Introduction of microfluidic mixing technique opens a new door for preparation of the liposomes and lipid-based nanoparticles by on-chip technologies that are applicable in a laboratory and industrial scale. This study demonstrates the role of phospholipid bilayer fragment as the key intermediate in the mechanism of liposome formation by microfluidic mixing in the channel with "herring-bone" geometry used with the instrument NanoAssemblr. The fluidity of the lipid bilayer expressed as fluorescence anisotropy of the probe N,N,N-Trimethyl-4-(6-phenyl-1,3,5-hexatrien-1-yl) was found to be the basic parameter affecting the final size of formed liposomes prepared by microfluidic mixing of an ethanol solution of lipids and water phase. Both saturated and unsaturated lipids together with various content of cholesterol were used for liposome preparation and it was demonstrated, that an increase in fluidity results in a decrease of liposome size as analyzed by DLS. Gadolinium chelating lipids were used to visualize the fine structure of liposomes and bilayer fragments by CryoTEM. Experimental data and theoretical calculations are in good accordance with the theory of lipid disc micelle vesiculation.

Central European Institute of Technology CEITEC Structural Virology Masaryk University Kamenice 753 5 62500 Brno Czech Republic

Department of Immunology Faculty of Medicine and Dentistry Palacky University Olomouc Hněvotínská 3 775 15 Olomouc Czech Republic

Department of Pharmacology and Immunotherapy Veterinary Research Institute v v i Hudcova 70 621 00 Brno Czech Republic

Institute of Physics Czech Academy of Sciences Na Slovance 2 Prague 8 Czechia

Malvern Panalytical Malvern Worcestershire United Kingdom

Mendel University in Brno Department of Chemistry and Biochemistry Zemedelska 1 61300 Brno Czech Republic

Regional Centre of Advanced Technologies and Materials Palacký University Šlechtitelů 11 78371 Olomouc Czech Republic

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