Arianna Bartolini1, Paolo Tempesti1, Ahmad F Ghobadi2, Debora Berti1, Johan Smets3, Yousef G Aouad4, Piero Baglioni5. 1. Department of Chemistry "Ugo Schiff" and CSGI, University of Florence, Via della Lastruccia 3, Sesto Fiorentino, 50019 Florence, Italy. 2. Computational Chemistry, Modeling and Data Sciences, The Procter & Gamble Company, Beckett Rd. 8611, West Chester, OH 45069, United States. 3. The Procter & Gamble Company, Temselaan 100, 1853 Strombeek Bever, Belgium. 4. The Procter & Gamble Company, Winton Hill Technical Center, 6100 Center Hill, Cincinnati, OH 45224, United States. 5. Department of Chemistry "Ugo Schiff" and CSGI, University of Florence, Via della Lastruccia 3, Sesto Fiorentino, 50019 Florence, Italy. Electronic address: baglioni@csgi.unifi.it.
Abstract
HYPOTHESIS: Liquid-liquid phase separation (LLPS) can provide micron-sized liquid compartments dispersed in an aqueous medium. This phenomenon is increasingly appreciated in natural systems, e.g., in the formation of intracellular membraneless organelles, as well as in synthetic counterparts, such as complex coacervates and vesicles. However, the stability of these synthetic phase-separated microstructures versus coalescence is generally challenged by the presence of salts and/or surfactants, which narrows the range of possible applications. We propose a new strategy to obtain micron-sized liquid domains via LLPS, by mixing an amphiphilic copolymer with surfactants and sodium citrate in water at room temperature. EXPERIMENTS: Combining Confocal Laser Scanning Microscopy (CLSM) and Differential Scanning Calorimetry (DSC) with Dissipative Particle Dynamics (DPD) simulations, we map the phase diagram to detect LLPS and address the presence and morphology of these microscopic domains. This mapping in turn provides a first mechanistic hypothesis for the formation of such confined polymer-rich microenvironments. FINDINGS: LLPS is driven by the phase behavior of the copolymer in water and by its associative interactions with surfactants, combined with the water-sequestering ability of salting-out electrolytes. The key factor for LLPS and formation of microdomains is the entropy-driven dehydration of the copolymer head groups, which can be quantified through the Free Water Content (FWC). Interestingly, the internal morphology of the LLPS microdomains is finely controlled by the ratio between nonionic and anionic surfactants. Beside its applicative potential, this approach represents a tool for designing synthetic mimics that improve our understanding of the occurrence of LLPS in cells.
HYPOTHESIS: Liquid-liquid phase separation (LLPS) can provide micron-sized liquid compartments dispersed in an aqueous medium. This phenomenon is increasingly appreciated in natural systems, e.g., in the formation of intracellular membraneless organelles, as well as in synthetic counterparts, such as complex coacervates and vesicles. However, the stability of these synthetic phase-separated microstructures versus coalescence is generally challenged by the presence of salts and/or surfactants, which narrows the range of possible applications. We propose a new strategy to obtain micron-sized liquid domains via LLPS, by mixing an amphiphilic copolymer with surfactants and sodium citrate in water at room temperature. EXPERIMENTS: Combining Confocal Laser Scanning Microscopy (CLSM) and Differential Scanning Calorimetry (DSC) with Dissipative Particle Dynamics (DPD) simulations, we map the phase diagram to detect LLPS and address the presence and morphology of these microscopic domains. This mapping in turn provides a first mechanistic hypothesis for the formation of such confined polymer-rich microenvironments. FINDINGS: LLPS is driven by the phase behavior of the copolymer in water and by its associative interactions with surfactants, combined with the water-sequestering ability of salting-out electrolytes. The key factor for LLPS and formation of microdomains is the entropy-driven dehydration of the copolymer head groups, which can be quantified through the Free Water Content (FWC). Interestingly, the internal morphology of the LLPS microdomains is finely controlled by the ratio between nonionic and anionic surfactants. Beside its applicative potential, this approach represents a tool for designing synthetic mimics that improve our understanding of the occurrence of LLPS in cells.