Cellular Uptake of His-Rich Peptide Coacervates Occurs by a Macropinocytosis-Like Mechanism

Abstract

Coacervates are dense microdroplets formed by liquid-liquid phase separation (LLPS) of macromolecules that have gained increasing attention as drug delivery vehicles. Recently, we have reported a new intracellular delivery system based on self-coacervating histidine (His)-rich beak peptides (HBpep and HBpep-SP) inspired by beak proteins of the Humboldt squid. These peptide microdroplets combine excellent encapsulation efficiency of therapeutics with high transfection rate and low cytotoxicity. However, the mechanism by which they cross the cell membrane remains elusive. Previous inhibitor studies provided incomplete clues into the detail uptake pathway, although they suggested a cholesterol-dependent and, possibly, an energy-independent non-classical mechanism of internalization. In this study, we improved our understanding of coacervates/cell membrane interactions using model membranes, namely Giant Unilamellar Vesicles (GUVs) and Giant Plasma Membrane Vesicles (GPMVs). We also employ a combination of electron microscopy techniques to gain detailed structural insights into the cell uptake of HBpep and HBpep-SP coacervates. We demonstrate that modulating lipid charge and cholesterol level influence coacervate attachment to GUVs. However, they are not able to cross the GUV's lumen in an energy-independent manner. We then show that the coacervates enter HeLa and HepG2 cells via a mechanism sharing morphological features of macropinocytosis and phagocytosis, in particular involving cytoskeleton rearrangement and capture by filipodia. Our study provides key insights into the interaction of HPpep and HBpep-SP coacervates with model membranes as well as their cellular uptake pathway.

Complex Coacervates as a Promising Vehicle for mRNA Delivery: A Comprehensive Review of Recent Advances and Challenges

“…Specific advantages of using polymers, − proteins, ,, and peptides ,,, as coacervate components for mRNA delivery, as well as the synergistic potential of combining them to form hybrid − and composite − ,, coacervates. By leveraging the unique properties of each component, coacervate formulations can be tailored to specific applications, offering enhanced delivery efficacy and versatility.…”

“…To minimize potential side effects, researchers should explore the use of biocompatible and non-immunogenic materials such as natural polysaccharides (e.g., chitosan or alginate) or coacervate functionalized nanoparticles with modifications to their physiochemical properties (e.g., PEG surface modification, hybrid structures) . Another challenge is the tendency of coacervates to fuse over time during storage, which could potentially be addressed by utilizing stabilizing agents, chemical cross-linking, or incorporating liposome or polymersome delivery systems. , Additionally, advanced imaging could enable real-time tracking to help elucidate the mechanisms of uptake …”

“…26,31 When coacervates are introduced into the cytosol, they have the potential to disrupt normal intracellular processes by interacting with the cell membrane. 33 This interaction holds significance in various cellular processes, such as signal transduction, long-distance RNA transport, waste disposal mechanisms, and protein storage. 34 Coacervates can influence the membrane by affecting its fluidity, inducing deformation, and causing budding.…”

“…33 However, the coacervates were not able to enter the interior of cells (modeled by giant unilamellar vesicles) without the use of energy, suggesting that an energy-dependent pathway is required for the coacervates to cross the cellular membrane. 33 Additional experiments revealed that the coacervates were taken up by the cells through a process called filipodia-mediated capture. 33 This mechanism involved rearrangement of the cytoskeleton, which is a network of protein filaments that helps to maintain the cell's shape and structure.…”

“…33 Additional experiments revealed that the coacervates were taken up by the cells through a process called filipodia-mediated capture. 33 This mechanism involved rearrangement of the cytoskeleton, which is a network of protein filaments that helps to maintain the cell's shape and structure. 33,53 The implications of this finding are significant because it provides insight into a specific pathway that can be targeted to improve the effectiveness and specificity of coacervate-based mRNA delivery.…”