Low pressure plasma methods for generating non-reactive hydrophilic and hydrogel-like bio-interface coatings

Review

Low-Pressure Plasma Methods for GeneratingNon-Reactive Hydrophilic and Hydrogel-LikeBio-Interface Coatings – A Review

Kim S. Siow,* Sunil Kumar, Hans J. Griesser

This review surveys low-pressure plasma-based m
ethods for producing hydrophilic andhydrogel-like bio-interface coatings without reactive functional groups in aqueousmedia. Themain focus of the review is one-step plasma polymerization; other plasma-based methods such as plasma with grafting are also discussedwithin the context of monomers used, processdevelopment, ageing properties, and interaction ofthese coatings with proteins and cells. Coatingscontaining polyethylene glycol (PEG) or polyethyl-ene oxide (PEO), acrylamides such as N-isopropyla-crylamide (NIPAM), and sulfonate (SO3) or sulfate(SO4) moieties are reviewed here.
K. S. Siow, S. Kumar,þ H. J. GriesserIan Wark Research Institute, University of South Australia,Mawson Lakes, SA 5095, AustraliaK. S. SiowInstitute of Microengineering and Nanoelectronics, UniversitiKebangsaan Malaysia, Bangi 43600, Selangor D.E., MalaysiaE-mail: [email protected], [email protected]. J. GriesserMawson Institute, University of South Australia, Mawson Lakes,SA 5095, AustraliaþPresent address: Coatings Mantra Science and TechnologyConsulting 11 Beresina Place, Greenwith, Adelaide SA 5125,Australia.

Plasma Process. Polym. 2014, DOI: 10.1002/ppap.201400116

� 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com

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CPS

174 172 170 168 166 164 162 160 Binding Energy (eV)

1,7 Octadiene-SO2 pp

S-S, S-C, S-H (3/2)

HA-SO2 pp

SO2, SO3 (3/2)

-O-SO3 (3/2) -O-O-SO3 (3/2)

1. Introduction and general term ‘‘biocompatibility’’) for the intended

The control of interfacial interactions between synthetic

materials and biological entities, such as proteins, cells,

or tissue, is of considerable interest for a number of

biomedical, bio-diagnostic, and biotechnological applica-

tions. It is often challenging to design new synthetic

materials such that they meet the ‘‘bulk’’ material require-

ments such as strength, elasticity, transparency as well as

the ‘‘surface’’ requirements of controlled bio-interfacial

interactions (often summarized under the rather vague

application. It has therefore become a popular strategy to

separate the two classes of requirements by fabricating

devices that contain a bulk material that meets the

mechanical and other bulk requirements, and either a

surface modification or a coating that governs the bio-

interfacial interactions.

Plasma-based approaches have attracted considerable

attention for the versatile and facile creation of bio-

interfaces. For some 20 years, a substantial body of

literature has reported a wide variety of approaches

utilizing many different plasma process vapors and

approaches. A popular strategy has been the use of

plasma-fabricated surfaces that carry reactive surface

groups, such as amines, followed by the subsequent

immobilization by a conventional chemical reaction of

a biologically active molecule. The literature on such

‘‘reactive’’ plasma surfaces and examples of bioactive

molecules, mostly proteins, immobilized thereon has been

reviewed in ref.[1] Other strategies have aimed to create

‘‘passive’’ or protective surfaces or coatings; such surfaces

are intended not to be able to participate in covalent

interfacial reactions with biological entities in aqueous

environments; instead, their purpose is the control of bio-

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Kim S. Siow, Ph.D. C.Eng. MIMMM is a ResearchFellow/Senior Lecturer at the Institute of Micro-engineering and Nanoelectronics (IMEN), theNational University of Malaysia. His researchinterests are related to surface modification usingplasma technologies for biomaterials, adhesion,and microfluidic applications, and joining technol-ogies using sintered silver. Prior to joining IMEN, hehas worked as a materials engineer at multi-national companies and the National University ofSingapore. His numerous publications receivemore than 470 citations, and his h-index is 7.

Sunil Kumar currently directs an independentscientific consulting business CoatingsMantrabased in Adelaide, Australia. Prior to directingCoatingsMantra, he held a professorship at theUniversity of South Australia, Adelaide. His re-search and consultancy are centred on investigat-ing plasma-based methods for depositing andprocessing biomedical coatings and biomaterials.Kumar’s peer-reviewed research publications haveattracted around2000 citations,withh-index�25.

Hans Griesser is Professor of Surface Science andDirector of the Mawson Institute at the Universityof South Australia, located in Adelaide, Australia.WithaPhDinphysical chemistry,hehasspecialisedin polymer surface science and engineering,supported by research in surface analysis method-ologies. His main interest is in plasma polymercoatings forbiomedicalandprotectiveengineeringapplications, and in using plasma polymers asinterlayer coatings for immobilising bioactive

K. S. Siow, S. Kumar, H. J. Griesser

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interfacial interactions via their physico-chemical surface

properties and surface forces. Moreover, some approaches

are specifically designed for minimal bio-interactiveness,

thereby aiming to produce surfaces that do not possess

attractive interfacial forces toward proteins and thus

should be ‘‘non-fouling.’’

It is these non-reactive, bio-passive surfaces that are

the subject of this review,which is intended to complement

and extend shorter, selective earlier reviews of plasma

approaches to bio-interfaces.[2–6] We aim to review the

literaturenot forcompleteness,whichwouldbe impractical

given the large body of literature and result in much

duplication, but, instead, with the purpose of highlighting

and discussing some of the main approaches toward

surfaces thatdonotengage incovalent reactions inaqueous

media but, instead, control interfacial interactions via

surface properties and interfacial forces. For those prefer-

ring the language of wettability, many of the approaches

produce surfaces that are either highly hydrophobic or

highly hydrophilic and thereby affect contacting biological

media or entities accordingly.

While low-pressure gas plasmamethods have been used

in biomaterials research for several decades, the use of

atmospheric pressure plasmas is more recent. Some

considerations are common to both whereas others differ;

such comparisons would be of interest but add a consider-

able volume of text. For the sake of keeping this review to

within manageable size and cohesive content, we will

concentrate on low-pressure plasma methods and will

leave it to leading researchers in atmospheric pressure

plasmas to review analogous literature.

In this review, we will concentrate on hydrophilic

coatings produced by various low-pressure plasma tech-

nologies. Among the hydrophilic non-reactive surfaces,

much interest has centered on plasma polymer coatings

that are intended to mimic the chemical composition and

properties of poly(ethylene oxide) (PEO) graft layers. PEO

hasbeenofparticular interest asaprotein-repelling coating

or graft layer.[7] Using suitable volatile monomers and

plasma conditions, PEO-like plasma coatings have been

fabricated for the purpose of achieving non-fouling coat-

ings. Similarly, the thermo-responsive polymer poly(N-isopropylacrylamide) (pNIPAM), which is protein repellent

in its hydrated state, has inspired work on producing

plasma NIPAM (ppNIPAM) coatings.

Amoreconventionalapproach,used inmanystudies,has

been the activation of a bulk material by plasma surface

treatment or plasma polymerization followed by solution

chemical grafting of PEO molecules to create a fouling-

resistant steric barrier layer. Due to the large number of

reports and the similarity of many approaches, we will

review this particular area selectively and only discuss

some of the approaches. Another biologically relevant

surface chemistry that will be reviewed is sulfur-based


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plasma polymers, which can be produced by commonly

employed plasma-based techniques.

2. PEO-Like Plasma Polymers

PEO coatings have shown considerable promise as steric

barrier layers for the purpose of preventing the adsorption

of proteins and cells.[7,8] Typically, such PEO graft layers

are produced by interfacial covalent attachment of end-

reactive PEO chains onto a suitably reactive polymer

surface, for instance a reactive plasma polymer, as

discussed in Section 3.2 below. The success of such

strategies in producing non-fouling surfaces stimulated

research into ways of producing analogously adhesion-

resistant surfaces by a one-step plasma approach and

hence, the name PEO-like coating is used in this review.

2.1. PEO-Like Coatings by Plasma Polymerization

2.1.1. Types of Monomers

The plasma-based approaches for fabricating PEO-like

coatings utilize volatile monomers that contain the

DOI: 10.1002/ppap.201400116

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Low Pressure Plasma to Generate Non-Reactive Hydrophilic Coatings

structural element —CH2CH2O—. An early attempt to

fabricate PEO-like surfaces involved the plasma polymeri-

zationofethyleneoxideontoglass, polytetrafluoroethylene

(PTFE) and polyethylene (PE) substrates using a liquid

nitrogen cooled electrode.[9] Although PEO-like XPS spectra

were obtained, non-uniformities across the sample and

signals from the substrates were also observed. Lopez and

Ratner[9] attributed the non-uniformity to the ‘‘relatively

slow rate of polymerization of the precursor compared to

the condensation rate’’. Rapid condensation of monomer

led to trapping of unreacted monomer inside the plasma

polymer, and thesemonomermolecules then volatilized at

ambient temperature. Oehr et al.[10] obtained similar

results when argon gas was added to the plasma

polymerization of ethylene oxide on siloxane precoated

substrate. Earlier, Inagaki and Suzuki[11] had demonstrated

that the surface energy of ethylene oxide plasma polymer

was very sensitive to theW/FM ratio, as assessed by curve-

fitted C1s XPS spectra (note: Power, W; flow rate, F;monomer molecular weight, M).

Others used ethylene glycol as monomer to produce a

PEO-like coating.[12,13] Similar to ethylene oxide-derived

plasma polymers, principal component analysis of ethyl-

ene glycol-derived plasma polymers confirmed that low

power deposition at 2W produced PEO-like coatings while

higher power deposition, between 5 and 20W, produced a

higher degree of aliphatic hydrocarbon nature.[12] Another

approach for producing PEO-like coatings is to thermally

degrade poly(ethylene oxide) into a plasma glow discharge

chamber.[14,15]

Most researchers have used ether-containing mono-

mers,[16–23] which are also commonly known as

glymes,[24–28] to formPEO-like coatings by plasmapolymer-

ization.Argonplasmatreatmentwasoften carriedout, prior

to plasma polymerization of glyme chemistry, to clean the

substrate and increase the density of interfacial bonds

Figure 1. XPS C1s signals recorded with plasma polymers from lindioxane, and crown ethers (reprinted with permission from ref.[26

American Chemical Society).



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between the polymeric substrate surface to enhance

adhesion of PEO-like plasma polymers.[17,24,29]

Thechoiceof theglyme-basedmonomersand theplasma

power density can affect the chemistry of the coatings.

When dioxane was used to produce plasma polymers, the

dominantmoietieswere hydroxyl groupswhilemonomers

such as monoglyme, diglyme, triglyme, and tetraglyme,

12-crown-4-ether and 15-crown-5-ether, produced a

more PEO-like structure.[25,26] Plasma polymers produced

from linear oligoglyme had better resistance to protein

adsorption than those deposited from cyclic monomers

even though the percentages of the C—O component in

the XPS C1s signals were quite similar (Figure 1).[26]

This findingwas attributed to differentmolecular arrange-

ments and mobility of the fragments at the outermost

regions of the various plasma polymers.[26] Similar results

were obtained by others, although it was not highlighted

in their research, which focused on bacterial attachment

and biofilm formation.[22]

The growth mechanisms of these glyme or PEO-like

coatings can be linked to the degree of saturation of the

monomers.[30] Unsaturated glyme monomers such as

diethylene glycol divinyl ether (DEGDVE) produced frag-

ments that can graft directly to surface radicals on the

plasma polymers in addition to the usual growth mecha-

nism of accumulation of incoming ions and radicals onto

the plasma polymers.[30]

2.1.2. Discharge Power

The formation of PEO-like structures from glyme or

ether containing monomers is favored at low values

of W/FM.[8,16–18,28,29,31] The XPS C1s peaks of PEO-like

coatings showedthathigherW/FMvaluesproducedplasma

polymerswith higher hydrocarbon content[28,29,31] and also

increased presence of carbonyl bonds.[17] These character-

ear oligoglymes,] Copyright 2005

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istics suggested increasedmonomer frag-

mentation; a conjecture that was con-

firmed by actinometric optical emission

spectroscopy studies with triglyme.[28] In

pulsed plasma polymerization, a longer

off time reduced fragmentation of the

monomerandallowed theethyleneoxide

group to be tethered more effectively to

the growing coatings.[17,20] This result

was analogous to the pulsed plasma

polymerization of allyl amine and allyl

alcohol, where the amounts of NH2[32]

and OH[33] groups increased with longer

off times. In the case of continuouswave

plasma polymerization of ether-based

monomers, increasing the deposition

time was shown to reduce the surface

ether component of these plasma

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polymers because of the higher degree of monomer

fragmentation for the longer polymerization times.[34]

2.2. Bio-Interfacial Performance of PEO-Like Coatings

As expected, PEO-like plasma polymer coatingswere found

toexhibit reducedadsorptionofproteins suchasfibrinogen,

albumin, and g-globulin compared to uncoated substrates

during in vitro studies.[10,17,20,24,26] However, the term

‘‘non-fouling’’ needs to be qualified with regard to the

sensitivity of the analytical technique employed. The

absence of an observable nitrogen peak in XPS analysis

can be interpreted as corresponding to less than 10ng cm�2

of adsorbed protein, while TOF-SIMS gives a sensitivity of

0.1 ng cm�2 of adsorbed fibrinogen depending on the

chemistry and type of the substrate and the organization

of adsorbed protein.[35] Another commonly accepted

standard for protein resistance is less than 5 ng cm�2 of

fibrinogen; this limit will prevent platelet activation in

static or flow conditions.[36] A good correlationwas found

between the percentage of ether content and protein

resistance; higher ether content will reduce protein

adsorption.[23,34] From curve-fitted C1s spectra, it was

concluded that aminimumof 70%of ether (assuming that

all the C—O component intensity is from ether groups)is

needed to prevent cells such as HeLa or fibroblasts from

attaching to these surfaces[37–39] though resistance to

protein is effective at 65% ether.[14]

2.2.1. Protein Characteristics

Besides the morphology of PEO coatings summarily

described earlier in Section 2.1.1, protein sizes and charges

can influence their adsorption behavior on PEO-like coat-

ings because of hydrophobic and electrostatic interactions

between proteins and surfaces.[23] Studies to analyze the

influence of electrostatic properties of PEO-like coatings

on protein adsorption can sometimes be confounded by

the presence of acidic groups on these coatings.[23] Other

mechanisms such as hydrophobic interactions have been

proposed to explain unexpected results such as higher

adsorption of negatively charged albumin than positively

charged lysozyme on PEO-like coatings.[23] The preferential

adsorption of positively charged polymers such as poly-

lysine on O2 modified PEO-like coatings has been exploited

to culture cells in a biochip.[40]

Plasma (protein) concentration also plays a role in

determining the non-fouling properties of PEO-like coat-

ings.[36] Unlike many synthetic surfaces that show a

Vroman effect (i.e., maximum protein adsorption occurring

at lower plasma concentration), PEO-like coatings from

tetraglyme showed a progressive increase of protein

adsorption with a maximum fibrinogen adsorption of

85ng cm�2whenexposedto100%plasmaconcentration.[36]




However, the highest fibrinogen adsorption does not

translate to highest platelet adhesion because of the lower

potency of densely adsorbed fibrinogen compared to that

of a lower density adsorbed layer.[36]

2.2.2. Cell Adhesion Studies

In cell adhesion studies, Lopez et al. showed that PEO-like

coatings created by plasma polymerization of tetraglyme

reduced the attachment of bovine aortic endothelial cells

to negligible levels and reduced the dynamic platelet

adhesion invitro.[24]However, caution isnecessarybecause

platelet activation by transient surface contact might not

be detected in dynamic platelet adhesion tests. Scanning

electron microscopy imaging is normally used to visualize

adhered platelets, though this technique may not be a

suitablemethod because of possible loss of platelets during

sample preparation and inability to detect non-adhering

activatedplatelets. Cell adhesionstudiesarealso influenced

by physical properties such as elasticity of the substrate

and flexibility of side chains,[41] which has not been

thoroughly investigated in early work.

Similarly, monocyte adhesion correlated linearly with

the amount of adsorbed protein.[42] While protein

adsorption and platelet adhesion could be correlated

with the content of ethylene oxide structures,[17,26,42,43]

bacterial adhesion depended on the outermost structure

of the plasma polymer in in vitro tests.[25] Others

showed significant reduction in biofilm formation and

adhesion of the bacterium Listeria monocytogenes[31,44]ormixed culture of Salmonella typhimurium, Staphylococ-cus epidermis, and Pseudomonas fluorescens[22] on PEO-

like surfaces. The reduction of protein adsorption likely

leads to reduction in bacterial colonization, though

there are other established mechanisms of bacterial

attachment.

However, these positive results of in vitro protein

adsorption and platelet adhesion tests failed to translate

to in vivo situations. Leukocyte adhesion to plasma

polymerized tetraglymewasmore severe than tountreated

FEP when samples were implanted into mice for four

weeks.[45] The same polymerized tetraglyme when pre-

adsorbed with fibronectin, fibrinogen, or blood plasma, or

tetraglymeplasmapolymerperse, showed lowermonocyte

adhesion than untreated FEP after 1 h and even 2d of in

vitro testing.[46] A further investigation suggested that

while complement component 3 (C3) adsorptionmay be as

low as 20ng cm�2, the retained C3 adsorbed strongly to the

PEO-like coating to produce complement activation.[47]

Similarly,polyethyleneglycol (PEG)graftedontoallylamine

polymers also failed to translate to good in vitro results

to contact lenses worn in a clinical study.[48]

Such differences between in vivo and in vitro test results

are common and probably reflect the complexity of real

DOI: 10.1002/ppap.201400116



biological systems that are very difficult to mimic in vitro,

thus preventing testing of all relevant interfacial inter-

actions. In the case of plasma polymerized tetraglyme, a

likely scenario is leukocytes secreting oxygen radicals to

degrade the tetraglyme plasma polymer coating and

therebymediate adhesionof leukocytes in vivo.[45] Another

likely scenario is increased biological activity of leukocyte

cells in a localized area that was not covered by the PEO

coating, because of the inability of protein to spread

evenly onto the substrate. This increased biological activity

resulted in higher leukocyte adhesion on tetraglyme

plasma polymers compared to the untreated substrate.

The amount of adsorbed fibrinogen to activate platelet

adhesion was less than 10ng cm�2 [49] but the amount of

protein to activate leukocyte adhesion in vivo is likely to

be lower than 10ng cm�2.[45]

2.3. Stability and Aging of PEO-Like Coatings

A main concern that arises particularly with plasma

polymers deposited at very low power levels, or low on-

off duty cycles when pulsing, is the solubility of material

from the plasma polymer in solvents. Another concern is

post-plasma oxidative reaction cycles that lead to changes

in surface compositions and properties.[50] Both issues are

well understood in principle, but the extents to which

they occur vary considerably not only with monomer and

deposition conditions but also with the plasma reactor

geometry and post-plasma environmental conditions.

The ability of solvents to extract material from plasma

polymers reflects the presence of relatively low molecular

weight components in the broad molecular weight

distribution of material that makes up the plasma

polymer[15]; in contrast to early assumptions plasma

polymers do not necessarily consist of a highly crosslinked

3D network of essentially infinite molecular weight.[14]

The stability of PEO-like plasma coatings in liquids has

been reported to be satisfactory.[17,20,24,31] Lopez et al.

soaked tetraglyme plasma polymers for one month in

water and did not detect any changes except for some

initial dissolution during the first minute of soaking[24]

although other types of PEO-like coatings showed a

reduction of their density,[38] which was likely caused by

water absorption.[51] There were no changes in the

chemical structures detectable by curve-fitting of XPS C1s

spectra within 5 d of incubation in water at 25 8C.[38]

Similar results were reported with other coatings and

testing conditions, including solvents such as acetone,[38]

DMSO,[17] and phosphate buffered saline (PBS)[20,52] though

with different soaking times.

In the pulsedplasmapolymerization of diethylene glycol

vinyl ether, the applied peak power influenced the stability

of the samples.[43] During 15d of soaking in PBS at 37 8C,the plasmapolymer deposited at 200Wpeak power did not




show any detectable changes while samples deposited at

50W peak power showed reductions of C—O components

with continuous washing in phosphate buffer solutions

with flow.[43] This result suggests that a minimum power

density must be used to achieve a sufficient density of

crosslinks in the plasma polymer to minimize the leaching

of low molecular weight material. Similar results on the

influenceofdischargepoweronaqueousstabilityofplasma

polymerized ‘‘glyme’’ have also been reported.[14,53]

PEO-like plasmapolymerswere also reported to be stable

upon three months of shelf storage, as assessed by the

absence of fibrinogen on this surface.[52] Another study on

PEO-like coatings showed that the ability to inhibit biofilm

formation was similar after 60 d storage in air compared

with that of freshly deposited coatings.[31] However, these

observations that the performance of the plasma polymers

was invariantwith timedonotmeanthatonecan infer that

their chemical composition also was invariant with time;

chemical changes might just not translate to altered bio-

interfacial responses. For many plasma polymers, it is

known that that study did not mention that the use of

low powers or pulsing leads to very low densities of

trapped radicals react with in-diffusing oxygen post-

plasma. The relatively soft nature of PEO-like films

suggests that they may have more internal segmental

mobility and thus radicals incorporated during deposition

might be sufficiently mobile to find another radical

relatively quickly and thus dissipate before significant

oxygen in-diffusion and addition takes place. However,

the issue should be investigated since PEO is known to

be very susceptible to degradation by radical reactions,

and hence trapped radicals might have detrimental effects

on PEO-like plasma coatings.

In more aggressive environments such as dry heat or

autoclaving, commonly used for sterilization, the PEO-like

coating exhibited reduction in thickness, lower concen-

trations of C—O—C ether groups in the coating, and

reduced ability to repel cell adhesion,[54] though the

correlation between the concentration of C—O—C and

resistance to cell adhesion was not clear because of

changes in the physical properties of the coating. This

aggressive environment is likely to cause faster diffusion

of the lower molecular weight oligomers containing

higher amounts of the C—O—C ether group than the

more crosslinked segments. On the other hand, UV

radiation did not produce any differences in thickness,

cell adhesion or C—O—C concentration compared to the

unsterilized PEO-coating.[54]

Air/water contact angle measurements may not be a

good way to detect any ageing behavior or other relevant

bio-interfacial factors; as plasma polymerized di(ethylene

glycol)dimethyl ether coatings produced by different

discharge powers showed different behaviors in resisting

protein adsorption although the coatings had similar


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air/water contact angles.[51] It has been shown that there is

no correlation between air/water contact angles and

proteinadsorption[34] orbacterial attachment[22] onplasma

polymerized ‘‘glyme’’ coatings.

In summary, it is clear that plasmapolymerization allows

thegenerationofPEO-likecoatingsthatshowhighresistance

to adsorption of proteins and attachment of mammalian

cells aswell asmicrobes. Optimal coatings require, however,

very low plasma power levels in order to maintain a high

extent of linear oligo-ethylene oxide structural elements,

and such low power levels lead to slowdeposition rates. It is

also clear that low plasma power levels lead to higher

amounts of lower molecular weight material that leaches

fromtheplasmapolymer inaqueoussolutions. It is therefore

unclear at present whether plasma polymerization of PEO-

like coatings is an industrially viable alternative to the rich

assortment of reported PEO graft coatings. While grafting

generally requires two processing steps, as opposed to the

single step of plasma polymerization of PEO-like coatings, a

number of practical considerations including process

efficiency, coating reproducibility and stability, and others,

will ultimately determine the selection of a suitable coating

methodology for specific products.

3. Grafting of PEO Layers onto PlasmaSurfaces

As shown in Figure 2, there are two main plasma grafting

methods; they are direct irradiation and post-irradiation,

respectively, to tether ethylene oxide groups to plasma

treatedorpolymerizedsurfaces. Thesegraftingmethodsare

discussed in the following paragraphs.

3.1. Plasma Grafting (Direct Irradiation)

In the ‘‘direct irradiation’’ technique, alkyl PEO com-

pounds are coated onto a substrate prior to argon

Plasma grafting (post irradiation): (1) PEGMA grafting after Ar plasma treated PU

(2) PEG grafting after water plasma treating the layer of deposited silica (3) PEO grafting after treating the amine/ hydroxyl coated PET with cyanuric chloride

(4) methoxy-PEG or dialdehyde PEG grafting to aminated FEP or PET (5) amino-terminated PEG to aldehyde coated FEP

Polyethylene Glycol- Polyethylene Oxide (PEG-PEO)

Plasma polymerization:ethylene oxide, ethylene glycol

and glyme family

Plasma grafting (direct irradiation): Ar plasma treatment on preadsorbed (1) oleyl

surfactant (2) PEO-Polypropylene oxide (PPO)- PEO (3) Poly(ethylene glycol) methyl ether

methacylate (PEGMA) (4) alkyl PEO

Figure 2. Plasma-based techniques to create PEO-like and PEGcoatings.




plasma treatment to ‘‘stitch’’ and crosslink segments

of PEO polymers to the substrate.[44,55–60] Often, Ar

plasma was used to achieve this covalent ‘‘stitching,’’

but other studies showed that PEG[61] could also be

directly immobilized onto e-PTFE in atmospheric pressure

glow discharges.

The exact mechanisms of immobilization of PEG to the

substrate are the subject of research. In one study, it has

been shown that argon plasma ‘‘stitched’’ and crosslinked

thehydrophobic PPO segments of thepoly(ethylene oxide)/

poly(propylene oxide)/poly(ethylene oxide) (PEO–PPO–

PEO) surfactants to the substrate.[55,56] Similar to immobi-

lization of PEO–PPO–PEO, alkyl PEOs of different lengths

or terminal groups such as hydroxyl, carboxy, and sulfate

(SO4) were also immobilized onto PE substrates via the

hydrophobic alkyl chain.[60] In another study, it has been

speculated that the formation of propylene centers from

C—O and C—C radicals of neighboring PEG molecules

led to dense crosslinking, which prevented dissolution of

PEG coatings on a silicon substrate.[58] Others proposed

that another alkyl PEG, polyethylene glycol methyl

ether methacrylate (PEGMA),was grafted onto a silicon

substrate via the carbon–carbon double bond instead

of cleavage and recombination of C—O—C functional

groups.[59]

3.2. Plasma Grafting (Post-Irradiation)

Compared to the direct irradiation approach, the post-

irradiation approach immobilizes PEG moieties via con-

trolledcovalent interfacial reactionsontoplasma-treatedor

plasma-polymerized substrates.[10,18,19,62–68] PEGMAcanbe

used for grafting onto a substrate in direct irradiation, as

described above, as well as with the post-irradiation

method; for the latter, PEGMA was grafted onto Ar

plasma-treated polyurethane (PU) utilizing radicals created

on the polymer surface.[62] Oehr et al.[10] also used argon

plasma treatment to immobilize ethylene oxide onto

siloxane plasma polymer substrates. Besides Ar plasma

treatment, oxygen plasma treatment has also been used to

generate peroxide radicals on polymeric substrate surfaces,

which can then be used to immobilize PEG by surface-

initiated simultaneous normal and reverse atom transfer

radical polymerization (s-ATRP) of poly(oligo(ethylene

glycol)methacrylate).[69]

Another approach utilized water plasma treatment to

increase the amount of silanol groups on deposited

amorphous silica to react with terminal —OH groups of

PEG.[63] Similar to the silanol group, hydroxyl groups on

substrates can be used to tether PEO moieties though other

chemistries such as cyanuric chloride[64] or with cerium

ammonium nitrate[68] as a catalyst to initiate the grafting.

In addition to peroxide and hydroxyl groups, amine

group can beused for grafting PEGmoieties.[64–66] Gombotz

DOI: 10.1002/ppap.201400116



et al. activated amine-coated PET with cyanuric chloride

before reacting with bis-amino PEO.[64,65] Others used

reductive amination to graft methoxy-terminated alde-

hyde PEG[18,19,66] or dialdehyde PEG[66] to amine plasma

polymer surfaces, which produced highly effective fouling-

resistant PEG graft layers. The converse chemistry scheme

has also been explored, where amino terminated PEO

was grafted onto an aldehyde plasma polymer by Schiff

base formation and reductive amination with sodium

cyanoborohydride.[67] Another alternative is to graftmono-

amino PEO on plasma polymers of acrylic acid.[70]

3.3. Stability and Aging of PEO Graft Coatings

Little information is available on the ageing behavior of PEO

graft coatings; available data suggest that they are sufficient-

ly stable for up to eight months[46] in air and manifest no

significant changes in air/water contact angles[62] (however,

air/water contact angles may not be a reliable technique

to monitor ageing behavior, as discussed in Section 2.3).

More detailed studies using appropriate combinations of

surface analysis techniques, such as XPS, FTIR, and ToF-SIMS,

are needed to verify chemical stability upon storage.

4. Acrylamide Plasma Polymers and TheirDerivatives

This section focuses on acrylamide-based coatings, with

primary interest on N-isopropylacrylamide (NIPAM) coat-

ings produced using plasma-based methods. Another

common acrylamide derivative, which will be briefly

mentioned is N,N diethylacrylamide (PDEAAm).

Plasma grafting (direct irradiation) Argon plasma treatment on precoated

N-Isopropylacrylamide with PEG as side groups

Plasma grafting (post irradiation): Argon plasma treatment before grafting with

N-Isopropylacrylamide

N-Isopropylacrylamide (NIPAM)

Plasma polymerization: N-Isopropylacrylamide

Figure 3. Plasma-based techniques to create pNIPAM coatings.

4.1. Acrlyamide Plasma Polymers

Primary interest in acrylamide coatings stems from their

useas separationmembranes in contactwithbloodorother

heterogenous phases such as mixtures of organic liquids.

Although acrylamide can be plasma polymerized readi-

ly,[71] in most bio-interface studies acrylamide coatings

weremade via grafting, by pre-treating polymeric surfaces

with argon plasma or oxygen plasma, followed by

immersion in acrylamide solution[72–75] or vapor.[76] The

reason for the predominance of grafting is the ability to

produce linear chains, as opposed to the crosslinked, less

hydrophilic and less hydrogel-like nature of acrylamide

plasma polymers. The use of vapor phase grafting has two

advantages over liquidphasegrafting:higher graftingyield

and absence of acrylamide homopolymer formation. The

solution and vapor phase grafting techniques are similar to

thosediscussed aboveasplasmagrafting (post-irradiation).

It has also been claimed that acrylamide can be grafted to

mulberry silk via plasma grafting (direct irradiation).[77]




The silk was immersed in aqueous acrylamide solution

before air plasma treatment, which activated the acrylic

bonds to achieve crosslinking.

4.2. N-Isopropyl Acrylamide Plasma Polymer

(ppNIPAM)

Due to their thermo-responsive behavior, pNIPAMcoatings

have shown potential for application as substrates for

growing cell sheets and in drug release and actuators.[78]

Similar to the lower critical solution temperature parame-

ter in linear polymers, pNIPAM hydrogels swell in aqueous

solutions below the volume phase transition temperature

(VPT). Above the VPT, some of thewater is expelled and the

hydrogel collapses because attractive intermolecular forces

arising from hydrophobic side groups of NIPAM overcome

hydrogen bonds between water and hydrophilic groups in

pNIPAM. This transition temperature can be tailored to

human physiological temperature; it depends on fabrica-

tion routes, parameters,[79] and typesof co-monomers.[80,81]

These factors will be discussed next.

4.2.1. Plasma Polymerized ppNIPAM

As shown in Figure 3, plasma polymerization[79,82–85] and

plasma grafting (post-irradiation[80] and direct irradia-

tion[81,86]) have emerged as three main plasmamethods to

produce NIPAM coatings. The plasma polymerization

method suffers from low volatility of the monomer, which

requires extreme care to avoid extraneous other gases such

asoutgassingwatervapor, andcausesavery lowdeposition

rate and difficulties in obtaining a stable plasma. However,

this can be mitigated somewhat by heating the NIPAM

monomer to above 70 8C,[79,84] but this is experimentally

demanding as it requires heating of the entire system to

isothermal conditions so as to avoid condensation. In order

to obtain good adhesion to the substrate, high discharge

power (close to 80W) was used at initial stages of the




8

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plasma polymerization, followed by progressive reduction

to low discharge power (to 1W) to impart thermo-

responsive behavior to the NIPAM plasma polymer

coating.[84] At high discharge powers, nitriles and imines

were detected while the quantity of amide groups was

reduced, attesting to significant molecular fragmenta-

tion.[79] The retention of NIPAM monomer structure at

low discharge power was confirmed by various analytical

tools such as ToF-SIMS,[83] XPS, and near-edge X-ray

absorption fine structure spectroscopy (NEXAFS).[79] A

similar thermo-responsive coatingwas reported for a lower

chamber temperature of 45 8C during deposition.[79] This

feature is likely caused by the different sticking coefficients

of the species present in the plasma, which have different

temperature dependences and consequently result in

different surface chemistries.[79]

An alternative approach to using low discharge power is

to use pulsed plasma polymerization of NIPAM, which led

to good thermo-responsiveness at duty cycles of 60ms.[87]

Figure 4 shows that the XPS C1s signal of pulsed NIPAM

plasmapolymer closely resembles theoreticalNIPAMwhile

continuously deposited NIPAM plasma polymer shows

structural differences due to fragmentation and rearrange-

ment of themonomer.[87] Such lowduty cycleswill result in

substantial lengths of linear polymer structural elements

Figure 4. XPS C1s signal of (a) conventional pNIPAM (b) pulsedNIPAM plasma coating (35W, ton¼60ms, toff¼ 2ms and (c)continuous wave NIPAM plasma coating (35W) (reprinted withpermission from ref.[87] Copyright 2005 American Chemical Society).




and reduced cross-linking, which provides an increased

degree of freedom for the polymer chain to change

conformation fromproteinadsorption toprotein resistance

across the transition temperature.[87]

Plasma polymers from NIPAM were shown not to be

cytotoxic in direct contact with cells.[88] However, in all

cases there is a tradeoff between deposition rate and the

desirable hydrogel quality of the NIPAM plasma polymer

coatings. The best coatings require very low power or

pulsing, both of which lead to very slow deposition rates. It

isnot clearwhetherNIPAMplasmapolymercoatingscanbe

produced in an industrially viable manner compared with

pNIPAM graft coatings.

4.2.2. Plasma Grafting (Post-Irradiation and Direct

Irradiation)

In the case of post-irradiation, the grafting of NIPAM

molecules is normally preceded by argon plasma treat-

ment.[80,89,90] If diethyleneglycol methacrylate (DEGMA) is

added as co-monomer, the transition temperature of the

coating can be increased from 32 8C to just below the

physiological value of 37 8C[89] andwith shorter duration of

incubation for cell detachment[90] than with pure NIPAM

grafted plasma polymer.

In the direct irradiation method, surfaces are pre-coated

with various derivatives of NIPAM molecules before

undergoing argon plasma treatment.[81,86] NIPAM deriva-

tive commonly used for post-irradiation grafting includes a

co-polymer with a poly(ethylene glycol) side chain,

maintaining thebackboneofNIPAMtoreducecell adhesion

during the lift-off of cell sheets.[86] At the same time,

NIPAM/PEG copolymers show sharper transition temper-

atures than pure poly(N,N-diethylacrylamide) because of

the strong interaction between NIPAM and ethylene glycol

at temperatures above the transition temperature, while

solubilization of ethylene glycol elements with water

occurs below this temperature.[81,86]

4.2.3. Ageing Properties of Acrylamide and N-IsopropylAcrylamide (NIPAM)

Ageing studies of grafted acrylamide coatings are almost

non-existent. The only report available in the open

literature suggested that this coating remainedhydrophilic

after three months of ageing in air if the grafting yield was

more than 100mg cm�2.[76] This result is to be expected

because similar behavior has been reported for acrylic acid

graft coatings.[91] Due to similarity in the processing steps

and the size of grafting molecules, the same mechanism

may be applicable here. The acrylic acid grafts form

domains that restrict the movement of hydrophilic seg-

ments at high density. The absence of acrylic acid domains

and interfacial tension of grafted acrylic acid creates the

DOI: 10.1002/ppap.201400116



impetus for thepolar chains tomove into the interior at low

grafting density.[1]

The stability of plasma polymerized NIPAM coatings in

water depends on a combination of high discharge power

and high chamber temperature used during the polymeri-

zation process.[79] While the effect of higher discharge

power was straightforward, the role of higher chamber

temperature could not be elucidated. High discharge power

resulted in increased fragmentation in thegasphase,which

produced highly crosslinked coatings that were relatively

stable in hydrated form. However, the process conditions

used did not produce NIPAMplasma polymers with awell-

defined VPT, which is to be expected when using plasma

conditions that lead to extensive monomer molecule

fragmentation and thereby to a more random and more

crosslinked chemical structure of the coating.

Based on observations with other plasma polymers, it

can also be expected that NIPAMplasma polymer coatings

produced under very low power conditions would exhibit

a high extent of dissolution of soluble lower molecular

weightmaterial. Thus, the viability of plasmadepositionof

NIPAM coatings is uncertain not only because of the low

deposition rates but also because of limited stability in

aqueous solutions; grafted coatings seemadvantageouson

both counts. A study involving direct comparison between

plasma-NIPAM coatings and grafted polyacrylamide coat-

ings would appear to be of interest, but would need to

assess awide rangeof parameters; in addition to switching

ability and stability, uniformity, and reproducibility,

efficiencyof processingandscale-up considerations should

also be assessed. It is not straightforward to compare

literature reports from different laboratories that often

use different test methodologies; side-by-side comparison

is required.

Figure 5. XPS S 2p binding energies of various organo-sulfurcompounds. As these studies used different binding energyreferences, all data have been re-normalized to a referencevalue of 285.0 eV for C—C.[94]

4.3. Cell Colonization on Acrylamide and NIPAM

Coatings

The presence of acrylamide coatings on porous polyether-

sulfone (PES) membrane[76] and PU[75] substrates has been

shown to lead to reduced protein adsorption, with the least

protein fouling observed with a specific optimal grafting

yield.[75] The mechanism is probably similar to that

discussed earlier for the PEO coatings, conferring steric

hindrance to prevent protein adsorption at an optimal

balance between hydration and polymer chain density.

Plasma polymerized NIPAM coatings have proven to be

effective as a platform to grow and detach a variety of cells

such as bovine endothelial cells,[82] bovine smooth muscle

cells[85] and human embryonic kidney cells (HEK-293),[85]

with considerably less damage than when cells are

detached by enzymatic digestion or mechanical dissocia-

tion. Biological tests such as immunoassays and surface

analytical investigations using techniques such as XPS




and ToF-SIMS showed that most of the underlying

extracellular matrix was present with the lifted-off cell

sheets and hence ensured its viability.[82] This shows that

proteins adsorb onto suitable plasma-NIPAM surfaceswith

low binding affinity and hence are easily desorbed again.

One notable advantage of plasma polymerized NIPAM is

the insensitivity of this coating to substrate effects in

producing confluent cell monolayers[92] compared to coat-

ings produced by the direct irradiation e-beam grafting

technique.[93]

5. Sulfonate–Sulfate (SO3–SO4) PlasmaPolymerized and Treated Surfaces

Sulfonate and SO4 groups at bio-interfaces are of consider-

able interest as those groups arewell hydrated and occur in

a variety of biomolecules, for example, in some glyco-

saminoglycans that form part of the extracellular matrix.

Thus, it is of interestwhether such groupsmight also confer

advantageous properties to synthetic polymeric biomate-

rials surfaces. In the field of biomaterials, SO3 and SO4 are

generally studied as one class of functionalities because

thebio-interfacial effects arising fromthepresenceof—SO3

groups on syntheticmaterials surfacesmay not differ from

effects from SO4 groups, as the fourth oxygen ‘‘behind’’

the S, linking sulfates to polymer surfaces, causes little

difference to properties such as electronegativity. Thus, it

appears reasonable to assume that SO3 and SO4 groupsmay

have very similar interfacial effects, and they will be

discussed together. The similarity of SO3 and SO4 groups are

also reflected by their overlapping XPS binding energies.

Figure 5 shows the XPS S 2p binding energies of various

organo-sulfur compounds generated by various plasma

and non-plasma techniques.[94]




10

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Interest in SO4–SO3 groups stems also from their ability

to delay the onset of thrombus formation, withmost of the

early coatings fabricated by acid etching techniques.[95]

End-sulfonated PEG graft coatings were also shown to

improve hemocompatibility.[96] These results were inter-

preted as sulfonated PEGs having better repellence of

negatively charged blood proteins, but it was shown later

that, for reasons unknown, sulfonated PEG coatings

attracted albumin,[97] thereby effectively passivating the

surfacesagainst thrombus-formingevents. Theseexamples

show that SO3 groups can cause substantial bio-interfacial

effects.

In recent years, there has been interest in exploring the

role of SO3–SO4 groups in mediating the adsorption of

proteins and cells. Interest in thesemoieties stems from the

ability of SO3 groups to separate mixtures of peptides in

microfluidic chips.[98]

5.1. Plasma Treatment with Sulfur Dioxide (SO2)

Sulfur dioxide (SO2) is a common process gas used to insert

sulfur groups into surfaces by plasma treatment.[98–108]

SO2 plasma treatment can produce a range of sulfur groups

of differentoxidation states,with S2pXPSbindingenergies

centered around 163–65 and 167–169 eV, which is

evidence for a considerable range of oxidation states. A

typical example is shown in Figure 6.[109] This multi-

functionality in the oxidation states of sulfur is common

amongst all reported plasma treatments and not unique to

SO2 plasma treatment. The success of SO2plasma treat-

ment depends on the balance between etching by the

oxygen ions from SO2 or residual air and implantation of

chemical functionalities such as SO, SO2, SO3, and SO4, on

the substrate. This balance depends on the discharge

power, reaction time, substrate type, hydrodynamic

x 10 2

2

4

6

8

10

12

CPS

174 172 170 168 166 164 162 160 Binding Energy (eV)

1,7 Octadiene-SO2 pp

S-S, S-C, S-H (3/2)

HA-SO2 pp

SO2, SO3 (3/2)

-O-SO3 (3/2) -O-O-SO3 (3/2)

Figure 6. XPS S2p signals recorded with heptylamine andoctadiene plasma polymers that were subjected to SO2 plasmatreatment.[109]




factors, and location of the substrate in the plasma

reactors.[100,104,107,110] Although it is not possible to

provide definitive rules for tailoring these factors so as

to produce specific desired sulfur oxidation states, there

are some factors that can be used to predict the oxidation

state of added sulfur.

5.1.1. Influence of Substrate

Generally, polymers that contain oxygen functionalities

such as carboxylic acids and ethers are more susceptible to

plasma treatment, particularly etching reactions, com-

pared with those without such groups.[111] In addition,

amorphous regions of polymers were found to be more

likely to react to plasma treatment than crystalline regions,

possibly because of higher efficiency of radical generation

in the former regions.[112] It has been reported that PE has

the highest sulfur concentration, followed by polypropyl-

ene and polyethylene terephthalate (PET), after SO2 plasma

treatment.[107] This difference in sulfur concentration is

likely to be related to the different degree of crystallinity of

these polymers based on the foregoing discussions.

This ease of oxidation in the amorphous regionwas used

to explain the preferential formation of SO4 groups in

polypropylene compared to SO3 groups in highly oriented

pyrolytic graphite.[104] In the case of SO2 plasma-treated

PTFE substrate, the fluorine has an electron withdrawing

effect, which would shift the S2p XPS binding energy to a

higher value.[102]

5.1.2. Influence of Co-Monomers

Similar to the influence of the substrate, an analogous

electron withdrawing effect was present during the co-

polymerization of SO2 with fluoro-benzene[113,114] and co-

polymerization of trifluoromethanesulfonic acid and chlor-

otrifluoroethylene.[115] The inclusion of perfluorobenzene

promoted the generation of higher oxidation state sulfur

moieties (168–169 eV)while pentafluorobenzene produced

sulfur moieties of lower binding energy (166 eV).[114]

However, secondary binding energy shifts fromF confound

interpretation of the oxidation states and thus identifica-

tion of sulfur compounds produced.

Since the desired treatment is to produce sulfur of

higher oxidation states (i.e., sulfonates and sulfates with

S2p XPS binding energies of 168–169 eV) for biomaterials

applications, one possible approach is to use a process gas

mixture of H2 and SO2 followed by ageing for six

weeks.[116] Oxygen could be added to suppress the

formation of sulfur with lower oxidation states such as

sulfide or sulfoxide, while hydrogenwas found to increase

the proportion of sulfur with lower oxidation states.[107]

This may seem contradictory to the previous report[116]

but ageing evidently played a key role there in achieving

DOI: 10.1002/ppap.201400116


SULFATE-SULFONATE

Plasma Polymerization SO2-monomers e.g. perfluorobenzene, C2H2, C2H4

Plasma grafting (post irradiation): 1) ClSO3 etching or Vinyl sulfonate after air plasma treatment 2) 1,3 propane sultone or SO2 plasma after NH3 or allyamine plasma treatment

Plasma grafting (direct irradiation) Ar plasma on preadsorbed SDS or SO4

2- terminated PEO or NaS11

Plasma Treatment SO2

Plasma Immersion Ion Implantation

Figure 7. Plasma-based techniques to create coatings with sulfate/sulfonate groups.


higher oxidation states. Other reports

indicated that carrier gases such as H2,

O2, andN2 could reduce the total amount

of incorporated sulfur, notably those of

higher oxidation states.[104] However,

with substrates such as polylactide,

added single oxidation state sulfur

groups (c.a. 168–169 eV) could be

achievedwithH2–SO2plasma treatment

after six weeks of ageing.[116] When

using electrical biasing, negative DC-

electrical bias could produce high ener-

getic ions to etch SO3 and SO4 groups

from the surface of a sulfated plasma

polymer.[104]

5.1.3. Limitations of SO2 Plasma Treatment

One major setback of SO2 plasma treatment is that

commercially pure SO2 gas (99.98 vol%) contains some

trace nitrogen, sometimes as high as 50ppm,[107] which

results in the effective addition in plasmas of nitrogen

moieties, as high as 1.7 at% implanted on the substrate.[108]

Suchnitrogen contamination has been reported[100,104] and

is also evident on closer examination of published XPS

spectra of SO2 plasma-treated substrates.[98] Further

analysis using optical emission spectroscopy (OES) con-

firmed the presence of activated N2 molecules during SO2

plasma treatment.[107,110] Deposited nitrogen moieties can

mask or synergistically accentuate the contribution of SO3–

SO4 groups during cell and protein adsorption studies.

5.2. Plasma Polymerization with Sulfur-Containing

Monomers

Early work to produce sulfur-containing plasma polymers

was hampered by a lack of suitable monomers, with issues

such as volatility and ready loss of the functional groups in

theplasma. The sulfonic groupof thebenzene sulfonic acids

were not stable in the plasma and the resulting plasma

polymer did not contain sulfonic groups.[111] Other mono-

mers such asmethyl vinyl sulfone, ethyl vinyl sulfone, and

vinyl sulfone had a very low density of SO3 or SO4

groups.[117] Allyl phenyl sulfone has low volatility though

its process window is relatively large.[100] Alternatively,

plasma co-polymerization of SO2 with unsaturated hydro-

carbon (C2H2 and C2H4) monomers[118] or hexamethyldisi-

loxane (HMDSO) gave more promising results.[101] During

co-polymerization of C2H2 or C2H4 and SO2 plasma, thiol

groups made up 70–80mol% of the total sulfur moieties

while the remaining sulfur groups were of a higher

oxidation state as shown by a signal at higher XPS S2p

binding energy of 168–169 eV.[118] The lack of specificity

and predominance of sulfur with low oxidation states




prompted the search for another approach to produce the

desired SO3/SO4 coatings for biomaterials applications.

5.3. Plasma Grafting (Post-Irradiation)

As shown in Figure 7, there are two plasma grafting

approaches, which can provide more specificity to the

modified surfaces. In the ‘‘post-irradiation’’ approach, one

of the reported schemes in the literature involved the

grafting of SO2molecules on NH3 plasma-treated PU.[99,119]

Although the intensity of sulfur, as measured by XPS, was

much lower than when using SO2 plasma treatment,[99] its

effectiveness in mediating protein adsorption was demon-

strated with protein adsorption studies in attenuated total

reflectance – Fourier transform infrared (ATR-FTIR) flow

cells.[119] Giroux and Cooper[99] did not propose any

mechanism for this grafting but Collaud Coen et al.,[104]

who used SO2–N2 plasma treatment, suggested that the

primary amine formed hydrogen bonds with SO4 and thus

stabilized it. In a similar approach, Klee et al.[120] saponified

a copolymer of PVC-poly(ethene-co-vinylacetate) to create

hydroxyl groups before treating them with SO2 plasma.

The bimolecular nucleophilic displacement reaction of

1,3-propane sultone on amine terminated substrates

also can be used to create specifically SO�3 group on the

substrate.[105,121] In other work, air plasma treatment of

PEU or PEUU produced different oxygen functionalities,

notably carbonyl and carboxyl groups, which were then

functionalized with vinyl SO3 to produce sulfonated

surfaces.[122]

5.4. Plasma Grafting (Direct Irradiation)

Besides the ‘‘post-irradiation’’ approach, plasma grafting

involving ‘‘direct irradiation’’ of adsorbed surfactants has

also been used successfully to functionalize surfaces with

SO4/SO3 groups. In this method, surfactants, which

contained SO4 functionalities such as sodium dodecyl

sulfate (SDS),[123,124] SO4-modified polyethylene oxide




12

REa

(PEO),[60] and sodium 10-undecene SO4[124] were adsorbed

on the polymer substrate prior to treatment with Ar

plasma. Although the quantity of the sulfur groups

decreased with longer Ar plasma treatment times due to

etching, its chemical state remained as SO4.[60,123,124] This

direct irradiation technique provided specific chemical

species but resulted in an immobilization efficiency as low

as 6% for SDS[124] or 25% for sodium 10-undecene SO4.[124]

This efficiency depended on optimizing the adsorption of

surfactants and the length of Ar plasma treatment time. To

maximize grafting efficiency, the adsorption of surfactants

depended on a range of issues such as the thickness of the

adsorbed surfactant layer,[124] critical micelle concentra-

tion,[123] length of alkyl chain, and degree of saturation.[60]

5.5. Plasma Immersion Ion Implantation of

Sulfur-Containing Monomers

As shown in Figure 7, another method for grafting sulfur

moieties to a substrate is plasma immersion ion implanta-

tion (PIII)[125] or its variants such as ionic cluster deposi-

tion.[126,127] Both techniques utilize high potential differ-

ences to embed the fragments of sulfur moieties and Arþ

ions into the substrates. In PIII, the sulfur was evaporated

into an ionization chamber[125] while ionic cluster deposi-

tion bombarded the substrateswith ammoniumsulfamate

clusters.[126,127] The ionic cluster deposition method

showed that sulfur with a S2p XPS binding energy of

167.6 eV was immobilized on the PE substrates in the

absence of Arþ ions.[126] When Arþ ions were introduced

into the reaction chamber, part of those sulfurmoietieswas

converted to groups with lower binding energies at

162.3 eV. A likely mechanism for this observation is that

Ar ions abstract oxygen ions from sulfamate groups.

0 2 4 6 8 10 12 14 16

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

Com

pone

nts

of S

2p &

Tot

al S

(at.

%)

Days of Air Ageing

S-S, S-O, S-C, S-H SO, SO2 SO3, SO4 total S

Figure 8. Fitted percentages of components in the S2p signals ofSO2 plasma treated heptylamine plasma polymer at differenttimes of air ageing, at a takeoff angle of 908.[109]

5.6. Ageing Behavior of SO3–SO4 Plasma-Modified

Surfaces

All functionalization approaches mentioned in Figure 7

produced similar results in that the surface energy and

hydrophilicity of the treated substrates increased after

surface modification. These changes were caused by the

incorporation of polar sulfur groups on the substrates.

However, the ageing properties of sulfur-containing

plasma polymers differed between reported studies. While

Collaud Coen et al.[104] reported that the sulfur content of

SO2 plasma treated highly oriented pyrolytic graphite was

reduced by 25% within 1h and the reduction was 40%

within 14h. A similar decrease in the sulfur content was

also reported for SO2 plasma-treated low density PE by Ko

et al. after 11d of air ageing.[100] Medium discharge power

(10 and 30W) could reduce this loss to approximately 10%

compared to more than 25% at 5 and 50W discharge

power.[100] Angle-resolved XPS did not detect any increase




of sulfur in the deeper region of the substrate during the

11d of ageing, thus indicating that the reduced surface

contentwasnot due to surface rearrangementmotionsbut,

instead, due to volatilization of sulfur-containingmoieties.

In the case of SO2 plasma-treated PU, the reduction of

sulfurwas24%within7d.[99] Theresults likewisesuggested

that evaporation of low molecular weight material

containing sulfur dominated the ageing process. This result

differed from that with 1,3-propane sultone grafted onto

NH3 plasma treated low density PE.[121] With discharge

powers of 10W, the sulfur to carbon (S/C) ratios showed a

slight increase at deeper regions, implying migration of

sulfurgroupsafter7 dofairageing.However, this trendwas

not observed for grafting onto amine plasma-treated

surfaces created at other discharge powers or onto amine

precursors. Based on these results, we conclude that the

ageing mechanism of sulfur containing plasma surfaces

depends on a specific combination of process conditions

and substrates; there are no clear guidelines that can be

derived.

In regard to post-plasma oxidation, angle-resolved XPS

showed an increase of oxygen/carbon ratios at a take-off

angle of 108 and 758 for the SO2 plasma treatment after 11 d

of air-ageing.[100] This result implied that the plasma

treatment created radicals that continued to be oxidized

throughout this ageing period. Progressive oxidation below

the surface can arise from in-diffusion of atmospheric

oxygenaswellasbymobilityofpolymerchainstransferring

surface oxygen groups to the deeper regions. Similar results

have also been reported by others.[109] Figure 8 shows the

initial reduction in S-containing groups in the top 2–3nm

with time. This appears to occur to a higher extent for the

groups with higher oxidation states, but subsequently the

DOI: 10.1002/ppap.201400116



concentration of the latter groups increases again as a

result of post-plasma oxidation.[109] Migration has also

been detected, albeit with mixtures of H2SO4 acid-oxidant

treated low-density PE.[95,128] The decrease of wettability

of H2SO4–KClO3 etched low density PE coincided with the

beginning of the melting transition temperature of the

polymer, obtained from differential thermal analysis.[128]

The increase of chain mobility led to the migration of the

surface polar groups into the bulk polymer.

5.7. Cell Colonization and Protein Adsorption on

SO3–SO4 Plasma-Modified Surfaces

Results for protein adsorption onplasma-modified surfaces

varied between studies; fibronectin adsorption in-

creased[120] while fibrinogen[103] decreased on SO2 plas-

ma-treated surfaces. These results concurred with adsorp-

tion of an albumin–fibrinogen–g-globulin mixture that

also decreased on vinyl SO3-grafted polyetherurethane

(PEU).[122] Human endothelial cells proliferated on SO2

plasma-treated surfaces[120] while murine fibroblast did

not show any inhibition of cell growth.[103] On the other

hand, mouse fibroblast STO cells neither attached nor

proliferated on vinyl SO3-grafted PEU at all levels of density

investigated.[122]

While somehemocompatibility tests showeda beneficial

effect from incorporating sulfur species,[100,103,118,120,126,129]

others have reported negligible effects with SO2-HMDSO

plasma copolymers[101] or even negative effects with SO2

plasma treatment[100,101] or sultone grafting techni-

ques.[121] Thesemixed resultswere reported at overlapping

levels of atomic percentages of sulfur, suggesting that the

nature of the S-containing groups (oxidation state) may be

more important than the absolute amount of S.

Two main possibilities that can account for these

contradicting results are the different types of hemocom-

patibility tests conducted by the investigators, varying

oxidation states and density of sulfur groups, and presence

of other (non-S) functionalities. This was clearly illustrated

by work with SO�3 -terminated SAMs. All work with such

SAMs consistently demonstrated that SO�3 containing

surfaces have higher platelet reactivity than OH-terminat-

ed SAMsbutwere comparablewithCOOH-SAMsbecauseof

the high density of SO�3 associated with SAM techni-

ques.[130,131] This argument was further strengthened by

Takaharaetal.[132]whocarriedoutanexvivoA-Vshunt test

with segmented PU containing SO3 groups of different

densities. These PUs showed improvement at low densities

but the performance quickly deteriorated at high densities.

In thecaseofpolylactidemembranes, trappedSO2gas in the

polylactide membrane might mask the positive effects of

surface SO3–SO4 groups.[116]

While this compares results from various techniques,

certain factors such as the density of functional groups and




the presence of other groups will influence the interfacial

responses. Plasma-based technologies are also known to

produce other groups, especially OH and COOH because of

subsequent oxidation by radicals. The influence of these

two groups is not always straightforward. While OH is

generally acknowledged to be beneficial for hemocompat-

ibility (though causing complement activation), COOH, at a

certain density reduced cell adhesion and related protein

adsorption.[1] SO2 plasma treatment has also been reported

to encourage incorporation of nitrogen moieties into

substrates via oxidation of nitrogen to nitric oxide, which

reacts with radicals on the surface.[108] The source of

nitrogen is impurity of processing gases or desorption from

walls of the plasma reactor or leaks.[108]

It can be challenging to compare reports as in addition to

obvious differences between various in vitro and ex vivo

tests,[101,122] various platelet adhesion tests have also been

used.[100,103,118,121,122,126,129] The type of blood platelets

(human vs. animal), presence and types of proteins, shear

rate of blood flow, and duration of tests can affect the

outcomes of the tests. These tests can only serve as

qualitative screening tests andusually cannot be compared

among laboratories, although a good correlation between

ex vivo and in vitro platelet adhesion studies had been

reported.[133] This difficulty was again demonstrated by

Silveretal.[134]whentheyfoundagoodcorrelationbetween

in vitro and ex vivo performance for non-sulfonated PU but

not for sulfonated PU. Correlation between ex vivo and in

vitro platelet adhesion was observed by Kiaei et al.[135]

for different types of plasma polymers and untreated

substrates.

Other in vitro tests such as activated partial thrombo-

plastin time (APTT),[103,105,122] prothrombin time

(PT),[103,105] thrombin time (TT)[105] and thrombus forma-

tion[122] have also been carried out to complement platelet

adhesion tests but these tests had different degrees of

sensitivity and targeted different steps of the coagulation

cascade. This was clearly illustrated by Lu et al.[106] when

their prothrombin time (PT) was hardly increased though

their APTT and TT increasedmore than four timeswith SO2

plasma treated copolymers of PU-acrylic acid.

In-depth analysis of protein adsorption and hemocom-

patibility tests with sulfonated plasma polymers were

not carried out in most reports. Instead, much of the

understandingwas derived from thework carried outwith

SO3-containing bulk polymers and liquid or gas sulfonated

copolymers. Depending on the phase mixture of the SO3-

containing PU, an increase in SO3 groups resulted in an

increase in fibrinogen adsorption that did not translate to

an increase of platelet reactivity.[136,137] Instead, the

conformation of the adsorbed protein appeared to play a

role in the subsequent platelet adhesion, as shown by

Hylton et al.[138] with their studies using circular dichroism

spectroscopy.




14

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Changes in protein conformation upon adsorption were

confirmed by Kowalczynska et al.[139–141] who utilized

radioisotope (125I-fibronectin) and enzyme-linked immu-

nosorbent assays on their SO3-treated polymer. The differ-

ences between leukemia L1210 cell adhesion on fibronec-

tin-coated sulfonated and non-sulfonated styrene-methyl

methacrylate copolymers were caused by changes in the

arrangement of fibronectin.[139,140] Analogous results were

obtained with 3T3 fibroblast cells in serum-containing

medium on the same substrate.[141]

While the influence of sulfurmoieties and the increase in

hydrophilicity could not be discounted for all of these

results, the surfacemorphology couldalsohave contributed

to different protein adsorption findings and cell adhesion

results on plasma-treated surfaces.[103] Unfortunately,

most researchers provided little or no roughness measure-

ment data.

6. Conclusions

This review focuses on immobilizing non-reactive, hydro-

philic, and hydrogel-likemoieties via low-pressure plasma-

based methods onto solid substrates in order to elicit bio-

specific interfacial responses. These coatings contain

moieties such as PEO, SO3–SO4 groups, acrylamide, and

NIPAM. PEO-like coatings have been fabricated by plasma

polymerization of ‘‘glyme’’ or ether-containing monomers,

and two plasma grafting techniques called direct- or post-

irradiation. These PEO coatings proved to be relatively

stable against ageing.

Acrylamide and NIPAM polymer chains were often

grafted onto argon plasma irradiated surfaces, though in

recent years, NIPAM has been plasma polymerized directly

onto various substrates. The stability of acrylamide-grafted

surfaces depends on their grafting density while NIPAM

plasmapolymers require some cross-linked structures to be

effective and stable in aqueous media. Acrylamide-grafted

plasma polymers have been reported to be resistant to

protein adsorption and stability is satisfactory.

Of five plasma-based methods to produce SO3–SO4

containing biomaterials surfaces, the most widespread

method used is SO2 plasma treatment. Analysis of SO2

plasma-treated surfaces revealed sulfur with various

oxidation states as well as nitrogen moieties; the latter is

caused by the ppm level impurities of nitrogen in the gas

supply. The ageing properties of SO3–SO4 containing

plasma polymers showed a strong dependence on fabrica-

tion routes and type of polymeric substrates, and hence no

clear design rules can be derived yet.

The review also surveys studies using hydrogel and

hydrophilic surfaces for cell colonization assays. PEO

coatings were relatively resistant to protein adsorption

and cell adhesion in vitro, but in vivo test results are less




convincing. With careful selection of process parameters,

NIPAM plasma polymers were shown to be effective as a

platform to grow and detach cell sheets, with their

effectiveness depending on the extent of retention of

NIPAM groups in the coatings while acrylamide coatings

exhibited similar protein and cell resistance as PEO-like

coatings. Surfaces containing SO3–SO4 showed contradic-

tory results for protein adsorption and cell proliferation

studies. Interpretation of these inconsistent results is

hampered by insufficient characterization of the oxidation

states of the sulfur groups, lack of data on surface

morphology, and different testing methods.

In summary, low-pressure plasma methods are capable

of producing hydrophilic, non-reactive surfaces suitable for

bio-interfacial applications. While the simplicity and

process scale-up of a one-step plasma treatment/coating

approach is attractive for industrial processing to generate

novel biomaterials surfaces, there are some concerns in

regard to the quality and the stability of PEO-like coatings

and NIPAM plasma coatings, in that the very low power

levels needed to generate hydrogel coatings akin to

conventional coatings also lead to the generation of

considerable amounts of soluble material that dissolves

from the plasma coatings with time. Likewise, plasma

approaches for SO4/SO3 surfaces have some drawbacks. In

the absence of direct comparisons with conventional

coatings such as those produced by grafting techniques

and an analysis of production costs, it is not clear whether

highly hydrophilic biomaterials surfaces produced by one-

step plasma approaches can provide clear benefits over

conventional approaches. On the other hand, plasma

surface functionalization approaches clearly are highly

suitable for preparation of materials surfaces for subse-

quent separate covalent linking or grafting of layers of PEG,

pNIPAM, and sulfated molecules.

Acknowledgements: This work was partially supported by theAustralian Research Council via the Special Research Centre forParticle and Material Interfaces, including a PhD scholarship forK.S.S. This work was also supported by the Malaysia Ministry ofEducation (Fundamental Research Grant Scheme FRGS/2/2013/SG06/UKM/02/3).

Received: June 23, 2014; Revised: July 14, 2014; Accepted: July 15,2014;DOI: 10.1002/ppap.201400116

Keywords: bio-interfaces; glyme; N-isopropylacrylamide; PEG;PEO; sulfate; sulfonate

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Engineering

Low pressure plasma methods for generating non-reactive hydrophilic and hydrogel-like bio-interface coatings