WO2025144993A1

WO2025144993A1 - Transdermal optical silk microneedle sensors for continuous monitoring for physiological analytes in interstitial fluid

Info

Publication number: WO2025144993A1
Application number: PCT/US2024/062043
Authority: WO
Inventors: David L. Kaplan; Sawnaz SHAIDANI; Jack LY; Thomas FALCUCCI; Kayla PRESLEY; Tod GRUSENMEYER
Original assignee: Tufts University; United States Department of the Air Force
Current assignee: Tufts University; United States Department of the Air Force
Priority date: 2023-12-27
Filing date: 2024-12-27
Publication date: 2025-07-03
Anticipated expiration: 2026-06-27

Abstract

Optical-based microneedle (MN) sensors to detect analytes in dermal interstitial fluid in a minimally invasive manner are described. Compared to electrochemical MN strategies, optical sensing is largely unexplored but has numerous potential benefits, including enhanced shelf-life, avoidance of drift, decreased impact of biofouling, and a smaller form factor when not in active use. This strategy also avoids the considerable challenges associated with both implantable optical sensors and sensors that employ skin interstitial fluid wicking/collection for downstream analysis.

Description

TRANSDERMAL OPTICAL SILK MICRONEEDLE SENSORS FOR CONTINUOUS MONITORING FOR PHYSIOLOGICAL ANALYTES IN INTERSTITIAL FLUID

CLAIM TO PRIORITY

[0001] This application relates to, incorporates by reference for all purposes, and claims priority to United States Provisional Application 63/615,081 filed on December 23, 2023.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under FA9550-20-1-0363 awarded by the Air Force Office of Scientific Research and P41EB027062 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0003] Current state-of-the-art technologies for electrolyte sensing include various sweat-based sensor patches. Sweat sensors rely on the patient sweating, which can be a hindrance to data collection in certain situations, particularly in clinical environments. Results from sweat sensors can also be affected by the rate of perspiration. Lastly, analyte concentrations in sweat are generally not directly related to blood serum concentrations, and typical levels of certain electrolytes are still under debate.

SUMMARY

[0004] Concentrations in interstitial skin fluid (ISF) are comparable to blood levels for most analytes. Thus, optical-based silk MN sensors to detect analytes in dermal ISF in a minimally invasive manner are described. Compared to electrochemical MN strategies, optical sensing is largely unexplored but has numerous potential benefits, including enhanced shelf-life, avoidance of drift, decreased impact of biofouling, and a smaller form factor when not in active use. This strategy also avoids the considerable challenges associated with both implantable optical sensors and sensors that employ ISF wicking/collection for downstream analysis. Silk fibroin, the primary material used, is a natural protein utilized in FDA approved medical devices that provides robust mechanical properties, and can slowly be safely broken down into amino acids and absorbed by the body, eliminating the need for surgical removal after use. These features are unlike other materials used for these applications. The biocompatibility of silk materials minimizes the risk of adverse immune responses, making it suitable for a wide range of biomedical applications.

[0005] 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. [0006] 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

[0007] The disclosure and the following detailed description of certain embodiments thereof may be understood by reference to the following figures:

[0008] Fig. 1 A depicts the distribution of SNPs loaded with a luminescent sensing chromophore in the tip of a microneedle.

[0009] Fig. IB depicts the distribution of chromophores that were loaded in the silk solution not attached to the SNPs and are throughout the MN. SNPs were used only as a structural component. Isolation of the dye to the tips of the needles can be adjusted via centrifuging more/less SNPs.

[0010] Fig. 1C depicts sensing chromophore located in the tip of the MNs only, loaded in the SNPs prior to casting in the molds.

[0011] Fig. ID depicts sensing chromophore loaded throughout the needles, loaded in the silk solution itself, with blank nanoparticles added to the solution prior to casting in the molds.

[0012] Fig. 2 depicts Silk MNs loaded with BTB demonstrated as both a qualitative (top images) and quantitative (bottom Figure) pH sensor.

[0013] Fig. 3 depicts 7% silk films with or without DMSO, dried overnight at 4 different temperatures: 4 °C, 20 °C, 40 °C, and 60 °C. Each photo has the blank film on the left and the DMSO film on the right. The blank films are prepared with 100 uL of 7% 30 min boil silk, and the DMSO films are 100 uL of 7% silk solution with 5% DMSO.

[0014] Fig. 4 depicts a schematic of the microneedle fabrication process.

DETAILED DESCRIPTION

[0015] 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.

[0016] 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.

[0017] 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).

[0018] Composition: as used herein, may be used to refer to a discrete physical entity that comprises one or more specified components. In general, unless otherwise specified, a composition may be of any form - e.g., gas, gel, liquid, solid, etc. In some embodiments, “composition” may refer to a combination of two or more entities for use in a single embodiment or as part of the same article. It is not required in all embodiments that the combination of entities result in physical admixture, that is, combination as separate co-entities of each of the components of the composition is possible; however many practitioners in the field may find it advantageous to prepare a composition that is an admixture of two or more of the ingredients in a pharmaceutically acceptable carrier, diluent, or excipient, making it possible to administer the component ingredients of the combination at the same time.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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 m ri. is the most common and represents an earth-friendly, renewable resource. For instance, silk fibroin used in a silk film may be attained by extracting sericin from the cocoons of B. mori. Organic silkworm cocoons are also commercially available. There are many different silks, however, including spider silk (e.g., obtained from Nephila clavipes), transgenic silks, genetically engineered silks, such as silks from bacteria, yeast, mammalian cells, transgenic animals, or transgenic plants, and variants thereof, that can be used. See, e.g., WO 97/08315 and U.S. Pat. No. 5,245,012, each of which is incorporated herein by reference in their entireties.

[0023] Disclosed herein are silk fibroin films and sponges loaded with a water insoluble oxygen sensing chromophore, which were cytocompatible in vitro, biocompatible upon subcutaneous implantation in rats, and maintained real-time oxygen-sensing functions throughout physiological states (hyperoxic normoxic, and hypoxic conditions). In the present disclosure, the use of silk fibroin microneedles (MNs) is expanded as a more minimally invasive version of this diagnostic tool. Silk fibroin overall serves as a stabilizing matrix for various cargo including chromophores, antibiotics, antibodies, growth factors, hormones and small molecules, and can be exploited in combination with the convenience of self-administered drug delivery via these MN patches to provide more accessible, safe, and ease-of-use systems that can be shelf-stored for extended time frames until used. Therefore, transdermal optical silk MN sensors for the continuous monitoring of physiological analytes is disclosed, such as (but not limited to) tissue oxygenation analytes, electrolytes (Na+, K+, Ca2+, CL), and other biomarkers like pH, lactate, or other metabolites for assessing physiological states. Disclosed herein is technology for monitoring hydration and electrolyte levels, identifying a metabolic status, real-time minimally invasive monitoring of tissue oxygenation, providing data on environmental conditions, monitoring food and/or beverage conditions such as acidity or oxygen levels, or the like.

[0024] In certain aspects, silk microneedles are used for diagnostic purposes.

[0025] In other aspects, a new understanding on how to reproducibly load silk MNs with chromophores that have sensing capabilities is gained. In some aspects, this disclosure provides loading chromophores into silk nanoparticles (SNPs) prior to inclusion in MNs.

[0026] In some aspects, disclosed herein is the achievement of mechanical integrity of the microneedles being enhanced/controlled via the inclusion of SNPs.

[0027] In other aspects, a new understanding on how to reproducibly load silk MNs with SNPs and the ability to control the position of silk nanoparticles within the microneedles, such as to optimize function is gained.

[0028] In certain aspects, the system can be configured into additional modes depending on the needs, e.g., hollow MNs to facilitate fluid sampling or integration with electronics to provide readouts.

[0029] Transdermal MN patches are a method of drug delivery that has gained traction in recent years as an alternative, painless, and thus minimally invasive route of administration when compared to standard injections or implantable drug delivery devices. MN patches can penetrate the skin and deliver peptides, vaccines, proteins, and other small molecules. However, in MN formulations using non-degradable systems or synthetic materials, issues of thermal processing of polymers, harsh chemical conditions, ultraviolet light, or other factors used in the fabrication process can negatively impact the functionality of the loaded cargo. An alternative material, silk fibroin, a biocompatible protein derived from the cocoons of Bombyx mori silkworms, has been established as a robust carrier material due to its excellent mechanical properties, biocompatibility, amphiphilic nature, tunable rates of biodegradability, and aqueous and ambient processing conditions, which protect and stabilize cargo functionality. Compared to MNs made using degradable synthetic materials, silk fibroin-based MNs are cleaved by proteolytic enzymes which are further broken down into amino acids and metabolized or absorbed by the body. MN patches using silk fibroin protein were previously developed and utilized systems for the incorporation of both water insoluble small molecules and water-soluble large molecules. Various drugs and complex proteins have also been incorporated into silk fibroin MNs and remain bioactive during shelf-storage due to the low moisture and stabilizing chemistry from the silk. Additionally, silk fibroin offers robust mechanical properties to satisfy the needs for skin penetration of MN patches. Microparticles or silk nanoparticles (SNPs) can also be added as structural components to fortify the needles or be loaded with cargos to provide more sustained release. [0030] While MNs, and more specifically silk fibroin MNs, have been used primarily for drug delivery applications, MNs have been gaining attention as a diagnostic tool for testing different analytes in the interstitial skin fluid (ISF). Disclosed herein are silk fibroin films and sponges that maintained real-time oxygen-sensing functions throughout physiological states, and the loaded chromophore retained functionality in silk fibroin substrates in vivo. Thus, the use of silk fibroin MNs is expanded as a more minimally invasive version of this diagnostic tool. SNPs were either utilized to fortify MNs, or they were loaded with chromophores and subsequently localized or stacked in specific portions of the MN array or needles themselves. For example, since transdermal MN arrays may be exposed to atmospheric oxygen, we loaded oxygen sensing chromophores inside nanoparticles, which we specifically positioned only in the tip of the needles to avoid atmospheric influence. By loading chromophores into nanoparticles prior to casting them into the microneedles, there is also loaded multiple chromophores in the same needles or different chromophores within different needles (arrays).

[0031] In some embodiments, the SNPs can be centrifuged and localized to the MN tip during fabrication, while the remainder of the needles are subsequently comprised of neat silk. This system provides a biodegradable platform that exhibits excellent tunability of degradation time and is environmentally friendly, and is designed to allow ISF diffusion into the dye-containing area to allow for analyte detection.

[0032] Optical sensing via MNs could employ a wide variety of analyte- sensing dyes and sensing strategies. Most demonstrated analyte-sensing dyes operate in the ultraviolet (UV) or visible (400- 700 nm) regime, precluding their use in implantable sensors. However, such dyes are compatible with a transparent MN approach. Optical sensing strategies for MNs could monitor changes in phosphorescence lifetime, fluorescence intensity, or absorption (i.e., change in color). Note that lifetime-sensing is also possible with implantable technologies as we previously demonstrated in rodent studies with bioresorbable tissue oxygen (O2) sensors. However, most analyte sensing-dyes require colorimetric- or intensity-based sensing mechanisms. Colorimetric-based detection is challenging with implantable sensors, while the correction factors required to account for light traveling through the skin present considerable engineering challenges for intensity-based methods. [0033] Focus was shifted to MN sensors to allow for multiple sensing approaches, enable the use of a wider variety of analyte-sensing dyes, and avoid the considerable synthetic efforts required to redshift dye absorption and emission into the optical tissue window. Furthermore, implantable sensors are invasive and (unless biodegradable) persist permanently or require retrieval surgery. Optical MNs could be a less invasive means to test concentrations of key analytes for physiological monitoring in ISF. Analytes of interest include tissue oxygenation, electrolytes (Na⁺, K⁺, Ca²⁺, Cl ) from the Chem 8 basic metabolic panel, as well as additional biomarkers such as lactate, pH, and other metabolites relevant to physiological and metabolic monitoring.

[0034] Loading nanoparticles with chromophores and then concentrating the nanoparticles in a certain location subsequently concentrates the chromophores in that location. The opposite is also possible, where concentrating nanoparticles reduces the concentration of chromophores in a region. To use one specific example, during the back-filling vacuum steps detailed, immediately after centrifugation of the needles where more chromophore may be loaded, it is possible to load the back half of the needle with more chromophore than the top half containing particles. Additionally, the system may be adaptable to utilize different particle sizes, different centrifuge times/forces to produce a “gradient” effect of SNPs loaded with chromophores, thus controlling particle location/chromophore concentration. It is contemplated that blank nanoparticles could be used with chromophores in the silk solution/matrix, the blank silk nanoparticles could be concentrated in a certain region (e.g., the tip), and then a higher concentration of chromophore could be present everywhere except the certain region (e.g., the tip).

[0035] Without wishing to be bound by any particular theory, nanoparticles with 120-200 nm size provides excellent transparency of resulting microneedles.

[0036] Without wishing to be bound by any particular theories, it is believed that the methods described herein are capable of forming hollow microneedles. Those hollow microneedles can optionally be back-filled (e.g., vacuum back-filling) with additional silk nanoparticles/solution. [0037] In certain aspects, a method of making a microneedle including a base, a tip, and a tapered section connecting the base of the tip may comprise the following steps a)-f). a) A plurality of silk fibroin nanoparticles in a mixture of water and dimethyl sulfoxide (DMSO) may be suspended to produce a first suspension, b) The first suspension may be diluted with water to produce a second suspension that is a dilution of the first suspension, wherein the DMSO may be present in the second suspension in a percent by volume of 25% or less, c) A concentrated silk solution may be added to the second suspension to produce a third suspension having a final silk fibroin concentration and a final silk fibroin nanoparticle concentration, wherein the final silk fibroin concentration by weight is between 3% and 11%, including but not limited to between 7% and 10%, and the final silk fibroin nanoparticle concentration by weight is between 0.01% and 5.0%, including but not limited to between 0.05% and 0.1%. d) The third suspension may be cast into a microneedle mold defining a negative shape of the microneedle to produce a casted suspension having a microneedle shape, e) The casted solution may be dried at a drying temperature between 4 °C and 90 °C (e.g., preferred ambient conditions) for a drying length of time between 2 hours and 24 hours, thereby producing a dried microneedle having the microneedle shape, f) The microneedle may be annealed, thereby forming the microneedle. A first chromophore of interest may be i) coated onto or distributed throughout the plurality of silk fibroin nanoparticles prior to step a) and/or dissolved in the DMSO prior to step a).

[0038] The plurality of silk fibroin nanoparticles may be present in the first suspension in an amount by weight of between 0.01% and 5%, or between 0.01% and 1.0%, and or between 0.05% and 0.1%. In step b), dilution of the first suspension may lead to a 2:1 dilution of concentration of silk fibroin nanoparticles in the second suspension relative to the first suspension, including but not limited to, at least a 3: 1 dilution or at least a 4:1 dilution. The concentrated silk fibroin solution may have a stock silk fibroin concentration by weight of between 8% and 20%.

[0039] After casting the third suspension into the microneedle mold in step d) and before drying the casted suspension in step e), the casted suspension may be subjected to a centrifugal force for a first centrifugal length of time, thereby concentrating the plurality of silk fibroin nanoparticles into a portion of the microneedle. The portion of the microneedle may be the tip. The first centrifugal force may be between 400 relative centrifugal force (ref) and 4000 ref. The centrifugal length of time may be between 5 minutes and 60 minutes. The mold may be adapted to be positioned within a centrifuge with the gravitational vector pointed towards a negative tip of the negative shape. The resulting microneedle may have a higher concentration of silk fibroin nanoparticles into the tip.

[0040] The method may further include adding additional silk fibroin solution optionally containing an additional chromophore to the mold, thereby backfilling the mold. The additional silk fibroin solution may contain an additional chromophore of interest that is the same as or different than the first chromophore of interest. The drying of step e) may by performed at a pressure of between 0.75 and 1.5 atm.

[0041] The annealing of step f) may include water-vapor annealing. The water- vapor annealing may be performed for an annealing length of time between 1 hour and 24 hours. The annealing step may be performed at a pressure relative to atmospheric pressure of between -15 inHg and -30 inHg or between -20 inHg and -25 inHg.

[0042] The first chromophore of interested may be coated onto or distributed throughout the plurality of silk fibroin nanoparticles prior to step a). The first chromophore of interest may be dissolved in the DMSO prior to step a).

[0043] In some aspects, a microneedle includes a base, a tip, and a tapered section connecting the base and the tip. The microneedle may be composed of a composite material comprising a plurality of silk fibroin nanoparticles embedded in a silk fibroin matrix. A first chromophore of interest may be either i) coated onto or distributed through the plurality of silk fibroin nanoparticles and absent from the silk fibroin matrix, or ii) distributed throughout the silk fibroin matrix and absent from the silk fibroin nanoparticles, wherein the plurality of silk fibroin nanoparticles are distributed throughout the silk fibroin matrix in a sensing pattern. As used herein, a “sensing pattern” refers to any pattern of distribution of chromophores intended for any given sensing purpose. Non-limiting examples of a sensing pattern include, but are not limited to, homogeneous distributions, nonhomogeneous distributions, concentrations at edges and/or tips, or other intended uniform or non- uniform distributions.

[0044] In the microneedle, the first chromophore of interest may be coated onto or distributed throughout the plurality of silk fibroin nanoparticles and absent from the silk fibroin matrix. The first chromophore of interest may also be coated onto the plurality of silk fibroin nanoparticles. The first chromophore of interest may be distributed throughout the plurality of silk fibroin nanoparticles. The tip may have a concentration of silk fibroin nanoparticles that is different than the base or the tapered section. The concentration of silk fibroin nanoparticles may be substantially uniform across the base, the tapered section, and the tip. The microneedle may have an obelisk shape (i.e., wherein a degree of taper changes part way up the microneedle).

[0045] The sensing pattern may be a homogenous distribution throughout the silk fibroin matrix. The sensing pattern may be a nonhomogenous distribution throughout the silk fibroin matrix. The sensing pattern may include a homogenous distribution of silk fibroin nanoparticles throughout the base, the tapered section, and the tip. The sensing pattern may include a higher concentration of silk fibroin nanoparticles within the tip than within the base or tapered section.

[0046] The first chromophore of interest within the microneedle may be hydrophobic (i.e., <0. 1 mg/mL solubility in water) or sparingly soluble (i.e., <10 mg/mL in water). The second chromophore of interest in the microneedle may be different from the first chromophore of interest. The first chromophore of interest may be a sensing chromophore, and the second chromophore of interest may be a reference chromophore. The first chromophore of interest may be localized in a separate location than the second chromophore of interest. The first chromophore of interest may be at least partly co-localized with the second chromophore of interest.

[0047] In some aspects, a microneedle array includes a plurality of microneedles of or made by the method described herein affixed to a transparent backing material, wherein the transparent backing material is optionally dissolvable. The transparent backing material may either be a transparent silk fibroin material or a transparent dissolvable blend of polyvinyl alcohol and sucrose.

[0048] In some aspects, a method of making a finished article includes the following steps a)-f). In step a) a plurality of silk fibroin nanoparticles may be suspended in a mixture of water and dimethyl sulfoxide (DMSO) to produce a first suspension. In step b), the first suspension may be diluted with water to produce a second suspension that is a dilution of the first suspension. In step c), a concentrated silk fibroin solution may be added to the second suspension to produce a third suspension having a final silk fibroin solution concentration and a final silk fibroin nanoparticle concentration, wherein the final silk fibroin concentration by weight is between 5% and 10% and the final silk fibroin nanoparticle concentration by weight is between 0.05% and 1.0%. In step d), the third suspension may be casted into a mold defining a volumetric shape to produce a casted suspension having the volumetric shape. In step e), the casted solution may be dried at a drying temperature of between 4 °C and 90 °C (e.g., preferred ambient conditions) for a drying length of time of between 2 hours and 24 hours, thereby producing a dried article having the volumetric shape. In step f), the dried article may be annealed, thereby forming the finished article. A first chromophore of interest may be i) coated onto or distributed throughout the plurality of silk fibroin nanoparticles prior to step a), and/or ii) dissolve din the DMSO prior to step a).

[0049] The plurality of silk fibroin nanoparticles may be present in the first suspension in an amount by weight of between 0.01% and 5%, or between 0.01% and 1.0%, and or between 0.05% and 0.1%. The diluting of step b) may be at least a 2:1 dilution of concentration of silk fibroin nanoparticles in the second suspension relative to the first suspension, including but not limited to, at least a 3:1 dilution or at least a 4: 1 dilution. The concentrated silk fibroin solution may have a stock silk fibroin concentration by weight of between 8% and 20%.

[0050] The method may further include subsequent to step d) and prior to step e) subjecting the casted suspension to a first centrifugal force for a first centrifugal length of time, thereby concentration the plurality of silk fibroin nanoparticles into a portion of the microneedle. The portion of the microneedle may be the tip. The first centrifigual force may be between 400 ref and 4000 ref. The first centrifugal length of time may be between 5 minutes and 60 minutes.

[0051] The method may further include adding additional silk fibroin solution optionally containing an additional chromophore to the mold, thereby back-filling the mold. The additional silk fibroin solution may contain an additional chromophore of interest that is the same as or different than the first chromophore of interest.

[0052] The drying of step e) may be performed at a pressure of between 0.75 and 1.5 atm. The annealing of step f) may include water-vapor annealing. The water-vapor annealing may be performed for an annealing length of time of between 1 hour and 24 hours. The annealing of step f) may be performed at a pressure relative to atmospheric pressure of between -15 inHg and -30 inHg or between -20 inHg and -25 inHg.

[0053] The first chromophore of interest may be coated onto or distributed throughout the plurality of silk fibroin nanoparticles prior to step a). The first chromophore of interest may be dissolved in the DMSO prior to step a). [0054] The article may include a second chromophore of interest that is different from the first chromophore of interest. The first chromophore of interest may be a sensing chromophore, and the second chromophore of interest may be a reference chromophore. The first chromophore of interest may be localized in a separate location than the second chromophore of interest. The first chromophore of interest may be at least partly co-localized with the second chromophore of interest. The first chromophore of interest may be hydrophobic (i.e., <0.1 mg/mL solubility in water) or sparingly soluble (i.e., < 10 mg/mL in water).

[0055] In some aspects, a silk microneedle may have oxygen-sensing chromophores concentrated at a tip of the microneedle.

[0056] In some aspects, a silk microneedle may have chromophores distributed throughout the microneedle or concentrated at a tip of the microneedle such that the presence of the chromophore allows for the sensing of a physiological analyte.

[0057] In some aspects, a silk microneedle may have chromophores distributed throughout the microneedle or concentrated at a tip such that the presence of the chromophores allows for sensing of an electrolyte or of pH.

[0058] According to various embodiments, a variety of functionalizing agents may be used with the silk-containing embodiments described herein (e.g., silk microneedle, 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 to any silk-containing embodiment, as applicable. In some embodiments, a functionalizing agent may be any compound or molecule that facilitates the attachment to and/or development (e.g., growth) of one or more endothelial cells on a silk membrane. In some embodiments, a functionalizing agent may be any compound or molecule that facilitates the attachment and/or development (e.g., growth) of one or more megakaryocytes and/or hematopoietic progenitor cells on a silk matrix and/or silk membrane. In some embodiments, a functionalizing agent may be or comprise an agent suitable for facilitating the production of one or more of white blood cells and red blood cells.

[0059] 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. [0060] 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).

[0061] According to some embodiments, a plurality of functionalizing agents may be used. For example, in some embodiments wherein production of platelets is desired, provided compositions may comprise the use of laminin, fibronectin and/or fibrinogen, and type IV collagen in order to facilitate the attachment and growth of endothelial cells on a silk membrane (e.g., a porous silk membrane) and/or attachment of megakaryocytes to a silk matrix.

[0062] In some embodiments, a functionalizing agent may be embedded or otherwise associated with a silk membrane and/or silk matrix such that at least a portion of the functionalizing agent is surrounded by a silk membrane and/or silk matrix as contrasted to a functionalizing agent simply being positioned along the surface of a silk membrane and/or silk matrix. In some embodiments, a functionalizing agent is distributed along and/or incorporated in substantially the entire surface area of a silk membrane/silk wall. In some embodiments, a functionalizing agent is distributed and/or incorporated only at one or more discrete portions of a silk membrane/wall and/or silk matrix. In some embodiments, a functionalizing agent is distributed in and/or along at least one of the lumenfacing side of a silk wall and the matrix-facing side of a silk wall.

[0063] According to various embodiments, any application- appropriate amount of one or more functionalizing agents may be used. In some embodiments, the amount of an individual functionalizing agent may be between about 1 pg/ml and 1,000 pg/ml (e.g., between about 2 and 1 ,000, 5 and 1 ,000, 10 and 1 ,000, 10 and 500, 10 and 100 pg/ml). In some embodiments, the amount of an individual functionalizing agent may be at least 1 pg/ml (e.g., at least 5, 10, 15, 20 25, 50, 100, 200, 300 400, 500, 600, 700, 800, or 900 pg/ml ). In some embodiments, the amount of an individual functionalizing agent is at most 1,000 pg/ml (e.g., 900, 800, 700, 600, 500, 400, 300 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 pg/ml ).

[0064] 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.

[0065] 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.

[0066] Exemplary pH sensitive dyes or agents include, but are not limited to, cresol red, methyl violet, crystal violet, ethyl violet, malachite green, methyl green, 2-(p- dimethylaminophenylazo) pyridine, paramethyl red, metanil yellow, 4-phenylazodiphenylamine, thymol blue, metacresol purple, orange IV, 4-o-Tolylazo-o-toluindine, quinaldine red, 2,4- dinitrophenol, erythrosine disodium salt, benzopurpurine 4B, N,N-dimethyl-p-(m-tolylazo) aniline, p- dimethylaminoazobenene, 4,4'-bis(2-amino-l-naphthylazo)-2,2'-stilbenedisulfonic acid, tetrabromophenolphthalein ethyl ester, bromophenol blue, Congo red, methyl orange, ethyl orange, 4-(4-dimethylamino-l-naphylazo)-3-methoxybenesulfonic acid, bromocresol green, resazurin, 4- phenylazo-l-napthylamine, ethyl red 2-(l-dimethylaminophenyazo) pyridine, 4-(p- ethoxypehnylazo)-m-phenylene-diamine monohydrochloride, resorcin blue, alizarin red S, methyl red, propyl red, bromocresol purple, chlorophenol red, p-nitrophenol, alizarin 2-(2,4- dinitrophenylazo) l-napthol-3,6-disulfonic acid, bromothymol blue, 6,8-dinitro-2,4-(lH) quinazolinedione, brilliant yellow, phenol red, neutral red, m-nitrophenol, cresol red, turmeric, metacresol purple, 4,4'-bis(3-amino-l-naphthylazo)-2,2'-stilbenedisulfonic acid, thymol blue, p- naphtholbenzein, phenolphthalein, o-cresolphthalein, ethyl bis(2,4-dimethylphenyl) ethanoate, thymolphthalein, nitrazine yellow, alizarin yellow R, alizarin, p-(2,4-dihydroxyphenylazo) benzenesulfonic acid, 5,5'-indigodisulfonic acid, 2,4,6-trinitrotoluene, 1,3,5-trinitrobenezne, and clay ton yellow.

[0067] 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. [0068] 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).

[0069] Exemplary temperature sensitive dyes or agents include, but are not limited to, thermochromic compounds or agents, such as thermochromic liquid crystals, leuco dyes, fluoran dyes, octadecylphosphonic acid.

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

[0071] 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.

[0072] In some aspects, the compositions comprise one or more additive, dopant, or biologically active agent suitable for a desired intended purpose. In some aspects, the additive or dopant may be present in the composition in an amount effective to impart an optical or organoleptic property to the composition. Exemplary additives or dopants that impart optical or organoleptic properties include, but are not limited to, dyes/pigments, flavorants, aroma compounds, granular or fibrous fillers.

[0073] 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). [0074] In some aspects, the additive or dopant comprises a flavoring agent or flavorant.

[0075] 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.

[0076] In some aspects, the additive or dopant comprises an aroma compound. Exemplary aroma compounds include ester aroma compounds, terpene aroma compounds, cyclic terpenes, and aromatic aroma compounds, such as, but not limited to, geranyl acetate, methyl formate, metyl acetate, methyl propionate, methyl butyrate, ethyl acetate, ethyl butyrate, isoamyl acetate, pentyl butrate, pentyl pentanoate, octyl acetate, benzyl acetate, methyl anthranilate, myrecene, geraniol, nerol, citral, cironellal, cironellol, linalool, nerolidol, limonene, camphor, menthol, carone, terpineol, alpha-lonone, thujone, eucalyptol, benzaldehyde, eugenol, cinnamaldehyde, ethyl maltol, vanillin, anisole, anethole, estragole, thymol.

[0077] In some aspects, the additive or dopant comprises a colorant, such as a dye or pigment. In some aspects, the dye or pigment imparts a color or grayscale to the composition. The colorant can be different than the sensing agents and/or sensing dyes below. Any organic and/or inorganic pigments and dyes can be included in the inks. Exemplary pigments suitable for use in the present disclosure include International Color Index or C.I. Pigment Black Numbers 1 , 7, 1 1 and 31 , C.I. Pigment Blue Numbers 15, 15 : 1 , 15 :2, 15 :3, 15 :4, 15 :6, 16, 27, 29, 61 and 62, C.I. Pigment Green Numbers 7, 17, 18 and 36, C.I. Pigment Orange Numbers 5, 13, 16, 34 and 36, C.I. Pigment Violet Numbers 3, 19, 23 and 27, C.I. Pigment Red Numbers 3, 17, 22, 23, 48: 1 , 48:2, 57: 1 , 81 : 1 , 81 :2, 81 :3, 81 :5, 101 , 1 14, 122, 144, 146, 170, 176, 179, 181 , 185, 188, 202, 206, 207, 210 and 249, C.I. Pigment Yellow Numbers 1 , 2, 3, 12, 13, 14, 17, 42, 65, 73, 74, 75, 83, 30, 93, 109, 1 10, 128, 138, 139, 147, 142, 151 , 154 and 180, D&C Red No. 7, D&C Red No. 6 and D&C Red No. 34, carbon black pigment (such as Regal 330, Cabot Corporation), quinacridone pigments (Quinacridone Magenta (228-0122), available from Sun Chemical Corporation, Fort Lee, N.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. [0078] Exemplary red acid dyes include Acid Red 1. (C.I. 18050); Acid Red 4 (C.I. 14710); Acid Red 18 (C.I. 16255), Acid Red 26 (C.I. 16150); Acid Red 2.7 (C.I. as Acid Red 51 (C.I. 45430, available from BASF Corporation, Mt. Olive, N.J.) Acid Red 52 (C.I. 45100); Acid Red 73 (C.I. 27290); Acid Red 87 (C. I. 45380); Acid Red 94 (C.I. 45440) Acid Red 194; and Food Red 1 (C.I. 14700). Exemplary violet acid dyes include Acid Violet 7 (C.I. 18055); and Acid Violet 49 (C.I. 42640). Exemplary blue acid dyes include Acid Blue 1 (C.I. 42045); Acid Blue 9 (C.I. 42090); Acid Blue 22 (C.I. 42755); Acid Blue 74 (C.I. 73015); Acid Blue 93 (C.I. 42780); and Acid Blue 158A (C.I. 15050). Exemplary green acid dyes include Acid Green 1 (C.I. 10028); Acid Green 3 (C.I. 42085); Acid Green 5 (C.I. 42095); Acid Green 26 (C.I. 44025); and Food Green 3 (C.I. 42053). Exemplary black acid dyes include Acid Black 1 (C.I. 20470); Acid Black 194 (Basantol® X80, available from BASF Corporation, an azo/1 :2 CR-complex.

[0079] 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). [0080] 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.

[0081] In addition to or in place of visible colorants, compositions provided herein can contain ETV fluorophores that are excited in the ETV range and emit light at a higher wavelength (typically 400 nm and above). Examples of ETV fluorophores include but are not limited to materials from the coumarin, benzoxazole, rhodamine, napthalimide, perylene, benzanthrones, benzoxanthones or benzothia- xanthones families. The addition of a UV fluorophore (such as an optical brightener for instance) can help maintain maximum visible light transmission. The amount of colorant, when present, generally is between 0.05% to 5% or between 0.1% and 1% based on the weight of the composition. [0082] For non-white compositions, the amount of pigment/dye generally is present in an amount of from at or about 0.1 wt% to at or about 20 wt% based on the weight of the composition. In some applications, a non-white ink can include 15 wt% or less pigment/dye, or 10 wt% or less pigment/dye or 5 wt% pigment/dye, or 1 wt% pigment/dye based on the weight of the composition. In some applications, a non-white ink can include 1 wt% to 10 wt%, or 5 wt% to 15 wt%, or 10 wt% to 20 wt% pigment/dye based on the weight of the composition. In some applications, a non-white ink can contain an amount of dye/pigment that is 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15%, 16 wt%, 17 wt%, 18 wt%, 19 wt% or 20 wt% based on the weight of the composition.

[0083] For white compositions, the amount of white pigment generally is present in an amount of from at or about 1 wt% to at or about 60 wt% based on the weight of the composition. In some applications, greater than 60 wt% white pigment can be present. Preferred white pigments include titanium dioxide (anatase and rutile), zinc oxide, lithopone (calcined coprecipitate of barium sulfate and zinc sulfide), zinc sulfide, blanc fixe and alumina hydrate and combinations thereof, although any of these can be combined with calcium carbonate. In some applications, a white ink can include 60 wt% or less white pigment, or 55 wt% or less white pigment, or 50 wt% white pigment, or 45 wt% white pigment, or 40 wt% white pigment, or 35 wt% white pigment, or 30 wt% white pigment, or 25 wt% white pigment, or 20 wt% white pigment, or 15 wt% white pigment, or 10 wt% white pigment, based on the weight of the composition. In some applications, a white ink can include 5 wt% to 60 wt%, or 5 wt% to 55 wt%, or 10 wt% to 50 wt%, or 10 wt% to 25 wt%, or 25 wt% to 50 wt%, or 5 wt% to 15 wt%, or 40 wt% to 60 wt% white pigment based on the weight of the composition. In some applications, a non-white ink can an amount of dye/pigment that is 5%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15%, 16 wt%, 17 wt%, 18 wt%,

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

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

[0084] 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.

[0085] In some aspects, the additive is a biologically active agent. The term “biologically active agent” as used herein refers to any molecule which exerts at least one biological effect in vivo. For example, the biologically active agent can be a therapeutic agent to treat or prevent a disease state or condition in a subject. Biologically active agents include, without limitation, organic molecules, inorganic materials, proteins, peptides, nucleic acids (e.g., genes, gene fragments, gene regulatory sequences, and antisense molecules), nucleoproteins, polysaccharides, glycoproteins, and lipoproteins. Classes of biologically active compounds that can be incorporated into the composition provided herein include, without limitation, anticancer agents, antibiotics, analgesics, antiinflammatory agents, immunosuppressants, enzyme inhibitors, antihistamines, anti-convulsants, hormones, muscle relaxants, antispasmodics, ophthalmic agents, prostaglandins, anti-depressants, anti-psychotic substances, trophic factors, osteoinductive proteins, growth factors, and vaccines. [0086] 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.

[0087] 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.

[0088] The active agent can be generally present in the silk matrix (e.g., a silk microsphere), composition, or the like in an amount of about 0.01% (w/w) to about 70% (w/w), or about 0.1% (w/w) to about 50% (w/w), or about 1% (w/w) to about 30% (w/w). The active agent can be present on a surface of the silk matrix (e.g., a silk microsphere), composition, or the like and/or encapsulated and dispersed in the silk matrix (e.g., a silk microsphere), composition, or the like homogeneously or heterogeneously or in a gradient. In some embodiments, the active agent can be added into the silk solution, which is then subjected to the methods described herein for preparing a silk matrix (e.g., a silk microsphere), composition, or the like. In some embodiments, the active agent can be coated on a surface of the silk matrix (e.g., a silk microsphere), composition, or the like. In some embodiments, the active agent can be loaded in a silk matrix (e.g., a silk microsphere), composition, or the like by incubating the silk microsphere in a solution of the active agent for a period of time, during which an amount of the active agent can diffuse into the silk matrix (e.g., a silk microsphere), composition, or the like, and thus distribute within the silk matrix (e.g., a silk microsphere), composition, or the like. [0089] 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.

[0090] The term “therapeutic agent” also includes an agent that is capable of providing a local or systemic biological, physiological, or therapeutic effect in the biological system to which it is applied. For example, the therapeutic agent can act to control infection or inflammation, enhance cell growth and tissue regeneration, control tumor growth, act as an analgesic, promote anti-cell attachment, and enhance bone growth, among other functions. Other suitable therapeutic agents can include anti-viral agents, hormones, antibodies, or therapeutic proteins. Other therapeutic agents include prodrugs, which are agents that are not biologically active when administered but, upon administration to a subject are converted to biologically active agents through metabolism or some other mechanism. Additionally, a silk-based drug delivery composition can contain one therapeutic agent or combinations of two or more therapeutic agents.

[0091] 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.

[0092] The term “bioactivity,” as used herein in reference to an active agent, generally refers to the ability of an active agent to interact with a biological target and/or to produce an effect on a biological target. For example, bioactivity can include, without limitation, elicitation of a stimulatory, inhibitory, regulatory, toxic or lethal response in a biological target. The biological target can be a molecule or a cell. For example, a bioactivity can refer to the ability of an active agent to modulate the effect/activity of an enzyme, block a receptor, stimulate a receptor, modulate the expression level of one or more genes, modulate cell proliferation, modulate cell division, modulate cell morphology, or any combination thereof. In some instances, a bioactivity can refer to the ability of a compound to produce a toxic effect in a cell. Exemplary cellular responses include, but are not limited to, lysis, apoptosis, growth inhibition, and growth promotion; production, secretion, and surface expression of a protein or other molecule of interest by the cell; membrane surface molecule activation including receptor activation; transmembrane ion transports; transcriptional regulations; changes in viability of the cell; changes in cell morphology; changes in presence or expression of an intracellular component of the cell; changes in gene expression or transcripts; changes in the activity of an enzyme produced within the cell; and changes in the presence or expression of a ligand and/or receptor (e.g., protein expression and/or binding activity). Methods for assaying different cellular responses are well known to one of skill in the art, e.g., western blot for determining changes in presence or expression of an endogenous protein of the cell, or microscopy for monitoring the cell morphology in response to the active agent, or FISH and/or qPCR for the detection and quantification of changes in nucleic acids. Bioactivity can be determined in some embodiments, for example, by assaying a cellular response.

[0093] In reference to an antibody, the term “bioactivity” includes, but is not limited to, epitope or antigen binding affinity, the in vivo and/or in vitro stability of the antibody, the immunogenic properties of the antibody, e.g., when administered to a human subject, and/or the ability to neutralize or antagonize the bioactivity of a target molecule in vivo or in vitro. The aforementioned properties or characteristics can be observed or measured using art-recognized techniques including, but not limited to, scintillation proximity assays, ELISA, ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence ELISA, competitive ELISA, SPR analysis including, but not limited to, SPR analysis using a BIAcore biosensor, in vitro and in vivo neutralization assays (see, for example, International Publication No. WO 2006/062685), receptor binding, and immunohistochemistry with tissue sections from different sources including human, primate, or any other source as needed. In reference to an immunogen, the “bioactivity” includes immunogenicity, the definition of which is discussed in detail later. In reference to a virus, the “bioactivity” includes infectivity, the definition of which is discussed in detail later. In reference to a contrast agent, e.g., a dye, the “bioactivity” refers to the ability of a contrast agent when administered to a subject to enhance the contrast of structures or fluids within the subject’s body. The bioactivity of a contrast agent also includes, but is not limited to, its ability to interact with a biological environment and/or influence the response of another molecule under certain conditions.

[0094] As used herein, the term “small molecule” can refer to compounds that are “natural productlike,” however, the term “small molecule” is not limited to “natural product-like” compounds. Rather, a small molecule is typically characterized in that it contains several carbon — carbon bonds, and has a molecular weight of less than 5000 Daltons (5 kDa), preferably less than 3 kDa, still more preferably less than 2 kDa, and most preferably less than 1 kDa. In some cases it is preferred that a small molecule have a molecular weight equal to or less than 700 Daltons.

[0095] Exemplary therapeutic agents include, but are not limited to, those found in Harrison’ s Principles of Internal Medicine, 13th Edition, Eds. T.R. Harrison et al. McGraw-Hill N.Y., NY; Physicians’ Desk Reference, 50th Edition, 1997, Oradell New Jersey, Medical Economics Co.; Pharmacological Basis of Therapeutics, 8th Edition, Goodman and Gilman, 1990; United States Pharmacopeia, The National Formulary, ETSP XII NF XVII, 1990, the complete contents of all of which are incorporated herein by reference.

[0096] Therapeutic agents include the herein disclosed categories and specific examples. It is not intended that the category be limited by the specific examples. Those of ordinary skill in the art will recognize also numerous other compounds that fall within the categories and that are useful according to the present disclosure. Examples include a radiosensitizer, a steroid, a xanthine, a beta- 2-agonist bronchodilator, an anti-inflammatory agent, an analgesic agent, a calcium antagonist, an angiotensin-converting enzyme inhibitors, a beta-blocker, a centrally active alpha- agonist, an alpha- 1 -antagonist, an anticholinergic/antispasmodic agent, a vasopressin analogue, an anti arrhythmic agent, an antiparkinsonian agent, an antiangina/antihypertensive agent, an anticoagulant agent, an antiplatelet agent, a sedative, an ansiolytic agent, a peptidic agent, a biopolymeric agent, an antineoplastic agent, a laxative, an antidiarrheal agent, an antimicrobial agent, an antifungal agent, a vaccine, a protein, or a nucleic acid. In a further aspect, the pharmaceutically active agent can be coumarin, albumin, steroids such as betamethasone, dexamethasone, methylprednisolone, prednisolone, prednisone, triamcinolone, budesonide, hydrocortisone, and pharmaceutically acceptable hydrocortisone derivatives; xanthines such as theophylline and doxophylline; beta-2- agonist bronchodilators such as salbutamol, fenterol, clenbuterol, bambuterol, salmeterol, fenoterol; antiinflammatory agents, including antiasthmatic anti-inflammatory agents, antiarthritis antiinflammatory agents, and non-steroidal antiinflammatory agents, examples of which include but are not limited to sulfides, mesalamine, budesonide, salazopyrin, diclofenac, pharmaceutically acceptable diclofenac salts, nimesulide, naproxene, acetaminophen, ibuprofen, ketoprofen and piroxicam; analgesic agents such as salicylates; calcium channel blockers such as nifedipine, amlodipine, and nicardipine; angiotensin converting enzyme inhibitors such as captopril, benazepril hydrochloride, fosinopril sodium, trandolapril, ramipril, lisinopril, enalapril, quinapril hydrochloride, and moexipril hydrochloride; beta-blockers (i.e., beta adrenergic blocking agents) such as sotalol hydrochloride, timolol maleate, esmolol hydrochloride, carteolol, propanolol hydrochloride, betaxolol hydrochloride, penbutolol sulfate, metoprolol tartrate, metoprolol succinate, acebutolol hydrochloride, atenolol, pindolol, and bisoprolol fumarate; centrally active alpha-2-agonists such as clonidine; alpha- 1 -antagonists such as doxazosin and prazosin; anticholinergic/antispasmodic agents such as dicyclomine hydrochloride, scopolamine hydrobromide, glycopyrrolate, clidinium bromide, flavoxate, and oxybutynin; vasopressin analogues such as vasopressin and desmopressin; antiarrhythmic agents such as quinidine, lidocaine, tocainide hydrochloride, mexiletine hydrochloride, digoxin, verapamil hydrochloride, propafenone hydrochloride, flecainide acetate, procainamide hydrochloride, moricizine hydrochloride, and disopyramide phosphate; antiparkinsonian agents, such as dopamine, L-Dopa/Carbidopa, selegiline, dihydroergocryptine, pergolide, lisuride, apomorphine, and bromocryptine; antiangina agents and antihypertensive agents such as isosorbide mononitrate, isosorbide dinitrate, propranolol, atenolol and verapamil; anticoagulant and antiplatelet agents such as Coumadin, warfarin, acetylsalicylic acid, and ticlopidine; sedatives such as benzodiazapines and barbiturates; ansiolytic agents such as lorazepam, bromazepam, and diazepam; peptidic and biopolymeric agents such as calcitonin, leuprolide and other LHRH agonists, hirudin, cyclosporin, insulin, somatostatin, protirelin, interferon, desmopressin, somatotropin, thymopentin, pidotimod, erythropoietin, interleukins, melatonin, granulocyte/macrophage-CSF, and heparin; antineoplastic agents such as etoposide, etoposide phosphate, cyclophosphamide, methotrexate, 5 -fluorouracil, vincristine, doxorubicin, cisplatin, hydroxyurea, leucovorin calcium, tamoxifen, 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.

[0097] Anti-cancer agents include alkylating agents, platinum agents, antimetabolites, topoisomerase inhibitors, antitumor antibiotics, antimitotic agents, aromatase inhibitors, thymidylate synthase inhibitors, DNA antagonists, farnesyltransferase inhibitors, pump inhibitors, histone acetyltransferase inhibitors, metalloproteinase inhibitors, ribonucleoside reductase inhibitors, TNF alpha agonists/antagonists, endothelinA receptor antagonists, retinoic acid receptor agonists, immunomodulators, hormonal and antihormonal agents, photodynamic agents, and tyrosine kinase inhibitors. [0098] 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.

[0099] Enzyme inhibitors are substances which inhibit an enzymatic reaction. Examples of enzyme inhibitors include edrophonium chloride, N-methylphysostigmine, neostigmine bromide, physostigmine sulfate, tacrine, tacrine, 1 -hydroxy maleate, iodotubercidin, p- bromotetramiisole, 10- (alpha-diethylaminopropionyl)-phenothiazine hydrochloride, calmidazolium chloride, hemicholinium-3,3,5-dinitrocatechol, diacylglycerol kinase inhibitor I, diacylglycerol kinase inhibitor II, 3-phenylpropargylamine, N°-monomethyl-Larginine acetate, carbidopa, 3- hydroxybenzylhydrazine, hydralazine, clorgyline, deprenyl, hydroxylamine, iproniazid phosphate, 6- MeO-tetrahydro-9H-pyrido-indole, nialamide, pargyline, quinacrine, semi carb azide, tranylcypromine, N,N-diethylaminoethyl-2,2-diphenylvalerate hydrochloride, 3 - isobutyl- 1- methylxanthne, papaverine, indomethacind, 2-cyclooctyl-2 -hydroxy ethylamine hydrochloride, 2,3- dichloro-a-methylbenzylamine (DCMB), 8,9-dichloro-2,3,4, 5 -tetrahydro- lH-2-benzazepine hydrochloride, p-amino glutethimide, p-aminoglutethimide tartrate, 3- iodotyrosine, alpha- methyltyrosine, acetazolamide, dichlorphenamide, 6-hydroxy-2- benzothiazolesulfonamide, and allopurinol.

[00100] Antihistamines include pyrilamine, chlorpheniramine, and tetrahydrazoline, among others. [00101] 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.

[00102] Muscle relaxants include mephenesin, methocarbomal, cyclobenzaprine hydrochloride, trihexylphenidyl hydrochloride, levodopa/carbidopa, and biperiden.

[00103] Anti-spasmodics include atropine, scopolamine, oxyphenonium, and papaverine.

[00104] Analgesics include aspirin, phenybutazone, idomethacin, sulindac, tolmetic, ibuprofen, piroxicam, fenamates, acetaminophen, phenacetin, morphine sulfate, codeine sulfate, meperidine, nalorphine, opioids (e.g., codeine sulfate, fentanyl citrate, hydrocodone bitartrate, loperamide, morphine sulfate, noscapine, norcodeine, normorphine, thebaine, nor- binaltorphimine, buprenorphine, chlomaltrexamine, funaltrexamione, nalbuphine, nalorphine, naloxone, naloxonazine, naltrexone, and naltrindole), procaine, lidocain, tetracaine and dibucaine. Ophthalmic agents include sodium fluorescein, rose bengal, methacholine, adrenaline, cocaine, atropine, alpha-chymotrypsin, hyaluronidase, betaxalol, pilocarpine, timolol, timolol salts, and combinations thereof.

[00105] 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.

[00106] Anti-depressants are substances capable of preventing or relieving depression.

[00107] Examples of anti-depressants include imipramine, amitriptyline, nortriptyline, protriptyline, desipramine, amoxapine, doxepin, maprotiline, tranylcypromine, phenelzine, and isocarboxazide. [00108] Trophic factors are factors whose continued presence improves the viability or longevity of a cell trophic factors include, without limitation, platelet-derived growth factor (PDGP), neutrophilactivating protein, monocyte chemoattractant protein, macrophage- inflammatory protein, platelet factor, platelet basic protein, and melanoma growth stimulating activity; epidermal growth factor, transforming growth factor (alpha), fibroblast growth factor, platelet- derived endothelial cell growth factor, insulin-like growth factor, glial derived growth neurotrophic factor, ciliary neurotrophic factor, nerve growth factor, bone growth/cartilage- inducing factor (alpha and beta), bone morphogenetic proteins, interleukins (e.g., interleukin inhibitors or interleukin receptors, including interleukin 1 through interleukin 10), interferons (e.g., interferon alpha, beta and gamma), hematopoietic factors, including erythropoietin, granulocyte colony stimulating factor, macrophage colony stimulating factor and granulocyte- macrophage colony stimulating factor; tumor necrosis factors, and transforming growth factors (beta), including beta-1, beta-2, beta-3, inhibin, and activin. [00109] Hormones include estrogens (e.g., estradiol, estrone, estriol, diethylstibestrol, quinestrol, chlorotrianisene, ethinyl estradiol, mestranol), anti-estrogens (e.g., clomiphene, tamoxifen), progestins (e.g., medroxyprogesterone, norethindrone, hydroxyprogesterone, norgestrel), antiprogestin (mifepristone), androgens (e.g, testosterone cypionate, fluoxymesterone, danazol, testolactone), anti- androgens (e.g., cyproterone acetate, flutamide), thyroid hormones (e.g., triiodothyronne, thyroxine, propylthiouracil, methimazole, and iodixode), and pituitary hormones (e.g., corticotropin, sumutotropin, oxytocin, and vasopressin). Hormones are commonly employed in hormone replacement therapy and / or for purposes of birth control. Steroid hormones, such as prednisone, are also used as immunosuppressants and anti-inflammatories. In some aspects, the additive is an agent that stimulates tissue formation, and/or healing and regrowth of natural tissues, and any combinations thereof. Agents that increase formation of new tissues and/or stimulates healing or regrowth of native tissue at the site of injection can include, but are not limited to, fibroblast growth factor (FGF), transforming growth factor-beta (TGF-beta, platelet-derived growth factor (PDGF), epidermal growth factors (EGFs), connective tissue activated peptides (CTAPs), osteogenic factors including bone morphogenic proteins, heparin, angiotensin II (A-II) and fragments thereof, insulin-like growth factors, tumor necrosis factors, interleukins, colony stimulating factors, erythropoietin, nerve growth factors, interferons, biologically active analogs, fragments, and derivatives of such growth factors, and any combinations thereof.

[00110] 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.

[00111] 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.

[00112] 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. [00113] 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.

[00114] In some aspects, the additive is a cell, e.g., a biological cell. Cells useful for incorporation into the composition can come from any source, e.g., mammalian, insect, plant, etc. In some aspects, the cell can be a human, rat or mouse cell. In general, cells to be used with the compositions provided herein can be any types of cells. In general, the cells should be viable when encapsulated within compositions. In some aspects, cells that can be used with the composition include, but are not limited to, mammalian cells (e.g. human cells, primate cells, mammalian cells, rodent cells, etc.), avian cells, fish cells, insect cells, plant cells, fungal cells, spore cells, bacterial cells, and hybrid cells. In some aspects, exemplary cells that can be 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.

[00115] 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.

[00116] Differentiated cells that have been reprogrammed into stem cells can also be used.

[00117] 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).

[00118] 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.” [00119] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term.

[00120] 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.

[00121] 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.

[00122] 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.

[00123] 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 abovedescribed elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[00124] While the invention has been illustrated and described in detail in the foregoing drawings and description, the same is to be considered as illustrative and not restrictive in character, it being understood that only illustrative embodiments thereof have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected. For example, any of the features or functions of any of the embodiments disclosed herein may be incorporated into any of the other embodiments disclosed herein.

[00125] 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.

[00126] EXAMPLES

[00127] Control of sensor loading and maintenance of sensor function

[00128] MNs are loaded with chromophores that have sensing capabilities and have developed several ways of loading the MNs with these chromophores, resulting in the chromophores being positioned in various, specific portions of the needles or the array. The positioning of the chromophores and isolation of the chromophores to certain areas of the needles are crucial for sensor functionality, depending on the intended analyte to be quantified. For example, for oxygen- sensing chromophores, it is important to isolate chromophores just to the tip of the needles to prevent noise from atmospheric oxygen, as the base of the needles will be exposed to atmospheric conditions. In contrast, for colorimetric chromophores, such as pH indicators (bromothymol blue for instance), needles should be maximally loaded throughout the needle to increase the intensity of colorimetric readouts for accurate quantification. This is controlled by incorporating chromophore-loaded SNPs in the silk solution used to create the microneedle arrays (which upon centrifugation of the silk solution and the SNPs into the microneedle mold would cause the SNPs to pellet to the tips of the needles (Fig 1A, Fig. 1C ), or by incorporating the chromophore in the silk solution itself, with blank, unloaded SNPs used as just a structural component (Fig IB, Fig. 1G, and Fig. 1 J). Control of how far the chromophore is in the needle can be done by increased centrifugation rounds, or increased concentrations of chromophore-loaded SNPs. This level of control also enables us to strategically load multiple chromophores within the same needle. The disclosure also describes how to load only certain regions of the array with various chromophores within the same patch by localizing where in the microneedle mold the silk and SNPs are centrifuged into. Post fabrication of SNPs or MNs loaded with chromophores allows for retention of the functionality of the chromophore (Fig. 1C, Fig. IE). pH, oxygen, and sodium at physiologically relevant levels may be detected. Because the chromophores are hydrophobic, they do not release out of the silk MNs at significant amounts post fabrication when tested in in vitro conditions, showing their stability in and affinity to the silk.

[00129] Optimization of sensor loading and MN formulation

[00130] Optimizing chromophore loading is a critical step in developing effective biosensor-based MN platforms. Achieving the right balance ensures minimal dye usage, which reduces costs, while maintaining sufficient signal intensity for accurate and reliable measurements. Furthermore, careful optimization ensures that all components in the sensor formulation are compatible (do not cause premature gelling or warping), promoting uniformity and consistency in performance across patches for more facile scalability. Loading hydrophobic chromophores into the “bulk” silk MNs required the use of DMSO; however, it was found that during the drying process, DMSO tends to warp silk (Fig. 3). Additionally, SNPs (which are included for either chromophores to be loaded into and/or for structural integrity) tend to cause silk solution to become more gel-like. This can cause needle deformation as well as nonuniform loading of hydrogel-like chromophore aggregates within the MNs. It was discovered that several factors influence the generation of more uniform MNs, including i) the order in which the stock silk solution used to form the MNs is added; ii) the DMSO content; and iii) the temperature at which the MNs are dried after centrifugation. Overall, ambient processing conditions, a 5% DMSO content, and order #7 in Table 1 resulted in reproducibly uniform needles as shown in Fig. 1. After optimizing the formulation, the effect of increasing SNP loading within the MNs was evaluated. This adjustment resulted in enhanced, tunable mechanical integrity, producing stronger MNs and increasing the likelihood for successful insertion into the skin, with higher amounts of SNPs leading to increased strength.

[00131] Table 1: Addition of silk solution components prior to casting into MNs, performed with all components scaled down to 25% of their original quantities to conserve materials. Parenthesis denote order of addition. Original volumes were decided based on: 5% DMSO content (10 mg/mL chromophore stock soln), 2 mg of SNPs, 7% silk (diluted from 10% silk, with volumes of other components accounted for to bring the solution to 7% silk).

[00132] Methods

[00133] Silk fibroin were isolated from Bombyx mori cocoons as previously described (Yavuz B, Chambre L, Harrington K, Kluge J, Valenti L, Kaplan DL. Silk Fibroin Microneedle Patches for the Sustained Release of Levonorgestrel. ACS Applied Bio Materials. 2020;3(8):5375-82. doi: 10.1021/acsabm.0c0067). Briefly, B. mori cocoons (Tajima Shoji Co., Japan) were boiled in a 0.02 M sodium carbonate solution for 30 minutes to remove sericin. The extracted silk fibroin was then dried for 12 hours in a chemical hood before being dissolved in a 9.3 M LiBr solution at 60 °C for 4 hours, yielding a 20% w/v solution. This solution was dialyzed against distilled water using Pierce Slide-aLyzer cassettes, MWCO 3,500 Da (Rockford, IL) to remove the LiBr. The solution was centrifuged (9,000 rpm, 4 °C, 20 min cycle), and a final concentration of the aqueous silk fibroin was acquired ~6-8% w/v.

[00134] Silk Nanoparticle Preparation

[00135] Nanoprecipitated silk nanoparticles were prepared as previously described, with modifications (Xiao L, Lu G, Lu Q, Kaplan DL. Direct Formation of Silk Nanoparticles for Drug Delivery. ACS Biomaterials Science & Engineering. 2016;2(l l):2050-7. doi: 10.1021/acsbiomaterials.6b00457). Briefly, a 5-6% w/v silk solution was added dropwise to acetone to generate a turbid solution that is >75% v/v acetone using a 60 nil addition funnel (Fisherscientific CG170401), while stirring (200 rpm, Cole Palmer UX-84003-80) in a 20 mL scintillation vial. The nanoparticle solution was stirred for >3 hours to evaporate the acetone. Once evaporated, the nanoparticle solution was sonicated with a Branson Ultrasonic Cell Disruptor for 30 seconds at 30% amplitude to yield particles of -150 nm, confirmed using dynamic light scattering (DLS). Blank, unloaded particles were incorporated into microneedles to fortify the MNs, or particles were loaded with chromophore via coatings or pre-mixing the chromophore with the silk solution prior to conducting the nanoprecipitation protocol (entrapment), and then ultracentrifuged at -180,000 x G for 30 minutes at 4 °C to remove unbound chromophore, similar to drug loading methods previously published (Wongpinyochit T, Johnston BF, Seib FP. Manufacture and Drug Delivery Applications of Silk Nanoparticles. J Vis Exp. 2016(116). Epub 20161008. doi: 10.3791/54669. PubMed PMID: 27768078; PMCID: PMC5092179). To entrap chromophores within the particles, chromophore was dissolved in DMSO at a concentration of 10 mg/mL. Per 100 mg of silk solution needed for the nanoprecipitation process, 0.1, 0.5 or 1 mg of chromophore was added to the silk solution from the stock solution. After adding the chromophore to the silk, and ensuring proper mixing, the nanoprecipitation process was conducted.

[00136] Microneedle Fabrication

[00137] MNs were prepared using poly(dimethylsiloxane) (PDMS) molds as previously described (Yavuz B, Chambre L, Harrington K, Kluge J, Valenti L, Kaplan DL. Silk Fibroin Microneedle Patches for the Sustained Release of Levonorgestrel. ACS Applied Bio Materials. 2020;3(8):5375- 82. doi: 10. 102 l/acsabm.0c00671). The molds were patterned with a 11 x 11 array of MNs, each MN with a 700 pm height, with a dose area of 1 cm². Using 3D printing, an insert that fits tightly in a 50 mL Falcon conical tube was used to allow the PDMS microneedle molds to be fixed during centrifugation and to maximize contact between the silk solution and the microneedle patch (Fig. 4). After placing the insert containing the PDMS mold into the Falcon tube, 2 mL of 7% silk solution, with or without chromophore and/or silk nanoparticles loaded with or without chromophore, were pipetted into the insert on top of the mold. For loading the chromophore into free silk solution, 100 pL of a stock solution of the chromophore dissolved in DMSO was added to the silk solution and accounted for in the total volume and concentration of the silk solution (2 mL and 7%, respectively). The 50 mL tube, containing the insert, silk fibroin chromophore solution, and microneedle mold, was then centrifuged at 3,220 ref for 20 minutes (10 minutes 2x, reversing the direction of the tube in the centrifuge after 10 minutes) at 4 °C, to allow silk solution to fill the microneedle molds fully (Fig. 4). The microneedle molds were removed from the 3D printed insert and allowed to dry for 8 hours in ambient conditions in the dark, followed by another 8 hours of being placed in a vapor annealer (Fig. 4). This process crystallizes the silk to maintain insolubility in aqueous systems. A backing was pipetted onto the mold, either using silk solution or another optically translucent material, and left to dry for 8 hours in ambient conditions. To maximize chromophore loading or filling of the needles, extra silk solution loaded with or without chromophore could be vacuumed into the molds using the vapor annealer immediately following the centrifugation step and prior to the ambient drying step.

[00138] EQUIVALENTS AND SCOPE

[00139] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combinations (or subcombinations) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

CLAIMS What is claimed is:

1. A method of making a microneedle comprising a base, a tip, and a tapered section connecting the base and the tip, the method comprising: a) suspending a plurality of silk fibroin nanoparticles in a mixture of water and dimethyl sulfoxide (DMSO) to produce a first suspension; b) diluting the first suspension with water to produce a second suspension that is a dilution of the first suspension, wherein the DMSO is present in the second suspension in a percent by volume of 25% or less; c) adding a concentrated silk fibroin solution to the second suspension to produce a third suspension having a final silk fibroin concentration and a final silk fibroin nanoparticle concentration, wherein the final silk fibroin concentration by weight is between 3% and 11%, including but not limited to between 7% and 10%, and the final silk fibroin nanoparticle concentration by weight is between 0.01% and 5.0%, including but not limited to between 0.05% and 0.1%; d) casting the third suspension into a microneedle mold defining a negative shape of the microneedle to produce a casted suspension having a microneedle shape; e) drying the casted suspension at a drying temperature of between 4 °C and 90 °C (e.g., preferred ambient conditions) for a drying length of time of between 2 hours and 24 hours, thereby producing a dried microneedle having the microneedle shape; f) annealing the dried microneedle, thereby forming the microneedle, wherein a first chromophore of interest is: i) coated onto or distributed throughout the plurality of silk fibroin nanoparticles prior to step a); and/or ii) dissolved in the DMSO prior to step a).

2. The method of claim 1 , wherein the plurality of silk fibroin nanoparticles is present in the first suspension in an amount by weight of between 0.01% and 5%, or between 0.01% and 1.0%, and or between 0.05% and 0.1%.

3. The method of any one of the preceding claims, wherein the diluting of step b) is at least a

2: 1 dilution of concentration of silk fibroin nanoparticles in the second suspension relative to the first suspension, including but not limited to, at least a 3:1 dilution or at least a 4: 1 dilution.

4. The method of any one of the preceding claims, wherein the concentrated silk fibroin solution has a stock silk fibroin concentration by weight of between 8% and 20%.

5. The method of any one of the preceding claims, the method further comprising: subsequent to step d) and prior to step e), subjecting the casted suspension to a first centrifugal force for a first centrifugal length of time, thereby concentrating the plurality of silk fibroin nanoparticles into a portion of the microneedle.

6. The method of the immediately preceding claim, wherein the portion of the microneedle is the tip.

The method of either of the two immediately preceding claims, wherein the mold is adapted to be positioned within a centrifuge with the gravitational vector pointed toward a negative tip of the negative shape.

8. The method of the immediately preceding claim, wherein the resulting microneedle has a higher concentration of silk fibroin nanoparticles in the tip.

9. The method of any one of claims 5 to the immediately preceding claim, wherein the first centrifugal force is between 400 relative centrifugal force (ref) and 4000 ref.

10. The method of any one of claims 5 to the immediately preceding claim, wherein the first centrifugal length of time is between 5 minutes and 60 minutes.

11. The method of any one of claims 5 to the immediately preceding claim, wherein the method further comprises adding additional silk fibroin solution optionally containing an additional chromophore to the mold, thereby back-filling the mold.

12. The method of the immediately preceding claim, wherein the additional silk fibroin solution contains an additional chromophore of interest that is the same as or different than the first chromophore of interest.

13. The method of any one of the preceding claims, wherein the drying of step e) is performed at a pressure of between 0.75 atm and 1.5 atm.

14. The method of any one of the preceding claims, wherein the annealing of step f) includes water-vapor annealing.

15. The method of the immediately preceding claim, wherein the water- vapor annealing is performed for an annealing length of time of between 1 hour and 24 hours.

16. The method of either of the two immediately preceding claims, wherein the annealing of step f) is performed at a pressure relative to atmospheric pressure of between -15 inHg and -30 inHg or between -20 inHg and -25 inHg.

17. The method of any one of the preceding claims, wherein the first chromophore of interest is coated onto or distributed throughout the plurality of silk fibroin nanoparticles prior to step a).

18. The method of any one of the preceding claims, wherein the first chromophore of interest is dissolved in the DMSO prior to step a).

19. A microneedle comprising a base, a tip, and a tapered section connecting the base and the tip, the microneedle composed of a composite material comprising a plurality of silk fibroin nanoparticles embedded in a silk fibroin matrix, wherein a first chromophore of interest is either: i) coated onto or distributed throughout the plurality of silk fibroin nanoparticles and absent from the silk fibroin matrix; or ii) distributed throughout the silk fibroin matrix and absent from the silk fibroin nanoparticles, wherein the plurality of silk fibroin nanoparticles are distributed throughout the silk fibroin matrix in a sensing pattern.

20. The microneedle of claim 19, wherein the first chromophore of interest is coated onto or distributed throughout the plurality of silk fibroin nanoparticles and absent from the silk fibroin matrix.

21. The microneedle of claim 20, wherein the first chromophore of interest is coated onto the plurality of silk fibroin nanoparticles.

22. The microneedle of claim 20 or 21, wherein the first chromophore of interest is distributed throughout the plurality of silk fibroin nanoparticles.

23. The microneedle of claim 19, wherein the first chromophore of interest is distributed throughout the silk fibroin matrix and absent from the plurality of silk fibroin nanoparticles.

24. The microneedle of claim 23, wherein the sensing pattern is a homogeneous distribution throughout the silk fibroin matrix.

25. The microneedle of claim 23, wherein the sensing pattern is a nonhomogeneous distribution throughout the silk fibroin matrix.

26. The microneedle of any one of claims 19 to 25, wherein the tip has a concentration of silk fibroin nanoparticles that is different than the base or the tapered section.

27. The microneedle of any one of claims 19 to 25, wherein a concentration of silk fibroin nanoparticles is substantially uniform across the base, the tapered section, and the tip.

28. The microneedle of any one of claims 19 to 25, wherein the sensing pattern includes a homogeneous distribution of silk fibroin nanoparticles throughout the base, the tapered section, and the tip.

29. The microneedle of any one of claims 19 to 25, wherein the sensing pattern includes a higher concentration of silk fibroin nanoparticles within the tip than within the base or tapered section.

30. The microneedle of or made by the method of any one of the preceding claims, wherein the first chromophore of interest is hydrophobic (i.e., <0.1 mg/mL solubility in water) or sparingly soluble (i.e., <10 mg/mL in water).

31 . The microneedle of or made by the method of any one of the preceding claims, the microneedle comprising a second chromophore of interest that is different from the first chromophore of interest.

32. The microneedle of or made by the method of the immediately preceding claim, wherein the first chromophore of interest is a sensing chromophore and the second chromophore of interest is a reference chromophore.

33. The microneedle of or made by the method of either of the two immediately preceding claims, wherein the first chromophore of interest is localized in a separate location than the second chromophore of interest.

34. The microneedle of or made by the method of either claim 31 or 32, wherein the first chromophore of interest is at least partly co-localized with the second chromophore of interest.

35. The microneedle of or made by the method of any one of the preceding claims, wherein the microneedle has an obelisk shape (i.e., wherein a degree of taper changes part way up the microneedle).

36. A microneedle array comprising a plurality of the microneedles of or made by the method of any one of the preceding claims affixed to a transparent backing material, wherein the transparent backing material is optionally dissolvable.

37. The microneedle array of the immediately preceding claim, wherein the transparent backing material is either a transparent silk fibroin material or a transparent dissolvable blend of polyvinyl alcohol and sucrose.

38. A method of making a finished article, the method comprising: a) suspending a plurality of silk fibroin nanoparticles in a mixture of water and dimethyl sulfoxide (DMSO) to produce a first suspension; b) diluting the first suspension with water to produce a second suspension that is a dilution of the first suspension; c) adding a concentrated silk fibroin solution to the second suspension to produce a third suspension having a final silk fibroin solution concentration and a final silk fibroin nanoparticle concentration, wherein the final silk fibroin concentration by weight is between 5% and 10% and the final silk fibroin nanoparticle concentration by weight is between 0.05% and 1.0%; d) casting the third suspension into a mold defining a volumetric shape to produce a casted suspension having the volumetric shape; e) drying the casted suspension at a drying temperature of between 4 °C and 90 °C (e.g., preferred ambient conditions) for a drying length of time of between 2 hours and 24 hours, thereby producing a dried article having the volumetric shape; f) annealing the dried article, thereby forming the finished article, wherein a first chromophore of interest is: i) coated onto or distributed throughout the plurality of silk fibroin nanoparticles prior to step a); and/or ii) dissolved in the DMSO prior to step a).

39. The method of claim 38, wherein the plurality of silk fibroin nanoparticles is present in the first suspension in an amount by weight of between 0.01% and 5%, or between 0.01% and 1.0%, and or between 0.05% and 0.1%.

40. The method of any one of claims 38 to the immediately preceding claim, wherein the diluting of step b) is at least a 2:1 dilution of concentration of silk fibroin nanoparticles in the second suspension relative to the first suspension, including but not limited to, at least a 3:1 dilution or at least a 4:1 dilution.

41. The method of any one of claims 38 to the immediately preceding claim, wherein the concentrated silk fibroin solution has a stock silk fibroin concentration by weight of between 8% and 20%.

42. The method of any one of claims 38 to the immediately preceding claim, the method further comprising: subsequent to step d) and prior to step e), subjecting the casted suspension to a first centrifugal force for a first centrifugal length of time, thereby concentrating the plurality of silk fibroin nanoparticles into a portion of the microneedle.

4 . The method of the immediately preceding claim, wherein the portion of the microneedle is the tip.

44. The method of either of the two immediately preceding claims, wherein the mold is adapted to be positioned within a centrifuge with the gravitational vector pointed toward a negative tip of the negative shape.

45. The method of the immediately preceding claim, wherein the resulting microneedle has a higher concentration of silk fibroin nanoparticles in the tip.

46. The method of any one of claims 42 to the immediately preceding claim, wherein the first centrifugal force is between 400 relative centrifugal force (ref) and 4000 ref.

47. The method of any one of claims 42 to the immediately preceding claim, wherein the first centrifugal length of time is between 5 minutes and 60 minutes.

48. The method of any one of claims 42 to the immediately preceding claim, wherein the method further comprising adding additional silk fibroin solution optionally containing an additional chromophore to the mold, thereby back-filling the mold.

49. The method of the immediately preceding claim, wherein the additional silk fibroin solution contains an additional chromophore of interest that is the same as or different than the first chromophore of interest.

50. The method of any one of claims 38 to the immediately preceding claim, wherein the drying of step e) is performed at a pressure of between 0.75 atm and 1.5 atm.

51 . The method of any one of claims 38 to the immediately preceding claim, wherein the annealing of step f) includes water- vapor annealing.

52. The method of the immediately preceding claim, wherein the water- vapor annealing is performed for an annealing length of time of between 1 hour and 24 hours.

53. The method of either of the two immediately preceding claims, wherein the annealing of step f) is performed at a pressure relative to atmospheric pressure of between -15 inHg and -30 inHg or between -20 inHg and -25 inHg.

54. The method of any one of claims 38 to the immediately preceding claim, wherein the first chromophore of interest is coated onto or distributed throughout the plurality of silk fibroin nanoparticles prior to step a).

55. The method of any one of claims 38 to the immediately preceding claim, wherein the first chromophore of interest is dissolved in the DMSO prior to step a).

56. The method of any one of claims 38 to the immediately preceding claim, the article comprising a second chromophore of interest that is different from the first chromophore of interest.

57. The method of the immediately preceding claim, wherein the first chromophore of interest is a sensing chromophore and the second chromophore of interest is a reference chromophore.

58. The method of either of claim 56 or 57, wherein the first chromophore of interest is localized in a separate location than the second chromophore of interest.

59. The method of either of claim 56 or 57, wherein the first chromophore of interest is at least partly co-localized with the second chromophore of interest.

60. The method of any one of claims 38 to the immediately preceding claim, wherein the first chromophore of interest is hydrophobic (i.e., <0.1 mg/mL solubility in water) or sparingly soluble (i.e., <10 mg/mL in water).

61 . A silk microneedle having oxygen-sensing chromophores concentrated at a tip of the microneedle.

62. A silk microneedle having chromophores distributed throughout the microneedle or concentrated at a tip of the microneedle such that the presence of the chromophore allows for sensing of a physiological analyte.

63. A silk microneedle having chromophores distributed throughout the microneedle or concentrated at a tip such that the presence of the chromophores allows for sensing of an electrolyte or of pH.