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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
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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
This journal is © The Royal Society of Chemistry 2015
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.
48742 | RSC Adv., 2015, 5, 48739–48759
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
This journal is © The Royal Society of Chemistry 2015
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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.
This journal is © The Royal Society of Chemistry 2015
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.
48744 | RSC Adv., 2015, 5, 48739–48759
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.
This journal is © The Royal Society of Chemistry 2015
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
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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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4.
View Article Online
http://dx.doi.org/10.1039/c4ra17244b
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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
RSC Advances Review
Publ
ishe
d on
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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
This journal is © The Royal Society of Chemistry 2015
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
48750 | RSC Adv., 2015, 5, 48739–48759
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
This journal is © The Royal Society of Chemistry 2015
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
48752 | RSC Adv., 2015, 5, 48739–48759
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
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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
This journal is © The Royal Society of Chemistry 2015
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