Day: April 9, 2022

Cell-Penetrating Peptides in Diagnosis and Treatment of Human Diseases: From Preclinical Research to Clinical Application

Cell-penetrating peptides (CPPs) are short peptides (fewer than 30 amino acids) that have been predominantly used in basic and preclinical research during the last 30 years. Since they are not only capable of translocating themselves into cells but also facilitate drug or CPP/cargo complexes to translocate across the plasma membrane, they have potential applications in the disease diagnosis and therapy, including cancer, inflammation, central nervous system disorders, otologic and ocular disorders, and diabetes. However, no CPPs or CPP/cargo complexes have been approved by the US Food and Drug Administration (FDA). Many issues should be addressed before translating CPPs into clinics. In this review, we summarize recent developments and innovations in preclinical studies and clinical trials based on using CPP for improved delivery, which have revealed that CPPs or CPP-based delivery systems present outstanding diagnostic therapeutic delivery potential.

Based on their origins, CPPs can be divided into protein-derived CPPs, synthetic CPPs, and chimeric CPPs. (1). Protein-derived CPPs, including the TAT protein (Vives et al., 1997) and penetratin (Derossi et al., 1994), can enter the cell because they have a specific motifs or helical structures (Traub, 2009). (2). Synthetic CPPs. In this group, polyarginine 8-10-mers are most widely studied because of their high efficiency in cellular uptake (Mitchell et al., 2000). (3). Chimeric CPPs. This group of CPPs is generally recognized as a transition from natural to synthetic CPPs since they contain sequences from two or more different naturally occurring proteins. Examples include amphipathic peptide (CADY) (20 amino acids) which combines aromatic residue (Tryptophan, W) and cationic residue (Arginine, R) (Crombez et al., 2009). (Front Pharmacol. 2020; 11: 697.)

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Protein Delivery to Cytosol by Cell-Penetrating Peptide Bearing Tandem Repeat Penetration-Accelerating Sequence

To facilitate the cytosolic delivery of larger molecules such as proteins, we developed a new cell-penetrating peptide sequence, named Pas2r12, consisting of a repeated Pas sequence (FFLIG-FFLIG) and d-dodeca-arginine (r12). This peptide significantly enhanced the cellular uptake and cytosolic release of enhanced green fluorescent protein and immunoglobulin G as cargos. We found that simply mixing Pas2r12 with cargos could generate cytosolic introducible forms. The cytosolic delivery of cargos by Pas2r12 was found to be an energy-requiring process, to rely on actin polymerization, and to be suppressed by caveolae-mediated endocytosis inhibitors (genistein and methyl-β-cyclodextrin) and small interfering RNA against caveolin-1. These results suggest that Pas2r12 enhances membrane penetration of cargos without the need for cross-linking and that caveolae-mediated endocytosis may be the route by which cytosolic delivery is enhanced.

Pas2r12 significantly enhances cytosolic delivery of cargo proteins, including enhanced green fluorescent protein and immunoglobulin G. Simply incubating Pas2r12 with cargo leads to their cytosolic tranlsocation. Cytosolic delivery of cargo by Pas2r12 involves caveolae-mediated endocytosis. In this chapter, we describe methods of cytosolic delivery of cargo using Pas2r12 and provide methods for investigating the cellular uptake pathway of cargo by Pas2r12. (Methods Mol Biol . 2022;2383:265-273.)