Compression and heat-assisted production of silk-based materials

Abstract

In some embodiments, the present disclosure provides a method comprising: (i) providing a silk fibroin material comprising a substantially amorphous structure; and (ii) applying at least one of high temperature and high pressure to the silk fibroin material to form a silk fibroin article, wherein the applying step induces fusion between at least a portion of the silk fibroin and a structural change of the fibroin in the silk fibroin material. In some embodiments, the present disclosure also provides a silk fibroin article made according to the method disclosed herein.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application is related to and claims priority based on U.S. Provisional Patent Application No. 62 / 743,975, filed on October 10, 2018, and the entire disclosure thereof is incorporated herein by reference for all purposes.

[0002] Statement Regarding Federally Sponsored Research This invention was made with government support under grants AR068048 and DE016525 awarded by the National Institutes of Health of the United States, and grant FA9550 - 17 - 1 - 0333 awarded by the United States Air Force. The government has certain rights in this invention.

Background Art

[0003] Background Silk is generally defined as a protein polymer naturally produced by the larvae of Lepidoptera such as silkworms, spiders, scorpions, and flies. Bombyx mori silk has been commercially used in textile production for centuries and is recognized as one of the most studied protein - based materials in history. Bombyx mori silk and spider silk are the most studied and widely used, and due to their biocompatibility, biodegradability, and exceptional mechanical properties, they are excellent materials for biomedical applications such as drug delivery and tissue engineering. Bombyx mori silk has been used as a suture material for centuries, but in recent years, it has attracted great attention as a biomaterial with various medical applications due to its adjustable degradation rate in vivo and its ability to be fabricated into multiple types of materials such as fibers, films, gels, and foams. However, compared to the conventional polymer industry, the technologies developed for silk processing and the production of silk - based materials are very limited.

Summary of the Invention

Means for Solving the Problems

[0004] Abstract The compositions and methods described herein relate, inter alia, to the production of silk-based materials using novel compression and heat-based methods. The silk-based materials herein include pure silk materials (e.g., silk films, silk monolith materials) and / or composite materials (e.g., silk-graphene composite materials, silk-enzyme composite materials). The processes described herein have been shown to produce a variety of silk materials with properties comparable to or even superior to those of materials produced by solution-based processes, such as transparent silk films, silk orthopedic devices, and functional silk patterns. Further, in some embodiments, these processes can be used to produce functional silk formats that cannot be achieved by solution-based processes, such as silk materials with controllable degradability by incorporating silk-degrading enzymes and conductive silk-graphene composite materials. In some embodiments, the manufacturing techniques described herein can be extended to other structural biopolymers such as collagen and recombinant silk proteins.

[0005] In some embodiments, the present disclosure provides a method comprising (i) providing a silk fibroin material comprising a substantially amorphous structure, and (ii) applying at least one high temperature and high pressure to the silk fibroin material to form a silk fibroin article, wherein the applying step induces a fusion between at least a portion of the silk fibroin and a structural change of the fibroin in the silk fibroin material. In some embodiments, the silk fibroin material is converted into a solid silk product that has undergone a conformational change of silk and comprises at least a portion of the silk fibroin fused together.

[0006] In some embodiments, the present disclosure provides a method comprising: (i) selecting high temperature and high pressure to produce a silk fibroin article having a desired crystallinity and desired material properties; and (ii) applying the high temperature and high pressure to a silk fibroin material having a substantially amorphous structure to form a silk fibroin article, the silk fibroin article having the desired crystallinity and desired material properties.

[0007] In some embodiments, the present disclosure also provides a silk fibroin article made according to the methods disclosed herein. In some embodiments, the silk fibroin article is or comprises a packaging material. In some embodiments, the packaging material is suitable for use in the manufacture of electronic devices, drug delivery systems, patterning, molding, and any combination thereof. In some embodiments, the silk fibroin article comprises semi-crystalline silk fibroin, and the glass transition temperature of the silk fibroin article is between about 40 °C and 135 °C. In some embodiments, the provided composition comprises both crystalline silk fibroin and amorphous silk fibroin. In some embodiments, the bending strength of the silk fibroin article is at least 5 MPa, and the substantially overall density of the silk fibroin article is at least 1.20 g / cm 3 3. In some embodiments, the silk fibroin article comprises silk in an amount of about 10% (w / w) or more. In some embodiments, the silk fibroin article degrades by at least 1 wt% after being exposed to an aqueous environment at 37 °C for 30 days.

[0008] In various embodiments, the present disclosure encompasses the discovery that the application of certain combinations of high temperature and / or high pressure can result in the crystallization of silk molecules in silk fibroin materials. In some embodiments, crystallization may involve a transition from an amorphous state to a semi-crystalline or crystalline structure and / or a β-sheet structure. In some embodiments, by applying high temperature and / or pressure, the amount of β-sheets in a silk fibroin article may increase by at least 1% compared to the level of β-sheets in the silk fibroin material. In some embodiments, the applying step causes the amount of β-sheets in the silk fibroin article to increase by at least 50% compared to the level of β-sheets in the silk fibroin material. As discussed herein, the present disclosure provides a series of new methods for providing silk fibroin articles that include the application of each of at least one period of exposing the silk fibroin material to high temperature and at least one period of exposing the silk fibroin material to high pressure. In some embodiments, the high temperature and high pressure are applied simultaneously. In some embodiments, the high temperature is applied to the silk fibroin material after the high pressure has been applied. In some embodiments, the applying step is performed, at least in part, within a mold. In some embodiments, the application is not performed within a mold.

[0009] As discussed herein, the present disclosure provides a series of new methods for providing silk fibroin articles that include the application of each of at least one period of exposing the silk fibroin material to high temperature and at least one period of exposing the silk fibroin material to high pressure. In some embodiments, the high temperature and high pressure are applied simultaneously. In some embodiments, the high temperature is applied to the silk fibroin material after the high pressure has been applied. In some embodiments, the applying step is performed, at least in part, within a mold. In some embodiments, the application is not performed within a mold.

[0010] In some embodiments, the applying step is or includes a hot press. In some embodiments, the hot press is performed at a pressure of at least 1 MPa. In some embodiments, the hot press is performed at a temperature between 25 °C and 200 °C.

[0011] In some embodiments, the silk fibroin article is substantially transparent. In some embodiments, the silk fibroin article is bioabsorbable. In some embodiments, the silk fibroin article has thermoforming properties and can be reshaped into a desired shape at high temperature or high pressure.

[0012] The present disclosure also encompasses, in some embodiments, compositions comprising one or more additives (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more additives). In some embodiments, the additives can enhance one or more properties of the provided composition (e.g., physical properties, mechanical properties, etc.). In some embodiments, prior to the applying step, the silk fibroin material is mixed with at least one additive to form a composite silk fibroin article. In some embodiments, the additives are small organic or inorganic molecules, organic polymers, inorganic polymers, biopolymers, such as peptides and proteins; conductive materials, carbon-based materials, antibodies and their antigen-binding fragments; antigens; nucleic acids; nucleic acid analogs and derivatives; saccharides; immunogens; natural compounds and extracts from biological systems such as cells, bacteria, or tissues; synthetic materials; metallic materials; alloys; hydrophobic materials; hydrophilic materials; nanomaterials; and at least one of, or including, any combination thereof. In some embodiments, the organic polymer is, or includes, at least one enzyme, such as a protease. In some embodiments, the activity of the enzyme is stabilized by silk under high temperature and pressure. In some embodiments, the protease is one or more of, or includes, protease XIV, proteinase K, α-chymotrypsin, collagenase, matrix metalloproteinase-1 (MMP-1) and MMP-2. In some embodiments, the conductive material is, or includes, an inorganic conductive material (e.g., silver, gold, iron oxide), an organic conductive material (e.g., graphene), a metal, an alloy, a semiconductor material and / or a conjugated polymer. In some embodiments, the additives are mixed with the silk fibroin material at a ratio between 0.001 wt% and 95.0 wt%.

[0013] The methods and compositions (e.g., articles) provided can include one or more physical and / or mechanical properties not observed when using conventional known methods. By way of example, in some embodiments, silk fibroin articles are substantially homogeneous. In some embodiments, a provided silk article is considered to be substantially homogeneous if the article exhibits a consistent structure throughout most of the article (e.g., at least 70%, 80%, 90%, 95% or more of the silk article exhibits a consistent structural pattern).

[0014] Further, in certain embodiments, the methods provided use multiple techniques to enable the production of complex articles that would result in articles significantly different (e.g., with respect to physical and / or mechanical properties) when applied using conventional methods. According to some embodiments, the methods provided can be used to produce any of a variety of silk fibroin articles. In some embodiments, the silk fibroin articles can be, or can include, films, fibers, meshes, needles, tubes, plates, screws, rods, or any desired shape.

[0015] Further, in certain embodiments, the methods provided use multiple techniques to enable the production of complex articles that would result in articles significantly different (e.g., with respect to physical and / or mechanical properties) when applied using conventional methods. According to some embodiments, the methods provided can be used to produce any of a variety of silk fibroin articles. In some embodiments, the silk fibroin articles can be, or can include, films, fibers, meshes, needles, tubes, plates, screws, rods, or any desired shape.

[0016] The methods and compositions provided enable the production of silk articles that exhibit enhanced properties of one or more characteristics. In some embodiments, the silk fibroin articles may have enhanced thermal or electrical properties. In some embodiments, the silk fibroin articles are conductive. According to various embodiments, the compositions provided may be suitable for patterning (e.g., on the surface of the composition).

[0017] In some embodiments, the compositions provided may be biocompatible and / or biodegradable. According to various embodiments, the methods and compositions provided enable any of a variety of degradation profiles, which in turn enable a wide variety of or potential applications. For example, in some embodiments, the silk article degrades by at least 50 wt% after being exposed to an aqueous environment at 37 °C for about 96 hours. As a further example, in some embodiments, the silk fibroin article does not degrade by more than 10% even after being exposed to in vivo environments or conditions for 6 months.

[0018] Any of a variety of forms of silk fibroin materials can be used according to the various methods and compositions provided. Generally, any silk fibroin material that contains a significant amount of amorphous silk can be used according to some embodiments. By way of non-limiting example, in some embodiments, the silk fibroin material may be, or may contain, particles, films, and / or fibers. In some embodiments, the particles may be, or may contain, at least one microparticle and nanoparticle.

[0019] In some embodiments, the silk article provided may be made by a process that includes the step of converting a silk fibroin material into a solid silk article that has undergone a conformational change of silk and includes at least a portion of the silk fibroin that has transitioned from an amorphous state to a semi-crystalline structure.

[0020] Citations to publications, patents, or patent applications in this specification are incorporated by reference in their entirety. The numbers used in this application are intended to cover normal variations understood by those of ordinary skill in the relevant art, regardless of whether they are accompanied by the words "about" or "approximately".

[0021] Other features, objects, and advantages of this specification will become apparent in the following detailed description. However, it should be understood that the detailed description is provided by way of illustration only and not by way of limitation, although it shows embodiments of the invention. Various changes and modifications within the scope of the invention will be apparent to those skilled in the art from the form for carrying out the invention.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0052] Detailed Description Definition

[0053] Unless otherwise apparent from the context, in this application: (i) the term "a" may be understood to mean "at least one"; (ii) the term "or" may be understood to mean "and / or"; (iii) the terms "comprising" and "including" may be understood to include the listed components or steps whether presented by themselves or in conjunction with one or more additional components or steps; (iv) the terms "about" and "approximately" are used interchangeably and may be understood to permit the standard variations understood by those of ordinary skill in the art; and (v) when ranges are recited, endpoints are included.

[0054] Approximately: As used herein, the term "approximately" or "about" when applied to one or more values of interest refers to a value similar to the recited reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either (greater than or less than) direction from the recited reference value, unless otherwise specified or apparent from the context (so long as such numbers are not greater than 100% of the possible value).

[0055] Biocompatible: As used herein, the term "biocompatible" refers to a material that, for example, does not cause significant harm to living tissue when placed in contact with such tissue in vivo. In certain embodiments, materials are "biocompatible" if they are not toxic to cells. In certain embodiments, a material is "biocompatible" if addition of the material to cells in vitro results in no more than 20% cell death and / or if administration of the material in vivo does not induce significant inflammation or other adverse effects.

[0056] Biodegradability: As used herein, the term "biodegradable" refers to a material that, when introduced into a cell, can be recycled or processed by the cell (e.g., by cell mechanisms such as enzymatic degradation, hydrolysis, and / or combinations thereof) without causing significant toxic effects to the cell. In certain embodiments, the components produced by the degradation of the biodegradable material are biocompatible and thus do not induce significant inflammation and / or other adverse effects in vivo. In some embodiments, the biodegradable polymer material is degraded into its constituent monomers. In some embodiments, the degradation of the biodegradable material (e.g., including a biodegradable polymer material) involves hydrolysis of ester bonds. Alternatively, or in addition thereto, in some embodiments, the degradation of the biodegradable material (e.g., including a biodegradable polymer material) involves cleavage of urethane bonds. Examples of biodegradable polymers include, for example, polymers of hydroxy acids such as lactic acid and glycolic acid, including, but not limited to, poly(hydroxy acid), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), and copolymers with PEG, polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(butyric acid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoate), poly(lactide-co-caprolactone), blends and copolymers thereof. Many naturally occurring polymers are also biodegradable, including, for example, albumin, collagen, gelatin and prolamins, such as proteins like zein, and polysaccharides such as alginates, cellulose derivatives and polyhydroxyalkanoates, such as polyhydroxybutyrate blends and their copolymers. One of ordinary skill in the art will be able to understand or determine that such polymers are biocompatible and / or their biodegradable derivatives (e.g., those related to the parent polymer with a substantially identical structure differing only by substitution or addition of certain chemical groups as known in the art).

[0057] Compression: As used herein, the term "compression" refers to the process by which a material gradually loses its porosity due to the influence of a load.

[0058] Composition: As used herein, may be used to refer to an individual physical entity that contains one or more specified components. Generally, unless otherwise specified, a composition may be in any form, such as a gas, gel, liquid, solid, etc. In some embodiments, "composition" may refer to a single embodiment or a combination of two or more entities for use as part of the same article. In all embodiments, it is not necessary for the combination of entities to result in a physical mixture, i.e., the components of the composition can be combined as separate co-entities. However, many practitioners in the art may consider it advantageous to prepare a composition by mixing two or more components with a pharmaceutically acceptable carrier, diluent, or excipient to enable the simultaneous administration of the combination of components.

[0059] Fusion: As used herein, the term "fusion" refers to the process of combining two or more separate entities into a new whole.

[0060] Hydrophilic: As used herein, the terms "hydrophilic" and / or "polar" refer to the tendency to mix readily with water or to dissolve easily in water.

[0061] Hydrophobic: As used herein, "hydrophobic" and / or "non-polar" refer to the tendency to repel water, not to bind to water, or not to dissolve easily in water.

[0062] Improve, increase, or decrease: As used herein or its grammatical equivalents, indicate a value relative to a baseline measurement, such as a measurement in a similar composition prepared according to a previously known method.

[0063] Macro - particles: As used herein, the term "macro - particles" refers to particles having a diameter of at least 1 millimeter. In some embodiments, macro - particles are micelles in that they contain an enclosed compartment separated from the bulk solution by a micelle membrane and are generally composed of amphiphilic entities that surround a space or compartment (e.g., to define a lumen). In some embodiments, the micelle membrane is composed of at least one polymer such as, for example, a biocompatible and / or biodegradable polymer. In some embodiments, a population of particles is considered a population of macro - particles if the average diameter of the population is equal to or greater than 1 millimeter.

[0064] Micro - particles: As used herein, the term "micro - particles" refers to particles having a diameter between 1 micrometer and 1 millimeter. In some embodiments, micro - particles are micelles in that they contain an enclosed compartment separated from the bulk solution by a micelle membrane and are generally composed of amphiphilic entities that surround a space or compartment (e.g., to define a lumen). In some embodiments, the micelle membrane is composed of at least one polymer such as, for example, a biocompatible and / or biodegradable polymer. In some embodiments, a population of particles is considered a population of micro - particles if the average diameter of the population is between 1 micrometer and 1 millimeter.

[0065] Nanoparticles: As used herein, the term "nanoparticle" refers to particles having a diameter of less than 1000 nanometers (nm). In some embodiments, the diameter of the nanoparticle is less than 300 nm according to the definition of the National Science Foundation of the United States. In some embodiments, the diameter of the nanoparticle is less than 100 nm according to the definition of the National Institutes of Health of the United States. In some embodiments, the nanoparticle is a micelle in that it contains an enclosed compartment separated from the bulk solution by a micelle membrane and is generally composed of an amphiphilic entity surrounding a space or compartment (e.g., to define a lumen). In some embodiments, the micelle membrane is composed of at least one polymer such as, for example, a biocompatible and / or biodegradable polymer. In some embodiments, a population of particles is considered a population of nanoparticles if the average diameter of the population is equal to or less than 1000 nm.

[0066] Physiological conditions: As used herein, it has the meaning understood in the art of conditions for cells and organisms to survive and / or reproduce. In some embodiments, the term refers to the conditions of the external or internal environment that can occur naturally in an organism or cell line. In some embodiments, physiological conditions are the conditions present in the body of a human or non-human animal, particularly at and / or within the surgical site. Physiological conditions generally include, for example, a temperature range of 20 to 40 °C, 1 atmospheric pressure, a pH of 6 to 8, a glucose concentration of 1 to 20 mM, an oxygen concentration at atmospheric pressure levels, and the gravity encountered on Earth. In some embodiments, laboratory conditions are manipulated and / or maintained under physiological conditions. In some embodiments, physiological conditions are found in organisms.

[0067] Pure: As used herein, a material, additive, and / or entity is “pure” if it substantially contains no other elements. For example, a preparation containing more than about 90% of a particular agent or entity is generally considered a pure preparation. In some embodiments, the agent or entity is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0068] Reference: As used herein, describes a standard or control against which a comparison is performed. For example, in some embodiments, a material, article, additive, entity or other sample, sequence or value of interest is compared to a reference or control material, article, additive, entity or other sample, sequence or value. In some embodiments, the reference or control is tested and / or determined substantially simultaneously with the test or determination of interest. In some embodiments, the reference or control is a historical reference or control and is embodied in a tangible medium as needed. Generally, as understood by one of ordinary skill in the art, the reference or control is determined or characterized under conditions or circumstances equivalent to those being evaluated. One of ordinary skill in the art will understand when there is sufficient similarity to justify reliance on and / or comparison to a particular possible reference or control.

[0069] Solid Forms: As is known in the art, many chemical substances (especially many organic molecules and / or many small molecules) can take on a variety of different solid forms, such as, for example, amorphous forms and / or crystalline forms (e.g., polymorphs, hydrates, solvates, etc.). In some embodiments, such an entity can be utilized as a single such form (e.g., as a pure preparation of a single polymorph). In some embodiments, such an entity can be utilized as a mixture of such forms.

[0070] Substantially: As used herein, the term "substantially" refers to a qualitative state indicating the entire or almost entire range or degree of a feature or characteristic of interest. Those skilled in the biological arts will understand that biological and chemical phenomena rarely, if ever, complete and / or proceed completely or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the lack of potential completeness inherent in many biological and chemical phenomena.

[0071] Compositions and Methods

[0072] This description includes, among other things, the recognition that applying, in certain ways, each of high temperature and high pressure for a period of one or more results in changes in the structure of silk fibroin in silk fibroin materials that were not previously known. In some embodiments, the disclosure includes the surprising discovery that silk fibroin articles can be made directly from amorphous silk fibroin materials without the need for solubilization of intermediates widely used in the art. Without wishing to be bound by any particular theory, it is specifically contemplated that the new structures enabled by the methods disclosed herein result, at least in part, from a combination of compression and fusion of silk fibroin materials.

[0073] As a natural protein-based biopolymer, silk in various material forms has promising features including biocompatibility and biodegradability in addition to excellent mechanical properties. Thus, silk has been utilized for decades as a material option for biomaterials and scaffolds in biomedical applications 1-4 including drug delivery, tissue engineering, and regenerative medicine. Natural silk is a semi-crystalline biopolymer material composed of nano-crystals of β-sheets embedded in an unorganized, low-crystallinity continuous phase due to the amphiphilic nature of protein chemistry. 5-7。The strong hydrogen-bond network of the nanocrystals of β-sheets contributes significantly to the stability and excellent mechanical properties of silk similar to cellulose, but this has been a challenge to the ability to thermally process silk-based materials without decomposing them. In fact, there are limited reports on the thermal melting and reconstitution of silk materials that require ultra-fast laser heating. 8、9 。Furthermore, historically, starting from Pauling's research on the basic structural features of silk fibroin, the antiparallel β-pleated sheet (antiparallel β-sheet) that forms the crystalline phase of silk has been very stable due to the well-aligned N-H-O hydrogen bonds. 6、10 。

[0074] Previously known methods of silk protein processing include solution-based processing, and otherwise the material often decomposed before melting. Over the past few decades, researchers have focused on extracting silk fibroin from fibers to make silk solutions and have put a significant amount of effort into the development of technologies for processing silk fiber materials. For example, silk fibers (degummed silk fibers) after removing the outer sericin can be dissolved in an aqueous solution of high salt concentration LiBr / CaCl 2 to produce a silk aqueous solution. Furthermore, degummed silk fibers are soluble in several organic solvents such as formic acid, trifluoroacetic acid, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), etc. These methods are aimed at downstream processing (solvent removal) to produce silk materials including gels, foams, films, new fiber forms, and related materials. 16-18 。The addition and removal of solvents related to the solubility limitations of proteins lead to new useful materials, but in addition to the limitations on material properties due to solubility limitations, it also incurs a great cost for various processing steps.

[0075] Prior to the present invention, methods using aqueous silk solutions as starting materials were preferred because they were more user-friendly and could be further used to create a variety of material forms such as films, foams, sponges, hydrogels, tubes, and bulk materials. However, due to the tendency of silk fibroin to self-organize in aqueous solutions, scale-up production and product quality control have become difficult challenges. Furthermore, in addition to solution-based processing methods, established techniques for the engineering and production of silk-based structural materials with tunable physical properties are very limited. To overcome these limitations and better process silk materials, a simple and effective strategy for directly converting amorphous natural silk powder into high-performance structural materials with tunable mechanical properties is disclosed herein.

[0076] In some embodiments, the methods disclosed herein include the production of amorphous silk nanomaterials (ASN) generated from aqueous silk fibroin solutions. The ASN can then be processed by hot pressing to effect the fusion and densification of silk (e.g., into silk articles). The resulting bulk silk material exhibits a higher specific strength than most natural structural materials and has been shown to be effective in the production of silk-based composite materials. Additionally, the engineered silk materials have been shown to have thermoforming properties and can be further deformed into a desired shape under appropriate conditions. FIG. 6 shows a schematic of a particular method provided that combines top-down and bottom-up approaches to convert natural silk fibers into bulk silk portions. In some embodiments, the compositions and methods described herein demonstrate a heat- and pressure-based, time-efficient, and controllable method for directly converting silk fibroin from silk fibroin materials containing a significant amount of amorphous silk fibroin (e.g., in powder form) into bulk structural materials. In some embodiments, the methods and compositions described herein may enable the application of more traditional processes and shaping techniques to silk materials that have not heretofore been successfully used with silk. Further, in some embodiments, the processing methods described herein avoid the need for solvent or aqueous approaches and provide a direct route for converting silk fibroin materials into parts. According to various embodiments, the methods described herein provide for the conversion of silk fibroin from an amorphous material into a semi-crystalline, high-performance structural material by the controlled application of heat and pressure. In some embodiments, the process provided induces a conformational transition of silk molecules from random coils to β-sheets. In some embodiments, the methods provided include degumming natural silk fibers, processing them by silk fibroin solubilization and lyophilization into amorphous silk materials (e.g., powders) to create suitable preformed materials; feeding the amorphous silk materials into a pre-designed mold; and inducing conformational and structural changes in the silk by applying heat and pressure.Furthermore, this method can also be processed with only silk, or an inorganic filler or a second polymer can be added to fabricate a composite device.

[0077] By using the processing utilizing the heat and pressure of the amorphous silk precursor material, various forms of silk materials such as plates, rods, screws, and tubes can be prepared with adjustable mechanical properties and thermoforming properties while maintaining the good biocompatibility and degradability characteristics of the material through the treatment using the heat and pressure of the amorphous silk precursor material. In some embodiments, the methods described herein enable an environmentally considerate and cost-effective method for converting natural fibers into silk monoliths compared to previously reported methods (Figure 27). This discovery greatly changes the landscape from the perspective of the processing methods of silk-based materials, enabling the application of conventional processing and forming techniques that have not been successfully adopted in silk to silk materials. The fabricated silk-based devices hold the potential for a variety of biomedical applications, including orthopedic implants. Furthermore, the heat and pressure-assisted method can be extended to other protein-based materials (such as recombinant proteins) for manufacturing protein-based monoliths.

[0078] In some examples, the methods described herein may include selecting high temperature and high pressure to produce a desired silk fibroin (fiborni) article with a desired crystallinity and desired material properties, and then applying the high temperature and high pressure to a silk fibroin material having a structure that is substantially amorphous. That is, the methods described herein can predictably select and apply temperature and pressure to produce an article having a desired crystallinity and material properties. This is different from other applications where heat and / or pressure can be applied to silk materials without pre-determining the desired results from the perspective of crystallinity and material properties.

[0079] The amount of plasticizer in the silk fibroin material can be adjusted to produce the desired crystallinity and material properties. This is particularly effective in low-temperature embodiments, and the amount of plasticizer is selected to produce the desired crystallinity and material properties. In some examples, the plasticizer is water.

[0080] Silk material

[0081] Any of a variety of silk materials can be used according to various embodiments. In some embodiments, the silk material may be silk fibroin (e.g., degummed or substantially sericin-free silk fibroin), or may include these. In some embodiments, the silk material may be silk powder (e.g., including a plurality of silk particles), or may include these.

[0082] In some embodiments, the silk material may be silk particles (e.g., microparticles or nanoparticles), or may include these. As used herein, the term "particle" includes spheres, rods, shells, prisms, and related structures. Although any particle size appropriate for the application is contemplated to be within the scope of the present disclosure, in some embodiments, the diameter of one silk particle is between 1 nm and 1,000 μm (e.g., between 1 nm and 1 μm, between 1 μm and 1,000 μm, etc.). In some embodiments, the diameter of the silk particle may be greater than 1,000 μm.

[0083] Various methods for producing silk particles (e.g., nanoparticles and microparticles) are known in the art. For example, a pulverizer (e.g., a Retsch planetary ball mill) can be used to produce silk powder. Generally, a ball mill consists of two or four sample cups arranged around a central axis, and each cup is adjusted to rotate both centrally and locally. Each ceramic cup is filled with small ceramic spheres. Various sizes are available. Balls with a diameter of 10 millimeters were used / are used in the grinding operations described in the present disclosure. As the cup rotates, the spheres grind the material within the cup into small characteristic sizes. Both degummed silk and non-degummed silk can be converted from the finely ground material into powder form within the ball mill.

[0084] In other embodiments, alternative powder forming techniques can be used (e.g., freeze drying or instant freezing and crushing). In other embodiments, an alternative grating of the pulverizer with larger holes can be used. This can result in the generation of larger silk particle sizes.

[0085] In some embodiments, silk particles can be produced using the lyophilization method described in U.S. Provisional Patent Application No. 61 / 719,146, filed October 26, 2012, the entire content of which is incorporated herein by reference in its entirety. Specifically, a silk foam can be produced by lyophilizing a silk solution. Next, the foam can be reduced to particles. For example, the silk solution can be cooled to a temperature at which the liquid carrier changes into a plurality of solid crystals or particles, and at least a portion of the plurality of solid crystals or particles is removed, leaving a porous silk material (e.g., a silk foam). After cooling, the liquid carrier can be removed, at least in part, by sublimation, evaporation, and / or lyophilization. In some embodiments, the liquid carrier can be removed under reduced pressure. After formation, the silk fibroin foam can be subjected to grinding, cutting, crushing, or any combination thereof to form silk particles. For example, the silk fibroin foam can be blended with a conventional blender or ground with a ball mill to form silk particles of a desired size.

[0086] In some embodiments, a silk fibroin material containing a substantial amount of amorphous structure is made from a silk solution and is composed of a nanostructure (as shown in FIG. 1). This may be referred to as nanosized silk powder (NSP) and can be part of a material called amorphous silk nanomaterial (ASN). In this specification, these terms are equivalent and can be used synonymously.

[0087] While not wishing to be bound by any particular theory, in some embodiments, the present disclosure encompasses the recognition that by using certain starting materials (e.g., a silk fibroin material containing a substantial amount of amorphous structure), it becomes possible to produce compositions that were not previously achievable. In some embodiments, the silk material is not made from solubilized silk. In some embodiments, the silk material may be lyophilized.

[0088] Silk fibroin

[0089] According to various embodiments, it can be used in a method of providing any silk fibroin. In some embodiments, the silk fibroin is selected from the group consisting of spider silk (e.g., from Nephila clavipes), silkworm silk (e.g., from Bombyx mori), and recombinant silk (e.g., silk produced / engineered from bacterial cells, yeast cells, mammalian cells, transgenic animals and / or transgenic plants). According to various embodiments, the silk used in the provided methods and compositions is degummed silk (i.e., silk fibroin from which at least a portion of the native sericin has been removed). Degummed silk can be made by conventional methods known to those skilled in the art. For example, the cocoons of B. mori are boiled in an aqueous solution for a predetermined time. Generally, as the degumming time increases, low molecular weight silk fibroin is produced. In some embodiments, the silk cocoons are boiled for at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes, at least 120 minutes, or more. Additionally or alternatively, in some embodiments, the silk cocoons can be heated or boiled at a warm temperature. For example, in some embodiments, the silk cocoons can be heated or boiled at about 101.0 °C, about 101.5 °C, about 102.0 °C, about 102.5 °C, about 103.0 °C, about 103.5 °C, about 104.0 °C, about 104.5 °C, about 105.0 °C, about 105.5 °C, about 106.0 °C, about 106.5 °C, about 107.0 °C, about 107.5 °C, about 108.0 °C, about 108.5 °C, about 109.0 °C, about 109.5 °C, about 110.0 °C, about 110.5 °C, about 111.0 °C, about 111.5 °C, about 112.0 °C, about 112.5 °C, about 113.0 °C, 113.5 °C, about 114.0 °C, about 114.5 °C, about 115.0 °C, about 115.5 °C, about 116.0 °C, about 116.5 °C, about 117.0 °C, about 117.5 °C, about 118.0 °C, about 118.5 °C, about 119.0 °C, about 119.5 °C, about 120.0 °C, or more.In some embodiments, such high temperatures can be achieved by performing at least some of the heating processes (e.g., boiling processes) under pressure. For example, suitable pressures at which the silk fibroin fragments described herein can be produced are generally between about 10 - 40 psi, such as about 11 psi, about 12 psi, about 13 psi, about 14 psi, about 15 psi, about 16 psi, about 17 psi, about 18 psi, about 19 psi, about 20 psi, about 21 psi, about 22 psi, about 23 psi, about 24 psi, about 25 psi, about 26 psi, about 27 psi, about 28 psi, about 29 psi, about 30 psi, about 31 psi, about 32 psi, about 33 psi, about 34 psi, about 35 psi, about 36 psi, about 37 psi, about 38 psi, about 39 psi, or about 40 psi.

[0090] In some embodiments, the aqueous solution used in the degumming process of silk cocoons contains about 0.02M of Na 2 CO 3 The cocoons are rinsed, for example, with water to extract the sericin protein. The degummed silk can be dried and used to make silk powder. Alternatively, the extracted silk can be dissolved in an aqueous salt solution. Salts useful for this purpose include lithium bromide, lithium thiocyanate, calcium nitrate, or other chemicals that can solubilize silk. In some embodiments, the extracted silk can be dissolved in an about 8M - 12M LiBr solution. The salt is then removed, for example, using dialysis.

[0091] In some embodiments, the silk fibroin has a substantially depleted natural sericin content (e.g., the residual sericin in the finally extracted silk is 5% (w / w) or less). In some embodiments, the silk fibroin contains no natural sericin content at all. As used herein, "contains no" (i.e., the term "consisting of") means that the substance cannot be detected or its presence cannot be confirmed within the detection range of the measuring instrument or process used. In some embodiments, the silk fibroin is essentially free of its natural sericin content. As used herein, the term "essentially free of" (or "consisting essentially of") means that only trace amounts of the substance can be detected, that it is present in amounts below the limit of detection, or that it is absent.

[0092] Optionally, the silk solution can be concentrated, for example, using dialysis against a hygroscopic polymer such as PEG, polyethylene oxide, amylose or sericin. In some embodiments, the PEG has a molecular weight of 8,000 to 10,000 g / mol and a concentration of about 10% to about 50% (w / v). A slide-a-lyzer dialysis cassette (Pierce, MW CO3500) can be used. However, any dialysis system may be used. Dialysis can be carried out for a time sufficient for the final concentration of the aqueous silk solution to be about 10% to about 30%. In most cases, dialysis for 2 to 12 hours may be sufficient. See, for example, International Patent Application Publication No. WO2005 / 012606, the contents of which are incorporated herein by reference in their entirety. Another method for producing a concentrated silk solution involves drying a dilute silk solution (e.g., by evaporation or lyophilization). The dilute solution can be partially dried to reduce its volume, thereby increasing the silk concentration. After the dilute solution is completely dried, the dried silk fibroin can be dissolved in a small amount of solvent compared to the dilute silk solution. In some embodiments, the silk fibroin solution can be filtered and / or centrifuged, if necessary, at an appropriate time. For example, in some embodiments, the silk fibroin solution can be filtered and / or centrifuged, if necessary, after a heating or boiling step. In some embodiments, the silk fibroin solution can be filtered and / or centrifuged, if necessary, after a dialysis step. In some embodiments, the silk fibroin solution can be filtered and / or centrifuged, if necessary, after a concentration adjustment step. In some embodiments, the silk fibroin solution can be filtered and / or centrifuged, if necessary, after a reconstitution step. In any of such embodiments, one or more filtration and / or centrifugation steps can be performed to remove insoluble materials. In any of such embodiments, one or more filtration and / or centrifugation steps can be performed to selectively concentrate silk fibroin fragments of a particular molecular weight.

[0093] In some embodiments, silk fibroin and / or silk fibroin articles may include a protein structure that substantially includes β-turn and / or β-strand regions. Without wishing to be bound by theory, the β-sheet content of silk may affect the gel function of the composition and its lifespan in vivo. It should be understood that compositions including non-β-sheet content (e.g., e-gels) may also be utilized. In some embodiments, silk fibroin has a protein structure that includes, for example, about 5% β-turn and β-strand regions, about 10% β-turn and β-strand regions, about 20% β-turn and β-strand regions, about 30% β-turn and β-strand regions, about 40% β-turn and β-strand regions, about 50% β-turn and β-strand regions, about 60% β-turn and β-strand regions, about 70% β-turn and β-strand regions, about 80% β-turn and β-strand regions, about 90% β-turn and β-strand regions, or about 100% β-turn and β-strand regions. In other aspects of these embodiments, silk fibroin has a protein structure that includes, for example, at least 10% β-turn and β-strand regions, at least 20% β-turn and β-strand regions, at least 30% β-turn and β-strand regions, at least 40% β-turn and β-strand regions, at least 50% β-turn and β-strand regions, at least 60% β-turn and β-strand regions, at least 70% β-turn and β-strand regions, at least 80% β-turn and β-strand regions, at least 90% β-turn and β-strand regions, or at least 95% β-turn and β-strand regions.In still other aspects of these embodiments, the silk fibroin has a protein structure comprising, for example, about 10% to about 30% beta-turn and beta-strand regions, about 20% to about 40% beta-turn and beta-strand regions, about 30% to about 50% beta-turn and beta-strand regions, about 40% to about 60% beta-turn and beta-strand regions, about 50% to about 70% beta-turn and beta-strand regions, about 60% to about 80% beta-turn and beta-strand regions, about 70% to about 90% beta-turn and beta-strand regions, about 80% to about 100% beta-turn and beta-strand regions, about 10% to about 40% beta-turn and beta-strand regions, about 30% to about 60% beta-turn and beta-strand regions, about 50% to about 80% beta-turn and beta-strand regions, about 70% to about 100% beta-turn and beta-strand regions, about 40% to about 80% beta-turn and beta-strand regions, about 50% to about 90% beta-turn and beta-strand regions, about 60% to about 100% beta-turn and beta-strand regions, or about 50% to about 100% beta-turn and beta-strand regions. In some embodiments, silk beta-sheet contents from less than 10% to about 55% can be used in the silk fibroin compositions disclosed herein.

[0094] In some embodiments, silk fibroin, or a silk fibroin article, has a protein structure that substantially does not contain α-helix and / or random coil regions. In aspects of these embodiments, silk fibroin has a protein structure that contains, for example, about 5% α-helix and / or random coil regions, about 10% α-helix and / or random coil regions, about 15% α-helix and / or random coil regions, about 20% α-helix and / or random coil regions, about 25% α-helix and / or random coil regions, about 30% α-helix and / or random coil regions, about 35% α-helix and / or random coil regions, about 40% α-helix and / or random coil regions, about 45% α-helix and / or random coil regions, or about 50% α-helix and / or random coil regions. In other aspects of these embodiments, silk fibroin has a protein structure that contains, for example, up to 5% α-helix and / or random coil regions, up to 10% α-helix and / or random coil regions, up to 15% α-helix and / or random coil regions, up to 20% α-helix and / or random coil regions, up to 25% α-helix and / or random coil regions, up to 30% α-helix and / or random coil regions, up to 35% α-helix and / or random coil regions, up to 40% α-helix and / or random coil regions, up to 45% α-helix and / or random coil regions, or up to 50% α-helix and / or random coil regions.In still other aspects of these embodiments, the silk fibroin has a protein structure that includes, for example, about 5% to about 10% alpha helix and / or random coil regions, about 5% to about 15% alpha helix and / or random coil regions, about 5% to about 20% alpha helix and / or random coil regions, about 5% to about 25% alpha helix and / or random coil regions, about 5% to about 30% alpha helix and / or random coil regions, about 5% to about 40% alpha helix and / or random coil regions, about 5% to about 50% alpha helix and / or random coil regions, about 10% to about 20% alpha helix and / or random coil regions, about 10% to about 30% alpha helix and / or random coil regions, about 15% to about 25% alpha helix and / or random coil regions, about 15% to about 30% alpha helix and / or random coil regions, or about 15% to about 35% alpha helix and / or random coil regions.

[0095] High temperature

[0096] As discussed herein, the provided methods and compositions include one or more exposures to high temperature. As used herein, the term "high temperature" refers to a temperature higher than standard room temperature (i.e., higher than 25°C). In some embodiments, the provided method or composition includes one exposure to high temperature. In some embodiments, the provided method or composition includes at least two (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) exposures to high temperature. In some embodiments where the method of the composition includes two or more high temperatures, at least two of those high temperatures are different from each other.

[0097] In some embodiments, the high temperature may be between 25°C and 200°C. As specific exemplary ranges, in some embodiments, the high temperature may be between 25°C and 150°C, between 25°C and 100°C, between 25°C and 95°C, between 25°C and 50°C, between 50°C and 200°C, between 50°C and 150°C, between 50°C and 100°C, between 25°C and 100°C, between 125°C and 200°C, or any other range between 125°C and 175°C.

[0098] In some embodiments, the high temperature may be at least 25°C. As further examples, in some embodiments, the high temperature may be at least 26°C, 27°C, 28°C, 29°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C or 100°C. In some embodiments, the enhancement of silk fibroin material crystallization is observed at a temperature of 95°C or higher.

[0099] In some embodiments, the high temperature may be at most 125°C. As further examples, in some embodiments, the high temperature may be at most 126°C, 127°C, 128°C, 129°C, 130°C, 135°C, 140°C, 145°C, 150°C, 155°C, 160°C, 165°C, 170°C, 175°C, 180°C, 185°C, 190°C, or 195°C.

[0100] The application of the high temperature one or more times to the provided composition or the provided method may be performed in a manner appropriate for any application. As non-limiting examples, in some embodiments, the application of the high temperature one or more times may be performed by thermal pressing via a heating device such as an oven, a heating stage, an exposed flame, or other mechanisms.

[0101] The application of high temperature one or more times may be performed at any of a variety of periods or over that period. For example, in some embodiments, the application of high temperature one or more times is performed substantially immediately (e.g., by placing on a flame or placing in an oven). In some embodiments, the application of high temperature one or more times is performed over a period of seconds, minutes, or hours. In some embodiments, the application of high temperature one or more times is performed over a period between 1 second and 1 hour.

[0102] High pressure

[0103] As discussed herein, the provided methods and compositions include exposure to high pressure one or more times. As used herein, the term "high pressure" refers to a pressure higher than standard atmospheric pressure (i.e., 1.013 bar). In some embodiments, the provided method or composition includes one exposure to high pressure. In some embodiments, the provided method or composition includes at least two (e.g., 3, 4, 5, 6, 7, 8, 9, 10 or more) exposures to high pressure. In some embodiments where the method of the composition includes two or more high pressures, at least two of those high pressures are different from each other.

[0104] Appropriate methods for any one or more applications can be used to generate the high pressure applied to the provided composition or the provided method. By way of non-limiting example, in some embodiments, high pressure can include the use of vacuum, press (e.g., heat press), and combinations thereof.

[0105] In some embodiments, the application of high pressure may be or include uniaxial compression. In some embodiments, the application of high pressure may be or include multiaxial compression (e.g., biaxial compression).

[0106] Any suitable level of high pressure may be used for any application, although in some embodiments, high pressure between 1 MPa and 1 GPa is used. As specific exemplary ranges, in some embodiments, the high pressure may be between 10 MPa and 1 GPa, between 50 MPa and 1 GPa, between 100 MPa and 1 GPa, between 200 MPa and 1 GPa, between 300 MPa and 1 GPa, between 400 MPa and 1 GPa, or between 500 MPa and 1 GPa. In some embodiments, the high pressure may be at least 1 MPa and may include these (e.g., at least 2 MPa, 3 MPa, 4 MPa, 5 MPa, 6 MPa, 7 MPa, 8 MPa, 9 MPa, 10 MPa, 20 MPa, 30 MPa, 40 MPa, 50 MPa, 60 MPa, 70 MPa, 80 MPa, 90 MPa, 100 MPa, 150 MPa, 200 MPa, 250 MPa, 300 MPa, 350 MPa, 400 MPa, 450 MPa, 500 MPa, 550 MPa, 600 MPa, 650 MPa, 700 MPa, or 750 MPa).

[0107] Silk articles

[0108] The methods and compositions provided enable the production of silk articles that were not previously achievable, as well as silk articles with enhanced properties. In some embodiments, the silk articles provided exhibit a substantially homogeneous structure (e.g., as shown in FIG. 25, Panel A). As used herein, "substantially homogeneous structure" means that the silk fibroin molecules are distributed and / or configured in a consistent manner throughout substantially all parts or the entirety of the article. Further, in some embodiments, the silk articles may exhibit a significant amount of silk fibroin in a semi-crystalline structure (see, e.g., FIG. 25, Panels C and E). In some embodiments, the production of silk articles according to the methods provided involves a structural transition of silk fibroin from a substantially amorphous state to a semi-crystalline state, as observed, for example, by X-ray diffraction.

[0109] In some embodiments, the silk article may exhibit a significant amount of β-sheet structure. For example, in some embodiments, the silk article may exhibit at least 10 wt% or more (e.g., at least 20 wt%, 30 wt%, 40 wt%) of β-sheet structure compared to the starting silk fibroin material. In some embodiments, the silk article may exhibit at least 50 wt% or more (e.g., at least 60 wt%, 70 wt%, 80 wt%, 90 wt%, 95 wt%) of β-sheet structure compared to the starting silk fibroin material.

[0110] In some embodiments, the crystallinity of the silk article can be controlled by the application of temperature and pressure. For example, in some embodiments, when amorphous silk is processed at a temperature in the range of about 25°C to 125°C, the silk article may contain about 10-15% of β-sheet structure. In some embodiments where amorphous silk is processed at a temperature in the range of about 125°C to 175°C, the silk article may contain, for example, about 20-35% of β-sheet structure, or, for example, more than 40% of β-sheet structure.

[0111] In some embodiments, the provided methods and compositions enable the production of a homogeneous silk article in which circular amorphous powders are filled together through the bonding between adjacent raw silk powders, for example, at a processing temperature of about 25°C to 95°C. In some embodiments, the provided methods and compositions enable the production of a homogeneous silk article in which the silk molecules of the amorphous powder obtain greater mobility by being heated above the glass transition temperature and self-organize into intertwined nano spheres, for example, at a processing temperature of about 125°C to 175°C.

[0112] In some embodiments, the provided methods and compositions enable the production of silk articles (e.g., thin films) that are thermally softened, bendable, and can be shaped into a desired shape. In some embodiments, the provided methods and compositions enable the production of silk articles that can be machined.

[0113] The methods and compositions provided enable the production of complex silk articles in ways not achievable by previous methods (e.g., silk screws that can resist torsional forces associated with in vivo use). By way of non-limiting example, in some embodiments, the methods and compositions provided may be used to produce silk articles such as films, fibers, meshes, needles, tubes, plates, screws, rods, and any combination thereof.

[0114] In some embodiments, the silk article may be suitable for one or more types of patterning. In some embodiments, the patterning may be macro-patterning and may include these. In some embodiments, the patterning may be micro-patterning (i.e., patterning having micro-scale features) and may include these. In some embodiments, the patterning may be nano-patterning (i.e., patterning having nano-scale features) and may include these. In some embodiments, the patterning may be etching, lithography-based patterning, curving, cutting, and any combination thereof and may include these.

[0115] In some embodiments, the silk article may be subjected to one or more types of processing (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). Within the scope of the present disclosure, any form of processing suitable for application is contemplated, but in some embodiments, the processing may be machining, rolling, drilling, milling, sanding, punching die cutting, extrusion, chemical etching, coating, molding, turning, thread rolling, and any combination thereof and may include these.

[0116] Examples of properties or characteristics of silk articles

[0117] In some embodiments, the provided composition (e.g., silk article) may be substantially transparent. In some embodiments, the provided composition (e.g., silk article) may be translucent. In some embodiments, the provided composition (e.g., silk article) may be substantially opaque. As used herein, the term "transparent" refers to the tendency of an object to transmit light (with or without scattering of the light). In some embodiments, a composition / article is said to be substantially transparent if it transmits more than 80% of the light exposed in the visible range (400 nm - 800 nm). In some embodiments, a composition / article is said to be translucent if it transmits 50% - 80% of the light exposed in the visible range (400 nm - 800 nm). In some embodiments, a composition / article is said to be substantially opaque if it transmits 50% or less of the light exposed in the visible range (400 nm - 800 nm).

[0118] In some embodiments, the provided composition may be biocompatible and / or biodegradable. In some embodiments, the provided composition may exhibit one or more specific degradation profiles. As a specific example, in some embodiments, the provided composition may degrade by at least 50 wt% after being exposed to an aqueous environment at 37°C for about 96 hours. In some embodiments, the provided composition may not degrade by more than 10% even after being exposed to in vivo environments or conditions for one month.

[0119] In some embodiments, the provided composition may exhibit one or more desirable properties including, but not limited to, conductivity, enhanced machinability, and / or enhanced thermoformability.

[0120] Additive

[0121] In some embodiments, the provided method or composition includes one or more additives (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more additives). In some embodiments, at least one additive may be mixed with the silk fibroin material or otherwise associated before the step of applying (e.g., exposure to high temperature and high pressure one or more times). In some embodiments, at least one additive may be mixed with the silk fibroin material or otherwise associated substantially simultaneously with the step of applying. In some embodiments, at least one additive may be mixed with the silk fibroin material or otherwise associated following the step of applying.

[0122] The provided methods and compositions are suitable for the addition of any of a variety of additives. By way of non-limiting example, in some embodiments, the additives may be small molecules, organic polymers, inorganic polymers, conductive materials, inorganic materials, hydrophobic materials, hydrophilic materials, nanomaterials, and any combination thereof, and may include these.

[0123] The processing of silk-based materials, including pure silk materials and silk-based composite materials, can be altered by adding one or more additives. In some embodiments, the function of the additive can be to adjust processing conditions and the properties of the product. In some embodiments, the additive may be selected from water; glycerol; saccharides; biopolymers such as peptides, proteins; antibodies and antigen-binding fragments; nucleic acids; immunogens; antigens; enzymes; synthetic polymers such as poly(ethylene) glycol, polylactic acid, poly(lactic-co-glycolic acid), etc., although additives suitable for the application are specifically contemplated as being within the scope of the present disclosure.

[0124] In some embodiments, for example, in some embodiments intended for in vivo use, the provided composition may contain one or more proteases. In some embodiments, the organic polymer is or comprises at least one protease. In some embodiments, the protease is one or more of, or comprises, proteinase XIV, proteinase K, α-chymotrypsin, collagenase, matrix metalloproteinase-1 (MMP-1), and MMP-2. In some embodiments, the protease may be useful for adjusting the degradation profile of the provided specific composition (e.g., in the in vivo environment).

[0125] In some embodiments, the conductive material may be an organic conductive material and / or an inorganic conductive material (e.g., a metal), and may include these. In some embodiments, the conductive material may be at least one of, or include, a conductive polymer, graphene, silver, gold, aluminum, copper, platinum, steel, brass, bronze, and iron oxide.

[0126] According to various embodiments, an amount of one or more additives appropriate for any application may be useful. By way of non-limiting example, in some embodiments, the additive may be present in the provided composition in an amount between 0.001 wt% and 95 wt%. In some embodiments, one or more additives may be mixed with the silk fibroin material in an amount ranging from 0.001 wt% to 95 wt% of the silk fibroin material.

Examples

[0127] Example 1 - Preparation of a Silk Aqueous Solution for the Production of Lyophilized Silk

[0128] The silk aqueous solution was prepared by dissolving degummed silk fibers (silk fibers after removing sericin on their outer surface) in a high-salt-concentration LiBr / CaCl 2 solution to produce a silk aqueous solution. The cocoons of Bombyx mori (B. mori) were cut into small pieces and 0.02M Na2 CO 3 was boiled in an aqueous solution (Sigma-Aldrich, USA) for 30 minutes, rinsed with distilled water, and then Na 2 CO 3 and sericin were removed. The degummed silk was dried overnight at room temperature. Next, 20 grams of the dried degummed silk was dissolved in 100 mL of 9.3 M LiBr solution at 60 °C for 3 - 4 hours. Subsequently, the solution was dialyzed in distilled water for 3 days using a Slide-a-Lyzer dialysis cassette (MWCO 3,500, Pierce, USA). During dialysis, the water was changed 5 times (1 hour, 4 hours, 8 hours, 24 hours, 48 hours). After dialysis, the solution was centrifuged at 13,000 rpm for 10 minutes to remove insoluble impurities. The concentration of the final silk solution was determined by drying a known amount of the solution and measuring the mass of the remaining solid (about 6 w / v%).

[0129] Example 2 - Characterization of Amorphous Silk Materials

[0130] Natural silk fibers derived from silkworm cocoons (Bombyx mori) contain silk fibroin, a natural protein with a heavy chain of approximately 390 kDa and a light chain of approximately 25 kDa 5、19 . Silk aqueous solution is obtained by cleaving the hydrogen bond network of degummed natural silk fibers with a high-concentration salt solution to form a regenerated silk aqueous solution. Next, the fresh solution is further lyophilized to obtain an amorphous silk material (Figure 1, panels D, E, F) in the solid state. This is more stable than the silk solution and can be stored under ambient conditions for several years without significant structural changes.

[0131] In this example, fresh solubilized silk was lyophilized, further processed into particles, and then formed into various final structures by heat and compression. To understand the properties of the final materials, the properties of the starting materials were evaluated and compared with the material properties of natural silk.

[0132] Fresh solubilized silk can be converted back into the form of solid silk by lyophilization to produce lyophilized silk. Here, it has been found that lyophilized silk has a fibroin structure (mainly random coil) very similar to that in the solution state. More importantly, the dried lyophilized silk can be stored under ambient conditions for years without significant structural changes. Comparing raw silk cocoon fibers and degummed silk fibers, lyophilized silk has no well-defined hierarchical structure composed of secondary structures (e.g., β-sheets). It is an amorphous material with a random coil structure. Due to such important structural differences, the thermal or mechanical behavior of lyophilized silk is different from that of raw silk cocoon fibers and degummed silk fibers.

[0133] Panel (A) of Figure 2 shows the thermal properties of degummed silk fibers and lyophilized silk powder. From thermogravimetric analysis (TGA), the lyophilized silk had a higher moisture content (5.0 wt%) than the degummed silk fibers (2.8 wt%).

[0134] Characterization by differential scanning calorimetry (DSC) further showed that the lyophilized silk had a glass transition related to water near 65 °C and a glass transition of pure silk fibroin at 178 °C, which were not observed in the degummed silk fibers (Panel B of Figure 2).

[0135] To understand the structural changes during heating, a more detailed DSC study of freeze-dried silk was carried out, and the results are shown in Figure 3. During heating, the silk-aqueous system undergoes dynamic conformational and compositional changes due to a combination of reconfiguration of silk fibroin chains and evaporation of bound water. Specifically, silk fibroin may first be reconfigured to form a new conformation using the bound water as a plasticizer, which was demonstrated in the low temperature range (35 °C to 80 °C). A well-resolved glass transition of the silk-aqueous system was shown at 65 °C. When the silk is further heated, the bound water molecules start to be released simultaneously. Two types of bound water molecules were found in this system, including weakly bound water molecules and strongly bound water molecules. The weakly bound water molecules started to evaporate around 35 °C, and the maximum evaporation rate was 95 °C, while the strongly bound water molecules started to evaporate around 55 °C, and the maximum evaporation rate was 125 °C. When the temperature reached 160 °C, all the bound water molecules had evaporated. Upon continuous heating, a stable glass transition of pure silk fibroin was shown to be around 178 °C. The heating rate in the case of TGA, DTG and DSC property evaluation was 10 °C / min.

[0136] DSC measurements were performed by enclosing the sample in an aluminum pan under a dry nitrogen gas flow rate of 50 mL / min. In the standard DSC measurement, the sample was heated from -50 °C to 200 °C at a heating rate of 10 °C / min. The moisture content of ASN is similar to the reported value for solution-cast silk fibroin with a less regular structure. 27、28 。

[0137] Thermal analysis showed that the water content of ASN was 5.0 ± 0.5 wt% (Figure 2, Panel A), which was significantly higher than that of degummed natural silk fibers (about 2.8 ± 0.3 wt%). Differential scanning calorimetry (DSC) showed that ASN had a glass transition related to water at 65 °C and a stable glass transition temperature of 178 °C (Figure 2, Panel B), which was in good agreement with previous studies on amorphous silk films (see Hu, X., Kaplan, D. & Cebe, P. Determining Beta-Sheet Crystallinity in Fibrous Proteins by Thermal Analysis and Infrared Spectroscopy. Macromolecules 39, 6161-6170 (2006)). The detailed stepwise scanning profile further showed two types of water bound to the amorphous silk material, namely water bound to the amorphous silk material with a maximum evaporation rate at 95 °C and water strongly bound with a maximum evaporation rate at 125 °C (Figure 2, Panel C). Characterization by differential scanning calorimetry (DSC) further showed that the lyophilized silk had a glass transition related to water near 71 °C (from TMDSC) and a glass transition of pure silk fibroin at 178 °C, which were not observed in degummed silk fibers (Figure 4). In this example, silk fibroin showed a well-resolved glass transition of the silk-water system at 71 °C. Upon continuous heating, the stable glass transition of pure silk fibroin was observed near 178 °C. The heating rate was 2 °C / min, the temperature amplitude was 0.318 K, and the period was 1 min.

[0138] X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and solid-state NMR spectroscopy showed that the silk fibroin of the amorphous silk material was mainly in an amorphous structure and had a low β-sheet structure content (about 1.0%) (Figure 1, Panel G, Panel H, Panel I, Panel J).

[0139] Example 3 - Engineering of Silk Monoliths Using Amorphous Silk Materials at Various Temperatures and High Pressures

[0140] Natural silk fibers have a very stable molecular structure that includes a strong hydrogen bond network under ambient and high-pressure conditions 26 which limits the direct thermal processing of raw silk fibers. By reconstructing natural silk into amorphous silk, the molecular structure of silk fibroin becomes less regular. At high pressure and high temperature, the free energy change of the material system (Gibbs free energy G = E + PV - TS) may provide new opportunities to adjust the phase or molecular structure of the material. Thus, the inventors decided to investigate how external pressure (or stress) and heat affect an assembly of silk proteins in the solid state.

[0141] Amorphous silk is obtained by lyophilization and used as a raw material for densification and thermal processing. The combination of molecular structure (XRD, FTIR) and morphological analysis (SEM) reveals that the self-organization of amorphous silk fibroin is promoted as the processing temperature increases during densification. The pressure-driven densification of raw ASN may promote intermolecular interactions including hydrogen bonds and van der Waals interactions, which causes the formation of a new β-sheet structure.

[0142] Regenerated amorphous silk prepared from silk solutions of various concentrations (<7 wt%) was used as a raw material for manufacturing silk materials by a heat / pressure process. In this example, regenerated amorphous silk powder prepared from a 1% silk solution was used as an exemplary starting material for the production of various forms of silk materials. In this example, the effects of heat and pressure on the silk structure were further investigated, and various processing routes for designing amorphous silk materials into functional devices (such as silk screws) using heat and pressure were further demonstrated (Figure 5). A collection of silk parts manufactured by various processing routes is shown in Figure 7.

[0143] The method used to engineer and induce structural changes in ASN is the control of the application of pressure and heat. The pressure applied ranges from 1 MPa to over 1 GPa, and the temperature ranges from 0 °C to 200 °C. Since silk fibroin begins to decompose near 200 °C, the temperature is controlled below 200 °C. ASN was filled into a pre-designed mold and compressed onto a silk plate at 632 MPa at various temperatures. As the temperature increased, the silk plate changed from opaque to transparent and its appearance changed to a pale yellowish color. Scanning electron microscope analysis showed significant differences in the internal structure of silk plates processed at low (25, 65, and 95 °C) and high (125, 145, and 175 °C) temperatures. At low processing temperatures, despite an increase in the packing density of the raw silk powder and a large plastic deformation as the temperature increased, features similar to the dimensions of the raw silk powder still remained (Figure 8 panel a: A2–C2, and A3–C3). Silk microspheres of approximately 30 nm were observed at high processing temperatures (Figure 8 panel a: D3–F3). This is similar to the globular structure of natural silkworm and spider silk fibers.

[0144] Fourier transform infrared (FTIR) analysis showed that a small amount of β-sheet structure (about 10 - 15%) was formed at low processing temperatures (25 °C, 65 °C, 95 °C, 125 °C), and a larger amount of β-sheet structure (more than 40%) was formed at high processing temperatures (145 °C, 175 °C) (Figure 8, Panel bB and Panel D). Furthermore, in the characterization of X-ray diffraction (XRD), it has been shown that the crystallinity of the densified silk plates increases slowly as the temperature rises in the temperature range of 25 - 95 °C. In contrast, when hot pressing is carried out above 125 °C, the crystallinity increases significantly with the temperature (Figure 8, Panel C and Panel E). At low temperatures (25, 65 and 95 °C), the silk amorphous powder is filled together through the bonds between adjacent particles, and the content of the β-sheet structure may be low; at high temperatures (125, 145 and 175 °C), the silk molecules acquire greater mobility when exceeding the glass transition temperature and may self-organize into a structure characterized by intertwined nano-spheres and moderate crystallinity (about 20 - 35%). Furthermore, when hot pressing is carried out above 95 °C, XRD shows the silk II structure (similar to natural silk fibers), indicating a structural transition during processing (from silk I to silk II) (Figure 8, Panel C). A simple mechanism has been proposed to show the structural transition of ASN during thermal processing (Figure 8, Panel F).

[0145] In one example, the mechanical properties of the thermally processed bulk silk material were characterized by a three-point bending test. As the processing temperature increased (25 °C - 145 °C), the strength of the bulk silk material increased (Figure 9, Panel A and Panel B). However, at 175 °C, the strength began to decrease. This may be due to high crystallinity and partial thermal decomposition. The maximum strength was achieved with a specific strength of 109 ± 10 MPa / g cm for the silk plates processed at 145 °C -3 This exceeds the strength of some natural structural materials such as CP nacre and wood (Figure 9, Panel C). In contrast, the densification of degummed silk powder with a high β-sheet content and low crystallinity has poor mechanical properties. This may indicate weak fusion of the degummed silk powder (Figure 10).

[0146] In another example, the role of pressure in the conversion from amorphous to silk-structured materials was investigated. At room temperature, pressure-driven densification of raw amorphous silk materials results in the formation of a β-sheet structure (β-sheet content is approximately 10% relative to approximately 1% of the amorphous silk material, Figure 8, panels B and D). When densification is carried out at elevated temperatures (65 - 175 °C), the density of the densified silk material reaches a constant value of approximately 1.35 g / cm 3 at high pressure (632 MPa) (Figure 11, panel A). Densification under low external pressure shows low flexural strength in a three-point bending test (Figure 11, panel B). A combination of molecular structure (XRD, FTIR) and morphological analysis (SEM) revealed that as the processing temperature during densification increases, self-organization of amorphous silk fibroin is promoted, as indicated by the formation of a significant β-sheet structure at elevated temperatures (>145 °C). The structural transition of amorphous silk induced by pressure and heat is thought to help fuse silk fibroin and transform amorphous silk nanomaterials into robust bulk materials.

[0147] Example 4 - Direct Molding of Amorphous Silk Nanomaterials into Designed Shapes

[0148] Amorphous silk materials can be loaded into a pre-designed mold for direct molding, as shown in Process B of Figure 5. After molding, the silk material is deformed into the designed shape. In this case, the amorphous silk material was filled into a screw-shaped mold, and subsequently, the material was molded into the shape at 120 °C and 800 MPa. The resulting silk screw is shown in Figure 12.

[0149] Example 5 - Thermoforming and Patterning

[0150] In another example, silk monoliths obtained by heat and pressure can be used for thermoforming and patterning. Standard DSC was performed on silk plates obtained at 125 °C and 632 MPa (Figure 13, Panel A). This material showed a glass transition temperature (Tg) of 97 °C, followed by an exothermic peak due to crystallization around 137 °C. The silk plates contain a significant amount of amorphous structure (about 79% non-crystalline structure) and become flexible when heated above the Tg of this plate. Utilizing this thermoplastic property, pre-pressed silk materials can be further shaped into desired shapes such as screws, patterns, and other complex shapes (Figure 13, Panels B - E). For example, to reproduce the features of a dime, first 200 mg of ASN is pressed in a 13 mm mold set at 700 MPa to create small pellets. Next, the pellet is placed on a dime and thermocompressed (100 MPa, 140 °C) to pattern the coin. Panel (D) of Figure 13 shows a photograph of the patterned silk coin. Due to the transparency of the silk plate, the patterns formed from both sides of the silk coin can be seen. Micro / nano-structured patterning can also be achieved using this method. Panel (E) of Figure 13 shows an SEM image of a nanostructured patterned silk film.

[0151] Example 6 - Engineering of Silk Monoliths for Machining into Designed Shapes

[0152] By applying different processing conditions, silk monoliths with adjustable molecular structures can be produced. More specifically, silk monoliths with a low β-sheet content can be prepared at 95 °C and 632 MPa, and silk monoliths with a high β-sheet content can be prepared at 145 °C and 632 MPa. Silk monoliths can be further machined into designed shapes as shown in Process A of Figure 14. In this example, silk screws and ear tubes were machined from silk monoliths. Additionally, a water absorption test was conducted to examine the swelling properties of silk screws with different molecular structures.

[0153] The swelling and degradability of these products can be controlled by manipulating the processing conditions. In one example, silk-based bone screws were manufactured by machining silk bars prepared under different processing conditions (95 °C and 632 MPa; 125 °C and 632 MPa; 145 °C and 632 MPa) to vary the characteristics of the internal molecular structure (β-sheet content and crystallinity). The degradation profile showed that processing at 145 °C provided the slowest degradation rate in the enzyme solution over time, indicating that the degradability of the silk screws can be adjusted (Figure 15). Silk screws processed at 95 °C and 125 °C were found to exhibit a weight loss of more than 60% in a 5 U / mL protease XIV solution and rapidly degrade within 30 days. Additionally, water absorption in PBS at 37 °C showed that silk screws machined from silk bars prepared at 95 °C and 125 °C rapidly absorbed water within 15 minutes, reaching a maximum of approximately 30% by weight (Figure 16, panel B). In comparison, silk screws machined from silk bars prepared at 145 °C showed a much slower water absorption, reaching a maximum of approximately 20% by weight. These differences reflect a fundamental control of the crystal content.

[0154] Furthermore, degradation tests showed that the degradability of silk screws can be adjusted when highly crystalline silk screws provide a low degradation rate in both PBS and the enzyme solution over 30 days (Figure 15).

[0155] Example 7 - Production of Composite Materials including Silk-Enzyme Composite Materials

[0156] The method described in the previous example can be extended to produce composite materials other than pure silk materials. Among the numerous composite materials, there may be included silk-small molecule systems, silk-polymer systems, silk-inorganic material systems, silk-hydrophobic / hydrophilic material systems, and / or silk-nanomaterial systems. In one example, dry silk powder was mixed with protease XIV powder, an enzyme that degrades silk, to produce a silk-enzyme composite material. The ratio of protease XIV was varied (0.2 wt%, 1.0 wt%, 5.0 wt%). Next, the mixture was pressed in a 6 mm mold set at 700 MPa to produce small pellets. The prepared pellets were incubated in methanol for 48 hours to induce a β-sheet structure in the silk, improving the mechanical properties and water resistance.

[0157] The degradation test was carried out in PBS solution at 37 °C. The results showed that the degradation of the silk material can be controlled by changing the ratio of protease XIV in the composite material. For example, when the loading rate of protease XIV is 5 wt%, the silk degrades by 60% after incubation in PBS solution at 37 °C for 4 days (Figure 17 panel (a)), while when the loading rate of protease XIV is 0.2 wt%, the silk degrades by approximately 30%. The degradation of silk screws containing 0.5% protease XIV, machined from silk bars pressed at 145 °C and 632 MPa, was observed (Figure 16 panel (b)). It is noteworthy that the activity of the enzyme, which would be lost in the absence of silk during the hot press (145 °C and 632 MPa), was maintained in the silk / enzyme system. This suggests that silk can stabilize the enzyme at high temperature and high pressure. These methods demonstrate the ability to produce silk-based bulk materials with controllable degradability.

[0158] The components added to the silk matrix / article may be organic or inorganic molecules. The components may be incorporated into the silk matrix by mixing the silk solution with the components and then lyophilizing the solution. The components may include, but are not limited to, micro / nano materials, such as metal micro / nano materials, graphene, graphene oxide, and / or hydroxyapatite.

[0159] Components may be added to create fiber-reinforced silk matrix / articles. Natural or synthetic fibers can be incorporated into the silk matrix by using the methods described. Natural or synthetic fibers may include raw silk fibers, synthetic nylon fibers, and / or synthetic polymer fibers.

[0160] Example 8 - Production of composite materials including silk-graphene composite materials

[0161] In this example, a silk-graphene composite material was produced using a lyophilized silk material. Graphene is hydrophobic and it is difficult to directly fabricate a silk-graphene composite material using a solution method. However, a silk-graphene composite material was formed by mixing graphene and silk powder in the solid state using the method described in the previous example. In one example, graphene was mixed with fine silk powder at a loading rate of 70.0 wt%. Next, the mixture was pressed in a 6 mm mold set at 700 MPa to produce a compressed silk-graphene film. The as-prepared silk-graphene film has high conductivity and shows great potential in the production of bioelectronics (Figure 18).

[0162] Example 9 - Investigation of the structural differences between lyophilized silk and solid silk articles

[0163] In this example, specific structural characteristics of lyophilized silk and solid silk articles subjected to the high temperature and high pressure described herein were investigated.

[0164] Panel (A) of FIG. 19 shows an SEM image of freeze-dried silk powder. The internal structure of the silk article pressed at 145° C. and 632 MPa for 15 minutes is shown in Panels (B) and (C) of FIG. 19. The loosely freeze-dried silk powder was transformed into a solid silk article. The enlarged view of the internal structure of the silk article (Panel (C) of FIG. 19) revealed spherical features in the nanometer range. These features show the similarity to the spherical structure of natural silkworm and spider silk fibers. The SEM images indicate that the freeze-dried raw silk material undergoes a fusion process, which is not a simple physical compression of the raw silk powder to form the pressed silk articles in Panels (B) and (C) of FIG. 19. The molecular conformation of silk has partially changed from a random coil / amorphous state to a crystalline β-sheet structure. The level of uniformity observed in the structure of these pressed silk articles seems to be possible only by using the method described herein, as it could not be achieved by using previous methods.

[0165] Example 10 - Method for Converting Silk into a New Material

[0166] Materials and Chemicals. Sodium carbonate, lithium bromide, protease XIV, and chymotrypsin were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO) and used as received.

[0167] Preparation of Regenerated Silk Fibroin. Cocoons of Bombyx mori (B. mori) were cut into small pieces and boiled in 0.02 M Na 2 CO 3 aqueous solution (Sigma-Aldrich, USA) for 30 minutes, then rinsed with distilled water to remove Na 2 CO 3Sericin was removed and degummed silk was dried overnight at room temperature. The dried silk was dissolved in 9.3 M LiBr solution at 60 °C for 3 - 4 hours. Subsequently, the solution was dialyzed against distilled water for 3 days using a Slide-A-Lyzer dialysis cassette (MWCO 3,500, Pierce, USA). During dialysis, the water was changed 5 times (1 hour, 4 hours, 8 hours, 24 hours, 48 hours). After dialysis, the solution was centrifuged at 13,000 rpm for 10 minutes to remove insoluble impurities. The concentration of the final silk solution was determined by measuring the volume of the solution and the final dry weight (about 6 w / v%). The solution was diluted with distilled water and frozen in liquid nitrogen. Next, the frozen silk was lyophilized at -80 °C until completely sublimated. The lyophilized silk was ground into fine powder and stored under ambient dry conditions to prevent rehydration of the lyophilized solid until use in the following processing steps.

[0168] Hot pressing of amorphous silk nanomaterial (ASN). ASN was filled into a pre-designed mold and subsequently hot pressed at 632 MPa and variable temperatures (25 °C, 65 °C, 95 °C, 125 °C, 145 °C, 175 °C) for 15 minutes. After hot pressing, the samples were cooled to room temperature and used for property evaluation.

[0169] Thermal analysis. The thermal decomposition of silk samples was measured by thermogravimetric analysis (TGA) in N 2 (99.99%) from 30 to 800 °C at a scanning rate of 5 °C / min. For all measurements, the samples were held in the furnace under N 2 to reach a stable weight before heating. Differential scanning calorimetry (DSC) measurements were performed on a TA Instruments Q100 DSC (TA Instruments, Newcastle, Delaware) with the samples encapsulated in aluminum pans under a dry nitrogen gas flow of 50 mL / min. Both standard DSC and temperature-modulated DSC (TMDSC) measurements were carried out. In the standard DSC measurement, the samples were heated from -50 °C to 200 °C at a heating rate of 10 °C / min. In the TMDSC measurement, the samples were heated from -50 °C to 200 °C at 10 °C / min with a modulation period of 60 seconds and a temperature amplitude of 1.59 °C.

[0170] Scanning electron microscope (SEM). The form of the stage was characterized by a scanning electron microscope (SEM) (Ultra 55 field emission scanning electron microscope, Carl Zeiss AG, Harvard University Nanoscale Systems Center). SEM images were collected at a voltage of 5 kV, and the samples were sputter-coated with a thin layer of gold.

[0171] Fourier transform infrared spectroscopy (FTIR). Fourier transform infrared spectroscopy (FTIR) was performed using a JASCO FTIR6200 spectrometer (JASCO, Tokyo, Japan) equipped with an MIRacle attenuated total reflectance (ATR) Ge crystal cell in absorbance mode. For each measurement, the spectra were recorded with 32 scans and a resolution of 4.0 cm -1 . The content of the secondary structure of the protein was determined by performing peak deconvolution in the amide I region (1600 - 1700 cm -1 ). The number and position of the peaks were defined from the results of the second derivative spectra and fixed during the deconvolution process. A Gaussian model was selected for the band shape and bandwidth and automatically adjusted by the software.

[0172] X-ray diffraction. X-ray diffraction was performed using a SAXSLAB small angle / wide angle X-ray scattering system (MIT Materials Science and Engineering Center). The wavelength of the X-ray beam was 1.5409 Å and the fixed energy was 45 kV. The distance from the sample to the detector was 109.1 mm, and an exposure time of 60 seconds was used in the experiment.

[0173] Three-point bending test. A three-point bending test was performed on unnotched dry and wet specimens at 25 °C and RH 50% using the bending test mode of an Instron 3366 testing machine (Instron, Norwood, USA) at loading rates of 0.2 mm / min and 2 mm / min, respectively. The length of the specimen was 12 mm, the width was 7 mm, and the thickness was 1 mm.

[0174] In vitro water supply and swelling test. Machined silk screws (n = 4 per condition) were placed in phosphate-buffered saline (PBS) at 37 °C for various times (0 min, 15 min, 60 min, 3 h, 24 h, 48 h, 72 h, 9 days, 15 days, and 30 days), and changes in weight and diameter indicating fluid uptake and swelling were observed. Surface moisture was removed from the samples by wiping with Kimwipes (Kimberly-Clark, USA), and the wet weight (Ws) and the head and screw diameters of the collected samples were measured. The water supply rate (%) was calculated.

[0175] Water absorption rate (%) = [(Ws - Wd) / Ws] × 100 (1)

[0176] In vitro enzymatic degradation test. Machined silk screws (n = 3 per condition) were incubated at 37 °C in 2 mL solutions of 5 U / mL protease XIV and 40 U / mL chymotrypsin in PBS or PBS as a negative control. The solutions were changed every 2 - 3 days. At the designated time points (2 days, 5 days, 10 days, 20 days, 30 days), the sample groups were rinsed with DI water, dried, and then weighed. The remaining mass of each sample was collected, and SEM images of the samples were collected.

[0177] NMR spectrum of the regenerated silk aqueous solution (6 wt%). Solution NMR experiments were carried out on a Bruker 850 MHz Avance III HD spectrometer equipped with a 5 mm cryogenic helium-cooled triple resonance TCL CryoProbe. The Larmor frequencies of 1H, 13C, and 15N were 850.28 MHz, 213.82 MHz, and 86.17 MHz, respectively. A sealed capillary containing D2O was used for NMR locking. NOESY was carried out with a mixing time of 150 ms, a spectral width of 11 ppm and in both dimensions of t1 and t2, 512 and 2048 complex points in the dimensions of t1 and t2, 16 scans and a relaxation delay of 1 s. TOCSY was carried out with a mixing time of 60 ms, a spectral width of 11 ppm and in both dimensions of t1 and t2, 512 and 2048 complex points in the dimensions of t1 and t2, 16 scans and a relaxation delay of 1 s. 1H-13C HSQC was collected with a spectral width of 165.8 ppm in t1, 11 ppm in the t2 dimension, 256 and 1024 complex points in t1 and t2, and 64 scans. 1H-15N HSQC was collected with a spectral width of 28.0 ppm in t1, 11 ppm in the t2 dimension, 512 and 1024 complex points in t1 and t2, and 32 scans. NMR data were processed and analyzed using MestReNova. Figure 20 shows the solution NMR spectrum of the regenerated silk aqueous solution (6 wt%).

[0178] of native degummed silk and freeze-dried silk 13 C cross-polarization magic angle spinning (CP-MAS) spectra. The spectra were collected on a Varian VNMRS 400 MHz spectrometer equipped with a 3.2 mm triple resonance probe operating in the double resonance ( 1 H / 13 C) mode. 1 Hπ 13 The CP conditions for the C CP-MAS experiment were 2.25 μs 1Composed of an Hπ / 2 pulse followed by a 1.0 ms ramp (3%) 1H spin-lock pulse of 70 kHz radio frequency (rf) electric field strength. The experiments were carried out at a 2-pulse phase modulation (TPPM) 1H decoupling level of 91 kHz with a sweep width of 25 kHz, a recycle delay of 3.0 s, 8192 scans, and a MAS speed of 20 kHz for all samples. Figure 23 shows the 13 13C cross-polarization magic angle spinning (CP-MAS) spectra of native degummed silk and freeze-dried silk.

[0179] Example 11 - In Vitro Degradation Analysis of Silk Ear Tubes with or without Protease XIV Doping

[0180] Using the method described in the previous example, ear tubes of pure silk and silk-protease XIV composites were fabricated. The ear tubes were machined from pure silk and silk-protease XIV composite bulk materials prepared under two different conditions: temperature 125°C and pressure 632 MPa; temperature 145°C and pressure 632 MPa.

[0181] The degradation tests were carried out in PBS solution at 37°C for various times of 5 minutes, 1 hour, 3 hours, 6 hours, 24 hours, 48 hours, and 72 hours. The results are shown in Figure 30.

[0182] References. The following list of references is identified by the number immediately preceding the reference, rather than by the paragraph number in parentheses, in the above disclosure.

[0183] 1. Altman, G. H. et al. Silk-based Biomaterials. Biomaterials 24, 401 - 416 (2003).

[0184] 2. Kundu, B., Rajkhowa, R., Kundu, S. C. & Wang, X. Silk fibroin biomaterials for tissue regenerations. Advanced Drug Delivery Reviews 65, 457-470 (2013).

[0185] 3. Koh, L.-D. et al. Structures, mechanical properties and applications of silk fibroin materials. Prog. Polym. Sci. 46, 86-110 (2015).

[0186] 4. Vepari, C. & Kaplan, D. L. Silk as a biomaterial. Prog. Polym. Sci. 32, 991-1007 (2007).

[0187] 5. Zhou, C. et al. Silk Fibroin: Structural Implications of a Remarkable Amino Acid Sequence. Proteins: Struct., Funct., Genet. 44, 119-122 (2001).

[0188] 6. Marsh, R. E., Corey, R. B. & Pauling, L. An Investigation of the Structure of Silk Fibroin. Biochim. Biophys. Acta 16, 1-34 (1955).

[0189] 7. Lefevre, T., Rousseau, M.-E. & Pezolet, M. Protein Secondary Structure and Orientation in Silk as Revealed by Raman Spectromicroscopy. Biophys. J. 92, 2885-2895 (2007).

[0190] 8. Cebe, P. et al. Beating the Heat - Fast Scanning Melts Silk Beta Sheet Crystals. Sci. Rep. 3, 741-7 (2013).

[0191] 9. Sidhu, M. S., Kumar, B. & Singh, K. P. The processing and heterostructuring of silk with light. Nat. Mater. 16, 938-945 (2017).

[0192] 10. Pauling, L. & Corey, R. B. Configurations of Polypeptide Chains with Favored Orientations Around Single Bonds: Two New Pleated Sheets. Proc. Natl. Acad. Sci. U.S.A. 37, 729-740 (1951).

[0193] 11. Cheng, G., Wang, X., Tao, S., Xia, J. & Xu, S. Differences in regenerated silk fibroin prepared with different solvent systems: From structures to conformational changes. Journal of Applied Polymer Science 132, n / a-n / a (2015).

[0194] 12. Um, I. C., Kweon, H., Park, Y. H. & Hudson, S. Structural characteristics and properties of the regenerated silk fibroin prepared from formic acid. Int. J. Biol. Macromol. 29, 91-97 (2001).

[0195] 13. Ha, S.-W., Tonelli, A. E. & Hudson, S. Structural studies of Bombyx mori silk fibroin during regeneration from solutions and wet fiber spinning. Biomacromolecules 6, 1722-1731 (2005).

[0196] 14. Trabbic, K. A. & Yager, P. Comparative structural characterization of naturally- and synthetically-spun fibers of Bombyx mori fibroin. Macromolecules 31, 462-471 (1998).

[0197] 15. Ha, S.-W., Park, Y. H. & Hudson, S. Dissolution of Bombyx mori silk fibroin in the calcium nitrate tetrahydrate-methanol system and aspects of wet spinning of fibroin solution. Biomacromolecules 4, 488-496 (2003).

[0198] 16. Rockwood, D. N. et al. Materials fabrication from Bombyx mori silk fibroin. Nat. Protoc. 6, 1612-1631 (2011).

[0199] 17. Perrone, G. S. et al. The use of silk-based devices for fracture fixation. Nat. Commun. 5, 1-9 (2014).

[0200] 18. Li, C. et al. Regenerated silk materials for functionalized silk orthopedic devices by mimicking natural processing. Biomaterials 110, 24-33 (2016).

[0201] 19. Yamaguchi, K. et al. Primary Structure of the Silk Fibroin Light Chain Determined by cDNA Sequencing and Peptide analysis. J. Mol. Biol. 210, 127-139 (1989).

[0202] 20. Wray, L. S. et al. Effect of processing on silk-based biomaterials: reproducibility and biocompatibility. J. Biomed. Mater. Res. Part B Appl. Biomater. 99, 89-101 (2011).

[0203] 21. Marelli, B. et al. Programming function into mechanical forms by directed assembly of silk bulk materials. Proc. Natl. Acad. Sci. USA 114, 451-456 (2017).

[0204] 22. Kluge, J. A., Kahn, B. T., Brown, J. E., Omenetto, F. G. & Kaplan, D. L. Optimizing molecular weight of lyophilized silk as a shelf-stable source material. ACS Biomater. Sci. Eng. 2, 595-605 (2016).

[0205] 23. Hu, X., Kaplan, D. & Cebe, P. Determining Beta-Sheet Crystallinity in Fibrous Proteins by Thermal Analysis and Infrared Spectroscopy. Macromolecules 39, 6161-6170 (2006).

[0206] 24. Lu, Q. et al. Silk Self-Assembly Mechanisms and Control From Thermodynamics to Kinetics. Biomacromolecules 13, 826-832 (2012).

[0207] 25. Koebley, S. R. et al. Silk Reconstitution Disrupts Fibroin Self-Assembly. Biomacromolecules 16, 2796-2804 (2015).

[0208] 26. Krywka, C., Krasnov, I., Figuli, R., Burghammer, M. & Mueller, M. Determination of silkworm silk fibroin compressibility using high hydrostatic pressure with in situ X-ray microdiffraction. Macromolecules 47, 7187-7193 (2014).

[0209] 27. Hu, X.; Kaplan, D.; Cebe, P. Thermochim. Acta 2007, 461 (1-2), 137-144.

[0210] 28. Hu, X.; Kaplan, D.; Cebe, P. Macromolecules 2008, 41, 3939-3948.

[0211] Equivalents and Scope. In any definition of a variable herein, the listing of a list of elements includes the definition of that variable as any single element or combination (or sub-combination) of the listed elements. In the listing of embodiments herein, the embodiments are included as any single embodiment or in combination with any other embodiment or part thereof. One of ordinary skill in the art will recognize many equivalents to the particular embodiments of the invention described herein or will be able to ascertain such equivalents using only routine experimentation. The scope of the invention is not intended to be limited to the above description but is as set forth in the following claims.

Claims

1. (i) providing a silk fibroin material comprising an amorphous structure; and (ii) applying to the silk fibroin material a temperature of greater than 95°C and less than 200°C and a pressure of at least 100 MPa to form a silk fibroin article, wherein the applying step induces fusion of at least a portion of the silk fibroin and induces a structural change of the fibroin in the silk fibroin material, and the temperature is applied to the silk fibroin material immediately after the pressure is applied.

2. (i) selecting a temperature and a pressure for producing a desired silk fibroin article having a desired crystallinity and desired material properties, wherein the temperature is from 125°C to less than 200°C and the pressure is at least 100 MPa; and (ii) applying the temperature and the pressure to a silk fibroin material comprising an amorphous structure to form a silk fibroin article, wherein the silk fibroin article has the desired crystallinity and the desired material properties, wherein the desired crystallinity is a semi-crystalline or crystalline structure, and the desired material properties are conductivity, machinability, and / or thermoformability, and the temperature is applied to the silk fibroin material immediately after the pressure is applied.

3. The method according to claim 2, wherein the applying step induces fusion of at least a portion of the silk fibroin and induces a structural change of the fibroin in the silk fibroin material.

4. The method according to any one of claims 1 to 3, wherein the silk fibroin article is homogeneous.

5. The method according to any one of claims 1 to 4, wherein the silk fibroin article comprises silk in an amount of 10% (w / w) or more.

6. The method according to any one of claims 1 to 5, further comprising selecting an amount of a plasticizer to be included in the silk fibroin material and including the plasticizer in the silk fibroin material in the selected amount to produce the desired crystallinity and the desired material properties, wherein the plasticizer is water as needed.

7. The method according to any one of claims 1 to 6, wherein the applying step is performed in a mold.

8. The method according to any one of claims 1 to 6, wherein the step of applying is not performed in the mold.

9. The method according to any one of claims 1 to 8, further comprising the step of processing the silk fibroin article.

10. The method according to claim 9, wherein the step of processing is at least one of machining, turning, rolling, screw forging, drilling, milling, sanding, punching, die cutting, chemical etching, coating, molding, and any combination thereof, or includes them.

11. The method according to any one of claims 1 to 10, wherein the silk fibroin article is a film, fiber, mesh, needle, tube, plate, screw, rod or any desired shape, or includes them.

12. The method according to any one of claims 1 to 11, wherein the step of applying results in a structural change of the silk fibroin and induces a β-sheet structure with an increase of at least 1% compared to the level of β-sheets in the silk fibroin material before the step of applying.

13. The method according to any one of claims 1 to 12, wherein the silk fibroin article is transparent.

14. The method according to any one of claims 1 to 13, wherein the silk fibroin article is subjected to at least one of macro-patterning, micro-patterning and nano-patterning.

15. The method according to any one of claims 1 to 14, wherein the silk fibroin article contains at least one additive.

16. The method according to claim 15, wherein the silk fibroin material is mixed with at least one additive before the step of applying to form a composite silk fibroin article.

17. The additive is at least one of organic or inorganic molecules, organic polymers, inorganic polymers, biopolymers; conductive materials, carbon-based materials, antibodies and their antigen-binding fragments; antigens; nucleic acids; nucleic acid analogs and derivatives; saccharides; immunogens; natural compounds, and extracts from biological systems, bacteria, or tissues; synthetic materials; metal materials; alloys; hydrophobic materials; hydrophilic materials; nanomaterials; and any combination thereof, or includes them. The method according to claim 15 or 16.

18. The method according to claim 17, wherein the organic polymer is at least one enzyme or comprises the same.

19. The method according to claim 18, wherein the activity of the enzyme is stabilized by silk under the temperature and the pressure.

20. The method according to claim 18 or 19, wherein the enzyme is a protease, and the protease is one or more of protease XIV, proteinase K, α-chymotrypsin, collagenase, matrix metalloproteinase-1 (MMP-1) and MMP-2, or comprises the same.

21. The method according to claim 17, wherein the conductive material is an inorganic conductive material, an organic conductive material, a metal, an alloy, a semiconductor material and / or a conjugated polymer, or comprises the same.

22. The method according to any one of claims 16 to 21, wherein the additive is mixed with the silk fibroin material at a ratio between 0.001% by weight and 95.0% by weight.

23. The method according to any one of claims 1 to 22, wherein the silk fibroin article decomposes by at least 1% by weight after being exposed to an aqueous environment at 37°C for 30 days.

24. The method according to any one of claims 1 to 23, wherein the silk fibroin article is bioabsorbable.

25. The method according to any one of claims 1 to 24, wherein the silk fibroin article has thermoforming properties and can be reshaped into a desired shape at the temperature or the pressure.

26. The method according to any one of claims 1 to 25, wherein the β-sheet content of the silk fibroin material containing the amorphous structure is 60% or less.

27. A silk fibroin article obtained by the method according to any one of claims 1 to 26.

28. The article according to claim 27, wherein the silk fibroin article is a packaging material or comprises the same.

29. The article according to claim 28, wherein the packaging material is suitable for use in the manufacture of electronic devices, drug delivery systems, patterning, shaping, and any combination thereof.

30. A silk fibroin article produced by a method comprising the steps of any one of claims 1 to 26; wherein the silk fibroin material is converted into a solid silk article that has undergone a conformational change of silk and contains at least some of the silk fibroin that is fused together.

31. The silk fibroin article according to any one of claims 27 to 30, wherein the silk fibroin article contains semi-crystalline silk fibroin and the glass transition temperature of the silk fibroin article is between 40°C and 120°C.

32. The silk fibroin article according to any one of claims 27 to 31, wherein the silk fibroin article is homogeneous.

33. The bending strength of the silk fibroin article is at least 5 MPa, and the overall density of the silk fibroin article is at least 1.20 g / cm ３ The silk fibroin article according to any one of claims 27 to 32, which is as described above.

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