Understanding the relationship between an amino acid sequence and its phase separation has important implications for analyzing cellular function, treating disease, and designing novel biomaterials. Several sequence features have been identified as drivers for protein liquid-liquid phase separation (LLPS), leading to the development of a “molecular grammar” for LLPS. In this work, we further probed how sequence modulates phase separation and the material properties of the resulting condensates. Specifically, we used a model intrinsically disordered polypeptide composed of an 8-residue repeat unit and performed systematic sequence manipulations targeting sequence features previously overlooked in the literature. We generated sequences with no charged residues, high net charge, no glycine residues, or devoid of aromatic or arginine residues. We report that all but one of the twelve variants we designed undergo LLPS, albeit to different extents, despite significant differences in composition. These results support the hypothesis that multiple interactions between diverse residue pairs work in tandem to drive phase separation. Molecular simulations paint a picture of underlying molecular details involving various atomic interactions mediated by not just a handful of residue types, but by most residues. We characterized the changes to inter-residue contacts in all the sequence variants, thereby developing a more complete understanding of the contributions of sequence features such as net charge, hydrophobicity, and aromaticity to phase separation. Further, we find that all condensates formed behave like viscous fluids, despite large differences in their viscosities. The results presented in this study significantly advance the current sequence-phase behavior and sequence-material properties relationships to help interpret, model, and design protein assembly.

Bicompartmental Janus particles have many advantages in drug delivery, including co-delivery of two compounds with varying solubilities, differential release kinetics, and two surfaces available for targeting ligands. We present a novel strategy using the double emulsion method for the coencapsulation and staggered release of a hydrophobic and hydrophilic drug from anisotropic PLGA/PCL Janus particles, as well as a UV detection method to measure the release of two different compounds from Janus particles. Curcumin and quercetin were chosen as the model hydrophobic compounds for drug loading studies, while acetaminophen (APAP) and naproxen were chosen as the model hydrophilic–hydrophobic drug pair for encapsulation methods and drug loading. Also, a similar double emulsion method was also applied for PLGA/Preicrol® Janus particles containing Doxorubicin and Curcumin. Hydrophobic drugs were encapsulated by the single O/W emulsion technique. Hydrophilic compounds required special modifications due to their poor oil solubility and tendency to escape to the outer aqueous phase during the emulsification and solvent evaporation steps. In total, three different strategies for incorporating hydrophilic drugs were employed: (1) O/W emulsion with partially water miscible solvent, (2) O/W emulsion with co-solvent (i.e. acetone, methanol, ethanol), or (3) W/O/W double emulsion. The encapsulation efficiencies and drug loading percentages were measured using UV/Vis spectroscopy and compared for the different synthesis methods. It was found that the double emulsion method resulted in the highest encapsulation efficiency and drug loading of the hydrophilic drug.

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