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www.biopharminternational.com Manufacturing and Facilities eBook November 2024 BioPharm International ® 23 Analytics Extracellular Vesicles TABLE I. Absorbance and fluorescence properties of extracellular vesicles tested at 5 ppm. Serotype Absorbance (280nm, unit: a.u.) Fluorescence (Ex280nm/ Em330nm, unit: RSU) Relative quantum efficiency HEK293 0.174 0.7842 4.50 MCF7 0.618 1.0727 1.73 PC3 0.615 2.1913 3.56 released by cells into the extracellular environment. These vesicles are involved in numerous physiologi- cal processes, including immune responses, cell sig- naling, and the transfer of genetic material. EVs have gained attention as potential biomarkers for various diseases, such as cancer, neurodegenerative disor- ders, and cardiovascular diseases, due to their cargo (i.e., proteins, lipids, and nucleic acids) (1). As mentioned earlier, the characterization of EVs is crucial for understanding their biological f unc- tions and potential therapeutic applications. Tra- dit iona l met hods, such as nanopar t icle track ing ana lysis (N TA) and electron microscopy, prov ide valuable information about EV size and mor phol- og y. Fluorescence spectroscopy, a technique that measures the emission of light by f luorescent mol- ecules within a sample, can offer a rapid and sen- sitive complementary alternative technique for EV characterization. This study aims to investigate the application of f luorescence spectroscopy for the analysis of EVs de- rived from different cell types. Fluorescence could be another powerful complementar y technique in EV research's toolbox. Materials and methods Materials and reagents Lyophilized human embryonic kidney 293 (HEK293) cell-derived exosomes were obtained from Hansa BioMed (Tallinn, Estonia). Additionally, lyophilized e xosome s t a nd a rd s, P C-3 (ab239689) a nd MCF 7 (ab239691), each at a concentration of 1 x 1012, were pu rcha sed f rom Abca m (Ca mbr idge, Ma ss.). A l l samples were stored at -80 °C. In addition, 1X phos- phate-buffered saline (PBS) was obtained from Corn- ing (Corning, NY). Excitation emission fluorescence spectroscopy analysis A l l sa mple mea s u rement s were m ade u si ng si- multaneous absor ption, transmittance, and exci- tation-emission matrix acquisition (HORIBA Scien- tific, Piscataway, NJ), with excitation wavelengths ranging from 220 to 450 nm. A blank measurement was collected with PBS buffer. PC-3 and MCF7 exo- somes were reconstituted to 5 µg/µL with PBS, then diluted an additional 200X and placed in a standard UV/Vis 1 cm path length cuvette for measurement. The PC-3/PBS solution was measured, as well as mix- tures with the MCF7/PBS solution in the following propor tions: 75%, 50%, and 25%. Pure MCF7/PBS solutions were also measured. Statistical analysis Data analysis employed multivariate methods. Par- allel factor analysis (PARAFAC) was utilized for both qualitative and quantitative purposes, allowing for the differentiation and quantification of the two exo- some types. Results and discussion EV serotype discrimination The f luorescence analysis revealed distinct optical properties for EVs derived from different cell types, including extinction coefficient and quantum yield, as summarized in Table I. The fluorescence excitation emission matrix fingerprint is shown in Figure 1. All three EV types exhibited a peak excitation/emission wavelength of 280 nm/330 nm, corresponding to the tr yptophan and tyrosine residues in their compo- The characterization of EVs is crucial for understanding their biological functions and potential therapeutic applications.