Encapsulation of Nucleic Acids into Giant Unilamellar Vesicles by Freeze-Thaw: a Way Protocells May Form.

Protocells are believed to consist of a lipid membrane and encapsulated nucleic acid. As the lipid membrane is impermeable to macromolecules like nucleic acids, the processes by which nucleic acids become encapsulated inside lipid membrane compartments are still unknown. In this paper, a freeze-thaw method was modified and applied to giant unilamellar vesicles (GUVs) and deoxyribonucleic acid (DNA) in mixed solution resulting in the efficient encapsulation of 6.4 kb plasmid DNA and similar length linear DNA into GUVs. The mechanism of encapsulation was followed by observing the effect of freeze-thaw temperatures on GUV morphological change, DNA encapsulation and ice crystal formation, and analyzing their correlation. Following ice crystal formation, the shape of spherical GUVs was altered and membrane integrity was damaged and this was found to be a necessary condition for encapsulation. Heating alone had no effects on DNA encapsulation, but was helpful for restoring the spherical shape and membrane integrity of GUVs damaged during freezing. These results suggested that freeze-thaw could promote the encapsulation of DNA into GUVs by a mechanism: the vesicle membrane was breached by ice crystal formation during freezing, DNA entered into damaged GUVs through these membrane gaps and was encapsulated after the membrane was resealed during the thawing process. The process described herein therefore describes a simple way for the encapsulation of nucleic acids and potentially other macromolecules into lipid vesicles, a process by which early protocells might have formed.

Focused characteristics and effects of light reflected from spherical lipid membrane of giant unilamellar vesicles.

Lipid vesicle is spherical membranous structure with a concave surface on the inside. When a beam of light illuminates a lipid vesicle, the light reflected from the vesicular concave membrane can be focused to have higher intensity and generate enhanced effects. By observing and simulating light reflected from giant unilamellar vesicles (GUVs), the intensity distribution of the light reflected from a spherical concave lipid membrane was investigated. The reflected light had focused characteristics. Its intensity was concentrated 10,000 times and even exceeded the intensity of incident light in a confined region, creating another effective light source in the lipid vesicle. The fluorescence quenching of sulfo-Cy5 encapsulated in spherical GUVs was stronger than that of the outside solution when irradiated by a 632.8 nm laser. When irradiated with ultraviolet light C (UVC), the damage to plasmid DNA encapsulated with spherical GUVs was greater than that of pure plasmid DNA solution and plasmid DNA mixed with lipid membrane fragments. Therefore, in addition to the effects of incident light, the focused light reflected from GUVs could generate incremental effects on encapsulated photoreactive materials if the spherical structure of the lipid membrane was maintained. These results proved that concave lipid membranes of spherical vesicles can focus light and utilize it to generate enhanced effects. The capability of light focusing and its influence on DNA may provide new insights for understanding the function of lipid membranes in cellular life.