Mechanistic insights into the antitumoral potential and in vivo antiproliferative efficacy of a silver-based core@shell nanosystem.

This study delves into the biomolecular mechanisms underlying the antitumoral efficacy of a hybrid nanosystem, comprised of a silver core@shell (Ag@MSNs) functionalized with transferrin (Tf). Employing a SILAC proteomics strategy, we identified over 150 de-regulated proteins following exposure to the nanosystem. These proteins play pivotal roles in diverse cellular processes, including mitochondrial fission, calcium homeostasis, endoplasmic reticulum (ER) stress, oxidative stress response, migration, invasion, protein synthesis, RNA maturation, chemoresistance, and cellular proliferation. Rigorous validation of key findings substantiates that the nanosystem elicits its antitumoral effects by activating mitochondrial fission, leading to disruptions in calcium homeostasis, as corroborated by RT-qPCR and flow cytometry analyses. Additionally, induction of ER stress was validated through western blotting of ER stress markers. The cytotoxic action of the nanosystem was further affirmed through the generation of cytosolic and mitochondrial reactive oxygen species (ROS). Finally, in vivo experiments using a chicken embryo model not only confirmed the antitumoral capacity of the nanosystem, but also demonstrated its efficacy in reducing cellular proliferation. These comprehensive findings endorse the potential of the designed Ag@MSNs-Tf nanosystem as a groundbreaking chemotherapeutic agent, shedding light on its multifaceted mechanisms and in vivo applicability.

Unraveling the Mechanisms of Ch-SeNP Cytotoxicity against Cancer Cells: Insights from Targeted and Untargeted Metabolomics.

Although chitosan-stabilized selenium nanoparticles (Ch-SeNPs) have emerged as a promising chemical form of selenium for anticancer purposes, gathering more profound knowledge related to molecular dysfunctions contributes significantly to the promotion of their evolution as a chemotherapeutic drug. In this sense, metabolites are the end products in the flow of gene expression and, thus, the most sensitive to changes in the physiological state of a biological system. Therefore, metabolomics provides a functional readout of the biochemical activity and cell state. In the present study, we evaluated alterations in the metabolomes of HepG2 cells after the exposure to Ch-SeNPs to elucidate the biomolecular mechanisms involved in their therapeutic effect. A targeted metabolomic approach was conducted to evaluate the levels of four of the main energy-related metabolites (adenosine triphosphate (ATP); adenosine diphosphate (ADP); nicotinamide adenine dinucleotide (NAD+); and 1,4-dihydronicotinamide adenine dinucleotide (NADH)), revealing alterations as a result of exposure to Ch-SeNPs related to a shortage in the energy supply system in the cell. In addition, an untargeted metabolomic experiment was performed, which allowed for the study of alterations in the global metabolic profile as a consequence of Ch-SeNP exposure. The results indicate that the TCA cycle and glycolytic pathways were impaired, while alternative pathways such as glutaminolysis and cysteine metabolism were upregulated. Additionally, increased fructose levels suggested the induction of hypoxia-like conditions. These findings highlight the potential of Ch-SeNPs to disrupt cancer cell metabolism and provide insights into the mechanisms underlying their antitumor effects.

Capabilities of Single Cell ICP-MS for the Analysis of Cell Suspensions from Solid Tissues.

Single cell elemental (SC) analysis of isogenic cell cultures can be done using inductively coupled plasma (ICP-MS) detection. However, 2D cell cultures are just models to simplify the complexity of real tissue samples. Here, we show for the first time the capabilities of the technique (SC-ICP-MS) to analyze single cell suspensions of isolated cells from tissues. An optimized cocktail of proteolytic and collagenolytic enzymes was applied in a single preparation step with cellular yields up to 28% using 0.5 g of fresh rat spleen and liver, respectively. The retrieved cells revealed adequate morphology and stability to be examined by SC-ICP-MS. Quantitative elemental analysis of P, S, Cu, and Fe from disaggregated cells from rat spleen and liver tissues revealed levels of Fe of 7-16 fg/cell in the spleen and 8-12 fg/cell in the liver, while Cu was about 3-5 fg/cell in the spleen and 1.5-2.5 fg/cell in the liver. Evaluation of the transmembrane protein transferrin receptor 1 (TfR1) expression levels in disaggregated cells was also conducted by using a Nd-labelled antibody against this cell surface biomarker. Quantitative results showed significantly lower expression in the disaggregated cells than in the cell model HepG2, in agreement with the overexpression of this biomarker in tumor cells. In this proof of concept study, the tissue disaggregation protocol has shown to maintain the elemental intracellular content of cells as well as the presence of relevant antigens. This opens a completely new area of research for SC-ICP-MS in tissue samples as a complementary strategy with validation capabilities.

Complementary techniques (spICP-MS, TEM, and HPLC-ICP-MS) reveal the degradation of 40 nm citrate-stabilized Au nanoparticles in rat liver after intraperitoneal injection.

BACKGROUND: Due to the increased use of engineered nanoparticles (NPs), their tracing in environmental and biological systems is of utmost importance. Besides their accumulation within a biological specimen, little is known about their degradation and transformation into corresponding low-molecular species that might influence any toxicological impact. ANALYTICAL METHODS: Wistar rats underwent intraperitoneal injections of 40 nm citrate-stabilized gold nanoparticles. Different liver samples were analysed for the occurrence of nanoparticles and potential degradation products by means of spICP-MS, TEM and HPLC-ICP-MS. MAIN FINDINGS: Studies using spICP-MS revealed the presence of the originally administrated Au NPs (40 nm diameter) and some evidences of other Au-containing species due to the increased background signal. Images obtained by transmission electron microscopy (TEM) showed the predominant presence of particles of significantly smaller diameter (6 ± 2 nm). As complementary method, HPLC-ICP-MS confirmed the presence of both particle types indicating a degradation of the Au NPs accompanied by detection of low-molecular Au species. CONCLUSIONS: This study underlines that degradation of gold nanoparticles to low-molecular gold species might have to be taken into account in future for studies on their toxicological behaviour and their potential use in clinical applications.

Cytosolic copper is a major modulator of germination, development and secondary metabolism in Streptomyces coelicolor.

Streptomycetes are important biotechnological bacteria with complex differentiation. Copper is a well-known positive regulator of differentiation and antibiotic production. However, the specific mechanisms buffering cytosolic copper and the biochemical pathways modulated by copper remain poorly understood. Here, we developed a new methodology to quantify cytosolic copper in single spores which allowed us to propose that cytosolic copper modulates asynchrony of germination. We also characterised the SCO2730/2731 copper chaperone/P-type ATPase export system. A Streptomyces coelicolor strain mutated in SCO2730/2731 shows an important delay in germination, growth and sporulation. Secondary metabolism is heavily enhanced in the mutant which is activating the production of some specific secondary metabolites during its whole developmental cycle, including germination, the exponential growth phase and the stationary stage. Forty per cent of the S. coelicolor secondary metabolite pathways, are activated in the mutant, including several predicted pathways never observed in the lab (cryptic pathways). Cytosolic copper is precisely regulated and has a pleiotropic effect in gene expression. The only way that we know to achieve the optimal concentration for secondary metabolism activation, is the mutagenesis of SCO2730/2731. The SCO2730/2731 genes are highly conserved. Their inactivation in industrial streptomycetes may contribute to enhance bioactive compound discovery and production.