Liquid–Liquid Phase‐Separated Systems from Reversible Gel–Sol Transition of Protein Microgels

Abstract

Liquid–liquid phase‐separated biomolecular systems are increasingly recognized as key components in the intracellular milieu where they provide spatial organization to the cytoplasm and the nucleoplasm. The widespread use of phase‐separated systems by nature has given rise to the inspiration of engineering such functional systems in the laboratory. In particular, reversible gelation of liquid–liquid phase‐separated systems could confer functional advantages to the generation of new soft materials. Such gelation processes of biomolecular condensates have been extensively studied due to their links with disease. However, the inverse process, the gel–sol transition, has been less explored. Here, a thermoresponsive gel–sol transition of an extracellular protein in microgel form is explored, resulting in an all‐aqueous liquid–liquid phase‐separated system with high homogeneity. During this gel–sol transition, elongated gelatin microgels are demonstrated to be converted to a spherical geometry due to interfacial tension becoming the dominant energetic contribution as elasticity diminishes. The phase‐separated system is further explored with respect to the diffusion of small particles for drug‐release scenarios. Together, this all‐aqueous system opens up a route toward size‐tunable and monodisperse synthetic biomolecular condensates and controlled liquid–liquid interfaces, offering possibilities for applications in bioengineering and biomedicine.

Biomaterials for microfluidic technology

“…Wongpinyochit et al [95] injected SF solution and organic solvent into two separate microfluidic channels, and then these two phases met and mixed at the junction, inducing the selfassembly of SF spheres. Microfluidic mixing was proved to not only allow the sphere size to be tuned by varying the flow Gelatin [116] Microsphere Physical crosslinking --Gelatin [117] Microsphere Enzymatic crosslinking --Gel-SH [119] Microsphere Chemical crosslinking BMSCs Cell BP-grafted GelMA [121] Microsphere Photo-crosslinking --…”

“…As one of the most popular thermosensitive hydrogels, gelatin assumes a solvated state when heated, and will gradually cure as the temperature decreases. In most cases, to produce microfluidic gelatin microspheres, a heating plate is placed at the inlet to liquefy the gelatin for injection into the channel, and then a cooling module is installed at the outlet to solidify the gelatin droplets [116]. Due to the weak physical crosslinking as well as the reversible phase transition of gelatin, the stabilization of gelatin microspheres is poor, limiting its application in cargo delivery.…”

Recent Advances in Microgels: From Biomolecules to Functionality

“…[1][2][3][4][5][6][7] Homogenous microgels have constant composition and gel density throughout the microgels; spatially inhomogeneous microgels have varying density of the gel materials; heterogeneous microgels have varied composition of gel materials. [3,4,8] While the homogenous microgels have been used as 3D scaffold models for cell growth, inhomogeneous and heterogeneous microgels can more accurately mimic the physiological environment in vivo. [3,[9][10][11] By exploiting microfluidic platforms, microgels can be produced with controlled size, shape, structures, and compositions.…”

“…[30] Scale bars = 200 µm (tear-drop), 500 µm (microrod), 0.3 µm (submicron, raspberry-like), 400 µm (compartmentalized), and 100 µm (all other). The microgel/environment interfaces can include gel/oil, [1,3] gel/sol, [8] gel/water, [3,4] gel/gel, [33] and gel/ tissue interfaces. [34,341] The internal structures of the building blocks can be random coils, [3] alpha helices, [3] beta sheets, [339] etc.…”

The chromatin regulator HMGA1a undergoes phase separation in the nucleus

“…2,[30][31][32] Within the LLPS framework for nuclear organization, 30,32,33 multivalent proteins (including RNAbinding proteins and proteins with low complexity domains), 24,[34][35][36] RNAs [37][38][39][40][41][42] and DNAs 32,33 undergo a concentration-dependent demixing to yield biomolecular condensates. [43][44][45] Accordingly, the formation of the nucleoli, 37,46,47 nuclear speckles, 48 PML bodies, 49 and several other nuclear compartments that lack physical membranes have been attributed to LLPS. Furthermore, chromatin proteins found within the heterchromatic environment, like H1 50 and the heterochromatin protein 1 (HP1), 2,32,33 have been shown to phase separate in vitro and in cells.…”