Duncan S. Sutherland

Toxicity of silver nanoparticles—Nanoparticle or silver ion?

The toxicity of silver nanoparticles (AgNPs) has been shown in many publications. Here we investigated to which degree the silver ion fraction of AgNP suspensions, contribute to the toxicity of AgNPs in A549 lung cells. Cell viability assays revealed that AgNP suspensions were more toxic when the initial silver ion fraction was higher. At 1.5μg/ml total silver, A549 cells exposed to an AgNP suspension containing 39% silver ion fraction showed a cell viability of 92%, whereas cells exposed to an AgNP suspension containing 69% silver ion fraction had a cell viability of 54% as measured by the MTT assay. In addition, at initial silver ion fractions of 5.5% and above, AgNP-free supernatant had the same toxicity as AgNP suspensions. Flow-cytometric analyses of cell cycle and apoptosis confirmed that there is no significant difference between the treatment with AgNP suspension and AgNP supernatant. Only AgNP suspensions with silver ion fraction of 2.6% or less were significantly more toxic than their supernatant as measured by MTT assays. From our data we conclude that at high silver ion fractions (≥5.5%) the AgNPs did not add measurable additional toxicity to the AgNP suspension, whereas at low silver ion fractions (≤2.6%) AgNP suspensions are more toxic than their supernatant.

Nanoscale features influence epithelial cell morphology and cytokine production

Available, easy and fast fabrication methods of nanostructured surfaces, and the knowledge that cells in vivo interacts with nanometer-sized structures/objects, led us to study the impact of nanotopography on cell morphology and cytokine production. Uroepithelial cells were seeded on three different substrate types: two with defined nanometer topographies and a flat control, all three having identical surface chemistry. The nanostructured substrates contained hemispherical pillars or step edges, the latter in the form of parallel grooves and ridges. Qualitative and quantitative analysis of cell morphology and cytokine production were studied. Both quantities were significantly different between cells cultured on hemispherically structured surfaces compared to flat control surfaces. Cells cultured on hemispherically structured surfaces showed a decrease in IL-6 and IL-8 production and were less spread, less round and more stellate (larger dispersion). Only cell morphology differed between cells cultured on grooved surfaces and flat control surfaces. These findings suggest that epithelial cell morphology and cytokine production are dependent on the underlying nanotopography.

Control of nanoparticle film structure for colloidal lithography

Colloidal lithography utilises nanoparticles’ ability to self-organise on surfaces, which make them suitable as lithographic masks for the production of nano sized surface features. Adsorption under the influence of electrostatic particle–particle interactions results in ordered structures with the particles separated an average distance described by the random sequential adsorption model (RSA). Large areas (cm2) with a high density of nanoparticles can be covered, which is very useful for application areas like biosensors, biomaterials and catalysis, where large numbers of nano sized features are often required. Feature size, shape and spacing can be systematically varied. In this work methods to control the deposition of films consisting of polystyrene particles on flat oxidised titanium surfaces for particle sizes between 20 and 500 nm and coverages of 0–0.45 are demonstrated and discussed. Experimental difficulties encountered were aggregation of particles at high coverage/large particles and a low coverage limit at low ionic strengths. Experimental solutions to overcome these limitations, maintaining the fast parallel processing advantage of colloidal lithography, are presented. They include heating to stabilize initial (i.e. after adsorption) particle arrangements, use of spacer silica particles, and plasma etching to reduce particle sizes.

Enhanced nanoplasmonic optical sensors with reduced substrate effect.

We present a straightforward method to double the refractive index sensitivity of surface-supported nanoplasmonic optical sensors by lifting the metal nanoparticles above the substrate by a dielectric nanopillar. The role of the pillar is to substantially decrease the spatial overlap between the substrate and the enhanced fields generated at plasmon resonance. Data presented for nanodisks and nanoellipsoids supported by pillars of varying heights are found to be in excellent agreement with electrodynamics simulations. The described concepts apply to multitude of plasmonic nanostructures, fabricated by top-down or bottom-up techniques, and are likely to further facilitate the development of novel nanooptical sensors for biomedicine and diagnostics.

Response of rat osteoblast-like cells to microstructured model surfaces in vitro

The role of surface microtopography in combination with different surface wettability for rat calvaria cell differentiation was examined. Mineralization and alkaline phosphatase (ALP) activity of rat calvaria cells on flat polydimethylsiloxane (PDMS) or PDMS contained pyramids which were either hydrophilic or hydrophobic were compared. ALP expressing cells were more frequent on hydrophilic PDMS contained pyramids. ALP activity, peaked at day 9, was highest for hydrophilic pyramids followed by hydrophobic pyramids and flat hydrophilic PDMS surfaces. A similar pattern was obtained with respect to mineralized nodules. These observations showed that micro-sized surface features promote differentiation of rat calvaria cells. Further, hydrophilic surfaces are more prone to stimulate differentiation in comparison with hydrophobic surfaces. The results suggest that both material surface chemistry and topography affect osteoblast differentiation.

Quantitative assessment of the response of primary derived human osteoblasts and macrophages to a range of nanotopography surfaces in a single culture model in vitro

The effect of nanotopography on a range of Ti oxide surfaces was determined. Flat Ti, 3%, 19%, 30% and 43% topography densities of 110 nm high hemispherical protrusions were cultured in contact with primary derived human macrophages and osteoblasts in single culture models. Prior to introduction of the test substrate the phenotype and optimum conditions for in vitro cell culture were established. The cellular response was investigated and quantified by assessments of cytoskeletal development and orientation, viable cell adhesion, cytokine production and release and RT-PCR analysis of osteogenic markers. The tested nanotopographies did not have a statistically significant effect on viable cell adhesion and subsequent cytoskeletal formation. Surface chemistry was the dominant factor as established via incorporation of a tissue culture polystyrene, TCPS, control. The topography surfaces induced a release of chemotactic macrophage activation agents at 1 day in conjunction with stress fibre formation and a subsequent fibronectin network formation. Osteoblasts migrated away from the topography surfaces to the exposed TCPS within the wells during the 7-day period.

Influence of systematically varied nanoscale topography on the morphology of epithelial cells

With the knowledge that cells can react to lithographically manufactured nanometer-sized surface objects, our interest concerned whether cells would respond to surface structures of systematically increasing size. Our approach to answer this question was to fabricate surfaces with the same surface chemistry and similar surface roughness but increasing size of structural features. To fabricate large areas of patterned surfaces, required for cell culture studies, we used colloidal lithography utilizing colloidal particles as a template for surface nanostructuring. The fabricated surfaces contained hemispherical nanopillars with diameters ranging from 60 to 170 nm. Changes in cell morphology of a pancreatic epithelial cell line (AR4-2J) were studied by evaluating cell area and cell shape. The latter was studied by applying the cell shape classification method using three shape descriptors. The pancreatic cells responded in a systematic way to the surface nanostructures. The cells spread more and became more nonround when cultured on surfaces with increasing size of the topographic features.

The effects of continuous and discontinuous groove edges on cell shape and alignment

Nanofabricated model surfaces and digital image analysis of cell shape were used to address the importance of a continuous sharp edge in the alignment of cells to shallow surface grooves. The grooved model surfaces had either continuous or discontinuous edges of various depths (40-400 nm) but identical surface chemistry and groove/ridge dimensions (15 microm wide). Epithelial cells were cultured on the model surfaces for 10 and 24 h. Fluorescence microscopy combined with image analysis were used to quantify cell area and alignment and to make cell shape classifications of individual cells. The degrees of alignment of cells and the percentages of elongated cell classes increased with groove depth on samples with continuous grooves. Two main differences, with regard to cell response, were observed between the continuous and discontinuous grooved surfaces. First, significantly fewer cells aligned to surface grooves with discontinuous edges than to grooves with continuous edges. Second, there were lower percentages of the elongated cell classes on discontinuous grooves than on continuous ones. We concluded that grooved surfaces with continuous edges are more potent in aligning and inducing elongated cells. The results from the present study suggest that a mechanism of alignment involving orientation along a continuous edge is likely.

Coherent imaging of nanoscale plasmon patterns with a carbon nanotube optical probe

We introduce a carbon nanotube as optical near-field probe and apply it to visualize the plasmon fields of metal nanostructures in both amplitude and phase at 30 nm resolution. With 91 nm Au disks designed for fundamental plasmon resonance, we observe the antiphase optical fields near two pole regions that are evidence of dipolar oscillation, in good agreement with theoretical field patterns. This opens the door to phase-sensitively map optical propagation and storage in photonic crystals and nanooptic resonators or circuits, in particular to verify coherent control of plasmon polaritons.

Nanomechanotransduction and interphase nuclear organization influence on genomic control.

The ability of cells to alter their genomic regulation in response to mechanical conditioning or through changes in morphology and the organization of the interphase nuclei are key questions in cell biology. Here, two nanotopographies have been used as a model surfaces to change cell morphology in order to investigate spatial genomic changes within the nuclei of fibroblasts. Initially, centromeres for chromosome pairs were labeled and the average distance on different substrates calculated. Further to this, Affymetrix whole genome GeneChips® were used to rank genomic changes in response to topography and plot the whereabouts on the chromosomes these changes were occurring. It was seen that as cell spreading was changed, so were the positions along the chromosomes that gene regulations were being observed. We hypothesize that as changes in cell and thus nuclear morphology occur, that this may alter the probability of transcription through opening or closing areas of the chromosomes to transcription factors. J. Cell. Biochem. 102: 1234–1244, 2007.