## 2 Keratin-Based Nanoparticles
### Wool Keratin-Based Nanoparticles
Similarly, for the treatment of breast cancer, we fabricated DOX-loaded KNPs through ionic gelation and aggregation techniques (DOX-KNPs_IG and DOX-KNPs_Agg, respectively). DOX-KNPs_IG allowed a higher drug loading (up to 30 wt%) and a pH-responsive release; most interestingly, they showed a similar antiproliferative activity compared to free DOX, both against MCF-7 and MDA-MB-231 cells, whereas DOX-KNPs_Agg induced lower antitumoral activity on both cell lines [60]. In 2012, keratin has been grafted with polyethylene glycol in different ratios to obtain three types of keratin-g-PEG copolymers. Indeed, according to the keratin content, ranging from 14 to 48% \(w/w\), the concentrations of the graft copolymers were 0.21, 0.27, and 0.71 mg/mL (\(\text{keratino}_{\text{0.25}}\text{-g-PEG}\), \(\text{keratino}_{\text{0.41}}\text{-g-PEG}\), and \(\text{keratino}_{\text{0.97}}\text{-g-PEG}\), respectively). Preliminary in vitro experiments demonstrated that blank graft copolymers have no cytotoxicity to the MCF-7 cell line, and the relative cell viability remained above 80% under different concentrations of keratin-g-PEG after 24 h incubation. All obtained copolymers were able to self-assemble into nanoparticles in aqueous medium and to load a significative content of DOX (up to 18.1 wt% in \(\text{keratino}_{\text{0.25}}\text{-g-PEG}\)). To increase nanoformulations stability for in vivo administration, KNPs were crosslinked DMSO (thiol oxidation into disulfide bonds). Release experiments revealed that \(\text{keratino}_{\text{0.25}}\text{-g-PEG}\) nanoparticles showed the most favorable release profile under higher GSH levels (10 mM), releasing about 50% of loaded DOX within 12 h [61]. On this basis, [PERSON] et al. grafted polyethylene glycol-40 stearate with keratin to produce redox-responsive polymersomes with a mean diameter of 134 nm and PDI lower than 0.25, and post-loaded with hydrophilic methotrexate (MTX@KerPs) and hydrophobic curcumin (CUR@KerPs) (entrapment efficiencies of 80% and 85%, respectively). MTX is a folate antagonist, interfering with DNA and protein synthesis, and cell replication; instead, CUR is a potent antioxidant and anti-inflammatory agent, suppressing tumor cells proliferation [62]. As shown in Figure 10a, nanocarriers' biocompatibility was investigated through MTT assay on Balb3T3 cells, revealing that cell viability was not compromised at any tested concentration (from 1.65 to 33 \(\upmu\)g/mL). The two different polymersomes were efficiently internalized via endocytosis by HeLa and CHO-K1 cells, exhibiting GSH-triggered release profiles (>80% within nine hours), and thus inhibited HeLa and CHO-K1 cancer cells proliferation (Figure 10b,c). Figure 10: (a) Balb3T3 cell viability after incubation with blank KNPs at different concentrations for 24 or 48 h. (b) HeLa and (c) CHO-K1 cells viability when exposed to blank and drug-loaded KNPs (33 \(\upmu\)g/mL) after 24 and 48 h. Adapted with permission from [62]. Copyright 2021 American Chemical Society. Novel keratin nano- and micro-particles (size ranging from 200 to 800 nm) were prepared from a blend of protein and poly(vinylalcohol) (I:2), followed by a simple and efficient loading of different kinds of drugs and nanotherapeutics: eosin Y, carmine red, methylene blue, curcumin, FITC-albumin, and gold nanoparticles (AuNPs, 5 wt%). In detail, the positive charged methylene blue and AuNPs attained the highest encapsulation efficiency (88% and 93%, respectively), confirming their remarkable affinity for keratin. It is worth mentioning that all these nanoformulations proved the establishment of a strong interaction with mucin, probably due to cysteine residues that confer to keratin tunable mucoadhesive properties. In fact, the loading of neutral hydrophobic entities strongly enhanced the interaction between keratin particles and mucosal tissues as compared to charged molecules [63].