Final Report Summary - GUVS-3G (Smart photo-activable devices based in plasmonic nanoparticles: Microfluidic-assisted engineering of a third generation of lipid vesicles)
The delicate control over the flow of fluids afforded by microfluidic technologies enables the fabrication of highly monodisperse emulsions, drop-by-drop, at typical rates of several hundreds per second. This technology not only enables the fabrication of single emulsions but also multiple emulsions with controlled dimensions and compositions. Concentric water-in-oil-in-water (w/o/w) double emulsion drops are particularly interesting to encapsulate payloads with high efficiency. Therefore, this project proposes the use of concentric water-in-oil-in-water (w/o/w) double emulsion drops with ultrathin oil shells as templates to form smart GUVs with controlled payload content, controlled membrane composition, controlled number of gold NPs and controlled distribution of these NPs in the GUV membrane. To achieve control over NP distribution, this project proposes the use of asymmetric membranes: Membranes with lateral asymmetries or domains that form through the phase separation of membrane lipids, and membranes with transversal asymmetries due to the asymmetric distribution of lipids in the two leaflets that constitute the bilayer membrane. Moreover, this project studies the impact of the concentration and distribution of NPs on the mechanical properties of the GUV membrane in the presence and absence of the external stimulus, which is light, at the adequate wavelength to excite the phonon modes of the membrane through plasmon-phonon energy transfer. Furthermore, this project proposes the development of a screening method based on GUV activity under irradiation: A change in GUV shape under flow can be used to separate active GUVs form inactive GUVs. This screening method may be applied to screen activity in living cells.
Lipid membranes have typical thicknesses of approximately 5 nm; this limits the size of the NPs that can be embedded in the membrane without membrane destabilization. We find that the GUV membrane ruptures if dodecanothiol surface coated gold NPs with a diameter that exceeds 7 nm are embedded in the membrane. Smaller NPs can be embedded at concentrations up to 0.5 wt.%, without membrane rupture using our microfluidic approach. This type of membranes is highly sensitive to osmotic pressure. We observe a drastic decrease in size of the GUV when exposed to a hypertonic solution. The presence of NPs in the membrane also affects lipid phase separations. For example, membranes that contain a ternary mixture of lipids that includes a saturated phospholipid, an unsaturated phospholipid and cholesterol phase separate into two single large domains: A liquid disordered and a liquid ordered domain. However, membranes of the same lipid composition that additionally have NPs embedded form smaller domains. Moreover, upon photo-irradiation, NPs tend to be expelled from the lipid membrane through drastic changes in the shape of the vesicles; this ultimately results in domain coalescence. After this coalescence process, the GUV phase separation resemble to that of a NP-free membrane.
To further modify the mechanical and dynamical properties of these GUVs, we develop a microfluidic approach for the production of asymmetric vesicles with a controlled content in NPs. Using water-in-oil-in-oil-in-water triple emulsion drops as templates for GUVs, we produce GUVs with two compositionally different leaflets. The degree of asymmetry that we get is limited by the diffusion of the lipids in the two oil phases and therefore is of approximately 70%. This is the first single-step approach that enables a continuous production of GUVs at typical rates of several hundreds per second. In addition, we have implemented these microfluidic approaches to GUV production in PDMS chips, which enables scaling up their production; this is a requirement in any industrial application.
The results obtained in GUVs-3G provide a quantitative description of the mechanical and dynamical properties of GUVs with NPs embedded in the membrane. GUVs with different compositions and asymmetry, containing NPs embedded in the membrane, that flow within a microfluidic channel can change in size and shape under photo-irradiation. We are currently testing if these changes can be detected using a contour detection algorithm, recently developed in our group, and used as an input to the computer that can activate an electrode, and sort active GUVs in a different channel than those unresponsive to light. This will provide a novel approach to screen activity in GUVs.