Surface coatings with covalently attached anidulafungin and micafungin prevent Candida albicans biofilm formation.

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.

Promiscuous hydrogen in polymerising plasmas.

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 mechanisms 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/H2O and ETMA/D2O 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.

Synthesis of highly functionalised plasma polymer films from protonated precursor ions via the plasma α-γ transition.

Chemically functionalized surfaces may be produced via plasma polymerization, however a high degree of functional group retention is often difficult to achieve. Here, the plasma polymerization of three structurally related ester precursors, ethyl isobutyrate (EIB), methyl isobutyrate (MIB) and ethyl trimethylacetate (ETMA) is compared at low and high pressure. In moving from a low pressure to higher pressure regime, significant changes in the plasma chemistry and resulting plasma polymer deposit were observed with much higher retention of chemical functionality at the higher pressure observed. Until now these changes would have been attributed to a decrease in the energy/molecule, however we show by direct measurement of the chemistry and physics of the plasma that there is fundamental shift in the properties of the plasma and surface interactions which explain the results. At low pressure (α regime) precursor fragmentation and neutral deposition dominate resulting in poor functional group retention. Increasing the pressure such that the sheath region close to surfaces becomes collisional (γ regime) favours production of protonated precursor ions which retain functionality and dominate the deposition process rather than radical species.

Combatting fungal biofilm formation by diffusive release of fluconazole from heptylamine plasma polymer coating.

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 against Candida albicans attachment and biofilm formation, and against the control heptylamine plasma polymer coating without drug loading, on which substantial fungal growth occurred. Fluconazole release led to marked antifungal activity in all assays, with log 1.6 reduction in CFUs/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.

The Physics of Plasma Ion Chemistry: A Case Study of Plasma Polymerization of Ethyl Acetate.

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 (<2 Pa) this assumption is valid; however, at elevated pressures ion-molecule collisions close to the deposition surface affect both the energy and chemistry of these ions. Smaller ions are formed close to the surface and have lower energy than larger ionic species which are formed in the bulk of the plasma. The changes in plasma chemistry therefore are closely linked to the physics of the plasma-surface interface.

Surface-grafted antimicrobial drugs: Possible misinterpretation of mechanism of action.

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.

Hyperthermal Intact Molecular Ions Play Key Role in Retention of ATRP Surface Initiation Capability of Plasma Polymer Films from Ethyl α-Bromoisobutyrate.

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 intact molecular 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 molecules can 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.

Comparison of Plasma Polymerization under Collisional and Collision-Less Pressure Regimes.

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.