Cellular Uptake of Phase‐Separating Peptide Coacervates

Author:

Shebanova Anastasia1,Perrin Quentin Moana1,Zhu Kexin2,Gudlur Sushanth1,Chen Zilin1,Sun Yue1,Huang Congxi1,Lim Zhi Wei1,Mondarte Evan Angelo1,Sun Ruoxuan3,Lim Sierin34,Yu Jing14,Miao Yansong24,Parikh Atul N.145,Ludwig Alexander26,Miserez Ali12

Affiliation:

1. Centre for Sustainable Materials, School of Materials Science and Engineering Nanyang Technological University (NTU) 50 Nanyang Avenue Singapore 637553 Singapore

2. School of Biological Sciences NTU 60 Nanyang Drive Singapore 637551 Singapore

3. School of Chemistry, Chemical Engineering and Biotechnology NTU 70 Nanyang Drive Singapore 637457 Singapore

4. Institute for Digital Molecular Analytics and Science (IDMxS) NTU 59 Nanyang Drive Singapore 636921 Singapore

5. Departments of Biomedical Engineering and Materials Science & Engineering University of California Davis CA 95616 USA

6. NTU Institute of Structural Biology NTU 59 Nanyang Drive Singapore 636921 Singapore

Abstract

AbstractPeptide coacervates self‐assembling via liquid‐liquid phase separation are appealing intracellular delivery vehicles of macromolecular therapeutics (proteins, DNA, mRNA) owing to their non‐cytotoxicity, high encapsulation capacity, and efficient cellular uptake. However, the mechanisms by which these viscoelastic droplets cross the cellular membranes remain unknown. Here, using multimodal imaging, data analytics, and biochemical inhibition assays, identify the key steps by which droplets enter the cell. find that the uptake follows a non‐canonical pathway and instead integrates essential features of macropinocytosis and phagocytosis, namely active remodeling of the actin cytoskeleton and appearance of filopodia‐like protrusions. Experiments using giant unilamellar vesicles show that the coacervates attach to the bounding membrane in a charge‐ and cholesterol‐dependent manner but do not breach the lipid bilayer barrier. Cell uptake in the presence of small molecule inhibitors – interfering with actin and tubulin polymerization – confirm the active role of cytoskeleton remodeling, most prominently evident in electron microscopy imaging. These findings suggest a peculiar internalization mechanism for viscoelastic, glassy coacervate droplets combining features of non‐specific uptake of fluids by macropinocytosis and particulate uptake of phagocytosis. The broad implications of this study will enable to enhance the efficacy and utility of coacervate‐based strategies for intracellular delivery of macromolecular therapeutics.

Funder

Ministry of Education - Singapore

Publisher

Wiley

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