Specificity of carbon nanotube accumulation and distribution in cancer cells revealed by K-means clustering and principal component analysis of Raman spectra.

Single-walled carbon nanotubes (SWCNTs) show great potential for their application as cancer therapeutic nanodrugs, but the efficiency and mechanism of their accumulation in the cell, the modulation of cell activity, and the strong dependence of the results on the type of capping molecule still hinder the transfer of SWCNTs to the clinic. In the present study, we determined the mechanism and sequence of accumulation, distribution and type discrimination of SWCNTs in glioma cells by applying K-means clustering and principal component analysis (PCA) of Raman spectra of cells exposed to SWCNTs capped with either DNA or oligonucleotides (ON). Based on the specific biochemical information uncovered by PCA and further applied to K-means, we show that the accumulation of SWCNT-DNA occurs in two phases. The first phase involves the transport of SWCNT-DNA through vesicles and its redistribution in the cytoplasm, which is reflected in two SWCNT-related clusters. The second phase begins after 18 hours of interaction between cells and SWCNT-DNA. PCA shows the appearance of two SWCNT-associated PC loadings, reflected by the addition of a new cluster of SWCNTs with a narrowed and shifted G-peak in the spectra. It is caused by the loss of DNA capping and clumping of SWCNTs and triggered by the acidic conditions in autolysosomes resulting from the fusion of transport vesicles with lysosomes. SWCNTs penetrate all cellular compartments after 42-66 hours and lead to cell death. The clumped SWCNTs are released to the outside. In contrast, SWCNT-ON is hardly accumulated in glioma cells and after 72 hours of exposure to SWCNT-ON, the accumulation of SWCNTs corresponds to the first stage without reaching the second. PCA made it possible to separate the characteristics of cellular components against the high-intensity Raman signal from nanotubes and, thus, to propose the mechanism of accumulation and metabolism of nanomaterials in living cells without the use of additional research approaches. Our results elucidate the time dependence of the accumulation of SWCNTs on the capping molecule. We expect that our results can make an important contribution to the use of these nanomaterials in the clinic.

Differential induction of C6 glioma apoptosis and autophagy by 3ß-hydroxysteroid-indolamine conjugates.

In a previous work, we reported the synthesis of four novel indole steroids and their effect on rat C6 glioma proliferation in vitro. The steroid derived from dehydroepiandrosterone and tryptamine (IS-1) was the most active (52 % inhibition at 10 µM), followed by one of the epimers derived from pregnenolone and tryptamine (IS-3, 36 % inhibition at 10 µM). By contrast, the steroid derived from estrone and tryptamine (IS-2) showed negligible activity at 10 µM. No necrosis, increase in intracellular calcium or ROS levels was observed. In this work, the effect of compounds on C6 glioma apoptosis and autophagy is examined by fluorimetry and fluorescent microscopy. The IS-3 epimers disrupt the mitochondrial membrane potential and induce apoptosis in vitro moderately whereas IS-1 and IS-2 do not. However, IS-1 produces a large increase in monodansylcadaverine-positive autophagic vesicles over 24 h. The antiproliferative effect of indole steroids is ameliorated by autophagy inhibitor hydroxychloroquine, suggesting an autophagy-dependent mechanism of cell death.

Photostability and phototoxicity of graphene quantum dots interacting with red blood cells.

Here we discuss fluorescent properties of graphene quantum dots (GQDs) interacting with the membranes of red blood cells. We report the results of spectroscopic, microscopic, and photon-counting measurements of the GQDs in different surroundings for uncovering specific features of the GQD fluorescence, and describe two observed phenomena important for implementation of the GQDs as fluorescent labels and agents for drug delivery. Firstly, the GQDs can suffer from photodegradation but also can be stabilized in the presence of antioxidants (reduced glutathione, N-acetylcysteine, or 1,4-hydroquinone). Secondly, GQDs can accumulate in red blood cell membranes without compromising the viability of the cells but also can induce hemolysis in the presence of visible light. We discuss mechanisms and regimes of the photodegradation, stabilization, interaction of the GQDs with red blood cell membranes, and hemolysis. Notably, photohemolysis for the case is dependent on the light dose and GQD concentration but not caused by the production of reactive oxygen species.

Stable and Reusable Lace-like Black Silicon Nanostructures Coated with Nanometer-Thick Gold Films for SERS-Based Sensing.

We propose a simple, fast, and low-cost method for producing Au-coated black Si-based SERS-active substrates with a proven enhancement factor of 106. Room temperature reactive ion etching of silicon wafer followed by nanometer-thin gold sputtering allows the formation of a highly developed lace-type Si surface covered with homogeneously distributed gold islands. The mosaic structure of deposited gold allows the use of Au-uncovered Si domains for Raman peak intensity normalization. The fabricated SERS substrates have prominent uniformity (with less than 6% SERS signal variations over large areas, 100 × 100 µm2). It has been found that the storage of SERS-active substrates in an ambient environment reduces the SERS signal by less than 3% in 1 month and not more than 40% in 20 months. We showed that Au-coated black Si-based SERS-active substrates can be reused after oxygen plasma cleaning and developed relevant protocols for removing covalently bonded and electrostatically attached molecules. Experiments revealed that the Raman signal of 4-MBA molecules covalently bonded to the Au coating measured after the 10th cycle was just 4 times lower than that observed for the virgin substrate. A case study of the reusability of the black Si-based substrate was conducted for the subsequent detection of 10-5 M doxorubicin, a widely used anticancer drug, after the reuse cycle. The obtained SERS spectra of doxorubicin were highly reproducible. We demonstrated that the fabricated substrate permits not only qualitative but also quantitative monitoring of analytes and is suitable for the determination of concentrations of doxorubicin in the range of 10-9-10-4 M. Reusable, stable, reliable, durable, low-cost Au-coated black Si-based SERS-active substrates are promising tools for routine laboratory research in different areas of science and healthcare.

Nanodiamond surface as a photoluminescent pH sensor.

A systematic spectroscopic characterization of highly homogeneous water suspensions of 'buckydiamonds' comprising sp3cubic nanodiamond (ND) core covered with disordered sp2shell densely decorated with oxygen-containing groups demonstrates the excitation-wavelength-dependent photoluminescence (PL) given by at least four types of specific structures on the ND surface (hydroxyl, C=O containing ketones, carboxylic anhydrides, and carboxyl groups). PL properties of NDs suspensions possess concentration-dependent behavior revealing tendency of NDs to agglomerate. PL of NDs has been found to be strongly sensitive to pH of the environment in wide range of pH values, i.e. 2-11. We disclosed the mechanisms of pH sensitivity of the 'buckydiamond' and proved that it can serve as all-optical sensor of tiny pH variations suitable for further exploitation for pH sensing locally in the area where NDs have been delivered for any purpose, e.g. bioimaging or therapeutic needs.

Hysteresis and Stochastic Fluorescence by Aggregated Ensembles of Graphene Quantum Dots.

"Blinking" behavior of fluorophores, being harmful for the majority of super-resolved techniques, turns into a key property for stochastic optical fluctuation imaging and its modifications, allowing one to look at the fluorophores already used in conventional microscopy, such as graphene quantum dots, from a completely new perspective. Here we discuss fluorescence of aggregated ensembles of graphene quantum dots structured at submicron scale. We study temperature dependence and stochastic character of emission. We show that considered quantum dots ensembles demonstrate rather complicated temperature-dependent intermittent emission, that is, "blinking" with a tendency to shorten "blinking" times with the increase of temperature. We verify "blinking" mechanism demonstrating hysteresis of the optical response under pulsed excitation timed to expected rates of dots transition to "dark" nonemitting states. Experimental results are well fitted by a simple qualitative model of transitions to the "dark" states. The obtained results suggest that this type of standardized quantum dots and even their submicron-size agglomerations can be useful as controlled fluorophores for super-resolution microscopy and, particularly, for SOFI-like microscopy.

Quantitative and qualitative analysis of pulmonary arterial hypertension fibrosis using wide-field second harmonic generation microscopy.

We demonstrated that wide-field second harmonic generation (SHG) microscopy of lung tissue in combination with quantitative analysis of SHG images is a powerful tool for fast and label-free visualization of the fibrosis pathogenesis in pulmonary arterial hypertension (PAH). Statistical analysis of the SHG images revealed changes of the collagen content and morphology in the lung tissue during the monocrotaline-induced PAH progression in rats. First order statistics disclosed the dependence of the collagen overproduction on time, the second order statistics indicated tightening of collagen fiber network around blood vessels and their spreading into the alveolar region. Fourier analysis revealed that enhancement of the fiber orientation in the collagen network with PAH progression was followed with its subsequent reduction at the terminating phase of the disease. Proposed approach has potential for assessing pulmonary fibrosis in interstitial lung disease, after lung(s) transplantation, cancer, etc.

Visualizing hypochlorous acid production by human neutrophils with fluorescent graphene quantum dots.

In living organisms, redox reactions play a crucial role in the progression of disorders accompanied by the overproduction of reactive oxygen and reactive chlorine species, such as hydrogen peroxide and hypochlorous acid, respectively. We demonstrate that green fluorescence graphene quantum dots (GQDs) can be employed for revealing the presence of the hypochlorous acid in aqueous solutions and cellular systems. Hypochlorous acid modifies the oxygen-containing groups of the GQD, predominantly opens epoxide ring C-O-C, forms excessive C=O bonds and damages the carbonic core of GQDs. These changes, which depend on the concentration of the hypochlorous acid and exposure time, manifest themselves in the absorbance and fluorescence spectra of the GQD, and in the fluorescence lifetime. We also show that the GQD fluorescence is not affected by hydrogen peroxide. This finding makes GQDs a promising sensing agent for selective detecting reactive chlorine species produced by neutrophils. Neutrophils actively accumulate GQDs allowing to visualize cells and to examine the redox processes via GQDs fluorescence. At high concentrations GQDs induce neutrophil activation and myeloperoxidase release, leading to the disruption of GQD structure by the produced hypochlorous acid. This makes the GQDs a biodegradable material suitable for various biomedical applications.

Rapid and delayed effects of single-walled carbon nanotubes in glioma cells.

Single-walled carbon nanotubes (SWCNTs) demonstrate a strong potential as an optically activated theranostic nano-agent. However, using SWCNTs in theranostics still requires revealing mechanisms of the SWCNT-mediated effects on cellular functions. Even though rapid and delayed cellular responses can differ significantly and may lead to undesirable consequences, understanding of these mechanisms is still incomplete. We demonstrate that introducing short (150-250 nm) SWCNTs into C6 rat glioma cells leads to SWCNT-driven effects that show pronounced time dependence. Accumulation of SWCNTs is carried out due to endocytosis with modification of the actin cytoskeleton but not accompanied with autophagy. Its initial stage launches a rapid cellular response via significantly heightened mitochondrial membrane potential and superoxide anion radical production, satisfying the cell demand of energy for SWCNT transfer inside the cytoplasm. In the long term, SWCNTs agglomerate to micron-sized structures surrounded by highly active mitochondria having parameters return to control values. SWCNTs postponed effects are also manifested themselves in the suppression of the cell proliferative activity with further restoration after five passages. These results demonstrate relative cellular inertness and safety of SWCNTs eliminating possible side effects caused by optically activated theranostic applications.

Surface-Enhanced Raman Spectroscopy of Organic Molecules and Living Cells with Gold-Plated Black Silicon.

Black silicon (bSi) refers to an etched silicon surface comprising arrays of microcones that effectively suppress reflection from UV to near-infrared (NIR) while simultaneously enhancing the scattering and absorption of light. This makes bSi covered with a nm-thin layer of plasmonic metal, i.e., gold, an attractive substrate material for sensing of bio-macromolecules and living cells using surface-enhanced Raman spectroscopy (SERS). The performed Raman measurements accompanied with finite element numerical simulation and density functional theory analysis revealed that at the 785 nm excitation wavelength, the SERS enhancement factor of the bSi/Au substrate is as high as 108 due to a combination of electromagnetic and chemical mechanisms. This finding makes the SERS-active bSi/Au substrate suitable for detecting trace amounts of organic molecules. We demonstrate the outstanding performance of this substrate by highly sensitive and specific detection of a small organic molecule of 4-mercaptobenzoic acid and living C6 rat glioma cell nucleic acids/proteins/lipids. Specifically, the bSi/Au SERS-active substrate offers a unique opportunity to investigate the living cells' malignant transformation using characteristic protein disulfide Raman bands as a marker. Our findings evidence that bSi/Au provides a pathway to the highly sensitive and selective, scalable, and low-cost substrate for lab-on-a-chip SERS biosensors that can be integrated into silicon-based photonics devices.

Macro-, Micro- and Nano-Roughness of Carbon-Based Interface with the Living Cells: Towards a Versatile Bio-Sensing Platform.

Integration of living cells with nonbiological surfaces (substrates) of sensors, scaffolds, and implants implies severe restrictions on the interface quality and properties, which broadly cover all elements of the interaction between the living and artificial systems (materials, surface modifications, drug-eluting coatings, etc.). Substrate materials must support cellular viability, preserve sterility, and at the same time allow real-time analysis and control of cellular activity. We have compared new substrates based on graphene and pyrolytic carbon (PyC) for the cultivation of living cells. These are PyC films of nanometer thickness deposited on SiO2 and black silicon and graphene nanowall films composed of graphene flakes oriented perpendicular to the Si substrate. The structure, morphology, and interface properties of these substrates are analyzed in terms of their biocompatibility. The PyC demonstrates interface biocompatibility, promising for controlling cell proliferation and directional intercellular contact formation while as-grown graphene walls possess high hydrophobicity and poor biocompatibility. By performing experiments with C6 glioma cells we discovered that PyC is a cell-friendly coating that can be used without poly-l-lysine or other biopolymers for controlling cell adhesion. Thus, the opportunity to easily control the physical/chemical properties and nanotopography makes the PyC films a perfect candidate for the development of biosensors and 3D bioscaffolds.

New 3ß-hydroxysteroid-indolamine conjugates: Design, synthesis and inhibition of C6 glioma cell proliferation.

Four novel indole steroids based on dehydroepiandrosterone (IS-1), estrone (IS-2) and pregnenolone (IS-3) were obtained and studied for their ability to inhibit C6 glioma proliferation. A reduction in cell proliferation by 52 ± 13% was observed for IS-1 at 10 µM, whereas IS-3 and abiraterone acetate at 10 µM caused a 36 ± 8% decrease. Surprisingly, the cellular effects reported for abiraterone, namely, cytochrome P450 CYP17A1 inhibition and endoplasmic reticulum stress were not detected for IS-1. However, both abiraterone and IS-1 significantly increased glutathione levels. Docking studies predicted good affinity of IS-1 to liver X receptors and regulatory protein Keap1, which are proposed to be involved in the compounds' antiproliferative activity.

Enhancement of single-walled carbon nanotube accumulation in glioma cells exposed to low-strength electric field: Promising approach in cancer nanotherapy.

The objective of the study is to determine the patterns of regulation of single-walled carbon nanotube accumulation, distribution, and agglomeration in glioma cells exposed to an external electric field. C6 glioma cells were treated with 5 µg/ml DNA wrapped single-walled carbon nanotubes and exposed to bi-phasic electric pulses (6.6 V/m, 200 Hz, pulse duration 1 ms). Nanotube accumulation was determined by Raman microspectroscopy and their intracellular local concentration was evaluated using the G-band intensity in Raman spectra of single-walled carbon nanotubes. It was revealed that the low-frequency and low-strength electric field stimulation of glioma cells exposed to single-walled carbon nanotubes led to facilitation and, thus, to amplification of nanotube accumulation inside the cells. The number of nanotubes in intracellular agglomerates increased from (28.8 ± 13.1) un./agglom. and (84.0 ± 28.7) un./agglom. in control samples to (60.6 ± 21.4) un./agglom. and (184.2 ± 53.4) un./agglom. for 1 h and 2 h stimulation, respectively. Thus, the tumor exposure to an external electric field makes it possible to more effectively regulate the accumulation and distribution of carbon nanotubes inside glioma cells allowing to reduce the applied therapeutic doses of carbon nanomaterial delivered anticancer drugs.

Morphometric characteristics of neutrophils stimulated by adhesion and hypochlorite.

The aim of this work was to compare cell form, size and volume as well as the locomotor activity of polymorphonuclear leukocytes (PMNLs) stimulated by adhesion to glass and exposed to hypochlorous acid at non-toxic dose. After 20min of adhesion to a glass surface, volume, cell surface area and projection area of PMNLs were equaled to 143.1±21.4µm3, 288.8±28.8µm2 and 248.3±32.3µm2, respectively. Projection area of PMNLs exposed to NaOCl was noticeably enlarged as compared with control samples. The cell volume of 20min adherent cells exposed to NaOCl was enlarged in comparison with both control cells and 5min adhered exposed to NaOCl cells. NaOCl exposure induced a degranulation of PMNLs as measured by lysozyme release. Granules could be found both above the cell surface and on the substratum near the cell. The S/V ratio for PMNLs increased (from 1.52 to 2.02µm-1) with the increasing of cell activation time. But at NaOCl addition the reverse tendency was observed (from 2.10 to 1.87µm-1). In cells exposed to NaOCl the redistribution and decrease of concentration of F-actin took place. This observation supports the hypothesis that the priming of PMNLs with hypochlorous acid modifies cell motility and morphology and reflects also on other functions.

Modification of redox processes in astroglial cells induced by microbial and viral infections.

BACKGROUND: The authors hypothesized that the cell redox state might be modified during microbial and viral infections. To detect and evaluate changes in astroglial cell redox state, rat C6 glioma cells after exposure to lipopolysaccharide (LPS) or after herpes simplex virus type 1 (HSV-1) inoculation were used. Redox state modification of glioma cells was determined by the change in menadione-induced superoxide yield. MATERIAL/METHODS: Menadione-induced superoxide formation was registered by the lucigenin-enhanced chemiluminescence (CL) method. RESULTS: The results demonstrate that exposure of C6 glioma cells to LPS for 24 hours resulted in a dose-dependent increase in the mitotic index and integral intensity of menadione-induced lucigenin-enhanced CL. Menadione-induced ROS generation in C6 cells during HSV-1 infection changed depending on the time after HSV-1 inoculation. CONCLUSIONS: The redox state of astroglial cells is modified during microbial and viral infections. The use of redox-active quinones is an informative model for determining cell redox state change and analyzing cells' functional state.

Effects of peroxynitrite and lipopolysaccharide on mitotic activity of C6 glioma cells.

Peroxynitrite is one of the most potent neurotoxic agents with multiple targets in neurons and glial cells. This study addressed a question of whether peroxynitrite-mediated cytotoxicity can be prevented by Escherichia coli lypopolisaccharide (LPS) due to its mitogenic activity towards C6 glioma cells. A number of characteristic morphological changes (processes impairments, nuclei modifications, cytoplasm vacuolization) and apoptotic cells were observed in the cell culture after 24-h treatment with 3-morpholinosyndnonimine (SIN-1), a well-known donor of peroxynitrite. These morphological changes were clearly associated with a SIN-1 dose-dependent increase in the number of pathological mitoses as well as with SIN-1 inhibition of the menadione-induced, lucigenin-enhanced chemiluminescence of C6 glioma cells, an independent indicator of mitotic activity of these cells. The mitotic index of C6 glioma cells increased in response to LPS and underwent non-uniform changes depending on SIN-1 concentrations. At a mitogenic concentration of 100 ng/ml, LPS reduced significantly the toxicity of SIN-1 determined as the accumulation of pathological mitoses, thus acting as a protective agent. Taken together, our findings indicate that SIN-1 specifically impairs the mitotic process in C6 glioma cells, and provide the first evidence that antimitotic effects of peroxynitrite can be restored by LPS.