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

Abstract

Advancements in nanoparticle characterization techniques are critical for improving the understanding of how biological nanoparticles (BNPs) contribute to different cellular processes, such as cellular communication, viral infection, as well as various drug-delivery applications. Since BNPs are intrinsically heterogeneous, there is a need for characterization methods that are capable of providing information about multiple parameters simultaneously, preferably at the singlenanoparticle level. In this work, fluorescence microscopy was combined with surface-based two-dimensional flow nanometry, allowing for simultaneous and independent determination of size and fluorescence emission of individual BNPs. In this way, the dependence of the fluorescence emission of the commonly used self-inserting lipophilic dye 3,3′-dioctadecyl-5,5′-di(4-sulfophenyl)oxacarbocyanine (SP-DiO) could successfully be correlated with nanoparticle size for different types of BNPs, including synthetic lipid vesicles, lipid vesicles derived from cellular membrane extracts, and extracellular vesicles derived from human SH-SY5Y cell cultures; all vesicles had a radius, r, of ∼50 nm and similar size distributions. The results demonstrate that the dependence of fluorescence emission of SP-DiO on nanoparticle size varies significantly between the different types of BNPs, with the expected dependence on membrane area, r 2 , being observed for synthetic lipid vesicles, while a significant weaker dependence on size was observed for BNPs with more complex composition. The latter observation is attributed to a size-dependent difference in membrane composition, which may influence either the optical properties of the dye and/or the insertion efficiency, indicating that the fluorescence emission of this type of selfinserting dye may not be reliable for determining size or size distribution of BNPs with complex lipid compositions.

Sizing Extracellular Vesicles Using Membrane Dyes and a Single Molecule-Sensitive Flow Analyzer

“…From our results, Di-8-ANEPPS provided the most efficient vesicle detection (as previously suggested by Stoner et al ), resulting from a higher staining efficiency and higher fluorogenic behavior (see Figures S3, S11 and Scheme S1 for a proposed mechanism of vesicle staining by Di-8-ANEPPS), and had a low tendency to aggregate. Furthermore, although Di-8-ANEPPS and derivatives are commonly used as excitation ratiometric dyes for probing their local microenvironment, both their absorption at 488 nm and emission spectra remain relatively insensitive to the local environment across a variety of lipid compositions and membrane dipole, − dye concentration (see Figure S12), and cholesterol content , (which already shows little variation∼10%in relative abundance among EVs derived from cells and biological fluids). − Another advantage of Di-8-ANEPPS is the absence of a net negative charge in the head group, which could reduce the efficiency of dye intercalation into EV membranes (typically negatively charged) due to repulsive forces . Therefore, Di-8-ANEPPS was selected as a suitable membrane.…”

Quantifying DNA-mediated liposome fusion kinetics with a fluidic trap

“…Current approaches in bulk for the detection of lipid membrane fusion are not easily implemented for biological membranes, which has driven the development of singleparticle assays with vesicles tethered to a surface for physical [32][33][34] or chemical characterization. 35 These assays monitor individual fusion events and can thus detect inhomogeneities in the fusion process.…”

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.…”

“…The POPC vesicles were prepared by the freeze–thaw extrusion method (Supporting Information, Section 1). Experimental details for the EV data are described in ref . The vesicles were subjected to a volumetric flow of TE buffer (50–200 mM NaCl, 10 mM TRIS, 1 mM Na 2 EDTA) at 30 μL/min.…”

“…The red dashed lines correspond to b ef = 4 nm (i.e., no slip with δ = 4 nm) and b ef = 100 nm. The EV data were adopted from ref .…”