Influence of titanium nanoscale surface roughness on fibrinogen and albumin protein adsorption kinetics and platelet responses.

Biomaterials with nanoscale topography have been increasingly investigated for medical device applications to improve tissue-material interactions. This study assessed the impact of nanoengineered titanium surface domain sizes on early biological responses that can significantly affect tissue interactions. Nanostructured titanium coatings with distinct nanoscale surface roughness were deposited on quartz crystal microbalance with dissipation (QCM-D) sensors by physical vapor deposition. Physico-chemical characterization was conducted to assess nanoscale surface roughness, nano-topographical morphology, wettability, and atomic composition. The results demonstrated increased projected surface area and hydrophilicity with increasing nanoscale surface roughness. The adsorption properties of albumin and fibrinogen, two major plasma proteins that readily encounter implanted surfaces, on the nanostructured surfaces were measured using QCM-D. Significant differences in the amounts and viscoelastic properties of adsorbed proteins were observed, dependent on the surface roughness, protein type, protein concentration, and protein binding affinity. The impact of protein adsorption on subsequent biological responses was also examined using qualitative and quantitative in vitro evaluation of human platelet adhesion, aggregation, and activation. Qualitative platelet morphology assessment indicated increased platelet activation/aggregation on titanium surfaces with increased roughness. These data suggest that nanoscale differences in titanium surface roughness influence biological responses that may affect implant integration.

Protein Adsorption on Chemically Modified Block Copolymer Nanodomains: Influence of Charge and Flow.

Understanding the interactions of biomacromolecules with nanoengineered surfaces is vital for assessing material biocompatibility. This study focuses on the dynamics of protein adsorption on nanopatterned block copolymers (BCPs). Poly(styrene)-block-poly(1,2-butadiene) BCPs functionalized with an acid, amine, amide, or captopril moieties were processed to produce nanopatterned films. These films were characterized using water contact angle measurements and atomic force microscopy in air and liquid to determine how the modification process affected. wettability and swelling. Protein adsorption experiments were conducted under static and dynamic conditions via a quartz crystal microbalance with dissipation. Proteins of various size, charge, and stability were investigated to determine whether their physical characteristics affected adsorption. Significantly decreased contact angles were caused by selective swelling of modified BCP domains. The results indicate that nanopatterned chemistry and experimental conditions strongly impact adsorption dynamics. Depending on the structural stability of the protein, polyelectrolyte surfaces significantly increased adsorption over controls. Further analysis suggested that protein stability may correlate with dissipation versus frequency plots.

Assessment of total silver and silver nanoparticle extraction from medical devices.

There is concern over the release of silver nanoparticles (AgNPs) from medical devices due to their potential toxicological consequences inside the body. Towards developing the exposure component of a risk assessment model, the purpose of this study was to determine the amount and physical form of silver released from medical devices. Scanning electron microscopy was used to confirm that three of five marketed medical devices contained nanosilver coatings (mean feature sizes 115-341 nm). Aqueous device extracts (water, saline and human plasma) were analyzed with inductively coupled plasma mass spectrometry, ultraviolet-visible spectroscopy, dynamic light scattering, transmission electron microscopy, and nanoparticle tracking analysis. The amount of silver extracted from the devices ranged from 1 × 10(-1) to 1 × 10(6) ng/cm(2) (conditions ranged from 37 to 50 °C, over one hour to seven days). The results further indicated that one of the five devices (labeled MD1) released significantly more AgNPs than the other devices. This data suggests that some but not all devices that are formulated with nanosilver may release detectable levels of AgNPs upon extraction. Further work is underway to quantitate the proportion of silver released as AgNPs and to incorporate this data into a risk assessment for AgNP exposure from medical devices.

Silver nanoparticles: correlating nanoparticle size and cellular uptake with genotoxicity.

The focus of this research was to develop a better understanding of the pertinent physico-chemical properties of silver nanoparticles (AgNPs) that affect genotoxicity, specifically how cellular uptake influences a genotoxic cell response. The genotoxicity of AgNPs was assessed for three potential mechanisms: mutagenicity, clastogenicity and DNA strand-break-based DNA damage. Mutagenicity (reverse mutation assay) was assessed in five bacterial strains of Salmonella typhimurium and Echerichia coli, including TA102 that is sensitive to oxidative DNA damage. AgNPs of all sizes tested (10, 20, 50 and 100nm), along with silver nitrate (AgNO3), were negative for mutagenicity in bacteria. No AgNPs could be identified within the bacteria cells using transmission electron microscopy (TEM), indicating these bacteria lack the ability to actively uptake AgNPs 10nm or larger. Clastogenicity (flow cytometry-based micronucleus assay) and intermediate DNA damage (DNA strand breaks as measured in the Comet assay) were assessed in two mammalian white blood cell lines: Jurkat Clone E6-1 and THP-1. It was observed that micronucleus and Comet assay end points were inversely correlated with AgNP size, with smaller NPs inducing a more genotoxic response. TEM results indicated that AgNPs were confined within intracellular vesicles of mammalian cells and did not penetrate the nucleus. The genotoxicity test results and the effect of AgNO3 controls suggest that silver ions may be the primary, and perhaps only, cause of genotoxicity. Furthermore, since AgNO3 was not mutagenic in the gram-negative bacterial Ames strains tested, the lack of bacterial uptake of the AgNPs may not be the major reason for the lack of genotoxicity observed.

Different cytotoxicity responses to antimicrobial nanosilver coatings when comparing extract-based and direct-contact assays.

This study was performed to understand how the choice of cytotoxicity assay format affects the observed biocompatibility of nanosilver (nAg). nAg coatings are physical coatings containing silver (Ag) that have feature sizes of 100 nm or less, often in the form of nanoparticles or grains. They are used on medical devices to prevent infection, but in spite of this intended benefit, observations of potential cytotoxicity from nAg have been reported in numerous published studies. For medical device regulation, cytotoxicity testing is part of a biocompatibility evaluation, in which specific test methods are chosen based on the technological characteristics and intended use of a device. For this study, nAg-coated tissue culture polystyrene surfaces were prepared using magnetron sputter coating, resulting in nAg films of 0.2 to 311 µg cm(-2) Ag. These coatings exhibited nanometer-scale morphologies and demonstrated a > 4log10 reduction in Escherichia coli viability. It was observed that extracts of nAg caused no cytotoxicity to L929 mouse fibroblasts, but cells cultured directly on nAg coatings (direct-contact assay format) showed a dose-dependent reduction in viability by up to 100% (P < 0.001). Results using inductively coupled plasma mass spectrometry to measure Ag release suggested that extracts of nAg are not toxic because the dissolved Ag in those samples becomes less cytotoxic over time, probably owing to the reaction with cell culture media and serum (six-fold cytotoxicity reductions observed over a 24-h period). These findings highlight the potential value of direct-contact cytotoxicity testing for nAg in predicting biological interactions with cells or tissue in vivo.

Assessment of titanium dioxide nanoparticle effects in bacteria: association, uptake, mutagenicity, co-mutagenicity and DNA repair inhibition.

Due to their unique properties, the use of nanoparticles (NPs) is expanding; these same properties may affect their potential risk to humans. However, standard methods for genotoxicity assessment may not be adequate for NPs; altered tests reported here have been developed to address perceived inadequacies. The bacterial reverse mutation assay is an essential part of the battery of tests to determine genotoxicity. The utility of this test for assessing NPs is currently questioned, due to negative results seemingly caused by failure of particle uptake. To probe uptake issues, we examined the physical state in different media, dose and time dependent association, uptake and mutagenicity of titanium dioxide (TiO2) NPs in Salmonella typhimurium and Escherichia coli. The NPs suspended in water were characterized using dynamic light scattering, NP tracking analysis and transmission electron microscopy. NP association with bacteria was assessed by flow cytometry. Association was found to be time and dose dependent, with maximal association by 60 min. Therefore mutagenicity was assessed after a 60 min pre-incubation in a miniaturized assay demonstrating enhanced sensitivity. To assess potential indirect effects on bacterial mutagenicity, the effect of TiO2 NPs on the action of standard mutagens or on DNA repair capability was also investigated. TiO2 NPs did not affect mutant yields in standard strains of S. typhimurium or E. coli, including those detecting oxidative damage, using the modified methods. Nor did TiO2 NPs affect the action of standard mutagens or DNA excision repair capability. Despite particle association with the bacteria, subsequent analysis using electron microscopy and energy dispersive x-ray spectroscopy indicated that the NPs were not internalized. This work demonstrates that additional studies, including flow cytometry, are valuable tools for understanding the action of NPs in biological systems.

Distribution of silver nanoparticles in pregnant mice and developing embryos.

The objective of this study was to evaluate the distribution of silver nanoparticles (NPs) in pregnant mice and their developing embryos. Silver NPs (average diameter 50 nm) were intravenously injected into pregnant CD-1 mice on gestation days (GDs) 7, 8, and 9 at dose levels of 0, 35, or 66 µg Ag/mouse. Mice were euthanised on GD10, and tissue samples were collected and analysed for silver content. Compared with control animals injected with citrate buffer vehicle, silver content was significantly increased (p < 0.05) in nearly all tissues from silver NP-treated mice. Silver accumulation was significantly higher in liver, spleen, lung, tail (injection site), visceral yolk sac, and endometrium compared with other organs from silver NP-treated mice. Furthermore, silver NPs were identified in vesicles in endodermal cells of the visceral yolk sac. In summary, the results demonstrated that silver NPs distributed to most maternal organs, extra-embryonic tissues, and embryos, but did not accumulate significantly in embryos.

The effect of substrate material on silver nanoparticle antimicrobial efficacy.

With the advent of nanotechnology, silver nanoparticles increasingly are being used in coatings, especially in medical device applications, to capitalize on their antimicrobial properties. The attractiveness of nanoparticulate silver systems is the expected increased antimicrobial efficacy relative to their bulk counterparts, which may be attributed to an increased silver ion (Ag+) solubility, and hence availability, that arises from capillarity effects in small, nanometer-sized particles. However, a change of the material upon which the antimicrobial nanoparticulate silver is deposited (herein called "substrate") may affect the availability of Ag+ ions and the intended efficacy of the device. We utilize both theory and experiment to determine the effect of substrate on ion release from silver particles in electrochemical environments and find that substrate surface charge, chemical reactivity or affinity of the surface for Ag+ ions, and wettability of the surface all affect availability of Ag+ ions, and hence antimicrobial efficacy. It is also observed that with time of exposure to deionized water, Ag+ ion release increases to a maximum value at 5 min before decreasing to undetectable levels, which is attributed to coarsening of the nanoparticles, and which subsequently reduces the solubility and availability of Ag+ ions. This coarsening phenomenon is also predicted by the theoretical considerations and has been confirmed experimentally by transmission electron microscopy.

Microstructure and elution of tetracycline from block copolymer coatings.

A critical metrology issue for pharmaceutical industries is the application of analytical techniques for the characterization of drug delivery systems to address interrelationships between processing, structure, and drug release. In this study, cast coatings were formed from solutions of poly(styrene-b-isobutylene-b-styrene) (SIBS) and tetracycline in tetrahydrofuran (THF). These coatings were characterized by several imaging modalities, including time-of-flight secondary ion mass spectrometry (TOF-SIMS) for chemical imaging and analysis, atomic force microscopy (AFM) for determination of surface structure and morphology, and laser scanning confocal microscopy (LSCM), which was used to characterize the three-dimensional structure beneath the surface. The results showed phase separation between the drug and copolymer regions. The size of the tetracycline phase in the polymer matrix ranged from hundreds of nanometers to tens of microns, depending on coating composition. The mass of drug released was not found to be proportional to drug loading, because the size and spatial distribution of the drug phase varied with drug loading and solvent evaporation rate, which in turn affected the amount of drug released.

Osmotic cavitation of elastomeric intraocular lenses.

In recent years, traditional rigid materials have been replaced with softer elastomers in intraocular lenses to minimize the size of the required surgical incision, thereby reducing patient recuperation time. However, water-filled cavities that may impact visual acuity are found in many of these new implants. We demonstrate that the cavitation observed in vivo can occur due to an osmotic pressure difference between the aqueous solution within the cavity and the external media in which the lens is immersed. By reducing the osmolarity of the external solution from 300 to 0mM, we observe an increase in cavity volume of almost a factor of 30. Further, we have developed a model for cavity growth assuming the controlling factor is diffusion of hydrophilic molecules from the polymer matrix into the cavity. We find that the experimental observations are consistent with the model and suggest that oligomeric species generated during polymerization are responsible for the observed cavitation.