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Plasma polymers from glycidol vapors are of interest for direct covalent grafting of molecules bearing amine or thiol groups. The question of whether pulsed plasma operation might lead to a higher surface density of epoxide groups and a higher density of grafted molecules is studied using the antifungal drug caspofungin. X-ray photoelectron spectroscopy and Time of flight-secondary ions mass spectrometry analysis followed by caspofungin grafting revealed that both continuous wave and pulsed plasmas led to surface epoxides but with higher densities upon pulsing. Investigations into stability suggested that glycidol plasma polymer coatings were still able to immobilize caspofungin after 2 years of storage, making them suitable for applications where grafting of molecules needs to be done immediately before usage of a device. Refereed/Peer-reviewed

Microbial pathogens use hydrolases as a virulence strategy to spread disease through tissues and colonize medical device surfaces; however, visualizing this process is a technically challenging problem. To better understand the role of secreted fungal hydrolases and their role in

Refereed/Peer-reviewed Plasma polymers have long been of interest as thin film coatings on biomedical devices and products, to generate desirable surface properties for favorable bio-interfacial interactions. Plasma polymers have also been used as platforms for the covalent immobilization of bioactive molecules. More recently, additional aspects have been investigated, such as selective prevention of adhesion of microbial pathogens, either via plasma polymers per se or including antimicrobial drugs. Plasma polymers have also been investigated for the release of silver ions and small organic molecules. Complementing low-pressure plasma approaches, processes at atmospheric pressure have attracted interest recently, including for nano/biocomposite coatings. This contribution reviews the use of plasma polymers for intended biomedical applications, with a focus on more recent topic areas.

Deposition chemistry from plasma is highly dependent on both the chemistry of the ions arriving at surfaces and the ion energy. Typically, when measuring the energy distribution of ions arriving at surfaces from plasma, it is assumed that the distributions are the same for all ionic species. Using ethyl acetate as a representative organic precursor molecule, we have measured the ion chemistry and ion energy as a function of pressure and power. We show that at low pressure (

Improved retention of desirable chemical structures during plasma polymerization requires rational tailoring of plasma-phase conditions. Using ethyl trimethylacetate, we studied the effects of pressure and power on the contribution of intact molecular ions to deposition and retention of ester groups. The abundance of protonated molecular ions in plasmas varies with pressure and power, but the functionality of plasma polymers, assessed by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry, is not correlated. Together with high ion flux, the ion energy distribution was found to be a key parameter and needs to be tailored to enable the soft landing of ions on the surface after traversing the sheath. The compromise between the abundance of ions and their energy distribution is optimal near the transition between the α and γ plasma phases. Refereed/Peer-reviewed

This study demonstrates the ability of Candida albicans, a medically significant human fungal pathogen, to minimize contact with an antifungal surface coating that on a flat surface is lethal on contact by growing on and between micron-sized surface topographical features, thus minimizing the contact area. Scanning electron microscopy showed that cells contacting the "floor" between microcones were killed, whereas cells attached to microcones survived and formed hyphal filaments. These spanned space between cones and avoided contact with the flat surface in-between cones. Thus, fungal cells managed to attach and grow despite the antifungal coating. This ability of Candida albicans to exploit topography features to minimize surface contact yet utilize the solid surface for anchoring reduces the effectiveness of the grafted antifungal surface coating. This suggests that biomedical devices with rough surfaces might be more challenging to protect against fungal biofilm formation via application of an antifungal coating. Refereed/Peer-reviewed

Candida auris is known to survive for weeks on solid material surfaces. Its longevity contributes to medical device contamination and spread through healthcare facilities. We fabricated antifungal surface coatings by coating plastic and glass surfaces with a thin polymer layer to which the antifungal drug caspofungin was covalently conjugated. Caspofungin-susceptible and -resistant C. auris strains were inhibited on these surfaces by 98.7 and 81.1%, respectively. Cell viability studies showed that this inhibition was fungicidal. Our findings indicate that C. auris strains can be killed on contact when exposed to caspofungin that is reformulated as a covalently-bound surface layer. Lay summary Candida auris is pathogenic, multidrug resistant yeast with the ability to survive on surfaces and remain transmissible for long periods of time in healthcare settings. In this study, we have prepared an antifungal surface coating and demonstrated its ability to kill adhering C. auris cells on contact.

Stable organic nitroxide radicals have been shown to exhibit similar cell biology signaling properties as the wellknown but short-lived small molecule nitric oxide, such as affecting intracellular redox states and cell proliferation behavior. Biological processes might thus be amenable to biointerfacial regulation via release of stable nitroxide molecules from coatings applied onto biomedical devices. In this study, we utilized the facile and technologically attractive process of plasma polymerization for the deposition of thin layers containing stable nitroxide radicals, using TEMPO (2,2,6,6-tetramethylpiperidin-1-yl)oxyl as the "monomer" for creating a thin polymeric film. Coatings (TEMPOpps) produced under various conditions were characterized by ellipsometry, XPS, ToF-SIMS, and EPR as well as in vitro biological effects on bacteria (Staphylococcus epidermidis), fungi (Candida albicans), and human cancer cells (KG1a). TEMPOpps were compared with plasma coatings from three structurally related precursors that lack nitroxide groups. Surface characterization by XPS and ToFSIMS confirmed the similarity of atomic composition and molecular fragments of the TEMPOpp films to the precursor molecule. Thin (241-312 nm) films were shown by EPR to contain stable nitroxide radicals, with a G-factor of 17 G typical of TEMPO. The plasma conditions modulated the density of radicals included in the films. On TEMPOpp surfaces, the microbial pathogens Staphylococcus epidermidis and Candida albicans exhibited reduced capacity to form biofilm, and fungal cells did not transition to hyphal forms. In addition, for the nonadherent human cancer cell line KG1a, we found that TEMPOpp coatings upregulated the cells' intracellular reactive oxygen species (ROS) but were not cytotoxic. Thus, we demonstrate that TEMPOpp films with nitroxide radicals possess versatile promising biological activities, such as for coating biomedical devices to prevent infections. Refereed/Peer-reviewed

In the oral cavity, dental implants are exposed to an environment rich in various microbes that can produce infectious biofilms on the implant surface. Here we report the development of two distinct antimicrobial coatings that prevent biofilm formation by fungi or bacteria. The antimicrobial peptides Mel4 and caspofungin were immobilized on titanium surfaces through reactions with radicals embedded within a mechanically robust, ion-assisted plasma polymerized (PP) film. The immobilization does not require additional chemical reagents and is achieved by simply incubating the surfaces at room temperature in a buffer solution containing the antimicrobial agent. The antibiotic-functionalized surfaces were rigorously washed with hot sodium dodecyl sulphate (SDS) to remove physisorbed molecules, and analyzed by time of flight secondary ion mass spectrometry (ToF-SIMS), which revealed characteristic fragments of the peptides and provided strong evidence for the covalent nature of the binding between the molecules and the PP coating. Both Candida albicans and Staphylococcus aureus pathogens were significantly inhibited in their ability to colonize the surfaces and form biofilms. Our findings suggest that antimicrobial surfaces fabricated using ion-assisted plasma polymerization have great potential for coatings on biomedical devices where activity against fungal and bacterial pathogens is required. Refereed/Peer-reviewed

A series of polymerized high internal phase emulsion (polyHIPE) materials have been prepared by using a water in oil emulsion stabilized by a macro-RAFT agent, 2-(butylthiocarbonothioylthio)-2-poly(styrene)-b-poly(acrylic acid), acting as a polymeric surfactant. The pore structures of the formed polyHIPEs were closed. By removing the RAFT-endgroup of the amphiphilic macro-RAFT agent, the obtained polyHIPEs possessed an open structure with voids. The effect of the RAFT-endgroup of the amphiphilic macro-RAFT agent on the surface chemistry of the polyHIPEs is discussed. The obtained polyHIPEs via this surfactant-assisted functionalization strategies were characterized by FTIR spectroscopy, FTIR mapping, SEM, SEM-EDX, TEM, XPS as well as synchrotron-based scanning transmission X-ray microscopy (STXM). The latter technique revealed the surface chemistry of the obtained polyHIPEs and macro-RAFT agent multicomponents with a surface spatial resolution of the order of 30-100 nm. Refereed/Peer-reviewed

In recent years, increasing evidence has been collated on the contributions of fungal species, particularly Candida, to medical device infections. Fungal species can form biofilms by themselves or by participating in polymicrobial biofilms with bacteria. Thus, there is a clear need for effective preventative measures, such as thin coatings that can be applied onto medical devices to stop the attachment, proliferation, and formation of device-associated biofilms. However, fungi being eukaryotes, the challenge is greater than for bacterial infections because antifungal agents are often toxic towards eukaryotic ho st cells. Whilst there is extensive literature on antibacterial coatings, a far lesser body of literature exists on surfaces or coatings that prevent attachment and biofilm formation on medical devices by fungal pathogens. Here we review strategies for the design and fabrication of medical devices with antifungal surfaces. We also survey the microbiology literature on fundamental mechanisms by which fungi attach and spread on natural and synthetic surfaces. Research in this field requires close collaboration between biomaterials scientists, microbiologists and clinicians; we consider progress in the molecular understanding of fungal recognition of, and attachment to, suitable surfaces, and of ensuing metabolic changes, to be essential for designing rational approaches towards effective antifungal coatings, rather than empirical trial of coatings. Refereed/Peer-reviewed

There are many reports of antimicrobial coatings bearing immobilized active agents on surfaces; however, strong analytical evidence is required to verify that the agents are indeed covalently attached to the surface. In the absence of such evidence, antimicrobial activity could result from a release of active agents. We report a detailed assessment of antifungal surface coatings prepared using covalent attachment chemistries, with the aim of establishing a set of instrumental and biological evidence required to convincingly demonstrate antimicrobial activity due to nonreleasing, surface active compounds and to exclude the alternate possibility of activity due to release. The strongest biological evidence initially supporting permanent antifungal activity was the demonstration of the ability to reuse samples in multiple, sequential pathogen challenges. However, additional supporting evidence from washing studies and instrumental analysis is also required to probe the possibility of gradual desorption of strongly physisorbed compounds versus covalently attached compounds. Potent antifungal surface coatings were prepared from approved pharmaceutical compounds from the echinocandin drug class (caspofungin, anidulafungin, and micafungin) and assessed by microbiological tests and instrumental methods. Carbonyl diimidazole linking chemistry enabled covalent attachment of caspofungin, anidulafungin, and micafungin to plasma polymer surfaces, with antifungal surface activity likely caused by molecular orientations that present the lipophilic tail toward interfacing fungal cells. This study demonstrates the instrumental and biological evidence required to convincingly ascertain activity due to nonreleasing, surface active compounds and summarize these as three criteria for assessing other reports on surface-immobilized antimicrobial compounds. Refereed/Peer-reviewed

Fungal hyphal growth and branching are essential traits that allow fungi to spread and proliferate in many environments. This sustained growth is essential for a myriad of applications in health, agriculture, and industry. However, comparisons between different fungi are difficult in the absence of standardized metrics. Here, we used a microfluidic device featuring four different maze patterns to compare the growth velocity and branching frequency of fourteen filamentous fungi. These measurements result from the collective work of several labs in the form of a competition named the “Fungus Olympics.” The competing fungi included five ascomycete species (ten strains total), two basidiomycete species, and two zygomycete species. We found that growth velocity within a straight channel varied from 1 to 4 μm/min. We also found that the time to complete mazes when fungal hyphae branched or turned at various angles did not correlate with linear growth velocity. We discovered that fungi in our study used one of two distinct strategies to traverse mazes: high-frequency branching in which all possible paths were explored, and low-frequency branching in which only one or two paths were explored. While the high-frequency branching helped fungi escape mazes with sharp turns faster, the low-frequency turning had a significant advantage in mazes with shallower turns. Future work will more systematically examine these trends.

We report a facile, one-step, aqueous surface bioconjugation approach for producing an antifungal surface coating that prevents the formation of fungal biofilms. By direct reaction between surface epoxide groups and amine groups on caspofungin, it avoids the use of secondary chemicals. The coating withstands washing with detergent and reduces the growth of the fungal pathogens Candida albicans by log 6 and Candida glabrata by log 3. Importantly, we show that surface adsorption of albumin does not inhibit the activity of this antifungal coating. Refereed/Peer-reviewed

Plant metabolites that have shown activity against bacteria and/or environmental fungi represent valuable leads for the identification and development of novel drugs against clinically important human pathogenic fungi. Plants from the genus Eremophila were highly valued in traditional Australian Aboriginal medicinal practices, and E. alternifolia was the most prized among them. As antibacterial activity of extracts from E. alternifolia has been documented, this study addresses the question whether there is also activity against infectious fungal human pathogens. Compounds from leaf-extracts were purified and identified by 1- and 2-D NMR. These were then tested by disk diffusion and broth microdilution assays against ten clinically and environmentally relevant yeast and mould species. The most potent activity was observed with the diterpene compound, 8,19-dihydroxyserrulat-14-ene against Cryptococcus gattii and Cryptococcus neoformans, with minimum inhibition concentrations (MIC) comparable to those of Amphotericin B. This compound also exhibited activity against six Candida species. Combined with previous studies showing an antibacterial effect, this finding could explain a broad antimicrobial effect from Eremophila extracts in their traditional medicinal usage. The discovery of potent antifungal compounds from Eremophila extracts is a promising development in the search for desperately needed antifungal compounds particularly for Cryptococcus infections.

Microbial pathogens use hydrolases as a virulence strategy to spread disease through tissues and colonize medical device surfaces; however, visualizing this process is a technically challenging problem. To better understand the role of secreted fungal hydrolases and their role in Candida albicans virulence, we developed an in situ model system using luminescent Re(I) and Ir(III) containing probe molecules embedded in a biodegradable (poly(lactic-co-glycolic acid), PLGA) polymer and tracked their uptake using epifluorescent imaging. We found that secretion of esterases can explain how physically embedded probes are acquired by fungal cells through the degradation of PLGA since embedded probes could not be liberated from nonbiodegradable polystyrene (PS). It was important to verify that epifluorescent imaging captured the fate of probe molecules rather than naturally occurring fungal autofluorescence. For this, we exploited the intense luminescent signals and long spectral relaxation times of the Re and Ir containing probe molecules, resolved in time using a gated imaging system. Results provide a visual demonstration of a key virulence trait of C. albicans: the use of hydrolases as a means to degrade materials and acquire hydrolysis products during fungal growth and hyphal development. These results help to explain the role of nonspecific hydrolases using a degradable material that is relevant to the study of fungal pathogenesis on biotic (tissues) surfaces. Additionally, understanding how fungal pathogens condition surfaces by using nonspecific hydrolases is important to the study of fungal attachment on abiotic surfaces, the first step in biofilm formation on medical devices. Refereed/Peer-reviewed

Aspergillus fumigatus infections are associated with high mortality rates and high treatment costs. Limited available antifungals and increasing antifungal resistance highlight an urgent need for new antifungals. Thioredoxin reductase (TrxR) is essential for maintaining redox homeostasis and presents as a promising target for novel antifungals. We show that ebselen [2-phenyl-1,2-benzoselenazol-3(2H)-one] is an inhibitor of A. fumigatus TrxR (K i 0.22 M) and inhibits growth of Aspergillus spp., with in vitro MIC values of 16 to 64 g/ml. Mass spectrometry analysis demonstrates that ebselen interacts covalently with a catalytic cysteine of TrxR, Cys148. We also present the X-ray crystal structure of A. fumigatus TrxR and use in silico modeling of the enzyme-inhibitor complex to outline key molecular interactions. This provides a scaffold for future design of potent and selective antifungal drugs that target TrxR, improving the potency of ebselen toward inhbition of A. fumigatus growth Refereed/Peer-reviewed

Controlling the release kinetics from a drug carrier is crucial to maintain a drug's therapeutic window. We report the use of biodegradable porous silicon microparticles (pSi MPs) loaded with the anticancer drug camphothecin, followed by a plasma polymer overcoating using a loudspeaker plasma reactor. Homogenous "Teflon-like" coatings were achieved by tumbling the particles by playing AC/DC's song "Thunderstruck". The overcoating resulted in a markedly slower release of the cytotoxic drug, and this effect correlated positively with the plasma polymer coating times, ranging from 2-fold up to more than 100-fold. Ultimately, upon characterizing and verifying pSi MP production, loading, and coating with analytical methods such as time-of flight secondary ion mass spectrometry, scanning electron microscopy, thermal gravimetry, water contact angle measurements, and fluorescence microscopy, human neuroblastoma cells were challenged with pSi MPs in an in-vitro assay, revealing a significant time delay in cell death onset. Refereed/Peer-reviewed

In this work, we aimed to understand the key properties that confer antibacterial activity to chlorinated plasma polymers, by investigating plasma polymers deposited from a series of chlorinated precursor molecules and also investigating the effect of plasma conditions. Results revealed that only precursors with a Cl/C ratio ≥1.5 generate effective antibacterial coatings, whereas the coating thickness is irrelevant. Furthermore there is a “sweet spot” in process conditions to generate coatings with optimal antimicrobial activity. Lastly, we investigated the release of chlorinated fragments and attenuation of the surface-active antibacterial properties of the chlorinated plasma polymers by overcoating them with a perfluorinated plasma polymer.

A drug-eluting coating applied onto biomedical devices and implants is an appropriate way to ensure that an inhibitory concentration of antimicrobial drugs is present at the device surface, thus preventing surface colonization and subsequent biofilm formation. In this study, a thin polymer coating was applied to materials, and it acted as a drug-delivery reservoir capable of surface delivery of the antifungal drug fluconazole to amounts up to 21 μg/cm2. The release kinetics into aqueous solution were quantified by UV spectroscopy and conformed to the Ritger–Peppas and Korsmeyer–Peppas model. Complementary microbiological assays were used to determine effectiveness againstCandida albicans attachment and biofilm formation, and against the control heptylamine plasma polymer coating without drug loading, onwhich substantial fungal growth occurred. Fluconazole release led to marked antifungal activity in all assays, with log 1.6 reduction inCFUs/cm2. Cell viability assays and microscopy revealed that fungal cells attached to the fluconazole-loaded coating remained rounded and did not form hyphae and biofilm. Thus, in vitro screening results for fluconazole-releasing surface coatings showed efficacy in the prevention of the formation of Candida albicans biofilm. Refereed/Peer-reviewed

Endothelialisation of implantable vascular grafts and stents is directed by the adsorbed protein layer. Chemical and mechanical cues sensed at the biomaterial surface by endothelial cells determine their attachment, survival and proliferation. Given the multiplicity of possible interactions we describe the novel application of live cell imaging, factorial analysis and single molecule imaging to investigate higher order interactions between surface chemistry and adsorbed proteins influencing endothelial cell (EC) adhesion dynamics. EC fates were tracked by time-lapse imaging of cell contact area on plasma polymer modified surfaces. The combinatorial effect of plasma polymer chemistry and adsorbed proteins was characterised by factorial experimental design and analysis. Single molecule imaging and counting was used for the first time to quantify binding of fluorescently labelled albumin to plasma polymerized surfaces: Modified glass surfaces with thin plasma polymer coatings rich in primary amine groups had a high affinity for albumin (~2, 650 molecules/μm2) while plasma polymer surfaces functionalised with hydroxyl groups had very low levels of albumin binding (22–30 molecules/μm2). Plasma polymer coatings rich in primary amine groups also promoted endothelial cell adhesion and was superior to tissue culture plastic. An interaction between albumin, heparin and fibronectin promoted adhesion to amine plasma polymer coated glass, while hydroxyl plasma polymer coated glass prevented EC attachment. Tracking cell-specific interactions with the adsorbed protein layer by time-lapse microscopy is more predictive of in vivo cellular responses to biomaterials compared to studies that only measure protein adsorption Refereed/Peer-reviewed

Aspergillus fumigatus infections are associated with high mortality rates and high treatment costs. Limited available antifungals and increasing antifungal resistance highlight an urgent need for new antifungals. Thioredoxin reductase (TrxR) is essential for maintaining redox homeostasis and presents as a promising target for novel antifungals. We show that ebselen [2-phenyl-1,2-benzoselenazol-3(2H)-one] is an inhibitor of A. fumigatus TrxR (K(i) = 0.22 μM) and inhibits growth of Aspergillus spp., with in vitro MIC values of 16 to 64 µg/ml. Mass spectrometry analysis demonstrates that ebselen interacts covalently with a catalytic cysteine of TrxR, Cys148. We also present the X-ray crystal structure of A. fumigatus TrxR and use in silico modeling of the enzyme-inhibitor complex to outline key molecular interactions. This provides a scaffold for future design of potent and selective antifungal drugs that target TrxR, improving the potency of ebselen toward inhbition of A. fumigatus growth.

Objectives: Fungal biofilms caused by Candida spp. are a major contributor to infections originating from infected biomaterial implants. Since echinocandin-class molecules interfere with the integrity of the fungal cell wall, it was hypothesized that surface-immobilized anidulafungin and micafungin could play a role in preventing fungal adhesion and biofilm formation on surfaces. Methods: Anidulafungin and micafungin were covalently coupled to biomaterial surfaces and washed. Surface-sensitive instrumental analysis quantitatively and qualitatively confirmed their presence. Analysis after washing experiments provided evidence of their covalent immobilization. The in vitro antifungal properties of surfaces were confirmed using static biofilm assays and fluorescence microscopy kinetic studies. Results: Antifungal surface coatings eliminated 106 cfu/cm2 inoculations of Candida albicans and prevented biofilm formation and hyphal development on coated surfaces. Surfaces were successively exposed to fresh inoculum and were effective for at least five challenges in eliminating adherent yeasts. Conclusions: We have observed antifungal and anti-biofilm activity of surfaces bearing conjugated echinocandins, which operate through surface contact. The analytical and biological evidence suggests an antifungal mechanism for echinocandins that does not rely upon freely diffusing molecules. Refereed/Peer-reviewed

Historically, there have been two opposing views regarding deposition mechanisms in plasma polymerisation, radical growth and direct ion deposition, with neither being able to fully explain the chemistry of the resultant coating. Deposition rate and film chemistry are dependent on the chemistry of the plasma phase and thus the activation mecha nisms of species in the plasma are critical to understanding the relative contributions of various chemical and physical routes to plasma polymer formation. In this study, we investigate the roles that hydrogen plays in activating and deactivating reactive plasma species. Ethyl trimethylacetate (ETMA) is used as a representative organic precursor, and additional hydrogen is added to the plasma in the form of water and deuterium oxide. Optical emission spectroscopy confirms that atomic hydrogen is abundant in the plasma. Comparison of the plasma phase mass spectra of ETMA/H 2 O and ETMA/D 2 O reveals that (1) proton transfer from hydronium is a common route to charging precursors in plasma, and (2) hydrogen abstraction (activation) and recombination (deactivation) processes are much more dynamic in the plasma than previously thought. Consideration of the roles of hydrogen in plasma chemistry may then provide a more comprehensive view of deposition processes and bridge the divide between the two disparate schools of thought. Refereed/Peer-reviewed

While plasma polymerization is used extensively to fabricate functionalized surfaces, the processes leading to plasma polymer growth are not yet completely understood. Thus, reproducing processes in different reactors has remained problematic, which hinders industrial uptake and research progress. Here we examine the crucial role pressure plays in the physical and chemical processes in the plasma phase, in interactions at surfaces in contact with the plasma phase, and how this affects the chemistry of the resulting plasma polymer films using ethanol as the gas precursor. Visual inspection of the plasma reveals a change from intense homogeneous plasma at low pressure to lower intensity bulk plasma at high pressure, but with increased intensity near the walls of the chamber. It is demonstrated that this occurs at the transition from a collision-less to a collisional plasma sheath, which in turn increases ion and energy flux to surfaces at constant RF power. Surface analysis of the resulting plasma polymer films show that increasing the pressure results in increased incorporation of oxygen and lower cross-linking, parameters which are critical to film performance. These results and insights help to explain the considerable differences in plasma polymer properties observed by different research groups using nominally similar processes. Refereed/Peer-reviewed

In this work we have prepared surface coatings formulated with the antifungal drug caspofungin, an approved pharmaceutical lipopeptide compound of the echinocandin drug class. Our hypothesis was to test whether an antifungal drug with a known cell-wall disrupting effect could be irreversibly tethered to surface coatings and kill (on contact) biofilm-forming fungal human pathogens from Candida spp. The first aim of the study was to use surface analysis to prove that the chemical binding to the surface polymer interlayer was through specific and irreversible bonds (covalent) and not due to non-specific adsorption through weak forces that could be later reversed (physisorption). Secondly, we quantified the antifungal nature of these coatings in a biological assay showing excellent killing against C. albicans and C. tropicalis and moderate killing against C. glabrata and C. parapsilosis. We concluded that caspofungin retains antifungal activity even when it is irreversibly immobilized on a surface, providing a new insight into its mechanism of action. Thus, surface coatings that have echinocandins permanently bound will be useful in preventing the establishment of fungal biofilms on materials. Refereed/Peer-reviewed

We here report two reactor designs for the efficient and uniform plasma treatment of particles: 1) retrofitting an existing plasma reactor with a loudspeaker for agitation (LPR) and 2) adapting a rotary evaporator by adding custom electrodes (RPR). Both designs offer versatility, low building cost, convenience of use, flexibility, facile vacuum sealing and achieve effective agitation and coating of particles. The latter was exemplified by plasma polymerizing perfluorooctane (PFO) onto micron-sized polystyrene particles. The coated particles were evaluated by XPS, ToF-SIMS and water contact angle measurement to compare and contrast the resulting PFO plasma coatings (PFOpp) between the two reactor designs. The highest fluorine to carbon ratio (1.85, Teflon ¼2) was achieved at a pressure of 100mTorr in the RPR.

Polyethyleneimine (PEI) is known to bind copper ions effectively and selectively. However, this is the first report on PEI-based materials for copper scavenging from ultra-low concentrations in seawater matrixes. The findings are relevant for water purification and sensing applications as well as extraction of copper from oceans. Refereed/Peer-reviewed

Antimicrobial surface coatings that act through a contact-killing mechanism (not diffusive release) could offer many advantages to the design of medical device coatings that prevent microbial colonization and infections. However, as the authors show here, to prevent arriving at an incorrect conclusion about their mechanism of action, it is essential to employ thorough washing protocols validated by surface analytical data. Antimicrobial surface coatings were fabricated by covalently attaching polyene antifungal drugs to surface coatings. Thorough washing (often considered to be sufficient to remove noncovalently attached molecules) was used after immobilization and produced samples that showed a strong antifungal effect, with a log 6 reduction in Candida albicans colony forming units. However, when an additional washing step using surfactants and warmed solutions was used, more firmly adsorbed compounds were eluted from the surface as evidenced by XPS and ToF-SIMS, resulting in reduction and complete elimination of in vitro antifungal activity. Thus, polyene molecules covalently attached to surfaces appear not to have a contact-killing effect, probably because they fail to reach their membrane target. Without additional stringent washing and surface analysis, the initial favorable antimicrobial testing results could have been misinterpreted as evidencing activity of covalently grafted polyenes, while in reality activity arose from desorbing physisorbed molecules. To avoid unintentional confirmation bias, they suggest that binding and washing protocols be analytically verified by qualitative/quantitative instrumental methods, rather than relying on false assumptions of the rigors of washing/soaking protocols. Published by the AVS. Refereed/Peer-reviewed

We report a systematic study of the plasma polymerization of ethyl α-bromoisobutyrate (EBIB) to produce thin film coatings capable of serving as ATRP initiation surfaces, for which they must contain α-bromoisobutyryl functional groups. In the deposition of polymeric coatings by plasma polymerization there generally occurs considerable fragmentation of precursor(“monomer”) molecules in the plasma; and the retention of larger structural elements is challenging, particularly when they are inherently chemically labile. Empirical principles such as low plasma power and low pressure are usually utilized. However, we show that the α-bromoisobutyryl structural moiety is labile in a plasma gas phase and in low pressure plasma conditions, below the collisional threshold, there is little retention. At higher pressure, in contrast, fragmentation of this structural motif appears to be reduced substantially, and coatings useful for ATRP initiation were obtained. Mass spectrometry analysis of the composition of the plasma phase revealed that the desired structural moiety can be retained through the plasma, if the plasma conditions are steered toward ions of the precursor molecule. Whereas at low pressure the plasma polymer assembles mainly from various neutral (radical) fragments, at higher pressure the deposition occurs from hyperthermal ions, among which the protonated intactmolecular ion is the most abundant. At higher pressure, a substantial population of ions has low kinetic energy, leading to “soft landing” and thus less fragmentation. This study demonstrates that relatively complex structural motifs in precursor molecule scan be retained in plasma polymerization if the chemical and physical processes occurring in the plasma phase are elucidated and controlled such that desirable larger structural elements play a key role in the film deposition. Refereed/Peer-reviewed

The attachment of pathogenic fungal cells onto materials surfaces, which is often followed by biofilm formation, causes adverse consequences in a wide range of areas. Here we have investigated the ability of thin film coatings from chlorinated molecules to deter fungal colonization of solid materials by contact killing of fungal cells reaching the surface of the coating. Coatings were deposited onto various substrate materials via plasma polymerization, which is a substrate-independent process widely used for industrial coating applications, using 1,1,2-trichloroethane as the process vapour. XPS surface analysis showed that the coatings were characterized by a highly chlorinated hydrocarbon polymer nature, with only a very small amount of oxygen incorporated. The activity of these coatings against human fungal pathogens was quantified using a recently developed, modified yeast assay and excellent antifungal activity was observed against Candida albicans and Candida glabrata. Plasma polymer surface coatings derived from chlorinated hydrocarbon molecules may therefore offer a promising solution to preventing yeast and mould biofilm formation on materials surfaces, for applications such as air conditioners, biomedical devices, food processing equipment, and others. Refereed/Peer-reviewed

Patient safety and well-being are under increasing threat from hospital-acquired infections [1]. The root cause of a large number of these infections arises from microbial biofilms that colonise on surfaces of medical devices such as the millions of catheters, endotracheal tubes, and prosthetics implanted every year [2]. Biofilm infections are accompanied by increased resistance to antimicrobial therapy and immune clearance, severely limiting treatment options and leading to life-threatening disease [3,4]. Device-associated infections are caused by both bacteria and fungi and, while most studies have focused on single-species biofilms, biofilm-related infections are often polymicrobial [5–8]. Multi-species biofilms, particularly those involving bacterial and fungal pathogens, are more challenging to treat, likely as a consequence of their combined architecture, protective extracellular matrix, and potential synergism in protecting against antimicrobials and host immunity [9–11]. Among the fungi, Candida species are the most important biofilm pathogens [12,13] and the fourth leading cause of blood-stream infections in United States hospitals [7]. Fungal diseases remain difficult to diagnose, mortality rates remain high, and antifungal drug resistance continues to limit therapeutic options [14,15]. We are in desperate need of innovative strategies that target the mechanisms of pathogenesis of polymicrobial biofilms on medical devices. This is a grand challenge because it requires multi-disciplinary collaboration and breakthrough research involving physical chemistry, materials science, and microbiology. Communication between these disciplines has not been common, but recent advances show greater convergence in the development of anti-infective devices. At this nexus, we outline the therapeutic promise of anti-infective coatings for medical devices and discuss pitfalls and strategies for overcoming them. Refereed/Peer-reviewed

Surface density gradients of streptavidin (SAV) were created on solid surfaces and demonstrated functionality as a bioconjugation platform. The surface density of immobilized streptavidin steadily increased in one dimension from 0 to 235 ng cm−2 over a distance of 10 mm. The density of coupled protein was controlled by its immobilization onto a polymer surface bearing a gradient of aldehyde group density, onto which SAV was covalently linked using spontaneous imine bond formation between surface aldehyde functional groups and primary amine groups on the protein. As a control, human serum albumin was immobilized in the same manner. The gradient density of aldehyde groups was created using a method of simultaneous plasma copolymerization of ethanol and propionaldehyde. Control over the surface density of aldehyde groups was achieved by manipulating the flow rates of these vapors while moving a mask across substrates during plasma discharge. Immobilized SAV was able to bind biotinylated probes, indicating that the protein retained its functionality after being immobilized. This plasma polymerization technique conveniently allows virtually any substrate to be equipped with tunable protein gradients and provides a widely applicable method for bioconjugation to study effects arising from controllable surface densities of proteins. Refereed/Peer-reviewed

Atom transfer radical polymerization (ATRP) of methacrylic acid (MAA) is challenging. Herein is reported a study of conditions for facile surface-initiated ATRP by activator regenerated electron transfer (SI-ARGET-ATRP) growth of poly-methacrylic acid (PMAA) chains from a plasma polymer surface bearing surface-immobilized α-bromoisobutyryl bromide, with no deoxygenation required. Factors that affect PMAA polymer growth off the surface under ARGET-ATRP conditions are systematically investigated, such as monomer/catalyst ratio, solvent, and, most importantly, addition of salts and change of pH. While the concentrations of the copper catalyst and acid affect grafting, the most pronounced effect arises from the concentration of chloride ions. Adding 0.1 m NaCl and acidifying the reaction solution to pH 3 offers the best trade-off between reaction rate and reproducibility; yielding ≈60 nm thick PMAA graft polymers in 1 h under ambient conditions. Using this easily scalable recipe and surface analysis, the grafted polymers are verified to be pure PMAA and the graft coatings to be homogenous across a substrate of 100 mm diameter. Refereed/Peer-reviewed

Entropic interaction chromatography (EIC) provides efficient size-based separation of protein mixtures through the entropy change associated with solute partitioning into a layer of hydrophilic homopolymer that has been end-grafted within the pores of a macroporous chromatography support. In this work, surface-initiated atom-transfer radical polymerization (ATRP) is used to prepare a library of EIC stationary phases covering a wide range of grafted-chain densities and molecular weights. Exhaustive chain cleavage and analysis by saponification and GPC-MALLS, respectively, show that the new ATRP synthesis procedure allows for excellent control over graft molecular weight and polydispersity. The method is used to prepare high-density grafts (up to 0.164 ± 0.005 chains/nm2) that extend the range of EIC applications to include efficient buffer-exchange and desalting of protein preparations. Reducing the graft density allows for greater partitioning of high molecular weight solutes, extending the linear range of the selectivity curve. Increasing graft molecular weight also alters selectivity, but more directly affects column capacity by increasing the volume of the grafted layer. Protein partitioning in high-density EIC columns is found to decrease with mobile-phase velocity (u). Although solute mass transfer resistances leading to an increase in plate height can explain this effect, pressure drop data across the column are indicative of weak convective flow through at least a fraction of the grafted architecture. Modeling of the grafted brush properties in the presence of solvent flow by subjecting a self-consistent-field theory representation of the brush to a viscous shear force predicts that the grafted chains will tilt and elongate in the direction of flow. The shear force may therefore act to reduce the number of conformations available to chains, increasing their rigidity without significantly altering the thickness of the grafted layer. A reduction in protein partitioning is then predicted when the dependence on u of the solute entropy loss is stronger than that of the grafted polymer, a condition met at high graft densities. Biotechnol. Bioeng. 2007;97: 574–587. © 2006 Wiley Periodicals, Inc.

A method is described that allows potentially any surface to be functionalized covalently with atom transfer radical polymerization (ATRP) initiators derived from ethyl-2-bromoisobutyrl bromide in a single step. In addition, the initiator surface density was variable and tunable such that the thickness of polymer chain grafted from the surface varied greatly on the surfaces providing examples, across the surface of a substrate, of increased chain stretching due to the entropic nature of crowded polymer chains leading towards polymer brushes. An initiator gradient of increasing surface density was deposited by plasma copolymerization of an ATRP initiator (ethyl 2-bromoisobutyrate) and a non-ATRP reactive diluent molecule (ethanol). The deposited plasma polymer retained its chemical ability to surface-initiate polymerization reactions as exemplified by N,N’-dimethyl acrylamide and poly(ethylene glycol) methyl ether methacrylate polymerizations, illustrating linear and bottle-brush-like chains, respectively. A large variation in graft thickness was observed from the low to high chain-density side suggesting that chains were forced to stretch away from the surface interface – a consequence of entropic effects resulting from increased surface crowding. The tert-butyl bromide group of ethyl 2-bromoisobutyrate is a commonly used initiator in ATRP, so a method for covalent linkage to any substrate in a single step desirably simplifies the multi-step surface activation procedures currently used. Refereed/Peer-reviewed

Novel, highly chlorinated surface coatings were produced via a one-step plasma polymerization (pp) of 1,1,1-trichloroethane (TCE), exhibiting excellent antimicrobial properties against the vigorously biofilm-forming bacterium Staphylococcus epidermidis.

A new method for generating a surface density gradient of polymer chains is presented. A substrate independent polymer deposition technique was used to coat materials with a chemical gradient based on plasma copolymerization of 1,7-octadiene and allylamine. This provided a uniform chemical gradient to which initiators for atom transfer radical polymerization (ATRP) were immobilized. After surface-initiated atom transfer radical polymerization (SI-ATRP), poly(2-hydroxyethyl methacrylate) (PHEMA) chains were grafted from the surface and the measured thickness profiles provided direct evidence for how surface crowding provides an entropic driving force resulting in chain extension away from the surface. Film thicknesses were found to increase with the position along the gradient surface, reflecting the gradual transition from collapsed to more extended surface-tethered polymer chains as the grafting density increased. The method described is novel in that the approach provides covalent linkages from the polymer coating to the substrate and is not limited to a particular surface chemistry of the starting material. Refereed/Peer-reviewed

Microbial attachment onto biomedical devices and implants leads to biofilm formation and infection; such biofilms can be bacterial, fungal, or mixed. In the past 15 years, there has been an increasing research effort into antimicrobial surfaces but the great majority of these publications present research on bacteria, with some reports also testing resistance to fungi. Very few studies have focused exclusively on antifungal surfaces. However, with increasing recognition of the importance of fungal infections to human health, particularly related to infections at biomaterials, it would seem that the interest in antifungal surfaces is disproportionately low. In studies of both bacteria and fungi, fungi tend to be the minor focus with hypothesized antibacterial mechanisms of action often generalized to also explain the antifungal effect. Yet bacteria and fungi represent two Distinct biological Domains and possess substantially different cellular physiology and structure. Thus it is questionable whether these generalizations are valid. Here we review the scientific literature focusing on surface coatings prepared with antifungal agents covalently attached to the biomaterial surface. We present a critical analysis of generalizations and their evidence. This review should be of interest to researchers of "antimicrobial" surfaces by addressing specific issues that are key to designing and understanding antifungal biomaterials surfaces and their putative mechanisms of action. Refereed/Peer-reviewed

Background: The ability to present signalling molecules within a low fouling 3D environment that mimics the extracellular matrix is an important goal for a range of biomedical applications, both in vitro and in vivo. Cell responses can be triggered by non-specific protein interactions occurring on the surface of a biomaterial, which is an undesirable process when studying specific receptor-ligand interactions. It is therefore useful to present specific ligands of interest to cell surface receptors in a 3D environment that minimizes non-specific interactions with biomolecules, such as proteins. Conclusion: Overall, the studies presented an effective platform for the preparation of 3D scaffolds which contain effective conjugation sites for attachment of specific bioactive signals of interest, as well as actively reducing non-specific protein interactions. Method: In this study, surface-initiated atom transfer radical polymerization (SI-ATRP) of poly(ethylene glycol)-based monomers was carried out from the surface of electrospun fibers composed of a styrene/vinylbenzyl chloride copolymer. Surface initiated radical addition-fragmentation chain transfer (SI-RAFT) polymerisation was also carried out to generate bottle brush copolymer coatings consisting of poly(acrylic acid) and poly(acrylamide). These were grown from surface trithiocarbonate groups generated from the chloromethyl styrene moieties existing in the original synthesised polymer. XPS was used to characterise the surface composition of the fibers after grafting and after coupling with fluorine functional XPS labels. Results: Bottle brush type coatings were able to be produced by ATRP which consisted of poly(ethylene glycol) methacrylate and a terminal alkyne-functionalised monomer. The ATRP coatings showed reduced non-specific protein adsorption, as a result of effective PEG incorporation and pendant alkynes groups existing as part of the brushes allowed for further conjugation of via azide-alkyne Huisgen 1,3-dipolar cycloaddition. In the case of RAFT, carboxylic acid moieties were effectively coupled to an amine label via amide bond formation. In each case XPS analysis demonstrated that covalent immobilisation had effectively taken place. Refereed/Peer-reviewed

A novel algorithm, Adjacent Pixel Temporal Intensity Correlation (APTIC), was developed to detect single fluorescent molecules by their stochastic emission patterns; photoblinking and photobleaching. The algorithm was evaluated using simulated image data and Total Internal Reflection Fluorescence Microscopy (TIRF-M) to count the number fluorescently labelled protein molecules adsorbed onto glass substrates modified by Radio Frequency Glow Discharge (RFGD) deposition. By selecting an appropriate correlation threshold, the algorithm was capable of detecting synthetic flashes with a signal-to-noise ratio (SNR) as low as 2.0 with 90% sensitivity. The methodology holds great promise for mapping the amount and distribution of biomolecules on surfaces.

Coated layers of biologically active molecules on synthetic biomaterials and biomedical devices can promote a variety of desirable biological reactions by the host body or the biological medium, such as cell and tissue attachment or deterring bacterial biofilm formation. Such coated layers should be immobilised covalently in order to avoid competitive displacement phenomena, and the use of surface-activating plasmas or plasma polymer interlayers with suitable chemical surface groups has proved to be very convenient means of grafting bioactive molecules onto solid materials surfaces. We review selected work on the covalent immobilisation of proteins and peptides onto solid biomaterial surfaces and describe efforts towards plasma methods that allow biomolecules to be covalently captured in a single step. After reviewing a number of approaches, we discuss in more detail the use of plasma polymer interlayers that possess aldehyde or epoxide surface groups; these groups react readily with amine groups on proteins and peptides without undesirable side reactions, and avoid other issues such as crosslinking. We also emphasise the importance of detailed surface analysis to verify that covalent grafting has indeed taken place, and to assess the surface density of grafted molecules. With suitably chosen peptides or proteins, such covalently grafted layers can support the surface attachment of delicate cells, or combat bacterial biofilm formation. Refereed/Peer-reviewed

We describe a method for grafting PEG-based polymer chains of variable surface density using a substrate independent approach, allowing grafting from virtually any material substrate. The approach relies upon initial coupling of a macroinitiator to plasma polymer treated surfaces. The macroinitiator is a novel random terpolymer containing ATRP initiator residues, strongly negatively charged groups, and carboxylic acid moieties that facilitate covalent surface anchoring. Surface-initiated ATRP (SI-ATRP) using polyethylene glycol methyl ether methacrylate (PEGMA) at different concentrations led to grafted surfaces of controlled thickness in either the “brush” or “mushroom” morphology, which was controlled by the abundance of initiator residues in the macroinitiator. Grafted polymer layer structure was investigated via direct interaction force measurements using colloid probe atomic force microscopy (AFM). Equilibrium, hydrated graft layer thicknesses inferred from the highly repulsive AFM force data suggest that the polymer brush graft layer contained polymer chains which were fully stretched. Since the degree of stretching resulted in layer thicknesses approaching the polymer contour length, the polymer brushes studied must be very close to maximum graft density. Grafted layers where the polymer molecules were in the mushroom regime resulted in much thinner layers but the chains had greater chain entropic freedom as indicated by strongly attractive bridging interactions between tethered chains and the silica colloid probe. Use of this experimental methodology would be suitable for preparing grafted polymer layers of a preferred density free from substrate-specific linking chemistries. Refereed/Peer-reviewed

In the development of bioactive coatings on biomaterials, it is essential to characterize the successful fabrication and the uniformity of intended coatings by sensitive surface analytical techniques, so as to ensure reliable interpretation of observed biointerfacial responses. This can, however, be challenging when small bioactive molecules are grafted onto biomaterials surfaces at sub-and near-monolayer densities. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) provides the required sensitivity, but ion signals from small grafted molecules may still be dominated by fragment ions from the underlying polymer. In such cases, multivariate analysis provides valuable enhancement of spectral data, as illustrated here by examples comprising the surface grafting of bioactive serrulatane molecules, the peptide GRGDSP, the oligonucleotide 15-thymidine, and the antifungal compound Amphotericin B. The authors also show how ToF-SIMS plus principal component analysis can distinguish between covalent grafting and physisorption of the antibiotics caspofungin and micafungin. (C) 2015 American Vacuum Society. Refereed/Peer-reviewed

Not only bacteria but also fungal pathogens, particularly Candida species, can lead to biofilm infections on biomedical devices. By covalent grafting of the antifungal drug caspofungin, which targets the fungal cell wall, onto solid biomaterials, a surface layer can be created that might be able to provide long-term protection against fungal biofilm formation. Plasma polymerization of propionaldehyde (propanal) was used to deposit a thin (~20 nm) interfacial bonding layer bearing aldehyde surface groups that can react with amine groups of caspofungin to form covalent interfacial bonds for immobilization. Surface analyses by x-ray photoelectron spectroscopy and timeof-flight secondary ion mass spectrometry confirmed the intended grafting and uniformity of the coatings, and durability upon extended washing. Testing for fungal cell attachment and ensuing biofilm formation showed that caspofungin retained activity when covalently bound onto surfaces, disrupting colonizing Candida cells. Mammalian cytotoxicity studies using human primary fibroblasts indicated that the caspofungin-grafted surfaces were selective in eliminating fungal cells while allowing attachment and spreading of mammalian cells. These in vitro data suggest promise for use as antifungal coatings, for example, on catheters, and the use of a plasma polymer interlayer enables facile transfer of the coating method onto a wide variety of biomaterials and biomedical devices. Refereed/Peer-reviewed

A general method for producing low-fouling biomaterials on any surface by surface-initiated grafting of polymer brushes is presented. Our procedure uses radiofrequency glow discharge thin film deposition followed by macro-initiator coupling and then surface-initiated atom transfer radical polymerization (SI-ATRP) to prepare neutral polymer brushes on planar substrates. Coatings were produced on substrates with variable interfacial composition and mechanical properties such as hard inorganic/metal substrates (silicon and gold) or flexible (perfluorinated poly(ethylene-co-propylene) film) and rigid (microtitre plates) polymeric materials. First, surfaces were functionalized via deposition of an allylamine plasma polymer thin film followed by covalent coupling of a macro-initiator composed partly of ATRP initiator groups. Successful grafting of a hydrophilic polymer layer was achieved by SI-ATRP of N,N0-dimethylacrylamide in aqueous media at room temperature. We exemplified how this method could be used to create surface coatings with significantly reduced protein adsorption on different material substrates. Protein binding experiments using labelled human serum albumin on grafted materials resulted in quantitative evidence for low-fouling compared to control surfaces. Refereed/Peer-reviewed