Mg²⁺ Regulated ATP Release From Coacervates In Vesicles And Its Applications

The membrane-less organelles in cells,such as nucleoli and stress granules,are generally coacervations by phase separation.The coacervations are relatively independent in cells with higher concentration of molecules to ensure that the reaction can be carried out smoothly without interference.The coacervations constructed in vitro can not only mimic organelles,but also act as microreactors for specific reactions.Coacervations can be disaggregated by the regulation of p H,temperature,inorganic salt concentration,polymer concentration and light.Phospholipids are the main component of cell membrane,and the vesicles formed by phospholipids can be used as the model of artificial cells.Herein,polylysine(Ply)/ATP coacervations were encapsulated in giant phospholipid vesicles(GUVs)by emulsion transfer method to construct a confined-space microreaction system.MgCl₂ was used to regulate the disaggregation of coacervations in GUVs to release ATP,which promoted the polymerization of actin.Firstly,Ply and ATP were mixed at room temperature to form Ply/ATP coacervate solution.The transmittance of solution at 550 nm was measured by UV-vis spectroscopy.The morphology of coacervate droplets was observed by fluorescence microscope and the corresponding particle size distribution from microscope image was calculated,determined that the optimum ratio of coacervations in pure water solution was[Ply]:[ATP]to be 1:3,while in the polymerization buffer of p H of 8.8it was[Ply]:[ATP]to be 4:1.The effects of time and concentration of salt solution on the stability of coacervations were investigated.The results showed that coacervations in aqueous solvents remained relatively stable in 60?180 min,followed by gradual disaggregation after 180 min.The process of disaggregation was completely finished in 24 h.The MgCl₂ concentrations for complete coacervations disaggregation in pure water and buffer solution(p H of 8.8)were 20 m M and 5 m M,respectively.Then,the Ply/ATP coacervate droplets were encapsulated in GUVs by emulsion transfer method in the physiological environment.By optimizing the process parameters,determined that the optimal parameters of the emulsion method process were as follows:vortex time to be 60 s,ATP to be 3m M,Ply to be 12 m M.the entrapment efficiency under such condition up to 70%.Nano-micropores were constructed on the surface of vesicles by adding external melittin,and the concentration of MgCl₂ outside the vesicles was adjusted to regulate the disaggregation of coacervations.The reaction process was in situ observed.The fluorescence microscope images showed the coacervations inside the vesicles could be disaggregated within 30 min.The assembly of actin monomers into actin filaments in the vesicles proved the successful release of ATP.In the system,the controllable disaggregation of coacervations releasing ATP in confined space was achieved,indicating the potentials of the constructed system mimicking artificial organelle for energy supply.