How to characterize exosomes?

Exosomes are vesicles with a diameter of 30-150 nm, containing various lipids, proteins, and nucleic acids. Increasing research highlights the potential of exosomes as biomarkers and therapeutic agents. Precise quantitative and qualitative characterization of exosomes is crucial to realizing this potential. This article summarizes the techniques for quantifying and characterizing the proteins, lipids, and nucleic acids in exosomes.

Quantitative Characterization

Total exosome count

• Nanoparticle tracking analysis (NTA): NTA uses laser scattering microscopy to track the motion of nanoparticles in suspension via Brownian motion, allowing measurement of particle size and concentration. It can analyze exosome populations of varying sizes and provides high-resolution size information, making it one of the most common methods for quantifying exosomes.
• Electron microscopy (EM): EM employs electron beams for imaging, enabling observation at the sub-nanometer level. Common types include transmission electron microscopy (TEM) and scanning electron microscopy (SEM). This technique provides ultrastructural information essential for identifying exosome morphology and size.
• Flow cytometry: This technique analyzes and sorts cells and particles in mixtures based on light scattering and fluorescence signals as they pass through a laser beam. Nano flow cytometry (NanoFCM) is specifically designed for exosome detection, offering high sensitivity and resolution, capable of detecting exosomes as small as 30 nm, while providing information on size, concentration, and purity.
• Fluorescence correlation spectroscopy (FCS): FCS is a fluorescence-based autocorrelation analysis method that measures molecular diffusion and concentration in solution, suitable for detecting low concentrations and small exosomes.
• Dynamic light scattering (DLS): DLS analyzes the scattering of light by particles much smaller than the wavelength of light to determine particle size based on fluctuations in scattering intensity over time. While useful for studying dynamic size distribution, it cannot differentiate particles of different sizes and may overestimate total particle counts.
• Resistive pulse sensing (RPS): RPS measures changes in resistance as particles pass through a small aperture, allowing high-throughput detection with single-particle resolution, unaffected by optical properties. However, it may also overestimate total counts, limiting its application.

Exosomal proteins

• Mass spectrometry: This method analyzes components by measuring the mass-to-charge ratio (m/z) of ions, and when combined with bioinformatics, it can systematically characterize exosome-specific proteins, offering high sensitivity and resolution.
• Enzyme-linked immunosorbent assay (ELISA): ELISA quantitatively detects specific markers on exosome surfaces through antigen-antibody interactions. It is simple, rapid, and sensitive, but requires specific antibodies and may be affected by non-specific proteins in samples.

Exosomal lipids

• Sulfobenzoyl phosphatidylserine assay: This method quantifies lipids by generating colored compounds through reactions between phosphatidylserine and lipid-derived carbon ions in sulfuric acid, offering high sensitivity.
• Fluorescence microscopy with lipophilic dyes: Lipids are stained with dyes (e.g., Nile Red) for observation under fluorescence microscopy, providing a straightforward method to visualize lipid distribution in exosomes, though dye stability may be affected by sample conditions.
• Fourier transform infrared spectroscopy (FTIR): FTIR infers chemical composition by measuring infrared light absorption, suitable for qualitative and quantitative analysis of exosomal lipids. It is non-destructive and highly sensitive but lacks sensitivity for cholesterol and other sterols.

Exosomal DNA/RNA

• Polymerase chain reaction (PCR): PCR amplifies DNA or RNA in exosomes, providing a sensitive nucleic acid detection method suitable for low-abundance targets.
• Microarray chips: This high-throughput technique simultaneously analyzes multiple nucleic acids, commonly used for gene expression profiling in exosomes.
• Next-generation sequencing (NGS): NGS comprehensively analyzes nucleic acids in exosomes, allowing in-depth sequencing for genomic, transcriptomic, and epigenomic analysis, yielding rich sequence information suitable for complex samples.

Qualitative Characterization

Exosomal proteins

Specific exosome subtypes can be identified by combining exosomal protein markers, using methods such as flow cytometry, mass spectrometry, and ELISA, along with techniques like western blotting (WB), stimulated emission depletion (STED) microscopy, and surface plasmon resonance microscopy (SPRM).

• Western blotting: WB separates proteins via electrophoresis and detects them with specific antibodies, a classic and sensitive method for protein qualitative analysis that reveals molecular weight information.
• STED microscopy: This technique visually identifies exosomal protein markers with ultra-high resolution by reducing fluorescence background interference through focused laser beams.
• Surface plasmon resonance microscopy: This real-time monitoring technology analyzes molecular interactions by detecting changes in surface plasmon resonance, providing data on exosomal membrane protein binding kinetics.

Exosomal Lipids

Lipids can serve as alternative markers for qualitative analysis of exosomes, commonly detected using Raman Spectroscopy and mass spectrometry.

• Raman spectroscopy: This scattering technique based on molecular vibrations detects chemical composition and structural information, being non-destructive and high-resolution, suitable for various lipid components without sample morphology constraints. Mass spectrometry is also frequently used for lipid analysis.

Exosomal DNA/RNA

Qualitative analysis of DNA and RNA in exosomes employs microarray technology, NGS, and qPCR for comprehensive assessment.

References

1. Lai JJ, Chau ZL, et al. Exosome Processing and Characterization Approaches for Research and Technology Development. Adv Sci (Weinh). 2022. 9(15), e2103222.
2. Schey KL, Luther JM, et al. Proteomics characterization of exosome cargo. Methods. 2015. 1, 87:75-82.
3. Huang LH, Rau CS, et al. Identification and characterization of hADSC-derived exosome proteins from different isolation methods. J Cell Mol Med. 2021. 25(15), 7436-7450.

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