Biophysical characterization and regulation of bioinspired peptide coacervates

Abstract

Peptide coacervates formed through liquid-liquid phase separation (LLPS) have emerged as versatile platforms for biomedical applications due to their tunable properties and responsiveness to environmental stimuli. This thesis systematically explores the design, structural and biophysical characterization, and functional applications of peptide-based coacervates, focusing on the relationships between peptide sequence and biophysical properties, and impacts on their applications as drug delivery vehicles.

The first study develops a model system of GY23 peptide variants, inspired by the Histidine-rich beak protein 1 (HBP-1) sequence, to investigate molecular structure-property relationships within coacervates. Through specific amino acid substitutions to modulate hydrophobicity, as well as adjustments in ionic strength and ion type, this study demonstrates how these factors govern coacervate biophysical properties, particularly in relation to β-sheet content. Additionally, GY23 coacervates exhibit aging phenomena, with enhanced physical properties over time that correlate with increased β-sheet content. These findings provide foundational insights for designing peptide-based coacervates with tailored biophysical properties, broadening their potential in bioadhesives, microreactors, and therapeutic delivery.

Building on these principles, the second study designs GW27-KSP peptide variants as optimized delivery vehicles for the intracellular delivery and release of functional biomacromolecules. Derived from Histidine-rich beak protein 2 (HBP-2), the GW27-KSP sequence incorporates lysine residues and a self-immolative modification, enabling pH- and redox-responsive cargo release. Systematic mutations at specific hydrophobic residues within the sequence allow for tunable coacervation behavior and biophysical properties. These optimized peptide coacervates demonstrate controlled cellular uptake and release kinetics for a variety of biomolecular cargo, which linked to molecular hydration of peptides. The study also introduces complex coacervation via cation-π interactions between Arg- and Tyr-containing peptide variants, presenting a novel mechanism for complex coacervate formation. This approach enables precise modulation of hydration and cargo release kinetics simply by adjusting the ratios of cationic to aromatic peptides, offering a flexible platform for customizable delivery systems.

The third study addresses the challenge of coacervate stability and size control, crucial for consistent therapeutic outcomes. Coacervate microdroplets often undergo spontaneous coalescence due to their dynamic, membraneless nature, leading to unstable size distributions. To mitigate this, GW27-KSP variants were modified with poly(ethylene glycol) (PEG), introducing steric hindrance to stabilize the coacervates and reduce their size from micrometers to controlled nanoscale dimensions. PEGylated coacervate nanodroplets favor clathrin-mediated endocytosis over macropinocytosis, potentially reducing macrophage clearance and enhancing in vivo delivery potentials. Additionally, PEGylated coacervates could coalesce into larger microdroplets upon heating to 50°C, enabling targeted mRNA release in diseased tissues through localized heat application.

In summary, this thesis presents a comprehensive approach to developing peptide coacervates with customizable properties, stability, and delivery efficiency, advancing their potential as bioinspired delivery systems. By linking molecular insights to practical applications, these studies contribute to the rational design of coacervates, bridging fundamental research with translational potential in drug delivery, bioadhesion, and beyond.