WO2014021954A2 - Matières à base de fibroïne de soie magnéto-sensible - Google Patents

Matières à base de fibroïne de soie magnéto-sensible Download PDF

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Publication number: WO2014021954A2
Authority: WO; WIPO (PCT)
Prior art keywords: silk fibroin; based material; fiber; composition; silk
Prior art date: 2012-04-13
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.): Ceased

Application number

PCT/US2013/036539

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English (en)

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WO2014021954A3 (fr

Inventor

David L. Kaplan

Gary G. Leisk

Berendien Jacoba PAPENBURG

Ethan GOLDEN

Nereus PATEL

Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

Tufts University

Original Assignee

Tufts University

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

2012-04-13

Filing date

2013-04-15

Publication date

2014-02-06

2013-04-15 Application filed by Tufts University filed Critical Tufts University

2014-02-06 Publication of WO2014021954A2 publication Critical patent/WO2014021954A2/fr

2014-03-27 Publication of WO2014021954A3 publication Critical patent/WO2014021954A3/fr

2014-10-13 Anticipated expiration legal-status Critical

Status Ceased legal-status Critical Current

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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
- A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/18—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing inorganic materials
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/02—Inorganic materials
- A61L27/04—Metals or alloys
- A61L27/042—Iron or iron alloys
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
- A61L27/446—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with other specific inorganic fillers other than those covered by A61L27/443 or A61L27/46

Definitions

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.
Electrospinning is a widely used processing technique to create small-diameter fibers from typically polymeric solutions or melts.
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.
magneto-sensitive polymeric particles like iron oxides, maghemite y-Fe20 3 and magnetite Fe 3 0 4 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.
MRI magnetic resonance imaging
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.
the ability to direct alignment and/or layering of silk fibroin fibers through the magnet technology can provide electrospun silk fibroin geometries with tailored physical and/or mechanical properties.
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.
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.
the magnetic particles can include any ferromagnetic material, ferrimagnetic material, paramagnetic material or any combinations thereof.
the Attorney Docket No. 700355-070131-PCT magnetic particles can include ferromagnetic particles, e.g., ferrous particles such as iron particles.
the iron particles can include carbonyl iron particles.
the magnetic particle embedded inside the silk fibroin-based material can be of any size.
the magnetic particles can have a diameter of about 1 nm to about 100 ⁇ . In some embodiments, the magnetic particles can have a diameter of about 5 nm to about 50 ⁇ . In some embodiments, the magnetic particles can have a diameter of about 10 nm to about 10 ⁇ . In some embodiments, the magnetic particles can have a diameter of about 10 nm to about 5 ⁇ .
the composition can comprise one or more magneto- responsive silk fibroin fibers.
the silk fibroin fibers can be arranged to form a network.
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.
the silk fibroin fiber can have a substantially circular cross-section.
the silk fibroin fiber can have a diameter of about 0.1 ⁇ to about 1 mm or about 0.5 ⁇ to about 500 ⁇ .
silk fibroin can generally stabilize active agents
some embodiments of the silk fibroin can be used to encapsulate and/or deliver an active agent.
at least one magneto-responsive silk fibroin-based material e.g., but not limited to, silk fibroin fibers, gels, foams, tubes, and/or films
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.
the magneto-responsive silk fibroin-based material can respond to an external magnetic field and/or gradient.
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.
the magneto-responsive silk fibroin-based material can further comprise a biopolymer.
a biopolymer can include, but are not limited to, polyethylene oxide Attorney Docket No. 700355-070131-PCT
PEO polyethylene glycol
PEG polyethylene glycol
collagen 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.
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.
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).
Different embodiments of the magneto-responsive silk fibroin-based materials 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.
applications and/or compositions can include, without limitations, medical implants, wound dressings, tissue engineering scaffolds, sensors, drug delivery devices, robotics, and separation membranes.
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
at least a portion of the silk fibroin fiber can align in the direction of, attract to, or repel from an applied magnetic field.
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.
the magnetic 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.
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.
a method of producing a magneto-responsive silk fibroin mat or mesh is provided herein.
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.
the concentration of the silk fibroin solution can be adjusted for each application, composition and/or product.
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%.
the silk fibroin solution can have a concentration of about 30 wt%.
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.
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.
the magnetic particles can have a diameter of about 0.01 ⁇ to about 100 ⁇ .
the magnetic particles can have a diameter of about 0.1 ⁇ to about 50 ⁇ . In some embodiments, the magnetic particles can have a diameter of about 0.5 ⁇ to about 10 ⁇ . In some embodiments, the magnetic particles can have a diameter of about 1 ⁇ to about 5 ⁇ .
any magnetic material can be used for the magnetic particles, including ferromagnetic material, ferrimagnetic material, paramagnetic material or any combinations thereof.
the magnetic particles can include ferromagnetic particles, e.g., ferrous particles such as iron particles.
the iron particles can include carbonyl iron particles.
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.
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.
ions e.g., water-soluble salts such as NaCl
a base e.g., sodium hydroxide
conducting polymers e.g., 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
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.
the silk fibroin solution can further comprise a porogen, e.g., a salt.
the silk fibroin solution can further comprise an active agent described herein.
the silk fibroin fiber can be formed from the silk fibroin solution with any methods known in the art
the silk fibroin fiber can be formed, at least partly, by electrospinning the silk fibroin solution comprising the plurality of magnetic particles.
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.
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.
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.
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.
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.
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.
post-treatment of the silk fibroin-based material can be employed.
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.
drying e.g., constraint-drying
mechanical stretching e.g., lyophilization
gas-drying e.g., solvent immersion (e.g., methanol and/or ethanol)
solvent immersion e.g., methanol and/or ethanol
water annealing e.g., water vapor annealing, heat annealing
shear stress e.g., ultrasound
ultrasound e.g., by
a magneto-responsive silk fibroin-based material e.g., but not limited to, silk fibroin fibers, gels, foams, tubes, and/or films
a magneto-responsive silk fibroin-based material e.g., but not limited to, silk fibroin fibers, gels, foams, tubes, and/or films
a magneto-responsive silk fibroin-based material e.g., a silk fibroin fiber or a network of silk fibroin fibers
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
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.
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.
Figures 4A-4B show front panel and block diagram for National Instruments LabVIEW program to control electromagnetically-assisted silk fibroin electrospinning, respectively.
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.
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.
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
an externally-applied energy source e.g., but not limited to, a magnetic field, ultrasound, radio frequency, and/or electromagnetic waves
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.
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.
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.
a coating see, e.g., International Patent Application Nos. WO 2007/016524; WO 2012/145652
a magnetic-responsive material see, e.g., International Patent Application No. WO 2012/054 82; a machinable material; or any combinations thereof.
a plurality of magnetic particles can be incorporated into a silk fibroin particle (see, e.g., U.S. Patent Application Nos. US
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).
an external energy source e.g., a magnetic field
at least a portion of a silk fiber comprising magnetic particles can align in the direction of an externally- applied magnetic field.
the magnetic particles or a magnetic fluid 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.
the magnetic particles or a magnetic fluid 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.
the magnetic particles or a magnetic fluid 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.
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)).
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).
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.
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.
silks 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.
silk fibroin can be derived from other sources such as spiders, other silkworms, bees, and bioengineered variants thereof.
silk fibroin can be extracted from a gland of silkworm or transgenic silkworms (see, e.g., WO 2007/098951).
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.
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.
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.
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.
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
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.
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.
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).
an additive e.g., an active agent
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.
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
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).
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.
the silk fibroin can be genetically modified to be fused with a protein, e.g., a therapeutic protein.
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 20140060600A1
a chemical such as glycerol
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.
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%.
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.
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,
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).
the silk fibroin/ PEO blend ratio in a magneto-responsive silk fibroin-based material can vary from about 1 : 100 to about 100: 1.
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.
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.
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.
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.
an active agent can be genetically fused to silk fibroin to form a fusion protein.
an active agent can be present in a magneto-responsive silk fibroin-based material.
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
the magneto-responsive silk fibroin-based material can further comprise at least one other additive.
an additive can alter flexibility, solubility, ease of processing, and/or enhanced stability of at least one property of the component distributed therein.
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).
vibrational sources e.g., ultrasound
electromagnetic waves or light e.g., electromagnetic waves or light, radio frequency, heat, and also any combinations thereof
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.
plasticizers e.g., glycerol
nanoparticles e.g., gold nanoparticles or plasmonic particles
optical particles e.g., fluorescent particles
adding an additive that can interact with an external energy source other than a magnetic field
an external energy source other than a magnetic field
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.
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.
magnetic particles can be about 1 nm to about 500 ⁇ in size, or about 5 nm to about 250 ⁇ in size, or about 50 nm to about 250 ⁇ in size.
magnetic particles can be about 0.01 ⁇ to about 100 ⁇ in size.
magnetic particles can be about 0.1 ⁇ to about 50 ⁇ in size.
magnetic particles can be about 0.5 ⁇ to about 10 ⁇ in size.
magnetic particles can be about 1 ⁇ to about 5 ⁇ 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.
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
the magnetic particles can include any ferromagnetic material, paramagnetic material, superparamagnetic material or any combinations thereof.
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.
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.
ferromagnetic (including ferrimagnetic) materials include, but are not limited to, iron, nickel and cobalt, Magnetite (Fe 3 0 4 ), maghemite (yFe 2 0 3 ), jacobsite (MnFe 2 0 4 ), trevorite (NiFe 2 0 4 ), magnesioferrite (MgFe 2 0 4 ), pyrrhotite (Fe 7 Ss), greigite (Fe 3 S 4 ), feroxyhyte (5FeOOH), awaruite (Ni 3 Fe), wairauite (CoFe), and any combinations thereof.
the magnetic particles embedded in a silk fibroin-based material can include ferromagnetic particles, e.g., ferrous particles such as iron particles.
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.
a silk fibroin-based material comprising carbonyl iron as magnetic particles can be used in vivo, e.g., for tissue engineering applications.
magnetic particles embedded in a silk fibroin-based material can include a paramagnetic material.
paramagnetic refers to materials having a small and positive susceptibility to magnetic fields, which are slightly attracted by a magnetic field.
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.
magnetic particles embedded in a silk fibroin-based material can include a superparamagnetic material.
superparamagnetic 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.
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.
the magnetic particles can be coated with a biocompatible polymer.
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.
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).
an agent e.g., an active agent and/or silk fibroin protein.
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, SO 2 , SO 2 NH, 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), S0 2 , NH, C(O).
Attorney Docket No. 700355-070131-PCT a unit such as NH
the organic moiety or functional group can be a branched moiety or functional group, which contains a branchpoint available for multiple valencies.
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.
the branchpoint is glycerol or derivative thereof.
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).
magnetic particles that can be used herein can be any DYNABEADS® magnetic particles (Invitrogen Inc.), depending on the substrate surface chemistry.
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.
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.
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.
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.
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.
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).
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.
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).
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.
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.
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.
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).
AMF alternating magnetic field
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.
the AMF can enhance the diffusion of water (and any active agent, if any) out of the gel.
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.
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.
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.
At least a portion of the magneto-responsive silk fibroin-based material 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 ⁇ , at least about 0.005 ⁇ , at least about 0.01 ⁇ , at least about 0.05 ⁇ , at least about 0.1 ⁇ , at least about 0.5 ⁇ , at least about 1 ⁇ , 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 750 ⁇ , at least about 1000 ⁇ , at least about 2 mm, at least about 3 mm, or more, in a direction toward or away from the external magnetic field source.
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.
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.
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.
deformation e.g., shape and/or volume change
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.
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.
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.
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.
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.
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.
a permanent magnet of any shape e.g., but not limited to, cylinders, spheres, squares
a permanent magnet of any shape 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).
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.
an electromagnet of any shape 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.
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.
magnetic field gradient refers to a variation in the magnetic field with respect to position.
a one-dimensional magnetic field gradient is a variation in the magnetic field with respect to one direction
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.
size e.g., volume
shape e.g., temperature
crystallinity of silk fibroin e.g., 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.
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.
the silk fibroin-based material e.g., but not limited to, a hydrogel, or a silk fiber
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.
a vibrational source e.g., ultrasound
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.
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).
the silk fibroin-based material comprising magnetic particles is a fiber or a network of fibers.
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.
the network of the silk fibroin fibers can be formed from a plurality of individual silk fibroin fibers.
the network of silk fibroin fibers can be formed from a single silk fibroin fiber wrapping and/or folding a plurality of times.
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.
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).
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.
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.
the silk fibroin fiber(s) are magneto-responsive.
a silk fibroin fiber or silk fibroin fibers can comprise sericin.
a silk fibroin fiber or silk fibroin fibers can exclude sericin.
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.
the silk fibroin fiber(s) can be substantially formed from silk fibroin.
the silk fibroin fiber(s) can be substantially formed from silk fibroin comprising at least one additive (e.g., an active agent).
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.
the silk fibroin fiber(s) can have a cross-section of any shape.
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.
the silk fibroin fiber(s) can have a substantially circular cross-section.
the cross-section of the silk fibroin fiber(s) can have an average size of any dimension.
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.
the silk fibroin fiber(s) can have an average cross-sectional dimension (e.g., diameter) of about 0.1 ⁇ to about 1 mm, about 0.5 ⁇ to about 500 ⁇ , about 0.75 ⁇ to about 250 ⁇ , about 1 ⁇ to about 100 ⁇ , about 5 ⁇ to about 50 ⁇ . 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.
average size and average cross-sectional dimension encompass a fiber having a constant or varying size or cross-sectional dimension across its length.
average size and average cross-sectional dimension can refer to the size or cross-sectional dimension averaged from numerous fibers of different sizes or cross-sectional dimensions.
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).
the silk fibroin/ PEO blend ratio in a silk fibroin fiber can vary from about 1 : 100 to about 100: 1.
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.
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.
the magnetic particles e.g., iron oxide particles
the magnetic 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.
the magneto-responsive silk fibroin-based material can be in the form of a film, e.g., a silk film.
a 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.
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
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.
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.
a silk particle e.g., a silk nanosphere or a silk microsphere.
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.
the term “microparticle” refers to a particle having a particle size of about 1 ⁇ to about 1000 ⁇ .
the term “nanoparticle” refers to particle having a particle size of about 0.1 nm to about 1000 nm.
the magneto-responsive silk fibroin based material can be in the form of a gel or hydrogel.
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.
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.
a magnetic fluid e.g., a ferro fluid
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.
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.
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.
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.
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.
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.
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.
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 ⁇ in size
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
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.
a composition e.g., a mat or a mesh
the silk fibroin fiber(s) can be arranged in a predetermined pattern by manipulation of the applied magnetic field.
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.
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%.
the silk fibroin solution can have a concentration of about 30 wt%.
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.
the solution can then be concentrated using, for example, dialysis against a hygroscopic polymer solution, for example, PEG, a polyethylene oxide, amylose or sericin.
a hygroscopic polymer solution for example, PEG, a polyethylene oxide, amylose or sericin.
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.
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%.
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.
the silk fibroin solution can be produced using organic solvents.
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,
an exemplary organic solvent that can be used to produce a silk solution includes, but is not limited to, hexafluoroisopropanol.
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.
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.
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.
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.
the magnetic particles can be present in a silk fibroin
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.
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).
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.
the magnetic particles can have a diameter of about 1 nm to about 1 mm.
magnetic particles can have a diameter of about 50 nm to about 250 ⁇ .
magnetic particles can Attorney Docket No. 700355-070131-PCT have a diameter of about 0.01 ⁇ to about 100 ⁇ .
magnetic particles can have a diameter of about 0.1 ⁇ to about 0 ⁇ . In some embodiments, magnetic particles can have a diameter of about 0.5 ⁇ to about 10 ⁇ . In some embodiments, magnetic particles can have a diameter of about 1 ⁇ to about 5 ⁇ . In some embodiments, magnetic particles can have a diameter of about 1 nm to about 10 ⁇ , or about 5 nm to about 5 ⁇ . 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.
any magnetic material can be used for the magnetic particles, including ferromagnetic material, ferrimagnetic material, paramagnetic material or any combinations thereof.
the magnetic particles can include ferromagnetic particles, e.g., ferrous particles such as iron particles.
the iron particles can include carbonyl iron particles described herein.
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.
the magnetic particles can be added into a silk fibroin solution as individual solid particles or powder.
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.
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.
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.
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.
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.
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.
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.
the active agent can be dispersed homogeneously or
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.
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.
at least one active agent described here can diffuse into the magneto- responsive silk fibroin-based material upon the contacting.
the magneto-responsive silk fibroin-based material can 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.
porosity 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.
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.
the pores of a magneto-responsive silk fibroin- based material can have a size distribution ranging from about 1 nm to about 100 ⁇ , from about 10 nm to about 50 ⁇ , from about 50 nm to about 25 ⁇ , from about 100 nm to about 20 ⁇ , from about 500 nm to about 10 ⁇ , or from about 1 ⁇ to about 5 ⁇ .
the term "pore size" refers to a diameter or an effective diameter of the cross-sections of the pores.
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.
post-treatment of the silk fibroin-based material can be employed.
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).
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));
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.
the silk in the magneto-responsive silk fibroin-based material is present completely in a silk II beta-sheet conformation.
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.
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.
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).
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
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).
short bursts of heat on very limited areas can be used to maximize permeability with minimal harmful effects on surrounding tissues.
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.
the silk fibroin matrices can include photothermal agents such as gold nanoparticles.
composition e.g., mat or mesh
a magneto- responsive silk fibroin fiber comprising a magneto- responsive silk fibroin fiber
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.
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.
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).
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.
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.
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.
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).
PEO poly(ethylene oxide)
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).
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.
the apparatus or setup needed to carry out the electrospinning includes a delivery point, an electric field, and a target solid substrate.
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.
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
the delivery point 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.
the delivery point (e.g., the tip of a needle) and target solid substrate can be conductive in order to create an electric field.
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.
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.
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 ⁇ / ⁇ to about 1000 ⁇ / ⁇ , about 0.05 ⁇ / ⁇ to about 750 ⁇ / ⁇ , about 0.1 ⁇ / ⁇ to about 500 ⁇ 7 ⁇ , about 0.5 ⁇ / ⁇ to about 250 ⁇ / ⁇ , about 1 ⁇ ⁇ to about 100 ⁇ 7 ⁇ , or about 2 ⁇ to about 75 ⁇ / ⁇ .
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 ⁇ to about 10 ⁇ ⁇ , or about 1 ⁇ 7 ⁇ to about 7 ⁇ / ⁇ .
the flow rate of the silk fibroin solution can be higher than 1000 ⁇ / ⁇ or lower than 0.01 ⁇ / ⁇ , 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.
the delivery point e.g., the tip of a needle
the delivery point can have an orifice capable of extruding a controlled amount of a silk fibroin solution to form a silk fibroin fiber.
the delivery point e.g., the tip of a needle
the delivery point 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.
a syringe with a needle is used to extrude a controlled amount of silk fibroin solution to form a silk fibroin fiber.
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.
the syringe needle can have an orifice of about gauge 18 to about gauge 16.
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.
the delivery means e.g., the needle
the target solid substrate can be grounded; and when the target solid substrate is electrically charged, the delivery means can be grounded.
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.
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.
the voltage applied to the delivery point including the delivery means is at least about 25 kV, while keeping the target solid substrate grounded.
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).
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
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.
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
the electrospinning rate 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.
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.
a conductivity enhancer agent e.g., electrolyte such as ions
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., CaCl 2 ), (Bu) 4 NCl, 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.
a sodium salt e.g., NaCl and NaBr
a base e.
the silk fibroin solution prepared in aqueous solution can also be prepared in organic solvents.
a salt that is soluble in an organic solvent can be used, e.g., a tetraalkylammonium triflate salt such as (Bu) 4 N(CF3SC>3) (tetrabutylammomum trifluoromethanesulphonate (triflate) or TBATFL due to its high solubility in organic solvents.
a sodium salt e.g., NaCl and NaBr
a base e.g., NaOH
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.
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.
the silk fibroin solution can contain a conductivity enhancer agent at a concentration of about 40 mM.
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
NiFe 2 0 4 See, e.g., Santala, 2009, Id.; Li, 2003], CoFe 2 0 4 (See, e.g., Santala, 2009, Id.), Fe 3 0 4 (See, e.g., Wang, 2009, Id.; Chung, 2007 et al, Id.; Yang, 2007, Id.), Fe/Co/Ni(No 3 ) 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.
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.
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.
the arrangement of the formed silk fibroin fiber can include aligning the formed silk fibroin fiber in a direction of the magnetic field pattern.
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.
a magnetic field can be generated on a target solid substrate by any methods known to one of ordinary skill in the art.
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.
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.
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.
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.
a magnetic field source can include one or more permanent magnets, one or more electromagnets (including electrically-polarizable elements), or a combination thereof.
a magnetic field can be generated on a target solid substrate by one permanent magnet, e.g., as shown in Figs. 5A-5B.
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).
a certain pattern including a 2-D or a 3-D pattern
about 80 to about 85 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.
the magnets can be joined such that the north pole of one magnet met the south pole of another magnet.
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.
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.
the spherical magnets can be arranged into a 3-D cylindrical pattern.
the spherical magnets can be arranged into two 3-D cylinders spaced apart by a distance.
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).
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.
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.
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.
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.
at least 2 permanent magnets of such dimension can be spaced apart on a target solid substrate to create a magnetic field pattern.
electromagnets can be an alternative to permanent magnets.
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.
an electromagnet of any shape 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.
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.
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).
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.
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.
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.
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).
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.
warp and weft courses can be generated with, e.g., zero and 90 degrees electromagnet coordination.
electromagnets can provide non-orthogonal directional control.
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.
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.
the magnetic field pattern can determine the arrangement of silk fibroin fibers or wrapping and/or folding of a silk fibroin fiber deposited on the target solid substrate.
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.
the strength of a magnetic field used during electrospinning can range from 0.01 Tesla to about 10 Tesla.
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)).
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.
a surface covering or protection sheet e.g., an aluminum foil
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.
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).
a metal disc e.g., an aluminum disc or a steel rod (e.g., a stationary or rotating steel rod).
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.
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.
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).
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
the diameter of the target solid substrate 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.
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.
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.
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.
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.
the electrospinning time can range from minutes, hours to days.
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.
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.
compositions comprising a magneto-responsive silk fibroin- based material
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.
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.
control of mechanical properties (e.g., loading conditions) transmitted through a silk fibroin network can help direct cell-driven tissue properties.
the magnetic responsiveness property of the silk fibroin-based materials can be used in applications where external manipulation via a magnetic field is desirable.
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.
the composition comprising the network of the silk fibroin fiber(s) described herein can be adapted for use as a separation membrane or filter.
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.
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.
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.
a drug delivery device e.g., an implantable microchip or scaffold, or an injectable drug depot
wound dressing e.g., a bandage or an adhesive
a magneto-responsive silk fibroin-based material e.g., a hydrogel and/or a network of the silk fibroin fiber(s) described herein
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.
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.
the drug delivery device can be implanted in vivo.
applying an energy source e.g., not necessarily limited to a magnetic field, e.g., ultrasound, radio frequency, heat, and/or electric fields
a magneto-responsive silk fibroin-based material e.g., a hydrogel
a desired a release profile if the silk fibroin-based material comprises an active agent, e.g., a pulsatile drug active agent release profile.
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
Alternating magnetic field can expand and contract a magneto- responsive silk fibroin-based material (e.g., a hydrogel).
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).
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.
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).
a magneto-responsive silk fibroin-based material e.g., a hydrogel
radio frequency 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.
electrogelated gel 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.
heat can be, additionally or alternatively, used to increase the degradation of a magneto-responsive silk fibroin-based material.
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
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.
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).
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
the network of the silk fibroin fiber(s) or any other silk fibroin-based material described herein 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).
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
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
one or a plurality of magneto-responsive silk fibroin-based materials 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.
a matrix material e.g., to form a composite or multilayered material
Such tunable reinforcement materials can be used in wound dressings, tissue engineering scaffolds and/or medical implants.
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
a piezomagnetic transducer e.g., 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.
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).
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.
a magneto-responsive silk fibroin-based material e.g., the network of the silk fibroin fiber(s), gels, scaffolds, and/or films
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).
the magneto-responsive silk fibroin-based materials e.g., the network of the silk fibroin fiber(s), gels, scaffolds, and/or films
a magneto-responsive silk fibroin-based material e.g., gels, scaffolds, and/or films
a target site comprising diseased cells, e.g., tumor or cancer cells
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.
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.
in situ mechanical deformation of a magneto-responsive silk fibroin-based material can promote growth and development.
placing cells of interest in a magneto-responsive silk fibroin-based material e.g., a scaffold
actuating it using one of the methods described herein e.g., magnetic field, AMF, ultrasound, RF, heat, electric fields, and any combinations thereof
one of the methods described herein e.g., magnetic field, AMF, ultrasound, RF, heat, electric fields, and any combinations thereof
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.
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.
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.
tissues e.g., connective tissues such as ligaments and muscles
localized tissue therapy e.g., muscle therapy
the magneto-responsive silk fibroin-based materials 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 spectacular 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).
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.
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
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.
the active agent can also be an organism such as a fungus, plant, animal, bacterium, or a virus (including bacteriophage).
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.
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.
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.
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.
modified amino acids e.g., phosphorylated, glycated, etc.
amino acid analogs regardless of its size or function.
peptide refers to peptides, polypeptides, proteins and fragments of proteins, unless otherwise noted.
protein and “peptide” are used interchangeably herein when referring to a gene product and fragments thereof.
exemplary peptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.
nucleic acids 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.
DNA deoxyribonucleic acid
RNA ribonucleic acid
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)).
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.
nucleic acid also encompasses modified RNA Attorney Docket No. 700355-070131-PCT
nucleic acid also encompasses siRNA, shR A or any combinations thereof.
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-.
siRNA short interfering RNA
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.
siRNA refers to small inhibitory RNA duplexes that induce the RNA interference (RNAi) pathway.
siRNA includes duplexes of two separate strands, as well as single strands that can form hairpin structures comprising a duplex region.
RNAi 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.
enzymes 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 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.
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.
antibody 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.
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.
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.
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.
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, orego
CDRs Complementarity Determining Regions
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.
a complementarity determining region can include amino acids from both a CDR region defined according to Kabat and a hypervariable loop.
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
Linear antibodies can be bispecific or monospecific.
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.
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.
diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH)
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.
organic or inorganic compounds i.e., including heteroorganic and organometallic compounds
an antibiotic is further intended to include an antimicrobial, bacteriostatic, or bactericidal agent.
antibiotics can include, but are not limited to, actinomycin; aminoglycosides (e.g., neomycin, gentamicin, tobramycin); ⁇ -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.,
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;
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.
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.
TCR T cell receptor
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.
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.
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.
DNA deoxyribonucleic acid
RNA ribonucleic acid
modified DNA or RNA or mixtures or combinations thereof, including, for example, DNA nanoplexes.
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.
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.
a silk- based composition can contain combinations of two or more therapeutic agents.
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.
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.
a composition comprising a silk fibroin-based material embedded with a plurality of magnetic particles.
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.
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.
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.
composition of paragraph 7 wherein the ferrous particles comprise iron particles.
composition of paragraph 9, wherein the silk fibroin fiber has a diameter of about 0.1 ⁇ ⁇ about 1 mm.
composition of any of paragraphs 9-10, wherein the silk fibroin fiber has a diameter of about 0.5 ⁇ to about 500 ⁇ .
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 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.
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.
composition of 14 wherein the external energy source comprises a magnetic field.
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.
PEO polyethylene oxide
PEG polyethylene glycol
collagen fibronectin
keratin polyaspartic acid
polylysine alginate
chitosan chitin
hyaluronic acid pectin
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;
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%.
ferrous particles comprise iron particles.
step (a) further comprises an agent to increase conductivity of the silk fibroin solution.
step (a) further comprises a biopolymer.
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,
PEO polyethylene oxide
PEG polyethylene glycol
collagen fibronectin
keratin polyaspartic acid
polylysine 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.
a strength of the electric field is at least about 15 kV.
telomeres 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.
composition comprising a silk fibroin fiber produced by the methods of any of
compositions, methods, and respective component(s) thereof are 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.
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.
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.
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
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.
the silk fibroin-based material can be substantially formed from silk fibroin.
the silk fibroin-based material can be substantially formed from silk fibroin and at least one active agent.
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.
Example 1 Exemplary materials and methods used for generating a composition comprising a magneto-responsive silk fiber Attorney Docket No. 700355-070131-PCT
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.
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 ⁇ IN NaOH was added to 2-2.5 ml concentrated silk solution. Approximately 10 vol% carbonyl iron particles (1-3 ⁇ diameter spheres, Alfa Aesar, MA, USA) were mixed into the solution by manual stirring.
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.
a 10 cm diameter aluminum disk or small diameter steel rod can be placed on Attorney Docket No. 700355-070131-PCT the scissor lift.
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:
Ball magnets - diameter 5 mm
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.
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.
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.
magnetic fields were created using electromagnets.
FIG 2 shows an exemplary hardware for such purpose.
a computer e.g., a laptop
a graphical development environment e.g., National Instruments' LabVIEW software
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.
the electromagnets e.g., Magnetic Sensor Systems E-66-38-37
the perforated steel plate can allow for rapid re-arrangement of the electromagnets.
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.
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
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.
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.
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.
FIGS 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
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.
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.
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.
lower electrical fields could cause the solution to form droplets and no continuous fibrous mat was formed.
the needle-target collector distance of 20 cm could give the best results with respect to fiber formation and mesh continuity.
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.
the magneto-sensitive silk fibroin fibers tended to anchor themselves around the periphery of the square magnet.
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.
FIGS 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.
axially magnetized Neodymium spheres (0.75 cm diameter) were used to create the flat hexagon pattern as shown in Figure 7B.
Figure 7A a silk fibroin fiber tower was formed, with the anchoring locations at the innermost coil of spherical magnets.
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.
the outer magnets can attract the silk fibroin fibers as well.
the fiber 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.
the silk fibroin fiber appears to be aligned locally with magnetic field generated by the pattern of the axially magnetized spheres.
Figure 8B 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.
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.
FIG. 9A 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
Permanent magnets generally (a) have a fixed magnetization level; and (b) are either stationary or need to be physically moved.
electromagnets were used in the control of electrospun magnetized silk fibroin.
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.
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.
the electromagnets can be individually turned on or off at different times.
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.
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.
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).
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.
electromagnets can offer a wider range of control options than permanent magnets.
An array of electromagnets of any sizes e.g., small electromagnets
an array of electromagnets can allow the controlled lay-up of a mesh with an alternating alignment in one direction or another.
warp and weft courses can be generated with zero and 90 degree electromagnet coordination.
Electromagnets can provide non- orthogonal directional control.
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.
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
a magnetic silk solution is prepared.
An exemplary protocol of preparing a silk fibroin solution from cocoons is described as follows:
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.
the resulting aqueous silk solution has a concentration between 7% and 9% silk.
Magnetic particles or a ferrofluid are then added to a silk fibroin solution to form a magnetic silk fibroin solution.
a water-based ferrofluid e.g., a ferrofluid from EMG series, Ferrotec (USA) Corporation, Bedford, NH, USA
EMG series EMG series
Ferrotec (USA) Corporation Bedford, NH, USA
aqueous silk fibroin solution can be added to the aqueous silk fibroin solution as described above.
the ferrofluid (e.g., EMG series obtained from Ferrotec) generally contains nano- particles (e.g., approximately lOnm in diameter) of iron oxides (Fe 2 0 3 and Fe 3 C>4).
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).
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.
the ferrofluid particles can be coated with a surfactant coating.
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.
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.
the magnetic silk fibroin solution can respond to an applied magnetic field, e.g., produced by both temporary and/or permanent magnets.
the magnetic silk fibroin solution can exhibit temporary magnetism an applied field.
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.
hydrogels e.g., self-assembled, electrogelated, vortex or shear stress-induced, pH-induced, particle-induced gels
foams e.g., self-assembled, electrogelated, vortex or shear stress-induced, pH-induced, particle-induced gels
foams e.g., foams, scaffolds, films, tubes, particles, electrospun geometries, and any combinations thereof.
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.
the magnetic silk fibroin solution can be used to form a hydrogel.
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).
the silk molecules cross- link, using the iron particles as an initiator or catalyst, resulting in the formation of a hydrogel.
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).
the degumming time of the silk fibroin e.g., lower degumming time results in shorter gelation time
the size of iron particles used e.g., the size and magnetization of the ferrofluid or magnetic particles used (e.g., higher concentration and/or magnetization results in shorter gelation time)
the ratio of ferrofluid/magnetic particles to silk solution e.g., higher ratio of ferrofluid/
electrogelation can be applied to a magnetic silk fibroin solution to form a magneto-responsive electrogelated silk fibroin gel.
a potential difference e.g., about 25V
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.
a regular silk fibroin solution without magnetic particles or a ferrofluid.
Figure 11 A shows a magnetic silk hydrogel being fabricated using an
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.

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