The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is a revolutionary tool for precise genome editing across various cell types. Ribonucleoproteins (RNPs), encompassing the Cas9 protein and guide RNA (gRNA), have emerged as a promising technique due to their increased specificity and reduced off-target effects. This method eliminates the need for plasmid DNA introduction, thereby preventing potential integration of foreign DNA into the target cell genome. Given the requirement for large quantities of highly purified protein in various Cas9 studies, we present an efficient and simple method for the preparation of recombinant Streptococcus pyogenes Cas9 (SpCas9) protein. This method leverages the Small Ubiquitin Like Modifier(SUMO) tag system, which includes metal-affinity chromatography followed by anion-exchange chromatography purification. Furthermore, we compare two methods of CRISPR-Cas9 system delivery into cells: transfection with plasmid DNA encoding the CRISPR-Cas9 system and RNP transfection with the Cas9-gRNA complex. We estimate the efficiency of genomic editing and protein lifespan post-transfection. Intriguingly, we found that RNP treatment of cells, even in the absence of a transfection system, is a relatively efficient method for RNP delivery into cell culture. This discovery is particularly promising as it can significantly reduce cytotoxicity, which is crucial for certain cell cultures such as induced pluripotent stem cells (iPSCs).

Chimeric antigen receptor (CAR) therapy represents a promising modality for treating cancer and autoimmune diseases, employing genetically engineered immune cells. Despite remarkable clinical outcomes, its broad implementation is constrained by significant challenges, including toxicity, limited specificity, and complexities associated with genetic material delivery. Biological nanoparticles, such as exosomes, virus-like particles, and biomimetic nanostructures, possess unique properties that can address these limitations. These nanoplatforms enable targeted delivery of genetic constructs, mitigate the risk of cytokine release syndrome, modulate CAR cell activity, and can function as biosensors. Furthermore, they facilitate non-viral, in vivo CAR cell engineering, streamlining the process compared to conventional ex vivo methods. The advancement of in vivo strategies underscores the critical need to overcome toxicity hurdles inherent to current CAR-T platforms. In this context, exosomes and biomimetic nanoparticles offer considerable potential due to their innate biocompatibility, programmability, and versatile cargo capacity for payloads like mRNA and circular RNA. This review comprehensively outlines contemporary genetic platforms for CAR expression and examines the opportunities presented by biological delivery vehicles. It focuses on recent achievements and revisits fundamental CAR principles through the lens of emerging technologies aimed at confronting persistent challenges in the field.

For over eight decades, antibiotics have been the cornerstone of treating bacterial infections. However, the rapid rise of antibiotic-resistant pathogens has created an urgent need for alternative therapeutic strategies. Advances in nanotechnology offer a promising solution through the development of bio-derived nanoparticles. This broad class includes extracellular vesicles such as exosomes and bacterial outer membrane vesicles (OMVs), as well as bioengineered cell membrane-coated nanoparticles (CMNPs) that combine synthetic cores with natural membranes from diverse source cells. These particles possess unique physicochemical and biological properties, such as intrinsic bioactivity, biocompatibility, and structural versatility, that can be harnessed for antimicrobial therapy. This review synthesizes recent progress in the design, characterization, and application of biological nanoparticles for combating bacterial infections. We place particular emphasis on their mechanisms of action, therapeutic potential, and key research directions that could accelerate their translation into clinical use.

Extracellular vesicles (EVs) represent a promising tool for diagnostics and therapy, but high heterogeneity limits their practical use. In this work, a comparative study of the physicochemical properties of native EVs secreted by HEK-293T cells and exosome-like nanovesicles (EMNVs) obtained by extrusion of whole HEK-293T cells was carried out. Using transmission electron microscopy, dynamic and electrophoretic light scattering, and surface-enhanced Raman spectroscopy, the authors found that EMNVs form a more homogeneous population with an average diameter of ~115 nm and a high negative charge (−22.9 mV), whereas native EVs are more heterogeneous in size and have a lower zeta potential (−12.1 mV). Spectral analysis revealed the presence of nucleic acid signals in native EVs and their absence in EMNVs, indicating differences in molecular composition due to different biogenesis mechanisms. The obtained data demonstrate that EMNVs are a more standardized and colloidally stable alternative to native EVs, making them a promising platform for the development of drug-delivery systems.