## 2 Keratin-Based Nanoparticles
### Chicken feathers Keratin-Based Nanoparticles
[PERSON] and collaborators designed and prepared reduction/pH dual-responsive keratin-based nanoparticles as a delivery system of DOX. In detail, DOX was covalently conjugated onto pegylated keratin through an acid-sensible hydrazone moiety or a reductive cleavable linker, allowing the tumor microenvironment-triggered drug release [73; 74]. Notably, in vitro release studies demonstrated a sustained and cumulative prodrug leakage up to 43% within 107 h in an acidic/reductive environment, while a negligible amount of free DOX was released under physiological conditions. After longer incubation time, prodrug-nanoparticles demonstrated a similar or enhanced antitumor efficacy compared to free DOX in HepG2 human liver and MCF-7 breast cancer cells, probably due to the long-term release in tumor intracellular microenvironment. Recently, keratin has been also investigated as a platform template for both MRI and therapeutic application [72]. Firstly, metal ions (e.g., Mn\({}^{2+}\) and Gd\({}^{3+}\)) were sequenced onto protein's backbone by exploiting interaction with thiol and carboxyl moieties. Then, a simple and straightforward mixing procedure provided monodisperse metal oxide nanoparticles, MnNPs@Keratin, and GdNPs@Keratin with an average size of \(\sim\)5 nm and \(\sim\)8 nm by TEM observation and a hydrodynamic diameter of \(\sim\)12 nm and \(\sim\)25 nm by DLS analysis. The successful loading of metal ions was confirmed through inductively coupled plasma atomic emission spectrometry (ICP-AES), indicating that the Mn element content in MnNPs@Keratin was 1.3%, and the Gd in GdNPs@Keratin was 3.8%. The _T1_ relaxivity (_r1_ value) of MnNPs@Keratin and GdNPs@Keratin reached up to 6.8 mM\({}^{-1}\) s\({}^{-1}\) and 7.8 mM\({}^{-1}\) s\({}^{-1}\), respectively, demonstrating concentration-dependent MRI performances. It is worth noting that the _T1_-weighted relaxivity of MnNPs@Keratin dramatically increased under acid and reducing conditions, due to the release of Mn\({}^{2+}\) after the reaction between MnO\({}_{2}\) and acids or GSH. Notably, at concentrations below 250 ug mL\({}^{-1}\), MnNPs@Keratin displayed nearly 100% viability in breast cancer 4T1 cells and a hemolysis percentage of 1.16%, while GdNPs@Keratin exhibited above 80% viability at a concentration of 1000 ug mL\({}^{-1}\) and a hemolysis percentage of less than 5% at 250 ug mL\({}^{-1}\), thus demonstrating low cytotoxicity and a negligible hemolytic effect of both formulations. The in vivo biodistribution of nanoparticles was investigated by measurement of the Mn and Gd content in the main organs of mice, indicating that, at seven days post-injection, most of the elements were mainly found in the liver, and almost all of the nanoparticles were gradually excreted at 15\({}^{\circ}\) day. The in vivo MR imaging on 4T1 tumor-bearing mice revealed an impressive MRI performance, and MnNPs@Keratin showed a maximum _T1_-weighted after 2 h of administration, thus offering a suitable time window for the observation via the MR scan. Based on these promising data, the authors loaded DOX into the metallic oxide nanoparticles by a simple and straightforward mixing procedure, showing excellent drug loading efficiency and capacity (86.8 and 8.7% for MnNPs@Keratin, and 90 and 9% for GdNPs@Keratin, respectively). In vitro and in vivo studies on 4T1 breast cancer cells confirmed the nanoparticles' remarkable MRI performance, a pH and redox-dual responsive DOX release profile, and a significative reduction in tumor growth, thus making these nanoparticles promising candidates for theranostic applications (Figure 12). Biodegradable KNPs made from chicken feathers have also been exploited to absorb, on their surface, antimicrobial metal ions, e.g., silver, copper, and zinc, aimed at inducing an antimicrobial effect against Gram+ and Gram- bacteria. All KNPs metal ion complexes showed an average hydrodynamic diameter of about 80 nm and demonstrated a strong bactericidal activity against foodborne pathogenic bacteria, _E. coli_ and _L. monocytogenes_[76].