pH-Reversible Leuco-BODIPY Probe for Live EV Secretion Imaging
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Solution Overview
Problem
Existing imaging tools are inefficient or infeasible for real-time, live visualization of extracellular vesicle (EV) secretion from living cells, as they often require cell fixation or suffer from nonuniform transfection and cytotoxicity.
Innovation Solution
Development of pH-reversible BODIPY-based probes that switch between fluorescent and non-fluorescent forms, allowing in situ visualization of EVs by transitioning between leuco-BODIPY and BODIPY forms based on pH changes, enabling selective detection of acidic EVs and intracellular precursor vesicles with minimal background interference.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If transmission electron microscopy or immunofluorescence is used to visualize EVs, then nanoscale visualization or real-time imaging of specific EV types is achieved, but cell fixation is required which prevents live imaging of exocytosis
Solution Approach 1:
The probe utilizes pH parameter changes to switch between fluorescent and non-fluorescent states. The probe is designed with a pKa tuned to approximately 6.5, allowing it to remain non-fluorescent in the neutral pH of culture medium and become fluorescent when entering the acidic environment of EVs during exocytosis, enabling live imaging without fixation
Solution Approach 2:
The pH-responsive BODIPY probe acts as an intermediary that responds to the pH gradient between the neutral culture medium and the acidic EV lumen. This intermediary mechanism allows the probe to selectively detect EVs in their native state during live imaging, bridging the gap between visualization precision and live imaging capability
2Adaptability or versatility
If cells are transfected with plasmids encoding EV markers fused with fluorescent reporters, then confocal imaging of EV secretion from living cells is enabled, but nonuniform transfection, prolonged protein expression, and cytotoxicity occur that may affect exocytosis
Solution Approach 1:
The probe is designed as a small molecule that can be added to the culture medium temporarily for imaging purposes. Unlike stable protein expression systems, the probe provides transient labeling that does not require genetic modification, avoiding the cytotoxicity and nonuniform transfection issues while maintaining imaging capability
Solution Approach 2:
The invention extracts the fluorescent labeling function from the cell's genetic machinery (plasmid transfection system) and places it in the extracellular medium as a free-floating pH-responsive probe. This separation eliminates the need for cellular uptake and protein expression, removing the sources of cytotoxicity and nonuniform transfection while preserving the ability to image EV secretion
3Measurement precision
If fluorescent probes are used to detect EVs in culture medium, then EV detection is achieved, but background interference from free dyes in the medium increases
Solution Approach 1:
The probe's fluorescence is gated by pH parameter changes. In the neutral pH of culture medium (pH 7.4), the probe remains in its non-fluorescent leuco form, eliminating background interference. Upon encountering acidic EVs (pH < 6.5), the probe switches to its fluorescent form, providing high-contrast detection with minimal background noise
Solution Approach 2:
The probe exhibits dynamic fluorescence switching based on local pH conditions. Rather than being constantly fluorescent, the probe dynamically transitions between fluorescent and non-fluorescent states, ensuring that fluorescence is generated only in the specific microenvironment of acidic EVs, thereby minimizing background interference from free dye in the medium
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables real-time, sensitive imaging of EV secretion with reduced background noise, obviating the need for EV isolation and providing fundamental insights into secretion mechanisms.
Implementation Method 1
The BODIPY core can be used for tuning of its pKa of about 4 to about 7 and hence its pH-dependent photophysical properties for visualizing cellular processes
Implementation Method 2
Nucleophilic attack of the central meso-carbon of the BODIPY form (e.g., by water, hydroxide ions, thiols, or amino acids in culture medium) can cause the loss of it-conjugation and >200 nm blueshift of its absorption peak to its leuco-BODIPY form
Data Source
AI summary
The subject invention pertains to compositions of a probe comprising two pH-reversible forms, including the boron dipyrromethene (BODIPY) fluorescent form and the non-fluorescent leuco-BODIPY form, for use in confocal imaging methods of intracellular vesicles as well as extracellular vesicles that are present in conditioned culture medium and/or secreted by living cells, without inducing severe cytotoxicity.