Low molecular weight silk compositions and stabilization of silk compositions

Abstract

To provide certain silk fibroin compositions with particular characteristics and / or properties.SOLUTION: In some embodiments, the disclosure provides a low molecular weight composition. In some embodiments, the disclosure provides a silk fibroin composition comprising an active (e.g., biologically active) agent or component. In some embodiments, the disclosure provides a low molecular weight silk fibroin composition comprising an active (e.g., biologically active) agent or component. In some embodiments, the active agent is stabilized in the silk composition for a certain period of time and / or against certain conditions or events. In some embodiments, the component in the silk fibroin composition may be subjected to analysis and / or characterization. In some embodiments, the component present in the silk fibroin composition may be recovered from the composition.SELECTED DRAWING: None

Description

Technical Field

[0001] Government Support This invention was made with government support under Award Number SB112 - 005 awarded by DARPA and Award Number P41 EB002520 awarded by NIH. The government has certain rights in this invention.

Background Art

[0002] Background The stabilization and subsequent recovery of active agents and / or biological samples are extremely important features for many applications because active agents and / or biological samples are typically unstable and sensitive to changes in ambient conditions such as temperature, humidity, and / or light. Even if an active agent or biological sample has been identified as useful for a given reaction, its application is often hindered by a lack of long - term stability under process conditions.

[0003] A variety of methods for stabilizing active agents (e.g., enzymes, therapeutic proteins including vaccines, and / or biological samples (e.g., blood and / or blood components)) have been utilized and studied, including cold - chain storage, lyophilization, covalent immobilization, and cellulose - based technologies. However, all of these methods have drawbacks such as extreme energy requirements or poor recovery rates of active agents and / or biological samples. These drawbacks further limit the options in on - demand or point - of - care applications. Therefore, there is a need for improved technologies and / or products, or new materials, that can stabilize active agents and / or biological samples (e.g., under ambient conditions) and enable the recovery of sufficient amounts of various analytes from the stabilized samples for treatment, diagnosis, detection, and / or analysis.

Summary of the Invention

Means for Solving the Problems

[0004] Summary The present disclosure provides certain silk fibroin compositions having novel and / or unexpected structural and / or functional characteristics and / or properties. The present disclosure provides methods of making and / or using such compositions, as well as articles comprising or consisting of these. In some embodiments, the compositions provided include an active (e.g., biological) agent or component. In some embodiments, the active agent or component is stabilized in the composition, for example, as compared to silk fibroin and / or other equivalent conditions lacking certain structural and / or functional characteristics and / or properties.

[0005] In particular, embodiments of the various aspects described herein are based on the discovery that low molecular weight silk fibroin-based silk materials provide unique material properties suitable for several applications. As described in detail below, the low molecular weight silk fibroin compositions described herein provide certain material properties that are clearly different or improved as compared to conventional silk fibroin-based materials. Thus, in one aspect, a low molecular weight silk fibroin composition comprising a population of silk fibroin fragments having a range of molecular weights, wherein no more than 15% of the total number of silk fibroin fragments in the population have a molecular weight greater than 200 kDa, and at least 50% of the total number of silk fibroin fragments in the population have a molecular weight within a specified range, the specified range being between about 3.5 kDa and about 120 kDa, or between about 5 kDa and about 125 kDa, is provided herein.

[0006] In other words, the low-molecular-weight silk fibroin composition described herein is a population of silk fibroin fragments having a range of molecular weights, wherein no more than 15% of the total number of moles of silk fibroin fragments in the population have a molecular weight greater than 200 kDa, and at least 50% of the total number of moles of silk fibroin fragments in the population have a molecular weight within a specified range, the specified range being between about 3.5 kDa and about 120 kDa, or between about 5 kDa and about 125 kDa, and may include a population of silk fibroin fragments characterized thereby.

[0007] The low-molecular-weight silk fibroin composition can exist in various forms. In some embodiments, the low-molecular-weight silk fibroin composition is an aqueous solution. The inventors have surprisingly discovered that an agent (e.g., but not limited to, a protein, nucleic acid such as DNA, RNA, and / or a modified form thereof, a therapeutic agent, a vaccine), or a sample (e.g., a biological sample) incorporated or mixed with the low-molecular-weight silk fibroin solution described herein can be stabilized over time at a wide range of temperatures. In some embodiments, an agent or sample incorporated or mixed with the low-molecular-weight silk fibroin solution can be stabilized for a period of time, e.g., at least 24 hours or more, under any of a variety of conditions, including, for example, ambient conditions (e.g., room temperature) and / or elevated temperature conditions (e.g., 60 °C).

[0008] In some embodiments, the low-molecular-weight silk fibroin composition can be in a solid state form, e.g., a silk fibroin article. Examples of silk fibroin articles can include, but are not limited to, films, sheets, gels or hydrogels, meshes, mats, non-woven mats, fabrics, scaffolds, tubes, slabs or blocks, fibers, particles, powders, three-dimensional constructs, implants, foams or sponges, needles, lyophilized articles, and any combination thereof. In some embodiments, the silk fibroin article can be a bioabsorbable implant, tissue scaffold, suture, reinforcement material, medical device, coating, constituent material, wound dressing, tissue sealant, fabric, textile product, and any combination thereof.

[0009] In some embodiments, the low molecular weight silk fibroin article is readily reconstituted into a low molecular weight silk fibroin solution for further processing. In some embodiments, the low molecular weight silk fibroin articles described herein may have a solubility of at least 5% or more in an aqueous solution. That is, at least 5% or more of the total weight or volume of the low molecular weight silk fibroin article can be dissolved or reconstituted in the aqueous solution. In some embodiments, the low molecular weight silk fibroin articles described herein may have a solubility of more than 5% or more, including, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more in an aqueous solution. In some embodiments, the low molecular weight silk fibroin articles described herein may be soluble or reconstitutable at room temperature. In some embodiments, the low molecular weight silk fibroin articles described herein may be soluble or reconstitutable in water such as deionized water at room temperature. In some embodiments, the low molecular weight silk fibroin article can be used for on-demand applications.

[0010] In some embodiments, the low molecular weight silk fibroin articles described herein may have enhanced solubility in an aqueous solution compared to a reference silk fibroin composition (e.g., a conventional silk fibroin-based material). In some embodiments, the solubility of the low molecular weight silk fibroin article in the aqueous solution can be enhanced by at least about 5% or more compared to the solubility of the reference silk fibroin composition. For example, the solubility of the low molecular weight silk fibroin article in the aqueous solution can be enhanced by, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or more than 5% or more including more than that compared to the solubility of the reference silk fibroin composition. In some embodiments, the solubility of the low molecular weight silk fibroin article in the aqueous solution can be enhanced by at least about 1.1-fold or more compared to the solubility of the reference silk fibroin composition. For example, the solubility of the low molecular weight silk fibroin article in the aqueous solution can be enhanced by, for example, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, or more than 1.1-fold or more including more than that compared to the solubility of the reference silk fibroin composition. In some embodiments, the aqueous solution can be water. In some embodiments, the aqueous solution can be a buffer solution, e.g., but not limited to, a phosphate buffer solution.

[0011] In some embodiments, the low molecular weight silk fibroin articles described herein can be used to encapsulate at least one active agent or sample (e.g., a biological sample) therein for the stabilization of the active agent or sample and subsequent recovery thereof. In some embodiments, a partial or original loading amount of the active agent or sample can be recovered from at least a portion or aliquot of the low molecular weight silk fibroin article by dissolving a portion or aliquot of the low molecular weight silk fibroin article in an aqueous solution. In some embodiments, a portion or aliquot of the low molecular weight silk fibroin article can be dissolved in an aqueous solution (e.g., but not limited to, water such as deionized water or a buffer) at room temperature. In some embodiments, the active agent or sample (e.g., a biological sample) encapsulated in the low molecular weight silk fibroin articles described herein can have a recovery rate of at least 5% or more in an aqueous solution. That is, at least 5% or more of the original loading amount of the active agent or sample can be recovered from the low molecular weight silk fibroin article in solution or liquid form. In some embodiments, the active agent or sample (e.g., a biological sample) encapsulated in the low molecular weight silk fibroin articles described herein can have a recovery rate of more than 5% or more, including, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or more in an aqueous solution.

[0012] In some embodiments, the low molecular weight silk fibroin article can enhance the recovery rate of an active agent or sample encapsulated therein as compared to the recovery rate of the active agent or sample encapsulated in a reference silk fibroin composition. In some embodiments, the recovery rate of the active agent or sample encapsulated in the low molecular weight silk fibroin article can be enhanced by at least about 5% or more as compared to the recovery rate of the active agent or sample encapsulated in the reference silk fibroin composition. For example, the recovery rate of the active agent or sample encapsulated in the low molecular weight silk fibroin article can be enhanced by, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or more than 5% including more than that as compared to the recovery rate of the active agent or sample encapsulated in the reference silk fibroin composition. In some embodiments, the recovery rate of the active agent or sample encapsulated in the low molecular weight silk fibroin article can be enhanced by at least about 1.1-fold or more as compared to the recovery rate of the active agent or sample encapsulated in the reference silk fibroin composition. For example, the recovery rate of the active agent or sample encapsulated in the low molecular weight silk fibroin article can be enhanced by, for example, at least about 1.5-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, or more than 1.1-fold including more than that as compared to the recovery rate of the active agent or sample encapsulated in the reference silk fibroin composition. In some embodiments, the aqueous solution can be a buffer solution, for example, but not limited thereto, a phosphate buffer solution. In some embodiments, using a buffer solution (e.g., but not limited thereto, a phosphate buffer solution) to reconstitute the low molecular weight silk fibroin article described herein can improve the recovery rate of the active agent or sample incorporated into the low molecular weight silk fibroin article as compared to using a non-buffered solution (e.g., water).

[0013] In various embodiments of the different aspects described herein, the reference silk fibroin composition can be a composition or mixture produced by degumming silk cocoons for about 60 minutes or less, such as less than 60 minutes, less than 50 minutes, less than 40 minutes, less than 30 minutes, less than 20 minutes, less than 10 minutes, or less, at atmospheric boiling point. In one embodiment, the reference silk fibroin composition can be a composition or mixture produced by degumming silk cocoons for about 60 minutes or less, such as less than 60 minutes, less than 50 minutes, less than 40 minutes, less than 30 minutes, less than 20 minutes, less than 10 minutes, or less, in an aqueous sodium carbonate solution at atmospheric boiling point.

[0014] Another aspect provided herein is a shelf-stable composition comprising a silk fibroin article, wherein the silk fibroin article comprises one or more embodiments of the low molecular weight silk fibroin compositions described herein, relating to the shelf-stable composition. In some embodiments, the shelf-stable composition is shelf-stable in that the material properties of the silk fibroin article remain stable for at least one month or more. In some embodiments, the shelf-stable composition can comprise a low molecular weight silk fibroin composition present in the form of particles including powders.

[0015] Generally, the present disclosure encompasses the recognition that certain silk fibroin compositions are particularly useful for the incorporation of active (e.g., biological) and / or unstable entities. In some embodiments, silk compositions are provided that contain active and / or unstable agents such as biological samples or their components. In some embodiments, the provided silk compositions (e.g., including low molecular weight silk fibroin compositions) stabilize active and / or unstable agents (e.g., stabilize biological samples or their components).

[0016] Low molecular weight silk fibroin particles, including powders, can be produced by any method recognized in the art. In some embodiments, the low molecular weight silk fibroin particles can be produced by a process that includes subjecting a low molecular weight silk fibroin solution to lyophilization, thereby forming a lyophilized low molecular weight silk fibroin material or particles. In some embodiments, the lyophilized low molecular weight silk fibroin material or particles can be further reduced to smaller particles. In one embodiment, the lyophilized low molecular weight silk fibroin material or particles can be further milled to produce smaller particles or powders. Accordingly, another aspect provided herein is a storage-stable reagent comprising a low molecular weight silk fibroin powder. In some embodiments, the low molecular weight silk fibroin powder can be a lyophilized powder. The storage-stable reagent can be provided as part of a kit or within a container, such as a vial, syringe, or sample collection container, such as a blood collection container, or a multiwell plate.

[0017] The various embodiments described herein provide storage-stable compositions comprising one or more embodiments of the low molecular weight silk fibroin compositions described herein and an agent that is desired to be stabilized, wherein the agent is associated with or incorporated within the low molecular weight silk fibroin composition. In some embodiments, the agent that is desired to be stabilized can be an active agent. For example, in some embodiments, proteins, peptides, nucleic acid molecules, and / or modified nucleic acid molecules can be stabilized in a low molecular weight silk fibroin composition. In some embodiments, the agent that is desired to be stabilized can be a therapeutic agent. In some embodiments, the agent that is desired to be stabilized can be a diagnostic marker for a disease or disorder. In some embodiments, the agent that is desired to be stabilized can be a sample that is assayed for one or more components present in the sample. In some embodiments, the sample can be a biological sample.

[0018] Yet another aspect provided herein relates to a fibroin article comprising one or more embodiments of the low molecular weight fibroin compositions described herein and a substrate. The low molecular weight fibroin composition can be dispersed in the substrate, deposited on the substrate, or a combination thereof.

[0019] Another aspect provided herein is a method of forming a fibroin solution, the method comprising dissolving one or more embodiments of the low molecular weight fibroin compositions described herein in a liquid, wherein the fibroin solution is a homogeneous solution. In one embodiment, the liquid is water, such as, but not limited to, deionized water. As used herein, the term "homogeneous solution" generally refers to a solution having a uniform appearance or composition throughout the solution. For example, the size of fibroin fragments or particles in a homogeneous fibroin solution is generally too small to be visible, for example, to the naked eye or detectable. Put another way, a homogeneous fibroin solution is substantially free of fibroin aggregates (e.g., insoluble fibroin fragments, fibroin particles, and / or clusters). Examples of fibroin aggregates can include, but are not limited to, full-length fibroin molecules, larger fibroin fragments, fibroin particles or clusters formed by the assembly or aggregation of smaller fibroin fragments, and any combination thereof.

[0020] Another aspect provided herein relates to a method of stabilizing an agent over a period of time under certain conditions in one or more embodiments of the fibroin compositions described herein. In some embodiments, the agent can be stabilized under ambient conditions.

[0021] A further aspect provided herein is a method for recovering at least one active agent, comprising: (a) preparing one or more embodiments of the low molecular weight silk fibroin compositions described herein, one or more embodiments of the storage stable compositions described herein, or one or more embodiments of the storage stable described compositions herein, wherein at least one active agent is stabilized in the composition; and (b) dissolving at least a portion of the composition in water to form a sample solution comprising silk fibroin and a detectable or measurable amount of at least one active agent.

[0022] Yet another aspect provided herein is a method for modulating at least one property of a silk fibroin composition, comprising varying the weight ratio of silk fibroin fragments in a composition having a molecular weight greater than 200 kDa to silk fibroin fragments in a composition having a molecular weight within a specified range, the specified range being between about 3.5 kDa and about 120 kDa, or between about 5 kDa and about 125 kDa, in the silk fibroin composition.

[0023] Still another aspect provided herein is a method for adjusting the size of silk fibroin particles, comprising two steps: (a) varying the weight ratio of silk fibroin fragments in a solution having a molecular weight greater than 200 kDa to silk fibroin fragments in a solution having a molecular weight within a specified range, the specified range being between about 3.5 kDa and about 120 kDa, or between about 5 kDa and about 125 kDa, in the silk fibroin solution; and (b) forming silk fibroin particles from the silk fibroin solution.

[0024] Silk fibroin is an example of a polypeptide having one or more portions of an amino acid sequence capable of adopting a beta-sheet secondary structure. For example, the silk fibroin structure generally includes an amino acid sequence in which one or more portions of the sequence are generally characterized by alternating glycine and alanine, or alanine alone. Without wishing to be bound by theory, such a configuration allows the fibroin molecules to self-assemble into a beta-sheet higher-order structure. Thus, in yet another aspect, provided herein is a composition comprising a population of polypeptide fragments having one or more portions of an amino acid sequence characterized by alternating glycine and alanine, or alanine alone, and having a molecular weight in a range between about 3.5 kDa and about 120 kDa, or between about 5 kDa and about 125 kDa. In some embodiments, the composition is characterized in that no more than 15% of the total number of polypeptide fragments in the population have a molecular weight greater than 200 kDa, and at least 50% of the total number of polypeptide fragments in the population have a molecular weight within a specified range, the specified range being between about 3.5 kDa and about 120 kDa, or between about 5 kDa and about 125 kDa.

[0025] In another aspect, the silk fibroin in the compositions and / or methods described herein can be replaced with, or used in combination with, other non-silk polypeptides that include a beta-sheet structure or have a tendency to form such a structure based on their amino acid sequence. Thus, provided herein are also polypeptide compositions comprising a population of beta-sheet-forming polypeptide fragments. In some embodiments, the population of beta-sheet-forming polypeptide fragments described herein can have a molecular weight in a range such that no more than 15% of the total number of beta-sheet-forming polypeptide fragments in the population have a molecular weight greater than 200 kDa, and at least 50% of the total number of beta-sheet-forming polypeptide fragments in the population have a molecular weight within a specified range, the specified range being between about 3.5 kDa and about 120 kDa, or between about 5 kDa and about 125 kDa.

[0026] As used herein, the term "beta-sheet forming polypeptide" refers to a polypeptide having one or more portions of an amino acid sequence that adopts a beta-sheet secondary structure. In some embodiments, the beta-sheet forming polypeptide can be selected based on having a beta-sheet structure or a tendency to form such a structure based on the amino acid sequence.

[0027] In some embodiments, the beta-sheet forming polypeptide can have an amphiphilic character (i.e., have both hydrophilic and hydrophobic properties and / or portions). Amphiphilic polypeptides can be obtained from a single source (e.g., a naturally occurring protein), can contain both hydrophobic modules or stretches and hydrophilic modules or stretches within the polypeptide, such that the single polypeptide itself is naturally amphiphilic. In some embodiments, hydrophobic modules or stretches and hydrophilic modules or stretches can be fused or coupled together to form an amphiphilic entity. Such "fusion" or "chimeric" polypeptides can be produced using recombinant techniques, chemical coupling, or both.

[0028] In some embodiments, the beta-sheet forming polypeptide can include one or more portions of the amino acid sequence of a polypeptide selected from the following list: fibroin, actin, collagen, catenin, claudin, coilin, elastin, elaunin, extensin, fibrillin, lamin, laminin, keratin, tubulin, viral structural proteins, zein proteins (seed storage proteins), and any combination thereof.

[0029] In some embodiments, the beta-sheet forming polypeptide can include a regenerated (e.g., purified) protein from a natural source, a recombinant protein produced in a heterologous system, a synthetic or chemically generated peptide, or a combination thereof.

[0030] In some embodiments, the beta sheet-forming polypeptide may include one or more portions of the amino acid sequence of a polypeptide corresponding to any one of the lists provided above, regardless of the presence or absence of one or more sequence variations compared to the native or wild-type counterpart. For example, in some embodiments, such variants may exhibit at least 85% overall sequence identity compared to the wild-type sequence, such as at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% overall sequence identity.

[0031] This patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee. In embodiments of the present invention, for example, the following items are provided. (Item 1) A low-molecular-weight silk fibroin composition comprising a population of silk fibroin fragments having a range of molecular weights, wherein no more than 15% of the total number of said silk fibroin fragments in said population have a molecular weight exceeding 200 kDa, wherein at least 50% of the total number of said silk fibroin fragments in said population have a molecular weight within a specified range, wherein said specified range is between a lower limit of about 3.5 kDa or more and an upper limit of about 120 kDa or less, a low-molecular-weight silk fibroin composition characterized thereby. (Item 2) wherein said lower limit of said specified range is 3.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, or 115 kDa, wherein said upper limit of said specified range is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 kDa, The low-molecular-weight silk fibroin composition according to item 1. (Item 3) The low molecular weight silk fibroin composition according to item 1 or 2, wherein the specified range is (i) between about 5 and 120 kDa; (ii) between about 10 and 120 kDa; (iii) between about 15 and 120 kDa; (iv) between 20 and 120 kDa; (v) between 20 and 110 kDa; (vi) between about 20 and 100 kDa; (vii) between about 20 and 90 kDa; (viii) between about 20 and 80 kDa; (ix) between about 30 and 120 kDa; (x) between about 30 and 100 kDa; (xi) between about 30 and 90 kDa; (xii) between about 30 and 80 kDa; (xiii) between about 40 and 100 kDa; and (xiv) between about 40 and 90 kDa. (Item 4) The low molecular weight silk fibroin composition according to any one of items 1 to 3, wherein not more than about 35% of the total silk fibroin fragment population has a molecular weight in the range of about 120 kDa to about 200 kDa. (Item 5) The low molecular weight silk fibroin composition according to any one of items 1 to 4, wherein at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85% of the total number of the silk fibroin fragments in the population has a molecular weight within the specified range. (Item 6) The low molecular weight silk fibroin composition according to any one of items 1 to 5, wherein the composition is a solution. (Item 7) The low molecular weight silk fibroin composition according to any one of items 1 to 5, wherein the composition is in solid form. (Item 8) The low molecular weight silk fibroin composition according to item 7, characterized in that the low molecular weight silk fibroin composition has a higher water solubility than that of the reference silk fibroin composition. (Item 9) The low molecular weight silk fibroin composition according to item 7, characterized in that the low molecular weight silk fibroin composition has a dissolution rate of about 1 mg / s to about 100 mg / s. (Item 10) An aqueous silk fibroin solution containing the low molecular weight silk fibroin composition according to any one of items 1 to 5. (Item 11) The aqueous fibroin solution according to item 10, wherein the fibroin is present in the solution at a concentration of about 0.1% wt / v to about 50% wt / v. (Item 12) The aqueous fibroin solution according to item 10, characterized in that the aqueous fibroin solution remains stable for at least 3 days, at least 7 days, or at least 2 weeks. (Item 13) A fibroin article comprising the low-molecular-weight fibroin composition according to any one of items 1 to 5, wherein the fibroin article is in a form selected from the group consisting of a film, a sheet, a gel or hydrogel, a mesh, a mat, a non-woven mat, a fabric, a scaffold, a tube, a block, a fiber, a particle, a powder, a three-dimensional construct, an implant, a foam, a needle, a lyophilized article, and any combination thereof. (Item 14) The article according to item 13, characterized in that the fibroin article has the ability to re-solubilize in water to form a fibroin solution substantially free of fibroin aggregates. (Item 15) A composition comprising fibroin particles, wherein the fibroin particles comprise the low-molecular-weight fibroin composition according to any one of items 1 to 5. (Item 16) The composition according to item 15, characterized in that the composition has storage stability for at least 1 month. (Item 17) A composition comprising the low-molecular-weight fibroin composition according to any one of items 1 to 5 and an active agent distributed in the low-molecular-weight fibroin composition. (Item 18) The composition according to item 17, wherein the composition is in a form selected from the group consisting of a solution, a film, a sheet, a gel or hydrogel, a mesh, a mat, a non-woven mat, a fabric, a scaffold, a tube, a block, a fiber, a particle, a powder, a three-dimensional construct, an implant, a foam, a needle, a lyophilized article, and any combination thereof. (Item 19) The composition according to item 17, wherein the composition is a solution. (Item 20) The composition according to item 17, wherein the composition is in solid form. (Item 21) The composition according to item 20, wherein the composition is characterized by the ability to re-solubilize in water to form a silk fibroin solution substantially free of silk fibroin aggregates. (Item 22) The composition according to item 21, wherein a detectable amount of the active agent can be recovered after re-solubilization of the composition. (Item 23) An article comprising the low molecular weight silk fibroin composition according to any one of items 1 to 5 and a substrate, wherein the silk fibroin fragment is dispersed in the substrate, deposited on the substrate, or a combination thereof. (Item 24) The article according to item 23, wherein the substrate is a solid porous substrate or comprises the same. (Item 25) The article according to item 24, wherein the solid porous substrate comprises paper. (Item 26) A method for forming a silk fibroin solution, comprising the step of dissolving the silk fibroin article according to item 14 or the composition according to item 21 at an unsaturated concentration in water, thereby forming a silk fibroin solution substantially free of silk fibroin aggregates. (Item 27) The method according to item 26, wherein the dissolved silk fibroin in the solution remains stable. (Item 28) A method for stabilizing an agent over a period of time, comprising the step of contacting the agent with the low molecular weight silk fibroin composition according to any one of items 1 to 5, whereby an agent-containing composition is formed. (Item 29) The method according to item 28, wherein the low molecular weight silk fibroin composition is substantially free of a solvent and / or the contacting step is carried out under conditions substantially free of a solvent. (Item 30) The method according to item 29, further comprising the step of forming a silk fibroin solution or a silk fibroin article comprising the low molecular weight silk fibroin composition and the agent distributed therein. (Item 31) The method according to item 26, further comprising the step of maintaining the silk fibroin solution for a period of time at room temperature or above. (Item 32) The method according to item 28, further comprising the step of maintaining the agent-containing composition for a period of time at room temperature or above. (Item 33) A method for stabilizing an agent in an aqueous solution over a period of time, the method comprising the step of contacting the agent with an aqueous silk fibroin solution according to any one of items 10 to 12. (Item 34) The method according to item 28, further comprising the step of forming a silk fibroin article comprising the low molecular weight silk fibroin composition and the agent distributed therein. (Item 35) The method according to item 34, further comprising the step of maintaining the agent in the silk fibroin solution or the silk fibroin article for a period of time at room temperature or above. (Item 36) The method according to item 31, wherein the time is about 24 hours or more, or about 1 week or more. (Item 37) The method according to item 32, wherein the time is about 24 hours or more, or about 1 week or more. (Item 38) The method according to item 35, wherein the time is about 24 hours or more, or about 1 week or more. (Item 39) A method for recovering at least one active agent, comprising the step of providing a solid silk fibroin composition comprising at least one active agent distributed therein, and Dissolving at least a portion of the composition in water, thereby forming a sample solution comprising silk fibroin and a detectable amount of the at least one active agent. (Item 40) The method according to item 39, wherein the at least one active agent is or comprises blood or a blood component. (Item 41) The low molecular weight silk fibroin composition, in that the silk fibroin composition is composed of a population of silk fibroin fragments having a range of molecular weights, wherein no more than 15% of the total number of the silk fibroin fragments in the population have a molecular weight exceeding 200 kDa, wherein at least 50% of the total number of the silk fibroin fragments in the population have a molecular weight within a specified range, The method according to item 39 or item 40, wherein the specified range is between a lower limit of about 3.5 kDa and an upper limit of about 120 kDa. (Item 42) The method according to item 41, further comprising subjecting the sample solution to at least one fluidics-based assay to detect the at least one active agent. (Item 43) The method according to item 41, wherein the at least one active agent is derived from a sample. (Item 44) The method according to item 43, wherein the sample is derived from a biological sample of a subject. (Item 45) A method of modulating at least one property of a silk fibroin composition, comprising changing the weight ratio of silk fibroin fragments in the composition having a molecular weight exceeding 200 kDa to silk fibroin fragments in the composition having a molecular weight within a specified range in the silk fibroin composition, the specified range being between about 3.5 kDa and about 120 kDa. (Item 46) A method for adjusting the size of silk particles, comprising: (a) changing the weight ratio of silk fibroin fragments in the solution having a molecular weight exceeding 200 kDa to silk fibroin fragments in the solution having a molecular weight within a specified range in a silk fibroin solution, wherein the specified range is between about 3.5 kDa and about 120 kDa; and (b) forming the silk particles from the silk fibroin solution. (Item 47) A composition comprising a silk-based material containing silk fibroin and a biological sample, wherein at least one property of at least one component of the biological sample is stabilized over a period of time and the at least one component of the biological sample is detectable after the period of time. (Item 48) The composition according to item 47, wherein the at least one property includes activity, integrity, quantity, physical or structural properties, chemical properties, biological properties, or any combination thereof. (Item 49) The composition according to item 47 or 48, wherein the period of time is at least about 24 hours or at least about one week. (Item 50) The composition according to item 47, wherein the silk-based material is in a form selected from the group consisting of a solution, a film, a fiber, a particle, a gel, a hydrogel, a foam, a sponge, a mat, a mesh, a fabric, a powder, a coating layer, freeze-dried forms thereof, and any combination thereof. (Item 51) The composition according to item 47, wherein the silk-based material is soluble. The silk-based material according to any one of items 1 to 6, wherein the biological sample is collected from a subject. (Item 52) The composition according to item 47, wherein the biological sample is pretreated before being incorporated into the silk-based material. (Item 53) The composition according to item 47, wherein the biological sample is cells, tissues, blood (including, for example, whole blood, plasma, cord blood, platelets, and serum), lactation products (such as milk), amniotic fluid, sputum, urine, saliva, mucus, semen, cerebrospinal fluid, bronchial aspirate, sweat, nasal discharge, vaginal fluid, liquefied feces, synovial fluid, lymph fluid, tears, tracheal aspirate, or fractions or combinations thereof, or contains them. (Item 54) The composition according to item 47, wherein the at least one component is selected from the group consisting of peptides, proteins, antibodies, enzymes, amino acids, nucleic acids (such as polynucleotides, oligonucleotides, genes, genes containing regulatory regions and termination regions, self-replicating systems such as viruses or plasmid DNA, genomic DNA, cDNA, mRNA, pre-mRNA, single-stranded and double-stranded siRNA and other RNA interference reagents (RNAi agents or iRNA agents), shRNA (short hairpin RNA), antisense oligonucleotides, aptamers, ribozymes, microRNA (miRNA), pre-miRNA, and modified RNA), nucleotides, metabolites, lipids, sugars, glycoproteins, peptidoglycans, microorganisms, cells, and any combination thereof. (Item 55) The composition according to item 47, wherein the ratio of the silk fibroin to the biological sample is about 1:1000 to about 1000:1, or about 1:1 to about 1000:1. (Item 56) The composition according to item 47, wherein the silk-based material contains at least about 50% by weight of silk fibroin. (Item 57) The composition according to item 47, wherein the silk-based material is prepared from a silk solution containing about 0.25% to about 50% (w / v) of silk fibroin, or about 0.5% to about 10% (w / v) of silk fibroin. (Item 58) The composition according to item 47, further comprising a substrate, wherein the silk-based material forms a layer on the surface of the substrate. (Item 59) The composition according to item 58, wherein the substrate is selected from the group consisting of a dipstick, a cellulose-based product, a microtiter plate, a specimen container, and any combination thereof. (Item 60) At least one constituent of the biological sample is subjected to at least one freeze-thaw cycle; a temperature above 0 °C; exposed to light (e.g., UV); a relative humidity of at least about 10%; or any combination thereof The composition according to item 47, which is subjected to at least one condition selected from the group consisting of. (Item 61) a. preparing a mixture comprising silk fibroin and a biological sample; and b. forming a silk-based material from the mixture, the silk-based material comprising silk fibroin and a biological sample; The method, which includes, wherein at least one property of at least one constituent of the biological sample is stabilized over a period of time and the at least one constituent of the sample is detectable after the period of time. (Item 62) The method according to item 61, further comprising contacting the biological sample with the silk fibroin to form the mixture. (Item 63) The method according to item 61, further comprising drying the mixture of step a. (Item 64) The method according to item 63, wherein the drying step includes freeze-drying. (Item 65) The method according to item 63, wherein the drying step includes air-drying. (Item 66) The method according to item 61, wherein the silk-based material is formed by applying an electric field to the mixture. (Item 67) The method according to any one of items 61, further comprising reducing the silk-based material to particles. (Item 68) The method according to item 61, wherein the silk-based material is prepared using a process comprising solution casting, salt leaching, freeze drying, gas formation, electrospinning, gelation, fiber drawing, coating, spraying, miniaturization, or any combination thereof. (Item 69) A biological sample collection device comprising a chamber for collecting a biological sample, wherein the chamber contains silk fibroin. (Item 70) A kit comprising a biological sample collection device containing a silk fibroin material. (Item 71) The kit according to item 70, further comprising at least one container containing a gelation inducer. (Item 72) The kit according to item 71, wherein the gelation inducer comprises a functionally activated PEG component, a pH reducing agent, or a combination thereof. (Item 73) The kit according to item 70, further comprising a container containing a silk solubilizer. (Item 74) The kit according to item 73, wherein the silk solubilizer comprises water, a buffer, or a combination thereof. (Item 75) The kit according to item 73, further comprising a container containing a stabilizer, wherein the stabilizer stabilizes at least one component of the biological sample. (Item 76) The kit according to item 75, wherein the stabilizer is selected from the group consisting of saccharides, sugar alcohols, ions, surfactants, amino acids, human serum albumin, bovine serum albumin, gelatin, and gelatin derivatives, antioxidants, or any combination thereof. (Item 77) The kit according to item 75, wherein the stabilizer is a nuclease or a protease inhibitor. (Item 78) The kit according to item 75, wherein the stabilizer is an RNase inhibitor. (Item 79) The kit according to any of item 71, further comprising a container containing an agent for detecting at least one constituent component of a biological sample. (Item 80) The kit according to item 79, wherein the agent is selected from the group consisting of a constituent component purifying agent, a nucleic acid amplifying agent, an immunoaffinity-based detecting agent, or any combination thereof. (Item 81) A method comprising the step of preparing the composition according to item 47, and the step of subjecting at least one constituent component of the biological sample to at least one analysis.

Brief Description of Drawings

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Mode for Carrying Out the Invention

[0062] Certain Definitions Unless otherwise stated or implicit from the context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise or not apparent from the context, the following terms and phrases do not exclude the meaning obtained in the technical field to which the term or phrase belongs. The definitions are provided to assist in explaining specific embodiments and are not intended to limit the invention claimed in the patent. The reason is that the scope of the present invention is limited only by the claims. Further, unless otherwise required by the context, singular terms shall include pluralities, and plural terms shall include the singular.

[0063] One (a): The singular terms “a (one),” “an (one),” and “the (the)” include the plural referents unless the context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise.

[0064] About: Except in the case of operating examples or as otherwise indicated, all numerical values representing amounts of components or reaction conditions used herein should be understood to be modified in all cases by the term “about.” The term “about” may mean ±5% of the value being referred to when used in relation to a percentage. For example, about 100 means 95 - 105.

[0065] Ambient: As used herein, the "ambient" conditions are the surrounding conditions without active cooling, heating, etc. Typically, this term refers to room temperature, which generally describes a typical indoor temperature and is often between 20 and 25 °C (68 and 77 °F). In some embodiments, the ambient temperature is between 0 °C and 60 °C, between 0 °C and 50 °C, or between 0 °C and 40 °C. In some embodiments, the ambient temperature is a refrigerator temperature (e.g., between 0 °C and 15 °C (including both ends)). In some embodiments, the ambient temperature is room temperature, e.g., between 20 °C and 35 °C, which can vary depending on geographical conditions. For example, the room temperature in warm climate regions, e.g., in Africa, can generally be warmer than that in cool climate regions, e.g., in the United States or the United Kingdom. In some embodiments, the storage temperature can be at least about 37 °C or above 37 °C.

[0066] Antigen: As used herein, the term "antigen" refers to a molecule or part of a molecule that can be bound by a selective binding agent such as an antibody and can further be used in an animal to induce the production of an antibody that can bind to an epitope of that antigen. An antigen can have one or more epitopes. The term "antigen" can also refer to a molecule that can be bound by a T cell receptor (TCR) when presented by an antibody or an MHC molecule. The term "antigen" includes T cell epitopes herein. An antigen can further be recognized by the immune system and / or can induce a humoral and / or cellular immune response, resulting in the activation of B and / or T lymphocytes. However, this can, at least in certain cases, require that the antigen contains or is linked to a Th cell epitope and is given with an adjuvant. An antigen can have one or more epitopes (B and T epitopes). The specific reaction referred to above means that the antigen preferably reacts with the corresponding antibody or TCR, typically in a highly selective manner, and does not react with a number of other antibodies or TCRs that can be induced by other antigens. An antigen can be a mixture of several individual antigens herein.

[0067] Bioactivity: As used herein with reference to an active agent, the term "bioactivity" generally refers to the ability of the active agent to interact with a biological target and / or to have an effect on a biological target. For example, bioactivity can include, without limitation, the induction of stimulatory, inhibitory, regulatory, toxic, or lethal responses in a biological target. The biological target can be a molecule or a cell. For example, 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 cases, bioactivity can refer to the ability of a compound to cause a toxic effect within a cell. Exemplary cellular responses include, but are not limited to, lysis, apoptosis, growth inhibition, and growth promotion; production, secretion, and surface expression of a protein or other molecule of interest by a cell; membrane surface molecule activation, including receptor activation; transmembrane ion transport; transcriptional control; changes in cell viability; changes in cell morphology; changes in the presence or expression of intracellular components of a cell; changes in gene expression or transcripts; changes in the activity of an enzyme produced intracellularly; and changes in the presence or expression (e.g., protein expression and / or binding activity) of a ligand and / or receptor. Methods for assaying various cellular responses, such as Western blot for determining changes in the presence or expression of endogenous proteins in a cell, or microscopy for monitoring cell morphology in response to an active agent, or FISH and / or qPCR for detecting and quantifying changes in nucleic acids, are well known to those of skill in the art. Bioactivity can, in some embodiments, be determined, for example, by assaying a cellular response.

[0068] In reference to an antibody, the term "biological activity" includes, but is not limited to, for example, epitope or antigen binding affinity, in vivo and / or in vitro stability of the antibody, immunogenicity of the antibody, and / or the ability to neutralize or antagonize the biological activity of a target molecule in vivo or in vitro when administered to a human subject. The above-described properties or characteristics can be observed and measured using techniques recognized in the art, including, but not limited to, scintillation proximity assay, ELISA, ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence ELISA, competitive ELISA, surface plasmon resonance (SPR) analysis including SPR analysis using a BIAcore biosensor, in vitro and in vivo neutralization assays (see, e.g., International Publication No. WO2006 / 062685), receptor binding, and immunohistochemistry using tissue sections from various sources including human, primate, or any other source as required. In reference to an immunogen, "biological activity" includes immunogenicity, the definition of which is discussed in detail later. In reference to a virus, "biological activity" includes infectivity, the definition of which is discussed in detail later. In reference to a contrast agent, for example, a dye, "biological activity" refers to the ability of the contrast agent to enhance the contrast of a structure or fluid in the body of a subject when administered to the subject. The biological activity of a contrast agent includes, but is not limited to, its ability to interact with the biological environment and / or affect the response of another molecule under certain conditions.

[0069] Cold Chain: The term "cold chain" refers to a temperature-controlled supply chain. An unbroken cold chain is a continuous series of storage and distribution activities that maintain a given temperature range. This is used to help extend and guarantee the shelf life of products such as chemicals, biologics, and pharmaceuticals. Cold chains are common in the food and pharmaceutical industries, as well as in some chemical shipments. The typical temperature range for a cold chain in the pharmaceutical industry is 2 - 8°C. In some cases, the specific temperature (and time at that temperature) tolerance depends on the actual product being stored and / or shipped.

[0070] Constituent: As used herein, the term "constituent" of a sample refers to a physical, chemical, or biological entity (e.g., but not limited to, proteins, peptides, nucleic acids, cells, growth factors, and / or therapeutic agents) present in the sample that can be detected or analyzed.

[0071] Comprises: Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The term "comprises" means "includes". The abbreviation "e.g." is derived from the Latin exempli gratia and is used herein to indicate non-limiting examples. Thus, the abbreviation "e.g." is synonymous with the term "for example".

[0072] Comprising: As used herein, the term "comprising" or "comprises" is used with reference to a composition, method, and each of their respective constituents useful in one embodiment, and moreover accepts the inclusion of unspecified elements whether or not they are useful.

[0073] "decrease": As used herein, the terms "decrease", "reduced", "reduction", "decrease", or "inhibit" are all generally used to mean a decrease by a statistically significant amount. However, to avoid doubt, "reduced", "reduction", or "decrease", or "inhibit" means a decrease of at least 10%, for example, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to 100% decrease and decreases including the same (e.g., non - existent level compared to a reference sample), or any decrease between 10 - 100% compared to a reference level.

[0074] "essentially": As used interchangeably herein, the terms "essentially" and "substantially" mean at least about 60%, or preferably at least about 70%, or at least about 80%, or at least about 90%, at least about 95%, at least about 97%, or at least about 99%, or more, or any integer ratio between 70% and 100%. In some embodiments, the term "essentially" means at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more, or any integer ratio between 90% and 100%. In some embodiments, the term "essentially" may include 100%.

[0075] Fibroin: As used herein, the term "fibroin" includes silkworm fibroin and silk proteins of insects or spiders (Lucas et al., Adv. Protein Chem, Vol. 13: 107-242 (1958)). Any type of silk fibroin can be used according to the embodiments of the present invention. There are many different types of silk produced by a wide variety of species, including, without limitation: Antheraea mylitta; Antheraea pernyi; Antheraea yamamai; Galleria mellonella; Bombyx mori; Bombyx mandarina; Galleria mellonella; Nephila clavipes; Nephila senegalensis; Gasteracantha mammosa; Argiope aurantia; Araneus diadematus; Latrodectus geometricus; Araneus bicentenarius; Tetragnatha versicolor; Araneus ventricosus; Dolomedes tenebrosus; Euagrus chisoseus; Plectreurys tristis; Argiope trifasciata; and Nephila madagascariensis. In some embodiments, the fibroin is obtained from a solution containing dissolved silkworm silk or spider silk. The silkworm silk protein is obtained, for example, from Bombyx mori, and the spider silk is obtained from Nephila clavipes. Other silks include transgenic silk, genetically engineered silk (recombinant silk), such as silk derived from bacteria, yeast, mammalian cells, transgenic animals, or transgenic plants, and variants thereof. See, for example, WO97 / 08315 and U.S. Patent No. 5,245,012. The entire contents of both are incorporated herein by reference. In some embodiments, the silk fibroin may be derived from other sources, such as spiders, other silkworms, honeybees, synthetic silk-like peptides, and their biotechnologically processed variants.In some embodiments, silk fibroin can be extracted from the glands of silkworms or transgenic silkworms. See, e.g., WO2007 / 098951, the content of which is incorporated herein by reference in its entirety. Different species of silk-producing organisms, and different types of silk, have different amino acid compositions, but various fibroin proteins share certain structural features. A common trend in the silk fibroin structure is an amino acid sequence characterized by alternating glycine and alanine, or alanine alone. Such a configuration allows the fibroin molecule to self-assemble into a beta-sheet higher-order structure. These "Ala-rich" and "Gly-rich" hydrophobic blocks are typically separated by segments of amino acids with bulky side groups (e.g., hydrophilic spacers). In some embodiments, the core repeat sequences of the hydrophobic blocks of fibroin can be represented by the following amino acid sequences and / or formulas: (GAGAGS)5-15 (SEQ ID NO: 1); (GX)5-15 (X = V, I, A) (SEQ ID NO: 2); GAAS (SEQ ID NO: 3); (S1-2A11-13) (SEQ ID NO: 4); GX1-4 GGX (SEQ ID NO: 5); GGGX (X = A, S, Y, R, D V, W, R, D) (SEQ ID NO: 6); (S1-2A1-4)1-2 (SEQ ID NO: 7); GLGGLG (SEQ ID NO: 8); GXGGXG (X = L, I, V, P) (SEQ ID NO: 9); GPX (X = L, Y, I); (GP(GGX)1-4 Y)n (X = Y, V, S, A) (SEQ ID NO: 10); GRGGAn (SEQ ID NO: 11); GGXn (X = A, T, V, S); GAG(A)6-7GGA (SEQ ID NO: 12); and GGX GX GXX (X = Q, Y, L, A, S, R) (SEQ ID NO: 13). In some embodiments, the fibroin peptide can contain multiple hydrophobic blocks within the peptide, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 hydrophobic blocks. In some embodiments, the fibroin peptide can contain between 4 and 17 hydrophobic blocks. In some embodiments of the present invention, the fibroin peptide includes at least one hydrophilic spacer sequence ("hydrophilic block") having a length of about 4 to 50 amino acids.Non-limiting examples of hydrophilic spacer sequences include TGSSGFGPYVNGGYSG (SEQ ID NO: 14); YEYAWSSE (SEQ ID NO: 15); SDFGTGS (SEQ ID NO: 16); RRAGYDR (SEQ ID NO: 17); EVIVIDDR (SEQ ID NO: 18); TTIIEDLDITIDGADGPI (SEQ ID NO: 19) and TISEELTI (SEQ ID NO: 20). In certain embodiments, the fibroin peptide may contain a hydrophilic spacer sequence that is a derivative of any one of the representative spacer sequences listed above. Such derivatives are at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identical to any one of the hydrophilic spacer sequences. In some embodiments, the fibroin peptides suitable for the present invention do not contain any spacers at all. Silk is generally a fibrous protein and is characterized by modular units that are linked together to form high molecular weight, highly repetitive proteins. These modular units or domains each have a specific amino acid sequence and chemical properties and are thought to confer specific functions. For example, sequence motifs such as poly-alanine (polyA) and poly-alanine-glycine (poly-AG) tend to be beta-sheet forming; the GXX motif contributes to 31 helix formation; the GXG motif provides rigidity; and GPGXX (SEQ ID NO: 22) contributes to beta helix formation. These are examples of different components in various silk structures, and the positioning and arrangement of these components lead to the final material properties of silk-based materials (reviewed in Omenetto and Kaplan (2010), Science, 329:528-531). See also WO2011 / 130335 (PCT / US2011 / 032195). These contents are incorporated herein by reference.

[0076] Increasing: The terms "increased", "increasing", "enhanced", or "activated" are all used herein to generally mean a statistically significant amount of increase. To avoid any doubt, the terms "increased", "increasing", or "enhanced", or "activated" mean an increase of at least 10% compared to a reference level, for example, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to a 100% increase, and including increases between these, or any increase between 10 - 100%, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold increase compared to the reference level, or any increase between 2-fold and 10-fold or more.

[0077] Inhibiting: As used herein, the term "inhibiting" means preventing something from occurring, delaying the occurrence of something, and / or reducing the degree or likelihood of something occurring.

[0078] Maintaining: As used herein, the terms "maintaining", "maintain", and "maintenance", when referring to a composition or an active agent, mean keeping, sustaining, or retaining the biological activity of at least one active agent in a silk fibroin matrix when the active agent is subjected to certain conditions. In some embodiments, one or more active agents distributed in the silk fibroin matrix retain at least about 30% of their original biological activity, including at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more of their original biological activity.

[0079] Nanopattern: As used herein, the term "nanopattern" or "nanopatterned" refers to small patterning introduced into a silk fibroin-based matrix, such as a film or foam, or a composition comprising such a silk fibroin-based matrix. Generally, it has a structural feature with a size that can be appropriately measured on the nanometer scale (i.e., 10 -9 meters), for example, a size in the range of 1 nanometer to several millimeters (including both ends).

[0080] Nucleic acid: As used herein, the term "nucleic acid" or "oligonucleotide", or grammatical synonyms thereof herein, means at least two nucleotides that are covalently linked together, including analogs or derivatives thereof. Exemplary nucleic acids include, but are not limited to, polynucleotides, oligonucleotides, genes, genes including regulatory and termination regions, self-replicating systems such as viral or plasmid DNA, genomic DNA, cDNA, mRNA, pre-mRNA, single-stranded and double-stranded siRNA and other RNA interference reagents (RNAi agents or iRNA agents), shRNA (short hairpin RNA), antisense oligonucleotides, aptamers, ribozymes, microRNA (miRNA), pre-miRNA, and modified RNAs (e.g., locked nucleic acids). Nucleic acids can be single-stranded or double-stranded. Nucleic acids can be DNA, RNA, or hybrids, in which case the nucleic acids contain any combination of deoxyribonucleotides and ribonucleotides, and any combination of uracil, adenine, thymine, cytosine, and guanine. Nucleic acids can include one or more backbone modifications, such as phosphoramidates (Beaucage et al., Tetrahedron, Vol. 49 (No. 10): 1925 (1993) and references therein; Letsinger, J. Org. Chem., Vol. 35: 3800 (1970)), phosphorothioates, phosphorodithioates, O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), or peptide nucleic acid linkages (see Egholm, J. Am. Chem. Soc., Vol. 114: 1895 (1992); Meier et al., Chem. Int. Ed. Engl., Vol. 31: 1008 (1992); and Nielsen, Nature, Vol. 365: 566 (1993)). The entire content of these references is incorporated herein by reference. Nucleic acids can also include modifications to the nucleobase and / or sugar moiety of the nucleotide.Exemplary sugar modifications in the sugar moiety include replacement of the 2'-OH with a halogen (e.g., fluoro), O-methyl (O-mehtyl), O-methoxyethyl, NH. 2 , SH, and S-methyl.

[0081] Polyethylene glycol: "PEG" means an ethylene glycol polymer containing from about 20 to about 2,000,000 linked monomers, typically from about 50 to 1000 linked monomers, usually about 100 to 300. PEGs include PEGs containing various numbers of linked monomers, such as PEG20, PEG30, PEG40, PEG60, PEG80, PEG100, PEG115, PEG200, PEG 300, PEG400, PEG500, PEG600, PEG1000, PEG1500, PEG2000, PEG3350, PEG4000, PEG4600, PEG5000, PEG6000, PEG8000, PEG11000, PEG12000, PEG2,000,000, and any mixture thereof.

[0082] Prevent: As used herein, the term "preventing" when used to refer to the action of an agent on a process (e.g., a causative agent, disease progression, etc.), means reducing the extent of such a process and / or delaying its onset when the agent (e.g., a therapeutic agent) is administered before one or more symptoms or characteristics associated with the process develop.

[0083] Ready-to-use: The phrase "ready-to-use" as in "ready-to-use formulation" refers to a composition or product that does not require further processing (e.g., steps during manufacture) before use by the end user. In some embodiments, ready-to-use formulations include formulations ready for injection (i.e., injectable pharmaceuticals). In the context of the present invention, ready-to-use formulations include concentrated formulations such as stock formulations designed to be diluted before administration.

[0084] Reference: The term "reference" as used herein refers to, for example, a reference composition or set of conditions. One of ordinary skill in the art will understand from the context what may be an appropriate "reference" in a particular context. In general, a "reference" shares sufficient similarity with the composition or set of conditions of interest to permit a meaningful comparison. In many embodiments, the "reference composition" herein is a conventional fibroin composition (e.g., not a low molecular weight fibroin composition).

[0085] Short hairpin RNA: The term "shRNA" as used herein refers to short hairpin RNA that functions as an RNAi and / or siRNA species, but the shRNA species differs in that it has a double-stranded hairpin-like structure for increased stability. The term "RNAi" as used herein refers to interfering RNA, or a nucleic acid molecule or analog thereof that inhibits gene expression, e.g., an RNA interference molecule that is an RNA-based molecule. RNAi refers to a means of selective post-transcriptional gene silencing. RNAi can destroy a specific mRNA or prevent the processing or translation of an RNA such as mRNA.

[0086] Short interfering RNA: The term "short interfering RNA" (siRNA), also referred to herein as "small interfering RNA", is defined as an agent that functions, for example, to inhibit the expression of a target gene by RNA interference (RNAi). siRNA can be chemically synthesized, can be produced by in vitro transcription, or can be produced within a host cell. siRNA molecules can also be generated by cleavage of double-stranded RNA in which one strand is identical to the inactivated message. The term "siRNA" refers to small inhibitory RNA duplexes that induce the RNA interference (RNAi) pathway. These molecules can vary in length (generally 18 - 30 base pairs) and can contain varying degrees of complementarity to their target mRNAs in the antisense strand. Some, but not all, siRNAs have unpaired overhanging bases at the 5' or 3' ends of the sense and / or antisense strands. The term "siRNA" includes duplexes of two separate strands and single strands that can form hairpin structures containing a duplex region.

[0087] Solution: The term "solution" broadly refers to a homogeneous mixture composed of a single phase. Typically, a solution contains one or more solutes dissolved in one or more solvents. It is characterized in that the properties of the mixture (such as concentration, temperature, and density) can be uniformly distributed throughout the volume. Thus, in the context of the present application, a "silk fibroin solution" refers to silk fibroin protein in a soluble form dissolved in a solvent such as water. In some embodiments, the silk fibroin solution can be prepared from a solid-state silk fibroin material (i.e., a silk matrix), such as a silk film and other scaffolds. Typically, the solid-state silk fibroin material is reconstituted with an aqueous solution such as water and buffer to form a silk fibroin solution. It should be noted that non-homogeneous liquid mixtures, such as colloids, suspensions, and emulsions, are not considered solutions. To give just one example, silk fibroin microspheres or particles suspended in a solution do not, by themselves, constitute a silk fibroin solution.

[0088] Stabilization: As used herein, “stabilization” of an agent provided by soluble silk fibroin refers to any effect of a silk fibroin polypeptide that supports, promotes, fosters, and / or maintains the integrity of the structure (e.g., higher-order structure) of the agent and the corresponding function or activity such that the agent is less susceptible to degradation, misfolding, denaturation, aggregation, and / or inactivation. A more detailed discussion of the stabilizing effects of silk fibroin matrices is provided in PCT / US12 / 34643 (filed Apr. 23, 2012) and Zhang et al., (2012), Proc. Nat’l. Acad. Sci. U.S.A., 109(30):11981-11986, the entire contents of each of which are incorporated herein by reference. According to embodiments of various aspects described herein, at least one property of one or more components of a biological sample can be stabilized within a low molecular weight silk fibroin composition over any period of time. As used herein, the terms “stabilize,” “stabilizing,” or “stabilization” refer to at least one property (e.g., activity, integrity, and / or amount) of a component of a biological sample that is at least partially or fully maintained or retained within a low molecular weight silk-based material over a period of time. With respect to stabilization of activity, in some embodiments, at least about 30% or more (including at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to 100%) of the components originally present in the biological sample can retain at least partial or full activity over a period of time. Stated another way, the components can retain at least about 30% or more (including at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to 100%) of their original activity over a period of time.With respect to integrity stabilization, at least about 30% or more (including at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to 100%) of the components originally present in the biological sample can maintain integrity over a period of time. With respect to quantity stabilization, at least about 30% or more (including at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to 100%) of the components originally present in the biological sample can be retained over a period of time, regardless of whether the component is active, inactive, intact, or non-intact. As used herein, the terms "originally" or "original" when used with reference to the original presence or original activity of a component refer to the level of the component measured immediately after the biological sample is obtained from the subject or, alternatively, immediately before or after the biological sample is mixed or encapsulated within the silk-based material. In some embodiments, the original presence or original activity of a component refers to the level of the component in a control, e.g., a control stabilized by a non-silk technique, e.g., a frozen control.

[0089] Statistically significant: The terms "statistically significant" or "significantly" refer to statistical significance and generally mean at least two standard deviations (2SD) away from a reference level. This term refers to the statistical evidence of a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true.

[0090] Subject: As used herein, "subject" means a human or an animal. Typically, the animal is a vertebrate such as a primate, rodent, domestic animal, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques such as rhesus monkeys. Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Domestic animals and game animals include cows, horses, pigs, deer, bison, buffalo, feline species such as domestic cats, canine species such as dogs, foxes, wolves, bird species such as chickens, emus, ostriches, and fish such as salmon, catfish, and trout. A patient or subject includes all of the above, excluding any subset thereof, such as one or more groups or species such as humans, primates, or rodents. In certain embodiments, the subject is a mammal such as a primate such as a human. The terms "patient" and "subject" are used interchangeably herein.

[0091] Susceptible: As used herein, the term "susceptible" means having an increased risk and / or tendency (typically based on genetic predisposition, environmental factors, personal history, or combinations thereof) for something such as a disease, disorder, or condition (such as cancer) as compared to what is observed in the general population as a whole. This term takes into account that an individual who is "susceptible" to a condition may never be diagnosed with that condition.

[0092] Tube: The term "tube" as used herein refers to an elongated shaft having a lumen therein. The tube can typically be an elongated hollow cylinder, but can also be a hollow shaft of other cross-sectional shapes.

[0093] Preferred embodiments have been described in detail herein, but various modifications, additions, substitutions, etc. can be made without departing from the spirit of the present invention, and thus it will be apparent to those skilled in the art that these are considered to be within the scope of the present invention as defined in the following claims. Furthermore, it will be understood by those skilled in the art that any one of the various embodiments described and illustrated herein can be further modified to incorporate features shown in any of the other embodiments disclosed herein to an extent not already shown.

[0094] The present disclosure is further illustrated by the following examples, which should not be construed as limiting. The examples are illustrative only and are not intended to limit any of the aspects described herein in any manner. The following examples do not limit the present invention in any way.

[0095] Detailed description of a particular embodiment The present invention is not limited to the specific methodologies, protocols, reagents, etc. described herein, and thus it should be understood that various alternatives are possible. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0096] As used in this specification and the claims, unless the context clearly dictates otherwise, the singular forms include references to the plural forms, and vice versa. All numerical values representing amounts of components or reaction conditions used herein, unless otherwise indicated in the operating examples or otherwise specified, are to be understood as being modified in all instances by the term "about".

[0097] All specific patents and other publications are hereby expressly incorporated by reference herein for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in connection with the present invention. These publications are provided solely because they were disclosed prior to the filing date of the present application. Nothing herein should be construed as an admission that the inventors have any right to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation of the content of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the date or content of these documents.

[0098] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any known methods, devices, and materials may be used in the practice or testing of the present invention, but the methods, devices, and materials in this regard are described herein.

[0099] Low molecular weight composition One aspect provided herein relates to a low molecular weight silk fibroin composition. According to one aspect of the present invention, the low molecular weight silk fibroin composition is a population of silk fibroin fragments having a range of molecular weights, wherein no more than 15% of the total number of silk fibroin fragments in the population have a molecular weight greater than 200 kDa, and at least 50% of the total number of silk fibroin fragments in the population have a molecular weight within a specified range, the specified range being between about 3.5 kDa and about 120 kDa, or between about 5 kDa and about 125 kDa, and comprising a population of silk fibroin fragments.

[0100] Stated another way, in some embodiments, the low molecular weight silk fibroin composition is a population of silk fibroin fragments having a range of molecular weights, wherein no more than 15% of the total molar number of silk fibroin fragments in the population has a molecular weight greater than 200 kDa, and at least 50% of the total molar number of silk fibroin fragments in the population has a molecular weight within a specified range, the specified range being between about 3.5 kDa and about 120 kDa, or between about 5 kDa and about 125 kDa, and includes a population of silk fibroin fragments.

[0101] In some embodiments, the low molecular weight silk fibroin composition is a population of silk fibroin fragments having a range of molecular weights, wherein no more than 15% of the total weight of silk fibroin fragments in the population has a molecular weight greater than 200 kDa, and at least 50% of the total weight of silk fibroin fragments in the population has a molecular weight within a specified range, the specified range being between about 3.5 kDa and about 120 kDa, or between about 5 kDa and about 125 kDa, and includes a population of silk fibroin fragments.

[0102] As used herein, the phrase "silk fibroin fragment" refers to a peptide chain or polypeptide having an amino acid sequence corresponding to a fragment derived from a silk fibroin protein or variant thereof. In the context of the present disclosure, silk fibroin fragments generally refer to silk fibroin peptide chains or polypeptides that are smaller than the naturally occurring full-length silk fibroin counterpart such that one or more of the silk fibroin fragments within the population or composition is less than 300 kDa, less than 250 kDa, less than 200 kDa, less than 175 kDa, less than 150 kDa, less than 120 kDa, less than 100 kDa, less than 90 kDa, less than 80 kDa, less than 70 kDa, less than 60 kDa, less than 50 kDa, less than 40 kDa, less than 30 kDa, less than 25 kDa, less than 20 kDa, less than 15 kDa, less than 12 kDa, less than 10 kDa, less than 9 kDa, less than 8 kDa, less than 7 kDa, less than 6 kDa, less than 5 kDa, less than 4 kDa, less than 3.5 kDa, etc.

[0103] In some embodiments, a "composition comprising a population of silk fibroin fragments" may include a composition comprising unfragmented (i.e., full-length) silk fibroin polypeptides in addition to shorter fragments of silk fibroin polypeptides. The silk fibroin fragments described herein can be produced as recombinant proteins or can be derived from or isolated (e.g., purified) from natural silk fibroin proteins or silk cocoons.

[0104] In some embodiments, silk fibroin fragments can be obtained by scouring silk cocoons under specified conditions selected to produce silk fibroin fragments having a desired range of molecular weights.

[0105] In some embodiments, silk fibroin fragments can be obtained by scouring silk cocoons for at least about 60 minutes or more, including at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes, at least about 120 minutes, or more at a temperature at or near the atmospheric boiling point (e.g., within about 5% of it). As used herein, the term "atmospheric boiling point" refers to the temperature at which a liquid boils under atmospheric pressure.

[0106] In some embodiments, silk fibroin fragments can be produced by scouring silk cocoons in an aqueous solution at about 90°C to about 110°C for at least 60 minutes or more, including at least 70 minutes or more. In some embodiments, silk fibroin fragments can be obtained by scouring silk cocoons for a longer time at a temperature below the atmospheric boiling point, e.g., more than 60 minutes or more, e.g., more than 70 minutes, more than 80 minutes, more than 90 minutes, more than 100 minutes, more than 110 minutes, more than 120 minutes, more than 130 minutes, more than 140 minutes, more than 150 minutes, or more.

[0107] While not wishing to be bound by theory, silk fibroin fragments can be produced by degumming silk cocoons at a temperature of about 70° C. for at least 60 minutes or more, including, for example, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, or more. In some embodiments, silk fibroin fragments can be produced by a process that includes the step of degumming silk cocoons under specified conditions and the step of further exposing the resulting silk fibroin solution to high temperature and / or high pressure. For example, silk fibroin fragments can be produced by a process that includes the step of degumming silk cocoons for about 10 minutes around the boiling point and then subjecting the resulting silk fibroin solution to high temperature and / or high pressure (e.g., autoclaving).

[0108] In one embodiment, an example of a low molecular weight silk fibroin composition can be produced as follows: at least 50 wt% of the total silk fibroin present in a silk fibroin preparation exhibits a reduction in molecular weight, as determined by, for example, SDS gel electrophoresis, i.e., is smaller than full-length silk fibroin (e.g., silk fibroin fragments), and the silk fibroin preparation is treated (e.g., heated and / or boiled) in an amount sufficient to effect fragmentation of the silk fibroin polypeptide. By way of illustration, for example, for a silk fibroin preparation containing a total of 1.0 gram of full-length silk fibroin as a starting material, after the step of heating and / or boiling, at least 0.5 gram of the silk fibroin is in a reduced form (smaller fragments) compared to the starting material (e.g., full-length polypeptide) at that time. As a result, the average molecular weight of the silk fibroin polypeptide in the preparation will decrease when a low molecular weight silk fibroin composition is formed.

[0109] In some embodiments, the silk fibroin fragments in the low molecular weight silk fibroin composition described herein can be substantially free of sericin. In some embodiments, the silk fibroin fragments can contain 5 wt% or less of sericin, or less, based on the total weight of the silk protein composition. For example, the silk fibroin fragments can contain 4 wt% or less, 3 wt% or less, 2 wt% or less, 1 wt% or less, 0.5 wt% or less of sericin, based on the total weight of the silk protein composition.

[0110] In some embodiments, the silk fibroin fragments can be modified. For example, in some embodiments, the silk fibroin fragments can be modified to include functional groups. In some embodiments, the silk fibroin fragments can be covalently or non-covalently linked or fused to an agent, including, but not limited to, for example, a peptide, protein, nucleic acid molecule, contrast agent, therapeutic agent, target binding ligand, cell binding ligand, amphiphilic peptide, or any combination thereof. In some embodiments, the amino acid sequence of the silk fibroin fragments can include a cell binding ligand and / or an amphiphilic peptide. In one embodiment, the amino acid sequence of the silk fibroin fragments can include an RGD sequence.

[0111] In embodiments of the low molecular weight silk fibroin composition described herein, 15% or less of the total number (or total moles) or total weight of the silk fibroin fragments in the population have a molecular weight greater than 200 kDa. As used herein, the phrase "greater than 200 kDa" refers to the molecular weight of silk fibroin fragments larger than 200 kDa. In some embodiments, the phrase "greater than 200 kDa" may include the molecular weight of silk fibroin fragments that are approximately 200 kDa. In some embodiments, silk fibroin fragments having a molecular weight greater than 200 kDa are present in the population in an amount of 10% or less of the total number (or total moles) or total weight of the silk fibroin fragments, including, for example, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less. In one embodiment, the low molecular weight silk fibroin composition is substantially free of silk fibroin fragments having a molecular weight greater than 200 kDa.

[0112] In the low molecular weight silk fibroin composition described herein, at least 50% of the total number (or total moles) or total weight of the silk fibroin fragments have a molecular weight within a specified range, where the specified range is between about 3.5 kDa and about 120 kDa, or between about 5 kDa and about 125 kDa. In some embodiments, more than 50% of the total number (or total moles) or total weight of the silk fibroin fragments, including, for example, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more, may have a molecular weight within the specified range. In one embodiment, the low molecular weight silk fibroin composition may have a population of silk fibroin fragments having a molecular weight substantially between about 3.5 kDa and about 120 kDa, or between about 5 kDa and about 125 kDa.

[0113] The specified range of the molecular weight distribution of fibroin fragments present in at least 50% of the total number (or total moles) or total weight of fibroin fragments in the group can vary between about 3.5 kDa and about 120 kDa, or between about 5 kDa and about 125 kDa. In some embodiments, the molecular weight distribution of at least about 50% of the total number (or total moles) or total weight of fibroin fragments can have a lower limit that is 3.5 kDa or greater, but less than 120 kDa. For example, the lower limit of the specified range can be 3.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, or 115 kDa. In some embodiments, the specified range of the molecular weight distribution of at least about 50% of the total number (or total moles) or total weight of fibroin fragments can have an upper limit that is 10 kDa or greater (including 120 kDa, up to 120 kDa). By way of example only, the upper limit of the specified range can be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120 kDa. Examples of the specified range, without limitation, include (i) between about 5 - 120 kDa; (ii) between about 10 - 120 kDa; (iii) between about 15 - 120 kDa; (iv) between 20 - 120 kDa; (v) between 20 - 110 kDa; (vi) between about 20 - 100 kDa; (vii) between about 20 - 90 kDa; (viii) between about 20 - 80 kDa; (ix) between about 30 - 120 kDa; (x) between about 30 - 100 kDa; (xi) between about 30 - 90 kDa; (xii) between about 30 - 80 kDa; (xiii) between about 40 - 120 kDa; (xiv) between about 40 - 110 kDa; (xv) between about 40 - 100 kDa; (xvi) between about 40 - 90 kDa, and (xvii) between about 40 - 80 kDa.

[0114] The silk fibroin fragments having a molecular weight distribution with the specified range described above can exhibit a continuous or discontinuous molecular weight distribution. As used herein, the term "continuous molecular weight distribution" refers to a distribution of molecular weights having any sub-range between the specified ranges. As used herein, the term "discontinuous molecular weight distribution" refers to a distribution of molecular weights having a specific sub-range between the specified ranges. By way of example only, a silk fibroin fragment having a discontinuous molecular weight distribution with a specified range between about 3.5 kDa and about 120 kDa, or between about 5 kDa and about 125 kDa, can refer to a population of silk fibroin fragments in which a portion of the silk fibroin fragments has a molecular weight between about 3.5 kDa and 10 kDa, and at least a portion or the remainder of the silk fibroin fragments has a molecular weight between about 110 kDa and about 120 kDa.

[0115] Thus, in some embodiments, at least about 50% or more of the total number (or total moles) or total weight of silk fibroin fragments in a population having a molecular weight within a specific range between 3.5 kDa and 120 kDa, or between about 5 kDa and about 125 kDa, is characterized as a population of silk fibroin fragments in which at least about 50% of the total number (or total moles) or total weight of silk fibroin fragments in a population having a molecular weight within a specific range is composed of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) of the following sub-ranges (i)-(x), which sub-ranges include: (i) silk fibroin having a molecular weight distribution of 20 kDa - 30 kDa; (ii) silk fibroin having a molecular weight distribution of 30 kDa - 40 kDa; (iii) silk fibroin having a molecular weight distribution of 40 kDa - 50 kDa; (iv) silk fibroin having a molecular weight distribution of 50 kDa - 60 kDa; (v) silk fibroin having a molecular weight distribution of 60 kDa - 70 kDa; (vi) silk fibroin having a molecular weight distribution of 70 kDa - 80 kDa; (vii) silk fibroin having a molecular weight distribution of 80 kDa - 90 kDa; (viii) silk fibroin having a molecular weight distribution of 90 kDa - 100 kDa; (ix) silk fibroin having a molecular weight distribution of 100 kDa - 110 kDa; and (x) silk fibroin having a molecular weight distribution of 110 kDa - 120 kDa.

[0116] The amount of fibroin fragments having molecular weight sub-ranges (i) to (x) can vary from 0% to 100% of the total number (or total moles) or total weight of all fibroin fragments in the composition described herein, provided that the total weight of fibroin fragments having molecular weight sub-ranges (i) to (x) constitutes at least 50% or more of the total number (or total moles) or total weight of all fibroin fragments in the composition. Thus, the low molecular weight fibroin composition described herein can be configured to have any combination of fibroin fragments having molecular weight sub-ranges (i) to (x). In some embodiments, the low molecular weight fibroin composition can have fibroin fragments corresponding to one specific molecular weight sub-range as defined herein. In other embodiments, the low molecular weight fibroin composition can have a mixture of fibroin fragments corresponding to two or more specific molecular weight sub-ranges as defined herein.

[0117] In some embodiments, the ratio of the fibroin fragment having a molecular weight of 76 kDa to the fibroin fragment having a molecular weight of 18 kDa is not from 5:1 to 1.5:1. Thus, in some embodiments, the low molecular weight fibroin composition is a population of fibroin fragments having a range of molecular weights, wherein no more than 15% of the total number (or total moles) or total weight of the fibroin fragments in the population has a molecular weight greater than 200 kDa, and at least 50% of the total number (or total moles) or total weight of the fibroin fragments in the population has a molecular weight within a specified range, the specified range being between about 3.5 kDa and about 120 kDa, or between about 5 kDa and about 125 kDa, provided that the ratio of the fibroin fragment having a molecular weight of 76 kDa to the fibroin fragment having a molecular weight of 18 kDa is not from 5:1 to 1.5:1.

[0118] In some embodiments, the low molecular weight silk fibroin composition may further comprise 35% or less of the total number (or total moles) or total weight of silk fibroin fragments having a molecular weight distribution between 120 kDa and 200 kDa. For example, in some embodiments, less than 35%, such as 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 3% or less, 1% or less of the total number (or total moles) or total weight of silk fibroin having a molecular weight distribution between 120 kDa and 200 kDa may be present in the compositions described herein. In one embodiment, the compositions described herein can be substantially free of silk fibroin fragments having a molecular weight distribution between 120 kDa and 200 kDa.

[0119] In some embodiments, when the composition comprises silk fibroin fragments having a molecular weight distribution between 120 kDa and 200 kDa, these silk fibroin fragments can be constituted by one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of the following sub-ranges (xi) to (xviii), which sub-ranges include: (xi) silk fibroin having a molecular weight distribution of 120 kDa to 130 kDa; (xii) silk fibroin having a molecular weight distribution of 130 kDa to 140 kDa; (xiii) silk fibroin having a molecular weight distribution of 140 kDa to 150 kDa; (xiv) silk fibroin having a molecular weight distribution of 150 kDa to 160 kDa; (xv) silk fibroin having a molecular weight distribution of 160 kDa to 170 kDa; (xvi) silk fibroin having a molecular weight distribution of 170 kDa to 180 kDa; (xvii) silk fibroin having a molecular weight distribution of 180 kDa to 190 kDa; and (xviii) silk fibroin having a molecular weight distribution of 190 kDa to 200 kDa.

[0120] In some embodiments, the compositions described herein can be enriched in at least one or more silk fibroin fragments within certain sub-ranges (i)-(xviii) as defined herein, compared to a reference silk fibroin composition. In some embodiments, the reference silk fibroin composition can be a composition or mixture produced by degumming silk cocoons at atmospheric boiling point for about 60 minutes. In one embodiment, the reference silk fibroin composition can be a composition or mixture produced by degumming silk cocoons in an aqueous sodium carbonate solution at atmospheric boiling point for about 60 minutes. In some embodiments, the compositions described herein can be enriched in at least two or more silk fibroin fragments within certain sub-ranges (i)-(xviii) as defined herein, compared to a reference silk fibroin composition (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, at least one or more of the silk fibroin fragments can be enriched in the composition by, for example, at least about 10% or more, including at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or more, compared to the reference silk fibroin composition. In some embodiments, at least one or more of the silk fibroin fragments can be enriched in the composition by, for example, at least about 1.1 times or more, including at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, or more, compared to the reference silk fibroin composition.

[0121] According to various embodiments described herein, the low molecular weight silk fibroin composition is different from so-called "hydrolyzed silk". Hydrolyzed silk is generally produced by hydrolyzing or degrading silk proteins into smaller peptide chains having a molecular weight of, for example, less than 1 kDa, and / or constituent amino acids such as glycine, alanine, and serine. Thus, the term "hydrolyzed silk" refers herein to silk peptide chains or amino acids having a molecular weight of less than 2 kDa, less than 1 kDa, less than 500 Da, or less. Put another way, hydrolyzed silk generally does not contain silk fibroin peptide chains having a molecular weight of at least 3.5 kDa or more, including, for example, at least 5 kDa, at least 10 kDa, at least 15 kDa, at least 20 kDa, at least 25 kDa, at least 30 kDa, at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, or more.

[0122] The molecular weight of the silk fibroin fragments described herein can be determined by any known method in the art, including, but not limited to, SDS-PAGE gels, size exclusion gel chromatography, mass spectrometry, or any combination thereof. In some embodiments, the molecular weight of the silk fibroin fragments referred to herein in the low molecular weight silk fibroin compositions described herein can be determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). By way of example only, for each low molecular weight silk fibroin composition, an appropriate amount (e.g., about 7.5 μg) of the composition or silk fibroin protein is prepared in solution and then loaded into a gel such as a 3-8% Tris-acetate gel (e.g., NuPAGE® Novex® 3-8% Tris-acetate gel obtained from Invitrogen). Protein standards or ladders are also loaded into the gel to provide reference molecular weights. Examples of protein standards include, but are not limited to, HiMark™ unstained protein standard (range 30-460 kDa, Invitrogen) and Mark12™ protein standard (range 30-200 kDa, Invitrogen). The gel can be run under reducing conditions according to the manufacturer's instructions. For example, in one embodiment, the gel is run at about 200 V for about 45 minutes and then stained to visualize the protein bands. In some embodiments, the gel can be stained, for example, with a colloidal blue staining kit obtained from Invitrogen. The molecular weight distribution of the silk fibroin fragments present in the low molecular weight silk fibroin composition can then be quantified using any method recognized in the art, for example, by performing densitometry measurements of the protein bands along the length of the sample lane of the gel. In one embodiment, ImageJ (NIH, Bethesda, MD) can be used to determine densitometry measurements of the protein bands on the gel. In one embodiment, the "Gel Analyzer" tool within the ImageJ software can be used to perform densitometry analysis.

[0123] Without limitation, the molecular weight, as used herein, is the peak average molecular weight (M p ), number average molecular weight (M n ), or weight average molecular weight (M w ).

[0124] In some embodiments, the low molecular weight silk fibroin composition may further comprise at least one or more active agents, examples of which are described below. The active agents can be dispersed into the compositions described herein by any known method in the art. For example, the active agents can be dispersed homogeneously or heterogeneously (e.g., forming a gradient of the active agent) in the compositions described herein. See U.S. Patent Application Publication No. US20070212730A1, the content of which is incorporated herein by reference in its entirety. In some embodiments, the agents contained in the low molecular weight silk fibroin composition can be stored in and / or released or recovered from such compositions whether in liquid or solid form. In some embodiments, the included agents can be analyzed, for example, before, during, or after such release or recovery.

[0125] Low molecular weight solution In some embodiments, the low molecular weight silk fibroin compositions provided herein can be solutions (optionally containing agents such as agents stabilized and / or analyzed as described herein). Thus, in another aspect, provided herein is an aqueous silk fibroin solution comprising one or more embodiments of the low molecular weight silk fibroin compositions described herein. In some embodiments, the aqueous silk fibroin solution can be formulated in water. In some embodiments, the aqueous silk fibroin solution can be formulated in a buffer. Examples of buffers include, but are not limited to, phosphate buffers.

[0126] Silk fibroin can be present in solution at any concentration suitable for the need, for example, injectability of the silk fibroin solution. In some embodiments, the aqueous silk fibroin solution can have silk fibroin at a concentration of about 0.1% wt / v to about 90% wt / v, 0.1% wt / v to about 75% wt / v, or 0.1% wt / v to about 50% wt / v. In some embodiments, the aqueous silk fibroin solution can have silk fibroin at a concentration of about 0.1% wt / v to about 10% wt / v, about 0.1% wt / v to about 5% wt / v, about 0.1% wt / v to about 2% wt / v, or about 0.1% wt / v to about 1% wt / v. In some embodiments, the silk fibroin solution can have silk fibroin at a concentration of about 10% wt / v to about 50% wt / v, about 20% wt / v to about 50% wt / v, about 25% wt / v to about 50% wt / v, or about 30% wt / v to about 50% wt / v.

[0127] In some embodiments, the silk aqueous solution can remain stable under certain conditions for at least about 3 days or more, including, for example, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, or more. The specific conditions can be characterized by one or more of environmental parameters including, but not limited to, temperature, light, humidity, pressure, and any combination thereof. In some embodiments, the silk aqueous solution can remain stable at about room temperature to at least about 37 °C or more, for example, for at least about 3 days or more, including at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, or more. In some embodiments, the silk aqueous solution does not gel when the aqueous fibroin solution is exposed to certain conditions for at least about 3 days or more, including, for example, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, or more. In some embodiments, the silk aqueous solution does not gel when the aqueous fibroin solution is exposed to a temperature of at least room temperature or more (e.g., about 15 °C, about 20 °C, about 25 °C, about 30 °C, about 35 °C, about 40 °C, or more) for at least about 3 days or more, including, for example, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, or more.

[0128] In some embodiments, the low molecular weight silk fibroin solution described herein gels slower than the reference silk fibroin solution defined herein. In some embodiments, the low molecular weight silk fibroin solution described herein gels slower than a conventional silk fibroin solution.

[0129] As used herein, the term "remain stable" refers to the material properties of an aqueous fibroin solution that remain substantially the same when exposed to certain conditions over a period of time. Examples of material properties include, but are not limited to, viscosity, particle size, opacity, and any combination thereof. In some embodiments, a stable aqueous fibroin solution can be characterized by having no substantial change in viscosity when exposed to certain conditions over a period of time. In some embodiments, a stable aqueous fibroin solution can be characterized as a homogeneous solution as defined herein. For example, a homogeneous aqueous fibroin solution can be characterized as an aqueous fibroin solution that substantially does not contain fibroin aggregates (e.g., visible insoluble fibroin fragments, fibroin particles, and / or clusters) when exposed to specified conditions over a period of time. Examples of fibroin aggregates include, but are not limited to, full-length fibroin molecules, larger fibroin fragments, fibroin particles or clusters formed by the assembly or aggregation of smaller fibroin fragments, and any combination thereof. In some embodiments, fibroin aggregates can include fibroin particles or clusters formed by processes including, but not limited to, precipitation, gelation, and / or clumping. In some embodiments, fibroin aggregates can be fibroin particles or clusters having a size of about 1 mm or greater. In one embodiment, fibroin particles or clusters can be formed by the assembly or aggregation of fibroin fragments present in the solution.

[0130] Low molecular weight article: In some embodiments, the low molecular weight silk fibroin compositions provided herein can form solid silk fibroin articles. Examples of such solid silk fibroin articles include, but are not limited to, films, sheets, gels or hydrogels, meshes, mats, non-woven mats, fabrics, scaffolds, tubes, blocks, fibers, particles, powders, three-dimensional constructs, implants, foams, needles, lyophilized articles, and any combination thereof. Methods for forming various forms of solid silk fibroin articles are known in the art, and some exemplary methods are described below in this specification.

[0131] In some embodiments, the low molecular weight silk fibroin articles can have storage stability for at least one month. The term "storage stability" as used herein refers to the material properties of silk fibroin particles that remain substantially the same when stored over a period of time. Examples of the material properties of silk fibroin articles include, but are not limited to, hardness, porosity, solubility, particle size, dryness, and any combination thereof.

[0132] In some embodiments, the low molecular weight silk fibroin articles can be re-solubilized in water to form a silk fibroin solution that is substantially free of silk fibroin aggregates. In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or more of the low molecular weight silk fibroin articles can be re-solubilized in water to form a silk fibroin solution that is substantially free of silk fibroin aggregates.

[0133] In some embodiments, heating and / or salt addition are not required to dissolve a low molecular weight silk fibroin article fabricated using one or more embodiments of the low molecular weight silk fibroin compositions described herein. Thus, in some embodiments, the silk fibroin article can be re-solubilized in an aqueous solution at room temperature to form a silk fibroin solution. In some embodiments, the low molecular weight silk fibroin article can completely dissolve in water (e.g., deionized water) at room temperature, provided that the concentration of the dissolved silk fibroin is below saturation. In some embodiments, the saturation point of the low molecular weight silk fibroin composition in an aqueous solution (e.g., water) can be at least 30% w / v, at least 40% w / v, at least 50% w / v, at least 60% w / v, at least 70% w / v, at least 80% w / v, or more.

[0134] In some embodiments, the saturation point of the low molecular weight silk fibroin composition in an aqueous solution (e.g., water) can range from about 30% w / v to about 80% w / v, from about 40% w / v to about 70% w / v, or from about 50% w / v to about 60% w / v. Thus, in some embodiments, the low molecular weight silk fibroin article can have a water solubility of at least about 10 mg / mL or more, including at least about 20 mg / mL, at least about 30 mg / mL, at least about 40 mg / mL, at least about 50 mg / mL, at least about 60 mg / mL, at least about 70 mg / mL, at least about 80 mg / mL, at least about 90 mg / mL, at least about 100 mg / mL, at least about 200 mg / mL, at least about 400 mg / mL, at least about 600 mg / mL, at least about 800 mg / mL, at least about 1000 mg / mL, or more.

[0135] In some embodiments, the low molecular weight silk fibroin article may have a dissolution rate of at least 0.01 mg / s to about 100 mg / s, or about 0.1 mg / s to about 90 mg / s, or about 1 mg / s to about 80 mg / s. In some embodiments, the low molecular weight silk fibroin article may dissolve in an aqueous solution (e.g., water such as deionized water) at a rate of about 0.01 mg / s to about 100 mg / s, or about 0.1 mg / s to about 90 mg / s, or about 1 mg / s to about 80 mg / s at room temperature.

[0136] In some embodiments, the low molecular weight silk fibroin article can be used in on-demand or point-of-care applications. For example, a user (e.g., a subject or a healthcare provider) can apply or self-administer the silk fibroin article anywhere (e.g., at home, in a remote village, and on the battlefield). For diagnostics, in some embodiments, the user can send the silk fibroin article to a hospital, clinic, or any analytical facility. Alternatively or additionally, in some embodiments, the low molecular weight silk fibroin article can be used to create a stored sample (e.g., a stored blood sample), which in some embodiments can be stable for at least a specified time (e.g., days, weeks, months, years, etc.) and / or can be retested or sampled over time (e.g., to enable comparison with later obtained samples, etc.).

[0137] While not wishing to be bound by theory, in some embodiments, the resolubilization of silk fibroin articles (i.e., the ability to dissolve or reconstitute in a solution or liquid form a low molecular weight silk fibroin composition in solid form) may be determined by the length and / or temperature of time for degumming silk cocoons. For example, in some embodiments, a resolubilizable silk fibroin article can be formed from a low molecular weight silk fibroin solution obtained by degumming silk cocoons for at least 60 minutes or more, such as at least 90 minutes or more, at or near atmospheric boiling temperature. In other embodiments, a resolubilizable silk fibroin article can be formed from a low molecular weight silk fibroin solution obtained by degumming silk cocoons for at least 90 minutes or more, such as at least 120 minutes or more, at a lower temperature, such as about 60-90 °C.

[0138] In some embodiments, the low molecular weight silk fibroin compositions described herein in solid form are highly soluble. Thus, in one aspect, the present invention provides a solid silk fibroin composition in a highly soluble form that comprises low molecular weight silk fibroin fragments. Compared to previously described conventional silk fibroin compositions, the solid silk fibroin compositions described herein in highly soluble form are characterized by their high water solubility, high dissolution rate, and biocompatibility with a wide range of agents (e.g., but not limited to, biological agents). From a materials science perspective, the low molecular weight silk fibroin compositions described herein result in greater handling flexibility, tunability, and ease, as well as diversity, due in part to the ability to better regulate liquid-solid or solid-liquid transitions. This is based at least in part on the finding that the solubility of silk fibroin can be precisely tuned when the composition is primarily composed of smaller fragments of silk fibroin (i.e., low molecular weight silk fibroin fragments). This added flexibility allows silk-based materials to be utilized in ways not possible with conventional silk materials that contain or are composed of higher molecular weight silk fibroin. Thus, low molecular weight silk fibroin materials with unique material properties include, but are not limited to, solid form silk fibroin compositions that can be readily dissolved in aqueous solution; and liquid form silk fibroin compositions (e.g., silk fibroin solutions) that provide a desired stabilizing effect on agents incorporated or associated therein while withstanding unwanted self-assembly or gelation.

[0139] Thus, in some embodiments, the low molecular weight silk fibroin composition can be formulated to behave as a sufficiently soluble form that can be readily and rapidly reconstituted in water (e.g., pure water or deionized water), or any other water-based solution suitable for a particular use, such as a buffer solution. In these embodiments, since the low molecular weight silk fibroin composition in the solid form described herein is readily soluble in an aqueous solution, one or more agents incorporated therein can be readily recovered from the soluble form of the solid low molecular weight silk fibroin composition. For example, a biological agent, such as a biological sample, can be "stored" with the low molecular weight silk fibroin composition in the solid form, which can then be released into an aqueous solution to achieve almost complete recovery of the agent. Once released, such an agent can be readily analyzed (e.g., detected, measured, assayed, identified, isolated, etc.) by any suitable means, such as an analytical tool. Alternatively or additionally, in some embodiments, the included agent (e.g., a biological agent) can be analyzed without being released from the silk fibroin composition.

[0140] Stabilization of the composition The stabilization and subsequent recovery of agents (e.g., active agents and / or biological samples) that are desired to be stabilized are extremely important features for many applications, because active agents and / or biological samples are typically unstable and sensitive to changes in ambient conditions, such as temperature, humidity, and / or light. In some previously described methods, even when an active agent or biological sample has been identified as useful for a given reaction, its application is often hampered by a lack of long-term stability and / or low recovery rates under process conditions. As further described below, various embodiments of the invention described herein address these shortcomings present in the prior art.

[0141] For example, a technique capable of protecting a stabilized sample against a harmful temperature profile can be extended to an encompassing scale within a centralized test system. To achieve this objective, the inventors have discovered, inter alia, that a particular silk-based material can protect an active agent (e.g., a biological sample or its constituents, a therapeutic and / or a diagnostic reagent, etc.) from the degradation of its structural and / or functional features. The term "active agent" is used herein to refer to a biological sample (e.g., a tissue or a sample of a fluid such as blood), or its constituents, and / or a biologically active entity or compound, and / or a structurally or functionally unstable entity.

[0142] In some embodiments, the stabilization of the biological activity of the active agent / biological sample is of utmost importance. In other embodiments, the stabilization of activity or biological activity is not always required. For example, the maintenance of biological activity is often not a requirement for assaying and quantifying a stabilized biological sample.

[0143] In some particular embodiments, the present disclosure demonstrates, inter alia, that a silk-based material can protect blood components such as human whole blood and RNA over a long duration and in the context of adverse environmental conditions. The present disclosure demonstrates, for example, that whole blood and / or plasma matched to a donor can be stabilized in a silk-based material, e.g., a film, by casting and air drying, and that each plasma protein can be recovered by a simple mixing protocol with water.

[0144] The present disclosure also specifically demonstrates various particular advantageous features of low molecular weight silk compositions for use in stabilizing included agents, as described herein. For example, among other things, the present disclosure demonstrates that low molecular weight silk compositions tend to gel more slowly than higher molecular weight silk compositions. In particular, such delayed gelling properties can, in some embodiments, contribute to an extended shelf life. Further, the present disclosure demonstrates that low molecular weight silk compositions protect against sample loss even when using autoclaving. In some embodiments, the present invention provides, for example, an autoclaved low molecular weight silk composition comprising an included agent. In some embodiments, the present invention is a system for reconstituting a low molecular weight silk composition (e.g., a solid form low molecular weight silk composition) comprising such an autoclaved composition, the composition being characterized by a high recovery rate of the included agent. In certain embodiments, such compositions and / or systems are suitable for large scale manufacture and / or processing (e.g., shipping) of the composition.

[0145] The demonstrated stabilizing effect of the silk fibroin compositions herein has broad implications, including, for example, for the maintenance, storage, and / or transport of samples obtained in the field (e.g., blood samples). Thus, in many embodiments, the techniques provided are particularly useful in settings where samples (e.g., blood draws) are obtained with limited resources and / or in situations where downstream analysis extends beyond the scope of point-of-case technology.

[0146] The present disclosure particularly demonstrates the breadth of clinically relevant analytical tools that can be used to measure the absolute abundance of blood analytes (or blood components) derived from a range of donors after being recovered from silk-based materials. The studies reported herein show that by using the compositions and methods of the various embodiments described herein, the analyte levels (or component levels recovered) match the measurements performed on fresh or frozen plasma matched to the donor, which is the current format used as the optimal standard for clinical assays. In some embodiments, the silk-stabilized analyte levels (or silk-stabilized component levels) are higher than their frozen counterparts, indicating that the silk-based material can exceed the current optimal standards for certain applications. The inventors have also demonstrated that different techniques such as lyophilization can be used in the presence of the same silk formulation to stabilize biological samples, such as RNA, and subsequently recovered using a simple aqueous reconstitution method. The recovered RNA was available at levels consistent with the frozen controls and was able to transfect model cell lines.

[0147] Silk-based materials in various forms can protect biological samples against widely varying temperature profiles and mechanical perturbations encountered during shipping. The silk-based material behaves as a fully solubilized system upon reconstitution in water (e.g., pure water or a buffer suitable for sample interaction) and can be specifically formulated such that the encapsulated components or biomarkers analyzed by some clinically relevant analytical tools are fully recovered. Prior to the present disclosure, there were no technologies that combined these unique properties, namely, i) ease of sample procurement, ii) excellent thermal / mechanical stability profiles, and iii) simplicity of reconstitution / recovery. However, as demonstrated herein, the present disclosure provides compositions and methods that result in, among other things, i) ease of sample procurement, ii) excellent thermal / mechanical stability profiles, and iii) simplicity of reconstitution / recovery.

[0148] Silk fibroin is highly resistant to enzymatic / thermal / UV degradation, so the encapsulated biological samples can be banked for long-term storage (i.e., months to years in duration) in a centralized facility without the need for refrigeration / freezing to collect time-series datasets of the entire population or to enhance individualized drugs.

[0149] Accordingly, in one aspect, the present disclosure provides the use of silk solutions (e.g., adjusted to produce silk-based materials with desirable solubility), and silk-based materials made from these solutions for encapsulating and stabilizing biological samples, such as whole blood and blood components. This silk purification and encapsulation scheme results in the ability to fully reconstitute encapsulated blood components (e.g., cells, and / or nucleic acids, such as circulating DNA or RNA, etc.). In one embodiment, a blood draw equivalent to a finger prick volume can be mixed with a silk solution, for example, as described in the Examples section herein, and then at least partially dried silk-based materials containing stabilized blood components can be readily procured.

[0150] The silk encapsulation / stabilization platform described herein is basic. For example, a liquid silk fibroin composition can be immediately applicable to routine liquid assays of biological samples in the composition and can be subjected to the same validation and quality control (QC) procedures used for fresh whole blood or frozen samples. The solution composition can be at least partially dried or formed into silk fibroin articles. The silk fibroin in the dried composition or article can be re-solubilized, thereby providing a solution applicable to routine liquid assays for detecting biological samples or their components.

[0151] In contrast, since DBS analytes require some level of chemical interaction with the paper substrate after drying in order to be protected, some sample retention and / or analyte damage in the matrix is expected with standard DBS recovery procedures. Consequently, this imposes unique validation protocols and QC metrics for DBS assays to monitor losses and inhomogeneities between extractions. Second, due to spot inhomogeneities across and through the thickness of the paper substrate (from user-to-user variability and hematocrit sample-to-sample variability), it can be difficult to define the sample volume and then aliquot the paper-based DBS samples accordingly. This is particularly problematic in patient populations with widely varying ages and those suffering from abnormal hematocrit levels (such as kidney dysfunction, cancer patients, etc.). In contrast, the silk-based materials described herein (e.g., low molecular weight silk fibroin compositions) can be fully recovered as needed (e.g., an entire volume such as 50 μL can be reconstituted), or aliquots can be easily prepared by weight since the silk / blood composite is homogeneously mixed and dried.

[0152] Accordingly, embodiments of the various aspects described herein relate to silk-based materials for stabilizing at least one component of a biological sample mixed with, encapsulated within, or otherwise associated with the silk-based material, enabling detection of the component at a later time, as well as methods of making and using such silk-based materials.

[0153] In one aspect, provided herein is a silk-based material comprising silk fibroin (e.g., a low molecular weight silk fibroin composition) and a biological sample / active agent, wherein at least one property of at least one component of the biological sample is stabilized over a period of time and / or at least one component of the biological sample / active agent is detectable after that period of time.

[0154] The properties of at least one active agent (e.g., a biological sample or a constituent) stabilized in a silk-based material can be physical or structural properties, chemical properties, and / or biological properties. In some embodiments, the at least one property can include activity or bioactivity, structural integrity, structural higher-order structure, and / or the amount of the agent. Depending on different applications (e.g., diagnostic purposes vs. therapeutic purposes), silk fibroin and / or silk-based materials can be processed to stabilize one or more properties of at least one constituent of a biological sample.

[0155] In some embodiments, it is not always required to stabilize the activity or bioactivity of the constituents of a biological sample. For example, for diagnostic applications, the activity of the detected constituent may not be as important as the integrity and / or amount of the constituent stabilized in the biological sample. In these embodiments, the detected constituent can be inactive as long as the constituent remains intact and / or the amount of the constituent is maintained. As used interchangeably herein, the terms “activity” and “bioactivity” refer to one or more structural and / or functional characteristics of a constituent, such as its ability to interact with a biological target and / or to produce an effect on a biological target. For example, bioactivity can include, without limitation, the induction of stimulatory, inhibitory, regulatory, toxic, or lethal responses in a biological target. The biological target can be a molecule or a cell. For example, bioactivity can refer to the ability of a constituent (e.g., a protein or a nucleic acid molecule) to modulate the effect / activity of a cell or 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, infect or transfect a cell, or any combination thereof. In some cases, bioactivity can refer to the ability of a constituent to produce a toxic effect inside a cell.

[0156] In some embodiments, it may be desirable to further stabilize the activity of the components of the biological sample. As a mere example, the proteins present in the biological sample can be in an active or inactive state. In this embodiment, the silk-based material can stabilize the proteins present in the biological sample in their active or native form, such as phosphorylated proteins or glycated proteins. This embodiment can be useful when the activity of the components plays a role in the diagnosis of diseases or disorders.

[0157] According to embodiments of the various aspects described herein, at least one property of one or more components of a biological sample can be stabilized within a silk-based material over any period of time. As used herein, the terms "stabilize", "stabilizing", or "stabilization" refer to at least one property (e.g., activity, integrity, and / or amount) of a component of a biological sample that is at least partially or fully maintained or retained within a silk-based material over a period of time. With respect to stabilization of activity, in some embodiments, at least about 30% or more (including at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to 100%) of the components originally present in the biological sample can retain at least partial or full activity over a period of time. In other words, the component can retain at least about 30% or more (including at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to 100%) of its original activity over a period of time. With respect to stabilization of integrity, at least about 30% or more (including at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to 100%) of the components originally present in the biological sample can retain integrity over a period of time. With respect to stabilization of amount, at least about 30% or more (including at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to 100%) of the components originally present in the biological sample can be retained over a period of time, regardless of whether the component is active, inactive, intact, or not intact. As used herein, the terms "originally" or "original" when used with reference to the original presence or original activity of a component refer to the level of the component measured immediately after the biological sample is obtained from the subject or, alternatively, measured immediately before or after the biological sample is mixed or encapsulated within the silk-based material.In some embodiments, the original presence or original activity of a component refers to the level of the component in a control, e.g., a control stabilized by a non-silk technique, e.g., a frozen control.

[0158] The property of at least one of one or more active agents / biological samples / these components can be stabilized within a silk-based material over a period of time, e.g., until the component is extracted or recovered for detection. In some embodiments, the at least one property can be stabilized for at least about 3 hours, at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, or more. In some embodiments, the at least one property can be stabilized for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, or more. In some embodiments, the at least one property can be stabilized for at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 2 months, at least about 3 months, at least about 6 months, at least about 9 months, at least about 1 year, at least about 2 years, at least about 3 years, at least about 6 years, or more.

[0159] As further discussed below, the silk-based materials described herein can exist in any form. For example, the silk-based material can be in the form of a solution, film, fiber, particle, gel, hydrogel, foam, sponge, mat, mesh, fabric, powder, coating layer, their lyophilized forms, or any combination thereof. In some embodiments, the silk-based material can be a thin film. In some embodiments, the silk-based material can be lyophilized.

[0160] One or more components of a biological sample can be detected without isolating them from the silk-based material, but components at a measurable or detectable level can instead be extracted or recovered from the silk-based material for subsequent processing, analysis, and / or characterization. As used herein, the term "measurable or detectable level" refers to the lower limit of detection, which generally depends on the detection sensitivity of the detection and / or characterization method or assay selected for a particular component. For example, if the component to be detected is a nucleic acid molecule (e.g., RNA or DNA) and an amplification-based detection method (e.g., but not limited to, polymerase chain reaction (PCR)) is selected to detect the component, about 0.0001% of the nucleic acid molecule (e.g., 1 copy of an RNA molecule) can be extracted or recovered from the silk-based material for amplification and detection.

[0161] In some embodiments, the silk-based material may be soluble such that measurable or detectable levels of components can become accessible or available for subsequent processing, analysis, and / or characterization. As used herein, the term "soluble" generally refers to the solubility, dissolution, or breakdown of the silk-based material in a fluid. A soluble silk-based material can partially or completely dissolve or break down in a fluid over a period of time. For example, at least about 5% or more of the silk-based material, including at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or up to 100% of the silk-based material, can dissolve or break down in a fluid (e.g., an aqueous fluid) over a time range of seconds, minutes, hours to days. In some embodiments, the silk-based material can partially or completely (e.g., at least about 5% or more of the silk-based material) dissolve or break down in a fluid (e.g., an aqueous fluid) within at least about 10 seconds, at least about 20 seconds, at least about 30 seconds, at least about 40 seconds, at least about 50 seconds, at least about 60 seconds, or more. In some embodiments, the silk-based material can partially or completely (e.g., at least about 5% or more of the silk-based material) dissolve or break down in a fluid (e.g., an aqueous fluid) within at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 30 minutes, at least about 1 hour, or more. In other words, a soluble silk-based material is a silk-based material that can dissolve or break down a sufficient portion of itself in a fluid (e.g., an aqueous fluid) to recover or extract at least one active agent / sample / component present therein.

[0162] The solubility or dissolution of the silk-based material can depend on several factors, such as, but not limited to, the composition of the silk-based material (e.g., the ratio of the biological sample to silk fibroin), the type of biological sample mixed or encapsulated in the silk fibroin (e.g., but not limited to, urine, blood, and / or earwax), and / or the type of buffer / excipient used to assist in stabilizing the biological sample and / or at least one or more of its components, the silk purification method (e.g., scouring conditions such as boiling time), the method for forming the silk-based material described herein, the sterilization method, and any combination thereof.

[0163] In some embodiments, the solubility or dissolution of the silk-based material can be regulated by the molecular weight / chain length of the silk. Generally, silk-based materials containing silk fibroin with a lower molecular weight / chain length can have higher solubility in aqueous solvents than those containing silk fibroin with a higher molecular weight / chain length. In one embodiment, the molecular weight / chain length of the silk fibroin can be regulated by scouring conditions. For example, the silk cocoon can be boiled in a salt solution (e.g., Na 2 CO 3 ) for a predetermined time in the range of, for example, about 1 minute to about 3 hours, about 5 minutes to about 2 hours, about 10 minutes to about 1.5 hours, or about 15 minutes to about 1 hour. In some embodiments, the silk cocoon is boiled in a salt solution (e.g., Na 2 CO 3)It can be boiled. Without wishing to be bound by theory, longer boiling times can result in silk fibroin with lower molecular weight / chain length. For details on the effect of degumming on silk material properties, including molecular weight, viscosity, diffusivity, and degradation behavior, see, for example, Pritchard EM et al., "Effect of Silk Protein Processing on Drug Delivery from Silk Films", Macromol Biosci., electronically published on January 24, 2013. Depending on the biological sample to be stabilized and the type(s) of these component(s), the optimal degumming time can be determined, for example, as described in the examples, with respect to the desired solubility of the silk-based material and thus the recovery rate of the components. In some embodiments, the silk-based stabilizing compositions described herein are low molecular weight silk compositions.

[0164] In fact, the inventors have discovered that various forms of low molecular weight silk fibroin compositions provide particularly advantageous properties for protecting and / or stabilizing agents (e.g., biological samples) associated with or incorporated therein. For example, the low molecular weight silk compositions described herein can act as "molecular stabilizers" to protect biological samples against widely varying environmental factors such as fluctuating temperature profiles and / or mechanical perturbations encountered, for example, during transportation, storage, and / or handling. The inventors have also discovered that the compositions containing the low molecular weight silk fibroin disclosed herein can be fully reconstituted in water. In contrast, high molecular weight silk fibroin is not fully reconstituted in water under similar conditions. High molecular weight forms form aggregates when reconstituted in water under similar conditions.

[0165] Heretofore, techniques that combine these unique properties, namely, i) ease of sample procurement, ii) excellent thermal / mechanical stability profiles, and iii) simplicity and yield of reconstitution / recovery, have not been described in the art. However, as demonstrated herein, the present disclosure provides compositions and methods that result in i) ease of sample procurement, ii) excellent thermal / mechanical stability profiles, and iii) simplicity and yield of reconstitution / recovery. Without wishing to be bound by theory, silk fibroin is highly resistant to enzymatic / thermal / UV degradation, and encapsulated agents (e.g., active agents and / or biological samples) in silk fibroin compositions can be banked for long-term storage (i.e., months to years in duration) in a centralized facility without the need for refrigeration / freeze, and can be readily recovered later whenever the agent is in a state to be used and / or analyzed. Accordingly, the silk fibroin compositions provided herein, particularly low molecular weight silk fibroin compositions, can enable the collection of time-series datasets for an entire population or the enhancement of individualized drugs.

[0166] In particular, the inventors have found that incorporating a biological sample into one or more embodiments of the low molecular weight silk fibroin compositions described herein can stabilize detectable moieties present in the biological sample over a period of time under certain conditions, which in turn can enable a higher recovery rate of subsequent detectable moieties for, e.g., subsequent analysis and / or characterization of the components, as compared to the recovery rate of detectable moieties from conventional silk fibroin-based materials.

[0167] In some embodiments, the present disclosure provides for the use of the low molecular weight silk fibroin compositions disclosed herein (e.g., various soluble or solid forms of low molecular weight silk fibroin-based materials with desirable properties such as enhanced solubility (compared to conventional silk fibroin-based materials), enhanced recovery rate of additives (compared to conventional silk fibroin-based materials), etc., such as solutions, films, particles or powders, scaffolds, meshes, and / or fibers, etc., adjusted to produce) for encapsulation, stabilization, and subsequent recovery of samples. In some embodiments, the sample is a biological sample.

[0168] The amount of silk fibroin relative to the active agent / biological sample in the silk fibroin compositions described herein can be adjusted for several factors, such as, but not limited to, for example, the volume and / or type of the biological sample, the silk fibroin concentration, the solubility of the resulting silk-based material, stabilization and / or the abundance of the target component detected, the recovery efficiency of the components present in the biological sample, and the detection sensitivity of the detection / characterization method selected for a particular component.

[0169] In some embodiments, the ratio of silk fibroin to the active agent / sample / component (e.g., mass ratio, volume ratio, or molar ratio) is in the range of about 1:10000 to about 10000:1 or about 1:1000 to about 1000:1. In some embodiments, the ratio (e.g., mass ratio, volume ratio, or molar ratio) is about 1:1 to about 1000:1. In some embodiments, the ratio is about 1:1000 to about 1000:1, about 1:500 to about 500:1, about 1:250 to about 250:1, about 1:125 to about 125:1, about 1:100 to about 100:1, about 1:50 to about 50:1, about 1:25 to about 25:1, about 1:10 to about 10:1, about 1:5 to about 5:1, about 1:3 to about 3:1, or about 1:1. By way of example only, in some embodiments, the volume ratio of silk fibroin to a blood sample can be in the range of about 1:1 to about 1000:1, or about 1:1 to about 100:1, or about 1:1 to about 50:1, or about 1:1 to about 25:1.

[0170] The ratio of the silk fibroin matrix to the active agent / sample / component can vary depending on several factors, including the selection of the active agent, storage conditions and duration, the concentration of the silk fibroin matrix, and the form of the silk matrix. One of ordinary skill in the art can determine the appropriate ratio of the silk fibroin matrix to the active agent, for example, by measuring the biological activity of the active agent maintained at various ratios described herein under defined conditions, such as at a temperature above 0°C, for a predetermined amount of time. Methods for measuring the biological activity of the various active agents described herein, such as enzymes, vaccines, proteins, antibodies, and nucleic acids, are well known in the art. By way of example, the stability or biological activity of a given active agent in silk fibroin can be determined based on a combination of time and temperature. For example, a stability study can be conducted over a period of six months. Activity assays can be performed, for example, after two weeks, four weeks, and then monthly. Samples can be prepared to result in N = 3 for each time point. The range of temperature storage conditions to be evaluated includes 4°C (refrigeration), 25°C (room temperature), 37°C (body temperature), 45°C, and / or 50°C (including everything). Alternatively or additionally, activity can be assayed after one, two, three, or more freeze-thaw cycles. These variables can be comprehensively combined to fully characterize the optimal formulation for the long-term stability of the active agent(s). In some embodiments, the results of silk-related active agent stability can be compared to lyophilized active agent preparations using the same storage conditions, for example, for the purpose of improving the stability of storage conditions (e.g., 4°C) recommended for the manufacture of lyophilized active agent preparations.

[0171] The stabilized silk fibroin composition described herein can have any amount of silk fibroin, provided that amount is sufficient to stabilize at least one property of at least one component of a biological sample over a period of time and to enable detection of the active agent / sample / component after that time. In some embodiments, the amount of silk fibroin in the composition varies depending on, for example, the type and / or volume size of the active agent / sample / component described herein, the form of the silk-based material (e.g., film vs. foam), the method of making any silk-based material formed (e.g., air drying vs. lyophilization), and / or the silk fibroin concentration used to form the initial silk fibroin composition.

[0172] By way of just a few examples, the silk-based stabilizing material can be prepared from a silk solution containing the active agent that contains from about 0.25% to about 50% (w / v) silk fibroin, or from about 0.5% to about 30% (w / v), or from about 0.5% to about 15% (w / v), or from about 0.5% to about 10% (w / v). In some embodiments, the silk-based material can be a silk solution containing a biological sample. For example, in one embodiment, the silk-based film can be prepared from a silk solution containing from about 1% to about 15% (w / v) silk fibroin and an active agent or biological sample / component. In another embodiment, the silk-based foam can be prepared from a silk solution containing from about 0.1% to about 5% (w / v) silk fibroin and an active agent / biological sample / component.

[0173] In some embodiments, the silk-based material can be a solid-state silk-based material formed from a silk solution containing a biological sample.

[0174] In some embodiments, the agent / sample / component can be stabilized in a silk composition (e.g., a low molecular weight based composition) over a range of storage temperatures over the time periods described herein, and can enable the recovery and / or detection of at least one component of a biological sample for analysis. Unlike the typical sub-zero storage temperatures required for biological samples (e.g., in an -80 °C freezer or in liquid nitrogen), the silk-based materials described herein can enable biological samples to be stabilized therein at storage temperatures above 0 °C or higher. In some embodiments, the storage temperature can range from about 0 °C to about ambient temperature. In some embodiments, the storage temperature can be at least about 40 °C, or above 40 °C. In some embodiments, the storage temperature can be at least about 45 °C, or above 45 °C.

[0175] In some embodiments, the agent / sample / component can be stabilized in a silk fibroin composition described herein under light exposure over the time periods described herein, and can enable the recovery and / or detection of at least one component of a biological sample for analysis. For example, in some embodiments, the agent / sample / component present in the composition can be exposed to light, e.g., light of different wavelengths and / or from different sources. In some embodiments, the agent / sample / component present in the silk-based material can be exposed to UV or infrared irradiation. In some embodiments, the agent / sample / component present in the silk-based material can be exposed to visible light.

[0176] In some embodiments, a biological sample can be stabilized in a silk fibroin composition provided over a range of relative humidities over the time periods described herein, and can enable the recovery and / or detection of at least one component of a biological sample for analysis. In some embodiments, the relative humidity can be at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or higher.

[0177] As used herein, the term "relative humidity" is a measure of the amount of water vapor in a mixture of air and water vapor. This is generally defined as the partial pressure of water vapor in the air-water mixture and is given as a percentage of the saturated vapor pressure under these conditions.

[0178] In some embodiments, the described stabilized silk fibroin compositions can be lyophilized, for example, to reduce residual moisture during storage. In some embodiments, the residual moisture is reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%.

[0179] In some embodiments, the compositions described herein can be maintained at any air pressure or exposed to any air pressure. In some embodiments, the compositions described herein can be maintained at or above about atmospheric pressure, for example, about 1 atmosphere, about 2 atmospheres, about 3 atmospheres, about 4 atmospheres, about 5 atmospheres, about 6 atmospheres, about 7 atmospheres, about 8 atmospheres, about 9 atmospheres, or about 10 atmospheres, or exposed to that air pressure. In some embodiments, the compositions described herein can be maintained under vacuum or exposed to vacuum.

[0180] In some embodiments, but not necessarily, when a silk fibroin composition (e.g., a silk-based solution) is stored at a temperature below zero degrees Celsius, the agent / sample / component can be stabilized in the composition after at least one or a plurality of freeze-thaw cycles (e.g., a frozen silk-based solution thawed from a frozen silk-based solution), enabling the recovery and / or detection of at least one component of a biological sample for analysis. The term "freeze-thaw cycle" is used herein to describe a series of alternating freezes and thaws, and also encompasses a series of alternating frozen (solid) and fluid states. For example, one freeze-thaw cycle involves a change in state between a frozen (solid) state and a fluid state. The time interval between freezing and thawing, or between the frozen state and the fluid state, can be any time, e.g., several hours, days, weeks, or months. For example, after a silk-based solution has been frozen or is in a frozen state, it can be continuously stored in a frozen state at a temperature below zero degrees Celsius, e.g., between about -20°C and -80°C, until it needs to be thawed for reuse. Freezing of the composition can be carried out rapidly, e.g., in liquid nitrogen, or slowly, e.g., at a freezing temperature, e.g., between about -20°C and -80°C. Thawing of the frozen composition can be carried out rapidly at any temperature above 0°C, e.g., at room temperature, or slowly, e.g., on ice. Typically, components of a biological sample in a non-silk fibroin matrix, e.g., proteins and / or nucleic acids, can lose their activity or integrity over one or more freeze-thaw cycles. As described herein, distributing a biological sample in a silk fibroin matrix increases the stability of at least one of its components, and thus its activity or integrity can be maintained during one or more freeze-thaw cycles.

[0181] In one embodiment, the agent / sample / component can be stabilized in the provided silk fibroin composition over the time described herein under two or more of the conditions described above, enabling the recovery and / or detection of at least one component of the agent / sample / component for analysis.

[0182] Another aspect provided herein relates to methods and compositions for maintaining or stabilizing the biological activity of an active agent. The method includes the step of maintaining a composition that includes a silk fibroin matrix and at least one active agent distributed, mixed, or encapsulated therein, wherein the at least one active agent retains or stabilizes at least about 30% of its original biological activity when the composition is subjected to specified conditions that inhibit or reduce the biological activity of the active agent over a period of time. Such conditions can include, but are not limited to, state change cycles, temperature, air pressure, humidity, and light exposure.

[0183] The term "state change cycle" as used herein refers to a change in the state of a material including, but not limited to, a change from a solid state to a fluid state or from a fluid state to a solid state. Fluid states can include, but are not limited to, liquids, gases, slurries, flowable pastes, plasmas, and any combination thereof. The solid state refers to a non-flowing state, which can also include semi-solids such as gels. The compositions described herein can be maintained in a particular state for any period of time, such as seconds, minutes, hours, weeks, months, or years, before changing to another state. State change cycles can result from at least one change in the environmental conditions described herein, such as a temperature change, ambient air pressure, light conditions, a change in humidity, or any combination thereof.

[0184] In one embodiment, the state change cycle refers to a freeze-thaw cycle. In such an embodiment, when the compositions described herein are stored or transported, they can be subjected to at least 1 freeze-thaw cycle, at least 2 freeze-thaw cycles, at least 3 freeze-thaw cycles, at least 4 freeze-thaw cycles, at least 5 freeze-thaw cycles, at least 6 freeze-thaw cycles, at least 7 freeze-thaw cycles, at least 8 freeze-thaw cycles, at least 9 freeze-thaw cycles, at least 10 freeze-thaw cycles, or more. The term "freeze-thaw cycle" is used herein to describe a series of alternating freezes and thaws and also encompasses a series of alternating frozen (solid) and fluid states. For example, one freeze-thaw cycle involves a change in state between a frozen (solid) state and a fluid state. The time interval between freezing and thawing, or between the frozen state and the fluid state, can be any time, such as hours, days, weeks, or months. For example, after the active agent composition has been frozen or is in a frozen state, it can be continuously stored in a frozen state at a temperature below zero, for example, between about -20°C and -80°C, until it needs to be thawed for reuse. Freezing of the composition can be carried out rapidly, for example, in liquid nitrogen, or gradually, for example, at a freezing temperature, for example, between about -20°C and -80°C. Thawing of the frozen composition can be carried out rapidly at any temperature above 0°C, for example, at room temperature, or gradually, for example, on ice. Typically, an active agent in a non-silk fibroin matrix can lose its biological activity over one or more freeze-thaw cycles. As described herein, distributing the active agent in a silk fibroin matrix increases the stability of the active agent and thus enables it to retain its biological activity during one or more freeze-thaw cycles.

[0185] Embodiments of the various aspects described herein result in a stabilized active agent, where stabilization of the active agent is achieved by distributing, mixing, or encapsulating the active agent in the silk fibroin compositions disclosed herein. The silk fibroin can be a silk fibroin solution or a solid-state silk fibroin matrix, such as a silk fibroin article. This approach results in the active agent retaining its biological activity regardless of the cold chain and / or environmental conditions under which the active agent is stored and / or transported. Exemplary environmental conditions include, but are not limited to, temperature, air pressure, humidity, and light exposure. For example, the cold chain is a standard practice for stabilizing active agents in the pharmaceutical industry, and maintaining the cold chain ensures that the active agent is transported and stored according to the temperature range recommended by the manufacturer (e.g., 2°C to 8°C, or temperatures below zero) until the time of use.

[0186] In some embodiments, the silk fibroin composition containing the active agent is in the form of an implant or an implantable drug delivery device. The active agent in such a composition can retain at least 30% (including at least about 40%, at least about 60%, at least about 80%, or more) of its original biological activity over a period of time, or more. In some embodiments, the active agent in the silk fibroin composition in the form of an implantable drug device can retain at least about 30% or more of its original biological activity at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, at least 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, or at least 1 year, or more, after implantation.

[0187] In some embodiments, one or more active agents encapsulated in the injectable silk fibroin composition can be administered to a subject as a depot of the active agent (e.g., a vaccine depot), for example, by injection such as subcutaneous injection, such that the active agent (e.g., a vaccine) can be continuously or intermittently released from the depot over a long period of time, for example, over a period of hours, days, weeks, or months. In some embodiments, the active agent (e.g., a vaccine) can be released at a rate such that at least about 1% (including at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or more) of the encapsulated active agent is released over a period of at least 1 hour, at least 2 hours, at least 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, or more. In some embodiments, the active agent (e.g., a vaccine) can be released at a rate such that at least about 10% (including at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or more) of the encapsulated active agent is released over a period of 5 days, 1 week, at least about 2 weeks, at least about 3 weeks, at least about 1 month, at least about 2 months, at least about 3 months, or more.

[0188] In some embodiments, the active agent in the silk fibroin composition retains at least about 30% of its original biological activity, such as at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more of its original biological activity for at least 6 months at about 4°C, about 25°C, about 37°C, about 45°C, or above. In some embodiments, the active agent retains at least about 8% of its original biological activity for at least 6 months at a temperature of about 37°C or above.

[0189] Certain aspects described herein are compositions that are stable upon storage, comprising a silk fibroin composition (e.g., a low molecular weight silk fibroin composition), and an active agent distributed, mixed, or encapsulated therein, wherein the active agent retains at least about 30% (e.g., at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, etc.) of its original biological activity when the composition is subjected to at least one state change cycle and / or maintained for a period of time under one or more of the conditions specified herein. In some embodiments, the active agent can be covalently or non-covalently fused or linked to silk fibroin fragments in the composition. In one embodiment, the state change cycle is a freeze-thaw cycle. In one embodiment, the time for maintaining the active agent is at least about 24 hours. In some embodiments, the specified conditions can be the environmental conditions under which the active agent is stored and / or transported. Non-limiting examples of environmental conditions are temperature, air pressure, humidity, and light exposure. In some embodiments, the compositions described herein can be immunogenic. In some such embodiments, the active agent is an immunogen. In some embodiments, the active agent is a vaccine.

[0190] In one aspect, as described herein, the present invention provides a method for stabilizing a biological sample or an active agent. Generally, the method involves contacting, combining, or mixing the biological sample or active agent with the silk fibroin composition disclosed herein, thereby preparing a mixture comprising silk fibroin and the biological sample or active agent. In some embodiments, the method further comprises forming a silk-based material (e.g., a silk fibroin article) from the mixture. Thus, in some embodiments, a method for stabilizing a biological sample or active agent comprises preparing a low molecular weight silk fibroin composition comprising the biological sample or active agent and forming a silk-based article from the composition. In some embodiments, when the silk fibroin composition comprising the active agent / sample / component is in a solid state, it can be further processed to induce the formation of a beta-sheet secondary structure in the silk fibroin.

[0191] In some embodiments, the provided method comprises contacting a biological sample or an active agent with a silk fibroin composition, wherein the composition is in the form of a powder. In some further embodiments thereof, the method further comprises forming a solution from the composition.

[0192] In some embodiments, the method comprises contacting a biological sample or an active agent with a silk fibroin composition, wherein the composition is in the form of a solution.

[0193] In some embodiments, a solution composition comprising a biological sample or an active agent can be formed into a silk fibroin article. The resulting article can be soluble in an aqueous solution (e.g., water, buffer, or a combination thereof).

[0194] The amount of silk fibroin for a biological sample within the composition described herein can be adjusted for several factors, such as, but not limited to, for example, the volume and / or type of the biological sample, the silk fibroin concentration, the solubility of the resulting silk-based material, the stabilization and / or abundance of the target component to be detected, the recovery efficiency of the components present in the biological sample, and the detection sensitivity of the detection / characterization method selected for a particular component. For example, as already described, in some embodiments, the ratio of silk fibroin to the biological sample (e.g., mass ratio, volume ratio, or molar ratio) can range from about 1:10000 to about 10000:1 or from about 1:1000 to about 1000:1. In some embodiments, the ratio of silk fibroin to the biological sample (e.g., mass ratio, volume ratio, or molar ratio) is from about 1:1 to about 1000:1. By way of mere example, in some embodiments, the volume ratio of silk fibroin to a blood sample can range from about 1:1 to about 1000:1, or from about 1:1 to about 100:1, or from about 1:1 to about 50:1, or from about 1:1 to about 25:1.

[0195] Active agent / component Silk compositions (e.g., the low molecular weight silk fibroin compositions described herein) can include / incorporate any of a variety of active and / or unstable agents.

[0196] Non-limiting examples of biological samples that can be stored, stabilized, analyzed, and / or recovered from the silk fibroin compositions described herein include, but are not limited to, for example, body fluid samples such as blood samples including whole blood samples, plasma samples, and serum samples; urine samples; cerebrospinal fluid samples, saliva samples, and any combination thereof. In some embodiments, such samples can be collected from a patient for medical or clinical purposes. In some embodiments, such samples can be collected for forensic purposes.

[0197] In some embodiments, for example, in relation to the incorporation, stabilization, and recovery of an active agent, the unique properties of the silk fibroin compositions provided herein (e.g., low molecular weight silk compositions) enable the adequate reconstitution of various encapsulated blood components (e.g., cells, circulating factors such as hormones, growth factors, cytokines, antibodies, and other proteins, nucleic acids such as DNA or RNA, etc.). For example, a blood draw equivalent to a finger prick volume can be mixed with a silk fibroin solution to form a silk-based material that is at least partially dried, and then an at least partially dried silk-based material containing stabilized blood components can be readily obtained, which can be stored until it is in a state for use and / or analysis.

[0198] In addition to blood samples, other biological samples used for diagnostic purposes, including but not limited to, for example, urine, blood, feces, earwax, nucleic acids (e.g., DNA / RNA, modified nucleic acids), antibodies, whole cells, therapeutic agents, can also be associated with silk fibroin in a similar manner depending on the performance requirements and sample availability. The provided silk fibroin compositions containing biological samples can stabilize one or more components of the biological sample over a period of time, thereby enabling subsequent analysis and / or characterization of the biological sample or its components. Accordingly, embodiments of the various aspects described herein relate to silk fibroin-based compositions for enabling the detection of components at a later time for the stabilization of at least one component of a biological sample mixed with or encapsulated therein, as well as methods of making and using such silk fibroin-based compositions.

[0199] According to various embodiments described herein, a biological sample mixed or encapsulated within a low molecular weight silk fibroin is intended to be stabilized with respect to at least one of its components and / or evaluated for the presence of components, and includes any fluid or specimen (treated or untreated) that meets this criterion. The biological sample can be a liquid, supercritical fluid, solution, suspension, gas, gel, slurry, solid, or a combination thereof.

[0200] In some embodiments, the biological sample can include an aqueous fluid. As used herein, the term "aqueous fluid" refers to any water-containing material having any fluidity.

[0201] In some embodiments, the biological sample can be obtained from a subject, such as a mammalian subject, for example, a human subject. Exemplary biological samples obtained from a subject include, but are not limited to, biological cells, tissues, blood (including whole blood, plasma, cord blood, and serum), lactation products (e.g., milk), amniotic fluid, sputum, saliva, urine, semen, cerebrospinal fluid, bronchial aspirate, sweat, mucus, liquefied feces, synovial fluid, lymph fluid, tears, tracheal aspirate, and fractions thereof. In some embodiments, the biological sample can include a homogenate of a tissue specimen (e.g., a biopsy) derived from a subject. In one embodiment, the biological sample can include a suspension obtained from the homogenization of a solid sample obtained from a solid organ or a fragment thereof. The biological sample can be obtained from a subject by any means known in the art, which can vary depending on the type of biological sample. By way of example only, a blood sample can be obtained from a subject by, for example, finger prick, fine needle, venipuncture, or any known method for collecting a blood sample.

[0202] In some embodiments, the biological sample can include biological cells selected from the group consisting of live cells or dead cells (prokaryotes and eukaryotes including mammals), viruses, bacteria, fungi, yeast, protozoa, microorganisms, and parasites. The biological cells can be normal cells or diseased cells, such as cancer cells. Mammalian cells include, without limitation, cells derived from primates, humans, and, without limitation, any animal of interest including mice, hamsters, rabbits, dogs, cats, birds, domestic animals such as horses, cows, sheep, dogs, and cats. In some embodiments, the cells can be derived from a human subject. In other embodiments, the cells can be derived from a domesticated animal, such as a dog or a cat. Exemplary mammalian cells include, but are not limited to, stem cells, cancer cells, progenitor cells, immune cells, blood cells, fetal cells, and any combination thereof. The cells can be derived from a wide variety of tissue types, such as hematopoietic, neural, mesenchymal, skin, mucosal, stromal, muscle, spleen, reticuloendothelial, epithelial, endothelial, liver, kidney, gastrointestinal, lung, cardiovascular, T cells, and fetal, without limitation. Also included are stem cells, embryonic stem (ES) cells, ES-derived cells, induced pluripotent stem cells, and stem cell progenitor cells, including, without limitation, hematopoietic, neural, mesenchymal, muscle, cardiovascular, liver, lung, and gastrointestinal stem cells. Yeast cells can also be used as cells in some embodiments described herein. In some embodiments, the cells can be ex vivo or cultured cells, such as in vitro. For example, for ex vivo cells, the cells can be obtained from a subject, where the subject is healthy and / or suffering from a disease.

[0203] In some embodiments, the biological sample can include animal parts, such as, without limitation, tissues derived from one or more organs (including skin), muscle, beak, claw, feather, wing, and / or tail.

[0204] In some embodiments, the biological sample can include a fluid or specimen derived from a source such as, but not limited to, a laboratory, an animal population, a crime scene, a cell bank or a contract organization, a blood bank, a tissue bank, a biorepository, a diagnostic testing facility, a clinical setting, and / or any combination thereof.

[0205] In some embodiments, the biological sample can include a fluid (e.g., a culture medium) and / or cells from a biological culture. Examples of fluids (e.g., culture media) and / or cells obtained from biological cultures include those obtained from the culture or fermentation of unicellular or multicellular organisms, including, for example, prokaryotes (e.g., bacteria) and eukaryotes (e.g., animal cells, plant cells, yeast, fungi), and including fractions thereof.

[0206] In some embodiments, the biological sample comprises at least one constituent or a mixture of constituents. Examples of constituents that can be stabilized for subsequent detection and / or characterization include, but are not limited to, peptides, proteins, antibodies, enzymes, antigens, amino acids, nucleic acids (e.g., DNA, RNA, siRNA, miRNA, non-coding RNA, or any variant of RNA that can be found endogenously in a subject), nucleotides, metabolites, lipids, sugars, glycoproteins, peptidoglycans, microorganisms, cells, and any combination thereof. Using the compositions and / or methods described herein, for example, at least one constituent, including at least two constituents, at least three constituents, at least four constituents, at least five constituents, or more, can be stabilized over a period of time and detected after that time. In some embodiments, the silk-based material can stabilize at least one or more (e.g., 1, 2, 3, 4, 5, or more) proteins in a biological sample (e.g., plasma or serum proteins in a blood sample), and the protein can be detected at a later time. In some embodiments, the silk-based material can stabilize at least one or more (e.g., 1, 2, 3, 4, 5, or more) cell-free or circulating DNA or RNA in a blood sample. In some embodiments, the silk-based material can stabilize at least one or more (e.g., 1, 2, 3, 4, 5, or more) diagnostic biomarkers present in a biological sample.

[0207] In some embodiments, the silk-based material can stabilize the activity and / or integrity of RNA, and it is contemplated that the silk fibroin compositions and methods described herein can be used to stabilize any nucleic acid, including but not limited to DNA, RNA, and modified DNA or RNA. Some exemplary nucleic acids include peptide plasmid DNA, genomic DNA, mRNA, siRNA, pre-miRNA, miRNA, antisense oligonucleotides, shRNA, activating RNA, decoy oligonucleotides, peptide nucleic acids (PNAs), oligonucleotides, and / or any nucleic acid that can be administered to a subject for therapeutic purposes.

[0208] In some embodiments, when incorporating a biological sample into one or more embodiments of the silk fibroin compositions described herein, it may be desirable to stabilize the state of at least one component of the biological sample (e.g., at least one detectable entity). By way of mere example, a protein present in a biological sample can be in an active or inactive state. In some embodiments, the active or native state of a protein of a biological sample, e.g., a phosphorylated protein, or a glycated protein, can be stabilized or maintained in the silk fibroin material. These embodiments can be useful, for example, when the activity or state of a component plays a role in the diagnosis of a disease or disorder. For example, a particular state or level of post-translational modification correlates with a disease state and, as a result, is useful in diagnosis. In some embodiments, post-translational modifications include, but are not limited to, phosphorylation, myristoylation, palmitoylation, isoprenylation or prenylation, farnesylation, geranylgeranylation, glypiation, glycosylphosphotidylinositol anchor formation, lipoylation, attachment of a flavin moiety, attachment of heme C, phosphopantetheinylation, retinylidene Schiff base formation, diphthamide formation, ethanolamine phosphoglycerol attachment, hypusine formation, acylation, acetylation, formylation, alkylation, methylation, amide bond formation, amidation at the C-terminus, amino acid addition, arginylation, polyglutamylation, polyglycylation, butyrylation, gamma-carboxylation, glycosylation, polysialylation, malonylation, hydroxylation, iodination, nucleotide addition, oxidation, phosphate ester or phosphoramidate formation, phosphorylation, adenylation, propionylation, pyroglutamic acid formation, S-glutathionylation, S-nitrosylation, succinylation, sulfation, selenoylation, glycation, biotinylation, pegylation, ISGylation, SUMOylation, ubiquitination, Neddylation, Pupylation, citrullination, amide hydrolysis, eliminylation, carbamylation, disulfide bridging, proteolytic cleavage, and proline racemization.

[0209] Generally, to incorporate a biological sample or an agent into a silk fibroin article, the biological sample or the active agent can be included in the silk fibroin solution used to produce the silk fibroin article. Alternatively or additionally, a pre-formed silk fibroin article can be added to a solution containing the biological sample or the active agent to absorb the biological sample (or its components) or the active agent into / onto the silk fibroin article.

[0210] In some embodiments, the biological sample / agent can be distributed homogeneously or heterogeneously (e.g., in a gradient) in the silk-based material. In some embodiments, the biological sample can be encapsulated or enclosed by silk fibroin in the silk-based material. In some embodiments, the biological sample can be mixed or blended with silk fibroin in the silk-based material.

[0211] After forming the silk-based material, the material can be treated, for example, to induce the formation of the beta-sheet secondary structure in the silk fibroin. Methods for inducing the formation of the beta-sheet secondary structure in silk fibroin are described elsewhere in this specification.

[0212] According to embodiments of the various aspects described herein, silk fibroin compositions containing a biological sample can stabilize at least one constituent of the biological sample, thereby enabling detection and / or analysis of the constituents at a later time. In some embodiments, these silk-based materials containing a biological sample can be stored and / or transported without the need for refrigeration or freezing, which is a typical method of currently storing and handling biological samples to maintain / hold sample quality for diagnostic evaluation of human health. Thus, in some embodiments, the silk-based materials and methods described herein may be useful for diagnostic applications. For example, the silk-based materials and methods can be used to maintain and / or hold the quality of a biological sample during storage and / or transportation, or in some developing countries or remote field conditions where there is no minimal infrastructure to support continuous cryogenic storage, such that at least one constituent of the biological sample can be assayed for diagnostic applications. In some embodiments, the silk-based materials and methods described herein can be used to maintain and / or hold the quality of a biological sample under at least one or any combination of the following conditions described above, namely: (a) temperatures above 0°C during storage and / or transportation (e.g., at least about room temperature or higher); (b) light exposure during storage and / or transportation (e.g., UV, infrared, and / or visible light); and (c) relative humidity of at least about 10% or higher during storage and / or transportation.

[0213] Accordingly, yet another aspect provided herein relates to a method for using the silk-based materials described herein. The method includes (a) preparing one or more embodiments of a silk fibroin (e.g., low molecular weight silk fibroin) composition containing an active agent / sample / constituent, and (b) dissolving at least a portion of the composition in water to form a sample solution containing silk fibroin and a detectable amount of at least one active agent.

[0214] In some embodiments, a subject or patient in need of diagnosing a disease or disorder can provide a biological sample, which can be contacted with a silk fibroin composition and then sent to a diagnostic laboratory for analysis. In some embodiments, the biological sample can be collected by a skilled practitioner at the point of care from the subject and then contacted there with silk fibroin (e.g., low molecular weight silk fibroin) and sent to a diagnostic laboratory for analysis.

[0215] In some embodiments, at least one constituent of the biological sample can be detected and / or analyzed without isolating the constituent from the biological sample, but in alternative embodiments, the constituent can be extracted or recovered from at least a portion of the silk fibroin composition prior to detection and / or analysis using any method known in the art. According to some embodiments of the silk-based materials described herein, the composition can be soluble in an aqueous solution (e.g., water, buffer, or a combination thereof). Unlike cellulose-based techniques, such as dried blood spots, where blood is absorbed onto a filter paper and then difficult to recover, at least a portion of the silk fibroin composition described herein can be solubilized in an aqueous solution (e.g., water, buffer, or a combination thereof). At this time, the final silk / biological sample solution can be applicable to routine liquid assays, such as the ELISA and Luminex™ assays described in the examples, without further purification. Thus, in some embodiments, the method can further include contacting at least a portion of the silk fibroin composition with an aqueous solution (e.g., water, buffer, or a combination thereof) before subjecting the at least one constituent of the biological sample to at least one analysis.

[0216] Various types of analysis can be performed on the target constituent of a biological sample, for example, according to the nature of the target constituent. Non-limiting examples of analysis include, but are not limited to, genotyping, nucleic acid sequencing, expression analysis (e.g., at the protein level or the transcriptional level), binding affinity, enzyme activity, transfection efficiency, cell counting, cell identification, cell viability, immunogenicity, infectivity, metabolite profiling, and any combination thereof. In some embodiments, at least one constituent of a biological sample can be subjected to at least one genotyping or nucleic acid sequencing analysis, expression analysis (e.g., at the protein level and / or the transcriptional level), metabolite profiling, or any combination thereof. Various methods for performing these analyses include, but are not limited to, polymerase chain reaction (PCR), real-time quantitative PCR, microarray, Western blot, immunohistochemical analysis, enzyme-linked immunosorbent assay (ELISA), mass spectrometry, nucleic acid sequencing, flow cytometry, gas chromatography, high-performance liquid chromatography, nuclear magnetic resonance (NMR) spectroscopy, or any combination thereof. Nucleic acid sequencing techniques are known in the art and can be used to assay constituents for determining nucleic acid or gene expression measurements, for example, but not limited to, DNA sequencing, RNA sequencing, de novo sequencing, next-generation sequencing such as massively parallel signature sequencing (MPSS), polony sequencing, pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, ion semiconductor sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, single molecule real-time (SMRT) sequencing, nanopore DNA sequencing, sequencing by hybridization, sequencing using mass spectrometry, microfluidic Sanger sequencing, microscopy-based sequencing techniques, RNA polymerase (RNAP) sequencing, or any combination thereof.

[0217] In some embodiments, at least one analysis can be performed in a format that can interface with a reading device or system. In some embodiments, at least one analysis can be performed on a system.

[0218] In some embodiments, prior to analysis or prior to contacting a composition containing a biological sample with an aqueous solution, the composition can be reduced or aliquoted into smaller portions, for example, some of these can be saved for later analysis and / or analyzed for different target components (e.g., proteins, nucleic acids, and / or metabolites). The aliquoting into smaller portions can be by weight or volume.

[0219] The recovery rate of at least one active substance from a silk fibroin composition generally depends in part on the solubility of the silk fibroin composition in which the active substance is encapsulated, the stability of the active substance when present in the silk fibroin composition, and / or the ease of processing the active substance-containing silk fibroin solution for subsequent analysis. As described herein, the inventors have found that, unlike other silk compositions in which a larger portion of silk fibroin fragments having a molecular weight greater than 200 kDa are present (e.g., silk compositions produced from silk solutions obtained by degumming silk cocoons for less than 30 minutes), the low molecular weight silk fibroin compositions described herein can be easily dissolved or re-solubilized in water at ambient temperature without the use of any additives (e.g., salts) or heating to form a silk fibroin solution. Surprisingly. For example, in some embodiments, the silk fibroin particles (including powders) and films described herein can be easily dissolved or re-solubilized in deionized water at room temperature. Thus, in some embodiments, the resulting silk fibroin solution can be in a state ready for use without the use of any subsequent dialysis, which is usually required otherwise. In some embodiments, the inventors have shown that silk fibroin solutions reconstituted from the low molecular weight silk fibroin compositions described herein can be directly injected into a fluidics-based analyzer for subsequent analysis. Further, the resulting silk fibroin solution can also be used to reform other low molecular weight silk fibroin compositions described herein.

[0220] In addition to its unique feature of re-solubility and the ability to reform articles from the resulting silk solution, the low molecular weight silk fibroin compositions described herein can also stabilize at least one or more active agents in a sample over time, as compared to the stability of an active agent in the absence of a silk fibroin composition, for example, as described in International Application No. PCT / US12 / 34643 and U.S. Provisional Application Nos. 61 / 792,161 and 61 / 830,950. Thus, in some embodiments, the low molecular weight silk fibroin compositions described herein are suitable for any use where it is desired that an active agent or sample be stabilized over time so that the active agent or sample can be recovered and / or retrieved for future use and / or analysis. By way of mere example, in some embodiments, one or more embodiments of a low molecular weight silk fibroin article, e.g., an active agent or sample encapsulated in a low molecular weight silk fibroin film or powder, can be stored at room temperature without refrigeration over time until it is in a state where it can be used and / or analyzed. In these embodiments, at least a portion of the low molecular weight silk fibroin article containing the active agent or sample can be dissolved in water at room temperature to recover the active agent or sample in a usable form, while the remainder of the sample can be saved for later use and / or other analysis.

[0221] In some embodiments, at least a desirable or detectable amount of an agent or sample encapsulated within the low molecular weight silk fibroin compositions described herein can be recovered in a useable form. As used herein, the term "useable form" refers to a form of an agent or sample that is applicable to a particular use. For example, the agent or sample can be recovered in solution for assay analysis. In some embodiments, the term "useable form" can include, for example, an agent or sample that is isolated from the solution by any purification method recognized in the art. Since the low molecular weight silk fibroin compositions can stabilize the agent and can be readily soluble, a smaller aliquot of the low molecular weight silk fibroin composition may generally be sufficient to yield a desirable or detectable amount of the agent or sample as compared to the recovery of the agent or sample from a non-silk fibroin composition. Stated another way, for an aliquot containing the same loading amount of an agent or sample, the recovery rate of the agent or sample from the low molecular silk fibroin compositions described herein can be increased by at least about 10% or more, including, for example, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more as compared to the recovery rate from a non-silk fibroin composition.

[0222] In some embodiments, at least about 50% or more of the original loading amount of an agent or sample in a silk fibroin composition (e.g., a low molecular weight composition) can be recovered. In some embodiments, more than 50% of the original loading amount of an agent or sample in a low molecular weight silk fibroin composition, such as at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, can be recovered. In some embodiments, the agent or sample can be recovered by dissolving the low molecular weight silk fibroin article encapsulating them in water (e.g., deionized water) at room temperature. In some embodiments, the dissolved silk fibroin article containing the agent or sample can be subjected to further processing, such as analysis of the agent and / or purification of the agent from the silk fibroin solution.

[0223] Due to the ease of manipulation and / or handling of the low molecular weight silk fibroin compositions described herein, the compositions described herein can be applied in battlefield settings, in clinic settings, during emergency transport, and / or for home use. By way of mere example, for instance, to stabilize a blood sample for transport from a home or remote site to a laboratory facility, a home user or a military person can reconstitute a storage-stable low molecular weight silk fibroin powder to make a silk fibroin solution and then mix it with the blood sample. Then, a sample solution mixture in which the components of the blood sample are stabilized in the presence of silk fibroin can be sent to a laboratory facility. In some embodiments, the silk fibroin solution containing the blood sample can be air-dried to form an article, such as a film, in which the components in the blood sample can be stabilized for a longer time at ambient temperature, such as for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or more. Preserving the activity and / or level of one or more desirable components in a sample using the low molecular weight silk fibroin compositions and / or methods described herein can aid in the discovery of new biomarkers for diseases or disorders, increase the sensitivity of diagnostic tests, and / or result in a more accurate diagnostic test reading.

[0224] Compositions and methods for stabilizing biological samples using silk fibroin are described in U.S. Provisional Application No. 61 / 792,161, filed March 15, 2013, and U.S. Provisional Application No. 61 / 830,950, filed June 4, 2013, both of which are incorporated herein by reference in their entirety.

[0225] Exemplary Composition Formats Silk fibroin compositions as described herein (e.g., low molecular weight silk fibroin compositions and / or silk fibroin compositions containing biological samples or active agents for stabilization), among others, can be in any form, shape, or size. For example, the composition can be a solution, fiber, film, sheet, fiber, mat, non-woven mat, mesh, fabric, sponge, foam, gel, hydrogel, tube, particle (e.g., nano or micro particle, gel-like particle), powder, scaffold, three-dimensional construct, coating layer on a substrate, or any combination thereof.

[0226] Methods for generating silk-based materials in different formats are known in the art, including, but not limited to, drying, solution casting, salt leaching, freeze drying, gas forming, electrospinning, gelation (e.g., electrical gelation), shear stress, sonication, pH reduction, water annealing, steam annealing, alcohol immersion, fiber drawing, coating, spraying, miniaturization, or any combination thereof. In some specific embodiments, silk fibroin compositions (e.g., low molecular weight compositions and / or stabilization compositions) are generated by a method involving drying a solution of silk fibroin, optionally with an active agent / sample / component. In some embodiments, the drying includes freeze drying or air drying.

[0227] In some embodiments, for example, depending on the moisture content remaining in silk-based materials, in some embodiments, silk-based materials (e.g., in a solid state) can contain at least about 10 wt% or more of silk fibroin. For example, silk-based materials (e.g., in a solid state) can contain at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more of silk fibroin by weight.

[0228] In some embodiments, silk fibroin compositions as described herein (e.g., low molecular weight silk fibroin compositions and / or stabilized silk compositions) are in the form of particles. For example, the silk fibroin article can be in the form of silk nanospheres or silk microspheres. As used herein, the term "particle" includes spheres; rods; shells; prisms; and powders. In some embodiments, these particles can be part of a network or aggregate. Without limitation, the particles can have any size from nanometers (nm) to millimeters (mm). In some embodiments, the particles can have a size in the range of about 0.01 μm to about 1000 μm, about 0.05 μm to about 500 μm, about 0.1 μm to about 250 μm, about 0.25 μm to about 200 μm, or about 0.5 μm to about 100 μm. As used herein, the term "nanoparticle" refers to particles having a particle size of about 0.1 nm to about 1000 nm. Certain embodiments and related techniques of silk fibroin particles from the microscale to the nanoscale are also provided in a U.S. Provisional Application filed concurrently with this specification entitled "SYNTHESIS OF SILK FIBROIN MICRO- AND SUBMICRON SPHERES USING A CO-FLOW METHOD".

[0229] Those skilled in the art will understand that particles typically exhibit a particle size distribution around the indicated "size". Unless otherwise specified, the term "particle size", as used herein, refers to the mode of the particle size distribution, i.e., the value that most frequently occurs in the size distribution. Methods for measuring particle size, such as by dynamic light scattering (e.g., photon correlation spectroscopy, laser diffraction, low-angle laser light scattering (LALLS), and medium-angle laser light scattering (MALLS)), light blocking methods (e.g., Coulter analysis method), or other techniques (e.g., rheology, and optical or electron microscopy), are known to those skilled in the art.

[0230] In some embodiments, the particles may be substantially spherical. "Substantially spherical" means that the ratio of the length of the longest vertical axis to the shortest vertical axis of the particle cross-section is equal to or less than about 1.5. Being substantially spherical does not require a center of symmetry. Further, the particles can have surface patterning, such as lines or cuts or protrusions, that are small in scale compared to the overall size of the particle and still be substantially spherical. In some embodiments, the ratio of the length between the longest and shortest axes of the particle is equal to or less than about 1.5, or equal to or less than about 1.45, or equal to or less than about 1.4, or equal to or less than about 1.35, or equal to or less than about 1.30, or equal to or less than about 1.25, or equal to or less than about 1.20, or equal to or less than about 1.15, or equal to or less than about 1.1. Without being bound by theory, surface contact is minimized in particles that are substantially spherical, thereby minimizing undesirable agglomeration of the particles during storage. Many crystals or flakes have flat surfaces that can allow for a large surface contact area, where agglomeration can occur by ionic or non-ionic interactions. Spheres allow for contact over a much smaller area.

[0231] In some embodiments, the particles have substantially the same particle size. Particles having a broad size distribution with both relatively large and small particles present can allow smaller particles to fill in the gaps between the larger particles, thereby creating new contact surfaces. A broad size distribution can result in larger spheres by creating many opportunities for the agglomerates to bond. The particles described herein are within a narrow size distribution, thereby minimizing the opportunity for contacting agglomerates. "Narrow size distribution" means a particle size distribution having a ratio of the 90th percentile volume diameter to the 10th percentile volume diameter of small spherical particles that is equal to or less than 5. In some embodiments, the 90th percentile volume diameter relative to the 10th percentile volume diameter of the small spherical particles is equal to or less than 4.5, equal to or less than 4, equal to or less than 3.5, equal to or less than 3, equal to or less than 2.5, equal to or less than 2, equal to or less than 1.5, equal to or less than 1.45, equal to or less than 1.40, equal to or less than 1.35, equal to or less than 1.3, equal to or less than 1.25, equal to or less than 1.20, equal to or less than 1.15, or equal to or less than 1.1.

[0232] The geometric standard deviation (GSD) can also be used to indicate a narrow size distribution. The GSD calculation involves the determination of effective cut-off diameters (ECDs) at cumulative percentages less than 15.9% and 84.1%. The GSD is equal to the square root of the ratio of the ECD less than 84.17% to the ECD less than 15.9%. The GSD has a narrow size distribution when GSD < 2.5. In some embodiments, the GSD is less than 2, less than 1.75, or less than 1.5. In one embodiment, the GSD is less than 1.8.

[0233] Although not bound by theory, the particle size can greatly determine the microscopic and macroscopic properties of the final product. The particle size is determined by several process parameters including, but not limited to, the size of the ceramic balls used, the amount of silk placed in each ball mill cup, the rotational speed (RPM) of the machine, and the duration of ball mill grinding. The particle size of the powder can be predicted based on some of these process parameters by, for example, mathematical modeling and / or experimental methods to determine correlations. For example, this can be done by milling a given volume of silk fibroin for varying ball mill speeds and durations. Scanning electron microscopy (SEM) can be performed on representative samples from each experiment to determine the particle size. Further tests can be performed on each sample to determine the effect of the process parameters on the color, molecular weight, viscosity in solution, and solubility in water of the constructs thus obtained.

[0234] Various methods for generating silk particles (e.g., nanoparticles and microparticles) are known in the art. In some embodiments, the silk particles can be generated by, for example, a polyvinyl alcohol (PVA) phase separation method as described in International Application No. WO2011 / 041395, the entire contents of which are incorporated herein by reference. Other methods for generating silk fibroin particles are described, for example, in U.S. Patent Application Publication No. US2010 / 0028451 and PCT Patent Application Publication No. WO2008 / 118133 (using lipids as templates for making silk microspheres or nanospheres), the entire contents of both of which are incorporated herein by reference, as well as in Wenk et al., J Control Release, 2008; 132: 26 - 34 (using a spraying method for generating silk microspheres or nanospheres).

[0235] These include, but are not limited to, at least six types of particles that can be formulated with silk fibroin and additives or active agents / samples, namely: (1) particles containing a core formed by silk fibroin, where the additive / agent / sample is absorbed / adsorbed in the core or forms a coating on the particle core; (2) particles containing a core formed by an additive / agent / sample and coated with one or more layers of silk fibroin; (3) particles containing a globally uniform mixture of silk fibroin and additive / agent / sample; (4) particles containing a core that includes a mixture of silk fibroin and additive / agent / sample with a coating on the silk fibroin core; (5) particles containing a core of a material other than silk fibroin or a biological sample, coated with one additional layer containing a biological sample or silk fibroin, or any combination of a biological sample and silk fibroin; and (6) particles that include any of the particles of (1) - (5) and further include one or more layers of a material other than silk fibroin or a biological sample, such as a polymer. Silk fibroin particles (e.g., microspheres, nanospheres, or gel-like particles) and methods for preparing them are described, for example, in U.S. Patent No. 8,187,616; and U.S. Patent Application Publication Nos. US2008 / 0085272, US2010 / 0028451, US2012 / 0052124, US2012 / 0070427, US2012 / 0187591, the entire contents of which are hereby incorporated by reference herein. These include, but are not limited to, biological samples that may be distributed in the silk fibroin matrix of the film, present on the surface of the film, coated by the film, or any combination thereof.

[0236] In some embodiments, the silk particles can be produced using a lyophilization method as described in U.S. Provisional Patent Application No. 61 / 719,146, filed October 26, 2012, the entire contents of which are incorporated herein by reference. Specifically, the silk foam can be produced by lyophilizing a silk solution. The foam can then be reduced to particles. For example, the silk solution can be cooled to a temperature at which the liquid carrier is converted into a plurality of solid crystals or particles, and at least some of the plurality of solid crystals or particles are removed, leaving a porous silk material (e.g., silk foam). After cooling, the liquid carrier can be at least partially removed by sublimation, evaporation, and / or lyophilization. In some embodiments, the liquid carrier can be removed under reduced pressure. After formation, the silk fibroin foam can be subjected to grinding, cutting, crushing, or any combination thereof to form silk particles. For example, the silk fibroin foam can be blended in a conventional blender or milled in a ball mill to form silk particles of a desired size. Thus, in some embodiments, the low molecular weight silk fibroin composition is in the form of a lyophilized powder.

[0237] In some embodiments, the silk fibroin composition provided (e.g., an article) may be in the form of a gel or hydrogel. The term "hydrogel" is used herein to mean a silk-based material that exhibits the ability to retain a substantial portion of water or other liquid within its structure without dissolution. Exemplary methods for preparing silk fibroin gels and hydrogels include, but are not limited to, sonication, vortexing, pH titration, exposure to an electric field, solvent immersion, water annealing, steam annealing, and the like. Exemplary methods for preparing silk fibroin gels and hydrogels are described, for example, in PCT Publications WO2005 / 012606, WO2008 / 150861, WO2010 / 036992, and WO2011 / 005381, the entire contents of each of which are incorporated herein by reference. Gels formed by exposure to an electric field are also referred to herein as e-gels. The method for forming e-gels is described, for example, in U.S. Patent Application Publication No. US2011 / 0171239, the entire contents of which are incorporated herein by reference. Without limitation, additives / agents / samples may be distributed within the silk fibroin matrix of the gel or hydrogel, absorbed onto the surface of the gel or hydrogel or sponge, present within the pores of the gel or hydrogel, or any combination thereof.

[0238] In some embodiments, the silk fibroin compositions (e.g., articles) described herein may be in the form of a sponge or foam. In some embodiments, the foam or sponge is a patterned foam or sponge, e.g., a nanopatterned foam or sponge. Exemplary methods for preparing silk foams and sponges are described in, for example, PCT Application Publications WO2004 / 000915, WO2004 / 000255, and WO2005 / 012606, the entire contents of each of which are incorporated herein by reference. Without limitation, additives / active substances / samples may be distributed within the silk fibroin matrix of the foam or sponge, absorbed onto the surface of the foam or sponge, present within the pores of the foam or sponge, or any combination thereof.

[0239] In some embodiments, the silk fibroin compositions (e.g., articles) as described herein are in the form of fibers. As used herein, the term "fiber" means a relatively flexible unit of matter having a high ratio of length to width across its cross-section perpendicular to its length. Methods for preparing silk fibroin fibers are well known in the art. Fibers can be prepared by electrospinning a silk solution, drawing a silk solution, etc. Electrospun silk materials, e.g., fibers, and methods for preparing them are described in, for example, WO2011 / 008842, the entire contents of which are incorporated herein by reference. Micron-sized silk fibers (e.g., sizes from 10 to 600 μm) and methods for preparing them are described in, for example, Mandal et al., PNAS, 2012, doi:10.1 073 / pnas.1119474109; U.S. Patent Provisional Application No. 61 / 621,209, filed April 6, 2012; and PCT Application No. PCT / US13 / 35389, filed April 5, 2013.

[0240] In some embodiments, a silk fibroin composition (e.g., an article) as described herein may be in the form of a film, e.g., a silk film. As used herein, the term "film" refers to a substantially flat structure and can also include structures formed from a substantially flat structure. For example, the term "film" can include a tubular structure, or any structure that can be formed by manipulating (e.g., winding and / or folding) a substantially flat film into a desired form. It should be noted that the term "film" is used in its general sense and can include webs, sheets, laminates, etc. In some embodiments, the film is a patterned film, e.g., a nanopatterned film. Exemplary methods for preparing silk fibroin films are described in PCT Publication Nos. WO2004 / 000915 and WO2005 / 012606, the entire contents of both of which are incorporated herein by reference. In some embodiments that include additives and / or agents / samples, these may be distributed within the film, present on the surface of the film, coated by the film, or any combination of these.

[0241] The film can have any desired thickness. For example, the film thickness can range from about 1 nm to about 10 mm. In some embodiments, the film has a thickness in the range of about 1 nm to about 1000 nm or about 1 μm to about 1000 μm.

[0242] In some embodiments, the silk fibroin composition as described herein may be in the form of a coating layer. In some embodiments, such a coating layer may be on a substrate surface. Without being limited thereto, the silk fibroin coating layer can include one or more layers. Further, if two or more layers are present, each layer may be a single layer or multiple layers containing a silk fibroin composition (e.g., low molecular weight silk fibroin), and at least one or more layers contain low molecular weight silk fibroin fragments. In some embodiments, the coating layer is an ultra-thin coating layer. In some embodiments, the silk fibroin composition disclosed herein can form a part of a substrate.

[0243] Examples of substrates can include, without being limited thereto, dipsticks, cellulose-based products, microtiter plates, specimen containers (e.g., blood collection containers, without being limited thereto), medical devices, implants, and any combination thereof. Methods of forming a coating layer containing silk fibroin are described, for example, in U.S. Patent Application No. 11 / 997,193, the entire contents of which are incorporated herein by reference.

[0244] In some embodiments, the silk fibroin compositions (e.g., articles) disclosed herein may be in the form of a cylindrical matrix, such as a silk tube. The silk tube can be made using any method known in the art. For example, the tube can be made using molding, dipping, electrospinning, gel spinning, etc. Gel spinning is described in Lovett et al., Biomaterials, 2008, Vol. 29 (No. 35): 4650 - 4657, and the construction of gel-spun silk tubes is described in PCT Application No. PCT / US2009 / 039870 filed on April 8, 2009 (the entire contents of both are incorporated herein by reference). The construction of silk tubes using the dip coating method is described in PCT Application No. PCT / US2008 / 072742 filed on August 11, 2008, the entire contents of which are incorporated herein by reference. The construction of silk fibroin tubes using the film spinning method is described in PCT Application No. PCT / US2013 / 030206 filed on March 11, 2013; U.S. Patent Provisional Application No. 61 / 613,185 filed on March 20, 2012; and PCT Application Publication No. WO2013126799, the entire contents of all of which are incorporated herein by reference. Without being bound by theory, it is believed that the inner and outer diameters of the silk tube can be more easily adjusted using film spinning or gel spinning rather than dip coating techniques.

[0245] In some embodiments, the provided silk-based material is in the form of a matrix that includes lumens or cavities therein. In some such embodiments, at least a portion of the additive and / or active agent / sample is distributed within the silk fibroin network. In some embodiments, this can be present within the lumen or cavity. In some embodiments, the silk fibroin is in the form of a matrix that includes lumens or cavities therein, at least a partial amount of the additive / agent / sample is present within the lumen or cavity, and at least a partial amount is distributed within the silk fibroin network itself. In some embodiments, when the matrix includes lumens or cavities, at least 5% of the additive or agent / (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) can be present within the lumen or cavity. In some embodiments, the entire amount is present within the lumen / cavity. In some embodiments, at least 5% of the / (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%) can be present within the silk fibroin network of the matrix (e.g., the non-lumen portion of the silk-based material).

[0246] In some embodiments, the silk fibroin compositions (e.g., articles) disclosed herein may be porous (e.g., a porous matrix or scaffold). For example, the porous scaffold can have a porosity of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more. As used herein, the term "porosity" is a measure of the void space in a material and is the fraction of the volume of voids over the total volume as a percentage from 0 to 100% (or 0 to 1). For example, the determination of porosity using standardized techniques such as mercury intrusion porosimetry and gas adsorption, e.g., nitrogen adsorption, is well known to those skilled in the art. As used herein, the term "porosity" is a measure of the void space in a material and is the fraction of the volume of voids over the total volume as a percentage from 0 to 100% (or 0 to 1). For example, the determination of porosity using standardized techniques such as mercury intrusion porosimetry and gas adsorption, e.g., nitrogen adsorption, is well known to those skilled in the art.

[0247] The porous scaffold can have any pore size. As used herein, the term "pore size" refers to the diameter or effective diameter of the cross-section of a pore. The term "pore size" can also refer to the average diameter or average effective diameter of the cross-section of pores based on the measurement of a plurality of pores. The effective diameter of a non-circular cross-section is equal to the diameter of a circular cross-section having the same cross-sectional area as the cross-sectional area of the non-circular cross-section.

[0248] In some embodiments, the pores of the silk-based material can have a size distribution in the range of about 50 nm to about 1000 nm, about 250 nm to about 500 nm, about 500 nm to about 250 nm, about 1 nm to about 200 nm, about 10 nm to about 150 nm, or about 50 nm to about 100 nm. In some embodiments, the silk-based material can be swellable when hydrated. Then, the pore size can vary depending on the moisture content in the silk-based material. In some embodiments, the pores can be filled with a fluid, e.g., water or air.

[0249] Methods for forming pores in silk fibroin-based scaffolds are known in the art and include, but are not limited to, porogen leaching methods, freeze-drying methods, and / or gas forming methods. Exemplary methods for forming pores in silk-based materials are described, for example, in U.S. Patent Application Publication Nos. US2010 / 0279112 and US2010 / 0279112, the entire contents of which are incorporated herein by reference; U.S. Patent No. 7,842,780; and WO2004062697.

[0250] Without being bound by theory, the porosity, structure, and mechanical properties of the porous scaffold can be adjusted by different post-spinning processes, such as vapor annealing, heat treatment, alcohol treatment, air drying, freeze-drying, etc. Furthermore, any desired release rate, profile, or kinetics of the molecules encapsulated in the matrix can be adjusted by varying processing parameters, such as matrix thickness, silk molecular weight, concentration of silk in the matrix, beta-sheet higher order structure, silk II beta-sheet crystallinity, or porosity and pore size. In some embodiments, low molecular weight silk fibroin scaffolds can allow for degradation of the scaffold over a desired period (e.g., one month or more), while achieving sufficient mechanical strength / stability at the implant site. Without being bound by theory, in some embodiments, the porosity of the silk-based material can be adjusted for a desired dissolution rate. For example, higher porosity of the silk-based material generally allows aqueous solutions to penetrate the silk-based material more quickly, thus accelerating the process of dissolution. One of ordinary skill in the art can, therefore, adjust the porosity based on several factors, including, but not limited to, the desired dissolution rate; the size and / or diffusion coefficient of the molecules of the components present in the silk-based material, and / or the concentration, amount, and / or desired physical or mechanical properties of the silk fibroin in the silk-based material.

[0251] To incorporate an additive / agent / sample into a silk fibroin matrix, it can be included in a silk fibroin solution used to produce a silk-based material. Alternatively, or additionally, a pre-formed silk-based material can be added to a solution containing the additive / agent / sample to absorb the additive / agent / sample into / onto the silk-based material.

[0252] In some embodiments, the additive / agent / sample can be distributed homogeneously or heterogeneously (e.g., in a gradient) in the silk-based material. In some embodiments, the additive / agent / sample can be encapsulated or trapped by silk fibroin in the silk-based material. In some embodiments, the additive / agent / sample can be mixed or blended with silk fibroin in the silk-based material.

[0253] In some embodiments, the silk fibroin compositions disclosed herein are osteoconductive. Osteoconductivity is generally defined as the ability of a material to promote the migration of osteogenic cells to the surface of a scaffold by a fibrin clot that is established immediately after implantation of the material. The porosity of the material affects the osteoconductivity of this material.

[0254] In some embodiments, the silk fibroin compositions disclosed herein are osteoinductive. Osteoinductivity is generally defined as the ability to induce undifferentiated stem cells or osteoprogenitor cells (osteoblasts), which are components of bone (osseous) tissue, to differentiate into osteoblasts. The simplest test of osteoinductivity is the ability to induce bone formation (ectopic osteogenesis) at a site of tissue that does not normally form bone, e.g., muscle. It is generally understood that the products described herein can be made osteoinductive by adding growth factors, e.g., rhBMP-2 (recombinant human bone morphogenetic protein-2), to them. Mineralization and the addition of growth factors can affect the osteoinductivity of the material.

[0255] In some embodiments, the silk fibroin compositions disclosed herein are osteogenic and exhibit new bone formation after an implant procedure in vivo. Bone formation is the process of anchoring new bone material using osteoblasts. Osteoblasts build bone by generating osteoid and forming an osteoid matrix composed mainly of type I collagen. Bone tissue contains the osteoid matrix and minerals (mostly with calcium phosphate), which form a chemical arrangement called calcium hydroxyapatite. Osteoblasts are typically the cause of mineralization of the osteoid matrix that forms bone tissue. Without being bound by theory, the osteoconductivity and osteoinductivity of materials have an impact on bone formation. The material can exhibit new bone formation within six months of an implant procedure in vivo. In some embodiments, the material exhibits new bone formation within eight weeks of an implant procedure in vivo.

[0256] In some embodiments, the silk fibroin articles disclosed herein can be sterilized using conventional sterilization processes, such as radiation-based sterilization (i.e., gamma rays), chemical-agent-based sterilization (ethylene oxide), autoclaving, or other suitable procedures. In some embodiments, the sterilization process may involve ethylene oxide at a temperature between about 52°C and about 55°C for 8 hours or less. In some embodiments, the silk fibroin articles described herein can also be processed aseptically. The aseptic silk fibroin articles described herein can be packaged in a suitable sterile, moisture-resistant package for shipping. In some embodiments, a silk fibroin composition containing an active agent is not subjected to sterilization that would significantly harm or degrade the active agent. In some embodiments, the silk fibroin composition protects the active agent from the harm of sterilization.

[0257] In some embodiments, the silk fibroin articles described herein are in the form of an implant or an implantable drug delivery device.

[0258] In some embodiments, the provided fibroin composition is in the form of an injectable composition. As used herein, the term "injectable composition" generally refers to a composition that can be delivered or administered into tissue with minimally invasive techniques. The term "minimally invasive technique" refers to a technique that is performed by entering the subject's body through the skin or through an opening in a body cavity or body structure, but with the smallest possible damage (e.g., small incision, injection) involved. In some embodiments, the injectable composition can be administered or delivered into tissue by injection. In some embodiments, the injectable composition can be delivered into tissue by a small incision on the skin followed by the insertion of a needle, cannula, and / or tubing, e.g., a catheter. Without limitation, the injectable composition can be administered or placed into tissue by surgery, e.g., an implant procedure. Some exemplary injectable compositions include, without limitation, solutions, hydrogels, gel-like particles, and / or microspheres.

[0259] For clarity, the term "injectable" as in "injectable formulation" and "injectable agent" refers to the physical properties of a solution (e.g., formulation) suitable for administration by injection such that there is a sufficient flow of the solution through a needle or any other suitable means and such flow occurs moderately easily by the user. A syringe is generally used to deliver an injectable drug to a subject. In some embodiments, the injectable formulation can be provided as a pre-filled syringe. In some embodiments, the injectable formulation can be provided as a ready-to-use formulation. In some embodiments, the injectable formulation can be provided as a kit.

[0260] In some embodiments, the provided composition, e.g., an injectable composition, can further comprise a pharmaceutically acceptable carrier. For example, in some embodiments, a composition suitable for injection comprises a sterile aqueous solution or dispersion. The carrier can be, for example, water, cell culture medium, buffer (e.g., phosphate buffered saline), polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), a solvent or dispersion medium containing suitable mixtures thereof. In some embodiments, the pharmaceutical carrier can be a buffered solution (e.g., PBS).

[0261] Alternatively or additionally, various additives can be added to enhance the stability, sterility, and isotonicity of the injectable composition, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. In many cases, it may be desirable to include an isotonic agent, such as sugar, sodium chloride, etc. The injectable composition can also contain auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, colors, etc., depending on the desired formulation.

[0262] The viscosity of the provided liquid composition (e.g., an injectable composition) can be modulated by adjusting the weight percentage of silk fibroin fragments having molecular weight sub-ranges (i) to (xviii). In some embodiments, the viscosity of the composition can be further maintained at a selected level using a pharmaceutically acceptable thickening agent. In one embodiment, methylcellulose can be used as it is readily and economically available and easy to handle. Other suitable thickening agents include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of the thickening agent can be determined by the selected agent and the desired viscosity for injection. The important point is to use an amount that achieves the selected viscosity, for example, by addition of such a thickening agent into some embodiments of the injectable composition.

[0263] In some embodiments, the provided silk fibroin composition is in the form of a sprayable composition.

[0264] In some embodiments, the provided silk fibroin composition (e.g., low molecular weight silk composition) described herein may be in the form of an aerogel or aerogel-like material. For example, methods of forming an aerogel or aerogel-like material comprising silk fibroin are described in U.S. Provisional Patent Application No. 61 / 9 02,145, filed November 8, 2013, entitled "PEPTIDE-BASED NANOFIBRILLAR MATERIALS", the entire contents of which are incorporated herein by reference.

[0265] Without being bound by theory, the properties (e.g., but not limited to, solubility) of silk-based materials (including those containing an active agent or biological sample as described herein) can be varied by changing the molecular weight of the silk fibroin fragments in the silk-based material. In some embodiments, silk fibroin of different molecular weights can be produced using different periods for degumming the cocoons to provide degummed fibroin. Thus, in some embodiments for producing a low molecular weight silk fibroin composition, the cocoons are boiled for a period of about 1 minute to 2 hours, about 5 minutes to about 2 hours, about 10 minutes to about 60 minutes, about 60 minutes to 4 hours, about 60 minutes to 3 hours, about 60 minutes to 2 hours, about 60 minutes to 90 minutes, or about 4 hours or more (e.g., in a salt solution, e.g., Na 2 CO 3 medium). In some embodiments, the cocoons can be boiled for about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 90 minutes, about 100 minutes or more (e.g., in a salt solution, e.g., Na 2 CO 3By adjusting the refining time, the solubility of silk-based materials (e.g., in an aqueous solution) can be optimized for the extraction or recovery of the agents present in the low molecular weight silk fibroin composition. Without being bound by theory, generally, longer boiling times result in silk fibroin with a lower molecular weight (MW) / chain length, and thus silk-based materials produced from such lower MW silk fibroin are generally more soluble (e.g., in an aqueous solution) than those produced from higher MW silk fibroin.

[0266] In some embodiments, the soluble silk-based material can be produced from a silk solution, and the cocoons have been boiled or refined for a period sufficient to produce lower MW silk fibroin, such as at least about 30 minutes, at least about 60 minutes, or at least about 90 minutes or more. In some embodiments, the soluble silk-based material is a freeze-dried silk-based material produced from a silk solution, and the cocoons have been boiled or refined for at least about 30 minutes, at least about 60 minutes, or at least about 90 minutes or more. In some embodiments, the soluble silk-based material is a silk-based film produced from a silk solution, and the cocoons have been boiled or refined for at least about 30 minutes, at least about 60 minutes, or at least about 90 minutes or more. The silk-based material can be in any other form as described herein, produced, for example, by electrospinning, gelling, or other rapid solidification techniques known in the art.

[0267] Other methods of changing the solubility of the silk-based materials described herein can also be used, either alone or in combination with adjustment of the refining time. For example, the solubility of silk-based materials can be improved by means of accelerating the drying rate of silk-based materials, such as by forced air, reduction of humidity (lower than ambient humidity), and / or increase of temperature. Additionally or alternatively, the solubility of silk-based materials can be improved by reducing the time of exposing silk-based materials to lyophilization conditions and / or conditions that can induce crystallinity in silk fibroin. In some embodiments, the solubility of silk-based materials can be improved by selectively removing high molecular weight fractions of silk (such as heavy chains and / or long hydrophobic sequences) during purification, such as via enzymatic digestion, filtration, chromatography, etc. In some embodiments, the solubility of silk-based materials can be improved by means of sterilization, including autoclaving and sterile filtration, which can serve to reduce the molecular weight and / or continue to remove insoluble microparticles.

[0268] In various embodiments, at least a portion of the silk fibroin compositions (such as in low molecular weight silk fibroin compositions and / or stabilizing compositions) as described herein can be modified for different uses, such as biomedical uses, and / or for different desired mechanical or chemical properties. Those skilled in the art can select appropriate methods for modifying silk fibroin, for example, based on the side groups of silk fibroin, the desired reactivity of silk fibroin, and / or the desired charge density on silk fibroin.

[0269] For example, when implanted in vivo for tissue engineering or drug delivery purposes, at least a portion of the silk fibroin can be genetically modified to maintain the stability of the active agent distributed within the silk fibroin composition, thereby enabling the inclusion of additional modifications of silk, such as fusion polypeptides containing fibrous protein domains and mineralization domains, which can be used to form organic-inorganic composites. See, for example, WO2006 / 076711, the entire contents of which are incorporated herein by reference. In some embodiments, the silk fibroin can be chemically modified, for example, by diazonium or carbodiimide coupling reactions, avidin-biotin interactions, or genetic modification, which changes the physical properties and functionality of the silk protein. Chemically modified silk fibroin and methods for their preparation are described, for example, in PCT Publication Nos. WO2011 / 011347 and WO2010 / 057142, the entire contents of which are incorporated herein by reference; and U.S. Patent Application No. 12 / 192,588.

[0270] In one embodiment, the modification of silk fibroin can use amino acid side chain chemistry, for example, chemical modification by covalent bonding or modification by charge-charge interaction. Exemplary chemical modification methods include, but are not limited to, carbodiimide coupling reactions (see, for example, U.S. Patent Application No. US2007 / 0212730), diazonium coupling reactions (see, for example, U.S. Patent Application No. US2009 / 0232963), avidin-biotin interactions (see, for example, International Application No. WO2011 / 011347), and pegylation with chemically active or activated derivatives of PEG polymers (see, for example, International Application No. WO2010 / 057142). Silk fibroin can also be modified by genetic modification that changes the functionality of the silk protein (see, for example, International Application No. WO2011 / 006133). For example, silk fibroin can be genetically modified such that it can contain additional modifications of silk, such as fusion polypeptides containing fibrous protein domains and mineralization domains, which can be used to form organic-inorganic composites. See WO2006 / 076711. In some embodiments, silk fibroin can be genetically modified to fuse with a protein, such as a therapeutic protein.

[0271] In some embodiments, at least a portion of the silk fibroin in the compositions described herein can be derivatized or modified by a positively charged / negatively charged molecule. In some embodiments, silk fibroin can be modified with a positively charged / negatively charged peptide or polypeptide, such as poly-lysine and poly-glutamic acid. While it is possible, it is not required that all single silk fibroin molecules in the composition be modified with a positively charged / negatively charged molecule. Methods for derivatizing or modifying silk fibroin with charged molecules are described, for example, in PCT Application Publication No. WO2011109691A2, the entire contents of which are incorporated herein by reference.

[0272] By adjusting the ratio of modified silk fibroin to unmodified silk fibroin, one or more desired properties of a low molecular weight silk fibroin composition or an article formed therefrom can be optimized. Thus, in some embodiments, the ratio of modified silk fibroin to unmodified silk fibroin in the composition can range from about 1000:1 (w / w) to about 1:1000 (w / w), about 500:1 (w / w) to about 1:500 (w / w), about 250:1 (w / w) to about 1:250 (w / w), about 200:1 (w / w) to about 1:200 (w / w), about 25:1 (w / w) to about 1:25 (w / w), about 20:1 (w / w) to about 1:20 (w / w), about 10:1 (w / w) to about 1:10 (w / w), or about 5:1 (w / w) to about 1:5 (w / w).

[0273] In some embodiments, the composition can include, for example, a molar ratio of modified silk fibroin to unmodified silk fibroin of at least 1000:1, at least 900:1, at least 800:1, at least 700:1, at least 600:1, at least 500:1, at least 400:1, at least 300:1, at least 200:1, at least 100:1, at least 90:1, at least 80:1, at least 70:1, at least 60:1, at least 50:1, at least 40:1, at least 30:1, at least 20:1, at least 10:1, at least 7:1, at least 5:1, at least 3:1, at least 1:1, at least 1:3, at least 1:5, at least 1:7, at least 1:10, at least 1:20, at least 1:30, at least 1:40, at least 1:50, at least 1:60, at least 1:70, at least 1:80, at least 1:90, at least 1:100, at least 1:200, at least 1:300, at least 1:400, at least 1:500, at least 1:600, at least 1:700, at least 1:800, at least 1:900, or at least 1:1000.

[0274] In some embodiments, the composition comprises a molar ratio of modified silk fibroin to unmodified silk fibroin of, for example, at most 1000:1, at most 900:1, at most 800:1, at most 700:1, at most 600:1, at most 500:1, at most 400:1, at most 300:1, at most 200:1, 100:1, at most 90:1, at most 80:1, at most 70:1, at most 60:1, at most 50:1, at most 40:1, at most 30:1, at most 20:1, at most 10:1, at most 7:1, at most 5:1, at most 3:1, at most 1:1, at most 1:3, at most 1:5, at most 1:7, at most 1:10, at most 1:20, at most 1:30, at most 1:40, at most 1:50, at most 1:60, at most 1:70, at most 1:80, at most 1:90, at most 1:100, at most 1:200, at most 1:300, at most 1:400, at most 1:500, at most 1:600, at most 1:700, at most 1:800, at most 1:900, or at most 1:1000.

[0275] In some embodiments, the composition comprises a modified silk fibroin and an unmodified silk fibroin having a molar ratio of, for example, from about 1000:1 to about 1:1000, from about 900:1 to about 1:900, from about 800:1 to about 1:800, from about 700:1 to about 1:700, from about 600:1 to about 1:600, from about 500:1 to about 1:500, from about 400:1 to about 1:400, from about 300:1 to about 1:300, from about 200:1 to about 1:200, from about 100:1 to about 1:100, from about 90:1 to about 1:90, from about 80:1 to about 1:80, from about 70:1 to about 1:70, from about 60:1 to about 1:60, from about 50:1 to about 1:50, from about 40:1 to about 1:40, from about 30:1 to about 1:30, from about 20:1 to about 1:20, from about 10:1 to about 1:10, from about 7:1 to about 1:7, from about 5:1 to about 1:5, from about 3:1 to about 1:3, or about 1:1. comprising a molar ratio.

[0276] In some embodiments, the silk fibroin has a substantially depleted natural sericin content (e.g., 5% (w / w) or less residual sericin in the final extracted silk). Instead, higher concentrations of residual sericin can remain on the silk following extraction, or the extraction step can be omitted. In some embodiments, the sericin-depleted silk fibroin has, for example, about 1% (w / w) residual sericin, about 2% (w / w) residual sericin, about 3% (w / w) residual sericin, about 4% (w / w), or about 5% (w / w) residual sericin. In some embodiments, the sericin-depleted silk fibroin has, for example, at most 1% (w / w) residual sericin, at most 2% (w / w) residual sericin, at most 3% (w / w) residual sericin, at most 4% (w / w), or at most 5% (w / w) residual sericin. In some other embodiments, the sericin-depleted silk fibroin has, for example, about 1% (w / w) to about 2% (w / w) residual sericin, about 1% (w / w) to about 3% (w / w) residual sericin, about 1% (w / w) to about 4% (w / w), or about 1% (w / w) to about 5% (w / w) residual sericin. In some embodiments, the silk fibroin is completely free of its natural sericin content. As used herein, the term "completely free" (i.e., the "consisting of" terminology) means that the substance cannot be detected or its presence confirmed within the detection range of the equipment or process being used. In some embodiments, the silk fibroin is essentially free of its natural sericin content. As used herein, the term "essentially free" (or "consisting essentially of") means that only trace amounts of the substance can be detected, are present in amounts below detection, or are absent.

[0277] Although not bound by theory, the properties of the provided silk fibroin compositions can be modified by the controlled partial removal of silk sericin or by the careful high-concentration of the source silk with sericin. This can be achieved by varying the conditions for the silk degumming process, such as time, temperature, concentration, and the like.

[0278] Refined silk can be prepared by any conventional method known to those skilled in the art. For example, B. mori cocoons are boiled in an aqueous solution for a predetermined period of time. Generally, a longer refining time results in low molecular weight silk fibroin. In some embodiments, the silk cocoons are boiled for at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes, at least 120 minutes, or more to produce low molecular weight silk fibroin fragments. Additionally or alternatively, in some embodiments, the silk cocoons can be heated or boiled while increasing the temperature. For example, in some embodiments, the silk cocoons are heated or boiled at about 101.0 °C, about 101.5 °C, about 102.0 °C, about 102.5 °C, about 103.0 °C, about 103.5 °C, about 104.0 °C, about 104.5 °C, about 105.0 °C, about 105.5 °C, about 106.0 °C, about 106.5 °C, about 107.0 °C, about 107.5 °C, about 108.0 °C, about 108.5 °C, about 109.0 °C, about 109.5 °C, about 110.0 °C, about 110.5 °C, about 111.0 °C, about 111.5 °C, about 112.0 °C, about 112.5 °C, about 113.0 °C, 113.5 °C, about 114.0 °C, about 114.5 °C, about 115.0 °C, about 115.5 °C, about 116.0 °C, about 116.5 °C, about 117.0 °C, about 117.5 °C, about 118.0 °C, about 118.5 °C, about 119.0 °C, about 119.5 °C, about 120.0 °C or more. In some embodiments, such high temperatures can be achieved by performing at least a portion of the heating process (e.g., boiling process) under pressure. For example, suitable pressures for generating the silk fibroin fragments described herein are typically between about 10 - 40 psi, such as about 11 psi, about 12 psi, about 13 psi, about 14 psi, about 15 psi, about 16 psi, about 17 psi, about 18 psi, about 19 psi, about 20 psi, about 21 psi, about 22 psi, about 23 psi, about 24 psi, about 25 psi, about 26 psi, about 27 psi, about 28 psi, about 29 psi, about 30 psi, about 31 psi, about 32 psi, about 33 psi, about 34 psi, about 35 psi, about 36 psi, about 37 psi, about 38 psi, about 39 psi or about 40 psi.

[0279] In one embodiment, the aqueous solution used in the process of refining silk cocoons is about 0.02M Na 2 CO 3 . The cocoons are rinsed, for example, with water to extract sericin protein. The refined silk can be dried and used to prepare silk powder. Alternatively, the extracted silk can be dissolved in an aqueous salt solution. Salts useful for this purpose include lithium bromide, lithium thiocyanate, calcium nitrate, or other chemicals that can solubilize silk. In some embodiments, the extracted silk can be dissolved in an about 8M - 12M LiBr solution. The salt is removed, for example, using dialysis.

[0280] Optionally, the solution can then be concentrated, for example, using dialysis against a hygroscopic polymer such as PEG, polyethylene oxide, amylose, or sericin. In some embodiments, the PEG has a molecular weight of 8,000 - 10,000 g / mol and has a concentration of about 10% - about 50% (w / v). A slide-a-lyzer dialysis cassette (Pierce, MW CO3500) can be used. However, any dialysis system can be used. Dialysis can be carried out for a period sufficient to result in a final concentration of the silk aqueous solution between about 10% - about 30%. In most cases, dialysis for 2 - 12 hours may be sufficient. See, for example, International Patent Application Publication No. WO2005 / 012606, the entire contents of which are incorporated herein by reference.

[0281] Another way to produce a concentrated silk solution involves drying a dilute silk solution (e.g., by evaporation or lyophilization). The dilute solution can be partially dried to reduce its volume, thereby increasing the silk concentration. The dilute solution can be completely dried, and then the dried silk fibroin can be dissolved in a smaller volume of solvent compared to the volume of the dilute silk solution. In some embodiments, the silk fibroin solution can optionally be filtered and / or centrifuged at an appropriate time. For example, in some embodiments, the silk fibroin solution can optionally be filtered and / or centrifuged following a heating or boiling step. In some embodiments, the silk fibroin solution can optionally be filtered and / or centrifuged following a dialysis step. In some embodiments, the silk fibroin solution can optionally be filtered and / or centrifuged following a step of adjusting the concentration. In some embodiments, the silk fibroin solution can optionally be filtered and / or centrifuged following a reconstitution step. In any of such embodiments, the filtration and / or centrifugation step(s) can be performed to remove insoluble materials. In any of such embodiments, the filtration and / or centrifugation step(s) can be performed to selectively enrich silk fibroin fragments of a certain molecular weight(s).

[0282] In some embodiments, the silk fibroin solution can be produced using an organic solvent. Such methods are described, for example, in Li, M. et al., J. Appl. Poly Sci., 2001, Vol. 79, pp. 2192-2199; Min, S. et al., Sen’I Gakkaishi, 1997, Vol. 54, pp. 85-92; Nazarov, R. et al., Biomacromolecules, 2004, Vol. 5, pp. 718-26, the entire contents of all of which are incorporated herein by reference. Exemplary organic solvents that can be used to produce the silk solution include, but are not limited to, hexafluoroisopropanol (HFIP). See, for example, International Application No. WO2004 / 000915, the entire contents of which are incorporated herein by reference. In some embodiments, the silk solution is completely free or essentially free of organic solvent. In some embodiments, the silk solution is substantially free of solvents other than water.

[0283] The silk fibroin composition disclosed in this specification can contain any amount / ratio of silk fibroin relative to the total volume / weight of the composition. Without being bound by theory, the properties of the silk fibroin composition can be changed by varying the amount of silk fibroin itself in the solution used to prepare the silk fibroin composition (e.g., low molecular weight silk fibroin composition and / or stabilizing composition). Generally, any amount of silk fibroin can be present in the solution used to prepare the silk fibroin composition. For example, the amount of silk fibroin in the solution can be from about 0.1% (w / v) to about 90% (w / v). In some embodiments, the amount of silk fibroin in the solution is from about 1% (w / v) to about 75% (w / v), from about 1% (w / v) to about 70% (w / v), from about 1% (w / v) to about 65% (w / v), from about 1% (w / v) to about 60% (w / v), from about 1% (w / v) to about 55% (w / v), from about 1% (w / v) to about 50% (w / v), from about 1% (w / v) to about 35% (w / v), from about 1% (w / v) to about 30% (w / v), from about 1% (w / v) to about 25% (w / v), from about 1% (w / v) to about 20% (w / v), from about 1% (w / v) to about 15% (w / v), from about 1% (w / v) to about 10% (w / v), from about 5% (w / v) to about 25% (w / v), from about 5% (w / v) to about 20% (w / v), from about 5% (w / v) to about 15% (w / v). In some embodiments, the silk fibroin in the solution is about 25% (w / v). In some embodiments, the silk fibroin in the solution is from about 0.5 (w / v) to about 30% (w / v), from about 4% (w / v) to about 16% (w / v), from about 4% (w / v) to about 14% (w / v), from about 4% (w / v) to about 12% (w / v), from about 4% (w / v) to about 0% (w / v), from about 6% (w / v) to about 8% (w / v). The exact amount of silk in the silk solution can be determined by drying a known amount of the silk solution, measuring the mass of the residue, and calculating the solution concentration.

[0284] The amount of silk fibroin in the provided composition or article (e.g., low molecular weight silk composition and / or stabilized composition) may be from about 1% (w / v) to about 90% (w / v). In some embodiments, the amount of silk fibroin in the silk fibroin composition is from about 0.1% (w / v) to about 75% (w / v), from about 1% (w / v) to about 70% (w / v), from about 1% (w / v) to about 65% (w / v), from about 1% (w / v) to about 60% (w / v), from about 1% (w / v) to about 55% (w / v), from about 1% (w / v) to about 50% (w / v), from about 1% (w / v) to about 45% (w / v), from about 1% (w / v) to about 40% (w / v), from about 1% (w / v) to about 35% (w / v), from about 1% (w / v) to about 30% (w / v), from about 1% (w / v) to about 25% (w / v), from about 1% (w / v) to about 20% (w / v), from about 1% (w / v) to about 15% (w / v), from about 1% (w / v) to about 10% (w / v), from about 5% (w / v) to about 25% (w / v), from about 5% (w / v) to about 20% (w / v), from about 5% (w / v) to about 15% (w / v). In some embodiments, the silk fibroin in the low molecular weight silk fibroin composition is about 25% (w / v). In some embodiments, the silk in the silk fibroin composition is from about 0.5 (w / v) to about 30% (w / v), from about 2% (w / v) to about 8% (w / v), from about 2% (w / v) to about 7% (w / v), from about 2% (w / v) to about 6% (w / v), from about 2% (w / v) to about 5% (w / v), from about 3% (w / v) to about 4% (w / v).

[0285] In some embodiments, the solution has a silk fibroin concentration of from about 0.25% to about 50% (w / v) or from about 0.5% to about 15% (w / v), or from about 0.5% to about 10% (w / v). In some embodiments, the silk fibroin solution has a silk fibroin concentration of from about 10% to about 40% or from 15% to about 35% (w / v). In one embodiment, the silk fibroin solution has a silk fibroin concentration of from about 20% to about 30% (w / v). In one embodiment, the silk fibroin solution has a silk fibroin concentration of about 30% (w / v). In some embodiments, the silk fibroin solution has a silk fibroin concentration of from about 0.1% to about 30% (w / v), from about 0.5% to about 15% (w / v), from about 1% to about 8% (w / v), or from about 1.5% to about 5% (w / v). In some embodiments, the silk fibroin solution has a silk fibroin concentration of from about 5% to about 30% (w / v), from about 10% to about 25% (w / v), or from about 15 to about 20% (w / v). In some embodiments, the silk solution has a silk fibroin concentration of from about 0.5% to 10% (w / v).

[0286] Depending on the use in some embodiments, high-order structural changes can be induced in the silk fibroin in the provided composition to regulate the solubility of the silk fibroin composition / article. In some embodiments, the high-order structural changes can induce the silk fibroin to be at least partially insoluble. Without being bound by theory, the induced high-order structural changes alter the crystallinity of the silk fibroin, e.g., the silk II beta-sheet crystallinity. The high-order structural changes can be induced by any method known in the art including, but not limited to, alcohol immersion (e.g., ethanol, methanol), water annealing, shear stress, ultrasound (e.g., by sonication), pH reduction (e.g., pH titration and / or exposure to an electric field), and any combination of these.For example, higher-order structural changes include, but are not limited to, controlled slow drying (Lu et al., Biomacromolecules, 2009, 10, 1032); water annealing (Jin et al., 15Adv. Funct. Mats., 2005, 15, 1241; Hu et al., Biomacromolecules, 2011, 12, 1686); stretching (Demura and Asakura, Biotech & Bioengin., 1989, 33, 598); compression; solvent immersion including methanol (Hofmann et al., J Control Release., 2006, 111, 219), ethanol (Miyairi et al., J. Fermen. Tech., 1978, 56, 303), glutaraldehyde (Acharya et al., Biotechnol J., 2008, 3, 226), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (Bayraktar et al., Eur J Pharm Biopharm., 2005, 60, 373); pH adjustment, e.g., pH titration and / or exposure to an electric field (see, e.g., U.S. Patent Application No. US2011 / 0171239); heat treatment; shear stress (see, e.g., International Application No. WO2011 / 005381), ultrasound, e.g., sonication (see, e.g., U.S. Patent Application Publication No. US2010 / 0178304 and International Application No. WO2008 / 150861); and can be induced by one or more methods including any combination of these. The entire contents of all of the references listed above are incorporated herein by reference in their entirety.

[0287] In some embodiments, the material can be treated by annealing. As used herein, the annealing process involves inducing the formation of a beta-sheet secondary structure in silk fibroin. This can be due to an increase in non-covalent interactions in silk fibroin. Such non-covalent interactions can include intramolecular interactions, intermolecular interactions, or both. Typically, the non-covalent interactions are mediated by hydrogen bonds, which result in an increase in beta-sheet formation. By reaching a certain critical level of the beta-sheet secondary structure, silk fibroin becomes insoluble, for example, in an aqueous environment. This phenomenon is generally referred to as crystallinity, and such a state of silk fibroin is referred to as silk II. Thus, “annealing” involves a change in the higher-order structure of silk fibroin to a higher-order structure dominated by beta-sheets (silk II). Without being bound by theory, this higher-order structure change is thought to be due to a structural shift of silk fibroin to a higher beta-sheet content mediated by hydrogen bonds and / or hydrophobic interactions.

[0288] In some embodiments, the higher-order structure of silk fibroin can be changed by water annealing. There are several different methods for water annealing. One method of water annealing involves treating a coagulated but soluble form of silk fibroin with steam. Without being bound by theory, water molecules act as plasticizers, which allows the chain mobility of fibroin molecules to promote the formation of hydrogen bonds, resulting in an increase in the beta-sheet secondary structure. This process is also referred to herein as “steam annealing”.

[0289] Although not bound by theory, physical temperature-controlled water vapor annealing (TCWVA) is thought to provide a simple and effective way to achieve fine control of the molecular structure of silk biocompatible materials. From low beta-sheet content (silk I structure with dominant alpha-helices) using conditions at 4 °C to higher beta-sheet content (silk II structure with dominant beta-sheets) with a crystallinity of about 60% at 100 °C, silk materials can be prepared by controlling crystallinity. This physical approach covers a series of structures previously reported to govern crystallization during silk material fabrication, yet still achieves a simpler environmentally friendly chemistry than approaches involving strict control of reproducibility. Water or water vapor annealing is described, for example, in PCT Application No. PCT / US2004 / 011199, filed Apr. 12, 2004, and PCT / US2005 / 020844, filed Jun. 13, 2005, the entire contents of both of which are incorporated herein by reference; and Jin et al., Adv. Funct. Mats., 2005, 15:1241, and Hu et al., Biomacromolecules, 2011, 12(5):1686 - 1696.

[0290] Another method of annealing is by the slow, controlled evaporation of water from silk fibroin in a silk material / matrix. Slow, controlled drying is described, for example, in Lu et al., Acta. Biomater., 2010, 6(4):1380 - 1387.

[0291] The annealing step can be performed for different periods within a water vapor environment, for example, within a chamber filled with water vapor. Without being bound by theory, the length of annealing gives rise to the amount of beta-sheet crystallinity obtained in the silk fibroin within the silk-based material. Thus, typical annealing periods can range from seconds to days. In some embodiments, the annealing is for a period of seconds to hours. For example, the annealing time can range from seconds (e.g., about 5 seconds, 10 seconds, 15 seconds, 20 seconds, 25 seconds, 30 seconds, 35 seconds, 40 seconds, 45 seconds, 50 seconds, 55 seconds, or 60 seconds) to about 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, or 48 hours.

[0292] The temperature of the water vapor used in the annealing process results in the amount of beta-sheet crystallinity obtained. See HU et al., Biomacromolecules, Volume 12: pages 1686 - 1696. Thus, annealing can be performed at any desired temperature. For example, annealing can be performed at a water vapor temperature of about 4°C to about 120°C. The optimal water vapor for obtaining the required amount of beta-sheet crystallinity in the silk fibroin within the silk-based material can be calculated based on Equation (I). C = a(1 - exp(-kT)) (I) Where C is the beta-sheet crystallinity, a is 62.59, k is 0.028, and T is the annealing temperature. See HU et al., Biomacromolecules, Volume 12: pages 1686 - 1696.

[0293] Without being bound by theory, the pressure at which annealing is performed can also affect the degree or amount of beta-sheet crystallinity. In some embodiments, the contact can be performed in a vacuum environment.

[0294] The relative humidity at which annealing is performed can also affect the degree or amount of beta-sheet crystallinity. The relative humidity at which the silk-based material is in contact with water or water vapor may range from about 5% to 100%. For example, the relative humidity may be from about 5% to about 95%, from about 10% to about 90%, or from about 15% to about 85%. In some embodiments, the relative humidity is 90% or more.

[0295] Another useful method for annealing silk fibroin is to subject the silk-based material to dehydration by using an organic solvent, such as an alcohol, such as methanol, ethanol, isopropyl, acetone, etc. Such solvents have the effect of dehydrating silk fibroin, which promotes the "packing" of silk fibroin molecules to form a beta-sheet structure. In some embodiments, the silk-based material can be treated with an alcohol, such as methanol, ethanol, etc. The alcohol concentration may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%. In some embodiments, the alcohol concentration is about 90%.

[0296] Thus, in some embodiments, changes in the higher-order structure of silk fibroin can be induced by immersion in an alcohol, such as methanol, ethanol, etc. The alcohol concentration may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%. In some embodiments, the alcohol concentration is 100%. When the change in the higher-order structure is due to immersion in a solvent, the silk composition can be washed, for example, with a solvent / water gradient to remove some of the residual solvent used for immersion. The washing can be repeated 1 time, for example, 1 time, 2 times, 3 times, 4 times, 5 times, or more.

[0297] Instead, changes in the higher-order structure of silk fibroin can be induced by shear stress. Shear stress can be applied, for example, by passing the silk composition through a needle. Other methods of inducing higher-order structure changes include applying an electric field, applying pressure, or changing the salt concentration.

[0298] The treatment time for inducing higher-order structure changes can be any period to achieve the desired silk II (beta-sheet crystallinity) content. In some embodiments, the treatment time can range from about 1 hour to about 12 hours, from about 1 hour to about 6 hours, from about 1 hour to about 5 hours, from about 1 hour to about 4 hours, or from about 1 hour to about 3 hours. In some embodiments, the sintering time can range from about 2 hours to about 4 hours or from 2.5 hours to about 3.5 hours.

[0299] When the induction of higher-order structure changes is by solvent immersion, the treatment time can range from a few minutes to several hours. For example, the immersion in the solvent can be for a period of at least about 15 minutes, at least about 30 minutes, at least about 1 hour, at least about 2 hours, at least 3 hours, at least about 6 hours, at least about 18 hours, at least about 12 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days. In some embodiments, the immersion in the solvent can be for a period of about 12 hours to about 7 days, from about 1 day to about 6 days, from about 2 to about 5 days, or from about 3 to about 4 days.

[0300] After a treatment that induces a higher-order structure change, silk fibroin can contain a silk II beta-sheet crystallinity content that is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, but not 100% (i.e., not all silk is present in the silk II beta-sheet higher-order structure). In some embodiments, the silk fibroin in the silk-based material is at least 10%, such as 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 70%, 85%, 90%, 95% or more, but not 100% (i.e., not all silk fibroin is in the beta-sheet higher-order structure) beta-sheet crystallinity. In some embodiments, the silk is present entirely in the silk II beta-sheet higher-order structure, i.e., 100% silk II beta-sheet crystallinity.

[0301] Regardless of the annealing method used, the final result of the annealing process will often be that the annealed silk fibroin has a high degree of crystallinity such that it becomes insoluble. In some embodiments, "high degree of crystallinity" refers to a beta-sheet content of about 20% to about 70%, such as about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65% and about 75%.

[0302] In some embodiments, the annealing process can result in a silk-based material that contains a silk II beta-sheet crystallinity content that is at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%, but not 100% (i.e., not all silk is present in the silk II beta-sheet higher-order structure). In some embodiments, the silk-based material can have 100% silk II beta-sheet crystallinity.

[0303] In some embodiments, the silk fibroin in the provided silk fibroin composition has a protein structure that substantially includes β-turn and β-strand regions. Without being bound by theory, the silk β-sheet content can affect the gel function of the composition and its long lifespan in vivo. It should be understood that compositions containing non-β-sheet content (e.g., e-gels) can also be utilized. In aspects of these embodiments, the silk fibroin in the provided composition has a protein structure that includes, for example, about 5% β-turn and β-strand regions, about 10% β-turn and β-strand regions, about 20% β-turn and β-strand regions, about 30% β-turn and β-strand regions, about 40% β-turn and β-strand regions, about 50% β-turn and β-strand regions, about 60% β-turn and β-strand regions, about 70% β-turn and β-strand regions, about 80% β-turn and β-strand regions, about 90% β-turn and β-strand regions, or about 100% β-turn and β-strand regions. In other aspects of these embodiments, the silk fibroin in the low molecular weight silk fibroin composition has a protein structure that includes, for example, at least 10% β-turn and β-strand regions, at least 20% β-turn and β-strand regions, at least 30% β-turn and β-strand regions, at least 40% β-turn and β-strand regions, at least 50% β-turn and β-strand regions, at least 60% β-turn and β-strand regions, at least 70% β-turn and β-strand regions, at least 80% β-turn and β-strand regions, at least 90% β-turn and β-strand regions, or at least 95% β-turn and β-strand regions.In still other aspects of these embodiments, the silk fibroin in the low molecular weight silk fibroin composition has a protein structure comprising, for example, about 10% to about 30% β-turn and β-strand regions, about 20% to about 40% β-turn and β-strand regions, about 30% to about 50% β-turn and β-strand regions, about 40% to about 60% β-turn and β-strand regions, about 50% to about 70% β-turn and β-strand regions, about 60% to about 80% β-turn and β-strand regions, about 70% to about 90% β-turn and β-strand regions, about 80% to about 100% β-turn and β-strand regions, about 10% to about 40% β-turn and β-strand regions, about 30% to about 60% β-turn and β-strand regions, about 50% to about 80% β-turn and β-strand regions, about 70% to about 100% β-turn and β-strand regions, about 40% to about 80% β-turn and β-strand regions, about 50% to about 90% β-turn and β-strand regions, about 60% to about 100% β-turn and β-strand regions, or about 50% to about 100% β-turn and β-strand regions. In some embodiments, a silk β-sheet content of less than 10% to about 55% can be used in the silk fibroin compositions disclosed herein.

[0304] In some embodiments, the silk fibroin in the silk fibroin composition provided herein has a protein structure that is substantially free of α-helix and random coil regions. In aspects of these embodiments, the silk fibroin has a protein structure that contains, for example, about 5% α-helix and random coil regions, about 10% α-helix and random coil regions, about 15% α-helix and random coil regions, about 20% α-helix and random coil regions, about 25% α-helix and random coil regions, about 30% α-helix and random coil regions, about 35% α-helix and random coil regions, about 40% α-helix and random coil regions, about 45% α-helix and random coil regions, or about 50% α-helix and random coil regions. In other aspects of these embodiments, the silk fibroin has a protein structure that contains, for example, at most 5% α-helix and random coil regions, at most 10% α-helix and random coil regions, at most 15% α-helix and random coil regions, at most 20% α-helix and random coil regions, at most 25% α-helix and random coil regions, at most 30% α-helix and random coil regions, at most 35% α-helix and random coil regions, at most 40% α-helix and random coil regions, at most 45% α-helix and random coil regions, or at most 50% α-helix and random coil regions.In still other aspects of these embodiments, the silk fibroin has a protein structure comprising, for example, about 5% to about 10% alpha helix and random coil regions, about 5% to about 15% alpha helix and random coil regions, about 5% to about 20% alpha helix and random coil regions, about 5% to about 25% alpha helix and random coil regions, about 5% to about 30% alpha helix and random coil regions, about 5% to about 40% alpha helix and random coil regions, about 5% to about 50% alpha helix and random coil regions, about 10% to about 20% alpha helix and random coil regions, about 10% to about 30% alpha helix and random coil regions, about 15% to about 25% alpha helix and random coil regions, about 15% to about 30% alpha helix and random coil regions, or about 15% to about 35% alpha helix and random coil regions.

[0305] In another aspect, the present disclosure provides a method for modulating at least one property of a silk fibroin article or manufactured article described herein. Generally, the method comprises varying the weight ratio of the various silk fibroin fragments in the low molecular weight silk in the silk fibroin article or manufactured article. In some embodiments, the method comprises varying the weight ratio of a silk fibroin fragment having a molecular weight within a first specified range between about 3.5 kDa and about 120 kDa and a silk fibroin fragment having a molecular weight within a second specified range between about 3.5 kDa and about 120 kDa, wherein the first and second specified ranges do not overlap. In some embodiments, the method comprises varying the weight ratio of a silk fibroin fragment having a molecular weight greater than 200 kDa and a silk fibroin fragment having a molecular weight within a specified range between about 3.5 kDa and about 120 kDa.

[0306] Without being limited to these, properties that can be modulated using the methods described herein can be selected from the group consisting of the release rate of an agent present in an article, the release kinetics of an agent present in an article, redissolution, degradation, mechanical properties, optical properties, porosity, pore size, viscosity, biocompatibility, bioabsorbability, the effective charge of an article, particle size, and any combination of these.

[0307] In another aspect, the present disclosure provides a method of adjusting the size of silk particles. The method generally includes varying the weight ratio of various silk fibroin fragments of low molecular weight silk in a solution to form silk particles from the solution. In some embodiments, the method includes varying the weight ratio of a silk fibroin fragment having a molecular weight within a first specified range between about 3.5 kDa and about 120 kDa and a silk fibroin fragment having a molecular weight within a second specified range between about 3.5 kDa and about 120 kDa, wherein the first and second specified ranges do not overlap. In some embodiments, the method includes varying the weight ratio of a silk fibroin fragment having a molecular weight greater than 200 kDa and a silk fibroin fragment having a molecular weight within a specified range between about 3.5 kDa and about 120 kDa. Methods of preparing silk particles are described, for example, in International Application No. WO2011 / 041395, the entire contents of which are incorporated herein by reference; International Patent Application Publication No. WO2008 / 118133; U.S. Patent Application Publication No. US2010 / 0028451; U.S. Patent Provisional Application No. 61 / 719,146, filed October 26, 2012; and Wenk et al., J Control Release, 2008; 132:26 - 34.

[0308] In any of the embodiments described herein, the low-molecular-weight silk fibroin fragment can be derived from one or more portions of the full-length silk fibroin polypeptide such that the amino acid sequence of the low-molecular-weight silk fibroin collectively represents less than 100% of the full-length silk fibroin polypeptide. For example, one or more recombinant techniques can be used to produce a silk fibroin polypeptide, and some embodiments of any of the aspects described herein can be performed using such a silk fibroin polypeptide. Such a recombinant silk fibroin polypeptide can contain fragments (s) of the full-length counterpart. In some embodiments, the silk fibroin polypeptide corresponding to the fragment (s) of the full-length counterpart can be produced from a transgenic organism carrying the transgene, and such fragments occur.

[0309] In any of the embodiments described herein, the low-molecular-weight silk fibroin fragment can include one or more mutations and / or modifications relative to the native (e.g., wild-type) sequence of silk fibroin. Such mutations and / or modifications in the silk fibroin fragment can occur spontaneously or can be introduced by design. For example, in some embodiments, such mutations and / or modifications in the silk fibroin fragment can be introduced using recombinant techniques, chemical modifications, and the like.

[0310] Silk fibroin is an example of a polypeptide having one or more portions of an amino acid sequence that can adopt a beta-sheet secondary structure. For example, the silk fibroin structure generally includes an amino acid sequence, and one or more portions of the sequence are generally characterized by alternating glycine and alanine, or alanine alone. Without being bound by theory, such an arrangement allows the fibroin molecules to self-assemble into a beta-sheet higher-order structure. Thus, in yet another aspect, provided herein is a composition comprising a population of polypeptide fragments having one or more portions of an amino acid sequence characterized by alternating glycine and alanine, or alanine alone, and having a molecular weight in the range of about 3.5 kDa to about 120 kDa or about 5 kDa to about 125 kDa. In some embodiments, the composition is characterized in that no more than 15% of the total number of polypeptide fragments in the population have a molecular weight greater than 200 kDa, and at least 50% of the total number of polypeptide fragments in the population have a molecular weight within a specified range, the specified range being about 3.5 kDa to about 120 kDa, or about 5 kDa to about 125 kDa.

[0311] In another aspect, the silk fibroin in the compositions and / or methods described herein can be replaced with, or used in combination with, other non-silk polypeptides that include a beta-sheet structure or have a propensity to form such a structure based on their amino acid sequence. Thus, provided herein is also a polypeptide composition comprising a population of beta-sheet-forming polypeptide fragments. In some embodiments, the population of beta-sheet-forming polypeptide fragments described herein can have a range of molecular weights, and no more than 15% of the total number of beta-sheet-forming polypeptide fragments in the population have a molecular weight greater than 200 kDa, and at least 50% of the total number of beta-sheet-forming polypeptide fragments in the population have a molecular weight within a specified range, the specified range being about 3.5 kDa to about 120 kDa or about 5 kDa to about 125 kDa.

[0312] As used herein, the term "beta-sheet forming polypeptide" refers to a polypeptide having one or more portions of an amino acid sequence that adopts a beta-sheet secondary structure. In some embodiments, the beta-sheet forming polypeptide can be selected based on having a beta-sheet structure or a propensity to form such a structure based on the amino acid sequence.

[0313] In some embodiments, the beta-sheet forming polypeptide can have an amphiphilic nature (i.e., having both hydrophilic and hydrophobic portions). Amphiphilic polypeptides can be obtained from a single source (e.g., a native protein), such that the single polypeptide itself contains both hydrophobic modules or stretches and hydrophilic modules or stretches within the polypeptide so as to be naturally amphiphilic. In some embodiments, the hydrophobic module or stretch, and the hydrophilic module or stretch can be fused or coupled together to form an amphiphilic entity. Such "fusion" or "chimeric" polypeptides can be produced using recombinant techniques, chemical coupling, or both.

[0314] In some embodiments, the beta-sheet forming polypeptide can include one or more portions of the amino acid sequence of a polypeptide selected from the following list, namely, fibroin, actin, collagen, catenin, chitosan, claudin, coilin, elastin, elaunin, extensin, fibrillin, lamin, laminin, keratin, tubulin, viral structural proteins, zein proteins (seed storage proteins), and any combination thereof.

[0315] In some embodiments, the beta-sheet forming polypeptide can include a protein regenerated (e.g., purified) from a natural source, a recombinant protein produced in a heterologous system, a synthetic or chemically produced peptide, or a combination thereof.

[0316] In some embodiments, the beta sheet forming polypeptide can include one or more portions of the amino acid sequence of a polypeptide corresponding to any one of the lists provided above, with or without one or more sequence variations compared to the native or wild-type counterpart. For example, in some embodiments, such variants can exhibit at least 85% overall sequence identity, e.g., at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% overall sequence identity compared to the wild-type sequence.

[0317] Certain exemplary preparation methods Without being bound by theory, treatment with heat or high pressure prior to processing to the final desired shape can reduce or limit some of the problems associated with forming articles containing silk fibroin. This enables the ability to produce articles of consistent geometric shape without significant bubbling, shrinkage, and deformation. This results in reproducible and consistent samples for testing and implant procedures. Further, the articles can be machined, allowing for computer numerical control (CNC) milling of silk blanks into exact replicas of biological geometric shapes, e.g., into the desired constructs. Methods including treatment with heat or high pressure prior to processing of silk fibroin articles to the final desired shape are described, for example, in U.S. Patent Application No. 61 / 808,768, filed Apr. 5, 2013, U.S. Patent Application No. 61 / 719,146, filed Oct. 26, 2012, U.S. Patent Application No. 61 / 809,535, filed Apr. 8, 2013, and U.S. Patent Application No. 61 / 881,653, filed Sep. 24, 2013, the entire contents of each of which are hereby incorporated by reference in their entirety.

[0318] Accordingly, in one aspect, the present disclosure provides a method for preparing a silk fibroin article. Generally, the method includes incubating a silk fibroin composition (e.g., a low molecular weight silk fibroin composition) under high temperature and / or high pressure. The silk fibroin in the composition may be at least partially in an insoluble state. After incubation, the composition can be processed into a desired final shape. Without limitation, the silk fibroin composition for preparing a manufactured article may be in the form of a solution, slurry, suspension, colloid, mixture, dispersion, paste, or the like.

[0319] As used herein, the term "insoluble state," when used with respect to silk fibroin, refers to the formation or state of a substantially amorphous, predominantly beta-sheet secondary structure. The term "formed in an insoluble state" is not intended to reflect the polymerization of silk monomers into silk polymers. Rather, it is intended to reflect the conversion of soluble silk fibroin into a water-insoluble state. As used herein, silk fibroin is in an "insoluble state" if it can be pelletized by centrifugation or if it cannot be dissolved by immersion in water or rinsing with water at 37°C or less.

[0320] In some embodiments, the silk fibroin composition can include an organic solvent. In one embodiment, the organic solvent may be hexafluoroisopropanol (HFIP). In some other embodiments, the silk fibroin composition is non-containing or essentially non-containing an organic solvent, i.e., non-containing a solvent other than water. Without being bound by theory, the organic solvent in the silk fibroin composition achieves a more uniform drying of the composition and results in more uniform mechanical and structural properties in the final silk fibroin manufactured article. Further, using an organic solvent also achieves better mechanical and structural properties for processing into the final shape.

[0321] In some embodiments, the method comprises: (i) providing a silk fibroin composition, wherein the silk fibroin is at least partially in an insoluble state; (ii) incubating the composition under high temperature and / or high pressure; (iii) optionally repeating step (ii); and (iv) processing the composition into a desired shape. As used herein, the term "incubating" means subjecting the composition to high temperature and / or pressure.

[0322] In some embodiments, the silk fibroin composition is within a mold. As used herein, the term "mold" is intended to encompass any mold, container, or substrate capable of shaping, holding, or supporting the silk fibroin composition. Thus, in its simplest form, the mold can simply comprise a support surface. The mold can be of any desired shape and can be made from any suitable material, including polymers (such as polysulfone, polypropylene, polyethylene, etc.), metals (such as stainless steel, titanium, cobalt chrome, etc.), ceramics (such as alumina, zirconia, etc.), glass ceramics, and glass (such as borosilicate glass, etc.). In some embodiments, the mold can provide a simple geometric shape scaffold that can be processed into the final desired shape, i.e., a blank can be provided using the mold, which can be processed into the final shape.

[0323] Accordingly, in some embodiments, the method comprises: (i) providing a mold containing a silk fibroin composition, wherein the silk fibroin in the composition is at least partially in an insoluble state; (ii) incubating the composition while raising the temperature or under pressure; (iii) optionally repeating step (ii) one or more times; and (iv) processing the composition into a desired shape.

[0324] In some embodiments, the step of providing a mold comprising a silk fibroin composition comprises transferring a solution comprising silk fibroin to the mold and inducing a conformational change in the silk fibroin.

[0325] As used herein, the term "high temperature" means a temperature higher than room temperature. Generally, high temperature is a temperature above about 25°C. For example, high temperature can be about 30°C or higher, about 35°C or higher, about 40°C or higher, about 45°C or higher, about 50°C or higher, about 55°C or higher, about 60°C or higher, about 65°C or higher, about 70°C or higher, about 75°C or higher, about 80°C or higher, about 85°C or higher, about 90°C or higher, about 95°C or higher, about 100°C or higher, about 105°C or higher, about 110°C or higher, about 115°C or higher, about 120°C or higher, about 125°C or higher, about 130°C or higher, about 135°C or higher, about 140°C or higher, about 145°C or higher, or about 150°C or higher. In some embodiments, high temperature is at least about 121°C.

[0326] As used herein, the term "high pressure" means a pressure of about 0.05 bar, about 0.1 bar, about 0.15 bar, about 0.2 bar, about 0.25 bar, about 0.3 bar, about 0.35 bar, about 0.4 bar, about 0.45 bar, about 0.5 bar, about 0.55 bar, about 0.6 bar, about 0.65 bar, about 0.7 bar, about 0.75 bar or more. For example, high pressure may be about 1 bar, 1.25 bar, 1.5 bar, 1.75 bar, 2 bar, 2.25 bar, 2.5 bar, 2.75 bar, 3 bar, 3.25 bar, 3.5 bar, 3.75 bar, 4 bar, 4.25 bar, 4.5 bar, 4.75 bar, 5 bar, 5.25 bar, 5.5 bar, 5.75 bar, 6 bar, 7.25 bar, 7.5 bar, 7.75 bar, 8 bar, 8.25 bar, 8.5 bar, 8.75 bar, 9 bar, 9.25 bar, 9.5 bar, 9.75 bar, 10 bar, or more. In some embodiments, high pressure is about 1 bar or more. In some embodiments, the incubation is under vacuum.

[0327] In some embodiments, the incubation is in the presence of water vapor.

[0328] Without limitation, the incubation may be for any desired period. For example, the incubation may be for a period of about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 8 minutes, about 10 minutes or more. In some embodiments, the incubation may be for a period of about 10 minutes to about 5 hours, about 15 minutes to about 2.5 hours, about 20 minutes to about 2 hours, about 25 minutes to about 1.5 hours. In some embodiments, the incubation is for about 25 minutes.

[0329] Some properties of the manufactured article (e.g., strength, molecular weight, degradation profile, swellability, density, color, etc.) can be adjusted by repeating the incubation step. Thus, in some embodiments, the incubation step can be repeated 1 time, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times.

[0330] In some embodiments, the silk fibroin composition can optionally be dried before repeating the incubation step. Without limitation, the drying can be for any desired period. For example, the drying can be for a period of about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes or more. In some embodiments, the incubation can be for a period of about 5 minutes to about 5 hours, about 10 minutes to about 2.5 hours, about 15 minutes to about 2 hours, about 20 minutes to about 1.5 hours. In some embodiments, the drying is for about 15 minutes. Further, the drying can be at room temperature or at an elevated temperature. For example, such drying can be at a temperature of about 4°C to about 100°C, about 10°C to about 95°C, about 15°C to about 90°C, about 20°C to about 85°C, about 25°C to about 80°C, about 30°C to about 75°C, about 35°C to about 60°C, or about 45°C to about 65°C.

[0331] In some embodiments, the incubation includes an autoclave treatment. The "autoclave treatment" means a heat treatment at superatmospheric pressure in the presence or absence of water vapor (e.g., saturated vapor). For the purposes of th...

Claims

[Claim 1] A low molecular weight silk fibroin composition as described in the drawings.

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