Phase-Diagram Observation of Liquid-Liquid Phase Separation in the Poly(l-lysine)/ATP System and a Proposal for Diagram-Based Application Strategy.

Liquid-liquid phase separation (LLPS) is essential to understanding the biomacromolecule compartmentalization in living cells and to developing soft-matter structures for chemical reactions and drug delivery systems. However, the importance of detailed experimental phase diagrams of modern LLPS systems tends to be overlooked in recent times. Even for the poly(l-lysine) (PLL)/ATP system, which is one of the most widely used LLPS models, any detailed phase diagram of LLPS has not been reported. Herein, we report the first phase diagram of the PLL/ATP system and demonstrate the feasibility of phase-diagram-based research design for understanding the physical properties of LLPS systems and realizing biophysical and medical applications. We established an experimentally handy model for the droplet formation-disappearance process by generating a concentration gradient in a chamber for extracting a suitable condition on the phase diagram, including the two-phase droplet region. As a proof of concept of pharmaceutical application, we added a human immunoglobulin G (IgG) solution to the PLL/ATP system. Using the knowledge from the phase diagram, we realized the formation of IgG/PLL droplets in a pharmaceutically required IgG concentration of ca. 10 mg/mL. Thus, this study provides guidance for using the phase diagram to analyze and utilize LLPS.

Marangoni Droplets of Dextran in PEG Solution and Its Motile Change Due to Coil-Globule Transition of Coexisting DNA.

Motile droplets using Marangoni convection are attracting attention for their potential as cell-mimicking small robots. However, the motion of droplets relative to the internal and external environments that generate Marangoni convection has not been quantitatively described. In this study, we used an aqueous two-phase system [poly(ethylene glycol) (PEG) and dextran] in an elongated chamber to generate motile dextran droplets in a constant PEG concentration gradient. We demonstrated that dextran droplets move by Marangoni convection, resulting from the PEG concentration gradient and the active transport of PEG and dextran into and out of the motile dextran droplet. Furthermore, by spontaneously incorporating long DNA into the dextran droplets, we achieved cell-like motility changes controlled by coexisting environment-sensing molecules. The DNA changes its position within the droplet and motile speed in response to external conditions. In the presence of Mg2+, the coil-globule transition of DNA inside the droplet accelerates the motile speed due to the decrease in the droplet's dynamic viscosity. Globule DNA condenses at the rear part of the droplet along the convection, while coil DNA moves away from the droplet's central axis, separating the dipole convections. These results provide a blueprint for designing autonomous small robots using phase-separated droplets, which change the mobility and molecular distribution within the droplet in reaction with the environment. It will also open unexplored areas of self-assembly mechanisms through phase separation under convections, such as intracellular phase separation.

Reentrant condensation of a multicomponent cola/milk system induced by polyphosphate.

Reentrant condensation (RC) is a protein behavior in which the protein solution shifts between the one- and two-phase state more than twice by increasing a single parameter. Although RC would be a candidate mechanism for the physicochemical design of food additives, no realistic model has been established under diverse contaminants like food materials. Here, we found that a mixture of cola and milk yielded RC. At pH 3.2-3.6, cola induced milk condensation at 30-40%, while lower or higher concentrations of cola did not. Furthermore, we reduced this cola/milk system to two pure components, casein in milk and polyphosphate (polyP) in cola, and investigated the characteristics of casein concentration and zeta potential. This was the first experimental demonstration of RC occurrence in a multicomponent system. The well-characterized cola/milk system would explore both the universal nature of proteins and the industrial application of RC.