Anti-bacterial surfaces: natural agents, mechanisms of ...researchonline.jcu.edu.au/40273/1/40273...

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Anti-bacterial sur

aSchool of Chemistry, Physics and Mechanic

Technology, Brisbane, Australia. E-mail:

[email protected] of Science, Technology and Enginee

Australia. E-mail: [email protected]

4781 5177; Tel: +61 7 4781 4494cFaculty of Pharmacy, University of Sydn

[email protected] Materials Science and Engineeri

[email protected]

DDBQnrtEAmAtA

Forestry, two Endeavour ResearcAdvanced Manufacturing CooperaPrize, an AINSE Gold Medal, and40 refereed journal papers. Herprocessing of materials and livinelectronic applications.

Cite this: RSC Adv., 2015, 5, 48739

Received 30th December 2014Accepted 21st May 2015

DOI: 10.1039/c4ra17244b

www.rsc.org/advances

This journal is © The Royal Society of C

faces: natural agents, mechanismsof action, and plasma surface modification

K. Bazaka,*ab M. V. Jacob,b W. Chrzanowskic and K. Ostrikovacd

Strategies that confine antibacterial and/or antifouling property to the surface of the implant, by modifying

the surface chemistry and morphology or by encapsulating the material in an antibiotic-loaded coating, are

most promising as they do not alter bulk integrity of the material. Among them, plasma-assisted

modification and catechol chemistry stand out for their ability to modify a wide range of substrates. By

controlling processing parameters, plasma environment can be used for surface nano structuring,

chemical activation, and deposition of biologically active and passive coatings. Catechol chemistry can

be used for material-independent, highly-controlled surface immobilisation of active molecules and

fabrication of biodegradable drug-loaded hydrogel coatings. In this article, we comprehensively review

the role plasma-assisted processing and catechol chemistry can play in combating bacterial colonisation

on medically relevant coatings, and how these strategies can be coupled with the use of natural

antimicrobial agents to produce synthetic antibiotic-free antibacterial surfaces.

al Engineering, Queensland University of

[email protected]; kostya.

ring, James Cook University, Townsville,

; [email protected]; Fax: +61 7

ey, Sydney, Australia. E-mail: wojciech.

ng, Sydney, Australia. E-mail: kostya.

r Kateryna Bazaka is an ARCECRA Fellow with Health andiomedical Technologies,ueensland University of Tech-ology, Australia. Kateryna is aecipient of the Australian Insti-ute of Nuclear Science andngineering Postgraduateward, the Queensland Govern-ent Smart Women Smart Stateward, the Science and Innova-ion Awards for Young People ingriculture Fisheries andh Fellowships, the Inauguraltive Research Centre Studentan author of 1 monograph andresearch focuses on nanoscaleg matter for biomedical and

hemistry 2015

1. Introduction

In the last twenty years, signicant progress has been made inthe development of biomaterials and implantable devices,which are characterised by superior biocompatibility, desiredintegration with peri-implant tissues, controlled fouling withhost cell and biomolecules, and which cause minimal acute orchronic inammation. Numerous modication techniqueshave been developed to ensure satisfactory clinical performanceof these devices by improving their biocompatibility with

Associate Professor Mohan Jacobis currently the Associate DeanResearch Education for theCollege of Science, Technologyand Engineering, James CookUniversity. University of Delhiawarded him PhD in 1999 inElectronic Science. He publishedover 150 peer reviewed articles.He developed methods toprecisely characterize thedielectric properties of materialsat cryogenic temperatures and

microwave frequencies. His main research interests also includesthe development of polymer thin lms and graphene fromsustainable sources using plasma enhanced chemical vapordeposition, environmentally friendly biomaterials and electronicand biomedical devices.

RSC Adv., 2015, 5, 48739–48759 | 48739

http://crossmark.crossref.org/dialog/?doi=10.1039/c4ra17244b&domain=pdf&date_stamp=2015-05-31http://dx.doi.org/10.1039/c4ra17244bhttp://pubs.rsc.org/en/journals/journal/RAhttp://pubs.rsc.org/en/journals/journal/RA?issueid=RA005060

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cells/tissues, by tailoring chemical composition andmechanicalproperties for specic application. To illustrate, metals such astitanium (Ti) are frequently modied by grain renement toimprove mechanical properties and enhance osteoblastattachment; abrasive-blasted to modify topography and thusimprove osseointegration; polished mechanically andchemically to achieve smooth surface morphology to reduceintegration with tissues and ease the removal of the devices(short-term implants); passivated/oxidised to improvecorrosion resistance and enhance bioinertness; and coatedwith biomolecules, e.g. proteins and DNA fragments, andother biologically active species for guided cell attachmentand integration with host tissues, to name but a few.

At the same time, the susceptibility of the implant surface tobacterial colonisation and biolm formation remains a majorproblem that is most commonly dealt with by means ofprophylaxis with systemic antibiotics. Although administrationof broad-spectrum systemic antibiotics is effective in preventingbiomaterial-associated infection arising from pathogens intro-duced into the peri-implant space in the course of surgery orpost-operative care, the practice is far less effective in dealingwith late haematogenous infections. In the case of the latter,bacteria from an inammation site elsewhere in the body canenter the blood stream and thus be transferred to the implantsurface. In the absence of antibacterial agent, there is little tostop the pathogen from attaching to the surface and initiating abiolm formation. The colonisation occurs quickly and is rarelydetected clinically in time to prevent biolm formation. Onceformed, the biolm affords the pathogenic cells necessaryprotection against ow detachment, opsonisation, and theharmful effects of host antimicrobial molecules and systemic

Dr Wojciech Chrzanowski joinedthe University of Sydney in 2010and he has established the Bio-interface group and Bio-nano-characterisation laboratorywithin the Faculty of Pharmacy.His research is balancedbetween basic and translationsciences. He develops multi-functional surfaces and newexamination approaches tointerrogate biological responsesto biomaterials at nanoscale.

Outcomes of his research inform the design of new biologicallyactive materials for implantable devices and drug delivery. For aseries of publications describing signicant advances in nano-biomedical sciences and biointerfaces he was invited to presentover 50 seminars, and lectures at Universities in the USA, Japan,Australia, UK, and Korea. He received three fellowships (UCL,Tokyo University and Chubu University). Dr Chrzanowski pub-lished over 100 peer-reviewed publications (last 10 years). Hispublications attracted 1300 citations and his h-index is 20. He isalso an inventor on 4 patents.

48740 | RSC Adv., 2015, 5, 48739–48759

antibiotics.1 In biolm state, the expression of genes andmetabolic activity in bacterial cells may also differ from that oftheir planktonic counterparts, which may lessen sensitivity ofsessile bacteria to certain antimicrobials designed to target thepathogen's metabolism.2–4 Oentimes, even signicantly higherdoses of systemic antimicrobials are insufficient to clear thebiolm, and implant replacement is required.

Although sound hospital practices ensure the rate ofimplant-associated infections remains relatively low, the everincreasing volume and variety of biomaterials and medicaldevices implanted globally results in a substantially largenumber of infections. Furthermore, increasing human lifeexpectancy and emphasis on active lifestyle is associated with agrowing number of revision surgeries, and these are known tohave a signicantly higher infection rate. With the growingissue of hospital acquired and multi-drug resistant microor-ganisms,5 there is a strong need to engineer biomaterials thatretard microorganism colonisation in the rst place.

2. Trends in surface modification

Microbial attachment can be effectively mitigated by intro-ducing an antimicrobial agent throughout the bulk of thematerial, e.g. silver can be blended into bulk polymeric mate-rials, alloyed into metallic biomaterials, or introduced intoglass/ceramic materials.6–8 While the nature of the resultantmaterial ensures the long-lasting antimicrobial effect, theaddition of the antibacterial agent may negatively impact on thefundamental properties, stability or processability of thematerial. In comparison, surface modication can be applied toexisting biomaterials, with little impact on such bulk properties

Professor Kostya (Ken) Ostrikovis a Science Leader, ARC FutureFellow, Chief Research Scientistwith CSIRO's ManufacturingFlagship and a Professor withthe Institutes of Future Envi-ronments and Health andBiomedical Technologies of theQueensland University of Tech-nology, Australia. His achieve-ments include the Pawsey (2008)medal of the AustralianAcademy of Sciences, the Walter

Boas (2010) medal of the Australian Institute of Physics, BuildingFuture Award (2012), the recent NSW Science and EngineeringAward (2014), 8 prestigious fellowships in 6 countries, 3 mono-graphs, and more than 430 refereed journal papers. His researchon nanoscale control of energy and matter contributes to thesolution of the grand challenge of directing energy and matter atthe nanoscale, a challenge that is critical for the development ofrenewable energy and energy-efficient technologies for a sustain-able future.

This journal is © The Royal Society of Chemistry 2015

http://dx.doi.org/10.1039/c4ra17244b

Fig. 1 (A) Antifouling strategies for biofilm management. (B)Commonly used hydrophilic chemistries, e.g. poly(ethylene glycol),

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as mechanical strength and stability under in vivo conditions,and is oen more cost and time effective.9,10

When selecting appropriate surface modication approach,there are many material- and application-based considerationsthat need to be addressed. From processing point of view, thechoice of the appropriate method is based on its compatibilitywith the type of biomaterial, i.e. polymeric, metallic, ceramic orcomposite; its stability, i.e. temperature sensitivity, solubility,mechanical robustness, etc.; its physical structure, e.g. porosity,and dimension, e.g. bulk or thin lm; to name a few.

Financial cost, ease of integration, and scalability of poten-tial modication techniques also need to be considered. Forinstance, chemical vapour deposition of vertically alignedcarbon nanotube forests consumes more time, energy andresources than template-based fabrication of polymer struc-tures. The former is also more difficult to scale up or translateinto continuing processing. At the same time, nanoscalematerials, such as nanotubes or graphene sheets offer uniqueand highly valuable properties, such as extreme mechanicalstrength and durability, electrical and thermal conductivities,and highly adjustable chemical reactivity. Indeed, althoughrelatively easy to fabricate, polymer structures are more fragileand fail easily under load or wear conditions.

From application perspective, general considerationsinclude the intended use, e.g. whether the surface will be sub-jected to load, wear, ow or harsh chemical environment, aswell as the length for which antimicrobial activity is required.The proposed application also places restrictions on the type ofantimicrobial activity, for example antibiofouling surfaces maybe desirable for urinary tract catheters, but they will not beappropriate for materials where tissue regeneration is required.In general, a biomaterial with excellent bactericidal activity butpoor compatibility with host biomolecules, cells and tissues isunlikely to nd broad clinical use.

Even a non-cytotoxic coating aimed at preventing bacterialadhesion may change the density or porosity of the underlyingmaterial, with signicant consequences for attachment, differ-entiation and metabolic activity of target mammalian cells.Changing surface topography of the biomaterial may alsochange the availability of specic chemical functionalities at itssurface, or recongure their 3D conrmation. It is thereforeimportant to understand the interdependence of surfacechemistry and physics in order to adequately predict theresultant biological performance with respect to bacteria andmammalian cells.11

The type of antimicrobial agent, its ability to withstandprocessing conditions, and maintain its antimicrobial potencyin the nal conformation under physiological conditions willalso affect the choice of modication methodology.

poly(methyl oxazoline), polyacrylamide, and zwitterionic poly-(carboxybetaine methacrylate) and poly(sulfobetaine methacrylate).(C–G) Natural and artificial superhydrophobic surfaces. (C) The hier-archical structure of Salvinia spp. hairs, composed of the multicellularhair with small rodlet-like wax crystals on top.15 (D) Macroporousgraphene oxide film (CA ¼ 152�).16 (E) Gecko-inspired setae made ofmicropatterned carbon nanotube bundles (CA ¼ 155�).17 (F) Per-fluoropolyether polymer hairs (CA ¼ 171�).18 (G) Epoxy/g-Al2O3nanoparticle composite (CA ¼ 160�).19 Reproduced with permissionfrom ref. 15–20.

2.1 Physico-chemical modications

For a number of years, control over the attachment and biolmformation of microorganisms was achieved using specicsurface chemistries. This is hardly surprising, as molecularrecognition is acknowledged as one of the key factors in deter-mining not only cell–surface interactions, but also many


biological functions within the cell itself. These chemistries canbe imparted onto the surface by a variety of means, includingplasma-assisted techniques, such as plasma (thermal) spraying,plasma immersion ion implantation, and plasma deposition,gas dynamic cold spraying, chemical and physical vapourdeposition, and sol–gel. Hydrotropic nanostructues, such ascarbon and halloysite nanotubes can also be used to controlfouling.12 The key challenge in using these methods is inensuring that the treatment process and/or the resultant surfacechemistry do not undermine the biocompatibility, performanceand degradation behaviour of the biomaterial in vitro and in vivo.

With the development of novel data acquisition, analysis andvisualisation tools, our understanding of cell–surface dynamicshave evolved to include the physical as well as the chemicalproperties of biomaterials as key factors that can regulate bio-logical responses of cells and tissues.13 As a result, severalmodication strategies have been developed that rely on thesynergistic effect of chemistry, e.g. hydrophobic moieties, andsurface morphology, e.g. hierarchical arrangement of nano- andmicro-features, to prevent microbial attachment and biolmformation (Fig. 1).14 Laser ablation, abrasive blasting, physicalvapour deposition, self-assembly, evaporation and ion assisteddeposition are among the frequently used physical modicationtechniques.

Nanostructured surfaces with surface chemistry-independent antimicrobial effect have also been reported.21

Fig. 2 shows the nanopattern on the surface of Clanger cicada

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(Psaltoda claripennis) wings which allows the surfaces to killbacteria on contact based solely on its physical surface struc-ture. Although unable to prevent microbial attachment,22 thedirect contact between the attached cells and highly orderedarrays of surface nanopillars resulted in cell membranestretching and eventual rupturing, where the adsorptionbehaviour of bacterial cells and their sensitivity to the materialsurfaces depended both on the geometry of the pillars and themechanical properties of the cells, especially cell rigidity.21 Inspite of substantial advances in our understanding of how thephysical properties of materials determine cell–surfacedynamics at nano-, molecular- and atomic scales, this eldrequires considerable further development.

Surface physical properties can be used to enhance theantimicrobial effect. For instance, physical disruption of cellmembranes have been demonstrated as an essential contrib-utor to antimicrobial efficacy of copper surfaces, where thedamage to cell envelope facilitated further damage by copper

Fig. 2 The unique surface morphology of wing surface enablesClanger cicada (A) to resist bacterial colonisation. (B) Proposedmechanism of chemistry-independent contact killing of bacteria oncicada wing surface. (C and D) SEM images of clinically relevantpathogens on the surface of a cicada wing. Pseudomonas aeruginosa(C) and Branhamella catarrhalis (D) cells are clearly penetrated by thenanopillar structures on the wing surface, with cells sinking betweenthe nanopillars (C, inset). On the wing surfaces, bactericidal effectobserved for all tested Gram-negative microorganisms, regardless ofcell morphology. On glass (D, inset) under equivalent incubationconditions, no killing effect was observed. Reproduced with permis-sion from ref. 21, 22 and 25.

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ions,23 and of copper containing nanoparticles, where particlesalso acted as physical carriers of copper into the cells.24

2.2 Biocide-based strategies

Broadly, the antimicrobial agent can either be entrapped in thecoating to be released in some predened fashion upon inter-action with its operational environment and/or stimuli, e.g.drug eluting hydrogels and coatings, or immobilised on thesurface of the implant to prevent bacterial attachment and/orkill the attached cells on contact, e.g. covalently attached poly-mer brushes, conventional antibiotics and antimicrobialpeptides.

There is merit to both strategies. Themain advantage of non-leaching systems is in the connement of the cytotoxic effect tothe surface of the implant, thus minimising the potentiallyharmful interactions between the agent and host tissues, e.g.damage to host cells in peri-implant milieu or accumulation ofthe antimicrobial liver, spleen, and brain.26,27 Given that theantimicrobial agent is not depleted over time, the effect issustained for longer. Furthermore, the issue of bacterial cellsbeing exposed to sub-inhibitory concentrations of the antibioticis avoided. This minimises the chance these bacteria willdevelop resistance to the drug in use.28

With the efficacy of many antimicrobials relying on acombination of chemical functionality and spatial conforma-tion, covalent immobilisation is more conducive to attainingspecic molecular orientation of the agent on the surface. Assuch, the availability of specic chemistries and structuralmotifs characteristic of the antimicrobial in suspension can bemaintained.29 Nevertheless, prolonged exposure to the physio-logical environment may result in the concealment of theantimicrobial chemical and physical features of the surface, e.g.through adsorption of host biomolecules or accumulation ofkilled bacterial cells and their fragments.30 Furthermore, abroader variety of antimicrobial agents can be entrapped in therelease- or leach-systems, and their concentration and releaserate controlled to ensure bacterial inhibition further away fromthe implant surface into the peri-implant space. The challengethat is common to drug-release and non-leaching antimicrobialsystems is the control over the quality of adhesion between theactive agent and the underlying biomaterial surface.

The example in Fig. 3 shows the use of traditional antibioticsas biocidal agents. For antibiotic-sensitive strains of bacteria,these coatings provide an effective means of combating infec-tion. However, a growing emergence of bacteria with antibioticresistance, particular in hospital settings, resulted in a growinginterest in alternative therapeutic concepts and agents. Ideally,these alternative agents should lead to the elimination ofbacteria, and have a mode of action that would be sufficientlydissimilar to systemic antibiotics to avoid promoting cross-resistance.

2.3 Aim and article organisation

The aim of this article is to review two types of highly-versatilemodication chemistries, namely (i) highly reactive plasmachemistry and (ii) catechol chemistry that can be applied to a



Fig. 3 Principles of bactericide contact and release coatings based onconventional antibiotics (penicillin, ampicillin, and gentamicin). Anti-biotics can be used individually or in combination. Active agents can bephysically adsorbed onto the surface or covalently conjugated to thepolymer chain (in this example, PEG).

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wide range of substrate materials to produce a variety of anti-fouling, biocide-releasing and contact kill surfaces. A particularfocus of this review is the potential use of plasma and catecholchemistries as enabling technologies for surface modicationbased on natural antimicrobial compounds.

Section 2 will provide a broad perspective on surface modi-cation of biomaterials for controlling microbial attachmentand biolm formation, giving examples of desirable surfaceproperties and the methods that are used to attain these prop-erties. Given the vast variety of materials, applications andsurface modication methodologies, the fully exhaustivecoverage of the relevant existing knowledge is outside the scopeof this review.

Section 3 will discuss two broad classes of natural antimi-crobial compounds, specically secondary plant metabolitesand antimicrobial peptides, as a viable alternative to conven-tional systemic antibiotics.

Section 4 will review catechol chemistries inspired by thedistinctive water-resistant, material-independent adhesiveabilities of many sessile aquatic organisms.31 This section willdiscuss the relevance of this chemistry to both the assembly ofwell-adhering releasing hydrogels and for the design ofsubstrate-independent adhesive coatings that can serve as abase layer for further functionalization, such as covalentbinding of natural antimicrobial agents.

Section 5 will review general principles of plasma assistedsurface modication, as a technique that can be used forsurface patterning, deposition of contact killing and drugeluting coatings and for surface functionalization that can besubsequently used for drug immobilisation in non-releasesystems. Section 5 will concentrate on select examples ofusing plasma environment to process natural antimicrobialagents into bioactive coatings.


3. Antimicrobials of natural origin

The use of systemic antibiotics has been challenged on manylevels, the key issue concerned with its contribution towards thedevelopment of microbial resistance. And while these agentsremain among the most potent weapons in treating advancedinfections, there has been an increasing interest in the use ofalternative, nature-derived antimicrobials, whose physico-chemical structure and mechanism of bioactivity are suffi-ciently dissimilar to those of currently used synthetic antibioticsto eliminate the possibility of bacterial cross-resistance. Devel-opment of cross-resistance is an important problem, andmethicillin resistant Staphylococcus aureus is one of the bestknown examples of microorganisms with multi-drug resistanceagainst most currently available antibiotics, including recentcases of vancomycin-resistant S. aureus.

Other notable clinically signicant drug-resistant pathogensinclude Acinetobacter baumannii, P. aeruginosa, E. coli andKlebsiella pneumoniae resistant to b-lactamases, and Mycobac-terium tuberculosis.32 Although some bacterial organisms areintrinsically resistant to some antimicrobials, excessive useand/or misadministration of antibiotics may select for patho-gens that acquired resistance by either de novo mutations or viagene transfer, conjugation, transformation, and transduction.32

These newly acquired genes can complement and thus enhancethe intrinsic resistance of the microorganism.

Phenotypically, the changes in genotype can manifest inmany ways, including synthesis of enzymes capable of deacti-vating antibacterial agent, physico-chemical changes to the sitetargeted by the antimicrobial, activation of an alternativemetabolic pathway to circumvent the activity of the drug, and tominimise the accessibility of internal drug targets via variousefflux mechanisms. Amongst numerous alternative antimicro-bials, metal ions, nitric oxide, antimicrobial peptides, andsecondary metabolites derived from plant organisms provide adiverse range of antimicrobial agents.

3.1 Antimicrobial peptides

Antimicrobial peptides are produced by all complex organismsas well as some microbes as part of innate immune response,and display diverse and complex antimicrobial activities againsta broad range of Gram negative and Gram positive bacteria,including those resistant to established antibiotic drug thera-pies, mycobacteria, enveloped viruses, parasites and fungi.35,36

Also known as host defense peptides, these are low molecularmass amphipathic molecules of 12–50 amino acids in length,and are secreted by many different cell types, either constitu-tively or in response to inammatory stimuli.37 These moleculestypically perform more than one function within the organism(Fig. 4). For instance, peptides produced by neurons, e.g.neurokinin-1, neuropeptide Y, orexins, function as both theneurotransmitters in the brain and the peripheral nervoussystem, and as immunomodulators, regulating immune func-tion and neurogenic inammatory responses through vasodi-latation, plasma extravasation, and recruitment ofimmunocompetent cells.35,38–40 Orexin B has been reported to

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affect the function of peritoneal macrophages via activation ofcalcium-dependent potassium channels and to facilitateenhancement of phagocytosis in mouse peritonealmacrophages.38,41

In plants, thionins, defensins, lipid transfer proteins, hevein-and knottin-like peptides, MBP1, IbAMP, and the recentlyreported snakins are the most commonly encountered antimi-crobial peptides.42 Structurally, these are small cationic peptideswith molecular masses of 2–10 kDa, with their structure stabi-lized via the formation of 2–6 disulde bridges. The antibacterialmechanism of thionins is through the binding of phospholipidsof the bacterial membrane which initiates a cascade of cyto-plasmic events leading to cell death.43,44 High positive charge,which renders them extremely soluble (>300 mg ml�1), and thephospholipid-binding specicity of thionin allows the agent tobind areas of negatively charged phospholipids, either

Fig. 4 (A) Overview of the biological activities of host defensepeptides (HDPs) and innate defense regulator (IDR) peptides. Directcytotoxic activities are shown in green, direct and indirect immuno-modulatory properties are in blue and pink, respectively. ROS, reactiveoxygen species; NO, nitric oxide.33 (B) Overview of the broad spectrumof cellular interactions associated with antimicrobial peptides.Peptides exert antimicrobial activity by disrupting bacterialmembranes, binding to specific target proteins within microbial cellsand activating the innate immune system.34 Reproduced withpermission from ref. 33 and 34.

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phosphatidic acid or phosphatidyl serine, and their subsequentwithdrawal. The segregation of phospholipids destabilizes themembrane, causing its solubilisation and lysis.

Defensins, typically 45–54 amino acids long cationicpeptides, display only modest antimicrobial activity, with soundefficacy against diverse fungi.45,46 Specic defensins have alsobeen reported to inhibit protein synthesis, protease trypsin, ora-amylase activity.45 Lipid transfer proteins range in sizebetween 7 and 10 kDa, and are typically of globular structurewith a large hydrophobic cavity. The cavity serves as a bindingsite for mono- or diacylated lipids and other hydrophobicmolecules, with a larger cavity of LTP2 allowing it to bind aplanar sterol.

In terms of the use of antimicrobial peptides as antibacterialsurface modication, numerous strategies have been tested todeliver these antimicrobials to the site of implantation. Theionic self-complementary of peptides allows for their use asbuilding blocks for self-assembly of nanostructures. Eachamphipathic molecule is comprised of distinct hydrophilic andhydrophobic regions. In aqueous environment, the hydro-phobic region tries to minimise its exposure to water, resultingin folding of the molecule. The hydrophilic domain iscomposed of alternating positively charged (e.g. arginine,lysine) and negatively charged (e.g. aspartate, glutamate) aminoacid residues, with various patterns of distribution of thecharged residues. These residues will engage in ionic interac-tions with the oppositely charged residues of the complimen-tary molecule, driving the self-assembly. Although non-covalentin nature, the interactions are sufficiently strong to supporthighly stable structures.

Hydrogels of b-hairpin peptides rich in arginine displayedstrong antibacterial activity against Gram-positive and Gram-negative bacteria, including multi-drug resistant P. aerugi-nosa.47 The fundamental and functional properties of thehydrogel, including killing efficacy, host cytocompatibility, bulkrheological properties and stimuli-responsiveness of this type ofhydrogel can be controlled via selection of the specic peptidesequence at the monomer level.48–50 Pre-functionalisation of theimplant surfaces, e.g. via plasma-assisted treatment, has beenused for UV immobilisation of 3-poly-L-lysine-gra-meth-acrylamide hydrogel thin lm.51 Coupled with excellent activityagainst bacteria and fungi, the low thickness of the coatingmakes it a good candidate for coating over medical devices andimplants.

A mode of delivery via loading of antimicrobial peptides intoa carrier platform has been trialled. Kazemzadeh-Narbat et al.used micro-porous octacalcium phosphate lms to load broadspectrum antimicrobial peptides for orthopaedic applications.52

Shukla et al. used thin layers of polyionic polymer lms tophysically entrap the antimicrobial agent, varying layer numberand composition for control over the amount of agent loadedinto the structure.53 It has been suggested that the antimicrobialagent may not be able to diffuse through the layers of thepolymer at a sufficient rate to ensure the steady level of theantimicrobial at the surface. In addition to the intrinsic prop-erties of the layers, through which the peptide is to diffuse, its



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release may also be limited by the bacterial cells accumulated atthe surface of the implant.54

Physical and chemical immobilisation of the peptide on thesurface of the implant can circumvent the issues with diffusion,although accumulation of bacterial debris may still remain achallenge.56 Although reported as an effective approach forpeptide surface immobilisation, non-specic physical adsorp-tion may compromise the availability of physico-chemicalparticulates of the peptide present in the soluble analogue,potentially rendering the coating inactive.55 Even specic,covalent attachment of the peptide is likely to affect the struc-tural exposure and exibility characteristic of the peptide.29 Forinstance, even though notably larger amounts of peptide perunit area can be conjugated to a surface via polymer brushcompared to the direct graing of peptides, a signicantportion of these peptides may not be available to interact withbiomembranes due to steric restrictions exerted by the polymerbrush structure.55 On the other hand, the surface-tetheredpeptides may be more effective in combating microbial colo-nisation due to higher concentration of appropriately struc-tured peptides in one location (Fig. 5).

To minimise the detrimental effects of the binding, manymethods have been developed, employing a variety of chemicalcoupling strategies, length of spacers, and peptide orientationand concentration.57,58 Many of these strategies focus on mini-mising nonspecic interactions between the peptide and thesubstrate.59 Another important consideration in using peptide

Fig. 5 Proposed mechanism of action of free (A) and polymer brush-immobilized host defense peptide (B). In (B), more peptides adopt astructure before they interact with the membrane, and therefore theremay be more structured peptides localized in one area when theybind/insert into the membrane. As a result, perturbation of themembrane may be more efficient. Reproduced with permission fromref. 55.


antimicrobials is their stability,60 as well as the stability of thecoating system as a whole under physiological conditions.61

3.2 Antimicrobial secondary plant metabolites

Plants produce a broad assortment of secondary metabolites,including tannins, terpenoids, alkaloids, polyphenols andavonoids, which have been found in vitro to have antimicrobialproperties against both Gram positive and Gram negativebacteria. Furthermore, these phytochemicals have been showntomodulate or modify resistance mechanisms in bacteria.62 Yet,since the discovery of penicillin in the 1950s, the medical worldhas relied on antibiotics derived from bacterial and fungalsources, with the use of plant derivatives as antimicrobialsbeing nearly non-existent.63 One of the possible reasons for thisis that the relatively higher minimum inhibitory concentrationslimited their utility as the sole agents, although certaincombination of phytochemicals with conventional antimicro-bial drugs demonstrated enhanced efficacy against methicillinresistant S. aureus.62 In that case, tannic acid was able toprolong and potentiate the bactericidal activity of fusidic acid,cefotaxime, minocycline and rifampicin, with a similar effectdemonstrated for combinations of quercetin with fusidic acid,minocycline and rifampicin.

Amongst the vast variety of phytochemicals, phenolics,terpenoids and other essential oils constituents, alkaloids, lec-tins and polypeptides, and polyacetylenes are most commonlyassociated with antimicrobial activity.32 These phytochemicalsplay other roles in plant physiological processes, e.g. avonoidsare the key pigments for plants that reproduce via biotic polli-nation; avonoids are also involved in UV ltration andsymbiotic nitrogen xation; and as chemical messengers,physiological regulators, and cell cycle inhibitors. The use ofthese antimicrobial agents has been limited to traditional andalternative medical domains, yet to be recognised by themainstream medical community as therapeutic agents. Asmentioned above, one of the main reasons lies in the relativelyweak and/or narrow spectrum of antimicrobial activity, andpotentially high toxicity associated with the administration ofsufficiently high antimicrobial doses. Indeed, the MICs typicallyreported for plant-derived antimicrobials are in the range of 100to 1000 mg ml�1, orders of magnitude weaker than MICs of 0.01to 10 mg ml�1 of antimicrobials synthesised by bacteria andfungi.64 It has been suggested that along with these antimicro-bials, plants may produce a range of other chemicals, e.g.inhibitors of bacterial multidrug resistance pumps, whichenhance permeation of the antimicrobials into the bacterialcells. Furthermore, there is a distinct lack of systematicdescription regarding the structure–property of antibacterialphytochemicals, potentially limiting their mainstreamadoption.

Essential oils are abundant in nature, and most commonlyassociated with the distinctive avours and aromas of manyplants.65 Commonly used herbs and spices such as garlic, blackcumin, cloves, cinnamon, thyme, bay leaves, mustard, androsemary have essential oils with demonstrated antimicrobialproperties.66 Garlic-derived allicin was found to exhibit

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antibacterial activity against a wide range of Gram-negative andGram-positive bacteria, including multidrug-resistant enter-otoxicogenic strains of Escherichia coli; antifungal activity,particularly against Candida albicans; antiparasitic activity,including some major human intestinal protozoan parasitessuch as Entamoeba histolytica and Giardia lamblia; and antiviralactivity.67 The crude methanolic extracts of such spices andherbs as cumin (Cuminum cyminum), fennel seed (Nigella sat-iva), anise (Pimpinella anisum), ajowan (Trachyspermum copti-cum), and ginger (Zingiber officinale) were demonstrated to beeffective against Gram-positive Bacillus amyloliquefaciens and S.aureus and Gram-negative E. coli and Pseudomonas aeruginosabacteria.68 Importantly, the extracts demonstrated similar orhigher broad-spectrum antimicrobial activity as compared withampicillin, erythromycin, and tetracycline.

Extracts of Nigella sativa were also effective against patho-genic yeast, C. albicans, and its diethyl ether extract was revealedto be similar in antibacterial activity to that of streptomycin andgentamicin. Traditionally used to treat urinary tract infections,a combination of garlic and black cumin has been reported asbeing more effective than Cefalexin, Cotrimoxazole, and Nali-dixic acid in the treatment of this infectious disease.66 Given themulticomponent nature of the extracts, which included carbo-hydrates, inulin, alkaloids, glycosides, avonoids, terpenoids,tannins, reducing sugars, soluble phenols, and saponin glyco-sides, it is difficult to attribute the observed antimicrobialactivity to a particular constituent (Table 1).68

The antibacterial and antifungal potency of caraway (Carumcarvi) oils are attributed to carvone, limonene and linalool,while antimicrobial activity of cumin is associated with thepresence of limonene, eugenol, pinene and minor constituents,and the effect is likely to be synergistic.73 Cumin essential oilwas found to the activity of the ciprooxacin against biolm-forming Klebsiella pneumoniae strains, although the oil on itsown was not able to induce plasmid DNA degradation.74 C.cyminum oil was also effective against Vibrio spp. strains.75

Essential oil from rosemary (Rosmarinus officinalis) was shownto be effective against E. coli, S. aureus and L. monocytogenes,although it was found less potent in comparison with Cu.Cyminum essential oil.76 Peppermint (Mentha piperita) oil wasdemonstrated to be more effective than chlorhexidine in pre-venting biolm formation by Streptococcus mutans and Strepto-coccus pyogenes, with potential to be used in therapies againstsupragingival dental plaque.77

A survey of 35 different Indian spices showed clove,cinnamon, bishop's weed, chili, horse radish, cumin, tamarind,black cumin, pomegranate seeds, nutmeg, garlic, onion, tejpat,celery, have potent antimicrobial activities against the testorganisms Bacillus subtilis, E. coli and Saccharomyces cer-evisiae.78 Oils of chilli, cinnamon, cloves, ginger, nutmeg,oregano, rosemary, sage, thyme demonstrated a range ofactivities against psychrotrophic Aeromonas hydrophila, Listeriamonocytogenes and Yersinia enterocolitica, from complete inhi-bition of growth in the case of cinnamon and cloves against A.hydrophila to no inhibition.79 The antimicrobial potency wasalso found to vary with the oil acquisition method, e.g. oilharvesting at different stages of plant development. Thyme

48746 | RSC Adv., 2015, 5, 48739–48759

(Thymus vulgaris) oil harvested at four ontogenetic stages had asignicant bacteriostatic activity against nine strains of Gram-negative bacteria and six strains of Gram-positive bacteria.However, the activity was the highest for the oil harvested fromthe plants in full ower.80

Although oils and their individual components, such asterpenoids, carvacrol, thymol, have been recognised as poten-tial antimicrobial agents, yet their exact mechanism of actionshas not been fully elucidated. In part, this may be due to thenumerous components that can potentially complement and/orenable the efficacy of the other component. For instance, Bro-phy et al. analysed over 800 samples of M. alternifolia essentialoil by gas chromatography and mass spectrometry and foundapproximately 100 components in oil ofM. alternifolia as well assignicant batch to batch variation.81 The comparison may becomplicated further by different methods used to quantifyantibacterial activity (which also oen differ from those used forevaluation of surface-immobilised antimicrobial agents).82–84

Oil from Australian native plant Melaleuca alternifolia hasbeen reported to have the broad-spectrum activity againstbacteria, including drug-resistant strains, fungi, viruses, andprotozoa,85–88 but similar to other phytochemicals, in vivo and invitro characterisation of tea tree oil thus far remains inade-quate. Nonetheless, various preparations that include tea treeoil are readily available commercially in many countries,including in Australia, Europe, and North America. Tea tree oilis composed of terpene hydrocarbons based on an isoprenestructure, mainly monoterpenes (C10H16), sesquiterpenes(C15H24), and their associated alcohols (terpenoids), with theantimicrobial activity of the oil is attributed mainly to terpinen-4-ol. The mechanism of action of terpenes is yet to be fullydescribed but is believed to involve membrane disruption by thelipophilic compounds.63,89 In the case of tea tree oil, the abilityof tea tree oil to disrupt the permeability barrier of cellmembrane structures and the accompanying loss of chemios-motic control were identied as the most likely source of itslethal action against E. coli, S. aureus, and Candida albicans.90

The predisposition to lysis, the loss of 260 nm-absorbingmaterial, the loss of tolerance to NaCl, and the alteredmorphology by S. aureus cells suggest that tea tree oil and itscomponents compromise the cytoplasmic membrane.91,92

Essential oils and their constituents are believed to interactwith the bacterial membrane, causing disruption throughlipophilic products (Fig. 6). These disruptions then lead tomembrane expansion, increase of membrane uidity andpermeability, disturbance of membrane embedded proteins,inhibition of respiration, and alteration of ion transportprocesses in both Gram-positive and Gram-negative bacteria.32

An analysis of the chemical structure of these herbs and spicesshows that the antimicrobial phytochemicals consist of phenolsand oxygen-substituted phenolic rings,63 with the inhibitoryaction associated with the –OH groups in phenolic compounds.

Garlic is different in such that it consists of non-aromaticsulfur compounds (thiosulnates) that carry the antimicrobialproperties. Diallyl thiosulnate (allicin), the phytochemicalagent found in garlic (Allium sativum) and believed to beresponsible for the antibacterial and antifungal activity of



Tab

le1

Minim

um

inhibitory

conce

ntrations(M

ICs)

ofselectedessential

oils

against

selectedclinically

relevantmicroorgan

isms6

9–72

Plan

tnam

eSp

ecies

Testorga

nism

Acinetob

acter

baum

anii

Aeromon

assobria

Can

dida

albicans

Enterococcus

faecalis

Escherichia

coli

Klebsiella

pneumon

iae

Pseudo

mon

asaerugino

saSa

lmon

ella

typh

imurium

Serratia

marcescens

Stap

hylococcus

aureus

MIC,%

v/v

Rosew

ood

Anibarosaeodo

ra0.1

0.1

0.3

0.5

0.1

0.5

>2.0

0.3

0.5

0.3

Celeryseed

Apium

graveolens

>2.0

1.0

1.0

2.0

2.0

>2.0

>2.0

>2.0

>2.0

1.0

Fran

kincense

Boswelliacarterii

1.0

1.0

1.0

2.0

1.0

>2.0

>2.0

>2.0

>2.0

1.0

Ylangylan

gCan

anga

odorata

1.0

0.5

1.0

2.0

2.0

>2.0

>2.0

>2.0

>2.0

1.0

Ced

arwoo

dCedrusatlantica

>2.0

>2.0

>2.0

0.5

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

Cinnam

onCinna

mom

umzeylan

icum

1.6

>2.0

0.8

>2.0

Lime

Citrusau

rantifolia

1.0

1.0

2.0

>2.0

1.0

>2.0

>2.0

>2.0

>2.0

2.0

Orange

Citrusau

rantium

>2.0

1.0

1.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

2.0

Petitgrain

Citrusau

rantium

0.5

0.5

0.3

2.0

0.3

>2.0

>2.0

>2.0

>2.0

0.5

Berga

mot

Citrusau

rantium

var.

bergam

ia2.0

2.0

1.0

>2.0

1.0

>2.0

>2.0

>2.0

>2.0

>2.0

Lemon

Citruslimon

>2.0

1.0

2.0

2.0

>2.0

>2.0

>2.0

>2.0

>2.0

2.0

Grape

fruit

Citrusxpa

radisi

>2.0

1.0

1.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

Man

darin

Citrusreticulata

var.

Mad

urensis

>2.0

>2.0

2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

Myrrh

Com

mipho

ramyrrha

>2.0

>2.0

>2.0

0.3

>2.0

>2.0

>2.0

>2.0

>2.0

0.5

Coriande

rCoriand

rum

sativum

0.3

0.3

0.3

1.0

0.3

0.5

>2.0

1.0

0.5

0.3

Pumpk

inCucurbita

pepo

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

Cyp

ress

Cup

ressus

sempervirens

>2.0

>2.0

>2.0

1.0

>2.0

>2.0

>2.0

>2.0

>2.0

2.0

Turmeric

CurcumalongaL.

0.02

0.02

0.01

Lemon

grass

Cym

bopo

goncitratus

0.0

0.1

0.1

0.1

0.1

0.3

1.0

0.3

0.3

0.1

Palm

arosa

Cym

bopo

gonmartinii

0.1

0.1

0.1

0.3

0.1

0.3

>2.0

0.5

0.3

0.1

Citronella

Cym

bopo

gonna

rdus

0.3

0.1

1.0

0.5

1.0

>2.0

>2.0

>2.0

0.3

Carrotseed

Dau

cuscarota

>2.0

>2.0

2.0

2.0

>2.0

>2.0

>2.0

>2.0

>2.0

1.0

Euc

alyp

tus

Eucalyptus

polybractea

1.0

0.5

1.0

2.0

1.0

2.0

>2.0

>2.0

1.0

2.0

Clove

Eugeniacaryop

hyllus

1.6

>2.0

1.6

>2.0

Fennel

Foeniculum

vulgare

1.0

0.5

0.5

>2.0

0.5

>2.0

>2.0

1.0

>2.0

0.3

Wintergreen

Gau

ltheriaprocum

bens

0.3

0.3

0.3

>2.0

0.5

1.0

>2.0

0.5

0.5

2.0

Juniper

Juniperuscommun

is>2

.01.0

2.0

2.0

>2.0

>2.0

>2.0

2.0

>2.0

2.0

Fren

chlavende

rLa

vand

ulaan

gustifolia

1.0

0.5

>2.0

0.5

2.0

>2.0

>2.0

>2.0

1.0

Tasman

ianlavender

Lavand

ulaan

gustifolia

0.5

0.5

0.3

2.0

0.3

>2.0

>2.0

>2.0

2.0

1.0

Tea

bush

Lipp

iachevalieri

1.0

>2.0

1.0

Tea

bush

Lipp

iamultiora

0.3

0.3

0.1

Macad

amia

Macad

amia

integrifolia

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

Tea

tree

Melaleuca

alternifolia

0.3

0.5

0.5

2.0

0.3

0.5

>2.0

0.5

0.5

0.5

Cajupu

tMelaleuca

cajupu

ti1.0

1.0

1.0

2.0

1.0

>2.0

>2.0

>2.0

2.0

1.0

Niaou

liMelaleuca

quinqu

enervia

0.3

0.3

0.3

1.0

0.3

0.5

>2.0

0.5

0.5

0.5

Pepp

ermint

Menthaxpiperita

0.5

0.5

2.0

0.5

1.0

>2.0

1.0

2.0

1.0

Spearm

int

Menthaspicata

0.3

0.3

0.1

2.0

0.3

0.5

>2.0

0.5

0.3

0.3

Basil

Ocimum

basilicum

0.5

0.5

0.5

>2.0

0.5

2.0

>2.0

2.0

>2.0

2.0

Eveningprim

rose

Oenothera

biennis

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

Marjoram

Origanu

mmajoran

a0.3

0.3

0.3

2.0

0.3

0.5

>2.0

0.5

0.5

0.5

This journal is © The Royal Society of Chemistry 2015 RSC Adv., 2015, 5, 48739–48759 | 48747

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Tab

le1

(Contd.)

Plan

tnam

eSp

ecies

Testorga

nism

Acinetob

acter

baum

anii

Aeromon

assobria

Can

dida

albicans

Enterococcus

faecalis

Escherichia

coli

Klebsiella

pneumon

iae

Pseudo

mon

asaerugino

saSa

lmon

ella

typh

imurium

Serratia

marcescens

Stap

hylococcus

aureus

MIC,%

v/v

Orega

no

Origanu

mvulgare

0.1

0.1

0.1

0.3

0.1

0.1

2.0

0.1

0.3

0.1

Geran

ium

Pelargon

ium

graveolens

0.3

0.3

0.1

0.5

0.3

>2.0

>2.0

>2.0

>2.0

0.3

Aniseed

Pimpinellaan

isum

0.5

0.3

0.5

2.0

0.5

>2.0

>2.0

2.0

1.0

0.3

Bay

Pimenta

racemosa

0.1

0.1

0.1

0.5

0.1

0.3

1.0

0.3

0.3

0.3

Pine

Pinu

ssylvestris

2.0

2.0

2.0

>2.0

2.0

>2.0

>2.0

>2.0

>2.0

>2.0

Black

pepp

erPipernigrum

>2.0

>2.0

>2.0

1.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

Patchou

liPo

gostem

onpa

tcho

uli

>2.0

>2.0

0.5

0.1

>2.0

>2.0

>2.0

>2.0

>2.0

0.3

Apricot

kernel

Prun

usarmeniaca

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

Sweetalmon

dPrun

usdu

lcis

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

Rosem

ary

Rosmarinus

officina

lis

1.0

0.5

1.0

>2.0

1.0

2.0

>2.0

>2.0

>2.0

1.0

Sage

Salvia

officina

lis

0.5

0.5

0.5

2.0

0.5

2.0

>2.0

2.0

1.0

1.0

Clary

sage

Salvia

sclarea

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

>2.0

Sandalwoo

dSa

ntalum

albu

m>2

.0>2

.00.1

0.3

>2.0

>2.0

>2.0

>2.0

>2.0

0.1

Clove

Syzygium

arom

aticum

0.3

0.1

0.5

0.3

0.3

>2.0

>2.0

0.3

0.3

Thym

eThymus

vulgaris

0.1

0.1

0.1

0.5

0.1

0.3

>2.0

>2.0

0.3

0.3

Vetiver

Vetiveriazizanioides

>2.0

>2.0

0.1

0.1

>2.0

>2.0

>2.0

>2.0

>2.0

0.1

Ginger

Zingiber

officina

le>2

.0>2

.0>2

.0>2

.0>2

.0>2

.0>2

.0>2

.0>2

.02.0

48748 | RSC Adv., 2015, 5, 48739–48759 This journal is © The Royal Society of Chemistry 2015

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Fig. 6 Proposed mechanism of action and target sites of secondaryplant metabolites on microbial cells. Reproduced with permissionfrom ref. 93.

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extracts of this plant is thought to interact with intracellularthiols and thiol containing enzymes, including alcohol dehy-drogenase, thioredoxin reductase and RNA polymerase.67 Thiscan affect essential metabolism of cysteine proteinase activityinvolved in the virulence of E. histolytica. The effect of bacte-riostatic concentrations of allicin (0.2 to 0.5 mM) on the growthof Salmonella typhimurium was characterised by a delayed andpartial inhibition of DNA and protein syntheses and immediateand total inhibition of RNA synthesis, suggesting that the latteris the primary target of allicin.94 Garlic extract has also beenshown to inhibit quorum sensing ability of biolm-residingPseudomonas aeruginosa, rendering the treated bacteriasusceptible to the bactericidal activity of tobramycin and poly-morphonuclear leukocytes.95,96

4. Nature-inspired catecholchemistry

In addition to nature-inspired antibiofouling and biocidalsurfaces, e.g. lotus leaf, buttery wing and shark skin-likebiomimetic surfaces, bio-inspired chemistries can inuencethe manner in which biomaterials and biomaterial coatings aresynthesized, functionalized and delivered in vivo.22,25,97,98 Thereare several methods of surface functionalisation that areinspired by the aquatic animals that are highly adept at fouling avariety of solid surfaces, both natural and man-made, inaqueous environments. Numerous sedentary marine organisms,including species of mussels, tubeworms, and barnacles attachto underwater surfaces by means of protein-rich adhesives.97,99

Adhesive proteins form approximately 70 wt% of the cementof Belanus crenatus, where the proteinaceous cement is releasedfrom the pores to ll the space between the base of theattachment disk of the animal and the solid surface to whichthe organism is attaching. The cement cures within several


hours, forming a high strength bond with attachment strengthof up to 9.3 � 105 N m�2. Where cement produced by adult andcyprid acorn barnacles (order Sessilia) solidies into a thin layerdirectly between the shell and the surface and is characterisedby either solid or reticulate structure,100 the cement produced byDosima fascicularis buoy barnacle is a gas-lled, foam-likestructure.101 Whereas the barnacle uses the cement for attach-ment to surfaces, the Phragmatopoma californica marine wormuses its glue to build its mineralized shell from sand grains andfragments of seashell collected from its environment.102 Settingwithin 30 s under, the glue forms a microporous water-lledfoam comprised of 50–80 nm spheres, and characterised by asharp gradient in porosity.

Water-resistant, material-independent adhesive abilities ofthe mollusc (Mytilus edulis) byssus, a proteinaceous liquid fromthe phenol gland in the mussel foot that forms an adhesiveholdfast, have been used to guide the development of substrate-independent adhesive hydrogels.31 Rapid solidication into ahardened adhesive and excellent adhesion to a variety ofsubstrates, including tissues, is attributed to reactivity of cate-chol side chains on 3,4-dihydroxy-L-phenylalanine (DOPA).103

Readily oxidised, catechol side chains form reactive species thatcan undergo Michael-type addition, Schiff base formation withnucleophiles, and radical coupling with other catechols (Fig. 7).They can also form coordination bonds with diverse metals andinorganic surfaces, hydrogen bonds, and p–p aromatic inter-actions. The mechanism by which 3,4-dihydroxy-L-phenylala-nine interacts with the wet surface depends on the state of themolecule.104 An atomic force microscopy (AFM) study of a singlemolecule immobilised on the scanning tip demonstrated highstrength yet fully reversible, non-covalent interaction with a wetmetal oxide surface, here titanium dioxide. Once 3,4-dihydroxy-L-phenylalanine was oxidised, the strength of this reversibleinteraction signicantly decreased, although a new, highstrength irreversible covalent bond was formed.

4.1 Catechol-based hydrogels

The incorporation of these catechol functionalities into watersoluble hydrogels, such as polyethylene glycol, ensures rapidcuring of these gels. The degradation properties of such adhe-sive hydrogel can be modied, by incorporating enzyme-degradable sites. For instance, a hydrogel based on poly-ethylene glycol functionalised by DOPA-mimetic catechol viabiodegradable linker, e.g. an Ala–Ala dipeptide substrate ofelastase, can be degraded by neutrophil elastase, the latterbeing a serine protease secreted by activated neutrophils as theresult of their recruitment to a wound or site of local inam-mation.108 The microstructure, composition and mechanicalproperties of the hydrogel can also be tuned by controlling theinput catechol, linker, and polymer backbone.

The hydrogel can be loaded with antibacterial agents forsustained release. For example, the use of silver nitrate tooxidize catechol-functionalised polyethylene glycol resulted incovalent cross-linking of the hydrogel and concomitant reduc-tion of Ag(I).31 The resultant bulk hydrogels demonstratedinhibition of Staphylococcus epidermidis and Pseudomonas

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Fig. 7 Catechols as versatile platforms in polymer chemistry. (A) Aphotograph of a mussel attached to the shell of another mussel. (B)Schematic representation of the adhesive plaque and byssal thread. (C)Chemical structure of the DOPA side chain found in mussel adhesiveproteins.105 (D) Illustration of the proposed binding mechanism ofDOPA to two types of surfaces, TiO2 and mica. DOPA andDOPAquinone, to a lesser extent, can form bidentate binuclearcomplexes with the TiO2 surface, whereas the interactions with micaare much less specific and may result from the hydrogen bonding ofthe phenolic OH groups to the oxygen atoms of the cleaved micasurface. DOPAquinone has no H to donate.106 (E) Possible reactionpathways of oxidized catechols with amines, thiols or imidazoleswhere R0 stands for a polymeric or peptidic backbone.107 Reproducedwith permission from ref. 105–107.

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aeruginosa due to sustained release of silver, with minimaldetriment to mammalian (3T3 broblast) cell viability.31 Whenused as a spin-cast 25 nm-thick coating over titanium dioxidesubstrate, the hydrogel resisted fouling by both bacterial andeukaryotic cells. Due to relatively low content of silver in the thinlm hydrogel, the non-fouling by mammalian cells was attrib-uted to the antifouling nature of the polyethylene glycol polymer,rather than cytotoxicity of released silver. However, the ndings

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that the toxic effect of silver ions and silver nanoparticles occursin a similar concentration range for Escherichia coli, Staphylo-coccus aureus, human mesenchymal stem cells, and peripheralblood mononuclear cells challenges this conclusion.109,110

4.2 Catechol adhesive layer

Dopamine has been demonstrated to possess the full adhesiveproperties of mussel adhesion protein, and can be used as athin highly adherent coating on a range of biomaterial surfaces,organic and inorganic alike. Such dopamine-based surfacecoatings are resistant to hydrolysis and provide chemical acti-vation on material surfaces for selective coupling of moleculesand layers.111 The coating is deposited as poly(dihydroxyindole),but undergoes oxidation to polyorthoquinoneindole uponexposure to basic (pH 8.5) conditions.112 To this layer, biomol-ecules containing amine moieties can be covalently bonded viaSchiff base type interactions, or Michael type reactions in thecase of those molecules with amine and thiol functionalities.

In addition to monolayers via self-assembly of long-chainmolecular building blocks, secondary reactions on thedopamine-modied surfaces can be used for deposition ofmetal lms by electroless metallization, and bioinert andbioactive surfaces via graing of macromolecules.113 Silvernanoparticles were immobilised onto ferromagnetic Fe2O3nanoparticles/brous bacterial cellulose nanocomposite bysoaking dopamine-treated composite in silver nitrate solu-tion.114 Dopamine coating was also shown to be a suitableplatform for fabrication of polymer brushes via atom transferradical polymerization.

The utility of barnacle cement for surface functionalisationhas been demonstrated on stainless steel, where the adhesivewas used as a surface anchor for coupling of functional polymerbrushes via “click” reactions in both “graing-to” and “graing-from” processes.115 A surface rich in thiol, alkyne, and azidegroups was obtained by rst depositing a thin layer of thecement onto the metallic surface. The reactive amine and/orhydroxyl groups on the surface100 were then allowed to reactwith ethylene sulde, propargyl carbonylimidazole, and azi-doethyl carbonylimidazole, respectively, to introduce thedesired functionality. Using these molecular anchors, a varietyof stable functional polymer brush coatings were developed,including antifouling zwitterionic 2-methacryloyloxyethylphosphorylcholine surfaces (via thiol photo polymerisation);protein-resistant hydrophilic poly(poly(ethylene glycol) methylether methacrylate) and protein-adsorbing hydrophobicpoly(2,3,4,5,6-pentauorostyrene) brushes (via azide–alkyneclick reaction); antifouling poly(N-hydroxyethyl acrylamide) andantimicrobial poly(2-(methacryloyloxy)ethyl trimethylammo-nium chloride) surfaces (via alkynyl–azide click chemistry).115

Of the developed coatings, the zwitterionic and hydrophilicsurfaces were most effective in reducing bovine serum albuminadsorption, with the zwitterionic, antifouling, and antimicro-bial surfaces inhibiting the adhesion of Gram-negative E. coliand Gram-positive S. epidermidis. Similar to hydrophilic poly(-ethylene glycol) and oligo(ethylene glycol), the antifoulingproperties of zwitterionic and polyampholyte polymer brushes



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rely on the formation of a strong hydrogen-bonded hydrationlayer, which limits protein interactions with the underlyingsurface.116 Surfaces rich in alkyl halide functionalities were alsoobtained by reacting the amine and hydroxyl moieties ofbarnacle cement with 2-bromoisobutyryl bromide.117 The initi-ator can be used for the surface-initiated atom transfer radicalpolymerization of 2-hydroxyethyl methacrylate, the hydroxylgroups of which can then be converted to carboxyl groups forcoupling of chitosan. Thus functionalised stainless steelsurfaces displayed antifouling property against bovine serumalbumin and antibacterial activity against E. coli.

Fig. 8 Examples of plasma-treated surfaces. (A) Petal- and tree-likegraphene networks. (B) Titanium pillars structured in bulk materialusing reactive ion etching with fluorine plasma. (C) Collagen-graftedtitanium surface via allylamine-glow discharge treatment and collagencrosslinking. Reprinted with permission from ref. 133–135.

5. Plasma-assisted nanofabrication

Plasma-assisted technologies, especially those based on low-temperature, non-equilibrium plasmas, have found numerousapplications in medicine, materials science, and biotech-nology.118,119 The ability to remove biomolecules, such asproteins, pyrogens or peptides, and bacterial spores frombiomaterial surfaces at high rates and low temperatures makeplasma-assisted treatment an effective and practical tool fordecontamination and sterilization of biomaterials andmedically-relevant devices and surfaces.120–122 The neutral andreactive species, particularly reactive oxygen species (ROS) andreactive nitrogen species (RNS), electric elds, charges, andphotons generated in low temperature ionized gas plasmas areresponsible for the well-documented antimicrobial activity ofthese plasmas when applied directly to media.123,124 The lowtemperature (at or below physiological level) of such plasmasallow for their application onto living tissues, e.g. a wound,where they can be used to sterilise, suppress inammation, andpromote healing.125,126

The unique chemistry of these plasmas also enables selectivebiomanipulation of the cells, where they can be used to increasecell proliferation, locally inuence cell adhesion withoutcausing necrosis or to initiate cell removal via induction ofapoptosis, the result dependent on the dose.127,128 The selectivitywhereby only one type of cells is affected, i.e. cancer cells andnot healthy cells in co-culture, has a clear potential as a safermeans for anti-cancer therapy.129–132

From biomaterials perspective, plasma-assisted techniquesare widely used for lasting, highly controlled modication of avariety of medically relevant surfaces.136–138 Indeed, over the last20 years, plasma-enabled nanoscale synthesis and modicationhave evolved from a relatively simple tool for materials scienceand microelectronics into a highly sophisticated instrument fordevelopment of a wide range of pure and hybrid nanoscaleobjects spanning across a vast number of materials systems andlength scales (Fig. 8).139 At the present level of development, low-temperature plasmas afford chemists andmaterial scientists thelevel of condence comparable to, and in many cases superiorto, conventional processing techniques, e.g. based on thermalchemical vapour deposition (CVD), wet chemistry-basedsynthesis and processing, laser-assisted microfabrication etc.139

Importantly, tailored plasmas enable the attainment of certainobjectives conventional fabrication methodologies fail to


achieve, such as providing the means for one-step greensynthesis of functional materials from natural precursors.140–144

5.1 Types of low-temperature plasma processing

Lower temperature processing suitable for temperature-sensitive biomaterials and implantable thin lm structuresand for production of polymer lms where the functionality ofthe monomer is retained can be attained in low pressure, lowenergy plasma systems. In non-equilibrium plasma processing,the substrate is exposed to a reactive environment of a partiallyionised gas comprising large concentrations of excited atomic,molecular, ionic, and free-radical species. The nature of theinteractions between the excited species and the solid surfacewill determine the type and the degree of the chemical andphysical modication that will take place, from lm deposition,substitution, cross-linking to ablation. Generally, polymerdeposition occurs when a monomer, either in vapour phase orat the surface, is fragmented into reactive species that thenrecombine and are deposited onto the surface of the substrate.As mentioned previously, even those monomers that do notcontain functionalities required for conventional polymerisa-tion, e.g. C]C or ring structures, can be deposited in this way.

When lm deposition is not desired, gases that do notfragment into polymerisable intermediates upon excitation areemployed. Air, nitrogen, argon, oxygen, nitrous oxide, helium,tetrauoromethane, water vapour, carbon dioxide, methane,and ammonia are amongst the most common gases used forsurface modication. Exposure to these plasmas may result inthe chemical functionalisation of the surface, with the degreeand nature of the functionalities being highly dependent on thechemical composition of the biomaterial and the process gas.Plasma-assisted surface oxidation, nitration, hydrolysation, oramination are commonly used to increase the surface energyand hydrophilicity of the biomaterial. Surface ablation can alsoresult from such plasma-exposure, whereby lower molecularweight species, such as volatile oligomers and monomers, are

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desorbed from the surface of the biomaterial. Cross-linkingoccurs when radicals from one chain on the surface of thepolymer combine with radicals from another polymer chain toform a bond, thus changing the mechanical surface propertiesof the material. When plasma-generated radicals recombinewith atoms or chemical groups that are different from thoseoriginally present at the surface of the biomaterial, surfaceactivation takes place. Surface activation can also take placethrough opening of dangling bonds on the surface.

The surface functionalities that arise as a result of plasmadeposition or functionalization can serve as a platform forfurther surface modication processes, such as the graing ofbiomolecules and other functional structures,61,136 and to tunethe properties of the biomaterial for a specic application.145

Surfaces coated with plasma polymers can be used to bindproteins specically via covalent linkages, e.g. streptavidinconjugation to aldehyde groups where the binding ability of theprotein is retained, or nonspecically through other irreversibleadsorption mechanisms, e.g. streptavidin binding to ethanolplasma polymer surfaces where protein denaturationoccurred.146 Chemical gradients with different density of aspecic functionality or with a changing concentration of twofunctional groups across the biomaterial surface can be fabri-cated via plasma polymerisation by using a mask.147 Suchgradients can be highly useful for investigations of microbialand eukaryotic cell response to variations in surface chemistry,with each sample serving as a platform for high-throughputtesting of a range of cell–surface interactions.148 Morpholog-ical gradients can also be obtained with the help of plasmapolymerization, whereby a surface is rst functionalised with aspecic moiety, e.g. amine, and then subjected to controlledimmersion into the solution of nanoparticles.149 The variationin nanoparticle density gives rise to differences in surfaceroughness, the effect of which on cell adhesion andmetabolismcan be investigated independently of surface chemistry with anaddition of thin plasma polymer top layer. Furthermore,biomolecules (e.g. proteins) that display selective attachment togiven nanoparticles can be immobilised on these surfaces.These surfaces can then be used to study the effect of biomol-ecule density on cell–surface interactions.

5.2 Controlling plasma-assisted surface modication

The processing conditions, such as power delivered to thereactor, pressure within the reactor, monomer molecularweight and ow rate, presence of feed gas, etc. will determineactivation, fragmentation, rearrangement and recombination ofthe monomer units in plasma. The key determinant of themodication outcome is the amount of energy delivered intothe chamber in relation to the building units (from whichpolymers and nanostructures are synthesised) or to substratematerial (in the case of etching).

Monomers do not always need to be fragmented; however, ina plasma environment, there are more options for monomerfragmentation. When fragmentation takes place, it typicallyinvolves the elimination of hydrogen atoms, and the scission ofC–C bonds. Retention of the original chemical functionality

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within the resultant polymer deposit is highly dependent on thedegree of monomer fragmentation. The technological challengehere lies in the ability to retain the desired chemistry and at thesame time attain sound mechanical properties, desired density,stability, and adhesion to substrate in the material.150,151 Toaddress this challenge, it is important to understand themechanisms implicated in the plasma-assisted deposition ofthe polymer onto the surface.

While surface radical–plasma radical interactions wereconsidered the primary route of polymer deposition for manyyears,152 recent ndings have implicated ion adsorption and/orneutral graing as potential drivers for plasma polymerformation at the surface.153–156 The mechanism to explain thesephenomena centred on the energies at which depositing speciesarrive at the surface of the substrate. Under low pressure, lowpower conditions commonly used for fabrication of functionalpolymer coatings, neutral species such as radicals and unfrag-mented precursor molecules reach the surface at nearlyambient temperature (0.03–0.05 eV). On the other hand, ionsare accelerated to the surface by the difference between therespective potentials of bulk plasma and the surface and thusarrive at the surface with much higher energies (15–20 eV).157

The higher energy of ions is sufficient to break chemical bondsat the biomaterial surface, leading to the formation of surfaceradicals. These radicals are then available for neutral graing asper surface radical–plasma radical model, and can also promotecross-linking within the plasma polymer.

There is a clear link between the process parameters, themechanism of lm growth, and the resultant chemical and phys-ical properties of the polymer structure.157 The chemical structureof the organic precursor was found to be critical, in particular, atlow powers. The increased monomer fragmentation at high powerreduces the ability of unsaturated monomers to grow via neutralgraing. For saturated monomers, there is a direct link betweenthe deposition rate and ion ux to the substrate, whereas forunsaturated monomers, the neutral ux also plays a role.153 Thematerial properties of these lms also varied signicantly. Poly-mers deposited from saturated monomers were characterised byhigher moduli, lower solubility, and lower density compared tothose grown from unsaturated precursors. As the utility of plasmacoatings is reliant on the combination of desirable chemistry andmorphology, as well as good substrate adhesion, controlledstability and suitable mechanical properties, understanding therelationship between the process parameters and material prop-erties is crucial in the design of plasma polymer lm processes tofully harness the unique plasma-specic chemistries and physicalphenomena of non-equilibrium plasmas (Fig. 9).

5.3 Pulsed plasma deposition

Although low-power, low-pressure and low-temperature depo-sition is more conducive to the fabrication of plasma polymerswith retained functionalities, the degree of fragmentation is stillrelatively high. As a result, polymers fabricated using thismethod retain only a fraction of the functionality present in theprecursor, and are typically highly cross-linked and amorphous.Lowering power and temperature may reduce fragmentation



Fig. 9 Processes that may take place during plasma treatment.

Fig. 10 (A) Pulsed plasma deposition allows for fabrication of poly-mers that consist of more chemically-regular products than thosefabricated by means of continuous wave plasma deposition, wherepredominantly random radical recombination occurs.159 (B) Abundantin functional groups, pulsed plasma treated surfaces can be used forcovalent immobilisation of polymer brushes.160 Reproduced withpermission from ref. 159 and 160.

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even further, preserving more of the chemical structure presentin the precursor. Yet, the utility of these coatings in vitro and invivo is limited by their poor mechanical and chemical stability,and oen unsatisfactory attachment to the substrate. Theseissues may be circumvented by pulsed plasma deposition,where two distinct regimes are employed (Fig. 10).

In contrast to continuous wave plasmas, monomer activa-tion and generation of reactive site on the surface occur onlyduring on-periods (typically microseconds) whereas polymeri-sation takes place during off-periods (usually milliseconds) inthe absence of UV-, ion-, or electron-induced damage to thegrowing lm.158

The resultant polymer is characterised by high retention oforiginal chemistries, good stability and covalent attachment of thegrown lm to the substrate at the free radical sites generatedduring the on-period. By controlling the input power,161 pulsingfrequency and the duration of the pulse it is possible to tunechemical functionality, surfacemorphology and density of desiredchemical functionality at the polymer surface.162,163 The process ishighly versatile in terms of resultant surface chemistry, withpyridine,158 anhydride,164 amine,165 ester,166 hydroxyl,167–169 sulfonicacid,170 carboxylic acid,171 cyano,172,173 epoxide,174 halide,175

thiol,137,176 and furan177 functionalised surfaces reported.

5.4 Plasma-assisted processing of essential oils

The limited understanding of the exact mechanism of anti-bacterial efficacy of the essential oils and their individualcompounds signicantly limited their potential clinical uses,especially as part of antimicrobial coatings for medicalimplants. Indeed, most in vitro and in vivo studies to dateemployed phytochemicals in their liquid or vapour, unboundform. Tea tree oil delivered into the cavities of prostaticabscesses in dogs in place of aspirated purulent matter resultedin the disappearance of the purulent matter in the cavities and amarked reduction in the volume of the cavities.178

The ability of using these antimicrobials for site-specicapplications, such as in release-based or non-leachingsurfaces remains largely undiscovered. A range of polymercoatings based on ultra-high molecular weight polyethylene,very high molecular weight polyethylene and latex compoundsand incorporating a wide range of biocidal phytochemicalagents, alone and in combinations, have been proposed, with


primary area of application beingmarine paints and coatings.179

In another patent, anti-fouling coating composition containingcapsaicin were proposed, although these were not designed formedical implantation applications.180 A polymer system loadedwith a variety of phytochemicals, phytonutrients, and chemicalreleasers has also been designed to inhibit the growth ofpathogenic bacteria associated with packaged foodstuff.181

Recently, a number of antibacterial coatings containing curcu-min have been developed. Sodium carboxymethyl cellulosesilver nanocomposite lms were loaded with curcumin bydiffusion mechanism, with higher encapsulation of the agentobserved in the lms with higher cellulose content.182 Silvernanoparticles also enhanced the encapsulation of curcumin,suggesting a degree of interaction between these two antimicro-bials. The synergistic effect between silver nanoparticles andcurcuminwas also observed in the antimicrobial activity against E.coli, with the activity being superior to either silver- or curcumin-only lms. Sustained release and sound antibacterial efficacy wasalso observed for silver/curcumin-containing hydrogels based onpoly(acrylamide)/poly(vinyl sulfonic acid sodium salt)183 and thosebased on chitosan–poly(vinyl alcohol) lms.184

Although promising, the aforementioned strategies relied onthe use of other polymers or chemical substances to produce a

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Fig. 11 SEM images of attachment and proliferation of Streptococcusepidermidis (left panel) and Staphylococcus aureus (right panel) after18 h incubation on surfaces subjected tomonoterpene alcohol plasmadeposition under varied input power conditions: (A and B) 10W; (C andD) 50W. Scale bar¼ 2 mm; 20 mm (inset).190 (E) Surface area covered byEscherichia coli biofilm formed on plasma polymerised 1,8-cineole(ppCo) and hydrophobic (ppOct) and hydrophilic (glass) controls.Samples were immersed in bacterial culture for 5 days (***p < 0.001,**p < 0.01, *p < 0.1).191 Reproduced with permission from ref. 190and 191.

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coating. Using non-equilibrium, low-temperature plasma poly-merisation, Jacob and Bazaka and their colleagues demonstratedthe possibility of producing solid polymer lms exclusively fromessential oils, including M. alternifolia and Lavandula angustifo-lia essential oils and their individual constituents.141,143 Fabri-cated over a wide range of processing parameters, these lmsvaried in terms of chemical composition, surface morphology,stability and mechanical properties, while displaying uniformcoverage and sound adhesion to a variety of substrates,including metals, ceramics, and polymers.185,186 Polymers fabri-cated from M. alternifolia oil and its major antimicrobialcomponent terpinen-4-ol was demonstrated to be cytocompat-ible with a number of host cells. In combination with biologicalactivity, their attractive optoelectronics properties maketerpinen-4-ol lms as potential candidates for inclusion inimplantable electronics, where they can be used as both thedevice components and protective encapsulating layers.144,187–189

Films fabricated at conditions that favoured preservation oforiginal functionalities of the monomer via limited

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fragmentation and incorporation of unfragmented species intothe polymer matrix were able to retard attachment and coloni-sation by such bacteria as P. aeruginosa, S. aureus, S. epidermidis,and E. coli.141,142,192 Fig. 11 shows the attachment preferences oftwo pathogens, S. aureus and S. epidermidis to polymers fabri-cated at three different input power levels.190 Surfaces rich inoxygen containing functional groups, particularly –OH, werecharacterised by higher antifouling and biocidal activitycompared to more hydrocarbon dense coatings. In addition toavailability of specic functionalities at the surface, it has beenspeculated that unfragmented monomer trapped within thepolymer during deposition may be eluting over time, thuscontributing to inhibition of biolm formation at the polymersurface. It is believed that just

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