**5. Established methods for peptide loaded NPs or conjugated biopolymers preparation**

We have mentioned that the peptides alone cannot produce an adequate immune response and also have poor stability with the internalization problem while crossing cell membranes. To solve all these limitations, peptides are loaded nanoparticle systems or conjugated biopolymers. Biopolymers are generally nontoxic products are generally preferred for producing continuously release systems with long term effect [34]. Here, the applicable and most common strategies for the synthesis of peptide-based NPs and encapsulation or conjugated methods of biopolymers are shown.

#### **5.1 Emulsification-solvent evaporation method**

The emulsion solvent evaporation technique is known as the most successful and useful method in the preparation of peptide loaded NPs and this technique is studied under two groups as single and double emulsion solvent evaporation methods [35].

#### **5.2 Conjugation methods**

Conjugation is a technique for achieving peptide and biopolymer complexes. The covalently linked peptide biopolymer conjugates can be linked using the water-soluble carbodiimide method as a cross-linker and synthesis with microwave energy methods [36]. Peptides conjugates biopolymers can be synthesized in organic media using microwave energy. Also, there are another methods, including complex formation of biopolymers and peptides and electrostatic complex formation and metal coordination via ion coordination [35]. Specific antibody titers were observed in mouse experiments against peptides containing polymeric conjugates and complexes. The molecular weights of these conjugates are also very important. Biopolymer conjugation is crucial to obtain a high immune response to antigens at low molecular weights [37–39].

## **5.3 Nanoprecipitation**

Nanoprecipitation is the most strategic method for the preparing of vaccine prototypes. Reducing the pH is very important to stabilization of system. Also, salt concentration under the solubility conditions is another important thing for the encapsulation method [70]. If the experiments cannot move on then adding a non-solvent phase in the quality of the solvent technique in which the parent compound of the NPs is dissolved can help [40]. Nanoprecipitation is frequently used in encapsulation of peptides. A pH-controlled precipitation rather than a non-solvent precipitation is a more preferred approach for passing the polymer to a non-dissolved phase with a simple pH change in the medium. For NPs or biopolymers prepared by nanoprecipitation, these solvents are known as the organic phase of acetone and ethanol [41].

## **5.4 Encapsulation of peptide**

Encapsulation is carried out simultaneously by synthesizing NPs and biopolymers in all of the methods mentioned the encapsulation method for peptides should be selected based on the hydrophobic or hydrophilic facilities of peptide. Using of peptide encapsulation is important because of


#### **5.5 Peptide characterization**

After purification of the peptides, they are commonly characterized by liquid chromatography-electrospray ionization-mass spectrometry (LS-ESI-MS), fluorescence spectroscopy and possible three-dimensional structures of the synthetic peptide (PEP-FOLD) server. It has validation since the chromatographic method has positive properties in terms of linearity, accuracy, precision and repeatability. Synthetic peptide vaccines are immunogens that can be used when creating vaccine

**33**

*New Generation Peptide-Based Vaccine Prototype DOI: http://dx.doi.org/10.5772/intechopen.89115*

is measured via size-exclusion chromatography (SEC) [44].

morphological examination of Polymers or bioconjugate [45].

**5.6 Characterization of peptide vaccine prototype**

**5.7 Toxicity studies**

prototypes, especially because of their lipophilic structure (which also allows cell permeability to pass easily) [43]. Methods such as Fourier transform infrared (FT-IR) and nuclear magnetic resonance (1H- and 31P-NMR) are frequently used to visualize the physical structure of the copolymer and peptide biopolymer conjugates and to perform characterization studies. The conjugation of molecular weights

Ultraviolet (UV) and FT-IR Spectrophotometers and ZetaSizer are used for studying the nanoparticles and Scanning Electron Microscope (SEM) is used for

Peptide-based vaccine prototypes need to be tested in a cell culture medium to be feasible because they may have physiological, biological and chemical effects, causing cytotoxicity. The method used to investigate the cytotoxic profiles of peptide-based vaccines is also called *in vitro* cytotoxicity assays or cell culture-based measurement methods [46, 47]. Tetrazolium salts are compounds used in cell lines to measure the metabolic pathways of cells of microbial origin. Tetrazolium salts are the heterocyclic organic structure of these compounds and their reduction to colorless or weak colored aqueous solutions known as formazans has been the basis of their use as vital dyes in redox chemistry, biological and chemical applications [46, 47]. The tetrazolium ring can only be broken by active mitochondria, so viable cells and dead cells can be distinguished by discoloration. The fact that this change can be made only by living cells in vitro has made tetrazolium compounds a highly biologically important to measure toxicity of peptide-based vaccine formulas. The mechanism of toxicity assays, such as 3-(4,5-dimethylthiazolyl)-2,5 diphenyltetrazolium bromide (MTT) [19], 3-(4,5-dimethylthiazol-2yl)-5-(3 carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), 2,3-bis(2 methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT), sodium 5-(2,4-disulfophenyl)-2-(4-iodophenyl)-3-(4 nitrophenyl)-2Htetrazolium inner salt (WST), 5-methyl-phenazinium methyl sulfate(PMS), 5-[3-(carboxymethoxy)phenyl]-3-(4,5-dimethyl-2-thiazo-

lyl)-2-(4- sulfophenyl)-2H-tetrazolium inner salt; (MTS) are used [49, 50] and in our studies, we generally use MTT analysis. For example, our technological vaccine prototype example is Zika peptide loaded PLGA nanoparticles which were determined on ECV304 human epithelial cells via MTT assay, which is the cytotoxicity test, was performed to determine the cytotoxic effects of the peptide, peptide loaded NPs [45]. The importance of toxicity studies is to determine the non-toxic

Live attenuated vaccine is highly immunogenic and considered as well-tolerant for healthy individuals. However, live attenuated vaccine should not be administered to immunocompromised individual as it would cause systemic infection. An alternative vaccine technology, subunit vaccine, is safer and more suitable for immunocompromised individual. It uses fragment of a pathogen (antigen) to trigger an immune response and stimulate immunity against the pathogen. However,

vaccine prototype and to switch to in vivo animal studies.

*5.8.1 Liposome based subunit vaccine*

**5.8 Contemporary advancements in peptide based vaccine**

*New Generation Peptide-Based Vaccine Prototype DOI: http://dx.doi.org/10.5772/intechopen.89115*

prototypes, especially because of their lipophilic structure (which also allows cell permeability to pass easily) [43]. Methods such as Fourier transform infrared (FT-IR) and nuclear magnetic resonance (1H- and 31P-NMR) are frequently used to visualize the physical structure of the copolymer and peptide biopolymer conjugates and to perform characterization studies. The conjugation of molecular weights is measured via size-exclusion chromatography (SEC) [44].

#### **5.6 Characterization of peptide vaccine prototype**

Ultraviolet (UV) and FT-IR Spectrophotometers and ZetaSizer are used for studying the nanoparticles and Scanning Electron Microscope (SEM) is used for morphological examination of Polymers or bioconjugate [45].
