Liver plays a central role in elimination of circulating extracellular vesicles (EVs), and it also significantly contributes to EV release. However, the involvement of the different liver cell populations remains unknown. Here, we investigated EV uptake and release both in normolipemia and hyperlipidemia. C57BL/6 mice were kept on high fat diet for 20–30 weeks before circulating EV profiles were determined. In addition, control mice were intravenously injected with ^99mTc-HYNIC-Duramycin labeled EVs, and an hour later, biodistribution was analyzed by SPECT/CT. In vitro, isolated liver cell types were tested for EV release and uptake with/without prior fatty acid treatment. We detected an elevated circulating EV number after the high fat diet. To clarify the differential involvement of liver cell types, we carried out in vitro experiments. We found an increased release of EVs by primary hepatocytes at concentrations of fatty acids comparable to what is characteristic for hyperlipidemia. When investigating EV biodistribution with ^99mTc-labeled EVs, we detected EV accumulation primarily in the liver upon intravenous injection of mice with medium (326.3 ± 19.8 nm) and small EVs (130.5 ± 5.8 nm). In vitro, we found that medium and small EVs were preferentially taken up by Kupffer cells, and liver sinusoidal endothelial cells, respectively. Finally, we demonstrated that in hyperlipidemia, there was a decreased EV uptake both by Kupffer cells and liver sinusoidal endothelial cells. Our data suggest that hyperlipidema increases the release and reduces the uptake of EVs by liver cells. We also provide evidence for a size-dependent differential EV uptake by the different cell types of the liver. The EV radiolabeling protocol using ^99mTc-Duramycin may provide a fast and simple labeling approach for SPECT/CT imaging of EVs biodistribution.

Recent studies showed an unexpected complexity of extracellular vesicle (EV) biogenesis pathways. We previously found evidence that human colorectal cancer cells in vivo release large multivesicular body-like structures en bloc. Here, we tested whether this large EV type is unique to colorectal cancer cells. We found that all cell types we studied (including different cell lines and cells in their original tissue environment) released multivesicular large EVs (MV-lEVs). We also demonstrated that upon spontaneous rupture of the limiting membrane of the MV-lEVs, their intraluminal vesicles (ILVs) escaped to the extracellular environment by a ‘torn bag mechanism’. We proved that the MV-lEVs were released by ectocytosis of amphisomes (hence, we termed them amphiectosomes). Both ILVs of amphiectosomes and small EVs separated from conditioned media were either exclusively CD63 or LC3B positive. According to our model, upon fusion of multivesicular bodies with autophagosomes, fragments of the autophagosomal inner membrane curl up to form LC3B positive ILVs of amphisomes, while CD63 positive small EVs are of multivesicular body origin. Our data suggest a novel common release mechanism for small EVs, distinct from the exocytosis of multivesicular bodies or amphisomes, as well as the small ectosome release pathway.

Collective cell migration is crucial in various biological processes, including tumor progression and metastasis. The widely used scratch assay (wound healing assay) has limitations in throughput, reproducibility, and data analysis. To overcome these challenges, we previously developed the Transient Agarose Spot (TAS) assay, which enhanced assay precision and reproducibility. In this study, we present an improved microplate-based TAS assay. By using a microplate reader, we automated data acquisition, enabling the detection of cell migration in a 96-well plate format with greater throughput and accuracy. The new method applies Hoechst staining to label viable cells, providing a stable signal for kinetic analysis without compromising cell viability. We validated this approach with fluorophore-expressing cancer cells and demonstrated its ability to monitor dose-dependent effects of fetal bovine serum on cell migration. Additionally, we applied the microplate-based TAS assay to assess the anti-migratory effects of kinase inhibitors and mesenchymal stem cell-derived extracellular vesicles (EVs) on lung cancer cells. The assay accurately quantified migration inhibition and revealed the concentration-dependent effects of EVs, highlighting their potential as therapeutic agents. This microplate-based TAS assay provides a scalable, efficient, and cost-effective platform for high-throughput screening of cell migration and drug discovery, offering a robust alternative to traditional microscopy-based methods.

Recent studies showed an unexpected complexity of extracellular vesicle (EV) biogenesis pathways. We previously found evidence that human colorectal cancer cells in vivo release large multivesicular body-like structures en bloc. Here, we tested whether this large extracellular vesicle type is unique to colorectal cancer cells. We found that all cell types we studied (including different cell lines and cells in their original tissue environment) released multivesicular large EVs (MV-lEVs). We also demonstrated that upon spontaneous rupture of the limiting membrane of the MV-lEVs, their intraluminal vesicles (ILVs) escaped to the extracellular environment by a ”torn bag mechanism”. We proved that the MV-lEVs were released by ectocytosis of amphisomes (hence, we termed them amphiectosomes). Both ILVs of amphiectosomes and small EVs separated from conditioned media were either exclusively CD63 or LC3B positive. According to our model, upon fusion of multivesicular bodies with autophagosomes, fragments of the autophagosomal inner membrane curl up to form LC3B positive ILVs of amphisomes, while CD63 positive small EVs are of multivesicular body origin. Our data suggest a novel common release mechanism for small EVs, distinct from the exocytosis of multivesicular bodies or amphisomes, as well as the small ectosome release pathway.

Getting through the cell membrane is challenging, and transporting a therapeutic agent while entering the cell is even more complicated. Cell-penetrating peptides (CPPs) are valuable tools for solving this problem, although they have drawbacks. In this work, the synthesis and investigation of efficient CPPs are described. We used an aromatic group, 1-pyrene carboxylic acid (PCA), to enhance internalization. We designed oligoarginines to investigate the effect of PCA in different positions at the N-terminus or in the side chain. Our novel peptide derivatives showed remarkable internalization on tumor cell lines, and more than one endocytic pathway plays a role in their internalization mechanism. With this modification, there is an opportunity to design short oligoarginines that can rival well-known CPPs like octaarginine in internalization.

Background/Objectives: Investigating the modified derivatives of known cell-penetrating peptides can highlight the important residues in the peptide sequence and help understand the cellular uptake mechanism better. Moreover, comparing peptides with different fluorescent-dye positions can highlight the importance of the conjugation site. Earlier, it was demonstrated that the fluorescence quencher 4-((4-(dimethylamino)phenyl)azo)benzoyl (Dabcyl) group can enhance the internalization efficiency of highly cationic oligoarginine peptides. However, its effect in the case of arginine-rich penetratin, a secondary amphipathic cationic CPP, remains undiscovered. Methods: Here, several penetratin derivatives were studied in which the aromatic residues were substituted and the effect of Dabcyl modification was also studied on the cellular uptake of peptides by flow cytometry. Results: The triple Nal-substituted penetratin and dodeca-penetratin with N-terminally positioned carboxyfluoresein (Cf) dye demonstrated remarkable internalization efficiency compared to penetratin. Moreover, almost all the Dabcyl-modified peptides were superior to penetratin except two peptides with C-terminal Cf-labelling. This result highlights the importance of the structure of the conjugate. The position of the cargo molecule may have a high impact on internalization ability. The relatively low cellular uptake of the Trp48 residue-substituted Dabcyl-Pen12 points to the importance of this residue in the cellular uptake of dodeca-penetratin. The confocal microscopic studies revealed that, besides the greater penetration efficiency of Dabcyl penetratin derivatives, these peptides enter the cytoplasm of cells in an increased manner. Conclusions: We identified several intriguing derivatives and expanded the applicability of Dabcyl, while also highlighting its limitations. Additionally, the critical role of Trp48 in the penetratin sequence was reaffirmed, along with the importance of the fluorescent molecule’s position.