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.

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.

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.