## 2 Keratin-Based Nanoparticles
### Human Hair Keratin-Based Nanoparticles
#### 2.1.1 Doxorubicin-Loaded Human Hair Keratin-Based Nanoparticles  
Over the past years, several research groups have focused their attention on the use of human hair keratin to deliver doxorubicin (DOX), intending to exploit both the intrinsic characteristics of this biomaterial (e.g., biodegradability, biocompatibility, and tumor responsiveness) and its high affinity with DOX. The following section will provide a brief overview of the most relevant results and references on this topic. Tea catechins are a group of polyphenic compounds extracted from green tea leaves known to possess preventive and therapeutic effects against cancer and many other diseases (Figure 7) [45,46]. Figure 6: (**A**) Mass of blood loss and (**B**) time to hemostasis in rat liver puncture and tail amputation models treated with keratin extracts and KNPs (\({}^{\star}\)_p_ < 0.01 and \({}^{\star}\)_p_ < 0.05 compared with the control group, \(n\) = 3). Reprinted with permission from [43]. In 2018, [PERSON] and coworker described a straightforward one-pot method to obtain size-controlled and covalently functionalized keratin-catechins nanoparticles, which were further post-loaded with DOX [46]. Through the setup of a systematic methodology, the authors were able to fine tune the ratio of keratin, catechins, and formaldehyde providing KNPs with the most suitable properties for drug delivery. The authors observed that the size of nanoparticles follows a formaldehyde-dependent concentration trend and argued that the morphological evolution of KNPs formation also depends on formaldehyde, which firstly promotes molecular interactions between keratin and catechins, yielding ultra-small nanoparticles of ca. 10 nm in size; subsequently, these particles act as seeds to continuously grow and finally evolve into spheric and stable nanoparticles. Among the different batches of KNPs, the authors selected the so-called np4 with a hydrodynamic diameter of 67 nm (PDI = 0.121) to test their ability to load DOX, and to kill cancer cells in vitro and in vivo. Their results confirmed that np4 effectively upload DOX, while release studies performed in the presence of GSH and trypsin confirmed that nanoparticles quickly disassembled, providing a nearly complete DOX release (ca. 75%) within six hours. Given the recognized anticancer capability of catechins, a comparative inhibitory study of free DOX and DOX loaded onto np4 (DOX/np4) was performed on colon cancer cell lines HT-29. The resulting IC\({}_{50}\) values were 0.66 mg/mL for DOX/np4 and 0.86 mg/mL for free DOX, indicating a possible synergistic/additive action of polyphenols to the anticancer effect. In vivo anticancer activities of DOX/np4 and np4 were performed and compared with pure catechins and free drug on HT-29 xenografted athymic nude mice models, using different administration routes. Results indicated that intraperitoneal (_i.p._) administration of pure catechins and np4 gives an 18.8% and 63.3% tumor growth inhibition rate, respectively. The tumor growth inhibition rate of intravenously injected (_i.v._) free DOX and DOX/np4 was 47.5 and 78.3%, respectively. Of note, the _i.p._ administration of pure np4 shows a significantly higher anticancer effect than _i.p._ administration of equivalent pure catechins, and higher anticancer effects than _i.v._ injection of free DOX, indicating that the molecular assembly of catechins and keratins into nanoparticles greatly increases the bioavailability and efficacy of tea polyphenols. MSNs@PDA@keratin nanoparticles from [PERSON] and colleagues, discussed in the previous section, were further loaded with DOX, displaying a loading content of 24.5% and an encapsulating efficiency of 71.3%. MSNs-DOX@PDA@keratin showed good pH and GSH responsiveness, as well as selective toxicity against A549 tumor cells as opposed to normal L929 cells (Figure 8), thus representing a promising approach to promote MSN-based drug delivery [26].