Adhesive sprayable wound dressings used in silk powders

Abstract

An adhesive wound dressing kit includes a silk fibroin powder and a polyphenol powder physically separated from one another and capable of independent solubilization, and present in amounts by weight such that adding a predetermined silk solubilization volume of water to the silk fibroin powder produces a silk fibroin solution and a predetermined polyphenol solubilization volume of water to the polyphenol powder produces a polyphenol solution. Rapidly mixing the silk fibroin solution and the polyphenol solution produces an adhesive wound dressing composition. The adhesive wound dressing composition has a storage modulus and a loss modulus, and the storage modulus is less than the loss modulus.

Description

PATENTAttorney Docket No. T002854 WO -2095.0711ADHESIVE SPRAYABLE WOUND DRESSINGS USED IN SILK POWDERSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 725,892 filed in the U.S. Patent and Trademark Office on November 27, 2024. The foregoing patent application is hereby incorporated by reference in its entirety for all purposes.STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under grants W911NF2120130 and W911NF2220213 awarded by the United States Department of Defense. The government has certain rights in the invention.BACKGROUND

[0003] The traditional method for spray -coating silk products is a solution-based process, in which the molecular interactions of hydrophobic and hydrophilic segments of silk with water in regenerated silk solutions are modified to promote the reassembly of silk protein chains through the aid of solvents such as polyethylene glycol (PEG). The process may result in spray formulations having a shelflife. A need exists for new processes overcoming these challenges.SUMMARY

[0004] In some aspects, the techniques described herein relate to an adhesive wound dressing kit including: a silk fibroin powder; and a polyphenol powder, wherein the silk fibroin powder and the polyphenol powder are physically separated from one another and capable of independent solubilization, wherein the silk fibroin powder and the polyphenol powder are present in amounts by weight such that adding a predetermined silk solubilization volume of water to the silk fibroin powder produces a silk fibroin solution and a predetermined polyphenol solubilization volume of water to the polyphenol powder produces a polyphenol solution, wherein rapidly mixing the silk fibroin solution and the polyphenol solution produces an adhesive wound dressing composition, wherein the adhesive wound dressing composition has a storage modulus and a loss modulus, and wherein the storage modulus is less than the loss modulus.PATENT Attorney Docket No. T002854 WO -2095.0711

[0005] In some aspects, the techniques described herein relate to a refill cartridge for a spray device that dispenses an adhesive wound dressing including: a silk fibroin reservoir containing a silk fibroin powder; and a polyphenol reservoir containing a polyphenol powder, wherein the silk fibroin powder and the polyphenol powder are present in amounts by weight such that adding a predetermined silk solubilization volume of water to the silk fibroin powder produces a silk fibroin solution and adding a predetermined polyphenol solubilization volume of water to the polyphenol powder produces a polyphenol solution, wherein the silk fibroin reservoir has sufficient volume to dissolve the silk fibroin powder in the predetermined silk solubilization volume of water, wherein the polyphenol reservoir has sufficient volume to dissolve the polyphenol powder in the predetermined polyphenol solubilization volume of water, wherein the silk fibroin powder and the polyphenol powder are capable of independent solubilization, wherein rapid mixing of the silk fibroin solution and polyphenol solution produces an adhesive wound dressing composition, wherein the adhesive wound dressing composition has a storage modulus and a loss modulus, and wherein the storage modulus is less than the loss modulus.

[0006] In some aspects, the techniques described herein relate to an adhesive wound dressing kit including: a silk fibroin solution; and a polyphenol solution, wherein the silk fibroin solution and the polyphenol solution are physically separated from one another, wherein rapidly mixing a predetermined silk volume of the silk fibroin solution and a predetermined polyphenol volume of the polyphenol solution produces an adhesive wound dressing composition, wherein the adhesive wound dressing composition has a storage modulus and a loss modulus, and wherein the storage modulus is less than the loss modulus.

[0007] In some aspects, the techniques described herein relate to an adhesive wound dressing spray device including: a silk fibroin reservoir containing a silk fibroin solution; a polyphenol reservoir containing a polyphenol solution; a fluid pathway including a rapid shear mixing chamber and a rapid expansion chamber; and a pump, wherein the pump is operatively coupled to the silk fibroin reservoir, the polyphenol reservoir, the shear mixing chamber, and the rapid expansion chamber, such that when the pump is activated, the spray device does the following: rapidly mixes the silk fibroin solution and the polyphenol solution to provide an adhesive wound dressing composition; sprays the adhesive wound dressing composition to a target location, wherein placing a wound at the target location results in application of the adhesive wound dressing composition to the wound, wherein the adhesive wound dressing composition has a storage modulus and a loss modulus, and wherein the storage modulus is less than the loss modulus.PATENT Attorney Docket No. T002854 WO -2095.0711

[0008] 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.

[0009] 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

[0010] The disclosure and the following detailed description of certain embodiments thereof may be understood by reference to the following figures:

[0011] Fig. 1 shows SDS polyacrylamide gel electrophoresis (PAGE) images of the silk fibroin dissolved with 30-, 60- and 120-minute boiled degummed silk fibers. The two wells for 30, 60 and 120 minutes in the gel corresponds to images at different concentrations.

[0012] Fig. 2A depicts the flow of adhesive silk / TA coatings prepared with LSP and TA powders using a double-chamber spray device.

[0013] Fig. 2B depicts adhesive silk / TA glue gels with high extensibility.

[0014] Fig. 3A-Fig. 3H shows rheology properties of silk / TA spray-on coatings. Viscosity measurements at the shear rate for silk / TA spray-on coatings with silk concentrations of 6% (Fig. 3 A) and 10% (Fig. 3E). Dynamic amplitude sweeps of the silk / TA spray-on coatings with silk concentrations of 6% (Fig. 3B) and 10% (Fig. 3F), sweeps were performed at 1 Hz with a strain range of 0. 1 to 1000%. Dynamic angular frequency sweeps of the silk / TA spray- on coatings with silk concentrations of 6% (Fig. 3C) and 10% (Fig. 3G), sweeps were performed at 1% strain with angular frequencies ranging from 0.1 to 10 rad / s. Time sweeps of the silk / TA spray-on coatings with silk concentrations of 6% (Fig. 3D) and 10% (Fig. 3H). Sweeps were performed with 1% strain at 1 Hz for 600 s. G' and G" represent storage modulus and loss modulus, respectively.

[0015] Fig. 4 shows an adhesion lap-shear curve of silk / TA spray-on coating.

[0016] Fig. 5 shows the solubility of lyophilized SF120 and SF30 powders with vary ing CaCh content.

[0017] Fig. 6 shows as-sprayed SF30 / TA spray-on coatings with varying CaCh content. The concentrations of CaC12-SF30 solution and TA solution were 5% and 10%, respectively.PATENT Attorney Docket No. T002854 WO -2095.0711

[0018] Fig. 7 shows a digital photograph of the crosslinked silk-TA spray-on hydrogel.

[0019] Fig. 8 shows a photographic flow of adhesion of the crosslinked silk-TA hydrogel to porcine skin (specimen width: 30 mm).

[0020] Fig. 9 shows adhesive performance of the crosslinked silk-TA hydrogel on moist porcine skin, including lap-shear and T-peel test configurations, representative forcedisplacement curves, and quantified shear strength and interfacial toughness (n = 5 independent samples).DETAILED DESCRIPTION

[0021] 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.

[0022] 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.

[0023] 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).

[0024] 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 thePATENTAttorney Docket No. T002854 WO -2095.0711 combination of entities result in physical admixture, that is, combination as separate coentities 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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. mon. Organic silkworm cocoons are also commercially available. There are many different silks, however, including spiderPATENTAttorney Docket No. T002854 WO -2095.0711 silk (e.g., obtained f omNephila clavipcs). 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.

[0029] The traditional method for spray-coating silk products is a solution-based process, in which the molecular interactions of hydrophobic and hydrophilic segments of silk with water in regenerated silk solutions are modified to promote the reassembly of silk protein chains through the aid of solvents such as polyethylene glycol (PEG). Described herein is a novel method to fabricate a silk-based spray-coating process starting from solid powders. This is a major improvement over prior methods, as silk powders are stable in storage for extended time frames, thus, the spray formulations can be utilized without concern for shelflife or environmental conditions. The materials can be constituted on-site in the field, by just adding water for on-demand use. Further, the use of tannic acid to drive the solidification process provides an antimicrobial feature. Finally, this is a method to provide readily sprayable coatings, bandages and dressings for wounds (internal and external), bums, tissue treatments, internal hemorrhage control, organ treatments and skin related coatings - with broad utility for consumer protection, professional field use, agricultural spray-on coatings, operating room use and many other indications.

[0030] Broadly, methods of making the adhesive spray able wound dressing described herein include three processing steps: 1) Preparation of lyophilized silk powders (LSPs); 2) Fabrication of silk spray product by using a two-chamber spray device with a combination of the LSPs and tannic acid (TA) powder; 3) simply and quickly dissolving LSPs and TA powder in distilled water before spraying. The properties of silk-based spray-coating process including the spray ability, adhesion, mechanical strength, and morphology, can be further optimized by modulating the concentrations or molecular weight of the dissolved silk solution and TA solution before spraying. This new approach allows the long-term shelfstorage of silk-based spray products during transportation, field carry and use, and other storage needs, meeting the requirements of emergency management for consumer or professional wounds and bums. The spray can consist of the silk matrices as the major dressing outcome, with antimicrobial features due to the presence of the TA. Other drugs (e.g., analgesics, vascular enhancers, wound promoting enhancers, etc.) can also be added into the powders for reconstitution on-site to provide additional functions to the dressings

[0031] The adhesive wound dressing may be stored in a solid powder state, which facilitates long-term transport or fieldable options at room temperature or related ambientPATENTAttorney Docket No. T002854 WO -2095.0711 temperatures. This new spray formulation consists of two powders, silk and tannic acid, which are stored separately in a traditional two-chamber spray bottle. Prior to use, these powders are simply dissolved in water to obtain a fresh solution of precursors to form an adhesive coating. In contrast to the previous method of storing the silk solution in spray bottles, which required refrigeration for preservation and posed transportation challenges, as well as limited the lifetime of use, the new formulation is not limited by any of these factors. Hence, the proposed new spray formulation can be stored at ambient temperature for long period of time. This additional feature facilitates field use, eliminates transportation and stability challenges, and reduces storage costs.

[0032] The composition is biocompatible, and the preparation process is mild. Conventional silk-based spraying methods contain regenerated silk solutions and polyethylene glycerol (PEG) solutions that require adjustment of pH and ionic strength to obtain sprayable, mechanically self-supporting hydrogels or fibers. In contrast, the present technology uses only biocompatible silk and plant-derived TA, followed by the addition of water as the solvent. This new technology has a neutral pH environment during the spraying process and does not require additional salt ions or PEG to induce gelation / fibrillation. Benefiting from the rich polyphenol content of TA, which is capable of binding with silk proteins by hydrogen bonding, this allows an adhesive to form during deposition of the spray, and forms into an adhesive gel-like coating by spraying.

[0033] The silk / TA spray shows an antimicrobial effect without the need for additional antibiotic additives, due to the antibacterial properties of TA. Thus, the new spray technology applicable to emergency antimicrobial management of wounds in the field.

[0034] The addition of calcium salts to the silk as a pre-treatment prior to lyophilization resulted in significantly improved resolubilization of the silk for deployment, resolving long term stability goals for the low-tech spray as it can be prepared in dry silk powder format for years of stability in the field.

[0035] Tannic acid (TA) may be used in hydrogel adhesives due to its poly phenolic structure, which provides strong adhesive properties, biocompatibility, and the ability to form hydrogen bonds. However, alternative compounds can be considered to replace or complement tannic acid in these applications, each offering unique benefits in the development of bio-adhesive materials. The use of tannic acid in hydrogel adhesives can be supplemented or replaced by a variety of other polyphenols, such as catecholamines, gallic acid, Epigallocatechin gallate (EGCG), urushiol, lignin, melanin, and synthetic polyphenols offer a range of adhesive strengths, biocompatibility', anti-bacterial ability', andPATENTAttorney Docket No. T002854 WO -2095.0711 functionalization options, making them versatile alternatives in the design of advanced biomaterials.

[0036] Gallic acid is a polyphenol with multiple hydroxyl groups that can form hydrogen bonds with silk fibroin, potentially offering similar adhesive and mechanical properties. Catecholamines (e.g., Dopamine, L-DOPA), particularly dopamine and L-DOPA, are known for their antibacterial performance and strong adhesive properties due to the presence of catechol groups. These groups can form hydrogen bonds, n-n stacking interactions, and coordination bonds with various surfaces. EGCG found in green tea, has been noted for its antioxidant and adhesive properties. It could be used as an alternative for providing strong binding and biocompatibility. Urushiol, which may be found in plants like poison ivy, contains catechol groups similar to dopamine and can form strong adhesive bonds. It also has the ability to polymerize, providing a durable adhesive matrix. Lignin is a natural, aromatic polymer found abundantly in plant cell walls, providing rich hydroxyl groups in its polyphenolic structure, enables it to form strong interactions with other polymers, making it an attractive candidate for enhancing the properties of hydrogel adhesives. Lignin is also noted for its antimicrobial and antioxidant properties, making it a valuable material in biobased applications. Melanin is a heterogeneous group of natural pigments, primarily composed of polymers derived from the oxidation of various precursor molecules like tyrosine, L-DOPA (L-3,4-dihydroxyphenylalanine). and other phenolic compounds, containing hydroxyl groups, amine groups, and carboxylic acid groups. These functional groups contribute to adhesive properties, radical scavenging ability, and metal -ion chelating capacity of the melanin. Melanin is a highly promising alternative to tannic acid in hydrogel adhesives. It offers strong adhesion, mechanical robustness, biocompatibility, antioxidant protection, and the ability to facilitate antibacterial therapy. Synthetic polyphenols are engineered to have specific functional groups and can be tailored for specific adhesive properties, making them a versatile alternative to tannic acid.

[0037] Aspects of the disclosure herein relate to a spray able adhesive comprising a silk fibroin powder and a polyphenol powder that can be stored in a solid powder state without a need for refrigeration for preservation, which facilitates long-term transport or fieldable options at room temperature or related ambient temperatures. An example of the disclosed spray formulation includes two powders, silk and tannic acid, which are stored separately, such as in a traditional two-chamber spray bottle. Prior to use, these powders are dissolved in an aqueous solvent, such as water, to obtain a fresh solution of precursors to form an adhesive coating. The new' spray formulation can be stored at ambient temperature for a long period ofPATENTAttorney Docket No. T002854 WO -2095.0711 time. This additional feature facilitates field use, eliminates transportation and stability challenges (which extends the lifetime of use), and also reduces storage costs.

[0038] The spray formulation is biocompatible, and the preparation process is mild. Conventional silk-based spraying methods contain regenerated silk solutions and polyethylene glycerol (PEG) solutions that require adjustment of pH and ionic strength to obtain sprayable, mechanically self-supporting hydrogels or fibers. In contrast, the present technology uses only biocompatible silk and plant-derived tannic acid, followed by the addition of water as the solvent. This new technology has a neutral pH environment during the spraying process and does not require additional salt ions or PEG to induce gelation / fibrillation. Benefiting from the rich polyphenol content of tannic acid, which is capable of binding with silk proteins by hydrogen bonding, this allows an adhesive to form during deposition of the spray, and forms into an adhesive gel-like coating by spraying.

[0039] The spray formulation possesses an effective antimicrobial effect without antibiotic additions. The silk / tannic acid spray shows an antimicrobial effect without the need for additional antibiotic additives, due to the antibacterial properties of tannic acid. Thus, the new spray technology may be applicable to emergency antimicrobial management of wounds in the field.

[0040] The spray formulation may be deployed without specialized equipment, electronics or related needs. In some aspects, only a simple pump action, as with a perfume bottle, is sufficient to generate the force to spray out the materials.

[0041] The addition of calcium salts to the silk as a pre-treatment prior to lyophilization resulted in significantly improved resolubilization of the silk for deployment, resolving long term stability goals for the spray able formulation as it can be prepared in dry silk powder format for years of stability in the field.

[0042] Prior work focused on directing mixing tannic acid solution with regenerated silk solution with higher molecular weight silk. Described herein is preparation of a spray able silk / TA solution to form coatings starting from powder format. This format transform utility into extended storage, low cost, easy to use format, versus the prior solution systems.

[0043] The lower MW silk can be readily reconstituted in water back to solution, including at higher concentrations (at least 15 wt%) as described in Example 1. In prior work with silk / tannic acid solutions, regenerated silk solutions was used with a 30-minute boiled degummed silk. They did not prepare materials in a spraying approach using silk powders; everything was solution-based. Higher molecular weight silk (30- and 60-minutes boiled silk) did not demonstrate ease of re-solubilization with low er solubility of 25%.PATENTAttorney Docket No. T002854 WO -2095.0711

[0044] The low molecular weight of silk powders in the present work also decreases clogging risks during the spray process when compared with the solution of silk / tannic acid materials prepared with high molecular weight of silk reported in prior work, thereby improving on-demand, instantaneous spray-ability.

[0045] Apart from the tannic acid as the additional component, PEG was also tested for its sprayabilily with the silk powder (as it was used in our earlier solution-based sprayable systems): however, the PEG system here required low pH, salts or buffers (to optimize ionic strength) to increase silk crosslinking and sprayability, adhesion and mechanical strength. Thus, the use of PEG in these new formulations was significantly less favorable than the use of the tannic acid.

[0046] The addition of calcium salts to the silk as a pre-treatment prior to lyophilization resulted in significantly improved resolubilization of the silk for deployment, resolving long term stability' goals for the low-tech spray as it can be prepared in dry silk powder format for years of stability in the field.

[0047] In an aspect, an adhesive wound dressing kit includes a silk fibroin powder and a polyphenol powder. The silk fibroin powder and the polyphenol powder are physically separated from one another and capable of independent solubilization. The silk fibroin powder and polyphenol powder may be present in amounts by weight such that adding a predetermined silk solubilization volume of water to the silk fibroin powder produces a silk fibroin solution and adding a predetermined polyphenol solubilization amount of water to the polyphenol powder produces a polyphenol solution. Rapid mixing of the silk fibroin solution and the polyphenol solution may produce an adhesive wound dressing composition. The adhesive wound dressing composition has a storage modulus and a loss modulus, and the storage modulus may be less than the loss modulus. Silk and tannic acid spray coatings with silk concentrations of 6% and 10% demonstrate a dominant viscosity behavior and exhibit shear-thinning characteristics. When the storage modulus is lower than the loss modulus which is typical for liquid-like materials, it facilitates efficient mixed-solution spraying.

[0048] In another aspect, a refill cartridge for a spray device that dispenses an adhesive wound dressing composition includes a silk fibroin reservoir containing a silk fibroin powder and a polyphenol reservoir containing a polyphenol powder. The silk fibroin powder and the polyphenol powder are present in amounts such that adding a predetermined silk solubilization volume of water to the silk fibroin powder produces a silk fibroin solution and adding a predetermined polyphenol solubilization volume of water to the polyphenol powder produces a polyphenol solution. The silk fibroin reservoir may have sufficient volume toPATENTAttorney Docket No. T002854 WO -2095.0711 dissolve the silk fibroin powder in the predetermined silk solubilization volume of water. The polyphenol reservoir may have sufficient volume to dissolve the polyphenol powder in the predetermined polyphenol solubilization volume of water. The silk fibroin powder and the polyphenol powder may be capable of independent solubilization. Rapid mixing of the silk fibroin solution and the polyphenol solution may produce an adhesive wound dressing composition. The adhesive wound dressing composition has a storage modulus and a loss modulus, and storage modulus may be less than the loss modulus.

[0049] The adhesive wound dressing kit or the refill cartridge may include an indication of the silk fibroin solubilization volume of water (e.g., a line showing how much water to add). The adhesive wound dressing kit or the refill cartridge may include an indication of the polyphenol solubilization volume of water (e.g.. a line showing how much water to add).

[0050] The adhesive wound dressing kit or the refill cartridge may be used by solubilizing the silk fibroin powder by a method comprising adding the predetermined silk solubilization volume of water to form the silk fibroin solution, solubilizing the polyphenol powder by adding the predetermined polyphenol solubilization volume of water to form the polyphenol solution, rapidly mixing the silk fibroin solution and the polyphenol solution to form the adhesive wound dressing composition, and applying the adhesive wound dressing composition to a wound. The rapid mixing may be under shear force conditions. The rapid mixing may be achieved by drawing a portion of the silk fibroin solution and a portion of the polyphenol solution into a mixing chamber under shear force conditions. The drawing the portion of the silk fibroin solution and the portion of the polyphenol solution may be achieved by pumping fluid out of the mixing chamber to produce a negative pressure therein to initiate the drawing. The pumping fluid out of the mixing chamber may be achieved by a manual pump or non-manual pump. The rapid mixing may be achieved by pushing a portion of the silk fibroin solution and a portion of the polyphenol solution into a mixing chamber under shear force conditions. The pushing the portion of the silk fibroin solution and the portion of the polyphenol solution may be achieved by elevating pressure within the silk fibroin solution and the polyphenol solution to initiate the pushing. Elevating the pressure may be done via a manual pump or a non-manual pump. The method may not use compressed gas or an applied electric field.

[0051] In the adhesive wound dressing kit, refill cartridge, or method of using, the silk fibroin pow der are present in an amount such that the silk fibroin solution includes silk fibroin in an amount by weight of between 2% and 15%. The silk fibroin may be present in an amount by weight of at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at leastPATENTAttorney Docket No. T002854 WO -2095.07117%, at least 8%, at least 9%, or at least 10%. The silk fibroin may be present in an amount by weight of at most 15%, at most 14%, at most 13%, at most 12%, at most 11%, at most 10%, at most 9%, at most 8%, at most 7%, at most 6%, or at most 5%.

[0052] In the adhesive wound dressing kit, refill cartridge, or method of using, the polyphenol power are present in an amount such that the polyphenol solution includes the polyphenol in an amount by weight of between 5% and 20% or between 7.5% and 15%. The polyphenol may be present in an amount by weight of at least 5%, at least 5.5%, at least 6%, at least 6.5%, at least 7%, at least 7.5%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, or at least 13%. The polyphenol may be present in an amount by weight of at most 20%, at most 19%, at most 18%. at most 17%, at most 16%, at most 15%, at most 14%, at most 13%, or at most 12%.

[0053] In the adhesive wound dressing kit, refill cartridge, or method of using, the silk fibroin powder includes one or more solubilizing salts. The solubilizing salts may be present in an amount and relative proportion to provide the solubilizing salt in the silk fibroin solution in an amount by weight of between 2.5% and 25% or between 5% and 20%. The solubilizing salts may be present in an amount by weight of at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, or at least 15%. The solubilizing salts may be present in an amount by weight of at most 25%. at most 24%, at most 23%, at most 22%. at most 21%. at most 20%. at most 19%, at most 18%, at most 17%, at most 16%, or at most 15%.

[0054] In another aspect, an adhesive wound dressing kit includes a silk fibroin solution and a polyphenol solution, the silk fibroin solution and the polyphenol solution are physically separated from one another, rapidly mixing a predetermined silk volume of the silk fibroin solution and a predetermined polyphenol volume of the polyphenol solution may produce an adhesive wound dressing composition, wherein the adhesive wound dressing composition may have a storage modulus and a loss modulus, and the storage modulus may be less than the loss modulus.

[0055] In another aspect, an adhesive wound dressing spray device includes a silk fibroin reservoir containing a silk fibroin solution, a polyphenol reservoir containing a polyphenol solution, a fluid pathway including a rapid shear mixing chamber and a rapid expansion chamber, and a pump. The pump may be operatively coupled to the silk fibroin reservoir, the shear mixing chamber, and the rapid expansion chamber, such that when the pump is activated, the spray device rapidly does the following: rapidly mix the silk fibroin solutionPATENT Attorney Docket No. T002854 WO -2095.0711 and the polyphenol solution to provide an adhesive wound dressing composition and spray the adhesive wound dressing composition to a target location, wherein placing a wound at the target location results in application of the adhesive wound dressing composition to the wound, wherein the adhesive wound dressing composition has a storage modulus and a loss modulus, and the storage modulus is less than the loss modulus.

[0056] The adhesive wound dressing kit comprising a silk fibroin solution and a polyphenol solution and the adhesive wound dressing spray device include a spray distance identifier indicating a desired spray distance or range of desired spraying distances for use of the adhesive wound dressing composition. The adhesive wound dressing kit comprising a silk fibroin solution and a polyphenol solution and the adhesive wound dressing spray device may be used by a method including rapidly mixing the silk fibroin solution and the polyphenol solution to produce an adhesive wound dressing composition and spraying the adhesive wound dressing composition on at least a portion of the wound, thereby forming an adhesive wound dressing on the at least a portion of the wound. Spraying is performed at a predetermined spraying distance from the wound. Rapid mixing is performed under shear force conditions. Drawing a portion of the silk fibroin solution and a portion of the polyphenol solution may be under shear force conditions, such as by pumping fluid out of the mixing chamber to produce a negative pressure therein to initiate the drawing. The pumping fluid out of the mixing chamber may be achieved by a manual pump or a non-manual pump. The rapid mixing may be achieved by pushing a portion of the silk fibroin solution and a portion of the polyphenol solution into a mixing chamber under shear force conditions. The pushing the portion of the silk fibroin solution and the portion of the polyphenol solution may be achieved by elevating the pressure within the silk fibroin solution and the polyphenol solution to initiate the pushing, such as via a manual pump or a non-manual pump. The method may not use compressed gas or an applied electric field.

[0057] In the adhesive wound dressing kit, the adhesive wound spray device, or the method of using, the silk fibroin solution includes silk fibroin in an amount by weight of between 2% and 15%. In the adhesive wound dressing kit, adhesive wound spray device, or method of using, the polyphenol powder may be present in an amount such that the polyphenol solution includes the polyphenol in an amount by weight of between 5% and 20% or between 7.5% and 15%. In the adhesive wound dressing kit, adhesive wound spray device, or method of using, the polyphenol may include a polyphenol selected from a group consisting of tannic acid, gallic acid, catecholamines (e.g.. dopamine and L-DOPA). epigallocatechin gallate, urushiol, lignin, melanin, a synthetic polyphenol (e.g., polyvinyl catechol), and combinationsPATENTAttorney Docket No. T002854 WO -2095.0711 thereof as described herein. The polyphenol powder and / or the polyphenol solution may include tannic acid.

[0058] In the adhesive wound dressing kit, the adhesive wound spray device, or the method of using, the silk fibroin powder and / or the silk fibroin solution includes silk fibroin having a molecular weight distribution consistent with a degumming boil time of between 15 minutes and 6 hours, including but not limited to, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 1 hour, at least 75 minutes, at least 90 minutes, or at least 2 hours and at most 6 hours, at most 5 hours, at most 4 hours, at most 3 hours, at most 2 hours, at most 90 minutes, at most 75 minutes, at most 70 minutes, at most 65 minutes, at most 1 hour, at most 55 minutes, at most 50 minutes, at most 45 minutes, at most 40 minutes, at most 35 minutes, or at most 30 minutes. In the adhesive wound dressing kit, adhesive wound spray device, or method of using, the silk fibroin powder includes silk fibroin having a molecular weight distribution consistent with a degumming boil time of between 15 and 60 minutes or between 15 minutes and 30 minutes. In the adhesive wound dressing kit. adhesive wound spray device, or method of using, the silk fibroin powder and / or silk fibroin solution includes a silk fibroin having a weight average molecular weight of 160 kDa or less, 150 kDa or less, 125 kDa or less, 120 kDa or less, 110 kDa or less, 100 kDa or less, 90 kDa or less, 80 kDa or less, 75 kDa or less, 60 kDa or less, 50 kDa or less, or 40 kDa or less. In the adhesive wound dressing kit. the adhesive wound spray device, or the method of using, the polyphenol powder and / or the polyphenol spray is antimicrobial. In the adhesive wound dressing kit, adhesive wound spray device, or method of using, the adhesive wound dressing composition is biocompatible. In the adhesive wound dressing kit, adhesive wound spray device, or method of using, the adhesive wound dressing composition may lack a (3-sheet inducing additive (e.g., polyethylene glycol). In the adhesive wound dressing kit, adhesive wound spray device, or method of using, the adhesive wound dressing composition may have a pH between 5 and 9 or between 6 and 8. The pH may be at least 5, at least 5.5, at least 6, or at least 6.5. The pH may be at most 9, at most 8.5. at most 8, or at most 7.5.

[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 silk-based films, etc.). It should be understood that the examples herein may recite one or a few silk-containing embodiments but are applicable toPATENTAttorney Docket No. T002854 WO -2095.0711 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 megakary ocytes 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 / w all and / or silk matrix. In some embodiments, a functionalizing agent is distributed in and / or along at least one of the lumen-facing side of a silk wall and the matrix-facing side of a silk wall.PATENT Attorney Docket No. T002854 WO -2095.0711

[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., at most 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,PATENTAttorney Docket No. T002854 WO -2095.0711N,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-methoxybenesulfbnic acid, bromocresol green, resazurin, 4- phenylazo-1- 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) 1- 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'-stilbenedisulfbnic 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] Exemplars- 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 opticalPATENT Attorney Docket No. T002854 WO -2095.0711 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, octy l acetate, benzy l 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 organicPATENTAttorney Docket No. T002854 WO -2095.0711 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.J.), 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 BluePATENT Attorney Docket No. T002854 WO -2095.071193 (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 (ty pically 400 nm and above). Examples of ETV fluorophores include but are not limited to materials from the coumarin, benzoxazole, rhodamine, napthalimide, peiylene, 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 / pigmentPATENTAttorney Docket No. T002854 WO -2095.0711 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 w eight 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% whi te pigment, or 15 wt% white pigment, or 10 wt% whi te pigment, based on the w eight of the composition. In some applications, a w hite 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 w eight of the composition. In some applications, a non-whi te 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 w t%, 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 w eight 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 compoundsPATENT Attorney Docket No. T002854 WO -2095.0711 that can be incorporated into the composition provided herein include, without limitation, anticancer agents, antibiotics, analgesics, anti-inflammatory 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 silkPATENT Attorney Docket No. T002854 WO -2095.0711 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 silkbased drug delivery' composition can contain one therapeutic agent or combinations of tw o or more therapeutic agents.PATENT Attorney Docket No. T002854 WO -2095.0711

[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. Exemplary7cellular 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 viability7of 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., w estern 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 / orPATENT Attorney Docket No. T002854 WO -2095.0711 the abi 1 i ty to neutralize or antagonize the bioacti vity 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 “bioacti vity ” includes immunogenicity7, the definition of which is discussed in detail later. In reference to a virus, the “bioactivity” includes infectivity7, 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 bioactivity7of 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 product-like,” however, the term “small molecule” is not limited to “natural product-like” compounds. Rather, a small molecule is ty pically 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 Formulary7, 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 ordinary7skill 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 enzy me inhibitors, a beta-blocker, aPATENT Attorney Docket No. T002854 WO -2095.0711 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; betablockers (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, acetvlsalicylic acid, and ticlopidine; sedatives such as benzodi azapines and barbiturates; ansiolytic agents such asPATENTAttorney Docket No. T002854 WO -2095.0711 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, flutamide. 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, famesyltransferase inhibitors, pump inhibitors, histone acetyltransferase inhibitors, metalloproteinase inhibitors, ribonucleoside reductase inhibitors, TNF alpha agonists / antagonists, endothelinA receptor antagonists, retinoic acid receptor agonists, immuno-modulators, 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, cioxacillin, 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 inhibitorPATENT Attorney Docket No. T002854 WO -2095.0711I, 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 - isobuty l- 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, pheny butazone, 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, amitripty line, nortripty line, protriptyline, desipramine, amoxapine, doxepin. maprotiline, tranylcypromine, phenelzine, and isocarboxazide.PATENTAttorney Docket No. T002854 WO -2095.0711

[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), neutrophil-activating 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 grow th 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 rcgrowth 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.PATENT Attorney Docket No. T002854 WO -2095.0711

[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.PATENT Attorney Docket No. T002854 WO -2095.0711 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 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 w hich are not clear to persons ofPATENT Attorney Docket No. T002854 WO -2095.0711 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 inPATENT Attorney Docket No. T002854 WO -2095.0711 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] Preparation of regenerated silk powder

[0130] Regenerated silk fibroin solution was prepared using the protocol described in Biomacromolecules 27, 7 (2020), 2829-2843, which is herein incorporated by reference in entirety. Briefly, B. mori cocoons (Tajima Shoji Co., Ltd, Yokohama, Japan) were degummed by boiling in 0.02 M sodium carbonate (NazCCh, Sigma-Aldrich, USA) solution for 120 minutes. The degummed silk fibers were rinsed in distilled water to remove the residual Na2COs and then dried at room temperature. The degummed silk fibers were dissolved in 9.3 M lithium bromide solution (LiBr, Sigma-Aldrich, USA) at 60 °C for 4 hours and then dialyzed in distilled water for 3 days (MWCO: 3,500 kDa, Spectra / Por3 Standard RC tubing, Spectrum Laboratories, USA). The dialyzed silk solution was then centrifuged twice at 9,000 rpm for 20 minutes each time to obtain a silk solution with a protein concentration of approximately 6% (wi / wt). The solution was diluted and frozen in liquid nitrogen. The frozen silk solution was then lyophilized at -80 °C and 0.006 bar until complete sublimation. The lyophilized silk was milled into fine pow ders using a high-speed analytical mill (20,000 rpm, 2 min, Col-Parmer), giving the product referred to as lyophilized silk powders (LSP). The LSPs were stored in ambient dry conditions to prevent any rehydration until use.

[0131] CaCh with varying concentrations was uniformly mixed with the silk fibroin solutions dissolved by using 30 min boiled degummed silk fibers to prepare CaC12-SF30 solutions. Similar to the LSP, frozen CaCh-SF30 solutions were lyophilized to prepare CaCh-SF30 powders.PATENT Attorney Docket No. T002854 WO -2095.0711

[0132] Crosslinked tannic-acid-modified silk powder (Silk-TA powder) was prepared starting from regenerated silk fibroin aqueous solutions. Regenerated silk fibroin solutions were first prepared as described above with concentrations in the range of 2-10 wt%. Tannic acid (TA) was dissolved in water at room temperature to obtain TA solutions with concentrations between 5-20 wt%. The silk and TA solutions w ere mixed at controlled volume ratios in the range of 1: 1 to 10: 1 (silk solution : TA solution) to obtain reaction mixtures. The mixed silk-TA solutions were incubated at an elevated temperature in the range of 40-80 °C for 2-24 h to form a crosslinked Silk-TA hydrogel or gel-like solid. After incubation, the Silk-TA gels were frozen and lyophilized to obtain dry Silk-TA solids. The dried material was subsequently milled using a mechanical mill to produce Silk-TA powder with particle sizes suitable for spraying.

[0133] Preparation of spray

[0134] A two-chamber spray bottle (volume 50 mL / chamber, material: polyethylene terephthalate (PET), 2 mm of inner diameter of rubber tube in each chamber was connected with 90° elbow connectors, diameter of spray nozzle: 2 mm) was used to perform silk-based spray able coatings. LSP and TA in distilled water, 6% and 10% (w / v) LPS solution and 10% (w / v) TA solution were obtained in each chamber, respectively. The adhesive silk / TA coatings w ere instantly fabricated upon pressing the atomizer cap of the spray device. Lyophilized CaCb-SF30 powders with various content of CaCh were dissolved in distilled water to generate the CaCh-SF30 solutions with concentrations of 5% (w / v). 5% (w / v) CaC12-SF30 solution and 10% (w / v) TA solution were prepared for the spray.

[0135] For preparation of the spray able product, the lyophilized crosslinked Silk-TA powder w as loaded into one chamber of a dual-chamber spray bottle. The second chamber was filled with water or an aqueous hybrid solution containing optional functional additives. The amounts of Silk-TA powder and aqueous solution were selected such that, upon actuation of the spray device, the mixed outlet stream provided an effective Silk-TA concentration appropriate for forming an adhesive coating on tissue or skin surfaces. The two chambers remained physically separated during storage, and mixing occurred only at the spray nozzle during device actuation.

[0136] Analysis of molecular weight (MW) distribution

[0137] MW distribution of the silk constructs w as semi-quantified with digital images of SDS-PAGE outcomes. A total of 20 pL of sample and 10 pL of NOVEX sharp pre-stained protein standard were run in NuPAGE 4-12% Bis-Tris gels (Invitrogen, Carlsbad, CA) at 200 V for 30 min and stained with colloidal blue stain solution for 4 hours. After rinsingPATENTAttorney Docket No. T002854 WO -2095.0711 overnight in DI water, gels were imaged and the molecular weight distributions were determined using ImageJ (1.48v, NIH. USA). Briefly, a calibration curve was prepared by the digital analysis of the reference ladder lane to obtain a linear equation for conversion of the distance from wells to MW values. Frequency of each MW was then calculated from the mean gray values, and MW distributions were plotted for each sample to determine the mean and peak molecular weights of the silk solutions.

[0138] Rheology

[0139] The rheology' measurements were performed using HR 20 rheometer (TA Instruments, USA), equipped with a parallel stainless-steel plate (20 mm diameter). The temperature was maintained at 25 °C. The rheological properties associated with angular frequencies ranging from 0. 1 to 10 rad s ' were investigated for stresses fixed at 1%. Straindependent oscillations were measured at 1 Hz over a range of shear strains from 0. 1% to 1000%. A dynamic time sweep was performed at 1 Hz with a 1% applied strain for 600 s to determine the stability of the sprayed materials. The viscosity used to characterize the adhesiveness of the materials was measured over a range of shear rates from 0. 1 to 1000 s '.

[0140] Adhesion strength

[0141] The in vitro tissue adhesion lap-shear strength (ASTM F2255-05) tests were performed using a universal testing machine (Instron 5565, USA). The fat under the porcine skins was removed before the measurements. Then, the silk / TA coatings were applied to the overlapping area of the two pieces of porcine skin (width of 15 mm, length of 10 mm) and allowed to be cured. All experiments were conducted in air at 25 °C with a constant stretching rate of 10 mm min The adhesion strength was calculated by dividing the pull-off force by the surface area of the tissue.

[0142] Water soluble fraction measurement

[0143] Twenty mg of silk fibroin powder was added into 2 mL of distilled water and incubated at room temperature for 1 h. Then the solutions were centrifuged to separate undissolved powder. The amount of silk protein dissolved in the solution w as w eighed after drying the solution at 60 °C.

[0144] Results

[0145] We obtained different MWs of silk by varying the duration of degumming. Silk solutions were prepared by silk fiber degummed for 30 min, 60 min, or 120 min and their MW distributions were digitally quantified on SDS-PAGE images, as shown in Figure 1. The peak MW of silk fibroin extracted by degumming for 30 min. 60 min, and 120 min were 160 kDa, 110 kDa, and 50 kDa approximately. We found that only LSPs prepared from 120-PATENTAttorney Docket No. T002854 WO -2095.0711 minute boiling degummed silk fibers could be readily dissolved in water at higher concentration due to their relatively low molecular weight compared to LSPs prepared from 30-minute and 60-minute boiled degummed fibers.

[0146] Silk can interact with the polyphenolic groups of TA through hydrogen bonding. Therefore, we investigated an instantaneous sprayable system containing dissolvable silk powder and the antimicrobial TA. As shown in Figure 2A, we developed an adhesive glue coating with interwoven fiber structure based on silk / TA by a facile method using a dual chamber spraying device (Figure 2A), featuring fast gelation, good adhesion, and favorable biocompatibility. The viscosity of the sprayed silk / TA coating was significantly enhanced, which was attributed to the formation of more intermolecular hydrogen bonds by the phenolic hydroxyl groups in TA, thus increasing its viscosity (Figure 2B).

[0147] The viscosity and mechanical properties of the silk / TA spray-on coatings were evaluated by rheological experiments (Figure 3). The viscosity of the silk-10% / TA coating was higher than that of the silk-6% / TA coating (Figure. 3 A, E). The viscosities of both the silk-6% / TA and silk-10% / TA coatings decreased with increasing shear rate, suggesting that silk / TA materials have shear-thinning properties. Additionally, the silk-10% / TA coating showed better stability at higher shear rates, indicating that the mechanical properties were enhanced with increasing silk concentration. Moreover, the storage modulus G' and loss modulus G” of silk-6% / TA (Figure 3B-D) and silk-10% / TA (Figure 3F-H) coatings were evaluated by dynamic frequency sweeps, strain sweeps, and time sweeps. The storage modulus (G) was lower than the loss modulus (G") for all samples, indicating the viscoelastic liquids rheology behavior of the silk / TA spray coatings. In conclusion, the rheology measurements show the shear-thinning and viscoelastic liquid properties of the silk / TA materials, suggesting that the new spray system formulated with silk and TA is sprayable and injectable. The tissue adhesion performance of the sprayed silk / TA hydrogel coating was evaluated using moist pig skin as model tissue. The as-sprayed silk / TA hydrogel coating w as placed between tw o pieces of pig skin, and after 10 minutes of curing, the adhesion strength was measured using lap shear (Figures 4). The shear strength between the SF120-6% / TA hydrogel coating and pig skin was approximately 49.3 kPa.

[0148] Alternative solutions for the solubility improvements of silk powders with high molecular weight

[0149] Previous studies have reported that calcium ions (Ca2+) form metal-chelate complexes with the carboxyl (-COO-), amino (-NH2), and imino (-NH) groups in silk fibroin. This chelation disrupts the regular (3-sheet structures typically observed in silk,PATENTAttorney Docket No. T002854 WO -2095.0711 thereby enhancing solubility by promoting the formation of more amorphous and random coil structures. Additionally, calcium ions exhibit a high affinity for water molecules, leading to increased hydration of the overall silk structure. This enhanced hydration interferes with the formation of hydrogen bonds between silk fibroin chains, thereby reducing the tendency for f> -sheet crystallization and increasing solubility7. Consequently, we added calcium chloride (CaCh) prior to lyophilization of high molecular weight SF30 to explore the potential that this type of pre-treatment would improve the resolubilization of lyophilized silk powders, thus, solubility changes of the dried silk powder.

[0150] We found that when the addition of CaCb reached 5%, the solubility of SF30 powder significantly increased from 20% to 85%. In addition, the solubility of SF30 powder increased gradually as the concentration of CaCb increased, reaching 95% at a CaCh concentration of 20%, which is comparable to that of SF120 without the addition of Cade. Additionally, we found that SF30 / TA and SF120 / TA had similar spraying ability and viscoelastic properties (Figure 6).

[0151] Example 2: Results

[0152] Materials were prepared according to the methods described in Example 1.

[0153] The tissue adhesion performance of the crosslinked Silk-TA sprayed hydrogel (Figure 7) was evaluated using moist porcine skin as a model substrate (Figure 8). The sprayed Silk-TA hydrogel was applied between two pieces of porcine skin, adhesion properties were quantified using standardized lap-shear and T-peel testing methods. As shown in Figure 9, the crosslinked Silk-TA hydrogel exhibited a shear adhesion strength of approximately 43.2 kPa and an interfacial toughness of approximately 408.4 J / m2(n = 5). These results demonstrate strong tissue adhesion and robust interfacial integrity under moist conditions, confirming the suitability of the Silk-TA hydrogel for use as an adhesive tissue coating or dressing.

[0154] EQUIVALENTS AND SCOPE

[0155] 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.PATENTAttorney Docket No. T002854 WO -2095.0711

[0156] In addition to the features described above and elsewhere herein, the present disclosure also includes the following clauses:1. An adhesive wound dressing kit comprising: a silk fibroin powder; and a polyphenol powder, wherein the silk fibroin powder and the polyphenol powder are physically separated from one another and capable of independent solubilization, wherein the silk fibroin powder and the polyphenol powder are present in amounts by weight such that adding a predetermined silk solubilization volume of water to the silk fibroin powder produces a silk fibroin solution and a predetermined polyphenol solubilization volume of water to the polyphenol powder produces a polyphenol solution, wherein rapidly mixing the silk fibroin solution and the polyphenol solution produces an adhesive wound dressing composition, wherein the adhesive wound dressing composition has a storage modulus and a loss modulus, and wherein the storage modulus is less than the loss modulus.2. A refill cartridge for a spray device that dispenses an adhesive wound dressing comprising: a silk fibroin reservoir containing a silk fibroin powder; and a polyphenol reservoir containing a polyphenol powder, wherein the silk fibroin powder and the polyphenol powder are present in amounts by weight such that adding a predetermined silk solubilization volume of water to the silk fibroin powder produces a silk fibroin solution and adding a predetermined polyphenol solubilization volume of water to the polyphenol powder produces a polyphenol solution, wherein the silk fibroin reservoir has sufficient volume to dissolve the silk fibroin powder in the predetermined silk solubilization volume of water, wherein the polyphenol reservoir has sufficient volume to dissolve the polyphenol powder in the predetermined polyphenol solubilization volume of water, wherein the silk fibroin powder and the polyphenol powder are capable of independent solubilization, wherein rapid mixing of the silk fibroin solution and polyphenol solution produces an adhesive wound dressing composition.PATENTAttorney Docket No. T002854 WO -2095.0711 wherein the adhesive wound dressing composition has a storage modulus and a loss modulus, and wherein the storage modulus is less than the loss modulus.3. The kit or refill cartridge of any one of the preceding clauses, wherein the kit or refill cartridge includes an indication of the silk fibroin solubilization volume of water (e g., a line showing how much water to add).4. The kit or refill cartridge of any one of the preceding clauses, wherein the kit or refill cartridge includes an indication of the polyphenol solubilization volume of water (e.g., a line showing how much water to add).5. A method of using the kit or refill cartridge of any one of clauses 1 to the immediately preceding clause, the method comprising: a) solubilizing the silk fibroin powder by adding the predetermined silk solubilization volume of water to form the silk fibroin solution; b) solubilizing the polyphenol powder by adding the predetermine polyphenol solubilization volume of water to form the polyphenol solution; c) rapidly mixing the silk fibroin solution and the polyphenol solution to form the adhesive wound dressing composition; and d) applying the adhesive wound dressing composition to a wound.6. The method of the immediately preceding clause, wherein the rapidly mixing is under shear force conditions.7. The method of clause 5 or 6, wherein the rapidly mixing is achieved by drawing a portion of the silk fibroin solution and a portion of the polyphenol solution into a mixing chamber under shear force conditions.8. The method of the immediately preceding clause, wherein drawing the portion of the silk fibroin solution and the portion of the polyphenol solution is achieved by pumping fluid out of the mixing chamber to produce a negative pressure therein to initiate the drawing.9. The method of clause 8. wherein pumping fluid out of the mixing chamber is via a manual pump.10. The method of clause 8, wherein pumping fluid out of the mixing chamber is via a non- manual pump.11. The method of clause 5 or 6, wherein rapidly mixing is achieved by pushing a portion of the silk fibroin solution and a portion of the polyphenol solution into a mixing chamber under shear force conditions.PATENTAttorney Docket No. T002854 WO -2095.071112. The method of the immediately preceding clause, wherein pushing is initiated by elevating pressure within the silk fibroin solution and the polyphenol solution.13. The method of clause 12, wherein elevating pressure is via a manual pump.14. The method of clause 12, wherein elevating pressure is via a non-manual pump.15. The method of any one of clauses 5 to the immediately preceding clause, wherein the method does not use compressed gas.16. The method of any one of clauses 5 to the immediately preceding clause, wherein the method does not use an applied electric field.17. The kit, refill cartridge, or method of any one of the preceding clauses, wherein the silk fibroin powder is present in an amount such that the silk fibroin solution includes silk fibroin in an amount by weight of between 2% and 15%.18. The kit, refill cartridge, or method of any one of the preceding clauses, wherein the polyphenol powder is present in an amount such that the polyphenol solution includes the polyphenol in an amount by weight of between 5% and 20% or between 7.5% and 15%.19. The kit. refill cartridge, or method of any one of the preceding clauses, wherein the silk fibroin powder comprises one or more solubilizing salts.20. The kit, refill cartridge, or method of the immediately preceding clause, wherein the one or more solubilizing salts includes CaCh.21. The kit, refill cartridge, or method of either of the two immediately preceding clauses, wherein the solubilizing salt is present in an amount and relative proportion to provide the solubilizing salt in the silk fibroin solution in an amount by weight of between 2.5% and 25% or between 5% and 20%.22. An adhesive wound dressing kit comprising: a silk fibroin solution; and a polyphenol solution, wherein the silk fibroin solution and the polyphenol solution are physically separated from one another, wherein rapidly mixing a predetermined silk volume of the silk fibroin solution and a predetermined polyphenol volume of the polyphenol solution produces an adhesive wound dressing composition, wherein the adhesive wound dressing composition has a storage modulus and a loss modulus, and wherein the storage modulus is less than the loss modulus.PATENTAttorney Docket No. T002854 WO -2095.071123. An adhesive wound dressing spray device comprising: a silk fibroin reservoir containing a silk fibroin solution; a polyphenol reservoir containing a polyphenol solution; a fluid pathway including a rapid shear mixing chamber and a rapid expansion chamber; and a pump, wherein the pump is operatively coupled to the silk fibroin reservoir, the polyphenol reservoir, the shear mixing chamber, and the rapid expansion chamber, such that when the pump is activated, the spray device does the following: rapidly mixes the silk fibroin solution and the polyphenol solution to provide an adhesive wound dressing composition; sprays the adhesive wound dressing composition to a target location, wherein placing a wound at the target location results in application of the adhesive wound dressing composition to the wound, wherein the adhesive wound dressing composition has a storage modulus and a loss modulus, and wherein the storage modulus is less than the loss modulus.24. The kit or spray device of either of the two immediately preceding clauses, wherein the kit or spray device includes a spray distance identifier indicating a desired spray distance or a range of desired spraying distances for use of the adhesive wound dressing composition.25. A method of using the kit or spray device of any one of clauses 22 to the immediately preceding clause, the method comprising: rapidly mixing the silk fibroin solution and the polyphenol solution to produce an adhesive wound dressing composition; and spraying the adhesive wound dressing composition on at least a portion of wound, thereby forming an adhesive wound dressing on the at least a portion of the wound.26. The method of the immediately preceding clause, wherein the spraying is performed at a predetermined spraying distance from the wound.27. The method of either of the two immediately preceding clauses, wherein the rapidly mixing is under shear force conditions.28. The method of any one of the three immediately preceding clauses, wherein the rapidly mixing is achieved by drawing a portion of the silk fibroin solution and a portion of the polyphenol solution into a mixing chamber under shear force conditions.PATENTAttorney Docket No. T002854 WO -2095.071129. The method of the immediately preceding clause, wherein drawing the portion of the silk fibroin solution and the portion of the polyphenol solution is achieved by pumping fluid out of the mixing chamber to produce a negative pressure therein to initiate the drawing.30. The method of clause 29, wherein the pumping fluid out of the mixing chamber is via a manual pump.31. The method of clause 29, wherein the pumping fluid out of the mixing chamber is via a non-manual pump.32. The method of any one of clauses 25 to 27, wherein the rapidly mixing is achieved by pushing a portion of the silk fibroin solution and a portion of the polyphenol solution into a mixing chamber under shear force conditions.33. The method of the immediately preceding clause, wherein the pushing the portion of the silk fibroin solution and the portion of the polyphenol solution is achieved by elevating pressure within the silk fibroin solution and the polyphenol solution to initiate the pushing.34. The method of clause 33, wherein the elevating pressure is via a manual pump.35. The method of clause 33, wherein the elevating pressure is via a non-manual pump.36. The method of any one of clauses 25 to the immediately preceding clause, wherein the method does not use compressed gas.37. The method of any one of clauses 25 to the immediately preceding clause, wherein the method does not use an applied electric field.38. The kit. spray device, or method of any one of clauses 22 to the immediately preceding clause, wherein the silk fibroin solution includes silk fibroin in an amount by weight of between 2% and 15%.39. The kit, spray device, or method of any one of clauses 22 to the immediately preceding clause, wherein the polyphenol powder is present in an amount such that the polyphenol solution includes the polyphenol in an amount by weight of between 5% and 20% or between 7.5% and 15%.40. The kit, refill cartridge, spray device, or method of any one of the preceding clauses, wherein the polyphenol comprises a polyphenol selected from a group consisting of tannic acid, gallic acid, catecholamines (e.g., dopamine and L-DOPA), epigallocatechin gallate, urushiol, lignin, melanin, a synthetic polyphenol (e g., polyvinyl catechol), and combinations thereof.41. The kit, refill cartridge, spray device, or method of the immediately preceding clause, wherein the polyphenol powder and / or the polyphenol solution comprises tannic acid.PATENT Attorney Docket No. T002854 WO -2095.071142. The kit, refill cartridge, spray device, or method of any one of the preceding clauses, wherein the silk fibroin powder and / or the silk fibroin solution comprises silk fibroin having a molecular weight distribution consistent with a degumming boil time of between 15 minutes and 6 hours, including but not limited to, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 1 hour, at least 75 minutes, at least 90 minutes, or at least 2 hours and at most 6 hours, at most 5 hours, at most 4 hours, at most 3 hours, at most 2 hours, at most 90 minutes, at most 75 minutes, at most 70 minutes, at most 65 minutes, at most 1 hour, at most 55 minutes, at most 50 minutes, at most 45 minutes, at most 40 minutes, at most 35 minutes, or at most 30 minutes.43. The kit, refill cartridge, spray device, or method of any one of the preceding clauses, wherein the silk fibroin powder comprises silk fibroin having a molecular weight distribution consistent with a degumming boil time of between 15 minutes and 60 minutes or between 15 minutes and 30 minutes.44. The kit, refill cartridge, spray device, or method of any one of the preceding clauses, wherein the silk fibroin powder and / or silk fibroin solution comprises silk fibroin having a weight average molecular weight of 160 kDa or less, 150 kDa or less, 125 kDa or less, 120 kDa or less, 110 kDa or less, 100 kDa or less, 90 kDa or less, 80 kDa or less, 75 kDa or less, 60 kDa or less, 50 kDa or less, or 40 kDa or less.45. The kit, refill cartridge, spray device, or method of any one of the preceding clauses, wherein the polyphenol powder and / or the polyphenol solution is antimicrobial.46. The kit, refill cartridge, spray device, or method of any one of the preceding clauses, wherein the adhesive wound dressing composition is biocompatible.47. The kit, refill cartridge, spray device, or method of any one of the preceding clauses, wherein the adhesive wound dressing composition does not contain a -sheet inducing additive (e.g., polyethylene glycol).48. The kit, refill cartridge, spray device, or method of any one of the preceding clauses, wherein the adhesive wound dressing composition has a pH of between 5 and 9 or between 6 and 8.

[0157] 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

PATENTAttorney Docket No. T002854 WO -2095.0711CLAIMSWhat is claimed is:

1. An adhesive wound dressing kit comprising: a silk fibroin powder; and a polyphenol powder, wherein the silk fibroin powder and the polyphenol powder are physically separated from one another and capable of independent solubilization, wherein the silk fibroin powder and the polyphenol powder are present in amounts by weight such that adding a predetermined silk solubilization volume of water to the silk fibroin powder produces a silk fibroin solution and a predetermined polyphenol solubilization volume of water to the polyphenol powder produces a polyphenol solution, wherein rapidly mixing the silk fibroin solution and the polyphenol solution produces an adhesive wound dressing composition, wherein the adhesive wound dressing composition has a storage modulus and a loss modulus, and wherein the storage modulus is less than the loss modulus.

2. A refill cartridge for a spray device that dispenses an adhesive wound dressing comprising: a silk fibroin reservoir containing a silk fibroin powder; and a polyphenol reservoir containing a polyphenol po der, wherein the silk fibroin powder and the polyphenol powder are present in amounts by weight such that adding a predetermined silk solubilization volume of water to the silk fibroin powder produces a silk fibroin solution and adding a predetermined polyphenol solubilization volume of water to the polyphenol powder produces a polyphenol solution, wherein the silk fibroin reservoir has sufficient volume to dissolve the silk fibroin powder in the predetermined silk solubilization volume of water, wherein the polyphenol reservoir has sufficient volume to dissolve the polyphenol powder in the predetermined polyphenol solubilization volume of water, wherein the silk fibroin powder and the polyphenol powder are capable of independent solubilization, wherein rapid mixing of the silk fibroin solution and polyphenol solution produces an adhesive wound dressing composition.PATENTAttorney Docket No. T002854 WO -2095.0711 wherein the adhesive wound dressing composition has a storage modulus and a loss modulus, and wherein the storage modulus is less than the loss modulus.

3. The kit or refill cartridge of any one of the preceding claims, wherein the kit or refill cartridge includes an indication of the silk fibroin solubilization volume of water (e g., a line showing how much water to add).

4. The kit or refill cartridge of any one of the preceding claims, wherein the kit or refill cartridge includes an indication of the polyphenol solubilization volume of water (e.g., a line showing how much water to add).

5. A method of using the kit or refill cartridge of any one of claims 1 to the immediately preceding claim, the method comprising: a) solubilizing the silk fibroin powder by adding the predetermined silk solubilization volume of water to form the silk fibroin solution; b) solubilizing the polyphenol powder by adding the predetermine polyphenol solubilization volume of water to form the polyphenol solution; c) rapidly mixing the silk fibroin solution and the polyphenol solution to form the adhesive wound dressing composition; and d) applying the adhesive wound dressing composition to a wound.

6. The method of the immediately preceding claim, wherein the rapidly mixing is under shear force conditions.

7. The method of claim 5 or 6, wherein the rapidly mixing is achieved by drawing a portion of the silk fibroin solution and a portion of the polyphenol solution into a mixing chamber under shear force conditions.

8. The method of the immediately preceding claim, wherein drawing the portion of the silk fibroin solution and the portion of the polyphenol solution is achieved by pumping fluid out of the mixing chamber to produce a negative pressure therein to initiate the drawing.

9. The method of claim 8, wherein pumping fluid out of the mixing chamber is via a manual pump.

10. The method of claim 8, wherein pumping fluid out of the mixing chamber is via a non- manual pump.

11. The method of claim 5 or 6, wherein rapidly mixing is achieved by pushing a portion of the silk fibroin solution and a portion of the polyphenol solution into a mixing chamber under shear force conditions.PATENTAttorney Docket No. T002854 WO -2095.071112. The method of the immediately preceding claim, wherein pushing is initiated by elevating pressure within the silk fibroin solution and the polyphenol solution.

13. The method of claim 12, wherein elevating pressure is via a manual pump.

14. The method of claim 12, wherein elevating pressure is via a non-manual pump.

15. The method of any one of claims 5 to the immediately preceding claim, wherein the method does not use at least one of compressed gas or an applied electric field.

16. The kit. refill cartridge, or method of any one of the preceding claims, wherein the silk fibroin powder is present in an amount such that the silk fibroin solution includes silk fibroin in an amount by weight of between 2% and 15%.

17. The kit, refill cartridge, or method of any one of the preceding claims, wherein the polyphenol powder is present in an amount such that the polyphenol solution includes the polyphenol in an amount by weight of between 5% and 20% or between 7.5% and 15%.

18. The kit, refill cartridge, or method of any one of the preceding claims, wherein the silk fibroin powder comprises one or more solubilizing salts, wherein the one or more solubilizing salts includes CaCh.

19. The kit, refill cartridge, or method of the immediately preceding claim, wherein the solubilizing salt is present in an amount and relative proportion to provide the solubilizing salt in the silk fibroin solution in an amount by weight of between 2.5% and 25% or between 5% and 20%.

20. An adhesive wound dressing kit comprising: a silk fibroin solution; and a polyphenol solution, wherein the silk fibroin solution and the polyphenol solution are physically separated from one another, wherein rapidly mixing a predetermined silk volume of the silk fibroin solution and a predetermined polyphenol volume of the polyphenol solution produces an adhesive wound dressing composition, wherein the adhesive wound dressing composition has a storage modulus and a loss modulus, and wherein the storage modulus is less than the loss modulus.PATENTAttorney Docket No. T002854 WO -2095.071121. The kit, refill cartridge, spray device, or method of any one of the preceding claims, wherein the polyphenol comprises a polyphenol selected from a group consisting of tannic acid, gallic acid, catecholamines (e.g., dopamine and L-DOPA), epigallocatechin gallate, urushiol, lignin, melanin, a synthetic polyphenol (e.g., polyvinyl catechol), and combinations thereof.

22. The kit, refill cartridge, spray device, or method of the immediately preceding claim, wherein the polyphenol powder and / or the polyphenol solution compnses tannic acid.

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