**6. Conclusion**

*Peptide Synthesis*

*5.4.3 Functional assays*

transfected HeLa pLuc 705 cells [96, 112].

*5.4.4 MALDI-TOF-MS assay*

*5.4.5 Membrane modeling assay*

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].

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

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].

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].

**20**

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 technology departments to be established academically in the next decade.
