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.

Isolation of Bacterial Extracellular Vesicles and Identification of Their Protein Cargo.

Bacterial membrane vesicles (BMVs) are important effectors in the pathogenesis, virulence, and biofilm formation during different bacterial infections. Because of their structure, BMVs can be applied as drug delivery systems (DDS) or in the production of immunogenic vaccines against different untreated diseases. In this sense, different antigens or immune stimulator molecules, such as proteins can be extracted for the development of such vaccines. Here, we describe a protocol adapted to be used in mycobacteria, Gram-positive, and Gram-negative bacteria for the isolation of BMVs, and further mass spectrometry-based characterization of their protein cargo.

Integrated transcriptomics and metabolomics analysis reveals the biomolecular mechanisms associated to the antitumoral potential of a novel silver-based core@shell nanosystem.

A combination of omics techniques (transcriptomics and metabolomics) has been used to elucidate the mechanisms responsible for the antitumor action of a nanosystem based on a Ag core coated with mesoporous silica on which transferrin has been anchored as a targeting ligand against tumor cells (Ag@MSNs-Tf). Transcriptomics analysis has been carried out by gene microarrays and RT-qPCR, while high-resolution mass spectrometry has been used for metabolomics. This multi-omics strategy has enabled the discovery of the effect of this nanosystem on different key molecular pathways including the glycolysis, the pentose phosphate pathway, the oxidative phosphorylation and the synthesis of fatty acids, among others.

Super-SILAC Quantitative Proteome Profiling of Zebrafish Larvae.

The super-SILAC approach enables the quantitative proteome profiling of highly complex samples such as biological tissues or whole organisms. In this approach, a super-SILAC mix consisting of heavy isotope-labeled cells representative of the tissue or organism to be analyzed is mixed with the unlabeled samples of interest, such that the labeled proteins act as a spike-in standard, thus allowing the relative quantification of proteins between the samples of interest. In this chapter, we thoroughly describe the protocol to carry out the super-SILAC approach using a common in vivo model such as zebrafish larvae.

A Multi-Omics Approach to Evaluate the Toxicity Mechanisms Associated with Silver Nanoparticles Exposure.

Silver nanoparticles (AgNPs) are currently used in many different industrial, commercial and health fields, mainly due to their antibacterial properties. Due to this widespread use, humans and the environment are increasingly exposed to these types of nanoparticles, which is the reason why the evaluation of the potential toxicity associated with AgNPs is of great importance. Although some of the toxic effects induced by AgNPs have already been shown, the elucidation of more complete mechanisms is yet to be achieved. In this sense, and since the integration of metabolomics and transcriptomics approaches constitutes a very useful strategy, in the present study targeted and untargeted metabolomics and DNA microarrays assays have been combined to evaluate the molecular mechanisms involved in the toxicity induced by 10 nm AgNPs. The results have shown that AgNPs induce the synthesis of glutathione as a cellular defense mechanism to face the oxidative environment, while inducing the depletion of relevant molecules implicated in the synthesis of important antioxidants. In addition, it has been observed that AgNPs completely impair the intracellular energetic metabolism, especially affecting the production of adenosine triphosphate (ATP) and disrupting the tricarboxylic acids cycle. It has been demonstrated that AgNPs exposure also affects the glycolysis pathway. The effect on such pathway differs depending on the step of the cycle, which a significant increase in the levels of glucose as way to counterbalance the depleted levels of ATP.

Strategies for Membrane Protein Analysis by Mass Spectrometry.

Membrane proteins are of utmost importance in different cellular processes including: cell signaling, substrate transport, homeostasis control, immune surveillance, etc. In addition, they represent between 60% and 70% of the therapeutic targets currently used. Therefore, the identification and characterization of these proteins is crucial in many fields of research. Although proteomics has undergone an extraordinary advance in recent years thanks to the development of mass spectrometry, the methods used for the identification and quantification of soluble proteins generally fail to be used for membrane proteins, mainly due to their hydrophobic character.In this chapter, we revised the different alternatives, modifications and improvements that have been developed over the years with the aim of adapting the methods used in proteomics to the particular study of membrane proteins, thus allowing to increase the number of membrane proteins identified, as well as their coverage.

Cancer cell targeting and therapeutic delivery of silver nanoparticles by mesoporous silica nanocarriers: insights into the action mechanisms using quantitative proteomics.

An approach for safely delivering AgNPs to cancer cells and the evaluation of the affected cellular mechanism are presented. The use of mesoporous silica nanoparticles (MSNs) as nanovehicles decorated with transferrin (Tf, targeting agent) provides a nanoplatform for the nucleation and immobilization of AgNPs (MSNs-Tf-AgNPs). We performed the physico-chemical characterization of the nanosystems and evaluated their therapeutic potential using bioanalytical strategies to estimate the efficiency of the targeting, the degree of cellular internalization in two cell lines with different TfR expression, and the cytotoxic effects of the delivered AgNPs. In addition, cellular localization of the nanosystems in cells has been evaluated by a transmission electron microscopy analysis of ultrathin sections of human hepatocarcinoma (HepG2) cells exposed to MSNs-Tf-AgNPs. The in vitro assays demonstrate that only the nanosystem functionalized with Tf is able to transport the AgNPs inside the cells which overexpress transferrin receptors. Therefore, this novel nanosystem is able to deliver AgNPs specifically to cancer cells overexpressing Tf receptors and offers the possibility of a targeted therapy using reduced doses of silver nanoparticles as cytotoxic agents. Then, a quantitative proteomic experiment validated through the analysis of gene expression has been performed to identify the molecular mechanisms of action associated with the chemotherapeutic potential of the MSNs-Tf-AgNP nanocarriers.