Fluorescent labelling and characterization of extracellular vesicles by time-resolved fluorescence techniques

Abstract

Extracellular vesicles (EV) are a heterogeneous group of phospholipid bilayer particles that are secreted by procaryotic and eukaryotic cells. They are potential drug delivery systems and therapeutic tools due to their biological functions. The opening of fluorescently labeled EVs can be studied with steady-state and time-resolved fluorescence spectroscopy.

In this master’s thesis, nanoerythrosome EVs were labeled with six different lipophilic dyes: DHPE-OG, tail-BODIPY, head-BODIPY, RB, tail-NBD, and head-NBD. DHPE-OG was used as a reference. This study aimed to find a fluorescence probe for which there would be a measurable change in the fluorescence signal when the EVs open in live cells. The EVs were labeled with each dye and purified with size-exclusion chromatography. The purification process was evaluated by comparing free dye control measurements to the results from labeled EVs. The labeling was deemed successful if 1) the SEC purification succeeded, i.e. there was a clear separation between EVs and free dye fractions, and 2) the labeled EV fraction had satisfactory fluorescence intensity. Based on the results of exclusively free dye controls only DHPE-OG and RB could be used for successful labeling.

The fluorescence properties of the dyes were studied with steady-state and time resolved fluorescence in different solutions: free dyes in methanol, Dulbecco’s phosphate buffered saline (DPBS) with and without 1 % Triton X. The labeled EVs were studied only in DPBS in the presence and absence of Triton X. For DHPE-OG the spectra showed monomeric characteristics in all solutions. For the rest of the free dyes, the presence of aggregates could be observed in DPBS. In the presence of EVs, the aggregation signs were observed only for head BODIPY suggesting that the dyes are solubilized into their monomeric form in the EV membrane. Thus, the conclusion was that all the dyes except head-BODIPY can be successfully used to label EVs. The lifetime measurements demonstrated the most significant changes in fluorescence lifetime together with high fluorescence intensity for DHPE-OG and tail-BODIPY labeled EVs. The results also indicated that RB is a potential acceptor for future Förster resonance energy transfer studies with DHPE-OG or tail-BODIPY as a donor. The most promising tail-BODIPY and DHPE OG were studied by steady-state and time-resolved anisotropy measurements. Labeled EV and free dye samples showed free movement of the dye whereas EV samples showed hindered movement of the dye. The hindered movement means that the dyes are attached to EVs and thus confirms the successful labeling of EVs with DHPE-OG and tail-BODIPY. In the presence of Triton X, both free dye samples and EV samples showed hindered movement of the dye. This is due to the relocation of the dyes into the Triton X micelles present in the solution. Thus, the opening of EVs could not be mimicked by the use of Triton X surfactant.