Polyethylene glycol-based isolation of urinary extracellular vesicles, an easily adoptable protocol.

Urine is a highly advantageous biological specimen for biomarker research and is a non-invasive source. Most of the urinary biomarkers are non-specific, volatile and need extensive validation before clinical adoption. Extracellular vesicles are secreted by almost all cells and are involved in homoeostasis, intercellular communication, and cellular processes in healthy and pathophysiological states. Urinary extracellular vesicles (UEVs) are released from the urogenital system and mirror the molecular processes of physiological and pathological states of their source cells. Therefore, UEVs serve as a valuable source of biomarkers for the non-invasive diagnosis of various pathologies. They hold a promising source of multiplex biomarkers suitable for prognosis, diagnosis, and therapy monitoring. UEVs are easily accessible, non-invasive, and suited for longitudinal sampling. Although various techniques are available for isolating UEVs, there is yet to be a consensus on a standard and ideal protocol. We have optimized an efficient, reliable, and easily adoptable polyethylene glycol (PEG) based UEV isolation technique following MISEV guidelines. The method is suitable for various downstream applications of UEVs. This could be a cost-effective, consistent, and accessible procedure for many clinical labs and is most suited for longitudinal analysis. Adopting the protocol will pave the way for establishing UEVs as the ideal biomarker source. •Urine can be collected non-invasively and repeatedly, hence a very useful specimen for biomarker discovery. Urinary EVs (UEVs), derived from urine, offer a stable diagnostic tool, but standardised isolation and analysis approaches are warranted.•To have enough UEVs for any study, large volumes of urine sample are necessary, which limits different isolation methods by cost, yield, and time.•The protocol developed could help researchers by offering a cost-effective and dependable UEV isolation method and may lay the foundation for UEVs adoption in clinical space.

Exploring urinary extracellular vesicles for organ transplant monitoring: A comprehensive study for detection of allograft dysfunction using immune-specific markers.

BACKGROUND: Allograft dysfunction (AGD) is a common complication following solid organ transplantation (SOT). This study leverages the potential of urinary extracellular vesicles (UEVs) for the non-invasive detection of AGD. AIM: We aimed to assess the diagnostic value of T-cell and B-cell markers characteristic of T-cell-mediated and antibody-mediated rejection in UEV-mRNA using renal transplantation as a model. MATERIALS AND METHODS: UEVs were isolated from 123 participants, spanning healthy controls, functional transplant recipients, and biopsy-proven AGD patients. T-cell and B-cell marker mRNA expressions were evaluated using RT-qPCR. RESULTS: We observed significant differences in marker expression between healthy controls and AGD patients. ROC analysis revealed an AUC of 0.80 for T-cell markers, 0.98 for B-cell markers, and 0.94 for combined markers. T-cell markers achieved 81.3 % sensitivity, 80 % specificity, and 80.4 % efficiency. A triad of T-cell markers (PRF1, OX40, and CD3e) increased sensitivity to 87.5 % and efficiency to 82.1 %. B-cell markers (CD20, CXCL3, CD46, and CF3) delivered 100 % sensitivity and 97.5 % specificity. The combined gene signature of T-cell and B-cell markers offered 93.8 % sensitivity and 95 % specificity. CONCLUSION: Our findings underscore the diagnostic potential of UEV-derived mRNA markers for T-cells and B-cells in AGD, suggesting a promising non-invasive strategy for monitoring graft health.

Recent Trends in the Use of Small Extracellular Vesicles as Optimal Drug Delivery Vehicles in Oncology.

Small extracellular vesicles (sEVs) are produced by most cells and play an important role in cell-to-cell communication and maintaining cellular homeostasis. Their ability to transfer biological cargo to target cells makes them a promising tool for cancer drug delivery. Advances in sEV engineering, EV mimetics, and ligand-directed targeting have improved the efficacy of anticancer drug delivery and functionality. EV-based RNA interference and hybrid miRNA transfer have also been extensively used in various preclinical cancer models. Despite these developments, gaps still exist in our understanding of using sEVs to treat solid tumor malignancies effectively. This article provides an overview of the last five years of sEV research and its current status for the efficient and targeted elimination of cancer cells, which could advance cancer research and bring sEV formulations into clinical use.

Identifying stable reference genes in polyethene glycol precipitated urinary extracellular vesicles for RT-qPCR-based gene expression studies in renal graft dysfunction patients.

BACKGROUND: Urinary extracellular vesicles (UEVs) hold RNA in their cargo and are potential sources of biomarkers for gene expression studies. The most used technique for gene-expression studies is quantitative polymerase chain reaction (qPCR). It is critical to use stable reference genes (RGs) as internal controls for normalising gene expression data, which aren't currently available for UEVs. METHODS: UEVs were precipitated from urine of graft dysfunction patients and healthy controls by Polyethylene glycol, Mn6000 (PEG6K). Vesicular characterisation confirmed the presence of UEVs. Gene expression levels of five commonly used RGs, i.e., Beta-2-Microglobulin (B2M), ribosomal-protein-L13a (RPL13A), Peptidylprolyl-Isomerase-A (PPIA), hydroxymethylbilane synthase (HMBS), and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) were quantified, and their stability was established through the RefFinder. The stability of identified RGs was validated by quantification of Perforin and granzyme B, signature molecules of renal graft dysfunction. RESULTS: Urine precipitated with 12% 6 K PEG yielded round and double-membraned UEVs of size ranging from 30 to 100 nm, as confirmed through transmission electron microscopy. Nanoparticle tracking analysis (59 ± 22 nm) and Dynamic-light-scattering (78 ± 56.5 nm) confirmed their size profile. Semi-quantitative Exocheck antibody array demonstrated the presence of EV protein markers in UEV. Using the comparative ΔCт method and RefFinder analysis, B2M (1.6) and RPL13A (1.8) genes emerged as the most stable reference genes. Validation of target gene expression in renal graft dysfunction patients confirmed the efficiency of B2M and RPL13A through significant upregulation compared to other RGs. CONCLUSIONS: Our study identified and validated B2M and RPL13A as optimal RGs for mRNA quantification studies in the UEVs of patients with renal graft dysfunction.