MXene Label-Free Detection and Immune-Based Applications

Two-dimensional (2D) transition metal carbides, nitrides, and carbonitrides (MXenes) are rapidly growing as nanoplatforms in biomedicine.[1-3] Considering the central importance of the immune response for any clinical translation of nanomaterials, we explored the potential biomedical applications of MXenes and their immune impact exploiting our expertise on the biological effects of 2D materials.[1-4]<br/>To satisfy a critical unmet need to detect 2D materials at the single-cell level while measuring multiple cell and tissue features, we proposed the “Label-free sINgle-cell tracKing of 2D matErials by mass cytometry and MIBI-TOF Design” (LINKED) strategy, enabling nanomaterial detection while surveying a high degree of information from single samples.[5] This strategy is based on single-cell mass cytometry (CyTOF) and ion-beam imaging by time-of-flight (MIBI-TOF). MXenes ensured mass detection within the cytometry range while avoiding overlap with more than 70 available tags. We demonstrated their detection in 15 human immune cell populations. We used mass cytometry to capture MXene biocompatibility and cytokine production after their uptake. <i>In vivo</i> biodistribution experiments using a mixture of MXenes in mice confirmed the versatility of our strategy and revealed MXene accumulation in the main organs.<br/>In addition, envisaging future MXene-based biomedical nanotools and drug nanoformulations we further explored MXene immune modulation and antiviral activity taking SARS-CoV-2 as a model.[6,7] We analyzed four SARS-CoV-2 genotypes. When viral inhibition was tested <i>in vitro</i>, Ti₃C₂, was able to reduce infection in SARS-CoV-2/clade GR-infected Vero E6 cells. Among the other MXenes tested, Mo₂Ti₂C₃ also showed antiviral properties. Proteomic and functional annotation analysis revealed that MXene-treatment exerts specific inhibitory mechanisms. The immune impact of MXenes was evaluated on human primary immune cells by flow cytometry and CyTOF on 17 distinct cell subpopulations. Moreover, 40 secreted cytokines were analyzed by Luminex technology. MXene immune profiling revealed i) the excellent bio and immune compatibility of the material, as well as the ability of MXene ii) to inhibit monocytes and iii) to reduce the release of pro-inflammatory cytokines, suggesting an anti-inflammatory effect elicited by MXene.<br/>Finally, we considered that although vanadium-based metallodrugs have been recently explored for their effective anti-inflammatory activity, they frequently cause undesired side effects. Therefore, we hypothesized that vanadium immune properties could be extended to MXene compounds.[8] Therefore, we synthesized V₄C₃, evaluating its biocompatibility and immunomodulatory effects. By combining multiple experimental approaches, we investigated the effects on hemolysis, apoptosis, necrosis, activation, and cytokine production. We demonstrated the ability to inhibit T cell-dendritic cell interactions, evaluating the modulation of CD40-CD40 ligand interaction, two key co-stimulatory molecules for immune activation. We confirmed the biocompatibility at the single-cell level on 17 human immune cell subpopulations by single-cell mass cytometry. Finally, we explored the molecular mechanism underlying V₄C₃immune modulation, demonstrating a MXene-mediated downregulation of antigen presentation-associated genes. Our findings set the basis for further V₄C₃investigation as a negative modulator of the immune response in inflammatory and autoimmune diseases. <br/>Taken together, our results provide a compendium of knowledge on the biocompatibility of MXenes for the safe development of multi-functioning nanosystems in biomedicine.<br/> <br/>References:<br/> <br/>1. Gogotsi Y & Anasori B. <i>ACS Nano</i>, 2019<br/>2. Fusco L<i> et al.</i> <i>Theranostics</i>, 2020<br/>3. Gazzi A <i>et al</i>. <i>Front Bioeng Biotechnol</i>, 2019<br/>4. Gazzi A <i>et al</i>. <i>J Phys materials</i>, 2020<br/>5. Fusco L <i>et al</i>. <i>Advanced Materials</i>, 2022<br/>6. Unal MA <i>et al. </i><i>Nano Today</i>, 2021<br/>7. Weiss C <i>et al</i>. <i>ACS Nano</i>, 2020<br/>8. Fusco L <i>et al</i>. <i>Small Methods</i>, 2023