), and poly(l-histidine) (PLH) furnished pH-responsive nanoparticles encapsulating vancomycin. At physiological pH, the nanoparticles are anionic. Lowering the pH to 6.0-6.5 protonates the imidazole groups on PLH, rendering the nanoparticles cationic for increased interaction with bacterial membranes and subsequent vancomycin-induced bacteria killing. d) Capping lysine amines of the AMP KLA on PEG-KLA conjugates with pH-responsive 2,3-dimethylmaleic anhydride (DA) yields anionic conjugates at physiological pH. In acidic environments (pH 5.5), the DA caps are cleaved to reveal cationic lysines that target bacterial membranes. Encapsulation of PDT agents in the PEG-KLA conjugates enables light-triggered bacteria killing.

Figure 9.

UV-induced polymerization of PEG-AMP from polydimethylsiloxane (PDMS) surfaces to generate tethered bottlebrush AMP-polymer conjugates. Adapted with permission from Gao et al., copyright © 2017 Elsevier.