MiR-21 and miR-375 have been reported as dysregulated in prostate cancer (PCa) in multiple previous studies. Still, variable or even opposing data for the expression of these microRNAs in PCa were found, and their potential biomarker properties remain elusive. In an attempt to clarify their significance as PCa biomarkers, as well as to compare different types of specimens as a source of relevant microRNAs, we used plasma and matching plasma-derived exosomes from patients with PCa and patients with benign prostatic hyperplasia (BPH). Plasma and exosomes were obtained from 34 patients with PCa and 34 patients with BPH, and their levels of expression of miR-21 and miR-375 were determined by RT-qPCR. We found no significant difference in the level of expression of these microRNAs in plasma and exosomes between patients with PCa and BPH. The level of exosomal miR-21 was elevated in PCa patients with high serum PSA values, as well as in patients with aggressive PCa, while for plasma samples, the results remained insignificant. For miR-375, we did not find an association with the values of standard prognostic parameters of PCa, nor with cancer aggressiveness. Therefore, our results support the potential prognostic role of exosomal miR-21 expression levels in PCa.

Prostate-specific membrane antigen (PSMA) and caveolin-1 are membrane proteins that are overexpressed in prostate cancer (PCa) and are involved in tumor growth and increase in aggressiveness. The aim of the present study is therefore to evaluate PSMA and caveolin-1 proteins from plasma exosomes as effective liquid biopsy biomarkers for PCa. This study included 39 patients with PCa and 33 with benign prostatic hyperplasia (BPH). The shape and size of the exosomes were confirmed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analysis. Immunogold analysis showed that PSMA is localized to the membrane of exosomes isolated from the plasma of both groups of participants. The relative protein levels of PSMA and caveolin-1 in the plasma exosomes of PCa and BPH patients were determined by Western blot analysis. The relative level of the analyzed plasma exosomal proteins was compared between PCa and BPH patients and the relevance of the exosomal PSMA and caveoin-1 level to the clinicopathological parameters in PCa was investigated. The analysis performed showed an enrichment of exosomal PSMA in the plasma of PCa patients compared to the exosomes of men with BPH. The level of exosomal caveolin-1 in plasma was significantly higher in PCa patients with high PSA levels, clinical-stage T3 or T4 and in the group of PCa patients with aggressive PCa compared to favorable clinicopathological features or tumor aggressiveness. Plasma exosomes may serve as a suitable object for the identification of potential biomarkers for the early diagnosis and prognosis of PCa as well as carriers of therapeutic agents in precision medicine of PCa treatment.

Urine, a common source of biological markers in biomedical research and clinical diagnosis, has recently generated a new wave of interest. It has recently become a focus of study due to the presence of its content of extracellular vesicles (EVs). These uEVs have been found to reflect physiological and pathological conditions in kidney, urothelial, and prostate tissue and can illustrate further molecular processes, leading to a rapid expansion of research in this field In this work, we present the advantages of an immunoaffinity-based method for uEVs’ isolation with respect to the gold standard purification approach performed by differential ultracentrifugation [in terms of purity and antigen presence. The immunoaffinity method was made feasible by combining specific antibodies with a functionalized polymethacrylate polymer. Flow cytometry indicated a significant fluorescence shift, validating the presence of the markers (CD9, CD63, CD81) and confirming the effectiveness of the isolation method. Microscopy evaluations have shown that the morphology of the vesicles remained intact and corresponded to the expected shapes and dimensions of uEVs. The described protocol is inexpensive, fast, easy to process, has good reproducibility, and can be applied to further biological samples.

Extracellular vesicles (EVs) are nanosized structures involved in intercellular communication that have high potential as disease biomarkers and for the delivery of therapeutic cargos. However, translation to the clinic is hampered by time-consuming, low-yield, and poorly reproducible EV isolation methods. We describe a cryogel-based immunoaffinity chromatography system that exploits single-domain VHH antibodies as capture elements for the selective isolation of EVs from human plasma. Supermacroporous cryogels functionalized with five unique anti-EV VHHs (total immobilization capacity ~500 µg/g) were prepared, yielding a highly permeable and hydrophilic support. They were captured and eluted under mild conditions, and their morphology and identity were confirmed by SEM, AFM, NTA, and flow cytometry. Proteomic profiling of the isolated samples identified 234 proteins, of which 63% were ExoCarta-listed exosomal proteins; contaminants such as albumin and apolipoproteins were also identified. The purification method provided samples with ~2 × 10⁹ EVs/mL, with EV median size of 135 nm and consistent protein-to-lipid ratio across three independent isolations (CV < 10%). This study demonstrates that VHH-functionalized cryogels (VHH-SMC) are a rapid and reproducible EV purification method that represents a promising alternative to conventional ultracentrifugation- or precipitation-based protocols. While optimization of nanobody density and reduction in plasma protein carryover are still necessary, the platform holds potential for scalable EV enrichment, a condition that can significantly speed up biomarker research and clinical diagnostics.

Malignant pleural effusion (MPE) is a common clinical manifestation of advanced non-small cell lung cancer (NSCLC) and represents a valuable source of tumor-derived components, including extracellular vesicles (EVs). The aim of this study was to compare three different EV enrichment methods with potential applicability in clinical practice: a commercial Norgen kit (NOR), immunoaffinity capture (IA), and ultracentrifugation (UC). Following EV characterization, IA demonstrated the best overall performance in terms of EV yield and purity, NOR showed intermediate efficiency, while UC was the least effective method. The IA method exhibited characteristics suitable for potential clinical implementation, whereas NOR and UC may require combination with additional enrichment approaches. Advancing pleural effusion–based liquid biopsy toward clinical application will depend on the development of robust, scalable, and user-friendly EV enrichment workflows, along with harmonized guidelines for sample collection, preprocessing, and data reporting. Establishing such standards will enhance reproducibility, enable meaningful cross-study comparisons, and accelerate the integration of EV-based biomarkers into precision oncology.

Extracellular vesicles (EVs) are membrane-enclosed nanoparticles involved in intercellular communication and numerous physiological and pathological processes. Their molecular cargo reflects the state of the cell of origin, making EVs promising sources of biomarkers and potential therapeutic agents. However, efficient and selective isolation of EVs from complex biological fluids remains challenging. Immunoaffinity-based approaches offer high selectivity through the recognition of EV-associated surface markers by specific affinity ligands. In this study, silica-coated magnetite nanoparticles were functionalized with a mixture of five VHH–eGFP constructs and evaluated as a solid phase for immunoaffinity isolation of EVs from human plasma. Surface modification of the nanoparticles was confirmed by FTIR spectroscopy, while protein-binding studies showed a maximum binding capacity (Qmax) of 118.2 mg/g. The developed material was then applied for EV isolation from plasma, and the resulting EV-enriched preparations were characterized by protein and lipid quantification, nanoparticle tracking analysis, flow cytometry, and atomic force microscopy. NTA revealed a median particle diameter of 124 nm and a particle concentration of 6.9 × 10⁹ particles/mL. The presence of the EV-associated markers CD9, CD63, and CD81, together with their reduced signal following Triton X-100 treatment, supported the vesicular nature of the isolated particles. The affinity material could be reused over five consecutive isolation cycles, and EVs could be detected from plasma volumes as low as 25 µL. These results demonstrate the potential of VHH-functionalized magnetic nanoparticles as a reusable and adaptable platform for immunoaffinity-based EV isolation from human plasma.