## 2 Keratin-Based Nanoparticles
### Human Hair Keratin-Based Nanoparticles  
Human hair is considered a useless material, being usually discarded as waste. In rural and in urban, highly populated areas, tons of hair are either disposed in the land or collected in the waste streams, posing several issues. In fact, due to its composition and slow degradation rate, hair remains for a long period of time, contributing to an increased nitrogen concentration in soil and water. At the same time, if disposed by incineration, hair produces toxic gases such as ammonia, carbonyl sulfides, hydrogen sulfides, sulfur dioxide, phenols, nitriles, pyrroles, and pyridines, that significantly increase pollution [22]. However, the unique properties of human hair, such as chemical composition, slow degradation rate, high tensile strength, thermal insulation, elastic recovery, and exceptional interaction with water and oils, make it a valuable material for different applications, such as production of fertilizers, hair extensions, musical instruments, hair brushes, etc. [10]. Besides the use of human hair per se, keratin, representing the hair's major constituent, has attracted increasing attention as a biomaterial for biomedical applications [7; 23]. In 2016, [PERSON] et al. studied the use of keratin extracted from hair, under reductive Shindai's conditions [24], that was further covalently functionalized with epigallocatechin (ECGG) trough formaldehyde bridges [25]. The effective EGCG-keratin conjugation was confirmed by the authors through different spectroscopic techniques, e.g., UV-Vis and IR spectroscopy, NMR (Figure 1), and XPS. EGCG-functionalized keratin was then assembled to form spherical nanoparticles, namely NANO, with a hydrodynamic diameter of 50 nm and a polydispersity index of 0.12, as potential antioxidant and radical scavenging materials. The scavenging ability of NANO was assessed by the authors with two different probes, e.g., 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and 2,2-azino-bis(3-ethylbenzothazo line-6-sulfonic acid) (ABTS). At the highest concentration, free EGCG, vitamin C, and NANO showed a comparable DPPH\({}^{\bullet}\) scavenging ability (ca. 93%) (Figure 2a); conversely, the ABTS5\({}^{\bullet+}\) scavenging rate of NANO was lower than free EGCG but much higher than vitamin C (Figure 2b). Indeed, at a concentration of 7.5 \(\upmu\)g/mL, the scavenging rate of NANO, EGCG, and vit C was 93.6%, 100%, and 67.1%, respectively. The toxicity of NANO on cell proliferation was evaluated on mouse fibroblast (L929) and on human umbilical vein endothelial (HUVEC) cell lines, showing that they are non-toxic for a concentration of up to 20 mg/mL. The authors also investigated the protective effect of NANO towards macrophages, indicating that nanoparticles efficiently reduce  
Figure 1: Comparative (**a**) UV-Vis spectra and (**b**) FTIR spectra of NANO, free EGCG, and keratin. (**c**) \({}^{1}\)HNMR spectra of EGCG and (**d**) of NANO. Reprinted and adapted with permission from [25]. Copyright 2021 American Chemical Society. excesses of reactive oxygen species (ROS) by increasing the activity of intracellular antioxidant enzymes, such as superoxide dismutase (SOD), and GSH. Importantly, keratin-EGCG nanoparticles displayed good anti-inflammatory effects by reducing the release of cellular nitric oxide (NO) and pro-inflammatory factors (TNF-\(\alpha\) and IL-1\(\beta\)), thus reducing the lipopolysaccharide-mediated inflammation. Overall, the work by [PERSON] and coworkers demonstrates that human hair keratin-EGCG nanoparticles are promising systems for the protection of cells from inflammation due to imbalanced ROS/NOS production. Human hair keratin intrinsic responsiveness to pH, GSH, and trypsin was exploited by [PERSON] and colleagues to fabricate drug carriers where keratin/poly-dopamine (PDA) complexes functioned as gatekeepers of mesoporous silica nanoparticles (MSNs) [26]. In more detail, PDA was self-polymerized and complexed with iron (III), followed by conjugation with keratin.