WO2014021954A2

WO2014021954A2 - Magneto-sensitive silk fibroin-based materials

Info

Publication number: WO2014021954A2
Application number: PCT/US2013/036539
Authority: WO
Inventors: David L. Kaplan; Gary G. Leisk; Berendien Jacoba PAPENBURG; Ethan GOLDEN; Nereus PATEL
Original assignee: Trustees Of Tufts College
Priority date: 2012-04-13
Filing date: 2013-04-15
Publication date: 2014-02-06
Also published as: WO2014021954A3

Abstract

Provided herein relates to processes of preparing magneto-responsive silk fibroin-based materials, and the resulting materials thereof, which can be used in various applications ranging from filtration to biomedical applications such as tissue engineering scaffolds.

Description

Attorney Docket No. 700355-070131-PCT

MAGNETO-SENSITIVE SILK FIBROIN-BASED MATERIALS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 1 19(e) of U.S. Provisional Application No. 61/623,982 filed April 13, 2012, the content of which is incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

[0002] This invention was made with government support under 2P41 EB002520 awarded by the National Institutes of Health (NIH) and W911SR08-C-0012 awarded by Defense Advanced Research Projects Agency (DARPA). The government has certain rights in the invention.

FIELD OF THE DISCLOSURE

[0003] The present invention relates to silk fibroin-based materials that are responsive to magnetic fields, compositions comprising the same and processes for preparing the same.

BACKGROUND OF THE DISCLOSURE

[0004] Electrospinning is a widely used processing technique to create small-diameter fibers from typically polymeric solutions or melts. In electrospinning, a polymer solution streaming through a metal needle is charged to a high voltage potential, typically between 10 kV and 30 kV. The solution, which attains a high surface charge, is attracted to any grounded surface, such as a vertically or horizontally mounted collector plate. The surface charge causes the stream to form a jet (Taylor cone) that is propelled at a high rate of speed and also causes instabilities, which forces the stream to bend as it streams toward a collector. The bending stream whips around as it moves quickly in air; activity which leads to the drying of the stream into a nano-sized fiber. Various flat meshes, mats, tubes, and other geometries have been produced and are being utilized in applications, such as the creation of tissue engineering scaffolds. Further, magneto-sensitive polymeric particles like iron oxides, maghemite y-Fe20₃ and magnetite Fe₃0₄ have distinct biomedical applications, e.g., cellular therapy, drug delivery, hyperthermia, tissue repair, contrast agents with magnetic resonance imaging (MRI), magnetofection and cell separation (See, e.g., Bahadur, D. and J. Giri, "Biomaterials and magnetism" Sadhana- Academy Proceedings in Engineering Sciences, 2003. 28: p. 639-656; Gupta, A.K. et al., "Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications" Biomaterials, 2005. 26(18): p. 3995-4021). However, there is still a lack of Attorney Docket No. 700355-070131-PCT sufficient control in the fabrication process over both fiber characteristics and structure/fiber orientation of fabricated geometries. Thus, the existing electrospun polymeric materials generally have poor mechanical properties. Hence, there is a need for new types of biomaterials and methods of making the same with improved mechanical properties and abilities to respond to an external stimulus that allows manipulation of the biomaterial, e.g., upon implantation in a subject.

SUMMARY

[0005] Embodiments provided herein generally relate to magnetic-responsive silk fibroin-based materials, compositions comprising the same, and methods of making the same. The inventors have demonstrated that manipulation of both electric and magnetic fields enables better control of processing silk fibroin solutions that contain magnetic particles such as iron particles into fibers. For example, the ability to direct alignment and/or layering of silk fibroin fibers through the magnet technology (e.g., non-contacting magnet technology) can provide electrospun silk fibroin geometries with tailored physical and/or mechanical properties. Further, the post-electrospun silk fibroin-based composition can also be mechanically manipulated with magnetic fields in various applications, e.g., tissue engineering. The ability to incorporate magnetic particles in a silk solution that can then be processed, e.g., through electrospinning, to make fibers and/or mats can be extended to fabrication of other magneto-responsive silk fibroin- based materials, e.g., but not limited to hydrogels, scaffolds, foams, tubes, films, and/or sheets. Utilization of magnetic particles in protein systems, such as silk fibroin, for biomaterial applications is novel as there are no existing protein systems or protein materials that are incorporated with magnetic particles and can thus respond to an externally-applied magnetic field, e.g., movement of the protein material such as contraction of the protein material in response to a magnetic field, and/or a change in the physical and/or mechanical property of the protein material in response to a magnetic field.

[0006] Accordingly, one aspect provided herein relates to compositions comprising a silk fibroin-based material embedded with a plurality of magnetic particles. The silk fibroin- based material can be present in any format, including, e.g., but not limited to, a film, a sheet, a gel or hydrogel, a mesh, a mat, a non-woven mat, a fabric, a scaffold, a tube, a slab or block, a fiber (e.g., a regenerated silk fiber), a particle, a 3-dimensional construct, an implant, a high- density material, a reinforced material, a foam or a sponge, a machinable material, a

microneedle, or any combinations thereof.

[0007] The magnetic particles can include any ferromagnetic material, ferrimagnetic material, paramagnetic material or any combinations thereof. In some embodiments, the Attorney Docket No. 700355-070131-PCT magnetic particles can include ferromagnetic particles, e.g., ferrous particles such as iron particles. In one embodiment, the iron particles can include carbonyl iron particles.

[0008] Depending on processing parameters and/or size of the silk fibroin-based material, the magnetic particle embedded inside the silk fibroin-based material can be of any size. In some embodiments, the magnetic particles can have a diameter of about 1 nm to about 100 μιη. In some embodiments, the magnetic particles can have a diameter of about 5 nm to about 50 μιη. In some embodiments, the magnetic particles can have a diameter of about 10 nm to about 10 μιη. In some embodiments, the magnetic particles can have a diameter of about 10 nm to about 5 μιη.

[0009] In some embodiments, the composition can comprise one or more magneto- responsive silk fibroin fibers. In such embodiments, the silk fibroin fibers can be arranged to form a network. In some embodiments, the silk fibroin fiber can have a cross section of any shape, e.g., circular, triangular, square, polygonal or irregular, and/or of any dimension. In some embodiments, the silk fibroin fiber can have a substantially circular cross-section. In these embodiments, the silk fibroin fiber can have a diameter of about 0.1 μηι to about 1 mm or about 0.5 μηι to about 500 μιη.

[0010] As silk fibroin can generally stabilize active agents, some embodiments of the silk fibroin can be used to encapsulate and/or deliver an active agent. In these embodiments, at least one magneto-responsive silk fibroin-based material (e.g., but not limited to, silk fibroin fibers, gels, foams, tubes, and/or films) can further comprise one or more active agents. Non- limiting examples of the active agents can include cells, proteins, peptides, nucleic acids, nucleic acid analogs, nucleotides or oligonucleotides, peptide nucleic acids, aptamers, antibodies or fragments or portions thereof, antigens or epitopes, hormones, hormone antagonists, growth factors or recombinant growth factors and fragments and variants thereof, cell attachment mediators, cytokines, enzymes, antibiotics or antimicrobial compounds, viruses, toxins, therapeutic agents and prodrugs thereof, small molecules, and any combinations thereof.

[0011] In some embodiments, at least a portion of the magneto-responsive silk fibroin- based material (e.g., but not limited to, silk fibroin fibers, gels, foams, tubes, and/or films) can respond to an external magnetic field and/or gradient. For example, where the magneto- responsive silk fibroin-based material is a silk fibroin fiber (e.g., a regenerated silk fibroin fiber), the silk fibroin fiber can deflect or contract in response to an external magnetic field and/or gradient.

[0012] In some embodiments, the magneto-responsive silk fibroin-based material (e.g., but not limited to, silk fibroin fibers, gels, foams, tubes, and/or films) can further comprise a biopolymer. Examples of the biopolymers can include, but are not limited to, polyethylene oxide Attorney Docket No. 700355-070131-PCT

(PEO), polyethylene glycol (PEG), collagen, fibronectin, keratin, polyaspartic acid, polylysine, alginate, chitosan, chitin, hyaluronic acid, pectin, polycaprolactone, polylactic acid, polyglycolic acid, polyhydroxyalkanoates, dextrans, polyanhydrides, polymer, PLA-PGA, polyanhydride, polyorthoester, polycaprolactone, polyfumarate, collagen, chitosan, alginate, hyaluronic acid, other biocompatible and/or biodegradable polymers and any combinations thereof.

[0013] In one embodiment, a magneto-responsive silk fibroin-based material (e.g., but not limited to, silk fibroin fibers, gels, foams, tubes, and/or films) comprises a silk fibroin-based material as a base or core material and a plurality of magnetic particles embedded in the silk fibroin-based material.

[0014] In some embodiments, when magnetic particles, such as small gold particles, are embedded in a silk fibroin-based material, a variety of external energy sources (in addition to a magnetic field) can be used to impart local vibration, which can induce local shearing and temperature increases. Such effects can lead to, for example, conformation changes, construct breakdown, and/or enhanced drug release. The external energy source can be a vibrational source (such as ultrasound), an external electrical stimulus, or other source, such as a radio frequency source (or other source that can excite the natural frequency of the particles, depending on size, mass, and wavelength).

[0015] Different embodiments of the magneto-responsive silk fibroin-based materials (e.g., but not limited to, silk fibroin fibers, gels, foams, tubes, and/or films) can be adapted for use in various applications and/or compositions, e.g., where external manipulation via an external energy source (e.g., but not limited to, a magnetic field) is desirable. Examples of such applications and/or compositions can include, without limitations, medical implants, wound dressings, tissue engineering scaffolds, sensors, drug delivery devices, robotics, and separation membranes. In some embodiments, at least a portion of the magneto-responsive silk fibroin- based material (e.g., but not limited to, silk fibroin fibers, gels, foams, tubes, and/or films), e.g., at least a portion of the silk fibroin fiber, can align in the direction of, attract to, or repel from an applied magnetic field.

[0016] Another aspect provided herein relates to methods of producing a magneto- responsive silk fibroin-based material. In general, the magneto-responsive silk fibroin-based material in any format can be produced from a silk fibroin solution comprising magnetic particles or a magnetic fluid (e.g., a ferrofluid). Surprisingly, the magnetic silk fibroin solution can then be processed into different formats (e.g., but not limited to, fibers, gels, foams, tubes, and/or films) in accordance with methods known to produce a regular silk fibroin-based material without magnetic particles. Without wishing to be bound by theory, the magnetic particles (e.g., iron oxide particles) can act as an initiator and/or catalyst, thus increasing the rate at which the Attorney Docket No. 700355-070131-PCT silk solution gets solidified. By way of example only, a magneto-responsive hydrogel can be produced by eletrogelation of a silk fibroin solution comprising magnetic particles or a magnetic fluid (e.g., a ferro fluid), or by altering the pH of the magnetic silk fibroin solution via the addition of an acidic or a basic solution to increase the rate of gelation. A magneto-responsive foam can be produced by using the freeze-drying process. Layered magneto-responsive foams can also be made by applying multiple layers of magnetic silk solution on top of other frozen layers, and allowing the newly applied layer to freeze. The final frozen structure can then be placed in a lyophilizer where the structure is freeze-dried and water molecules are extracted from the construct. A magneto-responsive film can be produced by drying a magnetic silk fibroin solution on a substrate, e.g. petri dish or a piece of acrylic. The resulting construct can be annealed, e.g., by water annealing, and the resulting film can then be removed.

[0017] In some embodiments, a method of producing a magneto-responsive silk fibroin mat or mesh is provided herein. In some embodiments, the method can include: (a) forming a silk fibroin fiber from a silk fibroin solution comprising a plurality of magnetic particles; (b) forming a magnetic field in a predetermined pattern on a target solid substrate receiving the silk fibroin fiber, wherein the magnetic field pattern can determine an arrangement or orientation of the formed silk fibroin fiber on the target solid substrate; and (c) depositing the formed silk fibroin fiber onto the target solid substrate; thereby producing a magneto -responsive silk fibroin- based material comprising a silk fibroin fiber embedded with magnetic particles.

[0018] The concentration of the silk fibroin solution can be adjusted for each application, composition and/or product. In some embodiments, the silk fibroin solution can range from 1 wt% to about 50 wt%, or from about 5 wt% to about 45 wt%, or from about 10 wt% to about 40 wt%, or from about 20 wt% to about 40 wt%. In some embodiments, the silk fibroin solution can have a concentration of about 30 wt%.

[0019] Depending on the properties of the magnetic particles and/or applications, the plurality of the magnetic particles added to the silk fibroin solution can be present in a concentration of about 1 vol% to about 30 vol% or about 5 vol% to about 20 vol%. In one embodiment, about 10 vol% magnetic particles can be added to the silk fibroin solution.

[0020] The magnetic particles added to the silk fibroin solution can be of any size, e.g., smaller than the shortest dimension of the resultant silk fibroin fiber. In some embodiments, the magnetic particles can have a diameter of about 0.01 μιτι to about 100 μηι. In some

embodiments, the magnetic particles can have a diameter of about 0.1 μιη to about 50 μπι. In some embodiments, the magnetic particles can have a diameter of about 0.5 μιτι to about 10 μιη. In some embodiments, the magnetic particles can have a diameter of about 1 μιη to about 5 μηι. Attorney Docket No. 700355-070131-PCT

[0021] Any magnetic material can be used for the magnetic particles, including ferromagnetic material, ferrimagnetic material, paramagnetic material or any combinations thereof. In some embodiments, the magnetic particles can include ferromagnetic particles, e.g., ferrous particles such as iron particles. In one embodiment, the iron particles can include carbonyl iron particles.

[0022] In some embodiments, the silk fibroin solution can further comprise an additive, e.g., but not limited to, a conductivity enhancer agent, a biopolymer, a porogen, an active agent as described herein or any combinations thereof. In some embodiments, the additive added to the silk fibroin solution can include a conductivity enhancer agent that can increase conductivity of the silk fibroin solution. An exemplary conductivity enhancer agent can include any ions, e.g., water-soluble salts such as NaCl; a base, e.g., sodium hydroxide; conducting polymers; carbon nanotubes or fullerenes and related materials; metals in various forms (e.g., but not limited to, nano- or micro-particles, nano- or micro-rods, nano- or micro-prisms, nano- or micro-discs); ionomers; and/or any other art-recognized conductive materials that can be added into a silk fibroin solution. In some embodiments, the silk fibroin solution can further comprise a biopolymer, e.g., without limitations, polyethylene oxide (PEO), polyethylene glycol (PEG), collagen, fibronectin, keratin, polyaspartic acid, polylysine, alginate, chitosan, chitin, hyaluronic acid, pectin, polycaprolactone, polylactic acid, polyglycolic acid, polyhydroxyalkanoates, dextrans, polyanhydrides, polymer, PLA-PGA, polyanhydride, polyorthoester, polycaprolactone, polyfumarate, collagen, chitosan, alginate, hyaluronic acid, other biocompatible and/or biodegradable polymers and any combinations thereof. In some embodiments, the silk fibroin solution can further comprise a porogen, e.g., a salt. In some embodiments, the silk fibroin solution can further comprise an active agent described herein.

[0023] While the silk fibroin fiber can be formed from the silk fibroin solution with any methods known in the art, in some embodiments, the silk fibroin fiber can be formed, at least partly, by electrospinning the silk fibroin solution comprising the plurality of magnetic particles. In such embodiments, a voltage applied during electrospinning can range from about 5 kV to about 50 kV, from about 8 kV to about 40 kV, or from about 10 kV to about 30 kV. In one embodiment, the voltage applied during electrospinning is about 25 kV.

[0024] In some embodiments, the arrangement or orientation of the formed silk fibroin fiber within the silk fibroin-based material can include aligning the formed silk fibroin fiber in a direction of the magnetic field pattern. As such, in order to control the arrangement and/or alignment of the formed silk fibroin fiber within the silk-fibroin based material, in some embodiments, one or more magnets can be arranged on the target solid substrate on where the silk fibroin fiber deposits during electrospinning, such that the generated pattern of the magnetic Attorney Docket No. 700355-070131-PCT field can guide the arrangement and/or alignment of the formed silk fibroin fiber. In some embodiments, the generated pattern of the magnetic field can guide the arrangement and/or alignment of a plurality of silk fibroin fibers with the silk fibroin-based material. For example, the plurality of silk fibroin fibers can be arranged or aligned in a certain pattern/configuration within the silk fibroin-based material according to a dynamic control (e.g., temporal and/or spatial control) of the magnetic field pattern applied during electrospinning. The magnet(s) can be a permanent magnet, an electromagnet, or any combinations thereof.

[0025] In some embodiments, the target solid substrate receiving the silk fibroin fiber can be conductive. In some embodiments, the target solid substrate receiving the silk fibroin fiber can be grounded.

[0026] To alter a property of the magneto-responsive silk fibroin-based material (e.g., but not limited to, fibers, gels, foams, tubes, and/or films), post-treatment of the silk fibroin- based material can be employed. For example, post-treatment methods can be applied to the silk fibroin-based material to induce beta-sheet conformational change and thus modulate physical properties of silk fibroin (e.g., mechanical strength, degradability and/or solubility). Examples of various post-treatments can include, without limitations, drying (e.g., constraint-drying), mechanical stretching, lyophilization, gas-drying, solvent immersion (e.g., methanol and/or ethanol), water annealing, water vapor annealing, heat annealing, shear stress, ultrasound (e.g., by sonication), pH reduction (e.g., pH titration and/or exposing a silk fibroin-based material to an electric field), or any combination thereof.

[0027] Different embodiments of the methods described herein can be used to produce a magneto-responsive silk fibroin-based material (e.g., but not limited to, silk fibroin fibers, gels, foams, tubes, and/or films), which can be employed and adapted for use in different applications and/or compositions including, but not limited to, medical implants, wound dressings, tissue engineering scaffolds, sensors, drug delivery devices and separation membranes. Accordingly, provided herein also relates to compositions comprising a magneto-responsive silk fibroin-based material (e.g., a silk fibroin fiber or a network of silk fibroin fibers) produced by the methods described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figures 1A-1C are microscope images of magneto-sensitive silk fibroin fibers in accordance with one or more embodiments of the invention. Figures 1A-1C in increasing magnification show even distribution of iron particles within the silk fibroin fibers. Attorney Docket No. 700355-070131-PCT

[0029] Figure 2 shows an exemplary electrospinning setup configured for operation with magnetic field control that can be used to generate one or more embodiments of the magneto- sensitive silk fibroin fibers described herein.

[0030] Figures 3A-3B are close-up images of representative electromagnet arrangements that can be used for electrospinning magnetized silk fibroin. Figure 3A shows an array of electromagnets arranged in a circular pattern. Figure 3B shows an array of electromagnets arranged in a linear configuration.

[0031] Figures 4A-4B show front panel and block diagram for National Instruments LabVIEW program to control electromagnetically-assisted silk fibroin electrospinning, respectively.

[0032] Figures 5A-5B are images showing magnetized silk fibroin fibers forming on flat, square Neodymium magnet placed on the aluminum target disk during electrospinning. The Neodymium magnet can be covered in aluminum foil.

[0033] Figures 6A-6C are images showing magnetized silk fibroin fibers forming on Neodymium bar magnets formed into a 5 -pointed star. Figure 6A shows the aluminum foil wrapped on top of the star pattern and a series of fibers that were anchoring to the bar magnet locations from the needle mounted within an aluminum counter electrode disk positioned under the top of the electrospinning chamber. Figure 6B shows the aluminum foil removed from the magnet star pattern. Figure 6C shows that magnetized silk fibroin has built up on the aluminum foil in locations where the magnets were located.

[0034] Figures 7A-7C are images showing magnetized silk fibroin fibers forming on Neodymium spherical magnets in a hexagon pattern. Figure 7A shows that a silk fibroin fiber tower was formed, with the anchoring locations at the innermost perimeter of the hexagon pattern. Figure 7B shows an image of axially magnetized Neodymium spheres arranged in a planar hexagon pattern, and a silk fibroin mesh generated by the arrangement of the spherical magnets. Figure 7C shows that magnetized silk fibroin fibers are aligned locally with the magnetic field generated by the pattern of the axially magnetized spheres as shown in Figure 7B.

[0035] Figures 8A-8C are images showing magnetized silk fibroin fibers forming on Neodymium spherical magnets in a 3D cylindrical pattern. Figure 8A shows the formation of magnetized silk fibroin fibers on the aluminum foil target collector before removal from the electrospinner. Figure 8B shows a three-dimensional cylindrical arrangement of axially magnetized Neodymium spherical magnets, and a silk fibroin mesh generated by the

arrangement of the spherical magnets. Figure 8C shows that electrospun silk fibroin fibers are concentrated at the spherical magnet locations and the fibers span the gaps between the spherical Attorney Docket No. 700355-070131-PCT magnets. Fiber alignment can be seen around or at the magnets and in the spans between the magnets.

[0036] Figures 9A-9E are images showing magnetized silk fibroin fibers forming on two cylindrical stacks of Neodymium spherical magnets. Figure 9A shows two cylindrical patterns of axially magnetized spherical Neodymium magnets. Figures 9B-9D shows that a larger silk fibroin tower was formed, which draped beyond the magnets and aluminum disk target collector. A significant amount of aligned fiber spanned between the two cylindrical patterns of spherical magnets. Figure 9E shows that the produced silk fibroin mesh was fairly touch and could be stretched by hand.

[0037] Figures 1 OA- IOC are images showing close-up views of magnetized silk fibroin fibers generated as illustrated in Figures 9A-9E. Figures 1 OA- IOC show the fiber alignment in at least one direction within the silk fibroin mesh or mat.

[0038] Figures 1 lA-1 1C are images showing magneto-responsive silk fibroin hydrogels and an exemplary method of making the same. Figure 11 A is an image showing a magnetic silk fibroin hydrogel was formed by electro gelation of a mixture of silk fibroin solution and ferrofluid (e.g., 10 % v/v). Figure 1 IB is an image showing a magnetic silk fibroin hydrogel responding to a permanent (e.g., neodymium) magnet. The weight loss on the scale in the image indicates the gel was attracted to the magnetic placed above the gel. Figure 11C is an image showing a magnetic silk fibroin hydrogel responding to an electromagnet.

DETAILED DESCRIPTION OF THE INVENTION

[0039] The existing electrospun polymeric materials generally have poor mechanical properties because there is still a lack of sufficient control in the fabrication process over both fiber characteristics and structure/fiber orientation of fabricated geometries. Thus, new types of biomaterials with improved mechanical properties and abilities to respond to an external stimulus that allows manipulation of the biomaterial during fabrication and/or post- fabrication, e.g., during application of the biomaterial, are desirable.

[0040] The inventors have demonstrated inter alia that manipulation of both electric and magnetic fields enables better control of processing silk fibroin solutions that contain magnetic particles such as iron particles into fibers. Embedding ferrous particles in an electrospun silk fibroin fiber can be useful from several perspectives: (1) creating a method to help direct the fiber formation and enabling a controlled layup through the use of permanent or electromagnets; (2) providing a fibrous construct that can be actuated post-fabrication to allow for mechano-transduction (e.g., mechanical trigger to encourage cell differentiation/tissue property development) using non-invasive energy input; (3) incorporating an ability for an external Attorney Docket No. 700355-070131-PCT energy source to enhance the degradation and resorption of a silk-based scaffold (in a tunable way); and (4) ability to provide tunable drug-release capability. These advantages possessed by a magneto-responsive electrospun silk fibroin fiber can be extended to other silk fibroin-based material formats, e.g., but not limited to films, hydrogels, foams, scaffolds, particles, tubes, and any combinations thereof.

[0041] In accordance with some embodiments described herein, a silk fibroin-based material incorporated with a plurality of magnetic particles or a magnetic fluid can be manipulated with an externally-applied energy source (e.g., but not limited to, a magnetic field, ultrasound, radio frequency, and/or electromagnetic waves), depending on types of magnetic particles and optionally additives. In some embodiments, at least one silk fibroin fiber incorporated with magnetic particles, e.g., produced by electrospinning an aqueous silk fibroin solution comprising magnetic particles in the presence of both electric and magnetic fields, can be manipulated with externally-applied magnetic fields in various applications, e.g., tissue engineering. Further, incorporation of magnetic particles into a silk fibroin solution allows the use of a magnetic field during processing, for example, electrospinning to facilitate the alignment of electrospun silk fibroin fiber containing magnetic particles such as iron particles, thus providing electrospun silk fibroin material with tailored physical and/or mechanical properties. The utilization of magnetic particles in protein systems such as silk fibroin creates a novel biomaterial and methods of making the same, because unlike polymer-based biomaterials, protein systems incorporated with magnetic particles do not currently exist.

A silk fibroin-based material embedded with magnetic particles

[0042] One aspect provided herein relates to compositions comprising a silk fibroin- based material embedded with a plurality of magnetic particles. The magnetic particles can be incorporated in any format of a silk fibroin-based material, including, but not limited to, a film (See, e.g., U.S. Patent Nos. 7,674,882; and 8,071,722); a sheet (see, e.g., PCT/US 13/24744 filed February 5, 2013); a gel (see, e.g., U.S. Patent No. 8,187,616; and U.S. Pat. App. Nos. US 2012/0070427; and US 201 1/0171239); a mesh or a mat (see, e.g., International Pat. App. No. WO 201 1/008842); a non- woven mat or fabric (see, e.g., International Pat. App. Nos. WO 2003/043486 and WO 2004/080346); a scaffold (see, e.g., U.S. Patent Nos. 7,842,780; and 8,361 ,617); a tube (see, e.g., U.S. Pat. App. No. US 2012/0123519; International Pat. App. No. WO 2009/126689; and International Pat. App. Serial No. PCT/US 13/30206 filed March 11, 2013); a slab or block; a fiber (see, e.g., U.S. Pat. App. No. US 2012/0244143); a 3 dimensional construct (see, e.g., International Pat. App. No. WO 2012/145594, including, but not limited to, an implant, a screw, a plate); a high-density material; a porous material (see, e.g., U.S. Patent Attorney Docket No. 700355-070131-PCT

Nos. 7,842,780; and 8,361 ,617); a coating (see, e.g., International Patent Application Nos. WO 2007/016524; WO 2012/145652); a magnetic-responsive material; a microneedle (see, e.g., International Patent Application No. WO 2012/054 82); a machinable material; or any combinations thereof. In some embodiments, a plurality of magnetic particles can be incorporated into a silk fibroin particle (see, e.g., U.S. Patent Application Nos. US

2010/0028451 ; and US 2012/0187591 for nanospheres and/or microspheres). The contents of all the aforementioned patent applications are incorporated herein by reference.

[0043] In accordance with various embodiments described herein, at least a portion of the silk fibroin-based material comprising magnetic particles can respond to or be actuated with an external energy source (e.g., a magnetic field). For example, in some embodiments, at least a portion of a silk fiber comprising magnetic particles can align in the direction of an externally- applied magnetic field.

[0044] In some embodiments, the magnetic particles or a magnetic fluid (e.g., a ferrofluid) can be present in the silk fibroin-based material in an amount of about 0.001 wt% to about 50 wt%, about 0.01 wt% to about 40 wt%, about 0.1 wt% to about 30 wt%, or about 1 wt% to about 20 wt%, of the total weight of the material. In some embodiments, the magnetic particles or a magnetic fluid (e.g., a ferrofluid) can be present in the silk fibroin-based material in an amount of about 0.001 vol% to about 50 vol%, about 0.01 vol% to about 40 vol%, about 0.1 vol% to about 30 vol%, or about 1 vol% to about 20 vol%, of the total weight of the material. In some embodiments, the magnetic particles or a magnetic fluid (e.g., a ferrofluid) can be present in the silk fibroin-based material in an amount of about 0.001 w/v to about 50 w/v, about 0.01 w/v to about 40 w/v, about 0.1 w/v to about 30 w/v, or about 1 w/v to about 20 w/v, of the total weight of the material.

[0045] Silk fibroin: Silk fibroin is a particularly appealing protein polymer candidate to be used for various embodiments described herein, e.g., because of its versatile processing e.g., all-aqueous processing (Sofia et al., 54 J. Biomed. Mater. Res. 139 (2001); Perry et al., 20 Adv. Mater. 3070-72 (2008)), relatively easy functionalization (Murphy et al, 29 Biomat. 2829-38 (2008)), and biocompatibility (Santin et al., 46 J. Biomed. Mater. Res. 382-9 (1999)). For example, silk has been approved by U.S. Food and Drug Administration as a tissue engineering scaffold in human implants. See Altman et al., 24 Biomaterials: 401 (2003).

[0046] As used herein, the term "silk fibroin" includes silkworm fibroin and insect or spider silk protein. See e.g., Lucas et al., 13 Adv. Protein Chem. 107 (1958). Any type of silk fibroin can be used according to different aspects described herein. Silk fibroin produced by silkworms, such as Bombyx mori, is the most common and represents an earth-friendly, renewable resource. For instance, silk fibroin used in a silk fibroin-based material can be Attorney Docket No. 700355-070131-PCT attained by extracting sericin from the cocoons of B. mori. Organic silkworm cocoons are also commercially available. There are many different silks, however, including spider silk (e.g., obtained from Nephila clavipes), transgenic silks, genetically engineered silks, such as silks from bacteria, yeast, mammalian cells, transgenic animals, or transgenic plants (see, e.g., WO 97/08315; U.S. Patent No. 5,245,012), and variants thereof, that can be used. In some embodiments, silk fibroin can be derived from other sources such as spiders, other silkworms, bees, and bioengineered variants thereof. In some embodiments, silk fibroin can be extracted from a gland of silkworm or transgenic silkworms (see, e.g., WO 2007/098951).

[0047] In some embodiments, the silk fibroin can include an amphiphilic peptide. In other embodiments, the silk fibroin can exclude an amphiphilic peptide. "Amphiphilic peptides" possess both hydrophilic and hydrophobic properties. Amphiphilic molecules can generally interact with biological membranes by insertion of the hydrophobic part into the lipid membrane, while exposing the hydrophilic part to the aqueous environment. In some embodiment, the amphiphilic peptide can comprise a RGD motif. An example of an amphiphilic peptide is a 23RGD peptide having an amino acid sequence: HOOC-Gly-ArgGly-Asp-Ile-Pro- Ala-Ser-Ser-Lys-Gly-Gly-Gly-Gly-SerArg-Leu-Leu-Leu-Leu-Leu-Leu-Arg-NH2. Other examples of amphiphilic peptides include the ones disclosed in the U.S. Patent App. No.: US 2011/0008406, the content of which is incorporated herein by reference.

[0048] Silk fibroin can be present in a magneto-responsive silk fibroin-based material described herein at any concentration. In some embodiments, silk fibroin can be present in the a magneto-responsive silk fibroin-based material in an amount of about 1 wt% to about 50 wt%, about 3 wt% to about 45 wt%, about 5 wt% to about 40 wt%, or about 10 wt% to about 35 wt%, of the total weight. In some embodiments, silk fibroin can be present in the magneto-responsive silk fibroin-based material in an amount of about 10 wt% to about 99 wt% or higher, about 40 wt% to about 95 wt%, about 50 wt% to about 90 wt%, of the total weight. In some

embodiments, silk fibroin can be present in the magneto-responsive silk fibroin-based material in an amount of at least about 10 wt%, at least about 15 wt%, at least about 20 wt%, at least about 25 wt%, at least about 30 wt%, at least about 35 wt%, at least about 40 wt%, at least about 45 wt%, at least about 50 wt%, at least about 60 wt%, at least about 70 wt%, at least about 80 wt%, at least about 90 wt%, at least about 95 wt% or higher, of the total weight. In one embodiment, a magneto-responsive silk fibroin-based material comprises a silk fibroin-based material as a base or core material and a plurality of magnetic particles embedded in the silk fibroin-based material.

[0049] In various embodiments, the silk fibroin can be modified for different applications and/or desired mechanical or chemical properties (e.g., to facilitate formation of a Attorney Docket No. 700355-070131-PCT gradient of an additive (e.g., an active agent) in silk fibroin-based materials). One of skill in the art can select appropriate methods to modify silk fibroins, e.g., depending on the side groups of the silk fibroins, desired reactivity of the silk fibroin and/or desired charge density on the silk fibroin. In one embodiment, modification of silk fibroin can use the amino acid side chain chemistry, such as chemical modifications through covalent bonding, or modifications through charge-charge interaction. Exemplary chemical modification methods include, but are not limited to, carbodiimide coupling reaction (see, e.g. U.S. Patent Application. No. US

2007/0212730), diazonium coupling reaction (see, e.g., U.S. Patent Application No. US

2009/0232963), avidin-biotin interaction (see, e.g., International Application No.: WO

2011/011347) and pegylation with a chemically active or activated derivatives of the PEG polymer (see, e.g., International Application No. WO 2010/057142). Silk fibroin can also be modified through gene modification to alter functionalities of the silk protein (see, e.g., International Application No. WO 201 1/006133). For instance, the silk fibroin can be genetically modified, which can provide for further modification of the silk such as the inclusion of a fusion polypeptide comprising a fibrous protein domain and a mineralization domain, which can be used to form an organic-inorganic composite. See WO 2006/076711. In some embodiments, the silk fibroin can be genetically modified to be fused with a protein, e.g., a therapeutic protein. Additionally, the silk fibroin-based material can be combined with a chemical, such as glycerol, that, e.g., affects flexibility of the material. See, e.g., WO

2010/042798, Modified Silk films Containing Glycerol. The contents of the aforementioned patent applications are all incorporated herein by reference.

[0050] In some embodiments, a magneto-responsive silk fibroin-based material can further comprise at least one biopolymer, including at least two biopolymers, at least three biopolymers or more. For example, a magneto-responsive silk fibroin-based material can comprise one or more biopolymers in a total concentration of about 0.5 wt% to about 70 wt%, about 5 wt% to about 60 wt%, about 10 wt% to about 50 wt%, about 15 wt% to about 45 wt% or about 20 wt% to about 40 wt%. In some embodiments, the biopolymer(s) can be incorporated homogenously or heterogeneously into the magneto-responsive silk fibroin-based material. In other embodiments, the biopolymer(s) can be coated on a surface of the magneto-responsive silk fibroin-based material. In any embodiments, the biopolymer(s) can be covalently or non- covalently linked to silk fibroin in a magneto-responsive silk fibroin-based material. In some embodiments, the biopolymer(s) can be blended with silk fibroin within a magneto-responsive silk fibroin-based material. Examples of the biopolymer can include biocompatible and/or biodegradable polymer, e.g., but are not limited to, polyethylene oxide (PEO), polyethylene glycol (PEG), collagen, fibronectin, keratin, polyaspartic acid, polylysine, alginate, chitosan, Attorney Docket No. 700355-070131-PCT chitin, hyaluronic acid, pectin, polycaprolactone, polylactic acid, polyglycolic acid,

polyhydroxyalkanoates, dextrans, polyanhydrides, polymer, PLA-PGA, polyanhydride, polyorthoester, polycaprolactone, polyfumarate, collagen, chitosan, alginate, hyaluronic acid, other biocompatible and/or biodegradable polymers and any combinations thereof. See, e.g., International Application Nos.: WO 04/062697; WO 05/012606. The contents of the international patent applications are all incorporated herein by reference. Depending on various applications (e.g., in a wound dressing), in some embodiments, a magneto -responsive silk fibroin-based material can include about 1% to about 50%, or about 2% to about 3% to about 10% polyethylene oxide (e.g., PEO with a molecular weight of about 500, 000 to about 1,500,000). In other embodiments, the silk fibroin/ PEO blend ratio in a magneto-responsive silk fibroin-based material can vary from about 1 : 100 to about 100: 1. In some embodiments, the silk fibroin/PEO blend ratio in a magneto-responsive silk fibroin-based material can vary from about 2: 1 to about 4: 1. See, e.g., International Application No.: WO 2011/008842, the content of which is incorporated herein by reference.

[0051] In some embodiments, a magneto-responsive silk fibroin-based material can further comprise at least one active agent as described below. The active agent can be dispersed homogeneously or heterogeneously within silk fibroin, or dispersed in a gradient, e.g., using the carbodiimide-mediated modification method described in the U.S. Patent Application No. US 2007/0212730. In some embodiments, the active agent can be coated on a surface of the magneto-responsive silk fibroin-based material, e.g., via diazonium coupling reaction (see, e.g., U.S. Patent Application No. US 2009/0232963), and/or avidin-biotin interaction (see, e.g., International Application No. : WO 201 1/011347). Non-limiting examples of the active agents can include cells, proteins, peptides, nucleic acids, nucleic acid analogs, nucleotides or oligonucleotides, peptide nucleic acids, aptamers, antibodies or fragments or portions thereof, antigens or epitopes, hormones, hormone antagonists, growth factors or recombinant growth factors and fragments and variants thereof, cell attachment mediators, cytokines, enzymes, antibiotics or antimicrobial compounds, viruses, toxins, therapeutic agents and prodrugs thereof, small molecules, and any combinations thereof. See, e.g., the International Patent Application No. WO/2012/145739 for compositions and methods for stabilization of active agents with silk fibroin. In some embodiments, an active agent can be genetically fused to silk fibroin to form a fusion protein. The contents of the aforementioned patent applications are incorporated herein by reference.

[0052] Any amounts of an active agent can be present in a magneto-responsive silk fibroin-based material. For example, in some embodiments, an active agent can be present in the magneto-responsive silk fibroin-based material at a concentration of about 0.001 wt% to about Attorney Docket No. 700355-070131-PCT

50 wt%, about 0.005 wt% to about 40 wt%, about 0.01 wt% to about 30 wt%, about 0.05 wt % to about 20 wt%, about 0.1 wt% to about 10 wt %, or about 0.5 wt% to about 5 wt%.

[0053] In some embodiments, the magneto-responsive silk fibroin-based material can further comprise at least one other additive. For example, an additive can alter flexibility, solubility, ease of processing, and/or enhanced stability of at least one property of the component distributed therein. Additionally or alternatively, an additive can interact with an external energy source (including, e.g., vibrational sources (e.g., ultrasound), electric field, electromagnetic waves or light, radio frequency, heat, and also any combinations thereof) to create a local effect that can change the material conformation and/or properties (e.g., use of external vibration to enhance crystallinity and thereby improving mechanical properties).

Examples of other additives can include, but are not limited to, plasticizers (e.g., glycerol); nanoparticles (e.g., gold nanoparticles or plasmonic particles); optical particles (e.g., fluorescent particles); and any combinations thereof. In one embodiment, adding an additive that can interact with an external energy source (other than a magnetic field), e.g., therapeutic ultrasound, external to the body can be used to create and/or enhance local vibration in an implanted magneto-responsive silk fibroin-based material.

[0054] Magnetic particles: The magnetic particles embedded in a magneto-responsive silk fibroin-based material can be of any shape, including but not limited to spherical, rod, elliptical, cylindrical, and disc. In some embodiments, magnetic particles having a substantially spherical shape and defined surface chemistry can be used to minimize chemical agglutination and non-specific binding, e.g., with an active agent. As used herein, the term "magnetic particles" can refer to a nano- or micro-scale particle that is attracted or repelled by a magnetic field gradient or has a non-zero magnetic susceptibility. Depending on processing parameters and/or size of the magneto-responsive silk fibroin-based material, the magnetic particle embedded inside the magneto-responsive silk fibroin-based material can be of any size. The magnetic particles can range in size from 1 nm to 1 mm. For example, magnetic particles can be about 1 nm to about 500 μηι in size, or about 5 nm to about 250 μιη in size, or about 50 nm to about 250 μηι in size. In some embodiments, magnetic particles can be about 0.01 μηι to about 100 μιη in size. In some embodiments, magnetic particles can be about 0.1 μιη to about 50 μιη in size. In some embodiments, magnetic particles can be about 0.5 μηι to about 10 μιη in size. In some embodiments, magnetic particles can be about 1 μιη to about 5 μιτι in size. In some embodiments, the magnetic particles can be about 1 nm to about 1000 nm, or about 5 nm to about 500 nm, or about 10 nm to about 250 nm in size.

[0055] Magnetic particles are a class of particles which can be manipulated using magnetic field and/or magnetic field gradient. Such particles commonly consist of magnetic Attorney Docket No. 700355-070131-PCT elements such as iron, nickel and cobalt and their oxide compounds. Magnetic particles

(including nanoparticles or microparticles) are well-known and methods for their preparation have been described in the art. See, e.g., U.S. Patents No. 6,878,445; No. 5,543,158; No.

5,578,325; No. 6,676,729; No. 6,045,925; and No. 7,462,446; and U.S. Patent Publications No. 2005/0025971; No. 2005/0200438; No. 2005/0201941; No. 2005/0271745; No. 2006/0228551; No. 2006/0233712; No. 2007/01666232; and No. 2007/0264199, the contents of which are incorporated herein by reference. Magnetic particles are also widely and commercially available.

[0056] The magnetic particles can include any ferromagnetic material, paramagnetic material, superparamagnetic material or any combinations thereof. As used herein, the term "ferromagnetic" refers to materials having large and positive susceptibility to an external magnetic field. Ferromagnetic materials have some unpaired electrons so their atoms have a net magnetic moment. They exhibit a strong attraction to magnetic fields and are able to retain their magnetic properties for at least a period of time after the external field has been removed. In some embodiments, a ferromagnetic material can include a ferrimagnetic material, which exhibits different hallmarks of ferromagnetic behavior, e.g., spontaneous magnetization, Curie temperatures, hysteresis, and remanence, but is different from ferromagnetism in terms of magnetic ordering. Examples of ferromagnetic (including ferrimagnetic) materials include, but are not limited to, iron, nickel and cobalt, Magnetite (Fe₃0₄), maghemite (yFe₂0₃), jacobsite (MnFe₂0₄), trevorite (NiFe₂0₄), magnesioferrite (MgFe₂0₄), pyrrhotite (Fe₇Ss), greigite (Fe₃S₄), feroxyhyte (5FeOOH), awaruite (Ni₃Fe), wairauite (CoFe), and any combinations thereof.

[0057] In some embodiments, the magnetic particles embedded in a silk fibroin-based material can include ferromagnetic particles, e.g., ferrous particles such as iron particles. In one embodiment, the iron particles can include carbonyl iron particles. Carbonyl iron is generally a highly pure iron (e.g., -97.5% for grade S, -99.5+% for grade R), prepared by chemical decomposition of purified iron pentacarbonyl. It is usually composed of spherical microparticles. Most of the impurities include, e.g., carbon, oxygen, and nitrogen. Carbonyl iron has also been used in pharmaceutical applications as iron supplements to treat iron deficiency. Even in non- anemic persons, high doses of carbonyl iron can be tolerated (Gordeuk, V et al., "Carbonyl iron therapy for iron deficiency anemia" Blood, 1986. 67 (3): 745-752). Thus, some embodiments of a silk fibroin-based material comprising carbonyl iron as magnetic particles can be used in vivo, e.g., for tissue engineering applications.

[0058] In some embodiments, magnetic particles embedded in a silk fibroin-based material can include a paramagnetic material. As used herein, the term "paramagnetic" refers to materials having a small and positive susceptibility to magnetic fields, which are slightly attracted by a magnetic field. In some embodiments, paramagnetic materials do not retain Attorney Docket No. 700355-070131-PCT magnetic properties when the external field is removed. These paramagnetic properties are due to the presence of some unpaired electrons and the realignment of the electron orbits caused by the external magnetic field. Examples of paramagnetic materials include, but are not limited to, magnesium, molybdenum, and lithium.

[0059] In some embodiments, magnetic particles embedded in a silk fibroin-based material can include a superparamagnetic material. The term "superparamagnetic" as used herein refers to the property of materials, which have no permanent (equiaxed) alignment of the elementary magnetic dipoles in the absence of the action of external magnetic fields. In the presence of an external magnetic field, however, superparamagnetic materials can have magnetic susceptibilities at a level similar to ferromagnetic materials. Superparamagnetism can occur when the diameter of the crystalline regions in a normally ferromagnetic substance falls below a particular critical value.

[0060] In some embodiments, magnetic particles can have a polymer shell and/or a surfactant coating, e.g., for inhibiting particle aggregation and/or for protecting silk fibroin and/or any active agent dispersed therein from exposure to iron provided that the polymer shell and/or the surfactant coating has no adverse effect on the magnetic property and/or silk fibroin composition. For example, the magnetic particles can be coated with a biocompatible polymer. Additionally or alternatively, the magnetic particles can be coated with a surfactant including, e.g., but not limited to, lipids, hydrocarbons, and/or any surfactant commonly used in a ferrofluid.

[0061] In some embodiments, magnetic particles distributed in a silk fibroin-based material for different applications can be functionalized with an organic moiety or functional group. Functionalized magnetic particles can allow interaction (e.g., binding) of the magnetic particles with an agent (e.g., an active agent and/or silk fibroin protein). By way of example only, the functionalized magnetic particles can be covalently or non-covalently linked to a silk fibroin protein and thus become immobilized at a certain location within a silk fibroin-based material, e.g., for better manipulation or actuation of the silk fibroin-based via an external magnetic field. Such organic moiety or functional groups can typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NH, C(O), C(0)NH, SO, SO₂, SO₂NH, SS, or a chain of atoms, such as substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted C5-C12 heteroaryl, substituted or unsubstituted C5-C12 heterocyclyl, substituted or unsubstituted C3-C12 cycloalkyl, where one or more methylenes can be interrupted or terminated by O, S, S(O), S0₂, NH, C(O). Attorney Docket No. 700355-070131-PCT

[0062] In some embodiments, the organic moiety or functional group can be a branched moiety or functional group, which contains a branchpoint available for multiple valencies. Examples of branchpoint can include, but not limited to, -N, -N( )-C, -O-C, -S-C, -SS-C, - C(0)N(R)-C, -OC(0)N(R)-C, -N(R)C(0)-C, or -N(R)C(0)0-C; wherein R is independently for each occurrence H or optionally substituted alkyl. In some embodiments, the branchpoint is glycerol or derivative thereof.

[0063] In some embodiments, the organic moiety or functional groups can include surface functional groups, e.g., amino groups, carboxylic acid groups, epoxy groups, tosyl groups, or silica-like groups. Suitable magnetic particles are commercially available such as from PerSeptive Diagnostics, Inc. (Cambridge, MA); Invitrogen Corp. (Carlsbad, CA); Cortex Biochem Inc. (San Leandro, CA); and Bangs Laboratories (Fishers, IN). In particular embodiments, magnetic particles that can be used herein can be any DYNABEADS® magnetic particles (Invitrogen Inc.), depending on the substrate surface chemistry.

[0064] In some embodiments, the organic moiety or functional group can be one member of an affinity binding pair that can facilitate the conjugation of the magnetic particles to an agent, e.g., an active agent and/or silk fibroin protein. The term "affinity binding pair" or "binding pair" refers to first and second molecules that specifically bind to each other. One member of the binding pair is conjugated with first part to be linked while the second member is conjugated with the second part to be linked. As used herein, the term "specific binding" refers to binding of the first member of the binding pair to the second member of the binding pair with greater affinity and specificity than to other molecules.

[0065] Exemplary binding pairs include any haptenic or antigenic compound in combination with a corresponding antibody or binding portion or fragment thereof (e.g., mouse immunoglobulin and goat antimouse immunoglobulin) and nonimmunological binding pairs (e.g., biotin-avidin, biotin-streptavidin, receptor-receptor agonist, receptor-receptor antagonist (e.g., acetylcholine receptor-acetylcholine or an analog thereof), IgG-protein A, IgG-protein G, IgG-synthesized protein AG, lectin-carbohydrate, enzyme-enzyme cofactor, enzyme-enzyme inhibitor, and complementary oligonucleotide pairs capable of forming nucleic acid duplexes), and the like. The binding pair can also include a first molecule which is negatively charged and a second molecule which is positively charged.

[0066] A plurality of magnetic particles can be distributed or embedded homogenously or heterogeneously within a silk fibroin-based material. Any number or concentration of the magnetic particles can be embedded in the silk fibroin-based material. In some embodiments, the magnetic particles can be present within the silk fibroin-based material in an amount or concentration of at least about 0.1% (v/v), at least about 0.5% (v/v), at least about 1% (v/v), at Attorney Docket No. 700355-070131-PCT least about 2% (v/v), at least about 3% (v/v), at least about 4% (v/v), at least about 5% (v/v), at least about 6% (v/v), at least about 7% (v/v), at least about 8% (v/v), at least about 9% (v/v), at least about 10% (v/v), at least about 15% (v/v), at least about 20% (v/v), at least about 30 % (v/v), at least about 40% (v/v), at least about 50% (v/v), at least about 60% (v/v), at least about 70% (v/v), at least about 80% (v/v), or higher. In one embodiment, the magnetic particles can be present within the silk fibroin-based material in an amount of about 5% (v/v) to about 15% (v/v). In one embodiment, the magnetic particles can be present within the silk fibroin-based material in an amount of about 0.1% (v/v) to about 10% (v/v). In one embodiment, the magnetic particles can be present within the silk fibroin-based material in an amount of about 10% (v/v).

[0067] In some embodiments, the magnetic particles can be present within a silk fibroin- based material in an amount of at least about 0.01 % (w/w), at least about 0.05% (w/w), at least about 0.1% (w/w), at least about 0.5% (w/w), at least about 1% (w/w), at least about 2% (w/w), at least about 3% (w/w), at least about 4% (w/w), at least about 5% (w/w), at least about 6% (w/w), at least about 7% (w/w), at least about 8% (w/w), at least about 9% (w/w), at least about 10%) (w/w), at least about 15% (w/w), at least about 20% (w/w), at least about 30 % (w/w), at least about 40% (w/w), at least about 50% (w/w), at least about 60% (w/w), at least about 70% (w/w), at least about 80%> (w/w), or higher. In one embodiment, the magnetic particles can be present within the silk fibroin-based material in an amount of about 5% (w/w) to about 15% (w/w). In one embodiment, the magnetic particles can be present within the silk fibroin-based material in an amount of about 0.1% (w/w) to about 10% (w/w). In one embodiment, the magnetic particles can be present within the silk fibroin-based material in an amount of about 0.1% (w/w) to about 20% (w/w).

[0068] In some embodiments, magnetic particles can be embedded in a silk fibroin- based material in any amount sufficient to allow the silk fibroin-based material respond to or be actuated with an external magnetic field and/or gradient. The phrase "respond to an external magnetic field" or "actuated with an external magnetic field" as used interchangeably herein and the terms "magneto-responsive" or "magnetic-responsive" as used interchangeably herein and below refer to a property of at least a portion of a silk fibroin-based material (comprising magnetic particles) that can produce any macroscopic or microscopic motion in response to an external magnetic field.

[0069] In some embodiments, a magneto-responsive silk fibroin-based material can change at least one property, e.g., but not limited to, size (e.g., volume), shape, temperature, mechanical (e.g., stiffness), crystallinity of silk fibroin, degradation rate of the material, release of an active agent (if any) from the material, orientation and/or arrangement, porosity and/or pore size of the material (for porous silk fibroin-based material), and any combinations thereof, Attorney Docket No. 700355-070131-PCT in the presence of a magnetic field. In some embodiments, at least a portion of a magneto- responsive silk fibroin-based material can be induced to microscopically or macroscopically move, e.g., vibrating, deflecting, bending, translating, contracting, stretching, and/or expanding, in the presence of a magnetic field and/or magnetic field gradient.

[0070] By way of example only, a magneto-responsive hydrogel can change its volume and/or size (e.g., deform, expand or contract) in the presence of a magnetic field, or an alternating magnetic field (AMF). As the gel expands and contracts in the presence of AMF, this allows water (and any active agent, if any) to release from the gel matrix via diffusion. Without wishing to be bound by theory, the AMF can enhance the diffusion of water (and any active agent, if any) out of the gel. In the absence of the AMF the hydrogel can reabsorb water from its surroundings, and upon reapplication of the AMF the gel expands and contracts thereby releasing water (and any active agent, if any) again.

[0071] In some embodiments where silk fibroin-based material is a fiber and/or a particle, at least a portion of the silk fibroin fiber and/or particle can align along the direction of an applied magnetic field. In some embodiments, in the presence of an external magnetic field, at least a portion of the silk fibroin fiber and/or particle can deform, vibrate, deflect, bend, contract, stretch, and/or translate (i.e., moving from one position to another) in a direction toward or away from the external magnetic field source. In some embodiments, at least a portion of the magneto-responsive silk fibroin-based material (e.g., but not limited to, a hydrogel, a film, a foam, a fiber and/or particle) can deflect, bend, contract, stretch, and/or translate (i.e., moving from one position to another) by a distance of at least about 0.5 nm, at least about 0.001 μιη, at least about 0.005 μηι, at least about 0.01 μιη, at least about 0.05 μηι, at least about 0.1 μιτι, at least about 0.5 μιη, at least about 1 μπι, at least about 5 μηι, at least about 10 μιτι, at least about 25 μιη, at least about 50 μηι, at least about 100 μηι, at least about 250 μιη, at least about 500 μηι, at least about 750 μιη, at least about 1000 μηι, at least about 2 mm, at least about 3 mm, or more, in a direction toward or away from the external magnetic field source.

[0072] In some embodiments where a composition comprises a network of silk fibroin fibers and/or silk fibroin particles, the motion of at least a portion of the silk fibroin fibers and/or silk fibroin particles in response to an external magnetic field can affect one or more properties of at least part of the network or the composition, e.g., porosity and/or pore size, size (e.g., volume), and/or mechanical properties of the network of fibers or composition.

[0073] In some embodiments, the magnetic responsiveness of at least a portion of the magneto-responsive silk fibroin-based material can produce a structural or physical modification of the material itself and/or the composition comprising the magneto-responsive silk fibroin- Attorney Docket No. 700355-070131-PCT based material. In some embodiments, the magnetic responsiveness of at least a portion of the magneto-responsive silk fibroin-based material can modulate the stiffness of the material itself and/or the composition comprising the magneto-responsive silk fibroin-based material. In other embodiments, the magnetic responsiveness of at least a portion of the magneto-responsive silk fibroin-based material can result in deformation (e.g., shape and/or volume change) of the material itself or a composition comprising the magneto-responsive silk fibroin-based material. By way of example only, in the presence of an external magnetic field, contraction of at least a portion of the magneto-responsive silk fibroin fibers and/or silk fibroin particles within the network can increase the bulk porosity and/or pore size of the network and/or the composition comprising the network of silk fibroin fibers and/or silk fibroin particles. In such embodiments, the composition comprising the network of silk fibroin fibers and/or silk fibroin particles embedded with magnetic particles can be used as a filter or drug reservoir, wherein changes in the bulk porosity and/or pore sizes of the composition (e.g., the network of fibers) in response to an external magnetic field can control the flow of a fluid through the filter or the release of a drug from the drug reservoir. In other embodiments, the combined motion of silk fibroin fibers and/or silk fibroin particles in response to an external magnetic field can actuate the network comprising the silk fibroin fibers and/or silk fibroin particles to produce a particular movement. In such embodiments, a composition comprising the network of silk fibroin fibers and/or silk fibroin particles embedded with the magnetic particles, e.g., when used as part of a robotic component, can be used as an actuator to control the movement of the robotic component.

[0074] In any embodiments described herein, the change in one or more properties of the silk fibroin-based material due to magnetic responsiveness of the silk fibroin-based material can be completely or at least partially reversible, i.e., one or more properties of the silk fibroin-based material can be restored to at least part of the original state after removal of the magnetic field and/or gradient.

[0075] The external magnetic field and/or gradient can be generated by a magnetic field and/or gradient source. A magnetic field and/or gradient source can be one or more permanent magnets, one or more electromagnets (including electrically-polarizable elements), one or more magnetic field concentrators and/or magnetic materials integrated as part of a composition described herein. In some embodiments, a permanent magnet of any shape (e.g., but not limited to, cylinders, spheres, squares) can be used to create a magnetic field and/or gradient. An exemplary permanent magnet that can be used as a magnetic field and/or gradient source can include, but not limited to, a neodymium magnet, which is a member of the rare earth magnet family and is generally referred to as a NdFeB magnet composed mainly of neodymium (Nd), iron (Fe) and boron (B). Additional examples of permanent magnet materials that can be used as Attorney Docket No. 700355-070131-PCT a magnetic field and/or gradient source for controlling a silk fibroin-based material and fabrication methods thereof described herein can include iron, nickel, cobalt, alloys of rare earth metals, naturally occurring minerals such as lodestone, and any combinations thereof.

[0076] In some embodiments, an electromagnet of any shape (e.g., but not limited to, cylinders, spheres, squares) can be used to create a magnetic field and/or gradient. An electromagnetic controller can be used to control and adjust the magnetic field and/or gradients thereof, and thus affect the alignment of a silk fibroin-based material (e.g., a silk fibroin fiber) and/or the response of the silk fibroin-based material (e.g., a silk fibroin fiber). The magnetic gradient can be produced at least in part according to a pre-programmed pattern. The magnetic gradient can have a defined magnetic field strength and/or spatial orientation. In some embodiments, the magnetic gradient has a defined magnetic field strength. An "electromagnet" is generally a type of magnet in which the magnetic field is produced by the flow of electric current. The magnetic field disappears when the current is turned off. The polarity of the electromagnet can be determined by controlling the direction of the electrical current in the wire.

[0077] As used herein, the term "magnetic field" generally refers to magnetic influences which create a local magnetic flux that flows through a composition and can refer to field amplitude, squared-amplitude, or time-averaged squared-amplitude. It is to be understood that a magnetic field and/or gradient can be created with a direct-current (DC) magnetic field or alternating-current (AC) magnetic field. The magnetic field strength can range from about 0.0001 Tesla to about 10 Tesla. In some embodiments, the magnetic field strength can be in the range from about 0.001 Tesla to about 5 Tesla. In some other embodiments, the magnetic field strength can be in the range from about 0.01 Tesla to about 2.5 Tesla. In some embodiments, the magnetic field strength can range from about 0.1 Tesla to about 2 Tesla.

[0078] The term "magnetic field gradient" as used herein refers to a variation in the magnetic field with respect to position. By way of example only, a one-dimensional magnetic field gradient is a variation in the magnetic field with respect to one direction, while a two- dimensional magnetic field gradient is a variation in the magnetic field with respect to two directions. The magnitude of the magnetic field gradient can be sufficient to cause at least a portion of the silk fibroin-based material to change in size (e.g., volume), shape, temperature, crystallinity of silk fibroin, mechanical property (e.g., stiffness), degradation rate, release of an active agent (if any) released from therein, or any combinations thereof; or to vibrate, deflect, bend, contract, stretch, and/or translate (i.e., moving from one position to another) in a direction of the magnetic field gradient. In some embodiments, the magnetic field gradient can cause at least a portion of the silk fibroin-based material (e.g., but not limited to, a hydrogel, or a silk fiber) to deform, expand, deflect, bend, contract, stretch, and/or translate (i.e., moving from one Attorney Docket No. 700355-070131-PCT position to another) by a distance of at least about 0.001 μηι, at least about 0.005 μηι, at least about 0.01 μη , at least about 0.05 μηι, at least about 0.1 μηι, at least about 0.5 μηι, at least about 1 μηι, at least about 5 μη , at least about 10 μηι, at least about 25 μηι, at least about 50 μηι, at least about 100 μηι, at least about 250 μηι, at least about 500 μπι, at least about 750 μηι, at least about 1000 μηι, or more, in a direction of the magnetic field gradient.

[0079] In some embodiments, magnetic particles incorporated into a silk fibroin-based material can be multifunctional, e.g., in addition to responding to a magnetic field, the magnetic particle can be also able to interact with, or be actuated and/or excited by, with at least one other energy source, e.g., a vibrational source (e.g., ultrasound), electromagnetic waves, light, radio frequency, electric fields, heat, and/or any combinations thereof. For example, in some embodiments, magnetic particles can comprise gold nanoparticles or other magnetic-plasmonic particles, which can also interact with light or electromagnetic waves. The other energy source described herein can impart local vibration and induce local shearing and/or temperature increase. In some embodiments, the local shearing and/or temperature increase can lead to, e.g., conformation changes, construct breakdown (or degradation), and/or enhanced release of an active agent (if any).

Magneto-responsive silk fibroin fibers

[0080] In some embodiments, the silk fibroin-based material comprising magnetic particles is a fiber or a network of fibers. Accordingly, in one aspect, provided herein is a composition comprising one or a network of silk fibroin fibers (e.g., a plurality of silk fibroin fibers formed into a network), wherein at least a portion of the silk fibroin fibers are embedded with a plurality of magnetic particles. In such embodiments, the network of the silk fibroin fibers can be formed from a plurality of individual silk fibroin fibers. In some embodiments, the network of silk fibroin fibers can be formed from a single silk fibroin fiber wrapping and/or folding a plurality of times. In certain embodiments, at least a portion of the silk fibroin fibers can be arranged or aligned in a desired pattern within the network, e.g., in response to an external magnetic field.

[0081] As used herein, the term "fiber" with respect to silk fibroin fiber(s) means a relatively flexible, unit of matter having a high ratio of length to width across its cross-sectional perpendicular to its length. A fiber used in reference to a silk fibroin fiber herein refers to a regenerated silk fiber or regenerated silk fibroin fiber (e.g., a silk fibroin fiber regenerated from a silk fibroin solution as described herein). Attorney Docket No. 700355-070131-PCT

[0082] The length of the silk fibroin fiber(s) described herein is not critical, inasmuch as the silk fibroin fiber can be kilometers in length, or can be produced in the range of micrometers, millimeters, centimeters, or meters. A skilled artisan will readily appreciate that a silk fibroin fiber having a shorter length, e.g., in the micrometer or millimeter range, can be produced by cutting a long silk fibroin fiber into shorter pieces.

[0083] As used herein, the phrases "silk fibroin fiber" and "silk fibroin fibers" generally refer to silk fibroin fiber(s) comprising silk fibroin and at least one or a plurality of magnetic particle embedded therein. In accordance with various aspects described herein, the silk fibroin fiber(s) are magneto-responsive. In some embodiments, a silk fibroin fiber or silk fibroin fibers can comprise sericin. In some embodiments, a silk fibroin fiber or silk fibroin fibers can exclude sericin. In some embodiments, the phrases "silk fibroin fiber" and "silk fibroin fibers" refer to fiber(s) in which silk fibroin constitutes at least about 30% of the total composition, including at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, up to and including 100% or any percentages between about 30%) and about 100%>, of the total composition. In certain embodiments, the silk fibroin fiber(s) can be substantially formed from silk fibroin. In various embodiments, the silk fibroin fiber(s) can be substantially formed from silk fibroin comprising at least one additive (e.g., an active agent).

[0084] In some embodiments, a unit length of a silk fibroin fiber can be embedded with at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 25, at least about 50, at least about 100, at least about 250, at least about 500, at least about 1000 or more magnetic particles.

[0085] The silk fibroin fiber(s) can have a cross-section of any shape. For example, the silk fibroin fiber(s) can have a cross-section in a shape of a circle, an oval, an ellipse, a triangle, a rectangular, a square, a polygon, or any irregular geometry. In some embodiments, the silk fibroin fiber(s) can have a substantially circular cross-section.

[0086] The cross-section of the silk fibroin fiber(s) can have an average size of any dimension. For example, in some embodiments, the silk fibroin fiber(s) can have an average cross-sectional dimension (e.g., diameter) of about 10 nm to 1000 nm, about 25 nm to about 750 nm, about 50 nm to about 500 nm, or about 75 nm to about 300 nm. In some embodiments, the silk fibroin fiber(s) can have an average cross-sectional dimension (e.g., diameter) of about 0.1 μηι to about 1 mm, about 0.5 μηι to about 500 μηι, about 0.75 μηι to about 250 μιη, about 1 μιη to about 100 μηι, about 5 μηι to about 50 μηι. In some embodiments, the silk fibroin fiber(s) can have an average cross-sectional dimension (e.g., diameter) of about 1 mm to about 5 mm, about 1.5 mm to about 4.5 mm, or about 2 mm to about 4 mm. In general, a silk fibroin fiber Attorney Docket No. 700355-070131-PCT comprising a plurality of magnetic particles described herein has a larger average cross-sectional dimension (e.g., diameter) than that of a silk fibroin fiber without magnetic particles. A skilled artisan will appreciate that the terms "average size" and "average cross-sectional dimension" as used herein encompass a fiber having a constant or varying size or cross-sectional dimension across its length. In some embodiments, the terms "average size" and "average cross-sectional dimension" as used herein can refer to the size or cross-sectional dimension averaged from numerous fibers of different sizes or cross-sectional dimensions.

[0087] Depending on various applications (e.g., in a wound dressing), in some embodiments, a silk fibroin fiber can include about 1% to about 50%, or about 2% to about 3% to about 10% polyethylene oxide (e.g., PEO with a molecular weight of about 500, 000 to about 1,500,000). In other embodiments, the silk fibroin/ PEO blend ratio in a silk fibroin fiber can vary from about 1 : 100 to about 100: 1. In some embodiments, the silk fibroin/PEO blend ratio in a silk fibroin fiber can vary from about 2: 1 to about 4: 1. See, e.g., International Application No.: WO 201 1/008842, the content of which is incorporated herein by reference.

Methods of producing a magneto-responsive silk fibroin-based material

[0088] Another aspect provided herein relates to methods of producing a magneto- responsive silk fibroin-based material. In general, the magneto-responsive silk fibroin-based material in any format can be produced from a silk fibroin solution comprising magnetic particles or a magnetic fluid (e.g., a ferrofluid). The magnetic silk fibroin solution can then be processed into different formats (e.g., but not limited to, fibers, gels, foams, tubes, and/or films) in accordance with methods known to produce a regular silk fibroin-based material without magnetic particles. Without wishing to be bound by theory, the magnetic particles (e.g., iron oxide particles) present in a silk fibroin solution can act as an initiator and/or catalyst, thus increasing the rate at which the silk solution gets solidified.

[0089] In some embodiments, the magneto-responsive silk fibroin-based material can be in the form of a film, e.g., a silk film. As used herein, the term "film" refers to a flat structure or a thin flexible structure that can be rolled to form a tube. Accordingly, in some embodiments, the term "film" also refers to a tubular flexible structure. It is to be noted that the term "film" is used in a generic sense to include a web, film, sheet, laminate, or the like. In some

embodiments, the film is a patterned film, e.g., nanopatterned film. Exemplary methods for preparing silk fibroin films are described in, for example, WO 2004/000915 and WO

2005/012606, content of both of which is incorporated herein by reference in its entirety. In some embodiments, a magneto-responsive film can be produced by drying a magnetic silk Attorney Docket No. 700355-070131-PCT fibroin solution on a substrate, e.g. petri dish or a piece of acrylic. The resulting construct can be annealed, e.g., by water annealing, and the resulting film can then be removed.

[0090] In some embodiments, the magneto-responsive silk fibroin-based material can be in the form of a silk particle, e.g., a silk nanosphere or a silk microsphere. As used herein, the term "particle" includes spheres; rods; shells; and prisms; and these particles can be part of a network or an aggregate. Without limitations, the particle can have any size from nm to millimeters. As used herein, the term "microparticle" refers to a particle having a particle size of about 1 μηι to about 1000 μιη. As used herein, the term "nanoparticle" refers to particle having a particle size of about 0.1 nm to about 1000 nm.

[0091] In some embodiments, the magneto-responsive silk fibroin based material can be in the form of a gel or hydrogel. The term "hydrogel" is used herein to mean a silk-based material which exhibits the ability to swell in water and to retain a significant portion of water within its structure without dissolution. Methods for preparing silk fibroin gels and hydrogels are well known in the art. Methods for preparing silk fibroin gels and hydrogels include, but are not limited to, sonication, vortexing, pH titration, exposure to electric field, solvent immersion, water annealing, water vapor annealing, and the like. Exemplary methods for preparing silk fibroin gels and hydrogels are described in, for example, WO 2005/012606, content of which is incorporated herein by reference in its entirety. By way of example only, a magneto-responsive hydrogel can be produced by eletrogelation of a silk fibroin solution comprising magnetic particles or a magnetic fluid (e.g., a ferro fluid), e.g., as shown in Example 4, or by altering the pH of the magnetic silk fibroin solution via the addition of an acidic or a basic solution to increase the rate of gelation.

[0092] In some embodiments, the magneto-responsive silk fibroin based material can be in the form of a foam or a sponge. Methods for preparing silk fibroin gels and hydrogels are well known in the art. In some embodiments, the foam or sponge is a patterned foam or sponge, e.g., nanopatterned foam or sponge. Exemplary methods for preparing silk foams and sponges are described in, for example, WO 2004/000915, WO 2004/000255, and WO 2005/012606, content of all of which is incorporated herein by reference in its entirety. In some embodiments, a magneto-responsive foam can be produced by using a freeze-drying process. Layered magneto-responsive foams can also be made by applying multiple layers of magnetic silk solution on top of other frozen layers, and allowing the newly applied layer to freeze. The final frozen structure can then be placed in a lyophilizer where the structure is freeze-dried and water molecules are extracted from the construct.

[0093] In some embodiments, a magneto-responsive silk fibroin-based material can be in the form of a cylindrical matrix, e.g., a silk tube. The silk tubes can be made using any method Attorney Docket No. 700355-070131-PCT known in the art. For example, tubes can be made using molding, dipping, electrospinning, gel spinning, and the like. Gel spinning is described in Lovett et al. (Biomaterials, 29(35):4650- 4657 (2008)) and the construction of gel-spun silk tubes is described in PCT application no. PCT/US2009/039870, filed April 8, 2009, content of both of which is incorporated herein by reference in their entirety. Construction of silk tubes using the dip-coating method is described in PCT application no. PCT/US2008/072742, filed August 11, 2008, content of which is incorporated herein by reference in its entirety. Construction of silk fibroin tubes using the film- spinning method is described in PCT application No. PCT/US2013/030206, filed March 11 , 2013 and US Provisional application No.61/613,185, filed March 20, 2012.

[0094] A magneto-responsive silk fibroin fiber can be formed from a silk fibroin solution with any methods known in the art, including, but not limited to, molding, machining, drawing, eletro gelation, electrospinning, or any combinations thereof. In some embodiments, a magneto-responsive silk fibroin fiber can be formed by drying (e.g., by freezing) a silk fibroin solution comprising magnetic particles in a mold that is in a form of an elongated tube. See, e.g., the International Patent Application No. WO 2012/145594, the content of which is incorporated herein by reference, for exemplary methods that can be modified to make a silk fibroin fiber described herein. In some embodiments, a magneto-responsive silk fibroin fiber can be formed by drawing a fiber from a viscous silk fibroin solution comprising magnetic particles that has been processed by electrogelation. See, e.g., the International Patent Application No. WO 2011/038401, the content of which is incorporated herein by reference, for exemplary methods that can be modified to making a silk fibroin fiber described herein. Electrospun silk materials, such as fibers, and methods for preparing the same are described, for example in

WO2011/008842, content of which is incorporated herein by reference in its entirety. Micron- sized silk fibers (e.g., 10-600 μιη in size) and methods for preparing the same are described, for example in Mandal et al., Proc Natl Acad Sci U S A. 2012 May 15;109(20):7699-704 "High- strength silk protein scaffolds for bone repair;" and PCT application no. PCT/US13/35389, filed April 5, 2013, content of all of which is incorporated herein by reference

[0095] In some embodiments, electrospinning a magnetic silk solution can be performed in the presence of a magnetic field to form a composition (e.g., a mat or a mesh) comprising a magneto-responsive silk fibroin fiber, wherein the silk fibroin fiber(s) can be arranged in a predetermined pattern by manipulation of the applied magnetic field. Such method is further described in detail below.

[0096] The concentration of the silk fibroin solution used to form a magneto-responsive silk fibroin-based material can be adjusted for each application and/or composition. In some embodiments, the silk fibroin solution can range from 1 wt% to about 50 wt%, or from about 5 Attorney Docket No. 700355-070131-PCT wt% to about 45 wt%, or from about 10 wt% to about 40 wt%, or from about 20 wt% to about 40 wt%. In some embodiments, the silk fibroin solution can have a concentration of about 30 wt%.

[0097] The silk fibroin solution can be prepared by any conventional method known to one skilled in the art. For example, B. mori cocoons are boiled for varying times (e.g., about 10 minutes to about 60 minutes, depending on the form of the silk fibroin-based material to be produced) in an aqueous solution. In one embodiment, the aqueous solution is about 0.02M Na2C03. The cocoons are rinsed, for example, with water to extract the sericin proteins and the extracted silk is dissolved in an aqueous salt solution. Salts useful for this purpose include lithium bromide, lithium thiocyanate, calcium nitrate or other chemicals capable of solubilizing silk. In some embodiments, the extracted silk is dissolved in about 8M -12 M LiBr solution. The salt is consequently removed using, for example, dialysis.

[0098] If necessary, the solution can then be concentrated using, for example, dialysis against a hygroscopic polymer solution, for example, PEG, a polyethylene oxide, amylose or sericin. In some embodiments, the PEG is of a molecular weight of 8,000-10,000 g/mol and has a concentration of 5 wt% - 50 wt% (e.g., about 15 wt%). A slide-a-lyzer dialysis cassette (Pierce, MW CO 3500) can be used. However, any dialysis system can be used. The dialysis can be performed for a time period sufficient to result in a final concentration of aqueous silk solution between about 10 wt% - about 50 wt%. In some embodiments, the dialysis can be performed for a time period sufficient to result in a final concentration of aqueous silk solution at about 30 wt%. In most cases dialysis for 5 - 20 hours (e.g., -14 hours) is sufficient and longer dialysis is also permitted. See, for example, International Application No. WO 2005/012606, the content of which is incorporated herein by reference.

[0099] Alternatively, the silk fibroin solution can be produced using organic solvents. Such methods have been described, for example, in Li, M., et al, J. Appl. Poly Sci. 2001, 79, 2192-2199; Min, S., et al. Sen'I Gakkaishi 1997, 54, 85-92; Nazarov, R. et al,

Biomacromolecules 2004 May-Jun;5(3):718-26. For example, an exemplary organic solvent that can be used to produce a silk solution includes, but is not limited to, hexafluoroisopropanol.

[00100] The amount of magnetic particles described herein added into the silk fibroin solution can vary with a number of factors, such as, but not limited to, desired magnetic sensitivity of the magneto-responsive silk fibroin-based material, magnetic field strength, concentration of the silk fibroin solution, applications of the magneto-responsive silk fibroin material, and/or properties of the magnetic particles. In some embodiments, the magnetic particles can be present in a silk fibroin solution at a concentration of at least about 0.1% (v/v), at least about 0.5% (v/v), at least about 1% (v/v), at least about 2% (v/v), at least about 3% (v/v), Attorney Docket No. 700355-070131-PCT at least about 4% (v/v), at least about 5% (v/v), at least about 6% (v/v), at least about 7% (v/v), at least about 8% (v/v), at least about 9% (v/v), at least about 10% (v/v), at least about 15% (v/v), at least about 20% (v/v), at least about 30 % (v/v), at least about 40% (v/v), at least about 50% (v/v), at least about 60% (v/v), at least about 70% (v/v), at least about 80% (v/v), or higher. In one embodiment, the magnetic particles can be present in a silk fibroin solution at a concentration of about 0.05 % (v/v) to about 30% (v/v), or about 0.1 % (v/v) to about 20 %(v/v) or about 0.1% (v/v) to about 10% (v/v). In one embodiment, about 10% (v/v) of magnetic particles can be added to the silk fibroin solution.

[00101] In some embodiments, the magnetic particles can be present in a silk fibroin solution in an amount of at least about 0.01 % (w/w), at least about 0.05% (w/w), at least about 0.1% (w/w), at least about 0.5% (w/w), at least about 1% (w/w), at least about 2% (w/w), at least about 3%) (w/w), at least about 4% (w/w), at least about 5% (w/w), at least about 6% (w/w), at least about 7% (w/w), at least about 8% (w/w), at least about 9% (w/w), at least about 10% (w/w), at least about 15% (w/w), at least about 20% (w/w), at least about 30 % (w/w), at least about 40%) (w/w), at least about 50% (w/w), at least about 60% (w/w), at least about 70% (w/w), at least about 80% (w/w), or higher. In one embodiment, the magnetic particles can be present in a silk fibroin solution in an amount of about 0.1% (w/w) to about 20% (w/w).

[00102] In some embodiments, the magnetic particles can be present in a silk fibroin solution in an amount of at least about 0.01 % (w/v), at least about 0.05% (w/v), at least about 0.1% (w/v), at least about 0.5% (w/v), at least about 1% (w/v), at least about 2% (w/v), at least about 3%) (w/v), at least about 4% (w/v), at least about 5% (w/v), at least about 6% (w/v), at least about 7% (w/v), at least about 8% (w/v), at least about 9% (w/v), at least about 10% (w/v), at least about 15% (w/v), at least about 20% (w/v), at least about 30 % (w/v), at least about 40% (w/v), at least about 50% (w/v), at least about 60% (w/v), at least about 70% (w/v), at least about 80%) (w/v), or higher. In one embodiment, the magnetic particles can be present in the silk fibroin solution in an amount of about 5% (w/v) to about 15% (w/v). In one embodiment, the magnetic particles can be present in the silk fibroin solution in an amount of about 0.1% (w/v) to about 10%) (w/v). In one embodiment, the magnetic particles can be present in the silk fibroin solution in an amount of about 0.1% (w/v) to about 20% (w/v).

[00103] As described earlier and herein, the magnetic particles can be of any shape, including but not limited to spherical, rod, elliptical, cylindrical, and disc. The magnetic particles added to the silk fibroin solution can be of any size, e.g., smaller than the shortest dimension of the resultant silk fibroin-based material. In some embodiments, the magnetic particles can have a diameter of about 1 nm to about 1 mm. For example, magnetic particles can have a diameter of about 50 nm to about 250 μιη. In some embodiments, magnetic particles can Attorney Docket No. 700355-070131-PCT have a diameter of about 0.01 μηι to about 100 μπι. In some embodiments, magnetic particles can have a diameter of about 0.1 μηι to about 0 μιη. In some embodiments, magnetic particles can have a diameter of about 0.5 μηι to about 10 μιη. In some embodiments, magnetic particles can have a diameter of about 1 μιη to about 5 μπι. In some embodiments, magnetic particles can have a diameter of about 1 nm to about 10 μηι, or about 5 nm to about 5 μηι. In some embodiments, the magnetic particles can have a diameter of about 1 nm to about 1000 nm, or about 5 nm to about 500 nm.

[00104] As described earlier and herein, any magnetic material can be used for the magnetic particles, including ferromagnetic material, ferrimagnetic material, paramagnetic material or any combinations thereof. In some embodiments, the magnetic particles can include ferromagnetic particles, e.g., ferrous particles such as iron particles. In one embodiment, the iron particles can include carbonyl iron particles described herein. In such embodiments, the silk fibroin material containing carbonyl iron particles are biocompatible and can be used in vivo, e.g., as a tissue engineering scaffold implanted in vivo.

[00105] In some embodiments, the magnetic particles can be added into a silk fibroin solution as individual solid particles or powder. In some embodiments, the magnetic particles can be added into a silk fibroin solution in a format of a magnetic fluid, e.g., a ferrofluid or a magnetorheological fluid (MR fluid). A ferrofluid is generally a colloidal suspension of magnetic nanoparticles in a liquid carrier. In some embodiments, the magnetic particles in a ferrofluid, e.g., having an average size of about 10 nm, can be coated with a surfactant or a stabilizing dispersing agent. The surfactant or stabilizing agent can be selected to prevent particle agglomeration even when a strong magnetic field gradient is applied to the ferrofluid.

[00106] In some embodiments, the silk fibroin solution comprising a plurality of magnetic particles can further comprise an agent, e.g., but not limited to, a biopolymer as described herein, a porogen (e.g., a water-soluble particle such as salt) for creating pores in a silk fibroin- based material, an active agent as described herein or any combinations thereof.

[00107] As silk fibroin can generally stabilize active agents, some embodiments of the silk fibroin-based material can be used to encapsulate and/or deliver an active agent. In these embodiments, at least one active agent can be dispersed into a silk fibroin solution. Non-limiting examples of the active agents can include cells, proteins, peptides, nucleic acids, nucleic acid analogs, nucleotides or oligonucleotides, peptide nucleic acids, aptamers, antibodies or fragments or portions thereof, antigens or epitopes, hormones, hormone antagonists, growth factors or recombinant growth factors and fragments and variants thereof, cell attachment mediators, cytokines, enzymes, antibiotics or antimicrobial compounds, viruses, toxins, therapeutic agents and prodrugs thereof, small molecules, and any combinations thereof. Attorney Docket No. 700355-070131-PCT

[00108] In some embodiments, at least one active agent described herein can be added to the silk fibroin solution before further processing into silk fibroin-based materials described herein. In some embodiments, the active agent can be dispersed homogeneously or

heterogeneously within the silk fibroin, dispersed in a gradient, e.g., using the carbodiimide- mediated modification method described in the U.S. Patent Application No. US 2007/0212730.

[00109] In some embodiments, the magneto-responsive silk fibroin-based material can be first formed and then contacted with (e.g., dipped into or incubated with) at least one active agent. In some embodiments, at least one active agent described herein can be coated on an exposed surface of the magneto-responsive silk fibroin-based material upon the contacting. In some embodiments, at least one active agent described here can diffuse into the magneto- responsive silk fibroin-based material upon the contacting.

[00110] In some embodiments, it can be desirable to have the magneto-responsive silk fibroin-based material to be porous, i.e., a silk fibroin-based material having a porosity of at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or higher. Too high porosity can generally yield a magneto-responsive silk fibroin-based material and thus the resulting network thereof with lower mechanical properties, but can allow a release of an active agent embedded therein, if any. One of skill in the art can adjust the porosity accordingly, based on a number of factors such as, but not limited to, desired release rates, molecular size and/or diffusion coefficient of the active agent, and/or concentrations and/or amounts of silk fibroin in a silk-based material. The term "porosity" as used herein is a measure of void spaces in a material, e.g., a silk fibroin-based material, and is a fraction of volume of voids over the total volume, as a percentage between 0 and 100% (or between 0 and 1). Determination of matrix porosity is well known to a skilled artisan, e.g., using standardized techniques, such as mercury porosimetry and gas adsorption, e.g., nitrogen adsorption.

[00111] The porous magneto-responsive silk fibroin-based material can have any pore size. However, in some embodiments, it can be desirable to have the pore size of the magneto- responsive silk fibroin-based material be small enough such that the magnetic particles embedded therein cannot leak or diffuse out from the silk fibroin-based material, but large enough for an active agent, if any, embedded therein to be released from the silk fibroin-based material, if desirable. In some embodiments, the pores of a magneto-responsive silk fibroin- based material can have a size distribution ranging from about 1 nm to about 100 μιτι, from about 10 nm to about 50 μιη, from about 50 nm to about 25 μηι, from about 100 nm to about 20 μιη, from about 500 nm to about 10 μιη, or from about 1 μιη to about 5 μιη. As used herein, the term "pore size" refers to a diameter or an effective diameter of the cross-sections of the pores. Attorney Docket No. 700355-070131-PCT

The term "pore size" can also refer to an average diameter or an average effective diameter of the cross-sections of the pores, based on the measurements of a plurality of pores. The effective diameter of a cross-section that is not circular equals the diameter of a circular cross-section that has the same cross-sectional area as that of the non-circular cross-section. Methods for forming pores in a silk-based material are known in the art, e.g., poro gen-leaching method, freeze-drying method, and/or gas-forming method. Such methods are described, e.g., in U.S. Pat. App. Nos.: US 2010/0279112, US 2010/02791 12, and US 7842780, the contents of which are incorporated herein by reference.

[00112] To alter a property of the silk fibroin-based material, post-treatment of the silk fibroin-based material can be employed. For example, post-treatment methods can be applied to the silk fibroin-based material to induce beta-sheet structure formation in silk fibroin and thus modulate physical properties of silk fibroin (e.g., mechanical strength, degradability and/or solubility). Further, such post-treatment to induce formation of beta- sheet conformation structure in silk fibroin can prevent a silk fibroin-based material from contracting into a compact structure and/or forming an entanglement. Examples of various post-treatments can include, without limitations, controlled slow drying (Lu et al., 10 Biomacromolecules 1032 (2009)); water annealing (Jin et al., Water-Stable Silk Films with Reduced Beta-Sheet Content, 15 Adv. Funct. Mats. 1241 (2005); Hu et al. Regulation of Silk Material Structure by Temperature- Controlled Water Vapor Annealing, 12 Biomacromolecules 1686 (201 1)); stretching (Demura & Asakura, Immobilization of glucose oxidase with Bombyx mori silk fibroin by only stretching treatment and its application to glucose sensor, 33 Biotech & Bioengin. 598 (1989));

compressing; solvent immersion, including methanol (Hofmann et al., Silk fibroin as an organic polymer for controlled drug delivery, 11 1 J Control Release. 219 (2006)), ethanol (Miyairi et al., Properties of b-glucosidase immobilized in sericin membrane. 56 J. Fermen. Tech. 303 (1978)), glutaraldehyde (Acharya et al., Performance evaluation of a silk protein-based matrix for the enzymatic conversion of tyrosine to L-DOPA. 3 Biotechnol J. 226 (2008)), and l-ethyl-3-(3- dimethyl aminopropyl) carbodiimide (EDC) (Bayraktar et al., Silk fibroin as a novel coating material for controlled release of theophylline. 60 Eur J Pharm Biopharm. 373 (2005)); pH adjustment, e.g., pH titration and/or exposing a silk-based material to an electric field (see, e.g., U.S. Patent App. No. US2011/0171239); heat treatment; shear stress (see, e.g., International App. No. : WO 2011/005381), ultrasound, e.g., sonication (see, e.g., U.S. Patent Application Publication No. U.S. 2010/0178304, and International Patent Application No.

WO2008/150861); constraint-drying (see, e.g., International Patent Application No. WO 2011/008842); and any combinations thereof. Content of all of the references listed above is incorporated herein by reference in their entirety. Attorney Docket No. 700355-070131-PCT

[00113] The magneto-responsive silk fibroin-based material can comprise a silk II beta- sheet crystallinity content of at least about 5%, for example, a silk II beta-sheet crystallinity content of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% but not 100% (i.e., all the silk is present in a silk II beta-sheet conformation. In some embodiments, the silk in the magneto-responsive silk fibroin-based material is present completely in a silk II beta-sheet conformation.

[00114] As used herein, the term "constraint-drying" refers to a process where the silk material is dried while being constrained, such that it dries while undergoing a drawing or stretching force. Without wishing to be bound by theory, as water molecules evaporate, hydrophobic domains at the surface substrate and throughout the bulk region of the protein can initiate the loss of free volume from the interstitial space of the non-woven cast and within bulk region of the material. The loss of free volume can thus cause the material to contract. An exemplary method of constraint-drying a silk fibroin-based material can employ a magnetic field to maintain a silk fibroin-based material being stretched until it becomes naturally or blown dry.

[00115] In some embodiments, the magneto-responsive silk fibroin-based matrices described herein can be sterilized. Sterilization methods for biomaterials are well known in the art, including, but not limited to, gamma or ultraviolet radiation, autoclaving (e.g., heat/ steam); alcohol sterilization (e.g., ethanol and methanol); and gas sterilization (e.g., ethylene oxide sterilization).

[00116] Further, the silk fibroin-based material described herein can take advantage of the many techniques developed to functionalize silk fibroin (e.g., active agents such as dyes and sensors). See, e.g., U.S. Patent No. 6,287,340, Bioengineered anterior cruciate ligament; WO 2004/000915, Silk Biomaterials & Methods of Use Thereof; WO 2004/001103, Silk

Biomaterials & Methods of Use Thereof; WO 2004/062697, Silk Fibroin Materials & Use Thereof; WO 2005/000483, Method for Forming inorganic Coatings; WO 2005/012606, Concentrated Aqueous Silk Fibroin Solution & Use Thereof; WO 2011/005381 , Vortex-Induced Silk fibroin Gelation for Encapsulation & Delivery; WO 2005/123114, Silk-Based Drug Delivery System; WO 2006/076711, Fibrous Protein Fusions & Uses Thereof in the Formation of Advanced Organic/Inorganic Composite Materials; U.S. Application Pub. No. 2007/0212730, Covalently immobilized protein gradients in three-dimensional porous scaffolds; WO

2006/042287, Method for Producing Biomaterial Scaffolds; WO 2007/016524, Method for Stepwise Deposition of Silk Fibroin Coatings; WO 2008/085904, Biodegradable Electronic Devices; WO 2008/118133, Silk Microspheres for Encapsulation & Controlled Release; WO 2008/108838, Microfluidic Devices & Methods for Fabricating Same; WO 2008/127404, Attorney Docket No. 700355-070131-PCT

Nanopatterned Biopolymer Device & Method of Manufacturing Same; WO 2008/1 1821 1 , Biopolymer Photonic Crystals & Method of Manufacturing Same; WO 2008/127402,

Biopolymer Sensor & Method of Manufacturing Same; WO 2008/127403, Biopolymer

Optofluidic Device & Method of Manufacturing the Same; WO 2008/127401 , Biopolymer Optical Wave Guide & Method of Manufacturing Same; WO 2008/140562, Biopolymer Sensor & Method of Manufacturing Same; WO 2008/127405, Microfluidic Device with Cylindrical MicroChannel & Method for Fabricating Same; WO 2008/106485, Tissue-Engineered Silk Organs; WO 2008/140562, Electroactive Biopolymer Optical & Electro-Optical Devices & Method of Manufacturing Same; WO 2008/150861 , Method for Silk Fibroin Gelation Using Sonication; WO 2007/103442, Biocompatible Scaffolds & Adipose-Derived Stem Cells; WO 2009/155397, Edible Holographic Silk Products; WO 2009/100280, 3-Dimensional Silk Hydroxyapatite Compositions; WO 2009/061823, Fabrication of Silk Fibroin Photonic

Structures by Nanocontact Imprinting; WO 2009/126689, System & Method for Making Biomaterial Structures.

[00117] In an alternative embodiment, the magneto-responsive silk fibroin-based material can include plasmonic nanoparticles to form photothermal elements, e.g., by adding plasmonic particles into a magnetic silk solution and forming a magneto-responsive silk fibroin-based material therefrom. This approach takes advantage of the superior doping characteristics of silk fibroin. Thermal therapy has been shown to aid in the delivery of various agents, see Park et al., Effect of Heat on Skin Permeability, 359 Intl. J. Pfiarm. 94 (2008). In one embodiment, short bursts of heat on very limited areas can be used to maximize permeability with minimal harmful effects on surrounding tissues. Thus, plasmonic particle-doped silk fibroin matrices can add specificity to thermal therapy by focusing light to locally generate heat only via the silk fibroin matrices. In some embodiments, the silk fibroin matrices can include photothermal agents such as gold nanoparticles.

An exemplary method of producing a composition (e.g., mat or mesh) comprising a magneto- responsive silk fibroin fiber

[00118] Another aspect provided herein relates to methods of producing a composition (e.g., mat or mesh) comprising a magneto-responsive silk fibroin fiber. In some embodiments, the method can include: (a) forming a silk fibroin fiber from a silk fibroin solution comprising a plurality of magnetic particles, wherein at least a subset of the plurality of magnetic particles are entrapped or embedded into the formed silk fibroin fiber; (b) forming a magnetic field in a predetermined pattern on a solid substrate receiving the silk fibroin fiber, wherein the magnetic field pattern can determine an arrangement of the formed silk fibroin fiber on the solid substrate; Attorney Docket No. 700355-070131-PCT and (c) depositing the formed silk fibroin fiber onto the solid substrate; thereby producing a magneto-responsive composition or silk fibroin-based material comprising a silk fibroin fiber embedded with magnetic particles. In some embodiments, the methods described herein can be used to produce a magneto-responsive composition or silk fibroin-based material comprising a plurality of silk fibroin fibers. Any methods described earlier for making magneto-responsive silk fibroin-based materials can be applicable to producing the composition comprising one or more magneto-responsive silk fibroin fibers described herein.

[00119] Electrospinning: In some embodiments, a silk fibroin fiber can be formed, at least partly, by electrospinning a silk fibroin solution comprising a plurality of magnetic particles (and optionally polyethylene oxide). In some embodiments, electrospinning of the magnetic particle-containing silk fibroin solution can generate an electrospun silk fibroin fiber comprising magnetic particles embedded therein, which is continuously layered on a target solid substrate to form a 2-D or 3-D structure or network. In some embodiments, electrospinning of the magnetic particle-containing silk fibroin solution can generate a plurality of electrospun silk fibroin fibers, which are deposited on a target solid substrate to form a 2-D or 3-D structure or network.

[00120] As a person having ordinary skill in the art can appreciate, the process of electrospinning generally creates a fine stream or jet of polymeric liquid that upon proper evaporation of a solvent can yield a fiber (e.g., a nanofiber). The fine stream of liquid is produced by pulling a small amount of fiber solution through space by using electrical forces. The process of electrospinning has been described in "Electrospinning Process and Applications of Electrospun Fibers" by Doshi and Reneker, Journal of Electrostatics, Vol. 35 (1995), pp. 151- 160, "Nanometer Diameter Fibres of Polymer, Produced by Electrospinning" by Reneker and Chun, Nanotechnology, Vol. 7 (1996), pp. 216-223, and "DNA Fibers by Electrospinning" by Fang and Reneker, Journal of Macromolecular Science and Physics, Vol. B36(2) (1997), pp. 169-173, which are incorporated herein by reference. Electrospun polymeric fiber

characteristics, such as fiber diameter and alignment, depend on the molecular weight of the polymer, solution concentration, viscosity, and flow rate, needle size, electric charge, and the distance between the needle and collector. {See, e.g., Bhardwaj, N. and S.C. Kundu, (2010) "Electrospinning: A fascinating fiber fabrication technique" Biotechnology Advances. 28(3): p. 325-347; Teo, W.E. and S. Ramakrishna, A review on electrospinning design and nanofibre assemblies. Nanotechnology, 2006. 17(14): p. R89; Theron, S.A., et al., "Experimental investigation of the governing parameters in the electrospinning of polymer solutions." Polymer, 2004. 45(6): p. 2017-2030; Huang, Z.-M., et al, A review on polymer nanofibers by

electrospinning and their applications in nanocomposites. Composites Science and Technology, Attorney Docket No. 700355-070131-PCT

2003. 63(15): p. 2223-2253.) Environmental variables such as temperature and humidity can also influence fiber features as well (See, e.g., Bhardwaj, N. and S.C. undu, (2010)

"Electrospinning: A fascinating fiber fabrication technique" Biotechnology Advances. 28(3): p. 325-347). Many biomaterials, mainly polymers in a solubilized or melt state, have been reported to be successfully electrospun (See, e.g., Bhardwaj, N. et al., 2010. Id; Teo, 2006, Id.; Huang, 2003, Id.], but none of the art teaches or suggests electrospinning in the presence of a magnetic field a protein solution (e.g., a silk fibroin solution) containing magnetic particles

[00121] Electrospinning of silk solutions with or without poly(ethylene oxide) (PEO) in the absence of a magnetic field has been reported (See, e.g., Wang, M., H.-J. Jin, et al. (2004). "Mechanical Properties of Electrospun Silk Fibers." Macromolecules 37(18): 6856-6864; Jin, H.-J., et al. (2004). "Human bone marrow stromal cell responses on electrospun silk fibroin mats." Biomaterials 25(6): 1039-1047; Jin, H.-J., et al. (2002). "Electrospinning Bombyx mori Silk with Poly(ethylene oxide)." Biomacromolecules 3(6): 1233-1239; Zhang, ., et al. (2010). "Electrospun scaffolds from silk fibroin and their cellular compatibility." Journal of Biomedical Materials Research Part A 93A(3): 976-983). For spinning pure silk and water solutions without PEO, concentrations of 20-35 wt% were required (See, e.g., Zhang, 2010, Id.). Electrospinning silk-PEO solutions in the absence of a magnetic field resulted in fiber mats with fibers ranging in nm-scale (700-800 nm) scale (See, e.g., Wang, 2004, Id.; Jin, 2004, Id. ; Jin 2002, Id.]. These fiber mats supported mesenchymal stem cell attachment, spreading and bone-like tissue formation in vitro (See, e.g., Jin, 2004, Id.). Electrospinning a silk fibroin solution comprising polyethylene oxide (PEO) to form a silk fibroin-based mat has also been described in the International Patent Application No. WO 201 1/008842, the content of which is incorporated herein by reference. However, none of the disclosure teaches or suggests electrospinning in the presence of a magnetic field (in addition to an electric field) a silk solution containing magnetic particles.

[00122] In general, the apparatus or setup needed to carry out the electrospinning includes a delivery point, an electric field, and a target solid substrate. In accordance with the methods described herein, unlike typical electrospinning process, a magnetic field is also needed during electrospinning a silk fibroin solution described herein to orient a silk fibroin fiber in a desired direction and/or to align a silk fibroin fiber or a plurality thereof into a desired network pattern or a 3-D structure. An exemplary electrospinning setup configured for operation with magnetic field control is shown in Fig. 2.

[00123] The delivery point is a place where at least one droplet of silk fibroin solution can be introduced or exposed to an electric field. The delivery point (e.g., the tip of a needle) can be oriented anywhere in space adjacent to the electric field; for example, the delivery point (e.g., Attorney Docket No. 700355-070131-PCT the tip of a needle) can be above the electric field, below the electric field, or within the electric field. The target solid substrate is a solid substrate where a silk fibroin fiber can be collected. In some embodiments, the delivery point (e.g., the tip of a needle) and target solid substrate can be conductive in order to create an electric field. In other embodiments, the delivery point (e.g., the tip of a needle) and target solid substrate can be non-conductive points as they can be placed within an electric field.

[00124] The silk fibroin solution can be extruded from the delivery point (e.g., the tip of a needle) at any flow rate by any art-recognized means, e.g., using a pump. In some embodiments, the silk fibroin solution can be extruded from the delivery point (e.g., the tip of a needle) at a flow rate of about 0.01 μί/ιτιίη to about 1000 μί/ηνίη, about 0.05 μί/ηνίη to about 750 μί/ηιίη, about 0.1 μί/ιηίη to about 500 μΙ7ηώι, about 0.5 μί/ιηίη to about 250 μί/ηώι, about 1 μΙ ιώι to about 100 μΙ7πιϊη, or about 2 μυηιίη to about 75 μί/ηιίη. In some embodiments, the silk fibroin solution can be extruded from the delivery point (e.g., the tip of a needle) at a flow rate of about 0.5 μυηιίη to about 10 μί ίη, or about 1 μΙ7ηήη to about 7 μί/ητίη. The flow rate of the silk fibroin solution can be higher than 1000 μί/ιηίη or lower than 0.01 μί/ηνίη, provided that a Taylor cone is formed from the delivery point. A Taylor cone generally refers to the cone observed in an electrospinning or electrospraying process from which a jet of charged particles emanates above a threshold voltage. Without wishing to be bound by theory, when a small volume of electrically conductive liquid is exposed to an electric field, the shape of liquid starts to deform from the shape caused by surface tension alone. As the voltage is increased, the effect of the electric field becomes more prominent such that the electric field exerts a similar amount of force on the droplet as the surface tension does, resulting in formation of a cone shape of the droplet.

[00125] Depending on the flow rate and properties of the silk fibroin solution (e.g., viscosity and/or concentrations), size of magnetic particles, and/or the desired surface tension of the droplet, the delivery point (e.g., the tip of a needle) can have an orifice capable of extruding a controlled amount of a silk fibroin solution to form a silk fibroin fiber. In some embodiments, the delivery point (e.g., the tip of a needle) can have an orifice of about 0.1 mm to about 5 mm, or about 0.5 mm to about 4 mm, or about 1 mm to about 3 mm. In one embodiment, a syringe with a needle is used to extrude a controlled amount of silk fibroin solution to form a silk fibroin fiber. In such embodiments, the syringe needle can have an orifice of about gauge 31 to about gauge 7, about gauge 25 to about gauge 9, or about gauge 19 to about gauge 11. In one embodiment, the syringe needle can have an orifice of about gauge 18 to about gauge 16. An Attorney Docket No. 700355-070131-PCT ordinary skill of the art can readily select delivery points or means other than a syringe with a needle to extrude a controlled amount of silk fibroin solution to form a silk fibroin fiber.

[00126] An electric field necessary to create a stream of the silk fibroin solution through space can be achieved, e.g., by electrically charging the delivery means (e.g., the needle) or the target solid substrate. When the delivery means (e.g., the needle) is electrically charged, the target solid substrate can be grounded; and when the target solid substrate is electrically charged, the delivery means can be grounded. For example, in some embodiments, an electric field for electrospinning can be generated by applying a voltage to the delivery point including the delivery means (e.g., a needle) ranging from about 5 kV to about 50 kV, from about 8 kV to about 40 kV, or from about 10 kV to about 30 kV, while keeping the target solid substrate grounded. In other embodiments, an electric field for electrospinning can be generated by applying a voltage to the target solid substrate ranging from about 5 kV to about 50 kV, from about 8 kV to about 40 kV, or from about 10 kV to about 30 kV, while keeping the delivery point including the delivery means (e.g., a needle) grounded. In one embodiment, the voltage applied to the delivery point including the delivery means (e.g., a needle) is at least about 25 kV, while keeping the target solid substrate grounded. Without wishing to be bound by theory, an applied electric field during electrospinning should be strong enough to overcome gravitational forces on the silk solution, overcome surface tension forces of the silk solution, provide enough force to form a stream or jet of solution in space, and accelerate that stream or jet across the electric field. Surface tension of a silk solution can be a function of a number of variables, including, but not limited to, silk fibroin solution properties (viscosity, conductivity, and/or concentration), orifice size of the delivery point, and processing conditions (e.g., temperature, flow rate).

[00127] The distance between the delivery point (e.g., the tip of a needle) and the target solid substrate can be varied to adjust for the fiber diameter and/or morphology. Without wishing to be bound by theory, too short distance between the delivery point (e.g., the tip of a needle) and the target solid substrate can prevent fiber formation and/or does not allow the fiber have sufficient time to dry before reaching the target solid substrate. Too long distance between the delivery point (e.g., the tip of a needle) and the target solid substrate can result in larger distribution of fiber diameters. An ordinary artisan can readily optimize the distance between the delivery point (e.g., the tip of a needle) and the target solid substrate, based on other

electrospinning parameters (e.g., but not limited to, electric field, flow rate, conductivity of the silk fibroin solution), desired fiber diameters and/or morphology. In some embodiments, the distance between the delivery point (e.g., the tip of the needle) and the target solid substrate can vary from about 5 cm to about 40 cm, or from about 10 cm to about 30 cm. In one embodiment, Attorney Docket No. 700355-070131-PCT the distance between the delivery point (e.g., the tip of the needle) and the target solid substrate can be about 20 cm.

[00128] In one embodiment, a silk fibroin solution of about 20-40 wt% (e.g., about 30 wt%), at room temperature and pressure, typically requires a voltage of about 25 kV applied to the delivery point including delivery means (e.g., a needle), and a distance of about 20 cm between the delivery point and the target solid substrate. The electrospinning rate (e.g., to control the fiber diameter) can be controlled by varying a number of factors, e.g., the flow rate of the fiber solution, the electric field, the distance between the delivery point and the target solid substrate, and/or the solution properties of silk fibroin.

[00129] Without wishing to be bound by theory, increasing conductivity of the electrospinning solution can generally facilitate generation of fiber(s) from the viscous electrospinning solution due to coulombic repulsion. Accordingly, in some embodiments, the silk fibroin solution to be electrospun can contain a conductivity enhancer agent (e.g., electrolyte such as ions) to increase conductivity and charge density. Examples of such conductivity enhancer agent that can be added into the silk fibroin solution for increasing its conductivity can include, but are not limited to, a sodium salt (e.g., NaCl and NaBr); a base (e.g., NaOH and KOH); a lithium salt (e.g., LiCl); a potassium salt (e.g., KC1); water-soluble calcium salts (e.g., CaCl₂), (Bu)₄NCl, and any water-soluble salts; conducting polymers; carbon nanotubes or fullerenes and related materials; metals in various forms (e.g., but not limited to, nano- or micro- particles, nano- or micro-rods, nano- or micro-prisms, nano- or micro-discs); ionomers; and/or any other art-recognized conductive materials that can be added into a silk fibroin solution. While the silk fibroin solution prepared in aqueous solution is more desirable, the silk fibroin solution can also be prepared in organic solvents. In such embodiments, a salt that is soluble in an organic solvent can be used, e.g., a tetraalkylammonium triflate salt such as (Bu)₄N(CF3SC>3) (tetrabutylammomum trifluoromethanesulphonate (triflate) or TBATFL due to its high solubility in organic solvents. In some embodiments, a sodium salt (e.g., NaCl and NaBr) can be added into the silk fibroin solution. In some embodiments, a base (e.g., NaOH) can be added into the silk fibroin solution.

[00130] However, without wishing to be bound by theory, too high conductivity of the silk fibroin solution can turn electrospinning into electrospraying and thus produce droplets and particles instead of a fiber. In some embodiments, the conductivity of the silk fibroin solution can be increased by having a conductivity enhancer agent present in the silk fibroin solution at a final concentration of about 1 mM to about 100 mM, about 5 mM to about 90 mM, about 10 mM to about 80 mM, about 20 mM to about 70 mM, about 25 mM to about 60 mM, or about 30 mM to about 50 mM. In some embodiments, the silk fibroin solution can contain a conductivity Attorney Docket No. 700355-070131-PCT enhancer agent at a concentration of about 30 mM to about 50 mM. In one embodiment, the silk fibroin solution can contain a conductivity enhancer agent at a concentration of about 40 mM.

[00131] The utilization of magnetic fields during electrospinmng of a synthetic polymer material has been previously reported, such as for stabilization of the polymeric solution jet and ensuring continuity of the resulting fibers. Numerical analysis of magnetic electrospinmng also reports that the magnetic field can lead to more stable polymeric fiber whipping; and the polymeric solution jet follows a stable pattern of parallel overlapping circles (See, e.g., Ren, Z.- F., et al., "Effect of magnetic force on stability of the electrospinmng process" Journal of the Textile Institute, 2010. 101(6): p. 571 - 574; Xu, L., et al., "Numerical study of magnetic electrospinning processes. Computers & Mathematics with Applications." 201 1 Computers & Mathematics with Applications 61(8) 21 16; Xu, L., "A mathematical model for electrospinning process under coupled field forces." Chaos, Solitons & Fractals, 2009. 42(3): p. 1463-1465; Wu, Y., et al., "Controlling stability of the electrospun fiber by magnetic field." Chaos, Solitons & Fractals, 2007. 32(1): p. 5-7). However, these reports do not describe the use of magnetic fields during electrospinning a protein solution (e.g., a silk fibroin solution). While it has been previously reported that applying a magnetic field during electrospinning can induce alignment of polymeric fibers, e.g., PEO fibers, produced from a spinning-solution containing no ferrite particles (See, e.g., Ajao, J., et al, "Electric-magnetic field-induced aligned electrospun poly (ethylene oxide) (PEO) nanofibers". Journal of Materials Science, 2010. 45(9): p. 2324-2329; Yarin, A.L. and E. Zussman, "Upward needleless electrospinning of multiple nanofibers." Polymer, 2004. 45(9): p. 2977-2980), the art does not describe that applying a magnetic field during electrospinning can induce alignment of one or more protein fibers (e.g., one or more silk fibroin fibers) produced from a silk fibroin solution containing magnetic particles (e.g., carbonyl iron particles). Electrospinning of synthetic polymer solutions with entrained ferrite particles has been previously reported (See, e.g., Santala, E. and et al., "The preparation of reusable magnetic and photocatalytic composite nanofibers by electrospinning and atomic layer deposition" Nanotechnology, 2009. 20(3): p. 035602; Wang, H., et al., "Fabrication of aligned ferrite nanofibers by magnetic-field-assisted electrospinning coupled with oxygen plasma treatment." Materials Research Bulletin, 2009. 44(8): p. 1676-1680; Chung, M. et al., "Electrospun

Magnetic Thin Film, in Electron Devices and Solid-State Circuits, 2007. IEEE Conference; Yang, D., et al., "Fabrication of Aligned Fibrous Arrays by Magnetic Electrospinning.

Advanced Materials," 2007. 19(21): p. 3702-3706; Wu, H., et al., "Electrospinning of Fe, Co, and Ni Nanofibers: Synthesis, Assembly, and Magnetic Properties." Chemistry of Materials, 2007. 19(14): p. 3506-351 1 ; Li, 2003). Such polymeric materials have included poly(vinyl pyrrolidone) (PVP) or poly(vinyl alcohol) (PVA) combined with magneto-sensitive particles Attorney Docket No. 700355-070131-PCT

NiFe₂0₄ (See, e.g., Santala, 2009, Id.; Li, 2003], CoFe₂0₄ (See, e.g., Santala, 2009, Id.), Fe₃0₄ (See, e.g., Wang, 2009, Id.; Chung, 2007 et al, Id.; Yang, 2007, Id.), Fe/Co/Ni(No₃)₃ (See, e.g., Wu, 2007, Id.), or iron oxide particles (See, e.g., Hu, F., et al., "Preparation of Biocompatible Magnetite Nanocrystals for In Vivo Magnetic Resonance Detection of Cancer" Advanced Materials, 2006. 18(19): p. 2553-2556). In some instances, it has been previously reported that removal of the polymer phase by heat/oxygen plasma treatments can led to pure ferrite nanof bers. The spinning of polypyrrole (PPy) into PVA, poly(ethylene oxide) (PEO) or polystyrene (PS) fibers to absorb Fe₃0₄ has been reported, but the PVA or PEO fibers did not form well (See, e.g., Sen, S. and et al, "Conducting nanofibres produced by electrospinning." Journal of Physics: Conference Series, 2009. 183(1): p. 012020). Nevertheless, none of the art has reported electrospinning a protein solution such as silk fibroin solution containing magnetic particles (e.g., carbonyl iron particles) in the presence of a magnetic field.

[00132] While electrospun silk has been previously reported, the previously-reported silk fibers do not contain any magnetic particles and are electrospun under sole influence of an electric field (without a magnetic field). In contrast, embodiments of the methods described herein further comprises depositing a magneto-responsive silk fibroin fiber on a target solid substrate under influences of both an electric field and a magnetic field. Thus, embodiments of the methods described herein includes forming a magnetic field in a predetermined pattern on a target solid substrate on which the silk fibroin fiber is deposited, wherein the magnetic field pattern determines an arrangement (e.g., alignment and/or orientation) of the formed silk fibroin fiber on the target solid substrate.

[00133] In some embodiments, the arrangement of the formed silk fibroin fiber can include aligning the formed silk fibroin fiber in a direction of the magnetic field pattern. As such, to control the arrangement and/or alignment of the formed silk fibroin fiber for formation of a silk-fibroin based material comprising the silk fibroin fiber, in some embodiments, one or more magnetic field sources can be arranged on the target solid substrate on where the silk fibroin fiber deposits during electrospinning, such that the pattern of the magnetic field generated corresponds to the desired arrangement and/or alignment of the formed silk fibroin fiber.

[00134] A magnetic field can be generated on a target solid substrate by any methods known to one of ordinary skill in the art. For example, a magnetic field can be generated on a target solid substrate by placing a magnetic field source on the top surface of the target solid substrate. In such embodiments, an aluminum foil can be used to wrap on top of the magnetic field source to keep the magnetic field source clean and to assist in removal of the post- electrospun silk fibroin material. Alternatively, a magnetic field can be generated on a target Attorney Docket No. 700355-070131-PCT solid substrate by placing a magnetic field source below the target solid substrate (e.g., beneath the bottom surface of the target solid substrate), or embedded within the target solid substrate, if the material of the target solid substrate is permeable to or penetrable by a magnetic field generated by the magnetic field source.

[00135] A magnetic field source can include one or more permanent magnets, one or more electromagnets (including electrically-polarizable elements), or a combination thereof. In some embodiments, a magnetic field can be generated on a target solid substrate by one permanent magnet, e.g., as shown in Figs. 5A-5B. In other embodiments, a magnetic field can be generated on a target solid substrate by a plurality of permanent magnets arranged in a certain pattern (including a 2-D or a 3-D pattern), including at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, or more permanent magnets arranged in a certain pattern (including a 2-D or a 3-D pattern). By way of example only, as shown in Fig. 6B, about 80 to about 85 square magnets (e.g., 5 x 5 mm square magnets) can be arranged to form a star pattern. Each square magnet can create a magnetic field in the same or a different direction. As shown in Fig. 6B, the magnets can be joined such that the north pole of one magnet met the south pole of another magnet. While Fig. 6B illustrates the use of square magnets to form a star pattern, it is not construed as limiting the methods described herein to certain magnets and/or magnetic field patterns. For example, in some embodiments, as shown in Fig. 7B, about 200-250 axially magnetized spherical magnets (e.g., spherical magnets with a diameter of about 0.5 -0.75 cm) can be arranged to form a 2-D hexagonal pattern. In other embodiments, as shown in Fig. 8B, the spherical magnets can be arranged into a 3-D cylindrical pattern. In some embodiments, as shown in Fig. 9 A, the spherical magnets can be arranged into two 3-D cylinders spaced apart by a distance.

[00136] Accordingly, one or more permanent magnets of any shape and/or of any size can be used to create any 2-D and/or 3-D magnetic field patterns. In some embodiments, a permanent magnet can be in a form of, e.g., but not limited to, cylinders, spheres, squares, prisms, pyramids, polyhedrons, rings, bars, blocks, discs, irregular shapes, and any combinations thereof. An exemplary permanent magnet that can be used as a magnetic field source can include, but not limited to, a neodymium magnet, which is a member of the rare earth magnet family and is generally referred to as a NdFeB magnet composed mainly of neodymium (Nd), iron (Fe) and boron (B). Additional examples of permanent magnet materials that can be used as a magnetic field source to create a magnetic field pattern can include iron, nickel, cobalt, alloys Attorney Docket No. 700355-070131-PCT of rare earth metals, naturally occurring minerals such as lodestone, and any combinations thereof.

[00137] Permanent magnets used to create a magnetic field pattern on a target solid substrate can be of any size, depending on, e.g., the surface area of the target solid substrate on which the silk fibroin fiber is deposited, the number of permanent magnets used to form a magnetic field pattern, and/or a desired area of magnetic field influence. In some embodiments, a permanent magnet can have a dimension of about 1 mm to about 10 mm, about 2 mm to about 8 mm, or about 4 mm to about 6 mm. A plurality of such small permanent magnets can be arranged to form a desired magnetic field pattern of any dimension. Generally, the larger an area of magnetic field influence, the more permanent magnets are needed to create a magnetic field pattern producing such area of influence. In some embodiments, a permanent magnet can have a dimension larger than 10 mm, e.g., from about 1 cm to 50 cm, from about 5 cm to about 25 cm, or from about 5 cm to about 15 cm. Such permanent magnet can be in a form of, e.g., but not limited to, a disc, a block or a bar. For example, at least 2 permanent magnets of such dimension can be spaced apart on a target solid substrate to create a magnetic field pattern.

[00138] Generally, permanent magnets have a fixed magnetization level and are stationary, and need to be physically removed, if not needed. To have a better control of electrospun magnetized silk, electromagnets can be an alternative to permanent magnets.

Accordingly, in some embodiments, a magnetic field can be generated on a target solid substrate by one or more electromagnets. An "electromagnet" is generally a type of magnet in which the magnetic field is produced by the flow of electric current. The magnetic field disappears when the current is turned off. The polarity of the electromagnet can be determined by controlling the direction of the electrical current in the wire.

[00139] In some embodiments, an electromagnet of any shape (e.g., but not limited to, cylinders, spheres, squares) can be used to create a magnetic field. An electromagnetic controller can be used to control and adjust the magnetic field of the electromagnets as a group or individually. Thus, the alignment of one or more silk fibroin fibers can be modulated transiently and/or spatially by independently varying a magnetic field of each individual electromagnet. For example, the use of an electromagnetic controller can allow all electromagnets to be on at the same time, thus mimicking permanent magnets; or can allow dynamic control of electromagnets (e.g., each electromagnet can have a variable magnetization and/or activated at different times). By way of example only, as shown in Figs. 3A-3B, the electromagnets can be arranged in a desired pattern such that it can allow localized magnetization to move along a circle (Fig. 3A), along a linear configuration (Fig. 3B), or along any specific pattern depending on the arrangement of the electromagnets. With dynamic control of more than one electromagnets, an Attorney Docket No. 700355-070131-PCT electrospun fiber, at one time point, can be attracted to an electromagnet that is on, but not to any other electromagnet that is off. When a neighboring electromagnet is turned on at the next time point, the electrospun silk fibroin fiber can then be attracted to the neighboring

electromagnet, creating a specific arrangement/alignment of the silk fibroin fiber based on dynamic magnetization control.

[00140] An array of electromagnets can be used to create numerous magnetic field with a computer-based programming interface, e.g., as shown in Fig. 4, or any art-recognized programming software. An array of electromagnets, including at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, or more electromagnets, can be arranged in a certain pattern (including a 2-D or a 3-D pattern). For example, an array of electromagnets can be arranged in a pattern such that a temporal and/or spatial change of magnetization in each electromagnet, controlled by a computer program, can produce a mesh of silk fibroin fiber(s) with alternating alignments in one direction or another. In one embodiment, for textile-like outcomes, warp and weft courses can be generated with, e.g., zero and 90 degrees electromagnet coordination.

Alternatively, electromagnets can provide non-orthogonal directional control. In some embodiments, circumferentially aligned silk fibroin fiber(s) can be created by sequentially activating a series of electromagnets that are positioned along the circumference of a circle. In other embodiments, a twisted electrospun silk fibroin fiber can be created by sequentially activating a series of electromagnets that are positioned along the circumference of a circle, thus moving the fiber anchoring point (e.g., moving the magnetization point) as the fiber is being formed. A skilled artisan can readily arrange an array of electromagnets in different patterns and/or dynamically control the magnetization produced by each electromagnet to create networks of silk fibroin fiber(s) in different structures (e.g., ropes, mesh, sheets, and fabrics) with desired alignment of silk fibroin fibers. Such approach can provide capabilities of making networks of silk fibroin fiber(s) with tailored mechanical performance, e.g., by varying the arrangement and/or alignment of silk fibroin fiber(s) in the presence of a controlled magnetic field.

[00141] Regardless of various types of a magnetic field source used during

electrospinning, e.g., permanent magnets and/or electromagnets, the magnetic field pattern, alone or in combination with dynamic control of magnetization, can determine the arrangement of silk fibroin fibers or wrapping and/or folding of a silk fibroin fiber deposited on the target solid substrate. In accordance with different embodiments described herein, the silk fibroin fiber Attorney Docket No. 700355-070131-PCT generally aligns along the direction of the magnetic field, and also tend to bridge the gaps between magnets, if any.

[00142] The strength of a magnetic field used during electrospinning can range from 0.01 Tesla to about 10 Tesla.

[00143] In the presence of a magnetic field, the electrospun silk fibroin fiber is deposited on the target solid substrate containing one or more magnetic field sources (e.g., permanent magnet(s) and/or electromagnet(s)). In some embodiments, the electrospun silk fibroin fiber is deposited on top of one or more magnetic field sources that are placed on the top surface of the target solid substrate. In such embodiments, a surface covering or protection sheet (e.g., an aluminum foil) can be used to cover or wrap over the magnetic field sources to keep them clean and/or to facilitate removal of the network of silk fibroin fiber(s). Non-limiting examples of a target solid substrate to capture one or more silk fibroin fibers can include, but are not limited to, a wire mesh, a polymeric mesh, a metal disc, a metal rod, or any combination thereof. In one embodiment, the electrospun silk fibroin fiber is deposited on a metal disc, e.g., an aluminum disc or a steel rod (e.g., a stationary or rotating steel rod). The skilled artisan will be able to readily select other target solid substrates that can be employed to capture the silk fibroin fiber as it travels through the electric field and deposits on the target solid substrate in the presence of a magnetic field. Typically, the target solid substrate is conductive, but need not be conductive as a non-conductive target solid substrate can be employed in conjunction with a conductive material, e.g., a non-conductive target solid substrate can be wrapped with a metal foil, e.g., an aluminum foil.

[00144] The size of the target solid substrate can vary with a desired dimension of the resulting network of silk fibroin fiber(s) produced by the methods described herein. Typically, the surface area of the target solid substrate required for deposition of silk fibroin fiber(s) can increase with the bulk surface area of the resulting network of silk fibroin fiber(s). By way of example only, if a resulting silk fibroin fiber mesh has a diameter of about 10 cm, the diameter of the target solid substrate to be used can be at least about 10 cm or larger. Accordingly, the dimension of the target solid substrate can range from centimeters to meters. In one

embodiment, the diameter of the target solid substrate (e.g., a target solid disc) is at least about 5 cm, at least about 10 cm, at least about 15 cm, at least about 20 cm, at least about 30 cm, at least about 40 cm, at least about 50 cm, or more. Further, the target solid substrate can have a flat surface (e.g., a disc) or a curved surface (e.g., a rod) for receiving the silk fibroin fiber.

[00145] Optionally, a silk fibroin fiber being electrospun can be placed into a solution bath, e.g., before reaching a target solid substrate. The solution bath can contain a solvent, e.g., Attorney Docket No. 700355-070131-PCT an alcohol-based solvent such as methanol and/or ethanol, that can induce formation of beta- sheet conformation structures in one or more silk fibroin fibers.

[00146] Without wishing to be bound, while Fig. 2 shows a vertical electrospinning setup configured for operation with magnetic field control, where the delivery point (e.g., the tip of a needle) and the target solid substrate (e.g., an aluminum disc) are vertically aligned, the electrospinning process of the methods described herein can also be performed horizontally, i.e., the delivery point (e.g., the tip of a needle) and the target solid substrate (e.g., an aluminum disc or a rotating structure such as a rotating mandrel) are aligned in a horizontal manner. Setups for horizontal electrospinning are known in the art, e.g., described in U.S. Pat. No. 6,110,590, the content of which is incorporated herein by reference.

[00147] The electrospinning time can vary with a number of factors, including but not limited to, the dimension, thickness and/or complexity of the network of silk fibroin fiber(s), the flow rate of the silk fibroin solution through the delivery point, and/or the volume of the silk fibroin solution to be electrospun. In some embodiments, the electrospinning time can range from minutes, hours to days. In some embodiments, the electrospinning time can range from about 30 mins to about 24 hours, about 1 hour to about 16 hours, about 2 hours to about 12 hours, about 3 hours to about 10 hours, or about 4 hours to about 8 hours. In one embodiment, it takes about 4-6 hours to electrospin a ~3mL of silk fibroin solution containing magnetic particles. Without wishing to be bound by theory, longer electrospinning time can generally produce a larger and/or thicker network of silk fibroin fiber(s) (e.g., in a form of a mesh), while shorter electrospinning time can usually generate a network of silk fibroin fiber(s) that is less mechanically strong, as compared to the one produced with a longer electrospinning time.

Exemplary applications and/or compositions comprising a magneto-responsive silk fibroin- based material

[00148] As the silk fibroin-based materials are magneto responsive and/or can be configured to have desired structural alignment (e.g., silk fibroin fiber(s) described herein can be configured to have desired fiber alignment), different embodiments of the silk fibroin-based materials described herein can be adapted for use in various applications and/or compositions. For example, desired fiber alignment in a network of silk fibroin fiber(s) can be beneficial in various applications such as formation of tissue engineering scaffolds with preferred fiber orientation for mechanical integrity and/or induction of certain cell responses due to controlled mechanotransduction. Additionally, control of mechanical properties (e.g., loading conditions) transmitted through a silk fibroin network can help direct cell-driven tissue properties. Further, the magnetic responsiveness property of the silk fibroin-based materials (e.g., silk fiber(s)) Attorney Docket No. 700355-070131-PCT described herein can be used in applications where external manipulation via a magnetic field is desirable. Examples of such applications and/or compositions can include, without limitations, medical implants, wound dressings, tissue engineering scaffolds, implants, sensors, drug delivery devices, robotics, and separation membranes or filters.

[00149] In one embodiment, the composition comprising the network of the silk fibroin fiber(s) described herein can be adapted for use as a separation membrane or filter. In such embodiments, the network of the silk fibroin fiber(s) can be placed inside a chamber comprising an inlet for introduction of a fluid, and an outlet for an exit of the fluid. For example, the magneto-responsive silk fibroin fiber(s) can be arranged in a network to form an impermeable barrier, when not under the influence of an external magnetic field. Upon exposure to an external magnetic field, the magneto-responsive silk fibrin fiber(s) can deform in a certain orientation, such that gaps or pores are created in the network, thereby introducing permeability of the silk fibroin fiber network, e.g., to facilitate passage of a fluid through the silk fibroin fiber network.

[00150] In alternative embodiments, a drug delivery device (e.g., an implantable microchip or scaffold, or an injectable drug depot) or wound dressing (e.g., a bandage or an adhesive) can comprise a magneto-responsive silk fibroin-based material (e.g., a hydrogel and/or a network of the silk fibroin fiber(s) described herein) such that the release of a drug from the delivery device or wound dressing can be controlled by manipulation of an external magnetic field to control the diffusivity of a drug through the material. In some embodiments where the silk fibroin-based material is a composition comprising a network of silk fibroin fibers, permeability of the network of the silk fibroin fiber(s) can be manipulated with an external magnetic field in a similar manner as described above. In some embodiments, the drug delivery device can be implanted in vivo.

[00151] In some embodiments, applying an energy source (e.g., not necessarily limited to a magnetic field, e.g., ultrasound, radio frequency, heat, and/or electric fields) to a magneto- responsive silk fibroin-based material (e.g., a hydrogel) can alter the degradation profile of a magneto-responsive silk fibroin-based material (e.g., a hydrogel), and/or create a desired a release profile (if the silk fibroin-based material comprises an active agent), e.g., a pulsatile drug active agent release profile. In some embodiments, the magneto-responsive silk fibroin- based material can be implanted in vivo, e.g., for drug delivery to a localized area, tissue repair and/or regeneration and/or localized muscle therapy in a subject. The controlled degradation of the magneto-responsive silk fibroin-based material and/or release of an active agent (e.g., a drug molecule) from the silk fibroin-based material can be controlled by any methods known in the art, some of which are further described below: Attorney Docket No. 700355-070131-PCT

[00152] Alternating magnetic field (AMF) can expand and contract a magneto- responsive silk fibroin-based material (e.g., a hydrogel). This allows water and the embedded drug molecules or active agents to release from the gel matrix via diffusion (where, without wishing to be bound by theory, the AMF enhances the diffusion of water and drug molecules out of the gel). In the absence of the AMF the hydrogel can reabsorb water from its surroundings, and upon reapplication of the AMF the gel can expand and contract thereby releasing water and drug molecules or active agents again.

[00153] In some embodiments, therapeutic ultrasound can be applied, additionally or alternatively, to a target area placed with a magneto-responsive silk fibroin-based material (e.g., magnetic hydrogel). The ultrasonic waves can generally penetrate a tissue (e.g., a subject's body) and cause the gel and tissues (e.g., muscles) to vibrate and heat up. This process can be used to enhance the release of the drug molecules or active agents from the magneto-responsive silk fibroin-based material (e.g., a hydrogel) due to the increase in energy being supplied to the molecules. The therapeutic ultrasound can also be used to increase the degradation rate of the magneto-responsive silk fibroin-based material (e.g., a hydrogel), thus releasing drug molecules or active agent distributed in the magneto-responsive silk fibroin-based material (e.g., a hydrogel).

[00154] In some embodiments, radio frequency (RF) can be, additionally or alternatively, used to increase the degradation rate of the magneto-responsive silk fibroin-based material (e.g., a hydrogel). RF can be used to vibrate the magneto-responsive silk fibroin-based material (e.g., a hydrogel) and/or the embedded magnetic particles at their resonance frequency which can cause the silk fibroin-based material to degrade at a faster rate and thus release drug molecules or active agents into the surrounding area.

[00155] In some embodiments where a magneto-responsive silk-based material is in a form of a gel, electric fields can be further used to reverse the gelation process that was initially used to create the hydrogel (referred to as electrogelated gel). Reversing the polarity of the voltage can cause the gel to degrade or disassemble, and release the drug molecules or active agents from the gel into the body. Controlling the time for which this voltage is applied can allow for controlled disassembly of the gel and hence controlled release of the drug or active agent from the gel matrix into the body. See, e.g., International Patent Application No.

WO/2010/036992, the content of which is incorporated herein by reference, for details on electrogelated gels and their properties thereof.

[00156] In some embodiments, heat can be, additionally or alternatively, used to increase the degradation of a magneto-responsive silk fibroin-based material. Attorney Docket No. 700355-070131-PCT

[00157] In one embodiment, a magneto-responsive silk fibroin-based material (e.g., but not limited to, a hydrogel or a composition comprising the network of the silk fibroin fiber(s) described herein) can be used as a sensor. For example, the sensor can take advantage of the deformation of one or more silk fibroin fibers or any other silk fibroin-based material described herein (e.g., but not limited to, a silk fibroin hydrogel) in response to an externally applied magnetic field. In some embodiments, the sensors can be utilized for remotely sensing the alternating currents (AC) in a set of substantially parallel conductors, where the alternating currents generate a magnetic field that can cause a deformation of one or more silk fibroin fibers or any other silk fibroin-based material described herein (e.g., but not limited to a silk fibroin hydrogel). Without limitations, a sensor comprising a magneto-responsive silk fibroin fiber or any other silk fibroin-based material described herein (e.g., but not limited to a silk fibroin hydrogel) can be used to detect the presence or generation of a magnetic field in any

circumstances. In some embodiments, in order to have a controlled sensing condition, the network of the silk fibroin fiber(s) or any other silk fibroin-based material described herein (e.g., but not limited to a silk fibroin hydrogel) can be housed inside or coated with a material through which a magnetic field can penetrate such that the network of the silk fibroin fibers or the silk fibroin-based material (e.g., but not limited to a silk fibroin hydrogel) is less likely affected by other external non-magnetic factors (e.g., temperature, and/or pressure).

[00158] In another embodiment, a magneto-responsive silk fibroin-based material (e.g., silk particles or a silk fibroin fiber or a network of silk fibroin fibers described herein) can be used as a tunable reinforcement material. In some embodiments, a magneto -responsive silk fibroin-based material (e.g., a silk fibroin fiber or a network of silk fibroin fibers, a hydrogel, or a foam) can be reduced (e.g., by milling or grinding) into magneto-responsive silk fibroin particles or powder. For example, one or a plurality of magneto-responsive silk fibroin-based materials (e.g., silk fibroin fiber or silk fibroin particles or powder) can be added into a matrix material, e.g., to form a composite or multilayered material, such that the composite or the multilayered material can be made stiffer when a magnetic field is turned on, with an increase in stiffness being higher when the magnetic field is higher and/or when the rate of deformation is faster. Such tunable reinforcement materials can be used in wound dressings, tissue engineering scaffolds and/or medical implants.

[00159] In another embodiment, a magneto-responsive silk fibroin-based material (e.g., but not limited to a network of the silk fibroin fiber(s), gels, scaffolds, and/or films) described herein can be used as a piezomagnetic transducer. For example, deformation of one or more magneto-responsive silk fibroin-based materials (e.g., but not limited to, silk fibroin fibers, gels, scaffolds, and/or films) can generate a magnetic field to sense or actuate other components. In Attorney Docket No. 700355-070131-PCT another embodiment, introduction of a magnetic field can result in deflection or deformation of the piezomagnetic silk fibroin-based material (e.g., silk fibroin fiber or the network thereof, gels, scaffolds, and/or films). Such piezomagnetic transducers can be used as an actuator in a robotic component, tissue engineering scaffolds and/or medical implants.

[00160] In some embodiments, a magneto-responsive silk fibroin-based material (e.g., the network of the silk fibroin fiber(s), gels, scaffolds, and/or films) can be implanted in a body, e.g., a tissue of a subject in vivo, a robotic body or any soft structures. External magnetic fields can then be used to effect deformation or contraction in the magneto-responsive silk fibroin- based materials (e.g., the network of the silk fibroin fiber(s), gels, scaffolds, and/or films), enabling controlled movement of soft structures including biological tissues or robotic bodies comprising the magneto-responsive silk fibroin-based materials (e.g., the network of the silk fibroin fiber(s), gels, scaffolds, and/or films).

[00161] In some embodiments, a magneto-responsive silk fibroin-based material (e.g., gels, scaffolds, and/or films) can be placed (e.g., surgically placed or injected) at a target site comprising diseased cells, e.g., tumor or cancer cells, for treatment, e.g., by actuating the silk fibroin-based material. For example, as described earlier, therapeutic ultrasound can be applied to the site placed with the magneto-responsive silk fibroin-based material. The ultrasonic waves can penetrate the body and cause the gel and tissue to vibrate and heat up. Accordingly, in some embodiments, hyperthermia treatment of cancer and/or tumors by placing a magneto-responsive silk fibroin-based material at a target site comprising cancer or tumors are also provided herein.

[00162] In some embodiments, in situ mechanical deformation of a magneto-responsive silk fibroin-based material (e.g., a scaffold) can promote growth and development. For example, placing cells of interest in a magneto-responsive silk fibroin-based material (e.g., a scaffold) and actuating it using one of the methods described herein (e.g., magnetic field, AMF, ultrasound, RF, heat, electric fields, and any combinations thereof) can encourage cell growth and development in a particular orientation

[00163] In some embodiments where a magneto-responsive silk fibroin-based material is a silk fiber (e.g., a regenerated silk fiber), the magneto-responsive silk fiber can be used a suture material. In one embodiment, regenerated silk fibers can be manufactured from a magneto- responsive silk hydrogel produced by electrogelation, e.g., by drawing fibers with the aid of steam or heat. These magneto-responsive regenerated silk fibers can be used as sutures, e.g., to close a wound, and/or in other implants. Using such sutures internally can also provide additional benefits to a subject as an external magnetic field can be used to actuate the sutures, and hence stimulate tissues (e.g., connective tissues such as ligaments and muscles) thereby providing localized tissue therapy (e.g., muscle therapy) to the subject. Attorney Docket No. 700355-070131-PCT

[00164] Without limitations, the magneto-responsive silk fibroin-based materials (e.g., but not limited to, silk fibroin fibers described herein, and/or hydrogels) can also be used in applications such as protective clothing, energy, immobilization of enzymes, cosmetics and affinity membranes (See, e.g., Bhardwaj, N. and S.C. Kundu, (2010) "Electrospinning: A fascinating fiber fabrication technique" Biotechnology Advances. 28(3): p. 325-347; Huang, Z.- M., et al., A review on polymer nanofibers by electrospinning and their applications in nanocomposites. Composites Science and Technology, 2003. 63(15): p. 2223-2253; Nisbet, D.R., et al., Review Paper: A Review of the Cellular Response on Electrospun Nanofibers for Tissue Engineering. Journal of Biomaterials Applications, 2009. 24(1): p. 7-29).

Exemplary active agents

[00165] An active agent that can be included in a magneto-responsive silk fibroin-based material can represent any material capable of being incorporated in a silk fibroin-based material. For example, the active agent can be a therapeutic agent, or a biological material, such as cells (including stem cells such as induced pluripotent stem cells), proteins, peptides, nucleic acids (e.g., DNA, RNA, siRNA), nucleic acid analogs, nucleotides, oligonucleotides, peptide nucleic acids (PNA), aptamers, antibodies or fragments or portions thereof (e.g., paratopes or complementarity-determining regions), antigens or epitopes, hormones, hormone antagonists, growth factors or recombinant growth factors and fragments and variants thereof, cell attachment mediators (such as RGD), cytokines, enzymes, small molecules, antibiotics or antimicrobial compounds, viruses, antivirals, toxins, therapeutic agents and prodrugs, small molecules and any combinations thereof. See, e.g., WO 2009/140588; U.S. Patent Application Ser. No. 61/224,618). The active agent can also be a combination of any of the above- mentioned agents. Encapsulating either a therapeutic agent or biological material, or the combination of them, is desirous because the encapsulated composition can be used for numerous biomedical purposes.

[00166] In some embodiments, the active agent can also be an organism such as a fungus, plant, animal, bacterium, or a virus (including bacteriophage). Moreover, the active agent may include neurotransmitters, hormones, intracellular signal transduction agents, pharmaceutically active agents, toxic agents, agricultural chemicals, chemical toxins, biological toxins, microbes, and animal cells such as neurons, liver cells, and immune system cells. The active agents may also include therapeutic compounds, such as pharmacological materials, vitamins, sedatives, hypnotics, prostaglandins and radiopharmaceuticals.

[00167] Exemplary cells suitable for use herein may include, but are not limited to, progenitor cells or stem cells, smooth muscle cells, skeletal muscle cells, cardiac muscle cells, Attorney Docket No. 700355-070131-PCT epithelial cells, endothelial cells, urothelial cells, fibroblasts, myoblasts, ocular cells, chondrocytes, chondroblasts, osteoblasts, osteoclasts, keratinocytes, kidney tubular cells, kidney basement membrane cells, integumentary cells, bone marrow cells, hepatocytes, bile duct cells, pancreatic islet cells, thyroid, parathyroid, adrenal, hypothalamic, pituitary, ovarian, testicular, salivary gland cells, adipocytes, and precursor cells. The active agents can also be the combinations of any of the cells listed above. See also WO 2008/106485; WO 2010/040129; WO 2007/103442.

[00168] As used herein, the terms "proteins" and "peptides" are used interchangeably herein to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "peptide", which are used interchangeably herein, refer to a polymer of protein amino acids, including modified amino acids (e.g., phosphorylated, glycated, etc.) and amino acid analogs, regardless of its size or function. Although "protein" is often used in reference to relatively large polypeptides, and "peptide" is often used in reference to small polypeptides, usage of these terms in the art overlaps and varies. The term "peptide" as used herein refers to peptides, polypeptides, proteins and fragments of proteins, unless otherwise noted. The terms "protein" and "peptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary peptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[00169] The term "nucleic acids" used herein refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA), polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.

Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer, et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka, et al, J. Biol. Chem. 260:2605-2608 (1985), and Rossolini, et al., Mol. Cell. Probes 8:91-98 (1994)). The term "nucleic acid" should also be understood to include, as equivalents, derivatives, variants and analogs of either RNA or DNA made from nucleotide analogs, and, single (sense or antisense) and double-stranded polynucleotides. The term "nucleic acid" also encompasses modified RNA Attorney Docket No. 700355-070131-PCT

(modR A). The term "nucleic acid" also encompasses siRNA, shR A or any combinations thereof.

[00170] The term "modified RNA" means that at least a portion of the RNA has been modified, e.g., in its ribose unit, in its nitrogenous base, in its internucleoside linkage group, or any combinations thereof. Accordingly, in some embodiments, a "modified RNA" may contain a sugar moiety which differs from ribose, such as a ribose monomer where the 2'-OH group has been modified. Alternatively, or in addition to being modified at its ribose unit, a "modified RNA" may contain a nitrogenous base which differs from A, C, G and U (a "non-RNA nucleobase"), such as T or MeC. In some embodiments, a "modified RNA" may contain an internucleoside linkage group which is different from phosphate (-0-P(0)₂-0- ), such as -O- P(0,S)-0-.

[00171] The term "short interfering RNA" (siRNA), also referred to herein as "small interfering RNA" is defined as an agent which functions to inhibit expression of a target gene, e.g., by RNAi. An siRNA can be chemically synthesized, it can be produced by in vitro transcription, or it can be produced within a host cell. siRNA molecules can also be generated by cleavage of double stranded RNA, where one strand is identical to the message to be inactivated. The term "siRNA" refers to small inhibitory RNA duplexes that induce the RNA interference (RNAi) pathway. These molecules can vary in length (generally 18-30 base pairs) and contain varying degrees of complementarity to their target mRNA in the antisense strand. Some, but not all, siRNA have unpaired overhanging bases on the 5 Or 3' end of the sense 60 strand and/or the antisense strand. The term "siRNA" includes duplexes of two separate strands, as well as single strands that can form hairpin structures comprising a duplex region.

[00172] The term "shRNA" as used herein refers to short hairpin RNA which functions as RNAi and/or siRNA species but differs in that shRNA species are double stranded hairpin-like structure for increased stability. The term "RNAi" as used herein refers to interfering RNA, or RNA interference molecules are nucleic acid molecules or analogues thereof for example RNA- based molecules that inhibit gene expression. RNAi refers to a means of selective post- transcriptional gene silencing. RNAi can result in the destruction of specific mRNA, or prevents the processing or translation of RNA, such as mRNA.

[00173] The term "enzymes" as used here refers to a protein molecule that catalyzes chemical reactions of other substances without it being destroyed or substantially altered upon completion of the reactions. The term can include naturally occurring enzymes and

bioengineered enzymes or mixtures thereof. Examples of enzyme families include, but are not limited to, peroxidase, lipase, amylose, organophosphate dehydrogenase, ligases, restriction endonucleases, ribonucleases, DNA polymerases, glucose oxidase, laccase, kinases, Attorney Docket No. 700355-070131-PCT dehydrogenases, oxidoreductases, GTPases, carboxyl transferases, acyl transferases, decarboxylases, transaminases, racemases, methyl transferases, formyl transferases, and a- ketodecarboxylases.

[00174] As used herein, the term "aptamers" means a single-stranded, partially single- stranded, partially double-stranded or double-stranded nucleotide sequence capable of specifically recognizing a selected non-oligonucleotide molecule or group of molecules. In some embodiments, the aptamer recognizes the non-oligonucleotide molecule or group of molecules by a mechanism other than Watson-Crick base pairing or triplex formation. Aptamers can include, without limitation, defined sequence segments and sequences comprising nucleotides, ribonucleotides, deoxyribonucleotides, nucleotide analogs, modified nucleotides and nucleotides comprising backbone modifications, branchpoints and nonnucleotide residues, groups or bridges. Methods for selecting aptamers for binding to a molecule are widely known in the art and easily accessible to one of ordinary skill in the art.

[00175] As used herein, the term "antibody" or "antibodies" refers to an intact immunoglobulin or to a monoclonal or polyclonal antigen-binding fragment with the Fc (crystallizable fragment) region or FcRn binding fragment of the Fc region. The term

"antibodies" also includes "antibody-like molecules", such as fragments of the antibodies, e.g., antigen-binding fragments. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. "Antigen-binding fragments" include, inter alia, Fab, Fab', F(ab')2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single domain antibodies, chimeric antibodies, diabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. Linear antibodies are also included for the purposes described herein. The terms Fab, Fc, pFc', F(ab') 2 and Fv are employed with standard immunological meanings (Klein, Immunology (John Wiley, New York, N.Y., 1982); Clark, W. R. (1986) The Experimental Foundations of Modern Immunology (Wiley & Sons, Inc., New York); and Roitt, I. (1991) Essential Immunology, 7th Ed.,

(Blackwell Scientific Publications, Oxford)). Antibodies or antigen-binding fragments specific for various antigens are available commercially from vendors such as R&D Systems, BD Biosciences, e-Biosciences and Miltenyi, or can be raised against these cell-surface markers by methods known to those skilled in the art.

[00176] Exemplary antibodies that may be incorporated in silk fibroin include, but are not limited to, abciximab, adalimumab, alemtuzumab, basiliximab, bevacizumab, cetuximab, certolizumab pegol, daclizumab, eculizumab, efalizumab, gemtuzumab, ibritumomab tiuxetan, infliximab, muromonab-CD3, natalizumab, ofatumumab omalizumab, palivizumab, Attorney Docket No. 700355-070131-PCT panitumumab, ranibizumab, rituximab, tositumomab, trastuzumab, altumomab pentetate, arcitumomab, atlizumab, bectumomab, belimumab, besilesomab, biciromab, canakinumab, capromab pendetide, catumaxomab, denosumab, edrecolomab, efungumab, ertumaxomab, etaracizumab, fanolesomab, fontolizumab, gemtuzumab ozogamicin, golimumab, igovomab, imciromab, labetuzumab, mepolizumab, motavizumab, nimotuzumab, nofetumomab merpentan, oregovomab, pemtumomab, pertuzumab, rovelizumab, ruplizumab, sulesomab, tacatuzumab tetraxetan, tefibazumab, tocilizumab, ustekinumab, visilizumab, votumumab, zalutumumab, and zanolimumab. The active agents can also be the combinations of any of the antibodies listed above.

[00177] As used herein, the term "Complementarity Determining Regions" (CDRs; i.e., CDR1 , CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain the presence of which are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3. Each complementarity determining region may comprise amino acid residues from a "complementarity determining region" as defined by Kabat ( i.e. about residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (HI), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al. , Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a "hypervariable loop" ( i.e. about residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (HI), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some instances, a complementarity determining region can include amino acids from both a CDR region defined according to Kabat and a hypervariable loop.

[00178] The expression "linear antibodies" refers to the antibodies described in Zapata et al. , Protein Eng., 8(10): 1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (VH -CH1-VH-CH1) which, together with complementary light chain

polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

[00179] The expression "single-chain Fv" or "scFv" antibody fragments, as used herein, is intended to mean antibody fragments that comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. (The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994)). Attorney Docket No. 700355-070131-PCT

[00180] The term "diabodies," as used herein, refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH)

Connected to a light-chain variable domain (VL) in the same polypeptide chain (VH - VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. (EP 404,097; WO 93/11 161; Hollinger et ah, Proc. Natl. Acad. Sd. USA, P0:6444-6448 (1993)).

[00181] As used herein, the term "small molecules" refers to natural or synthetic molecules including, but not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, aptamers, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1 ,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[00182] The term "antibiotics" or" antimicrobial compound" is used herein to describe a compound or composition which decreases the viability of a microorganism, or which inhibits the growth or reproduction of a microorganism. As used in this disclosure, an antibiotic is further intended to include an antimicrobial, bacteriostatic, or bactericidal agent. Exemplary antibiotics can include, but are not limited to, actinomycin; aminoglycosides (e.g., neomycin, gentamicin, tobramycin); β-lactamase inhibitors (e.g., clavulanic acid, sulbactam); glycopeptides (e.g., vancomycin, teicoplanin, polymixin); ansamycins; bacitracin; carbacephem; carbapenems; cephalosporins (e.g., cefazolin, cefaclor, cefditoren, ceftobiprole, cefuroxime, cefotaxime, cefipeme, cefadroxil, cefoxitin, cefprozil, cefdinir); gramicidin; isoniazid; linezolid; macrolides (e.g., erythromycin, clarithromycin, azithromycin); mupirocin; penicillins (e.g., amoxicillin, ampicillin, cloxacillin, dicloxacillin, flucloxacillin, oxacillin, piperacillin); oxolinic acid;

polypeptides (e.g., bacitracin, polymyxin B); quinolones (e.g., ciprofloxacin, nalidixic acid, enoxacin, gatifloxacin, levaquin, ofloxacin, etc.); sulfonamides (e.g., sulfasalazine,

trimethoprim, trimethoprim-sulfamethoxazole (co-trimoxazole), sulfadiazine); tetracyclines (e.g., doxycyline, minocycline, tetracycline, etc.); monobactams such as aztreonam;

chloramphenicol; lincomycin; clindamycin; ethambutol; mupirocin; metronidazole; pefloxacin; pyrazinamide; thiamphenicol; rifampicin; thiamphenicl; dapsone; clofazimine; quinupristin; metronidazole; linezolid; isoniazid; piracil; novobiocin; trimethoprim; fosfomycin; fusidic acid; or other topical antibiotics. Optionally, the antibiotic agents may also be antimicrobial peptides Attorney Docket No. 700355-070131-PCT such as defensins, magainin and nisin; or lytic bacteriophage. The antibiotic agents can also be the combinations of any of the agents listed above. See also PCT/US2010/026190.

[00183] As used herein, the term "antigens" refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to elicit the production of antibodies capable of binding to an epitope of that antigen. An antigen may have one or more epitopes. The term "antigen" can also refer to a molecule capable of being bound by an antibody or a T cell receptor (TCR) if presented by MHC molecules. The term "antigen", as used herein, also encompasses T- cell epitopes. An antigen is additionally capable of being recognized by the immune system and/or being capable of inducing a humoral immune response and/or cellular immune response leading to the activation of B- and/or T-lymphocytes. This may, however, require that, at least in certain cases, the antigen contains or is linked to a Th cell epitope and is given in adjuvant. An antigen can have one or more epitopes (B- and T-epitopes). The specific reaction referred to above is meant to indicate that the antigen will preferably react, typically in a highly selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs which may be evoked by other antigens. Antigens as used herein may also be mixtures of several individual antigens.

[00184] As used herein, the term "therapeutic agent" generally means a molecule, group of molecules, complex or substance administered to an organism for diagnostic, therapeutic, preventative medical, or veterinary purposes. As used herein, the term "therapeutic agent" includes a "drug" or a "vaccine." This term include externally and internally administered topical, localized and systemic human and animal pharmaceuticals, treatments, remedies, nutraceuticals, cosmeceuticals, biologicals, devices, diagnostics and contraceptives, including preparations useful in clinical and veterinary screening, prevention, prophylaxis, healing, wellness, detection, imaging, diagnosis, therapy, surgery, monitoring, cosmetics, prosthetics, forensics and the like. This term can also be used in reference to agriceutical, workplace, military, industrial and environmental therapeutics or remedies comprising selected molecules or selected nucleic acid sequences capable of recognizing cellular receptors, membrane receptors, hormone receptors, therapeutic receptors, microbes, viruses or selected targets comprising or capable of contacting plants, animals and/or humans. This term can also specifically include nucleic acids and compounds comprising nucleic acids that produce a bioactive effect, for example deoxyribonucleic acid (DNA), ribonucleic acid (RNA), modified DNA or RNA, or mixtures or combinations thereof, including, for example, DNA nanoplexes.

[00185] The term "therapeutic agent" also includes an agent that is capable of providing a local or systemic biological, physiological, or therapeutic effect in the biological system to Attorney Docket No. 700355-070131-PCT which it is applied. For example, the therapeutic agent can act to control infection or inflammation, enhance cell growth and tissue regeneration, control tumor growth, act as an analgesic, promote anti-cell attachment, and enhance bone growth, among other functions. Other suitable therapeutic agents can include anti-viral agents, hormones, antibodies, or therapeutic proteins. Other therapeutic agents include prodrugs, which are agents that are not biologically active when administered but, upon administration to a subject are converted to biologically active agents through metabolism or some other mechanism. Additionally, a silk- based composition can contain combinations of two or more therapeutic agents.

[00186] In some embodiments, different types of therapeutic agents that can be encapsulated or dispersed in a silk fibroin-based material can include, but not limited to, proteins, peptides, antigens, immunogens, vaccines, antibodies or portions thereof, antibody-like molecules, enzymes, nucleic acids, modified RNA, siRNA, shRNA, aptamers, small molecules, antibiotics, and any combinations thereof.

[00187] Exemplary therapeutic agents include, but are not limited to, those found in Harrison 's Principles of Internal Medicine, 13^th Edition, Eds. T.R. Harrison et al. McGraw-Hill N.Y., NY; Physicians Desk Reference, 50^th Edition, 1997, Oradell New Jersey, Medical Economics Co.; Pharmacological Basis of Therapeutics, 8^th Edition, Goodman and Gilman, 1990; United States Pharmacopeia, The National Formulary, USP XII NF XVII, 1990, the complete contents of all of which are incorporated herein by reference.

[00188] Embodiments of various aspects described herein can be defined in any of the following numbered paragraphs:

1. A composition comprising a silk fibroin-based material embedded with a plurality of magnetic particles.

2. The composition of paragraph 1 , wherein the silk fibroin-based material is in a form of a film, a sheet, a gel or hydrogel, a mesh, a mat, a non- woven mat, a fabric, a scaffold, a tube, a slab or block, a fiber, a particle, a 3-dimensional construct, an implant, a high- density material, a reinforced material, a foam or a sponge, a machinable material, a microneedle, or any combinations thereof.

3. The composition of paragraph 1 , wherein the silk fibroin based material is in a form of a fiber, a mesh, a mat, a non-woven mat, a fabric, or any combinations thereof.

4. The composition of paragraph 1 , wherein the silk fibroin based material is in a form of a hydrogel, a foam or sponge, a film, a scaffold, or any combinations thereof.

5. The composition of any of paragraphs 1-4, wherein the magnetic particles have a

diameter of about 1 nm to about 10 μιη. Attorney Docket No. 700355-070131-PCT The composition of any of paragraphs 1-5, wherein the magnetic particles have a diameter of about 5 nm to about 5 μηι.

The composition of any of paragraphs 1-6, wherein the magnetic particles comprise ferrous particles.

The composition of paragraph 7, wherein the ferrous particles comprise iron particles. The composition of any of paragraphs 1-8, wherein the silk fibroin based material is a silk fibroin fiber.

The composition of paragraph 9, wherein the silk fibroin fiber has a diameter of about 0.1 μηι ΐο about 1 mm.

The composition of any of paragraphs 9-10, wherein the silk fibroin fiber has a diameter of about 0.5 μπι to about 500 μηι.

The composition of any of paragraphs 1-11, wherein the silk fibroin based material further comprises at least one active agent.

The composition of paragraph 12, wherein the active agent is selected from the group consisting of cells, proteins, peptides, nucleic acids, nucleic acid analogs, nucleotides or oligonucleotides, peptide nucleic acids, aptamers, antibodies or fragments or portions thereof, antigens or epitopes, hormones, hormone antagonists, growth factors or recombinant growth factors and fragments and variants thereof, cell attachment mediators, cytokines, enzymes, antibiotics or antimicrobial compounds, viruses, toxins, therapeutic agents and prodrugs thereof, small molecules, and any combinations thereof. The composition of any of paragraphs 1-13, wherein the plurality of magnetic particles is present in an amount sufficient to allow at least a portion of the silk fibroin based material to respond to an external energy source.

The composition of paragraph 14, wherein the external energy source comprises a magnetic field, ultrasound, electromagnetic waves, radio frequency, heat, an electric field, or any combinations thereof.

The composition of 14, wherein the external energy source comprises a magnetic field. The composition of any of paragraphs 14-16, wherein the response of the silk fibroin based material comprises deflection or contraction.

The composition of any of paragraphs 1-17, wherein the silk fibroin based material further comprises a biopolymer.

The composition of paragraph 18, wherein the biopolymer is selected from the group consisting of polyethylene oxide (PEO), polyethylene glycol (PEG), collagen, fibronectin, keratin, polyaspartic acid, polylysine, alginate, chitosan, chitin, hyaluronic acid, pectin, polycaprolactone, polylactic acid, polyglycolic acid, Attorney Docket No. 700355-070131-PCT polyhydroxyalkanoates, dextrans, polyanhydrides, polymer, PLA-PGA, polyanhydride, polyorthoester, polycaprolactone, polyfumarate, collagen, chitosan, alginate, hyaluronic acid, and any combinations thereof.

The composition of any of paragraphs 1-19, wherein the composition is adapted for use as a medical implant.

The composition of any of paragraphs 1-19, wherein the composition is adapted for use as a wound dressing.

The composition of any of paragraphs 1-19, wherein the composition is adapted for use as a tissue engineering scaffold.

The composition of any of paragraphs 1-19, wherein the composition is adapted for use as a sensor.

The composition of any of paragraphs 1-19, wherein the composition is adapted for use as a drug delivery device.

The composition of any of paragraphs 1-19, wherein the composition is adapted for use as a separation membrane.

The composition of any of paragraphs 1-19, wherein the silk fibroin based material comprises a silk fibroin fiber and at least a portion of the silk fibroin fiber aligns in the direction of the external magnetic field.

A method of producing a magneto-responsive silk fibroin-based material comprising: a. forming a silk fibroin fiber from a silk fibroin solution comprising a plurality of magnetic particles;

b. forming a magnetic field in a predetermined pattern on a target solid substrate receiving the silk fibroin fiber, wherein the magnetic field pattern determines an arrangement or orientation of the formed silk fibroin fiber on the target solid substrate; and

c. depositing the formed silk fibroin fiber onto the target solid substrate, thereby producing a magneto-responsive silk fibroin-based material comprising a silk fibroin fiber embedded with magnetic particles.

The method of paragraph 27, wherein the silk fibroin solution is at a concentration of about 1 wt% to about 50 wt% , about 10 wt% to about 40 wt%, or about 20 wt% to about 40 wt%.

The method of paragraph 27 or 28, wherein the plurality of magnetic particles are present in a concentration of about 1 vol% to about 30 vol%, or about 5 vol% to about 20 vol%. The method of paragraph 29, wherein the plurality of magnetic particles are present in a concentration of no greater than 10 vol%. Attorney Docket No. 700355-070131-PCT The method of any of paragraphs 27-30, wherein the magnetic particles have a diameter of about 1 nm to about 10 μηι.

The method of paragraph 31, wherein the magnetic particles have a diameter of about 5 nm to about 5 μηι.

The method of any of paragraphs 27-32, wherein the magnetic particles comprise ferrous particles.

The method of paragraph 33, wherein the ferrous particles comprise iron particles.

The method of any of paragraphs 27-34, wherein the silk fibroin solution of step (a) further comprises an agent to increase conductivity of the silk fibroin solution.

The method of paragraph 35, wherein the agent comprises sodium hydroxide.

The method of any of paragraphs 27-36, wherein the silk fibroin solution of step (a) further comprises a biopolymer.

The method of paragraph 37, wherein the biopolymer is selected from the group consisting of polyethylene oxide (PEO), polyethylene glycol (PEG), collagen, fibronectin, keratin, polyaspartic acid, polylysine, alginate, chitosan, chitin, hyaluronic acid, pectin, polycaprolactone, polylactic acid, polyglycolic acid,

polyhydroxyalkanoates, dextrans, polyanhydrides, polymer, PLA-PGA, polyanhydride, polyorthoester, polycaprolactone, polyfumarate, collagen, chitosan, alginate, hyaluronic acid, and any combinations thereof.

The method of any of paragraphs 27-38, wherein the forming of the silk fibroin fiber comprises electrospinning the silk fibroin solution of step (a).

The method of paragraph 39, wherein a strength of the electric field is at least about 15 kV.

The method of paragraph 40, wherein the strength of the electric field is at least about 25 kV.

The method of any of paragraphs 27-41, wherein the silk fibroin fiber has a diameter of about 0.1 μιη to about 1 mm.

The method of any of paragraphs 27-42, wherein the silk fibroin fiber has a diameter of about 0.5 μιη to about 500 μιη.

The method of any of paragraphs 27-43, wherein the forming of a magnetic field on the target solid substrate in a predetermined pattern comprises placing at least one magnet on a top surface of the target solid substrate, wherein an arrangement of the at least one magnet on the target solid substrate determines the magnetic field pattern. Attorney Docket No. 700355-070131-PCT

4 . The method of paragraph 44, wherein the at least one magnet is a permanent magnet, an electromagnet or a combination thereof.

46. The method of any of paragraphs 27-45, wherein the arrangement or orientation of the formed silk fibroin fiber includes aligning the formed silk fibroin fiber in a direction of the magnetic field pattern.

47. The method of any of paragraphs 27-46, wherein the target solid substrate is conductive.

48. The method of any of paragraphs 27-47, wherein the target solid substrate is grounded.

49. The method of any of paragraphs 27-48, further comprising drying the silk fibroin fiber or the silk fibroin-based material.

50. The method of paragraph 27-49, wherein the drying comprises constraint-drying the silk fibroin fiber or the silk fibroin-based material.

51. The method of any of paragraphs 27-50, further comprising post-treating the silk fibroin fiber or the silk fibroin-based material.

52. The method of paragraph 51, wherein the post-treating comprises contacting the silk fibroin fiber or the silk fibroin-based material with methanol or ethanol.

53. The method of paragraph 51, wherein the post-treating comprises subjecting the silk fibroin fiber or the silk fibroin-based material to water annealing or water vapor annealing.

54. The method of any of paragraphs 27-53, further comprising embedding at least one

active agent in the silk fibroin fiber or in the silk fibroin-based material.

55. The method of paragraph 54, wherein the active agent is selected from the group

consisting of cells, proteins, peptides, nucleic acids, nucleic acid analogs, nucleotides or oligonucleotides, peptide nucleic acids, aptamers, antibodies or fragments or portions thereof, antigens or epitopes, hormones, hormone antagonists, growth factors or recombinant growth factors and fragments and variants thereof, cell attachment mediators, cytokines, enzymes, antibiotics or antimicrobial compounds, viruses, toxins, therapeutic agents and prodrugs thereof, small molecules, and any combinations thereof.

56. A composition comprising a silk fibroin fiber produced by the methods of any of

paragraphs 27-55.

Some selected definitions

[00189] Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing Attorney Docket No. 700355-070131-PCT particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[00190] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.

[00191] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise.

[00192] The term "a plurality of as used herein refers to 2 or more, including, e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 100 or more, 500 or more, 1000 or more, 5000 or more, or 10000 or more.

[00193] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages may mean ±5% of the value being referred to. For example, about 100 means from 95 to 105.

[00194] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[00195] The term "statistically significant" or "significantly" refers to statistical significance and generally means at least two standard deviation (2SD) away from a reference level. The term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true.

[00196] As used interchangeably herein, the term "substantially" means a proportion of at least about 60%, or preferably at least about 70% or at least about 80%, or at least about 90%, at least about 95%, at least about 97% or at least about 99% or more, or any integer between 70% and 100%. In some embodiments, the term "substantially " means a proportion of at least about 90%, at least about 95%, at least about 98%, at least about 99% or more, or any integer between 90%) and 100%. In some embodiments, the term "substantially" can include 100%. Attorney Docket No. 700355-070131-PCT

[00197] As used herein, the phrase "silk fibroin-based material" refers to a material in which the silk fibroin constitutes at least about 10% of the total material, including at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%), at least about 80%, at least about 90%, at least about 95%, up to and including 100% or any percentages between about 30% and about 100%, of the total material. In certain embodiments, the silk fibroin-based material can be substantially formed from silk fibroin. In various embodiments, the silk fibroin-based material can be substantially formed from silk fibroin and at least one active agent. In some embodiments where the silk fibroin constitute less than 100%) of the total material, the silk fibroin-based material can comprise a different material and/or component including, but not limited to, a metal, a synthetic polymer, e.g., but not limited to, poly(vinyl alcohol) and poly(vinyl pyrrolidone), a hydrogel, nylon, an electronic component, an optical component, an active agent, any additive described herein, and any combinations thereof.

[00198] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow. Further, to the extent not already indicated, it will be understood by those of ordinary skill in the art that any one of the various embodiments herein described and illustrated may be further modified to incorporate features shown in any of the other embodiments disclosed herein.

[00199] The disclosure is further illustrated by the following examples which should not be construed as limiting. The examples are illustrative only, and are not intended to limit, in any manner, any of the aspects described herein. The following examples do not in any way limit the invention.

EXAMPLES

[00200] The following examples illustrate some embodiments and aspects of the invention. It will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be performed without altering the spirit or scope of the invention, and such modifications and variations are encompassed within the scope of the invention as defined in the claims which follow. The following examples do not in any way limit the invention.

Example 1. Exemplary materials and methods used for generating a composition comprising a magneto-responsive silk fiber Attorney Docket No. 700355-070131-PCT

[00201] Silk fibroin solution. Silkworm Bombyx mori cocoons were degummed through an extraction process as described in Sofia S et al. (2001) Journal of Biomedical Materials Research; 54: 139-148. The cocoons were cut into pieces and boiled for 30 min in a 0.02 M sodium carbonate (Sigma Aldrich, MO, USA, ACS grade >99.5%) aqueous solution. The degummed silk fibroin was rinsed thoroughly in Milli-Q water and dried in air (both room temperature). The silk fibroin was then dissolved at 60° C at a 20 wt/vol % concentration in a 9.3 M lithium bromide solution (Sigma Aldrich, MO, USA, ReagentPlus >99%) and dialyzed against Milli-Q water (Slide-a-Lyzer dialysis cassettes, Thermo Scientific, IL, USA, MWCO 3,500) for 2 days, refreshing the water every 6 hours. The resulting aqueous silk solution was centrifuged twice (1,400 rpm, 4°C) to achieve a final concentration of 8-9 wt% silk in aqueous solution.

[00202] Electrospinning solution. To increase the concentration for electrospinning, the 8-9 wt% silk solution was dialyzed against a 15 wt% PEG solution (Sigma Aldrich, MO, USA) for 14 hours to withdraw water and obtain a ~30 wt% silk solution. In some embodiments, concentration of the silk solution was performed overnight with avoidance of premature gelation. The concentrated silk solution was transferred to 5 ml syringes. To increase the conductivity of the solution, about 100 μΐ IN NaOH was added to 2-2.5 ml concentrated silk solution. Approximately 10 vol% carbonyl iron particles (1-3 μηι diameter spheres, Alfa Aesar, MA, USA) were mixed into the solution by manual stirring. Before starting the electrospinning, the solution was allowed to sit in air at room temperature to allow entrapped bubbles to escape. The particles appeared well distributed throughout the silk fibroin solution. Microscopic images of the resulting spun fibers, as shown in Figures 1A-1C, showed that there was no significant aggregation of particles; rather, particles were evenly distributed.

[00203] Electrospinning apparatus. An exemplary electrospinner used to generate the magneto-sensitive silk fibroin fibers described herein is shown in Figure 2. Any other art- recognized electrospinners can also be used for generating the magneto-sensitive silk fibroin fibers described herein. An 18 gauge needle is mounted within an aluminum counter electrode disk adapted to be placed under the top of the chamber in Figure 2. The counter electrode can help to shape and/or modulate the electric field and propel the silk solution downward toward the target collector. A Gamma High Voltage Research ES30P-5W high voltage power supply can be used to provide between 10 kV and 30 kV to the needle and counter electrode disk. The silk fibroin solution flow rate can be controlled by a Thermo Electron Corporation Orion Sage M362 syringe pump, shown on top of the chamber in Figure 2. A manual scissor lift can allow fairly accurate control of needle-to-target spacing. In some embodiments of the methods described herein, a 10 cm diameter aluminum disk or small diameter steel rod can be placed on Attorney Docket No. 700355-070131-PCT the scissor lift. When the conductive geometry is properly grounded (grounding wire is shown in Figure 2 attached to an aluminum disk), it can act as the target collector for the spun material. An exemplary electrospinning set-up can include, but not limited to:

Brand of pump - M362 Orion Sage syringe pump, Thermo Scientific, MA, USA

Needle - 16 gauge

Square magnets - 5x5 mm per side

Ball magnets - diameter = 5 mm

Target collector - diameter = 10 cm

[00204] Magnetic field hardware. Magnetic fields were created, e.g., using permanent magnets and/or electromagnets. Neodymium rare earth magnets (K&J Magnetics, Inc.) of various sizes and magnetic strength were used. In some embodiments of the methods described herein, the magnets were placed on the aluminum target collector (e.g., on the adjustable scissor lift) in various configurations, depending on the application goals. Aluminum foil can be wrapped on top of the magnets to keep the magnets clean and to assist in post-spinning material removal. The foil was grounded using the grounding wire to assist in propelling the silk fibers toward the magnetized target area. In some embodiments, the magnets were placed in the space between the needle and target collector, along the spinning direction, e.g., to study the influence of the magnetic field on directional control of electrospinning.

[00205] In some embodiments, magnetic fields were created using electromagnets.

Figure 2 shows an exemplary hardware for such purpose. A computer (e.g., a laptop) can be used to run a graphical development environment (e.g., National Instruments' LabVIEW software) and to interface with a chassis (e.g., National Instruments NI cDAQ-9172 8-slot chassis with an NI 9485 8-channel Solid State Relay module). The purpose of the computer- controlled relay system is to manipulate electromagnets as a group or individually for generating controlled motion and lay-up of magnetic electrospun silk fibroin fibers. Power was provided to the electromagnets, e.g., using a Tenma 72-2085 Laboratory DC power supply. Figures 3A-3B shows a close-up of two typical arrangements of electromagnets used. In Figure 3A, the electromagnets (e.g., Magnetic Sensor Systems E-66-38-37) are arranged in a circular pattern. The perforated steel plate can allow for rapid re-arrangement of the electromagnets. Note that the aluminum foil cover is not shown in these images. Figure 3B shows larger-diameter electromagnets (e.g., Magnetic Sensor Systems E-66-75-32) arranged in a linear configuration. All of the electromagnetics were wired through an opening in the electrospinning chamber to the NI 9485 Solid State Relay module, drawing power from the Tenma DC power supply.

[00206] Electromagnet control program. When electromagnets were used instead of permanent magnets, a graphical development environment, e.g., National Instruments' Attorney Docket No. 700355-070131-PCT

LabVIEW software, was used for control. Figures 4A-4B show both the main user interface (Front Panel) and logic (Block Diagram) for operation. The relay system could be operated by sending a series of Boolean on/off commands to as many channels as there were electromagnets connected. If 5 electromagnets were to be turned on, then "on" or "true" states would be sent to the 5 channels where the electromagnets were connected. To dynamically turn on various electromagnets, a While loop structure was used that would turn on a sequence of

electromagnets one at a time, with a specified time interval (in seconds) between.

[00207] Fabrication of magneto-sensitive silk fibroin fibers. Silk fibroin solution including ferrous particles was first prepared. The solution was then stored in a syringe and mounted to the syringe pump. The adjustable scissor table was set such that the distance between the needle tip and target collector was approximately 20 cm. Depending upon different embodiments, either permanent magnets or electromagnets were then placed on an aluminum target disk. When an electromagnet was used, one or more electromagnets could be connected to a DC power supply and the relay hardware. In addition, the LabVIEW control program could be made ready. Aluminum foil could be wrapped on top of the magnets and grounded. The syringe pump could be set to between 0.002 ml/min-0.005 ml/min (standard about 0.004 ml/min) to start the silk fibroin solution flowing. Once solution began dripping from the needle, the high voltage power supply could be set to 25 kV and turned on (the electrospinning chamber door should be closed whenever the high voltage charge is applied for safety reasons). The needle tip could be monitored to watch for the formation of the Taylor cone and first evidence of successful spinning. If too much solution dropped from the needle, the flow rate could be decreased and if no drops formed at the tip, the flow rate could be increased. Once the Taylor cone formed and material was seen to deposit on the grounded aluminum foil, the electromagnet control program would be activated. In one embodiment of the method described herein, spinning could be continued under monitoring for 4-6 hours. Figures 5A-5B shows one embodiment of the methods described herein in which a flat, square Neodymium magnet is mounted on the aluminum target disk collector (which can be covered in aluminum foil).

Example 2. Use of a permanent magnet for generation of magneto-sensitive silk fibroin fibers

[00208] Optimal settings for silk fibroin and carbonyl iron concentrations in the solutions, solution flow rate, electrical field strength and needle-collector distance were determined. In some embodiments, one or more of these processing parameters could be fixed during generation of magneto-sensitive silk fibroin fibers. In some embodiments, the carbonyl iron concentration that can be used in the method described herein, e.g., without having the solution clog the tubing and/or solidifying in the needle, can be 10 vol% or lower. In other embodiments, Attorney Docket No. 700355-070131-PCT higher than 10 vol% carbonyl iron concentration can be used. Generally, the higher the iron concentration is used, the higher a magnetic field can be achieved. In some embodiments, a relatively slow solution flow rate in a range of 0.002 ml/min -0.005 ml/min was used; higher flow rates could led to droplet formation and even slower rates could led to solution drying in the needle. A relatively high voltage level of 25 kV was used for spinning the silk-iron solution. In some embodiments, lower electrical fields could cause the solution to form droplets and no continuous fibrous mat was formed. In some embodiments, the needle-target collector distance of 20 cm could give the best results with respect to fiber formation and mesh continuity. In some embodiments, the spinning time for a mesh was about 4-6 hours and around 3 ml of silk-iron solution was used to generate the mesh during this time frame. In certain embodiments, while it is not necessary, shorter time frames may not generate a full mesh that would support itself.

[00209] As shown in Figures 5A-5B, a flat, square Neodymium rare earth magnet, with north-south pole aligned in the mesh thickness direction, was used. Upon starting the electrospinning process, the silk fibroin fiber comprising magnetic particles became much more visible than what has been seen with electrospinning of virgin silk solution. Since silk solution alone produces fibers with a diameter on the order of 50 nm-100 nm, the fibers become impossible to detect with the naked eye. The fibers formed from magneto-sensitive silk fibroin are generally much larger in diameter, e.g., in the 1-20 micron range, and contain iron particles which make the fibers visible to the eye. After about 15 minutes of spinning, the magneto- sensitive fibers tended to anchor themselves to the aluminum foil on top of the magnet.

Subsequent fibers could continue to build up a tower-like structure spanning from the electrospinning needle to the aluminum foil. In some embodiments, the magneto-sensitive silk fibroin fibers tended to anchor themselves around the periphery of the square magnet. When the high voltage supply was turned off, the silk fibroin fibrous tower stayed in place. There was vertical alignment of the fibers along the tower and because the fibers generally anchored around the periphery of the magnet, these areas generally had the largest amount of silk fibroin fibers.

[00210] As shown in Figures 6A-6C, a series of Neodymium bar magnets were arranged into a star pattern. Each magnet was magnetized in the length- wise direction. Therefore, the magnets were joined where the north pole on one magnet met the south pole of another. Figure 6A shows the aluminum foil wrapped on top of the star pattern and a series of fibers that are anchoring to the bar magnet locations. On close inspection, it can be seen that the fibers are anchoring above where the magnets are located. After several hours of electrospinning, sufficient silk fibroin material had built up and the electrospinner was stopped. The fiber tower that formed was disconnected from the needle and allowed to collapse down onto the aluminum foil. Figure 6B shows the aluminum foil removed from the magnet star pattern with silk fibroin Attorney Docket No. 700355-070131-PCT fiber deposited as a mesh on the aluminum foil. When the silk fibroin mesh was held up to light, as in Figure 6C, it is shown that magnetized silk fibroin fibers have built up in locations where magnets were located. While the star pattern was aligned approximately under the needle, not all magnets necessarily had the same thickness of silk fibroin fibers. Other factors, such as slight air currents, viscosity variations, or even electrical fluctuations can contribute to the non- uniformity in the thickness of the formed mesh. On close inspection, magneto-sensitive silk fibroin fibers appear to align in the direction of the magnetic field created by the magnets.

[00211] In other embodiments, axially magnetized Neodymium spheres (0.75 cm diameter) were used to create the flat hexagon pattern as shown in Figure 7B. As shown in Figure 7A, a silk fibroin fiber tower was formed, with the anchoring locations at the innermost coil of spherical magnets. Without wishing to be bound by theory, the fiber naturally wants to anchor at a ground location most aligned with the needle and the inner coil of spherical magnets has the same level of magnetism as the others; all else being equal, the fiber therefore anchors at these magnets. There is also a large amount of silk fibroin fiber lying across all of the magnets. While silk fibroin fibers form from the needle, the outer magnets can attract the silk fibroin fibers as well. After initial anchoring of the silk fibroin fiber to a magnetized collector and as more fiber is ejected from the needle, the fiber lays flat on the magnets and continues to build the tower at the inner periphery. On closer inspection of the magneto-sensitive silk fibroin mat shown in Figure 7C, the silk fibroin fiber appears to be aligned locally with magnetic field generated by the pattern of the axially magnetized spheres.

[00212] In some embodiments, a three-dimensional cylindrical arrangement of axially magnetized Neodymium spherical magnets was created, as shown in Figure 8B. Figure 8 A shows the formation of magnetized silk fibroin fiber on the aluminum foil target collector before removal from the electrospinner. When the magnetized silk fibroin fiber mat or mesh was held up to light (Figure 8C), the electrospun fibers appear to be concentrated at spherical magnet locations and that fibers span the gaps between spherical magnets. Alignment of the silk fibroin fibers can be observed in both locations - at the magnets and in the spans between magnets.

[00213] To further evaluate the alignment of the silk fibroin fibers by spanning multiple magnets, in some embodiments, two cylindrical patterns of axially magnetized spherical Neodymium magnets were created, as shown in Figure 9A. Electrospinning outcomes are shown in Figures 9B-9D. A large silk fibroin fiber tower was formed, which draped beyond the magnets and aluminum disk target collector. A significant amount of silk fibroin fiber spanned between the two cylindrical patterns of spherical magnets. As shown in Figure 9E, the fiber mesh created was fairly tough and could be stretched out by hand. Figures 1 OA- IOC show Attorney Docket No. 700355-070131-PCT closer views of the silk fibroin fiber mesh as generated in Figures 9A-9E, in which the silk fibroin fibers are aligned in at least one direction.

Example 3. Use of an electromagnet for generation of magneto-sensitive silk fibroin fibers [00214] Permanent magnets generally (a) have a fixed magnetization level; and (b) are either stationary or need to be physically moved. Thus, in alternative embodiments, electromagnets were used in the control of electrospun magnetized silk fibroin. For example, two electromagnet sizes were utilized, with a control strategy of switching on or off the electromagnets with a fixed voltage. Figures 3A-3B show the ability to place electromagnets in various configurations, thus creating a magnetic field in various patterns. Along with a computer-controlled relay system, all electromagnets, in some embodiments, can be turned on at the same time, behaving as permanent magnets. Computer control can also allow for dynamic manipulation of the electromagnets. Thus, in other embodiments, the electromagnets can be individually turned on or off at different times. For example, the electromagnets in Figure 3B can be controlled to be active in a sequential order such that local magnetization can be effectively created and moved in a linear fashion in one direction. A circular pattern of electromagnets, as shown in Figure 3A, can allow localized magnetization to be created and moved along a circle. Electrospun silk fibroin fiber is attracted to electromagnets that are on and not to electromagnets that are off. Additionally, when a neighboring electromagnet is turned active, the stream of silk fibroin fiber produced from the needle can move to that neighboring position.

[00215] Presented herein relates to compositions comprising a magneto-sensitive silk fibroin-based material and methods of making the same, e.g., by electrospinning a silk fibroin solution containing iron particles. As shown herein, carbonyl iron particles can distribute well in silk fibroin solution and resulting electrospun fibers. The resulting electrospun fibers can be visualized by naked eye and tend to form straighter fibers than what is normally seen in silk electrospinning (without iron particles). In the presence of a magnetic field, e.g., generated by permanent magnets or electromagnets, a silk fibroin solution that contains iron particles can be drawn to magnets during electrospinning. The generated magneto-sensitive silk fibroin fibers can anchor locally to magnets and also tend to bridge the gaps between magnets. Alignment of the magneto-sensitive silk fibroin fibers can be observed along magnetic field lines. Various electromagnetic patterns can be created using one or more magnets arranged in different configurations to impart desired alignment in electrospun fibers. Such capabilities can be beneficial in various applications, such as formation of tissue engineering scaffolds with a preferred orientation of fibers, e.g., for mechanical integrity, and/or certain cell responses due to Attorney Docket No. 700355-070131-PCT controlled mechanotransduction. For example, loading conditions transmitted through a fiber network can help direct cell-driven tissue properties.

[00216] Without wishing to be bound by theory, electromagnets can offer a wider range of control options than permanent magnets. An array of electromagnets of any sizes (e.g., small electromagnets) can be used to create numerous magnetic field patterns with a simple computer- based programming interface. For example, an array of electromagnets can allow the controlled lay-up of a mesh with an alternating alignment in one direction or another. To produce textilelike magneto-sensitive silk fibroin sheets or meshes, warp and weft courses can be generated with zero and 90 degree electromagnet coordination. Electromagnets can provide non- orthogonal directional control. For example, circumferentially aligned magneto-sensitive silk fibroin fibers can be created by sequentially firing on electromagnets that are positioned circumferentially around a circular pattern. A similar setup can be used to create a twisted electrospun magneto-sensitive silk fibroin fiber by quickly moving the fiber anchoring point as the fiber is being formed. Such approaches can greatly improve mechanical performance of fibers and existing mechanically-poor electrospun mats, as the existing electrospun fibers and mats do not contain iron particles which can enable control of fiber alignment using a magnet, and thus fiber alignment within the existing mesh or mats can be more random.

[00217] The benefits of incorporating iron particles in silk fibroin solution extend beyond the ability to better control electrospinning. The electrospun silk fibroin materials containing iron particles described herein can respond to magnetic fields after removal from the electrospinner. Magnetic sensitivity of the silk fibroin materials described herein can be beneficial in many applications. For example, if such magneto-sensitive silk fibroin meshes are implanted in the body, external magnetic fields can be used to effect contraction in these meshes. Additionally, these magneto-sensitive silk fibroin materials can be embedded in soft structures, e.g., hydrogels or soft tissues, or robotic bodies to allow for controlled movement through external magnetic manipulation.

Example 4. Magneto-responsive hydrogels and exemplary methods of making the same

[00218] In order to generate a magneto-responsive hydrogel, a magnetic silk solution is prepared. An exemplary protocol of preparing a silk fibroin solution from cocoons is described as follows:

Cut cocoons and remove the pupae, pupae skins and any other dirt from the inside of the cocoon;

Degum the cut cocoons in a sodium carbonate (Na₂CC>3) solution. Boil time in the sodium carbonate solution can vary from about 10 minutes to about 30 minutes, Attorney Docket No. 700355-070131-PCT depending on the form of the silk fibroin-based material to be formed. Without wishing to be bound by theory, as boil time increases, the silk fibroin degrades and this can affect the physical and mechanical properties of the final silk construct or silk fibroin-based material (e.g., an electro gelated silk hydrogel vs. a film).

Rinse the degummed silk fibroin in water (e.g., Milli-Q water) at least thrice, for at least half an hour each time.

Air dry the rinsed silk fibroin.

Dissolve the silk fibroin in a 9.3 M lithium bromide solution (Sigma Aldrich, MO, USA, ReagentPlus > 99%) at 60°C and dialyze against water (e.g., Milli-Q water), e.g., with Slide-a-Lyzer dialysis cassettes (Thermo Scientific, IL, USA, MWCO 3,500) for about 2 days, regularly changing the water, e.g., every 6 hours.

Centrifuge the resulting aqueous silk solution twice, at approximately 1 l,000rpm, for 20 minutes each time.

The resulting aqueous silk solution has a concentration between 7% and 9% silk.

[00219] Magnetic particles or a ferrofluid are then added to a silk fibroin solution to form a magnetic silk fibroin solution. In some embodiments, a water-based ferrofluid (e.g., a ferrofluid from EMG series, Ferrotec (USA) Corporation, Bedford, NH, USA) can be added to the aqueous silk fibroin solution as described above.

Table 1. Ferrofluid (EMG series) properties and interaction with silk solution

[00220] The ferrofluid (e.g., EMG series obtained from Ferrotec) generally contains nano- particles (e.g., approximately lOnm in diameter) of iron oxides (Fe₂0₃ and Fe₃C>4). In some embodiments, water-based ferrofluid is used to increase the miscibility of the ferrofluid with the Attorney Docket No. 700355-070131-PCT aqueous silk fibroin solution. The iron oxides in a ferrofluid are generally coated with surfactants to prevent aggregation and agglomeration of the particles, and these coated particles are suspended in water. These surfactant coatings vary in nature with each ferrofluid sample (e.g. hydrocarbons, lipids).

[00221] Ferrofluid can also be manufactured using any art-recognized process. One of skill in the art can tune the average size of the ferrofluid particles to a desired one by changing the processing conditions, depending on the manufacturing process. In some embodiments, the ferrofluid particles can be coated with a surfactant coating.

[00222] The ferrofluid can be added to the silk solution in various concentrations, e.g., about 0.1% v/v to about 10% v /v or more; or about 0.1 % w/v to about 10% w/v or more; or about 0.1%) w/w to about 10% w/w or more. In one embodiment, the ferrofluid is added to the silk fibroin solution at a concentration of about 0.1% v/v to about 10% v/v. The mixture can be stirred vigorously either by hand (e.g., using a stirrer) or by using a vortex mixer.

[00223] The magnetic silk fibroin solution can respond to an applied magnetic field, e.g., produced by both temporary and/or permanent magnets. In some embodiments, the magnetic silk fibroin solution can exhibit temporary magnetism an applied field.

[00224] The magnetic silk fibroin solution can be used to create a variety of silk constructs, e.g., but not limited to, hydrogels (e.g., self-assembled, electrogelated, vortex or shear stress-induced, pH-induced, particle-induced gels), foams, scaffolds, films, tubes, particles, electrospun geometries, and any combinations thereof.

[00225] Without wishing to be bound by theory, the iron oxide particles in the ferrofluid can act as an initiator and/or catalyst, increasing the rate at which the silk fibroin molecules cross-link with each other. The characteristic size of micelles is approximately lOnm to lOOnm (Kevin Letchford, Helen Burt. "A Review of the Formation and Classification of Amphiphilic Block Copolymer Nanoparticulate Structures: Micelles, Nanospheres, Nanocapsules and Polymersomes." European Journal of Pharmaceutics and Biopharmaceutics 65 (2007): pp 259- 269). Since the ferrofluid contains particles in this range, the iron oxide particles can enhance the formation of micelles which in turn allows the silk solution to self-assemble and form a gel.

[00226] In some embodiments, the magnetic silk fibroin solution can be used to form a hydrogel. For example, formation of a magnetic silk fibroin hydrogel can be time-dependent, e.g., by subjecting a magnetic silk fibroin solution at an ambient temperature for a period of time. The magnetic silk fibroin solution can be subjected to various temperatures, e.g., room temperature, low temperatures (e.g., fridge temperatures), or elevated temperatures (e.g., in an oven at low heat). Without wishing to be bound by theory, the silk molecules cross- link, using the iron particles as an initiator or catalyst, resulting in the formation of a hydrogel. The time it Attorney Docket No. 700355-070131-PCT takes to gel can, in part, depend on the temperature of the solution and its surroundings, the degumming time of the silk fibroin (e.g., lower degumming time results in shorter gelation time), the size of iron particles used, the concentration and magnetization of the ferrofluid or magnetic particles used (e.g., higher concentration and/or magnetization results in shorter gelation time), and/or the ratio of ferrofluid/magnetic particles to silk solution (e.g., higher ratio of ferrofluid/magnetic particles: silk solution results in shorter gelation time).

[00227] In some embodiments, electrogelation can be applied to a magnetic silk fibroin solution to form a magneto-responsive electrogelated silk fibroin gel. In these embodiments, a potential difference (e.g., about 25V) can be applied across 2 electrodes (such as copper or platinum (more inert electrode material is desirable to prevent corrosion and/or leaching of the electrodes into the solution) placed in a magnetic silk fibroin solution, and a current is passed through the magnetic silk solution for a given amount of time. The magnetic silk fibroin gel is formed on the positive electrode (cathode) while hydrogen gas is formed on the negative electrode (anode). The process of gelation can be enhanced due to the presence of the iron particles (as described above) and as a result a larger volume of gel can be obtained than the one obtained from a regular silk fibroin solution without magnetic particles or a ferrofluid. See, e.g., International Patent Application No. WO/2010/036992, the content of which is incorporated herein by reference, for details of eletrogelation of a silk fibroin solution.

[00228] Figure 11 A shows a magnetic silk hydrogel being fabricated using an

electrogelation process and a ferrofluid (10% v/v). When a permanent magnet is brought in close proximity to the gel, there is a force of attraction (Figure 1 IB). This attraction can be created with an electromagnet as well (Figure 11C).

[00229] In some embodiments, the pH of the magnetic silk fibroin solution can be altered, e.g., via the addition of an acidic or a basic solution to modulate (e.g., increase) the rate of gelation. See, e.g., U.S. Patent Application No. US 2011/0171239, the content of which is incorporated herein by reference, for details of formation of a pH-induced silk fibroin gel.

[00230] All patents and other publications identified in the specification and examples are expressly incorporated herein by reference for all purposes. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

Claims

Attorney Docket No. 700355-070131-PCT CLAIMS What is claimed is:

2. The composition of claim 1, wherein the silk fibroin-based material is in a form of a film, a sheet, a gel or hydrogel, a mesh, a mat, a non- woven mat, a fabric, a scaffold, a tube, a slab or block, a fiber, a particle, a 3-dimensional construct, an implant, a high- density material, a reinforced material, a foam or a sponge, a machinable material, a microneedle, or any combinations thereof.

3. The composition of claim 1, wherein the silk fibroin based material is in a form of a fiber, a mesh, a mat, a non-woven mat, a fabric, or any combinations thereof.

4. The composition of claim 1, wherein the silk fibroin based material is in a form of a hydrogel, a foam or sponge, a film, a scaffold, or any combinations thereof.

5. The composition of any of claims 1-4, wherein the magnetic particles have a diameter of about 1 nm to about 10 μηι.

6. The composition of any of claims 1-5, wherein the magnetic particles have a diameter of about 5 nm to about 5 μηι.

7. The composition of any of claims 1-6, wherein the magnetic particles comprise ferrous particles.

8. The composition of claim 7, wherein the ferrous particles comprise iron particles.

9. The composition of any of claims 1-8, wherein the silk fibroin based material is a silk fibroin fiber.

10. The composition of claim 9, wherein the silk fibroin fiber has a diameter of about 0.1 μιη to about 1 mm.

11. The composition of any of claims 9-10, wherein the silk fibroin fiber has a diameter of about 0.5 μηι to about 500 μηι.

12. The composition of any of claims 1-11, wherein the silk fibroin based material further comprises at least one active agent.

13. The composition of claim 12, wherein the active agent is selected from the group

consisting of cells, proteins, peptides, nucleic acids, nucleic acid analogs, nucleotides or oligonucleotides, peptide nucleic acids, aptamers, antibodies or fragments or portions thereof, antigens or epitopes, hormones, hormone antagonists, growth factors or recombinant growth factors and fragments and variants thereof, cell attachment Attorney Docket No. 700355-070131-PCT mediators, cytokines, enzymes, antibiotics or antimicrobial compounds, viruses, toxins, therapeutic agents and prodrugs thereof, small molecules, and any combinations thereof.

14. The composition of any of claims 1-13, wherein the plurality of magnetic particles is present in an amount sufficient to allow at least a portion of the silk fibroin based material to respond to an external energy source.

15. The composition of claim 14, wherein the external energy source comprises a magnetic field, ultrasound, electromagnetic waves, radio frequency, heat, an electric field, or any combinations thereof.

16. The composition of 14, wherein the external energy source comprises a magnetic field.

17. The composition of any of claims 14-16, wherein the response of the silk fibroin based material comprises deflection or contraction.

18. The composition of any of claims 1-17, wherein the silk fibroin based material further comprises a biopolymer.

19. The composition of claim 18, wherein the biopolymer is selected from the group

consisting of polyethylene oxide (PEO), polyethylene glycol (PEG), collagen, fibronectin, keratin, polyaspartic acid, polylysine, alginate, chitosan, chitin, hyaluronic acid, pectin, polycaprolactone, polylactic acid, polyglycolic acid,

20. The composition of any of claims 1-19, wherein the composition is adapted for use as a medical implant.

21. The composition of any of claims 1-19, wherein the composition is adapted for use as a wound dressing.

22. The composition of any of claims 1-19, wherein the composition is adapted for use as a tissue engineering scaffold.

23. The composition of any of claims 1-19, wherein the composition is adapted for use as a sensor.

24. The composition of any of claims 1-19, wherein the composition is adapted for use as a drug delivery device.

25. The composition of any of claims 1-19, wherein the composition is adapted for use as a separation membrane.

26. The composition of any of claims 1-19, wherein the silk fibroin based material comprises a silk fibroin fiber and at least a portion of the silk fibroin fiber aligns in the direction of the external magnetic field. Attorney Docket No. 700355-070131-PCT

27. A method of producing a magneto-responsive silk fibroin-based material comprising: a. forming a silk fibroin fiber from a silk fibroin solution comprising a plurality of magnetic particles;

28. The method of claim 27, wherein the silk fibroin solution is at a concentration of about 1 wt% to about 50 wt% , about 10 wt% to about 40 wt%, or about 20 wt% to about

40 wt%.

29. The method of claim 27 or 28, wherein the plurality of magnetic particles are present in a concentration of about 1 vol% to about 30 vol%, or about 5 vol% to about 20 vol%.

30. The method of claim 29, wherein the plurality of magnetic particles are present in a concentration of no greater than 10 vol%.

31. The method of any of claims 27-30, wherein the magnetic particles have a diameter of about 1 nm to about 10 μηι.

32. The method of claim 31 , wherein the magnetic particles have a diameter of about 5 nm to about 5 μπι.

33. The method of any of claims 27-32, wherein the magnetic particles comprise ferrous particles.

34. The method of claim 33, wherein the ferrous particles comprise iron particles.

35. The method of any of claims 27-34, wherein the silk fibroin solution of step (a) further comprises an agent to increase conductivity of the silk fibroin solution.

36. The method of claim 35, wherein the agent comprises sodium hydroxide.

37. The method of any of claims 27-36, wherein the silk fibroin solution of step (a) further comprises a biopolymer.

38. The method of claim 37, wherein the biopolymer is selected from the group consisting of polyethylene oxide (PEO), polyethylene glycol (PEG), collagen, fibronectin, keratin, polyaspartic acid, polylysine, alginate, chitosan, chitin, hyaluronic acid, pectin, polycaprolactone, polylactic acid, polyglycolic acid, polyhydroxyalkanoates, dextrans, polyanhydrides, polymer, PLA-PGA, polyanhydride, polyorthoester, polycaprolactone, Attorney Docket No. 700355-070131-PCT polyfumarate, collagen, chitosan, alginate, hyaluronic acid, and any combinations thereof.

39. The method of any of claims 27-38, wherein the forming of the silk fibroin fiber

comprises electrospinning the silk fibroin solution of step (a).

40. The method of claim 39, wherein a strength of the electric field is at least about 15 kV.

41. The method of claim 40, wherein the strength of the electric field is at least about 25 kV.

42. The method of any of claims 27-41, wherein the silk fibroin fiber has a diameter of about 0.1 μιη ίο about 1 mm.

43. The method of any of claims 27-42, wherein the silk fibroin fiber has a diameter of about 0.5 μιη to about 500 μιη.

44. The method of any of claims 27-43, wherein the forming of a magnetic field on the target solid substrate in a predetermined pattern comprises placing at least one magnet on a top surface of the target solid substrate, wherein an arrangement of the at least one magnet on the target solid substrate determines the magnetic field pattern.

45. The method of claim 44, wherein the at least one magnet is a permanent magnet, an

electromagnet or a combination thereof.

46. The method of any of claims 27-45, wherein the arrangement or orientation of the

formed silk fibroin fiber includes aligning the formed silk fibroin fiber in a direction of the magnetic field pattern.

47. The method of any of claims 27-46, wherein the target solid substrate is conductive.

48. The method of any of claims 27-47, wherein the target solid substrate is grounded.

49. The method of any of claims 27-48, further comprising drying the silk fibroin fiber or the silk fibroin-based material.

50. The method of claim 27-49, wherein the drying comprises constraint-drying the silk fibroin fiber or the silk fibroin-based material.

51. The method of any of claims 27-50, further comprising post-treating the silk fibroin fiber or the silk fibroin-based material.

52. The method of claim 51 , wherein the post-treating comprises contacting the silk fibroin fiber or the silk fibroin-based material with methanol or ethanol.

53. The method of claim 51 , wherein the post-treating comprises subjecting the silk fibroin fiber or the silk fibroin-based material to water annealing or water vapor annealing.

54. The method of any of claims 27-53, further comprising embedding at least one active agent in the silk fibroin fiber or in the silk fibroin-based material.

55. The method of claim 54, wherein the active agent is selected from the group consisting of cells, proteins, peptides, nucleic acids, nucleic acid analogs, nucleotides or Attorney Docket No. 700355-070131-PCT oligonucleotides, peptide nucleic acids, aptamers, antibodies or fragments or portions thereof, antigens or epitopes, hormones, hormone antagonists, growth factors or recombinant growth factors and fragments and variants thereof, cell attachment mediators, cytokines, enzymes, antibiotics or antimicrobial compounds, viruses, toxins, therapeutic agents and prodrugs thereof, small molecules, and any combinations thereof.

56. A composition comprising a silk fibroin fiber produced by the methods of any of claims 27-55.