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

Saho Furukawa1, Kyoka Kawaguchi1, Kotomi Chikama1

  • 1Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.

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Summary

New fluorescent probes, GIF-2250 and GIF-2276, enable precise analysis of milk exosomes. These probes reveal that high-temperature sterilization eliminates exosomes, impacting their biological activity and immunomodulatory proteins.

Keywords:
BTN1A1Fluorescent compoundGel-filtrationImagingMilk exosomepIgR

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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.