Cytosolic Protein Delivery for Intracellular Antigen Targeting Using Supercharged Polypeptide Delivery Platform

Abstract

Despite the well-recognized clinical success of therapeutic proteins, especially antibodies, their inability to penetrate the cell membrane restricts them to secretory extracellular or membrane-associated targets. Developing a direct cytosolic protein delivery system would offer unique opportunities for intracellular target-related therapeutic proteins. Here, we generated a supercharged polypeptide (SCP) with high cellular uptake efficiency, endosomal escape ability, and good biosafety and developed an SCP with an unnatural amino acid containing the phenylboronic acid (PBA) group, called PBA-SCP. PBA-SCP is capable of potently delivering proteins with various isoelectric points and molecular sizes into the cytosol of living cells without affecting their bioactivities. Importantly, cytosolically delivered antibodies remain functional and are capable of targeting, labeling, and manipulating diverse intracellular antigens. This study demonstrates an efficient and versatile intracellular protein delivery platform, especially for antibodies, and provides new possibilities for expanding protein-based therapeutics to intracellular “undruggable” targets.

Protein Delivery to Insect Epithelial Cells In Vivo: Potential Application to Functional Molecular Analysis of Proteins in Butterfly Wing Development

“…However, the obstacle that proteins are often incorporated into a cell by endocytosis and are confined within endosomes has not been completely overcome. Several different peptides and their modified versions have been tested for more efficient endosomal escape [ 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 ]. Due to the technological demands for quick protein delivery to cells with fewer technical requirements, membrane-lytic peptides are receiving more attention from researchers in the twenty-first century.…”

Enrichment and delivery of target proteins into the cell cytosolviaOuter Membrane Vesicles

“…6 As the significance of intracellular protein delivery has been well recognized, multiple methods were developed for cellular protein delivery including physical methods, cell-penetrating peptides, polyethyleneimine, supercharged molecules, and nanocarriers in the past decades. [7][8][9][10][11] Most of these transportation systems have remained limitations in different aspects. Physical methods such as electroporation, microinjection, or microinjection, mainly use physical forces to temporally generate pores on the cell membrane to allow proteins to transport into cells, but they often induce damage to cell membranes and have limited throughput.…”

Robust Reversible Cross-Linking Strategy for Intracellular Protein Delivery with Excellent Serum Tolerance

“…Protein-based therapeutics have achieved great success in the drug markert. , Proteins such as insulin, growth factors, interferon and monoclonal antibodies have been approved for the treatment of various diseases, , and they have emerged as high potent and selective alternatives to small-molecule drugs . However, the clinically approved protein drugs are limited to extracellular targets and suffered from poor proteolytic stability and membrane impermeability. , Efficient intracellular protein delivery technologies could promote the development of protein drugs acting on intracellular targets. − Over the past decades, various strategies have been developed for intracellular protein delivery, − especially lipid nanoparticles, − peptides, − polymers, − and inorganic nanomaterials. − However, these delivery systems are usually associated with poor serum tolerance due to the competitive binding of serum proteins to the vectors during delivery.…”