**3. Silk biocompatibility**

Silk fibroin is an attractive material for numerous biomedical applications as, due to its mechanical and physicochemical properties, it encompasses applications such as drug delivery, tissue engineering, and implantable devices. However, in addition to the functionalities necessary for specific applications, a key factor necessary for the clinical success of any biomaterial is the appropriate in vivo interactions with the body or biocompatibility. Among them, (i) the immune and inflammatory response and (ii) the biodegradability can be studied.

#### **3.1 Immune and inflammatory response**

As already mentioned, silk fiber is essentially made up of two proteins, fibroin and sericin. While fibroin is highly biocompatible [3, 4] with a low immune response [35, 36], sericin can present unwanted adverse allergic reactions [37, 38]. For this reason, sericin is normally removed by different procedures, known as degumming [39]. Depending on the format of the material and the location of implantation, silk fibroin can induce a mild inflammatory response that diminishes within a few hours/days after implantation [40]. The response involves the recruitment and

activation of macrophages and may include the activation of a mild foreign body response with the formation of multinucleated giant cells, again depending on the format of the material and the location of implantation [36]. The number of immune cells decreases with time, and granular tissue, if formed, is replaced by endogenous non-fibrous tissue, although these responses are reserved for films, hydrogels, and bone implants [36].

The study carried by Meinel et al. [40] indicated that collagen films implanted in rats produce a greater inflammatory reaction in the tissue than equivalent films prepared with fibroin after 6 weeks. In another study comparing fibroin membranes and poly (styrene) and poly (2-hydroxyethyl methacrylate) membranes, Santin et al. [41] demonstrated that the former has a milder immune response than the latter. The results indicated that lower levels of fibrinogen were bound to the fibroin membrane than to the two synthetic polymers, while the same amounts of C3 human plasma complement fragment and adsorbed IgG were detected. The activation of mononuclear cells by fibroin, measured as production of interleukin 1β, was lower than that of synthetic materials. Another study indicated that the braided silk fibroin used for the reconstruction of the anterior cruciate ligament produces a mild inflammatory response after seven days of implantation in vivo, while an equivalent implant made with the biodegradable polymer polyglycolic acid (PGA) produced a more acute response [42]. In this case, although the breaking load for the PGA implant was twice that for the fibroin graft, the initial attachment and growth of cells in the prosthetic ligament was higher in the latter.

In the case of silk fibroin nanoparticles, the literature is not as extensive as for other formats of the same material. Tan et al. [43] showed that nanoparticles coated with fibroin hardly produced an immune response and the adaptive immune system was not activated. In another study, Totten et al. [44] used in vitro and nuclear magnetic resonance-based metabolomics assays to examine the inflammatory phenotype and metabolic profiles of macrophages after exposure to PEGylated and unmodified silk fibroin nanoparticles. The macrophages internalized both types of nanoparticles but showed different phenotypic and metabolic responses to each type of nanoparticle. Unmodified silk fibroin nanoparticles induced upregulation of several processes, including the production of proinflammatory mediators (such as cytokines), the release of nitric oxide, and the promotion of antioxidant activity. These responses were accompanied by changes in macrophage metabolomic profiles that were consistent with a pro-inflammatory state and indicated an increase in glycolysis and reprogramming of the tricarboxylic acid cycle and the creatine kinase/phosphocreatine pathway. In contrast, PEGylated silk fibroin nanoparticles induced milder changes in inflammatory and metabolic profiles, suggesting that immunomodulation of macrophages with silk fibroin nanoparticles is dependent on PEGylation. This would indicate that the PEGylation of silk fibroin nanoparticles reduces the inflammatory and metabolic responses initiated by macrophages. In the case of silk fibroin microparticles (10–200 μm) prepared by enzymatic digestion, Panilaitis et al. [38] found that suspension of the particles induced a significant release of TNF cytokines. In contrast, macrophages grown in the presence of silk fibroin fibers did not upregulate transcription levels for a wide range of pro-inflammatory cytokines to a significant degree. The combination of results from these two studies could indicate that the immune response is dependent on the size of the biomaterial, excluding materials at the nanoscale and macroscale, but not at the microscale. In a recent study carried out in our research group [45], the HeLa and EA.hy926 cell lines were incubated with up to 250 μg/mL of silk fibroin nanoparticles in vitro. Viability was studied by MTT tests, and the results did not show significant variations (p < 0.05) with respect to the controls.

*Silk Fibroin Nanoparticles: Synthesis and Applications as Drug Nanocarriers DOI: http://dx.doi.org/10.5772/intechopen.100386*

Recent studies indicated that nanoparticles loaded with resveratrol have shown immunomodulatory properties and anti-inflammatory effects in murine models with inflammatory bowel disease [46] and periodontal infections [47]. In another similar study [48], treatments with RGD linear peptide-functionalized silk fibroin nanoparticles were performed and were found to improve colonic damage in rats, reduce neutrophil infiltration, and improve the compromised oxidative state of the colon. It was also found that only rats treated with RGD-silk fibroin nanoparticles showed a significant reduction in the expression of different pro-inflammatory cytokines (interleukin-1β, IL-6 and IL-12) and inducible nitric oxide synthase compared to the control group. Furthermore, the expression of both cytokineinduced neutrophil chemoattractant-1 and monocyte chemotactic protein-1 was significantly decreased with RGD-silk fibroin nanoparticle treatment.

#### **3.2 In vivo degradation of silk fibroin**

Fibroin fibers implanted in the human body retain more than 50% of their mechanical properties after 60 days, which is why the North American Pharmacopeia classifies this material as non-biodegradable [49]. However, the rate at which fibroin degrades depends on the size of the implanted material, its morphology, mechanical and biological conditions at the implantation site, the secondary structure of the protein, and the molecular weight distribution of the fibroin chains. In particular, for the application of silk fibroin nanoparticles to drug transport, three of these parameters must be taken into account mainly: (i) size, (i) molecular weight distribution and (iii) secondary structure. But before analyzing each of them, the possible degradation pathways of fibroin will be discussed to later mention how these parameters are able to influence degradation.

As a protein, fibroin exhibits degradation against proteases capable of degrading amide bonds including α-chymotrypsin, collagenase IA, protease XIV, and metalloproteases [50–52]. The residues of the degradation process are the corresponding amino acids of the proteins, so they are easily absorbed in vivo and do not generate toxicity. The partial hydrolysis of the protein by enzymes into small fragments is not a problem either, since these can be easily phagocytosed by macrophages [38]. Li et al. [51] observed that the mean molecular weight of fibroin film products after degradation with the three enzymes followed the order of protease XIV > collagenase IA > α-chymotrypsin. The degradation mechanism is based on a two-stage process, based on enzymes finding binding domains on the surface of materials and their subsequent hydrolysis [53]. In this manner, different enzymes have different results for the degradation of different structures within fibroin. For example, chymotrypsin has been used to degrade the amorphous regions of fibroin to obtain highly crystallized fibroin [51]. Collagenase preferentially degrades the content of β sheets in hydrogels [38]. On the other hand, after incubation of fibroin with protease XIV, it was found that the mass was significantly reduced [51]. Brown et al. [38] concluded that the ability of enzymes to break down a biomaterial not only depends on the cleavage sites being present in the primary structure of the protein but also the secondary structure and the format of the material play a fundamental role. This indicates that the degradability of fibroin can be modulated by controlling the relative abundance of its secondary structures. In this way, for example, by reducing the content of highly crystalline structures in stacked β-sheets, degradation can be accelerated, since both protease XIV and chymotrypsin can act simultaneously in these areas.

Horan et al. [49] concluded that the degradation of electrospun fibers exhibited a predictable degradation dependent on the diameter of the fibers. As expected, as the diameter decreases and, therefore, the surface/volume ratio increases, the

degradation occurs at a higher rate. Decreasing the size from macroscopic fibers to nanoparticles will clearly increase this ratio, allowing greater access to enzymatic degradation and phagocytosis by macrophages. The degradation of PEG-functionalized and non-functionalized silk fibroin nanoparticles by proteolytic enzymes (protease XIV and α-chymotrypsin) and papain, as well as cysteine protease, were studied by Wongpinyochit et al. [54]. Both classes of particles presented similar degradation patterns in a period of 20 days, establishing the order of degradation of the particles by means of enzymes such as: Protease XIV > papain > > α-chymotrypsin. The authors reported that, after 1 day, silk fibroin nanoparticles and PEG-silk fibroin nanoparticles incubated with protease XIV lost 60 and 40% of their mass, respectively, a reduction in the amorphous content of the secondary structure and an increase in diameter. In contrast, 10 days of incubation were required for similar degradations with papain and 20 with α-chymotrypsin. Silk fibroin nanoparticles were also exposed to a complex mixture of rat liver lysosomal enzymes ex vivo, finding that they lost 45% of their mass in 5 days.

Lastly, it should be noted that modifications in the molecular weight distribution of fibroin chains can alter the rate of degradation [55]. A decrease in this can alter the order of crosslinking between polymers and potentially result in faster degradation [56]. For this reason, the purification and subsequent processing of fibroin not only affects the mechanical and physicochemical properties of the resulting biomaterials [57] but can also be used to modulate their biodegradability.
