## 2 Keratin-Based Nanoparticles
### Human Hair Keratin-Based Nanoparticles
#### 2.1.1 Doxorubicin-Loaded Human Hair Keratin-Based Nanoparticles
Figure 7: Chemical structures of (+)-Catechin and four major green tea catechins: (-)-Epicatechin, (-)-Epigallocatechin, (-)-Epicatechin gallate, and (-)-Epigallocatechin gallate (EGCG). In a previous paper, the same authors further deepened the production of DOX-loaded human hair KNPs with high GSH and pH responsiveness [47]. As opposed to other literature reports, this work describes the preparation of KNPs from reduced human hair keratin in the presence of H2O2. As claimed, this procedure allows for nanoparticles' stabilization by a \"green\" self-crosslinking approach that encompasses the oxidation of thiols to disulfide bridges. Post-loading with DOX provided KNPs of around 130 nm with a 11.5% loading content and an encapsulation efficiency of 49.4%. The authors further demonstrated that, under increasing GSH concentrations, the zeta potential of KNPs changed from negative (\(-\)9.5 mV) to positive (4.86 and 5.1 mV), and their size significantly increased, indicating a marked disassembly. In vitro cytotoxicity studies revealed that KNPs loaded with DOX reduced tumor cells viability (A549) in the low micromolar range, despite being less potent than free DOX. On the same topic, [PERSON] and co-workers reported the preparation of keratin-PDA conjugates for the production of biodegradable nanoparticles carrying DOX [48] by exploiting the ability of PDA to polymerize under mild conditions. For their work, the authors extracted keratin from hair in the form S-carboxymethyl keratin that was subsequently reacted with dopamine under EDC-HCl/NHS standard conditions. Afterward, the dopamine pendant of the synthesized conjugate was self-polymerized under mild alkaline conditions to afford blank keratin/PDA nanoparticles, which were further loaded with DOX through electrostatic interaction and hydrogen bonding. DOX-loaded nanoparticles displayed good pH and GSH responsiveness and a higher inhibitory effect on tumor cells than normal cells. [PERSON] and collaborators described the use of human hair keratin for the production of pH-sensitive DOX-loaded nanoparticles (KDNPs) able to release the drug at the tumor site [19]. To this purpose, S-carboxymethyl keratin was extracted from human hair and modified with iodoacetic acid; afterwards, keratin-DOX conjugate was prepared into nanoparticles by desolvation using genipin as a green cross linker [49]. Release of DOX from KNPs was carried out at pH 7.4 and pH 5.0 to simulate a normal physiological condition and the acidic tumor microenvironment, respectively, showing that the cumulative release of DOX over a period of 270 h was 5% and 78% at pH 7.4 and at pH 5.0, respectively. The authors claim that the pH-sensitive release characteristics of KDNPs were mainly ascribable to the break of the hydrazone bonds between keratin and DOX at pH 5.0, resulting in an enhanced release. In vitro cytotoxicity studies on A549 cells showed that the IC50 of free DOX (0.8 ug/mL) was considerably lower than that of KDNPs (5.0 ug/mL), probably due to the faster uptake of free DOX into 2D cells system. Indeed, in vivo evaluation showed that animals in the KDNPs group had higher body weight, indicating lower toxicity, whereas tumor volume and weight for the free DOX and control groups were both larger and higher than those of the KDNPs group, demonstrating a better in vivo performance of KDNPs as opposed to free DOX both in terms of systemic toxicity and antitumor efficacy. Figure 8: Toxicity of MSNs-DOX@PDA@keratin on L929 cells (**a**); toxicity of MSNs-DOX@PDA@keratin and free DOX against A549 cells (**b**). Keratin-hyaluronic acid nanogels were reported by [PERSON] and collaborators as drug carriers with high a DOX content and targeting ability, thanks to the presence of hyaluronic acid [50]. The CD44-targeted nanogels displayed greater DOX tumor uptake as opposed to the free drug and high tumor microenvironment responsiveness.