Lipid vesicle composition influences the incorporation and fluorescence properties of the lipophilic sulphonated carbocyanine dye SP-DiO

Abstract

Lipid membrane composition influences insertion efficiency and photophysical properties of lipophilic membrane-inserting dyes.

Independent Size and Fluorescence Emission Determination of Individual Biological Nanoparticles Reveals that Lipophilic Dye Incorporation Does Not Scale with Particle Size

“…In fact, Lubart et al recently demonstrated, using absorbance, fluorescence emission, and fluorescence lifetime measurements, that the incorporation efficiency of SP-DiO is highly dependent on lipid composition and that addition of cholesterol has a significant impact on the photophysical properties of the dye, making such effects very likely also in native lipid membranes of the type investigated in this work. 50 However, irrespective of the actual reason to the deviation from the expected dependence between emission intensity and size, our results demonstrate that membrane dyes of this type do not necessarily serve as reliable markers for the size of small-scale membrane structures with complex biomolecular composition. This insight has impact in several biological processes membrane curvature has been shown to play a significant role, including curvature-selective protein binding and sorting, 28 the activity of membrane-digesting enzymes, 52 and the action of antiviral peptides, 53 to mention a few examples.…”

“…al. which suggest that the photophysics of SP-DiO depends strongly on the membrane composition of synthetic lipid vesicles 50. …”

“…The lower incorporation efficiency of cholesterol-DNA in NMVs and EVs compared with synthetic lipid vesicles agrees with the observation of a lower fluorescence emission intensity of SP-DiO (a reduction in signal-to-background by a factor 3 to 5 compared to synthetic lipid vesicles of comparable dimension and imaged under otherwise identical conditions). Although the fluorescence intensity of SP-DiO is known to be sensitive to membrane composition, 50 the fact that also the efficiency of cholesterol-DNA self-insertion was reduced suggests that this observation reflects a lower lipid content in the NMVs and EVs and possibly also interference from solutes present in the complex media of these suspensions. Additionally, EVs isolated directly from FBS-supplemented cell medium and labeled with SP-DiO through standard mixing did not yield any detectable fluorescence (data not shown).…”

Diffusion of Lipid Nanovesicles Bound to a Lipid Membrane Is Associated with the Partial-Slip Boundary Condition

“…20 The possibility to analyze wide size distributions on the individual nanoparticle level without relying on the particle signal−size relation is a key asset for the analysis of biological nanoparticles, such as EVs, exosomes, and viruses, since their size distribution is typically broad, and the fluorescence signal depends strongly on the highly variable membrane composition. 25,32 The identification of tether subpopulations combined with measurements of both size and diffusivity enabled a direct quantitative comparison with theoretical expressions of the size-dependent mobility. The measurements for POPC and EVs were found not to be welldescribed by the equations obtained using the conventional no-slip boundary condition.…”

A New Lipid‐Droplets‐Targeted Fluorescence Probe with Dual‐Reactive Sites for Specific Detection of ClO− in Living Cells

“…[32][33][34] However, there is a main issue for lipophilic organic dyes is their low cell penetrability, which can be excluded from cells due to the absence of cell interface-targeting segments, such as cationic charges, which is detrimental for designing specific fluorescent probes, as it has a tendency to provoke attraction toward negatively charged biomolecules (proteins and biomembranes) by opposite charge attractions. [35][36][37] Therefore, the imbalance between hydrophobicity and cell penetrability should be considered in the context of probe designing.…”