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Basic building block ensuring and dictating resulting biophysicochemical properties of the fiber
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Cooperative hydrogen bonds improve crystal toughness via self-healing capacity of hydrogen bonding providing the basis for larger-scale toughness and strength (Keten et al, 2010) The interplay between β-sheet crystals and amorphous domains ensures strength and extensibility of the nanocomposite structure. The two domains establish a collaborative interaction contributing complementary function: strength and extensibility (Gosline et al, 1999; Keten and Buehler, 2010; Nova et al, 2010) The network-like structure within the fibril leads to uniform deformation of all protein domains within the fibril, enhancing strength and toughness due to the contribution of many protein components acting together The binding of several co-directional fibrils into a single fiber increases the properties of the material by achieving a uniform deformation state (flaw tolerance) (Giesa et al, 2011).
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in the laboratory, perfect replication of the natural spinning process is not currently possible (Blamires et al, 2012). These recent reviews expound on the subtleties and complexities of the bioengineering and biomimetics of spider silk (Andersson et al, 2016; Blamires et al, 2020).
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As is reflected in the poor mechanical performance of artificially synthesized fibers, they are quite distinct from the true highly ordered hierarchical structure of natural spider silk (Teulé et al, 2007; Tucker et al, 2014). Even when full or nearly full sequences of genes are replicated, the quality of the fibers differ greatly from that of naturally spun spider silks. It can be concluded that there are biochemical (secretory) and physiological (assembly and spinning) functions, and the possible contribution of less prominent compounds and synergic effects, that we do not seem to fully understand. Science still has not decoded all the secrets of natural spider silk biosynthesis that spiders have used every day for millions of years. A better understanding of the key features of this process allows for the development of more promising biomimetic protocols that can then generate ideas with broad and important implications for more accessible and durable advanced material production.
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By deciphering the secrets of natural spider silk synthesis, science can gain a lot of inspiration for high-performance silk replicating materials. The absolute uniqueness of natural spider silk biosynthesis lies in its transformation of watersoluble spidroins into solid eco-friendly high-performance fibers at ambient temperature and moderate pressure (Andersson et al, 2016). There are numerous studies documenting how spiders adapt their fibers, from the amino acid sequence to the nano- and microscale organization employed during spinning, which allows for them to tune silk properties by adjusting the fibers to various environmental conditions (Saravanan, 2006; Sponner et al, 2007; Pechmann et al, 2010). This is accomplished by reorienting the peptide chains of liquid spinning dope (the liquid material from which silks are formed) through the structure of the spinning gland and the effect of the spidroin terminal domains (Andersson et al, 2013). The spinning glands regulate the concentration of numerous ions, mainly hydrogen ions, in the spinning dope to regulate pH (Dicko et al, 2004). As the dope passes through the narrowing spinning channel, it is acidified and exposed to a high degree of shear (Jin and Kaplan, 2003; Breslauer et al, 2009). During this process, the terminal domains of spidroin amino acid sequences act as molecular regulators to improve alignment via physiologically controlled homodimerization (Hagn et al, 2010; Eisoldt et al, 2012; Kronqvist et al, 2014). Due to their less hydrophilic nature and in contrast to repetitive domains, activated terminals retain their nature to initiate the assembly of spidroins via the formation of micelles (Jin and Kaplan, 2003; Rising et al, 2006; Gaines et al, 2010). During extrusion, the micelles form globule-like secondary structures that change from a helical and/or random coil to a predominantly β-sheet structure when exposed to shear force (Kenney et al, 2002; Dicko et al, 2004).
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The self-assembly and rearrangement of silk peptide chains at the molecular level results in a high degree of β-sheet stacking in the fibers, accounting for their excellent mechanical properties. The strength of spider silk fibers can also be largely related to the presence of more mechanically stable β-sheets that possess a high density of hydrogen bonds due to the alanine-rich repeating domains of its amino acid sequence (this mechanism was described earlier). Moreover, it was found that, depending on the size of nanocrystals in dragline silk, the fiber exhibits varying ultimate strength and viscosity (Nova et al, 2010). Notably, the mechanical properties of spider silk depend not only on the composition of the amino acid sequences of spidroins or structural organization of the fiber, but also on the reeling speed of silk fibers during extrusion. Thus, faster silk spinning produces a stiffer fiber, while a decrease of the spinning rate leads to a fiber with higher elastic properties being formed (Vollrath et al, 2001; Perez-Rigueiro, 2005; Wu et al, 2009). This is also proven by the fact that spiders can greatly manipulate the material properties of their fibers by adjusting the size of the β-sheet nanocrystals through changing the reeling speed of the fiber (Du et al, 2006). Consequently, spider silk represents a comprehensive composite material. By altering its composition and organization, a spider can accomplish improved silk functionality and adapt spinning to changing environmental conditions.
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Another example of the amazing natural adaptation of spider silk is the supercontraction of fibers when exposed to water in the form of steam or liquid (Liu et al, 2005, 2019; Blackledge et al, 2009). Consistent with this observation, the diameter of the fiber increases while the fiber compresses up to 50% of its original length (Guinea et al, 2003; Pérez-Rigueiro et al, 2003). Supercontraction is most pronounced in natural dragline silk fibers, as opposed to tubuliform silks (Vierra et al, 2011). This phenomenon can initiate a mechanism for creating tension in webs when they are loaded with dew or rain (Guinea et al, 2003). Additionally, during supercontraction, the stiffness of the fiber reduces due to the loss of molecular structural order along the fiber axis (Yang et al, 2000). Therefore, supercontraction provides an important mechanism for adapting the properties of fibrous materials.
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Based on studies on the natural silk adaptation process, it is possible to provide a basis for potentially new bioprospective and biomimetic programs to fabricate highly adaptive fibrous materials (Agnarsson et al, 2010). Using these data, material scientists can learn how to better control the variability of flexible physicochemical properties of innovative smart and responsive materials.
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Spider silk is of practical interest because of its enormous functional potential. Understanding the connection between the morphology of the structural elements of spider silk proteins and their biochemical composition, physicochemical properties, and their supramolecular organization opens up opportunities for the production of new high-performance silk-based materials. The combination of nanostructures with biomaterials offers great prospects for constructing innovative functional devices for a wide range of applications. Therefore, the purpose of this section is to provide information on the possibilities and modern approaches to managing spider silk properties regarding possible ways to improve its functional properties. This section also aims to explain experimental observations and to lend scientific credence to the use of modeling approaches for the development of new biocompatible silk-based materials. To rationalize information on possible approaches to the modification of spider silk, namely combining techniques for manipulating natural or recombinant spider silk, this section was divided into sub-sections.
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In spider silk, hierarchical protein assembly over several orders of magnitude is observed (Reches and Gazit, 2006; Knowles et al, 2010). The formation of hierarchical fibrous arrays is feasible via protein intra- and intermolecular interactions, suggesting that these micro-architectures can be used to integrate inorganic components within silk fibers to produce biopolymer hybrids with increased mineral loadings. Nevertheless, functional diversity can be achieved using the structural design of spider silk building blocks in combination with chemical approaches. Motivated by these innate natural nano-sized structures and designs, a series of fabrication strategies have been promoted to create hybrid organic-inorganic materials using spider silk proteins. These include biomineralization, impregnation, nanoparticle synthesis, bio-integration, and genetic fusions that are adopted to modify spider silks (Figure 3). These compositions have resulted in the development of functionally advanced hybrid materials for various applications.
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Hybrid materials are widely used. For example, the improved biological and therapeutic properties of spider silk-based hybrid materials make it well-used in drug delivery, cancerfighting therapies, as well as designing antimicrobial agents and osseous tissue replacement materials. The enhanced mechanical characteristics introduced by the inorganic phase generate prospects for using spider silk–inorganic particlebased hybrids as bone grafts or extra durable materials. Using optically active nanoparticles during the fabrication of functional spider silk-based hybrids provides more opportunities for their use in biomolecular detection, bioimaging, and in optoelectronic nanodevices. Noteworthy, spider silk supports electron transport between conductive nanoparticles in hybrid materials, which allows for the application of the resulting hybrids in sensing and energy spheres as bio- and vaporsensors, energy harvesters, and microelectronic devices. Various methods of inorganic nanoparticles application to create said hybrid materials are discussed in the following sections in detail.
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It is important to mention active studies on the strength characteristics and specific properties introduced by the doping phase of the obtained hybrid materials. The precentage of the doping inorganic phase in spider silk-based hybrids is often determined through differential scanning calorimetry (DSC) tests (Farokhi et al, 2014). Specific biological tests usually include cell adhesion and proliferation studies (Allmeling et al, 2008; Wohlrab et al, 2012) alongside the determination of antimicrobial ability (demarcating its inhibition zone by way of the agar diffusion method) (Wright and Goodacre, 2012). The mechanical properties of spider silk fibers, namely strength, extensibility, and toughness are extensively investigated using tensile tests (Giesa et al, 2011; Madurga et al, 2017). The impact of the structural organization within the silk fibers on FIGURE 3 | Summary of techniques for creating spider silk-based hybrid materials.
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their mechanical performance is also evaluated through their glass transition temperature (Tg) (Guan et al, 2013, 2016). Tg is defined as the temperature range of glass transition, which is a reversible process occurring in material when its amorphous regions convert between glassy and rubbery states. The glass transition of silk-based materials directly correlates with the hydrogen-bond density and disordered fraction within silk (Hu et al, 2006). Thus, high Tg values typically display low extensibility yet high Young’s modulus (comparison of the strength and stiffness) and breaking strength of the material (Wang Y. et al, 2014).
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Atomic force (AFM) microscopy allows for surface structure, interactions and local mechanical properties to be investigated (Li et al, 1994; Kane et al, 2010; Brown et al, 2012; Menezes et al, 2013; Wang and Schniepp, 2018). The most straightforward charaterization methods for understanding the successful modification of silk fibers, however, are optical microscopy (Sponner et al, 2007; Zhao et al, 2017) and electron microscopy (Frische et al, 1998; Augsten et al, 2000; Du et al, 2006; Rousseau et al, 2007).
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The relationship between the macroscopic properties and the microscopic structure of spider silks have been adressed in several works (Grubb and Jelinski, 1997; Glišovicet al., 2008; Plaza et al, 2012), providing an explanation of the structure-function correlation of the material. For instance, to study the protein backbone solid 13C nuclear magnetic resonance (NMR) spectroscopy (Hronska et al, 2004; Holland et al, 2008; Wang et al, 2018b; Craig et al, 2019) and Raman spectroscopy (Sirichaisit et al, 2003; Colomban and Dinh, 2012) are frequently used. These methods are used to determine spidroin’s secondary structure. Fourier-transform infrared (FTIR) spectroscopy and X-ray diffraction (XRD) analysis are two frequently used techniques when studying protein configuration and crystalline structure in silk-based materials. FTIR spectroscopy is a well-established experimental method used to study protein and polypeptide conformation, while synchrotron radiation FTIR (S-FTIR) microspectroscopy is succesfully applied in the investigation of silk spidroin conformation in single silk fibers (Ling et al, 2011). According to the method described by Madurga et al (2017), FTIR spectroscopy provides information on spidroin secondary structure. The contribution of the secondary structures is attained from the mathematical processing of the amide I band (a characteristic band of polypeptide absorbance at about 1,650 cm−1). This method is extensively used for silk-based hybrid materials as it reveals the influence of the doping phases on the secondary sructure content in the protein backbone of the material. Both XRD and wide-angle XRD (WAXD) are also widely applied to study the crystalline structure, crystallite size and orientation in the materials (Sheu et al, 2004; Trancik et al, 2006; Sampath et al, 2012; Jenkins et al, 2013).
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Hybrid materials based on spider silk proteins and metal nanoparticles for the immobilization of nanoobjects on biomolecules for use in biochemistry, biotechnology, and medicine are currently being extensively studied. Many proteins can be conjugated to metallic colloids by simply mixing them with a pre-synthesized metallic colloidal sol. The metal is usually bonded to the amines found along the protein backbone. Complementing at least two different types of materials causes the resulting hybrid material to display extraordinary properties. In this context, both natural and recombinant spider silk materials show outstanding mechanical and biocompatible properties. For high-performance fibers, natural spider silks are used to make composites with inorganic nanoparticles. Combination of the inorganic nanostructures and the biomaterials offers great opportunities in engineering innovative functional devices such as biosensors and actuators.
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Fiber Coating and Impregnation Approaches One of the most interesting approaches of spider silk coating was developed by Lee et al (2009). In their work, spider dragline silk was used as a template for the deposition of zinc (Zn), titanium (Ti), and aluminum (Al) by multiple pulsed vaporphase infiltration. Notably, the authors report the formation of metal-protein complexes with long exposure to water vapor resulting in the breaking of inner hydrogen bonds of the silk
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via bombardment from water vapor molecules. Thus, after longterm exposure to the metal precursor vapor, metal ions tend to bind to the broken bonding sites, resulting in the formation of metal-coordinated or covalent bonds. Due to this treatment, the toughness of hybrid spider silk fibers increases (Lee et al, 2009).
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Another possible straightforward approach for obtaining natural spider silk fibers-based hybrid materials without significant changes to the protein backbone is by dipcoating natural spider silk fibers in nanoparticle suspensions. Natural spider silk–magnetite (Fe3O4) hybrids created via the aforementioned method have well-defined relatively stable coatings, possibly due to hydrogen bonding interactions at the oxide-silk interface. By exploiting the magnetic properties of these nanoparticles, such hybrid functional fibrous materials could be integrated into audio devices, where durable fabrics responding to a magnetic field are required (Mayes et al, 1998). The method is easy to perform and environmentally neutral, providing an opportunity for the routine fabrication of a variety of spider silk hybrids (Figure 4). As an example of the versatility of this approach, natural spider silk fibers dipcoated with hydrophobically functionalized gold nanoparticles were successfully fabricated in a similar way (Mayes et al, 1998). Additionally, a recent study on the fabrication of an optically active spider silk hybrid reports the successful attachment of ZrO2 and HfO2 upconversion nanoparticles to natural spider silk fibers by a simple and straightforward impregnation procedure. Namely, hybrids were formed by stirring of spider silk fibers in nanoparticle alcohol solution. It was shown that the upconversion luminescent properties of these nanoparticles could be extended to the macroscale components of the fibrous hybrid while the spidroin backbone remains unaffected. Such spider silk–inorganic nanoparticle-based hybrid materials may have prospective bio-applications in the fields of biosensing and bioimaging in a non-invasive and real-time manner (Kiseleva et al, 2019).
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Another interesting approach to fabricating hybrid materials is the layer-by-layer electrostatic absorption method. For
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instance, spider silks were successfully coated with CdTe quantum dots by consecutively assembling CdTe nanocrystals and polyelectrolyte macromolecules onto spider silk fibers. The spider silk–CdTe hybrids exhibited core–shell structure characteristics and emitted extremely bright fluorescence while spider silk’s mechanical properties were unaffected. This fluorescent spider silk may find applications in microelectronics and biomedicine (Chu and Sun, 2007).
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Understanding the compatibility between spider silk and conducting materials is essential to further applications of spider silk in electronics. Therefore, noteworthy is the water-based and shear-assisted coating method of fabricating tough, versatile, flexible, and multi-functional spider silk–carbon nanotube hybrid fibers. Recent research has shown that the strong affinity of amine-functionalized multi-walled carbon nanotubes for spider silk is due to the structural changes in the carboxylic acid of spider silk. The charge carrier transport in those hybrids was primarily driven by inter-tube charge hopping. The conductivity of hybrid fibers was reversibly sensitive to strain and humidity, leading to custom-shapeable sensors and actuating devices (Steven et al, 2013). In the previously named study, the same group deposited a thin metallic film of gold nanoparticles to obtain sufficiently flexible natural spider silk– gold hybrid fibers to be used as electrodes in microelectronics (Steven et al, 2011). Additionally, multifunctional hybrid fibers with outstanding flexibility and conductivity were fabricated by wrapping a thin film of carbon nanotubes on natural spider silk. The fabrication of carbon nanotube-wrapped spider silk was done using the dry-coating and wet-collapsing method. The carbon nanotube film was adhered to the spider silk surface through ethanol action. The hybrid spider silk–carbon nanotube fiber could direct cell growth and simultaneously record signals evoked from cell beating without degradation over an extended period. Moreover, such a thin coating did not radically influence the mechanical properties of spider silks (Hou et al, 2018).
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Nanoparticles Synthesis Using Natural Spider Silk Spider silk has been used as a template in the synthesis of nanoparticles. This nanoparticle synthesis approach is of interest because of the simplicity of the process, its eco-friendliness, and the reduced amount of chemicals used (Salem et al, 2014). The utilization of biological substances and macromolecules during the synthesis of inorganic nanoparticles allows to avoid the production of unwanted or harmful by-products. Spider silk was recently reported to serve as an appropriate biological capping and stabilization agent that contributes to the steady rise of the synthesis of nanoparticles. In this technique, spider silk acts as an excellent scaffold for one-step nanoparticle synthesis (Figure 5). This approach results in the formation of environmentally friendly hybrids based on nanoparticles and spider silk. It has been reported that naturally spun spider silk fibers were used in the synthesis of gold nanoparticle bioconjugates via the spontaneous reduction of gold ions at spider silk (Singh et al, 2007). Here, spider silk served as a template for fiber incubation in aqueous chloroauric acid. Apparently, the binding of the gold nanoparticles to the spider silk was due to the strong interaction of spidroin amine groups with the gold surface (Selvakannan et al, 2003). This approach illustrates that spider silk modulates electron transport between nanoparticles on the hybrid surface, making it a promising candidate for the development of materials for vapor-sensing applications (Singh et al, 2007).
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Hydrolyzed natural spider silk is also possible to be used for the synthesis of nanoparticles. In this context, spider silk was hydrolyzed in sodium hydroxide (NaOH) and added to FIGURE 6 | Schematic illustration of spider silk-based hybrid material production via biomineralization approach.
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silver nitrate (AgNO3) solution for the reduction of silver ions. Spidroins served as capping and stabilization molecules during the synthesis of silver nanoparticles as the carboxylate groups obtained from the alkaline degradation of spider silk served as a reducing agent in the generation of silver nanoparticles, while COO− and NH2+ groups stabilized the silver nanoparticles and prevented their precipitation. The resulting hybrid solution exhibited antimicrobial activities against several multi-drug resistant clinical bacteria. This approach can be used to fabricate materials that protect against a microbial attack (Lateef et al, 2016). The same group of scientists later documented anticoagulant and thrombolytic properties of these hybrid materials (Lateef et al, 2017). Another example of regenerated (or in other words dissolved) natural spider silk application for the preparation of spider silk fiber hybrids is the synthesis of magnetite (Fe3O4) nanoparticles. Magnetite nanoparticles were successfully attached to spider silk hydrated in tetrabutylammonium hydroxide (C16H37NO). The authors note the preservation of the chemical amino acid block structures of the spidroins during synthesis. The materials generated were biocompatible and showed antibacterial properties, proving their potential therapeutic applications (Singh et al, 2015).
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Recently, a carbon nanofiber synthesis method was also demonstrated via simple pyrolysis, using natural spider silk as a precursor. The resulting materials exhibited superior oxygen reduction reaction activity compared to that of carbon nanofibers prepared using metal-free carbon catalysts in alkaline conditions, thus providing opportunities for unconventional microbial energy harvesting (Zhou et al, 2016).
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Biomineralization Recently, progress in biology has enabled the proteins and peptides responsible for the precipitation of inorganic materials within cells and controlling their nucleation and crystal growth (Figure 6) to be distinguished. This can be exploited for the mineralization of inorganic particles. The mechanism underlying this biomineralization approach of semiconductor metal oxides use hydrolyzed spider silk peptides, which include nucleophilic hydroxyls of cysteine, aspartic acid, and histidine amines. These specific groups bind, for example, zinc oxide (ZnO) nanoparticles and promote the crystal growth of hierarchical ZnO particles via the aggregation-driven mineralization with spidroin peptides under mild conditions. Such biomineralized ZnO materials coupled with spider silk may be used as biosensors for biomolecular detection or as optoelectronic nanodevices (Huang et al, 2008). Similarly, the biomineralization of natural spider silk fibers resulted in the crystallization of calcite on the spider silk substrate (Mehta and Hede, 2005). Likewise, spider silk–calcite hybrids were obtained with a pure calcite phase on the spider silk surface. The mechanical properties of spider silk complemented the inelastic ones of calcite, which is promising in the design of bone grafts for osseous tissue replacement materials (Dmitrovicet al., 2016).
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In the same way, it is possible to biomineralize spider silks with hydroxyapatite (HAP) via repetitive cycles of silk incubation in calcium phosphate rich solutions and washing with water. Notably, the biomineralization of natural spider silk fibers with hydroxyapatite yielded in the aligned orientation of c-axis of hydroxyapatite crystals along the spider silk fiber axis. This can be explained by the interactions between hydroxyapatite crystals and spidroins aligned along the long axis of the fiber by elongational flow during the natural silk spinning process. Moreover, hydroxyapatite crystal growth was consistent with the orientation of β-sheet crystals in the silk fibers (Cao and Mao, 2007). Both spider silk–calcium carbonate hybrids and spider silk–hydroxyapatite hybrids allowed for the production
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of new scaffold materials for bone tissue engineering or bone replacement materials (Mehta and Hede, 2005; Cao and Mao, 2007). In this context, a similar approach to creating spider silk–silica hybrids is to coat spider silk with silica precursors [such as tetraethylorthosilicate (TEOS)] and then subsequently heat it at 105◦C. Later, the silk template can be removed via calcination at 600◦C, resulting in the formation of materials with their pore structure determined by the silk template (Huang et al, 2003).
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Bio-integration of Nanoparticles Into Natural Spider Silk The evolution of biominerals in the protein matrix of natural materials is known to enhance their mechanical properties (Zhang, 2002). In this regard, it is possible to artificially incorporate diverse nanomaterials in spider silk protein structures aimed at improving the mechanical properties of the material. Thus, a method for producing spider silk fibers directly spun by spiders and reinforced by carbon nanotubes and graphene has been documented (Figure 7). Here, having been fed appropriate aqueous dispersions, spiders spun graphene and carbon nanotube incorporated silk. This approach yields fibers with greatly enhanced mechanical properties surpassing those of synthetic polymeric high-performance fibers. This observation indicates that the nanomaterials can be successfully inserted into the spider silk fibers. This approach of the natural integration of reinforcements in biological structural materials can be extended to other biological systems and lead to a new class of bionic hybrid materials (Lepore et al, 2017).
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Bioengineered spider silk can be functionalized to create hybrid materials with new functions or modified properties. Genetic modification of silk increases the ability of silk nanoparticles to bind and accumulate inside cells, which in turn improves the efficiency of drug delivery.
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Hybrid Formation via Blending Nanoparticles With Spider Silk Proteins Advanced antimicrobial hybrid materials were designed for sustainable drug release when combatting bacterial and fungal infections by combining the engineered eADF4(C16) spider silk protein and antimicrobial loaded silica nanoparticles. The silica– eADF4(C16) hybrids were made into different morphologies. The resulting hybrids showed excellent performance in terms of antimicrobial properties (Kumari et al, 2020).
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Kucharczyk et al (2019) used three bioengineered spider silk proteins with different amino acid compositions derived from dragline silk proteins for hybrid production. These proteins were blended with iron oxide nanoparticle suspensions and formed spheres (Figure 8). The spider silk–iron oxide spheres were fabricated by salting the silk proteins–iron oxide nanoparticle suspension with a potassium phosphate solution. The resulting hybrid spheres showed promise to transport and release drugs. These nanomaterials have great potential in both magnetic resonance imaging applications and hyperthermia combined with drug delivery therapy against cancer cells (Kucharczyk et al, 2019). Another example is the fabrication of hybrid films made from recombinant spider silk proteins and singlewalled carbon nanotubes. These materials exhibited exceptional mechanical properties due to stress transfer in the silk protein matrix to the inorganic filler and the potential for extensive matrix reorganization under applied stress (Blond et al, 2007).
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Additionally, ceria (CeO) nanoparticles were added in situ to a recombinant spider silk solution and electrospun into hybrid nanofibers forming mats. In the hybrids, the embedded ceria nanoparticles introduced new mechanical and optical properties to spider silk nanofibers due to optical trivalent cerium ions, associated with the oxygen vacancies formed. Thus, the synthesized hybrids can be applied in different biomedical, sensing, energy spheres (Kandas et al, 2018).
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Biomineralization and Genetic Fusion Spider silk can assemble with organic and inorganic structures at different levels. It was shown that engineered spider silk binds with other structures via click chemistry and biotechnological approaches. In terms of biomineralization, the modification of FIGURE 8 | Schematic illustration of hybrid formation via blending nanoparticles with bioengineered spider silk proteins.
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recombinant spider silk proteins with specific binding motifs for hydroxyapatite (Huang et al, 2007), titanium dioxide, germania, and gold leads to various morphologies and allows for the control of organic-inorganic interfaces and structural features (Foo et al, 2006; Mieszawska et al, 2010a; Belton et al, 2012). The incubation of genetically engineered chimeric protein β-sheet
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rich films based on dragline spidroin and dentin matrix protein 1 in the simulated body fluid caused the growth of hydroxyapatite crystals on the film surface (Huang et al, 2007).
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Silaffin proteins (known as R5 peptide), responsible for silica mineralization in nature, are used for specific purposes. The genetically engineered chimeric spider silk proteins and R5 peptide have been reported to promote both self-assembly and biomineralization, as well as the biomimetic synthesis of spider silk–silica fusion proteins through combining the selfassembling domains of spider dragline silk and R5 peptides (Foo et al, 2006; Mieszawska et al, 2010b). With genetic control over nanodomain sizes and chemistry, as well as modification of synthetic conditions for silica formation, silk proteins self-assemble into highly stable β-sheet structures. The sizes and distributions of the silica components are controlled during the bioengineering process. The presence of silica in the silk films influenced osteogenic gene expression. These results indicate the potential use of these new silk–silica hybrid systems for bone regeneration. This approach can be extended to introduce alternative fusions of inorganic phases for other applications (Foo et al, 2006; Mieszawska et al, 2010b) (Figure 9).
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An aqueous sol-gel process combined with microwaveassisted dissolution of hydrophobic synthetic spider silk was also shown to fabricate silk–silica hybrid particles. The incorporation of spider silk into the sol-gel process resulted in relatively spherical silk hybrids with tunable sizes and morphologies (Yang et al, 2010). The organo- and fluoro-silanes influence the spider silk secondary structures through varying β-structures in silk– silica hybrids. The ability to induce the defined secondary structures in the silk protein–silane hybrid particles may allow for bottom-up design of bioactive materials, where subsequent epitaxial growth and biomineralization can be tuned (Giasuddin and Britt, 2019). Additionally, thick homogeneous biomimetic crystalline calcium phosphate coatings were deposited onto the bioengineered spider silk fibers (produced from recombinant minispidroins) in a supersaturated simulated body fluid via the mineralization process. These hybrid fibers supported the attachment and growth of human mesenchymal stem cells, indicating prospective biomedical use of these functional materials (Yang et al, 2010).
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Notably, two different biogenic materials, spider silk and magnetosomes, can be genetically combined, thereby generating a new hybrid composite with novel properties and enhanced application potential. In this respect, magnetosomes, which are natural magnetic nanoparticles with exceptional properties synthesized in magnetotactic bacteria via biomineralization, were used for encapsulation in biocompatible polymers to enhance their usability (Mickoleit et al, 2018). The genetic fusion of spider silk proteins with magnetosome membrane proteins were reported to enhance magnetite biomineralization and even cause the formation of a proteinaceous capsule, increasing the colloidal stability of the isolated particles. Furthermore, spider silk peptides fused to a magnetosome membrane protein can guide silk fibril growth on the magnetosome surface. This combination of two different biogenic materials generated a genetically encoded hybrid composite with new engineerable properties for various biotechnological and biomedical applications (Mickoleit et al, 2018).
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