Open Access

Andrej Kamenac

• The scope of this thesis is to consider phase transitions acting on lipid membranes and the implications on biological processes. To account for the diversity of fields involved, the projects range from transmembrane processes, such as permeabilization - to lateral, in-plane processes, such as enzymatic activity on membranes. The science conducted on these fields study fundamentals to applications and finally achieve the development of a biomedical tool. Firstly, we studied the membrane phase transition temperature as function of shear flow. Optical measurements of vesicles in microfluidic systems revealed a shift in chain order. The same effect was observed for another lipid type, which is a powerful reference. These results should be considered in the design of therapeutic temperature-sensitive liposomes encapsulating drugs to ensure precise control of the drug release. Secondly, we studied the nanoparticle uptake of vesicles as function of temperature. The micrographs time seriesThe scope of this thesis is to consider phase transitions acting on lipid membranes and the implications on biological processes. To account for the diversity of fields involved, the projects range from transmembrane processes, such as permeabilization - to lateral, in-plane processes, such as enzymatic activity on membranes. The science conducted on these fields study fundamentals to applications and finally achieve the development of a biomedical tool. Firstly, we studied the membrane phase transition temperature as function of shear flow. Optical measurements of vesicles in microfluidic systems revealed a shift in chain order. The same effect was observed for another lipid type, which is a powerful reference. These results should be considered in the design of therapeutic temperature-sensitive liposomes encapsulating drugs to ensure precise control of the drug release. Secondly, we studied the nanoparticle uptake of vesicles as function of temperature. The micrographs time series revealed that nanoparticle uptake was a complex function of temperature. Local maxima of the uptake as function of temperature do exist and do correlate with the phase transition. However, - contrary to expectations - the uptake peaks are located around T = Tm + 3 K. As the uptake is driven by adhesion, we conducted force spectroscopy on the adhesion, which turned out to be a non-linear function of temperature. Adding literature into consideration, we concluded that tension must exhibit a minimum at the nanoparticle uptake maximum. These results might become relevant for the design of porous silica nanoparticles, which are commonly used as a carrier material for drug delivery. Thirdly, in addition to carrier-centered nanoparticle permeability, we studied general permeability as a function of shear rate and temperature for vesicles. Unexpectedly, we observed all-or-nothing-like permeabilization behavior - entirely different from reports in literature - just by adding shear flow. All-or-nothing behavior is well-known from biology, like in triggering of action potentials. The all-or-nothing-like behavior is triggered at the lipid membrane phase transition temperature. This finding is particularly intriguing, as a macroscopic system acts probabilistic. Fourthly, after gaining expertise in shearing and permeabilization, we built a novel acoustofluidic permeabilization tool for suspended living cells. Acoustofluidic permeabilization is a vector-free method, where surface acoustic waves couple into the fluid of a microfluidic channel to trap cells in the vortices that are created by the acoustic wave application. The permeabilization is enhanced by more than an order of magnitude for a large spectrum of cargo sizes, ranging from a fluorescent molecule, to sugars and even proteins. Fifthly, we studied the correlation of permeability and shear forces in a viscosimeter. The cone-plate shearing of suspended cells revealed that the maximum permeability temperature is a linear function of shear rate. However, it was the plate-plate viscosimeter experiments on adherent cells, which brought a real leap forward in terms of robustness, visualization, and spatial information. Thanks to the continuous and linear shear profile, we were able to measure the whole shear dimension at once with only one cell passage. Furthermore, adherent cells allowed for spatial information on the single cell level. Thanks to the spatial information, the results revealed cell de-adhesion to be a non-linear function of shear rate, which is entirely new in literature. On top, we found permeability to be a non-linear function of shear rate - supporting prior findings in the form of fascinating fluorescent micrographs. Finally, we added biochemistry to the equation and studied membrane phase transition acting on the enzyme activity. The results revealed a correlation of the membrane-associated enzyme activity with the excess heat capacity of the system. In detail, we observed Anti-Arrhenius behavior in a temperature interval above the phase transition temperature - several Kelvin wide. This result resolved a dispute in literature from the 1970’s, where non-Arrhenius membrane-associated enzymes could not be accommodated by any theory. However, our results support a thermodynamic theory by Kaufmann. Concerning applications, this result proves the plausibility of the trigger-and-detection principle of the soliton nerve signalling theory.…