Lipids are integral to all forms of life. Both cells and the majority of particles involved with living systems are enveloped by a lipid membrane, which protects their content from the external environment while simultaneously controlling molecular transport using membrane-embedded proteins. A subset of these particles are known as biological nanoparticles (BNPs), including extracellular vesicles, exosomes and viruses. BNPs are known to transfer genetic material during cellular communication, but many aspects of the mechanisms regulating their various functions remain unknown. Progress within this scientific discipline is hampered by their small size (between 50 and 200 nm in diameter) and significant biomolecular heterogeneity, making both quantitative nanoparticle analytics and functional characterization, highly demanding tasks.
To overcome the challenges of BNP characterization, we constructed an approach to identify the mechanism by which lipid vesicles are spontaneously converted into a planar supported lipid bilayer (SLB) on glass surfaces (Paper I). Total internal reflection fluorescence (TIRF) microscopy was used to track and temporally resolve the rate of vesicle adsorption, the onset of supported lipid bilayer (SLB) formation and the kinetics of their growth into a continuous SLB, through the use of a small fraction (1/100) of labelled lipid vesicles. It was found that the SLB formation processes was initiated by the merger of multiple small SLB patches at appreciably high vesicle coverage. In addition, the subsequent growth of SLB patches was, for the first time, shown to occur via a gradual increase in the average front velocity. Paper II focuses on quantifying both the size and molecular content of different types of BNPs. This was accomplished by tethering BNPs of varying complexity, to a fluid SLB formed on the floor of a microfluidic channel. By moving the BNPs with an applied hydrodynamic shear flow and by determining both the Brownian and directed motion using single particle tracking analysis, it was possible to determine the hydrodynamic radii for each BNP. Furthermore, imaging the BNPs using TIRF or epi-microscopy, made it possible to simultaneously determine the extent of fluorescent label attachment, which was specifically used to address how the incorporation efficiency of the membrane-staining dye spDIO depends on the size of the BNP.
The insights gained on the SLB formation processes and BNP characterization are fundamental for the future development and advancement of novel techniques aimed at probing the molecular interaction between BNPs and cellular membranes.
Keywords: supported lipid bilayer, TIRF microscopy, diffusion, size determination, lipophilic dyes, microfluidics
Graduate school: Material Science
Examiner and main Supervisor: Fredrik Höök
Reviewer: Andreas Dahlin
PJ, lecture hall,
12 October, 2018, 15:00
12 October, 2018, 17:00