Simple methods for measuring milk exosomes using fluorescent compound GIF-2250/2276

Area of Science:

• Biochemistry
• Nanotechnology
• Immunology

Background:

• Exosomes, small extracellular vesicles, are crucial in intercellular communication but challenging to analyze due to their size.
• Current methods for exosome analysis are limited, necessitating novel approaches for accurate quantification and functional assessment.
• Milk exosomes possess potential biological activities and immunomodulatory properties that are sensitive to processing methods.

Purpose of the Study:

• To evaluate GIF-2250 and GIF-2276 as fluorescent probes for milk exosome analysis.
• To investigate the impact of heat sterilization on the integrity and biological activity of milk exosomes.
• To characterize the protein content and immunomodulatory functions of milk exosomes.

Main Methods:

• Synthesis and application of nitrobenzoxadiazole (NBD)-conjugated probes GIF-2250 and GIF-2276 for exosome labeling.
• Gel filtration chromatography for separating labeled milk exosomes.
• Correlation analysis of probe intensity, exosome quantity, size, and biological activity (NO synthesis suppression).
• Proteome analysis of labeled exosomes and assessment of heat denaturation effects.
• Transmission electron microscopy (TEM) for visualizing exosome integrity.

Main Results:

• A correlation was found between GIF-2250 intensity and protein markers in bovine milk exosomes.
• GIF-2276 successfully labeled and separated milk exosomes, with chromatographic peaks indicating >3 ng protein.
• Exosome quantity and size, measured by GIF-2276, correlated with NO synthesis suppression; heat denaturation disrupted these indicators.
• Proteome analysis identified immunomodulators (e.g., butyrophilin subfamily 1 member A1) in GIF-2276-labeled exosomes, with reduced immunogenicity after heat treatment.
• Raw and low-temperature sterilized milk contained exosomes, while high-temperature sterilized milk did not, confirmed by TEM.

Conclusions:

• GIF-2250 and GIF-2276 are valuable tools for evaluating milk exosomes, offering precise quantification and functional assessment.
• High-temperature sterilization significantly compromises milk exosome integrity and biological functions.
• Understanding exosome content and activity is crucial, especially concerning their immunomodulatory roles, and is affected by processing conditions.