## 2 Keratin-Based Nanoparticles
### Wool Keratin-Based Nanoparticles  
About two million tons of wool are produced yearly, Australia being the major contributor (over 25%), and wool wastes from textile industries have gained a growing relevance as a renewable source of keratin. Indeed, wool consists of up to 95% of keratin that can be obtained from the hairy part of the hide of sheep, goats, pigs, rabbits, and camelids [8]. To reduce the environmental impact of textile waste, several keratin extraction procedures have been applied, and the extracted protein has found wide application in the drug delivery field. It is worth noticing that wool-derived keratin seems highly compatible with human biological systems, possessing the same key cell-binding motifs of human keratin that are crucial to support and promote cellular adhesion [55]. In 2013, [PERSON] et al. demonstrated, for the first time, the feasibility of producing biocompatible keratin-based microparticles starting from wool wastes, and further confirmed particles' safety on human monocytic cell line [56]. In detail, the authors prepared an aqueous solution of regenerated keratin (RK) with a final concentration of 4.5% \(w/w\), via sulfitolysis procedure. The FTIR analysis revealed that RK was mainly organized in the disordered \(\alpha\)-helix structure, thus resulting in a remarkable water-solubility. After acidic treatment (pH 3.5) followed by washing with water, the RK solution was spray-dried, providing microparticles with a mean diameter of 6 um, a narrow size distribution, and an irregular and smooth surface. Overall, the in vitro biocompatibility assays, performed on human monocytic cell lines at increasing microparticle concentrations (from 1 ug/mL up to 1000 ug/mL for 24 or 72 h), confirmed the particles' safety. Based on this evidence, various straightforward and water-based procedures have been envisioned which are aimed at obtaining KPNs with tunable morphological, physical, and chemical properties. As previously mentioned, the sulphitolysis procedure allows for the obtainment of water-soluble and full-length keratin, that, most importantly, preserves the naive protein primary and secondary structure. In the past few years, several kinds of antitumoral therapeutics (chlorin e6, paclitaxel, doxorubicin, and 9(_R_)-9-hydroxystearic acid) have been loaded into keratin nanocarriers. In this regard, our research group described, for the first time, the covalent attachment of the photosensitizer chlorin e6 (ce6) on the keratin backbone via EDCI/NHS coupling, followed by nanoparticle formation, e.g., KNPs@Ce6, by self-assembly and desolvation techniques [57]. Both the self-assembly and desolvation methods provided stable nanoparticles with comparable hydrodynamic diameters (148 vs. 147 nm) and Ce6 loading ratios (23 vs. 45%), with a monodisperse distribution (PDI: 0.17 vs. 0.13), a negative zeta potential (\(-\)53 vs. \(-\)68 mV), and with a final yield of 24% and 30%, respectively. The loading of Ce6 on KNPs@Ce6 suspensions (0.1 mg/mL) ranged between 35 and 38 ug/mg of keratin, and was confirmed by UV-vis spectroscopy, displaying a broadened Soret band (400 nm) and a bathochromic shift from 650 nm to 665 nm of the Q-band. After a 24 h incubation period at different concentrations (e.g., 0.5, 2.5, 5.0 ug/mL), KNPs@Ce6 showed a superior internalization compared to free Ce6 on osteosarcoma (U2 OS) and glioblastoma (U87) cell lines, and upon light irradiation they produced a large amount of reactive oxygen species (ROS), triggering cell death in a concentration and irradiation-time dependent manner. We further exploited KNPs for delivering paclitaxel (PTX), an anticancer agent widely used in breast and ovarian cancers. KER-NPs-PTX were tested against human breast cancer MCF-7 and MDA MB 231 cell lines in two-dimensional (2D) cultures and in a perfused three-dimensional (3D) model [58]. Primarily, keratin showed an extraordinary PTX loading ratio up to sevenfold higher as compared to those of albumin, e.g., 43% vs. 6.5% (_w/w_).