# Nanoformulation of Peptides for Pharmaceutical Applications: In Vitro and In Vivo Perspectives
## 4 In Vitro Analysis of Nanoformulated Peptides  
Recently, various peptide encapsulated nanoparticles were developed for anti-tumor applications with less/no toxicity toward normal or noncancerous cells, as listed in Table 3. [PERSON] et al. (2007) synthesized nucleolin-encapsulated fluorescent silver nano clusters. Nucleolin acted as a protective layer to prevent silver clusters from rapid oxidization. In the study, the fibroblast cell line was utilized to observe the delivery of the encapsulant. The results revealed that the nucleolin can enter the cells via the nuclear membrane for a strong nuclear staining application [109]. Likewise, [PERSON] et al. (2013) developed chitosan nanoparticles, which are encapsulated by siRNA for anticancer applications. The resultant siRNA was identified to silence the overexpression of the anti-apoptotic Bcl-2 gene. The size ranges of the siRNA-encapsulated chitosan nanoparticles were identified to be 190-340 nm. The study showed that the encapsulation efficiency of the chitosan nanoparticles was ~80%. The cytotoxicity results showed that the cell viability was 95% after 48 h. After 72 h, 85% of cells were viable, which revealed that the encapsulated nanoparticles do not exhibit toxic reactions toward noncancerous cells [110]. Further, [PERSON] et al. (2012) compared the anticancer activities of the nano- and micro-sized formulations of a peptide drug named ellipticine, which is an anticancer drug. The particle size distributions of the peptide formulations were in the range of 40-300 nm due to the agglomeration of particles. The cytotoxicity results demonstrated that nanoformulated peptides possess enhanced cancer cell inhibition properties toward the human carcinoma cell line (A549) after 24 h via improved endocytosis-mediated cellular uptake [111]. Furthermore, [PERSON] et al. (2013) prepared a novel nanoemulsion with a peptide containing essential oil extracted from _Dodonaea angustifolia_ Miers (DA) and _D. brasiliensis_ Miers (DB). Transmission electron microscope (TEM) results showed homogeneous droplets of nanoemulsions with sizes less than 200 nm. The cytotoxicity analysis was performed toward human glioma (U-138 MG) and human bladder carcinoma (T24) cell lines. The results revealed that the essential oil from both DA and DB reduced the cell viability at a 500 \(\upmu\)g mL\({}^{-1}\) concentration, while the nanoemulsion containing 4% essential oil possesses the ability to reduce the cell viability at 250 \(\upmu\)g mL\({}^{-1}\). Thus, nanoemulsion has been identified to act as a promising candidate for cancer treatment at lower concentrations, compared to free essential oils [112]. Moreover, [PERSON] et al. (2013) synthesized doxorubicin (DOX)-coated gelatin nanoparticles, which are encapsulated with a specific peptide for targeting breast cancer cells. DOX is a chemotherapy drug that leads to certain side effects, such as vomiting, diarrhea, eye redness, and darkening of the skin. The goal of the study is to minimize the side effects of DOX via controlled drug delivery into cancer cells. Three cell lines were used for the in vitro analysis of the novel formulation, namely MCF7 mammary adenocarcinoma cells, 4T1 mouse mammary carcinoma cells, and 3T3 mouse fibroblasts. The results showed that the viability of tumor cells decreased to 50% after 5 h due to the targeted delivery of DOX. Meanwhile, the increase of the 3T3 mouse fibroblasts cell growth indicate that the peptide drug possesses the ability to selectively inhibit the tumor cells [113]. [PERSON] et al. (2009) successfully coated the (AG)-30 angiogenic peptide on gelatin nanoparticles (AG-30/gelatin NPs) to be utilized for the treatment of ischemic diseases. The cytotoxicity analysis of AG-30/gelatin NPs using HAECs (human aortic endothelial cells) and HASMCs (human aortic smooth muscle cells) revealed that the AG-30/gelatin NPs possess dose-dependent toxic effects [114]. Further, [PERSON] et al.