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.

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.

Plasma polymerization of (2,2,6,6-tetramethylpiperidin-1-yl)oxyl in a collisional, capacitively coupled radio frequency discharge.

Plasma polymerization of (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) yields thin films containing stable nitroxide radicals that have properties analogous to that of nitric oxide (NO) without short lifetimes. This property gives TEMPO films a wide variety of potential applications. Typically, control of the final film chemistry is difficult and the plasma discharge conditions must be tailored to in order to maximize the retention of these nitroxide groups during the polymerization and deposition process. In this study, plasma diagnostics and surface analysis of the deposited films were carried out to determine the optimal plasma conditions for the retention of nitroxide groups. These techniques included energy-resolved mass spectrometry, heated planar probe ion current measurements, deposition rate measurements, and x-ray photoelectron spectroscopy (XPS). Results show that operating the plasma with a combination of low input powers and high pressures produces a collisional discharge in which fragmentation of the TEMPO molecule is suppressed, leading to good retention of nitroxide groups. Ion energy distribution functions and quartz crystal microbalance measurements support the soft landing theory of ion deposition on the substrate within this γ-mode, in which the flux of low energy, soft landed ions form the primary contribution to film growth. XPS analysis of deposited polymers shows 75.7% retention of N-O groups in the polymer films deposited in a 25 Pa 5 W discharge.

Phenolic-Enriched Collagen Fibrillar Coatings on Titanium Alloy to Promote Osteogenic Differentiation and Reduce Inflammation.

The adsorption of biomolecules on biomaterial surfaces can promote their integration with surrounding tissue without changing their bulk properties. For biomaterials in bone reconstruction, the promotion of osteogenic differentiation and reduction of inflammation are desirable. Fibrillar coatings are interesting because of fibrils' high surface area-volume ratio, aiding adsorption and adhesion. Fibrils also serve as a matrix for the immobilization of biomolecules with biological activity, such as the phenolic compound phloroglucinol (PG), the subunit of marine polyphenols. The aim of this work was to investigate the influence of PG coatings on fibroblast- and osteoblast-like cells to increase the osseointegration of titanium implants. Collagen fibril coatings, containing PG at low and high concentrations, were produced on titanium alloy (Ti6Al4V) scaffolds generated by additive manufacturing (AM). These coatings, especially PG-enriched coatings, reduced hydrophobicity and modulated the behavior of human osteosarcoma SaOS-2 and mouse embryonic fibroblast 3T3 cell lines. Both osteoblastic and fibroblastic cells spread and adhered well on PG-enriched coatings. Coatings significantly reduced the inflammatory response. Moreover, osteogenic differentiation was promoted by collagen coatings with a high PG concentration. Thus, the enrichment of collagen fibril coatings with PG is a promising strategy to improve Ti6Al4V implants for bone contact in orthopedics and dentistry and is worthy of further investigation.

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.

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.