WO2025193717A1 - Sound mediated assembly of biopolymers - Google Patents

Abstract

A method includes applying a predetermined acoustic pattern to a silk fibroin solution and solidifying the silk fibroin solution into a silk fibroin article while maintaining application of the predetermined acoustic pattern having a protein alignment that mimics the predetermined acoustic pattern. The method may further include depositing a second silk fibroin solution on top of the first and repeating the first two steps to form an expanded silk fibroin article. The article may have acoustically-mediated features or a birefringence pattern.

Description

SOUND MEDIATED ASSEMBLY OF BIOPOLYMERS

CLAIM TO PRIORITY

[0001] This application relates to, incorporates by reference for all purposes, and claims priority to United States Provisional Application Serial Number 63/563,788 filed March 11, 2024.

BACKGROUND

[0002] Many efforts are being made to manipulate the structural properties of biomaterials, like silk, through various artificial schemes. However, as a result of the regeneration process, the structural organization that promotes silk’ s superior mechanical performance is lost. In this aqueous state, the proteins are predominantly in a random-coil or alpha-helix configurations within a micellular structure due to intramolecular hydrophobic interactions. The transition to the beta- sheet conformation is primarily concentration dependent as decreased water content allows for intermolecular hydrogen bonds to form between protein chains. Strategies for manipulating this assembly process, at both the molecular and macro-length scale presents a significant challenge, where control at the molecular level often imposes constraints on the control achievable at larger scales.

[0003] Among all material formats achievable using silk, thin films are one of the most well studied. Solvent evaporation is typically considered the simplest strategy for forming thin film structures from aqueous solutions. The method of dispensing a solution onto a substrate and allowing the solvent to evaporate is known as drop casting. As the solvent evaporates, a convective flow is generated that drags solutes from the bulk solution towards an ordered crystalline assembly. Experimentally, drop casting is commonly used with silk fibroin to assess the effects of physical factors on the protein assembly processes20. This technique can successfully form films over small areas but struggles to achieve uniformity at larger scales due to spatial variations in the drying rate. Specifically, 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.

SUMMARY

[0004] These and other systems, methods, objects, features, and advantages of the present disclosure will be apparent to those skilled in the art from the following detailed description of the preferred embodiment and the drawings.

[0005] All documents mentioned herein are hereby incorporated in their entirety by reference. References to items in the singular should be understood to include items in the plural, and vice versa, unless explicitly stated otherwise or clear from the text. Grammatical conjunctions are intended to express any and all disjunctive and conjunctive combinations of conjoined clauses, sentences, words, and the like, unless otherwise stated or clear from the context.

BRIEF DESCRIPTION OF THE FIGURES

[0006] The disclosure and the following detailed description of certain embodiments thereof may be understood by reference to the following figures:

[0007] Fig. 1 depicts a graphic representation of the effects of echoes on the self-assembly of silk fibroin solution.

[0008] Fig. 2A depicts a graphic representation of the side view of acoustic fabrication strategy to form films.

[0009] Fig. 2B depicts a graphic representation of the top view of acoustic fabrication strategy to form films.

[0010] Fig. 3A depicts a bottom-mounted piezoelectric element and reflector inducing bulk acoustic waves in a Petri dish.

[0011] Fig. 3B depicts a bottom mounted frequency generator inducing a standing wave in an opentop Petri dish.

[0012] Fig. 4A depicts a scanning electron micrograph of a control silk fibroin film cross section. Scale bar is 50 pm.

[0013] Fig. 4B depicts a scanning electron micrograph of a control silk fibroin film cross section. Scale bar is 4 pm.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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. [0018] 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. 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. [0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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. [0029] 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.

[0030] 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.

DETAILED DESCRIPTION

[0031] Before the present disclosure is described in further detail, it is to be understood that the disclosure is not limited to the particular embodiments described. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The scope of the present disclosure will be limited only by the claims. As used herein, the singular forms "a", "an", and "the" include plural embodiments unless the context clearly dictates otherwise.

[0032] In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and/or”; (iii) the terms “comprising” and “including” may be understood to 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.

[0033] Approximately: as used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0034] Composition: as used herein, may be used to refer to a discrete physical entity that comprises one or more specified components. In general, unless otherwise specified, a composition may be of any form - e.g., gas, gel, liquid, solid, etc. In some embodiments, “composition” may refer to a combination of two or more entities for use in a single embodiment or as part of the same article. It is not required in all embodiments that 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.

[0035] Improve, increase, or reduce: as used herein or grammatical equivalents thereof, indicate values that are relative to a baseline measurement, such as a measurement in a similar composition made according to previously known methods.

[0036] Substantially: as used herein, 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.

[0037] It should be apparent to those skilled in the art that many additional modifications beside those already described are possible without departing from the inventive concepts. In interpreting this disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. Variations of the term "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, so the referenced elements, components, or steps may be combined with other elements, components, or steps that are not expressly referenced. Embodiments referenced as "comprising" certain elements are also contemplated as "consisting essentially of" and "consisting of" those elements. When two or more ranges for a particular value are recited, this disclosure contemplates all combinations of the upper and lower bounds of those ranges that are not explicitly recited. For example, recitation of a value of between 1 and 10 or between 2 and 9 also contemplates a value of between 1 and 9 or between 2 and 10.

[0038] Acoustic manipulation (AM) has emerged as a promising technology for the precise and controlled distribution of mechanical forces to manipulate cells and particles using sound waves. Operating as a non-contact method, it has shown the ability to move, sort, and pattern microscale objects over one, two, and three dimensions. This offers a targeted and customizable application in various fields, from facilitating single-cell analysis in biological research to assisting in the separation and assembly of micro-nano particles in manufacturing processes.

[0039] 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.

[0040] 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. However, it is limited spatial resolution when imaging beyond a few centimeters, making it unable to image density-homogenous structures, such as vasculature.

[0041] 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. By utilizing standing wave fields during material fabrication, this approach allows us to tune the organization of material properties, such as compressibility, to optimize the sound-structure interactions leading to structural echogenicity. [0042] 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. In this design, the standing wave takes the shape of modal patterns that are based on the vibration frequency and geometry of the container.

[0043] Acoustic manipulation (AM) 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. In single-element designs, 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. In the case of a standing wave, a stationary pattern with regions of constructive interference, known as antinodes, and regions of destructive interference, known as nodes, is formed.

[0044] Acoustic manipulation via standing wave fields is explored herein as a tool to direct protein assembly and structural organization in a silk film. The acoustic field is used in combination with protein self-assembly to determine the effect of discrete mechanical forces on protein assembly processes. The spatial distribution of secondary structure as well as the optical properties will be evaluated for evidence of anisotropy as they relate to the pattern of the sound field.

[0045] 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.

[0046] 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 . 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.

[0047] Many factors such as pH, temperature, ion concentration, and shear forces promote secondary structure transitions in silk fibroin materials. In particular, shear stress may induce structural changes leading to the formation of P-sheet-rich and stable fibril networks through shear-induced ordering. Mechanistically, it is thought that the shear forces induce -sheet formation through the physical disruption of the micellular structures, altering the hydrophobic state of the protein chains. Subsequently, these mechanical forces accelerate the rate of intermolecular hydrogen bonding between proteins by allowing for more protein-protein interactions. Several physical factors related to 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.

[0048] In one aspect, 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 silk fibroin article may have birefringence at locations corresponding to the antinodes and may lack birefringence at locations corresponding to the nodes. The predetermined acoustic pattern may be generated by a wave driver. The wave driver may have vibrational damping mounting. The applying of step a) may include applying the predetermined acoustic pattern from beneath the silk fibroin solution.

[0049] 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.

[0050] The temperature during the solidifying of step b) may be maintained between 0 °C and 80 °C. In some cases, 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. In some cases, 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] In some aspects, a silk fibroin article is made by the methods described herein.

[0055] In other aspects, a silk fibroin article has acoustically-mediated features.

[0056] In some aspects, a silk fibroin article has a birefringence pattern that mimics a predetermined acoustic pattern.

[0057] 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.

[0058] As used herein, "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. For instance, 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. 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.

[0059] According to various embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[0060] In some embodiments, 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.

[0061] In some embodiments, 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).

[0062] According to some embodiments, a plurality of functionalizing agents may be used. For example, in some embodiments wherein production of platelets is desired, provided 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.

[0063] In some embodiments, 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. In some embodiments, a functionalizing agent is distributed along and/or incorporated in substantially the entire surface area of a silk membrane/silk wall. In some embodiments, a functionalizing agent is distributed and/or incorporated only at one or more discrete portions of a silk membrane/wall and/or silk matrix. In some embodiments, 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.

[0064] According to various embodiments, any application-appropriate amount of one or more functionalizing agents may be used. In some embodiments, 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). In some embodiments, 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 ). In some embodiments, 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 ).

[0065] In some aspects, 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. In some aspects, 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). In some cases, 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. [0066] In some aspects, 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.

[0067] 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-(4-dimethylamino-l-naphylazo)-3-methoxybenesulfonic acid, bromocresol green, resazurin, 4- phenylazo-l-napthylamine, ethyl red 2-(l-dimethylaminophenyazo) pyridine, 4-(p- ethoxypehnylazo)-m-phenylene-diamine monohydrochloride, resorcin blue, alizarin red S, methyl red, propyl red, bromocresol purple, chlorophenol red, p-nitrophenol, alizarin 2-(2,4- dinitrophenylazo) l-napthol-3,6-disulfonic acid, bromothymol blue, 6,8-dinitro-2,4-(lH) quinazolinedione, brilliant yellow, phenol red, neutral red, m-nitrophenol, cresol red, turmeric, metacresol purple, 4,4'-bis(3-amino-l-naphthylazo)-2,2'-stilbenedisulfonic acid, thymol blue, p- naphtholbenzein, phenolphthalein, o-cresolphthalein, ethyl bis(2,4-dimethylphenyl) ethanoate, thymolphthalein, nitrazine yellow, alizarin yellow R, alizarin, p-(2,4-dihydroxyphenylazo) benzenesulfonic acid, 5,5'-indigodisulfonic acid, 2,4,6-trinitrotoluene, 1,3,5-trinitrobenezne, and clayton yellow.

[0068] 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.

[0069] 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).

[0070] 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. [0071] Exemplary pressure or strain sensitive dyes or agents include, but are not limited to, spiropyran compounds and agents.

[0072] Exemplary 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.

[0073] In some aspects, the compositions comprise one or more additive, dopant, or biologically active agent suitable for a desired intended purpose. In some aspects, 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.

[0074] Additionally or alternatively, 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 (viruses and viral particles; prokaryotic cells such as bacteria; eukaryotic cells such as mammalian cells and plant cells; fungi). [0075] In some aspects, the additive or dopant comprises a flavoring agent or flavorant.

[0076] 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.

[0077] In some aspects, the additive or dopant comprises an aroma compound. Exemplary 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, benzaldehyde, eugenol, cinnamaldehyde, ethyl maltol, vanillin, anisole, anethole, estragole, thymol.

[0078] In some aspects, the additive or dopant comprises a colorant, such as a dye or pigment. In some aspects, 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. Pigment Orange Numbers 5, 13, 16, 34 and 36, C.I. Pigment Violet Numbers 3, 19, 23 and 27, C.I. Pigment Red Numbers 3, 17, 22, 23, 48: 1 , 48:2, 57: 1 , 81 : 1 , 81 :2, 81 :3, 81 :5, 101 , 1 14, 122, 144, 146, 170, 176, 179, 181 , 185, 188, 202, 206, 207, 210 and 249, C.I. Pigment Yellow Numbers 1 , 2, 3, 12, 13, 14, 17, 42, 65, 73, 74, 75, 83, 30, 93, 109, 1 10, 128, 138, 139, 147, 142, 151 , 154 and 180, D&C Red No. 7, D&C Red No. 6 and D&C Red No. 34, 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 Chemical Corporation); and phthalocyanine blue pigment (such as Blue 15 :3 (294-1298) available from Sun Chemical Corporation). 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. 18965); Acid Yellow 23 (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. [0079] Exemplary red acid dyes include Acid Red 1. (C.I. 18050); 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; and Food Red 1 (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. 42045); Acid Blue 9 (C.I. 42090); Acid Blue 22 (C.I. 42755); Acid Blue 74 (C.I. 73015); Acid Blue 93 (C.I. 42780); and Acid Blue 158A (C.I. 15050). 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.

[0080] Exemplary direct dyes for use in the present disclosure include Direct Blue 86 (C.I. 74180); Direct Blue 199; Direct Black 168; Direct Red 253; and Direct Yellow 107/132 (C.I. Not Assigned). [0081] Exemplary natural dyes for use in the present disclosure include Alkanet (C.I. 75520,75530); Annafto (C.I. 75120); Carotene (C.I. 75130); Chestnut; Cochineal (C.I.75470); Cutch (C.I. 75250, 75260); Divi-Divi; Fustic (C.I. 75240); Hypemic (C.I. 75280); Logwood (C.I. 75200); Osage Orange (C.I. 75660); Paprika; Quercitron (C.I. 75720); Sanrou (C.I. 75100) ; Sandal Wood (C.I. 75510, 75540, 75550, 75560); Sumac; and Tumeric (C.I. 75300). 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.

[0082] In addition to or in place of visible colorants, 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). Examples of ETV fluorophores include but are not limited to materials from the coumarin, benzoxazole, rhodamine, napthalimide, perylene, benzanthrones, benzoxanthones or benzothia- xanthones families. The addition of a UV fluorophore (such as an optical brightener for instance) can help maintain maximum visible light transmission. 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.

[0083] For non-white compositions, 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. In some applications, 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. In some applications, 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. In some applications, 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.

[0084] For white compositions, 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. In some applications, 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. In some applications, 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. In some applications, 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%,

19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%, 24 wt%, 25%, 26 wt%, 27 wt%, 28 wt%, 29 wt%, 30 wt%, 31 wt%, 32 wt%, 33 wt%, 34 wt%, 35%, 36 wt%, 37 wt%, 38 wt%, 39 wt%, 40 wt%, 41 wt%,

42 wt%, 43 wt%, 44 wt%, 45%, 46 wt%, 47 wt%, 48 wt%, 49 wt%, 50 wt%, 51 wt%, 52 wt%, 53 wt%, 54 wt%, 55%, 56 wt%, 57 wt%, 58 wt%, 59 wt% or 60 wt% based on the weight of the composition.

[0085] In some aspects, 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. In some aspects, the conductive additive is biocompatible and non-toxic.

[0086] In some aspects, the additive is a biologically active agent. The term “biologically active agent” as used herein refers to any molecule which exerts at least one biological effect in vivo. For example, 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. [0087] The term “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.

[0088] Exemplary active agents include, but are not limited to, therapeutic agents, diagnostic agents (e.g., contrast agents), and any combinations thereof. In some embodiments, 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. In some embodiments, the active agent present in the silk matrix (e.g., a silk microsphere), composition, or the like 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.

[0089] 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. In some embodiments, 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. In some embodiments, the active agent can be coated on a surface of the silk matrix (e.g., a silk microsphere), composition, or the like. In some embodiments, 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. [0090] In some aspects, the additive is a therapeutic agent. As used herein, the term “therapeutic agent” means a molecule, group of molecules, complex or substance administered to an organism for diagnostic, therapeutic, preventative medical, or veterinary purposes. As used herein, the term “therapeutic agent” includes a “drug” or a “vaccine.” This term include externally and internally administered topical, localized and systemic human and animal pharmaceuticals, treatments, remedies, nutraceuticals, cosmeceuticals, biologicals, devices, diagnostics and contraceptives, including preparations useful in clinical and veterinary screening, prevention, prophylaxis, healing, wellness, detection, imaging, diagnosis, therapy, surgery, monitoring, cosmetics, prosthetics, forensics and the like. This term can also be used in reference to agriceutical, workplace, military, industrial and environmental therapeutics or remedies comprising selected molecules or selected nucleic acid sequences capable of recognizing cellular receptors, membrane receptors, hormone receptors, therapeutic receptors, microbes, viruses or selected targets comprising or capable of contacting plants, animals and/or humans. This term can also specifically include nucleic acids and compounds comprising nucleic acids that produce a 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. Generally, any therapeutic agent can be included in the composition provided herein.

[0091] 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 which it is applied. For example, the therapeutic agent can act to control infection or inflammation, enhance cell growth and tissue regeneration, control tumor growth, act as an analgesic, promote anti-cell attachment, and enhance bone growth, among other functions. Other suitable therapeutic agents can include anti-viral agents, hormones, antibodies, or therapeutic proteins. Other therapeutic agents include prodrugs, which are agents that are not biologically active when administered but, upon administration to a subject are converted to biologically active agents through metabolism or some other mechanism. Additionally, a silk-based drug delivery composition can contain one therapeutic agent or combinations of two or more therapeutic agents.

[0092] 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. In some aspects, the therapeutic agent is a small molecule.

[0093] The term “bioactivity,” as used herein in reference to an active agent, 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. For example, 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. For example, 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. In some instances, 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). 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.

[0094] In reference to an antibody, the term “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. In reference to an immunogen, the “bioactivity” includes immunogenicity, the definition of which is discussed in detail later. In reference to a virus, the “bioactivity” includes infectivity, the definition of which is discussed in detail later. In reference to a contrast agent, e.g., a dye, 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.

[0095] As used herein, the term “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.

[0096] 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.

[0097] 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, a laxative, an antidiarrheal agent, an antimicrobial agent, an antifungal agent, a vaccine, a protein, or a nucleic acid. In a further aspect, 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, ibuprofen, ketoprofen and piroxicam; analgesic agents such as salicylates; calcium channel blockers such as nifedipine, amlodipine, and nicardipine; angiotensin converting enzyme inhibitors such as captopril, benazepril hydrochloride, fosinopril sodium, trandolapril, ramipril, lisinopril, enalapril, quinapril hydrochloride, and moexipril hydrochloride; beta-blockers (i.e., beta adrenergic blocking agents) such as sotalol hydrochloride, timolol maleate, esmolol hydrochloride, carteolol, propanolol hydrochloride, betaxolol hydrochloride, penbutolol sulfate, metoprolol tartrate, metoprolol succinate, acebutolol hydrochloride, atenolol, pindolol, and bisoprolol fumarate; centrally active alpha-2-agonists such as clonidine; alpha- 1 -antagonists such as doxazosin and prazosin; anticholinergic/antispasmodic agents such as dicyclomine hydrochloride, scopolamine hydrobromide, glycopyrrolate, clidinium bromide, flavoxate, and oxybutynin; vasopressin analogues such as vasopressin and desmopressin; antiarrhythmic agents such as quinidine, lidocaine, tocainide hydrochloride, mexiletine hydrochloride, digoxin, verapamil hydrochloride, propafenone hydrochloride, flecainide acetate, procainamide hydrochloride, moricizine hydrochloride, and disopyramide phosphate; antiparkinsonian agents, such as dopamine, L-Dopa/Carbidopa, selegiline, dihydroergocryptine, pergolide, lisuride, apomorphine, and bromocryptine; antiangina agents and antihypertensive agents such as isosorbide mononitrate, isosorbide dinitrate, propranolol, atenolol and verapamil; anticoagulant and antiplatelet agents such as Coumadin, warfarin, acetylsalicylic acid, and ticlopidine; sedatives such as benzodiazapines and barbiturates; ansiolytic agents such as lorazepam, bromazepam, and diazepam; peptidic and biopolymeric agents such as calcitonin, leuprolide and other LHRH agonists, hirudin, cyclosporin, insulin, somatostatin, protirelin, interferon, desmopressin, somatotropin, thymopentin, pidotimod, erythropoietin, interleukins, melatonin, granulocyte/macrophage-CSF, and heparin; antineoplastic agents such as etoposide, etoposide phosphate, cyclophosphamide, methotrexate, 5 -fluorouracil, vincristine, doxorubicin, cisplatin, hydroxyurea, leucovorin calcium, tamoxifen, fhitamide, asparaginase, altretamine, mitotane, and procarbazine hydrochloride; laxatives such as senna concentrate, casanthranol, bisacodyl, and sodium picosulphate; antidiarrheal agents such as difenoxine hydrochloride, loperamide hydrochloride, furazolidone, diphenoxylate hdyrochloride, and microorganisms; vaccines such as bacterial and viral vaccines; antimicrobial agents such as penicillins, cephalosporins, and macrolides, antifungal agents such as imidazolic and triazolic derivatives; and nucleic acids such as DNA sequences encoding for biological proteins, and antisense oligonucleotides.

[0098] 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. [0099] 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, clarithromycin), lincomyan, nitrofurantoin, sulfonamides, tetracyclines (e.g., tetracycline, doxycycline, minocycline, demeclocyline), and trimethoprim. Also included are metronidazole, fluoroquinolones, and ritampin.

[0100] Enzyme inhibitors are substances which inhibit an enzymatic reaction. Examples of 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, iproniazid phosphate, 6- MeO-tetrahydro-9H-pyrido-indole, nialamide, pargyline, quinacrine, semi carb azide, tranylcypromine, N,N-diethylaminoethyl-2,2-diphenylvalerate hydrochloride, 3 - isobutyl- 1- methylxanthne, papaverine, indomethacind, 2-cyclooctyl-2 -hydroxy ethylamine hydrochloride, 2,3- dichloro-a-methylbenzylamine (DCMB), 8,9-dichloro-2,3,4, 5 -tetrahydro- lH-2-benzazepine hydrochloride, p-amino glutethimide, p-aminoglutethimide tartrate, 3- iodotyrosine, alpha- methyltyrosine, acetazolamide, dichlorphenamide, 6-hydroxy-2- benzothiazolesulfonamide, and allopurinol.

[0101] Antihistamines include pyrilamine, chlorpheniramine, and tetrahydrazoline, among others. [0102] 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.

[0103] Muscle relaxants include mephenesin, methocarbomal, cyclobenzaprine hydrochloride, trihexylphenidyl hydrochloride, levodopa/carbidopa, and biperiden.

[0104] Anti-spasmodics include atropine, scopolamine, oxyphenonium, and papaverine. [0105] 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, naltrexone, and naltrindole), procaine, lidocain, tetracaine and dibucaine. Ophthalmic agents include sodium fluorescein, rose bengal, methacholine, adrenaline, cocaine, atropine, alpha-chymotrypsin, hyaluronidase, betaxalol, pilocarpine, timolol, timolol salts, and combinations thereof.

[0106] 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.

[0107] Anti-depressants are substances capable of preventing or relieving depression.

[0108] Examples of anti-depressants include imipramine, amitriptyline, nortriptyline, protriptyline, desipramine, amoxapine, doxepin, maprotiline, tranylcypromine, phenelzine, and isocarboxazide. [0109] 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, granulocyte colony stimulating factor, macrophage colony stimulating factor and granulocyte- macrophage colony stimulating factor; tumor necrosis factors, and transforming growth factors (beta), including beta-1, beta-2, beta-3, inhibin, and activin. [0110] 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 (e.g., corticotropin, sumutotropin, oxytocin, and vasopressin). 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. In some aspects, 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.

[0111] In some aspects, 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.

[0112] In some aspects, the additive is a wound healing agent. As used herein, a “wound healing agent" is a compound or composition that actively promotes wound healing process.

[0113] 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; amino acids, such as arginine and lysine; peptides such as the bradykinins, substance P and calcium gene-related peptide (CGRP); nitric oxide; and any combinations thereof. [0114] In certain aspects, the active agents provided herein are immunogens. In one aspect, 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. Many vaccines (e.g., BCG, MMR, Varicella, Meningococcal C Conjugate, and most DTaP-containing vaccines) are light sensitive. See, e.g., Galazka et al., Thermostability of vaccines, in Global Programme for Vaccines & Immunization (World Health Organization, Geneva, 1998); Peetermans et al., Stability of freeze-dried rubella virus vaccine (Cendehill strain) at various temperatures, 1 J. Biological Standardization 179 (1973). Thus, the compositions and methods provided herein also provide for stabilization of vaccines regardless of the cold chain and/or other environmental conditions.

[0115] In some aspects, the additive is a cell, e.g., a biological cell. Cells useful for incorporation into the composition can come from any source, e.g., mammalian, insect, plant, etc. In some aspects, the cell can be a human, rat or mouse cell. In general, cells to be used with the compositions provided herein can be any types of cells. In general, the cells should be viable when encapsulated within compositions. In some aspects, cells that can be used with the composition include, but are not limited to, mammalian cells (e.g. human cells, primate cells, mammalian cells, rodent cells, etc.), avian cells, fish cells, insect cells, plant cells, fungal cells, spore cells, bacterial cells, and hybrid cells. In some aspects, 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. In some aspects, exemplary cells that can be encapsulated within compositions include, but are not limited to, primary cells and/or cell lines from any tissue. For example, 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. Those skilled in the art will recognize that the 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.

[0116] In some aspects, 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. Methods of genetically modifying cells for expressing and secreting compounds of interest are known in the art and easily adaptable by one of skill in the art.

[0117] Differentiated cells that have been reprogrammed into stem cells can also be used.

[0118] For example, human skin cells reprogrammed into embryonic stem cells by the transduction of Oct3/4, Sox2, c-Myc and Klf4 (Junying Yu, et. ah, Science, 2007, 318 , 1917-1920 and Takahashi K. et. ah, Cell, 2007, 131 , 1-12). [0119] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.” [0120] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[0121] As used herein, 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.

[0122] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0123] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0124] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. [0125] While the invention has been illustrated and described in detail in the foregoing drawings and description, the same is to be considered as illustrative and not restrictive in character, it being understood that only illustrative embodiments thereof have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected. For example, any of the features or functions of any of the embodiments disclosed herein may be incorporated into any of the other embodiments disclosed herein.

[0126] The following examples illustrate some embodiments and aspects of the invention. It will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be performed without altering the spirit or scope of the invention, and such modifications and variations are encompassed within the scope of the invention as defined in the claims which follow. The following examples do not in any way limit the invention.

[0127] EXAMPLES

[0128] Example 1

[0129] Experiments were performed using 7.5% w/w silk solution dried under three acoustic frequencies (1.0, 1.5, 2.0 kHz) at various sound powers (0, 10, 13, 15, 17, 20, 25, 30 W). Scanning electron microscopy (SEM) is used to examine film thickness and microfibrillar structure in crosssection. Fig. 4A and Fig. 4B show cross-section SEM images of a control silk-fibroin film. It should be noted that the maximum acoustic power was constrained by fluid surface stability, where splattering would occur above the maximum power used for each frequency. Bright field reflection (BFR) and cross-polarized macroscopic images are shown in Figure 5. All films appear optically clear in BFR with few observable defects, with the exception of films dried under 1.5 kHz, which formed three-dimensional structures on the surface. Polarized light microscopy (BFT-XP) was performed to examine the optical properties and organization of the fibril networks. As expected, the control film did not display any birefringence. Films formed under acoustic frequencies between 1.0 - 2.0 kHz showed distinct areas of low-level birefringence, indicating that the protein assembly in these regions was significantly affected as a result of the acoustic wave, causing them to assemble into a minimally aligned structure with a variation in refractive index. At low relative acoustic powers, all three frequency groups appeared grayish blue and semi-opaque with no distinct pattern apparent. At increased powers, all films became optically clear along the outer region with low-level birefringence (grayish blue) at the center of the film. Additionally, these regions in birefringence occur in a pattern that closely resembles the modal pattern of the standing wave, suggesting that the microstructure in these regions is weakly aligned around the antinodes of the wave field, and otherwise un-ordered in the nodes. Interestingly, the shape of these birefringent regions become more defined with increased relative powers. This can be seen in the 2.0 kHz frequency group, where the birefringent pattern is maintained over a larger area at 30 W power.

[0130] To investigate the secondary structure, FTIR mapping was conducted. The patterns of the high-power films seen under cross-polarized light can be recognized in the secondary structure distribution, however it does not clearly follow the specific pattern, as shown in Fig. 6A, Fig. 6B, and Fig. 6C. It should be noted that this observation may be due to an insufficient resolution of the heat map. As expected, the control fdm had the lowest average P-sheet content (16 ±1.6%). The secondary structure measurements of birefringent regions were determined by superimposing each heat map over each BFT-XP image and using the data that corresponded to the grayish blue regions. [0131] For the 2.0 kHz group, 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%). For the 1.5 kHz group, 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.

[0132] However, examination of the secondary structure distribution shows a heterogenous distribution of crystallinity. Films in the 2.0 kHz group exhibiting birefringent patterns (17 W, 20 W, 25 W) showed the most distinct localized regions of higher P-sheet content (22 ± 3.7%, 22 ± 3.2%, 24 ± 3.1%) in the region of birefringence, while the regions not exhibiting birefringence showed lower P-sheet content (16 ± 1.6%, 19 ± 1.5%, 18 ± 1.65%). Films in the 1.5 kHz group exhibiting birefringent patterns (13 W, 15 W, 17 W) did not show a significant difference in P-sheet content in the region exhibiting birefringence (28 ± 2.4%, 24 ± 1.8%, 24 ± 2.6%) in comparison to regions not exhibiting birefringence (27 ± 1.5%, 24 ± 1.1%, 23 ± 1.7%). Films in the 1.0 kHz group exhibiting regions of birefringence (13 W, 15 W) also showed increased P-sheet content (25 ± 2.6%, 23 ± 2.3%) in the region of birefringence, while non-birefringent regions showed lower P-sheet content (21 ± 1.5%, 20 ± 2.2%). This suggests that the assembly processes within the antinode of the acoustic field led to ordered crystalline structures, while the nodal regions maintained a random less ordered state.

[0133] Cross-section SEM images of a film dried under an acoustic field (2.0 kHz, 20 W) show a spatial variation in morphology, shown in Fig. 7A - Fig. 7E. The birefringent region is thicker than the nonbirefringent region (~64 pm, ~92 pm, respectively), suggesting an uneven evaporation rate that was higher toward the center. This is likely due to increased fluid surface area caused by the deformation of the standing wave. Additionally, the non-birefringent area exhibits a uniform structure through the depth of the film, assuming the irregularities are a result of the fracture process. In contrast, the birefringent region exhibits two zones of distinct morphologies that change within the depth of the film. The bottom of the film that was in contact with the Petri dish shows a smooth and homogenous structure, similar to the control film, while the top of the film that was exposed to air has a non-uniform “skin” layer that is ~ 10 pm thick.

[0134] Broadly, the antinode regions of a standing wave are spatially locked but experience high temporal displacement and large mechanical stresses in those localized regions. During the sound- mediated assembly process, 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.

[0135] Acoustically directed secondary structure formation

[0136] 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. To further evaluate how acoustic field exposure affects the assembly of silk films, the 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.

[0137] Bright field reflection microscopy, polarized light microscopy, and macroscopic images were used as described previously to assess the structure and optical properties of silk films with different concentrations. All films appear optically clear in BFR; however, it is important to note that the high concentration films (11% w/w) experienced significant edge contraction during drying, causing the formation of wrinkles on the surface of the film. The reason for this contraction was attributed to the variation in drying rate between the center and outer edges.

[0138] As expected, the control films do not display birefringence under polarized light. In contrast, 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. At low concentrations, films dried under low power exhibit no birefringence. However, at increased powers, 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. Similar birefringent patterns, as well as increased pattern size and brightness, were observed in 9.0% w/w films fabricated under the acoustic field. When the highest concentration solution was exposed to the acoustic field, the birefringent area becomes both brighter and encompasses a majority of the film. This indicates that the pattern of acoustic field was maintained throughout the fabrication process and had a similar effect on the assembly organization across these concentrations. Closer proximity of the proteins as a result of the increased concentration may have allowed for more protein - protein interactions within the antinodes of the field, leading to variations in size and brightness of the pattern.

[0139] 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%). These results support the notion that concentration (water content) is a primary driver of P-sheet formation, as a consequence of stronger hydrophilic interactions. 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.

[0140] Conclusions

[0141] This strategy provides considerable evidence for the ability of acoustic fields to spatially direct the self-assembly of silk fibroin at both the nano and micro scales. Protein assembly in the presence of acoustic wave fields led to spatial variations in fibril network organization, secondary structure, and thickness that were not observed in control films. Specifically, these effects were localized to a distinct region of each film and varied with the mode and power of the standing wave, especially when the solution experienced maximum displacement at high acoustic powers.

[0142] Changing the initial concentration of silk films modified by an acoustic field provides an additional degree of freedom to control conformational protein structure while maintaining the spatial organization induced by the acoustic field. These changes in secondary structure were not observed in control films up to 9.0% w/w, suggesting that the presence of the acoustic field spatially facilitates the formation of intramolecular beta sheet structures. Additionally, the variation crystallinity observed between the regions of birefringence suggests that the molecular mobility of the proteins is increased by the constructive interference in the antinodes (center) but is not increased in the regions of the nodes (outer boundary). This local increase in [3-sheet content may be a result of a similar mechanism to that of shear induced ordering.

[0143] The ability to spatially control protein assembly through the manipulation of acoustic waves presents an innovative and promising pathway to the design and fabrication of hierarchal materials with unique spatial organization. This demonstrates a method to generate ordered crystalline films, with control over nano, micro, and macro scale assembly processes.

[0144] Materials and Methods

[0145] Regeneration of Silk Fibroin

[0146] 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. A 9.3 M lithium bromide (LiBr) solution was added to dissolve the fibers using a ratio of 1 :4 and kept in an oven at 60° C for 1 hour or until the silk was completely dissolved. This solution is then dialyzed in DI water for 48 hours using dialysis tubes (Fisherbrand, MWCO 3.5K) to remove the LiBr salt over 6 DI water changes. Solid impurities were then removed from the aqueous fibroin solution through two rounds of centrifugation at 10,200 rpm for 20 minutes. The concentration of the solution was varied through evaporation or DI water dilution to final concentrations between 6% - 11 % w/w before it was stored in a refrigerator at 4 °C. [0147] Film Fabrication in Acoustic Standing Wave Field

[0148] The film fabrication method using an acoustic wave field is shown in Figure 2A and Fig. 2B. To induce an acoustic standing wave in the silk solution, a wave driver (PUI Audio, ASX05408-HD- R) was mounted face up on a vibration-damping platform. A 3.5 cm polystyrene Petri dish was mounted to the wave driver using thin high-performance adhesive tape (2-5-F9473PC, 3M™) 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.

[0149] Optical Characterization

[0150] Bright field reflection microscopy (BFR) and polarized light microscopy (BFT-XP) were performed with an upright microscope (BX51 , Olympus) that was equipped with a polarizer and an analyzer. 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. For optical characterization, 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. It is a useful tool to infer molecular rearrangement and fibril alignment, as higher orders are attributed to increased fibril orientation. Polarized light is extinguished in optically isotropic materials and double refracted into ordinary and extraordinary rays in anisotropic materials. As polarized white light passes through an anisotropic sample, the phase difference between the fast and slow directions produces interference colors. The localization and quantification of birefringence may infer the spatial distribution of the acoustic forces and its effect on fibril network formation.

[0151] Secondary Structure Analysis

[0152] Fourier-transform infrared spectroscopy (FTIR) analysis was performed with an FTIR microscope (Hyperion 2000, Bruker), equipped with a diamond attenuated total reflectance (ATR) crystal. For each measurement, 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).

[0153] Morphological Characterization

[0154] Scanning electron microscopy (SEM) is used to examine the film thickness and microfibrillar structure in cross-section. Fracture of the samples allows exposure of the cross-section. Each film is sputtered with a 5-10 nm thick gold layer using an EMS 300T D Dual Head Sputter Coater. SEM was performed using a scanning electron microscope (EVO MAIO, Zeiss, Germany) at EHT = 10 kV and work distance ranging from 5 mm to 10 mm.

[0155] Preparation and Fabrication of Silk Fibroin Films Using Acoustic Waves

[0156] 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.

[0157] EQUIVALENTS AND SCOPE

[0158] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combinations (or subcombinations) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

CLAIMS What is claimed is:

1. A method of acoustically-mediated assembly of silk fibroin materials, the method comprising the following steps: a) applying a predetermined acoustic pattern to a silk fibroin solution; and b) solidifying the silk fibroin solution into a silk fibroin article while maintaining application of the predetermined acoustic pattern, wherein the silk fibroin article has a pattern of protein alignment that mimics the predetermined acoustic pattern.

2. The method of claim 1 , wherein the predetermined acoustic pattern has nodes and antinodes, wherein the silk fibroin article has birefringence at locations corresponding to the antinodes and lacks birefringence at locations corresponding to the nodes.

3. The method of claim 1 or 2, wherein the predetermined acoustic pattern is generated by a wave driver.

4. The method of the immediately preceding claim, wherein the wave driver has vibrational damping mounting.

5. The method of any one of the preceding claims, wherein the applying of step a) including applying the predetermined acoustic pattern from beneath the silk fibroin solution.

6. The method of any one of the preceding claims, wherein the predetermined acoustic pattern has an acoustic intensity below a solution disruption threshold, above which the predetermined acoustic pattern disrupts a surface of the silk fibroin solution.

7. The method of any one of the preceding claims, wherein the predetermined acoustic pattern is a standing wave.

8. The method of any one of the preceding claims, wherein the predetermined acoustic pattern is applied at a frequency of between 10 Hz and 100 kHz, including but not limited to, between 500 Hz and 10 kHz.

9. The method of any one of the preceding claims, wherein temperature is maintained between 20 °C and 30 °C during the solidifying of step b).

10. The method of any one of the preceding claims, wherein the silk fibroin solution includes silk fibroin in a weight ratio of between 6% (w/w) and 11% (w/w).

11. The method of any one of the preceding claims, the method further comprising the following steps: c) depositing a second silk fibroin solution atop the silk fibroin article; d) applying a second predetermined acoustic pattern to the second silk fibroin solution; e) 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.

12. The method of the immediately preceding claim, wherein the second silk fibroin solution has a different silk fibroin concentration than the first silk fibroin solution.

13. The method of either two of the immediately preceding claims, wherein the second predetermined acoustic pattern is different than the first predetermined acoustic pattern.

14. The method of any one of the preceding claims, wherein the silk fibroin solution is positioned atop a partial silk fibroin article and solidifying the silk fibroin into the silk fibroin article includes expanding the partial silk fibroin article to form the silk fibroin article.

15. A silk fibroin article made by the method of any one of the preceding claims.

16. A silk fibroin article having acoustically-mediated features.

17. A silk fibroin article having a birefringence pattern that mimics a predetermined acoustic pattern.

18. The method of claim 1, wherein the predetermined acoustic pattern has nodes and antinodes, wherein the silk fibroin solution solidifies in regions of high density and low density following the predetermined acoustic pattern.

19. The method of the immediately preceding claim, wherein the predetermined pattern is a standing wave.

20. A method of acoustically-mediated assembly of silk fibroin materials, the method comprising the following steps: a) applying a silk fibroin solution to an article to form a silk fibroin solution coated article; b) applying a predetermined acoustic pattern to the silk fibroin solution coated article; and c) solidifying the silk fibroin solution to form a silk fibroin coated article while maintaining application of the predetermined acoustic pattern, wherein the silk fibroin coated article has a pattern of protein alignment that mimics the predetermined acoustic pattern.

21. The method of any one of claims 1 to 7, wherein the predetermined acoustic pattern is applied at an ultrasound frequency or an ultra low frequency.

22. The method of any one of claims 1 to 14 or 18 to 21, wherein the silk solution is a silk foam.