*5.3.3 Role of arginine-rich residues*

Structure-activity relationship studies show how important it is to identify the single residues in the CPP structure. When the CPPs are rich in arginine (especially for its guanidinium group), they can compose hydrogen bonds with polar lipid groups. The presence of arginines in the CPP has been related with a better uptake efficiency. In addition, replacement or wiping of arginines may reduce the cellular uptake [93, 104, 106].

#### *5.3.4 Role of chirality*

*Peptide Synthesis*

*5.2.2.3 Caveolin-mediated endocytosis*

*5.2.2.4 Escape from endosomes*

*5.3.1 Role of glycosaminoglycans*

*5.3.2 Peptide secondary structure*

on the cell line used, and decreased cell viability [96, 107].

better escape from the endosomal membrane [93, 95].

**5.3 Factors affecting the mechanism of cellular uptake**

CPPs after inhibition by hyperosmolar media [96]. The mechanism starts with the creation of a vesicle that is covered by a crystal on the interior surface of the cell membrane. After the coating of clathrin proteins by vesicles, it combines with an early endosome which moves via microtubules from the cell into the nucleus. Macromolecules carried on late endosome are fused with vesicles of the Golgi apparatus, which include pioneer of lysosomal hydrolase. After combining, lysosomal hydrolase enzymes are activated, and the late endosome turns into active lysosome. In the lysosome, the endocytosis substance is decomposed [105].

Caveolin-mediated endocytosis which starts with a flask-shaped pit in a cavelike membrane is a specific uptake of extracellular molecules. The CPP with its cargo is bound to the membrane, trapped in caveolae which are linked to actinic cytoskeletal elements, and then a protein tyrosine phosphorylation and actin depolymerization are supported. Dynamin is the type of a GTPase liable for endocytosis in the eukaryotic cells that define other actin polymerization on the patch. Finally, cargo-loaded pouches are released inside the cytosol [106]. The various endocytic paths mentioned above have disadvantages such as low specificity, high dependence

Endocytosis of CPPs happens in two stages as endocytic entry and endosomal escape. Endosomal escape is a major step to prevent disruption of the cargo in the lysosomes and to allow the cargo to reach the extra-endosomal aim and to achieve its biological activity. Several models have been proposed to prevent endosomal escapes of CPPs. For instance, as a result of the electrical interaction between the negative charges of the endosomal membrane and the positively charged parts of the CPPs, the membrane that triggers the release of the vesicle content leads to stiffing and rupture. According to the other example, reduced pH develops the capability of CPPs to attach to the endosomal membrane and then raises intracellular circulation; as a result of which, the increment of endocytic vesicles may ensure

Initially the CPPs and the cell membrane interact via electrostatic linkage to proteoglycans called as glycosaminoglycans (GAGs) such as chondroitin sulfate B. Since these glycoproteins are present all over the cell membrane, they make the cell membrane negative charges, and they compose a platform that connects the CPPs or CPP/cargo binding to the cellular matrix. Recent studies have shown that GAGs are less critical for CPP uptake than the previous opinions [93, 103, 108].

The secondary structure of amphipathic and tachycardic peptides, the CPP conformation, and the length of the CPP sequence are very related to cellular uptake

**18**

mechanisms.

Although L amino acid and D amino acid peptides have similar effects against heparin, cell binding affinities of CPPs containing with amino acids in the L amino acid peptides are higher than D amino acid peptides. On the other hand, studies show that using of the D amino acid peptides in terms of increasing cell sensitivity and decreasing enzyme degradation gives better results [93, 101].

### *5.3.5 Role of concentration*

It has been understood from the result of thermodynamic analyses that primary and secondary amphipathic CPPs can pass directly through the cell membrane at low micromolar concentrations, but non-amphipathic CPPs often use endocytosis even at low concentrations [109]. The concentration threshold of direct penetration depends on the type of CPPs, cargo, and cell lines [93].

#### *5.3.6 Role of cargos*

The existence of the cargo could affect the CPP uptake pathway. In addition, dimension and binding methodology are also shown by CPP translocation mechanism.

#### **5.4 Molecular detection of CPPs' cellular uptake**

#### *5.4.1 In cellular assay*

The cellular researches largely propose to follow the CPP and/or the cargo uptake or to elicit the molecular mechanisms of the internalization. As a direct method, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been improved, and quantitatively determination of the amount of intact CPPs in the cells or in the cellular membranes can be provided [110]. Electron microscopy and Raman spectroscopy methods are also other biophysical methods used on cells [111].

#### *5.4.2 Fluorescence-based assays*

The covalent binding of a peptide to the fluorophore and the measurement of the fluorescence (fluorimetry) of the treated cells are the most common methods used to detect both CPP uptake and localization. In this approach, while confocal microscopy provides location information, the probes in the living cells allow indirect quantification of the peptides. This method cannot show the molecular integrity of internalized entities, so it cannot prove whether the peptide is still attached to the fluorescent probe in the cells. Furthermore, fluorescence quenching can cause false-positive outcomes to detect the involvement in intracellular parts and transitory binding areas [94, 96, 111]. Also, CPPs are known as cationic peptides, and they tend to link to the external of the cell membrane and may consequently lead to false-positive results [96]. Fluorescence-activated cell sorting (FACS) is another fluorescence approach used for quantitative measurement uptake of labeled CPPs. The cellular uptake of CPPs is measured by sorting cells which relied on fluorescence intensity by a cell sorter equipment [94]. The advantage of using the aforementioned fluorescence-based protocols in the confocal microscopy process is that it allows distinguishing between internalized and extracellular peptides [94, 96].

#### *5.4.3 Functional assays*

These experiments, which are really suitable for biotechnological and therapeutic applications, can be carried out to determine the biological responses of linked molecules or cargos and to assess their mechanisms of uptake [1, 94]. These systems are based on internalizing antisense oligonucleotides (ONs) and upregulation of luciferase gene expression [96, 112]. One of the functional assays is the splicing redirection assay. Due to elucidation of the mechanism of CPP-based cell internalization, cell lines are transfected by molecular methods with a plasmid carrying a luciferase gene and having their abnormal splice junction. Technically, the pre-mRNA of luciferase is correctly working, producing the expression of the protein whose activity can be utilized by luminescence when the abnormal joint site is blocked by antisense ONs [94, 96, 112]. Another system takes advantage of Cre-mediated recombination of an improved green fluorescent protein reporter gene causing EGFP expression [94, 96]. Since abnormal splicing of the luciferase pre-mRNA inhibits the translation of luciferase, if this junction is masked with antisense ON, translation of luciferase will result in the expression of activity of the protein, which can be assessed by luminescence. For the investigation of CPP-based transfer of different ONs, their internalization systems have been studied with transfected HeLa pLuc 705 cells [96, 112].

#### *5.4.4 MALDI-TOF-MS assay*

This approach is based on the quantification of internalization of peptides in living cells [111]. Furthermore, the quantity of both plasma membrane-bound and internalized peptides is accessible. In this method, an internal standard is needed for the ionization and the detection in MALDI which extremely depends on the structure of the molecule. Information about both the amount and molecular status (degraded or intact) of the internalized peptide can be obtained by using this method. The disadvantage of this method is that no data about the intracellular localization of the peptide can be obtained. Nevertheless this method can get a knowledge about mechanisms of internalization (endocytosis versus direct translocation) and compare the relevant internalization affinities of CPP [96].

#### *5.4.5 Membrane modeling assay*

The assay often prefers to simulate the interaction, internalization, and direct translocation of CPPs through the inert plasma membrane [94, 96]. Membrane models are used in other techniques such as 31P-NMR, small-angle X-ray scattering, calcein or fluorescein-entrapped liposome leakage, and differential scanning calorimetry. These methods are not based on labeling of the CPP sequences with fluorescence agents [1]. Various researches have been performed by using giant or large unilamellar vesicles (GUV and LUV) to examine the translocation qualities of CPPs [96].

**21**

provided the original work is properly cited.

\*, Tayfun Acar1

1 Yildiz Technical University, Istanbul, Turkey

\*Address all correspondence to: brccr87@gmail.com

2 Koc University Hospital, Istanbul, Turkey

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

**6. Conclusion**

**Conflict of interest**

**Author details**

and Zeynep Mustafaeva1

Burcu Ucar1

Unfortunately, the precise mechanism of CPP internalization is still not fully elucidated. CPPs are internalized into the cells by direct or endosomal mechanisms, and these uptake mechanisms have been demonstrated by various molecular techniques. Though many recent studies have provided strong data for translocation of CPPs into cells, a better physical definition of these entry mechanisms in cells by molecular techniques is still an open challenge for the future.

Consequently, this chapter provides a brief manual for anyone in the fields of solid-phase peptide synthesis, peptide vaccines, peptide theranostics, and cell-penetrating peptides. While the solid-phase peptide synthesis is the most convenient way to synthesize the peptides, the need for peptides with longer chain structure and peptides including unnatural amino acids has emerged with the identification of the pathological and physiological functions of the peptides through improvements in the molecular biology, bioengineering, and medical imaging areas. Synthetic chemistry will overcome these challenges, and peptides produced for different purposes, such as drugs, vaccines, and therapeutics, will be the solution for incurable diseases such as lots of epidemic disease and some cancer types with available materials and methods. It has been foreseen that the abovementioned researches can be investigated within the peptide science and technol-

ogy departments to be established academically in the next decade.

The authors declare no conflict of interest.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Pelin Pelit Arayici1

, Melis Sen2

, Serap Derman1

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

Unfortunately, the precise mechanism of CPP internalization is still not fully elucidated. CPPs are internalized into the cells by direct or endosomal mechanisms, and these uptake mechanisms have been demonstrated by various molecular techniques. Though many recent studies have provided strong data for translocation of CPPs into cells, a better physical definition of these entry mechanisms in cells by molecular techniques is still an open challenge for the future.
