Candida auris susceptibility on surfaces coated with the antifungal drug caspofungin.

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.

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.

Interfacial Forces at Layered Surfaces: Substrate Electrical Double-Layer Forces Acting through Ultrathin Polymer Coatings.

Manipulating the surface properties of materials via the application of coatings is a widely used strategy to achieve desired interfacial interactions, implicitly assuming that the interfacial forces of coated samples are determined exclusively by the surface properties of the coatings. However, interfacial interactions between materials and their environments operate over finite length scales. Thus, the question addressed in this study is whether interactions associated with bulk substrate materials could act through thin coatings or, conversely, how thick a coating needs to be to completely screen subsurface forces contributed by underlying substrates. Plasma polymer layers were deposited on silicon wafer substrates from ethanol vapor, with identical chemical composition, ultrasmooth surfaces, and varying thicknesses. Using colloid-probe atomic force microscopy, electrical double-layer forces were determined in solutions of various ionic strengths and fitted using the Derjaguin-Landau-Verwey-Overbeek theory. For the thicker ethanol plasma polymers, the fitted surface potentials reflected the presence of surface carboxylate groups and were invariant with thickness. In contrast, for coatings <18 nm thick, the surface potentials increased steadily with decreasing film thickness; the measured electrical double-layer forces contained contributions from both the coating and the substrate. Theoretical calculations were in agreement with this model. Thus, our observations indicate that the higher surface potential of the underlying SiO2 surface can influence the interactions between a colloid particle and the multilayer structure if coatings are sufficiently thin. Such superposition needs to be factored into the design of coatings aimed at the control of material interactions via surface forces.

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.

Affinity Binding of EMR2 Expressing Cells by Surface-Grafted Chondroitin Sulfate B.

The propensity of glycosaminoglycans to mediate cell-cell and cell-matrix interactions opens the door to capture cells, including circulating blood cells, onto biomaterial substrates. Chondroitin sulfate (CS)-B is of particular interest, since it interacts with the receptor (EGF)-like module-containing mucin-like hormone receptor-like 2 precursor (EMR2) displayed on the surface of leukocytes and endothelial progenitor cells. Herein, CS-B and its isomer CS-A were covalently immobilized onto heptylamine plasma polymer films via three different binding chemistries to develop platform technology for the capture of EMR2 expressing cells onto solid carriers. Surface characterization verified the successful immobilization of both glycosaminoglycans. The EMR2 expressing human myeloid cell line U937 preferentially bound onto CS-B-modified substrates, and U937 cells preincubated with CS-B in solution exhibited reduced affinity for the substrate. The direct capture of hematopoietic and blood-circulating endothelial cell types via a glycosaminoglycan-binding surface receptor opens an unexplored route for the development of biomaterials targeted at these cell types.

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.

Cellular micromotion monitored by long-range surface plasmon resonance with optical fluctuation analysis.

Long-range surface plasmon resonance (LRSPR) is a powerful biosensing technology due to a substantially larger probing depth into the medium and sensitivity, compared with conventional SPR. We demonstrate here that LRSPR can provide sensitive noninvasive measurement of the dynamic fluctuation of adherent cells, often referred to as the cellular micromotion. Proof of concept was achieved using confluent layers of 3T3 fibroblast cells and MDA-MB-231 cancer cells. The slope of the power spectral density (PSD) of the optical fluctuations was calculated to determine the micromotion index, and significant differences were measured between live and fixed cell layers. Furthermore, the performances of LRSPR and conventional surface plasmon resonance (cSPR) were compared with respect to micromotion monitoring. Our study showed that the micromotion index of cells measured by LRSPR sensors was higher than when measured with cSPR, suggesting a higher sensitivity of LRSPR to the micromotion of cells. To investigate further this finding, simulations were conducted to establish the relative sensitivities of LRSPR and cSPR to membrane fluctuations. Increased signal intensity was predicted for LRSPR in comparison to cSPR, suggesting that membrane fluctuations play a significant role in the optical micromotion measured in LRSPR. Analogous to cellular micromotion measured using impedance techniques, LRSPR micromotion has the potential to provide important biological information on the metabolic activity and viability of adherent cells.

Colloid-probe AFM studies of the interaction forces of proteins adsorbed on colloidal crystals.

In recent years, colloid-probe AFM has been used to measure the direct interaction forces between colloidal particles of different size or surface functionality in aqueous media, as one can study different forces in symmerical systems (i.e., sphere-sphere geometry). The present study investigates the interaction between protein coatings on colloid probes and hydrophilic surfaces decorated with hexagonally close packed single particle layers that are either uncoated or coated with proteins. Controlled solvent evaporation from aqueous suspensions of colloidal particles (coated with or without lysozyme and albumin) produces single layers of close-packed colloidal crystals over large areas on a solid support. The measurements have been carried out in an aqueous medium at different salt concentrations and pH values. The results show changes in the interaction forces as the surface charge of the unmodified or modified particles, and ionic strength or pH of the solution is altered. At high ionic strength or pH, electrostatic interactions are screened, and a strong repulsive force at short separation below 5 nm dominates, suggesting structural changes in the absorbed protein layer on the particles. We also study the force of adhesion, which decreases with an increment in the salt concentration, and the interaction between two different proteins indicating a repulsive interaction on approach and adhesion on retraction.

Serrulatane Diterpenoid from Eremophila neglecta Exhibits Bacterial Biofilm Dispersion and Inhibits Release of Pro-inflammatory Cytokines from Activated Macrophages.

The purpose of this study was to assess the biofilm-removing efficacy and inflammatory activity of a serrulatane diterpenoid, 8-hydroxyserrulat-14-en-19-oic acid (1), isolated from the Australian medicinal plant Eremophila neglecta. Biofilm breakup activity of compound 1 on established Staphylococcus epidermidis and Staphylococcus aureus biofilms was compared to the antiseptic chlorhexidine and antibiotic levofloxacin. In a time-course study, 1 was deposited onto polypropylene mesh to mimic a wound dressing and tested for biofilm removal. The ex-vivo cytotoxicity and effect on lipopolysaccharide-induced pro-inflammatory cytokine release were studied in mouse primary bone-marrow-derived macrophage (BMDM) cells. Compound 1 was effective in dispersing 12 h pre-established biofilms with a 7 log10 reduction of viable bacterial cell counts, but was less active against 24 h biofilms (approximately 2 log10 reduction). Compound-loaded mesh showed dosage-dependent biofilm-removing capability. In addition, compound 1 displayed a significant inhibitory effect on tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) secretion from BMDM cells, but interleukin-1 beta (IL-1ß) secretion was not significant. The compound was not cytotoxic to BMDM cells at concentrations effective in removing biofilm and lowering cytokine release. These findings highlight the potential of this serrulatane diterpenoid to be further developed for applications in wound management.

Study of the Structure of Hyperbranched Polyglycerol Coatings and Their Antibiofouling and Antithrombotic Applications.

While blood-contacting materials are widely deployed in medicine in vascular stents, catheters, and cannulas, devices fail in situ because of thrombosis and restenosis. Furthermore, microbial attachment and biofilm formation is not an uncommon problem for medical devices. Even incremental improvements in hemocompatible materials can provide significant benefits for patients in terms of safety and patency as well as substantial cost savings. Herein, a novel but simple strategy is described for coating a range of medical materials, that can be applied to objects of complex geometry, involving plasma-grafting of an ultrathin hyperbranched polyglycerol coating (HPG). Plasma activation creates highly reactive surface oxygen moieties that readily react with glycidol. Irrespective of the substrate, coatings are uniform and pinhole free, comprising O─C─O repeats, with HPG chains packing in a fashion that holds reversibly binding proteins at the coating surface. In vitro assays with planar test samples show that HPG prevents platelet adhesion and activation, as well as reducing (>3 log) bacterial attachment and preventing biofilm formation. Ex vivo and preclinical studies show that HPG-coated nitinol stents do not elicit thrombosis or restenosis, nor complement or neutrophil activation. Subcutaneous implantation of HPG coated disks under the skin of mice shows no evidence of toxicity nor inflammation.

Antimicrobial properties of 8-hydroxyserrulat-14-en-19-oic acid for treatment of implant-associated infections.

Treatment options are limited for implant-associated infections (IAI) that are mainly caused by biofilm-forming staphylococci. We report here on the activity of the serrulatane compound 8-hydroxyserrulat-14-en-19-oic acid (EN4), a diterpene isolated from the Australian plant Eremophila neglecta. EN4 elicited antimicrobial activity toward various Gram-positive bacteria but not to Gram-negative bacteria. It showed a similar bactericidal effect against logarithmic-phase, stationary-phase, and adherent Staphylococcus epidermidis, as well as against methicillin-susceptible and methicillin-resistant S. aureus with MICs of 25 to 50 µg/ml and MBCs of 50 to 100 µg/ml. The bactericidal activity of EN4 was similar against S. epidermidis and its Δica mutant, which is unable to produce polysaccharide intercellular adhesin-mediated biofilm. In time-kill studies, EN4 exhibited a rapid and concentration-dependent killing of staphylococci, reducing bacterial counts by >3 log(10) CFU/ml within 5 min at concentrations of >50 µg/ml. Investigation of the mode of action of EN4 revealed membranolytic properties and a general inhibition of macromolecular biosynthesis, suggesting a multitarget activity. In vitro-tested cytotoxicity on eukaryotic cells was time and concentration dependent in the range of the MBCs. EN4 was then tested in a mouse tissue cage model, where it showed neither bactericidal nor cytotoxic effects, indicating an inhibition of its activity. Inhibition assays revealed that this was caused by interactions with albumin. Overall, these findings suggest that, upon structural changes, EN4 might be a promising pharmacophore for the development of new antimicrobials to treat IAI.

Grafting of poly(ethylene glycol) on click chemistry modified Si(100) surfaces.

Poly(ethylene glycol) (PEG) is one of the most extensively studied antifouling coatings due to its ability to reduce protein adsorption and improve biocompatibility. Although the use of PEG for antifouling coatings is well established, the stability and density of PEG layers are often inadequate to provide optimum antifouling properties. To improve on these shortcomings, we employed the stepwise construction of PEG layers onto a silicon surface. Acetylene-terminated alkyl monolayers were attached to nonoxidized crystalline silicon surfaces via a one-step hydrosilylation procedure with 1,8-nonadiyne. The acetylene-terminated surfaces were functionalized via a copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction of the surface-bound alkynes with an azide to produce an amine terminated layer. The amine terminated layer was then further conjugated with PEG to produce an antifouling surface. The antifouling surface properties were investigated by testing adsorption of human serum albumin (HSA) and lysozyme (Lys) onto PEG layers from phosphate buffer solutions. Detailed characterization of protein fouling was carried out with X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) combined with principal component analysis (PCA). The results revealed no fouling of albumin onto PEG coatings whereas the smaller protein lysozyme adsorbed to a very low extent.

Guanylated polymethacrylates: a class of potent antimicrobial polymers with low hemolytic activity.

We have synthesized a series of copolymers containing both positively charged (amine, guanidine) and hydrophobic side chains (amphiphilic antimicrobial peptide mimics). To investigate the structure-activity relationships of these polymers, low polydispersity polymethacrylates of varying but uniform molecular weight and composition were synthesized, using a reversible addition-fragmentation chain transfer (RAFT) approach. In a facile second reaction, pendant amine groups were converted to guanidines, allowing for direct comparison of cation structure on activity and toxicity. The guanidine copolymers were much more active against Staphylococcus epidermidis and Candida albicans compared to the amine analogues. Activity against Staphylococcus epidermidis in the presence of fetal bovine serum was only maintained for guanidine copolymers. Selectivity for bacterial over mammalian cells was assessed using hemolytic and hemagglutination toxicity assays. Guanidine copolymers of low to moderate molecular weight and hydrophobicity had high antimicrobial activity with low toxicity. Optimum properties appear to be a balance between charge density, hydrophobic character, and polymer chain length. In conclusion, a suite of guanidine copolymers has been identified that represent a new class of antimicrobial polymers with high potency and low toxicity.

Parvifloranines A and B, two 11-carbon alkaloids from Geijera parviflora.

Two novel alkaloids (parvifloranines A and B), possessing an unusual 11-carbon skeleton linked with amino acids, were isolated from Geijera parviflora, an endemic Australian Rutaceae. Their structures were elucidated by extensive spectroscopic measurements including 2D NMR analyses. Parvifloranine A was found to be a mixture of two enantiomers, (S)-1 and (R)-1, in a ratio of 1:4, based on their separation using a chiral column. Parvifloranine B is also believed to be a mixture of enantiomers. Proposed biosynthetic pathways are discussed. Parvifloranine A inhibited the synthesis of nitric oxide in LPS-stimulated RAW 264.7 macrophages with an IC50 value of 23.4 µM.

Immobilized streptavidin gradients as bioconjugation platforms.

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.

Biologically active dibenzofurans from Pilidiostigma glabrum, an endemic Australian Myrtaceae.

In an effort to identify new anti-inflammatory and antibacterial agents with potential application in wound healing, five new dibenzofurans, 1,3,7,9-tetrahydroxy-2,8-dimethyl-4,6-di(2-methylbutanoyl)dibenzofuran (1), 1,3,7,9-tetrahydroxy-2,8-dimethyl-4-(2-methylbutanoyl)-6-(2-methylpropionyl)dibenzofuran (2), 1,3,7,9-tetrahydroxy-2,8-dimethyl-4,6-di(2-methylpropionyl)dibenzofuran (3), 1,3,7,9-tetrahydroxy-4,6-dimethyl-2-(2-methylbutanoyl)-8-(2-methylpropionyl)dibenzofuran (4), and 1,3,7,9-tetrahydroxy-4,6-dimethyl-2,8-di(2-methylpropionyl)dibenzofuran (5), were isolated from the leaves of Pilidiostigma glabrum together with one previously described dibenzofuran. Structure elucidation was achieved by way of spectroscopic measurements including 2D-NMR spectroscopy. Compounds with 2,8-acyl substitutions had potent antibacterial activity against several Gram-positive strains (MIC in the low micromolar range), while compounds with 4,6-acyl substitutions were less active. All compounds except 3 inhibited the synthesis of nitric oxide in RAW264 macrophages with IC(50) values in the low micromolar range. Compounds with 2,8-acyl substitutions also inhibited the synthesis of PGE(2) in 3T3 cells, whereas 4,6-acyl-substituted compounds were inactive. None of the compounds inhibited the synthesis of TNF-α in RAW264 cells. The compounds showed variable but modest antioxidant activity in the oxygen radical absorbance capacity assay. These findings highlight that much of the Australian flora remains unexplored and may yet yield many new compounds of interest. Initial clues are provided on structure/activity relationships for this class of bioactives, which may enable the design and synthesis of compounds with higher activity and/or selectivity.

Attachment of endothelial colony-forming cells onto a surface bearing immobilized anti-CD34 antibodies: Specific CD34 binding versus nonspecific binding.

Cardiovascular disease is a leading cause of death worldwide; however, despite substantial advances in medical device surface modifications, no synthetic coatings have so far matched the native endothelium as the optimal hemocompatible surface for blood-contacting implants. A promising strategy for rapid restoration of the endothelium on blood-contacting biomedical devices entails attracting circulating endothelial cells or their progenitors, via immobilized cell-capture molecules; for example, anti-CD34 antibody to attract CD34+ endothelial colony-forming cells (ECFCs). Inherent is the assumption that the cells attracted to the biomaterial surface are bound exclusively via a specific CD34 binding. However, serum proteins might adsorb in-between or on the top of antibody molecules and attract ECFCs via other binding mechanisms. Here, we studied whether a surface with immobilized anti-CD34 antibodies attracts ECFCs via a specific CD34 binding or a nonspecific (non-CD34) binding. To minimize serum protein adsorption, a fouling-resistant layer of hyperbranched polyglycerol (HPG) was used as a "blank slate," onto which anti-CD34 antibodies were immobilized via aldehyde-amine coupling reaction after oxidation of terminal diols to aldehydes. An isotype antibody, mIgG1, was surface-immobilized analogously and was used as the control for antigen-binding specificity. Cell binding was also measured on the HPG hydrogel layer before and after oxidation. The surface analysis methods, x-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry, were used to verify the intended surface chemistries and revealed that the surface coverage of antibodies was sparse, yet the anti-CD34 antibody grafted surface-bound ECFCs very effectively. Moreover, it still captured the ECFCs after BSA passivation. However, cells also attached to oxidized HPG and immobilized mIgG1, though in much lower amounts. While our results confirm the effectiveness of attracting ECFCs via surface-bound anti-CD34 antibodies, our observation of a nonspecific binding component highlights the importance of considering its consequences in future studies.

Tunable antibacterial coatings that support mammalian cell growth.

Bacterial infections present an enormous problem causing human suffering and cost burdens to healthcare systems worldwide. Here we present novel tunable antibacterial coatings which completely inhibit bacterial colonization by Staphylococcus epidermidis but allow normal adhesion and spreading of osteoblastic cells. The coatings are based on amine plasma polymer films loaded with silver nanoparticles. The method of preparation allows flexible control over the amount of loaded silver nanoparticles and the rate of release of silver ions.

Assessment of nonreleasing antifungal surface coatings bearing covalently attached pharmaceuticals.

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.