Synthesis, Self-Assembly, and Biomedical Applications of

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Synthesis, Self-Assembly and Biomedical Applications of Antimicrobial Peptide-Polymer Conjugates Hui Sun, Yuanxiu Hong, Yuejing Xi, Yijie Zou, Jingyi Gao, and Jianzhong Du Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00208 • Publication Date (Web): 14 Mar 2018 Downloaded from http://pubs.acs.org on March 15, 2018

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Synthesis, Self-Assembly and Biomedical Applications of Antimicrobial Peptide-Polymer Conjugates Hui Sun†, Yuanxiu Hong†, Yuejing Xi†, Yijie Zou†, Jingyi Gao† and Jianzhong Du*,†,‡ †

Department of Polymeric Materials, School of Materials Science and Engineering, Tongji

University, 4800 Caoan Road, Shanghai 201804, China. E-mail: [email protected]; Tel: +8621-6958 0239 ‡

Department of Orthopedics, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai 200072, China

ABSTRACT: Antimicrobial peptides (AMPs) have been attracting much attention due to their excellent antimicrobial efficiency and low rate in driving antimicrobial resistance (AMR) which has been increasing globally to alarming levels. Conjugation of AMPs into functional polymers not only preserves excellent antimicrobial activities but reduces the toxicity and offers more functionalities, which brings new insight towards developing multifunctional biomedical materials such as hydrogels, polymer vesicles, polymer micelles, etc. These nanomaterials have been exhibiting excellent antimicrobial activity against a broad-spectrum of bacteria including multi-drug resistant (MDR) ones, high selectivity and low cytotoxicity, suggesting promising potentials in wound dressing, implant coating, anti-biofilm, tissue engineering, etc. This

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Perspective seeks to highlight the state-of-the-art strategy for the synthesis, self-assembly and biomedical applications of AMP-polymer conjugates and explore the promising directions for future research ranging from synthetic strategies, multistage and stimuli-responsive antibacterial activities, anti-fungi applications and potentials in elimination of inflammation during medical treatment. It also will provide perspectives on how to stem the remaining challenges and unresolved problems in combating bacteria including MDR ones.

INTRODUCTION Antibiotics have been used to inhibit and kill bacteria for several decades. However, AMR is becoming more and more serious due to the increasing in global antibiotics use, inappropriate use of antibiotics in medical practice and the widespread and uncontrolled use in animals to increase meat production.1-4 Consequently, drug-resistant pathogens such as MDR bacteria have been increasing globally to alarming levels.5-7 AMPs represent a wide range of short, cationic, gene-encoded peptide antibiotics that can be found in virtually every organism, which have been regarded as a promising solution to combat MDR bacteria.8-10 The bacterial AMR induced by antibiotics and antimicrobial mechanism of AMPs are illustrated in Scheme 1. Different from traditional antibiotics which act on specific intracellular targets,11 AMPs interact with microbial membranes through electrostatic interactions and physically damage the bacterial morphology.12 Several mechanisms were proposed to explain the AMP-mediated membrane disruption of bacteria, including barrel-stave pore,13 carpet mechanism,14 toroidal pore15 and disordered toroidal pore,16 and the nature of these antimicrobial mechanisms renders bacteria less likely to develop resistance to AMPs. Besides, AMPs possess excellent anti-fungal activities that are difficult to accomplish by traditional antibiotics due to the eukaryotic cellular structure of


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fungi.17,18 However, AMPs had limited success in clinical trials due to their poor in vivo stability, salt sensitivity and high toxicity to mammalian cells caused by the lack of selectivity, etc.19-25

Scheme 1. Bacterial Antimicrobial Resistance by Antibiotics and Antimicrobial Mechanism of Peptides a)

a)

(A) Molecular mechanisms of antibiotic resistance. Antibiotics either kill bacteria or stop them from growing. Antibiotic mechanisms are based on (i) prevention of DNA or RNA synthesis, (ii) prevention of folate synthesis, thereby blocking nucleic acid synthesis, (iii) destruction of the cell wall/membrane and (iv) prevention of protein synthesis by interfering with ribosome function. Resistance mechanisms can be acquired by horizontal transfer of plasmids or other genetic elements, removal of the antibiotic by efflux pumps and the modification and degradation of antibiotics. Adapted by permission from Springer Nature: ref 4, Copyright 2018 Nature America, Inc. https://www.nature.com/nm/. (B) Mechanism of membrane-active AMPs. The positively charged AMPs contact with the negatively charged bacterial membrane via electrostatic interactions. Then the AMPs disrupt the cell membrane of bacteria by different processes. Adapted by permission from Springer Nature: ref 10, Copyright 2009 Macmillan Publishers Ltd. https://www.nature.com/nrmicro/.

To solve the above problems, AMPs have been conjugated with functional polymers. For example, poly(ethylene glycol) (PEG) can shield the positive charges of AMPs, which helps them escape the attack of immune system and prolonging the circulation time in blood, although the mechanism of the immune escape is not clear yet.26-28 Moreover, the PEGylation of AMPs


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offers improved water solubility and stability as well as reduced clearance through kidney, leading to a longer circulation time.27,29 The conjugation of functional polymers to AMPs can be achieved either by coupling30-33 or polymerization.7,34,35 The coupling method can be applied under mild conditions and in aqueous solutions to afford tailored architectures with high yield.36-38 This method is especially useful for linkage of AMPs with low molar mass polymers. However, to obtain a high coupling efficiency, excess amount of AMPs over polymer is necessary, followed by the complicated purification after the reaction.39-41 Alternatively, the ring-opening polymerization (ROP) of α-amino acid N-carboxyanhydrides (NCAs) is a versatile method for synthesizing polymers with long polypeptide chains.42-44 For example, a range of AMP-polymer conjugates with various macromolecular architectures have been synthesized by the combination of ROP of NCAs with other living polymerization methods, such as atom transfer radical polymerization (ATRP), reversible addition-fragmentation chain transfer (RAFT) polymerization, and ROP of other monomers, etc.7,34,44-51 This approach can afford a variety of AMP-polymer conjugates with controlled chain lengths, low polydispersity, and high degree of amine functionalization, etc.28,34,48 Self-assembly of amphiphilic polymers can afford nano-objects with different morphologies and functions.52-57 Recently, self-assembly of polymers using AMPs as building blocks has drawn much attention,34,47,48,58-62 which is either of benefit from the synergistic behavior of both components or to overcome shortcomings inherent to the components alone.63 The nanostructures of AMP-polymer conjugates have shown promising potentials in antibacterial applications, anti-biofilm, wound dressing, implant coating and drug delivery, etc.35,45,46,48,64-66

SYNTHESIS OF AMP-POLYMER CONJUGATES


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Coupling Methods. The covalent conjugations between the AMPs and functional polymers can be performed by a variety of reactions, in which the coupling method is very important. AMPs and polymers should have functional groups that can react with each other. Amide bond formation and click chemistry are popular for conjugation. In addition, oxidation reaction, photo cross-linking and Michael addition/Schiff base reaction are used to immobilize AMPs onto the polymers. The chemical functional groups for the conjugation of polymers with AMPs by coupling methods are shown in Error! Reference source not found., while the corresponding description of the chemical reactions is presented in Table 1. Amidation

CuAAC O

NH2

O

O or

N O

+

N3

+

HOOC

Cu (I)

N N

N H

Formation of disulfide bond

O NHS esters

Formation of urethane bond

SH

+

SH

S

S

NH2

or

O N

C

O

+

N

OH

O

Nucleophilic addition

N H

OH

O OH

Michael addition

O +

SH

O SH +

N

or

or

o-quinone

catechol

O

N

O or

OH OH

OH

HN

S

S N

OH O

S

or

or

or

S

O

Figure 1. Functional groups for the synthesis of AMP-polymer conjugates via coupling method. Fast coupling between amines and N-hydroxysuccinimide (NHS) esters in neutral aqueous media has proven to be efficient in many biomedical applications. For example, Song and coworkers

fabricated

hydrogels

via

cross-linking

polypeptides

poly(lysine)x(alanine)y

[poly(Lys)x(Ala)y, x + y = 100] bearing amine groups with multi-arm NHS ester terminated PEG (PEG-ASG). NHS groups can completely react with amines in polypeptides, which proved the


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high efficiency of this reaction. However, most of the amine groups in polypeptides will convert to amide groups after cross-linking, leading to the decrease in the hydrophilicity, positive charges and antibacterial properties.67 Muszanska et al. activated PF127-COOH with trifluoroacetic acid NHS ester and reacted with the AMP (ILPWRWPWWPWRR-NH2) to synthesize AMP-functionalized anti-adhesive polymer brush coatings. The in vitro experimental results demonstrated the excellent performance of the coatings in reducing biofilm formation and enhancing tissue integration.68 Many other functional groups are also used to couple AMPs and polymers via amidation.30,31,39 For instance, Liu and coworkers conjugated polymaleic anhydride (PMA) with the N-termini of monomeric peptides RWRW-NH2 and RRWW-NH2, in which all side chains were protected to avoid cross-linking. The enhancement in the antimicrobial activity was observed against Gramnegative Escherichia coli (E. coli) and Gram-positive Bacillus subtilis (B. subtilis), with a concomitant increase in hemolytic activity.30 As shown in Figure 2, our group synthesized an antibacterial polypeptide poly(phenylalanine12-stat-lysine15) (poly(Phe12-stat-Lys9)) and poly(εcaprolactone)19 (PCL19) via ROP. The antibacterial polypeptide poly(Phe12-stat-Lys9) was modified with hexamethylene diisocyanate (HDI) to form poly(Phe12-stat-Lys9)-NCO. And then AMP-polymer conjugate PCL19-b-poly(Phe12-stat-Lys15) was obtained with an efficiency of 54% by the coupling reaction of the –NCO group with the –OH end group of PCL using dibutyltin dilaurate as the catalyst. Besides, the cancer cell targeting molecule folic acid (FA) can be linked to the AMP-polymer conjugate to afford PCL19-b-poly[Phe12-stat-Lys9-stat-(Lys-FA)6] .39


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Figure 2. Synthetic strategy toward antibacterial polypeptide-PCL copolymer. Reprinted with permission from ref 39. Copyright 2015 American Chemical Society. Cysteines are attractive targets for conjugation of polymers and AMPs.36,37,40,69,70 One of the widely used chemistry is maleimide-thiol addition reaction, which is conducted in the pH range of 6.5-7.5. For example, Gao et al. conjugated cysteine-functionalized cationic AMP Te213


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(KRWWKWWRRC) to poly(N,N-dimethylacrylamide-stat-N-(3-aminopropyl)-methacrylamide hydrochloride) (poly(DMA-stat-APMA)) copolymer brushes on titanium surface using a maleimide-thiol addition reaction after initial modification of the grafted chains by 3maleimidopropionic acid NHS ester.37 They also synthesized peptide-conjugated copolymer brushes by tethering one end of IDR-1010cys to poly(DMA-stat-APMA) brushes again via a maleimide-thiol addition reaction.40 Kumar and coworkers modified hyperbranched polyglycerol (HPG) with 3-(maleimido)propionic acid NHS ester to introduce the maleimide groups and then reacted with sulfhydryl containing peptide (GLFDIVKKVVGALC-CONH2), as shown in Figure 3.69 A natural AMP, magainin I, was grafted onto the copolymer brushes of poly(2-(2methoxyethoxy)ethyl methacrylate-stat-hydroxyl-terminated oligo(ethylene glycol) methacrylate) (poly(MEO2MA-stat-HOEGMA)) via a N-(p-maleimidophenyl) isocyanate (PMPI) hetero linker, which can react with not only the hydroxyl groups on HOEGMA but also the additional Cterminal cysteine residue on magainin I derivatives MAG-Cys and Biotinyl-MAG-Cys via the maleimide group.70


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Figure 3. Conjugation of AMP with HPG via maleimide-thiol addition reaction. Reprinted with permission from ref 69. Copyright 2017 American Chemical Society. Thiol-ene and thiol-yne click reactions are extensively used to link the AMPs and polymers.64,71-73 For example, poly(eugenyl methacrylate) (PEgMA) provided abundant pendant double bonds for covalently linking cysteine-terminated CREDV peptide by photo-initiated thiol-ene click chemistry to prepare a series of REDV peptide functionalized surfaces.71 Cai et al. prepared a PEG-based hydrogel via nucleophilic thiol-yne addition between a 4-arm PEG functionalized with thiols and an electron-deficient alkyne. The as-fabricated hydrogels possess residual functionalities, enabling a second nucleophilic thiol-yne addition on the gel matrix. Then, a thiol-containing AMP CLATTLTIAT-NH2 was embedded into the gel matrix via nucleophilic thiol-yne addition.64 In addition, haloacetyl derivatives are thiol-reactive reagents. In Yu’s work, APMA based copolymer brushes with amine group were modified with iodoacetic acid NHS ester to afford an iodacyl group, which was then reacted with the thiol group of the cysteine residue at the C-terminus of AMPs E6 and Tet20.72


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Table 1. AMP-Polymer Conjugates Synthesized by Coupling Methods AMP

Reactive group

Polymer

Reactive group

Coupling reaction

Efficiency

Feature

Ref.

RWRW-NH2 and RRWW-NH2

–NH2

PMA

Maleic anhydride moieties

Amide bond formation

~80%

Moderate yield, excess – NH2, water sensitive

30

Poly(Lys)x(Ala)y

–NH2

6-arm PEG

NHS activated – COOH

Amide bond formation

High

High efficiency, aqueous solution, fast, pH 7~8

67

Poly(Phe12-statLys9)

-NCO

PCL

-OH

Urethane bond formation

54%

Organic solvent, catalyst

39

Cecropin B

–NH2

PVDMA

Azlactone group

Coupling reaction

-

PBS, aqueous solution, non-toxicity

74

CWR11

–NH2, – SH

Polydopami ne

Catechol groups

Nucleophilic addition

-

Tris buffer, pH 8.8, slow

51

Te213(KRWWKW WRRC)

–SH

Poly(DMA- Maleimide stat-APMA)

Thiol-maleimide 20%~70% reaction

Inert atmosphere, pH 7, side reaction

37

RRWRIVVIRVRR C

–SH

Poly(DMA- Iodacyl group stat-APMA)

Coupling chemistry

-

PBS, catalyst free

72

CLATTLTIATNH2

-SH

PEG-based hydrogels

Electron-deficient alkynyl

Thiol-yne click reaction

81%

Catalyst free, PBS, fast

64

Anoplin

Alkynyl

Chitosan

–N3

CuAAC click reaction

> 95%

Cu(I) catalyst, inert atmosphere, pH 6

75

PLL

Allyl group

PEGDA

Allyl group

Photo crosslinking

48, 55 and 70%

UV irradiation, photoinitiator, inert atmosphere

76


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Cycloaddition click reaction between alkynes and azides has been very useful in the synthesis of AMP-polymer bioconjugates.75,77,78 Copper-catalyzed alkyne-azide coupling (CuAAC) 79 is the most widely used method, which is highly specific, regioselective and can be performed in physiological solutions. Santos et al. prepared a thin film by the strong noncovalent fluorous interaction between alkynyl-terminated fluorocarbon and the fluoropolymer substrate. Subsequently, the attachment of the peptide was accomplished by CuAAC between the alkynyl group on the surface and the azido-OEG tag at the N-terminus of IG-25, using stabilizing ligands to improve the reaction outcome in aqueous solution. They also find that in comparison to binding IG-25 randomly via carbodiimide chemistry, tethering IG-25 onto the surface via click chemistry displayed higher antibacterial activities.77 Sahariah et al. acylated chitosan with in situ generated 2-azidoacetic acid using a carbodiimide coupling reagent, and then anoplin peptides were grafted onto chitosan polymers through azido moieties anchored on the 2-amino groups via CuAAC chemistry. When using CuSO4/sodium ascorbate in excess in MES which can produce Cu(I) in situ, 95% conversion was obtained after 1h at room temperature.75 Formed from the oxidation of sulfhydryl (−SH) groups, disulfide bonds is advantageous in linking AMPs and polymers.80 For instance, the immobilization of AMP hLF1-11 (GRRRRSVQWCA) on chitosan thin films was performed by forming a disulfide bridge between free sulfhydryl groups presented in the peptides cysteine side chain and in chitosan, which was pre-functionalized with N-acetyl cysteine (NAC) via carbodiimide chemistry.81 The immobilization of Dhvar5 (LLLFLLKKRKKRKY) on chitosan thin film was also performed via disulfide bridge formation between the side chain thiol of the terminal cysteine of the peptide and sulfhydryl groups on pre-functionalized chitosan.32


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Photo cross-linking is a convenient method for conjugating AMPs with polymers for engineering antibacterial hydrogels. For instance, the ROP of Z-L-Lysine NCA was initiated by allylamine to get poly(L-lysine) (PLL) end-capped with a reactive allyl group, which can be further covalently linked into PEG diacrylate (PEGDA) networks via photo-cross-linking.58 Annabi et al. obtained an antimicrobial composite hydrogels via simple visible light-induced crosslinking. Two extracellular matrix (ECM)-derived biopolymers, gelatin methacryloyl (GelMA) and methacryloyl-substituted recombinant human tropoelastin (MeTro) biopolymers and AMP Tet213 were dispersed together with co-initiator triethanolamine (TEA) and comonomer N-vinylcaprolactam (VC) in distilled water. The biopolymers/AMP/TEA/VC solution was then mixed with Eosin Y. Under the irradiation of visible light, dye molecules of Eosin Y will be excited into a triplet state, which abstracts hydrogen atoms from TEA. The deprotonated radicals initiate vinyl-bond crosslinking with VC via chain polymerization reactions, leading to the accelerated gelation and conjugation of Tet213 into the hydrogel.48 Bioinspired polydopamine (PD)-based coating is an attractive approach that can be easily adopted for AMP grafting. The reactive catechol groups exposed on the PD coating can be covalently grafted with thiols or amine groups, which increases the coupling specificity. For instance, Xu and coworkers proposed a PD film coated titanium substrates as an intermediate layer for the immobilization of a cationic peptide cecropin B (CecB). Michael addition and Schiff-base reactions take place between the amino groups in the peptide and the catechol/quinine groups in the PD, which can improve the cytocompatibility and reduce inflammation responses of the AMP alone.82 Lim et al. reported that a layer of PD was coated on the polydimethylsiloxane (PDMS) slide by dip coating, and potent synthetic AMP CWR11 (CWFWKWWRRRRR-NH2) was subsequently tethered onto catheter-relevant surfaces via


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Michael addition/Schiff base reaction of the inherent thiol and/or amine group of CWR11 and the catechol/quinine groups in PD.51 Other reactions are also used for coupling AMPs, such as specific condensation reaction between silane and silanol83 and the ring-opening reaction of azlactone group which is susceptible to attack from nucleophiles.84 For example, Coline et al. reported the synthesis of hybrid peptide building blocks bearing a hydroxysilane groups, which were immobilized via SiO-Si bonds with silanol groups generated by oxygen plasma treatment on silicone surfaces.85 The process is simple and requires neither any chemical reagent, nor toxic solvent. Although this specific approach based on Si-O-Si bonds is useful to silicone surfaces, it may be not very common for immobilizing peptide on other polymer surfaces. In Yan’s work, the azlactone functional group on the poly(2-vinyl-4,4-dimethyl azlactone) (PVDMA) can serve as convenient reactive surfaces for post-fabrication because they do not need any activation or pretreatment and can retain high activity of the bound biomolecules, which can react with the N-terminal Cecropin B.74 Polymerization Methods. Conjugation by polymerization method involves polymer macroinitiators, peptide-based initiators and peptide-based macromonomers. This method can afford AMP-polymer conjugates in a step-growth way, and may achieve various topological structures of the conjugates comparing to the coupling method. NCA-ROP is most widely applied

for

preparing

AMP-polymer

conjugates

using

amine-terminated

polymer

macroinitiators.86 For example, Deming and coworkers synthesized polypeptides of well-defined sequences using transition metal initiators.87-91 The most extensively used NCAs are derived from ʟ-lysine, phenylalanine or

D,

ʟ-valine.7,44,45,49 The sequence and the ratio of the positively

charged moieties to the hydrophobic moieties play important roles in determining the


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antibacterial activity of the AMPs.8,22,92 The chain segments of peptides can be designed under the guidance of AMP designing principles and controlled by stoichiometry using NCA-ROP initiated by polymer initiators. Besides, the polymerization can be easily conducted under mild reaction conditions. The kinds of polymerization methods for the synthesis of AMP-polymer conjugates are shown in Figure 4.

Figure 4. Synthesis of AMP-polymer conjugates by polymerization methods. Advances in NCA-ROP have provided a facile route to the synthesis of well-defined AMPpolymer conjugates with diverse macromolecular architectures, such as linear, hyperbranched and star polymers. Our group synthesized an antimicrobial polypeptide-based copolymer poly(ʟlactide)-b-poly(phenylalanine-stat-lysine), PLLA31-b-poly(Phe24-stat-Lys36), using NCA-ROP method. This copolymer can self-assemble into antibacterial micelles which are stable in aqueous solution.47 We also synthesized an AMP-grafted hyperbranched polymer which can self-


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assemble into nanosheets with weak positive charge and a “wrapping and penetrating” antibacterial mechanism.45 This hyperbranched polymer was synthesized by sequential Michael addition-based thiol-ene and free radical mediated thiol-ene reactions. A ring-opening reaction occurred between

D,

ʟ-homocysteinethiolactone hydrochloride and 2-propynylamine to afford

thiol-ene monomer, which could undergo click reaction upon the irradiation of UV light due to its alkynyl and sulfydryl. Since an alkynyl group can react with two sulfydryl groups, this process would lead to a hyperbranched structure. After that, the remaining amino group in the hyperbranched polymer can initiate the ROP of Z-Lys-NCA and Phe-NCA monomers to form randomly grafted antibacterial side chains. The click reactions in this study ensure a relative high efficiency of polymerization and quick reaction rate (4 h) under mild conditions. Qiao et al. reported a star-shaped peptide polymer nanoparticle, which was synthesized via NCA-ROP initiated from the terminal amines of poly(amido amine) (PAMAM) dendrimers (Figure 5).7 In this study, PAMAM-(NH2)16 and PAMAM-(NH2)32 with 16 and 32 peripheral primary amines were selected to be dendritic core to form 16- and 32-arm star peptide polymer nanoparticles. Their monomeric NCA derivatives were randomly polymerized from a PAMAM dendritic core to form the star arms with a theoretical lysine-to-valine ratio of 2:1. It is simple to synthesize AMP-polymer nanoparticles with precise number of arms and high branching density using dendrimer initiators, and this method can lead to predesigned topological structure.


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Figure 5. Synthesis of star-shaped AMP-polymer nanoparticles. Reprinted by permission from Springer

Nature:

ref

7,

Copyright

2016

Macmillan

Publishers

Ltd.

https://www.nature.com/nmicrobiol/. AMPs can be used as macroinitiators by coupling with initiating moieties in the synthesis of AMP-polymer conjugates, in which ATRP is widely used due to its high tolerance towards functional groups and the diversity of monomers polymerizable.93 Other controlled polymerization techniques have also been applied for AMP-polymer conjugates such as singleelectron-transfer living radical polymerization (SET-LRP)94 and nitroxide-mediated radical polymerization (NMP).95 Wooley and coworkers described a macroinitiator procedure to synthesize AMP-polymer conjugates, in which the poly(acrylic acid-b-styrene) (PAA-b-PS) block copolymer was functionalized with tritrpticin.34 The AMP tritrpticin was synthesized on Wang’s resin via solid-phase method,96 and then the terminal amino-group of this peptide reacted with initiating moieties, making it an AMP-based macroinitiator. Reactions with glutaric anhydride linker and living free radical polymerization initiator 2-bromo-isobutyryl bromide to form ATRP macroinitiator and N-tert-butyl-O-[1-(4-aminomethylphenyl)-ethyl]-N-(2-methyl-1(4-fluorophenyl)propyl)hydroxylamine to form NMP macroinitiator. Afterwards, NMP and ATRP methodologies are employed in the polymerization of tert-butyl acrylate and styrene


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monomers, facilitating the synthesis of amphiphilic block copolymer with the tritrpticin terminal. Subsequently, the peptide-polymer conjugates are cleaved from the resin and form micelle with enhanced antimicrobial activity and reduced cytotoxicity. AMP-polymer conjugates can also be prepared using peptide-based macromonomers.97-99 In principle, AMPs need to be functionalized with a polymerizable group. This method leads to the formation of polymer brushes grafted with AMP moieties. Ma and coworkers developed an antimicrobial and antifouling coating by surface polymerization of AMP macromonomers (Figure 6).100 In this study, cationic antimicrobial polypeptides were synthesized by NCA-ROP and then coupled with hetero-functionalized PEG with different lengths (methacrylate-PEGntosyl, n = 10/45/90). The methacrylate-PEGn-tosyl molecule was site-specific conjugated with polypeptide to form diblock amphiphiles, forming AMP-based macromonomers (MA-PEGn-bAMP). These copolymers were further grafted onto the surface of silicone rubber via plasma/UV-induced surface polymerizations to form bottlebrush-like coating. It is noteworthy that the polymer brush coatings can be attached to the surface of most kind of materials after decorating a polydopamine layer.49 Unlike most reported conjugates which are not site-specific and unfixed grafting density of polymer chains, and also require multi-step synthesis to provide the anchors, the polymerization of AMP macromonomers in the study of Ma’s group requires no decoration of anchors, but can achieve high fixity in the surface, relative high grafting density and homogeneity of polymer chains.


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Figure 6. (A) Synthesis of PEGn-b-AMP macromonomers. (B) Schematic illustration of the preparation of antimicrobial bottlebrush coatings on the polymeric substrate surface via plasma/UV-induced surface polymerization. Reprinted from ref 100, Copyright 2017, with permission from Elsevier. BIOMEDICAL APPLICATIONS OF AMP-POLYMER CONJUGATES Broad-Spectrum Antibacterial Activity of AMP-Polymer Conjugates. The conjugation of AMPs and functional polymers affords the AMP-polymer conjugates enhanced antimicrobial activity, stability and selectivity. The summary of the antibacterial and hemolytic activities of AMP-polymer conjugates are presented in Table 2. The initial attachment of AMPs to bacterial surfaces can be mediated by weak electrostatic interactions.25,101 Based on that mechanism, some polymers in conjugates are used to tether a number of monomeric peptides to enhance antibacterial activity.102,103 Liu and coworkers synthesized antimicrobial agents using the reactive PMA chain to link antimicrobial tetrapeptides, RRWW-NH2 and RWRW-NH2.30 Due to the


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increase in overall cationic charge of antibacterial polymeric peptides, the conjugates improved around 10-fold antibacterial effect compared to free peptides while remaining roughly constant hemolytic index (HI) value. Many soluble AMPs are random coil in aqueous solution, whereas they display α-helical structure upon interaction with a lipid membrane.18,92,101 The structure change is crucial for antimicrobial activity of AMPs.104,105 Recently, some polymers, such as HPG,69 are found to have an influence on antibacterial potency of conjugates by affecting the structure change of AMPs. For instance, Sahariah et al. reported anoplin-chitosan conjugates, grafting of short AMPs to biocompatible chitosan polymers, not only lead to an enhancement of antimicrobial potency, but also are essentially non-hemolytic.75 Moreover, the conjugates exhibited higher selectivity (HC50/MIC) towards E. coli (1000-4000-fold) or Staphylococcus aureus (S. aureus) (50-500 fold) than the parent peptide displaying only an 8-fold selectivity against E. coli or a 4-fold selectivity against S. aureus, respectively. Here HC50 is the concentration that causes 50% hemolytic; MIC is minimal inhibitory concentration. Circular dichroism (CD) spectroscopy assays were used to further confirm the formation and mechanism of α-helical structure of the conjugates induced by chitosan. The formation of α-helicity structure of peptides could be facilitated in the conjugate structure, indicating that the improvement on antibacterial activity was related to the ability of the conjugates to form intramolecular, pore-forming clusters of αhelical anoplin.106 Besides, they found that the orientation of the peptides relative to the chitosan backbone might play a role in antibacterial activity of conjugates. For example, N-terminal linkage was more active than C-terminal linkage in this case. This study presents an approach for enhancing the antibacterial activity and selectivity of AMPs at the same time by the formation of AMP-chitosan conjugates.


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Table 2. Antibacterial and Hemolytic Activities of AMP-Polymer Conjugates Name

AMP

Bacteria

MIC (µg/mL)

HC50 (µg/mL)

Selectivitya)

AMP

Conjugate

AMP

Conjugate

AMP

Conjugate

674b)

45.3b)

23

63

-

53

3.4

44

3.4

44

-

25

-

25

-

> 10000

-

> 10000

Feature

Ref.

Polypeptideg-PAMAM dendrimer

Poly(Lys2x E. coli 29.5b) -stat-Valx) CMDR A. ~40 baumannii fold

0.72b)

Polypeptideg-chitosan

Poly(Lys11 S. aureus -statE. coli Phe10)

32

16

32

16

Polypeptideg-HBP

Poly(Phe4- S. aureus stat-Lys5) E. coli

-

16

-

16

Chitosan-gpolylysine

Polylysine

S. aureus

> 1000

10

E. coli

> 1000

10

AMP-gchitosan

KLAK

S. aureus

150b)

40b)

-

> 400b)

-

10

Nanoparticle, 62 long-term activity

AMP-g-HPG

Aurein 2.2∆3-cys

S. aureus

32

85

32

> 250

1.0

> 2.9

Resistant proteolysis

Anoplin-gchitosan

Anoplin

S. aureus

128

64

512

> 4096

4.0

> 64

Enhanced activity, selectivity

a)

E. coli

64

0.85b)

110

-

-

700

400

> 100000

4

8.0

> 1024

Gram-negative 7 selectivity, less resistance to CMDR bacteria Vesicle, enhanced selectivity Nanosheet, activity selectivity

35

high 45 and

Ultrahigh selectivity

50

to 69 75 high

Selectivity: HC50/MIC; b) The unit is µM; c) The sequence is CLATTLTIAT-NH2.


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Considering that peptidoglycan is a feature component of the bacterial wall, peptidoglycanmimicry which consists of polymers and peptides might achieve high antibacterial activity with low hemolytic activity.107 Li et al. reported cationic peptidopoly-saccharides such as chitosan and polylysine based copolymer (CS-g-K16) showed excellent antibacterial activity and high selectivity.50 The peptidoglycan-mimicry was effective against Gram-negative and Grampositive bacteria (MIC, 5-20 µg mL–1), high selectivity (> 5000-10000-fold) and low toxicity to mammalian cells. Besides, polymers such as PMA and HOEGMA are used to immobilize peptides on the surface to form antibacterial brush coatings.30,70 Such polymers not only enhance the long-term antibacterial ability of peptides on the surface, but also affect the antibacterial activity of peptides. It has been demonstrated that the flexibility of polymer brush linker,40,77,108 linker chemistry,81 local charge density of peptide tethering-surface,40 conformational restrictions of polymer chains in the brush,72,108 graft density of brush37 and intrinsic properties of polymer, such as non-adhensive,70,81,109 may contribute to the antibacterial ability of the conjugates. Moreover, peptides tethered to functional polymers, like pH-sensitive74 and temperatureresponsive36 polymers were capable of reversibly switching between bactericidal and bacteriarepellent properties as the environmental conditions change. The smart surfaces provide a strategy to improve the selectivity of conjugates. Overall, the optimization of polymer structure, grafting density of peptides and linker chemistry is important for the development of highly effective AMP-polymer conjugates. Due to their excellent antimicrobial activity and biocompatibility, the AMP-polymer conjugates are expected to have potential applications in varied fields, such as anti-biofilm, wound dressing, implant coating and tissue engineering. Exploration of the Applications of AMP-Polymer Conjugates in Wound Dressing, Implant Coating and Tissue Engineering. Antibacterial polymeric hydrogels prepared from


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AMP-polymer conjugates are promising candidates for wound closure and treatment due to the following advantages: (i) good biocompatibility and biodegradability, (ii) high water absorption, (iii) rapid cross-linking, (iv) strong adhesion, (v) broad-spectrum antibacterial property and (vi) low susceptibility to AMR.9,67,110-112 For instance, Song et al. synthesized a cell-adhesive antibacterial hydrogel for wound healing based on a biodegradable antimicrobial polypeptide and 6-arm PEG-ASG.67 The polypeptide was synthesized via ROP of alanine- or lysine-derived NCA monomers. Different ratios of the monomers led to discrepant antibacterial activities (against both E. coli and S. aureus) due to the varied cationic/hydrophobic balance. Furthermore, besides the essential principles of nontoxicity and good biocompatibility, materials with fluidity and high air permeability are preferred as an ideal wound dressing.113,114 Thus, flexible repeat units,115,116 low solid concentration117 and low cross-linking density115,117 are often considered. Annabi and co-workers48 synthesized a hydrogel based on two extracellular matrix derived biopolymers, gelatin methacryloyl (GelMA) and methacryloyl-substituted recombinant human tropoelastin (MeTro). An AMP Tet213 was conjugated to the hydrogel to provide antimicrobial activity against Gram-positive and Gramnegative bacteria. The ratio of MeTro/GelMA was optimized to tune the physical properties including porosity, swelling ability and adhesive properties. When the ratio of MeTro/GelMA is 30/70, the 3D encapsulation of 3T3 fibroblasts was performed using composite hydrogels. As illustrated in Figure 7, Cell-laden MeTro/GelMA (Figure 7a, b) and MeTro/GelMA-AMP (Figure 7c, d) hydrogels were comprised of predominantly viable and proliferating cells over 5 days of culture. Similarly, Actin/DAPI staining revealed that 3T3 fibroblasts could proliferate and spread throughout MeTro/GelMA (Figure 7e, f) and MeTro/GelMA-AMP (Figure 7g, h) hydrogels. Furthermore, cell viability in MeTro/GelMA-AMP hydrogels and controls remained >


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85% (Figure 7i), and the metabolic activity increased consistently until day 5 post encapsulation (Figure 7j). However, the factors such as cross-linking chemistry, cross-linking degree and solid concentration, etc. should be considered for better applicability in the future.

Figure 7. In vitro 3D cell encapsulation in MeTro/GelMA and MeTro/GelMA-AMP (0.1% (w/v) AMP) hydrogels using 3T3 cells (scale bar = 200 µm). Data is represented as mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001, n ≥ 3). Reprinted from ref 48, Copyright 2017, with permission from Elsevier. Biomedical implants such as metallic biomaterials have been clinically used for bone repairing and regeneration, etc.118,119 However, how to prevent bacterial infection and subsequent inflammation is an important challenge for long-term use.82,120 AMP-polymer conjugates have been utilized to prepare implant coatings to combat bacteria-related inflammation that may lead to delayed wound healing and tissue necrosis in clinic implantations.32,60,82,85,120,121 Compared to other antibacterial agents, AMP-polymer conjugates in the coatings afford excellent antibacterial


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activity and good biocompatibility.122 For example, Xu et al. immobilized cecropin B (a cationic peptide) decorated PD film onto the surfaces of titanium substrates to reduce inflammation responses of orthopedic implantations.82 Pinese and coworkers modified medical silicone catheter activated by plasma treatment, with a hybrid silylated analogue of the short cationic AMP Palm-Arg-Arg-NH2.85 Costa et al. covalently immobilized an AMP, Dhvar5 (LLLFLLKKRKKRKY), onto chitosan coatings and successfully decreased Methicillin-resistant S. aureus (MRSA) colonization. Furthermore, recent progress demonstrated that such coatings of implant materials is one promising approach to prevent biofilm formation and thus to prolong the antimicrobial effect.41,123 As the formation of biofilm is related to the surface-associated bacterial communities encapsulated in a protective matrix, bacteria residing in biofilms are protected by isolating with the conventional antibiotics, resulting in concerns such as antibiotic resistance and short-time antimicrobial activity.124 Coatings by AMP-polymer conjugates can theoretically reduce or prevent bacterial attachment by altering surface properties.100,122 For example, Gao et al. synthesized a cationic AMP by NCA-ROP and then conjugated it to PEG chains to afford antibacterial and antifouling properties.100 A number of E. coli cells were adhering and growing on the surface of the pristine implant, but no visible bacteria were found on the surface of the PEG45-b-AMP coated implant. Furthermore, such coating greatly reduced non-specific protein adsorption and platelet adhesion in vitro. The later work of the same group proposed a polymer brush coating prepared based on methacrylate-ended polypeptides (Ppep) for antibacterial application and polysarcosine (Psar) for antifouling.49 The results in Figure 8 showed that the coating was capable of preventing biofilm formation by S. aureus and E. coli up to day 7. More importantly, after cross-linking or formation of coatings, the antibacterial activity of the


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polymers may be tuned, mostly influenced by the grafting density,68 cross-linking degree,32 brush length41,100 and charge distribution on the surface.

Figure 8. Formation of the biofilm by S. aureus (left) and E. coli (right) on the uncoated, poly(Ppep) and poly(Ppep/Psar) coatings, observed with (A) FE-SEM (scale bar = 10 µm), and (B) LIVE/DEAD bacterial viability assay (scale bar = 20 µm). Reproduced from ref 49 with permission of The Royal Society of Chemistry. http://dx.doi.org/10.1039/C7PY01495C. AMP-polymer

conjugates

are

also

suitable

to

synthesize

scaffolds

for

tissue

engineering.36,58,76,125 Hydrogels composed of these bioconjugates have been used as matrix for cell encapsulation. Generally, the biodegradable and non-toxic polymers like PCL and PEG are chosen as hydrogel matrix.58,123 Compared with dressing wounds, scaffolds for cell encapsulation are demanded to be stiffer, which is similar to the extracellular matrix.58 AMP chains are introduced by either chemical covalence or physical encapsulation to give the matrix with antibacterial property and bioactivity.76 Cai et al. grafted PLL onto a PEGDA hydrogel for nerve repair and regeneration. The result showed that PLL grafting significantly increased pheochromocytoma (PC12) viability by 20% in 7 days. Furthermore, compared with another


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positively charged small molecular MATC, PLL still promoted cell attachment, proliferation and neurite outgrowth.58 Their subsequent research further verified the dominant role of AMP chains in promoting cell proliferation as PEGDA matrix with different PLL grafting densities demonstrated different capability of neural progenitor cell differentiation and gene expression.76 Li et al. constructed a dual-responsive biointerface based on both antifouling polymeric layers (hexaethylene glycol, HEG) and cell adhesive tripeptide (Arg-Gly-Asp) binds for infectious prevention and tissue self-healing. The modified surfaces exhibited excellent cell adhesion, spreading, proliferation, and anti-biofilm formation.125 As for applications in tissue engineering, bioactive signals are essential for cell adhesion. AMP-polymer conjugates mainly serve as the scaffolds for cell adhesion due to bioactivity of the peptides, but it is still a tough task to remain the antibacterial activity after their modification into hydrogels. In the future, it is expected to fabricate ideal scaffolds for tissue engineering with selective bactericidal properties and good biocompatibility toward mammalian cells. SELF-ASSEMBLY: NEW INSIGHT OF AMP-POLYMER CONJUGATES Similar to the widely studied self-assembly behaviors of traditional amphiphilic copolymers,54 AMP-polymer conjugates can self-assemble into a range of nanostructures such as nanoparticles, nanosheets, micelles and vesicles by tuning the composition and structure of AMP-polymer conjugates.28,34,45,60,66,67 The nanostructures composed of AMPs and polymers take advantages of both components. For instance, the biocompatibility and stability of the AMPs can be improved, and the AMP segments endow the nanostructures excellent antimicrobial activities.27,126,127 Most importantly, the antimicrobial nano-objects exhibit great potentials in combating MDR bacteria.7 Besides, the involvement of functional polymers broadens the potential applications of AMPsbased nano-objects that are not only limited to antibacterial applications, such as tissue


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engineering, anticancer and gene delivery.58,76,128,129 In the meanwhile, the highly “armed” scaffolds or carriers that self-assembled from AMP-polymer conjugates effectively prevent the targets from the attacking of bacteria since the targets are always vulnerable. Micelles: Enhanced Antibacterial Activity and Selectivity. The amphiphilic AMP-polymer conjugates can self-assemble into micelles either with the AMPs forming the core or the coronas.28,34,46 In principle, the formation of micelles would enhance the antibacterial activity of the AMPs due to locally concentrated AMP chains,130 which is the same as the enhanced antibacterial mechanism of other antimicrobial micelles and vesicles with cationic polymer coronas reported by our group.131-134 Wooley and coworkers reported a functionalized micellar assembly prepared via an AMP (tritrpticin) conjugated amphiphilic block copolymer (PAA-bPS).34 The hydrodynamic diameter of the micelle was 51 ± 5 nm with PS segments forming the core and tritrpticin distributed on the surface. The MIC of the micelle was 13 µg/mL versus both of S. aureus and E. coli, while the MICs of the tritrpticin against S. aureus and E. coli were 16 and 32 µg/mL, respectively. Cai et al. reported a micelle formed by PEG-b-polylysine-bpoly(lysine-stat-phenylalanine)

[PEG-b-PLys-b-poly(Lys-stat-Phe)].46

The

micelle

has

comparable antimicrobial activity to AMP magainin II against the tested strains. The antibacterial micelle is highly selective against Gram-positive bacteria, including clinicallyrelated MDR Staphylococcus epidermidis. However, the mechanism of membrane disruption of bacteria and selectivity of the micelle need to be explored in further studies. Very recently, our group probed into the antibacterial mechanism of an antimicrobial micelle self-assembled from PLLA31-b-poly(Phe24-stat-Lys36).47 TEM studies were performed to monitor the membrane disruption process of E. coli and S. aureus caused by the antibacterial micelle, as shown in Figure 9. The structures of the E. coli and S. aureus were preserved well during the TEM sample


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preparation process (Figure 9A and E), then the micelles were attached to the surface of the bacteria (Figure 9B and F), leading to the disruption of the membranes and outflow of the contents of E. coli and S. aureus, as illustrated in Figure 9C, D, G and H. This is similar to the bactericidal mechanism of AMPs.13-16 The schematic illustration of the proposed antibacterial mechanism is shown in Figure 9I. However, the detailed interactions between antibacterial micelles and the membrane of bacteria are still not clear. More efforts need to be made to establish a systemic antibacterial mechanism of antimicrobial nanostructures in the further.

Figure 9. Comparative TEM images of E. coli (A-D) and S. aureus (E-H) in the absence (A and E) and presence (B, C, D, F, G and H) of antibacterial micelles from PLLA31-b-poly(Phe24-statLys36 and the schematic illustration of the antibacterial mechanism (I). The micelles which


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adhered to E. coli 1 surface and S. aureus 7 surface were highlighted by the yellow circles in TEM images (B) and (F). The rupture of E. coli 2-6 was highlighted by the red circles in TEM images (C, D), and the rupture of S. aureus 8-11 was highlighted by the red circles in TEM images (G, H). All the samples are stained by 1% phosphotungstic acid. Reprinted with permission from ref 47. Copyright 2016 American Chemical Society. Vesicles: “Armed” Nanocarriers. Polymer vesicles are promising candidates for biomedicine applications, especially as drug delivery vehicles because of the efficient loading of various cargos in the cavities and easy modification on the surfaces.54,135 The AMP-polymer conjugates as building blocks of vesicles make them “armed” drug carriers, which may benefit cancer patients who are administrating multiple drugs at the same time.35,136 For instance, our group proposed a highly anti-inflammatory antibacterial polypeptide-grafted chitosan-based polymer vesicle for simultaneously delivering anticancer and antiepileptic drugs, as shown in Figure 10.35 The synthesized vesicles have excellent antibacterial activity against E. coli and S. aureus with an MIC of 16 µg/mL based on poly(Lys11-stat-Phe10), which reduced half of the MIC of poly(Lys11-stat-Phe10) chains (32 µg/mL). Besides, the increasing of hydrophobic segments and reducing of negative charges by esterification of –COOH groups in the acid-functionalized chitosan can facilitate the antibacterial activity of vesicles. Furthermore, the antibacterial vesicle exhibits excellent blood compatibility with a HC50 of 700 µg/mL, as confirmed by the erythrocyte lysis experiment. This means the selectivity to E. coli is 44 (700/16), much higher than the poly(Lys11-stat-Phe10) chains (3.4 (110/31)). The incorporation of biocompatible acidfunctionalized chitosan into the self-assembled structure significantly lowers its toxicity, but maintains the antibacterial activity of poly(Lys11-stat-Phe10). Furthermore, the anticancer (doxorubicin) and antiepileptic (Dilantin) drugs can be efficiently loaded in the antibacterial


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vesicle and undergoes fast release triggered by the degradation of AMP segment catalyzed by trypsin. The exploration of antibacterial vesicles with stimuli-responsive antibacterial activities and high selectivity over mammalian cells and bacteria provide inspirations in developing smart drug carriers. He and coworkers functionalized a pH-responsive AMP, [D]-H6L9, on the surface of a polymer vesicle to construct a pH-responsive vesicle.137 The AMP is negatively charged at pH 7.4, making the vesicle nontoxic, serum protein resistant and long-termed circulation in blood. While the pH decreases to 6.3, the vesicle is positively charged owing to the protonation of histidines in the sequence of [D]-H6L9, leading to the enhanced cellular uptake and tumor spheroids uptake. Considering the acidic microenvironment at tumor sites, the AMP modified vesicles can be selectively accumulated in tumors and taken up by cancer cells promoted by the positively charged surface. The macropinocytosis and caveolae-mediated endocytosis by tumor cells induced by the positively charged AMP on the surface of the vesicles facilitate the lysosomes escape of the AMP modified vesicle,138,139 promoting the targeting of anticancer drugs to the nucleus of cancer cells. Nevertheless, the direct combatting of cancer cells with high selectivity by membrane disruption caused by the stimuli-responsive AMP modified vesicles will be of great significance in the treatment of cancers, since the membrane disruption of cancer cells induced by AMPs barely drives drug-resistance.


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Figure 10. Antibacterial polypeptide-grafted chitosan-based vesicle as an “armed” carrier of drugs. Reprinted with permission from ref 35. Copyright 2013 American Chemical Society. Other Nanoparticles as Antimicrobial Agents. Nanoparticles self-assembled from AMPpolymer conjugates show superior antibacterial performance over AMPs and their linear analogues.7,60,129,140 The transformation of nanoparticles obtained from AMP-polymer conjugates into fibers can accomplish the targeted treatment of bacterial infections in vivo.62 Very recently, Wang and coworkers reported an “on-site transformation” strategy for designing antibacterial agents with site-specific targeting, accumulation, and retention, which achieved precise control on the morphology of superstructures in infectious sites.62 As shown in Figure 11, the antibacterial nanoparticles were self-assembled from chitosan-peptide conjugates (CPC) that composed of a chitosan backbone and two functional peptides, e. g., an AMP and a PEG-tethered enzyme-cleavable peptide. The CPC initially self-assembles into nanoparticles with PEGylated coronas, which exhibits excellent compatibility and stability, low toxicity and long circulation in normal tissues. Once arriving at the infectious microenvironment, the protecting PEG layer was peeled off through cutting off enzyme-cleavable peptides by the gelatinase secreted by a broad spectrum of bacterial species, resulting in disruption of hydrophobic/hydrophilic balance that


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spontaneously promoted the reorganization of nanoparticles into fibrous structures via chainchain interaction of chitosan (Figure 11a). The exposure of the α-helical structures of the AMP (KLAK) leads to multivalent cooperative electrostatic interactions with bacteria and disruption of cell membranes. Intriguingly, the in situ morphological transformation also improves the accumulation and retention of CPC in infectious sites in vivo, which exhibits highly efficient antibacterial activity (Figure 11b). The in vivo experiments implied that the half retention time (τ1/2) of transformable CPC was up to 4 days and τ1/2 of morphology-unchanging analogues was only less than 1 day. Besides, the CPC showed that the female BALB/c mice injected with CPC were not noticed any obvious sign of side effects at the dose of 300 µM within 60 d, giving inspirations to develop well-engineered AMPs contained nanoparticles with stimuli-responsive antibacterial activities.

Figure 11. Illustration of the self-assembly of CPCs and the principle of enzyme-induced morphology transformation. Reprinted with permission from ref 62. Copyright 2017 WILEYVCH Verlag GmbH & Co. KGaA, Weinheim. Nanoparticles composed of AMP-polymer conjugates have also been revealed excellent antimicrobial activity in combating MDR bacteria. For instance, Qiao et al.7 proposed a class of


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antimicrobial agents, termed “structurally nanoengineered AMP polymers” (SNAPPs), which were composed of 16- and 32-arm star-shaped AMP-polymer nanoparticles synthesized by NCA-polymerization of NCA-lysine and NCA-valine initiated by a PAMAM dendritic core. The SNAPPs exhibit sub-µM activity (minimum bactericidal concentration (MBC)) against all Gramnegative bacteria tested that absent in their linear analogue, including ESKAPE (ESKAPE refers to a small group of drug-resistance bacteria including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa, and Enterobacter.) and colistin-resistant and MDR (CMDR) pathogens, while demonstrating high selectivity of killing Gram-negative bacteria over mammalian cells (> 100). The star-shaped structure is ascribed to the excellent antibacterial performance due to the high local charges and AMP mass afforded by the star architecture, which is consistent with previous studies.131,141 Cryo-TEM and optical microscope experimental 3D structured illumination microscopy (3DSIM) images (Figure 12) revealed that the antimicrobial activity of SNAPPs proceeds via a multimodal mechanism of bacterial cell death by outer membrane destabilization, unregulated ion movement across the cytoplasmic membrane and induction of the apoptotic-like death pathway, which has great differences with that of AMPs. The equipotency of SNAPPs against all of the Gram-negative bacteria tested suggested that the multimodal mechanism of action is nonspecific, and this is why bacteria did not acquire resistance, even after 600 generations growth of CMDR pathogens in the presence of the agent. At low concentration, the nanoparticles bind via electrostatic interactions with lipopolysaccharide and outer membrane of Gram-negative bacteria, leading to destabilized/fragmented area. They then assemble and traverse the cell envelope driven by the transmembrane electrical potential, which causes membrane perturbations that result in unregulated transmembrane ion movement in the cytoplasmic membrane, inducing the


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apoptotic-like death of bacteria. At high concentration, the nanoparticles rapidly cause cell lysis by direct disruption of the outer membrane and cytoplasmic membrane. Following the previous study, the same group investigated the bionano interaction of SNAPPs by determining the antimicrobial activity against different Gram-negative bacteria in complex biological media (e. g., simulated body fluid and animal serum).142 It was found that the presence of divalent cations reduced the antimicrobial activity of SNAPPs toward E. coli, Pseudomonas aeruginosa and Klebsiella pneumoniae because of the decrease in the ability of SNAPPs to cause membrane disruptions. However, the potency of SNAPPs could be re-established with the co-administration of a chelating agent such as ethylenediaminetetraacetic acid. Considering the difficulty and challenges in inhibiting CMDR pathogens, the resistance-free SNAPPs will have significant potentials in treatment of bacterial infections caused by CMDR pathogens. But the degradation, metabolism and safety of the SNAPPs need to be evaluated considering the demand for clinical applications.


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Figure 12. Optical Microscope eXperimental 3D-SIM images of E. coli before and after treatment with AF488-tagged SNAPP S16 in Mueller–Hinton broth. Z-projection images of E. coli before (a) and after incubation with AF488-S16 at 0.5 × MBCtagged (b), 1 × MBCtagged (c-e) and 2 × MBCtagged (f-h). The E. coli cell membrane was stained with FM4-64FX (red) and S16 with AF488 (green) in all images. All images are representative of three independent experiments. Reprinted by permission from Springer Nature: ref 7, Copyright 2016 Macmillan Publishers Ltd. https://www.nature.com/nmicrobiol/. CONCLUSIONS AND FUTURE PERSPECTIVES In this Perspective, we have highlighted recent advances in the synthesis, self-assembly and applications of AMP-polymer conjugates, which are synthesized by tethering AMPs with functional polymers either by coupling methods or polymerization methods. The conjugates preserve the functions of both components, and may create new properties that are absent individually. The incorporation of biocompatible polymers with AMPs can reduce cytotoxicity and improve in vivo stability, promoting their potentials in broad-spectrum antimicrobial applications, anti-biofilm, wound dressing, and implant coating, etc. Besides, the nanostructures such as micelles, vesicles, hydrogels, and other nanoparticles have shown advantages in cell culture, combatting MDR bacteria and fungi, and drug and gene delivery, etc. Despite the significant progress in the synthesis, self-assembly and exploration of biomedical applications of AMP-polymer conjugates as summarized above, this field is in a phase of rapid growth and discovery, and we end our Perspective here by discussing the scientific challenges to be conquered while proposing some directions for future research. It is difficult to control the specific sequences of the AMPs in AMP-polymer conjugates synthesized by NCA-ROP method, while the sequences may play an important role in


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determining the antimicrobial activity. The sequences of the AMP segments may be quite different from batch to batch since the AMP segments are usually synthesized by the random copolymerization of the hydrophobic and positively charged NCA-monomers. That is why it is difficult to reproduce the AMP-polymer conjugates with excellent antibacterial activity even at the same conditions. Fortunately, recent advances in the synthesis of sequence-controlled polymers may help to solve this problem.143-145 For example, our group synthesized a series of antibacterial peptide-mimetic alternating copolymers (PMACs) with excellent antibacterial activity against both Gram-positive and Gram-negative bacteria.146 The MICs of the PMACs against E. coli and S. aureus are only 8.0 µg/mL. The PMACs can self-assemble into vesicles in pure water with extremely low cytotoxicity (IC50 > 1000 µg/mL), which can encapsulate growth factors in aqueous solution and release them during long-term antibacterial process for facilitating bone repair. It would be very useful in the treatment of infections caused by multi-bacteria, fungi or drug resistant bacteria by synergistic antibacterial effect of antibiotics and AMP-polymer conjugate assemblies since most of traditional antibiotics are not sensitive to fungi and drug resistant bacteria. Considering broad-spectrum antibiotics are sensitive to most of the bacteria in short time, the AMP-polymer conjugate assemblies can be designed to kill the rest of bacteria including MDR bacteria. This strategy may effectively prevent bacterial antimicrobial resistance, and might enhance the antibacterial efficacy compared to individual components due to the synergistic antibacterial effect of antibiotic and AMP-polymer conjugate assemblies. Considering the different antibacterial mechanism of antibiotics and AMP-polymer conjugate assemblies, the membrane perturbation and destabilization caused by AMP-polymer conjugate assemblies would promote the interactions between antibiotics and bacteria, especially for those


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aiming at blocking the replication of generic materials of bacteria. Furthermore, the AMPpolymer conjugate assemblies have been proven to exhibit excellent antibacterial activity against MDR bacteria.7 The multistage antibacterial capability of the complex composed of different assemblies (e. g., nanoparticles embedded in hydrogels) would be advantageous in the treatment of MDR-bacteria-caused infections. Thus, nanostructures from self-assembly of AMP-polymer conjugates may contribute to antibacterial properties. In this case, the antimicrobial mechanism of nanostructures should be studied in more detail. Different from AMPs, the antimicrobial mechanism of nanostructures is more complicated and related to their morphologies, even concentrations.7 Therefore, there should be sufficient data and discussions from TEM, atomic force microscopy (AFM), dynamic light scattering (DLS) and static light scattering (SLS), which can support well with each other to illustrate the morphology of conjugates. The understanding of the antibacterial mechanism of different nanostructures in the absence and presence of antibiotics would be valuable for designing next-generation antibacterial agents. Conjugation of AMP to stimuli-responsive polymers can afford nanostructures with responsiveness to pH, redox, enzyme and temperature, etc., leading to “smart” antimicrobial behaviors. The antimicrobial activity can be “switched on” or “off” by the input stimuli or the difference of microenvironments between normal tissues and infected parts, which endows the antibacterial nanostructures targeting capability. Additionally, the cleavage of biocompatible polymer layers by removing vulnerable linkage such as enzyme-cleavable, pH or redox cutting off covalent bonds enables the antibacterial nano-objects with passive targeting ability. It would also be a promising design strategy to use AMP-polymer conjugates as building blocks for construction of nano-carriers, especially those for anticancer drug delivery. The “armed” carriers can kill cancer cells and eliminate the inflammation at the same time,35 which


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would be very beneficial for the patients since the necrosis of cancer cells always causes inflammation that promotes the regeneration of tumors. Moreover, some AMPs exhibit anticancer capability and high selectivity of killing cancer cells but nontoxicity to normal cells. The nano-carriers constructed by the conjugates of those AMPs and functional polymers (e. g., biocompatible, biodegradable, and stimuli-responsive polymers) would help to escape from the immune system and accumulate at the tumors. Subsequently, the release of those AMPs triggered by the dissociation of the nanostructures or the cleavage of the vulnerable bonds would bring long-term anticancer capability but no drug-resistance. Most promisingly, the generation of secondary structures of the AMPs by the in situ self-assembly might reveal enhanced accumulation and retention, as well as antibacterial and anticancer activities. We are confident that the AMP-polymer conjugates especially the nanostructures from self-assembly will exhibit unique properties and promising potentials in biomedical applications. However, overengineered structures should be avoided as too much chemical modification may reduce the biocompatibility and activity. An ideal antimicrobial drug should be of eligible physicochemical and biochemical structures to fulfill the demands on multidimensional spaces including antimicrobial activity, in vivo safety, druggability, pharmacokinetics and bioaccumulation, etc. Anti-fungal effects of AMP-polymer conjugates can be taken into consideration to develop drugs with broad-spectrum antimicrobial properties. Anti-fungi is a tough challenge because fungi are eukaryotic which means that on a cellular level they are closely related to other eukaryotic kingdoms such as human, animals and plants, leading to poor selectivity for most conventional antibiotics and AMPs. Liu et al. reported a family of nylon-3 polymer (poly-βpeptide), which displayed significant selective toxicity toward the fungal pathogen.147-149 Furthermore, the nylon-3 polymers have strong activity against spore outgrowth150 and fungal


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biofilms.151 Considering that some nylon-3 polymers have excellent antifungal activity but relatively poor antibacterial activity,147 one of future directions in AMP-polymer conjugates could be conjugation of antifungal polymers with antibacterial peptides. As a result, this kind of AMP-polymer conjugates will provide not only better antibacterial properties but also excellent antifungal and even multifunctional properties for a range of biomedical applications. The in vitro evaluation of antimicrobial effects should follow standard protocols and be comparable between different publications. MIC and MBC are factors to determine the susceptibility of bacteria to antimicrobial agents and to quantitatively evaluate their antimicrobial activity.47 Therefore, the definition of MIC and MBC should be expressed clearly to establish a unified standard, which is also convenient to compare the antibacterial ability with other work. MBC is complementary to the MIC as the MIC test demonstrates the bacteriostatic activity of antimicrobial agents while the MBC demonstrates the bactericidal activity.152,153 The following issues should be kept in mind for the in vitro evaluation: (1) Agar dilution and broth dilution are the most common methods utilized to give MIC values of the antimicrobial agents.154 Broth dilution method conducted on 96-well microtiter plate is easy and valid to get MIC by turbidity or redox-indicators.155 According to Clinical & Laboratory Standards Institute (CLSI), turbidity can be easily distinguished by unaided eyes or obtained more accurately by measuring the optical density at 405 nm or 600 nm.33,156-158 However, it’s better to use redox indicators to avoid interference when test samples are not fully soluble.159 Multiple assays can be conducted simultaneously for better comparison of the activities of different agents. (2) As for the determination of MBC, the most widely used method is to subculture the samples of completely inhibited dilution cultures from the MIC tests on agar plates.160 After


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incubation, the growth of the live organisms can be quantitatively obtained by counting colonies and MBC was taken as the minimum concentration with no visible bacterial colony on agar.155,156 Besides, some researchers have employed an alternative method to monitor the kinetic growth of bacteria by UV-vis spectrophotometer.161-163 According to this method, the optical density (OD) is measured every two hours and the MIC is the minimal concentration where no more than half of the largest OD value (control) is observed.163 However, this method remains controversial as the OD value is not linear with the bacterial quantity. Furthermore, incomplete inhibition is not scientifically valid in terms of microbiology as the survivals are selectionpressure induced phenotypes and probably bring out drug resistance, which might cause an even worse condition. Nevertheless, it can serve to further confirm the antibacterial activity and monitor the constant bacteria growth, thus benefiting the evaluation of their dynamic activities and providing insights into the design of highly efficient antimicrobial agents. In future work, multiple methods can be employed to comprehensively evaluate the antimicrobial activities. (3) In some cases, MIC50 or MIC90 was defined as the minimum concentration required for 50% or 90% bacterial growth inhibition from one isolate of a species.164-166 However, in terms of microbiology, MIC50 and MIC90 were derived from percentile calculations of individual MIC values conducted on a panel of isolates (approximately 10-50 isolates) of a bacterial species.164168

As described above, the definitions and the testing methods of MIC50 and MIC90 are totally

different, which may cause confusions between people with different academic background. It’s noteworthy that MIC50 or MIC90 is frequently used to indicate the susceptibility of pathogens in microbiology, which is time- and resource-consuming.164,165 However, most research on antibacterial materials are still at the proof-of-concept stage, where it is more easily to evaluate the antibacterial activity via MIC. Therefore, MIC50 and MIC90 are not essential at the


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preliminary stage, but can be expected to be added in the further work if necessary. In general, it is suggested to take microbiological standards as reference, which is more convenient to compare the results from different work. The in vivo testing of antimicrobial activity have been conducted by different rodent animal infection models, including mostly used subcutaneous infection model where specific bacterial strains are cultured in broth medium to prepare for the skin inoculation. In some cases, the antibacterial materials will be first implanted into dorsal side of the rats123 or some injectable hydrogels will be injected into the subcutaneous pocket.169,170 After that, the bacterial strains solution will be directly injected to the implants or hydrogel regions to make contact with each other. In other cases, the implants with pre-seeded bacterial strains will be simultaneously implanted171 or the prepared wound will be subsequently covered with the dressing.172,173 Significantly, this kind of subcutaneous infection model is not very complex by the direct injection of the bacterial strains. However, the experiments need exquisite skills to make sure the injection is in the right position because a slight deviation of injection may leads to the failure. Furthermore, large dose of bacterial strains will cause the skin burst while the small dose is not conducive to the observation of results. Therefore, the dose of the bacterial strains should be strictly controlled according to the weight of the mice and many other factors. Also, due to the AMR-polymer conjugates with promising potential in eradicating biofilms,41,49,68,100 more in vivo biofilm models need to be established for evaluation on antibiofilm activity. There still seems to be a long way to go for real world applications for the AMP-polymer conjugates and their corresponding nanostructures. There are several important challenges should be addressed before clinical trials, for example:


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(1) The biocompatibility testing should be more comprehensive. According to international standard ISO 10993 (or other similar standards), the following testing should be tested to claim a material

‘biocompatible’:

cytotoxicity,

sensitization,

hemocompatibility,

pyrogenicity,

implantation, genotoxicity, carcinogenicity, reproductive and developmental toxicity, and degradation assessments. However, in many cases, especially in polymer-based research articles, biocompatibility was simply considered as cytotoxicity. Also, the antibacterial selectivity of AMP-polymer conjugates and their assemblies in most systems are insufficient to meet the strict criteria in clinical settings. The incorporation of specific targeting biomolecules such as aptamers may be an option to solve this problem. (2) In vivo studies are necessary to evaluate the biological activities of AMP-polymer conjugates and their assemblies, as in vitro conditions do not necessarily reflect the true outcomes of nanomaterials. In addition, the in vivo stability, circulation, accumulation and metabolism should be evaluated very systematically. (3) The immunomodulatory responses of AMP-polymer conjugates and their assemblies need to be explored. Some synthetic antimicrobial polymers have demonstrated additional capability to invoke immune responses in vivo to combat bacterial infection. The understanding of the role of AMP-polymer conjugates in immune responses, and the controlling of the immune responses via the molecular structure of AMP-polymer conjugates and the corresponding morphology of the assemblies would bring new insights in the antibacterial field. (4) The antibacterial mechanism of AMPs or AMP-polymer conjugates should be investigated very clearly, which is important to their translation into clinical applications. As mentioned in the Introduction, AMPs can kill bacteria via the membrane disruption mechanism. However, the actual antibacterial process may be much more complicated. For example, membrane


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perturbation induced by charged lipid clustering is gradually accepted as an alternative way to kill bacteria.7,14,16 Therefore, efforts should be made to investigate the antibacterial mechanism of AMPs with an open mind. In conclusion, an important principle for designing AMP-polymer conjugates is to take advantage of the intrinsical toxicity of AMPs to kill microorganisms, while the polymer segments can neutralize the toxicity and improve the biocompatibility. Therefore, the balance between the ratio of AMPs and polymers is critical in designing the AMP-polymer conjugates. Based on the designing principle of AMP-polymer conjugates, the design and preparation of antimicrobial materials including nano-objects such as micelles and vesicles, as well as macroscopic antibacterial hydrogels, surfaces and coatings would be of particular concern, since those antimicrobial materials may improve antimicrobial property and biocompatibility and afford new functions, providing promising potentials for biomedical research and clinic applications.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT


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J.D. is supported by NSFC (21674081), Shanghai International Scientific Collaboration Fund (15230724500), the Fundamental Research Fund for the Central Universities (1500219107), and Shanghai 1000 Talents Plan (SH01068). REFERENCES (1) Pitout, J. D. D.; Laupland, K. B. Extended-Spectrum Beta-Lactamase-Producing Enterobacteriaceae: An Emerging Public-Health Concern. Lancet Infect. Dis. 2008, 8, 159-166. (2) Boucher, H. W.; Talbot, G. H.; Bradley, J. S.; Edwards, J. E., Jr.; Gilbert, D.; Rice, L. B.; Scheld, M.; Spellberg, B.; Bartlett, J. Bad Bugs, No Drugs: No ESKAPE! An Update from the Infectious Diseases Society of America. Clin. Infect. Dis. 2009, 48, 1-12. (3) Kumarasamy, K. K.; Toleman, M. A.; Walsh, T. R.; Bagaria, J.; Butt, F.; Balakrishnan, R.; Chaudhary, U.; Doumith, M.; Giske, C. G.; Irfan, S.; Krishnan, P.; Kumar, A. V.; Maharjan, S.; Mushtaq, S.; Noorie, T.; Paterson, D. L.; Pearson, A.; Perry, C.; Pike, R.; Rao, B.; Ray, U.; Sarma, J. B.; Sharma, M.; Sheridan, E.; Thirunarayan, M. A.; Turton, J.; Upadhyay, S.; Warner, M.; Welfare, W.; Livermore, D. M.; Woodford, N. Emergence of a New Antibiotic Resistance Mechanism in India, Pakistan, and the Uk: A Molecular, Biological, and Epidemiological Study. Lancet Infect. Dis. 2010, 10, 597-602. (4) Jansen, K. U.; Knirsch, C.; Anderson, A. S. The Role of Vaccines in Preventing Bacterial Antimicrobial Resistance. Nat. Med. 2018, 24, 10-19. (5) Rice, L. B. Federal Funding for the Study of Antimicrobial Resistance in Nosocomial Pathogens: No ESKAPE. J. Infect. Dis. 2008, 197, 1079-1081. (6) Taubes, G. The Bacteria Fight Back. Science 2008, 321, 356-361.


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Biomacromolecules

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Synthesis, Self-Assembly, and Biomedical Applications of

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