Expanding the Scope of Reporting Nanoparticles: Sensing of Lipid Phase Transitions and Nanoviscosities in Lipid Membranes

Abstract

Biological membrane fluidity and thus the local viscosity in lipid membranes are of vital importance for many life processes and implicated in various diseases. Here, we introduce a novel viscosity sensor design for lipid membranes based on a reporting nanoparticle, a sulfated dendritic polyglycerol (dPGS), conjugated to a fluorescent molecular rotor, indocarbocyanine (ICC). We show that dPGS-ICC provides high affinity to lipid bilayers, enabling viscosity sensing in the lipid tail region. The systematic characterization of viscosity-and temperature-dependent photoisomerization properties of ICC and dPGS-ICC allowed us to determine membrane viscosities in different model systems and in living cells using fluorescence lifetime imaging (FLIM). dPGS-ICC distinguishes between ordered lipids and the onset of membrane defects in small unilamellar single lipid vesicles and is highly sensitive in the fluid phase to small changes in viscosity introduced by cholesterol. In microscopy-based viscosity measurements of large multilamellar vesicles, we observed an order of magnitude more viscous environments by dPGS-ICC, lending support to the hypothesis of heterogeneous nanoviscosity environments even in single lipid bilayers. The existence of such complex viscosity structures could explain the large variation in the apparent membrane viscosity values found in the literature, depending on technique and probe, both for model membranes and live cells. In HeLa cells, a tumor-derived cell line, our nanoparticle-based viscosity sensor detects a membrane viscosity of ∼190 cP and is able to discriminate between cell membrane and intracellular vesicle localization. Thus, our results show the versatility of the dPGS-ICC nano-conjugate in physicochemical and biomedical applications by adding a new analytical functionality to its medical properties.

QuasAr Odyssey: the origin of fluorescence and its voltage sensitivity in microbial rhodopsins

“…DSPE-Biotin was used for anchoring the proteoliposomes at the surface of the biotin–neutravidin-functionalized FLIM glass slide; for the latter, we adapted a protocol by Götz et al 67 . For vesicle formation after the thin film method, 1 mg lipids were prepared in 1 ml chloroform at a concentration ratio of 1:1000 (DSPE-Biotin:DMPC) using established procedures to form multilamellar vesicles (MLVs) 68 . To the preformed MLVs in 5 mM Tris pH 7.5, 10 mM KCl, 140 mM tetraethylammonium chloride at a concentration of 1.5 mM lipid, 160 nM solubilized Archon1 proteins were added directly before the FLIM measurements to yield the desired final protein-to-LUV ratio of 5–10 Archons/vesicle.…”

A Dual Fluorescence–Spin Label Probe for Visualization and Quantification of Target Molecules in Tissue by Multiplexed FLIM–EPR Spectroscopy

“…Here, the data reveal a very good agreement between EPR and FLIM in the viable epidermis for all three donors (Figure 4 I). We estimated that minimum concentrations in the lower μM to 100 nM range can be determined from EPR and FLIM using the DL [9e, 25] …”

“…We present the design, synthesis and photophysical properties of DL. The major challenge for a dual fluorescence–spin label is fluorescence deactivation by the unpaired electron of the spin label, a process which is usually employed in fluorescence quenching studies [9e, 16] . We show that in contrast to other tested spin label–fluorescent dye combinations, such as fluorescein and 3‐Carboxy‐Proxyl (PCA), the combination of Rhodamine B (RhoB) and PCA (Figure 2) yields high fluorescence and spin probe stability.…”

“…[2,10,11] Thechanges in fluorescence lifetime offer insight into the microenvironment of the fluorophore,including pH, polarity,viscosity,b iomolecular interactions,orreactive oxygen species. [6,9] However,t his environmental sensitivity may also interfere with concentration determination in FLIM.…”

Micelle Heterogeneity from the 2D Kinetics of Solute Rotation

“…In soft, microstructured materialsmicelles, vesicles, gels, star polymers, polymer nanoparticles, and so onthere are intertwined questions of where a solute resides and what local properties it sees. The photophysics of a solute that is also a chromophore are often used to gain information. − Static (0D) measurements, for example, the fluorescence quantum yield or Stokes’ shift, give a spatial average of static properties, such as hydrogen-bond availability or polarity. Time-resolved measurements with one time dimension (1D) give rates that characterize dynamic properties.…”