## 4 Advances in Nanotechnology towards Development of Nano-Based Therapies
### AuNPs in the Targeted Treatment of Obesity and Cancer
#### 4.1.2 PHB-Targeted AuNPs for Colon Cancer Treatment  
CRC is the second and fourth most commonly diagnosed cancer and ranked the fifth and sixth leading cause of death among South African females and males, respectively [9]. In recent years, the correlation between obesity and CRC has been established, and the relative risk associated with obesity is higher in men than in women [10]. This suggests that the therapeutic agents that are used in the treatment of obesity could also aid in the treatment of CRC.  
In the search for PHB-expressing cell lines for use as in vitro models for AHP targeting, Western blotting and immunocyt techniques were employed. As shown in Figure 4A, the Western blot revealed that only the Caco-2 (colon cancer) and the MCF-7 (breast cancer) cells expressed PHB protein; while the CHO-K1 (Chinese Hamster Ovary), HeLa (cervical cancer), HT-29 (colon cancer), and KMT (skin fibroblast) cell lines did not.  
Figure 2: Biodistribution of AuNPs in obese Wistar rats. Reproduced with permission from [PERSON] [24]. Abbreviations: SQ—subcutaneous, Mes—mesenteric, Epi—epididymal, Retro—retroperitoneal, Peri—perirenal.  
Figure 4: Western blot (**A**) and cytochemistry (**B**) analyses of PHB expression [10] in humans (Caco-2, HeLa, MCF-7, HT-29, and KMST-6) and Chinese Hamster Ovary cells. Expression of PHB was detected only in Caco-2 and MCF-7 cells (**A**). Fluorescent microscopy revealed uptake of AHP-Fluorescein isothiocyanate (AHP-FITC) by Caco-2 cells, indicating expression of PHB on the cell surface. No AHP-FITC fluorescent was observed in MCF-7 cells, which indicates that the PHB receptor is not on the cell surface. Reproduced with permission from Future Medicine Ltd. [10].  
Figure 3: Anti-obesity mechanism of PHB-targeted AuNPs in obese rats. AGK targets and induce endothelial cell death in the WATs, resulting in body weight loss.  
Immunocytochemistry analysis proved that the Caco-2 cells expressed PHB on the cell surface, while the MCF-7 cells expressed it in the cytoplasm (Figure 4B). The Caco-2 cells are human epithelial colorectal adenocarcinoma cells, while the MCF-7 cells are epithelial luminal breast cancer cells [10]. The BRN used the same AuNPs that were used in the obesity studies to test their ability to recognize and bind to the PHB-expressing cells. HT-29 cells are also colon cancer cells and were included as a negative control since they do not express the PHB receptor (Figure 4A). As shown in Figure 5, the treatment of the Caco-2, MCF-7, and HT-29 cells with the four variants of the AuNPs validated the specificity and biocompatibility of the AHP-targeted AuNPs.  
The anti-proliferative activity of AGK was more pronounced on cells that expressed the PHB receptor on the cell surface (Caco-2 cells). The bi-functionalized AuNPs (AGK) induced Caco-2 cell death by KLA-mediated apoptosis [10]. Figure 5 also shows the specificity of the AGK treatment on cancer cells, where it significantly reduced the viability of the cells that express PHB receptor on the cell surface only, with no effect on the cells that express PHB in the cytoplasm and those that do not express PHB. The cAuNPs with AHP (AG) and non-specific peptides lacking the KLA peptide (AGS) and AuNPs with KLA without AHP (GK) were non-toxic to the three cell lines. Although further in vivo investigations still need to be conducted to further elucidate the mechanism of action, these studies served as a proof-of-concept that AHP-targeted AuNPs can serve as effective drug-delivery systems that can potentially be used in the treatment of both obesity and colon cancer [10].