Systems and methods of dry bioprinting with dry mixed biopolymers

Abstract

Methods of laser printing include xerographic printing, printing via patterns of electrostatic charge, and / or laser printing onto a substrate with a dry mixed silk fibroin toner to produce a printed article. The dry mixed silk fibroin toner is formed by dry mixing a silk fibroin powder with a pigment powder and / or a dye powder. Laser printing can also be with a silk fibroin biotoner powder formed by mixing a silk fibroin powder and an enzyme solution, and optionally a pigment powder and / or a dye powder, to form a biotoner solution, wherein the biotoner solution is spray dried to form the silk fibroin biotoner powder. Laser printing cartridges may be loaded with a dry mixed silk fibroin toner or a silk fibroin biotoner powder.

Description

PATENTAttorney Docket No. T002905 WO - 2095.0714SYSTEMS AND METHODS OF DRY BIOPRINTING WITH DRY MIXED BIOPOLYMERSCLAIM TO PRIORITY

[0001] This application relates to, incorporates by reference for all purposes, and claims priority to United States Application Serial Number 63 / 735,763, filed on December 18, 2024.BACKGROUND

[0002] Laser printing falls into the umbrella category of xerography, or dry printing methods. Xerography is performed by applying a uniform static electrical charge to a plate or roller made of materials like selenium or organic photoconductor (OPC), which becomes electrically conductive under illumination. A pattern of light is applied to the charged plate or roller with either a projected image or a scanned laser or LED array. This light causes certain regions to become conductive and dissipate their static charge while others remain charged, thus creating a latent image. A fine powder of dry particles is applied to the plate or roller, which adheres to the charged regions via electrostatic attraction. This roller or plate is then pressed up against the substrate to be printed on, and an electrostatic charge is applied uniformly to or behind the substrate to enable transfer of the material. Any residual material left on the roller or plate is collected for recycling, then the roller is uniformly discharged by flood illumination, and the process is ready to begin again.

[0003] Xerographic printing, more commonly called laser printing, is nearly ubiquitous in commercial use but also finds industrial uses as well. These printers are the go printing method for on-demand printing of text documents in commercial, academic, and government spaces due to the quality and durability of prints they produce as well as the improved cost-efficiency they offer over ink-jet style printers. Industrially they are used to produce items such as labels, barcodes and graphics, and are the method of choice when a label must be resilient to aqueous conditions.

[0004] While laser printing solves many commercial issues, it has created some issues of its own. Firstly, the primary component of laser printer toner is a petrochemical plastic (typically polystyrenebutadiene copolymer) that is used to house the chromic components (often carbon black or ferric oxide for black pigments, organic pigments for the colored toner), all of which are made from petrochemical feedstocks. While laser toner is theoretically recyclable, the cost and complexity of recycling make it economically infeasible and therefore the toner is produced almost exclusively from virgin stocks. Waste toner is also a contributor to environmental microplastics, nanoparticles, and airborne particulate matter in office spaces.Secondly, the market for on-demand printing (both toner-based and otherwise) is shrinking as digital distribution and storage of documents is becoming standard. While there are emerging opportunities, polymer-based toner systems will not be able to meet their demands.PATENTAttorney Docket No. T002905 WO - 2095.0714SUMMARY

[0005] In some aspects, the techniques described herein relate to a method of laser printing, the method including: xerographic printing, printing via patterns of electrostatic charge, and / or laser printing onto a substrate with a dry mixed silk fibroin toner to produce a printed article, the dry mixed silk fibroin toner formed by dry mixing a silk fibroin powder with a pigment powder and / or a dye powder.

[0006] In some aspects, the techniques described herein relate to a method of laser printing, the method including: xerographic printing, printing via patterns of electrostatic charge, and / or laser printing onto a substrate with a silk fibroin biotoner powder to produce a printed article, the silk fibroin biotoner powder formed by mixing a silk fibroin powder and an enzyme solution, and optionally a pigment powder and / or a dye powder, to form a biotoner solution, wherein the biotoner solution is spray dried to form the silk fibroin biotoner powder.

[0007] In some aspects, the techniques described herein relate to a laser printing cartridge loaded with a dry mixed silk fibroin toner formed by dry mixing a silk fibroin powder with a pigment powder and / or a dye powder.

[0008] In some aspects, the techniques described herein relate to a laser printing cartridge loaded with a silk fibroin biotoner powder formed by mixing a silk fibroin powder and an enzyme solution, and optionally a pigment or dye powder, to form a biotoner solution, wherein the biotoner solution is spray dried to form the silk fibroin biotoner powder.

[0009] In some aspects, the techniques described herein relate to a method of making a silk toner, the method including: ball-milling a porous silk fibroin article having amorphous crystalline structure and / or a silk I structure for a length of time sufficient to generate a population of silk fibroin particles that are suitable for xerographic printing, printing via patterns of electrostatic charge, and / or laser printing onto a substrate.

[0010] In some aspects, the techniques described herein relate to a method including: a) ball-milling a silk fabric, thereby producing a milled output including a mixture of soluble silk fibroin, silk fibroin particles, and silk fibroin fibers; b) isolating a portion of the milled output (e.g., removing particles of a specific size, solubilizing the soluble silk fibroin, etc.).

[0011] 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.PATENTAttorney Docket No. T002905 WO - 2095.0714BRIEF DESCRIPTION OF THE FIGURES

[0012] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0013] The disclosure and the following detailed description of certain embodiments thereof may be understood by reference to the following figures:

[0014] Figure 1 A - Figure 1C. Silk fibroin inks printed on (Fig. 1A) paper, (Fig. IB) cellulose acetate films, and (Fig. 1C) silk cloth.

[0015] Figure 2A - Figure 2B. Life cycle analysis of toners. (Fig. 2A) the linear cradle to grave life cycle of polymer-based toners and (Fig. 2B) the circular life cycle of biodegradable toners like silk fibroin.

[0016] Figure 3. The silk xerography process. 1) The OPC drum is uniformly charged by a charge roller, 2) the surface charge is patterned by a scanned laser, 3) the charged areas of the surface collect silk toner, 4) the toner is transferred to the substrate, 5) the toner is set by reflowing with water vapor, and 6) the toner is (optionally) made water-insoluble by high temperature vapor annealing.

[0017] Figure 4. Silk xerography in personalized medicine. (Left) Medical printers could use a medicinal toner comprised of therapeutics encapsulated in silk fibroin microparticles could be used as a xerographic toner. This toner then doses the powder by printing defined areas on an edible film, and afterwards the film can be consumed directly or loaded into an enteric capsule. (Right) The medical printer enables dynamic redosing of medicines to meet personalized medical outcomes in a fully automated feedback loop. This loop is mediated by biomedical data that is being continuously acquired by a wearable sensor.

[0018] Figure 5A - Figure 5B. Morphology of commercial toners shown via scanning electron microscopy. Scale bars are (Fig. 5A) 10 pm and (Fig. 5B) 5 pm.

[0019] Figure 6A - Figure 6D. Laser-printed dry mixed silk fibroin toner morphology. Dry mixed silk fibroin toner laser printed on acetate is viewed under optical (Fig. 6A) and SEM (Fig. 6B - Fig. 6D) under various magnifications. Scale bars: Fig. 6B 400 pm; Fig. 6C 20 pm; Fig. 6D 100 pm.

[0020] Figure 7A - Figure 7B. Effects of inlet temperature on spray dried dry mixed silk fibroin toner particle size. (Fig. 7A) graph of outlet temperature resulting from varying inlet temperatures. (Fig. 7B) average particle diameter from silk spray dried at various temperatures.

[0021] Figure 8A - Figure 8F. Study of spray dried dry mixed silk fibroin toner morphology. Shown are SEM micrographs of silk powders resulting from solutions spray dried with varying inlet temperatures. (Fig. 8A) 105 °C; (Fig. 8B) 120 °C; (Fig. 8C) 140 °C; (Fig. 8D) 160 °C; (Fig. 8E) 120 °C (HRP, bulk); (Fig. 8F) 120 °C (HRP, scrapings).PATENT Attorney Docket No. T002905 WO - 2095.0714

[0022] Figure 9A - Figure 91. Silk particles and fibers result from electrospray drying. Silk particles on aluminum resulting from varying (top row) solution concentration, (middle row) emitter voltage, and (bottom row) solution conductivity with varying concentrations of added table salt. (Fig. 9A) 8% SF, 1 kV / cm; (Fig. 9B) 6% SF, 1 kV / cm; (Fig. 9C) 4% SF, 1 kV / cm; (Fig. 9D) 4% SF, 1 kV / cm; (Fig. 9E) 4% SF, 1.25 kV / cm; (Fig. 9F) 4% SF, 1.5 kV / cm; (Fig. 9G) 8% SF + 1 mM NaCl, 1 kV / cm; (Fig. 9H) 8% SF + 5 mM NaCl, 1 kV / cm; (Fig. 91) 8% SF + 10 mM NaCl, 1 kV / cm.

[0023] Figure 10A - Figure 10D. Microscopy of silk grinded by various methods. Silk milled by cryo-blade milling as seen under SEM (Fig. 10A) and optical microscopy (Fig. 10B). Silk milled by room temperature ball milling as seen under SEM (Fig. 10C) and optical microscopy (Fig. 10D).

[0024] Figure 11 A - Figure 1 IB. Laser prints from toners made by cryo-blade milling (Fig. 11 A) and room temperature ball milling (Fig. 1 IB).

[0025] Figure 12A - Figure 12C. Test of printer fidelity and alignment using a bullseye pattern to illustrate interlayer alignment. (Fig. 12A) Test bullseye; (Fig. 12B) results from HP M118; (Fig. 12C) results from HL-L2300D.

[0026] Figure 13A - Figure 13C. Resolution tests of different printers. (Top) a target pattern at the approximate resolution limit of both printers, with 9.2 lines per mm. (Below) A target pattern well beyond the resolution limits of the printers with 18.4 lines per mm. All scale bars are 500 pm. (Fig. 13A) test patterns; (Fig. 13B) from printer HP Ml 18; (Fig. 13C) from printer HL-L2300D.

[0027] Figure 14. Illustration of surface coverage by number of layers. (Left) a typical laser print of black commercial toner on white copy paper, showing complete coverage of the printed regions.(Upper right) Dry mixed silk fibroin toner printed on acetate and imaged against a dark background, showing improving coverage with multiple layers of silk printed. (Lower left) Prints of dyed silk (added thermochromic pigment) on white copy paper, showing large improvements to coverage with multiply layers. All scale bars are 5 cm.

[0028] Figure 15A - Figure 15B. Mass analysis of printed layers. (Fig. 15A) graph of total mass of copy paper and acetate as multiple layers of silk are printed and set on them. (Fig. 15B) The average density of each layer is calculated by dividing the printed area by the mass difference before and after printing.

[0029] Figure 16A - Figure 16D. Measurements of layer height of multilayer silk prints. (Fig. 16A) A pattern of 12 mm circles surrounding a 10 mm disk of printed silk, external silk ring is 1 layer thick, internal disk is between 0 and 10 layers thick, as indicated by the number. (Fig. 16B) micrograph of silk printed on acetate with red line indicating the measurement area of profilometer, starting in the outer ring, through the intermediate gap and onto the central disk, scale bar is 1 mm.PATENTAttorney Docket No. T002905 WO - 2095.0714(Fig 16C) A chart of disk height by number of layers, line of best fit indicates an average layer thickness of 2. 14 jam. (Fig. 16D) A profilometer measurement of a five-layer disk.

[0030] Figure 17. Analysis of silk prints before and after setting. (Top) Silk printed on acetate showing (left) what scattering due to individual particles, and (middle and right) particles that resemble crumpled spheres loosely adhered to an acetate film. (Bottom) silk printed on acetate that has been set by short steam treatment, (left) showing the disappearance of the scatting due to homogenous film formation and (middle and right) almost total disappearance of the collapsed spheroid particles.

[0031] Figure 18. Amide I peak of films annealed at 45 °C for 0-7 minutes, showing a gradual, time-dependent shift from random coils (peak centered at 1640 cm1) to anti -parallel beta sheets (peak centered at 1620 cm1).

[0032] Figure 19A - Figure 19C. Analysis of pH indicating dry mixed silk fibroin toner. (Fig. 19A) Dry mixed silk fibroin toner printed on filter paper and exposed to solutions of sodium carbonate (dark blue regions) citric acid (yellow regions) or mists of both (mottled regions). (Fig. 19B) SEM micrograph of near bromophenol blue crystals showing their distinctive rectangular crystal morphology. (Fig. 19C) EDX of near bromophenol blue show characteristic peaks from bromine and sulfur, allowing elemental contrast with silk.

[0033] Figure 20. SEM and EDX analysis of pH indicating prints. (Top) Dry mixed silk fibroin toner crumpled spheres and bromophenol blue rectangular crystals under SEM and isolated by characteristic x-rays from (blue) bromine in bromophenol blue and (yellow) nitrogen in silk. (Bottom) A pH indicating print after setting showing entrapment and partial dissolution of the bromophenol blue crystals in a smooth and uniform silk film.

[0034] Figure 21 A - Figure 2 IB. SEM images of (Fig. 21 A) neat thermochromic pigment and (Fig. 2 IB) thermochromic pigment entrapped in a laser printed and steam-set silk matrix.

[0035] Figure 22A - Figure 22D. Dry peroxide sensing form laser printed HRP-dry mixed silk fibroin toner. (Fig. 22A) Illustration of the DoE sheet with top 36 cells filled with reaction mixture. (Fig. 22B) The raw fluorescence data for each cell vs how many layers of silk were applied to the surface. (Fig. 22C) A closer photo showing the individual reaction sites on the DoE sheet. (Fig. 22D) The averaged fluorescence data by layer.

[0036] Figure 23. Effects of mechanical regeneration on silk cloths. Elements described from top left to lower right. Silk cloth waste is coarsely shredded with scissors then placed into a ball mill to grind for multiple days. Recovered materials is sorted into several sizes including macro particles that could not suspend in water, large micro particles (10 pm) that formed an unstable suspension,PATENTAttorney Docket No. T002905 WO - 2095.0714 medium scale microparticles (0.5-10 pm) that formed a semi-stable suspension, and fully soluble silk fibroin.

[0037] Figure 24. Molecular weight analysis of mechanically regenerated silk fibroin. (Left) The raw GPC chromatographs of 30 minute -boiled silk, chemically dissolved cloth, and mechanically regenerated silk after 1 and 2 weeks of grinding. (Center) Results of GPC analysis and calculated weight average molecular weights Mw. (Right) The mechanically regenerated solution was still able to form hydrogels.DETAILED DESCRIPTION

[0038] 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.

[0039] In this application, unless otherwise clear from context, (i) the tenn “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.

[0040] 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%, 1 1%, 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).

[0041] 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;PATENT Attorney Docket No. T002905 WO - 2095.0714 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.

[0042] Dry: as used herein, the term “dry” refers to the lack of moisture in a tactile sense, not in an absolute sense, so water that is trapped within a crystal structure can be present within dry particles. In some cases, a dry composition is free of bulk water. In general, a powder form is considered dry, so long as neighboring particles do not adhere to one another without additional components.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] As used herein, "silk fibroin" refers to silk fibroin protein whether produced by silkworm, spider, or other insect, or otherwise generated (Lucas et al., Adv. Protein Chem., 13: 107-242 (1958)). Any type of silk fibroin can be used in different embodiments described herein. Silk fibroin produced by silkworms, such as Bombyx mori, is the most common and represents an earth-friendly, renewable resource. For instance, silk fibroin used in a silk film may be attained by extracting sericin from the cocoons of B. mori. Organic silkworm cocoons are also commercially available. There arePATENTAttorney Docket No. T002905 WO - 2095.0714 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.

[0047] Laser printing is a staple of commercial and industrial printing. There is a need to revitalize the market and establish a line of products that fulfil not only current printing needs but opens the door to new avenues of usage in the fields of medical diagnostics, biomaterial fabrication, personalized medicine, and much more. Beyond the expanded markets, these printers also offer the capability to fully circularize the laser printing economy, with recycled materials (from both within and without the laser printing ecosystem) easily being incorporated into virgin processing chains with minimal added complexity. Described herein is an outline the current technological state of laser printing and how the incorporation of silk fibroin as a toner material could open new avenues of production, reduce logistical overhead for existing manufacturing, and negate the environmental impact of laser printing by using biofriendly materials in a highly circularizable economy.

[0048] Laser printing falls into the umbrella category of xerography, or dry printing methods. Xerography is performed by applying a uniform static electrical charge to a plate or roller made of materials like selenium or organic photoconductor (OPC), which becomes electrically conductive under illumination. A pattern of light is applied to the charged plate or roller with either a projected image or a scanned laser or LED array. This light causes certain regions to become conductive and dissipate their static charge while others remain charged, thus creating a latent image. A fine powder of dry particles is applied to the plate or roller, which adheres to the charged regions via electrostatic attraction. This roller or plate is then pressed up against the substrate to be printed on and an electrostatic charge is applied uniformly to or behind the substrate to enable transfer of the material. Any residual material left on the roller or plate is collected for recycling, then the roller is uniformly discharged by flood illumination, and the process is ready to begin again.

[0049] One of the opportunities unreachable by petrochemical toners is printing on biodegradable or edible packaging, for which the current petrochemical polymer toners are unsuitable. Another is the printing of active materials such as distributed sensing patches or laminar flow devices, which would benefit from a biochemically inert and hydratable encapsulant to maintain their function after printing.

[0050] Silk micropowders may serve as a replacement for traditional petrochemical toners, which can enable a large suite of new capabilities for the printed material and reduce the environmental impact of toner production. Laser printer toner is typically made from virgin petrochemical stocksPATENTAttorney Docket No. T002905 WO - 2095.0714 and is largely used in a linear cradle-to-grave economy, resulting in microplastic waste. Silk materials used for toners can participate in a fully circular economy and introduce many new functions for the toner. Silk-based toners can be used for edible printing, point-of-care diagnostic fabrication in addition to traditional printing applications.

[0051] Explored herein are a variety of strategies for toner fabrication, including electrospraying, spray drying, cryo-blade milling and ball milling. Of the two spraying approaches, spray drying was the superior technique, yielding usable particle sizes and morphologies in a more scalable fabrication approach. Of the two grinding techniques investigated, ball milling was superior due to the rounded final morphologies and finer particle sizes. The inventors surprisingly discovered that extended ball milling could be used to decrease the molecular weight of silk materials and render insoluble silk-II materials into water-soluble silk-I solids. These solids had a dramatically lower molecular weight but still had sufficient structure to form hydrogels.

[0052] A skilled artisan will appreciate that many factors play a role in suitability of a printer for printing with the dry mixed silk fibroin toners described herein. Finer particle sizes and interlayer repeatability are two important metrics for silk printing. The silk prints themselves were found to have difficulty in achieving uniform coverage in a single coating -likely due to the flow characteristics of the toner- but this deficit could be overcome by multiple layers. Each layer added approximately 230 pg / cm2of toner and applied a layer about 2.1 pm thick after setting. Setting via short exposure to wet-steam caused the toner to reflow into a smooth and largely uniform film. This film could then be water annealed by heated water-vapor exposure for differing lengths of time to achieve differing levels of crystallinity.

[0053] Dry mixed silk fibroin toners were shown to successfully incorporate dyes by simple dry mixing. These dyes remain discrete in the dry phase but become entrapped and incorporated after setting. Despite their water solubility, the described dyes do not effectively reflow to the same extent as the silk, leaving identifiable microscopic inclusions in the resulting films. Both pH indicators and thermochromics may retain their function through printing and onto the final substrate, allowing the direct fabrication of functional surfaces.

[0054] Methods of Making Dry Mixed Silk Fibroin Toners

[0055] The spray drying process was found to be compatible with labile biomolecules like HRP, which may retain a portion of their activity after the spraying process. The high surface area of the particles, however, seems to introduce difficulty with environmental oxygen, leading to a new degradation mechanism. The enzyme survives printing and may retain sensitivity in the dry state. The oxygen barrier provided by high numbers of silk layers has a demonstrable protective effect on the sensitive Amplex red redox sensor.PATENTAttorney Docket No. T002905 WO - 2095.0714

[0056] An alternative method for creating silk-I powders involves drying solution to an bulk solid using either lyophilization, drop casting, or other bulk drying methods, then grinding those solids into fine particulate. One method for creating fine powders in the biochemical laboratory is cryomilling. This technique uses cryogenic liquids to thermally embrittle the material to enable it to break instead of deforming during the grinding process. It also has the secondary benefit of preserving biological materials, as the cold temperatures are ideal for preventing degradation of sensitive compounds.

[0057] To perform cryomilling, the silk sample is first lyophilized to remove water while maintaining a silk-I conformation. The lyophilized silk is recovered in the form of a sponge, which is then frozen by immersion in liquid nitrogen and subjected to up to 5 minutes of blade grinding in a pre-chilled milling machine. Any longer is not feasible as the heat of the milling process warms the sample outside of the needed temperature range. The samples are then immersed in liquid nitrogen again to displace any water condensate and allowed to come up to room temperature in a container that allows gas to egress but not ingress. If the latter steps are not taken, the powder will condense water out of the air, begin to agglomerate, and ultimately become a solution if sufficient water is allowed to accumulate.

[0058] Methods of Laser Printing Dry’ Mixed Silk Fibroin Toner

[0059] One method of laser printing includes xerographic printing, printing via patterns of electrostatic charge, and / or laser printing onto a substrate with a dry mixed silk fibroin toner to produce a printed article. The dry mixed silk fibroin toner is formed by dry mixing silk fibroin powder with a pigment powder and / or a dye powder.

[0060] The method may further include setting the dry mixed silk fibroin toner using mist and / or low temperature steam, resulting in set dry mixed silk fibroin toner. The method may further include water annealing the set dry mixed silk fibroin toner.

[0061] The dry mixed silk fibroin toner may be produced by at least one of spray drying, ball milling, electrospraying, lyophilization and grinding, drop casting and grinding, or cryomilling. The method may further include curing, crosslinking, and / or recrystallizing the silk fibroin.

[0062] The dry mixed silk fibroin toner may include amorphous silk fibroin nanoparticles and / or silk fibroin particles. The dry mixed silk fibroin toner may be dry mixed with the pigment powder and / or the dye powder. The dry mixed silk fibroin toner may include a photochemical additive.

[0063] The method may further include two iterations: one iteration laser printing with the dry mixed silk fibroin toner and another iteration laser printing with a biopolymer toner. The two iterations may occur in either order relative to one another (e.g., either biopolymer or dry mixed silk fibroin first). The method may further include a third iteration subsequent to the second laser printing iteration.PATENTAttorney Docket No. T002905 WO - 2095.0714

[0064] Methods of Laser Printing Silk Fibroin Biotoner Powders

[0065] Another method of laser printing includes xerographic printing, printing via patterns of electrostatic charge, and / or laser printing onto a substrate with a silk fibroin biotoner powder to produce a printed article. The silk fibroin biotoner powder may be formed by mixing a silk fibroin powder and an enzyme solution to form a biotoner solution. The biotoner solution may include a pigment powder or a dye powder. The biotoner solution is spray dried to form the silk fibroin biotoner powder.

[0066] The silk fibroin powder may include amorphous silk fibroin nanoparticles and / or silk fibroin particles. The enzyme in the enzyme solution may be a biosensing enzyme.

[0067] The method may further include setting the silk fibroin biotoner powder using mist and / or low temperature steam. The silk fibroin powder may be processed by at least one of spray drying, ball milling, electrospraying, lyophilization and grinding, drop casting and grinding, or cryomilling. The method may further include curing, crosslinking, and / or recrystallizing the silk fibroin.

[0068] The silk fibroin biotoner powder may be dry mixed with a dye powder and / or pigment powder. The silk fibroin biotoner powder may include a photochemical additive.

[0069] The method may further include two iterations: one iteration laser printing with the dry mixed silk fibroin toner and another iteration laser printing with a biopolymer toner. The two iterations may occur in either order relative to one another (e.g., either biopolymer or dry mixed silk fibroin first). The method may further include a third iteration subsequent to the second laser printing iteration.

[0070] Common to Methods of Laser Printing Both Dry Mixed Silk Fibroin Toner and Silk Fibroin Biotoner Powder

[0071] In methods of laser printing both dry mixed silk fibroin toner and silk fibroin biotoner powder, referred to hereinafter as ‘both methods’, the laser printing may be to a resolution of at least 2 lines / mm, at least 3 lines / mm, at least 4 lines / mm, at least 5 lines / mm, at least 6 lines / mm, at least 7 lines / mm, at least 8 lines / mm, at least 9 lines / mm, or at least 10 lines / mm.

[0072] Laser printing of both methods may deposit between 100 pg / cm3and 400 pg / cm3, or between 200 pg / cm3and 300 pg / cm3. Laser printing may deposit at least 100 pg / cm3, at least 125 pg / cm3, at least 150 pg / cm3, at least 175 pg / cm3, at least 200 pg / cm3, at least 225 pg / cm3, or at least 250 pg / cm3. Laser printing may deposit at most 400 pg / cm3, at most 375 pg / cm3, at most 350 pg / cm3, at most 325 pg / cm3, at most 300 pg / cm3, at most 275 pg / cm3, or at most 250 pg / cm3.

[0073] The silk fibroin powder of both methods may be produced by spray drying with an inlet temperature and / or outlet temperature may be between 75 °C and 200 °C. The inlet temperature and / or outlet temperature may be at least 75 °C, at least 100 °C, at least 125 °C, or at least 150 °C.PATENTAttorney Docket No. T002905 WO - 2095.0714The inlet temperature and / our outlet temperature may be at most 200 °C, at most 175 °C, at most 150°C, or at most 125 °C.

[0074] The silk fibroin powder of both methods may include silk fibroin in a concentration (w / v) of between 1% and 10%. Silk fibroin may be present in a concentration of at least 1%, at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, at least 4%, at least 4.5%, at least 5%, at least 5.5%, or at least 6%. Silk fiborin may be present in a concentration of at most 10%, at most 9.5%, at most 9%, at most 8.5%, at most 8%, at most 7.5%, at most 7%, at most 6.5%, at most 6%, at most 5.5%, or at most 5%.

[0075] Methods of Making Silk Toners

[0076] In one example, a silk toner may be made by a method including ball-milling a porous silk fibroin article having amorphous crystalline structure and / or a silk I structure for a length of time sufficient to generate a population of silk fibroin particles that are suitable for xerographic printing, printing via patterns of electrostatic charge, and / or laser printing onto a substrate.

[0077] In another example, silk fabric may be ball-milled to produce a milled output including a mixture of soluble silk fibroin, silk fibroin particles, and silk fibroin fibers. A portion of the milled output may be isolated, such as by removing particles of a specific size, solubilizing the soluble silk fibroin, etc.

[0078] Incorporating Active Enzymes

[0079] One goal of the disclosure herein is the encapsulation of bioactive materials in the toner to create a print-on-demand diagnostic. Dry state biomanufacturing has several potential advantages to conventional water-based methods, including improved stability of the compounds, enabling multiple component co-fabrication without activating unwanted chemical reactions, and very high dosage precision thanks to the spatial precision of the printers. These factors could both reduce costs of both research and industrial scale biomanufacturing and enable the fabrication of heretofore infeasible devices entirely with only minimal modification to existing laser-printing hardware.

[0080] In modern colorimetric sensors, HRP is often coupled with Amplex Red to produce a colorimetric and fluorescent signal. Amplex red (AmpR) is a colorless and non-fluorescent small molecule, that upon oxidation converts into the compound called resorufin. Resorufin is a potent fluorophore and appears pink to the naked eye, with concentration in the low micromolar range producing a distinct visual signal. Coupled with HRP, this enzyme / substrate pair can be used to sense nanomolar concentrations of peroxide, or when coupled with peroxide can be used to detect microunits of HRP. While fantastically sensitive, AmpR is unfortunately sensitive to both environmental oxygen and ultraviolet light, thus requiring laboratory conditions to produce reliablePATENTAttorney Docket No. T002905 WO - 2095.0714 results. Stabilizing AmpR in a dry room temperature state would unlock a new generation of highly sensitive biosensors.

[0081] Laser Printing Cartridges

[0082] Laser printing cartridges may be loaded with the dry mixed silk fibroin toner formed by dry mixing silk fibroin powder with a pigment powder and / or a dye powder as described herein. Alternatively, laser printing cartridges may be loaded with a silk fibroin biotoner powder formed by mixing a silk fibroin powder and an enzyme solution, and optionally a pigment or dye powder, to form a biotoner solution. The biotoner solution may be spray dried to form the silk fibroin biotoner powder.

[0083] Silk, a dielectric particle, can be used as a toner for white color or as a carrier for compounds that provide functionality such as color, etc. Disclosed herein are silk powders that have been functionalized and loaded into toner cartridges to be used in a laser printing platform to print sensors by the sheet. Silk powder is a stable form for storing as a raw material. Eliminating the liquid format of silk enables a reinterpretation of the design and process to make sensors and functional devices.

[0084] By virtue of silk being a carrier, a suite of functional toners are enabled. Certain categories of inks include thermochromic inks, thermo-reactive inks, biologically active, scented, edible, and color. This enables a wide array of potential uses. One example includes functional printing for medical applications to make assays using biofunctional inks (proteins, fluorophores, conductive inks, thermoreactive inks), functionalized microfluidics (prepared sheets and then put them in a in a wax printer). Disclosed herein is an ad hoc laser printer that is sterile (i.e., is medical grade) and has sterile printing cartridges. This might be a small laser printer with miniaturized toner that then can be used to print assays for disease detection using particular reagents.

[0085] Another example includes patterned printing of artwork or even sensors using patterns, origami folds, photochromic or thermochromics inks, vanishing codes that can be reconstructed upon reaction with the suitable developer ink.

[0086] Another example includes sustainable printing. Toner waste is a big byproduct of paper recycling and often ends up in waste sludge. Using a biodegradable material such as silk for toner could significantly reduce this problem. Moreover, silk-based colored inks could be obtained from silk textile waste streams. Dry mixed silk fibroin toner by itself will be a very good white color because of light scattering by the material. Colored toners can be achieved by using a sequence of additives (e.g., anthocyanine and red beet red toner ink). Greys could be obtained through the use of squid ink attached to silk. Black will be complex one to obtain, and an exemplary approach is to use a post-process to carbonize the silk and turn the white to black. This also enables light browns during partial charring of the silk protein. This process may use a local temperature of 400 degrees.PATENTAttorney Docket No. T002905 WO - 2095.0714Potentially, powders from crushed optical films that retained optical properties could be used to offer iridescent or opalescent colors.

[0087] Advantages of Biotoners

[0088] An advantage of biotoners is the dielectric attraction that the toner particles have to adhere. Silk is a very negatively charged molecule and its end material as seen in its fine formats ends up being really sticky and dielectric, which is typical of a lot of structural proteins including cellulose. Cellulose powder with the proper polarity could be accelerated under the voltages used in a laser printer in such a way that it sticks together. The dielectric nature of the material and this net charge in the powder is conducive to its use as a toner.

[0089] Another advantage is the setting or curing properties of the toner material to bond with the underlying substrate. One would think that if the printed silk particles aren’t set or cured using vapor (moisture) the bond with the underlying substrate may be weak. Surprisingly, in this case, the laser printed silk is solidly and firmly attached to the substrate. Without wishing to be bound by any particular theory, it appears that with silk, the setting process is through cross-linking precluding the post-processing step. Modulating the crosslinking will facilitate tunable binding of the ink to the substrate thus offering both permanent and nonpermanent binding to the substrate. The latter could be useful for a range of fields including aesthetic patterns and others.

[0090] In examples, the structure size and the crystallinity of silk toners and dry mixed silk fibroin toners can be matched to the particle size of existing toners. Depending on the size range, suitable processes such as spray drying, lyophilization and so on may be specified.

[0091] Another area of growth is in printing materials such as sensors or devices that manage fluid flow. These products would benefit from a coating that is both non-reactive and capable of retaining moisture to ensure proper functionality after printing.

[0092] Silk Xerography

[0093] Replacing the polymeric component of toner with silk fibroin can transform the laser printing process from one that creates health and environmental hazards to one that enables health and reduces the environmental impact of other sectors of the economy. Silk fibroin microparticles are readily created by a variety of processes including spray drying, ball milling and electro-spraying. These particles are transferrable by the electrostatic charges on the OPC roller with no additional modification and can form patterns on a variety of substrates (Fig. 1 A, Fig IB, and Fig. 1C). These particles can then be set by reflowing with water aerosol, after which they form highly transparent films. These films can be made water-insoluble by exposure to heated water vapor or can be left in a dissolvable state for transient applications.PATENT Attorney Docket No. T002905 WO - 2095.0714

[0094] Disclosed herein are silk fibroin inks as a 1-for-l replacement for traditional toners. For black inks, composites of fibroin and natural chromophores such as melanin, fulvic acid, and activated charcoal are edible, ecofriendly alternatives. These compounds can provide a deep black color and are already FDA approved for use in foods, seen in products like BLK. For colored toners, silk is replete with tyrosine functional groups and is thus an excellent substrate for azo-dyes. These dyes directly bind to the fibroin polymer and are thus immune from leaching and washing out, increasing the durability of the final prints.

[0095] Together, these could be used to laser-print on edible or biodegradable items. Next generation green packaging is composed of biopolymers like proteins, polysaccharides, chitin, and others that are fully biodegradable. These could not be printed upon with petrochemical toners, as they would introduce non-degradable microplastics into the system. Fibroin toners on the other hand are fully degradable in both at-home and industrial composting systems and have no microplastic components. Beyond that, some fully edible packaging exists, such as rice-paper, which would require food-safe toners to print on. Fibroin is already used as an edible food-additive and fibroin toners would be well-suited for printing on edible packaging or directly onto food items.

[0096] While fibroin is a completely renewable resource, it can also be economically advantageous to be able to draw from recycled feedstocks in addition to virgin sources. Fibroin has a large (and untapped) supply of recyclable fibers from the textile industry, which produces over 11 million tons of silk waste per year. During growth, cocoons are often discarded for physical damage from either rough handling or the hatching of the moth. During fabric weaving, large sections are trimmed from rolls and weaves and discarded. During the manufacture of textiles, large off-cuts are discarded if they cannot be incorporated into the clothing pattern. Finally after being used damaged or worn-out silk garments are often discarded. All these sources represent potential feedstocks for toner creation. Those early in production can be directly intermixed with virgin stocks for the regeneration process. Those later in production can be used differently. Fibroin regenerated from discarded silk garments often retains the dye components from the original clothing and can be made directly into colored toners. Referring to Fig. 2A and Fig. 2B, life cycles of conventional polymer toner (Fig. 2A) and silk toner (Fig. 2B) are shown for comparison.

[0097] Silk-based toners could also reduce the overhead of producing point-of-care diagnostic devices, such as pregnancy tests, COVID-19 tests, and similar consumable testing devices. Firstly, the dry format of xerographic printing provides numerous benefits, as biomolecules are substantially more stable in the dry state and thus are often shipped and stored as lyophilized powder. Xerographic printing maintains that dry state throughout the printing process, thus eliminating solubilization, liquid handling, and drying from the manufacturing scheme. Secondly, fibroin acts to activelyPATENTAttorney Docket No. T002905 WO - 2095.0714 stabilize labile biomolecules and enables the elimination of refrigeration and cold chains in reagent storage and shipping. The final device will also benefit from this stability as the labile components are continuously encapsulated in fibroin throughout the entire life cycle of the product.

[0098] Silk fibroin has also been proven to facilitate advanced optical and chemical functionalities. Due to the high visible light transparency of silk fibroin, it can also incorporate optically active additives like gold nanoparticles to exploit their plasmonic resonance. Silk can also house photochemical additives such as tetrazole-ene photoclick pairs, facilitating the printing of photoreactive and fluorescent materials.

[0099] Finally, in much the same way that silk stabilizes enzymes, it can also stabilize antibiotics like Rifampicin, enabling the creation of antibiotic-resistance test strips that can be stored at room temperature.

[0100] How Silk Xerography Works

[0101] Silk xerographic printing resembles xerography with polymer toners. The light-reactive roller or plate is charged, the charge photographical patterned, and the powder is applied in a similar manner (Fig. 3). This means that existing technology can be applied without modification. Silk toner is different in how it is set compared to ordinary' toner, as it displays little in the way of thermoplastic properties. Instead, the reflow of the toner is accomplished by water vapor that partially dissolves the toner, achieving setting by either wetting out on smooth surfaces, like cellulose acetate films, or infiltrating porous surfaces, like paper or cloth. Once set, the fibroin remains water soluble, but that can be altered by tuning the secondary structure of the silk protein.

[0102] Silk fibroin has two primary solid states, amorphous and crystalline, characterized by the secondary structural organization of the protein. In the amorphous state, the protein has no consistent secondary structure and exists largely as random coils. In the crystalline state, the protein adopts a regular crystalline structure that substantially alters the physical and mechanical properties of the bulk material. In the amorphous state, the silk has a lower melting point, modulus, and is, crucially, still water soluble. This final trait enables the prints to be set by using water vapor to reflow the silk and cause it to bind to the printing substrate. However, once set, the final use of the item will determine the ideal secondary structure to use.

[0103] The annealing process with silk fibroin adds secondary flexibility to prints and larger constructs made with this method. Prints may be left intentionally unannealed for transient or edible applications, where remaining water-soluble is a desirable feature. In cases where the print must be permanent, annealing is readily achievable with exposure to high humidity. Water insolubility can be achieved in as little as 5 minutes at 45 °C and 90% relative humidity. This can be further accelerated by using higher temperatures, with the limit being high-pressure steam annealing, which can achievePATENTAttorney Docket No. T002905 WO - 2095.0714P-sheet structures in seconds. Alternatively, if the silk is loaded with a thermolabile compound, lower temperatures can be used to more gently anneal the print at the cost of longer residence times.

[0104] Fabrication of Dry Mixed Silk Fibroin Toners

[0105] The fabrication of dry mixed silk fibroin toners, which may be distinct from the silk toners described herein, requires the processing of fibroin into microscopic (<10 pm) particles while retaining a silk-I conformation. The microscopic size is the commercial standard for traditional toners and must be accommodated to use commercially available hardware for the patterning application. The silk must be in the silk-I conformation in order to be set by water-vapor reflow, as silk-II is water-insoluble and while it may be electrostatically patterned, it cannot be set. This combination of attributes restricts a large portion of the established methods for microparticle formation (such as solvent precipitation or co-flow fluidics) as these produce either chemically crosslinked or silk-II particles unsuitable for toner applications.

[0106] Spraying Methods

[0107] Spray drying is a method of solution powderization by aerosolizing a solution of dissolved solids into a hot air vortex that rapidly evaporates the solvent, leaving a finely divided powder. This method is advantageous as it is readily scalable, particle morphology can be controlled by drying conditions, and rapidly dried aqueous fibroin adopts silk-I morphology.

[0108] When dried with a lab-scale spray drier, fibroin efficiently forms particles of the correct size and dimension to be used with commercial laser printers. When loaded into a cleaned toner cartridge with no modification, the dry mixed silk fibroin toners produced well defined and mostly uniform prints. The prints appear white to the naked eye (Fig. 6A), but microscopy reveals the prints are composed a tight agglomeration of particles in the 1-10 pm range (Fig. 6B, Fig. 6C, and Fig. 6D). Closer inspection of these particles reveals them to have a collapsed-sphere shape that likely originated from the rapid drying in the spray-drying process. It is known for other varieties of solute and particle dispersions that spray drying conditions can have a dramatic impact on morphology, so described in the examples are various conditions to ascertain their effect on the resulting silk powder.

[0109] While spray drying has already proven to produce particles in the desired size and shape range, it is not ideal as it involves high temperatures that can damage thermally labile materials that are co-dissolved with the silk. An alternative method to spray drying that can potentially yield similar particles is electrospraying. Unlike spray drying, electrospraying is usually carried out at room temperature and particle morphology may be controlled by varying process parameters like acceleration voltage, solution conductivity, and concentration. This has been used to fabricate particles ranging from spheres, toroids, beaded strings, and uniform strands.

[0110] Setting and AnnealingPATENTAttorney Docket No. T002905 WO - 2095.0714

[0111] Laser toner prints are typically set (i.e. the toner fixed to the paper) by rapid melting and reflow of the thermoplastic toner, which bonds tightly to the fibers of the paper. Silk fibroin is not thermoplastic in the same temperature range that fixes commercial toners and has a small operational window between melting and polymer degradation. Compression at high pressures ~5 MPa and heating to 150 °C did not substantially reflow the dry mixed silk fibroin toners, even with extended periods of compression. Thus, thermal reflow is not a viable option. Instead, we have found the most effective way to reflow the dry mixed silk fibroin toner is to introduce water vapor in the form of mist or low temperature (wet) steam.

[0112] Prior to setting, dry mixed silk fibroin toners are loosely adhered to the substrate and can be easily removed by gentle rubbing of the surface. The dry mixed silk fibroin toner also has many ridges and air gaps, effectively scattering light and appearing as an opaque white print, despite the individual particles being largely transparent. Upon reflow, the silk absorbs the water and reflows into droplets or confluent films, drastically reducing scattering of the print and causing the printed regions to become transparent. These films are well adhered to the surface of both paper and plastics like cellulose acetate and have similar resistance to abrasion as thermoplastic toners.

[0113] Annealing is the process of causing the silk toner to transition from a water-soluble amorphous crystal structure to a water-insoluble beta-sheet rich conformation. There are many ways this can be accomplished, but the most amenable to printing is water-vapor annealing. This process involves exposing the prints to water vapor, which enables dynamic molecular movement and allows spontaneous reorganization of the protein into the more thermodynamically favorable annealed conformation. This process is distinct from setting as the macroscale reflow process is significantly faster and has little temperature dependance. Annealing requires either longer vapor exposure or substantially higher temperatures.

[0114] Incorporating Dyes

[0115] Like current commercial toners, dry mixed silk fibroin toners need added coloring agents in order to become visible on the page. The thermoplastic toners used by commercial laser printers are supplemented with pigments to give them the desired hue. These take the form of either carbon black or iron oxide for black pigments and organic pigments for colored toners. These dyes can equally be used with dry mixed silk fibroin toners as well, but the wettable nature of dry mixed silk fibroin toners open many new avenues of exploration that were unavailable to thermoplastic toners.

[0116] Exemplary colorants that may be incorporated include carbon black, iron oxide, organic pigments, thermochromic dyes, pH indicator dyes, acid dyes, natural dyes, direct dyes (either cationic or anionic), basic dyes, azo-dyes, and reactive dyes. Additional exemplary colorants are described in further sections herein.PATENTAttorney Docket No. T002905 WO - 2095.0714

[0117] pH Indicator Toners

[0118] One advantage of silk based toners is their ability to interact with water-based compounds. The thermoplastics of commercial toners render them water-incompatible, thus any cargo will not interact with the aqueous environment. Silk by contrast will swell with water even when annealed and thus can be used to house water-reactive compounds. A prime example of this are pH indicator pigments that change color with environmental pH. These pigments are crucial to both laboratory and biochemical assays, as many assays behave differently at different environmental pHs.

[0119] Exemplary pH indicators that may be incorporated are described in further sections herein.

[0120] Production of Toner

[0121] One technique for the production of silk toners, which are distinct from the dry mixed silk fibroin toners described herein, may be performed with standard industrial powder fabrication and handling techniques. Spray drying is a well-established method for creating fine, free-flowing powders with particulate on the micron scale. Here, aqueous fibroin solution is aerosolized into a stream of hot air that quickly disperses the liquid into fine droplets and removes the water in a single step, resulting in a fine powder. Control of this technique has been well characterized in the literature and the control parameters to alter for creating particles of different sizes and morphologies is well understood (See Lintingre, E., Lequeux, F., Talini, L. & Tsapis, N. Control of particle morphology in the spray drying of colloidal suspensions. Soft Matter 12, 7435-7444 (2016), which is incorporated herein in its entirety by reference for all purposes). This technique can produce toner in bulk, but the temperatures involved may be too high for certain thermally sensitive components.

[0122] An alternate technique for creating silk toners is fluidized bed grinding. In this method, large fibroin granules would be loaded into a container that forces air through the mixture to cause constant collisions between the particles and results in mutual abrasive action that reduces the size of the granules over time. This technique is already commonplace in the fabrication of toners, so existing infrastructure can be used with minimal modification to workflow.

[0123] A final option for small batch processes or highly sensitive compounds is electrospraying. This technique involves placing a large electrical potential (5-50 kV) between a conductive emitter needle and a grounded collector to electrically accelerate the solution. The high fields involved can create droplets smaller than a nebulizing nozzle and without the associated clogging issues. Further, this can be combined with an air stream for continuous powder collection similar to spray drying. Like spray drying, this technique has established control parameters to create particles of desired size and morphology (See Gao, Y. et al. Morphology control of electrosprayed core-shell particles via collection media variation. Materials Letters 146, 59-64 (2015), which is incorporated herein in itsPATENT Attorney Docket No. T002905 WO - 2095.0714 entirety by reference for all purposes). This technique similarly avoids high temperatures and so can be used with temperature sensitive cargo encapsulated within the silk fibroin.

[0124] Silk Xerography for Personalized Medicine

[0125] Each layer of a laser print deposits micrograms of toner per square centimeter and dosing can be controlled to an exquisitely fine degree with spatial coverage and multilayer deposition. One could conceive of this being used to help dose therapeutics on an as-need basis. With many people trying many kinds of diets, there are often micronutrient and vitamin deficiencies that occur from the exclusion of certain foods. These are often rectified by taking multi-vitamins, but this in turn comes with the risk of vitamin overdose, as these multivitamins often contain fat-soluble vitamins that can easily accumulate and cause harm through regular multivitamin consumption. In a data-driven health plan, vitamins and micronutrients can be tracked at home with either wearable or home-use sensors, but require the patient to constantly monitor and change dosages manually. This automated feedback loop can be closed by automating the dosing of vitamins by printing vitamin loaded fibroin onto consumable soluble films (Figure 4). In this scheme a medical toner is made containing and stabilizing the medicinal compounds. This is loaded into a xerographic printer that doses these compounds onto an edible substrate. For vitamins this substrate could be eaten directly, but other compounds may be foul tasting or need to be protected from stomach acid, so this strip could be rolled and loaded into an enteric capsule for delivery.

[0126] Beyond vitamins the same scheme could be extended into drugs with narrow therapeutic windows, which are drugs that are efficacious very near the dosage they become toxic. These drugs are often those used to treat intractable diseases such as metastatic cancer and antibiotic resistant pathogens. Complicating dosage beyond merely the mass and blood volume of a patient is the fact that medicines are metabolized at different rates by different individuals. Even for two people of the same stature, one may find a dose ineffective while the other would suffer overdose effects from the same amount. This is currently handled by regular visits with a managing physician and numerous blood tests to slowly close in on the correct dosage. By carefully monitoring drug concentrations in the blood with a wearable device, the drug could be metered for every dose in order to maximize clinical effect while simultaneously minimizing side effects and dosage-based toxicity. This process would therefore allow medications dosages to be tailored to each patient’s metabolism and response to the treatment.

[0127] While the market for laser printing of text documents is being eroded by the digital revolution, there are certain aspects of our life that cannot be digitized. Packaging, food, medical diagnostic devices and scientific assays will always be needed in physical form, but could also benefit from greener methods of manufacturing. Xerographic printing with silk fibroin offers anPATENTAttorney Docket No. T002905 WO - 2095.0714 avenue to repurpose existing infrastructure and technical knowledge to tackle these problems in a way that is green and renewable. The unique properties of silk also enable the fabrication of futuristic medical devices that could fill the gap in the feedback cycle that currently separates constant diagnostic monitoring and tailored therapeutic delivery.

[0128] Wherever laser printing is articulated herein, xerographic printing and printing via patterns of electrostatic charge are expressly contemplated as being disclosed as alternatives.

[0129] Disclosed herein is an example method of laser printing including xerographic printing, printing via patterns of electrostatic charge, and / or laser printing onto a substrate with a silk fibroin particle -based toner including silk fibroin particles to produce a printed article. In the example method, the laser printing sequentially includes a first laser printing iteration with the silk fibroin particle-based toner, a first intermediate printing iteration, and a second laser printing iteration with the silk firoin particle-based toner. The laser printing can further sequentially include, following the second laser printing iteration, a second intermediate printing iteration, and a third laser printing iteration with the silk fibroin particle-based toner.

[0130] Populations of bioprinting protein particles and / or silk fibroin particles with varying properties can be generated to provide a catalog of printable resources, with pixel level control. Powders can be mixed in various proportions and deposited with unique proportions at unique pixels, thus sampling a large number of variables in a single printing process.

[0131] The printing process may enable very accurate (and adjustable) dosing of therapeutic agents to achieve certain therapeutic effects while avoiding side effects that result from too-high dosage. When paired with a continuous monitoring device, it could allow for continuously varied dosages to hit specified therapeutic targets.

[0132] The precise spatial and compositional control will allow for implementation of large-scale samplings, where multiple variables are simultaneously sampled across a variety of unique printed environments.

[0133] Dry bioprinting allows the integration of bioactive printing with the underlying paper substrate, bringing paper to life without the usual requirement of wet printing.

[0134] Dry bioprinting allows an integrated sensor to be prepared by a dry bioprinting process, where the sensing portion in the form of dried particles can be shelf-stabilized and / or inactive in a dehydrated state, extending shelf life and allowing on-demand activation of various capabilities, including but not limited to, assays, medical screening devices, combination chemistry, displays, biological displays, security tags, bio-barcodes, aesthetic articles, time-varying images with timevarying tunable colors, flavor profiles, scent profiles, biological activity, fluorescence, chemistry, and the like.PATENT Attorney Docket No. T002905 WO - 2095.0714

[0135] In some cases, the methods described herein can be used to print onto existing devices, such as a previously-printed device. The methods described herein can be utilized to layer biologically active systems onto existing systems. The methods described herein can be utilized to prepare layered capabilities, such as those described above.

[0136] The example method further includes printing a second biopolymer prior to or subsequent to the laser printing and / or post-processing the silk fibroin particle-based toner following the laser printing.

[0137] In the example method, the first intermediate printing iteration includes printing with a biopolymer ink including lignin, alginate, chitin, keratin, chitosan, or a combination thereof.

[0138] As described herein, an intermediate printing iteration can include the laser printing and / or dry bioprinting methods described herein or they can include other printing approaches, including conventional printing, wet printing, inkjet printing, aqueous bioprinting, combinations thereof, and the like. For the avoidance of doubt, the inventive nature of the printing can be combined with more conventional printing steps to produce complex structures that include one or more of the properties described herein as related to the laser printing and / or dry bioprinting of the disclosure.

[0139] Without wishing to be bound by any particular theory, it is believed that the printing methods described herein can be broadly applicable across multiple levels of a complex material structure, with printed articles serving as components that are assembled into a larger structure. The present disclosure provides the capability of printing unique combinations of materials by mixing particles to provide a specific chemical signature to the toner composition. The present disclosure also provides the capability of providing unique physical arrangements of the combinations of materials.

[0140] In certain cases, the methods can include printing two different toners that are incompatible with one another (e.g., reactive with one another), with physical isolation from one another and separation by one or more layers of a different material.

[0141] In the example method, laser printing may be to a resolution of at least 2 lines / mm, at least 3 lines / mm, at least 4 lines / mm, at least 5 lines / mm, at least 6 lines / mm, at least 7 lines / mm, at least 8 lines / mm, at least 9 lines / mm, or at least 10 lines / mm. In the example method, laser printing deposits between 200 and 300 pg / cm3of toner per layer.

[0142] Disclosed herein is an example dry bioprinting process including selecting a bioprinting protein, a desired particle physical dimension, a desired particle electrostatic property, and optionally a desired particle residual moisture or a desired setting process (e.g., parameters to be utilized in a setting process), and laser printing with a bioprinting protein particle based toner onto a substrate to produce a printed article, the bioprinting protein particle based toner including bioprinting proteinPATENT Attorney Docket No. T002905 WO - 2095.0714 particles having the desired particle physical dimension, the desired particle electrostatic property, and optionally the desired particle residual moisture or the desired setting process.

[0143] In the example dry bioprinting process, the bioprinting protein is a structural protein, a globular protein, a fibrillar protein, an amphiphilic protein, or a combination thereof. The bioprinting protein is albumin, sericin, collagen, casein, gluten, keratin, zein, or silk fibroin. In specific instances, the bioprinting protein is silk fibroin.

[0144] In the example dry bioprinting process, the desired particle physical dimension is selected from a physical dimension disclosed in any one of US Pat. App. Pub. Nos. 2023 / 0039387 Al, 2023 / 0045444 Al, 2023 / 0051936 Al, 2023 / 0080900 Al, 2023 / 0091090 Al, 2023 / 0116451 Al, 2023 / 0117448 Al, 2023 / 0161277 Al, 2023 / 0176495 Al, Korean Patent No. 10-2517821 Bl, each of which is incorporated herein in its entirety by reference for all purposes.

[0145] In the example dry bioprinting process, the desired particle physical dimension is between 1 pm and 10 pm or between 3 pm and 5 pm.

[0146] In the example dry bioprinting process, the desired particle electrostatic property is selected from an electrostatic property disclosed in any one of US Pat. App. Pub. Nos. 2023 / 0039387 Al, 2023 / 0045444 Al, 2023 / 0051936 Al, 2023 / 0080900 Al, 2023 / 0091090 Al, 2023 / 0116451 Al, 2023 / 0117448 Al, 2023 / 0161277 Al, 2023 / 0176495 Al, Korean Patent No. 10-2517821 Bl, each of which is incorporated herein in its entirety by reference for all purposes.

[0147] In the example dry bioprinting process, laser printing may be to a resolution of at least 2 lines / mm, at least 3 lines / mm, at least 4 lines / mm, at least 5 lines / mm, at least 6 lines / mm, at least 7 lines / mm, at least 8 lines / mm, at least 9 lines / mm, or at least 10 lines / mm. In the example dry bioprinting process, laser printing deposits between 200 and 300 pg / cm3of toner per layer.

[0148] In examples disclosed herein, the bioprinting protein particles or the silk fibroin particles may have certain particle physical dimensions, particle electrostatic properties, physical properties, or certain formulations for printing.

[0149] An average particle diameter of a toner matrix particle may be in the range of 4 to 10 pm in median diameter on a volume basis, or may be in the range of 6 to 9 pm. In some examples, a typical particle diameter of is 3 pm to 10 pm. Some example toners have an average particle diameter of 7 pm.

[0150] For some example toners, an average circularity, calculated from the formula (Circularity=Perimeter of circle calculated from the circle equivalent diameter / Perimeter of projected image of particles), may be in the range of 0.930 to 1.000, or may be in the range of 0.950 to 0.995, or in the range of 0.94 to 0.99, or in the range of 0.97 to 0.99, or in the range from 0.920 to 0.965, or in the range from 0.940 to 0.962, or in the range from 0.945 to 0.955.PATENTAttorney Docket No. T002905 WO - 2095.0714

[0151] An example toner formulation may include toner particles that contain a binder resin and a coloring agent is used as the toner. Another example formulation includes a toner particle including a toner base particle containing a binder resin and a protruded portion on a surface of the toner base particle; the protruded portion including an organosilicon polymer and a polyhydric acid metal salt; and the polyhydric acid metal salt is present on a surface of the protruded portion. Yet another example formulation includes a toner particle containing a binder resin, and an external additive; the toner particle has at least one multivalent metal element selected from the group consisting of aluminum, magnesium, calcium and iron. Still another example formulation includes a toner particle including a binder resin and an ester compound, wherein: the binder resin comprises a resin A comprising a specific amount of a long-chain acrylate unit and a styrene-based monomer unit, and a resin B comprising a specific monomer unit in a specific amount; the ester compound has an alkyl chain with a specific chain length. In yet another formulation, an example polymerized toner includes a binder resin, and toner particles including a pigment or carbon black dispersed in the binder resin, a charge control agent, and a wax.

[0152] Some example toners have a Martens hardness of 200 MPa or more and 1100 MPa or less as measured at a maximum load of 2.0 x 10-4[N] .

[0153] An example toner has a loss elastic modulus G" at 100° C of preferably not more than 3.0x10’ (dyn / cm2) or not more than 7.0xl04(dyn / cm2), with a lower limit greater than or equal to 2.0xl04(dyn / cm2) or 4.0xl04(dyn / cm2).

[0154] In the example dry bioprinting process, the desired electrostatic property is dielectric.

[0155] In the example dry bioprinting process, the desired residual moisture is 20% or less, 15% or less, or 10% or less.

[0156] Disclosed herein is an example dry bioprinting process including administering a bioprinting protein particle-based toner comprising bioprinting protein particles to a substrate, and sintering the bioprinting protein particles.

[0157] In the example dry bioprinting process, the bioprinting protein is a structural protein, a globular protein, a fibrillar protein, an amphiphilic protein, or a combination thereof. The bioprinting protein is silk fibroin.

[0158] In some cases, the bioprinting protein particles and / or the silk fibroin particles can be non- porous. In some cases, the bioprinting protein particles and / or the silk fibroin particles can be monolithic.

[0159] In the example dry bioprinting process, the administering forms an image and the sintering sets the image. In the example dry bioprinting process, the administering includes xerography.PATENTAttorney Docket No. T002905 WO - 2095.0714

[0160] In the example dry bioprinting process, the sintering includes contacting the bioprinting protein particles with water vapor. Sintering includes contacting the bioprinting protein particles with water vapor at two different water vapor temperatures. Contacting the bioprinting protein particles with water vapor includes maintaining the bioprinting protein particles and the water vapor at a temperature of less than 75 °C, less than 70 °C, less than 65 °C, less than 60 °C, less than 55 °C, or less than 50 °C.

[0161] Disclosed herein is an example silk fibroin particle-based toner including silk fibroin particles.

[0162] Disclosed herein is an example laser printing cartridge loaded with silk fibroin particlebased toner including silk fibroin particles.

[0163] The disclosed methods, processes, particles, and / or cartridges can include post-processing, as would be appreciated by a person having ordinary skill in the art. Post-processing can involve a setting and / or sintering process, which may depend on the substrate. Post-processing can involve a curing, crosslinking, and / or recrystallizing process, which may depend on the substrate. In some cases, the setting and / or sintering can be achieved in the same basic process steps as the curing, crosslinking and / or recrystallizing. Without wishing to be bound by any particular theory, it is believed that water vapor reflow is a process that encompasses setting and / or sintering. Water vapor reflow can be achieved by contacting particles with water vapor for seconds at a temperature from room temperature up to 90 °C.

[0164] Without wishing to be bound by any particular theory, it is believed that annealing is a process that encompasses curing, crosslinking, and / or recrystallizing. Annealing can be achieved by exposing particles to water vapor at elevated temperatures. For example, the annealing process is explained in detail in Hu et al. “Regulation of Silk Material Structure by Temperature-Controlled Water Vapor Annealing’’, Biomacromolecules 2011, 12, 1686-1696, which is incorporated herein in its entirety by reference. Without being limiting, annealing of particles can be achieved within 5 minutes with exposure to 100% relative humidity at 45 °C, can be achieved within seconds using steam above 100 °C, or other conditions as would be appreciated by a skilled artisan.

[0165] In some cases, any of the methods or process described herein, or the products used in methods, can include setting and / or sintering. In some cases, any of the methods or process described herein, or the products used in methods, can include curing, crosslinking, and / or recrystallizing. In some cases, the setting and / or sintering is separate and distinct from the curing, crosslinking, and / or recrystallizing. In some cases, the setting and / or sintering is achieved using the same basic method steps as the curing, crosslinking, and / or recrystallizing.PATENTAttorney Docket No. T002905 WO - 2095.0714

[0166] In the example methods, processes, particles, or cartridges disclosed herein, the silk fibroin particle-based toner further includes filler fine particles. In some examples, the filler fine particles include inorganic fine particles. The inorganic fine particles include silica, titania, alumina, or a combination thereof. In some examples, the filler fine particles include organic fine particles. In some examples, the filler fine particles include organic-inorganic composite fine particles.

[0167] In the example methods, processes, particles, or cartridges disclosed herein, a number average particle diameter of the silk fibroin particles is 20 nm or more and 1000 pm or less, including but not limited to, 1 pm or more and 10 pm or less, or 3 pm or more and 5 pm or less.

[0168] In the example methods, processes, particles, or cartridges disclosed herein, the silk fibroin particles have one or more physical properties or physical dimensions, have one or more electrostatic properties, or are formulated for printing as recited in any one of US Pat. App. Pub. Nos.2023 / 0039387 Al, 2023 / 0045444 Al, 2023 / 0051936 Al, 2023 / 0080900 Al, 2023 / 0091090 Al, 2023 / 0116451 Al, 2023 / 0117448 Al, 2023 / 0161277 Al, 2023 / 0176495 Al, Korean Patent No. 10- 2517821 Bl, each of which is incorporated herein in its entirety by reference for all purposes.

[0169] In the example methods, processes, particles, or cartridges disclosed herein, the silk fibroin particles further include an additive.

[0170] In the example methods, processes, particles, or cartridges disclosed herein, the silk fibroin particles include a functional additive, wherein the functional additive is a selective protein, a fluorophore, a conductive material, a thermoreactive material, a sensing additive, a biologically active agent, or the like.

[0171] In the example methods, processes, particles, or cartridges disclosed herein, the silk fibroin particles include a medical diagnostic additive.

[0172] In the example methods, processes, particles, or cartridges disclosed herein, a functional additive is covalently linked to the silk fibroin. In the example methods, processes, particles, or cartridges disclosed herein, the silk fibroin particles and / or the bioprinting protein particles include one or more nutritional supplements. The functional additive is mixed with the silk fibroin within the silk fibroin particles.

[0173] In the example methods, processes, particles, or cartridges disclosed herein, the silk fibroin particles include a non-functional additive, wherein the non-functional additive is a non-selective protein, a colorant (e.g., a dye), an opacifying agent, an iridescent agent, a flavorant, an aroma, or the like.

[0174] In the example methods, processes, particles, or cartridges disclosed herein, the silk particles include a colorant, wherein the colorant is optionally a dye. The colorant is a naturally-occurring colorant, wherein the colorant is optionally anthocyanin.PATENTAttorney Docket No. T002905 WO - 2095.0714

[0175] In the example methods, processes, particles, or cartridges disclosed herein, the silk fibroin particles include silk fibroin that is at least partly sourced from recycled silk fabric.

[0176] In the example methods, processes, particles, or cartridges disclosed herein, the silk fibroin particles are made by spray drying.

[0177] In the example methods, processes, particles, or cartridges disclosed herein, the silk fibroin particles are made by powdering or lyophilization.

[0178] In the example methods, processes, particles, or cartridges disclosed herein, the silk fibroin particles have a beta-sheet crystallinity degree of greater than 50% of a maximum achievable betasheet crystallinity degree. In some cases, the silk fibroin particles have a beta-sheet crystallinity of greater than 60%, greater than 70%, greater than 80%, or greater than 90% of a maximum achievable beta sheet crystallinity. In some cases, the silk fibroin particles have the maximum achievable beta sheet crystallinity.

[0179] In the example methods, processes, particles, or cartridges disclosed herein, the silk fibroin particles have a beta-sheet crystallinity degree of less than 50% of a maximum achievable beta-sheet crystallinity degree. In some cases, the silk fibroin particles have a beta-sheet crystallinity of less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, or less than 5% or a maximum achievable beta-sheet crystallinity. In some cases, the silk fibroin particles have 0% of the maximum achievable beta-sheet crystallinity.

[0180] In the example methods, processes, particles, or cartridges disclosed herein, the silk fibroin particles have a minimum achievable beta-sheet crystallinity degree.

[0181] In the example methods, processes, particles, or cartridges disclosed herein, the substrate is a paper, a biodegradable substrate, or an edible substrate. Without wishing to be bound by any particular theory, it is believed that the disclosed methods may have advantages in printing onto biodegradable substrates and / or edible substrates.

[0182] In some cases, the substrate is conventional paper, a clear sheet, or the like. In some cases, the substrate is cellulose paper. In some cases, the substrate is cellulose acetate.

[0183] In the example methods, processes, particles, or cartridges disclosed herein, the substrate includes silk fibroin.

[0184] In the example methods and / or processes, the method and / or process includes sintering (or setting / water vapor reflowing) the silk fibroin particles and / or the bioprinting protein particles. Sintering can include contacting the silk fibroin particles and / or the bioprinting protein particles with water vapor. Sintering can include applying heat to the silk fibroin particles and / or the bioprinting protein particles. Applying heat can include a temperature of at least 30 °C, at least 35 °C, at least 40 °C, at least 45 °C, at least 50 °C, at least 55 °C, at least 60 °C, at least 65 °C, at least 70 °C, at leastPATENTAttorney Docket No. T002905 WO - 2095.071475 °C, at least 80 °C, at least 85 °C, at least 90 °C, or at least 95 °C and optionally at most 100 °C. Sintering can include administering a protease, applying pressure, or applying heat and pressure to the silk fibroin particles and / or the bioprinting protein particles.

[0185] In the example methods and / or processes, the method and / or process includes annealing (or curing / crosslinking / recrystallizing) the silk fibroin particles and / or the bioprinting protein particles. The annealing can include applying higher humidities and / or higher temperatures and / or longer exposures than the sintering / setting / water vapor reflow step.

[0186] In the example methods and / or processes, the method and / or process includes simultaneously setting (sintering / water vapor reflowing) and annealing (curing / crosslinking / recrystallizing) the silk fibroin particles and / or the bioprinting protein particles.

[0187] In the example methods and / or process, the method can include post-processing of contacting the silk fibroin particles and / or the bioprinting protein particles with water vapor.

[0188] In the example methods and / or process, the method can include post-processing of applying elevated temperature the silk fibroin particles and / or the bioprinting protein particles with water vapor.

[0189] In the example methods and / or process, the method can include post-processing of contacting the silk fibroin particles and / or the bioprinting protein particles with water vapor at elevated temperature.

[0190] The relative humidity of the water vapor can be at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or approximately 100%.

[0191] The elevated temperature of the water vapor can be at least 30 °C, at least 35 °C, at least 40 °C, at least 45 °C, at least 50 °C. at least 55 °C, at least 60 °C, at least 65 °C, at least 70 °C, at least 75 °C, at least 80 °C, at least 85 °C, at least 90 °C, or at least 95 °C. The elevated temperature of the water vapor can be at least 100 °C or higher.

[0192] A skilled artisan will recognize that relative humidity, temperature, and exposure time can be adjusted to achieve a desired setting and optionally annealing process.

[0193] In some cases, the methods and / or processes can result in a printed article that has silk fibroin particles that have amorphous crystallinity. Without wishing to be bound by any particular theory, it is believed that a printing process that retains silk fibroin particles with amorphous crystallinity can have some advantages when it comes to particular applications. In some cases, the final printed article has silk fibroin particles with minimum achievable beta sheet crystallinity.

[0194] In some cases, the methods and / or process can result in a printed article that has silk fibroin particles with significant beta sheet crystallinity. Without wishing to be bound by any particularPATENT Attorney Docket No. T002905 WO - 2095.0714 theory, it is believed that a printing process that maximizes the beta sheet crystallinity in silk fibroin particles can have some advantages when it comes to particular applications. In some cases, the final printed article has silk fibroin particles with maximum achievable beta sheet crystallinity.

[0195] In the example methods and / or processes, the method further includes activating the silk fibroin particle based toner and / or the bioprinting protein particle based toner by hydrating the silk fibroin particles and / or the bioprinting protein particles. At least one property of the silk fibroin particle based toner and / or the bioprinting protein particle based toner is changed by the activating.

[0196] In examples, the laser printing and / or dry bioprinting generates a personalized drug discovery platform.

[0197] In examples, the laser printing and / or dry bioprinting generates a spatially-varying sensing platform, a conductive pathway, or light-emitting compounds.

[0198] In examples, the laser printing and / or dry bioprinting includes one or more lasing media, one or more conductive molecules, one or more photoconducting materials (e.g., perovskites), or a natural dye (e.g., anthocyanin).

[0199] In examples, the printed article includes a physically unclonable function.

[0200] Physically unclonable functions (PUFs) are cryptographic tools that exploit the inherent randomness of various physical fabrication processes to make unique object identifiers that are easy to fabricate but exceptionally difficult to reproduce. A classical example of a PUF is glitter embedded in clear polymer resin. The fabrication of such an object is trivial, but each contains an exceptionally high number of degrees of freedom. Patterns of colors, the reflectivity at various angles of illumination and other properties are easily measured for any individually created piece but would be extraordinarily difficult to reproduce exactly. Thus, each piece acts as a unique identifier that cannot be readily copied by a counterfeiter.

[0201] Electrostatic silk printing is well suited to fabricating PUFs thanks to the inherent randomness of the shape and distribution of toner particulate and the ease of loading active cargo into the toner. Silk toner is fabricated by a grinding process giving a distribution of sizes and shapes to the toner particles that can be readily viewed under a microscope. The distribution of these shapes has the high degree of freedom needed for simple PUFs. Recently active PUFs have been created with challenge-response pairing, adding yet more degrees of freedom and further complicating duplication. These include optical responses (structural color, fluorescence, up-conversion, random lasing, thermochromics, plasmonics, etc.), chemical responses (pH, enzyme catalyzed reactions, affinity-pairs, etc), and physical responses (patterned magnetism, etc), all of which can be readily loaded into the silk during the formulation process. Lastly, silk is entirely biocompatible and edible, opening the use of these PUFs on medical implants and high-value pharmaceuticals.PATENT Attorney Docket No. T002905 WO - 2095.0714

[0202] According to various embodiments, a variety of functionalizing agents may be used with the silk-containing embodiments described herein (e.g., silk membrane, silk composition, silk articles, silk matrix, silk foam, silk microsphere, liquid composition, whipped silk cream, silk meringue, compressed silk meringue, hot-pressed silk meringue, silk leather, silk powder, silk toner, edible silkbased films, etc.). It should be understood that the examples herein may recite one or a few silkcontaining embodiments but are applicable to any silk-containing embodiment, as applicable. In some embodiments, a functionalizing agent may be any compound or molecule that facilitates the attachment to and / or development (e.g., growth) of one or more endothelial cells on a silk membrane. In some embodiments, a functionalizing agent may be any compound or molecule that facilitates the attachment and / or development (e.g., growth) of one or more megakaryocytes and / or hematopoietic progenitor cells on a silk matrix and / or silk membrane. In some embodiments, a functionalizing agent may be or comprise an agent suitable for facilitating the production of one or more of white blood cells and red blood cells.

[0203] 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.

[0204] 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).

[0205] 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.

[0206] 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. InPATENTAttorney Docket No. T002905 WO - 2095.0714 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.

[0207] 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 pg / mL and 1,000 pg / mL, 5 pg / mL and 1,000 pg / mL, 10 pg / mL and 1,000 pg / mL, 10 pg / mL and 500 pg / mL, 10 pg / mL 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 pg / mL, 10 pg / mL, 15 pg / mL, 20 pg / mL, 25 pg / mL, 50 pg / mL, 100 pg / mL, 200 pg / mL, 300 pg / mL, 400 pg / mL, 500 pg / mL, 600 pg / mL, 700 pg / mL, 800 pg / mL, or 900 pg / mL). In some embodiments, the amount of an individual functionalizing agent is at most 1,000 pg / mL (e.g., 900 pg / mL, 800 pg / mL, 700 pg / mL, 600 pg / mL, 500 pg / mL, 400 pg / mL, 300 pg / mL, 200 pg / mL, 100 pg / mL, 90 pg / mL, 80 pg / mL, 70 pg / mL, 60 pg / mL, 50 pg / mL, 40 pg / mL, 30 pg / mL, 20 pg / mL, 10 pg / mL, or 5 pg / mL).

[0208] 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.

[0209] In some aspects, the first and second chemical-physical state may be a physical property of the composition, such as a 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.PATENTAttorney Docket No. T002905 WO - 2095.0714

[0210] 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- 1 -naphthylazo)-2,2'-stilbenedisulfonic acid, tetrabromophenolphthalein ethyl ester, bromophenol blue, Congo red, methyl orange, ethyl orange, 4-(4-dimethylamino-l-naphylazo)-3-methoxybenzenesulfonic acid, bromocresol green, resazurin, 4- phenylazo-l-napthylamine, ethyl red 2-(l-dimethylaminophenyazo) pyridine, 4-(p- ethoxyphenylazo)-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-lH-quinazoline-2,4- dione, 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-trinitrobenzene, and clayton yellow.

[0211] Exemplary light responsive dyes or agents include, but are not limited to, photochromic compounds or agents, such as triaryhnethanes, stilbenes, azastilbenes, nitrones, fulgides, spiropyrans, napthopyrans, spiro-oxazines, quinones, derivatives, and combinations thereof.

[0212] 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).

[0213] 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.

[0214] Exemplary pressure or strain sensitive dyes or agents include, but are not limited to, spiropyran compounds and agents.

[0215] 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.

[0216] 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 thePATENTAttorney Docket No. T002905 WO - 2095.0714 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.

[0217] 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).

[0218] In some aspects, the additive or dopant comprises a flavoring agent or flavorant.

[0219] 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, acetyl propionyl, 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.

[0220] 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, methyl acetate, methyl propionate, methyl butyrate, ethyl acetate, ethyl butyrate, isoamyl acetate, pentyl butyrate, pentyl pentanoate, octyl acetate, benzyl acetate, methyl anthranilate, myrcene, geraniol, nerol, citral, citronellal, cironellol, linalool, nerolidol, limonene, camphor, menthol, carone, terpineol, alpha-ionone, thujone, eucalyptol, benzaldehyde, eugenol, cinnamaldehyde, ethyl maltol, vanillin, anisole, anethole, estragole, thymol.

[0221] 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 presentPATENTAttorney Docket No. T002905 WO - 2095.0714 disclosure include International Color Index or C.I. Pigment Black Numbers 1, 7, 11, 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, 114, 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.

[0222] 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 27 (C.I. 16185); 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.PATENTAttorney Docket No. T002905 WO - 2095.0714

[0223] 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).

[0224] Exemplary natural dyes for use in the present disclosure include Alkanet (C.I. 75520,75530); Annatto (C.I. 75120); Carotene (C.I. 75130); Chestnut; Cochineal (C.I.75470); Cutch (C.I. 75250, 75260); Divi-Divi; Fustic (C.I. 75240); Brazilin (C.I. 75280); Logwood (C.I. 75200); Osage Orange (C.I. 75660); Paprika; Quercitron (C.I. 75720); Saffron (C.I. 75100); Sandal Wood (C.I. 75510, 75540, 75550, 75560); Sumac; and Turmeric (C.I. 75300). Exemplary reactive dyes for use in the present disclosure include Reactive Yellow 37 (monoazo dye); Reactive Black 31 (diazo 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.

[0225] 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.

[0226] 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 wt%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15 wt%, 16 wt%, 17 wt%, 18 wt%, 19 wt% or 20 wt% based on the weight of the composition.

[0227] 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, althoughPATENT Attorney Docket No. T002905 WO - 2095.0714 any of these can be combined with calcium carbonate. In some applications, a white ink can include 60 wt% or less white pigment, 55 wt% or less white pigment, 50 wt% white pigment, 45 wt% white pigment, 40 wt% white pigment, 35 wt% white pigment, 30 wt% white pigment, 25 wt% white pigment, 20 wt% white pigment, 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%, 5 wt% to 55 wt%, 10 wt% to 50 wt%, 10 wt% to 25 wt%, 25 wt% to 50 wt%, 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 wt%, 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.

[0228] 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.

[0229] 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.

[0230] 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.

[0231] 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,PATENTAttorney Docket No. T002905 WO - 2095.0714 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.

[0232] 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), 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, 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.

[0233] 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 includes 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 andPATENTAttorney Docket No. T002905 WO - 2095.0714 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.

[0234] 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.

[0235] 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; saccharides; 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.

[0236] 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, andPATENTAttorney Docket No. T002905 WO - 2095.0714 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.

[0237] 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.

[0238] 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 has a molecular weight equal to or less than 700 Daltons.PATENTAttorney Docket No. T002905 WO - 2095.0714

[0239] 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.

[0240] 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 / anti spasmodic agent, a vasopressin analogue, an antiarrhythmic agent, an antiparkinsonian agent, an antiangina / antihypertensive agent, an anticoagulant agent, an antiplatelet agent, a sedative, an anxiolytic 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, and salmeterol; antiinflammatory agents, including antiasthmatic anti-inflammatory agents, antiarthritis antiinflammatory agents, and non-steroidal anti-inflammatory agents, examples of which include but are not limited to sulfides, mesalamine, budesonide, salazopyrin, diclofenac, pharmaceutically acceptable diclofenac salts, nimesulide, naproxen, 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,PATENTAttorney Docket No. T002905 WO - 2095.0714 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 bromocriptine; 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 benzodiazepines and barbiturates; anxiolytic 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 difenoxin hydrochloride, loperamide hydrochloride, furazolidone, diphenoxylate hydrochloride, 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.

[0241] 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, endothelin A receptor antagonists, retinoic acid receptor agonists, immunomodulators, hormonal and antihormonal agents, photodynamic agents, and tyrosine kinase inhibitors.

[0242] Antibiotics include aminoglycosides (e.g., gentamicin, tobramycin, netilmicin, streptomycin, amikacin, neomycin), bacitracin, carbapenems (e.g., imipenem / cilastatin), cephalosporins, colistin, methenamine, monobactams (e.g., aztreonam), penicillins (e.g., penicillin G, penicillin V, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, carbenicillin, ticarcillin, piperacillin, mezlocillin, azlocillin), polymyxin B, quinolones, and vancomycin; and bacteriostatic agents such as chloramphenicol, clindamycin, macrolides (e.g., erythromycin, azithromycin, clarithromycin), lincomycin, nitrofurantoin, sulfonamides, tetracyclines (e.g.,PATENTAttorney Docket No. T002905 WO - 2095.0714 tetracycline, doxycycline, minocycline, demeclocy cline), and trimethoprim. Also included are metronidazole, fluoroquinolones, and rifampin.

[0243] Enzyme inhibitors are substances which inhibit an enzymatic reaction. Examples of enzyme inhibitors include edrophonium chloride, N-methylphysostigmine, neostigmine bromide, physostigmine sulfate, tacrine, 1-hydroxymaleate, iodotubercidin, p-bromotetranisole, 10-(alpha- diethylaminopropionyl)-phenothiazine hydrochloride, calmidazolium chloride, hemicholinium-3,3,5- dinitrocatechol, diacylglycerol kinase inhibitor I, diacylglycerol kinase inhibitor II, 3- phenylpropargylamine, N°-monomethyl-L-arginine acetate, carbidopa, 3-hydroxybenzylhydrazine, hydralazine, clorgyline, deprenyl, hydroxylamine, iproniazid phosphate, 6-MeO-tetrahydro-9H- pyrido-indole, nialamide, pargyline, quinacrine, semicarbazide, tranylcypromine, N,N- diethylaminoethyl-2,2-diphenylv alerate hydrochloride, 3-isobutyl-l-methylxanthine, papaverine, indomethacin, 2-cyclooctyl-2-hydroxyethylamine hydrochloride, 2,3-dichloro-a-methylbenzylamine (DCMB), 8,9-dichloro-2,3,4,5-tetrahydro-lH-2-benzazepine hydrochloride, p-aminoglutethimide, p- aminoglutethimide tartrate, 3-iodotyrosine, alpha-methyltyrosine, acetazolamide, dichlorphenamide. 6-hydroxy-2-benzothiazolesulfonamide, and allopurinol.

[0244] Antihistamines include pyrilamine, chlorpheniramine, and tetrahydrozoline, among others.

[0245] 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.

[0246] Muscle relaxants include mephenesin, methocarbamol, cyclobenzaprine hydrochloride, trihexyphenidyl hydrochloride, levodopa / carbidopa, and biperiden.

[0247] Anti-spasmodics include atropine, scopolamine, oxyphenonium, and papaverine.

[0248] Analgesics include aspirin, phenylbutazone, indomethacin, sulindac, tolmetin, 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, funaltrexamine, nalbuphine, nalorphine, naloxone, naloxonazine, naltrexone, and naltrindole), procaine, lidocaine, tetracaine and dibucaine. Ophthalmic agents include sodium fluorescein, rose bengal, methacholine, adrenaline, cocaine, atropine, alphachymotrypsin, hyaluronidase, betaxolol, pilocarpine, timolol, timolol salts, and combinations thereof.

[0249] 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.

[0250] Anti -depressants are substances capable of preventing or relieving depression.PATENTAttorney Docket No. T002905 WO - 2095.0714

[0251] Examples of anti-depressants include imipramine, amitriptyline, nortriptyline, protriptyline, desipramine, amoxapine, doxepin, maprotiline, tranylcypromine, phenelzine, and isocarboxazid.

[0252] 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.

[0253] Hormones include estrogens (e.g., estradiol, estrone, estriol, diethylstilbestrol, 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, somatotropin, 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.

[0254] 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(methylPATENTAttorney Docket No. T002905 WO - 2095.0714 methacrylate) microspheres, hydroxyapatite, 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.

[0255] 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.

[0256] 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; analgesics; opioids; aminoxyls; furoxans; nitrosothiols; nitrates and anthocyanins; nucleosides, such as adenosine; and nucleotides, such as adenosine diphosphate (ADP) and adenosine triphosphate (ATP); neurotransmitter / 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.

[0257] 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, 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.

[0258] 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.),PATENTAttorney Docket No. T002905 WO - 2095.0714 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.

[0259] 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.

[0260] Differentiated cells that have been reprogrammed into stem cells can also be used.

[0261] 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. al, Cell, 2007, 131, 1-12).

[0262] 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.”

[0263] 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.

[0264] 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 thosePATENT Attorney Docket No. T002905 WO - 2095.0714 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.

[0265] 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.

[0266] 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.

[0267] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0268] 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.EXAMPLES

[0269] Example 1

[0270] Spray-dryingPATENTAttorney Docket No. T002905 WO - 2095.0714

[0271] Various spray drying conditions were screened by inlet temperature. These resulted in a variety of outlet temperatures from 160 °C to 105 °C and all yielded a fine, free-flowing powder upon completion of drying. Particle size and outlet temperature were measured and outlet temperature was found to be closely related to inlet temperature, while resulting particle size was loosely correlated (Fig. 7A and Fig. 7B).

[0272] The various temperatures yielded slightly different particle sizes, with generally smaller particles resulting from lower drying temperatures. Particle morphology remained largely the ‘collapsed sphere’ morphology (Fig. 8A, Fig. 8B, Fig. 8C, Fig. 8D, Fig. 8E, and Fig. 8F), resulting from a rapidly formed spherical shell that quickly becomes incapable of reflow then collapses upon depletion of the material in the interior. Occasional intact or nearly intact spherical shells were observed (Fig. 8C and Fig. 8D), and their frequency increased with the temperature, perhaps hinting that more rapid drying produces more stable spherical shells. SEM micrographs of commercial toners are shown in Fig. 5A and Fig. 5B for comparison.

[0273] Electrospraying

[0274] An electrospraying setup was constructed with a syringe pump, a high voltage power supply and a controlled humidity chamber. The emitter needle was a 26 gauge stainless steel blunt needle connected directly to the high voltage supply and the collector was a grounded aluminum plate coated with aluminum foil. The concentration, accelerating voltage, emitter / collector distance, solution conductivity were varied and electrospraying was performed for 2 minutes to collect a thin layer particulate on the aluminum foil. The particulate was then imaged by SEM to examine particle morphology.

[0275] At high solution concentrations the silk adopted a beaded-string morphology but as solution concentration dropped the interconnections became thinner and fewer, instead adopting a highly prolate spheroid morphology (Fig. 9A, Fig. 9B). When solution concentrations dropped below 4.0 w / v% the solution failed to spray (Fig. 9C), so this was taken as the minimum viable spraying concentration. Surprisingly, increasing accelerating voltage had the opposite anticipated effect, moving from prolate spheroid (Fig. 9D) back to beads-on-a-string morphology as the accelerating voltage increased (Fig. 9E, Fig. 9F). Increasing solution conductivity also caused a transition from beads-on-a-string (Fig. 9G) to prolate spheroids (Fig. 9H), but once saline concentrations reached 10 mM, spinning once again failed (Fig. 91). None of these methods produced a particle morphology desirable for laser printing and the labor included in the process was substantial, so it was discontinued in favor of more scalable methods.

[0276] Grinding MethodsPATENTAttorney Docket No. T002905 WO - 2095.0714

[0277] Cryo-blade milling produced many jagged fragments from the brittle fracture of the lyophilized sponge (Fig. 10A, Fig. 10B). The mill could not break down fragments to the required diameter and the jagged morphology caused dramatic clumping and difficulty flowing. While longer periods of grinding may have fixed the issues, the grinder was not able to run for sufficient time before overheating. Thus, the resulting powders were poorly suited to laser printing (Fig. 11 A).

[0278] Ball milling is another common method of grinding macroscale powders. This method relies on the continuous impact of solid ball bearings to produce powders. This method generally requires longer periods of time but the constant abrasion and impact tends to produce more rounded particulate that are more suitable for printing. When a lyophilized sponge was added to the ball mill and allowed to grind for 24 hours, the resulting powder was finely divided, free flowing and adopted a rounded morphology (Fig. 10C and Fig. 10D). It printed reliably and uniformly in an unmodified laser printer cartridge (Fig. 1 IB).

[0279] While performing ball milling on water-insoluble fibroin materials we noticed that they slowly became water soluble over the course of one week of grinding. We then attempted to use this as a method of solubilizing fibroin from waste silk cloth. To attempt this silk cloth was manually shredded into 1 cm2fragments and placed into a rubber milling cylinder with approximately lOOx 8 mm diameter steel ball bearings. This was placed on a continuously circulating base and left to grind for 1 week. After 1 week, 25 mL of DI water was added to the cylinder and wet ground for 1 hour. Afterwards the solution was filtered through a coarse (1 mm mesh) sieve to remove the macro-scale fragments. These were dried, weighed, and returned to dry grinding the next day. The remaining filtrate was filter sequentially through Whatman grade 1 paper to remove course debris, then a nitrocellulose filter with 0.45 pm pores to remove fine debris, leaving a solution of soluble silk fibroin. The solids were dried and collected from the various filtration steps, with fine solids forming a stable suspension in DI water while coarse solids formed unstable suspension that settled out after several hours of storage (Fig. 23). The same recovery procedure was repeated for ground fibers removed from grinding after a further week of milling.

[0280] The recovery at 1 week was minimal with only 5.4% of the total mass being recovered as soluble or suspended particles (Table 1). Of these, 81.1% of the total mass was soluble fibroin. At two weeks increased substantially but in the form of very large (>10 pm) suspended particles. While soluble fibroin represented less of the total recovered mass, it made up about the same absolute mass. This indicates that there are varied milling modes by population. This means that for more complete conversion to soluble fibroin, the large suspended population should be recovered and readmitted to the grinding to participate in the fine-grinding mode to produce more soluble powder.PATENTAttorney Docket No. T002905 WO - 2095.0714

[0281] Without wishing to be bound by any particular theory, it is believed that the method of solubilization is molecular level degradation that converted the long intact fibroin chains into low molecular weight segments that favored dissolution. Gel permeation chromatography was performed to ascertain the molecular weight distribution of recovered soluble fragments (Fig. 24). A portion of the silk cloth was processed to solution via the lithium bromide chemical dissolution method to compare molecular weights before and after grinding. Before grinding the fiber had very similar molecular weight to 30 minute boiled virgin silk, while after grinding it showed a sharp decrease in molecular weight in the first week. Interestingly, while the bulk molecular weight did not shift much during the second week of grinding, there is a clear shift towards a lower weight population on average, indicating further degradation of the fibroin molecules. Given the large loss of molecular weight, it was a concern that perhaps the silk would be unable to form gels, however the material spontaneously gelled during storage after several months, similar to chemically regenerated fibroin.

[0282] Characterization of Printing

[0283] Laser printers are highly engineered devices, designed to print with exceptionally high quality within very narrow performance constraints. Toner sizes and energy are well controlled to remain as a free-flowing powder in the reservoir yet cling tightly to transfer and imaging rollers. The particle size is set to match the cartridge to provide uniform particle monolayers for transfer to the OPC drum. The particles must also be removeable from the OPC drum by both the paper and cleaning wipers without residue. All these constraints are designed into the printer as much as the toner itself.

[0284] As such, introducing an entirely novel type of toner material will likely exceed those constraints in numerous and perhaps unexpected ways, leading to suboptimal performance. The effects of the printer must be decoupled from the influence of the toner whenever possible to allow the toner to be optimized for the printing hardware. In this section we choose from two different printers that are optimized for different toner profiles to see which is most closely matched to our current toners. After selection, we perform various characterizations of the print itself to determine ofPATENTAttorney Docket No. T002905 WO - 2095.0714 the toners interact with both the printer and substrate. Finally, we explore the novel setting process for dry mixed silk fibroin toners, which differs completely from traditional thermoplastic toners.

[0285] The selection of the commercial printer must be considered in tandem with the specifications of the toner that will be placed into the repurposed cartridge. Different printers are optimized to work with different toner compositions and thus may display differential compatibility with dry mixed silk fibroin toners. Two printers were selected for testing. The HP Laserjet Pro Ml 18 and the Brother HL-L2300D laser printers. Both are consumer grade black and white laser printers with comparable nominal specifications. Clean cartridges were loaded with ~10 mL of spray dried silk powder and printing was executed on the highest resolution mode with the setting temperature.

[0286] Resolution, alignment and print fidelity were tested by printing a resolution test pattern (Fig. 12A) onto a cellulose acetate transparency. The printed film was set with 2 seconds of wet-steam exposure, allowed to fully dry, then another layer was printed directly atop the previous print. Printed patterns were imaged under macro photograph and optical microscopy, with a portion of the test pattern (e.g., the bullseye portion) and print results displayed in Fig. 12B and Fig. 12C. The results described herein relate to a standard full resolution test pattern (not shown, see Ronnekleiv, Erlend. Erlend R nnekleivs Websider) which includes lines of different thicknesses, dots, curves, patterns, and characters, as well as the bullseye pattern shown in Fig. 12A, 12B, and 12C. On high level inspection, the Ml 18 had a lower level of vertical striations than the L2300D, resulting in a more uniform appearance to the printed regions of the film. Conversely, in the areas that should not contain any powder, the L2300D showed minimal off-target printing while the Ml 18 showed significant non-specific deposition. Together these indicate that the Ml 18 is likely tuned for larger toner granules and is thus more permissive of particles, while the L2300D is likely tuned for smaller toner particles and is thus more restrictive of flow.

[0287] Interlayer alignment was more consistent with the L2300D than the Ml 18, which produced visible misalignment between layers. Attempts were made to mitigate this by carefully positioning the print paper in the feed trays or using the alternate feed tray but neither reliably improved interlayer alignment. The L2300D by contrast had reliable interlayer alignment and was not substantially affected by discrepancies in the position of the paper during loading. The Ml 18 printed reliably and never produced any feed errors while the L2300D did encounter multiple feed errors resulting in paper jams. It was found these jams could be avoided by opening the rear door and bypassing the rollers that reorient the paper to dispense on the top of the printer.

[0288] Nominal print resolution was the same for both printers in their highest quality setting, and this was also observed in the printed test pattern. Both printers were able to print down to resolutions of 9.2 lines / mm but beyond that were constrained by compression artefacts (Fig. 13 A, Fig. 13B, andPATENTAttorney Docket No. T002905 WO - 2095.0714Fig. 13C). These compression artefacts were the same in both cases and were therefore likely to originate from either the encoding of the original file or by the printer drivers themselves. Neither printer was found to be superior in resolution as they were constrained by limitations in the software not the hardware.

[0289] We conclude from this that the L2300D printer has been engineered to dispense toner that is smaller in diameter and is therefore better suited to the particle diameters that silk naturally adopts during spray drying. The L2300D did experience more technical faults, but these could be overcome by simple modifications to the operating procedure, while no modifications were found to circumvent the challenges with the Ml 18. We therefore proceeded to use the L2300D for all subsequent experiments noted here.

[0290] Toner Performance Characterization

[0291] A well-engineered toner should show uniform and complete coverage of the printed surface, ideally in a single print. We define coverage here as the portion of area that is covered by toner, which is measured visually as the average optical density of the defined regions. Commercial toners are well matched to their printer and achieve 99.5% ± 2.0% on a single print (Figure 14). Spray dried silk was only able to achieve 57.1% ± 6.3% on a single print, with coverage rising to 69.3% ± 6.2% after 5 subsequent prints. Dyed silk on copy paper performed worse initially with 17.3% ± 7.4% coverage on a single layer, but rising to 79.6% ± 8.3% after 5 layers of deposition. The low coverage of dry mixed silk fibroin toners was largely due to two principal macroscopic defects, traceable to feed errors in the cartridge. There were large horizontal defects, likely due to bulk feed issues inside the cartridge failing to fully cover the transfer rollers. Vertical defects were also present but were traced back to toner clumping on the transfer rollers. Both defects can likely be remedied by process aids like anti-caking agents.

[0292] We next quantified the amount of silk deposited on each layer by printing the same test pattern seen above for coverage testing. Silk was printed on both copy paper and cellulose acetate and weighed before and after each print layer (Fig. 15A and Fig. 15B). The mass trends were seen to be identical on both substrates, thus illustrating the deposition is agnostic towards the receiving substrate. When the mass differential was divided by the printed area, we arrive at an average deposition density of 230 pg / cm2. This compares favorably to commercial black toner that applies 290 pg / cm2per layer.

[0293] The thickness of each layer was measured by scanning probe profilometry. Large sheets were printed with a multi-cell pattern we refer to as the DoE pattern (Figure 16A). Each cell has a 10 mm diameter disk surrounded by a 12 mm diameter ring with a 250 pm gap between them. The outer ring is always 1 layer thick while the inner ring differs in thickness from 0 to 10 layers, as indicatedPATENT Attorney Docket No. T002905 WO - 2095.0714 by the number to the lower right of the cell. The cells are randomly distributed across an entire lettersized piece of substrate with 16 replicates to minimize the effect local variance in printing performance has on the overall result. A DoE pattern of 0-10 layers was printed on an acetate sheet (Fig. 16B) and randomly selected samples of different layer height were excised and measured by a scanning probe profilometer (Fig. 16C and Fig. 16D). The probe was positioned in the bottom-most region of the outer ring and scanned upward so that it traversed both the ring / disk gap and the edge of the disk. The height of the disk was then measured vs the gap.

[0294] The height of subsequent printed layers was found to increase approximately linearly with 2.14 pm of additional height measured per layer. When the density of the layer is back calculated we find that we have approximately 80% bulk density in each printed layer. This density figure is likely dependent on the particle morphology and the allowed packing density. Without wishing to be bound by any particular theory, it is hypothesized that particles that are more spherical may improve packing density and thus the deposition density may be increased nearer to 100%. Higher bulk densities are likely preferable to achieve the most uniform behavior of the printed segments and highest delivery of encapsulated cargo.

[0295] While working with printed materials for active sensors, it was noticed that filter paper with higher numbers of layers began to resist wetting with aqueous solutions. To quantify this behavior we made a DoE pattern of differing layer count on Ahlstrom filter paper and placed the samples in a goniometer setup monitored with a Canon DLSR camera with a custom lens array for focus and magnification. The drops were applied while taking video and the absorption time was calculated by counting the number of frames the drop was visible after initial wetting and dividing that by the frame rate. The wetting time of the filter paper was found to follow an approximately exponential trend despite a nearly linear increase in bulk height with layer count. With 10 layers the wetting time was found to increase by approximately a factor of 10, but we hypothesize this trend will eventually plateau and the paper will not become a full hydrophobic surface.

[0296] Setting and Annealing

[0297] Here we demonstrate the setting and annealing process works for dry mixed silk fibroin toners in an identical manner to bulk silk constructs. Fig. 17 shows dry mixed silk fibroin toners before (top) and after (bottom) setting. Set prints were exposed to water vapor (90% relative humidity) for times ranging from 1 to 7 minutes. We can see be the evolution of the Amide I / II peaks in FTIR that we are getting time dependent structural rearrangement of the silk structures (Fig. 18). These results show that printed silk behaves in a highly similar manner to bulk silk at a molecular level, and thus previously applied processing techniques will likely produce very similar results to printed layers as they would to any other silk structure.PATENT Attorney Docket No. T002905 WO - 2095.0714

[0298] When testing printers to find the most suitable one for our application, we determined the HL2300D was better tuned for finer particle sizes and interlayer repeatability, two metrics that are crucially important for silk printing. The silk prints themselves were found to have difficulty in achieving uniform coverage in a single coating -likely due to the flow characteristics of the toner- but this deficit could be overcome by multiple layers. Each layer added approximately 230 pg / cm2of toner and applied a layer about 2.1 pm thick after setting. Setting via short exposure to wet-steam caused the toner to reflow into a smooth and largely uniform film. This film could then be water annealed by heated water-vapor exposure for differing lengths of time to achieve differing levels of crystallinity.

[0299] Dry mixed silk fibroin toners have proven capable of being used in existing commercial printers but would benefit from custom hardware designed specifically for their use. Specifically, interlayer repeatability is not a high priority for commercial printers as multiple layers are rarely printed on the same sheet, however in silk printing it is significant. Thus, while existing hardware is serviceable, it is not ideal. The silk toner itself exhibits unique qualities such as the ability to build prints layer by layer and to affect a wettability difference on certain substrates.

[0300] Incorporating Dyes

[0301] To understand the baseline chromaticity needed to achieve the vivid coloration of commercial printer toners, samples of toners were dissolved at concentrations of 1 mg / mL in isopropyl alcohol. From the stock solution a factor 2 serial dilution was made of each toner and the full dilution set was pipetted into a multi well plate and analyzed by a UV-Vis plate reader.Extinction coefficients were calculated from the wavelengths 420, 530 and 560 nm (Table 2). These wavelengths were chosen as they are the peak wavelengths detected by the human eye and are therefore the most important when designing visible colors.Table 2: Mass extinction coefficients of commercial toners

[0302] pH Indicator TonersPATENTAttorney Docket No. T002905 WO - 2095.0714

[0303] A standard example of a pH indicator is bromophenol blue, which changed between a blue and yellow color at basic and acid pHs respectively (Fig. 19A). This pigment is a small organic molecule that is sold in solid crystalline form (Fig. 19B). Energy dispersive x-ray (EDX) elemental analysis confirms that this pigment contains bromine and sulfur atoms (Fig. 19C), which can be used in combination with the crystal morphology to distinguish these crystals from dry mixed silk fibroin toner under SEM / EDX analysis.

[0304] Dry mixed silk fibroin toners with pH indicating capability were prepared by dry mixing spray -dried silk powder with bromophenol blue in a 5:1 mass ratio. Despite the crystal often being above 10 pm on their major axis, the high aspect ratio of the crystal allows them to print in an unmodified cartridge. These powders were loaded into a cleaned toner cartridge and printed onto Ahlstrom filter paper to produce pH test strips. Prints showed good fidelity, but required thorough setting for proper color development, which was otherwise obscured by the scattering from the silk particles despite the high extinction coefficient of the pigment (s @ 600 nm is up to 200 g L-l cm-1 depending on pH).

[0305] Under SEM / EDX analysis of the unset prints, the silk particles and pigment crystals can be easily distinguished both morphologically and by elemental composition (Fig. 20). After printing but before setting the particles remain distinct and isolated, with bromophenol blue showing strong characteristic emissions from bromine but lacking nitrogen, while silk particles show strong emissions from nitrogen but lack bromine. After setting, the silk becomes a relatively uniform and evenly distributed film, while bromophenol blue crystals show partial dissolution (as seen by the increase in background emission from bromine) and entrapment under the silk film layer.

[0306] Thermochromic Toners

[0307] Another pigment of interest are thermochromics, which undergo a reversible color shift when at certain temperature thresholds. Temperature is yet another crucial parameter for many biochemical assays and on-assay temperature monitoring can act as a reliable double-check that the assay is functioning as desired.

[0308] Thermochromic dry mixed silk fibroin toners were made by dry mixing spray dried silk powder with thermochromic pigment (red-yellow at 34 °C, Atlanta Chemical Engineering company) in a ratio of 2:1 by mass. The chosen thermochromic pigment is water-soluble to enable comingling of the pigment with the silk while setting. The toner was loaded into a cleaned toner cartridge and printed on a variety of substrates including copy paper, acetate films and filter paper. Upon printing and setting the color change was activated but reverted as soon as the paper cooled to room temperature, demonstrating the reactions remained fully reversible.PATENTAttorney Docket No. T002905 WO - 2095.0714

[0309] Under SEM / EDX analysis the pigment was found to adopt a spherical morphology with a large size distribution from 5 pm to sub-micron particles (Fig. 21 A). Elemental analysis revealed it was composed entirely of carbon, nitrogen, and oxygen, and as such is indistinguishable from silk elementally, thus we must rely entirely on morphological analysis. We see that after setting the silk toner again reflows to form a continuous film (Fig. 21B). Pigment granules can be seen below the film in some regions, indicating that reflow did not completely dissolve the pigment, so partial entrapment is observed similar to the pH indicator inks.

[0310] Dry mixed silk fibroin toners were shown to successfully incorporate dyes by simple dry mixing. These dyes remain discrete in the dry phase but become entrapped and incorporated after setting. Despite their water solubility, the included dyes do not effectively reflow to the same extent as the silk, leaving identifiable microscopic inclusions in the resulting films. Both pH indicators and thermochromics retain their function through printing and onto the final substrate, allowing the direct fabrication of functional surfaces.

[0311] Dry mixing presents an interesting possibility for the quick formulation of arbitrary printer toners. This first requires that the dye conforms to the specifications of the printer, which was an issue here. The thermochromic dyes were well within tolerance and printed without additional difficulties, while bromophenol blue were only in tolerance on their minor axis, causing substantial defects in the prints.

[0312] Fabrication of HRP / AmpR Biosensors with Dry mixed silk fibroin toners

[0313] To validate that silk fibroin was compatible with HRP / AmpR system, 0.5-2.0 w / v% fibroin was added to a solution of HRP (20 mU / mL) and AmpR (100 pM) in phosphate buffered saline (IX, pH 7.4). To this solution was added an equal volume of 0-10 pM hydrogen peroxide in PBS to create 0-5 pM final solution concentrations. These solutions were pipetted into a multi well plate and allowed to incubate in the dark for 30 minutes at room temperature. After incubation the plate was read by measuring fluorescence with an excitation of 530 nm and an emission of 590 nm. Results show that silk concentrations of up to 2.0 SN!N% do not have any significant impact on sensitivity (slope of line of best fit) in the 1-5 pM range. Going forward, volumes will be adjusted such that, in the liquid state, silk will be at 1 w / v% when the analyte is added. This concentration is sufficient to produce cohesive films, yet low enough to enable solutions to be stored for several weeks without fear of gelation if needed.

[0314] Seeing that the HRP / AmpR system is compatible with silk fibroin, we began attempting to make a printable enzyme-laden toner. Attempts to do this at the lab scale were stymied by equipment failure, eir lab-scale spray drying system was attempted to encapsulate the enzyme during the spray drying process. A solution of 5 w / v% silk fibroin with 5 U / mL HRP was spray dried. Powders werePATENT Attorney Docket No. T002905 WO - 2095.0714 fabricated with a 120 °C inlet temperature, which resulted in a 59 °C outlet temperature, which HRP is known to tolerate when encapsulated in silk fibroin.

[0315] Upon receipt of the encapsulated enzyme it was subjected to a quick, low sensitivity activity test. A small aliquot of the toner was dissolved in PBS (IX, pH 7.4), while enzyme-free silk was dissolved in a different vial and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was added to a final concentration of 1 mg / mL to both. ABTS is a chromogenic compound that goes from a light green color to dark purple when oxidized by HRP in the presence of silk. Hydrogen peroxide was then added to both vials to a final concentration of 0. 1 v / v%. Within seconds of addition of the peroxide the HRP-laden toner produced a vivid purple color while the enzyme -free silk did not produce a noticeable color change even after 30 minutes of incubation at 37 °C. This assay clearly indicated that the HRP contained in the enzyme-laden toner was still active and capable of transducing color changes from hydrogen peroxide.

[0316] While HRP survived the process of spray drying, we next needed to test if it could survive the laser printing process. The enzyme-laden toner was loaded into the print cartridge and 0-10 layers were printed on Ahlstrom filter paper, followed by a 2 second steam reflow to set the print. These layers were printed in the “DoE” pattern (Fig. 22A, Fig. 22C), which is a randomized pattern of 180x 10 mm circles that enables high-throughput screening. In this case the number to the left of the circle on the DoE pattern indicates the number of layers of HRP-silk toner that have been applied to that location. This thus enables us to simultaneously test the effect multiple layers have on the sensing of peroxide in the solid state. This DoE pattern was applied to the filter paper prior to Silk-HRP printing with a Xerox ColorQube wax printer, and the prints were set by heating the paper to 150 °C for 2 minutes to reflow the wax. This enclosed the circular regions, ensuring a uniform and isolated reaction environment for each sample.

[0317] The ability of dry HRP to sense peroxide was then tested with the printed silk by applying 5 uL of a solution containing 10 pM hydrogen peroxide, 5 mM AmpR, and IX PBS to each the top 36 sectors in the DoE pattern. The pattern was incubated in the dark for 30 minutes, the reacted regions were photographed then cut out by biopsy punch. The punched regions were loaded into a microwell plate to perform a fluorescence readout to quantify the amount of reaction products (Fig. 22B). Higher numbers of silk layers produced a distinctly stronger response (Fig. 22D), indicating the greater amount of enzyme produced more reaction product. As these layers have been treated to most often with both the heat of the printing process and steam reflow did not substantially impact the performance of the enzyme. Interestingly the lowest signal was not observed in the HRP-free controls but in the regions with 3 layers of HRP-silk applied. Indeed there is a downward trend in the opposite anticipated direction for the first 3 layers. We believe this to be an indication of a parasiticPATENT Attorney Docket No. T002905 WO - 2095.0714 oxidation process due to atmospheric oxygen. Here the additional silk served as a protective coating, shielding the AmpR from environmental oxygen, while later layers produced a stronger oxidative effect due to the higher amount of active HRP. Thus, the silk is performing multiple roles, both as enzyme carrier and fixing agent but also as an oxygen barrier protecting the sensitive AmpR dye.

[0318] The exact activity of the HRP was measured upon arrival and upon noticing a seeming drop in the activity of the HRP-dry mixed silk fibroin toner. Activity was also measured again after two weeks of dark room temperature storage. Activity was measured by a reverse AmpR assay, where a standard curve of hydrogen peroxide (0.05-1.00 mU / mL) was prepared and reacted against solutions of 100 pM AmpR and 10 mM hydrogen peroxide. This produced a highly linear calibration curve within the defined activity range. Silk-HRP toner was dissolved in IX PBS to a final concentration of 10 mg / mL and a factor-10 serial dilution was made down to 0.001 mg / mL. These were then also treated with the same AmpR and peroxide solution and incubated for 30 minutes. After incubation, the fluorescence was measured and the dilution within the correct activity range was quantified with the bulk activity back calculated with the dilution factor. An aliquot of the original solution used to make the HRP-laden toner was saved, stored at -80 °C and was similarly tested for activity.

[0319] The activity of the saved sample showed the anticipated activity of about 100 mU / mg, which was the amount loaded into the solution. The activity of the spray dried toner was much lower, at about 6.4 mU / mg, indicating substantial amounts of activity were lost in the spray drying and passage of time. Interestingly, at two weeks the activity continued to drop, indicating ongoing degradation of the HRP. This was not anticipated, as silk-stabilized HRP has been known to retain near total activity with room temperature storage for months at a timel O. One difference between our current results and the previous results from the literature is the form factor. The spray dried silk has a very high surface area and thus a great deal of exposure to atmospheric oxygen. In this form, the oxygen barrier provided by silk is insufficient to protect the cargo with such a small surface are to volume ratio.

[0320] After confirming we had sufficient sensitivity to attain a measurable signal, we measured the concentration of HRP in solution by creating 2 DoE patterns with either 1 or 10 layers of HRP silk printed in every sector. Solution containing 5 mM Amplex Red, PBS and between 10 pM and 300 mM hydrogen peroxide. A drop of 5 pL of the corresponding concentration was spotted on every cell of the two DoE sheets and allowed to incubate in the dark. Fluorescence was measured at 5 and 30 minutes.

[0321] At 5 minutes we observed the characteristic zig-zag shape of Amplex Red solutions measuring high concentration peroxide in the 10-layer DoE sheet. The signal rises at low linearly increasing concentrations, drops at intermediate concentrations due to side-reactions, then the sidePATENTAttorney Docket No. T002905 WO - 2095.0714 reactions are overwhelmed at high concentrations! 1. At 30 minutes we observed saturation at all concentrations save the lowest, indicating sensitive range is in the low to mid micromolar range for peroxide concentrations. These results indicate that we have successfully fabricated toner and printed the enzymatic component of a biosensor.

[0322] The 1 -layer DoE sheet produced a similar but less precise trend with concentration dependent fluorescence. An interesting observation was made after the sheets were stored at room temperature overnight after the reactions. All of the cells in the 1 -layer sheet had completely oxidized, while those in the 10-layer sheet retained the same oxidation pattern as observed the previous night. This yet again reinforces the hypothesis that the higher number of silk layers provide protection for oxidatively sensitive compounds, like AmpR, by providing a thickness-dependent oxygen barrier.

[0323] Here we proved the spray drying process is compatible with labile biomolecules like HRP, retaining a portion of their activity after the spraying process. The high surface area of the particles however, seem to introduce difficulty with environmental oxygen, leading to a new degradation mechanism. The enzyme survives printing and retains sensitivity in the dry state. The oxygen barrier provided by high numbers of silk layers has a demonstrable protective effect on the sensitive Amplex red redox sensor. The results here are highly encouraging, showing device fabrication in a dry state that can actively exploit the advantages of the system to achieve greater stability while retaining sensitivity.

[0324] EQUIVALENTS AND SCOPE

[0325] 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.

[0326] The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

PATENTAttorney Docket No. T002905 WO - 2095.0714CLAIMSWhat is claimed is:

1. A method of laser printing, the method comprising: xerographic printing, printing via patterns of electrostatic charge, and / or laser printing onto a substrate with a dry mixed silk fibroin toner to produce a printed article, the dry mixed silk fibroin toner formed by dry mixing a silk fibroin powder with a pigment powder and / or a dye powder.

2. The method of claim 1 , further comprising setting the dry mixed silk fibroin toner on the printed article using at least one of mist or low temperature steam.

3. The method of claim 2, further comprising water annealing the set dry mixed silk fibroin toner.

4. The method of claim 1 , wherein the dry mixed silk fibroin powder is produced by at least one of spray drying, ball milling, electrospraying, lyophilization and grinding, drop casting and grinding, or cryomilling.

5. The method of claim 1, wherein the dry mixed silk fibroin powder comprises at least one of amorphous silk fibroin nanoparticles and silk fibroin particles.

6. The method of claim 1 , wherein the dry mixed silk fibroin powder is dry mixed with the pigment powder.

7. The method of claim 1, wherein the dry mixed silk fibroin powder is dry mixed with the dye powder.

8. The method of claim 1, wherein the laser printing sequentially comprises a first laser printing iteration with a biopolymer toner and a second laser printing iteration with the dry mixed silk fibroin toner.

9. The method of claim 1 , wherein the laser printing sequentially comprises a first laser printing iteration with the dry mixed silk fibroin toner and a second laser printing iteration with a biopolymer toner.

10. The method of any one of claims 7 or 8, wherein the method further comprises a third printing iteration subsequent to the second laser printing iteration.

11. A method of laser printing, the method comprising:PATENTAttorney Docket No. T002905 WO - 2095.0714 xerographic printing, printing via patterns of electrostatic charge, and / or laser printing onto a substrate with a silk fibroin biotoner powder to produce a printed article, the silk fibroin biotoner powder formed by mixing a silk fibroin powder and an enzyme solution, and optionally a pigment powder and / or a dye powder, to form a biotoner solution, wherein the biotoner solution is spray dried to form the silk fibroin biotoner powder.

12. The method of claim 11, wherein the silk fibroin powder comprises at least one of amorphous silk fibroin nanoparticles and silk fibroin particles.

13. The method of claim 11, wherein the enzyme is a biosensing enzyme.

14. The method of claim 11, further comprising setting the silk fibroin biotoner powder on the printed article using at least one of mist or low temperature steam.

15. The method of claim 11, wherein the silk fibroin powder is processed by at least one of spray drying, ball milling, electrospraying, lyophilization and grinding, drop casting and grinding, or cryomilling.

16. The method of claim 11, wherein the silk fibroin biotoner powder is dry mixed with the dye powder and / or the pigment powder.

17. The method of any of claims 1 - 10, or 16, wherein the pigment powder is selected from the group consisting of carbon black, iron oxide, and organic pigments.

18. The method of any of claims 1 - 10, or 16, wherein the dye is selected from the group consisting of thermochromic dyes, pH indicator dyes, acid dyes, natural dyes, direct dyes (either cationic or anionic), basic dyes, azo-dyes, and reactive dyes.

19. The method of claim 11, wherein the laser printing sequentially comprises a first laser printing iteration with a biopolymer toner and a second laser printing iteration with the silk fibroin biotoner powder.

20. The method of claim 11, wherein the laser printing sequentially comprises a first laser printing iteration with the silk fibroin biotoner powder and a second laser printing iteration with a biopolymer toner.

21. The method of any one of claims 8, 9, 19, or 20, wherein the method further comprises a third printing iteration subsequent to the second laser printing iteration.

22. The method of any one of the preceding claims, wherein the laser printing comprises setting and / or sintering the silk fibroin.PATENT Attorney Docket No. T002905 WO - 2095.071423. The method of any one of the preceding claims, wherein the laser printing comprises curing, crosslinking, and / or recrystallizing the silk fibroin.

24. The method of any one of claims 8, 9, 19, or 20, wherein the biopolymer toner comprises lignin, alginate, chitin, keratin, chitosan, or a combination thereof.

25. The method of any of the preceding claims, wherein the silk fibroin biotoner powder or the dry mixed silk fibroin toner further comprises a photochemical additive.

26. The method of any one of the preceding claims, wherein the laser printing is to a resolution of at least 5 lines / mm.

27. The method of any one of the preceding claims, wherein the laser printing is to a resolution of at least 10 line / mm.

28. The method of any one of the preceding claims, wherein the laser printing deposits between 200 and 300 pg / cm3of toner per layer.

29. The method of any one of the preceding claims, wherein the silk fibroin powder is produced by spray drying, and wherein the inlet temperature or the outlet temperature is between 100 °C and 175 °C.

30. The method of claim 29, wherein the inlet temperature is between 100 °C and 175 °C.

31. The method of claim 29, wherein the outlet temperature is between 100 °C and 175 °C.

32. The method of any one of the preceding claims, wherein the silk fibroin powder comprises silk fibroin in a concentration (w / v) of between 4% and 8%.

33. A laser printing cartridge loaded with a dry mixed silk fibroin toner formed by dry mixing a silk fibroin powder with a pigment powder and / or a dye powder.

34. A laser printing cartridge loaded with a silk fibroin biotoner powder formed by mixing a silk fibroin powder and an enzyme solution, and optionally a pigment or dye powder, to form a biotoner solution, wherein the biotoner solution is spray dried to form the silk fibroin biotoner powder.

35. A method of making a silk toner, the method comprising: ball-milling a porous silk fibroin article having amorphous crystalline structure and / or a silk I structure for a length of time sufficient to generate a population of silk fibroin particles that are suitable for xerographic printing, printing via patterns of electrostatic charge, and / or laser printing onto a substrate.PATENTAttorney Docket No. T002905 WO - 2095.071436. A method comprising: a) ball-milling a silk fabric, thereby producing a milled output including a mixture of soluble silk fibroin, silk fibroin particles, and silk fibroin fibers: b) isolating a portion of the milled output (e.g., removing particles of a specific size, solubilizing the soluble silk fibroin, etc.).

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