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WO2025193717A1 - Assemblage de biopolymères provoqué par le son - Google Patents

Assemblage de biopolymères provoqué par le son

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Publication number
WO2025193717A1
WO2025193717A1 PCT/US2025/019398 US2025019398W WO2025193717A1 WO 2025193717 A1 WO2025193717 A1 WO 2025193717A1 US 2025019398 W US2025019398 W US 2025019398W WO 2025193717 A1 WO2025193717 A1 WO 2025193717A1
Authority
WO
WIPO (PCT)
Prior art keywords
silk fibroin
silk
predetermined acoustic
article
acoustic pattern
Prior art date
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.)
Pending
Application number
PCT/US2025/019398
Other languages
English (en)
Inventor
Fiorenzo G. Omenetto
Connor Mikael MERINDER
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.)
Filing date
Publication date
Application filed by Tufts University filed Critical Tufts University
Publication of WO2025193717A1 publication Critical patent/WO2025193717A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J5/00—Manufacture of articles or shaped materials containing macromolecular substances
    • C08J5/18—Manufacture of films or sheets
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
    • C08H1/00—Macromolecular products derived from proteins
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
    • C08J7/04—Coating
    • C08J7/0427—Coating with only one layer of a composition containing a polymer binder
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L89/00—Compositions of proteins; Compositions of derivatives thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2389/00—Characterised by the use of proteins; Derivatives thereof

Definitions

  • the solution nearest to the outer boundary which has a greater surf ace-to- volume ratio, evaporates more quickly than the rest.
  • This uneven evaporation rate results in a higher concentration at the periphery and a lower concentration at the center, often referred to as the coffee ring effect.
  • Fig. 1 depicts a graphic representation of the effects of echoes on the self-assembly of silk fibroin solution.
  • Fig. 2A depicts a graphic representation of the side view of acoustic fabrication strategy to form films.
  • Fig. 2B depicts a graphic representation of the top view of acoustic fabrication strategy to form films.
  • Fig. 3A depicts a bottom-mounted piezoelectric element and reflector inducing bulk acoustic waves in a Petri dish.
  • Fig. 3B depicts a bottom mounted frequency generator inducing a standing wave in an opentop Petri dish.
  • Fig. 4A depicts a scanning electron micrograph of a control silk fibroin film cross section. Scale bar is 50 pm.
  • Fig. 4B depicts a scanning electron micrograph of a control silk fibroin film cross section. Scale bar is 4 pm.
  • Fig. 5A depicts bright field reflection (BFR) and cross-polarized (BFT-XP) images with a graphic representation of the acoustic field pattern (mode) for 2.0 kHz at various acoustic powers. Scale bar is 3 cm.
  • Fig. 5B depicts BFR and BFT-XP images with a graphic representation of the mode for 1.5 kHz at various acoustic powers. Scale bar is 3 cm.
  • Fig. 5C depicts BFR and BFT-XP images with a graphic representation of the mode for 1.0 kHz at various acoustic powers. Scale bar is 3 cm.
  • Fig. 6A depicts BFT-XP and spatially-resolved FTIR spectra (P-sheet map) of films fabricated at 2.0 kHz at various acoustic powers. Red to white gradient represents P-sheet portion of secondary structure. The red box indicates the area of the film that was measured. Scale bar 1 cm.
  • Fig. 6B depicts BFT-XP and spatially-resolved FTIR spectra (P-sheet map) of films fabricated at 1.5 kHz at various acoustic powers. Red to white gradient represents P-sheet portion of secondary structure. The red box indicates the area of the film that was measured. Scale bar 1 cm. [0019] Fig.
  • FIG. 6C depicts BFT-XP and spatially-resolved FTIR spectra (P-sheet map) of films fabricated at 1.0 kHz at various acoustic powers. Red to white gradient represents P-sheet portion of secondary structure. The red box indicates the area of the film that was measured. Scale bar 1 cm.
  • Fig. 7A depicts a cross-section SEM image of silk film dried under 2.0 kHz at 20 W power on a non-birefringent area. Scale bar is 50 pm.
  • Fig. 7B depicts a cross-section SEM image of silk film dried under 2.0 kHz frequency at 20 W power on a birefringent area. Scale bar is 50 pm.
  • Fig. 7C depicts a cross-section SEM image of silk film dried under 2.0 kHz frequency at 20 W power on a non-birefringent area. Scale bar is 4 pm.
  • Fig. 7D depicts a cross-section SEM image of silk film dried under 2.0 kHz frequency at 20 W power on a birefringent area. Scale bar is 4 pm.
  • Fig. 7E depicts a cross-section SEM image of silk film dried under 2.0 kHz frequency at 20 W power on a birefringent area. Scale bar is 4 pm.
  • Fig. 8A depicts BFR and BFT-XP images of fdms modified by 2.0 kHz frequency at 6.0% w/w at various acoustic powers. Scale bar is 1 cm.
  • Fig. 8B depicts BFR and BFT-XP images of films modified by 2.0 kHz frequency at 9.0% w/w at various acoustic powers. Scale bar is 1 cm.
  • Fig. 8C depicts BFR and BFT-XP images of films modified by 2.0 kHz frequency at 11.0% w/w at various acoustic powers. Scale bar is 1 cm.
  • Fig. 9A depicts BFT-XP and spatially-resolved FTIR spectra of 6.0% w/w films modified by 2.0 kHz frequency at various acoustic powers. Red to white gradient represents P-sheet portion of secondary structure. The red box indicates the area of the film that was measured. Scale bar 1 cm.
  • Fig. 9B depicts BFT-XP and spatially-resolved FTIR spectra of 9.0% w/w films modified by 2.0 kHz frequency at various acoustic powers. Red to white gradient represents P-sheet portion of secondary structure. The red box indicates the area of the film that was measured. Scale bar 1 cm.
  • Fig. 9C depicts BFT-XP and spatially-resolved FTIR spectra of 11.0% w/w films modified by 2.0 kHz frequency at various acoustic powers. Red to white gradient represents P-sheet portion of secondary structure. The red box indicates the area of the film that was measured. Scale bar 1 cm.
  • the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and/or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” are used as equivalents and may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.
  • composition as used herein, may be used to refer to a discrete physical entity that comprises one or more specified components.
  • a composition may be of any form - e.g., gas, gel, liquid, solid, etc.
  • composition may refer to a combination of two or more entities for use in a single embodiment or as part of the same article.
  • the combination of entities result in physical admixture, that is, combination as separate co-entities of each of the components of the composition is possible; however many practitioners in the field may find it advantageous to prepare a composition that is an admixture of two or more of the ingredients in a pharmaceutically acceptable carrier, diluent, or excipient, making it possible to administer the component ingredients of the combination at the same time.
  • the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
  • the term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
  • A Acoustic manipulation
  • Directed self-assembly of structural proteins provides the opportunity to fabricate materials with spatially controlled organization and functional structures.
  • the controlled dimensions and arrangement of these structures can be designed to interact with environmental stimuli, such as mechanical forces, to elicit a programmed response - providing additional utility for advanced material applications.
  • Ultrasound imaging is a widely available, non-invasive, and fast method of imaging inside the body.
  • Image contrast is generated from echoes based on changes in tissue density and elasticity.
  • it is limited spatial resolution when imaging beyond a few centimeters, making it unable to image density-homogenous structures, such as vasculature.
  • Described herein is a method of imparting organization and structure into silk- fibroin based materials using sound, and in turn, investigate how the sound-mediated organization impacts the material echogenicity using ultrasound imaging.
  • this approach allows us to tune the organization of material properties, such as compressibility, to optimize the sound-structure interactions leading to structural echogenicity.
  • Sound as mechanical energy, is a series of periodic compressions moving through a medium, causing particles to bunch together and space apart as they oscillate.
  • the standing wave takes the shape of modal patterns that are based on the vibration frequency and geometry of the container.
  • Acoustic manipulation operates as a non-contact method, with the ability to move, soft, and pattern microscale objects over one, two, and three dimensions.
  • Acoustic transducers typically piezoelectric devices, are used to generate sound waves at specific frequencies.
  • the frequency generator is attached to the bottom of a fluid-filled container.
  • the sound waves travel through the fluid and reflect off the boundaries of the container, creating pressure gradients that act on the suspended particles.
  • the distribution of pressure gradients can be designed through superposition, where the displacements of the incoming waves and reflected waves combine, forming an interference pattern.
  • a stationary pattern with regions of constructive interference, known as antinodes, and regions of destructive interference, known as nodes, is formed.
  • One of the simplest methods to do this is to use an acoustic frequency that matches the natural frequency of a system, known as resonance, causing the amplitude of the standing wave to increase dramatically.
  • Different frequencies can cause different mode patterns to emerge, creating complex distribution of nodes and antinodes.
  • Lower frequencies typically establish a pattern with one node at the center (representing a single standing wave), while higher frequencies result in more intricate patterns with multiple nodes and antinodes, representing multiple waves existing within the same area.
  • the mechanical forces associated with the sound field act on objects suspended in the fluid through variations in pressure. These forces tend to move particles towards nodes or antinodes of the wave, depending on the properties of the particles and the fluid.
  • the main factors governing this interaction are the interface of the liquid (compressibility, viscosity, density) to the particles (size, compressibility, density, rigidity, morphology, impedance, wettability), as well as the applied frequency, amplitude, and duration.
  • Fig. 1 By locking the state of the medium in a Petri dish using a standing wave, the silk fibroin proteins are forced to assemble in higher and lower density environments as shown in Fig. 1 .
  • FIG. 2A and Fig. 2B An overview of the process is shown in Fig. 2A and Fig. 2B. Briefly, a silk fibroin solution is dropcast in a Petri dish. Standing waves are produced under the Petri dish until a dry film is formed. Schematics of the two main setups used for acoustic manipulation using standing wave fields are shown in Fig 3A and Fig. 3B.
  • shear stress may induce structural changes leading to the formation of P-sheet-rich and stable fibril networks through shear-induced ordering.
  • shear forces induce -sheet formation through the physical disruption of the micellular structures, altering the hydrophobic state of the protein chains.
  • these mechanical forces accelerate the rate of intermolecular hydrogen bonding between proteins by allowing for more protein-protein interactions.
  • sonication including local temperature increases, mechanical/shear forces, and increased airliquid interfaces, affect the self-assembly process. In the case of hierarchal assembly and anisotropic structures, sonication alone is rather constrained, as it affects the solution homogeneously and offers little spatial control, limiting the construction of highly ordered materials.
  • a method of acoustically-mediated assembly of silk fibroin materials includes applying an acoustic pattern to a silk fibroin solution and solidifying the silk fibroin solution in steps a) and b).
  • Step a) entails applying a predetermined acoustic pattern to a silk fibroin solution.
  • Step b) entails solidifying the silk fibroin solution into a silk fibroin article while maintaining application of the predetermined acoustic pattern.
  • the silk fibroin article has a pattern of protein alignment that mimics the predetermined acoustic pattern.
  • the predetermined acoustic pattern may have nodes and antinodes.
  • the predetermined acoustic pattern may have an acoustic intensity below a solution disruption threshold, above which the predetermined acoustic pattern disrupts a surface of the silk fibroin solution.
  • the predetermined acoustic pattern may be a standing wave.
  • the predetermined acoustic pattern may be applied at a frequency between 10 Hz and 100 kHz. In some cases, the frequency can be between 500 Hz and 10 kHz. In some cases, the frequency can be at least 10 Hz, at least 100 Hz, at least 1 kHz, or at least 10 kHz. In some cases, the frequency can be at most 100 kHz, at most 50 kHz, at most 5 kHz, at most 500 Hz, or at most 50 Hz.
  • the temperature during the solidifying of step b) may be maintained between 0 °C and 80 °C.
  • the temperature can be at least 0 °C, at least 10 °C, at least 20 °C, at least 30 °C, at least 40 °C, at least 50 °C, at least 60 °C, or at least 70 °C.
  • the temperature can be at most 80 °C, at most 70 °C, at most 60 °C, at most 50 °C, at most 40 °C, at most 30 °C, or at most 20°C.
  • the silk fibroin solution may include silk fibroin in a weight ratio of between 6% (w/w) and 11% (w/w). In some cases, the solution includes silk fibroin in a weight ratio of at least 6% (w/w), at least 7% (w/w), at least 8% (w/w). In some cases, the solution includes silk fibroin in a weight ratio of at most 11 % (w/w), at most 10% (w/w), or at most 9% (w/w).
  • the silk fibroin solution may be a degassed liquid solution, a non-degassed liquid solution, a gassed liquid solution, a foam, a whipped silk cream, or a combination thereof.
  • the method may further include steps c), d), and e).
  • Step c) may entail depositing a second silk fibroin solution atop the silk fibroin article.
  • Step d) may entail applying a second predetermined acoustic pattern to the second silk fibroin solution.
  • Step e) may entail solidifying the second silk fibroin solution while maintaining application of the predetermined acoustic pattern, thereby forming a layer of an expanded silk fibroin article atop the silk fibroin article.
  • the second silk fibroin solution may have a different silk fibroin concentration than the first silk fibroin solution.
  • the second predetermined acoustic pattern may be different than the first predetermined acoustic pattern. It should be appreciated that these steps may be repeated an additional number of times to produce more complex structures.
  • the silk fibroin solution may be positioned atop a partial silk fibroin article.
  • Solidifying the second silk fibroin solution into the silk fibroin article may include expanding the partial silk fibroin article to form the silk fibroin article.
  • a silk fibroin article is made by the methods described herein.
  • a silk fibroin article has acoustically-mediated features.
  • a silk fibroin article has a birefringence pattern that mimics a predetermined acoustic pattern.
  • these methods may be applicable to objects that have characteristic vibration patterns, such as an engine. It is contemplated that the methods disclosed herein may encompass a method of contacting, coating, and/or enveloping an object, such as an engine, with a silk fibroin solution, such as a silk foam and/or a whipped silk cream, and solidifying the silk fibroin solution to form a solidified article that has within it physical properties associated with the characteristic vibration patterns.
  • a silk fibroin solution such as a silk foam and/or a whipped silk cream
  • silk fibroin refers to silk fibroin protein whether produced by silkworm, spider, or other insect, or otherwise generated (Lucas et al., Adv. Protein Chem., 13: 107-242 (1958)). Any type of silk fibroin can be used in different embodiments 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 film may be 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, and variants thereof, that can be used. See, e.g., WO 97/08315 and U.S. Pat. No. 5,245,012, each of which is incorporated herein by reference in their entireties.
  • spider silk e.g., obtained from Nephila clavipes
  • transgenic silks e.g., obtained from Nephila clavipes
  • genetically engineered silks such as silks from bacteria, yeast, mammalian cells, transgenic animals, or transgenic plants, and variants thereof, that can be used. See, e.g., WO 97/08315 and U.S. Pat. No. 5,245,012, each of which is incorporated herein by reference in their entireties.
  • a variety of functionalizing agents may be used with the silk-containing embodiments described herein (e.g., silk membrane, silk composition, silk articles, silk matrix, silk foam, silk microsphere, liquid composition, whipped silk cream, silk meringue, compressed silk meringue, hot-pressed silk meringue, silk leather, silk powder, silk toner, edible silkbased films, etc.). It should be understood that the examples herein may recite one or a few silkcontaining embodiments but are applicable to any silk-containing embodiment, as applicable.
  • a functionalizing agent may be any compound or molecule that facilitates the attachment to and/or development (e.g., growth) of one or more endothelial cells on a silk membrane.
  • a functionalizing agent may be any compound or molecule that facilitates the attachment and/or development (e.g., growth) of one or more megakaryocytes and/or hematopoietic progenitor cells on a silk matrix and/or silk membrane.
  • a functionalizing agent may be or comprise an agent suitable for facilitating the production of one or more of white blood cells and red blood cells.
  • a functionalizing agent may be or comprise a cell attachment mediator and/or an extracellular matrix protein, for example: collagen (e.g., collagen type I, collagen type III, collagen type IV or collagen type VI), elastin, fibronectin, vitronectin, laminin, fibrinogen, von Willebrand factor, proteoglycans, decorin, perlecan, nidogen, hyaluronan, and/or peptides containing known integrin binding domains e.g. “RGD” integrin binding sequence, or variations thereof, that are known to affect cellular attachment.
  • collagen e.g., collagen type I, collagen type III, collagen type IV or collagen type VI
  • elastin e.g., fibronectin, vitronectin, laminin, fibrinogen, von Willebrand factor, proteoglycans, decorin, perlecan, nidogen, hyaluronan
  • peptides containing known integrin binding domains e
  • a functionalizing agent may be any soluble molecule produced by endothelial cells.
  • Non-limiting examples include fibroblast growth factor- 1 (FGF1) and vascular endothelial growth factors (VEGF).
  • compositions may comprise the use of laminin, fibronectin and/or fibrinogen, and type IV collagen in order to facilitate the attachment and growth of endothelial cells on a silk membrane (e.g., a porous silk membrane) and/or attachment of megakaryocytes to a silk matrix.
  • a silk membrane e.g., a porous silk membrane
  • a functionalizing agent may be embedded or otherwise associated with a silk membrane and/or silk matrix such that at least a portion of the functionalizing agent is surrounded by a silk membrane and/or silk matrix as contrasted to a functionalizing agent simply being positioned along the surface of a silk membrane and/or silk matrix.
  • a functionalizing agent is distributed along and/or incorporated in substantially the entire surface area of a silk membrane/silk wall.
  • a functionalizing agent is distributed and/or incorporated only at one or more discrete portions of a silk membrane/wall and/or silk matrix.
  • a functionalizing agent is distributed in and/or along at least one of the lumenfacing side of a silk wall and the matrix-facing side of a silk wall.
  • any application-appropriate amount of one or more functionalizing agents may be used.
  • the amount of an individual functionalizing agent may be between about 1 pg/ml and 1,000 pg/ml (e.g., between about 2 and 1,000, 5 and 1,000, 10 and 1,000, 10 and 500, 10 and 100 pg/ml).
  • the amount of an individual functionalizing agent may be at least 1 pg/ml (e.g., at least 5, 10, 15, 20 25, 50, 100, 200, 300 400, 500, 600, 700, 800, or 900 pg/ml ).
  • the amount of an individual functionalizing agent is at most 1,000 pg/ml (e.g., 900, 800, 700, 600, 500, 400, 300 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 pg/ml ).
  • the composition comprises one or more sensing agents, such as a sensing dye.
  • the sensing agents/sensing dyes are environmentally sensitive and produce a measurable response to one or more environmental factors.
  • the environmentally- sensitive agent or dye may be present in the composition in an effective amount to alter the composition from a first chemical -physical state to a second chemical -physical state in response to an environmental parameter (e.g., a change in pH, light intensity or exposure, temperature, pressure or strain, voltage, physiological parameter of a subject, and/or concentration of chemical species in the surrounding environment) or an externally applied stimulus (e.g., optical interrogation, acoustic interrogation, and/or applied heat).
  • an environmental parameter e.g., a change in pH, light intensity or exposure, temperature, pressure or strain, voltage, physiological parameter of a subject, and/or concentration of chemical species in the surrounding environment
  • an externally applied stimulus e.g., optical interrogation, acoustic interrogation, and/or applied heat.
  • the sensing dye is present to provide one optical appearance under one given set of environmental conditions and a second, different optical appearance under a different given set of environmental conditions.
  • Suitable concentrations for the sensing agents described herein can be the concentrations for the colorants and additives described elsewhere herein.
  • a person having ordinary skill in the chemical sensing arts can determine a concentration that is appropriate for use in a sensing application of the inks described herein.
  • the first and second chemical-physical state may be a physical property of the composition, such as mechanical property, a chemical property, an acoustical property, an electrical property, a magnetic property, an optical property, a thermal property, a radiological property, or an organoleptic property.
  • Exemplary sensing dyes or agents include, but are not limited to, a pH sensitive agent, a thermal sensitive agent, a pressure or strain sensitive agent, a light sensitive agent, or a potentiometric agent.
  • Exemplary pH sensitive dyes or agents include, but are not limited to, cresol red, methyl violet, crystal violet, ethyl violet, malachite green, methyl green, 2-(p- dimethylaminophenylazo) pyridine, paramethyl red, metanil yellow, 4-phenylazodiphenylamine, thymol blue, metacresol purple, orange IV, 4-o-Tolylazo-o-toluindine, quinaldine red, 2,4- dinitrophenol, erythrosine disodium salt, benzopurpurine 4B, N,N-dimethyl-p-(m-tolylazo) aniline, p- dimethylaminoazobenene, 4,4'-bis(2-amino-l-naphthylazo)-2,2'-stilbenedisulfonic acid, tetrabromophenolphthalein ethyl ester, bromophenol blue, Congo red, methyl orange, ethyl orange, 4-
  • Exemplary light responsive dyes or agents include, but are not limited to, photochromic compounds or agents, such as triarylmethanes, stilbenes, azasilbenes, nitrones, fulgides, spiropyrans, napthopyrans, spiro-oxzines, quinones, derivatives and combinations thereof.
  • photochromic compounds or agents such as triarylmethanes, stilbenes, azasilbenes, nitrones, fulgides, spiropyrans, napthopyrans, spiro-oxzines, quinones, derivatives and combinations thereof.
  • Exemplary potentiometric dyes include, but are not limited to, substituted amiononaphthylehenylpridinium (ANEP) dyes, such as di-4-ANEPPS, di-8-ANEPPS, and N-(4- Sulfobutyl)-4-(6-(4-(Dibutylamino)phenyl)hexatrienyl)Pyridinium (RH237).
  • ANEP substituted amiononaphthylehenylpridinium
  • Exemplary temperature sensitive dyes or agents include, but are not limited to, thermochromic compounds or agents, such as thermochromic liquid crystals, leuco dyes, fluoran dyes, octadecylphosphonic acid.
  • Exemplary pressure or strain sensitive dyes or agents include, but are not limited to, spiropyran compounds and agents.
  • chemi-sensitive dyes or agents include, but are not limited to, antibodies such as immunoglobulin G (IgG) which may change color from blue to red in response to bacterial contamination.
  • IgG immunoglobulin G
  • the compositions comprise one or more additive, dopant, or biologically active agent suitable for a desired intended purpose.
  • the additive or dopant may be present in the composition in an amount effective to impart an optical or organoleptic property to the composition.
  • Exemplary additives or dopants that impart optical or organoleptic properties include, but are not limited to, dyes/pigments, flavorants, aroma compounds, granular or fibrous fillers.
  • the additive, dopant, or biologically active agent may be present in the composition in an amount effective to "functionalize” the composition to impart a desired mechanical property or added functionality to the composition.
  • exemplary additive, dopants, or biologically active agent that impart the desired mechanical property or added functionality include, but are not limited to: environmentally sensitive/sensing dyes; active biomolecules; conductive or metallic particles; micro and nanofibers (e.g., silk nanofibers for reinforcement, carbon nanofibers); nanotubes; inorganic particles (e.g., hydroxyapatite, tricalcium phosphate, bioglasses); drugs (e.g., antibiotics, small molecules or low molecular weight organic compounds); proteins and fragments or complexes thereof (e.g., enzymes, antigens, antibodies and antigen-binding fragments thereof);
  • DNA/RNA e.g., siRNA, miRNA, mRNA
  • cells and fractions thereof viruseses and viral particles; prokaryotic cells such as bacteria; eukaryotic cells such as mammalian cells and plant cells; fungi).
  • the additive or dopant comprises a flavoring agent or flavorant.
  • Exemplary flavorants include ester flavorants, amino acid flavorants, nucleic acid flavorants, organic acid flavorants, and inorganic acid flavorants, such as, but not limited to, diacetyl, acetylpropionyl, acetoin, isoamyl acetate, benzaldehyde, cinnamaldehyde, ethyl propionate, methyl anthranilate, limonene, ethyl decadienoate, allyl hexanoate, ethyl maltol, ethylvanillin, methyl salicylate, manzanate, glutamic acid salts, glycine salts, guanylic acids salts, inosinic acid salts, acetic acid, ascorbic acid, citric acid, fumaric acid, lactic acid, malic acid, phosphoric acid, tartaric acid, derivatives, and mixtures thereof.
  • diacetyl acetylpropion
  • the additive or dopant comprises an aroma compound.
  • aroma compounds include ester aroma compounds, terpene aroma compounds, cyclic terpenes, and aromatic aroma compounds, such as, but not limited to, geranyl acetate, methyl formate, metyl acetate, methyl propionate, methyl butyrate, ethyl acetate, ethyl butyrate, isoamyl acetate, pentyl butrate, pentyl pentanoate, octyl acetate, benzyl acetate, methyl anthranilate, myrecene, geraniol, nerol, citral, cironellal, cironellol, linalool, nerolidol, limonene, camphor, menthol, carone, terpineol, alpha-lonone, thujone, eucalyptol, benzaldehy
  • the additive or dopant comprises a colorant, such as a dye or pigment.
  • the dye or pigment imparts a color or grayscale to the composition.
  • the colorant can be different than the sensing agents and/or sensing dyes below. Any organic and/or inorganic pigments and dyes can be included in the inks.
  • Exemplary pigments suitable for use in the present disclosure include International Color Index or C.I. Pigment Black Numbers 1 , 7, 1 1 and 31 , C.I. Pigment Blue Numbers 15, 15 : 1 , 15 :2, 15 :3, 15 :4, 15 :6, 16, 27, 29, 61 and 62, C.I. Pigment Green Numbers 7, 17, 18 and 36, C.I.
  • carbon black pigment such as Regal 330, Cabot Corporation
  • quinacridone pigments Quinacridone Magenta (228-0122), available from Sun Chemical Corporation, Fort Lee, N.I.
  • diarylide yellow pigment such as AAOT Yellow (274- 1788) available from Sun
  • the classes of dyes suitable for use in present invention can be selected from acid dyes, natural dyes, direct dyes (either cationic or anionic), basic dyes, and reactive dyes.
  • the acid dyes also regarded as anionic dyes, are soluble in water and mainly insoluble in organic solvents and are selected, from yellow acid dyes, orange acid dyes, red acid dyes, violet acid dyes, blue acid dyes, green acid dyes, and black acid dyes.
  • European Patent 0745651 incorporated herein by reference, describes a number of acid dyes that are suitable for use in the present disclosure.
  • Exemplary yellow acid dyes include Acid Yellow 1 International Color Index or C.I. 10316); Acid Yellow 7 (C.I. 56295); Acid Yellow 17 (C.I.
  • Exemplary orange acid dyes include Acid Orange 1 (C.I. 13090/1); Acid Orange 10 (C.I. 16230); Acid Orange 20 (C.I. 14603); Acid Orange 76 (C.I. 18870); Acid Orange 142; Food Orange 2 (C.I. 15980); and Orange B. [0079] Exemplary red acid dyes include Acid Red 1. (C.I. 19140); Acid Yellow 29 (C.I. 18900); Acid Yellow 36 (C.I. 13065); Acid Yellow 42 (C.I. 22910); Acid Yellow 73 (C.I. 45350); Acid Yellow 99 (C.I. 13908); Acid Yellow 194; and Food Yellow 3 (C.I. 15985).
  • Exemplary orange acid dyes include Acid Orange 1 (C.I. 13090/1); Acid Orange 10 (C.I. 16230); Acid Orange 20 (C.I. 14603); Acid Orange 76 (C.I. 18870); Acid Orange 142; Food Orange 2 (C.I. 15980); and Orange B. [00
  • Acid Red 4 C.I. 14710
  • Acid Red 18 C.I. 16255
  • Acid Red 26 C.I. 16150
  • Acid Red 2.7 C.I. as Acid Red 51 (C.I. 45430, available from BASF Corporation, Mt. Olive, N.J.)
  • Acid Red 52 C.I. 45100
  • Acid Red 73 C.I. 27290
  • Acid Red 87 C. I. 45380
  • Acid Red 94 C.I. 45440
  • Acid Red 194 C.I. 14700
  • Exemplary violet acid dyes include Acid Violet 7 (C.I. 18055); and Acid Violet 49 (C.I. 42640).
  • Exemplary blue acid dyes include Acid Blue 1 (C.I.
  • Exemplary green acid dyes include Acid Green 1 (C.I. 10028); Acid Green 3 (C.I. 42085); Acid Green 5 (C.I. 42095); Acid Green 26 (C.I. 44025); and Food Green 3 (C.I. 42053).
  • Exemplary black acid dyes include Acid Black 1 (C.I. 20470); Acid Black 194 (Basantol® X80, available from BASF Corporation, an azo/1 :2 CR-complex.
  • Exemplary reactive dyes for use in the present disclosure include Reactive Yellow 37 (monoazo dye); Reactive Black 31 (disazo dye); Reactive Blue 77 (phthalo cyanine dye) and Reactive Red 180 and Reactive Red 108 dyes. Suitable also are the colorants described in The Printing Ink Manual (5th ed., Leach et al. eds. (2007), pages 289-299. Other organic and inorganic pigments and dyes and combinations thereof can be used to achieve the colors desired.
  • compositions provided herein can contain ETV fluorophores that are excited in the ETV range and emit light at a higher wavelength (typically 400 nm and above).
  • ETV fluorophores include but are not limited to materials from the coumarin, benzoxazole, rhodamine, napthalimide, perylene, benzanthrones, benzoxanthones or benzothia- xanthones families.
  • a UV fluorophore such as an optical brightener for instance
  • the amount of colorant, when present, generally is between 0.05% to 5% or between 0.1% and 1% based on the weight of the composition.
  • the amount of pigment/dye generally is present in an amount of from at or about 0.1 wt% to at or about 20 wt% based on the weight of the composition.
  • a non-white ink can include 15 wt% or less pigment/dye, or 10 wt% or less pigment/dye or 5 wt% pigment/dye, or 1 wt% pigment/dye based on the weight of the composition.
  • a non-white ink can include 1 wt% to 10 wt%, or 5 wt% to 15 wt%, or 10 wt% to 20 wt% pigment/dye based on the weight of the composition.
  • a non-white ink can contain an amount of dye/pigment that is 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15%, 16 wt%, 17 wt%, 18 wt%, 19 wt% or 20 wt% based on the weight of the composition.
  • the amount of white pigment generally is present in an amount of from at or about 1 wt% to at or about 60 wt% based on the weight of the composition. In some applications, greater than 60 wt% white pigment can be present.
  • Preferred white pigments include titanium dioxide (anatase and rutile), zinc oxide, lithopone (calcined coprecipitate of barium sulfate and zinc sulfide), zinc sulfide, blanc fixe and alumina hydrate and combinations thereof, although any of these can be combined with calcium carbonate.
  • a white ink can include 60 wt% or less white pigment, or 55 wt% or less white pigment, or 50 wt% white pigment, or 45 wt% white pigment, or 40 wt% white pigment, or 35 wt% white pigment, or 30 wt% white pigment, or 25 wt% white pigment, or 20 wt% white pigment, or 15 wt% white pigment, or 10 wt% white pigment, based on the weight of the composition.
  • a white ink can include 5 wt% to 60 wt%, or 5 wt% to 55 wt%, or 10 wt% to 50 wt%, or 10 wt% to 25 wt%, or 25 wt% to 50 wt%, or 5 wt% to 15 wt%, or 40 wt% to 60 wt% white pigment based on the weight of the composition.
  • a non- white ink can an amount of dye/pigment that is 5%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15%, 16 wt%, 17 wt%, 18 wt%,
  • the additive or dopant comprises a conductive additive.
  • exemplary conductive additives include, but are not limited to graphite, graphite powder, carbon nanotubes, and metallic particles or nanoparticles, such as gold nanoparticles.
  • the conductive additive is biocompatible and non-toxic.
  • the additive is a biologically active agent.
  • biologically active agent refers to any molecule which exerts at least one biological effect in vivo.
  • the biologically active agent can be a therapeutic agent to treat or prevent a disease state or condition in a subject.
  • Biologically active agents include, without limitation, organic molecules, inorganic materials, proteins, peptides, nucleic acids (e.g., genes, gene fragments, gene regulatory sequences, and antisense molecules), nucleoproteins, polysaccharides, glycoproteins, and lipoproteins.
  • Classes of biologically active compounds that can be incorporated into the composition provided herein include, without limitation, anticancer agents, antibiotics, analgesics, antiinflammatory agents, immunosuppressants, enzyme inhibitors, antihistamines, anti-convulsants, hormones, muscle relaxants, antispasmodics, ophthalmic agents, prostaglandins, anti-depressants, anti-psychotic substances, trophic factors, osteoinductive proteins, growth factors, and vaccines.
  • active agent may also be used herein to refer to a biological sample (e.g., a sample of tissue or fluid, such as for instance blood) or a component thereof, and/or to a biologically active entity or compound, and/or to a structurally or functionally labile entity.
  • Exemplary active agents include, but are not limited to, therapeutic agents, diagnostic agents (e.g., contrast agents), and any combinations thereof.
  • the active agent present in a silk matrix e.g., a silk microsphere), composition, or the like can include a labile active agent, e.g., an agent that can undergo chemical, physical, or biological change, degradation and/or deactivation after exposure to a specified condition, e.g., high temperatures, high humidity, light exposure, and any combinations thereof.
  • a labile active agent e.g., an agent that can undergo chemical, physical, or biological change, degradation and/or deactivation after exposure to a specified condition, e.g., high temperatures, high humidity, light exposure, and any combinations thereof.
  • the active agent present in the silk matrix can include a temperature-sensitive active agent, e.g., an active agent that will lose at least about 30% or more, of its original activity or bioactivity, upon exposure to a temperature of at least about 10° C. or above, including at least about 15° C. or above, at least about room temperature or above, or at least about body temperature (e.g., about 37° C.) or above.
  • a temperature-sensitive active agent e.g., an active agent that will lose at least about 30% or more, of its original activity or bioactivity, upon exposure to a temperature of at least about 10° C. or above, including at least about 15° C. or above, at least about room temperature or above, or at least about body temperature (e.g., about 37° C.) or above.
  • the active agent can be generally present in the silk matrix (e.g., a silk microsphere), composition, or the like in an amount of about 0.01% (w/w) to about 70% (w/w), or about 0.1% (w/w) to about 50% (w/w), or about 1% (w/w) to about 30% (w/w).
  • the active agent can be present on a surface of the silk matrix (e.g., a silk microsphere), composition, or the like and/or encapsulated and dispersed in the silk matrix (e.g., a silk microsphere), composition, or the like homogeneously or heterogeneously or in a gradient.
  • the active agent can be added into the silk solution, which is then subjected to the methods described herein for preparing a silk matrix (e.g., a silk microsphere), composition, or the like.
  • the active agent can be coated on a surface of the silk matrix (e.g., a silk microsphere), composition, or the like.
  • the active agent can be loaded in a silk matrix (e.g., a silk microsphere), composition, or the like by incubating the silk microsphere in a solution of the active agent for a period of time, during which an amount of the active agent can diffuse into the silk matrix (e.g., a silk microsphere), composition, or the like, and thus distribute within the silk matrix (e.g., a silk microsphere), composition, or the like.
  • the additive is a therapeutic agent.
  • therapeutic agent 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.
  • nucleic acids and compounds comprising nucleic acids that produce a therapeutic effect for example deoxyribonucleic acid (DNA), ribonucleic acid (RNA), nucleic acid analogues (e.g., locked nucleic acid (LNA), peptide nucleic acid (PNA), xeno nucleic acid (XNA)), or mixtures or combinations thereof, including, for example, DNA nanoplexes, siRNA, microRNA, shRNA, aptamers, ribozymes, decoy nucleic acids, antisense nucleic acids, RNA activators, and the like.
  • any therapeutic agent can be included in the composition provided herein.
  • 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 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.
  • 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 drug delivery composition can contain one therapeutic agent or combinations of two or more therapeutic agents.
  • a therapeutic agent can include a wide variety of different compounds, including chemical compounds and mixtures of chemical compounds, e.g., small organic or inorganic molecules; saccharines; oligosaccharides; polysaccharides; biological macromolecules, e.g., peptides, proteins, and peptide analogs and derivatives; peptidomimetics; antibodies and antigen binding fragments thereof; nucleic acids; nucleic acid analogs and derivatives; an extract made from biological materials such as bacteria, plants, fungi, or animal cells; animal tissues; naturally occurring or synthetic compositions; and any combinations thereof.
  • the therapeutic agent is a small molecule.
  • bioactivity generally refers to the ability of an active agent to interact with a biological target and/or to produce an effect on a biological target.
  • bioactivity can include, without limitation, elicitation of a stimulatory, inhibitory, regulatory, toxic or lethal response in a biological target.
  • the biological target can be a molecule or a cell.
  • a bioactivity can refer to the ability of an active agent to modulate the effect/activity of an enzyme, block a receptor, stimulate a receptor, modulate the expression level of one or more genes, modulate cell proliferation, modulate cell division, modulate cell morphology, or any combination thereof.
  • a bioactivity can refer to the ability of a compound to produce a toxic effect in a cell.
  • exemplary cellular responses include, but are not limited to, lysis, apoptosis, growth inhibition, and growth promotion; production, secretion, and surface expression of a protein or other molecule of interest by the cell; membrane surface molecule activation including receptor activation; transmembrane ion transports; transcriptional regulations; changes in viability of the cell; changes in cell morphology; changes in presence or expression of an intracellular component of the cell; changes in gene expression or transcripts; changes in the activity of an enzyme produced within the cell; and changes in the presence or expression of a ligand and/or receptor (e.g., protein expression and/or binding activity).
  • a ligand and/or receptor e.g., protein expression and/or binding activity
  • Methods for assaying different cellular responses are well known to one of skill in the art, e.g., western blot for determining changes in presence or expression of an endogenous protein of the cell, or microscopy for monitoring the cell morphology in response to the active agent, or FISH and/or qPCR for the detection and quantification of changes in nucleic acids.
  • Bioactivity can be determined in some embodiments, for example, by assaying a cellular response.
  • bioactivity includes, but is not limited to, epitope or antigen binding affinity, the in vivo and/or in vitro stability of the antibody, the immunogenic properties of the antibody, e.g., when administered to a human subject, and/or the ability to neutralize or antagonize the bioactivity of a target molecule in vivo or in vitro.
  • the aforementioned properties or characteristics can be observed or measured using art-recognized techniques including, but not limited to, scintillation proximity assays, ELISA, ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence ELISA, competitive ELISA, SPR analysis including, but not limited to, SPR analysis using a BIAcore biosensor, in vitro and in vivo neutralization assays (see, for example, International Publication No. WO 2006/062685), receptor binding, and immunohistochemistry with tissue sections from different sources including human, primate, or any other source as needed.
  • the “bioactivity” includes immunogenicity, the definition of which is discussed in detail later.
  • the “bioactivity” includes infectivity, the definition of which is discussed in detail later.
  • the “bioactivity” refers to the ability of a contrast agent when administered to a subject to enhance the contrast of structures or fluids within the subject's body.
  • the bioactivity of a contrast agent also includes, but is not limited to, its ability to interact with a biological environment and/or influence the response of another molecule under certain conditions.
  • small molecule can refer to compounds that are “natural productlike,” however, the term “small molecule” is not limited to “natural product-like” compounds. Rather, a small molecule is typically characterized in that it contains several carbon — carbon bonds, and has a molecular weight of less than 5000 Daltons (5 kDa), preferably less than 3 kDa, still more preferably less than 2 kDa, and most preferably less than 1 kDa. In some cases it is preferred that a small molecule have a molecular weight equal to or less than 700 Daltons.
  • Exemplary therapeutic agents include, but are not limited to, those found in Harrison’s Principles of Internal Medicine, 13th Edition, Eds. T.R. Harrison et al. McGraw-Hill N.Y., NY; Physicians’ Desk Reference, 50th Edition, 1997, Oradell New Jersey, Medical Economics Co.; Pharmacological Basis of Therapeutics, 8th Edition, Goodman and Gilman, 1990; United States Pharmacopeia, The National Formulary, ETSP XII NF XVII, 1990, the complete contents of all of which are incorporated herein by reference.
  • Therapeutic agents include the herein disclosed categories and specific examples. It is not intended that the category be limited by the specific examples. Those of ordinary skill in the art will recognize also numerous other compounds that fall within the categories and that are useful according to the present disclosure. Examples include a radiosensitizer, a steroid, a xanthine, a beta- 2-agonist bronchodilator, an anti-inflammatory agent, an analgesic agent, a calcium antagonist, an angiotensin-converting enzyme inhibitors, a beta-blocker, a centrally active alpha- agonist, an alpha- 1 -antagonist, an anticholinergic/antispasmodic agent, a vasopressin analogue, an anti arrhythmic agent, an antiparkinsonian agent, an antiangina/antihypertensive agent, an anticoagulant agent, an antiplatelet agent, a sedative, an ansiolytic agent, a peptidic agent, a biopolymeric agent, an antineoplastic agent,
  • the pharmaceutically active agent can be coumarin, albumin, steroids such as betamethasone, dexamethasone, methylprednisolone, prednisolone, prednisone, triamcinolone, budesonide, hydrocortisone, and pharmaceutically acceptable hydrocortisone derivatives; xanthines such as theophylline and doxophylline; beta-2- agonist bronchodilators such as salbutamol, fenterol, clenbuterol, bambuterol, salmeterol, fenoterol; antiinflammatory agents, including antiasthmatic anti-inflammatory agents, antiarthritis antiinflammatory agents, and non-steroidal antiinflammatory agents, examples of which include but are not limited to sulfides, mesalamine, budesonide, salazopyrin, diclofenac, pharmaceutically acceptable diclofenac salts, nimesulide, naproxene, acetaminophen,
  • steroids such as beta
  • Anti-cancer agents include alkylating agents, platinum agents, antimetabolites, topoisomerase inhibitors, antitumor antibiotics, antimitotic agents, aromatase inhibitors, thymidylate synthase inhibitors, DNA antagonists, farnesyltransferase inhibitors, pump inhibitors, histone acetyltransferase inhibitors, metalloproteinase inhibitors, ribonucleoside reductase inhibitors, TNF alpha agonists/antagonists, endothelinA receptor antagonists, retinoic acid receptor agonists, immunomodulators, hormonal and antihormonal agents, photodynamic agents, and tyrosine kinase inhibitors.
  • Antibiotics include aminoglycosides (e.g., gentamicin, tobramycin, netilmicin, streptomycin, amikacin, neomycin), bacitracin, corbapenems (e.g., imipenem/cislastatin), cephalosporins, colistin, methenamine, monobactams (e.g., aztreonam), penicillins (e.g., penicillin G, penicillinV, methicillin, natcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, carbenicillin, ticarcillin, piperacillin, mezlocillin, azlocillin), polymyxin B, quinolones, and vancomycin; and bacteriostatic agents such as chloramphenicol, clindanyan, macrolides (e.g., erythromycin, azithromycin, clarithro), macrol
  • Enzyme inhibitors are substances which inhibit an enzymatic reaction.
  • enzyme inhibitors include edrophonium chloride, N-methylphysostigmine, neostigmine bromide, physostigmine sulfate, tacrine, tacrine, 1 -hydroxy maleate, iodotubercidin, p- bromotetramiisole, 10- (alpha-diethylaminopropionyl)-phenothiazine hydrochloride, calmidazolium chloride, hemicholinium-3,3,5-dinitrocatechol, diacylglycerol kinase inhibitor I, diacylglycerol kinase inhibitor II, 3-phenylpropargylamine, N°-monomethyl-Larginine acetate, carbidopa, 3- hydroxybenzylhydrazine, hydralazine, clorgyline, deprenyl, hydroxylamine,
  • Antihistamines include pyrilamine, chlorpheniramine, and tetrahydrazoline, among others.
  • Anti-inflammatory agents include corticosteroids, nonsteroidal anti-inflammatory drugs (e.g., aspirin, phenylbutazone, indomethacin, sulindac, tolmetin, ibuprofen, piroxicam, and fenamates), acetaminophen, phenacetin, gold salts, chloroquine, D-Penicillamine, methotrexate colchicine, allopurinol, probenecid, and sulfinpyrazone.
  • nonsteroidal anti-inflammatory drugs e.g., aspirin, phenylbutazone, indomethacin, sulindac, tolmetin, ibuprofen, piroxicam, and fenamates
  • acetaminophen phenacetin
  • gold salts chloroquine
  • Muscle relaxants include mephenesin, methocarbomal, cyclobenzaprine hydrochloride, trihexylphenidyl hydrochloride, levodopa/carbidopa, and biperiden.
  • Anti-spasmodics include atropine, scopolamine, oxyphenonium, and papaverine.
  • Analgesics include aspirin, phenybutazone, idomethacin, sulindac, tolmetic, ibuprofen, piroxicam, fenamates, acetaminophen, phenacetin, morphine sulfate, codeine sulfate, meperidine, nalorphine, opioids (e.g., codeine sulfate, fentanyl citrate, hydrocodone bitartrate, loperamide, morphine sulfate, noscapine, norcodeine, normorphine, thebaine, nor- binaltorphimine, buprenorphine, chlomaltrexamine, funaltrexamione, nalbuphine, nalorphine, naloxone, naloxonazine, n
  • Ophthalmic agents include sodium fluorescein, rose bengal, methacholine, adrenaline, cocaine, atropine, alpha-chymotrypsin, hyaluronidase, betaxalol, pilocarpine, timolol, timolol salts, and combinations thereof.
  • Prostaglandins are art recognized and are a class of naturally occurring chemically related long-chain hydroxy fatty acids that have a variety of biological effects.
  • Anti-depressants are substances capable of preventing or relieving depression.
  • anti-depressants examples include imipramine, amitriptyline, nortriptyline, protriptyline, desipramine, amoxapine, doxepin, maprotiline, tranylcypromine, phenelzine, and isocarboxazide.
  • Trophic factors are factors whose continued presence improves the viability or longevity of a cell trophic factors include, without limitation, platelet-derived growth factor (PDGP), neutrophilactivating protein, monocyte chemoattractant protein, macrophage- inflammatory protein, platelet factor, platelet basic protein, and melanoma growth stimulating activity; epidermal growth factor, transforming growth factor (alpha), fibroblast growth factor, platelet- derived endothelial cell growth factor, insulin-like growth factor, glial derived growth neurotrophic factor, ciliary neurotrophic factor, nerve growth factor, bone growth/cartilage- inducing factor (alpha and beta), bone morphogenetic proteins, interleukins (e.g., interleukin inhibitors or interleukin receptors, including interleukin 1 through interleukin 10), interferons (e.g., interferon alpha, beta and gamma), hematopoietic factors, including erythropoietin,
  • Hormones include estrogens (e.g., estradiol, estrone, estriol, diethylstibestrol, quinestrol, chlorotrianisene, ethinyl estradiol, mestranol), anti-estrogens (e.g., clomiphene, tamoxifen), progestins (e.g., medroxyprogesterone, norethindrone, hydroxyprogesterone, norgestrel), antiprogestin (mifepristone), androgens (e.g, testosterone cypionate, fluoxymesterone, danazol, testolactone), anti- androgens (e.g., cyproterone acetate, flutamide), thyroid hormones (e.g., triiodothyronne, thyroxine, propylthiouracil, methimazole, and iodixode), and pituitary hormones
  • Hormones are commonly employed in hormone replacement therapy and / or for purposes of birth control. Steroid hormones, such as prednisone, are also used as immunosuppressants and anti-inflammatories.
  • the additive is an agent that stimulates tissue formation, and/or healing and regrowth of natural tissues, and any combinations thereof.
  • Agents that increase formation of new tissues and/or stimulates healing or regrowth of native tissue at the site of injection can include, but are not limited to, fibroblast growth factor (FGF), transforming growth factor-beta (TGF-beta, platelet-derived growth factor (PDGF), epidermal growth factors (EGFs), connective tissue activated peptides (CTAPs), osteogenic factors including bone morphogenic proteins, heparin, angiotensin II (A-II) and fragments thereof, insulin-like growth factors, tumor necrosis factors, interleukins, colony stimulating factors, erythropoietin, nerve growth factors, interferons, biologically active analogs, fragments, and derivatives of such growth factors, and any combinations thereof.
  • FGF fibroblast growth factor
  • TGF-beta transforming growth factor-beta
  • PDGF platelet-derived growth factor
  • EGFs epidermal growth factors
  • CTAPs connective tissue activated peptides
  • osteogenic factors
  • the silk composition can further comprise at least one additional material for soft tissue augmentation, e.g., dermal filler materials, including, but not limited to, poly(methyl methacrylate) microspheres, hydroxylapatite, poly(L-lactic acid), collagen, elastin, and glycosaminoglycans, hyaluronic acid, commercial dermal filler products such as BOTOX® (from Allergan), DYSPORT®, COSMODERM®, EVOLENCE®, RADIESSE®,RESTYLANE®, JUVEDERM® (from Allergan), SCULPTRA®, PERLANE®, and CAPTIQEIE®, and any combinations thereof.
  • dermal filler materials including, but not limited to, poly(methyl methacrylate) microspheres, hydroxylapatite, poly(L-lactic acid), collagen, elastin, and glycosaminoglycans, hyaluronic acid, commercial dermal filler products such as BOTOX® (from
  • the additive is a wound healing agent.
  • a wound healing agent is a compound or composition that actively promotes wound healing process.
  • Exemplary wound healing agents include, but are not limited to dexpanthenol; growth factors; enzymes, hormones; povidon-iodide; fatty acids; anti-inflammatory agents; antibiotics; antimicrobials; antiseptics; cytokines; thrombin; angalgesics; opioids; aminoxyls; furoxans; nitrosothiols; nitrates and anthocyanins; nucleosides, such as adenosine; and nucleotides, such as adenosine diphosphate (ADP) and adenosine triphosphate (ATP); neutotransmitter/neuromodulators, such as acetylcholine and 5 -hydroxy tryptamine (serotonin/5- HT); histamine and catecholamines, such as adrenalin and noradrenalin; lipid molecules, such as 5 sphingosine- 1 -phosphate and lysophosphatidic acid;
  • the active agents provided herein are immunogens.
  • the immunogen is a vaccine.
  • Most vaccines are sensitive to environmental conditions under which they are stored and/or transported. For example, freezing may increase reactogenicity (e.g., capability of causing an immunological reaction) and/or loss of potency for some vaccines (e.g., HepB, and DTaP/IPV/FQB), or cause hairline cracks in the container, leading to contamination. Further, some vaccines (e.g., BCG, Varicella, and MMR) are sensitive to heat.
  • compositions and methods provided herein also provide for stabilization of vaccines regardless of the cold chain and/or other environmental conditions.
  • exemplary cells that can be can be used with the compositions include platelets, activated platelets, stem cells, totipotent cells, pluripotent cells, and/or embryonic stem cells.
  • exemplary cells that can be encapsulated within compositions include, but are not limited to, primary cells and/or cell lines from any tissue.
  • cardiomyocytes myocytes, hepatocytes, keratinocytes, melanocytes, neurons, astrocytes, embryonic stem cells, adult stem cells, hematopoietic stem cells, hematopoietic cells (e.g. monocytes, neutrophils, macrophages, etc.), ameloblasts, fibroblasts, chondrocytes, osteoblasts, osteoclasts, neurons, sperm cells, egg cells, liver cells, epithelial cells from lung, epithelial cells from gut, epithelial cells from intestine, liver, epithelial cells from skin, etc., and/or hybrids thereof, can be included in the silk/platelet compositions disclosed herein.
  • Cells listed herein represent an exemplary, not comprehensive, list of cells.
  • Cells can be obtained from donors (allogenic) or from recipients (autologous). Cells can be obtained, as a non-limiting example, by biopsy or other surgical means known to those skilled in the art.
  • the cell can be a genetically modified cell.
  • a cell can be genetically modified to express and secrete a desired compound, e.g. a bioactive agent, a growth factor, differentiation factor, cytokines, and the like.
  • a desired compound e.g. a bioactive agent, a growth factor, differentiation factor, cytokines, and the like.
  • Differentiated cells that have been reprogrammed into stem cells can also be used.
  • the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.”
  • the terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims.
  • the terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims.
  • the term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.
  • the three films dried under low ( ⁇ 17 W) acoustic power had a higher average P-sheet content (24 ⁇ 1.4%, 25 ⁇ 1.0%, 24 ⁇ 1.7%) than the three films dried at high ( ⁇ 17 W) acoustic power (18 ⁇ 3.6%, 20 ⁇ 2.6%, 19 ⁇ 3.1%).
  • the two films dried under low ( ⁇ 15 W) acoustic power had a higher average P-sheet content (25 2.8%, 27 2.8%) than the two films dried at high ( ⁇ 15 W) acoustic power (24 ⁇ 1.4%, 24 ⁇ 2.1%).
  • the 1.0 kHz group showed decreased average P-sheet content (26 ⁇ 0.9%, 23 ⁇ 2.5%, 21 ⁇ 2.5%) at increased acoustic powers. Overall, the average P-sheet content decreases with acoustic power, with a distinct threshold around 50% the maximum power used for maintaining fluid surface stability.
  • the antinode regions of a standing wave are spatially locked but experience high temporal displacement and large mechanical stresses in those localized regions.
  • these localized variations in mechanical stress seem to induce P-sheet conformation, resulting in radially and axially localized increased crystallinity.
  • Radial distribution of crystallinity can be seen using FTIR secondary structure mapping while axial distribution can be inferred from SEM cross section morphology.
  • An additional step of varying the initial concentration of the silk solution allows for tuning of the evaporation rate and overall crystallinity of the films.
  • concentration of the silk solution varied between 6% - 11% w/w at a constant frequency (2.0 kHz). This frequency is chosen because it produced the most distinct pattern as previously observed and is expected to generate the same acoustic field as seen using 7.5% w/w silk solution.
  • the control films do not display birefringence under polarized light.
  • the films dried under an acoustic field display regions of birefringence emanating from the center that varied with acoustic power.
  • This birefringent pattern became more pronounced with increased power and silk concentration, shown in Fig. 8A, Fig. 8B, and Fig. 8C.
  • films dried under low power exhibit no birefringence.
  • these films exhibit a birefringent four lobe pattern that increases in size with increased acoustic power, with a dimmer birefringence than that observed in 7.5% w/w films.
  • FTIR mapping was used to analyze secondary structure distribution as shown in Fig. 9A, Fig. 9B, and Fig. 9C.
  • the control films (6.0% w/w, 9.0% w/w) showed a homogenous distribution of random coil dominant secondary structure, indicated by an average P-sheet content ⁇ 20% (17 ⁇ 2.9%, 14 ⁇ 2.0%, respectively).
  • the high concentration control film (11.0% w/w) showed a homogenous distribution of P-sheet dominant secondary structure, indicated by an average P-sheet content > 30% (31 ⁇ 1.2%).
  • the birefringent region can be recognized in the distribution of secondary structure of the films modified by the acoustic field, consistently showing increased P-sheet content within the birefringent region than the surrounding non-birefringent region.
  • the low concentration films (6.0%; 17 W, 20 W) had an average P-sheet content (19 ⁇ 5.6%, 18 ⁇ 4.7%) with significantly higher P-sheet content (24 ⁇ 6.2%, 22 ⁇ 4.9%) in the region of birefringence, than the regions not exhibiting birefringence (17 ⁇ 0.01%, 16 ⁇ 2.9%).
  • the 9.0% w/w films (17 W, 20 W) had a higher average P-sheet content (22 ⁇ 5.6%, 26 ⁇ 6.3%), with higher P-sheet content (31 ⁇ 5.1%, 25 ⁇ 5.23%) in the region of birefringence, while the regions not exhibiting birefringence showed lower P-sheet content (22 ⁇ 4.3%, 21 ⁇ 5.45%).
  • the 11.0% w/w film (20 W) had the highest average P-sheet content (31 ⁇ 2.6%), however there was little difference between the regions of birefringence (33 ⁇ 2.0%) and nonbirefringence (31 ⁇ 2.6%). Overall, this heterogenous distribution of secondary structure agrees well with the observed birefringence. Center localization of increased crystallinity was achieved up to 9.0% w/w and was uniformly higher at 11.0% w/w concentration.
  • Regenerated silk fibroin (SF) solution is extracted from Bombyx mori cocoons, prepared following an established protocol from Rockwood, D., Preda, R., Yiicel, T. et al. Materials fabrication from Bombyx mori silk fibroin. Nat Protoc 6, 1612-1631 (2011). Briefly, shredded cocoons were boiled in 0.02 M sodium carbonate (Na2CO3) solution for 30 minutes in order to remove the sericin that binds the fibroin fibers together. The extracted silk fibroin was then rinsed with deionized (DI) water and dried overnight.
  • DI deionized
  • LiBr lithium bromide
  • FIG. 2A The film fabrication method using an acoustic wave field is shown in Figure 2A and Fig. 2B.
  • a wave driver PI Audio, ASX05408-HD- R
  • a 3.5 cm polystyrene Petri dish was mounted to the wave driver using thin high-performance adhesive tape (2-5-F9473PC, 3MTM) so that the sound propagation axis was perpendicular to the bottom of the dish.
  • a minimum volume (1.5 mL) of silk fibroin solution of 7.5% w/w concentration was drop-cast onto the Petri dish and spread to coat the entire area of the dish using a rubber spatula.
  • This solution volume is favorable to minimize the change in mass of the system during evaporation while ensuring that the entire surface of the Petri dish is sufficiently covered.
  • the vibration frequency and amplitude of the sound waves were controlled through function generator software connected to an amplifier.
  • the standing wave field was generated by continuously driving the wave driver close to the fundamental ( ⁇ 1.0 kHz) and harmonic frequencies of the Petri dish until a dry film was formed (about 8 - 12 hours).
  • the temperature of the solution was monitored with an infrared gun to ensure the solution did not exceed 30°C.
  • Acoustic power was varied across three frequencies to produce different mode patterns of the standing wave (1.0 kHz, 1 .5 kHz, and 2.0 kHz). It should be noted that the associated power level is relative to the amplifier and not the acoustic power delivered to the sample.
  • BFR Bright field reflection microscopy
  • BFT-XP polarized light microscopy
  • BX51 upright microscope
  • Macroscopic crossed-polarized images were taken using polarized film and a diffuse light source. By introducing polarization filters, the light passing through the sample is linearly polarized.
  • polarized light microscopy is used to examine optical anisotropy (birefringence) by placing the films flat between two crossed polarizers. Birefringence is a measure of the difference of the two independent refractive indices of anisotropic crystals.
  • FTIR Fourier-transform infrared spectroscopy
  • FTIR Fourier-transform infrared spectroscopy
  • ATR diamond attenuated total reflectance
  • a bright- field composite image of the sample was obtained using a 4x objective and a 10x10 grid was generated for a total of 100 IR spectra per film.
  • Each measurement was collected using an ATR crystal in the range of 4000 - 600 cm' 1 at a resolution of 4 cm' 1 with an average of 32 scans performed on the air-exposed side of the film.
  • the beta-sheet crystallinity portion is calculated by analysis of the Amide I (1595-1705 cm 1 ) peak position and area, performing Fourier self-deconvolution and peak fitting algorithms from Opus 5.0 software (Bruker).
  • SEM Scanning electron microscopy
  • Regenerated silk fibroin (7.5% w/w) was prepared as previously described. The concentration was decreased through water dilution (6% w/w) and increased through controlled evaporation (9%, 1 1% w/w). Briefly, to increase the concentration, 7.5% w/w silk fibroin solution was added to a 3.5 kDa cellulose dialysis tube (Spectra/Por 3, Fisher Scientific) and placed in a drying rack until the desired concentration was reached. Silk films were fabricated under a 2.0 kHz frequency, following the same method as previously described.

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Abstract

L'invention concerne un procédé comprenant l'application d'un motif acoustique prédéterminé à une solution de fibroïne de soie et la solidification de la solution de fibroïne de soie en un article de fibroïne de soie, tout en maintenant l'application du motif acoustique prédéterminé, ayant un alignement de protéines qui imite le motif acoustique prédéterminé. Le procédé peut en outre comprendre le dépôt d'une seconde solution de fibroïne de soie au-dessus de la première et la répétition des deux premières étapes pour former un article de fibroïne de soie expansé. L'article peut avoir des caractéristiques provoquées acoustiquement ou un motif de biréfringence.
PCT/US2025/019398 2024-03-11 2025-03-11 Assemblage de biopolymères provoqué par le son Pending WO2025193717A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012054121A2 (fr) * 2010-07-30 2012-04-26 Tufts University/Trustees Of Tufts College Capteurs biophotoniques à base de soie
US20150164117A1 (en) * 2012-07-13 2015-06-18 Tufts University Encapsulation of fragrance and/or flavors in silk fibroin biomaterials
US20150202304A1 (en) * 2012-07-13 2015-07-23 Tufts University Encapsulation of immiscible phases in silk fibroin biomaterials
US20160263228A1 (en) * 2013-03-15 2016-09-15 Trustees Of Tufts College Low molecular weight silk compositions and stabilizing silk compositions

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012054121A2 (fr) * 2010-07-30 2012-04-26 Tufts University/Trustees Of Tufts College Capteurs biophotoniques à base de soie
US20150164117A1 (en) * 2012-07-13 2015-06-18 Tufts University Encapsulation of fragrance and/or flavors in silk fibroin biomaterials
US20150202304A1 (en) * 2012-07-13 2015-07-23 Tufts University Encapsulation of immiscible phases in silk fibroin biomaterials
US20160263228A1 (en) * 2013-03-15 2016-09-15 Trustees Of Tufts College Low molecular weight silk compositions and stabilizing silk compositions

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ARMSTRONG JAMES P., PCHELINTSEVA EKATERINA, TREUMUTH SIRLI, CAMPANELLA CRISTIANA, MEINERT CHRISTOPH, KLEIN TRAVIS J., HUTMACHER DI: "Tissue Engineering Cartilage with Deep Zone Cytoarchitecture by High‐Resolution Acoustic Cell Patterning", ADVANCED HEALTHCARE MATERIALS, vol. 11, no. 24, 1 December 2022 (2022-12-01), DE , pages 1 - 10, XP093359605, ISSN: 2192-2640, DOI: 10.1002/adhm.202200481 *
GUEX A.G., DI MARZIO N., EGLIN D., ALINI M., SERRA T.: "The waves that make the pattern: a review on acoustic manipulation in biomedical research", MATERIALS TODAY BIO, vol. 10, 1 March 2021 (2021-03-01), pages 100110, XP093029599, ISSN: 2590-0064, DOI: 10.1016/j.mtbio.2021.100110 *

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