**Abstract**

The use of nanoparticles in biomedical fields is a very promising scientific area and has aroused the interest of researchers in the search for new biodegradable, biocompatible and non-toxic materials. This chapter is based on the features of the biopolymer silk fibroin and its applications in nanomedicine. Silk fibroin, obtained from the *Bombyx mori* silkworm, is a natural polymeric biomaterial whose main features are its amphiphilic chemistry, biocompatibility, biodegradability, excellent mechanical properties in various material formats, and processing flexibility. All of these properties make silk fibroin a useful candidate to act as nanocarrier. In this chapter, the structure of silk fibroin, its biocompatibility and degradability are reviewed. In addition, an intensive review on the silk fibroin nanoparticle synthesis methods is also presented. Finally, the application of the silk fibroin nanoparticles for drug delivery acting as nanocarriers is detailed.

**Keywords:** silk fibroin, structure, biocompatibility, nanoparticle, synthesis, nanocarrier

### **1. Introduction**

Silk is an ancestral material used since 2450 BC [1] for making fabrics. After having been an economic engine of several empires and coining the name to the trade route that linked Asia, Europe and Africa, silk suffered a debacle in the early 20th century when the much cheaper synthetic polymers derived from hydrocarbons were introduced. However, today, motivated by its biocompatibility and excellent mechanical properties, researchers around the world are trying to produce biomaterials based on this biopolymer for a variety of biomedical applications: films with a surface roughness that increase cell adhesion, 3D structures for bone implants, hydrogels for wound protection and nanoparticles for drug delivery, among others [2–5].

Silk fibroin probably receives a lot of attention from the general public due to its mechanical properties, so a brief text will be devoted to comparing them with other natural fibers and engineering materials. **Table 1** shows values of stress at break, elasticity and percentage of nominal deformation at the break of silk fibroin together with the values of other biomaterials and synthetic materials. Excluding

#### *21st Century Nanostructured Materials – Physics, Chemistry, Classification, and Emerging…*


*a Type I collagen fibers extruded from rat tail tested after stretching from 0–50%.*

*b Cross-linked rat tail collagen tested after stretching from 0–50%.*

*c Polylactic acid with molecular weights ranging from 50,000 to 300,000 units.*

*Adapted from reference (5) with permission of Elsevier.*

#### **Table 1.**

*Comparison of the mechanical properties of different natural and synthetic fibers.*

mineralized biomaterials (bones), Kevlar and carbon fibers, *Bombyx mori* silk fibroin together with *Nephila clavipes* spider silk are the biomaterials with the highest stress at break. While the list of biomaterials is incomplete, it is fair to say that fibroins are among the strongest polymeric biomaterials known. However, the tensile strength of fibroin is substantially lower than that of Kevlar and carbon fiber, engineering materials that are commonly used to transmit and support tensile forces. At first glance, we could infer that fibroin is superior to other biomaterials, such as collagen, but not as "good" as Kevlar and carbon fibers. However, this interpretation is based on the assumption that "good" means stiff and strong. Looking closely at **Table 1**, it can be seen that fibroin is quite extensible, presenting a maximum deformation of approximately 18%, while engineering materials fail in deformations of the order of 1–3%. The great extensibility of fibroin makes it more resistant than engineering materials.

It is especially notable that silkworms can produce strong and stiff fibers at room temperature and from an aqueous solution, while synthetic materials with comparable properties must be processed at elevated high temperatures and/or with less benign solvents. Furthermore, synthetic polymer fibers typically require post-spin stretching to ensure the necessary degree of molecular orientation in their structure [11]. On the other hand, this is not necessary for silk in the natural spinning process. This is due to its impressive amino acid sequence which gives rise to an extraordinary polymorphic secondary structure that will be discussed below.

#### **2. Silk structure**

Silk is a protein biopolymer synthesized by a wide variety of lepidoptera and arachnids in specialized glands. However, it is important to note that this chapter focuses on silk fibroin from the silkworm *Bombyx mori* of the *Bombycidae* family, fed only with mulberry leaves (*Morus alba L*). This is important because the amino acid sequence

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

#### **Figure 1.**

*Diagram of the silk fibroin gland. From reference [12] with permission of public library of science (PLOS).*

varies from species to species and with it its mechanical and physicochemical properties, which can have great implications in different applications.

Silk proteins are synthesized in a gland that extends through the abdomen of the worm and is divided into three sections, posterior, middle and anterior, as illustrated in **Figure 1**. The posterior gland cells secrete silk fibroin, reaching a concentration of approximately 12% by weight. At this time, the protein is in a water-soluble state [13], with a partially ordered secondary structure composed of irregular structures and type II β-turns [14], commonly known as *silk I*. This protein is pushed into the gland media, where cells lining the lumen secrete sericin along with other flavonoids (assimilated by worms in the diet) [15]. Fibroin and sericin are concentrated to 25% by weight and are driven to the anterior gland where they experience pH gradients (maintained through the secretion of carbonic anhydrase) and ionic strength gradients. These factors contribute to the elongation of fibroin (at this point 30% by weight) into two thin filaments and promote the crystallization of repetitive domains. Lastly, during the spinning process, the non-Newtonian protein solution is subjected to crystallization induced by changes in pH and ionic strength through the gland [16] and by the shear stress generated by an opposing pressure front to the flow, generating a velocity gradient from the inlet (0.334 mm/s) to the outlet (13.8 mm/s) of the spinning organ [17]. Throughout the process, the silk fibroin initially secreted with a partially ordered structure (*silk I*) undergoes a transition to one composed mainly (58%) [18] by antiparallel β sheets, adopting an insoluble crystalline structure known as *silk II* [18, 19].

To understand the formidable properties of this biopolymer, its structure must be studied in detail. A silk cocoon is composed of a single silk fiber between 1000 and 1500 m in length with a diameter of between 10 and 25 μm [20]. This fiber is composed of a core of two fibroin filaments, each one of approximately 10 μm covered by a layer of sericin that hold the fibers together, as illustrated in **Figure 2** left, providing greater resistance to the assembly of the fiber. In turn, fibroin fibers are

#### **Figure 2.**

*Left, scanning electron microscope image (2,000X magnification) of silk fiber, containing two fibroin fibers coated by sericin. From reference [21] with permission of Elsevier. Right, schematic representation of the structure of silk fibroin. Insets show the general structure of the fibrils and the alignment of the antiparallel*  β*-sheets. From reference [22] with permission of John Wiley and Sons.*

composed of coiled nanofibrils of between 20 and 25 nm, which gives them greater tensile strength (**Figure 2**, right) [23].

Fibroin, representing approximately 75% of the weight of the cocoon, is a linear, water-insoluble protein with high tensile strength. On the other hand, sericin represents approximately 25% of the cocoon weight, it is a globular, water-soluble protein whose function is to keep the fibroin fibers together [24]. Silk fibroin is made up of three components, a heavy chain (391 kDa) and a light chain (26 kDa) linked by a disulfide bridge, and a glycoprotein, P25 (25–30 kDa) in a 6: 6: 1 molar ratio to yield a 6.3 MDa megastructure [25]. The primary structure of the silk fibroin heavy chain is schematically represented in **Figure 3A**. This chain is composed of 5,263 amino acids divided into N- and C-terminal domains, both hydrophilic, and

#### **Figure 3.**

*Organization of the amino acid sequence within silk fibroin. A) In green and magenta, the N- and C-terminal domains are shown, respectively. The repetitive sequences of the GAGAGS type that give rise to the crystalline domains of the silk fibroin are represented by orange cylinders and in blue, the hydrophilic sequences that flank the crystalline regions. B) Diagram representing the transition from water-soluble silk (silk I) to crystalline silk fibers after the bio-spinning process. In silk fiber, amorphous regions (44%) and crystalline regions rich in antiparallel* β *sheets (56%) are represented. Reprinted with permission from reference [26]. Copyright 2020 American Chemical Society.*

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

12 hydrophobic domains flanked by 11 short and hydrophilic domains. The hydrophobic domains contain highly conserved and repetitive sequences of the GAGAGS, GAGAGY, and GAGAGVGY types that form the β-sheet structures that, in addition, are packaged in crystalline areas [16]. 86% of the amino acids of the heavy chain of fibroin are Glycine (45%), Alanine (29%) and Serine (12%) [27], which are mostly found in the hydrophobic and highly repetitive regions. The great bias that the primary sequence of fibroin presents towards amino acids with small residues such as Glycine and Alanine, promotes the formation of antiparallel β-sheets, which are mostly packed in the crystalline areas.

The secondary structure of fibroin contains approximately a total of 58% of β-sheet [28], of which approximately 33% correspond to antiparallel β-sheets organized in crystalline structures. Fibroin fibers are generally described as a matrix of disordered structures with β-sheet crystals embedded in it [26], as represented in **Figure 3B**. The intra and intermolecular hydrogen bond network provide strength and tensile strength to the biopolymer, while amorphous regions provide flexibility and elasticity [29]. In the literature, there is great variability in the sizes reported for these crystals [30–33]. To illustrate the size of these, reference can be made to X-ray diffraction measurements and low voltage transmission electron microscopy performed by Drummy et al. [30]. They have determined that the crystals within the fibroin fibers have dimensions of 21 x 6 x 2 nm and their major axis is aligned parallel to the axis of the fibers.

The secondary structure of fibroin with *silk II* conformation is extremely stable thanks to a large number of hydrogen bonds which makes it insoluble in most solvents, including under moderate acidic and alkaline conditions. As the content of acidic and basic groups is low in fibroin, the electrostatic factor is not decisive in the formation of the secondary structure, however, it can be decisive in the dissolution of fibroin. The secondary structure of fibroin is not only relevant in the biomaterial synthesis process due to the need for its dissolution, but also due to its influence on the mechanical and physicochemical properties of the resulting biomaterials. For example, Wang et al. [34] prepared silk and polyvinylpyrrolidone micro- and silk fibroin nanoparticles for controlled drug release and concluded that release profiles can be adjusted by modulating the number of β-sheets in the secondary structure of fibroin.
