Au nanoparticles for SERS: Temperature-controlled nanoparticle morphologies and their Raman enhancing properties.

Quasi-fractal gold nanoparticles can be synthesized via a modified and temperature controlled procedure initially used for the synthesis of star-like gold nanoparticles. The surface features of nanoparticles lead to improved enhancement of Raman scattering intensity of analyte molecules due to the increased number of sharp surface features possessing numerous localized surface plasmon resonances (LSPR). The LSPR is affected by the size and shape of surface features as well as inter-nanoparticle interactions, as these affect the oscillation modes of electrons on the nanoparticle surfaces. The effect of the particle morphologies on the localized surface plasmon resonance (LSPR) and on the surface-enhancing capabilities of these nanoparticles is explored by comparing different nanoparticle morphologies and concentrations. We show that in a fixed nanoparticle concentration regime, quasi-fractal gold nanoparticles (gold nanocaltrop) provide the highest level of surface enhancement, whereas spherical nanoparticles provide the largest enhancement in a fixed gold concentration regime. The presence of highly branched features enables these nanoparticles to couple with a laser wavelength, despite having no strong absorption band and hence no single surface plasmon resonance. This cumulative LSPR may allow these nanoparticles to be used in a variety of applications in which laser wavelength flexibility is beneficial, such as in medical imaging applications where fluorescence at short laser wavelengths may be coupled with non-fluorescing long laser wavelengths for molecular sensing.

Raman Signal Enhancement by Quasi-Fractal Geometries of Au Nanoparticles.

The synthesis of star-like gold nanoparticles (SGNs) in a temperature-controlled environment allows for temperature modulation and facilitates the growth of highly branched nanoparticles. By increasing the synthesis temperature, the level of branching increases as well. These highly branched features represent a distinctly novel, quasi-fractal nanoparticle morphology, referred to herein as gold nano caltrops (GNC). The increased surface roughness, local curvature and degree of inhomogeneity of GNC lend themselves to generating improved enhancement of the scattering signals in surface-enhanced Raman spectroscopy (SERS) via a mechanism in which the localized surface plasmon sites, or "hot spots," provide the engine for the signal amplification, rather than the more conventional surface plasmon. Here, the synthesis procedure and the surface-enhancing capabilities of GNC are described and discussed.

Gold Nanoparticles and Radio Frequency Field Interactions: Effects of Nanoparticle Size, Charge, Aggregation, Radio Frequency, and Ionic Background.

In this study, we investigated experimentally the dependency of radio frequency (rf) absorption by gold nanoparticles (AuNPs) on frequency (10 kHz to 450 MHz), NP size (3.5, 17, and 36 nm), charge of the ligand shell (positive amino and negative carboxylic functional groups), aggregation state, and presence of electrolytes (0-1 M NaCl). In addition, we examined the effect of protein corona on the rf absorption by AuNPs. For the first time, rf energy absorption by AuNPs was analyzed in the 10 kHz to 450 MHz rf range. We have demonstrated that the previously reported rf heating of AuNPs can be solely attributed to the heating of the ionic background and AuNPs do not absorb noticeable rf energy regardless of the NP size, charge, aggregation, and presence of electrolytes. However, the formation of protein corona on the AuNP surface resulted in rf energy absorption by AuNP-albumin constructs, suggesting that protein corona might be partially responsible for the heating of AuNPs observed in vivo. The optimal frequency of rf absorption for the AuNP-albumin constructs is significantly higher than conventional 13.56 MHz, suggesting that the heating of AuNPs in rf field should be performed at considerably higher frequencies for better results in vivo.

Cell-based cytotoxicity assays for engineered nanomaterials safety screening: exposure of adipose derived stromal cells to titanium dioxide nanoparticles.

BACKGROUND: Increasing production of nanomaterials requires fast and proper assessment of its potential toxicity. Therefore, there is a need to develop new assays that can be performed in vitro, be cost effective, and allow faster screening of engineered nanomaterials (ENMs). RESULTS: Herein, we report that titanium dioxide (TiO2) nanoparticles (NPs) can induce damage to adipose derived stromal cells (ADSCs) at concentrations which are rated as safe by standard assays such as measuring proliferation, reactive oxygen species (ROS), and lactate dehydrogenase (LDH) levels. Specifically, we demonstrated that low concentrations of TiO2 NPs, at which cellular LDH, ROS, or proliferation profiles were not affected, induced changes in the ADSCs secretory function and differentiation capability. These two functions are essential for ADSCs in wound healing, energy expenditure, and metabolism with serious health implications in vivo. CONCLUSIONS: We demonstrated that cytotoxicity assays based on specialized cell functions exhibit greater sensitivity and reveal damage induced by ENMs that was not otherwise detected by traditional ROS, LDH, and proliferation assays. For proper toxicological assessment of ENMs standard ROS, LDH, and proliferation assays should be combined with assays that investigate cellular functions relevant to the specific cell type.

Exposure to TiO2 nanoparticles increases Staphylococcus aureus infection of HeLa cells.

BACKGROUND: Titanium dioxide (TiO2) is one of the most common nanoparticles found in industry ranging from food additives to energy generation. Approximately four million tons of TiO2 particles are produced worldwide each year with approximately 3000 tons being produced in nanoparticulate form, hence exposure to these particles is almost certain. RESULTS: Even though TiO2 is also used as an anti-bacterial agent in combination with UV, we have found that, in the absence of UV, exposure of HeLa cells to TiO2 nanoparticles significantly increased their risk of bacterial invasion. HeLa cells cultured with 0.1 mg/ml rutile and anatase TiO2 nanoparticles for 24 h prior to exposure to bacteria had 350 and 250 % respectively more bacteria per cell. The increase was attributed to bacterial polysaccharides absorption on TiO2 NPs, increased extracellular LDH, and changes in the mechanical response of the cell membrane. On the other hand, macrophages exposed to TiO2 particles ingested 40 % fewer bacteria, further increasing the risk of infection. CONCLUSIONS: In combination, these two factors raise serious concerns regarding the impact of exposure to TiO2 nanoparticles on the ability of organisms to resist bacterial infection.

The impact of TiO2 nanoparticle exposure on transmembrane cholesterol transport and enhanced bacterial infectivity in HeLa cells.

We have previously shown that exposure to TiO2 nanoparticles (NPs) reduces the resistance of HeLa cells to bacterial infection. Here we demonstrate that the increased infectivity is associated with enhanced asymmetry in the cholesterol distribution. We applied a live cell imaging method which uses tunable orthogonal cholesterol sensors to visualize and quantify in-situ cholesterol distribution between the two leaflets of the plasma membrane (PM). In the control culture, we found marked transbilayer asymmetry of cholesterol, with the concentration in the outer plasma membrane (OPM) being 13 ± 2-fold higher than that in the inner plasma membrane (IPM). Exposure of the culture to 0.1 mg/mL of rutile TiO2 NPs increased the asymmetry such that the concentration in the OPM was 51 ± 10 times higher, while the total cholesterol content increased only 21 ± 2%. This change in cholesterol gradient may explain the increase in bacterial infectivity in HeLa cells exposed to TiO2 NPs since many pathogens, including Staphylococcus aureus used in the present study, require cholesterol for proper membrane attachment and virulence. RT-PCR indicated that exposure to TiO2 was responsible for upregulation of the ABCA1 and ABCG1 mRNAs, which are responsible for the production of the cholesterol transporter proteins that facilitate cholesterol transport across cellular membranes. This was confirmed by the observation of an overall decrease in bacterial infection in ABCA1 knockout or methyl-ß-cyclodextrin-treated HeLa cells, as regardless of TiO2 NP exposure. Hence rather than preventing bacterial infection, TiO2 nanoparticles upregulate genes associated with membrane cholesterol production and distribution, hence increasing infectivity. STATEMENT OF SIGNIFICANCE: A great deal of work has been done regarding the toxicology of the particles, especially focusing on detrimental outcomes associated with reactive oxygen species (ROS) production. In this paper we show unambiguously a very surprising result, namely the ability of these particles to enhance bacterial infection even at very small exposure levels, where none of the deleterious effects of ROS products can yet be detected. Using a new imaging technique, we are able to demonstrate, in operando, the effect of the particles on cholesterol generation and distribution in live HeLa cells. This paper also represents the first in a series where we explore other consequences of increased membrane cholesterol, due to particle exposure, which are known to have multiple other consequences on human tissue function and development.

Surface-Enhanced Raman Spectroscopy Characterization of Breast Cell Phenotypes: Effect of Nanoparticle Geometry.

The use of surface-enhanced Raman spectroscopy (SERS) to delineate between the breast epithelial cell lines MCF10A, SK-BR-3, and MDA-MB-231 is explored utilizing varied morphologies of gold nanoparticles. The nanoparticles studied had spherical, star-like, and quasi-fractal (nanocaltrop) morphologies and possessed varying degrees of surface inhomogeneity and complexity. The efficacy of Raman enhancement of these nanoparticles was a function of their size, their surface morphology, and the associated density of "hot spots," as well as their cellular uptake. The spherical and star-like nanoparticles provided strong signal enhancement that allowed for the discernment among the three cell phenotypes based solely on the acquired Raman spectra. The presence of overlapping Raman band spectral regions, as well as unique spectral bands, suggests that the underlying biological differences between these cells can be accessed without the need for tagging the nanoparticles or for specific cell targeting, demonstrating the potential ubiquity of this technique in imaging any cancer. This work provides clear evidence for the potential application of SERS as a tool for mapping cancerous lesions, possibly during surgery and under histopathological analysis.

Gold nanoparticles cellular toxicity and recovery: adipose Derived Stromal cells.

Gold nanoparticles (AuNPs) are currently used in numerous medical applications. Herein, we describe their in vitro impact on human adipose-derived stromal cells (ADSCs) using 13 nm and 45 nm citrate-coated AuNPs. In their non-differentiated state, ADSCs were penetrated by the AuNPs and stored in vacuoles. The presence of the AuNPs in ADSCs resulted in increased population doubling times, decreased cell motility and cell-mediated collagen contraction. The degree to which the cells were impacted was a function of particle concentration, where the smaller particles required a sevenfold higher concentration to have the same effect as the larger ones. Furthermore, AuNPs reduced adipogenesis as measured by lipid droplet accumulation and adiponectin secretion. These effects correlated with transient increases in DLK1 and with relative reductions in fibronectin. Upon removal of exogenous AuNPs, cellular NP levels decreased and normal ADSC functions were restored. As adiponectin helps regulate energy metabolism, local fluctuations triggered by AuNPs can lead to systemic changes. Hence, careful choice of size, concentration and clinical application duration of AuNPs is warranted.

Platinum folate nanoparticles toxicity: cancer vs. normal cells.

Almost for two decades metallic nanoparticles are successfully used for cancer detection, imaging and treatment. Due to their high electron density they can be easily observed by electron microscopy and used in laser and radiofrequency therapy as energy releasing agents. However, the limitation for this practice is an inability to generate tumor-specific heating in a minimally invasive manner to the healthy tissue. To overcome this restraint we proposed to use folic acid coated metallic nanoparticles and determine whether they preferentially penetrate cancer cells. We developed technique for synthesizing platinum nanoparticles using folic acid as stabilizing agent which produced particles of relatively narrow size distribution, having d=2.3 ± 0.5 nm. High resolution TEM and zeta potential analysis indicated that the particles produced by this method had a high degree of crystalline order with no amorphous outer shell and a high degree of colloidal stability. The keratinocytes and mammary breast cells (cancer and normal) were incubated with platinum folate nanoparticles, and the results showed that the IC50 was significantly higher for the normal cells than the cancer cells in both cases, indicating that these nanoparticles preferentially target the cancer cells. TEM images of thin sections taken from the two types of cells indicated that the number of vacuoles and morphology changes after incubation with nanoparticles was also larger for the cancer cells in both types of tissue studied. No preferential toxicity was observed when folic acid receptors were saturated with free folic acid prior to exposure to nanoparticles. These results confirm our hypothesis regarding the preferential penetration of folic acid coated nanoparticles to cancer cells due to receptor mediated endocytosis.

The effects of UV emission from compact fluorescent light exposure on human dermal fibroblasts and keratinocytes in vitro.

Compact fluorescent light (CFL) bulbs can provide the same amount of lumens as incandescent light bulbs, using one quarter of the energy. Recently, CFL exposure was found to exacerbate existing skin conditions; however, the effects of CFL exposure on healthy skin tissue have not been thoroughly investigated. In this study, we studied the effects of exposure to CFL illumination on healthy human skin tissue cells (fibroblasts and keratinocytes). Cells exposed to CFLs exhibited a decrease in the proliferation rate, a significant increase in the production of reactive oxygen species, and a decrease in their ability to contract collagen. Measurements of UV emissions from these bulbs found significant levels of UVC and UVA (mercury [Hg] emission lines), which appeared to originate from cracks in the phosphor coatings, present in all bulbs studied. The response of the cells to the CFLs was consistent with damage from UV radiation, which was further enhanced when low dosages of TiO(2) nanoparticles (NPs), normally used for UV absorption, were added prior to exposure. No effect on cells, with or without TiO(2) NPs, was observed when they were exposed to incandescent light of the same intensity.

Gold nanoparticles cellular toxicity and recovery: effect of size, concentration and exposure time.

Gold nanoparticles (AuNPs) are used in many applications; however, their interactions with cells and potential health risk(s) are not fully known. In this manuscript, we describe the interactions of AuNPs with human dermal fibroblasts and show that they can penetrate the plasma membrane and accumulate in large vacuoles. We also demonstrate that the uptake of the AuNPs is a function of time, their size and concentration. Specifically, we demonstrate that 45 nm AuNPs penetrate cells via clathrin-mediated endocytosis, while the smaller 13 nm enter mostly via phagocytosis. Furthermore, we provide evidence of cytoskeleton filament disruption as a result of AuNPs exposure and reconstitution during recovery (following AuNP removal), despite no changes in actin or beta-tubulin protein levels. In contrast, the expression of the extracellular matrix (ECM) proteins, collagen and fibronectin, was diminished in the cells exposed to AuNPs. We also examined the proliferation rates of cells exposed to AuNPs and show that its diminution is a function of apoptosis and speculate that apoptosis results from the number of vacuoles present in the cells, which is probably the main factor that disrupts the cytoskeleton causing cell area contraction and decreases in motility. Lastly, we also present data that indicates that AuNPs' damage to cells is not permanent and that the cells can completely recover as a function of AuNPs' size, concentration and exposure time. Taken together, our data suggest that AuNPs exert detrimental effects on cell function that could reverse following AuNPs removal.