Recent Developments in Antimicrobial-Peptide-Conjugated Gold

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Review

Recent Developments in Antimicrobial Peptide Conjugated Gold Nanoparticles Urawadee Rajchakit, and Vijayalekshmi Sarojini Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00368 • Publication Date (Web): 11 Sep 2017 Downloaded from http://pubs.acs.org on September 12, 2017

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Recent Developments in Antimicrobial Peptide Conjugated Gold Nanoparticles

Urawadee Rajchakit and Vijayalekshmi Sarojini* School of Chemical Sciences, The University of Auckland, Private Bag, 92019 Auckland, New Zealand

*Author for correspondence Vijayalekshmi Sarojini Phone: + 64 9 9233387 E-mail: [email protected]

Abstract The escalation of multidrug-resistant pathogens has created a dire need to develop novel ways of addressing this global therapeutic challenge. Because of their antimicrobial activities, combination of antimicrobial peptides (AMPs) and nanoparticles is a promising tool to kill drug resistant pathogens. In recent years, several studies using AMP-nanoparticle conjugates, especially metallic nanoparticles, as potential antimicrobial agents against drug resistant pathogens have been published. Amongst these, antimicrobial peptide conjugated gold nanoparticles (AMP-AuNPs) are particularly attractive because of the non-toxic nature of gold and the possibility of fine tuning the AMP-NP conjugation chemistry. The following review discusses recent developments in the synthesis and antimicrobial activity studies of AMP-AuNPs. Classification of AMPs, their mechanisms of action, methods used for


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functionalizing AuNPs with AMPs and the antimicrobial activities of the conjugates are discussed.

1. Introduction

The alarming increase in antibiotic resistance has become a serious global health issue threatening the achievements of modern medicine.1-3 The World Health Organisation warns that the 21st century may be seeing the beginning of a pre-antibiotic era.4,

5

Fatality from

antibiotic resistant infections are predicted to rise into the millions by 2050.6 While the development of new antibiotic drugs has been dramatically slow, the resistance rate has increased rapidly. Drug resistant bacteria often exist as biofilms protected by extracellular polymer matrices, making them impervious to conventional antibiotics. Biofilm formation is a major problem in various scenarios including hospital settings, food processing plants and marine installations to name a few.7, 8 There is an urgent need to develop novel antibiotics with less chance of resistance development in order to combat the threat from multidrug resistant (MDR) pathogens. Antimicrobial peptides (AMPs) are considered as attractive alternatives to conventional antibiotics in the fight against MDR pathogens.9 AMPs are emerging as essential tools for killing pathogenic bacteria, protozoa, and fungi alike because of their broad-spectrum of activity and low rate of resistance development.10-13 Despite these desirable features AMPs have limitations in therapeutic development due to their poor enzymatic stability and low permeability across biological barriers.14-16 Chemical modifications such as peptide cyclisation, use of non-protein amino acids, peptidomimetics, lipidation etc. are often used to overcome these drawbacks, particularly to enhance enzymatic stability.17-21



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Nanoparticles provide another potential solution to combat multidrug resistant pathogens. Nanoparticles by themselves (e.g. silver, other metal oxides such as titanium, copper, zinc, iron etc.) have been known to possess antimicrobial activities and work through numerous modes of action.22-26 Nanoparticles can disrupt the bacterial cell membrane causing cell penetration, react with intracellular targets and cause toxicity.22-27 Thus, immobilization of AMPs to metallic nanoparticles might represent an alternative solution in the fight against antibiotic resistant pathogens and also help to enhance the antimicrobial activity of both components.28 Additionally immobilization to nanoparticles could also help to overcome some of the inherent drawbacks of AMPs such as susceptibility to proteases and poor permeability across biological barriers, since nanoparticles are excellent target specific drug delivery systems.14 Despite significant progress made in nanoparticle research, concerns about their potential toxicity still persist and need to be fully addressed in order to realise their clinical potential.29-32 Lack of efficient clearance from the body is one of the major factors that contribute to the toxicity of nanoparticles.33 However, significant progress is being made in fine tuning the size, shape, surface properties, clearance attributes and biodegradation properties of inorganic NPs thus helping to address the current concerns and providing promise for the future.34-37

This review focusses on antimicrobial peptide conjugated gold nanoparticles (AMP-AuNPs). After presenting a brief introduction to and general properties of both antimicrobial peptides and nanoparticles in general, we discuss AuNP toxicity issues followed by results from the literature on AMP-AuNP conjugates. In recent years, significant progress has been made in their development allowing us to present the most recent examples from the literature where this approach has shown promise in the fight against MDR pathogens. Given the inherent


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antimicrobial properties of AgNPs, we have included selected examples on AMP-AgNP conjugates as well.

2. Overview of Antimicrobial Peptides (AMPs) 2.1 Classification of AMPs AMPs are produced by the innate immune system of all types of organisms, from microbes to mammals to defend themselves against pathogens.38,

39

Based on their biosynthetic origin,

AMPs can be divided into two groups: non-ribosomal and ribosomal peptides.9 Nonribosomally synthesized peptides which contain at least two moieties acquired from amino acids, are mostly produced by bacteria and significantly modified.9 Lipopeptide antibiotics, briefly described below, form an important sub-class within this group.

Lipopeptides are composed of long-chain fatty acids linked to short linear or cyclic peptides (about 15 residues).40 Natural lipopeptides are largely produced by fungi e.g. Aspergillus and bacteria e.g. Streptomyces, Pseudomonas, and Bacillus. Daptomycin40, 41 and polymyxins.42, 43

are examples of FDA approved commercial lipopeptide antibiotics. Daptomycin produced

by Streptomyces roseosporus is used against Gram-positive bacterial infections while the polymyxin family, produced by Bacillus polymyxa, is used against multidrug-resistant Gramnegative bacteria. Chemically synthesized lipopeptides with the ability to kill bacteria and eradicate bacterial biofilms have been reported and are gaining importance as promising antimicrobials in the post-antibiotic era.44-47

Ribosomally synthesized peptides are produced by both prokaryotic and eukaryotic organisms.9 Based on their secondary structure, AMPs can be classified into four groups (Figure 1).10,

48, 49

The first group is composed of α-helical peptides, such as Magainins,



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Cecropins, and LL-37.50-53 The second group includes cationic peptides that contain two to four disulfide bridges that form β-sheet structures including β-defensins, plectasin, and protegrins.49, 54-56 The third group, extended AMPs, contains cationic peptides which are rich in proline, tryptophan, arginine or histidine.57, 58 The loop peptides are the smallest group of AMPs that form loop structures due to interlinking by at least one disulfide bridge such as the dodecapeptides59 and tachyplesins.60, 61

Figure 1. Structural classification of AMPs. (A) α-helical (PDB ID: 2MAG), (B) β-sheet (PDB ID: 2LXZ), (C) extended (PDB ID: 1QXQ), and (D) loop (PDB ID: 1S7P) peptides.

2.2 Antimicrobial Peptides: Modes of action

Majority of AMPs are short to medium sized and have a high proportion of basic residues such as lysine and arginine making them positively charged at physiological pH.39 Perturbation or complete lysis of bacterial membranes is the most common and generally accepted mechanism of action of AMPs.39 The property of bacterial membrane lysis is related to the net positive charge and amphipathic structure of AMPs. The outer leaflets of bacterial cell membranes are characterised by the presence of negatively charged molecules such as lipopolysaccharides and lipoteichoic acids62-64 as opposed to mammalian cell membranes where zwitterionic phospholipids, sphingomyelin and cholesterol predominate.65,

66

AMPs

are especially attracted to the negative charges found on the outer bacterial membrane. The differences in membrane compositions between bacteria and eukaryotic cells allow AMPs to


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become selectively toxic to bacteria.38,67,68 The fundamental difference between bacterial and mammalian cell membranes is that negatively charged glyco and phospholipids predominate the outer leaflet of bacterial membranes, whereas in mammalian cell membranes the lipids with the negatively charged head groups are present on the inner leaflet.38 Nevertheless, compromise to the degree of selectivity between bacterial and host cell membranes cannot be completely ruled out and could lead to potential toxicity to mammalian cells from cationic molecules such as cationic AMPs and NPs (see later). Physiochemical factors such as hydrophobicity, net charge and helicity of AMPs and their influence on bacterial membrane lysis have been extensively investigated.69-72 It has been suggested that chemical modification of AMPs is a promising strategy for their future clinical applications.73-76

AMPs pose well-defined secondary structures when in contact with phospholipid membranes.48,

77-79

Upon binding of the AMPs onto the target membrane, membrane

permeabilization occurs, leading to leakage of cellular components and eventually cell death.80-82 Several models have been proposed to explain the mechanism of membrane disruption by AMPs (Figure 2). In the toroidal-pore model, the peptides accumulate on the membrane surface causing continuous bending of the lipid monolayers through the pore, resulting in the inserted peptides as well as lipid head groups to build up within the pore.49, 83 The barrel-stave model hypothesizes that the attached peptides first accumulate on the outer membrane and, as a result, insert into the cell membrane.15, 48, 84, 85 The hydrophilic peptide parts form the interior region of the core, and the hydrophobic region is positioned towards the cell membrane lipids.84,

86

In the carpet-like model, no pore formation takes place.

Peptides aggregate parallel to the bacterial membrane and cover the membrane surface like a carpet.48, 78, 87 Peptides that are attached on the surface trigger membrane permeabilization,



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causing the membrane to disrupt in a detergent-like manner finally resulting in micelle formation.48, 77, 88, 89

A

B

C

Figure 2. Modes of action of AMPs. (A) toroidal-pore model, (B) barrel-stave model, and (C) carpet-like model

3. Nanoparticles

Generally, particles having at least one dimension ranging from 1 to 100 nm are considered as nanoparticles. In 1959, the Nobel laureate, Richard P. Feynman, was the first to introduce the concept of nanotechnology in the meeting of American Physical Society entitled ‘There’s plenty of room at the bottom’.90 Feynman’s idea about the possibilities of manipulating matter at the atomic scale led to numerous revolutionary developments in multidisciplinary science.91 In 1974, the term ‘nanotechnology’ was coined by Professor Norio Taniguchi of


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Tokyo University of Science, who tried to engineer different materials on the nanoscale level.92, 93

Nano-scale materials have attracted the attention of researchers due to the unique physical, electronic, and magnetic properties of their miniscule size.94-96 Moreover, the large surface area to volume ratio of nanoparticles provides a high loading of coated molecules.97 Nanotechnology is emerging as a useful tool for various applications in biomedical devices, waste management, material science and electronics.98-101

3.1 Toxicity of Gold Nanoparticles

Due to their small size, NPs, in general, can affect the biochemical environment of the cell especially if they are smaller than 10 nm.102, 103 NPs have been shown to distribute to various organs including liver, heart, spleen, brain, lungs and gastrointestinal tract after inhalation and ingestion in both animal and human studies.104-106 The toxic effects of metallic NPs vary depending upon their concentration and composition. Smaller NPs have higher toxicities.107109

Gold NPs are considered to be comparatively safer than other metallic NPs because of the

inert and non-toxic nature of gold.31, 110 AuNPs can enter the cell via the pinocytosis pathway and localise themselves in the lysosomes without entering the nucleus111-113, which potentially helps to minimize their toxicity. Additionally, the redox nature of Au is a benefit in reducing the level of reactive oxygen species produced during exposure to nanoparticles. The fact that colloidal gold has been used for therapeutic purposes for centuries indicates that gold has high biocompatibility. Nevertheless, for biomedical applications, it is important to conduct thorough cytotoxicity assessment of AuNPs first using in vitro studies followed by further studies in vivo. Below, we discuss examples from the literature on gold NPs where



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their toxicity, or lack of it has been investigated. Majority of published studies focus on shortterm toxicity effects and therefore, actual experimental data on long-term toxicity effects of NPs is scarce. In a short-term toxicity study, Connor et al.

114

described that none of the

spherical 4, 12 and 18 nm AuNPs capped with citrate, cysteine, glucose, biotin or cetyltrimethylammonium bromide (CTAB) were toxic to human leukemia cells K562 up to 100 µM of AuNPs concentration based on results from the MTT assay. However, in contrast to the capped nanoparticles, the starting material salt HAuCl4 was found to be toxic to the cell at 10 nM concentration.114 Similarly Shukla et al.113 reported no significant effect of 3.5 nm AuNPs on the immune system of the cells. In addition to the size, shape and surface charge, the toxic effects of AuNPs also vary depending upon the capping agent.

A study by

Goodman et al.115 using cationic AuNPs functionalized with quaternary ammonium groups (Figure 3, MMPC 1) and anionic AuNPs functionalized with carboxylate groups (Figure 3, MMPC 2) showed that cationic AuNPs are more toxic than the anionic AuNPs. Toxicity of the cationic AuNPs has been correlated to the strong electrostatic attraction between their surface positive charges and the negatively charged molecules on the cell membrane.


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Figure 3. Schematic diagram of the structures of MMPCs 1 and 2. Redrawn with permission from.115 Copyright 2004 American Chemical Society. Cytotoxicity of NPs is mainly caused by the overproduction of reactive oxygen species (ROS), which induces oxidative stress leading to cellular damage.116 Even though, the redox nature of AuNPs, referred to above, can certainly help to quench, and thus reduce the level of, ROS species produced, the net effect of this process can have variations depending on the size of the NPs. The smaller sized NPs have higher surface area to volume ratio in comparison to the larger sized ones, which leads to higher surface reactivity inducing more ROS production.117 Pan et al.118 reported that 1.4 nm AuNPs capped with triphenylphosphine monosulfonate are more toxic than 15 nm AuNPs capped with the same agents. The cytotoxic effect of 1.4 nm AuNPs is caused by significantly more oxidative stress, than the larger sized NPs, leading to mitochondrial damage. Results from this study showed size dependency of toxicity from AuNPs, and are supported by a similar study Turner et al. 119 The smaller sized AuNPs (1.4 nm) were found to be efficient catalysts for the selective oxidation of styrene by dioxygen while those 2 nm and above were not. Turner et al.119 suggested that chemical reactivity of AuNPs is related to the altered intrinsic electronic structure of the smaller-sized AuNPs. Cytotoxicity from AuNPs is also believed to vary with the type of cells being studied. Petra et al. found that 33 nm diameter citrate capped AuNPs cause toxicity to human carcinoma lung cell line A549 while they were non-toxic to human carcinoma liver cell line.120 Some antimicrobial peptides with inherently high level of cytotoxicity have been conjugated to AuNPs in an effort to minimize the cytotoxicity of the peptide component. Pal et al. found that immobilization of sugar amino acid containing cyclic cationic peptides onto AuNPs show significantly reduced cytotoxicity than that exhibited by the free peptide. In this study,



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cytotoxicity was determined by hemolytic activity against human red blood cells (hRBCs).121 Rai et al. showed that CM-SH conjugated AuNPs are non-cytotoxic to several human cells (endothelial cells, fibroblasts and macrophages) at the same cytotoxic concentration as free CM-SH. Cellular uptake of NPs was monitored by inductively coupled plasma mass spectrometry (ICP-MS) for 24 h. Cytotoxicity was determined by hemo-compatibility testing. The biological effect of NPs including cell metabolism, cell viability and pro-inflammatory effects on macrophages was determined by ATP production, annexin V/PI staining and qRTPCR, respectively.122 Casciaro et al. reported that AuNPs@Esc(1-21) are non-toxic to human keratinocytes, as determined by the MTT assay.123 To investigate long-term toxicity, the same protocols used in short-term study can be followed, but extending the duration of the study. Hainfeld et al.124 showed enhancement of mice survival rates bearing EMT-6 mammary carcinomas after radiotherapy with Au clusters 1.9 nm compared to treatment with x-ray therapy or AuNPs alone for one year. Au clusters were found to be largely eliminated from the body through kidneys. Although other reports showed AuNPs tend to accumulate in the major organs during the study time frame, AuNPs were found to be less toxic to the other organs.125, 126 Clearance from the body is an important factor used to assess the cytotoxicity of NPs. Clearance of AuNPs from the body has been addressed based on two aspects; i) clearance from the blood stream and ii) from the body.127 Clearance from the blood stream is more frequently studied as it determines the biodistribution, phamacokinetics and toxicity. Clearance from the body is less well studied since Au cannot be digested by enzymes in the body which is a major limitation for the clinical use of AuNPs. Renal filtration of NPs is highly size and charge dependent. Size-dependent renal clearance has been investigated for other types of NPs (discussed below) but similar investigations on


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AuNPs are rare. Choi et al.128 demonstrated the size dependence of renal clearance by using different hydrodynamic diameter of PEGylated quantum dots. The result showed that NPs with hydrodynamic size less than 6 nm were rapidly eliminated while the 8 nm particles were not easy to eliminate. The charge of particles also affects the renal clearance. The negatively charged particles can interact with glomerular capillary wall by electrostatic interactions resulting in their decreased elimination compared to the positively charged particles. Biodegradable AuNPs is emerging as a promising strategy to incorporate into AuNP design to facilitate easy renal elimination of particles having sizes smaller than the threshold for renal elimination.36,37 Biliary clearance normally associated with the reticuloendothelial system (RES), involves a far more complex mechanism relative to renal clearance. Thus, biliary clearance studies are not the primary targeting mechanism for elimination of NPs. Better understanding of renal and biliary modes of NP clearance will be required before these can proceed to the clinic.

4. Nanoparticles: Potential Candidates for Conjugation to AMPs Several recent studies have shown that nanoparticles provide an efficient way to kill bacterial pathogens, including drug resistant bacteria, and have low probability for resistance development by the pathogens.24,

129-133

transporting drugs to their targets.30,

134

Nanoparticles are excellent candidates for

The use of nanoparticles in combination with

antibiotics makes it possible to decrease the toxicity from both agents toward human cells because of the synergistically enhanced antimicrobial activities and the reduced requirement for high dosages.28 Nanoparticles as antibiotic carriers to the site of infection are emerging as a promising strategy in antibiotic therapy.135,

136

Additionally, since AuNPs can undergo a

strong plasmon resonance with light, they are used for photo-activated drug release.137 (for recent reviews on gold NPs, see 31, 110).



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Although other metallic nanoparticles also display antimicrobial potential, their toxicity towards mammalian cells is concerning when it comes to biomedical applications.138-140 However, results from recent research where nanoparticles have been combined with antimicrobial peptides are promising and show an increasing trend towards a safer profile of the conjugates towards mammalian cells.121, 141, 142

4.1. Nanoparticles in Biomedical Applications Because of the unique properties of nanoparticles, functionalizing them with chemical or biological groups lead to more optimized biomedical applications. Table 1 provides a summary of the well-researched nanoparticles and their biomedical applications.

Table 1. Commonly used Nanoparticles and their Biomedical Applications95, 143, 144-150 Types of Nanomaterial Metallic nanoparticles like Au, Ag, Ti, Zn, Cu etc. Carbon nanotubes

Common Applications/Potential Effects Antimicrobial, delivery of peptides, proteins, nucleic acids and drugs, diagnostic assays, thermal ablation and radiotherapy enhancement Enhanced solubility of the drug, vaccine and gene delivery, peptide transporter, near-infrared photothermal agent, cancer therapy 145

Dendrimer

Enhanced drug bioavailability, targeted drug delivery for therapeutic and diagnostic agents and imaging, imaging probes 146


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Liposome

Enhanced drug bioavailability, delivery of drugs and diagnostic agents 147

Quantum dots

Biological system imaging including in vitro imaging of fixed cells and in vivo targeting for diagnosis or therapy 148

Polymeric micelles

Enhanced drug bioavailability, target specific drug delivery especially for anticancer drugs, diagnostic agents 149

Polymeric nanoparticles

Nanocarrier for biomolecules, targeted therapy to solid tumors 150

Some nanoparticles are already approved by FDA for clinical applications.32, 151-158 Amongst the various metallic nanoparticles, gold nanoparticles have been considered to be ideal candidates for loading small organic ligands and biomolecules due to their biocompatibility and relative stability of the ligand-bound gold nanoparticle.31, 110 As stated above, the major limitation of using gold and other nanoparticles is their size-dependent toxicity. Thus, nanoparticles must be synthesized via precise protocols to derive well-defined shapes and sizes for specific applications.31 (Table 2).

Table 2. Types of Au Nanoparticles and Their Applications. Shape

Schematic

Size

Application

drawing Clusters

Reference *

2 nm

Drug delivery, cell imaging, in

162-164

vivo imaging and cancer therapy Nanoshells

10-400 nm

In vitro assays, in vivo imaging,

165-168

cancer therapy, drug delivery Nanocage

20-200 nm Photothermal cancer therapy

169

(edge length)

*The references cited relate mainly to the applications of the various shapes.

5. Methods for Conjugating AMPs to Metallic Nanoparticles Peptide conjugation can be carried out on a pre-formed nanoparticle or the peptide can be conjugated to the components prior to nanoparticle formation in a single step.170 It has been found that the single-step process has clear advantages for the final product in terms of uniform particle size, higher AMP concentration and therefore higher antimicrobial activity.122, 170

The methods used for immobilization of biomolecules onto metallic nanoparticles involve either the process of physisorption or chemisorption.171 Typically, physisorption is mediated mainly through intermolecular forces (van der Waals forces). Metallic nanoparticles can also be functionalized with biomolecules through covalent bonding via different conjugation chemistries including metal thiolate bonds e.g. thiol-containing peptides can be directly


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conjugated to gold or silver nanoparticles172,

173

, activation using N-hydroxysuccinimide

(NHS) reactive groups, or thiol-reactive maleimide groups, as well as click chemistry based on copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC).174-176 The covalent conjugation approaches used to synthesize AMP-AuNP conjugates, with specific examples from the literature, are elaborated below.

Even though both covalent and non-covalent methods can be used for the biofunctionalization of metallic nanoparticles, for cationic AMPs, covalent conjugation is more reliable in terms of product quality and homogeneity. Covalent conjugation of AMPs to metallic nanoparticles has been successfully used in the literature to not only to enhance the antimicrobial properties of both components but also to minimize the toxicities of NPs.121, 122 The most common approach for AMP conjugation to gold nanoparticles involves the formation of the Au-S coordinate covalent bond. When alkane thiols react with Au, the S-H bond is broken first which is followed by the formation of the strong Au-S coordinate bond.177,

178

For this reason, peptides for the conjugation reaction are synthesized with a

terminal (N- or C-terminal) cysteine which facilitates conjugation to gold in a pH controlled manner. The possibility of using suitable (bifunctional) linkers is an attractive feature of the covalent conjugation strategy. The functional groups on either ends of linker provide selectivity to each step of the conjugation process. Additionally, use of a linker in between the peptide and gold enables control over peptide orientation on the surface of the nanoparticles. It is important to ensure that antimicrobial activity is maintained after immobilization to the surface. Peptide orientation on the surface plays a crucial role in maintaining activity. The bifunctional linkers almost exclusively have a thiol group on one end which forms the Au-S bond. The other end of the linker carries an appropriate functionality such as a carboxylic acid for coupling to the amino terminus of the peptide.



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Because of the poor electrophilic nature of the carboxyl group, it is important to activate the carboxyl function of the incoming amino acid to ensure efficient formation of the peptide bond. Several commonly used coupling reagents in peptide chemistry such as the carbodiimides (dicyclohexyl carbodiimide – DCC or its derivatives; Figure 4), aminium/uronium salts (HBTU, HATU etc.; Figure 4), and phosphonium salts (PyBOP, PyAOP; Figure 4) can be used to for this purpose. These coupling reagents convert the

carboxylic acid group into reactive esters that readily undergo nucleophilic attack by the free N-terminal amino group in the peptide thus forming the peptide bond. Succinimide ester, referred to above, is also used as a reactive group which can readily react with amino terminus of the peptide.

Figure 4. Chemical structures of commonly used peptide coupling reagents.

Majority of literature examples have used a fully deprotected peptide chain in the conjugation reaction (Figure 5. route A). However, unspecific amide bond formation to other free amines such as those arising from the side chains of lysine residues present in the peptide cannot be


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ruled out. While on the other hand, using the peptide in the fully deprotected form avoids exposing the peptide-nanoparticle conjugate to the harsh conditions of TFA mediated deprotection generally used in peptide synthesis.

Figure 5. Schematic diagram for the synthesis of AMP-SH conjugated AuNPs. Route (A) Fully deprotected peptide with thiol group at the N-terminus directly conjugated to gold nanoparticles via Au-S bond in a suitable buffer (e.g. HEPES). Route (B) Side chain protected peptide cleaved from the resin under mild cleavage conditions (1% TFA), followed by conjugation of the protected peptide and the gold nanoparticle performed in a suitable organic solvent. NaBH4 can be used to reduce the gold ion. After conjugation, side chain



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deprotection is performed using 95% TFA followed by solvent extraction to remove any unbound peptide.

However, in a recent example, it was shown that the fully protected peptide can be conjugated to gold via the Au-S bond followed by protecting group removal using TFA without damaging the peptide-AuNP conjugate (Figure 5. route B).179 In this particular example though, no linker was used between the peptide and the AuNP and it was shown that the peptides after conjugation to Au were resistant to trypsin cleavage and maintained their original antimicrobial activity. The ability to use a protected peptide for conjugation to gold avoids non-specific reaction with the side chain amino groups of the peptide and ensures that the peptide is exclusively linked to the AuNP via its N-terminus. However, fully protected peptides can be poorly soluble in the reaction medium, often aqueous, which could hamper efficient coupling. Therefore, the use of completely deprotected peptides in a step-wise conjugation process is the most suitable, as has been reported in one of the recent literature examples.180 The step-wise strategy essentially ensures homogenous coupling during each step of the process. Additionally, the intermediates can be fully characterized before proceeding to the next step of the reaction. In the example cited above180, the authors used circular glass slides coated with AuNPs which were functionalised with a shorter spacer, cystamine or thiol-PEG-NH2, to link to the surface of gold chemoselectively using the stable Au-S bond. This was followed by coupling the amine end of spacer to the hydroxysuccinimide group of a bifunctional linker with a short spacer arm. The final step involved the chemoselective reaction of the maleimide group on the other end of the linker to amino terminal cysteine of the AMP. As is obvious, this strategy requires the use of two different linkers. The length of the spacer part of the linker is another factor that will need to be optimised to sufficiently extend the peptide away from the surface of the nanoparticle and


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provide necessary flexibility resulting in the right orientation of the peptide to enable efficient interaction with the pathogens.

The glycopeptide antibiotic vancomycin has also been conjugated to gold following covalent chemistry as bis vancomycin cystamide (Figure 6A).181 Thorough investigations using the cecropin-melittin peptide as a model with and without a terminal cysteine has been reported.122 Different synthetic conditions, including reaction time, peptide concentration, conjugation buffer, effect of C-terminal cysteine on the peptide and HEPES mediated Au reduction with and without adding the peptide were investigated in an attempt to produce high-density peptide conjugated AuNPs with low polydispersity. Their results showed that the presence of cysteine and a one step process of formation of AMP-AuNP have significant advantages in terms of uniform particle size, aggregation behaviour and AMP density.

In the most recent example the very potent AMP esculentin-1a (1-21)-NH2 was conjugated to AuNP using a bifunctional PEG linker, without the use of an additional shorter spacer.123 In the first step, the thiol end of the PEG linker formed the Au-S bond with the nanoparticle. This was followed by coupling of the fully deprotected peptide to the COOH group of the PEG following carbodiimide chemistry (Figure 7 A). However, and as mentioned above, it is to be noted that some non-specific amide bond formation could have occurred to the side chain amines of the lysine residues present in the peptide. Nevertheless, results showed that the AuNP-peptide conjugate possessed more than twelve times antimicrobial potency than the non-conjugated peptide (discussed in the next section).

To the best of our knowledge, there is only one example in the literature where covalent conjugation of an AMP to gold nanoparticle has been achieved via an Au-O bond.182 In this



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example the glycopeptide antibiotic vancomycin was conjugated to gold nanoparticles in a one-step reaction where the glycoside part of vancomycin was used to reduce the Au ions and also trap the Au NPs. The reaction mechanism has been proposed as the reduction of Au ions by vancomycin at alkaline pH followed by acetal formation of vancomycin with gold.

The field of AMP conjugation to AuNPs is still at its infancy, with only very few examples (discussed above) reported in the literature. The different methods discussed above each have their own merits and demerits. Nevertheless, in our opinion, a fully deprotected peptide conjugated in a step-wise manner with two spacers to provide the necessary chemoselectivity to each step of the reaction is the most desirable in terms of product homogeneity. The following section discusses the effects on antimicrobial activity of the AMPs after conjugation to AuNPs, the chemistry of which has been discussed in the current section.

6. Effect of AMP-AuNP Conjugation on Antimicrobial Activity (summarised in Table 3) One of the earliest reports on AMPs attached to metallic nanoparticle was by Xu and coworkers which showed that vancomycin conjugated to AuNPs (Au@Van) via Au-S bonds can be active against vancomycin-resistant enterococci (VRE) (Figure 6B).181 Au@Van showed enhanced activity against VRE with minimum inhibitory concentration (MICs) of 2-4 µg/ml (based on vancomycin molecules) while the MIC of free vancomycin was much higher (> 64 µg/ml).


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Figure 6. (A) Schematic diagram of vancomycin capped AuNP synthesis (B) The multivalent interaction between vancomycin conjugated AuNPs and vancomycin-resistant enterococci. Reprinted with permission from.181 Copyright 2003 American Chemical Society.

Similarly, Fayaz et al. showed that vancomycin-capped AuNPs by electrostatic interaction, has enhanced activity against vancomycin-resistant Staphylococcus aureus (VRSA).183 Modified AuNPs showed approximately six times more potency (MIC-8 µg/ml) over freeform vancomycin (50 µg/ml). However, immobilization of vancomycin by this method



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resulted in precipitation and provided less activity enhancement compared with the method mentioned above by Xu et al., highlighting the advantage of the covalent conjugation strategy. Chen and colleagues demonstrated that MICs of vancomycin-immobilized AuNPs via Au-O bond (chemistry discussed in the previous section), were not only significantly lower than the free-form vancomycin but also inhibited the growth of vancomycin-resistant strains

including

VRE1

(E.

faecalis),

VRE4

(E.

faecium),

methicillin-resistant

Staphylococcus aureus (MRSA) and PDRAB.182 Rai et al. demonstrated that a one-pot reaction of immobilization of modified cecropin-melittin (CM-SH) onto AuNPs (CM-SH AuNPs) through Au-S bond, showed higher antimicrobial activity and higher stability in media compared to soluble CM-SH in vitro and in vivo in an infection animal model.122 The MICs of CM-SH AuNPs were four times lower than those of free CM-SH. Moreover, CMSH AuNPs were more efficient in inducing the permeabilization of bacterial cell membranes compared to free CM-SH. Only a small (< 5) percentage of the administered AMP-AuNP accumulated in the internal organs such as spleen, liver or kidney. Rai also reported that simple conjugated CM-SH by ligand exchange, even though exhibited slightly enhanced activity, was not stable after one hour. Recently, Casciaro et al. reported that covalent immobilization of AMPs onto AuNPs, via bifunctional thiol PEG carboxylic acid linker, significantly increased the antibacterial and anti-biofilm activities against P. aeruginosa (Figure 7 B-D).123 P. aeruginosa is a Gram negative pathogen notoriously resistant to conventional antibiotics. Infections from P. aeruginosa biofilms in cystic fibrosis patients is difficult to eradicate and can be fatal. There is an urgent need to develop novel anti-biofilm therapies against P. aeruginosa and other ESCKAPE pathogens. Hence these recent results provide significant boost to the field of AMP conjugated NPs and warrants further research and development.


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Figure 7. (A) Schematic diagram of step-wise synthesis of AMP-NH2 conjugated Au-NPs. (B) Esculentin-1a (1-21)-NH2 coated Au-NPs and TEM micrograph of its interaction with P. aeruginosa. (C) TEM micrographs of P. aeruginosa cells after 15 min treatment with (A, left) AuNPs@Esc(1-21) or (B, right) buffer. (D) SEM of P. aeruginosa cells treatment with AuNPs@Esc(1-21), AuNPs@PEG and buffer control (Ctrl). Reprinted with permission from.123

AuNPs can act as potential candidates for AMP delivery. Zhang and co-workers designed bacterial toxin triggerred drug release fromAuNP-stabilized liposomes (AuChi-liposome).184 In this study, vancomycin was used as anti-MRSA antibiotic. In the presence of MRSA, AuChi-liposomes released encapsulated vancomycin which significantly inhibited bacterial



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growth.184 Yeom and co-workers reported that conjugation of AuNPs with DNA aptamer (AuNP-Apt) successfully transferred AMPs into mammalian living system resulting in the inhibition of S. typhimurium colonization in the mice organs.185 This study is the most advanced regarding clinical applications of AMP immobilized AuNPs by using infected animal as a model study. Moreover, Peng and colleagues showed that AMPs conjugated with AuNPs can introduce genes into stem cells with antimicrobial activity (Figure 8).186

Figure 8. Schematic diagram of PEP and/or TAT peptide immobilized AuNPs and their interaction with pDNAs and cells. Reprinted with permission from.186

Besides AuNPs, AgNPs are also interesting candidates for immobilizing AMPs. The increase in antimicrobial activity of AMPs conjugated AgNPs compared to free AMPs has been well established.142, 187-189 Ruden et al. were the first to demonstrate that immobilizing AgNPs with peptide increases antibacterial activity.187 Although AgNPs can be toxic to mammalian cells190, as we have already stated, one way to reduce their toxicity is through immobilization of AgNPs with AMPs.142 Liu and co-workers showed that immobilization of the twenty residue cationic peptide with AgNPs, via ligand exchange enhanced the antimicrobial activity compared to the unbound peptide and also minimized toxicity compared to using nanoparticles alone.142 This study showed that weak interactions between AMPs and


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nanoparticles are adequate to decrease the toxicity of the nanoparticles. A weaker interaction is beneficial to ensure activity of the peptides which is dependent upon their overall charge and structure. Too strong electrostatic interaction between AMPs and AgNPs through positive charges on peptides can reduce their antimicrobial activity after conjugation. A recent study by Pal et al. demonstrated that AMP conjugation using the Ag-S bonds is better in terms of increased stability and enhanced antimicrobial activity than conjugation using electrostatic interactions.141 This study brings out the possibility to reuse AMPs immobilized AgNPs without losing activity.

Furthermore, AMP conjugated magnetic nanoparticles191, dendrimeric peptides192-194, and polymeric nanoencapsulation14 of AMPs also represent ways of improving the antimicrobial activity and stability of AMPs. Xian et al. reported that immobilization of iron oxide (Fe3O4) nanoparticles with bacitracin by click chemistry shows better antimicrobial activity against both Gram-positive (S. aureus) and Gram-negative (E. coli) compared to bacitracin itself.191



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Table 3. Summary of AMPs Conjugated AuNPs AMPs

bis(Vancomycin)

Diameter

Activity of molecule

Activity of Conjugated

of AuNPs

alone

AuNPs

4-5 nm

cystamide

> 64 µg/ml against

2-4 µg/ml against VRE

vancomycin-resistant

and 8 µg/ml against E.

enterococci (VRE) or

coli

Ref.

181

E. coli Vancomycin

15 nm

50 µg/ml against

8 µg/ml against VRSA

183

vancomycin-resistant Staphylococcus aureus (VRSA) Vancomycin

8.4 nm

64 - >128 µg/ml against 8-32 µg/ml against vancomycin-resistant

VRE1, VRE4, MRSA

strains including VRE1

and PDRAB

182

(E. faecalis), VRE4 (E. faecium), MRSA and PDRAB Cecropin-melittin

14 nm

5 µg/ml against E. coli

derivative

1.25 µg/ml against E.

122

coli

(cysteine at Cterminus)

Esculentin-1a (1-

14 nm

MBC50: 1 µM

MBC50: 0.08 µM


123

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21) NH2

Biofilm MBEC50: 3µM

Biofilm MBEC50: 0.17

against P. aeruginosa

µM against P. aeruginosa

PEP

Integrated AMP-AuNP with better antibacterial

186

activity and as an efficient carrier for gene delivery A3-APOHis

15 nm

deliver AMPs into mammalian living systems,

185

enhance stability, increase antimicrobial activity

7. Possible Mechanism of AMP-AuNP Conjugates Against Bacteria The possible reason for the increased antimicrobial activity of AMP-AuNPs over the individual components alone is that AuNPs facilitate achieving a higher concentration of the drug at the site of action. Additionally, the AuNPs can interact with lipopolysaccharide (LPS) and proteins in the outer membrane of bacteria and get deposited on the membrane.195-199 The deposited AuNPs can penetrate bacterial membrane through porin channel171 or diffuse through the phospholipid bilayer. This can facilitate better interaction of the therapeutic such as the conjugated antimicrobial peptide with the inner membrane thus making the conjugate more active than the non-conjugated peptide itself. This is particularly beneficial against biofilms.

Biofilm formation plays a crucial role in antibiotic resistance.200 Bacterial biofilms have an ability to tolerate several cycles of antibiotic treatments and colonize new surfaces after the treatment. Three main features that contribute to the high antibiotic resistance of biofilms are the extracellular polymeric substrates (EPS), heterogenic architecture of biofilms, and the presence of persisters.200-202 The main component of biofilm matrix is EPS which acts as a



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barrier to protect bacteria inside from the action of antibiotic and also harsh environmental conditions.203 In addition, EPS can potentially localise enzymes that can modify or deactivate antibiotics.201, 204 Since biofilm producing bacteria can survive under low nutrient conditions and live in a metabolically inactive state, antibiotic treatments, which target the cellular activity of bacteria, are not usually effective.201, 202 Nanoparticles are gaining importance as antibiofilm therapy rapidly.131, 205 As stated above a very recent example123 from the literature has shown the ability of AMP-AuNPs to disrupt the biofilms of P.aeruginosa which indicates that the AMP-AuNPs are able to penetrate the biofilm EPS.

8. Conclusions

Conjugation of AMPs with nanoparticles helps to increase serum stability of peptides and provides significant benefits in terms of antimicrobial activity, than free AMPs, against a broad range of drug resistant bacterial pathogens. Promising results from recent literature presented in this review provides increasing evidence on the potential of using AMP-NP conjugates to tackle the global health issue of alarming increase in antibiotic resistant bacteria. However, interactions between nanoparticles and bacterial membranes and mammalian cell need more investigations, particularly to address toxicity concerns before these conjugates can become common practice in the clinic.


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Recent Developments in Antimicrobial-Peptide-Conjugated Gold

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