*5.3.2 Peptide secondary structure*

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

**19**

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

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

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

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

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

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

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

and decreasing enzyme degradation gives better results [93, 101].

depends on the type of CPPs, cargo, and cell lines [93].

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

biophysical methods used on cells [111].

*5.4.2 Fluorescence-based assays*

*5.3.3 Role of arginine-rich residues*

uptake [93, 104, 106].

*5.3.4 Role of chirality*

*5.3.5 Role of concentration*

*5.3.6 Role of cargos*

*5.4.1 In cellular assay*

mechanism.

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