Phase-diagram observation of liquid–liquid phase separation in the poly-L-lysine/ATP system and diagram-based application strategy

Liquid–liquid phase separation (LLPS) is essential to understand biomacromolecule compartmentalization in living cells and to form soft-matter structures for chemical reactions and drug delivery systems. However, the importance of detail experimental phase diagrams of modern LLPS systems tend to be overlooked nowadays. Even for poly-L-lysine (PLL)/ATP system, one of the most widely used LLPS models, detailed phase diagram of LLPS have not been obtained. Herein, we report the first phase diagram of the (PLL)/ATP system and demonstrate the feasibility of phase-diagram based research design not only for understanding the physical properties of LLPS systems but also for realizing biophysical and medical applications. We established an experimental handy model of the droplet formation/disappearance process by forming a concentration gradient in a chamber as 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 PLL/ATP system. Using the knowledge of the phase diagram, we enabled to form IgG/PLL droplets clearly in the pharmaceutically required IgG concentration of ca. 10 mg/mL. This study provides a guidance for using the phase diagram to analyze and utilize LLPS