Extracellular vesicles (EVs) consist of lipid bilayers, occur in various biofluids, and are invaluable in biomarker screening. Liquid chromatography coupled with high-resolution mass spectrometry (LC-MS) was recently used to study comprehensive EV lipid profiles in vitro. The aim of this study was to establish a lipidomics platform for human plasma and serum EVs for comprehensive characterization of their lipid profiles, and to compare them with those of other lipid-containing particles, such as high-density lipoproteins (HDL), and low/very low-density lipoproteins (LDL/VLDL). Isolation was validated by specific protein markers; CD9 and MHC class I for EVs, apoA-I for HDL, and apoB-100 for LDL/VLDL. Lipidomics identified 264 lipids from isolated plasma EVs, HDL, and LDL/VLDL. The absolute lipid levels per unit protein content in the EVs were more than eight times lower than those of the lipoproteins. Moreover, the EVs had higher lysoglycerophospholipid levels than HDL or LDL/VLDL. Similar profiles were also determined for human serum. The present study found that the lipid profiles of EVs are unique and distinctly different from those of lipoproteins. The lipidomics platform applied to human plasma and serum EVs could generate important information for the exploration and qualification of biomarkers in disease diagnosis.

Background: Ion-pairing reverse-phase LC coupled with high-resolution mass spectrometry (IP-LC/HRMS) has gained attention in oligonucleotide therapeutic bioanalyses owing to its high sensitivity an...

Background: Extracellular vesicles (EVs), including exosomes, are promising pharmaceutical modalities. They are purified from cell culture supernatant; however, the preparation may contain EVs with the desired therapeutic effects and different types of EVs, lipoproteins, and soluble proteins. Evaluating the composition of particulate impurities and the levels of protein impurities in final preparations is critical for quality control. However, few analytical methods can detect these impurities. Methods: We established and evaluated an analytical method using size-exclusion chromatography–multi-angle light scattering (SEC-MALS) for particle and protein impurity analyses of EV samples. Results: In the particle size distribution analysis of EV samples, SEC-MALS showed higher resolution compared with nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS). MALS showed comparable accuracy and precision to that of other methods for particle size evaluation using polystyrene standard beads with 60, 100, or 200 nm diameter. Coupling SEC-MALS with UV detection quantitatively evaluated soluble protein impurities. Proteomic analysis on the SEC-MALS-fractionated samples identified different EV and lipoprotein marker proteins in different fractions. Conclusions: SEC-MALS can characterize EV preparations obtained from human adipose-derived mesenchymal stem cells, suggesting that it can evaluate the particle component composition in various EV samples and therapeutic exosome preparations.

Conventional anti-cancer therapies based on chemo- and/or radiotherapy represent highly effective means to kill cancer cells but lack tumor specificity and, therefore, result in a wide range of iatrogenic effects. A promising approach to overcome this obstacle is spliceosome-mediated RNA trans-splicing (SMaRT), which can be leveraged to target tumor cells while leaving normal cells unharmed. Notably, a previously established RNA trans-splicing molecule (RTM44) showed efficacy and specificity in exchanging the coding sequence of a cancer target gene (Ct-SLCO1B3) with the suicide gene HSV1-thymidine kinase in a colorectal cancer model, thereby rendering tumor cells sensitive to the prodrug ganciclovir (GCV). In the present work, we expand the application of this approach, using the same RTM44 in aggressive skin cancer arising in the rare genetic skin disease recessive dystrophic epidermolysis bullosa (RDEB). Stable expression of RTM44, but not a splicing-deficient control (NC), in RDEB-SCC cells resulted in expression of the expected fusion product at the mRNA and protein level. Importantly, systemic GCV treatment of mice bearing RTM44-expressing cancer cells resulted in a significant reduction in tumor volume and weight compared with controls. Thus, our results demonstrate the applicability of RTM44-mediated targeting of the cancer gene Ct-SLCO1B3 in a different malignancy.

Background: Many bioanalytical methods for antisense oligonucleotides (ASOs) using LC–MS have been reported. However, no data have been available on the reproducibility and robustness of a single b...