Copper-containing glass ceramic with high antimicrobial efficacy.

Hospital acquired infections (HAIs) and the emergence of antibiotic resistant strains are major threats to human health. Copper is well known for its high antimicrobial efficacy, including the ability to kill superbugs and the notorious ESKAPE group of pathogens. We sought a material that maintains the antimicrobial efficacy of copper while minimizing the downsides - cost, appearance and metallic properties - that limit application. Here we describe a copper-glass ceramic powder as an additive for antimicrobial surfaces; its mechanism is based on the controlled release of copper (I) ions (Cu1+) from cuprite nanocrystals that form in situ in the water labile phase of the biphasic glass ceramic. Latex paints containing copper-glass ceramic powder exhibit ≥99.9% reduction in S. aureus, P. aeruginosa, K. aerogenes and E. Coli colony counts when evaluated by the US EPA test method for efficacy of copper-alloy surfaces as sanitizer, approaching that of benchmark metallic copper.

Enzymes produced by autoactivation of blood factor XII in buffer: A contribution from the Hematology at Biomaterial Interfaces Research Group.

High-resolution electrophoresis of FXII-derived proteins produced by contact activation of FXII in buffer solutions (i.e. in absence of plasma proteins) with hydrophilic and silanized-glass activators spanning the observable range of water wettability (hydrophilic to hydrophobic), shows no evidence of proteolytic cleavage of FXII into αFXIIa or ßFXIIa. The autoactivation mixture contains only a single-chain protein with a molecular weight of ∼80 kDa, confirming Oscar Ratnoff's previous finding of a single-chain activated form of FXII that he called 'HFea'. Functional assays have shown that these autoactivation products exhibit procoagulant potential (protease activity inducing clotting of blood) or amidolytic potential (cleaves amino bonds in s-2302 chromogen but do not cause coagulation of plasma) or both amidolytic potential and procoagulant potential. Some of these proteins also have the remarkable potential to 'suppress autoactivation' (i.e. suppress creation of enzymes with procoagulant potential). It is thus hypothesized that autoactivation of FXII in the absence of plasma proteins generates not just a single type of activated conformer, as suggested by previous researchers, but rather an ensemble of conformer products with collective activity that varies with activator surface energy used in contact activation of FXII. Furthermore, reaction of αFXIIa with FXII in buffer solution does not produce additional αFXIIa by the putative autoamplification reaction FXIIa + FXII â†’ 2FXIIa as has been proposed in past literature to account for the discrepancy between chromogenic and plasma-coagulation assays for αFXIIa in buffer solution. Instead, net procoagulant activity measured directly by plasma-coagulation assays, decreases systematically with increasing FXII solution concentration. Under the same reaction conditions, chromogenic assay reveals that net amidolytic activity increases with increasing FXII solution concentration. Thus, although autoamplification does not occur it appears that there is some form of "FXII self reaction" that influences products of αFXIIa reaction with FXII. Electrophoretic measurements indicate that no proteolytic cleavage takes in this reaction leading us to conclude that change in activity is most likely due to change(s) in FXII conformation (with related change in enzyme activity).

A comparison of blood factor XII autoactivation in buffer, protein cocktail, serum, and plasma solutions.

Activation of blood plasma coagulation in vitro by contact with material surfaces is demonstrably dependent on plasma-volume-to-activator-surface-area ratio. The only plausible explanation consistent with current understanding of coagulation-cascade biochemistry is that procoagulant stimulus arising from the activation complex of the intrinsic pathway is dependent on activator surface area. And yet, it is herein shown that activation of the blood zymogen factor XII (Hageman factor, FXII) dissolved in buffer, protein cocktail, heat-denatured serum, and FXI deficient plasma does not exhibit activator surface-area dependence. Instead, a highly-variable burst of procoagulant-enzyme yield is measured that exhibits no measurable kinetics, sensitivity to mixing, or solution-temperature dependence. Thus, FXII activation in both buffer and protein-containing solutions does not exhibit characteristics of a biochemical reaction but rather appears to be a "mechanochemical" reaction induced by FXII molecule interactions with hydrophilic activator particles that do not formally adsorb blood proteins from solution. Results of this study strongly suggest that activator surface-area dependence observed in contact activation of plasma coagulation does not solely arise at the FXII activation step of the intrinsic pathway.

Contact activation of blood plasma and factor XII by ion-exchange resins.

Sepharose ion-exchange particles bearing strong Lewis acid/base functional groups (sulfopropyl, carboxymethyl, quaternary ammonium, dimethyl aminoethyl, and iminodiacetic acid) exhibiting high plasma protein adsorbent capacities are shown to be more efficient activators of blood factor XII in neat-buffer solution than either hydrophilic clean-glass particles or hydrophobic octyl sepharose particles (FXII (activator)→(surface) FXIIa; a.k.a autoactivation, where FXII is the zymogen and FXIIa is a procoagulant protease). In sharp contrast to the clean-glass standard of comparison, ion-exchange activators are shown to be inefficient activators of blood plasma coagulation. These contrasting activation properties are proposed to be due to the moderating effect of plasma-protein adsorption on plasma coagulation. Efficient adsorption of blood-plasma proteins unrelated to the coagulation cascade impedes FXII contacts with ion-exchange particles immersed in plasma, reducing autoactivation, and causing sluggish plasma coagulation. By contrast, plasma proteins do not adsorb to hydrophilic clean glass and efficient autoactivation leads directly to efficient activation of plasma coagulation. It is also shown that competitive-protein adsorption can displace FXIIa adsorbed to the surface of ion-exchange resins. As a consequence of highly-efficient autoactivation and FXIIa displacement by plasma proteins, ion-exchange particles are slightly more efficient activators of plasma coagulation than hydrophobic octyl sepharose particles that do not bear strong Lewis acid/base surface functionalities but to which plasma proteins adsorb efficiently. Plasma proteins thus play a dual role in moderating contact activation of the plasma coagulation cascade. The principal role is impeding FXII contact with activating surfaces, but this same effect can displace FXIIa from an activating surface into solution where the protease can potentiate subsequent steps of the plasma coagulation cascade.

Amidolytic, procoagulant, and activation-suppressing proteins produced by contact activation of blood factor XII in buffer solution.

The relative proportions of enzymes with amidolytic or procoagulant activity produced by contact activation of the blood zymogen factor XII (FXII, Hageman factor) in buffer solution depends on activator surface chemistry/energy. As a consequence, chromogenic assay of amidolytic activity (cleavage of amino acid bonds in s-2302 chromogen) does not correlate with the traditional plasma coagulation time assay for procoagulant activity (protease activity inducing clotting of blood plasma). Amidolytic activity did not vary significantly with activator particle surface energy, herein measured as water adhesion tension τ(o)=γ(lv)(o)cosθ(a) ; where γ(lv)(o) is pure buffer interfacial tension and θ(a) is the advancing contact angle. By contrast, procoagulant activity varied as a parabolic-like function of τ(o), high at both hydrophobic and hydrophilic extremes of activator surface energy and falling through a broad minimum within a 20<τ(o)<40 mJ/m(2) (55°<θ(a) < 75°) range, corroborating and expanding previously-published work. It is inferred from these functional assays that an unknown number of protein fragments are produced by contact activation of FXII (a.k.a. autoactivation) rather than just αFXIIa and ßFXIIa as popularly believed. Autoactivation products produced by activator particles within the 20<τ(o)<40 mJ/m(2) (55°<θ(a) < 75°) surface-energy range suppresses production of procoagulant enzymes by activators selected from the hydrophobic or hydrophilic surface-energy extremes through as-yet unknown biophysical chemistry. Suppression proteins may be responsible for the experimentally-observed autoinhibition of the autoactivation reaction.

Surface-energy dependent contact activation of blood factor XII.

Contact activation of blood factor XII (FXII, Hageman factor) in neat-buffer solution exhibits a parabolic profile when scaled as a function of silanized-glass-particle activator surface energy (measured as advancing water adhesion tension tau(a)(o)=gamma(lv)(o)cos theta in dyne/cm, where gamma(lv)(o) is water interfacial tension in dyne/cm and theta is the advancing contact angle). Nearly equal activation is observed at the extremes of activator water-wetting properties -36

Volumetric interpretation of protein adsorption: capacity scaling with adsorbate molecular weight and adsorbent surface energy.

Silanized-glass-particle adsorbent capacities are extracted from adsorption isotherms of human serum albumin (HSA, 66 kDa), immunoglobulin G (IgG, 160 kDa), fibrinogen (Fib, 341 kDa), and immunoglobulin M (IgM, 1000 kDa) for adsorbent surface energies sampling the observable range of water wettability. Adsorbent capacity expressed as either mass-or-moles per-unit-adsorbent-area increases with protein molecular weight (MW) in a manner that is quantitatively inconsistent with the idea that proteins adsorb as a monolayer at the solution-material interface in any physically-realizable configuration or state of denaturation. Capacity decreases monotonically with increasing adsorbent hydrophilicity to the limit-of-detection (LOD) near tau(o) = 30 dyne/cm (theta approximately 65 degrees) for all protein/surface combinations studied (where tau(o) identical with gamma(lv)(o) costheta is the water adhesion tension, gamma(lv)(o) is the interfacial tension of pure-buffer solution, and theta is the buffer advancing contact angle). Experimental evidence thus shows that adsorbent capacity depends on both adsorbent surface energy and adsorbate size. Comparison of theory to experiment implies that proteins do not adsorb onto a two-dimensional (2D) interfacial plane as frequently depicted in the literature but rather partition from solution into a three-dimensional (3D) interphase region that separates the physical surface from bulk solution. This interphase has a finite volume related to the dimensions of hydrated protein in the adsorbed state (defining "layer" thickness). The interphase can be comprised of a number of adsorbed-protein layers depending on the solution concentration in which adsorbent is immersed, molecular volume of the adsorbing protein (proportional to MW), and adsorbent hydrophilicity. Multilayer adsorption accounts for adsorbent capacity over-and-above monolayer and is inconsistent with the idea that protein adsorbs to surfaces primarily through protein/surface interactions because proteins within second (or higher-order) layers are too distant from the adsorbent surface to be held surface bound by interaction forces in close proximity. Overall, results are consistent with the idea that protein adsorption is primarily controlled by water/surface interactions.

Volumetric interpretation of protein adsorption: kinetics of protein-adsorption competition from binary solution.

The standard solution-depletion method is implemented with SDS-gel electrophoresis as a multiplexing, separation-and-quantification tool to measure competition between two proteins (i and j) for adsorption to the same hydrophobic adsorbent particles (either octyl sepharose or silanized glass) immersed in binary-protein solutions. Adsorption kinetics reveals an unanticipated slow protein-size-dependent competition that controls steady-state adsorption selectivity. Two sequential pseudo-steady-state adsorption regimes (State 1 and State 2) are frequently observed depending on i, j solution concentrations. State 1 and State 2 are connected by a smooth transition, giving rise to sigmoidally-shaped adsorption-kinetic profiles with a downward inflection near 60 min of solution/adsorbent contact. Mass ratio of adsorbed i, j proteins (m(i)/m(j)) remains nearly constant between States 1 and 2, even though both m(i) and m(j) decrease in the transition between states. State 2 is shown to be stable for 24 h of continuous-adsorbent contact with stagnant solution whereas State 2 is eliminated by continuous mixing of adsorbent with solution. In sharp contrast to binary-competition results, adsorption to hydrophobic adsorbent particles from single-protein solutions (pure i or j) exhibits no detectable kinetics within the timeframe of experiment from either stagnant or continuously mixed solution, quickly achieving a single steady-state value in proportion to solution concentration. Comparison of binary competition between dissimilarly-sized protein pairs chosen to span a broad molecular-weight (MW) range demonstrates that selectivity between i and j scales with MW ratio that is proportional to protein-volume ratio (ubiquitin, Ub, MW=10.7 kDa; human serum albumin, HSA, MW=66.3 kDa; prothrombin, FII, 72 kDa; immunoglobulin G, IgG, MW=160 kDa; fibrinogen, Fib, MW=341 kDa). Results are interpreted in terms of a kinetic model of adsorption that has protein molecules rapidly diffusing into an inflating interphase that is spontaneously formed by bringing a protein solution into contact with a physical surface (State 1). State 2 follows by rearrangement of proteins within this interphase to achieve the maximum interphase concentration (dictated by energetics of interphase dehydration) within the thinnest (lowest volume) interphase possible by ejection of interphase water and initially-adsorbed proteins. Implications for understanding biocompatibility are discussed using a computational example relevant to the problem of blood-plasma coagulation.