**5. Cell-penetrating peptides as molecular carries**

The selective permeability and hydrophobic profile of the cellular membranes provide strict control of the molecular changes between the cytosol and the extracellular environment [93–95]. Generally, peptides are selective and effective signaling molecules which bind to specific cell surface receptors that are involved in physiological mechanisms such as peptides, hormones, neurotransmitters, growth factors, G-cell receptors (GPCRs), and ion channel ligands [96]. This characteristic of the peptides mentioned above and their attractive pharmacological profile represent a new starting point in the redesign and in-cell recruitment of molecules for therapeutic purposes [93–97]. Prior to the discovery of cell-penetrating peptides (CPPs), various methods have been used for cellular uptake of therapeutic agents and drugs, such as microinjection, electroporation, and liposome- and viral-based vectors, but these have disadvantages such as restricted bioavailability, low productivity, high toxicity, and low specificity [95]. After all these developments, in the late 1980s, a group of short peptides, such as the protein translocation site, membrane translocation sequence, Trojan peptide, or most commonly CPP, which serve as cellular uptake and delivery vectors of large molecules for therapeutic purposes, were identified [94, 96].

#### **5.1 Definition and classification of CPP**

CPPs are mostly defined as the short (containing less than 40 amino acid residues) partially hydrophobic and/or polybasic natural and synthetic peptides [94, 97]. With the discovery of CPPs, it has emerged as a new tool that allows cell membrane

translocation without significant membrane damage and at low concentrations without using any chiral receptors. Due to the penetrating properties of these effective cellpenetrating peptides, it opened new opportunities for medical treatment and delivery of therapeutic agents across cells in vivo. Furthermore, and even more importantly, interdisciplinary studies show that CPPs are able to assist in internalizing covalently or electrostatically conjugated bioactive cargos such as nucleic acids, polymers, liposomes, nanoparticles, and low-molecular-weight drugs, with dose-dependent efficiency, cheapness, degradability inside cells, and low toxicity [94, 95, 97]. There are various classifications that rely on CPPs' qualities. A categorization based on the origin of the peptide is one of the classifications. The first subgroup is also called protein transduction domains (PTDs); they are including protein-derived peptides such as TAT and penetratin. The second subgroup peptides called chimeric peptides may occur two or more motifs from other peptides. And the last subgroup in this category is synthetic peptides such as the polyarginine family [94, 98]. In addition, CPPs are categorized according to peptide sequences and binding abilities to the lipids, including non-amphipathic (naCPPs), primary amphipathic (paCPPs), and secondary amphipathic (saCPPs) CPPs. R9 and TAT (48–60) are examples of naCPPs that have a high cationic amino acid ingredient, and they are bound to the cell membrane with a high consideration of anionic lipids. The second subgroup of paCCPs such as transportan or TP10 have hydrophobic and hydrophilic residues straight from their primary structure. The third subgroup in this categorization is saCPPs such as penetratin, pVEC, and M918. Their alpha-helix or beta-sheet patterns compose amphipathic properties [94].

In summary, in vitro cell and membrane modeling studies have shown that paCPPs are more toxic than naCPP and saCPPs.

#### **5.2 Cellular uptake mechanism of CPP**

#### *5.2.1 Direct penetration*

Direct penetration of CPPs eventuates along the membrane lipid bilayers without the contribution of receptors in low temperatures via energy-independent mechanism [93, 99]. There are three different types for the internalization pathway of CPPs by direct penetration [93].

#### *5.2.1.1 Barrel-stave model*

In the barrel-stave model, a different number of peptides are placed in the inside part of the membrane to give a hoop appearance. Each of these peptide molecules that provide the formation of membrane channels is likened to the boards that make up the barrel. Due to the appearance of the membrane, this model is called as the barrel-stave model. The barrel-stave model is carried out by an accumulation of peptides consisting of cationic peptides in the amphipathic helix structure. In this model, the hydrophilic sides of the peptides are contacted with the solvent, and the hydrophobic sides of the peptide come into contact with the lipid bilayer of the membrane, resulting in channels or pores in the membrane. The critical step in the barrel-stave model is that the peptides recognize each other while they are attached to the membrane. The peptide molecules then interact with the lipid molecules present in the membrane, and the peptides pass to the hydrophobic side of the membrane. By adding new monomers or small oligomers to the resulting structure, the width of the pores increases. The peptides act in this way even at very low concentrations once they penetrate the phospholipid membrane in the target cell [93].

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*Synthesis and Applications of Synthetic Peptides DOI: http://dx.doi.org/10.5772/intechopen.85486*

According to the carpet-like model, firstly an electrostatic interaction occurs between the phospholipid groups on the negatively charged cell membrane bilayer and the positively charged peptide monomers. The hydrophilic sides of the peptide monomers are bound to the surface of the target cell in such a way that they overlap the phospholipid groups in the membrane and cover the cell as a carpet. Once the peptides in this state have reached a sufficient concentration, the peptide molecules rotate, reorganizing the hydrophobic portion of the membrane, causing a similar effect to the detergents and resulting in digestion of the membrane. On the contrary to the model of the barrel-stave model in the carpet model, the positively charged peptides cannot penetrate the hydrophobic parts of the membrane but instead are in a group with the hydrophilic parts facing one another. According to this mechanism, CPPs can be pres-

ent in various secondary structures, sizes, and linear or cyclic form [93].

In toroidal model, after the halezonal peptides are parallel to the membrane, the polar groups of the membrane are replaced by the hydrophobic amino acid groups of the peptides. This leads to the formation of cleft that is vertical to the membrane in the hydrophobic part of the membrane, extending along the peptide and lipid groups. The difference of the toroidal model and the barrel-stave model is that the peptides are present vertically in the lipid membrane and always form the pores in

Direct penetration pathway results in an irreversible membrane destabilization and can cause artifact distribution in cells [93, 99]. Therefore, it has been shown that CPP-cargo complexes can be taken into the cell by a mechanism known as

Endocytosis is a natural process that occurs in all cells via energy-dependent mechanism. Different types of pinocytic pathways have been identified, since they vary according to the access of different CPPs and conjugated cargo com-

Macropinocytosis, which is related to the folding of the outer surface of the plasma membrane, is a nonspecific uptake of the extracellular compounds. It starts with an invasion of a membrane promoted by actinic cytoskeleton elements to create a pocket and large endocytic vesicles containing different types of cargoes, and it finishes by the occurrence of vesicles called macropinosomes. Macropinocytosis has critical importance in the uptake of TAT and polyarginine [93, 102]. TAT uptake happens when using macropinocaytosis without the need for GATs or sialic acids, while plasma membrane-active proteins must be present [103]. Passive transport does not have actin remodeling, so passive transport is recommended for CPP

Clathrin-mediated endocytosis is the specific uptake of CPPs that have been shown to have significance in the uptake of penetratine, TAT peptide, and other

*5.2.1.2 Carpet-like model*

*5.2.1.3 Toroidal model*

endocytosis [93, 101].

plexes of CPPs [96].

*5.2.2.1 Macropinocytosis*

*5.2.2 Endocytosis*

combination with the lipid groups [93, 100].

penetration instead of macropinocytosis [104].

*5.2.2.2 Clathrin-mediated endocytosis*
