Cell-penetrating peptides (CPPs) are a class of small-molecule peptides with the function of penetrating cell membranes, which can carry a variety of biologically active molecules into cells. With the in-depth study of CPP, it has been found that it has problems such as poor stability against proteolytic hydrolysis, low endosomal escape rate and lack of targeting, which seriously limit its application as a drug carrier.

What are cell-penetrating peptides?

20 years ago, the FRANKEL team extracted the activating transcriptional activator (TAT) protein from human immunodeficiency virus 1 and found that it has the activity of penetrating cell membranes. Since then, a large number of short peptides with similar activities have been discovered. Short peptides are vividly called cell penetrating peptides (CPP). Compared with electroporation, magnetic transfection, lipofection and viral vectors, CPP is characterized by its high penetrating efficiency, low cytotoxicity, The advantages of being able to carry various biologically active molecules (including nanoparticles) have attracted extensive attention from researchers.Such penetrating peptides can currently be synthesized with specific sequences by custom peptide synthesis in peptide company.

How many types of penetrating peptides are there?

There are thousands of CPPs that have been discovered before, and they can be divided into 3 types according to their physical and chemical properties:

(1) Cationic type, such as TAT, Penetratin, Polyarginine, etc.;

(2) Amphiphilic type, such as model amphiphilic peptide (MAP);

(3) Hydrophobic type, such as fibroblast growth factor

Penetrating peptides need improvement:

1. When applied in vivo, it is found that CPP marked by fluorescence and other technologies is partially or even mostly degraded due to poor resistance to proteolytic hydrolysis in vivo.

2. After CPP endocytosis and transmembrane, most of them are bound in vesicles (endosomes) and cannot reach the cytoplasm and nucleus due to the low escape rate of endosomes.

3. Most critically, CPP can pass through the cell membrane of any mammalian cell, resulting in extremely poor targeting of unmodified CPP, which greatly limits its clinical application as a drug carrier.

The above problems have caused the amount of CPP to reach the target tissue or target cells to be greatly reduced, resulting in a decrease in the effect of the drug

3 ideas to improve cell-penetrating peptides:

1. Stability against protease hydrolysis:

The SARKO team compared 10 commonly used CPPs, including penetratin, TAT, hepatitis B virus surface protein PreS2 translocation motif, MAP, nona-arginine, etc. and found that arginine-arginine bond (RR bond) is the most important CPP is a key factor for stability, and its clearance can be reduced by attaching CPP to macromolecular carriers such as nanoparticles or liposomes; in addition, its stability can be increased by modifying the penetrating peptide itself. WOLFE et al. proposed that the stability can be increased by bicyclizing the CPP backbone. A more useful strategy to increase the stability of CPP is to use D-type unnatural amino acids to replace L-type amino acids in the penetrating peptide sequence to increase its stability, but the disadvantage is that unnatural amino acids will bring higher toxicity.

We can choose a corresponding number of D-type amino acids (the number of D-type arginines in the penetrating peptide chain is less than 3) that does not significantly increase toxicity to increase the stability of CPP.

2. Mechanism of membrane penetration of CPP

The specific membrane penetration mechanism of CPP has been debated for a long time, and it is generally believed that there are two main ways of membrane penetration: endocytosis and direct non-endocytosis. At present, most experimental studies support endocytosis to penetrate the membrane, but the cargo molecules (CPP and bioactive molecules) that enter the cell through endocytosis are bound in vesicles (endosomes) and cannot function, and are long-term Trapped in vesicles, the acidic environment of the vesicles and lysosomes and the various enzymes they contain can further inactivate or degrade cargo molecules. The main solution to this problem is to rupture the endocytosed vesicles to release the cargo molecules by appropriate methods, such as by adding additional vesicle leakage-promoting agents or modifying penetrating peptides with membrane-destroying vesicles. structure, etc.

3. Targeting of CPP

Penetrating peptides are considered as promising potential drug carriers, but their lack of selectivity hinders their clinical application. At present, the research on targeted drug-loaded CPP mainly focuses on tumors, and most of the research ideas are to use different "stimulus-response" strategies to control the entry of drug-loaded CPP into tumor cells. Under the promotion of tumor tissue-specific factors, drug-loaded CPP without membrane-penetrating activity becomes drug-loaded CPP with membrane-penetrating activity, which enables it to selectively enter tumor cells. Thereby reducing adverse reactions and improving anti-tumor efficacy.