## 2 Keratin-Based Nanoparticles
### Wool Keratin-Based Nanoparticles
Moreover, KER-NPs-PTX exerted effective anticancer activities on both 2D and 3D models, leading to a significant increase in the expression of the proapoptotic \(BAX\) gene and cleaved caspase 3 (CC3) protein, and an increment in apoptotic cell percentages, following 24 h treatment. Although monotherapy remains a widely used approach for cancer treatment, its application is strictly limited by severe toxicity, as well as the development of resistance and recurrence phenomena. Therefore, a bimodal therapy combining photo- and chemotherapy could represent an encouraging strategy to overcome these limitations by reducing the effective drug dose and synergistically targeting different pathways. In this regard, we employed keratin for preparing nanoparticles co-loaded with PTX and Ce6 via drug-induced aggregation and desolvation procedures (PTX-Ce6@kerag and PTX-Ce6@kerag) as an innovative approach for the pharmacological treatment of osteosarcoma (OS) [18]. Both methods provided stable nanoparticles with a negative zeta potential (around \(-\)46 mV). As expected, PTX-Ce6@ker\({}_{\text{ds}}\) resulted as smaller and more mono-disperse than PTX-Ce6@kerag (hydrodynamic diameter 120 vs. 150 nm, and PDI 0.067 vs. 0.22, respectively), most probably due to the cross linking mediated by glutaraldehyde. In vitro cytotoxicity assays performed on three osteosarcoma cell lines (MG63, SaOS-2, U-2 OS) indicated that PTX-Ce6@kerag displayed a pharmacokinetic profile similar to free PTX, resulting in a significant decrease in cell viability. On the other hand, PTX-Ce6@kerd\({}_{\text{ds}}\) achieved the same cytotoxic effect at higher concentrations; indeed, the crosslinking process may affect the release of PTX, strongly reducing its efficacy. Therefore, PTX-Ce6@kerag was selected as the nanoformulation more suitable for further in vitro investigations both in 2D and 3D OS models. Remarkably, cells loaded with PTX-Ce6@kerag, at a final Ce6 and PTX concentration of 3.35 mM and 6.52 mM, respectively, upon light irradiation, showed a dramatic decrease in cell viability, as well as in chemoresistant SaOS-2 cells and in 3D OS models, thus accounting for an additive action of chemotherapy and photoactivation (Figure 9). 9(R)-9-hydroxystearic acid (9 HSA) is an endogenous lipid, known to inhibit histone deacetylase (HDAC1) at micromolar concentration. 9-HSA induces a cell cycle arrest in the G0/G1 phase and activates the p21 WAF1 gene, therefore representing a promising and non-toxic option for cancer treatment. However, 9-HSA is poorly water-soluble and its encapsulation within a nanocarir could improve its pharmacokinetic and pharmacodynamic profile. To this end, our group exploited the high affinity of keratin towards hydrophobic molecules to produce 9-HSA KNPs (9R@ker) through an in-water drug-induced aggregation method. Optimization studies allowed for the establishment of the optimal parameters for obtaining stable and highly reproducible NPs with an average diameter of 162 nm (PDI! 0.2), a negative zeta-potential of \(-\)43 mV, and a drug loading of 10 wt%/kerwt, reaching a higher 9R loading ratio with respect to those obtained with hydroxyapatite, e.g., 10 vs. 8.6%. Therefore, 9R@ker nanoparticles were investigated for in vitro antitumor activity compared with free 9R and 9R albumin-loaded NPs towards three human colon cancer cell lines, i.e., HT29, Caco-2, and HCT116. In vitro antiproliferative data indicated that 9R@ker induced a higher antiproliferative effect than the free drug, especially on HT29 cells (49 vs. 26%). Noteworthy, is that after incubation with nanoparticles, HT29 cells showed an increased cell membrane fluidity, and a greater intracellular ROS production, leading to cell death due to the unexpected S phase cell cycle arrest (25% increase as compared to the control) [59]. Figure 9: Impact of PTX-Ce6@kerag on 2D and 3D OS tumor model. Reprinted under Creative Common CC BY license from [18]. *** \(p\)! 0.001.