Cell-penetrating peptides are a class of short peptides with a length of 5~30 amino acids, which can carry peptides, nucleic acids, small molecules of drugs, and virus particles through cell membranes into cells. People use it as a carrier to transport objects into cells. Past research has proven effective in treating mouse models of cancer and inflammatory diseases with cells that carry proteins and peptides through peptides. Based on animal studies, it is thought that it will be possible for cells to penetrate peptides carrying DNA or siRNA to treat disease.


Cell-penetrating peptides can also improve the efficiency of viral transfection. Therefore, this can help treat the disease, but also may facilitate the spread of the virus, a double-edged sword, people need to increase research efforts to avoid harm. In addition, cell-penetrating peptides can carry fluorescent or radioactive reagents for imaging applications. In conclusion, cell-penetrating peptides carrying therapeutic genes or drug molecules into cells could have a very broad clinical application prospect.


The molecular design of conventional cell-penetrating peptides cannot be separated by three key parameters: guanidinium content (or cationic amino acid content), hydrophobicity, and amphipathicity. Cell-penetrating peptides rich in arginine (arginine with a guanidinium side chain, pKa of approximately 12, and protonated guanidinium cation at physiological pH) have been intensively studied in the field of primary structure-potency.


Membrane models, membrane extracts, and in vivo studies have played a key role in investigating the amino acid sequence and the contribution of individual amino acid residues (e.g. positive charge) of cell-penetrating peptides, these studies have guided the molecular design of cell-penetrating peptides. Despite these theoretical guidelines, it is still very difficult to predict the membrane penetration of a peptide from its sequence alone.


A variety of chemical and physicochemical properties, such as charge, chirality, aromaticity, and hydrophobicity, and their interactions are often important driving forces for the internalization of cell-penetrating peptides. It can be said that the peptide sequence is the basis on which the intermolecular interactions determine the efficacy of the cell-penetrating peptide. To design efficient cell-penetrating peptides, numerous parameters including charge, guanidine groups, chirality, hydrophobicity, aromaticity, and their interactions, need to be further investigated in depth.


Significance of cation enrichment for cell-penetrating peptides

Peptide sequences rich in side chain cationic amino acids are important in the field of cellular penetration of peptides. The discovery of the Tat peptide was a landmark event in the field of cell-penetrating peptides. Tat (Transactivating transcriptional activator) peptide is a peptide fragment of residues 48 to 60 of the original transcriptional activator of the human immunodeficiency virus (HIV-1). Tat's modified gene delivery system shows enhanced transport across multiple biofilms, such as cell membranes, endosomal membranes, and nuclear membranes.


Tat peptide contains six arginine and two lysine residues, all of which belong to basic amino acids. The side chains are guanidine and amino respectively, and all of them are positively charged due to protonation under physiological pH conditions. Given the significant guiding importance of Tat, it is not surprising that the simplest cell-penetrating peptide mimic was designed as an oligoarginine.


Cysteine cell-penetrating peptide

Advances have been made in the design of cell-penetrating peptides in response to challenges in the realization of their function, such as internalization efficiency, endosomal escape efficiency, cycle time, specificity, and selectivity (for cells, tissues, and diseases). One example of this is the cysteine-rich (Cys) cell-penetrating peptide.


The molecular design was inspired by Crotamine, a toxin found in snake venom, which contains two nuclear localization domains (crot(2-18) and crot(27-39)). By examining crot (27-39) (sequence: KMDCRWRWKCCKK), the researchers used systematic substitution and/or omission of amino acid residues, as well as in-depth conformational studies, to design a cysteine-rich decapeptide (CRWRWKCCKK) molecule. This potential cell-penetrating peptide has enhanced internalization efficiency.


Monocyclic, bicyclic, and tricyclic cell-penetrating peptides

Cell-penetrating peptides have become a popular area of research for intracellular therapeutic agents. Because of the natural limitations of straight-chain cell-penetrating peptides, such as endosomal encapsulation, toxicity, poor cell specificity, poor stability, and degradability, as well as imperfect cell penetration, modified cell-penetrating cyclic peptides have emerged. Compared to their straight-chain precursors, cell-penetrating cyclic peptides offer enhanced cell penetration and improved physical and chemical properties, as well as stability against hydrolytic degradation. Some cell-penetrating cyclic peptides can exhibit endosome-independent uptake, and nuclear targeting properties have been reported for some cell-penetrating cyclic peptides.


The two cell-penetrating cyclic peptide structural units [WR]4 and [WR]5 are characterized by the presence of alternating positively charged (arginine) and hydrophobic amino acids (tryptophan) in the sequence. Monocyclic cell-penetrating cyclic peptides containing tryptophan and arginine residues can also be coupled to potential therapeutic agents. For example, monocyclic peptides are coupled to doxorubicin, paclitaxel, and camptothecin, where the doxorubicin-cyclic peptide adduct demonstrates the effect of internalization.


In addition to this, several monocyclic peptides containing cysteine and arginine residues significantly enhanced the uptake of the impermeable cellular phosphopeptide (F')-Gly-(pTyr)-Glu-Glu-Ile (F'-GpYEEI). Cyclic decapeptides containing tryptophan and histidine effectively increased the intracellular delivery of the cell impermeable phosphopeptide with the anti-HIV drug emtricitabine (emtricitabine). The [WR]4-[WR]4-[WR]4 tricyclic peptide containing alternating arginine and tryptophan residues increased the cellular uptake of F'-GpYEEI and fluorescently labeled anti-HIV drugs (lamivudine (3TC), emtricitabine (FTC) and siRNA) in the breast cancer cell line MDA-MB-231.


In the above cell-penetrating cyclic peptides, successive combinations of tryptophan and arginine residues give rise to different types of cell-penetrating cyclic peptides with different cellular delivery properties. These data reveal the molecular transport potential of monocyclic, bicyclic, and tricyclic cell-penetrating peptides and provide a good basis for the design of next-generation drug delivery peptides.



Although the molecular design of cell-penetrating peptides is currently reported in the literature in great detail, translating them into the clinical setting remains a formidable challenge. The field of application of cell-penetrating peptides is vast and evolving, and deeper mechanistic exploration has led to an increase in the complexity of the molecular design. This includes the development of stable and multi-domain cyclic or self-assembled nanostructures. In addition, further developments are needed in terms of selectivity, targeting, and efficiency. Many researchers have proposed controlled spatial folding, cyclization, dimerization, stapling, self-assembly, and even peptide mimics with different backbones to achieve improved stability and activity of cell-penetrating peptides.