**4. Synthesis of silk fibroin nanoparticles**

Silk fibroin particles can be produced from the top-down as well as the bottomup approach. The first of these involves grinding the fibroin fibers to reduce their size. This can be achieved through ball milling [58, 59], bead milling [60], air-jet [61] as well as irradiating the material with an electron beam [62]. However, these tend to produce particles in the micrometer range and with a wide size distribution, so in the rest of this section, we will focus on the bottom-up approach which offers more control over the particles produced. This approach is based on the self-assembly of the smallest units that constitute a nanoparticle. In the particular case of silk fibroin nanoparticles, the fibroin fibers are firstly dissolved to obtain their individual constituent units and subsequently regenerated into nanoparticle format. This is normally achieved through a desolvation process, which can be achieved in different ways that will be discussed in this section along with some examples; but first, the dissolution of silk fibroin will be discussed, which is not an easy task due to its high structural stability and deserves a detailed analysis that will be exposed below.

#### **4.1 Solubilization of silk fibroin**

The preparation of most biomaterials depends on achieving a complete dissolution of fibroin. This process is referred to by some authors as reverse engineering [63], since it attempts to obtain water-soluble fibroin with *silk I* structure, starting from fibers with *silk II* structure. However, only a limited number of solvents are able to dissolve silk fibroin. Examples of these are strong acidic solutions (phosphoric, formic, sulfuric, hydrochloric) or aqueous/organic solutions concentrated in salts (LiCNS, LiBr, CaCl2, Ca (CNS)2, ZnCl2, NH4CNS, CuSO4, NH4OH, Ca(NO3)2) those that are capable of completing the dissolution of fibroin fibers [64–70]. This is due to the presence of the great network of intra- and intermolecular hydrogen bonds and the high crystallinity derived from its secondary structure.

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

In general, the literature describes the dissolution of fibroin in concentrated salt solutions as a two-step process [64, 67]. In the first step, the compact, crystalline structure of fibroin fibers swells due to the diffusion of solvent molecules. In the second, the dispersion of the silk fibroin molecules begins due to the collapse of the intermolecular interaction and their consequent dissolution [67]. During dissolution, amorphous sections with a higher content of massive amino acid residues or polar groups are firstly dissolved. The cations (Ca2+, Zn2+, Cu2+, NH4 + , Li+ ) form stable chelate complexes with hydroxyl groups of the serine and tyrosine side chains and also with the oxygen of the carbonyls, breaking the hydrogen bonds and the van der Waals forces between polypeptide chains resulting in the dissolution of the protein that adopts *silk I* structure [68, 70, 71].

The described dissolution process depends on reaching a high concentration of salts. This entails certain disadvantages since extensive dialysis (72 h) is required to eliminate these, which requires 6 liters of distilled water for every 12 mL of solution [64]. Furthermore, the solutions obtained are unstable and tend to gel within a period of days. Alternatively, when long-term storage of fibroin is desired, aqueous fibroin solutions can be lyophilized and subsequently dissolved in organic solvents such as hexafluoroisopropanol [64] at the time they are to be used. However, these solvents are toxic and extremely corrosive so they require special care when handling [72].

Recently, ionic liquids have emerged as an alternative for the dissolution of silk fibroin [73] providing numerous advantages over traditional methods [64, 65]. Firstly, the negligible vapor pressure and easy recyclability of ionic liquids make them a more "green" alternative to organic solvents [74–76]. Secondly, the possibility of obtaining high concentrations of fibroin in a stable solution (up to 25% w/w in some ionic liquids [77]). Fibroin solutions in ionic liquids are more stable because the hydrophobic regions (highly conserved GAGAGS or GAGAGAGS sequences) are stabilized by the alkyl chains of cations such as imidazolium [78]. On the other hand, the bulky charged imidazolium ring is oriented outwards providing electrostatic repulsive forces between the hydrophobic blocks and preventing the transition from *silk I* to *silk II* through the formation of the β sheets. According to Wang et al. [78], solutions of silk fibroin in 1-allyl-3-methylimidazolium chloride can be stable for periods longer than one and a half years. Thirdly, the ease with which silk fibroin can be dissolved. According to the method described by Lozano-Pérez et al. [77], by means of high-power ultrasound, the complete dissolution of fibroin can be achieved in a few minutes compared to several hours with traditional methods [64, 65].

### **4.2 Regeneration of fibroin into nanoparticles**

Nanoparticle synthesis processes in a bottom-up approach involve the dissolution of fibroin in its constituent polymer units and its subsequent regeneration into nanoparticles. This regeneration is normally carried out by means of a desolvation process in an "antisolvent", a process commonly referred to as antisolvation. As shown in **Figure 4**, in the nanoparticle synthesis process by antisolvation there are three key components, the polymer, the solvent and the antisolvent. The necessary conditions are that: (i) the solvent and the antisolvent are miscible under the process conditions, while (ii) the solute must be insoluble in the solvent/antisolvent mixture. In this way, when mixing the polymer solution, the antisolvent will seize the molecules that solvate it, leading to their aggregation. Employing kinetic and thermodynamic controls, a limited number of polymer units can be made to aggregate, thus forming nanoparticles. In practice, the preparation of nanoparticles by antisolvation can be achieved by different techniques that vary in the methodology

**Figure 4.** *Key components in the antisolvation process.*

of mixing the solution of the polymer and the antisolvent or the nature of the latter. We will describe some of the most representative methods in the literature below.

#### *4.2.1 Antisolvation in organic solvents*

Probably one of the most widely used methods due to its simplicity and good results is the addition of the silk fibroin solution to water-miscible polar organic solvents, which act as an antisolvent when initiating the transition from *silk I* to *silk II* through the β-sheet formation [44, 77, 79–83]. It should be noted that the inverse variant, where the antisolvent is added to the fibroin solution, is also frequently found in the literature [84–86].

As an example, Wongpinyochit et al. [83] dissolved the fibroin fibers in a 9.3 M LiBr solution, keeping them stirred for 4 hours at 60°C. Subsequently, the solution is dialyzed for 72 hours and centrifuged to remove insoluble residues. Then, the fibroin concentration is adjusted to 5% w/v and added dropwise (10 μl/drop at a speed of 50 drops/min) to acetone under strong agitation, the volume of acetone being greater than 75% of the final volume of both liquids. A white suspension is immediately formed upon contact of both liquids, marking the formation of the nanoparticles. The particles are washed and collected by centrifugation. The overall process is illustrated in **Figure 5**. An average diameter of ca. 100 nm and a Z-potential of -50 mV (in distilled water) are provided for the particles obtained.

One way to optimize the nanoparticle synthesis process is to reduce the mixing time between the fibroin solution and the antisolvent [87]. This can be achieved by reducing the size of the fibroin solution droplets (increased surface/volume ratio) that come into contact with the antisolvent, favoring mass transfer [87]. In our research group, a method has been developed that uses a coaxial injector where the fibroin solution flows through the center and nitrogen under pressure runs through the concentric cylinder, this manages to produce an aerosol with very small droplets [45, 88].

#### *4.2.2 Antisolvation in supercritical fluids*

A supercritical fluid is any substance that is under conditions of pressure and temperature above its critical point. Under these conditions, the substance has hybrid properties between a liquid and a gas, that is, it can diffuse like a gas, and dissolve substances like a liquid [89]. Assuming that the compound to be used as a supercritical fluid meets the conditions described at the beginning of this section, it can act as an antisolvent in an antisolvation process. In this case, the process is known as supercritical antisolvation (SAS). In particular, carbon dioxide (CO2) is one, if not the most used substance as a supercritical fluid due to its moderate critical conditions (T = 304 K and P = 7.38 MPa), harmless to the operator and the environment, as well as its economic obtaining and operation [89].

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

**Figure 5.**

*Scheme illustrating the key steps to generate a solution and obtain silk fibroin nanoparticles described by Wongpinyochit et al. [83]. Reprinted with permission from reference (83). Copyright 2016 MyJoVE corporation.*

The SAS process is well known and has been used for the preparation of silk fibroin nanoparticles [90–92]. SAS has some variants by other acronyms: Aerosol Solvent Extraction System (ASES), Solution Enhanced Dispersion by Supercritical Fluids (SEDS), Supercritical AntiSolvent with Enhanced Mass transfer (SAS-EM). The main difference between these processes is in the device for injecting the solution and CO2. In the case of the SAS and ASES processes, the liquid solution is injected into the precipitation reactor through a micrometric nozzle, in the case of the SEDS process, the nozzle is coaxial; whereas, the SAS-EM process uses a baffle surface that vibrates at ultrasonic frequencies to improve the atomization of the solution [93].

This method can be exemplified by the process outlined by Xie et al. [90] for the preparation of curcumin-loaded silk fibroin nanoparticles by SEDS. Briefly, lyophilized silk fibroin in the *silk I* state is dissolved in hexafluoroisopropanol together with curcumin. The solution is subsequently injected into a precipitation reactor containing CO2 at 20 MPa which will act as an antisolvent for fibroin. After the complete injection of the solution, a constant flow of CO2 is maintained to remove the hexafluoroisopropanol from the precipitation reactor. Finally, the reactor is depressurized and opened for the collection of the nanoparticles. The process produces nanoparticles with a mean diameter of less than 100 nm.

#### *4.2.3 Electrospray*

Electrospray is a technique in which an electrical potential difference is applied between the nozzle of an injector and a manifold, which can contain a liquid that acts as an antisolvent [94]. In this technique, the surface of the liquid emerging from a capillary subjected to electrical stress is deformed into an elongated injector that initially produces a series of micrometer-sized drops. Because the drops are charged, the repulsive forces break each drop into a group of smaller drops in a process called coulombic explosion [94]. Using this technique to spray a silk fibroin solution onto

#### **Figure 6.**

*Microfluidic chip made of glass. Channels are 50* μ*m deep and 150* μ*m wide (image by IX-factory STK, 2014, CC BY-SA 3.0; no changes were made to the original image).*

aluminum foil, Gholami et al. [95] succeeded in synthesizing silk fibroin nanoparticles of up to 80 nm on average. The authors showed that lower concentrations, lower feed rates, and longer distances between the needle and the collector led to a decrease in mean particle size. Increasing the voltage to 20 kV decreased the size of the particles but voltages higher than this produced an increase in the particle size.

#### *4.2.4 Microfluidics*

Microfluidic equipment can also be used for the preparation of nanoparticles. Microfluidic kits are devices, generally the size of millimeters/centimeters, that contain microcapillaries specially designed for mixing fluids [96, 97]. A representative image of these is shown in **Figure 6**. These types of equipment allow precise manipulation of liquids (like the dissolution of silk and the antisolvent) by means of the control of the parameters of the process such as the total flow, the relations of speed between different lines of injection, etc. The greatest advantages provided by this equipment are the possibility of producing particles in continuous flow and with a narrow size distribution.

Wongpinyochit et al. [63] have used a commercial microfluidic kit to mix a 3% wt solution of silk fibroin with acetone or isopropanol as an antisolvent. Through the use of different mixing conditions, the authors were able to control the final size of the nanoparticles obtained, which varied between 110 and 310 nm, with polydispersity and Z-potential indices between 0.1/0.25 and − 20/−30 mV, respectively.

#### *4.2.5 Salting-out*

Phase separation has also been used for the preparation of nanoparticles using the salting-out method. For example, by adding potassium phosphate to a solution of silk fibroin, Lammel et al. [98] prepared fibroin particles with sizes varying between 500 and 2000 nm depending on the initial concentration of fibroin in solution. The authors revealed that, for the formation of nanoparticles, a salt concentration greater than 750 mM is required, otherwise, the solution gels.

#### **5. Mechanism of nanoparticle formation in antisolvation processes**

After having explained some of the synthesis methods, it is appropriate to mention the mechanism by which antisolvation can generate nano-sized particles. Upon

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

mixing the fibroin solution and the antisolvent, supersaturation occurs, leading to phase separation (precipitation). The mechanism can be divided into two steps, with a nucleation stage driven by supersaturation occurring first and then a growth stage. Growth can occur by two coagulation mechanisms: i) the nuclei of particles converge to form a larger particle or condensation and ii) the polymer units add to growing nuclei. This is exemplified in **Figure 7**. Condensation decreases supersaturation by reducing the mass of solute in the mixture and therefore competes with nucleation. Coagulation can reduce the rate of condensation by reducing the total number of particles and therefore the surface area [99]. Supersaturation influences nucleation and growth rates to different degrees. The nucleation rate depends more strongly on supersaturation than the condensation rate. High nucleation rates offer the potential to produce a large number of submicron particles in the final suspension if growth can be controlled. This process can be compared with the formation of crystals in the order of millimeters for X-ray crystallography when it is sought to obtain a low number of nuclei and greater growth in slow nucleation processes.

The key to generate rapid nucleation is to achieve rapid supersaturation. This process will be directly influenced by mixing and phase separation, which can be represented by the Damkohler number (Da) defined as the relationship between the mixing time ( *mix* τ ) and the total precipitation time ( . τ):

$$Da = \frac{\pi\_{\text{mke}}}{\pi}. \tag{1}$$

Under poor mixing conditions, *mix* τ is large (as is Da) and the nucleation rate is slow relative to the growth rate, resulting in large particles and wide size distributions. As the *mix* τ is reduced with respect to the . τ , greater supersaturation and faster nucleation are achieved, resulting in smaller particles with narrow size distribution [87].

The *mix* τ can be reduced by reducing the size of the droplets (increasing the surface/volume ratio) of fibroin solution that meet the antisolvent, favoring mass transfer. Reduction in droplet size is typically achieved by increasing Reynolds number which produces turbulent flow and thus results in dissolution jet

#### **Figure 7.**

*Scheme representing the mechanism of precipitation by nucleation and growth of particles by coagulation and condensation.*

fragmentation. In fact, this is the goal of previously proposed methods such as the coaxial injector [45, 88], SEDS [90], electrospray [95] and microfluidic equipment [63].

According to Eq. (1), another strategy to reduce Da is to increase the . τ . This can be achieved by adding stabilizers that interact with the polymer units generating steric hindrances that retard growth by condensation and coagulation [87]. According to Matteucci et al. [87], adding the stabilizers to the antisolvent is more effective in preventing the growth of the particles than adding them to the polymer solution. This is because, when placed in the antisolvent, the stabilizing agents are more available to interact with the polymeric units, as they do not need to diffuse across the interface from the overall aqueous phase to the organic phase.

Temperature is another important thermodynamic control factor for the preparation of nanoparticles by antisolvation that affects the formation of particles in different ways. Firstly, an increase in temperature leads to an increase in the solubility of the polymer and therefore reduces the degree of supersaturation when mixing the solutions, favoring slow nucleation. Secondly, elevated temperatures increase diffusion and growth kinetics at the interface of the particle boundary layer. And thirdly, higher solubility also increases Oswald's maturation rate [100]. For these reasons, a reduced temperature in the precipitation stage is preferable for the formation of small nanoparticles.

#### **6. Nanoparticle drug loading**

The loading of drugs can be carried out mainly by two approaches, (i) during the nanoparticle formation process or (ii) a posteriori, by adsorption of the drug on the surface of the nanoparticle. The first approach can be achieved by adding the drug to the polymer solution (nanoparticle matrix) [88] or the antisolvent [101] before mixing both. This approach is often referred to as coprecipitation because the polymer and drug precipitate together. In contrast to this method, adsorption of the drug to the surface of the nanoparticle can be achieved by incubating the nanoparticles in a solution of the drug. These methods have advantages and disadvantages. On the one hand, the first method is usually simpler, since the loading and preparation of the nanoparticle are carried out in a single step. However, this process could affect the formation of the nanoparticle and therefore the second method could be preferential. On the other hand, drug release profiles must be considered. As demonstrated by Montalbán et al. [88], particles charged by coprecipitation have slower release profiles compared to particles charged by absorption. This is to be expected since, in the latter, the drug is on the surface of the nanoparticle and readily available to the medium.

Drug loading and encapsulation efficiency depend on drug-polymer interactions and the presence of functional groups (i.e, hydroxyl, carboxyl, etc.) in both. Montalbán et al. [102] used computational methods such as blind docking and molecular dynamics simulations to study the interactions of different drugs with silk fibroin nanoparticles. The authors found a strong correlation between drugfibroin interactions and their loading content. Similarly, drugs with weaker interactions had a higher release rate and a higher percentage of loaded drug release.

### **7. Conclusions**

Silk fibroin of the Bombyx mori silkworm is a natural, protein polymer that presents an interesting combination of mechanical properties, such as flexibility

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

and resistance which are still difficult to achieve with synthetic polymers. Furthermore, fibroin is biodegradable and biocompatible, which makes it an excellent material for the production of nanoparticles for drug delivery.

Although the remarkable mechanical resistance of fibroin is one of the attractive properties of the biomaterial, the nanoparticle synthesis process is hampered by its high stability, due to the high number of hydrogen bonds in its secondary structure, mostly in the form of antiparallel β-sheets. A recent method, developed by our research group, is based on the use of ionic liquids to dissolve native fibroin and has allowed the production of nanoparticles in an easy and scalable process for industry.

The silk fibroin nanoparticle synthesis process comprises several stages. Firstly, the fibroin is purified by removing the sericin (a method known as degumming). Secondly, fibroin must be dissolved in its monomeric units and, subsequently, regenerated into nanoparticles. Each of these steps can have significant effects on the secondary structure of the protein and given the implications that it has on the resistance, degradability and biocompatibility of the final product, their study is essential.
