*5.2.1.2 Carpet-like model*

*Peptide Synthesis*

properties [94].

*5.2.1 Direct penetration*

*5.2.1.1 Barrel-stave model*

paCPPs are more toxic than naCPP and saCPPs.

**5.2 Cellular uptake mechanism of CPP**

of CPPs by direct penetration [93].

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

In summary, in vitro cell and membrane modeling studies have shown that

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

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

**16**

the target cell [93].

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