Endogenous Labelling of Extracellular Vesicles and Image Capture of Their Interactions With Acceptor Cells

Area of Science:

• Biochemistry
• Cell Biology
• Nanotechnology

Background:

• Extracellular vesicles (EVs) play crucial roles in physiological and pathological processes.
• Mechanisms of EV uptake and cytosolic cargo delivery are not fully understood.
• Accurate tracking of EVs in biological systems is essential for studying intercellular communication.

Purpose of the Study:

• To develop a novel near-infrared (NIR) fluorophore for endogenous labeling of EVs.
• To enable real-time visualization of EV-acceptor cell interactions using fluorescence microscopy.
• To establish a robust platform for studying EV uptake and cargo delivery.

Main Methods:

• Development of a BF2-azadipyrromethene-based NIR fluorophore.
• Endogenous labeling of EVs with the developed fluorophore.
• Isolation and characterization of labeled EVs based on photophysical properties (emission wavelength and lifetime).
• Observation of EV-acceptor cell interactions using intensity and fluorescence lifetime imaging microscopy at different temperatures (4°C and 37°C).

Main Results:

• The developed fluorophore enabled efficient endogenous labeling of EVs.
• Labeled EVs exhibited distinct emission wavelengths (λmax 720 nm) and lifetimes (2.7 ns).
• Distinct photophysical properties allowed discrimination between aggregated and disaggregated fluorophore forms.
• Real-time imaging confirmed EV-plasma membrane interactions at 4°C and efficient fluorophore transfer into acceptor cells at 37°C.

Conclusions:

• Endogenous labeling with the novel NIR fluorophore is an efficient method for EV lumen modification.
• This technique provides a robust platform for real-time visualization of EV communication.
• The study advances the understanding of EV uptake and cargo delivery mechanisms.