# Nanoformulation of Peptides for Pharmaceutical Applications: In Vitro and In Vivo Perspectives
## 2 Overview of Peptides
### Membrane Filtration  
Membrane filtration is one of the prevalent purification methods. UF is an efficient and low cost purification technique to separate peptides with distinct molecular weight from crude hydrolyses [70]. Most studies identified that peptides with less than 3 kDa molecular weights exhibited the greatest antioxidant activities. For instance, [PERSON] et al. (2016) compared sandfish protein hydrolyses (SPH) with four different degrees of molecular weights (>10 kDa, 5-10 kDa, 5-5 kDa, and <3 kDa), which are separated using UF. The study showed that the protein hydrolysate with a molecular weight of <3 kDa showed a higher degree of hydrolysis (DH) and the highest DPPH radical scavenging activity [71]. Further, [PERSON] et al. (2007) compared the performance of UF with two different molecular weight cut-offs (MWCO) and NF. UF with high MWCO (20 kDa) is suitable for the separation of a peptide and a non-hydrolyzed protein, while UF with intermediate MWCO (4 and 8 kDa) is more befitting for peptide fractionation to the satisfactory flux and retention. The NF membrane is efficient for concentrated peptides for both the flux and recovery rate. The disadvantage of NF is a possible risk of a high salt concentration and, hence, an additional desalination step is required [72]. Furthermore, [PERSON] et al. (2000) demonstrated the effect of fractionation using NF membranes on the peptide distribution by comparing tryptic (TH) and chymotryptic (CH) hydrolyses with whey protein isolates. The study revealed that the peptides from TH with a lower negative surface charge is permeable, compared to the positive and neutral charged peptides. In addition, no significant changes in the CH \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Method** & **Advantages** & **Limitation** & **References** \\ \hline Enzymatic hydrolysis & Formation of specific chemical bonds, suitable for reverse-phase chromatography & Long sample treatment time (5–12 h) and low analyte recovery & [67] \\ \hline Microbial fermentation & Few unexpected metabolites, lower energy consumption, avoids degradation by endogenous proteases, improves productivity & Fails to induce biological effects in the consumer and poor reproducibility, highly dependent on purification and identification & [68] \\ \hline Chemical synthesis & Less expensive; requires being non-expensive & Production of intermediates, lengthy process, insolubility of the resultant peptides & [69] \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Advantages and limitations of protein/peptide extractions and formation methods. fractionation due to the lower content in each peptide sequence and, hence, permeation or retention of CH, were always missing, and the sign of the charge was not in accordance with the peptide transmission. Surface charges and properties of different short peptides may affect the intensities of both the convective and electro-migrative fluxes across NF membranes, which can later lead to poorer separation between the cation and counterion. Thus, the study emphasized that an inaccurate result and high purification cost is due to the fouling issues in the filtration membrane [73]. High Performance Liquid Chromatography  
High-performance liquid chromatography (HPLC) is preparative and extremely flexible for the purification of a wide range of peptides. The three separation modes of HPLC are size exclusion HPLC (SEC), ion exchange HPLC (IEX), and reverse phase HPLC (RP-HPLC). SEC separates peptides based on the size differences of the peptides and IEX allows fractionation to occur based on the net surface charges of the peptides. The RP-HPLC isolates the peptides according to their hydrophobicity [74]. SEC is also known as gel permeation chromatography/gel filtration chromatography, where fractionation is based on the molecular size or molecular weight [75]. Sephadex is a common material used in the stationary phase; it is available on the market.