Accurate Quantification and Imaging of Cellular Uptake Using Single-Particle Surface-Enhanced Raman Scattering.

Understanding the uptake, distribution, and stability of gold nanoparticles (NPs) in cells is of fundamental importance in nanoparticle sensors and therapeutic development. Single nanoparticle imaging with surface-enhanced Raman spectroscopy (SERS) measurements in cells is complicated by aggregation-dependent SERS signals, particle inhomogeneity, and limited single-particle brightness. In this work, we assess the single-particle SERS signals of various gold nanoparticle shapes and the role of silica encapsulation on SERS signals to develop a quantitative probe for single-particle level Raman imaging in living cells. We observe that silica-encapsulated gap-enhanced Raman tags (GERTs) provide an optimized probe that can be quantifiable per voxel in SERS maps of cells. This approach is validated by single-particle inductively coupled mass spectrometry (spICP-MS) measurements of NPs in cell lysate post-imaging. spICP-MS also provides a means of measuring the tag stability. This analytical approach can be used not only to quantitatively assess nanoparticle uptake on the cellular level (as in previous digital SERS methods) but also to reliably image the subcellular distribution and to assess the stability of NPs in cells.

Comparison of trace element concentrations in paired formalin-fixed paraffin-embedded and frozen human placentae.

INTRODUCTION: There is increasing interest in measuring metals concentrations in human placentas to better understand physiology, disease, and toxic and diagnostic exposures. For these purposes, formalin-fixed paraffin embedded (FFPE) tissues obtained at clinical pathology examination represent a valuable potential store of well-characterized tissues for analysis. However, the limited data that exist comparing metal concentrations in FFPE tissue to recently collected frozen tissues paints a confusing picture, and there is no published data directly comparing frozen and FFPE placental villus tissues. METHODS: Paired samples of fresh frozen and FFPE tissue from 22 rapidly processed human singleton placentae were weighed and digested using standard clean laboratory procedures and subsequently analyzed for a suite of 13 metals using a PerkinElmer DRC II ICP-MS. The analytical results were compared using either a paired t-test or a sign test depending on data normality. RESULTS: Concentrations of metals (aluminum (Al), arsenic (As), barium (Ba), cadmium (Cd), chromium (Cr), copper (Cu), iron (Fe), gadolinium (Gd), mercury (Hg), manganese (Mn), lead (Pb), strontium (Sr), and zinc (Zn)) measured in both types of tissue preparations (frozen and FFPE) displayed a consistent range with other studies and did not display significantly different values from each of the paired specimens for any of the 13 specific metals analyzed. DISCUSSION: Within placentae, metals concentrations of measured trace, toxic and diagnostic elements (Al, As, Ba, Cd, Cr, Cu, Fe, Gd, Hg, Mn, Pb, Sr, and Zn) are consistent between FFPE and fresh placental villus tissue, without indications of systematic element loss or bias. FFPE from archived pathology specimens may offer an important and convenient alternative for measuring trace metals in human frozen placental tissues.

ICP-MS measurements of elemental release from two palladium alloys into a corrosion testing medium for different solution volumes and agitation conditions.

STATEMENT OF PROBLEM: The in vivo release of Pd from palladium alloys into the oral environment and sensitivity reactions by patients has been of concern. However, little information is available about the variation in elemental release from different palladium alloys. PURPOSE: The purpose of this in vitro study was to compare the elemental release into a corrosion-testing medium from a high-palladium alloy (Freedom Plus, 78Pd-8Cu-5Ga-6In-2Au) and a Pd-Ag alloy (Super Star, 60Pd-28Ag-6In-5Sn) under different conditions. MATERIAL AND METHODS: Alloys were cast into Ø12×1-mm-thick disks, subjected to simulated porcelain-firing heat treatment, polished, and ultrasonically cleaned in ethanol. Three specimens of each alloy were immersed for 700 hours in a solution for in vitro corrosion testing (ISO Standard 10271) that was maintained at 37 °C. Two solution volumes (125 mL and 250 mL) were used, and the solutions were subjected to either no agitation or agitation. Elemental compositions of the solutions were analyzed by using inductively coupled plasma-mass spectroscopy (ICP-MS). Concentrations of released elements from each alloy for the 2 solution volumes and agitation conditions were compared by using the restricted maximum likelihood estimation method with a 4-way repeated-measures ANOVA, the Satterwhite degrees of freedom method, a lognormal response distribution, and the covariance structure of compound symmetry. RESULTS: For the 4 combinations of solution volume and agitation conditions, the mean amount of palladium released was 3 orders of magnitude less for the Pd-Ag alloy (0.009 to 0.017 µg/cm2 of alloy surface) compared with the Pd-Cu-Ga alloy (17.9 to 28.7 µg/cm2). Larger mean amounts of Sn, Ga, Ag, and In (0.29 to 0.39, 0.57 to 0.83, 0.71 to 1.08, and 0.91 to 1.25 µg/cm2, respectively) compared with Pd were released from the Pd-Ag alloy. Smaller amounts of Cu, Ga, and In (4.8 to 9.9, 5.9 to 12.8, and 4.2 to 9.5 µg/cm2, respectively) compared with Pd were released from the Pd-Cu-Ga alloy. The Ru released was much lower for the Pd-Ag alloy (0.002 µg/cm2) than the Pd-Cu-Ga alloy (0.032 to 0.053 µg/cm2). Statistically significant differences (P<.001) in elemental release were found for the factors of alloy and element and the alloy×element interaction. Significant differences were found for the solution volume (P=.022), solution volume×element interaction (P=.022), and alloy×solution volume×element interaction (P=.004). No significant effect was found for agitation condition. CONCLUSIONS: The relative amounts of released elements from each alloy were not proportional to the relative amounts in the composition. The amounts of Pd and Ga released from the Pd-Cu-Ga alloy were consistent with the breakdown of a Pd2Ga microstructural phase and perhaps some dissolution of the palladium solid solution matrix. Precipitates, rather than the palladium solid solution matrix, appeared to undergo greater dissolution in the Pd-Ag alloy. The Pd-Ag alloy should have lower risk of adverse biological reactions than the Pd-Cu-Ga alloy.

Dissipation of transmembrane potassium gradient is the main cause of cerebral ischemia-induced depolarization in astrocytes and neurons.

Membrane potential (VM) depolarization occurs immediately following cerebral ischemia and is devastating for the astrocyte homeostasis and neuronal signaling. Previously, an excessive release of extracellular K+ and glutamate has been shown to underlie an ischemia-induced VM depolarization. Ischemic insults should impair membrane ion channels and disrupt the physiological ion gradients. However, their respective contribution to ischemia-induced neuronal and glial depolarization and loss of neuronal excitability are unanswered questions. A short-term oxygen-glucose deprivation (OGD) was used for the purpose of examining the acute effect of ischemic conditions on ion channel activity and physiological K+ gradient in neurons and glial cells. We show that a 30 min OGD treatment exerted no measurable damage to the function of membrane ion channels in neurons, astrocytes, and NG2 glia. As a result of the resilience of membrane ion channels, neuronal spikes last twice as long as our previously reported 15 min time window. In the electrophysiological analysis, a 30 min OGD-induced dissipation of transmembrane K+ gradient contributed differently in brain cell depolarization: severe in astrocytes and neurons, and undetectable in NG2 glia. The discrete cellular responses to OGD corresponded to a total loss of 69% of the intracellular K+ contents in hippocampal slices as measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). A major brain cell depolarization mechanism identified here is important for our understanding of cerebral ischemia pathology. Additionally, further understanding of the resilient response of NG2 glia to ischemia-induced intracellular K+ loss and depolarization should facilitate the development of future stroke therapy.

Single Particle ICP-MS: Advances toward routine analysis of nanomaterials.

From its early beginnings in characterizing aerosol particles to its recent applications for investigating natural waters and waste streams, single particle inductively coupled plasma-mass spectrometry (spICP-MS) has proven to be a powerful technique for the detection and characterization of aqueous dispersions of metal-containing nanomaterials. Combining the high-throughput of an ensemble technique with the specificity of a single particle counting technique and the elemental specificity of ICP-MS, spICP-MS is capable of rapidly providing researchers with information pertaining to size, size distribution, particle number concentration, and major elemental composition with minimal sample perturbation. Recently, advances in data acquisition, signal processing, and the implementation of alternative mass analyzers (e.g., time-of-flight) has resulted in a wider breadth of particle analyses and made significant progress toward overcoming many of the challenges in the quantitative analysis of nanoparticles. This review provides an overview of spICP-MS development from a niche technique to application for routine analysis, a discussion of the key issues for quantitative analysis, and examples of its further advancement for analysis of increasingly complex environmental and biological samples. Graphical Abstract Single particle ICP-MS workflow for the analysis of suspended nanoparticles.

Uptake of bright fluorophore core-silica shell nanoparticles by biological systems.

Nanoparticles are used in a variety of consumer applications. Silica nanoparticles in particular are common, including as a component of foods. There are concerns that ingested nano-silica particles can cross the intestinal epithelium, enter the circulation, and accumulate in tissues and organs. Thus, tracking these particles is of interest, and fluorescence spectroscopic methods are well-suited for this purpose. However, nanosilica is not fluorescent. In this article, we focus on core-silica shell nanoparticles, using fluorescent Rhodamine 6G, Rhodamine 800, or CdSe/CdS/ZnS quantum dots as the core. These stable fluorophore/silica nanoparticles had surface characteristics similar to those of commercial silica particles. Thus, they were used as model particles to examine internalization by cultured cells, including an epithelial cell line relevant to the gastrointestinal tract. Finally, these particles were administered to mice by gavage, and their presence in various organs, including stomach, small intestine, cecum, colon, kidney, lung, brain, and spleen, was examined. By combining confocal fluorescence microscopy with inductively coupled plasma mass spectrometry, the presence of nanoparticles, rather than their dissolved form, was established in liver tissues.

Impaired myocardial perfusion reserve and fibrosis in Friedreich ataxia: a mitochondrial cardiomyopathy with metabolic syndrome.

AIMS: Cardiomyopathy produces significant mortality in patients with Friedreich ataxia (FA), a genetic disorder that produces intra-mitochondrial iron accumulation. We sought to test the hypothesis that abnormal myocardial perfusion reserve and fibrosis represent early manifestations of cardiomyopathy. METHODS AND RESULTS: Twenty-six patients with genetically proven FA ages 36 ± 12 years without cardiomyopathy and eight controls underwent cardiac magnetic resonance with adenosine. Precontrast imaging for myocardial iron estimation was performed. Myocardial perfusion reserve index (MPRI) was quantified using the normalized upslope of myocardial enhancement during vasodilator stress vs. rest. Left ventricular (LV) mass and volumes were computed from short-axis cine images. Serologies included lipids, and platelets were isolated for iron quantification using inductively coupled plasma mass spectrometry. Left ventricular ejection fraction and mass averaged 64.1 ± 8.3% and 62.7 ± 16.7 g/m², respectively, indicating preserved systolic function and absence of significant hypertrophy. Myocardial perfusion reserve index quantification revealed significantly lower endocardial-to-epicardial perfusion reserve in patients vs. controls (0.80 ± 0.18 vs. 1.22 ± 0.36, P = 0.01). Lower MPRI was predicted by increased number of metabolic syndrome (met-S) features (P < 0.01). Worse concentric remodelling occurred with increased GAA repeat length (r = 0.64, P < 0.001). Peripheral platelet iron measurement showed no distinction between patients and controls (5.4 ± 8.5 × 10⁻7 vs. 5.5 ± 2.9 × 10⁻7 ng/platelet, P = 0.88), nor did myocardial T2* measures. CONCLUSIONS: Patients with FA have abnormal myocardial perfusion reserve that parallels met-S severity. Impaired perfusion reserve and fibrosis occur in the absence of significant hypertrophy and prior to clinical heart failure, providing potential therapeutic targets for stage B cardiomyopathy in FA and related myocardial diseases.

Inductively coupled plasma-mass spectroscopy measurements of elemental release from 2 high-palladium dental casting alloys into a corrosion testing medium.

STATEMENT OF PROBLEM: The biocompatibility of high-palladium alloy restorations has been of some concern due to the release of palladium into the oral environment and sensitivity reactions in patients. PURPOSE: This study measured the in vitro elemental release from a Pd-Cu-Ga alloy and a Pd-Ga alloy into a corrosion testing medium. MATERIAL AND METHODS: Both alloys were cast into 12-mm-diameter x 1-mm-thick disks, subjected to heat treatment that simulated porcelain firing cycles, polished to a 0.05-mm surface finish, and ultrasonically cleaned in ethanol. Two specimens of each alloy were immersed 3 times (at 7, 70, and 700 hours) in an aqueous lactic acid/NaCl solution used for in vitro corrosion testing and maintained at 37 degrees C. The specimens were removed after each immersion time, and the elemental compositions of the solutions were analyzed with inductively coupled plasma-mass spectroscopy (ICP-MS). Elemental concentrations for the 2 alloys at each immersion time were compared with Student t test (alpha=.05). RESULTS: No significant differences in palladium release were found for the 7- and 70-hour solutions, but significant differences were found for the 700-hour solutions. Mean concentrations of palladium and gallium in the 700-hour solutions, expressed as mass per unit area of alloy surface, were 97 (Pd) and 46 (Ga) microg/cm(2) for the Pd-Cu-Ga alloy and 5 (Pd) and 18 (Ga) microg/cm(2) for the Pd-Ga alloy. CONCLUSION: Relative proportions of the elements in the solutions were consistent with the release of palladium and breakdown of microstructural phases found in the alloys. The results suggest that there may be a lower risk of adverse biological reactions with the Pd-Ga alloy than with the Pd-Cu-Ga alloy tested.