Lethal Interactions of Atomically Precise Gold Nanoclusters and Pseudomonas aeruginosa and Staphylococcus aureus Bacterial Cells.

Ultrasmall metal nanoclusters (NCs) are employed in an array of diagnostic and therapeutic applications due to their tunable photoluminescence, high biocompatibility, polyvalent effect, ease of modification, and photothermal stability. However, gold nanoclusters' (AuNCs') intrinsically antimicrobial properties remain poorly explored and are not well understood. Here, we share an insight into the antimicrobial action of atomically precise AuNCs based on their ability to passively translocate across the bacterial membrane. Functionalized by a hydrophilic modified-bidentate sulfobetaine zwitterionic molecule (AuNC-ZwBuEt) or a more hydrophobic monodentate-thiolate, mercaptohexanoic acid (AuNC-MHA) molecule, 2 nm AuNCs were lethal to both Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus bacteria. The bactericidal efficiency was found to be bacterial strain-, time-, and concentration-dependent. The direct visualizations of the translocation of AuNCs and AuNC-cell and subcellular interactions were investigated using cryo-soft X-ray nano-tomography, transmission electron microscopy (TEM), and scanning TEM energy-dispersive spectroscopy analyses. AuNC-MHA were identified in the bacterial cytoplasm within 30 min, without evidence of the loss of membrane integrity. It is proposed that the bactericidal effect of AuNCs is attributed to their size, which allows for efficient energy-independent translocation across the cell membrane. The internalization of both AuNCs caused massive internal damage to the cells, including collapsed subcellular structures and altered cell morphology, leading to the eventual loss of cellular integrity.

Tunable morphological changes of asymmetric titanium nanosheets with bactericidal properties.

HYPOTHESIS: Titanium and titanium alloys are often the most popular choice of material for the manufacture of medical implants; however, they remain susceptible to the risk of device-related infection caused by the presence of pathogenic bacteria. Hydrothermal etching of titanium surfaces, to produce random nanosheet topologies, has shown remarkable ability to inactivate pathogenic bacteria via a physical mechanism. We expect that systematic tuning of the nanosheet morphology by controlling fabrication parameters, such as etching time, will allow for optimisation of the surface pattern for superior antibacterial efficacy. EXPERIMENTS: Using time-dependent hydrothermal processing of bulk titanium, we fabricated bactericidal nanosheets with variable nanoedge morphologies according to a function of etching time. A systematic study was performed to compare the bactericidal efficiency of nanostructured titanium surfaces produced at 0.5, 1, 2, 3, 4, 5, 6, 24 and 60 h of hydrothermal etching. FINDINGS: Titanium surfaces hydrothermally treated for a period of 6 h were found to achieve maximal antibacterial efficiency of 99 ±â€¯3% against Gram-negative Pseudomonas aeruginosa and 90 ±â€¯9% against Gram-positive Staphylococcus aureus bacteria, two common human pathogens. These surfaces exhibited nanosheets with sharp edges of approximately 10 nm. The nanotopographies presented in this work exhibit the most efficient mechano-bactericidal activity against both Gram-negative and Gram-positive bacteria of any nanostructured titanium topography reported thus far.

The Fate of Osteoblast-Like MG-63 Cells on Pre-Infected Bactericidal Nanostructured Titanium Surfaces.

Biomaterials that have been newly implanted inside the body are the substratum targets for a "race for the surface", in which bacterial cells compete against eukaryotic cells for the opportunity to colonize the surface. A victory by the former often results in biomaterial-associated infections, which can be a serious threat to patient health and can undermine the function and performance of the implant. Moreover, bacteria can often have a 'head start' if implant contamination has taken place either prior to or during the surgery. Current prevention and treatment strategies often rely on systemic antibiotic therapies, which are becoming increasingly ineffective due to a growing prevalence of antibiotic-resistant bacteria. Nanostructured surfaces that kill bacteria by physically rupturing bacterial cells upon contact have recently emerged as a promising solution for the mitigation of bacterial colonization of implants. Furthermore, these nanoscale features have been shown to enhance the adhesion and proliferation of eukaryotic cells, which is a key to, for example, the successful osseointegration of load-bearing titanium implants. The bactericidal activity and biocompatibility of such nanostructured surfaces are often, however, examined separately, and it is not clear to what extent bacterial cell-surface interactions would affect the subsequent outcomes of host-cell attachment and osseointegration processes. In this study, we investigated the ability of bactericidal nanostructured titanium surfaces to support the attachment and growth of osteoblast-like MG-63 human osteosarcoma cells, despite them having been pre-infected with pathogenic bacteria. MG-63 is a commonly used osteoblastic model to study bone cell viability, adhesion, and proliferation on the surfaces of load-bearing biomaterials, such as titanium. The nanostructured titanium surfaces used here were observed to kill the pathogenic bacteria, whilst simultaneously enhancing the growth of MG-63 cells in vitro when compared to that occurring on sterile, flat titanium surfaces. These results provide further evidence in support of nanostructured bactericidal surfaces being used as a strategy to help eukaryotic cells win the "race for the surface" against bacterial cells on implant materials.

PC 12 Pheochromocytoma Cell Response to Super High Frequency Terahertz Radiation from Synchrotron Source.

High frequency (HF) electromagnetic fields (EMFs) have been widely used in many wireless communication devices, yet within the terahertz (THz) range, their effects on biological systems are poorly understood. In this study, electromagnetic radiation in the range of 0.3⁻19.5 × 1012 Hz, generated using a synchrotron light source, was used to investigate the response of PC 12 neuron-like pheochromocytoma cells to THz irradiation. The PC 12 cells remained viable and physiologically healthy, as confirmed by a panel of biological assays; however, exposure to THz radiation for 10 min at 25.2 ± 0.4 °C was sufficient to induce a temporary increase in their cell membrane permeability. High-resolution transmission electron microscopy (TEM) confirmed cell membrane permeabilization via visualisation of the translocation of silica nanospheres (d = 23.5 ± 0.2 nm) and their clusters (d = 63 nm) into the PC 12 cells. Analysis of scanning electron microscopy (SEM) micrographs revealed the formation of atypically large (up to 1 µm) blebs on the surface of PC 12 cells when exposed to THz radiation. Long-term analysis showed no substantial differences in metabolic activity between the PC 12 cells exposed to THz radiation and untreated cells; however, a higher population of the THz-treated PC 12 cells responded to the nerve growth factor (NGF) by extending longer neurites (up to 0⁻20 µm) compared to the untreated PC12 cells (up to 20 µm). These findings present implications for the development of nanoparticle-mediated drug delivery and gene therapy strategies since THz irradiation can promote nanoparticle uptake by cells without causing apoptosis, necrosis or physiological damage, as well as provide a deeper fundamental insight into the biological effects of environmental exposure of cells to electromagnetic radiation of super high frequencies.

Exposure to high-frequency electromagnetic field triggers rapid uptake of large nanosphere clusters by pheochromocytoma cells.

BACKGROUND: Effects of man-made electromagnetic fields (EMF) on living organisms potentially include transient and permanent changes in cell behaviour, physiology and morphology. At present, these EMF-induced effects are poorly defined, yet their understanding may provide important insights into consequences of uncontrolled (e.g., environmental) as well as intentional (e.g., therapeutic or diagnostic) exposure of biota to EMFs. In this work, for the first time, we study mechanisms by which a high frequency (18 GHz) EMF radiation affects the physiology of membrane transport in pheochromocytoma PC 12, a convenient model system for neurotoxicological and membrane transport studies. METHODS AND RESULTS: Suspensions of the PC 12 cells were subjected to three consecutive cycles of 30s EMF treatment with a specific absorption rate (SAR) of 1.17 kW kg-1, with cells cooled between exposures to reduce bulk dielectric heating. The EMF exposure resulted in a transient increase in membrane permeability for 9 min in up to 90 % of the treated cells, as demonstrated by rapid internalisation of silica nanospheres (diameter d ≈ 23.5 nm) and their clusters (d ≈ 63 nm). In contrast, the PC 12 cells that received an equivalent bulk heat treatment behaved similar to the untreated controls, showing lack to minimal nanosphere uptake of approximately 1-2 %. Morphology and growth of the EMF treated cells were not altered, indicating that the PC 12 cells were able to remain viable after the EMF exposure. The metabolic activity of EMF treated PC 12 cells was similar to that of the heat treated and control samples, with no difference in the total protein concentration and lactate dehydrogenase (LDH) release between these groups. CONCLUSION: These results provide new insights into the mechanisms of EMF-induced biological activity in mammalian cells, suggesting a possible use of EMFs to facilitate efficient transport of biomolecules, dyes and tracers, and genetic material across cell membrane in drug delivery and gene therapy, where permanent permeabilisation or cell death is undesirable.

Pheochromocytoma (PC12) Cell Response on Mechanobactericidal Titanium Surfaces.

Titanium is a biocompatible material that is frequently used for making implantable medical devices. Nanoengineering of the surface is the common method for increasing material biocompatibility, and while the nanostructured materials are well-known to represent attractive substrata for eukaryotic cells, very little information has been documented about the interaction between mammalian cells and bactericidal nanostructured surfaces. In this study, we investigated the effect of bactericidal titanium nanostructures on PC12 cell attachment and differentiation—a cell line which has become a widely used in vitro model to study neuronal differentiation. The effects of the nanostructures on the cells were then compared to effects observed when the cells were placed in contact with non-structured titanium. It was found that bactericidal nanostructured surfaces enhanced the attachment of neuron-like cells. In addition, the PC12 cells were able to differentiate on nanostructured surfaces, while the cells on non-structured surfaces were not able to do so. These promising results demonstrate the potential application of bactericidal nanostructured surfaces in biomedical applications such as cochlear and neuronal implants.

Bactericidal activity of self-assembled palmitic and stearic fatty acid crystals on highly ordered pyrolytic graphite.

The wings of insects such as cicadas and dragonflies have been found to possess nanostructure arrays that are assembled from fatty acids. These arrays can physically interact with the bacterial cell membranes, leading to the death of the cell. Such mechanobactericidal surfaces are of significant interest, as they can kill bacteria without the need for antibacterial chemicals. Here, we report on the bactericidal effect of two of the main lipid components of the insect wing epicuticle, palmitic (C16) and stearic (C18) fatty acids. Films of these fatty acids were re-crystallised on the surface of highly ordered pyrolytic graphite. It appeared that the presence of two additional CH2 groups in the alkyl chain resulted in the formation of different surface structures. Scanning electron microscopy and atomic force microscopy showed that the palmitic acid microcrystallites were more asymmetric than those of the stearic acid, where the palmitic acid microcrystallites were observed to be an angular abutment in the scanning electron micrographs. The principal differences between the two types of long-chain saturated fatty acid crystallites were the larger density of peaks in the upper contact plane of the palmitic acid crystallites, as well as their greater proportion of asymmetrical shapes, in comparison to that of the stearic acid film. These two parameters might contribute to higher bactericidal activity on surfaces derived from palmitic acid. Both the palmitic and stearic acid crystallite surfaces displayed activity against Gram-negative, rod-shaped Pseudomonas aeruginosa and Gram-positive, spherical Staphylococcus aureus cells. These microcrystallite interfaces might be a useful tool in the fabrication of effective bactericidal nanocoatings. STATEMENT OF SIGNIFICANCE: Nanostructured cicada and dragonfly wing surfaces have been discovered to be able physically kill bacterial cells. Here, we report on the successful fabrication of bactericidal three-dimensional structures of two main lipid components of the epicuticle of insect wings, palmitic (C16) and stearic (C18) acids. After crystallisation onto highly ordered pyrolytic graphite, both the palmitic and stearic acid films displayed bactericidal activity against both Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus cells. The simplicity of the production of these microcrystallite interfaces suggests that a fabrication technique, based on solution deposition, could be an effective technique for the application of bactericidal nanocoatings.