# Origin and Composition of Exosomes as Crucial Factors in Designing Drug Delivery Systems
## 3 Methods for Exosomes Characterization  
Characterization of the exosomes structure in terms of drug delivery systems (DDSs) is crucial because it sets the further properties of DDS, e.g., cells/tissues affinity, stress response, absorption routes, and drug release. The International Society for Extracellular Vesicles in 2014 and 2018 (MISEV2018) presented guidelines for basic requirements that all exosomes including studies and exosomes preparation should meet [13]. Parameters such as amount, size, morphology, membrane composition, and proteins including receptors must be considered while developing exosome-based DDS. These parameters are characterized by techniques divided into optical, non-optical, and microfluidics [6]. Currently, the most widely utilized methods for exosomes characterization are optical, namely dynamic light scattering (DLS), multi-angle light scattering (MALS), and nanoparticle tracking analysis (NTA) [14,15]. These methods allow for high-resolution  
Figure 1: Scheme of the exosomes formation, release, and internalization by cells. measurement of size, (DLS 0.5-200 nm; MALS 10-500 nm; NTA 10-1000 nm), size distribution, and concentration. The combination of DLS and MALS greatly increases the range (0.5-500 nm) and precision of measurements. Thus, it leads to the detection of three subsequent exosomes populations, described as small (Exo-S), large (Exo-L), and non-membranous exomeres [16]. In the NTA method also, the inclusion of fluorescent dyes enhances the resolution and allows the characterization of the surface immunophenotype by the use of fluorescence labeling [17; 18]. Optical methods are the most popular but the downsides are low sensitivity, high reagent consumption, and the requirement for specialized equipment. Non-optical methods include scanning electron microscopy (SEM), transmission electron microscopy (TEM), cryo-electron microscopy (Cryo-EM), atomic force microscopy (AFM), immunodetection methods (ELISA), Fourier transform infrared spectroscopy (FTIR), tunable resistive pulse sensing (TRPS), and the single-particle interferometric reflectance imaging sensor (SP-IRIS) [19; 20; 21]. SEM, TEM, and AFM methods are routinely utilized for direct membrane structure and morphology determination, while ELISA assays provide detection and quantification of various structural particles, mostly proteins and receptors, e.g., for exosome markers presence confirmation [22; 23; 24; 20; 25; 26]. FTIR spectroscopy and attenuated total reflection-FTIR (ATR-FTIR) are proposed for exosomes quality quantification and the overall estimation of lipid and protein content [20]. With TRPS, the simultaneous measurement of size, concentration, and zeta potential is possible. This method is rapidly growing in the field of exosomes research [21]. A single-particle interferometric reflectance imaging sensor (SP-IRIS) is also used for exosomes quantification but can also be utilized for the detection of specific markers and the determination of exosomes subpopulation [26; 27]. Non-optical methods also include the use of specialized equipment and the need for laborious sample preparation but provide high-quality surface, structure, and composition data. Further development of exosomes investigation methods leads to the use of microfluidics methods in exosomes research. These methods provide high-quality, high-specificity data with subsequent low reagent consumption and high throughput [6; 28; 29]. Microfluidics-based methods are methods modified to suit the nanoscale for proper exosomes determination. These methods require specialized, prefabricated microchips, generally designed and prepared exclusively for a specific application in exosomes research. Microfluidics chips are generally fabricated out of a glass base and polydimethylsiloxane (PDMS) membrane and contain a lot of microchannels whose size is suited for the analyzed sample.