WO2025231379A1 - Solid-state coacervate adhesives and methods of making and using the same - Google Patents

Solid-state coacervate adhesives and methods of making and using the same

Info

Publication number
WO2025231379A1
WO2025231379A1 PCT/US2025/027523 US2025027523W WO2025231379A1 WO 2025231379 A1 WO2025231379 A1 WO 2025231379A1 US 2025027523 W US2025027523 W US 2025027523W WO 2025231379 A1 WO2025231379 A1 WO 2025231379A1
Authority
WO
WIPO (PCT)
Prior art keywords
solid state
state composition
silk fibroin
coacervate
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/027523
Other languages
French (fr)
Inventor
Fiorenzo G. Omenetto
Marco LO PRESTI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tufts University
Original Assignee
Tufts University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tufts University filed Critical Tufts University
Publication of WO2025231379A1 publication Critical patent/WO2025231379A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09J—ADHESIVES; NON-MECHANICAL ASPECTS OF ADHESIVE PROCESSES IN GENERAL; ADHESIVE PROCESSES NOT PROVIDED FOR ELSEWHERE; USE OF MATERIALS AS ADHESIVES
    • C09J189/00—Adhesives based on proteins; Adhesives based on derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans

Definitions

  • One particularly challenging adhesive task is the fast labeling of marine animals.
  • shark skin is a very challenging surface for adhesion and current adhesives do not provide adequate adhesive performance for tagging purposes.
  • the techniques described herein relate to a solid state composition including a coacervate-forming pair of materials.
  • the coacervate-forming pair of materials include silk fibroin and a silk-complementary material that is capable of forming a coacervate with the silk fibroin.
  • the solid state composition includes a mixture of silk fibroin powder, which includes the silk fibroin, and complementary powder, which includes the silk-complementary material.
  • the silk fibroin and the silk-complementary material are present in the solid state composition in a weight ratio of between 1:10 and 100:1.
  • the techniques described herein relate to a solid state composition including a coacervate-forming pair of materials.
  • the coacervate-forming pair of materials consist of a first material and a complementary material that is capable of forming a coacervate with the first material.
  • the solid state composition includes or consists essentially of a mixture of a first powder, which includes the first material, and a second powder, which includes the complementary material. Hydrating and agitating the solid state composition forms the coacervate.
  • the techniques described herein relate to a solid state composition including: a solid-state coacervate formed by grinding a combination of a freeze-dried silk fibroin and tannic acid in a ratio by weight of silk fibroin protein to tannic acid of between 1 :10 and 100 : 1.
  • the techniques described herein relate to a method of making a solid state composition, the method including: a) grinding together a freeze-dried silk fibroin and tannic acid in a ratio by weight of silk fibroin protein to tannic acid of between 1 :10 and 100:1, thereby forming a solid-state coacervate.
  • Fig. 1 depicts a general preparation method for solid-state coacervates as underwater adhesives.
  • FIG. 2 depicts an overview of the coacervation process.
  • Fig. 3A depicts a schematic representation of the microscopic observation setup for the coacervate.
  • Fig. 3B depicts photographs (top) and micrographs (bottom) of the adhesive at room temperature (left) and after exposure to 60 °C for 15 minutes in a water bath (right).
  • Fig. 3C depicts photographs of the adhesive confined between two coverslips and heated on a hot plate, showing macroscopic water expulsion as a result of heating.
  • Fig. 3D depicts (Left) the experimental setup of a metal soldering probe applied to the adhesive under a microscope and (Right) video frames capturing water expulsion from the adhesive due to localized heating.
  • Fig. 3E depicts a proposed schematic illustrating the potential mechanism of underwater adhesion, emphasizing bulk water removal facilitated by the adhesive’s melting properties and hydration layer removal enabled by the coacervate’s internal water management.
  • Fig. 4A depicts stress-strain curves from tensile testing of the plain coacervate, where the slope indicates the Young’s modulus.
  • Fig. 4B depicts complex modulus of the same material measured through rheological analysis.
  • Fig. 4C depicts long-term tensile performance of the plain coacervate stored in seawater at 8 °C and 20 °C.
  • Fig. 4D depicts one-month tensile performance of a FeCh (10%) coacervate stored in seawater at 8 °C.
  • Fig. 4E depicts schematics (left) and underwater application (right) of a solid-state adhesive tablet.
  • Fig. 5A depicts the performance of SFDA-TA coacervate in dry and underwater conditions.
  • Fig. 5B depicts the performance of SF-TA coacervate in dry and underwater conditions.
  • Fig. 6B depicts the performance of SF-TA solid-state coacervate after 1 day in seawater.
  • Fig. 6C depicts the performance of SF-TA solid-state coacervate after 1 week in seawater.
  • Fig. 6D depicts the performance of SF-TA solid-state coacervate at 60 °C.
  • Fig. 6E summarizes the performance of SF-TA solid-state coacervates after application, after heating, after 1 day in seawater, and after 1 week in seawater.
  • Fig. 7A depicts maximum shear stress and toughness of the solid SF-TA coacervate with 5% CaCE
  • Fig. 7B depicts maximum shear stress and toughness of the solid SF-TA coacervate with 10% CaCE
  • Fig. 7C depicts maximum shear stress and toughness of the solid SF-TA coacervate with 20% CaCE
  • Fig. 7D summarizes maximum shear stress and toughness of the solid SF-TA coacervate with 5% CaCE 10% CaCE and 20% CaCE
  • Fig. 8A depicts performance of SF-TA with 5% dopamine solid-state coacervate after its application.
  • Fig. 8B depicts performance of SF-TA with 5% dopamine after 1 day immersed in artificial seawater.
  • Fig. 8C depicts performance of SF-TA with 5% dopamine after 1 week immersed in artificial sea water.
  • Fig. 9A is a schematic representation of silk fibroin highlighting its
  • Fig. 9B depicts the chemical structure of tannic acid.
  • Fig. 9C depicts iron-catechol complexes and their characteristic light absorption spectra depending on the type of complex.
  • Fig. 9D depicts the process of complex coacervation, where two solutions undergo phase separation to form a dense, immiscible, viscous adhesive fluid.
  • Fig. 9E depicts a photograph showing the adhesion of silk-based coacervates in an underwater environment.
  • Fig. 9F depicts photographs of solid-state silk-based coacervates obtained with different iron compounds and concentrations.
  • Fig. 9G depicts schematics for the production of water-triggered adhesive tablets.
  • Fig. 9H depicts melting behavior of a silk-based coacervate between 25 °C and 45 °C.
  • Fig. 91 depicts temperature-resolved performance of silk-based coacervates.
  • Fig. 9J depicts a plot of tensile stress versus displacement for silk-based coacervates containing FeCh, demonstrating the modulation of performance at specific temperatures.
  • Fig. 9K depicts photographs illustrating the transition of the coacervate from highly cohesive at 25 °C to highly adhesive at 45 °C.
  • Fig. 10A shows adhesion in dry and underwater environments.
  • Fig. 10B shows a photo of lap-shear testing.
  • Fig. 11 shows sea water lap-shear testing of iron (III) complexed coacervates.
  • Fig. 12A shows the mechanical tuning of underwater adhesion with iron (III) compounds.
  • Fig 12B shows the tensile strength of control, 5 pm FezOa, 50 nm Fe2O3, and FeCh adhesives.
  • Fig. 12C shows the flexibility of control, 5 pm Fe ⁇ Oa, 50 nm FeaOa, and FeCh adhesives.
  • Fig. 13A shows underwater adhesion as a function of temperature for SF-TA coacervates.
  • Fig. 13B depicts a photograph of probe tack testing.
  • Fig. 14 shows adhesion of iron (III) coacervate at a) 25 °C, b) 35 °C, and c) under tensile stress.
  • Fig. 15a shows an image of plain coacervate in solution at 37 °C.
  • Fig. 15b shows FeCh coacervate in solution at 37 °C.
  • Fig. 15c shows temperature-resolved tensile testing of the plain coacervate.
  • Fig. 15d shows temperature-resolved tensile testing of the FeCh coacervate.
  • Fig. 16b shows images of 10% FeCh adhesion at different temperatures.
  • Fig. 16c plots a comparison of control, 0.1% FeCh coacervate, and 10% FeCh coacervate adhesion testing at different temperatures.
  • Fig. 16d is a plot showing performance of the coacervates as a function of time.
  • Fig. 17a depicts adhesion of the SF-TA coacervate on various materials.
  • Fig. 17b quantifies adhesion of the SF-TA coacervate on various materials.
  • Fig. 18 shows a schematic to produce water-triggered adhesive tablets.
  • Fig. 19a shows the experimental setup and results of a constant buoyant stress experiment, with CAD design of the GPS holder.
  • Fig. 19b shows the experimental setup and results of a constant buoyant stress experiment, with silicone GPS holder prototype stuck to glass wall with adhesive under constant water flow of 5.3 m/s.
  • Fig. 19c shows the experimental setup and results of a constant buoyant stress experiment, with adhesives used in the experiment.
  • Fig. 19d is a plot showing detachment time under buoyant stress.
  • the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and/or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” are used as equivalents and may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.
  • composition as used herein, may be used to refer to a discrete physical entity that comprises one or more specified components.
  • a composition may be of any form - e.g., gas, gel, liquid, solid, etc.
  • composition may refer to a combination of two or more entities for use in a single embodiment or as part of the same article.
  • the combination of entities result in physical admixture, that is, combination as separate co-entities of each of the components of the composition is possible; however many practitioners in the field may find it advantageous to prepare a composition that is an admixture of two or more of the ingredients in a pharmaceutically acceptable carrier, diluent, or excipient, making it possible to administer the component ingredients of the combination at the same time.
  • the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
  • the term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
  • compositions that undergo some chemical transformation during their use can be described in various ways. For instance, dissolving NaCl in water can be described as water having an NaCl concentration or water having a concentration of Na + and CF ions.
  • components of chemical compositions can be described either as the form they take prior to any chemical transformation or the form they take following the chemical transformation.
  • the assumption should be that the component is being described in the context of the particular composition being described (i.e., if describing a finished product or an intermediary after a given chemical transformation, then the chemically transformed entity is being described, and if describing a starting product or intermediary prior to the chemical transformation, then the untransformed entity is being described.
  • the present disclosure relates to adhesive compositions.
  • adhesive compositions are applied in thin layers or films.
  • film refers to a layer of material, either solid or liquid, which has a thickness suitable for use in an adhesive application.
  • Adhesive materials have been integral to the earliest tools crafted by humans, and their significance persists in our market, technology, and society. Throughout history, humans have sought inspiration from the biological realm, observing how animals and plants have developed adhesive mechanisms for survival. Examples include mussel-inspired polymers, gecko feet, and Velcro. Despite significant technological advancements, there remains a notable gap in the field of underwater adhesives. Many marine organisms have evolved the remarkable ability to produce and secrete adhesives derived from water-soluble biological molecules, allowing them to firmly attach to even the hulls of moving ships. Among these organisms, a mechanism that has independently evolved in various species involves the generation of adhesives through complex coacervation.
  • Coacervates are dense, liquid droplets formed when certain colloidal solutions undergo phase separation. This occurs when a solution containing two or more components, such as polymers or macromolecules, initially dissolved or dispersed homogeneously, separate into distinct phases. This phase transition can also be induced with concentrated solutions of biopolymers, which precipitate in an aqueous environment, producing a highly viscous fluid with adhesive properties. This approach has recently gained traction in the scientific community for producing coacervate adhesives using various polymers.
  • the present disclosure identified two primary challenges in developing underwater adhesives: i) avoiding dilution of materials used and ii) accessing surfaces to establish adhesive interactions.
  • Coacervate-based materials were identified as an ideal candidate due to avoiding dilution by leveraging liquid-liquid phase separation to form a macromolecule-rich adhesive material. Additionally, coacervates typically exhibit remarkably low surface tension with water ( ⁇ 1 mJ/m 2 ), enabling them to displace water from surfaces effectively, thus establishing strong adhesive interactions.
  • a mixture of silk fibroin powder and tannic acid powder was selected to formulate the adhesive composition based on biomolecules.
  • An exemplary overview of the process is shown in Fig- 1-
  • the present disclosure provides a solid state composition.
  • the solid state composition includes a coacervate-forming pair of materials including silk fibroin and a silk-complementary material.
  • the silk-complementary material is capable of forming a coacervate with the silk fibroin.
  • the silk fibroin includes silk fibroin powder, and the complementary powder includes the complementary material.
  • the silk fibroin and the silk- complementary material are present in the solid state composition in a weight ratio of between 1 :10 and 100:1. In some cases, the silk fibroin and the silk-complementary material are present in the solid state composition in a weight ratio of between 1:10 and 1:1.
  • the silk fibroin and the silk-complementary material are present in the solid state composition in a weight ratio of between 1 : 1 and 20:1. In some cases, the silk fibroin and the silk-complementary material are present in the solid state composition in a weight ratio of between 20: 1 and 40: 1 . In some cases, the silk fibroin and the silk-complementary material are present in the solid state composition in a weight ratio of between 40:1 and 60: 1. In some cases, the silk fibroin and the silk-complementary material are present in the solid state composition in a weight ratio of between 60: 1 and 80: 1. In some cases, the silk fibroin and the silk-complementary material are present in the solid state composition in a weight ratio of between 80:1 and 100:1.
  • the coacervate may be formed by hydrating and agitating the solid state composition. Hydrating the solid state composition may include mixing with water or an aqueous solution.
  • the aqueous solution may be one of fresh water or sea water. The mixing may occur within a syringe.
  • the silk fibroin powder and the complementary powder may independently have an average particle diameter (D50) of between 1 pm and 100 pm. In some cases, the D50 may be between 1 pm and 10 pm. In some cases, the D50 may be between 10 pm and 25 pm. In some cases, the D50 may be between 25 pm and 50 pm. In some cases, the D50 may be between 50 and 100 pm.
  • the silk fibroin may be a dopamine-substituted silk fibroin.
  • the hydrating and mixing to form the coacervate may be completed in under 5 seconds. In some cases, the hydrating and mixing may be completed in under 4 seconds. In some cases, the hydrating and mixing may be completed in under 3 seconds. In some cases, the hydrating and mixing may be completed in under 2 seconds. In some cases, the hydrating and mixing may be completed in under 1 second.
  • the solid state composition may retain the capacity to form the coacervate after storage for a predetermined storage time and upon contact with an aqueous solvent after the predetermined storage time. The predetermined storage time may be 1 month or more or 1 year or more.
  • the silk fibroin may be freeze-dried silk fibroin.
  • the freeze-dried silk fibroin may be dopamine- substituted silk fibroin.
  • the dopamine substitution may include polydopamine substitution.
  • the present disclosure provides a coacervate-based adhesive composition.
  • the coacervate-based adhesive composition comprises a dense phase of a coacervate.
  • the dense phase of a coacervate is formed by mixing a dopamine-substituted silk fibroin solution and tannic acid.
  • the light phase of the coacervate-based adhesive can be optionally an aqueous solution that contains an unreacted dopamine-substituted silk fibroin and unreacted tannic acid.
  • the coacervate-based adhesive composition wherein the coacervate-based adhesive is optionally substantially free of the light phase.
  • the light phase of the coacervate-based adhesive is the aqueous phase. In certain cases, the coacervate-based adhesive is substantially free of the light phase.
  • This complexing occurs in a fashion understood by those having ordinary skill in the art. A non-limiting description of this complexing and crosslinking is provided in Example 1.
  • the dense phase of a coacervate can be formed by mixing a dopamine- substituted silk fibroin and tannic acid in a ratio by weight between 1:10 and 100:1.
  • the ratio by weight of dopamine-substituted silk fibroin to tannic acid can be at least 1 : 10.
  • the ratio may be at least 1 :5.
  • the ratio may be at least 1:4.
  • the ratio may be at least 1:3.
  • the ratio may be at least 1:2.
  • the ratio may be at least 2:3.
  • the ratio may be at least 3:4.
  • the ratio may be at least 4:5.
  • the ratio may be at least 1:1. In some cases, the ratio may be at least 5:1. In some cases, the ratio may be at least 10: 1. In some cases, the ratio may be at least 15:1. In some cases, the ratio may be at least 20: 1. In some cases, the ratio may be at least 25:1. In some cases, the ratio may be at least 30: 1. In some cases, the ratio may be at least 40: 1. In some cases, the ratio may be at least 50:1. In some cases, the ratio may be or at least 60: 1. In some cases, the ratio by weight of dopamine-substituted silk fibroin to tannic acid can be at most 100: 1. In some cases, the ratio may be at most 90:1. In some cases, the ratio may be at most 80:1.
  • the ratio may be at most 75:1. In some cases, the ratio may be at most 70: 1. In some cases, the ratio may be at most 65:1. In some cases, the ratio may be at most 60: 1. In some cases, the ratio may be at most 50:1. In some cases, the ratio may be at most 45:1. In some cases, the ratio may be at most 40:1. In some cases, the ratio may be at most 30: 1. In some cases, the ratio may be at most 25:1. In some cases, the ratio may be at most 22: 1. In some cases, the ratio may be at most 20: 1. In some cases, the ratio may be at most 15: 1. In some cases, the ratio may be at most 10:1. In some cases, the ratio may be at most 7:1.
  • the ratio may be at most 6:1. In some cases, the ratio may be at most 5:1. In some cases, the ratio may be at most 3 : 1. In some cases, the ratio may be at most 1 : 1. In some cases, the ratio may be at most 1:2.
  • the tannic acid can be present in the adhesive composition in a dry-solids- basis amount by weight relative to the dry-solid-basis amount by weight of the silk fibroin protein and the dopamine of between 0.001% and 1.0%. In some cases, the tannic acid can be present in an amount between 0.005% and 0.9%. In some cases, the tannic acid can be present in an amount between 0.01 % and 0.75%. In some cases, the tannic acid can be present in an amount between 0. 1 % and 0.5%. In some cases, the tannic acid can be present in an amount between 0.025% and 0.25%. In some cases, the tannic acid can be present in an amount between 0.05% and 0.1%.
  • the tannic acid can be present in an amount between 0.25% and 0.85%. In some cases, the tannic acid can be present in an amount between 0.002% and 0.05%. In some cases, the tannic acid can be present in an amount of at least 0.005 mg per 1 mg of dopamine-modified silk fibroin.
  • the coacervate-based adhesive composition contains components that are present in naturally occurring organisms. These components may be covalently or ionically or otherwise linked to one another. In other words, two natural components that present in naturally occurring organisms can be covalently bound to one another and still be defined as a component that is present in naturally occurring organisms. Dopamine-substituted silk fibroin is not known to be present in naturally occurring organisms, but it’s made of components that are present in naturally occurring organisms.
  • the present disclosure provides a method of making coacervate-based adhesive.
  • the method includes mixing a dopamine-substituted silk firoin solution and tannic acid in a ratio by weight for dopamine-substituted silk fibroin to tannic acid between 1: 10 and 100:1 or one of the aforementioned ratios identified above.
  • the mixing thereby forming a coacervate.
  • the coacervate can comprise a dense phase and a light phase.
  • the dense phase of a coacervate is formed by mixing a dopamine-substituted silk fibroin solution and tannic acid and the light phase of the coacervate-based adhesive can be optionally an aqueous solution that contains an unreacted dopamine-substituted silk fibroin and unreacted tannic acid.
  • the light phase of the coacervate-based adhesive is the aqueous phase.
  • the coacervate-based adhesive is substantially free of the light phase.
  • the method includes removing at least a portion of the light phase from the coacervate. In some cases, the method includes optionally isolating at least a portion of the dense phase from the light phase.
  • a method of making a solid state composition includes grinding together a freeze-dried silk fibroin and tannic acid in a ratio by weight of silk fibroin protein to tannic acid of between 1 :10 and 100:1 or one of the aforementioned ratios identified above, thereby forming a solid-state coacervate.
  • the method may also include contacting the solid state composition with an aqueous solvent to obtain the coacervate-based adhesive.
  • the ratio of silk fibroin protein to tannic acid may be substantially 1:1.
  • the solid state composition may be preheated prior to its application.
  • the method of making the coacervate-based adhesive includes a mass ratio of tannic acid to silk fibroin that is substantially 1 :1.
  • the coacervate-based adhesive or the method of forming or using the coacervate-based adhesive comprises a dry adhesive strength that is greater than a first comparison dry adhesive strength.
  • the first comparison dry adhesive strength is from a first comparison coacervate-based adhesive that replaces the dopamine-substituted silk fibroin solution with an unmodified silk fibroin solution and is otherwise identical to the coacervate-based adhesive.
  • the dry adhesive strength can be 5% greater.
  • the dry adhesive strength can be 10% greater than the first comparison dry adhesive strength.
  • the dry adhesive strength can be 25% greater than the first comparison dry adhesive strength.
  • the dry adhesive strength can be 50% greater than the first comparison dry adhesive strength. In some aspects, the dry adhesive strength can be 75% greater than the first comparison dry adhesive strength. In some aspects, the dry adhesive strength can be 100% greater than the first comparison dry adhesive strength. In some cases, the dry adhesive strength is at least 500 kPa. In some cases, the dry adhesive strength is at least 750 kPa. In some cases, the dry adhesive strength is at least 1 MPa. In some cases, the dry adhesive strength is at least 2 MPa. In some cases, the dry adhesive strength is at least 3 MPa. In some cases, the dry adhesive strength is at least 5 MPa.
  • the composition or method comprises the coacervate-based adhesive which has a wet adhesive strength that is greater than a first comparison wet adhesive strength.
  • the first comparison wet adhesive strength is from a first comparison coacervate-based adhesive that replaces the dopamine-substituted silk fibroin solution with an unmodified silk fibroin solution and is otherwise identical to the coacervate-based adhesive.
  • the wet adhesive strength can be 5% greater than the first comparison wet adhesive strength.
  • the wet adhesive strength can be 10% greater than the first comparison wet adhesive strength.
  • the wet adhesive strength can be 25% greater than the first comparison wet adhesive strength.
  • the wet adhesive strength can be 50% greater than the first comparison wet adhesive strength. In some cases, the wet adhesive strength can be 75% greater than the first comparison wet adhesive strength. In some cases, the wet adhesive strength can be 100% greater than the first comparison wet adhesive strength. In some cases, the wet adhesive strength can be at least 200 kPa. In some cases, the wet adhesive strength can be at least 500 kPa. In some cases, the wet adhesive strength can be at least 750 kPa. In some cases, the wet adhesive strength can be at least 1 MPa. In some cases, the wet adhesive strength can be at least 2 MPa.
  • the composition or method comprises the coacervate-based adhesive which has a dry adhesive strength that is greater than a second comparison dry adhesive strength.
  • the second comparison dry adhesive strength is from a first comparison coacervate-based adhesive that replaces the dopamine-substituted silk fibroin solution with a polyethylene glycol solution and is otherwise identical to the coacervate-based adhesive.
  • the dry adhesive strength can be at least 25% of the second comparison dry adhesive strength.
  • the dry adhesive strength can be at least 50% of the second comparison dry adhesive strength.
  • the dry adhesive strength can be at least 75% of the second comparison dry adhesive strength.
  • the dry adhesive strength can be at least 100% of the second comparison dry adhesive strength.
  • the dry adhesive strength can be at least 125% of the second comparison dry adhesive strength.
  • the composition or method comprises the coacervate-based adhesive which has a wet adhesive strength that is greater than a second comparison wet adhesive strength.
  • the second comparison wet adhesive strength is from a first comparison coacervate-based adhesive that replaces the dopamine-substituted silk fibroin solution with a polyethylene glycol solution and is otherwise identical to the coacervate-based adhesive.
  • the wet adhesive strength can be at least 25% of the second comparison wet adhesive strength.
  • the wet adhesive strength can be at least 50% of the second comparison wet adhesive strength.
  • the wet adhesive strength can be at least 75% of the second comparison wet adhesive strength.
  • the wet adhesive strength can be at least 100% of the second comparison wet adhesive strength.
  • the wet adhesive strength can be at least 125% of the second comparison wet adhesive strength.
  • the second comparison wet adhesive strength is from a first comparison solid state composition that has not been immersed in an aqueous environment for at least 18 hours and is otherwise identical to the solid state composition.
  • the wet adhesive strength may be at least 100 times greater than the second comparison wet adhesive strength.
  • the wet adhesive strength may be at least 75 times greater than the second comparison wet adhesive strength.
  • the wet adhesive strength may be at least 50 times greater than the second comparison wet adhesive strength.
  • the wet adhesive strength may be at least 20 times greater than the second comparison wet adhesive strength.
  • the wet adhesive strength may be at least 10 times greater than the second comparison wet adhesive strength.
  • the solid state composition includes dopamine and has a wet adhesive duration that may be longer than a second comparison wet adhesive duration.
  • the second comparison wet adhesive duration may be from a first comparison solid state composition that does not include dopamine and is otherwise identical to the solid state composition.
  • the wet adhesive duration may be at least 10 times longer than the second comparison wet adhesive strength.
  • the wet adhesive duration may be at least 7 times longer than the second comparison wet adhesive strength.
  • the wet adhesive duration may be at least 5 times longer than the second comparison wet adhesive strength.
  • the wet adhesive duration may be at least 3 times longer than the second comparison wet adhesive strength.
  • the wet adhesive duration may be at least 2 times longer than the second comparison wet adhesive strength.
  • these comparisons are believed to be applicable across a broad range of surfaces for adhering together, but they are particularly true for surfaces that are challenging for adhesion (e.g., adhering to shark skin).
  • the comparative values it is believed that the values hold for the vast majority of surfaces and the compositions disclosed herein provide superior performance over the comparison compositions.
  • the absolute adhesion strength values it is possible that the values may vary based on the surfaces or articles being adhered and a skilled artisan would recognize that the disclosed absolute values may be for a more limited set of surfaces.
  • the adhesive strength may vary depending on the specific aqueous conditions and a skilled artisan would recognize that the presence of certain ions may enhance the adhesive strength.
  • the adhesive strength can be much higher than those discussed elsewhere herein, including peel strengths of greater than 1 N/mm, greater than 2 N/mm, or higher, with as high as 5 N/mm expected to be achievable with certain surfaces.
  • compositions described herein provided other unexpected results.
  • two-component epoxy resins are the state-of-the-art for repairing cracks in marine environments (e.g., swimming pools and the like).
  • these products have a slow setting time and become rigid after curing.
  • they have been shown ineffective on biological tissues (by way of experiments on sharks and manatees), potentially due to the skin irritation.
  • the inventors have discovered a composition that does not require curing time to be adhesive.
  • inventive compositions described herein can remain flexible long after application, which makes them compatible with an animal’s movements while maintaining adhesion.
  • the solid-state format of the coacervate offers several advantages. It provides a significantly extended shelf life, with minimal storage requirements, mainly protection from water.
  • the coacervate can be conveniently stored in a device, such as a syringe, and is readily accessible for use without the need for specialized training, approaching commercial product standards.
  • the powder format both allows easy handling of the adhesive and permits the inclusion of other reactive additives that can enhance or adjust the adhesive properties of the coacervate.
  • the coacervate itself is dried and stored as a powder it crumbles, rendering it ineffective for dry applications.
  • the composition or method of making the coacervate-based adhesive comprises the polydopamine-substituted silk fibroin.
  • the polydopamine-substituted silk fibroin has a degree of polydopamine-substitution of between 5% and 50%. In some cases, the degree of polydopamine-substitution may be between 10% and 40%. In some cases, the degree of polydopamine-substitution may be between 25% and 35%. The degree of polydopamine-substitution is measured as a percentage by weight of polydopamine substituents related to the weight of the silk fibroin backbone.
  • the polydopamine-substituted silk fibroin is polydopamine- substituted at one or more target amino acids.
  • the one or more target amino acids are selected from the group consisting of cysteine, tyrosine, arginine, lysine, histidine, phenylalinine, proline, and combinations thereof.
  • the polydopamine-substituted silk fibroin is polydopamine- substituted at one or more tyrosines.
  • polydopamine-substituted silk fibroin is the substituted with poly dopamine having 15 polymeric units or less. In some cases, the polydopamine has 13 polymeric units or less.
  • the polydopamine has 12 polymeric units or less. In some cases, the polydopamine has 10 polymeric units or less. In some cases, the polydopamine has 9 polymeric units or less. In some cases, the polydopamine has 8 polymeric units or less.
  • the silk fibroin backbone of the polydopamine-substituted silk fibroin can have a weight average molecular weight of between 25 kDa and 150 kDa.
  • the silk fibroin backbone can have a weight average molecular weight of between 30 kDa and 125 kDa.
  • the silk fibroin backbone can have a weight average molecular weight of between 35 kDa and 100 kDa.
  • the silk fibroin backbone can have a weight average molecular weight of between 25 kDa and 75 kDa.
  • the silk fibroin backbone can have a weight average molecular weight of between 100 kDa and 150 kDa. In some cases, the silk fibroin backbone can have a weight average molecular weight of between 50 kDa and 125 kDa. In some cases, the silk fibroin backbone can have a weight average molecular weight of between 40 kDa and 90 kDa. In some cases, the silk fibroin backbone can have a weight average molecular weight of between 55 kDa and 105 kDa. In some cases, the silk fibroin backbone can have a weight average molecular weight of between 45 kDa and 65 kDa.
  • the silk fibroin backbone of the polydopamine-substituted silk fibroin can have a number average molecular weight of between 25 kDa and 150 kDa.
  • the silk fibroin backbone can have a number average molecular weight of between 30 kDa and 125 kDa.
  • the silk fibroin backbone can have a number average molecular weight of between 35 kDa and 100 kDa.
  • the silk fibroin backbone can have a number average molecular weight of between 25 kDa and 75 kDa.
  • the silk fibroin backbone can have a number average molecular weight of between 100 kDa and 150 kDa. In some cases, the silk fibroin backbone can have a number average molecular weight of between 50 kDa and 125 kDa. In some cases, the silk fibroin backbone can have a number average molecular weight of between 40 kDa and 90 kDa. In some cases, the silk fibroin backbone can have a number average molecular weight of between 55 kDa and 105 kDa. In some cases, the silk fibroin backbone can have a number average molecular weight of between 45 kDa and 65 kDa.
  • the composition or method of making the coacervate-based adhesive comprises the polydopamine-substituted silk fibroin which is made by mixing silk fibroin with soluble dopamine in an aqueous solution.
  • the polydopamine-substituted silk fibroin has a structure that is indistinguishable from a comparison structure that is made by mixing comparison silk fibroin with comparison soluble dopamine in a comparison aqueous solution.
  • the soluble dopamine and/or the comparison soluble dopamine is dopamine hydrochloride.
  • the composition or method of making the coacervate-based adhesive comprises mixing silk fibroin with soluble dopamine in an aqueous solution.
  • the mixing of silk fibroin with soluble dopamine in an aqueous solution uses a ratio by weight of silk fibroin to soluble polydopamine of between 1 :2 and 10: 1.
  • the ratio may be between 1:1 and 5:1.
  • the ratio may be between 1.25:1 and 3:1.
  • the ratio may be at least 1:2.
  • the ratio may be at least 1.5:1.
  • the ratio may be at least 1:1.
  • the ratio may be at least 1.1: 1.
  • the ratio may be at least 1.2: 1.
  • the ratio may be at least 1.25: 1. In some aspects, the ratio may be at least 1.3:1. In some aspects, the ratio may be at least 1.4: 1. In some aspects, the ratio may be at least 1.5:1. In some aspects, the ratio may be at least 1.75:1. In some aspects, the ratio may be at least 2:1. In some aspects, the ratio may be at least 3:1. In some aspects, the ratio may be or at least 4:1. In some aspects, the ratio may be at most 10: 1. In some aspects, the ratio may be at most 9: 1. In some aspects, the ratio may be at most 8:1. In some aspects, the ratio may be at most 7:1. In some aspects, the ratio may be at most 6:1. In some aspects, the ratio may be at most 5:1. In some aspects, the ratio may be at most 4:1. In some aspects, the ratio may be at most 3:1. In some aspects, the ratio may be at most 2: 1. In some aspects, the ratio may be or at most 1 :1.
  • the composition or method of making the coacervate-based adhesive comprises mixing comparison silk fibroin with comparison soluble polydopamine in a comparison aqueous solution.
  • the mixing of comparison silk fibroin with comparison soluble polydopamine uses a ratio by weight of comparison silk fibroin to comparison soluble polydopamine of between 1 :2 and 10:1.
  • the ratio may be between 1 :1 and 5:1.
  • the ratio may be between 1.25:1 and 3:1.
  • the ratio may be at least 1 :2.
  • the ratio may be at least 1.5:1.
  • the ratio may be at least 1:1.
  • the ratio may be at least 1.1:1.
  • the ratio may be at least 1.2: 1. In some aspects, the ratio may be at least 1.25: 1. In some aspects, the ratio may be at least 1.3:1. In some aspects, the ratio may be at least 1.4: 1. In some aspects, the ratio may be at least 1.5:1. In some aspects, the ratio may be a least 1.75:1. In some aspects, the ratio may be at least 2:1. In some aspects, the ratio may be at least 3 : 1. In some aspects, the ratio may be at least 4: 1. In some aspects, the ratio may be at most 10: 1. In some aspects, the ratio may be at most 9:1. In some aspects, the ratio may be at most 8:1. In some aspects, the ratio may he at most 7:1. In some aspects, the ratio may he at most 6:1. In some aspects, the ratio may be at most 5:1. In some aspects, the ratio may be at most 4:1. In some aspects, the ratio may be at most 3:1. In some aspects, the ratio may be at most 2:1. In some aspects, the ratio may be at most 1:1.
  • the solid state composition (e.g., powder) may be compressed into a tablet form, solid state tablet, or similar solid individual unit form using a mold and applying a pressure.
  • the pressure can be applied at between 0.001 MPa and 5 MPa. In some cases, the applied pressure is at least 0.001 MPa. In some cases, the applied pressure is at least 0.01 MPa. In some cases, the applied pressure is at least 0. 1 MPa. In some cases, the applied pressure is at or at least 1 MPa. In some cases, the applied pressure is at most 5 MPa. In some cases, the applied pressure is at most 2 MPa. In some cases, the applied pressure is at most 0.5 MPa. In some cases, the applied pressure is at most 0.05 MPa. In some cases, the applied pressure is at most 0.005 MPa.
  • additives may be added to the solid state composition (e.g., powder) at between 10% and 80% of tablet weight.
  • the additive can be present in the solid state composition in an amount by weight of at least 10%.
  • the additive can be present in the solid state composition in an amount by weight of at least 30%.
  • the additive can be present in the solid state composition in an amount by weight of at least 50%.
  • the additive can be present in the solid state composition in an amount by weight of at least 70%.
  • the additive can be present in the powder in an amount by weight of at most 80%.
  • the additive can be present in the solid state composition in an amount by weight of at most 60%.
  • the additive can be present in the solid state composition in an amount by weight of at most 40%. In some cases, the additive can be present in the solid state composition in an amount by weight of at most 20%.
  • the additives may accelerate the dissolution of the tablet into adhesive upon contact with fresh or salt water.
  • the additive can be or can include one or more gas-evolving molecules.
  • suitable gas-evolving molecules include, but are not limited to succinic acid, sodium carbonate, carbonic acid, bicarbonates, sulfites, bisulfites, ammonium chloride, nitrites, chlorates, peroxides, oxalic acid, or azides.
  • the additive can be or can include one or more salts that produce exothermic reactions with water.
  • suitable salts that produce exothermic reactions with water include, but are not limited to, magnesium chloride, calcium chloride, aluminum chloride, sodium hydroxide, potassium hydroxide.
  • the additive can be or can include one or more hydrogen bonding disrupters.
  • suitable hydrogen bonding disruptors include, but are not limited to, urea, 1 ,6-hexanediol, ethanol or guanidium chloride. Without wishing to be bound by any particular theory, it is believed that the hydrogen bonding disrupters would lower the glass transition temperature of the adhesive material, lowering the temperature of adhesion.
  • the present disclosure provides a method of using the coacervate-based adhesive.
  • the method comprises contacting a first article and a second article together with an adhesive amount of the coacervate-based adhesive, thereby adhering the first and second articles to one another.
  • the first article can be an animal, such as a marine animal, such as a fish or a marine mammal.
  • the second article can be a tag, such as a numbered tag, an electronic tag (e.g., radio transmitters), a water-permeable tag, or another sensor that can be usefully applied to a marine animal or other article.
  • the first and/or second article can be water permeable, thereby allowing water to interact with the adhesive via penetration of the second article.
  • the second article can include an electronic component.
  • a delivery device or a syringe can include the solid state composition of or made by the methods described herein.
  • the present disclosure provides a method of using the solid state composition including contacting the solid state composition with an aqueous solvent within a delivery device or syringe to obtain a coacervate-based adhesive and dispensing the coacervate-based adhesive from the delivery device or syringe onto at least one surface of a first or second article and contacting the first and second articles together with the coacervate-based adhesive on the at least one surface, thereby adhering the first and second articles to one another.
  • the contacting can be completed in under 5 seconds. In some cases, the contacting can be completed in under 4 seconds.
  • the solid state composition may further include an additive.
  • the additive mass may be between 10% and 80% of the mass of the solid state composition. In some cases, the additive mass may be at least 10% of the mass of the solid state composition. In some cases, the additive mass may be at least 30% of the mass of the solid state composition. In some cases, the additive mass may be at least 50% of the mass of the solid state composition. In some cases, the additive mass may be at least 70% of the mass of the solid state composition. In some cases, the additive mass may be at most 80% of the mass of the solid state composition.
  • the additive mass may be at most 60% of the mass of the solid state composition. In some cases, the additive mass may be at most 40% of the mass of the solid state composition. In some cases, the additive mass may be at most 20% of the mass of the solid state composition.
  • the additive may comprise a gas-evolving molecule or a salt that produces an exothermic reaction as described herein.
  • the adhesives described herein can be used to adhere tags to marine animals, such as sharks.
  • marine animals such as sharks.
  • the examples below show testing to establish adequate adhesion with the very challenging surface of shark skin. Without wishing to be bound by any particular theory, it is believed that achieving adequate adhesion to shark skin is a baseline adhesion performance that can be broadly applied to other marine species with reasonable predictability (i.e., if it works on shark skin, it likely works on the exterior surface of most other species).
  • compositions could be useful as drug delivery systems for antibiotics or medications. They could be used to treat wounds in at-risk species, allowing for controlled release of therapeutic agents embedded therein. As yet another example, the compositions could be useful in fish farms.
  • the method of using can provide an adhesive strength that withstands shear forces and does not require curing.
  • the method of using can provide instantaneous adhesion following underwater application. In some cases, the adhesive strength grows over time following application to a final adhesive strength.
  • the solid state composition provides adhesion between two articles when submerged in at least one of simulated sea water or distilled water.
  • the method of using the coacervate-based adhesive includes maintaining the adhesive at a low temperature (e.g., below 15 °C, below 10 °C, below 5 °C, etc.) to control the viscosity of the adhesive and retain structural integrity of the adhesive in a desired shape. This lowered temperature may reduce the surface tackiness of the adhesive making it easier to handle.
  • the method can include raising the temperature of the adhesive following the contacting.
  • silk fibroin powder and tannic acid powder may be combined to form an adhesive coacervate. The powders may be ground together or otherwise combined with reduced particle sizes relative to the starting material. The coacervate may absorb between 25% and 200% of its weight in water.
  • the coacervate may absorb at least 25% of its weight in water. In some cases, the coacervate may absorb at least 50% of its weight in water. In some cases, the coacervate may absorb at least 100% of its weight in water. In some cases, the coacervate may absorb at least 150% of its weight in water. In some cases, the coacervate may absorb at most 200% of its weight in water. In some cases, the coacervate may absorb at most 175% of its weight in water. In some cases, the coacervate may absorb at most 125% of its weight in water. In some cases, the coacervate may absorb at most 100% of its weight in water.
  • the coacervate may absorb at most 75% of its weight in water. In some cases, the coacervate may absorb or at most 50% of its weight in water.
  • the adhesive may have vesicles between 10 pm and 100 pm when placed underwater. The vesicles may contain water. The vesicles may be expelled upon heating and reabsorbed upon cooling. The mechanical performance of the adhesive may change with changing conditioning temperatures. The adhesive strength of the adhesive may increase over time underwater. [0131] It should be apparent to those skilled in the art that many additional modifications beside those already described are possible without departing from the inventive concepts. In interpreting this disclosure, all terms should be interpreted in the broadest possible manner consistent with the context.
  • silks there are many different silks, however, including spider silk (e.g., obtained from Nephila clavipes), transgenic silks, genetically engineered silks, such as silks from bacteria, yeast, mammalian cells, transgenic animals, or transgenic plants, and variants thereof, that can be used. See, e.g., WO 97/08315 and U.S. Pat. No. 5,245,012, each of which is incorporated herein by reference in their entireties.
  • spider silk e.g., obtained from Nephila clavipes
  • transgenic silks e.g., obtained from Nephila clavipes
  • genetically engineered silks such as silks from bacteria, yeast, mammalian cells, transgenic animals, or transgenic plants, and variants thereof, that can be used. See, e.g., WO 97/08315 and U.S. Pat. No. 5,245,012, each of which is incorporated herein by reference in their entireties.
  • a variety of functionalizing agents may be used with the silk-containing embodiments described herein (e.g., silk membrane, silk composition, silk articles, silk matrix, silk foam, silk microsphere, liquid composition, whipped silk cream, silk meringue, compressed silk meringue, hot-pressed silk meringue, silk leather, silk powder, silk toner, edible silkbased films, etc.). It should be understood that the examples herein may recite one or a few silkcontaining embodiments but are applicable to any silk-containing embodiment, as applicable.
  • a functionalizing agent may be any compound or molecule that facilitates the attachment to and/or development (e.g., growth) of one or more endothelial cells on a silk membrane.
  • a functionalizing agent may be or comprise a cell attachment mediator and/or an extracellular matrix protein, for example: collagen (e.g., collagen type I, collagen type III, collagen type IV or collagen type VI), elastin, fibronectin, vitronectin, laminin, fibrinogen, von Willebrand factor, proteoglycans, decorin, perlecan, nidogen, hyaluronan, and/or peptides containing known integrin binding domains e.g. “RGD” integrin binding sequence, or variations thereof, that are known to affect cellular attachment.
  • collagen e.g., collagen type I, collagen type III, collagen type IV or collagen type VI
  • elastin e.g., fibronectin, vitronectin, laminin, fibrinogen, von Willebrand factor, proteoglycans, decorin, perlecan, nidogen, hyaluronan
  • peptides containing known integrin binding domains e
  • a functionalizing agent may be any soluble molecule produced by endothelial cells.
  • Non-limiting examples include fibroblast growth factor- 1 (FGF1) and vascular endothelial growth factors (VEGF).
  • compositions may comprise the use of laminin, fibronectin and/or fibrinogen, and type IV collagen in order to facilitate the attachment and growth of endothelial cells on a silk membrane (e.g., a porous silk membrane) and/or attachment of megakaryocytes to a silk matrix.
  • a silk membrane e.g., a porous silk membrane
  • a functionalizing agent may be embedded or otherwise associated with a silk membrane and/or silk matrix such that at least a portion of the functionalizing agent is surrounded by a silk membrane and/or silk matrix as contrasted to a functionalizing agent simply being positioned along the surface of a silk membrane and/or silk matrix.
  • a functionalizing agent is distributed along and/or incorporated in substantially the entire surface area of a silk membrane/silk wall.
  • a functionalizing agent is distributed and/or incorporated only at one or more discrete portions of a silk membrane/wall and/or silk matrix.
  • a functionalizing agent is distributed in and/or along at least one of the lumenfacing side of a silk wall and the matrix-facing side of a silk wall.
  • any application-appropriate amount of one or more functionalizing agents may be used.
  • the amount of an individual functionalizing agent may be between about 1 pg/ml and 1,000 pg/ml (e.g., between about 2 and 1,000, 5 and 1,000, 10 and 1,000, 10 and 500, 10 and 100 pg/ml).
  • the amount of an individual functionalizing agent may be at least 1 pg/ml (e.g., at least 5, 10, 15, 20 25, 50, 100, 200, 300 400, 500, 600, 700, 800, or 900 pg/ml ).
  • the composition comprises one or more sensing agents, such as a sensing dye.
  • the sensing agents/sensing dyes are environmentally sensitive and produce a measurable response to one or more environmental factors.
  • the environmentally- sensitive agent or dye may be present in the composition in an effective amount to alter the composition from a first chemical-physical state to a second chemical-physical state in response to an environmental parameter (e.g., a change in pH, light intensity or exposure, temperature, pressure or strain, voltage, physiological parameter of a subject, and/or concentration of chemical species in the surrounding environment) or an externally applied stimulus (e.g., optical interrogation, acoustic interrogation, and/or applied heat).
  • an environmental parameter e.g., a change in pH, light intensity or exposure, temperature, pressure or strain, voltage, physiological parameter of a subject, and/or concentration of chemical species in the surrounding environment
  • an externally applied stimulus e.g., optical interrogation, acoustic interrogation, and/or applied heat.
  • Exemplary light responsive dyes or agents include, but are not limited to, photochromic compounds or agents, such as triarylmethanes, stilbenes, azastilbenes, nitrones, fulgides, spiropyrans, napthopyrans, spiro-oxazines, quinones, derivatives, and combinations thereof.
  • Exemplary potentiometric dyes include, but are not limited to, substituted amiononaphthylehenylpridinium (ANEP) dyes, such as di-4-ANEPPS, di-8-ANEPPS, and N-(4- Sulfobutyl)-4-(6-(4-(Dibutylamino)phenyl)hexatrienyl)Pyridinium (RH237).
  • ANEP substituted amiononaphthylehenylpridinium
  • Exemplary temperature sensitive dyes or agents include, but are not limited to, thermochromic compounds or agents, such as thermochromic liquid crystals, leuco dyes, fluoran dyes, octadecylphosphonic acid.
  • Exemplary pressure or strain sensitive dyes or agents include, but are not limited to, spiropyran compounds and agents.
  • chemi-sensitive dyes or agents include, but are not limited to, antibodies such as immunoglobulin G (IgG) which may change color from blue to red in response to bacterial contamination.
  • IgG immunoglobulin G
  • Exemplary flavorants include ester flavorants, amino acid flavorants, nucleic acid flavorants, organic acid flavorants, and inorganic acid flavorants, such as, but not limited to, diacetyl, acetyl propionyl, acetoin, isoamyl acetate, benzaldehyde, cinnamaldehyde, ethyl propionate, methyl anthranilate, limonene, ethyl decadienoate, allyl hexanoate, ethyl maltol, ethylvanillin, methyl salicylate, manzanate, glutamic acid salts, glycine salts, guanylic acids salts, inosinic acid salts, acetic acid, ascorbic acid, citric acid, fumaric acid, lactic acid, malic acid, phosphoric acid, tartaric acid, derivatives, and mixtures thereof.
  • diacetyl acetyl propion
  • the additive or dopant comprises an aroma compound.
  • aroma compounds include ester aroma compounds, terpene aroma compounds, cyclic terpenes, and aromatic aroma compounds, such as, but not limited to, geranyl acetate, methyl formate, methyl acetate, methyl propionate, methyl butyrate, ethyl acetate, ethyl butyrate, isoamyl acetate, pentyl butyrate, pentyl pentanoate, octyl acetate, benzyl acetate, methyl anthranilate, myrcene, geraniol, nerol, citral, citronellal, cironellol, linalool, nerolidol, limonene, camphor, menthol, carone, terpineol, alpha-ionone, thujone, eucalyptol, benzaldehy
  • the additive or dopant comprises a colorant, such as a dye or pigment.
  • the dye or pigment imparts a color or grayscale to the composition.
  • the colorant can be different than the sensing agents and/or sensing dyes below. Any organic and/or inorganic pigments and dyes can be included in the inks.
  • Exemplary pigments suitable for use in the present disclosure include International Color Index or C.I. Pigment Black Numbers 1 , 7, 1 1 and 31, C.I. Pigment Blue Numbers 15, 15:1, 15:2, 15:3, 15:4, 15:6, 16, 27, 29, 61 and 62, C.I. Pigment Green Numbers 7, 17, 18 and 36, C.I. Pigment Orange Numbers 5, 13, 16, 34 and 36, C.I.
  • the classes of dyes suitable for use in present invention can be selected from acid dyes, natural dyes, direct dyes (either cationic or anionic), basic dyes, and reactive dyes.
  • the acid dyes also regarded as anionic dyes, are soluble in water and mainly insoluble in organic solvents and are selected, from yellow acid dyes, orange acid dyes, red acid dyes, violet acid dyes, blue acid dyes, green acid dyes, and black acid dyes.
  • European Patent 0745651 incorporated herein by reference, describes a number of acid dyes that are suitable for use in the present disclosure.
  • Exemplary yellow acid dyes include Acid Yellow 1 International Color Index or C.I. 10316); Acid Yellow 7 (C.I. 56295); Acid Yellow 17 (C.I. 18965); Acid Yellow 23 (C.I. 19140); Acid Yellow 29 (C.I. 18900); Acid Yellow 36 (C.I. 13065); Acid Yellow 42 (C.I.
  • Exemplary orange acid dyes include Acid Orange 1 (C.I. 13090/1); Acid Orange 10 (C.I. 16230); Acid Orange 20 (C.I. 14603); Acid Orange 76 (C.I. 18870); Acid Orange 142; Food Orange 2 (C.I. 15980); and Orange B.
  • Exemplary red acid dyes include Acid Red 1 (C.I. 18050); Acid Red 4 (C.I. 14710); Acid Red 18 (C.I. 16255); Acid Red 26 (C.I. 16150); Acid Red 27 (C.I.
  • Acid Red 51 (C.I. 45430, available from BASF Corporation, Mt. Olive, N.J.); Acid Red 52 (C.I. 45100); Acid Red 73 (C.I. 27290); Acid Red 87 (C.I. 45380); Acid Red 94 (C.I. 45440) Acid Red 194; and Food Red 1 (C.I. 14700).
  • Exemplary violet acid dyes include Acid Violet 7 (C.I. 18055); and Acid Violet 49 (C.I. 42640).
  • Exemplary blue acid dyes include Acid Blue 1 (C.I. 42045); Acid Blue 9 (C.I. 42090); Acid Blue 22 (C.I. 42755); Acid Blue 74 (C.I. 73015); Acid Blue 93 (C.I.
  • Exemplary green acid dyes include Acid Green 1 (C.I. 10028); Acid Green 3 (C.I. 42085); Acid Green 5 (C.I. 42095); Acid Green 26 (C.I. 44025); and Food Green 3 (C.I. 42053).
  • Exemplary black acid dyes include Acid Black 1 (C.I. 20470); Acid Black 194 (Basantol® X80, available from BASF Corporation, an azo/1 :2 CR-complex.
  • Exemplary direct dyes for use in the present disclosure include Direct Blue 86 (C.I. 74180); Direct Blue 199; Direct Black 168; Direct Red 253; and Direct Yellow 107/132 (C.I. Not Assigned).
  • Exemplary natural dyes for use in the present disclosure include Alkanet (C.I.
  • Exemplary reactive dyes for use in the present disclosure include Reactive Yellow 37 (monoazo dye); Reactive Black 31 (diazo dye); Reactive Blue 77 (phthalo cyanine dye) and Reactive Red 180 and Reactive Red 108 dyes. Suitable also are the colorants described in The Printing Ink Manual (5th ed., Leach et al. eds. (2007), pages 289-299). Other organic and inorganic pigments and dyes and combinations thereof can be used to achieve the colors desired.
  • the amount of pigment/dye generally is present in an amount of from at or about 0.1 wt% to at or about 20 wt% based on the weight of the composition.
  • a non-white ink can include 15 wt% or less pigment/dye, or 10 wt% or less pigment/dye or 5 wt% pigment/dye, or 1 wt% pigment/dye based on the weight of the composition.
  • a non-white ink can include 1 wt% to 10 wt%, or 5 wt% to 15 wt%, or 10 wt% to 20 wt% pigment/dye based on the weight of the composition.
  • a non-white ink can contain an amount of dye/pigment that is 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15%, 16 wt%, 17 wt%, 18 wt%, 19 wt% or 20 wt% based on the weight of the composition.
  • the amount of white pigment generally is present in an amount of from at or about 1 wt% to at or about 60 wt% based on the weight of the composition. In some applications, greater than 60 wt% white pigment can be present.
  • Preferred white pigments include titanium dioxide (anatase and rutile), zinc oxide, lithopone (calcined coprecipitate of barium sulfate and zinc sulfide), zinc sulfide, blanc fixe and alumina hydrate and combinations thereof, although any of these can be combined with calcium carbonate.
  • a white ink can include 60 wt% or less white pigment, 55 wt% or less white pigment, 50 wt% white pigment, 45 wt% white pigment, 40 wt% white pigment, 35 wt% white pigment, 30 wt% white pigment, 25 wt% white pigment, 20 wt% white pigment, 15 wt% white pigment, or 10 wt% white pigment, based on the weight of the composition.
  • a white ink can include 5 wt% to 60 wt%, 5 wt% to 55 wt%, 10 wt% to 50 wt%, 10 wt% to 25 wt%, 25 wt% to 50 wt%, 5 wt% to 15 wt%, or 40 wt% to 60 wt% white pigment based on the weight of the composition.
  • a non-white ink can an amount of dye/pigment that is 5%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%, 24 wt%, 25%, 26 wt%, 27 wt%, 28 wt%, 29 wt%, 30 wt%, 31 wt%, 32 wt%, 33 wt%, 34 wt%, 35%, 36 wt%, 37 wt%, 38 wt%, 39 wt%, 40 wt%, 41 wt%, 42 wt%, 43 wt%, 44 wt%, 45%, 46 wt%
  • the additive or dopant comprises a conductive additive.
  • exemplary conductive additives include, but are not limited to graphite, graphite powder, carbon nanotubes, and metallic particles or nanoparticles, such as gold nanoparticles.
  • the conductive additive is biocompatible and non-toxic.
  • the additive is a biologically active agent.
  • biologically active agent refers to any molecule which exerts at least one biological effect in vivo.
  • the biologically active agent can be a therapeutic agent to treat or prevent a disease state or condition in a subject.
  • Biologically active agents include, without limitation, organic molecules, inorganic materials, proteins, peptides, nucleic acids (e.g., genes, gene fragments, gene regulatory sequences, and antisense molecules), nucleoproteins, polysaccharides, glycoproteins, and lipoproteins.
  • Classes of biologically active compounds that can be incorporated into the composition provided herein include, without limitation, anticancer agents, antibiotics, analgesics, antiinflammatory agents, immunosuppressants, enzyme inhibitors, antihistamines, anti-convulsants, hormones, muscle relaxants, antispasmodics, ophthalmic agents, prostaglandins, anti-depressants, anti-psychotic substances, trophic factors, osteoinductive proteins, growth factors, and vaccines.
  • active agent may also be used herein to refer to a biological sample (e.g., a sample of tissue or fluid, such as for instance blood) or a component thereof, and/or to a biologically active entity or compound, and/or to a structurally or functionally labile entity.
  • Exemplary active agents include, but are not limited to, therapeutic agents, diagnostic agents (e.g., contrast agents), and any combinations thereof.
  • the active agent present in a silk matrix e.g., a silk microsphere), composition, or the like can include a labile active agent, e.g., an agent that can undergo chemical, physical, or biological change, degradation and/or deactivation after exposure to a specified condition, e.g., high temperatures, high humidity, light exposure, and any combinations thereof.
  • a labile active agent e.g., an agent that can undergo chemical, physical, or biological change, degradation and/or deactivation after exposure to a specified condition, e.g., high temperatures, high humidity, light exposure, and any combinations thereof.
  • the active agent present in the silk matrix can include a temperature-sensitive active agent, e.g., an active agent that will lose at least about 30% or more, of its original activity or bioactivity, upon exposure to a temperature of at least about 10 °C. or above, including at least about 15 °C. or above, at least about room temperature or above, or at least about body temperature (e.g., about 37 °C.) or above.
  • a temperature-sensitive active agent e.g., an active agent that will lose at least about 30% or more, of its original activity or bioactivity, upon exposure to a temperature of at least about 10 °C. or above, including at least about 15 °C. or above, at least about room temperature or above, or at least about body temperature (e.g., about 37 °C.) or above.
  • the active agent can be added into the silk solution, which is then subjected to the methods described herein for preparing a silk matrix (e.g., a silk microsphere), composition, or the like.
  • the active agent can be coated on a surface of the silk matrix (e.g., a silk microsphere), composition, or the like.
  • the active agent can be loaded in a silk matrix (e.g., a silk microsphere), composition, or the like by incubating the silk microsphere in a solution of the active agent for a period of time, during which an amount of the active agent can diffuse into the silk matrix (e.g., a silk microsphere), composition, or the like, and thus distribute within the silk matrix (e.g., a silk microsphere), composition, or the like.
  • the additive is a therapeutic agent.
  • therapeutic agent means a molecule, group of molecules, complex or substance administered to an organism for diagnostic, therapeutic, preventative medical, or veterinary purposes.
  • the term “therapeutic agent” includes a “drug” or a “vaccine.” This term includes externally and internally administered topical, localized and systemic human and animal pharmaceuticals, treatments, remedies, nutraceuticals, cosmeceuticals, biologicals, devices, diagnostics and contraceptives, including preparations useful in clinical and veterinary screening, prevention, prophylaxis, healing, wellness, detection, imaging, diagnosis, therapy, surgery, monitoring, cosmetics, prosthetics, forensics and the like.
  • This term can also be used in reference to agriceutical, workplace, military, industrial and environmental therapeutics or remedies comprising selected molecules or selected nucleic acid sequences capable of recognizing cellular receptors, membrane receptors, hormone receptors, therapeutic receptors, microbes, viruses or selected targets comprising or capable of contacting plants, animals and/or humans.
  • nucleic acids and compounds comprising nucleic acids that produce a therapeutic effect for example deoxyribonucleic acid (DNA), ribonucleic acid (RNA), nucleic acid analogues (e.g., locked nucleic acid (LNA), peptide nucleic acid (PNA), xeno nucleic acid (XNA)), or mixtures or combinations thereof, including, for example, DNA nanoplexes, siRNA, microRNA, shRNA, aptamers, ribozymes, decoy nucleic acids, antisense nucleic acids, RNA activators, and the like.
  • any therapeutic agent can be included in the composition provided herein.
  • therapeutic agent also includes an agent that is capable of providing a local or systemic biological, physiological, or therapeutic effect in the biological system to which it is applied.
  • the therapeutic agent can act to control infection or inflammation, enhance cell growth and tissue regeneration, control tumor growth, act as an analgesic, promote anti-cell attachment, and enhance bone growth, among other functions.
  • suitable therapeutic agents can include anti-viral agents, hormones, antibodies, or therapeutic proteins.
  • Other therapeutic agents include prodrugs, which are agents that are not biologically active when administered but upon administration to a subject are converted to biologically active agents through metabolism or some other mechanism.
  • a silk-based drug delivery composition can contain one therapeutic agent or combinations of two or more therapeutic agents.
  • a therapeutic agent can include a wide variety of different compounds, including chemical compounds and mixtures of chemical compounds, e.g., small organic or inorganic molecules; saccharides; oligosaccharides; polysaccharides; biological macromolecules, e.g., peptides, proteins, and peptide analogs and derivatives; peptidomimetics; antibodies and antigen binding fragments thereof; nucleic acids; nucleic acid analogs and derivatives; an extract made from biological materials such as bacteria, plants, fungi, or animal cells; animal tissues; naturally occurring or synthetic compositions; and any combinations thereof.
  • the therapeutic agent is a small molecule.
  • bioactivity generally refers to the ability of an active agent to interact with a biological target and/or to produce an effect on a biological target.
  • bioactivity can include, without limitation, elicitation of a stimulatory, inhibitory, regulatory, toxic or lethal response in a biological target.
  • the biological target can be a molecule or a cell.
  • a bioactivity can refer to the ability of an active agent to modulate the effect/activity of an enzyme, block a receptor, stimulate a receptor, modulate the expression level of one or more genes, modulate cell proliferation, modulate cell division, modulate cell morphology, or any combination thereof.
  • a bioactivity can refer to the ability of a compound to produce a toxic effect in a cell.
  • exemplary cellular responses include, but are not limited to, lysis, apoptosis, growth inhibition, and growth promotion; production, secretion, and surface expression of a protein or other molecule of interest by the cell; membrane surface molecule activation including receptor activation; transmembrane ion transports; transcriptional regulations; changes in viability of the cell; changes in cell morphology; changes in presence or expression of an intracellular component of the cell; changes in gene expression or transcripts; changes in the activity of an enzyme produced within the cell; and changes in the presence or expression of a ligand and/or receptor (e.g., protein expression and/or binding activity).
  • a ligand and/or receptor e.g., protein expression and/or binding activity
  • Methods for assaying different cellular responses are well known to one of skill in the art, e.g., western blot for determining changes in presence or expression of an endogenous protein of the cell, or microscopy for monitoring the cell morphology in response to the active agent, or FISH and/or qPCR for the detection and quantification of changes in nucleic acids.
  • Bioactivity can be determined in some embodiments, for example, by assaying a cellular response.
  • bioactivity includes, but is not limited to, epitope or antigen binding affinity, the in vivo and/or in vitro stability of the antibody, the immunogenic properties of the antibody, e.g., when administered to a human subject, and/or the ability to neutralize or antagonize the bioactivity of a target molecule in vivo or in vitro.
  • the aforementioned properties or characteristics can be observed or measured using art-recognized techniques including, but not limited to, scintillation proximity assays, ELISA, ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence ELISA, competitive ELISA, SPR analysis including, but not limited to, SPR analysis using a BIAcore biosensor, in vitro and in vivo neutralization assays (see, for example, International Publication No. WO 2006/062685), receptor binding, and immunohistochemistry with tissue sections from different sources including human, primate, or any other source as needed.
  • the “bioactivity” includes immunogenicity, the definition of which is discussed in detail later.
  • the “bioactivity” includes infectivity, the definition of which is discussed in detail later.
  • the “bioactivity” refers to the ability of a contrast agent when administered to a subject to enhance the contrast of structures or fluids within the subject’s body.
  • the bioactivity of a contrast agent also includes, but is not limited to, its ability to interact with a biological environment and/or influence the response of another molecule under certain conditions.
  • small molecule can refer to compounds that are “natural productlike,” however, the term “small molecule” is not limited to “natural product-like” compounds. Rather, a small molecule is typically characterized in that it contains several carbon — carbon bonds and has a molecular weight of less than 5000 Daltons (5 kDa), preferably less than 3 kDa, still more preferably less than 2 kDa, and most preferably less than 1 kDa. In some cases, it is preferred that a small molecule has a molecular weight equal to or less than 700 Daltons.
  • Exemplary therapeutic agents include, but are not limited to, those found in Harrison’ s Principles of Internal Medicine, 13th Edition, Eds. T.R. Harrison et al. McGraw-Hill N.Y., NY; Physicians’ Desk Reference, 50th Edition, 1997, Oradell New Jersey, Medical Economics Co.; Pharmacological Basis of Therapeutics, 8th Edition, Goodman and Gilman, 1990; United States Pharmacopeia, The National Formulary, ETSP XII NF XVII, 1990, the complete contents of all of which are incorporated herein by reference.
  • Therapeutic agents include the herein disclosed categories and specific examples. It is not intended that the category be limited by the specific examples. Those of ordinary skill in the art will recognize also numerous other compounds that fall within the categories and that are useful according to the present disclosure. Examples include a radiosensitizer, a steroid, a xanthine, a beta- 2-agonist bronchodilator, an anti-inflammatory agent, an analgesic agent, a calcium antagonist, an angiotensin-converting enzyme inhibitors, a beta-blocker, a centrally active alpha- agonist, an alpha - 1 -antagonist, an anticholinergic/antispasmodic agent, a vasopressin analogue, an antiarrhythmic agent, an antiparkinsonian agent, an antiangina/antihypertensive agent, an anticoagulant agent, an antiplatelet agent, a sedative, an anxiolytic agent, a peptidic agent, a biopolymeric agent, an antineoplastic agent
  • the pharmaceutically active agent can be coumarin, albumin, steroids such as betamethasone, dexamethasone, methylprednisolone, prednisolone, prednisone, triamcinolone, budesonide, hydrocortisone, and pharmaceutically acceptable hydrocortisone derivatives; xanthines such as theophylline and doxophylline; beta-2- agonist bronchodilators such as salbutamol, fenterol, clenbuterol, bambuterol, and salmeterol; antiinflammatory agents, including antiasthmatic anti-inflammatory agents, antiarthritis antiinflammatory agents, and non-steroidal anti-inflammatory agents, examples of which include but are not limited to sulfides, mesalamine, budesonide, salazopyrin, diclofenac, pharmaceutically acceptable diclofenac salts, nimesulide, naproxen, acetaminophen, ibuprofen
  • Anti-cancer agents include alkylating agents, platinum agents, antimetabolites, topoisomerase inhibitors, antitumor antibiotics, antimitotic agents, aromatase inhibitors, thymidylate synthase inhibitors, DNA antagonists, farnesyltransferase inhibitors, pump inhibitors, histone acetyltransferase inhibitors, metalloproteinase inhibitors, ribonucleoside reductase inhibitors, TNF alpha agonists/antagonists, endothelin A receptor antagonists, retinoic acid receptor agonists, immunomodulators, hormonal and antihormonal agents, photodynamic agents, and tyrosine kinase inhibitors.
  • Antibiotics include aminoglycosides (e.g., gentamicin, tobramycin, netilmicin, streptomycin, amikacin, neomycin), bacitracin, carbapenems (e.g., imipenem/cilastatin), cephalosporins, colistin, methenamine, monobactams (e.g., aztreonam), penicillins (e.g., penicillin G, penicillin V, methicillin, nafcillin, oxacillin, cioxacillin, dicloxacillin, ampicillin, amoxicillin, carbenicillin, ticarcillin, piperacillin, mezlocillin, azlocillin), polymyxin B, quinolones, and vancomycin; and bacteriostatic agents such as chloramphenicol, clindamycin, macrolides (e.g., erythromycin, azithromycin, clar
  • Enzyme inhibitors are substances which inhibit an enzymatic reaction.
  • enzyme inhibitors include edrophonium chloride, N-methylphysostigmine, neostigmine bromide, physostigmine sulfate, tacrine, 1-hydroxymaleate, iodotubercidin, p-bromotetranisole, 10-(alpha- diethylaminopropionyl)-phenothiazine hydrochloride, calmidazolium chloride, hemicholinium-3,3,5- dinitrocatechol, diacylglycerol kinase inhibitor I, diacylglycerol kinase inhibitor II, 3- phenylpropargylamine, N°-monomethyl-L-arginine acetate, carbidopa, 3 -hydroxybenzylhydrazine, hydralazine, clorgyline, deprenyl, hydroxylamine, iproniazi
  • Antihistamines include pyrilamine, chlorpheniramine, and tetrahydrozoline, among others.
  • Anti-inflammatory agents include corticosteroids, nonsteroidal anti-inflammatory drugs (e.g., aspirin, phenylbutazone, indomethacin, sulindac, tolmetin, ibuprofen, piroxicam, and fenamates), acetaminophen, phenacetin, gold salts, chloroquine, D-Penicillamine, methotrexate colchicine, allopurinol, probenecid, and sulfinpyrazone.
  • nonsteroidal anti-inflammatory drugs e.g., aspirin, phenylbutazone, indomethacin, sulindac, tolmetin, ibuprofen, piroxicam, and fenamates
  • acetaminophen phenacetin
  • gold salts chloroquine
  • Muscle relaxants include mephenesin, methocarbamol, cyclobenzaprine hydrochloride, trihexyphenidyl hydrochloride, levodopa/carbidopa, and biperiden.
  • Anti-spasmodics include atropine, scopolamine, oxyphenonium, and papaverine.
  • Analgesics include aspirin, phenylbutazone, indomethacin, sulindac, tolmetin, ibuprofen, piroxicam, fenamates, acetaminophen, phenacetin, morphine sulfate, codeine sulfate, meperidine, nalorphine, opioids (e.g., codeine sulfate, fentanyl citrate, hydrocodone bitartrate, loperamide, morphine sulfate, noscapine, norcodeine, normorphine, thebaine, nor-binaltorphimine, buprenorphine, chlomaltrexamine, funaltrexamine, nalbuphine, nalorphine, naloxone, naloxonazine, naltrexone, and naltrindole), procaine, lidocaine, tetracaine and dibucaine.
  • Ophthalmic agents include sodium fluorescein, rose bengal, methacholine, adrenaline, cocaine, atropine, alphachymotrypsin, hyaluronidase, betaxolol, pilocarpine, timolol, timolol salts, and combinations thereof.
  • Prostaglandins are art recognized and are a class of naturally occurring chemically related long-chain hydroxy fatty acids that have a variety of biological effects.
  • Anti-depressants are substances capable of preventing or relieving depression.
  • anti-depressants examples include imipramine, amitriptyline, nortriptyline, protriptyline, desipramine, amoxapine, doxepin, maprotiline, tranylcypromine, phenelzine, and isocarboxazid.
  • Trophic factors are factors whose continued presence improves the viability or longevity of a cell trophic factors include, without limitation, platelet-derived growth factor (PDGP), neutrophilactivating protein, monocyte chemoattractant protein, macrophage- inflammatory protein, platelet factor, platelet basic protein, and melanoma growth stimulating activity; epidermal growth factor, transforming growth factor (alpha), fibroblast growth factor, platelet- derived endothelial cell growth factor, insulin-like growth factor, glial derived growth neurotrophic factor, ciliary neurotrophic factor, nerve growth factor, bone growth/cartilage-inducing factor (alpha and beta), bone morphogenetic proteins, interleukins (e.g., interleukin inhibitors or interleukin receptors, including interleukin 1 through interleukin 10), interferons (e.g., interferon alpha, beta and gamma), hematopoietic factors, including erythropoietin,
  • Hormones include estrogens (e.g., estradiol, estrone, estriol, diethylstilbestrol, quinestrol, chlorotrianisene, ethinyl estradiol, mestranol), anti-estrogens (e.g., clomiphene, tamoxifen), progestins (e.g., medroxyprogesterone, norethindrone, hydroxyprogesterone, norgestrel), antiprogestin (mifepristone), androgens (e.g., testosterone cypionate, fluoxymesterone, danazol, testolactone), anti- androgens (e.g., cyproterone acetate, flutamide), thyroid hormones (e.g., triiodothyronne, thyroxine, propylthiouracil, methimazole, and iodixode), and pituitary hormones (e
  • Hormones are commonly employed in hormone replacement therapy and/or for purposes of birth control. Steroid hormones, such as prednisone, are also used as immunosuppressants and anti-inflammatories.
  • the additive is an agent that stimulates tissue formation, and/or healing and regrowth of natural tissues, and any combinations thereof.
  • Agents that increase formation of new tissues and/or stimulates healing or regrowth of native tissue at the site of injection can include, but are not limited to, fibroblast growth factor (FGF), transforming growth factor- beta (TGF-beta, platelet-derived growth factor (PDGF), epidermal growth factors (EGFs), connective tissue activated peptides (CTAPs), osteogenic factors including bone morphogenic proteins, heparin, angiotensin II (A-II) and fragments thereof, insulin-like growth factors, tumor necrosis factors, interleukins, colony stimulating factors, erythropoietin, nerve growth factors, interferons, biologically active analogs, fragments, and derivatives of such growth factors, and any combinations thereof.
  • FGF fibroblast growth factor
  • TGF-beta transforming growth factor- beta
  • PDGF platelet-derived growth factor
  • EGFs epidermal growth factors
  • CTAPs connective tissue activated peptides
  • osteogenic factors including bone morph
  • the silk composition can further comprise at least one additional material for soft tissue augmentation, e.g., dermal filler materials, including, but not limited to, poly(methyl methacrylate) microspheres, hydroxyapatite, poly(L-lactic acid), collagen, elastin, and glycosaminoglycans, hyaluronic acid, commercial dermal filler products such as BOTOX® (from Allergan), DYSPORT®, COSMODERM®, EVOLENCE®, RADIESSE®,RESTYLANE®, JUVEDERM® (from Allergan), SCULPTRA®, PERLANE®, and CAPTIQEIE®, and any combinations thereof.
  • dermal filler materials including, but not limited to, poly(methyl methacrylate) microspheres, hydroxyapatite, poly(L-lactic acid), collagen, elastin, and glycosaminoglycans, hyaluronic acid, commercial dermal filler products such as BOTOX® (from
  • the additive is a wound healing agent.
  • a wound healing agent is a compound or composition that actively promotes wound healing process.
  • the active agents provided herein are immunogens.
  • the immunogen is a vaccine.
  • Most vaccines are sensitive to environmental conditions under which they are stored and/or transported. For example, freezing may increase reactogenicity (e.g., capability of causing an immunological reaction) and/or loss of potency for some vaccines (e.g., HepB, and DTaP/IPV/FQB), or cause hairline cracks in the container, leading to contamination. Further, some vaccines (e.g., BCG, Varicella, and MMR) are sensitive to heat.
  • compositions and methods provided herein also provide for stabilization of vaccines regardless of the cold chain and/or other environmental conditions.
  • the additive is a cell, e.g., a biological cell.
  • Cells useful for incorporation into the composition can come from any source, e.g., mammalian, insect, plant, etc.
  • the cell can be a human, rat or mouse cell.
  • cells to be used with the compositions provided herein can be any types of cells.
  • the cells should be viable when encapsulated within compositions.
  • cells that can be used with the composition include, but are not limited to, mammalian cells (e.g.
  • exemplary cells that can be used with the compositions include platelets, activated platelets, stem cells, totipotent cells, pluripotent cells, and/or embryonic stem cells.
  • exemplary cells that can be encapsulated within compositions include, but are not limited to, primary cells and/or cell lines from any tissue.
  • cardiomyocytes myocytes, hepatocytes, keratinocytes, melanocytes, neurons, astrocytes, embryonic stem cells, adult stem cells, hematopoietic stem cells, hematopoietic cells (e.g. monocytes, neutrophils, macrophages, etc.), ameloblasts, fibroblasts, chondrocytes, osteoblasts, osteoclasts, neurons, sperm cells, egg cells, liver cells, epithelial cells from lung, epithelial cells from gut, epithelial cells from intestine, liver, epithelial cells from skin, etc., and/or hybrids thereof, can be included in the silk/platelet compositions disclosed herein.
  • Cells listed herein represent an exemplary, not comprehensive, list of cells.
  • Cells can be obtained from donors (allogenic) or from recipients (autologous). Cells can be obtained, as a non-limiting example, by biopsy or other surgical means known to those skilled in the art.
  • the cell can be a genetically modified cell.
  • a cell can be genetically modified to express and secrete a desired compound, e.g. a bioactive agent, a growth factor, differentiation factor, cytokines, and the like.
  • a desired compound e.g. a bioactive agent, a growth factor, differentiation factor, cytokines, and the like.
  • Differentiated cells that have been reprogrammed into stem cells can also be used.
  • Example 1 SF and TA Powders
  • Freeze-dried silk fibroin powder and TA powder are ground in a 1 :1 mass ratio. Grinding using a mortar and pestle achieves material homogenization and reduces particle sizes, enhancing dissolution of the powder in water. A ball mill or cryomill may be more effective than manual grinding. This resulting powder can be easily transferred into a syringe, compressed using the plunger, and subsequently deployed in underwater environments. The syringe allows recovery of either fresh or seawater, which can then be mixed with the powder using the plunger. This process enables preparation and delivery of the adhesive within a matter of seconds. Based on weight analysis described herein, the coacervate absorbs approximately its own mass in water during mixing. Therefore, it is more accurate to describe the coacervate composition as 25% silk, 25% tannic acid, and 50% water. An overview of the coacervation process is shown in Fig. 2.
  • Fig. 4a presents the stress-strain curves for the plain coacervate (four measurements) after one hour of application at 20 °C in seawater. These curves were used to calculate the Young’s modulus (E) in the initial linear region (strain ⁇ 10%) of the curve, yielding 120.82 ⁇ 64.56 kPa.
  • Fig. 4b shows the complex modulus (G) in the plateau region (strain ⁇ 10%) for the same material from an amplitude sweep test at 20 °C, with a value of 39.95 ⁇ 37.2 kPa.
  • Figs. 4e-4g illustrate several possible application methods for this adhesive.
  • Fig. 4e demonstrates the potential use of a solid adhesive tablet. In this approach, the tablet is confined within a rigid support (such as a glass coverslip), and frictional heat is applied to locally melt the adhesive, anchoring it to the base.
  • Fig. 4f shows the application of a flexible tag onto a submerged substrate. The coacervate was spread onto a polyurethane tag, which was pre-attached to the exterior of a Ziplock bag using double-sided tape. Filling the bag with hot water (40 °C - 50 °C) keeps the adhesive in a softened, active state, allowing it to bond to a surface upon application of pressure.
  • hot water 40 °C - 50 °C
  • This method enables adhesion to curved or irregular surfaces (e.g., a wrist), and the Ziplock bag can be easily removed as the double-sided tape has low water resistance.
  • the adhesive provides strong immediate adhesion, securing the tag in place even under vigorous mechanical agitation.
  • Fig. 4g presents a device incorporating a USB heating pad embedded within an epoxy resin plate, designed for controlled adhesive applications.
  • the subsequent images show the bottom view of the plate positioned inside a glass container before activation of the USB heater and after cooling. Upon activation, the heater melts the adhesive, which then flows spontaneously onto the bottom of the glass tank. Once cooled, the adhesive solidifies, forming a secure bond without requiring external pressure.
  • a preliminary modification of SF with polydopamine (PDA) results in a longer-lasting adhesion of the coacervates as shown in Fig. 5.
  • the modified silk displayed a gradual reduction in adhesive performance over time, generally remaining below 100 kPa.
  • the DA-modified silk exhibited higher adhesion strength, around 300 kPa, and maintained more consistent performance over three weeks underwater.
  • the control solid coacervate obtained by grinding 350 mg of SF powder and 350 mg of TA powder, was activated by adding the same mass (700 pL) of seawater, mixed, and then directly spread on glass adherents over a 10 x 25 mm area. It was then tested in lap shear measurements.
  • the control coacervate displayed very low values of initial adhesion (6.4 + 2 kPa), which can be increased by pre-heating the coacervate to 60 °C prior to its application (23.4 ⁇ 10.5 kPa).
  • the increased adhesion value after heating is because the coacervate phase consists of a hydrogen-bonded network. Increasing the temperature breaks some of the electrostatic interactions, causing the coacervate to melt.
  • the coacervate reforms the electrostatic bonds, leading to higher cohesion and improved adhesive performance.
  • the shear stress increases to 269.4 ⁇ 149.1 kPa. This increase could also be attributed to the reactive nature of TA, which can undergo covalent cross-linking with free amino groups via a Michael addition reaction.
  • the solid format allows for the inclusion of solid-state reactive additives into the mixture.
  • These additives react with the silk-TA matrix only when the coacervate is activated or applied with water or seawater.
  • One of these additives is calcium chloride (Fig. 7), which acts as a crosslinker for silk fibroin. Adding calcium chloride to the powder in a range between 5% and 20% (w/w%) leads to a change in the adhesive's mechanical behavior. With low calcium content, there is low shear stress (the peak value) and high toughness (area below the curve), while increasing the calcium content reduces toughness and increases the maximum shear stress. This modulation allows for control over the adhesive’s properties, allowing adjustments for either more rigid or plastic behavior as needed.
  • Fig. 9a shows a schematic representation of silk fibroin with abundant P-sheet content.
  • Fig. 9b shows the structure of the polyphenol tannic acid. Introducing iron compounds (FeCh or Fe ⁇ Od to these powders enhances material cohesion through complexation with the tannic acid (TA) (Fig. 9c). Combination of silk fibroin and tannic acid solutions generates coacervates that display immediately underwater adhesion, forming a flexible adhesive (Fig. 9d). Given the high solubility of silk fibroin and tannic acid, the powders can be ground together to create a solid-state material that can also be compressed in a tablet format (Fig. 9g).
  • the silk-based coacervates are applied in a viscous solid form, which can be dispensed from a syringe or similar system.
  • the viscosity is contingent on the composition and temperature of the coacervates. A lower viscosity facilitates the coacervate to fill the irregularities of a rough surface while displacing water. Conversely, higher viscosity is a factor in preventing cohesive failure, ensuring optimal adhesive performance. From our current measurements, we observe that salinity does not impact adhesion performance, and the coacervate in powder form can be activated with either distilled or seawater, yielding consistent results. When heated before application, they attain strengths of up to 500 kPa at 20 °C and remain stable for up to one month.
  • silk and tannic acid can be transformed into a solid-state tablet that transitions into an underwater adhesive upon contact with water or seawater.
  • Silk-based coacervates exhibit melting upon healing and solidification upon cooling. Viscosity at a specific temperature can be regulated by adjusting the silk’s molecular weight or iron content.
  • Fig. 10 shows the coacervate adhesive strengths of up to 150 kPa initially and up to 500 kPa after a few hours underwater, ensuring prolonged adhesion on the order of months.
  • adhesion is between 300 kPa and 400 kPa and is also stable for months.
  • Fig. 11 Iron (III) compounds can be used as crosslinkers as they form complexes with tannic acid’s galloyl moieties. The formation of iron complexes is confirmed by the change of color of the coacervate. Overall, iron (III) compounds improve initial adhesion and durability.
  • iron (III) compounds allows adjustment of the mechanical properties of the adhesive as shown in Fig. 12.
  • the size of iron (III) additives modulates the mechanical properties of the adhesive in underwater environments by changing the behavior of the adhesive from liquid like pressure sensitive adhesive to elastic solids.
  • Iron (III) compounds can be used to finely tune the mechanical behavior of the adhesive by controlling particle size.
  • SF-TA coacervates are hydrogen bonded networks, their viscosity is affected by temperature (Fig. 13a).
  • a probe tack test is depicted in Fig. 13B. Below 10 °C, the SF-TA coacervates are rigid and do not display adhesive behavior, but the SF-TA coacervates melt and are sticky if heated to 15 °C to 30 °C. Above this temperature the SF-TA coacervates behave more like a liquid, reducing their adhesion performance. To extend the temperature range of successful adhesion, iron compounds were incorporated into the coacervates to influence their glass transition temperature, enabling them to exhibit adhesion at higher temperatures.
  • Fig. 14c shows the adhesion of iron oxide coacervate underwater under tensile stress.
  • Fig. 15a-d compares 10% FeCh coacervate to plain coacervate.
  • the plain coacervate melts at a lower temperature and overall exhibits lower adhesion at all temperatures.
  • the temperature dependence of adhesion can be tailored. At low temperature, the 10% FeCI-, coacervate is too rigid and cannot interact with the target surface which results in adhesive failure as shown in Fig. 16a, Fig. 16b, and Fig. 16c.
  • Fig. 16c shows highest adhesion for 0.1 % FeCh at 24 °C while 10% FeCh is highest at 44 °C.
  • Fig 16d shows performance of the adhesives immediately, 1 day after application, and 1 week after application in seawater.
  • Fig. 17 shows (Fig. 17a) and quantifies (Fig. 17b) adhesion on aluminum, glass, polycarbonate, wood, PLA, polystyrene, and Teflon.
  • Fig. 18 shows an overview of an adhesive tablet form of coacervates made of silk fibroin powder, iron (III) compound powder, and tannic acid powder. Briefly, silk fibroin powder, an iron (III) compound powder, and tannic acid powder are mixed and ground together to form a solid-state coacervate powder. The solid-state coacervate powder is then compressed into dry tablets, which activate upon contact with water or seawater to form an underwater viscous adhesive, proving the versatility of this material.
  • Fig. 19 summarizes the experimental setup and results of a constant buoyant stress study.
  • the GPS holder design shown in Fig. 19a, fits the SPOT-253 tag by Wildlife Computers®.
  • the versatility of the coacervate adhesive allows for free selection of the material to be used in the GPS adapter.
  • a silicone GPS holder prototype stuck to a glass wall with the coacervate adhesive under constant water flow of 5.3 m/s is shown in Fig. 19b.
  • a mold was created and cast in flexible material.
  • flow testing was conducted under a constant water stream.
  • the adapter material was tested. Temperature and longevity were characterized.
  • Fig. 19c shows various adhesives used in the experiment.
  • Fig. 19d shows the time to detach under buoyant stress of the adhesives tested.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Adhesives Or Adhesive Processes (AREA)

Abstract

A solid state composition comprising a coacervate-forming pair of materials consisting of silk fibroin and a silk-complementary material and methods of making and using the same are disclosed. A coacervate-based adhesive composition and methods of making and using the same are disclosed. The composition includes a coacervate formed by mixing a freeze-dried silk fibroin solution and tannic acid. The tannic acid is present in an amount sufficient to initiate complexing and/or cross-linking of the adhesive composition. The adhesive has many useful applications, but one particularly valuable application is expected to involve adhesive application directly to the skin or outer surface of marine animals, such as sharks.

Description

SOLID-STATE COACERVATE ADHESIVES AND METHODS OF MAKING AND USING THE SAME
CLAIM TO PRIORITY
[0001] This application is related to, claims priority to, and incorporates herein by reference for all purposes U.S. Provisional Pat. App. No 63/641,806 filed on May 2, 2024.
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
[0002] This invention was made with government support under GLUE program HR0011-24-3- 0363 awarded by DARPA. The government has certain rights in the invention.
BACKGROUND
[0003] In the field of silk-based adhesives, synthetic derivatives have long been favored due to the improved performance that can be achieved. More recently, however, biocompatible products are increasingly preferred. Some attempts at making biocompatible adhesives from natural products exist, but the performance of the biocompatible adhesives has not matched the performance of synthetic derivatives.
[0004] One particularly challenging adhesive task is the fast labeling of marine animals. In particular, shark skin is a very challenging surface for adhesion and current adhesives do not provide adequate adhesive performance for tagging purposes.
[0005] Thus, there remains a need for improved adhesives that are biocompatible with improved performance. A need exists for improved curable adhesives for both dry and underwater use. A need exists for adhesives that are suitable for fast tagging of marine animals.
SUMMARY
[0006] In some aspects, the techniques described herein relate to a solid state composition including a coacervate-forming pair of materials. The coacervate-forming pair of materials include silk fibroin and a silk-complementary material that is capable of forming a coacervate with the silk fibroin. The solid state composition includes a mixture of silk fibroin powder, which includes the silk fibroin, and complementary powder, which includes the silk-complementary material. The silk fibroin and the silk-complementary material are present in the solid state composition in a weight ratio of between 1:10 and 100:1.
[0007] In some aspects, the techniques described herein relate to a solid state composition including a coacervate-forming pair of materials. The coacervate-forming pair of materials consist of a first material and a complementary material that is capable of forming a coacervate with the first material. The solid state composition includes or consists essentially of a mixture of a first powder, which includes the first material, and a second powder, which includes the complementary material. Hydrating and agitating the solid state composition forms the coacervate.
[0008] In some aspects, the techniques described herein relate to a solid state composition including: a solid-state coacervate formed by grinding a combination of a freeze-dried silk fibroin and tannic acid in a ratio by weight of silk fibroin protein to tannic acid of between 1 :10 and 100 : 1. [0009] In some aspects, the techniques described herein relate to a method of making a solid state composition, the method including: a) grinding together a freeze-dried silk fibroin and tannic acid in a ratio by weight of silk fibroin protein to tannic acid of between 1 :10 and 100:1, thereby forming a solid-state coacervate.
[0010] These and other systems, methods, objects, features, and advantages of the present disclosure will be apparent to those skilled in the art from the following detailed description of the preferred embodiment and the drawings.
[0011] All documents mentioned herein are hereby incorporated in their entirety by reference. References to items in the singular should be understood to include items in the plural, and vice versa, unless explicitly stated otherwise or clear from the text. Grammatical conjunctions are intended to express any and all disjunctive and conjunctive combinations of conjoined clauses, sentences, words, and the like, unless otherwise stated or clear from the context.
BRIEF DESCRIPTION OF THE FIGURES
[0012] The disclosure and the following detailed description of certain embodiments thereof may be understood by reference to the following figures:
[0013] Fig. 1 depicts a general preparation method for solid-state coacervates as underwater adhesives.
[0014] Fig. 2 depicts an overview of the coacervation process.
[0015] Fig. 3A depicts a schematic representation of the microscopic observation setup for the coacervate.
[0016] Fig. 3B depicts photographs (top) and micrographs (bottom) of the adhesive at room temperature (left) and after exposure to 60 °C for 15 minutes in a water bath (right).
[0017] Fig. 3C depicts photographs of the adhesive confined between two coverslips and heated on a hot plate, showing macroscopic water expulsion as a result of heating.
[0018] Fig. 3D depicts (Left) the experimental setup of a metal soldering probe applied to the adhesive under a microscope and (Right) video frames capturing water expulsion from the adhesive due to localized heating. [0019] Fig. 3E depicts a proposed schematic illustrating the potential mechanism of underwater adhesion, emphasizing bulk water removal facilitated by the adhesive’s melting properties and hydration layer removal enabled by the coacervate’s internal water management.
[0020] Fig. 4A depicts stress-strain curves from tensile testing of the plain coacervate, where the slope indicates the Young’s modulus.
[0021] Fig. 4B depicts complex modulus of the same material measured through rheological analysis.
[0022] Fig. 4C depicts long-term tensile performance of the plain coacervate stored in seawater at 8 °C and 20 °C.
[0023] Fig. 4D depicts one-month tensile performance of a FeCh (10%) coacervate stored in seawater at 8 °C.
[0024] Fig. 4E depicts schematics (left) and underwater application (right) of a solid-state adhesive tablet.
[0025] Fig. 4F depicts schematics (left) and underwater application (right) of a flexible tag coated with the adhesive.
[0026] Fig. 4G depicts photographs of a "thermo anchor" before (left) and after (right) being loaded with the adhesive, followed by heating and cooling to ensure adhesion.
[0027] Fig. 5A depicts the performance of SFDA-TA coacervate in dry and underwater conditions.
[0028] Fig. 5B depicts the performance of SF-TA coacervate in dry and underwater conditions.
[0029] Fig. 6A depicts the performance of SF-TA solid-state coacervate after its application.
[0030] Fig. 6B depicts the performance of SF-TA solid-state coacervate after 1 day in seawater.
[0031] Fig. 6C depicts the performance of SF-TA solid-state coacervate after 1 week in seawater.
[0032] Fig. 6D depicts the performance of SF-TA solid-state coacervate at 60 °C.
[0033] Fig. 6E summarizes the performance of SF-TA solid-state coacervates after application, after heating, after 1 day in seawater, and after 1 week in seawater.
[0034] Fig. 7A depicts maximum shear stress and toughness of the solid SF-TA coacervate with 5% CaCE
[0035] Fig. 7B depicts maximum shear stress and toughness of the solid SF-TA coacervate with 10% CaCE
[0036] Fig. 7C depicts maximum shear stress and toughness of the solid SF-TA coacervate with 20% CaCE
[0037] Fig. 7D summarizes maximum shear stress and toughness of the solid SF-TA coacervate with 5% CaCE 10% CaCE and 20% CaCE [0038] Fig. 8A depicts performance of SF-TA with 5% dopamine solid-state coacervate after its application.
[0039] Fig. 8B depicts performance of SF-TA with 5% dopamine after 1 day immersed in artificial seawater.
[0040] Fig. 8C depicts performance of SF-TA with 5% dopamine after 1 week immersed in artificial sea water.
[0041] Fig. 9A is a schematic representation of silk fibroin highlighting its |3-sheet secondary structure.
[0042] Fig. 9B depicts the chemical structure of tannic acid.
[0043] Fig. 9C depicts iron-catechol complexes and their characteristic light absorption spectra depending on the type of complex.
[0044] Fig. 9D depicts the process of complex coacervation, where two solutions undergo phase separation to form a dense, immiscible, viscous adhesive fluid.
[0045] Fig. 9E depicts a photograph showing the adhesion of silk-based coacervates in an underwater environment.
[0046] Fig. 9F depicts photographs of solid-state silk-based coacervates obtained with different iron compounds and concentrations.
[0047] Fig. 9G depicts schematics for the production of water-triggered adhesive tablets.
[0048] Fig. 9H depicts melting behavior of a silk-based coacervate between 25 °C and 45 °C.
[0049] Fig. 91 depicts temperature-resolved performance of silk-based coacervates.
[0050] Fig. 9J depicts a plot of tensile stress versus displacement for silk-based coacervates containing FeCh, demonstrating the modulation of performance at specific temperatures.
[0051] Fig. 9K depicts photographs illustrating the transition of the coacervate from highly cohesive at 25 °C to highly adhesive at 45 °C.
[0052] Fig. 10A shows adhesion in dry and underwater environments.
[0053] Fig. 10B shows a photo of lap-shear testing.
[0054] Fig. 11 shows sea water lap-shear testing of iron (III) complexed coacervates.
[0055] Fig. 12A shows the mechanical tuning of underwater adhesion with iron (III) compounds.
[0056] Fig 12B shows the tensile strength of control, 5 pm FezOa, 50 nm Fe2O3, and FeCh adhesives.
[0057] Fig. 12C shows the flexibility of control, 5 pm Fe^Oa, 50 nm FeaOa, and FeCh adhesives.
[0058] Fig. 13A shows underwater adhesion as a function of temperature for SF-TA coacervates.
[0059] Fig. 13B depicts a photograph of probe tack testing. [0060] Fig. 14 shows adhesion of iron (III) coacervate at a) 25 °C, b) 35 °C, and c) under tensile stress.
[0061] Fig. 15a shows an image of plain coacervate in solution at 37 °C.
[0062] Fig. 15b shows FeCh coacervate in solution at 37 °C.
[0063] Fig. 15c shows temperature-resolved tensile testing of the plain coacervate.
[0064] Fig. 15d shows temperature-resolved tensile testing of the FeCh coacervate.
[0065] Fig. 16a is a plot of tensile testing with 10% FeCh.
[0066] Fig. 16b shows images of 10% FeCh adhesion at different temperatures.
[0067] Fig. 16c plots a comparison of control, 0.1% FeCh coacervate, and 10% FeCh coacervate adhesion testing at different temperatures.
[0068] Fig. 16d is a plot showing performance of the coacervates as a function of time.
[0069] Fig. 17a depicts adhesion of the SF-TA coacervate on various materials.
[0070] Fig. 17b quantifies adhesion of the SF-TA coacervate on various materials.
[0071] Fig. 18 shows a schematic to produce water-triggered adhesive tablets.
[0072] Fig. 19a shows the experimental setup and results of a constant buoyant stress experiment, with CAD design of the GPS holder.
[0073] Fig. 19b shows the experimental setup and results of a constant buoyant stress experiment, with silicone GPS holder prototype stuck to glass wall with adhesive under constant water flow of 5.3 m/s.
[0074] Fig. 19c shows the experimental setup and results of a constant buoyant stress experiment, with adhesives used in the experiment.
[0075] Fig. 19d is a plot showing detachment time under buoyant stress.
DETAILED DESCRIPTION
[0076] Before the present disclosure is described in further detail, it is to be understood that the disclosure is not limited to the particular embodiments described. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. The scope of the present disclosure will be limited only by the claims. As used herein, the singular forms "a", "an", and "the" include plural embodiments unless the context clearly dictates otherwise.
[0077] In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and/or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) the terms “about” and “approximately” are used as equivalents and may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.
[0078] Approximately: as used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 1 1%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
[0079] Composition: as used herein, may be used to refer to a discrete physical entity that comprises one or more specified components. In general, unless otherwise specified, a composition may be of any form - e.g., gas, gel, liquid, solid, etc. In some embodiments, “composition” may refer to a combination of two or more entities for use in a single embodiment or as part of the same article. It is not required in all embodiments that the combination of entities result in physical admixture, that is, combination as separate co-entities of each of the components of the composition is possible; however many practitioners in the field may find it advantageous to prepare a composition that is an admixture of two or more of the ingredients in a pharmaceutically acceptable carrier, diluent, or excipient, making it possible to administer the component ingredients of the combination at the same time.
[0080] Improve, increase, or reduce: as used herein or grammatical equivalents thereof, indicate values that are relative to a baseline measurement, such as a measurement in a similar composition made according to previously known methods.
[0081] Substantially: as used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
[0082] It should be appreciated that compositions that undergo some chemical transformation during their use can be described in various ways. For instance, dissolving NaCl in water can be described as water having an NaCl concentration or water having a concentration of Na+ and CF ions. In the present disclosure, components of chemical compositions can be described either as the form they take prior to any chemical transformation or the form they take following the chemical transformation. If there is any ambiguity to a person having ordinary skill in the art, the assumption should be that the component is being described in the context of the particular composition being described (i.e., if describing a finished product or an intermediary after a given chemical transformation, then the chemically transformed entity is being described, and if describing a starting product or intermediary prior to the chemical transformation, then the untransformed entity is being described.
[0083] The present disclosure relates to adhesive compositions. Typically, adhesive compositions are applied in thin layers or films. As used here, the term "film" refers to a layer of material, either solid or liquid, which has a thickness suitable for use in an adhesive application.
[0084] Adhesive materials have been integral to the earliest tools crafted by humans, and their significance persists in our market, technology, and society. Throughout history, humans have sought inspiration from the biological realm, observing how animals and plants have developed adhesive mechanisms for survival. Examples include mussel-inspired polymers, gecko feet, and Velcro. Despite significant technological advancements, there remains a notable gap in the field of underwater adhesives. Many marine organisms have evolved the remarkable ability to produce and secrete adhesives derived from water-soluble biological molecules, allowing them to firmly attach to even the hulls of moving ships. Among these organisms, a mechanism that has independently evolved in various species involves the generation of adhesives through complex coacervation. Coacervates are dense, liquid droplets formed when certain colloidal solutions undergo phase separation. This occurs when a solution containing two or more components, such as polymers or macromolecules, initially dissolved or dispersed homogeneously, separate into distinct phases. This phase transition can also be induced with concentrated solutions of biopolymers, which precipitate in an aqueous environment, producing a highly viscous fluid with adhesive properties. This approach has recently gained traction in the scientific community for producing coacervate adhesives using various polymers.
[0085] Using silk fibroin, these mechanisms can be replicated in a solid-state adhesive that is activated upon contact with water and can be applied immediately. Designing a successful waterbased adhesive for use in water poses significant issues, including, but not limited to, surface wetting, swelling of materials when placed in water, erosion, degradation, and hydrolysis. The present disclosure explores using water to activate adhesion even though water is a well-known obstacle to adhesion. Overcoming these issues requires a combination of making a surface act as if it were dry while underwater, designing a system whose adhesion is activated on command while surrounded by water, ensuring the material is wet and adhesive when the material is submerged, and controlling the material’s water content and phase transitions, among others. [0086] The present disclosure identified two primary challenges in developing underwater adhesives: i) avoiding dilution of materials used and ii) accessing surfaces to establish adhesive interactions. Coacervate-based materials were identified as an ideal candidate due to avoiding dilution by leveraging liquid-liquid phase separation to form a macromolecule-rich adhesive material. Additionally, coacervates typically exhibit remarkably low surface tension with water (~1 mJ/m2), enabling them to displace water from surfaces effectively, thus establishing strong adhesive interactions.
[0087] A mixture of silk fibroin powder and tannic acid powder was selected to formulate the adhesive composition based on biomolecules. An exemplary overview of the process is shown in Fig- 1-
[0088] In an aspect, the present disclosure provides a solid state composition.
[0089] In an aspect, the solid state composition includes a coacervate-forming pair of materials including silk fibroin and a silk-complementary material. The silk-complementary material is capable of forming a coacervate with the silk fibroin. The silk fibroin includes silk fibroin powder, and the complementary powder includes the complementary material. The silk fibroin and the silk- complementary material are present in the solid state composition in a weight ratio of between 1 :10 and 100:1. In some cases, the silk fibroin and the silk-complementary material are present in the solid state composition in a weight ratio of between 1:10 and 1:1. In some cases, the silk fibroin and the silk-complementary material are present in the solid state composition in a weight ratio of between 1 : 1 and 20:1. In some cases, the silk fibroin and the silk-complementary material are present in the solid state composition in a weight ratio of between 20: 1 and 40: 1 . In some cases, the silk fibroin and the silk-complementary material are present in the solid state composition in a weight ratio of between 40:1 and 60: 1. In some cases, the silk fibroin and the silk-complementary material are present in the solid state composition in a weight ratio of between 60: 1 and 80: 1. In some cases, the silk fibroin and the silk-complementary material are present in the solid state composition in a weight ratio of between 80:1 and 100:1.
[0090] The coacervate may be formed by hydrating and agitating the solid state composition. Hydrating the solid state composition may include mixing with water or an aqueous solution. The aqueous solution may be one of fresh water or sea water. The mixing may occur within a syringe. The silk fibroin powder and the complementary powder may independently have an average particle diameter (D50) of between 1 pm and 100 pm. In some cases, the D50 may be between 1 pm and 10 pm. In some cases, the D50 may be between 10 pm and 25 pm. In some cases, the D50 may be between 25 pm and 50 pm. In some cases, the D50 may be between 50 and 100 pm. The silk fibroin may be a dopamine-substituted silk fibroin. The hydrating and mixing to form the coacervate may be completed in under 5 seconds. In some cases, the hydrating and mixing may be completed in under 4 seconds. In some cases, the hydrating and mixing may be completed in under 3 seconds. In some cases, the hydrating and mixing may be completed in under 2 seconds. In some cases, the hydrating and mixing may be completed in under 1 second. The solid state composition may retain the capacity to form the coacervate after storage for a predetermined storage time and upon contact with an aqueous solvent after the predetermined storage time. The predetermined storage time may be 1 month or more or 1 year or more.
[0091] The solid state composition may include an additive. The additive may be at least one of calcium chloride, a metal ion, a nanoparticle, iron oxide, or iron chloride.
[0092] The silk fibroin may be freeze-dried silk fibroin. The freeze-dried silk fibroin may be dopamine- substituted silk fibroin. The dopamine substitution may include polydopamine substitution.
[0093] In an aspect, the present disclosure provides a coacervate-based adhesive composition. The coacervate-based adhesive composition comprises a dense phase of a coacervate. The dense phase of a coacervate is formed by mixing a dopamine-substituted silk fibroin solution and tannic acid. The light phase of the coacervate-based adhesive can be optionally an aqueous solution that contains an unreacted dopamine-substituted silk fibroin and unreacted tannic acid. The coacervate-based adhesive composition wherein the coacervate-based adhesive is optionally substantially free of the light phase. In certain cases, the light phase of the coacervate-based adhesive is the aqueous phase. In certain cases, the coacervate-based adhesive is substantially free of the light phase. This complexing occurs in a fashion understood by those having ordinary skill in the art. A non-limiting description of this complexing and crosslinking is provided in Example 1.
[0094] In some aspects, the dense phase of a coacervate can be formed by mixing a dopamine- substituted silk fibroin and tannic acid in a ratio by weight between 1:10 and 100:1. In some cases, the ratio by weight of dopamine-substituted silk fibroin to tannic acid can be at least 1 : 10. In some cases, the ratio may be at least 1 :5. In some cases, the ratio may be at least 1:4. In some cases, the ratio may be at least 1:3. In some cases, the ratio may be at least 1:2. In some cases, the ratio may be at least 2:3. In some cases, the ratio may be at least 3:4. In some cases, the ratio may be at least 4:5. In some cases, the ratio may be at least 1:1. In some cases, the ratio may be at least 5:1. In some cases, the ratio may be at least 10: 1. In some cases, the ratio may be at least 15:1. In some cases, the ratio may be at least 20: 1. In some cases, the ratio may be at least 25:1. In some cases, the ratio may be at least 30: 1. In some cases, the ratio may be at least 40: 1. In some cases, the ratio may be at least 50:1. In some cases, the ratio may be or at least 60: 1. In some cases, the ratio by weight of dopamine-substituted silk fibroin to tannic acid can be at most 100: 1. In some cases, the ratio may be at most 90:1. In some cases, the ratio may be at most 80:1. In some cases, the ratio may be at most 75:1. In some cases, the ratio may be at most 70: 1. In some cases, the ratio may be at most 65:1. In some cases, the ratio may be at most 60: 1. In some cases, the ratio may be at most 50:1. In some cases, the ratio may be at most 45:1. In some cases, the ratio may be at most 40:1. In some cases, the ratio may be at most 30: 1. In some cases, the ratio may be at most 25:1. In some cases, the ratio may be at most 22: 1. In some cases, the ratio may be at most 20: 1. In some cases, the ratio may be at most 15: 1. In some cases, the ratio may be at most 10:1. In some cases, the ratio may be at most 7:1. In some cases, the ratio may be at most 6:1. In some cases, the ratio may be at most 5:1. In some cases, the ratio may be at most 3 : 1. In some cases, the ratio may be at most 1 : 1. In some cases, the ratio may be at most 1:2.
[0095] In some aspects, the tannic acid can be present in the adhesive composition in a dry-solids- basis amount by weight relative to the dry-solid-basis amount by weight of the silk fibroin protein and the dopamine of between 0.001% and 1.0%. In some cases, the tannic acid can be present in an amount between 0.005% and 0.9%. In some cases, the tannic acid can be present in an amount between 0.01 % and 0.75%. In some cases, the tannic acid can be present in an amount between 0. 1 % and 0.5%. In some cases, the tannic acid can be present in an amount between 0.025% and 0.25%. In some cases, the tannic acid can be present in an amount between 0.05% and 0.1%. In some cases, the tannic acid can be present in an amount between 0.25% and 0.85%. In some cases, the tannic acid can be present in an amount between 0.002% and 0.05%. In some cases, the tannic acid can be present in an amount of at least 0.005 mg per 1 mg of dopamine-modified silk fibroin.
[0096] In some aspects, the coacervate-based adhesive composition contains components that are present in naturally occurring organisms. These components may be covalently or ionically or otherwise linked to one another. In other words, two natural components that present in naturally occurring organisms can be covalently bound to one another and still be defined as a component that is present in naturally occurring organisms. Dopamine-substituted silk fibroin is not known to be present in naturally occurring organisms, but it’s made of components that are present in naturally occurring organisms.
[0097] The present disclosure provides a method of making coacervate-based adhesive. The method includes mixing a dopamine-substituted silk firoin solution and tannic acid in a ratio by weight for dopamine-substituted silk fibroin to tannic acid between 1: 10 and 100:1 or one of the aforementioned ratios identified above. The mixing thereby forming a coacervate. The coacervate can comprise a dense phase and a light phase.
[0098] In some aspects, the dense phase of a coacervate is formed by mixing a dopamine-substituted silk fibroin solution and tannic acid and the light phase of the coacervate-based adhesive can be optionally an aqueous solution that contains an unreacted dopamine-substituted silk fibroin and unreacted tannic acid. The coacervate-based adhesive composition wherein the coacervate-based adhesive is optionally substantially free of the light phase. In certain cases, the light phase of the coacervate-based adhesive is the aqueous phase. In certain cases, the coacervate-based adhesive is substantially free of the light phase. In certain cases, the method includes removing at least a portion of the light phase from the coacervate. In some cases, the method includes optionally isolating at least a portion of the dense phase from the light phase.
[0099] In some aspects, a method of making a solid state composition includes grinding together a freeze-dried silk fibroin and tannic acid in a ratio by weight of silk fibroin protein to tannic acid of between 1 :10 and 100:1 or one of the aforementioned ratios identified above, thereby forming a solid-state coacervate. The method may also include contacting the solid state composition with an aqueous solvent to obtain the coacervate-based adhesive. The ratio of silk fibroin protein to tannic acid may be substantially 1:1.
[0100] In some aspects, the solid state composition may be preheated prior to its application.
[0101] In some aspects, the method of making the coacervate-based adhesive includes a mass ratio of tannic acid to silk fibroin that is substantially 1 :1.
[0102] In some aspects, the coacervate-based adhesive or the method of forming or using the coacervate-based adhesive comprises a dry adhesive strength that is greater than a first comparison dry adhesive strength. In some cases, the first comparison dry adhesive strength is from a first comparison coacervate-based adhesive that replaces the dopamine-substituted silk fibroin solution with an unmodified silk fibroin solution and is otherwise identical to the coacervate-based adhesive. In some aspects, the dry adhesive strength can be 5% greater. In some aspects, the dry adhesive strength can be 10% greater than the first comparison dry adhesive strength. In some aspects, the dry adhesive strength can be 25% greater than the first comparison dry adhesive strength. In some aspects, the dry adhesive strength can be 50% greater than the first comparison dry adhesive strength. In some aspects, the dry adhesive strength can be 75% greater than the first comparison dry adhesive strength. In some aspects, the dry adhesive strength can be 100% greater than the first comparison dry adhesive strength. In some cases, the dry adhesive strength is at least 500 kPa. In some cases, the dry adhesive strength is at least 750 kPa. In some cases, the dry adhesive strength is at least 1 MPa. In some cases, the dry adhesive strength is at least 2 MPa. In some cases, the dry adhesive strength is at least 3 MPa. In some cases, the dry adhesive strength is at least 5 MPa.
[0103] In some aspects, the composition or method comprises the coacervate-based adhesive which has a wet adhesive strength that is greater than a first comparison wet adhesive strength. In some aspects, the first comparison wet adhesive strength is from a first comparison coacervate-based adhesive that replaces the dopamine-substituted silk fibroin solution with an unmodified silk fibroin solution and is otherwise identical to the coacervate-based adhesive. In some cases, the wet adhesive strength can be 5% greater than the first comparison wet adhesive strength. In some cases, the wet adhesive strength can be 10% greater than the first comparison wet adhesive strength. In some cases, the wet adhesive strength can be 25% greater than the first comparison wet adhesive strength. In some cases, the wet adhesive strength can be 50% greater than the first comparison wet adhesive strength. In some cases, the wet adhesive strength can be 75% greater than the first comparison wet adhesive strength. In some cases, the wet adhesive strength can be 100% greater than the first comparison wet adhesive strength. In some cases, the wet adhesive strength can be at least 200 kPa. In some cases, the wet adhesive strength can be at least 500 kPa. In some cases, the wet adhesive strength can be at least 750 kPa. In some cases, the wet adhesive strength can be at least 1 MPa. In some cases, the wet adhesive strength can be at least 2 MPa.
[0104] In some aspects, the composition or method comprises the coacervate-based adhesive which has a dry adhesive strength that is greater than a second comparison dry adhesive strength. In some aspects, the second comparison dry adhesive strength is from a first comparison coacervate-based adhesive that replaces the dopamine-substituted silk fibroin solution with a polyethylene glycol solution and is otherwise identical to the coacervate-based adhesive. In some cases, the dry adhesive strength can be at least 25% of the second comparison dry adhesive strength. In some cases, the dry adhesive strength can be at least 50% of the second comparison dry adhesive strength. In some cases, the dry adhesive strength can be at least 75% of the second comparison dry adhesive strength. In some cases, the dry adhesive strength can be at least 100% of the second comparison dry adhesive strength. In some cases, the dry adhesive strength can be at least 125% of the second comparison dry adhesive strength.
[0105] In some aspects, the composition or method comprises the coacervate-based adhesive which has a wet adhesive strength that is greater than a second comparison wet adhesive strength. In some aspects, the second comparison wet adhesive strength is from a first comparison coacervate-based adhesive that replaces the dopamine-substituted silk fibroin solution with a polyethylene glycol solution and is otherwise identical to the coacervate-based adhesive. The wet adhesive strength can be at least 25% of the second comparison wet adhesive strength. The wet adhesive strength can be at least 50% of the second comparison wet adhesive strength. The wet adhesive strength can be at least 75% of the second comparison wet adhesive strength. The wet adhesive strength can be at least 100% of the second comparison wet adhesive strength. The wet adhesive strength can be at least 125% of the second comparison wet adhesive strength. In some aspects, the second comparison wet adhesive strength is from a first comparison solid state composition that has not been immersed in an aqueous environment for at least 18 hours and is otherwise identical to the solid state composition. The wet adhesive strength may be at least 100 times greater than the second comparison wet adhesive strength. The wet adhesive strength may be at least 75 times greater than the second comparison wet adhesive strength. The wet adhesive strength may be at least 50 times greater than the second comparison wet adhesive strength. The wet adhesive strength may be at least 20 times greater than the second comparison wet adhesive strength. The wet adhesive strength may be at least 10 times greater than the second comparison wet adhesive strength.
[0106] In some aspects, the solid state composition includes dopamine and has a wet adhesive duration that may be longer than a second comparison wet adhesive duration. The second comparison wet adhesive duration may be from a first comparison solid state composition that does not include dopamine and is otherwise identical to the solid state composition. The wet adhesive duration may be at least 10 times longer than the second comparison wet adhesive strength. The wet adhesive duration may be at least 7 times longer than the second comparison wet adhesive strength. The wet adhesive duration may be at least 5 times longer than the second comparison wet adhesive strength. The wet adhesive duration may be at least 3 times longer than the second comparison wet adhesive strength. The wet adhesive duration may be at least 2 times longer than the second comparison wet adhesive strength.
[0107] Without wishing to be bound by any particular theory, these comparisons are believed to be applicable across a broad range of surfaces for adhering together, but they are particularly true for surfaces that are challenging for adhesion (e.g., adhering to shark skin). With respect to the comparative values, it is believed that the values hold for the vast majority of surfaces and the compositions disclosed herein provide superior performance over the comparison compositions. With respect to the absolute adhesion strength values, it is possible that the values may vary based on the surfaces or articles being adhered and a skilled artisan would recognize that the disclosed absolute values may be for a more limited set of surfaces. The adhesive strength may vary depending on the specific aqueous conditions and a skilled artisan would recognize that the presence of certain ions may enhance the adhesive strength. For some surfaces that are easier to adhere and some adhesion environments, the adhesive strength can be much higher than those discussed elsewhere herein, including peel strengths of greater than 1 N/mm, greater than 2 N/mm, or higher, with as high as 5 N/mm expected to be achievable with certain surfaces.
[0108] In addition to the impressive performance capabilities, the compositions described herein provided other unexpected results. Specifically, currently on the market, two-component epoxy resins are the state-of-the-art for repairing cracks in marine environments (e.g., swimming pools and the like). However, these products have a slow setting time and become rigid after curing. Moreover, they have been shown ineffective on biological tissues (by way of experiments on sharks and manatees), potentially due to the skin irritation. The inventors have discovered a composition that does not require curing time to be adhesive. Moreover, the inventive compositions described herein can remain flexible long after application, which makes them compatible with an animal’s movements while maintaining adhesion.
[0109] Furthermore, the solid-state format of the coacervate offers several advantages. It provides a significantly extended shelf life, with minimal storage requirements, mainly protection from water. The coacervate can be conveniently stored in a device, such as a syringe, and is readily accessible for use without the need for specialized training, approaching commercial product standards. Additionally, the powder format both allows easy handling of the adhesive and permits the inclusion of other reactive additives that can enhance or adjust the adhesive properties of the coacervate. However, if the coacervate itself is dried and stored as a powder it crumbles, rendering it ineffective for dry applications.
[0110] In some aspects, the composition or method of making the coacervate-based adhesive comprises the polydopamine-substituted silk fibroin. In some aspects, the polydopamine-substituted silk fibroin has a degree of polydopamine-substitution of between 5% and 50%. In some cases, the degree of polydopamine-substitution may be between 10% and 40%. In some cases, the degree of polydopamine-substitution may be between 25% and 35%. The degree of polydopamine-substitution is measured as a percentage by weight of polydopamine substituents related to the weight of the silk fibroin backbone. In some aspects, the polydopamine-substituted silk fibroin is polydopamine- substituted at one or more target amino acids. The one or more target amino acids are selected from the group consisting of cysteine, tyrosine, arginine, lysine, histidine, phenylalinine, proline, and combinations thereof. In some aspects, the polydopamine-substituted silk fibroin is polydopamine- substituted at one or more tyrosines. In some aspects, polydopamine-substituted silk fibroin is the substituted with poly dopamine having 15 polymeric units or less. In some cases, the polydopamine has 13 polymeric units or less. In some cases, the polydopamine has 12 polymeric units or less. In some cases, the polydopamine has 10 polymeric units or less. In some cases, the polydopamine has 9 polymeric units or less. In some cases, the polydopamine has 8 polymeric units or less.
[0111] In some aspects, the silk fibroin backbone of the polydopamine-substituted silk fibroin can have a weight average molecular weight of between 25 kDa and 150 kDa. For example, the silk fibroin backbone can have a weight average molecular weight of between 30 kDa and 125 kDa. In some cases, the silk fibroin backbone can have a weight average molecular weight of between 35 kDa and 100 kDa. In some cases, the silk fibroin backbone can have a weight average molecular weight of between 25 kDa and 75 kDa. In some cases, the silk fibroin backbone can have a weight average molecular weight of between 100 kDa and 150 kDa. In some cases, the silk fibroin backbone can have a weight average molecular weight of between 50 kDa and 125 kDa. In some cases, the silk fibroin backbone can have a weight average molecular weight of between 40 kDa and 90 kDa. In some cases, the silk fibroin backbone can have a weight average molecular weight of between 55 kDa and 105 kDa. In some cases, the silk fibroin backbone can have a weight average molecular weight of between 45 kDa and 65 kDa.
[0112] In some aspects, the silk fibroin backbone of the polydopamine-substituted silk fibroin can have a number average molecular weight of between 25 kDa and 150 kDa. For example, the silk fibroin backbone can have a number average molecular weight of between 30 kDa and 125 kDa. In some cases, the silk fibroin backbone can have a number average molecular weight of between 35 kDa and 100 kDa. In some cases, the silk fibroin backbone can have a number average molecular weight of between 25 kDa and 75 kDa. In some cases, the silk fibroin backbone can have a number average molecular weight of between 100 kDa and 150 kDa. In some cases, the silk fibroin backbone can have a number average molecular weight of between 50 kDa and 125 kDa. In some cases, the silk fibroin backbone can have a number average molecular weight of between 40 kDa and 90 kDa. In some cases, the silk fibroin backbone can have a number average molecular weight of between 55 kDa and 105 kDa. In some cases, the silk fibroin backbone can have a number average molecular weight of between 45 kDa and 65 kDa.
[0113] In some aspects, the composition or method of making the coacervate-based adhesive comprises the polydopamine-substituted silk fibroin which is made by mixing silk fibroin with soluble dopamine in an aqueous solution. In some aspects, the polydopamine-substituted silk fibroin has a structure that is indistinguishable from a comparison structure that is made by mixing comparison silk fibroin with comparison soluble dopamine in a comparison aqueous solution. In some aspects, the soluble dopamine and/or the comparison soluble dopamine is dopamine hydrochloride.
[0114] In some aspects, the composition or method of making the coacervate-based adhesive comprises mixing silk fibroin with soluble dopamine in an aqueous solution. In some aspects, the mixing of silk fibroin with soluble dopamine in an aqueous solution uses a ratio by weight of silk fibroin to soluble polydopamine of between 1 :2 and 10: 1. In some aspects, the ratio may be between 1:1 and 5:1. In some aspects, the ratio may be between 1.25:1 and 3:1. In some aspects, the ratio may be at least 1:2. In some aspects, the ratio may be at least 1.5:1. In some aspects, the ratio may be at least 1:1. In some aspects, the ratio may be at least 1.1: 1. In some aspects, the ratio may be at least 1.2: 1. In some aspects, the ratio may be at least 1.25: 1. In some aspects, the ratio may be at least 1.3:1. In some aspects, the ratio may be at least 1.4: 1. In some aspects, the ratio may be at least 1.5:1. In some aspects, the ratio may be at least 1.75:1. In some aspects, the ratio may be at least 2:1. In some aspects, the ratio may be at least 3:1. In some aspects, the ratio may be or at least 4:1. In some aspects, the ratio may be at most 10: 1. In some aspects, the ratio may be at most 9: 1. In some aspects, the ratio may be at most 8:1. In some aspects, the ratio may be at most 7:1. In some aspects, the ratio may be at most 6:1. In some aspects, the ratio may be at most 5:1. In some aspects, the ratio may be at most 4:1. In some aspects, the ratio may be at most 3:1. In some aspects, the ratio may be at most 2: 1. In some aspects, the ratio may be or at most 1 :1.
[0115] In some aspects, the composition or method of making the coacervate-based adhesive comprises mixing comparison silk fibroin with comparison soluble polydopamine in a comparison aqueous solution. In some aspects, the mixing of comparison silk fibroin with comparison soluble polydopamine uses a ratio by weight of comparison silk fibroin to comparison soluble polydopamine of between 1 :2 and 10:1. In some aspects, the ratio may be between 1 :1 and 5:1. In some aspects, the ratio may be between 1.25:1 and 3:1. In some aspects, the ratio may be at least 1 :2. In some aspects, the ratio may be at least 1.5:1. In some aspects, the ratio may be at least 1:1. In some aspects, the ratio may be at least 1.1:1. In some aspects, the ratio may be at least 1.2: 1. In some aspects, the ratio may be at least 1.25: 1. In some aspects, the ratio may be at least 1.3:1. In some aspects, the ratio may be at least 1.4: 1. In some aspects, the ratio may be at least 1.5:1. In some aspects, the ratio may be a least 1.75:1. In some aspects, the ratio may be at least 2:1. In some aspects, the ratio may be at least 3 : 1. In some aspects, the ratio may be at least 4: 1. In some aspects, the ratio may be at most 10: 1. In some aspects, the ratio may be at most 9:1. In some aspects, the ratio may be at most 8:1. In some aspects, the ratio may he at most 7:1. In some aspects, the ratio may he at most 6:1. In some aspects, the ratio may be at most 5:1. In some aspects, the ratio may be at most 4:1. In some aspects, the ratio may be at most 3:1. In some aspects, the ratio may be at most 2:1. In some aspects, the ratio may be at most 1:1.
[0116] In some aspects, the solid state composition (e.g., powder) may be compressed into a tablet form, solid state tablet, or similar solid individual unit form using a mold and applying a pressure. The pressure can be applied at between 0.001 MPa and 5 MPa. In some cases, the applied pressure is at least 0.001 MPa. In some cases, the applied pressure is at least 0.01 MPa. In some cases, the applied pressure is at least 0. 1 MPa. In some cases, the applied pressure is at or at least 1 MPa. In some cases, the applied pressure is at most 5 MPa. In some cases, the applied pressure is at most 2 MPa. In some cases, the applied pressure is at most 0.5 MPa. In some cases, the applied pressure is at most 0.05 MPa. In some cases, the applied pressure is at most 0.005 MPa.
[0117] In other aspects, additives may be added to the solid state composition (e.g., powder) at between 10% and 80% of tablet weight. In some cases, the additive can be present in the solid state composition in an amount by weight of at least 10%. In some cases, the additive can be present in the solid state composition in an amount by weight of at least 30%. In some cases, the additive can be present in the solid state composition in an amount by weight of at least 50%. In some cases, the additive can be present in the solid state composition in an amount by weight of at least 70%. In some cases, the additive can be present in the powder in an amount by weight of at most 80%. In some cases, the additive can be present in the solid state composition in an amount by weight of at most 60%. In some cases, the additive can be present in the solid state composition in an amount by weight of at most 40%. In some cases, the additive can be present in the solid state composition in an amount by weight of at most 20%. The additives may accelerate the dissolution of the tablet into adhesive upon contact with fresh or salt water.
[0118] In certain aspects, the additive can be or can include one or more gas-evolving molecules. Examples of suitable gas-evolving molecules include, but are not limited to succinic acid, sodium carbonate, carbonic acid, bicarbonates, sulfites, bisulfites, ammonium chloride, nitrites, chlorates, peroxides, oxalic acid, or azides.
[0119] In certain aspects, the additive can be or can include one or more salts that produce exothermic reactions with water. Examples of suitable salts that produce exothermic reactions with water include, but are not limited to, magnesium chloride, calcium chloride, aluminum chloride, sodium hydroxide, potassium hydroxide.
[0120] In certain aspects, the additive can be or can include one or more hydrogen bonding disrupters. Examples of suitable hydrogen bonding disruptors include, but are not limited to, urea, 1 ,6-hexanediol, ethanol or guanidium chloride. Without wishing to be bound by any particular theory, it is believed that the hydrogen bonding disrupters would lower the glass transition temperature of the adhesive material, lowering the temperature of adhesion.
[0121] The present disclosure provides a method of using the coacervate-based adhesive. The method comprises contacting a first article and a second article together with an adhesive amount of the coacervate-based adhesive, thereby adhering the first and second articles to one another.
[0122] In some cases, the first article can be an animal, such as a marine animal, such as a fish or a marine mammal. In some cases, the second article can be a tag, such as a numbered tag, an electronic tag (e.g., radio transmitters), a water-permeable tag, or another sensor that can be usefully applied to a marine animal or other article. In some cases, the first and/or second article can be water permeable, thereby allowing water to interact with the adhesive via penetration of the second article. In some cases, the second article can include an electronic component.
[0123] In some cases, a delivery device or a syringe can include the solid state composition of or made by the methods described herein. [0124] The present disclosure provides a method of using the solid state composition including contacting the solid state composition with an aqueous solvent within a delivery device or syringe to obtain a coacervate-based adhesive and dispensing the coacervate-based adhesive from the delivery device or syringe onto at least one surface of a first or second article and contacting the first and second articles together with the coacervate-based adhesive on the at least one surface, thereby adhering the first and second articles to one another. The contacting can be completed in under 5 seconds. In some cases, the contacting can be completed in under 4 seconds. In some cases, the contacting can be completed in under 3 seconds. In some cases, the contacting can be completed in under 2 seconds. In some cases, the contacting can be completed in under 1 second. The solid state composition may further include an additive. The additive mass may be between 10% and 80% of the mass of the solid state composition. In some cases, the additive mass may be at least 10% of the mass of the solid state composition. In some cases, the additive mass may be at least 30% of the mass of the solid state composition. In some cases, the additive mass may be at least 50% of the mass of the solid state composition. In some cases, the additive mass may be at least 70% of the mass of the solid state composition. In some cases, the additive mass may be at most 80% of the mass of the solid state composition. In some cases, the additive mass may be at most 60% of the mass of the solid state composition. In some cases, the additive mass may be at most 40% of the mass of the solid state composition. In some cases, the additive mass may be at most 20% of the mass of the solid state composition. The additive may comprise a gas-evolving molecule or a salt that produces an exothermic reaction as described herein.
[0125] In one particular use case, the adhesives described herein can be used to adhere tags to marine animals, such as sharks. The examples below show testing to establish adequate adhesion with the very challenging surface of shark skin. Without wishing to be bound by any particular theory, it is believed that achieving adequate adhesion to shark skin is a baseline adhesion performance that can be broadly applied to other marine species with reasonable predictability (i.e., if it works on shark skin, it likely works on the exterior surface of most other species).
[0126] Other non- limiting examples of methods of using the adhesive are contemplated. As one example, during harvesting of fish, off-species catch could be tagged and then identified for isolation, with the disclosed adhesive providing the ability to rapidly adhere tags to marine species without the need for curing and containing only naturally-occurring components in many cases. As another example, swimming competitions in the wild could use the disclosed adhesive to adhere race numbers or other identifiers to racers, with the disclosed adhesive providing impressive adhesive strength despite the significant agitation that results from competitive swimming. As another example, the compositions could be useful as drug delivery systems for antibiotics or medications. They could be used to treat wounds in at-risk species, allowing for controlled release of therapeutic agents embedded therein. As yet another example, the compositions could be useful in fish farms. The issues with antibiotic use and release in fish farms (e.g., salmon farms) can lead to accumulation of antibiotics in the environment and development of antibiotic -resistant bacteria (e.g., as shown in studies involving the Chilean salmon industry). An antibiotic that is locally deployed in the compositions disclosed herein could be one useful step toward a more sustainable solution.
[0127] In general, the method of using can provide an adhesive strength that withstands shear forces and does not require curing. The method of using can provide instantaneous adhesion following underwater application. In some cases, the adhesive strength grows over time following application to a final adhesive strength.
[0128] In some aspects, the solid state composition provides adhesion between two articles when submerged in at least one of simulated sea water or distilled water.
[0129] In some cases, the method of using the coacervate-based adhesive includes maintaining the adhesive at a low temperature (e.g., below 15 °C, below 10 °C, below 5 °C, etc.) to control the viscosity of the adhesive and retain structural integrity of the adhesive in a desired shape. This lowered temperature may reduce the surface tackiness of the adhesive making it easier to handle. In these cases, the method can include raising the temperature of the adhesive following the contacting. [0130] In some aspects, silk fibroin powder and tannic acid powder may be combined to form an adhesive coacervate. The powders may be ground together or otherwise combined with reduced particle sizes relative to the starting material. The coacervate may absorb between 25% and 200% of its weight in water. In some cases, the coacervate may absorb at least 25% of its weight in water. In some cases, the coacervate may absorb at least 50% of its weight in water. In some cases, the coacervate may absorb at least 100% of its weight in water. In some cases, the coacervate may absorb at least 150% of its weight in water. In some cases, the coacervate may absorb at most 200% of its weight in water. In some cases, the coacervate may absorb at most 175% of its weight in water. In some cases, the coacervate may absorb at most 125% of its weight in water. In some cases, the coacervate may absorb at most 100% of its weight in water. In some cases, the coacervate may absorb at most 75% of its weight in water. In some cases, the coacervate may absorb or at most 50% of its weight in water. The adhesive may have vesicles between 10 pm and 100 pm when placed underwater. The vesicles may contain water. The vesicles may be expelled upon heating and reabsorbed upon cooling. The mechanical performance of the adhesive may change with changing conditioning temperatures. The adhesive strength of the adhesive may increase over time underwater. [0131] It should be apparent to those skilled in the art that many additional modifications beside those already described are possible without departing from the inventive concepts. In interpreting this disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. Variations of the term "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, so the referenced elements, components, or steps may be combined with other elements, components, or steps that are not expressly referenced. Embodiments referenced as "comprising" certain elements are also contemplated as "consisting essentially of" and "consisting of" those elements. When two or more ranges for a particular value are recited, this disclosure contemplates all combinations of the upper and lower bounds of those ranges that are not explicitly recited. For example, recitation of a value of between 1 and 10 or between 2 and 9 also contemplates a value of between 1 and 9 or between 2 and 10.
[0132] As used herein, "silk fibroin" refers to silk fibroin protein whether produced by silkworm, spider, or other insect, or otherwise generated (Lucas et al., Adv. Protein Chem., 13: 107-242 (1958)). Any type of silk fibroin can be used in different embodiments described herein. Silk fibroin produced by silkworms, such as Bombyx mori, is the most common and represents an earth-friendly, renewable resource. For instance, silk fibroin used in a silk film may be attained by extracting sericin from the cocoons of B. mori. Organic silkworm cocoons are also commercially available. There are many different silks, however, including spider silk (e.g., obtained from Nephila clavipes), transgenic silks, genetically engineered silks, such as silks from bacteria, yeast, mammalian cells, transgenic animals, or transgenic plants, and variants thereof, that can be used. See, e.g., WO 97/08315 and U.S. Pat. No. 5,245,012, each of which is incorporated herein by reference in their entireties.
[0133] According to various embodiments, a variety of functionalizing agents may be used with the silk-containing embodiments described herein (e.g., silk membrane, silk composition, silk articles, silk matrix, silk foam, silk microsphere, liquid composition, whipped silk cream, silk meringue, compressed silk meringue, hot-pressed silk meringue, silk leather, silk powder, silk toner, edible silkbased films, etc.). It should be understood that the examples herein may recite one or a few silkcontaining embodiments but are applicable to any silk-containing embodiment, as applicable. In some embodiments, a functionalizing agent may be any compound or molecule that facilitates the attachment to and/or development (e.g., growth) of one or more endothelial cells on a silk membrane. In some embodiments, a functionalizing agent may be any compound or molecule that facilitates the attachment and/or development (e.g., growth) of one or more megakaryocytes and/or hematopoietic progenitor cells on a silk matrix and/or silk membrane. In some embodiments, a functionalizing agent may be or comprise an agent suitable for facilitating the production of one or more of white blood cells and red blood cells. [0134] In some embodiments, a functionalizing agent may be or comprise a cell attachment mediator and/or an extracellular matrix protein, for example: collagen (e.g., collagen type I, collagen type III, collagen type IV or collagen type VI), elastin, fibronectin, vitronectin, laminin, fibrinogen, von Willebrand factor, proteoglycans, decorin, perlecan, nidogen, hyaluronan, and/or peptides containing known integrin binding domains e.g. “RGD” integrin binding sequence, or variations thereof, that are known to affect cellular attachment.
[0135] In some embodiments, a functionalizing agent may be any soluble molecule produced by endothelial cells. Non-limiting examples include fibroblast growth factor- 1 (FGF1) and vascular endothelial growth factors (VEGF).
[0136] According to some embodiments, a plurality of functionalizing agents may be used. For example, in some embodiments wherein production of platelets is desired, provided compositions may comprise the use of laminin, fibronectin and/or fibrinogen, and type IV collagen in order to facilitate the attachment and growth of endothelial cells on a silk membrane (e.g., a porous silk membrane) and/or attachment of megakaryocytes to a silk matrix.
[0137] In some embodiments, a functionalizing agent may be embedded or otherwise associated with a silk membrane and/or silk matrix such that at least a portion of the functionalizing agent is surrounded by a silk membrane and/or silk matrix as contrasted to a functionalizing agent simply being positioned along the surface of a silk membrane and/or silk matrix. In some embodiments, a functionalizing agent is distributed along and/or incorporated in substantially the entire surface area of a silk membrane/silk wall. In some embodiments, a functionalizing agent is distributed and/or incorporated only at one or more discrete portions of a silk membrane/wall and/or silk matrix. In some embodiments, a functionalizing agent is distributed in and/or along at least one of the lumenfacing side of a silk wall and the matrix-facing side of a silk wall.
[0138] According to various embodiments, any application-appropriate amount of one or more functionalizing agents may be used. In some embodiments, the amount of an individual functionalizing agent may be between about 1 pg/ml and 1,000 pg/ml (e.g., between about 2 and 1,000, 5 and 1,000, 10 and 1,000, 10 and 500, 10 and 100 pg/ml). In some embodiments, the amount of an individual functionalizing agent may be at least 1 pg/ml (e.g., at least 5, 10, 15, 20 25, 50, 100, 200, 300 400, 500, 600, 700, 800, or 900 pg/ml ). In some embodiments, the amount of an individual functionalizing agent is at most 1,000 pg/ml (e.g., 900, 800, 700, 600, 500, 400, 300 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 pg/ml).
[0139] In some aspects, the composition comprises one or more sensing agents, such as a sensing dye. The sensing agents/sensing dyes are environmentally sensitive and produce a measurable response to one or more environmental factors. In some aspects, the environmentally- sensitive agent or dye may be present in the composition in an effective amount to alter the composition from a first chemical-physical state to a second chemical-physical state in response to an environmental parameter (e.g., a change in pH, light intensity or exposure, temperature, pressure or strain, voltage, physiological parameter of a subject, and/or concentration of chemical species in the surrounding environment) or an externally applied stimulus (e.g., optical interrogation, acoustic interrogation, and/or applied heat). In some cases, the sensing dye is present to provide one optical appearance under one given set of environmental conditions and a second, different optical appearance under a different given set of environmental conditions. Suitable concentrations for the sensing agents described herein can be the concentrations for the colorants and additives described elsewhere herein. A person having ordinary skill in the chemical sensing arts can determine a concentration that is appropriate for use in a sensing application of the inks described herein.
[0140] In some aspects, the first and second chemical-physical state may be a physical property of the composition, such as mechanical property, a chemical property, an acoustical property, an electrical property, a magnetic property, an optical property, a thermal property, a radiological property, or an organoleptic property. Exemplary sensing dyes or agents include, but are not limited to, a pH sensitive agent, a thermal sensitive agent, a pressure or strain sensitive agent, a light sensitive agent, or a potentiometric agent.
[0141] Exemplary pH sensitive dyes or agents include, but are not limited to, cresol red, methyl violet, crystal violet, ethyl violet, malachite green, methyl green, 2-(p- dimethylaminophenylazojpyridine, paramethyl red, metanil yellow, 4-phenylazodiphenylamine, thymol blue, metacresol purple, orange IV, 4-o-Tolylazo-o-toluindine, quinaldine red, 2,4- dinitrophenol, erythrosine disodium salt, benzopurpurine 4B, N,N-dimethyl-p-(m-tolylazo) aniline, p-dimethylaminoazobenene, 4,4’-bis(2-amino-l-naphthylazo)-2,2’-stilbenedisulfonic acid, tetrabromophenolphthalein ethyl ester, bromophenol blue, Congo red, methyl orange, ethyl orange, 4-(4-dimethylamino-l-naphylazo)-3-methoxybenesulfonic acid, bromocresol green, resazurin, 4- phenylazo-l-napthylamine, ethyl red 2-(l-dimethylaminophenyazo) pyridine, 4-(p-ethoxyphenylazo)- m-phenylene-diamine monohydrochloride, resorcin blue, alizarin red S, methyl red, propyl red, bromocresol purple, chlorophenol red, p-nitrophenol, alizarin, 2-(2,4-dinitrophenylazo)-l-napthol-
3.6-disulfonic acid, bromothymol blue, 6, 8-dinitro-lH-quinazoline-2, 4-dione, brilliant yellow, phenol red, neutral red, m-nitrophenol, cresol red, turmeric, metacresol purple, 4,4'-bis(3-amino-l- naphthylazo)-2,2'-stilbenedisulfonic acid, thymol blue, p-naphtholbenzein, phenolphthalein, o- cresolphthalein, ethyl bis(2,4-dimethylphenyl) ethanoate, thymolphthalein, nitrazine yellow, alizarin yellow R, alizarin, p-(2,4-dihydroxyphenylazo) benzenesulfonic acid, 5,5'-indigodisulfonic acid,
2.4.6-trinitrotoluene, 1,3,5-trinitrobenezne, and clayton yellow. [0142] Exemplary light responsive dyes or agents include, but are not limited to, photochromic compounds or agents, such as triarylmethanes, stilbenes, azastilbenes, nitrones, fulgides, spiropyrans, napthopyrans, spiro-oxazines, quinones, derivatives, and combinations thereof.
[0143] Exemplary potentiometric dyes include, but are not limited to, substituted amiononaphthylehenylpridinium (ANEP) dyes, such as di-4-ANEPPS, di-8-ANEPPS, and N-(4- Sulfobutyl)-4-(6-(4-(Dibutylamino)phenyl)hexatrienyl)Pyridinium (RH237).
[0144] Exemplary temperature sensitive dyes or agents include, but are not limited to, thermochromic compounds or agents, such as thermochromic liquid crystals, leuco dyes, fluoran dyes, octadecylphosphonic acid.
[0145] Exemplary pressure or strain sensitive dyes or agents include, but are not limited to, spiropyran compounds and agents.
[0146] Exemplary chemi-sensitive dyes or agents include, but are not limited to, antibodies such as immunoglobulin G (IgG) which may change color from blue to red in response to bacterial contamination.
[0147] In some aspects, the compositions comprise one or more additive, dopant, or biologically active agent suitable for a desired intended purpose. In some aspects, the additive or dopant may be present in the composition in an amount effective to impart an optical or organoleptic property to the composition. Exemplary additives or dopants that impart optical or organoleptic properties include, but are not limited to, dyes/pigments, flavorants, aroma compounds, granular or fibrous fillers.
[0148] Additionally or alternatively, the additive, dopant, or biologically active agent may be present in the composition in an amount effective to "functionalize" the composition to impart a desired mechanical property or added functionality to the composition. Exemplary additive, dopants, or biologically active agent that impart the desired mechanical property or added functionality include, but are not limited to: environmentally sensitive/sensing dyes; active biomolecules; conductive or metallic particles; micro and nanofibers (e.g., silk nanofibers for reinforcement, carbon nanofibers); nanotubes; inorganic particles (e.g., hydroxyapatite, tricalcium phosphate, bioglasses); drugs (e.g., antibiotics, small molecules or low molecular weight organic compounds); proteins and fragments or complexes thereof (e.g., enzymes, antigens, antibodies and antigen-binding fragments thereof); DNA/RNA (e.g., siRNA, miRNA, mRNA); cells and fractions thereof (viruses and viral particles; prokaryotic cells such as bacteria; eukaryotic cells such as mammalian cells and plant cells; fungi).
[0149] In some aspects, the additive or dopant comprises a flavoring agent or flavorant.
[0150] Exemplary flavorants include ester flavorants, amino acid flavorants, nucleic acid flavorants, organic acid flavorants, and inorganic acid flavorants, such as, but not limited to, diacetyl, acetyl propionyl, acetoin, isoamyl acetate, benzaldehyde, cinnamaldehyde, ethyl propionate, methyl anthranilate, limonene, ethyl decadienoate, allyl hexanoate, ethyl maltol, ethylvanillin, methyl salicylate, manzanate, glutamic acid salts, glycine salts, guanylic acids salts, inosinic acid salts, acetic acid, ascorbic acid, citric acid, fumaric acid, lactic acid, malic acid, phosphoric acid, tartaric acid, derivatives, and mixtures thereof.
[0151] In some aspects, the additive or dopant comprises an aroma compound. Exemplary aroma compounds include ester aroma compounds, terpene aroma compounds, cyclic terpenes, and aromatic aroma compounds, such as, but not limited to, geranyl acetate, methyl formate, methyl acetate, methyl propionate, methyl butyrate, ethyl acetate, ethyl butyrate, isoamyl acetate, pentyl butyrate, pentyl pentanoate, octyl acetate, benzyl acetate, methyl anthranilate, myrcene, geraniol, nerol, citral, citronellal, cironellol, linalool, nerolidol, limonene, camphor, menthol, carone, terpineol, alpha-ionone, thujone, eucalyptol, benzaldehyde, eugenol, cinnamaldehyde, ethyl maltol, vanillin, anisole, anethole, estragole, thymol.
[0152] In some aspects, the additive or dopant comprises a colorant, such as a dye or pigment. In some aspects, the dye or pigment imparts a color or grayscale to the composition. The colorant can be different than the sensing agents and/or sensing dyes below. Any organic and/or inorganic pigments and dyes can be included in the inks. Exemplary pigments suitable for use in the present disclosure include International Color Index or C.I. Pigment Black Numbers 1 , 7, 1 1 and 31, C.I. Pigment Blue Numbers 15, 15:1, 15:2, 15:3, 15:4, 15:6, 16, 27, 29, 61 and 62, C.I. Pigment Green Numbers 7, 17, 18 and 36, C.I. Pigment Orange Numbers 5, 13, 16, 34 and 36, C.I. Pigment Violet Numbers 3, 19, 23 and 27, C.I. Pigment Red Numbers 3, 17, 22, 23, 48:1 , 48:2, 57:1 , 81 :1 , 81 :2, 81 :3, 81:5, 101, 114, 122, 144, 146, 170, 176, 179, 181, 185, 188, 202, 206, 207, 210 and 249, C.I. Pigment Yellow Numbers 1, 2, 3, 12, 13, 14, 17, 42, 65, 73, 74, 75, 83, 30, 93, 109, 1 10, 128, 138, 139, 147, 142, 151, 154 and 180, D&C Red No. 7, D&C Red No. 6 and D&C Red No. 34, carbon black pigment (such as Regal 330, Cabot Corporation), quinacridone pigments (Quinacridone Magenta (228-0122), available from Sun Chemical Corporation, Fort Lee, N.J.), diarylide yellow pigment (such as AAOT Yellow (274- 1788) available from Sun Chemical Corporation); and phthalocyanine blue pigment (such as Blue 15:3 (294-1298) available from Sun Chemical Corporation). The classes of dyes suitable for use in present invention can be selected from acid dyes, natural dyes, direct dyes (either cationic or anionic), basic dyes, and reactive dyes. The acid dyes, also regarded as anionic dyes, are soluble in water and mainly insoluble in organic solvents and are selected, from yellow acid dyes, orange acid dyes, red acid dyes, violet acid dyes, blue acid dyes, green acid dyes, and black acid dyes. European Patent 0745651, incorporated herein by reference, describes a number of acid dyes that are suitable for use in the present disclosure. Exemplary yellow acid dyes include Acid Yellow 1 International Color Index or C.I. 10316); Acid Yellow 7 (C.I. 56295); Acid Yellow 17 (C.I. 18965); Acid Yellow 23 (C.I. 19140); Acid Yellow 29 (C.I. 18900); Acid Yellow 36 (C.I. 13065); Acid Yellow 42 (C.I. 22910); Acid Yellow 73 (C.I. 45350); Acid Yellow 99 (C.I. 13908); Acid Yellow 194; and Food Yellow 3 (C.I. 15985). Exemplary orange acid dyes include Acid Orange 1 (C.I. 13090/1); Acid Orange 10 (C.I. 16230); Acid Orange 20 (C.I. 14603); Acid Orange 76 (C.I. 18870); Acid Orange 142; Food Orange 2 (C.I. 15980); and Orange B. [0153] Exemplary red acid dyes include Acid Red 1 (C.I. 18050); Acid Red 4 (C.I. 14710); Acid Red 18 (C.I. 16255); Acid Red 26 (C.I. 16150); Acid Red 27 (C.I. 16185); Acid Red 51 (C.I. 45430, available from BASF Corporation, Mt. Olive, N.J.); Acid Red 52 (C.I. 45100); Acid Red 73 (C.I. 27290); Acid Red 87 (C.I. 45380); Acid Red 94 (C.I. 45440) Acid Red 194; and Food Red 1 (C.I. 14700). Exemplary violet acid dyes include Acid Violet 7 (C.I. 18055); and Acid Violet 49 (C.I. 42640). Exemplary blue acid dyes include Acid Blue 1 (C.I. 42045); Acid Blue 9 (C.I. 42090); Acid Blue 22 (C.I. 42755); Acid Blue 74 (C.I. 73015); Acid Blue 93 (C.I. 42780); and Acid Blue 158A (C.I. 15050). Exemplary green acid dyes include Acid Green 1 (C.I. 10028); Acid Green 3 (C.I. 42085); Acid Green 5 (C.I. 42095); Acid Green 26 (C.I. 44025); and Food Green 3 (C.I. 42053). Exemplary black acid dyes include Acid Black 1 (C.I. 20470); Acid Black 194 (Basantol® X80, available from BASF Corporation, an azo/1 :2 CR-complex.
[0154] Exemplary direct dyes for use in the present disclosure include Direct Blue 86 (C.I. 74180); Direct Blue 199; Direct Black 168; Direct Red 253; and Direct Yellow 107/132 (C.I. Not Assigned). [0155] Exemplary natural dyes for use in the present disclosure include Alkanet (C.I.
75520,75530); Annatto (C.I. 75120); Carotene (C.I. 75130); Chestnut; Cochineal (C.1.75470); Cutch (C.I. 75250, 75260); Divi-Divi; Fustic (C.I. 75240); Brazilin (C.I. 75280); Logwood (C.I. 75200); Osage Orange (C.I. 75660); Paprika; Quercitron (C.I. 75720); Saffron (C.I. 75100) ; Sandal Wood (C.I. 75510, 75540, 75550, 75560); Sumac; and Turmeric (C.I. 75300). Exemplary reactive dyes for use in the present disclosure include Reactive Yellow 37 (monoazo dye); Reactive Black 31 (diazo dye); Reactive Blue 77 (phthalo cyanine dye) and Reactive Red 180 and Reactive Red 108 dyes. Suitable also are the colorants described in The Printing Ink Manual (5th ed., Leach et al. eds. (2007), pages 289-299). Other organic and inorganic pigments and dyes and combinations thereof can be used to achieve the colors desired.
[0156] In addition to or in place of visible colorants, compositions provided herein can contain ETV fluorophores that are excited in the ETV range and emit light at a higher wavelength (typically 400 nm and above). Examples of ETV fluorophores include but are not limited to materials from the coumarin, benzoxazole, rhodamine, napthalimide, perylene, benzanthrones, benzoxanthones or benzothia-xanthones families. The addition of a UV fluorophore (such as an optical brightener for instance) can help maintain maximum visible light transmission. The amount of colorant, when present, generally is between 0.05% to 5% or between 0.1% and 1% based on the weight of the composition.
[0157] For non-white compositions, the amount of pigment/dye generally is present in an amount of from at or about 0.1 wt% to at or about 20 wt% based on the weight of the composition. In some applications, a non-white ink can include 15 wt% or less pigment/dye, or 10 wt% or less pigment/dye or 5 wt% pigment/dye, or 1 wt% pigment/dye based on the weight of the composition. In some applications, a non-white ink can include 1 wt% to 10 wt%, or 5 wt% to 15 wt%, or 10 wt% to 20 wt% pigment/dye based on the weight of the composition. In some applications, a non-white ink can contain an amount of dye/pigment that is 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15%, 16 wt%, 17 wt%, 18 wt%, 19 wt% or 20 wt% based on the weight of the composition.
[0158] For white compositions, the amount of white pigment generally is present in an amount of from at or about 1 wt% to at or about 60 wt% based on the weight of the composition. In some applications, greater than 60 wt% white pigment can be present. Preferred white pigments include titanium dioxide (anatase and rutile), zinc oxide, lithopone (calcined coprecipitate of barium sulfate and zinc sulfide), zinc sulfide, blanc fixe and alumina hydrate and combinations thereof, although any of these can be combined with calcium carbonate. In some applications, a white ink can include 60 wt% or less white pigment, 55 wt% or less white pigment, 50 wt% white pigment, 45 wt% white pigment, 40 wt% white pigment, 35 wt% white pigment, 30 wt% white pigment, 25 wt% white pigment, 20 wt% white pigment, 15 wt% white pigment, or 10 wt% white pigment, based on the weight of the composition. In some applications, a white ink can include 5 wt% to 60 wt%, 5 wt% to 55 wt%, 10 wt% to 50 wt%, 10 wt% to 25 wt%, 25 wt% to 50 wt%, 5 wt% to 15 wt%, or 40 wt% to 60 wt% white pigment based on the weight of the composition. In some applications, a non-white ink can an amount of dye/pigment that is 5%, 6 wt%, 7 wt%, 8 wt%, 9 wt%, 10 wt%, 11 wt%, 12 wt%, 13 wt%, 14 wt%, 15%, 16 wt%, 17 wt%, 18 wt%, 19 wt%, 20 wt%, 21 wt%, 22 wt%, 23 wt%, 24 wt%, 25%, 26 wt%, 27 wt%, 28 wt%, 29 wt%, 30 wt%, 31 wt%, 32 wt%, 33 wt%, 34 wt%, 35%, 36 wt%, 37 wt%, 38 wt%, 39 wt%, 40 wt%, 41 wt%, 42 wt%, 43 wt%, 44 wt%, 45%, 46 wt%, 47 wt%,
48 wt%, 49 wt%, 50 wt%, 51 wt%, 52 wt%, 53 wt%, 54 wt%, 55%, 56 wt%, 57 wt%, 58 wt%, 59 wt% or 60 wt% based on the weight of the composition.
[0159] In some aspects, the additive or dopant comprises a conductive additive. Exemplary conductive additives include, but are not limited to graphite, graphite powder, carbon nanotubes, and metallic particles or nanoparticles, such as gold nanoparticles. In some aspects, the conductive additive is biocompatible and non-toxic. [0160] In some aspects, the additive is a biologically active agent. The term “biologically active agent” as used herein refers to any molecule which exerts at least one biological effect in vivo. For example, the biologically active agent can be a therapeutic agent to treat or prevent a disease state or condition in a subject. Biologically active agents include, without limitation, organic molecules, inorganic materials, proteins, peptides, nucleic acids (e.g., genes, gene fragments, gene regulatory sequences, and antisense molecules), nucleoproteins, polysaccharides, glycoproteins, and lipoproteins. Classes of biologically active compounds that can be incorporated into the composition provided herein include, without limitation, anticancer agents, antibiotics, analgesics, antiinflammatory agents, immunosuppressants, enzyme inhibitors, antihistamines, anti-convulsants, hormones, muscle relaxants, antispasmodics, ophthalmic agents, prostaglandins, anti-depressants, anti-psychotic substances, trophic factors, osteoinductive proteins, growth factors, and vaccines. [0161] The term “active agent” may also be used herein to refer to a biological sample (e.g., a sample of tissue or fluid, such as for instance blood) or a component thereof, and/or to a biologically active entity or compound, and/or to a structurally or functionally labile entity.
[0162] Exemplary active agents include, but are not limited to, therapeutic agents, diagnostic agents (e.g., contrast agents), and any combinations thereof. In some embodiments, the active agent present in a silk matrix (e.g., a silk microsphere), composition, or the like can include a labile active agent, e.g., an agent that can undergo chemical, physical, or biological change, degradation and/or deactivation after exposure to a specified condition, e.g., high temperatures, high humidity, light exposure, and any combinations thereof. In some embodiments, the active agent present in the silk matrix (e.g., a silk microsphere), composition, or the like can include a temperature-sensitive active agent, e.g., an active agent that will lose at least about 30% or more, of its original activity or bioactivity, upon exposure to a temperature of at least about 10 °C. or above, including at least about 15 °C. or above, at least about room temperature or above, or at least about body temperature (e.g., about 37 °C.) or above.
[0163] The active agent can be generally present in the silk matrix (e.g., a silk microsphere), composition, or the like in an amount of about 0.01% (w/w) to about 70% (w/w), about 0.1% (w/w) to about 50% (w/w), or about 1% (w/w) to about 30% (w/w). The active agent can be present on a surface of the silk matrix (e.g., a silk microsphere), composition, or the like and/or encapsulated and dispersed in the silk matrix (e.g., a silk microsphere), composition, or the like homogeneously, heterogeneously, or in a gradient. In some embodiments, the active agent can be added into the silk solution, which is then subjected to the methods described herein for preparing a silk matrix (e.g., a silk microsphere), composition, or the like. In some embodiments, the active agent can be coated on a surface of the silk matrix (e.g., a silk microsphere), composition, or the like. In some embodiments, the active agent can be loaded in a silk matrix (e.g., a silk microsphere), composition, or the like by incubating the silk microsphere in a solution of the active agent for a period of time, during which an amount of the active agent can diffuse into the silk matrix (e.g., a silk microsphere), composition, or the like, and thus distribute within the silk matrix (e.g., a silk microsphere), composition, or the like. [0164] In some aspects, the additive is a therapeutic agent. As used herein, the term “therapeutic agent” means a molecule, group of molecules, complex or substance administered to an organism for diagnostic, therapeutic, preventative medical, or veterinary purposes. As used herein, the term “therapeutic agent” includes a “drug” or a “vaccine.” This term includes externally and internally administered topical, localized and systemic human and animal pharmaceuticals, treatments, remedies, nutraceuticals, cosmeceuticals, biologicals, devices, diagnostics and contraceptives, including preparations useful in clinical and veterinary screening, prevention, prophylaxis, healing, wellness, detection, imaging, diagnosis, therapy, surgery, monitoring, cosmetics, prosthetics, forensics and the like. This term can also be used in reference to agriceutical, workplace, military, industrial and environmental therapeutics or remedies comprising selected molecules or selected nucleic acid sequences capable of recognizing cellular receptors, membrane receptors, hormone receptors, therapeutic receptors, microbes, viruses or selected targets comprising or capable of contacting plants, animals and/or humans. This term can also specifically include nucleic acids and compounds comprising nucleic acids that produce a therapeutic effect, for example deoxyribonucleic acid (DNA), ribonucleic acid (RNA), nucleic acid analogues (e.g., locked nucleic acid (LNA), peptide nucleic acid (PNA), xeno nucleic acid (XNA)), or mixtures or combinations thereof, including, for example, DNA nanoplexes, siRNA, microRNA, shRNA, aptamers, ribozymes, decoy nucleic acids, antisense nucleic acids, RNA activators, and the like. Generally, any therapeutic agent can be included in the composition provided herein.
[0165] The term “therapeutic agent” also includes an agent that is capable of providing a local or systemic biological, physiological, or therapeutic effect in the biological system to which it is applied. For example, the therapeutic agent can act to control infection or inflammation, enhance cell growth and tissue regeneration, control tumor growth, act as an analgesic, promote anti-cell attachment, and enhance bone growth, among other functions. Other suitable therapeutic agents can include anti-viral agents, hormones, antibodies, or therapeutic proteins. Other therapeutic agents include prodrugs, which are agents that are not biologically active when administered but upon administration to a subject are converted to biologically active agents through metabolism or some other mechanism. Additionally, a silk-based drug delivery composition can contain one therapeutic agent or combinations of two or more therapeutic agents. [0166] A therapeutic agent can include a wide variety of different compounds, including chemical compounds and mixtures of chemical compounds, e.g., small organic or inorganic molecules; saccharides; oligosaccharides; polysaccharides; biological macromolecules, e.g., peptides, proteins, and peptide analogs and derivatives; peptidomimetics; antibodies and antigen binding fragments thereof; nucleic acids; nucleic acid analogs and derivatives; an extract made from biological materials such as bacteria, plants, fungi, or animal cells; animal tissues; naturally occurring or synthetic compositions; and any combinations thereof. In some aspects, the therapeutic agent is a small molecule.
[0167] The term “bioactivity,” as used herein in reference to an active agent, generally refers to the ability of an active agent to interact with a biological target and/or to produce an effect on a biological target. For example, bioactivity can include, without limitation, elicitation of a stimulatory, inhibitory, regulatory, toxic or lethal response in a biological target. The biological target can be a molecule or a cell. For example, a bioactivity can refer to the ability of an active agent to modulate the effect/activity of an enzyme, block a receptor, stimulate a receptor, modulate the expression level of one or more genes, modulate cell proliferation, modulate cell division, modulate cell morphology, or any combination thereof. In some instances, a bioactivity can refer to the ability of a compound to produce a toxic effect in a cell. Exemplary cellular responses include, but are not limited to, lysis, apoptosis, growth inhibition, and growth promotion; production, secretion, and surface expression of a protein or other molecule of interest by the cell; membrane surface molecule activation including receptor activation; transmembrane ion transports; transcriptional regulations; changes in viability of the cell; changes in cell morphology; changes in presence or expression of an intracellular component of the cell; changes in gene expression or transcripts; changes in the activity of an enzyme produced within the cell; and changes in the presence or expression of a ligand and/or receptor (e.g., protein expression and/or binding activity). Methods for assaying different cellular responses are well known to one of skill in the art, e.g., western blot for determining changes in presence or expression of an endogenous protein of the cell, or microscopy for monitoring the cell morphology in response to the active agent, or FISH and/or qPCR for the detection and quantification of changes in nucleic acids. Bioactivity can be determined in some embodiments, for example, by assaying a cellular response.
[0168] In reference to an antibody, the term “bioactivity” includes, but is not limited to, epitope or antigen binding affinity, the in vivo and/or in vitro stability of the antibody, the immunogenic properties of the antibody, e.g., when administered to a human subject, and/or the ability to neutralize or antagonize the bioactivity of a target molecule in vivo or in vitro. The aforementioned properties or characteristics can be observed or measured using art-recognized techniques including, but not limited to, scintillation proximity assays, ELISA, ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence ELISA, competitive ELISA, SPR analysis including, but not limited to, SPR analysis using a BIAcore biosensor, in vitro and in vivo neutralization assays (see, for example, International Publication No. WO 2006/062685), receptor binding, and immunohistochemistry with tissue sections from different sources including human, primate, or any other source as needed. In reference to an immunogen, the “bioactivity” includes immunogenicity, the definition of which is discussed in detail later. In reference to a virus, the “bioactivity” includes infectivity, the definition of which is discussed in detail later. In reference to a contrast agent, e.g., a dye, the “bioactivity” refers to the ability of a contrast agent when administered to a subject to enhance the contrast of structures or fluids within the subject’s body. The bioactivity of a contrast agent also includes, but is not limited to, its ability to interact with a biological environment and/or influence the response of another molecule under certain conditions.
[0169] As used herein, the term “small molecule” can refer to compounds that are “natural productlike,” however, the term “small molecule” is not limited to “natural product-like” compounds. Rather, a small molecule is typically characterized in that it contains several carbon — carbon bonds and has a molecular weight of less than 5000 Daltons (5 kDa), preferably less than 3 kDa, still more preferably less than 2 kDa, and most preferably less than 1 kDa. In some cases, it is preferred that a small molecule has a molecular weight equal to or less than 700 Daltons.
[0170] Exemplary therapeutic agents include, but are not limited to, those found in Harrison’ s Principles of Internal Medicine, 13th Edition, Eds. T.R. Harrison et al. McGraw-Hill N.Y., NY; Physicians’ Desk Reference, 50th Edition, 1997, Oradell New Jersey, Medical Economics Co.; Pharmacological Basis of Therapeutics, 8th Edition, Goodman and Gilman, 1990; United States Pharmacopeia, The National Formulary, ETSP XII NF XVII, 1990, the complete contents of all of which are incorporated herein by reference.
[0171] Therapeutic agents include the herein disclosed categories and specific examples. It is not intended that the category be limited by the specific examples. Those of ordinary skill in the art will recognize also numerous other compounds that fall within the categories and that are useful according to the present disclosure. Examples include a radiosensitizer, a steroid, a xanthine, a beta- 2-agonist bronchodilator, an anti-inflammatory agent, an analgesic agent, a calcium antagonist, an angiotensin-converting enzyme inhibitors, a beta-blocker, a centrally active alpha- agonist, an alpha - 1 -antagonist, an anticholinergic/antispasmodic agent, a vasopressin analogue, an antiarrhythmic agent, an antiparkinsonian agent, an antiangina/antihypertensive agent, an anticoagulant agent, an antiplatelet agent, a sedative, an anxiolytic agent, a peptidic agent, a biopolymeric agent, an antineoplastic agent, a laxative, an antidiarrheal agent, an antimicrobial agent, an antifungal agent, a vaccine, a protein, or a nucleic acid. In a further aspect, the pharmaceutically active agent can be coumarin, albumin, steroids such as betamethasone, dexamethasone, methylprednisolone, prednisolone, prednisone, triamcinolone, budesonide, hydrocortisone, and pharmaceutically acceptable hydrocortisone derivatives; xanthines such as theophylline and doxophylline; beta-2- agonist bronchodilators such as salbutamol, fenterol, clenbuterol, bambuterol, and salmeterol; antiinflammatory agents, including antiasthmatic anti-inflammatory agents, antiarthritis antiinflammatory agents, and non-steroidal anti-inflammatory agents, examples of which include but are not limited to sulfides, mesalamine, budesonide, salazopyrin, diclofenac, pharmaceutically acceptable diclofenac salts, nimesulide, naproxen, acetaminophen, ibuprofen, ketoprofen and piroxicam; analgesic agents such as salicylates; calcium channel blockers such as nifedipine, amlodipine, and nicardipine; angiotensin converting enzyme inhibitors such as captopril, benazepril hydrochloride, fosinopril sodium, trandolapril, ramipril, lisinopril, enalapril, quinapril hydrochloride, and moexipril hydrochloride; beta-blockers (i.e., beta adrenergic blocking agents) such as sotalol hydrochloride, timolol maleate, esmolol hydrochloride, carteolol, propanolol hydrochloride, betaxolol hydrochloride, penbutolol sulfate, metoprolol tartrate, metoprolol succinate, acebutolol hydrochloride, atenolol, pindolol, and bisoprolol fumarate; centrally active alpha-2-agonists such as clonidine; alpha- 1 -antagonists such as doxazosin and prazosin; anticholinergic/antispasmodic agents such as dicyclomine hydrochloride, scopolamine hydrobromide, glycopyrrolate , clidinium bromide, flavoxate, and oxybutynin; vasopressin analogues such as vasopressin and desmopressin; antiarrhythmic agents such as quinidine, lidocaine, tocainide hydrochloride, mexiletine hydrochloride, digoxin, verapamil hydrochloride, propafenone hydrochloride, flecainide acetate, procainamide hydrochloride, moricizine hydrochloride, and disopyramide phosphate; antiparkinsonian agents, such as dopamine, L-Dopa/Carbidopa, selegiline, dihydroergocryptine, pergolide, lisuride, apomorphine, and bromocriptine; antiangina agents and antihypertensive agents such as isosorbide mononitrate, isosorbide dinitrate, propranolol, atenolol and verapamil; anticoagulant and antiplatelet agents such as coumadin, warfarin, acetylsalicylic acid, and ticlopidine; sedatives such as benzodiazepines and barbiturates; anxiolytic agents such as lorazepam, bromazepam, and diazepam; peptidic and biopolymeric agents such as calcitonin, leuprolide and other LHRH agonists, hirudin, cyclosporin, insulin, somatostatin, protirelin, interferon, desmopressin, somatotropin, thymopentin, pidotimod, erythropoietin, interleukins, melatonin, granulocyte/macrophage-CSF, and heparin; antineoplastic agents such as etoposide, etoposide phosphate, cyclophosphamide, methotrexate, 5 -fluorouracil, vincristine, doxorubicin, cisplatin, hydroxyurea, leucovorin calcium, tamoxifen, flutamide, asparaginase, altretamine, mitotane, and procarbazine hydrochloride; laxatives such as senna concentrate, casanthranol, bisacodyl, and sodium picosulphate; antidiarrheal agents such as difenoxin hydrochloride, loperamide hydrochloride, furazolidone, diphenoxylate hydrochloride, and microorganisms; vaccines such as bacterial and viral vaccines; antimicrobial agents such as penicillins, cephalosporins, and macrolides, antifungal agents such as imidazolic and triazolic derivatives; and nucleic acids such as DNA sequences encoding for biological proteins, and antisense oligonucleotides.
[0172] Anti-cancer agents include alkylating agents, platinum agents, antimetabolites, topoisomerase inhibitors, antitumor antibiotics, antimitotic agents, aromatase inhibitors, thymidylate synthase inhibitors, DNA antagonists, farnesyltransferase inhibitors, pump inhibitors, histone acetyltransferase inhibitors, metalloproteinase inhibitors, ribonucleoside reductase inhibitors, TNF alpha agonists/antagonists, endothelin A receptor antagonists, retinoic acid receptor agonists, immunomodulators, hormonal and antihormonal agents, photodynamic agents, and tyrosine kinase inhibitors. [0173] Antibiotics include aminoglycosides (e.g., gentamicin, tobramycin, netilmicin, streptomycin, amikacin, neomycin), bacitracin, carbapenems (e.g., imipenem/cilastatin), cephalosporins, colistin, methenamine, monobactams (e.g., aztreonam), penicillins (e.g., penicillin G, penicillin V, methicillin, nafcillin, oxacillin, cioxacillin, dicloxacillin, ampicillin, amoxicillin, carbenicillin, ticarcillin, piperacillin, mezlocillin, azlocillin), polymyxin B, quinolones, and vancomycin; and bacteriostatic agents such as chloramphenicol, clindamycin, macrolides (e.g., erythromycin, azithromycin, clarithromycin), lincomycin, nitrofurantoin, sulfonamides, tetracyclines (e.g., tetracycline, doxycycline, minocycline, demeclocy cline), and trimethoprim. Also included are metronidazole, fluoroquinolones, and rifampin.
[0174] Enzyme inhibitors are substances which inhibit an enzymatic reaction. Examples of enzyme inhibitors include edrophonium chloride, N-methylphysostigmine, neostigmine bromide, physostigmine sulfate, tacrine, 1-hydroxymaleate, iodotubercidin, p-bromotetranisole, 10-(alpha- diethylaminopropionyl)-phenothiazine hydrochloride, calmidazolium chloride, hemicholinium-3,3,5- dinitrocatechol, diacylglycerol kinase inhibitor I, diacylglycerol kinase inhibitor II, 3- phenylpropargylamine, N°-monomethyl-L-arginine acetate, carbidopa, 3 -hydroxybenzylhydrazine, hydralazine, clorgyline, deprenyl, hydroxylamine, iproniazid phosphate, 6-MeO-tetrahydro-9H- pyrido-indole, nialamide, pargyline, quinacrine, semicarbazide, tranylcypromine, N,N- diethylaminoethyl-2,2-diphenylvalerate hydrochloride, 3-isobutyl-l-methylxanthine, papaverine, indomethacin, 2-cyclooctyl-2-hydroxyethylamine hydrochloride, 2,3-dichloro-a-methylbenzylamine (DCMB), 8,9-dichloro-2,3,4,5-tetrahydro-lH-2-benzazepine hydrochloride, p- aminoglutethimide, p- aminoglutethimide tartrate, 3-iodotyrosine, alpha-methyltyrosine, acetazolamide, dichlorphenamide, 6-hydroxy-2-benzothiazolesulfonamide, and allopurinol.
[0175] Antihistamines include pyrilamine, chlorpheniramine, and tetrahydrozoline, among others. [0176] Anti-inflammatory agents include corticosteroids, nonsteroidal anti-inflammatory drugs (e.g., aspirin, phenylbutazone, indomethacin, sulindac, tolmetin, ibuprofen, piroxicam, and fenamates), acetaminophen, phenacetin, gold salts, chloroquine, D-Penicillamine, methotrexate colchicine, allopurinol, probenecid, and sulfinpyrazone.
[0177] Muscle relaxants include mephenesin, methocarbamol, cyclobenzaprine hydrochloride, trihexyphenidyl hydrochloride, levodopa/carbidopa, and biperiden.
[0178] Anti-spasmodics include atropine, scopolamine, oxyphenonium, and papaverine.
[0179] Analgesics include aspirin, phenylbutazone, indomethacin, sulindac, tolmetin, ibuprofen, piroxicam, fenamates, acetaminophen, phenacetin, morphine sulfate, codeine sulfate, meperidine, nalorphine, opioids (e.g., codeine sulfate, fentanyl citrate, hydrocodone bitartrate, loperamide, morphine sulfate, noscapine, norcodeine, normorphine, thebaine, nor-binaltorphimine, buprenorphine, chlomaltrexamine, funaltrexamine, nalbuphine, nalorphine, naloxone, naloxonazine, naltrexone, and naltrindole), procaine, lidocaine, tetracaine and dibucaine. Ophthalmic agents include sodium fluorescein, rose bengal, methacholine, adrenaline, cocaine, atropine, alphachymotrypsin, hyaluronidase, betaxolol, pilocarpine, timolol, timolol salts, and combinations thereof. [0180] Prostaglandins are art recognized and are a class of naturally occurring chemically related long-chain hydroxy fatty acids that have a variety of biological effects.
[0181] Anti-depressants are substances capable of preventing or relieving depression.
[0182] Examples of anti-depressants include imipramine, amitriptyline, nortriptyline, protriptyline, desipramine, amoxapine, doxepin, maprotiline, tranylcypromine, phenelzine, and isocarboxazid. [0183] Trophic factors are factors whose continued presence improves the viability or longevity of a cell trophic factors include, without limitation, platelet-derived growth factor (PDGP), neutrophilactivating protein, monocyte chemoattractant protein, macrophage- inflammatory protein, platelet factor, platelet basic protein, and melanoma growth stimulating activity; epidermal growth factor, transforming growth factor (alpha), fibroblast growth factor, platelet- derived endothelial cell growth factor, insulin-like growth factor, glial derived growth neurotrophic factor, ciliary neurotrophic factor, nerve growth factor, bone growth/cartilage-inducing factor (alpha and beta), bone morphogenetic proteins, interleukins (e.g., interleukin inhibitors or interleukin receptors, including interleukin 1 through interleukin 10), interferons (e.g., interferon alpha, beta and gamma), hematopoietic factors, including erythropoietin, granulocyte colony stimulating factor, macrophage colony stimulating factor and granulocyte-macrophage colony stimulating factor; tumor necrosis factors, and transforming growth factors (beta), including beta-1, beta-2, beta-3, inhibin, and activin. [0184] Hormones include estrogens (e.g., estradiol, estrone, estriol, diethylstilbestrol, quinestrol, chlorotrianisene, ethinyl estradiol, mestranol), anti-estrogens (e.g., clomiphene, tamoxifen), progestins (e.g., medroxyprogesterone, norethindrone, hydroxyprogesterone, norgestrel), antiprogestin (mifepristone), androgens (e.g., testosterone cypionate, fluoxymesterone, danazol, testolactone), anti- androgens (e.g., cyproterone acetate, flutamide), thyroid hormones (e.g., triiodothyronne, thyroxine, propylthiouracil, methimazole, and iodixode), and pituitary hormones (e.g., corticotropin, somatotropin, oxytocin, and vasopressin). Hormones are commonly employed in hormone replacement therapy and/or for purposes of birth control. Steroid hormones, such as prednisone, are also used as immunosuppressants and anti-inflammatories. In some aspects, the additive is an agent that stimulates tissue formation, and/or healing and regrowth of natural tissues, and any combinations thereof. Agents that increase formation of new tissues and/or stimulates healing or regrowth of native tissue at the site of injection can include, but are not limited to, fibroblast growth factor (FGF), transforming growth factor- beta (TGF-beta, platelet-derived growth factor (PDGF), epidermal growth factors (EGFs), connective tissue activated peptides (CTAPs), osteogenic factors including bone morphogenic proteins, heparin, angiotensin II (A-II) and fragments thereof, insulin-like growth factors, tumor necrosis factors, interleukins, colony stimulating factors, erythropoietin, nerve growth factors, interferons, biologically active analogs, fragments, and derivatives of such growth factors, and any combinations thereof.
[0185] In some aspects, the silk composition can further comprise at least one additional material for soft tissue augmentation, e.g., dermal filler materials, including, but not limited to, poly(methyl methacrylate) microspheres, hydroxyapatite, poly(L-lactic acid), collagen, elastin, and glycosaminoglycans, hyaluronic acid, commercial dermal filler products such as BOTOX® (from Allergan), DYSPORT®, COSMODERM®, EVOLENCE®, RADIESSE®,RESTYLANE®, JUVEDERM® (from Allergan), SCULPTRA®, PERLANE®, and CAPTIQEIE®, and any combinations thereof.
[0186] In some aspects, the additive is a wound healing agent. As used herein, a “wound healing agent" is a compound or composition that actively promotes wound healing process.
[0187] Exemplary wound healing agents include, but are not limited to dexpanthenol; growth factors; enzymes; hormones; povidon-iodide; fatty acids; anti-inflammatory agents; antibiotics; antimicrobials; antiseptics; cytokines; thrombin; angalgesics; opioids; aminoxyls; furoxans; nitrosothiols; nitrates and anthocyanins; nucleosides, such as adenosine; and nucleotides, such as adenosine diphosphate (ADP) and adenosine triphosphate (ATP); neurotransmitter/neuromodulators, such as acetylcholine and 5 -hydroxy tryptamine (serotonin/5-HT); histamine and catecholamines, such as adrenalin and noradrenalin; lipid molecules, such as 5 -sphingosine- 1 -phosphate and lysophosphatidic acid; amino acids, such as arginine and lysine; peptides such as the bradykinins, substance P and calcium gene-related peptide (CGRP); nitric oxide; and any combinations thereof. [0188] In certain aspects, the active agents provided herein are immunogens. In one aspect, the immunogen is a vaccine. Most vaccines are sensitive to environmental conditions under which they are stored and/or transported. For example, freezing may increase reactogenicity (e.g., capability of causing an immunological reaction) and/or loss of potency for some vaccines (e.g., HepB, and DTaP/IPV/FQB), or cause hairline cracks in the container, leading to contamination. Further, some vaccines (e.g., BCG, Varicella, and MMR) are sensitive to heat. Many vaccines (e.g., BCG, MMR, Varicella, Meningococcal C Conjugate, and most DTaP-containing vaccines) are light sensitive. See, e.g., Galazka et al., Thermostability of vaccines, in Global Programme for Vaccines & Immunization (World Health Organization, Geneva, 1998); Peetermans et al., Stability of freeze-dried rubella virus vaccine (Cendehill strain) at various temperatures, J. Biological Standardization 179 (1973). Thus, the compositions and methods provided herein also provide for stabilization of vaccines regardless of the cold chain and/or other environmental conditions.
[0189] In some aspects, the additive is a cell, e.g., a biological cell. Cells useful for incorporation into the composition can come from any source, e.g., mammalian, insect, plant, etc. In some aspects, the cell can be a human, rat or mouse cell. In general, cells to be used with the compositions provided herein can be any types of cells. In general, the cells should be viable when encapsulated within compositions. In some aspects, cells that can be used with the composition include, but are not limited to, mammalian cells (e.g. human cells, primate cells, mammalian cells, rodent cells, etc.), avian cells, fish cells, insect cells, plant cells, fungal cells, spore cells, bacterial cells, and hybrid cells. In some aspects, exemplary cells that can be used with the compositions include platelets, activated platelets, stem cells, totipotent cells, pluripotent cells, and/or embryonic stem cells. In some aspects, exemplary cells that can be encapsulated within compositions include, but are not limited to, primary cells and/or cell lines from any tissue. For example, cardiomyocytes, myocytes, hepatocytes, keratinocytes, melanocytes, neurons, astrocytes, embryonic stem cells, adult stem cells, hematopoietic stem cells, hematopoietic cells (e.g. monocytes, neutrophils, macrophages, etc.), ameloblasts, fibroblasts, chondrocytes, osteoblasts, osteoclasts, neurons, sperm cells, egg cells, liver cells, epithelial cells from lung, epithelial cells from gut, epithelial cells from intestine, liver, epithelial cells from skin, etc., and/or hybrids thereof, can be included in the silk/platelet compositions disclosed herein. Those skilled in the art will recognize that the cells listed herein represent an exemplary, not comprehensive, list of cells. Cells can be obtained from donors (allogenic) or from recipients (autologous). Cells can be obtained, as a non-limiting example, by biopsy or other surgical means known to those skilled in the art.
[0190] In some aspects, the cell can be a genetically modified cell. A cell can be genetically modified to express and secrete a desired compound, e.g. a bioactive agent, a growth factor, differentiation factor, cytokines, and the like. Methods of genetically modifying cells for expressing and secreting compounds of interest are known in the art and easily adaptable by one of skill in the art.
[0191] Differentiated cells that have been reprogrammed into stem cells can also be used.
[0192] For example, human skin cells reprogrammed into embryonic stem cells by the transduction of Oct3/4, Sox2, c-Myc and Klf4 (Junying Yu, et. ah, Science, 2007, 318, 1917-1920 and Takahashi K. et. al, Cell, 2007, 131, 1-12).
[0193] Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules”.
[0194] As used herein, “about”, “approximately”, “substantially”, and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus <10% of the particular term and “substantially” and “significantly” will mean plus or minus > 10% of the particular term.
[0195] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.
[0196] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
[0197] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.
[0198] Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.
[0199] While the invention has been illustrated and described in detail in the foregoing drawings and description, the same is to be considered as illustrative and not restrictive in character, it being understood that only illustrative embodiments thereof have been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected. For example, any of the features or functions of any of the embodiments disclosed herein may be incorporated into any of the other embodiments disclosed herein.
[0200] The following examples illustrate some embodiments and aspects of the invention. It will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be performed without altering the spirit or scope of the invention, and such modifications and variations are encompassed within the scope of the invention as defined in the claims which follow. The following examples do not in any way limit the invention.
[0201] EXAMPLES
[0202] Example 1: SF and TA Powders
[0203] Freeze-dried silk fibroin powder and TA powder are ground in a 1 :1 mass ratio. Grinding using a mortar and pestle achieves material homogenization and reduces particle sizes, enhancing dissolution of the powder in water. A ball mill or cryomill may be more effective than manual grinding. This resulting powder can be easily transferred into a syringe, compressed using the plunger, and subsequently deployed in underwater environments. The syringe allows recovery of either fresh or seawater, which can then be mixed with the powder using the plunger. This process enables preparation and delivery of the adhesive within a matter of seconds. Based on weight analysis described herein, the coacervate absorbs approximately its own mass in water during mixing. Therefore, it is more accurate to describe the coacervate composition as 25% silk, 25% tannic acid, and 50% water. An overview of the coacervation process is shown in Fig. 2.
[0204] Preliminary microscopic analyses were conducted to examine the adhesive's structure and behavior when heated. The material was confined between two glass coverslips (Fig. 3a) and analyzed under a reflected light upright microscope before and after conditioning it in a water bath at 60 °C. As shown in Fig. 3b, the adhesive exhibits an internal architecture composed of vesicles of varying sizes (10 pm - 100 pm). Upon heating, the number and size of these vesicles generally increase, and internal vacuolization becomes evident. Given that the material consists of 50% water, we initially hypothesized that these vesicles contain water. To verify this, the adhesive was heated on a hot plate at 60 °C outside the water bath, revealing that water is expelled from the edges of the sample (Fig. 3c).
[0205] To further investigate this phenomenon at a microscopic level, the adhesive was locally heated using a metal soldering probe while being observed under a microscope (Fig. 3d). The dark spot in the micrographs represents the tip of the soldering probe, set to a localized temperature of 70 °C. As shown in the images, water is visibly expelled from the material in the region exposed to heat. A similar experiment was conducted using a coacervate containing 10% CuCh, which imparts a blue color when released into solution, making the water exchange process more apparent. Notably, when the heat source is removed, the vesicles are reabsorbed by the material as it cools.
[0206] These analyses have led to a more detailed hypothesis regarding the adhesive’s mechanism of action, particularly during its setting process. As illustrated in Fig. 3e, heating both induces water release from the material into the surrounding environment and reduces its viscosity. This allows the adhesive to spread over the submerged target surface, physically displacing the bulk water layer. Given the material’s water management properties, it appears capable of reabsorbing surrounding water upon cooling, potentially removing the monomolecular hydration layer on a submerged surface. We believe this feature is a factor explaining the adhesive’s strong underwater adhesion. A comprehensive demonstration of this mechanism could have an impact on the development of underwater adhesives.
[0207] Fig. 4a presents the stress-strain curves for the plain coacervate (four measurements) after one hour of application at 20 °C in seawater. These curves were used to calculate the Young’s modulus (E) in the initial linear region (strain < 10%) of the curve, yielding 120.82 ± 64.56 kPa. Fig. 4b shows the complex modulus (G) in the plateau region (strain < 10%) for the same material from an amplitude sweep test at 20 °C, with a value of 39.95 ± 37.2 kPa.
[0208] The relationship between shear modulus (6), Young’s modulus (E), and Poisson’s ratio (v) is given by:
[0209] Assuming a Poisson’s ratio of ~ 0.5 for coacervates, the modulus obtained from rheological measurements (G = 39.95 ± 37.2 kPa) closely matches those determined experimentally through tensile testing (E/2(l+ v) = 40.27 ± 21.52 kPa). This suggests that the mechanical performance of a given coacervate composition at a specific temperature can be predicted from its rheological properties, at least in terms of initial adhesion performance within the first few hours of application. [0210] As shown in Fig. 4c, which illustrates the mechanical performance of the plain coacervate in seawater over one week at 8 °C and 20 °C, adhesive strength increases over time, particularly when conditioned at lower temperatures. This suggests that the adhesive undergoes a form of curing influenced by its surrounding environment. The effect is even more pronounced in coacervates modified with ferric compounds, as shown in Fig. 4d, where a coacervate containing 10% FeCF reaches adhesive strengths of up to 800 kPa after four weeks in seawater at 8 °C. While rheological measurements can provide insight into the initial adhesion properties of a specific formulation, they are unlikely to predict long-term performance after curing.
[0211] Figs. 4e-4g illustrate several possible application methods for this adhesive. Fig. 4e demonstrates the potential use of a solid adhesive tablet. In this approach, the tablet is confined within a rigid support (such as a glass coverslip), and frictional heat is applied to locally melt the adhesive, anchoring it to the base. Fig. 4f shows the application of a flexible tag onto a submerged substrate. The coacervate was spread onto a polyurethane tag, which was pre-attached to the exterior of a Ziplock bag using double-sided tape. Filling the bag with hot water (40 °C - 50 °C) keeps the adhesive in a softened, active state, allowing it to bond to a surface upon application of pressure.
This method enables adhesion to curved or irregular surfaces (e.g., a wrist), and the Ziplock bag can be easily removed as the double-sided tape has low water resistance. The adhesive provides strong immediate adhesion, securing the tag in place even under vigorous mechanical agitation.
[0212] Fig. 4g presents a device incorporating a USB heating pad embedded within an epoxy resin plate, designed for controlled adhesive applications. The subsequent images show the bottom view of the plate positioned inside a glass container before activation of the USB heater and after cooling. Upon activation, the heater melts the adhesive, which then flows spontaneously onto the bottom of the glass tank. Once cooled, the adhesive solidifies, forming a secure bond without requiring external pressure.
Example 2: PDA-Functionalized SF
[0213] A preliminary modification of SF with polydopamine (PDA) results in a longer-lasting adhesion of the coacervates as shown in Fig. 5. The modified silk displayed a gradual reduction in adhesive performance over time, generally remaining below 100 kPa. In contrast, the DA-modified silk exhibited higher adhesion strength, around 300 kPa, and maintained more consistent performance over three weeks underwater. [0214] The control solid coacervate, obtained by grinding 350 mg of SF powder and 350 mg of TA powder, was activated by adding the same mass (700 pL) of seawater, mixed, and then directly spread on glass adherents over a 10 x 25 mm area. It was then tested in lap shear measurements. [0215] As shown in Fig. 6, the control coacervate displayed very low values of initial adhesion (6.4 + 2 kPa), which can be increased by pre-heating the coacervate to 60 °C prior to its application (23.4 ± 10.5 kPa). The increased adhesion value after heating is because the coacervate phase consists of a hydrogen-bonded network. Increasing the temperature breaks some of the electrostatic interactions, causing the coacervate to melt.
[0216] Decreasing the coacervate viscosity prior to its application will facilitate its spreading on the target surface and adaptation on the surface roughness. With a decrease in temperature, the coacervate will reform the electrostatic bonding increasing higher cohesion and improving the adhesive performance. Indeed, after 1 day immersed in sea water, the shear stress rises to 269.4 + 149.1 kPa. This increase might also be due to the reactive nature of TA which can undergo in covalent cross-linking with free amino groups via Michael addition reaction. Decreasing the coacervate viscosity before its application will facilitate its spreading on the target surface and enhance its adaptation to surface roughness. As the temperature decreases, the coacervate reforms the electrostatic bonds, leading to higher cohesion and improved adhesive performance. After one day of immersion in seawater, the shear stress increases to 269.4 ± 149.1 kPa. This increase could also be attributed to the reactive nature of TA, which can undergo covalent cross-linking with free amino groups via a Michael addition reaction.
[0217] The solid format allows for the inclusion of solid-state reactive additives into the mixture. These additives react with the silk-TA matrix only when the coacervate is activated or applied with water or seawater. One of these additives is calcium chloride (Fig. 7), which acts as a crosslinker for silk fibroin. Adding calcium chloride to the powder in a range between 5% and 20% (w/w%) leads to a change in the adhesive's mechanical behavior. With low calcium content, there is low shear stress (the peak value) and high toughness (area below the curve), while increasing the calcium content reduces toughness and increases the maximum shear stress. This modulation allows for control over the adhesive’s properties, allowing adjustments for either more rigid or plastic behavior as needed. [0218] Dopamine was also added to the powder (5%, w/w). While this addition doesn’t increase the initial adhesion values (since it's added as a monomer and is not yet covalently linked to SF), it is effective in ensuring long-lasting adhesion underwater. After one week, the coacervate with dopamine displayed 100 kPa of adhesion, whereas the plain coacervate was found to be already detached before the measurement (Fig. 8).
Example 3: Fe (III) Coacervates [0219] Fig. 9a shows a schematic representation of silk fibroin with abundant P-sheet content. Fig. 9b shows the structure of the polyphenol tannic acid. Introducing iron compounds (FeCh or Fe^Od to these powders enhances material cohesion through complexation with the tannic acid (TA) (Fig. 9c). Combination of silk fibroin and tannic acid solutions generates coacervates that display immediately underwater adhesion, forming a flexible adhesive (Fig. 9d). Given the high solubility of silk fibroin and tannic acid, the powders can be ground together to create a solid-state material that can also be compressed in a tablet format (Fig. 9g). Upon contact with water, the tablets transform into the fluid adhesive coacervate (Fig. 9e). Even in the solid state, mixing silk fibroin and tannic acid powders induces a color change, signifying molecular binding with tannic acid’s galloyl functionalities (Fig. 9f).
[0220] As these composites constitute a hydrogen-bonded network, their viscosity and adhesive behavior are temperature-sensitive and begin to melt at 35 °C and 45 °C (Fig. 9h). Depending on the type and concentration of the iron compound in the mixture, the coacervates exhibit different optimal application temperatures, with an initial underwater adhesion strength of approximately -150 kPa and no curing time (Fig. 9i, Fig. 9j, Fig. 9k).
[0221] The silk-based coacervates are applied in a viscous solid form, which can be dispensed from a syringe or similar system. The viscosity is contingent on the composition and temperature of the coacervates. A lower viscosity facilitates the coacervate to fill the irregularities of a rough surface while displacing water. Conversely, higher viscosity is a factor in preventing cohesive failure, ensuring optimal adhesive performance. From our current measurements, we observe that salinity does not impact adhesion performance, and the coacervate in powder form can be activated with either distilled or seawater, yielding consistent results. When heated before application, they attain strengths of up to 500 kPa at 20 °C and remain stable for up to one month.
[0222] In conclusion, silk and tannic acid can be transformed into a solid-state tablet that transitions into an underwater adhesive upon contact with water or seawater. Silk-based coacervates exhibit melting upon healing and solidification upon cooling. Viscosity at a specific temperature can be regulated by adjusting the silk’s molecular weight or iron content.
[0223] Fig. 10 shows the coacervate adhesive strengths of up to 150 kPa initially and up to 500 kPa after a few hours underwater, ensuring prolonged adhesion on the order of months. In buffered distilled water, adhesion is between 300 kPa and 400 kPa and is also stable for months. In sea water, the initial performance and endurance of plain coacervate is reduced (Fig. 11). Iron (III) compounds can be used as crosslinkers as they form complexes with tannic acid’s galloyl moieties. The formation of iron complexes is confirmed by the change of color of the coacervate. Overall, iron (III) compounds improve initial adhesion and durability. [0224] Using alternative iron (III) compounds allows adjustment of the mechanical properties of the adhesive as shown in Fig. 12. The size of iron (III) additives modulates the mechanical properties of the adhesive in underwater environments by changing the behavior of the adhesive from liquid like pressure sensitive adhesive to elastic solids. Iron (III) compounds can be used to finely tune the mechanical behavior of the adhesive by controlling particle size.
[0225] As silk fibroin-tannic acid (SF-TA) coacervates are hydrogen bonded networks, their viscosity is affected by temperature (Fig. 13a). A probe tack test is depicted in Fig. 13B. Below 10 °C, the SF-TA coacervates are rigid and do not display adhesive behavior, but the SF-TA coacervates melt and are sticky if heated to 15 °C to 30 °C. Above this temperature the SF-TA coacervates behave more like a liquid, reducing their adhesion performance. To extend the temperature range of successful adhesion, iron compounds were incorporated into the coacervates to influence their glass transition temperature, enabling them to exhibit adhesion at higher temperatures. This leads to an increase in rigidity, rendering the iron-based composition less adhesive at lower temperatures as shown in Fig. 14 and Fig. 15. Fig. 14c shows the adhesion of iron oxide coacervate underwater under tensile stress. Fig. 15a-d compares 10% FeCh coacervate to plain coacervate. The plain coacervate melts at a lower temperature and overall exhibits lower adhesion at all temperatures. [0226] The temperature dependence of adhesion can be tailored. At low temperature, the 10% FeCI-, coacervate is too rigid and cannot interact with the target surface which results in adhesive failure as shown in Fig. 16a, Fig. 16b, and Fig. 16c. Increasing temperature induces behavior like a viscoelastic fluid capable of interacting with underwater surfaces (cohesive failure). Underwater adhesion can be tuned for a specific temperature window - Fig. 16c shows highest adhesion for 0.1 % FeCh at 24 °C while 10% FeCh is highest at 44 °C. Fig 16d shows performance of the adhesives immediately, 1 day after application, and 1 week after application in seawater.
Example 4: Adhesion on Different Surfaces
[0227] The initial performance of the adhesive is not greatly affected by the interacting surface energy and shows bonding energy between 20 J/m2 and 80 I/m2. Even on Teflon, while underwater, the coacervate shows an initial bonding energy of 50 I/m2 without any curing. Fig. 17 shows (Fig. 17a) and quantifies (Fig. 17b) adhesion on aluminum, glass, polycarbonate, wood, PLA, polystyrene, and Teflon.
Example 5: Adhesive Tablets
[0228] Fig. 18 shows an overview of an adhesive tablet form of coacervates made of silk fibroin powder, iron (III) compound powder, and tannic acid powder. Briefly, silk fibroin powder, an iron (III) compound powder, and tannic acid powder are mixed and ground together to form a solid-state coacervate powder. The solid-state coacervate powder is then compressed into dry tablets, which activate upon contact with water or seawater to form an underwater viscous adhesive, proving the versatility of this material.
Example 6: Constant Buoyant Stress
[0229] Fig. 19 summarizes the experimental setup and results of a constant buoyant stress study. The GPS holder design, shown in Fig. 19a, fits the SPOT-253 tag by Wildlife Computers®. The versatility of the coacervate adhesive allows for free selection of the material to be used in the GPS adapter. A silicone GPS holder prototype stuck to a glass wall with the coacervate adhesive under constant water flow of 5.3 m/s is shown in Fig. 19b. A mold was created and cast in flexible material. Next, flow testing was conducted under a constant water stream. The adapter material was tested. Temperature and longevity were characterized. Fig. 19c shows various adhesives used in the experiment. Fig. 19d shows the time to detach under buoyant stress of the adhesives tested. Overall, performance under constant fluid flow or high-load stress is limited. There was no distinct trend observed under constant stress between iron compound additives at any concentration. As described herein, the adhesive works effectively on a wide range of material substrates. The addition of iron compounds manipulates the working temperature of the adhesive. The adhesive maintains its strength over time when stored underwater.
REFERENCE
[0230] U.S. Pat. App. No. 18/967,019 is hereby incorporated herein in its entirety by reference for all purposes.
[0231] EQUIVALENTS AND SCOPE
[0232] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combinations (or subcombinations) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

CLAIMS What is claimed is:
1. A solid state composition comprising a coacervate-forming pair of materials consisting of silk fibroin and a silk-complementary material that is capable of forming a coacervate with the silk fibroin, the solid state composition comprising a mixture of silk fibroin powder comprising the silk fibroin and complementary powder comprising the silk-complementary material, wherein the silk fibroin and the silk-complementary material are present in the solid state composition in a weight ratio of between 1:10 and 100:1.
2. The solid state composition of claim 1, wherein hydrating and agitating the solid state composition forms the coacervate.
3. The solid state composition of claim 1, wherein the silk fibroin powder and the complementary powder each independently have an average particle diameter (D50) of between 1 pm and 100 microns.
4. The solid state composition of the immediately preceding claim, wherein D50 is between 1 pm and 10 pm, between 10 pm and 25 pm, between 25 pm and 50 pm, or between 50 pm and 100 pm.
5. The solid state composition of claim 1, wherein the silk fibroin is a dopamine-substituted silk fibroin.
6. The solid state composition of claim 2, wherein the hydrating and mixing to form the coacervate can be completed in under 5 seconds.
7. The solid state composition of claim 1, wherein the solid state composition retains capacity to form the coacervate after storage for a predetermined storage time and upon contact with an aqueous solvent after the predetermined storage time.
8. The solid state composition of the immediately preceding claim, wherein the predetermined storage time is 1 month or more.
9. The solid state composition of the immediately preceding claim, wherein the predetermined storage time is 1 year or more.
10. The solid state composition of any one of the preceding claims, further comprising an additive.
11. The solid state composition of the immediately preceding claim, wherein the additive is at least one of calcium chloride, a metal ion, a nanoparticle, iron oxide, or iron chloride.
12. The solid state composition of any one of the preceding claims, wherein the silk fibroin is dopamine-substituted silk fibroin.
13. A solid state composition comprising a coacervate-forming pair of materials consisting of a first material and a complementary material that is capable of forming a coacervate with the first material, the solid state composition comprising or consisting essentially of a mixture of a first powder comprising the first material and a second powder comprising the complementary material, wherein hydrating and agitating the solid state composition forms the coacervate.
14. A method of using the solid state composition of any one of the preceding claims, the method comprising hydrating and agitating the solid state composition, thereby forming the coacervate.
15. The method of the immediately preceding claim, wherein hydrating comprises mixing with water or an aqueous solution.
16. The method of the immediately preceding claim, wherein the mixing occurs within a syringe.
17. The method of any of claims 14 to 16, further comprising adhering two surfaces to one another with a dense phase of the coacervate.
18. A solid state composition comprising: a solid-state coacervate formed by grinding a combination of a freeze-dried silk fibroin and tannic acid in a ratio by weight of silk fibroin protein to tannic acid of between 1:10 and 100: 1.
19. The solid state composition of claim 18, further comprising an aqueous solvent, wherein contacting the solid-state coacervate with the aqueous solvent obtains a coacervate-based adhesive.
20. The solid state composition of claim 18, wherein the solid state composition contains only components that are present in naturally-occurring organisms.
21. The solid state composition of any one of the preceding claims, wherein the solid-state coacervate retains capacity to form the solid state composition after storage for a predetermined storage time and upon contact with an aqueous solvent after the predetermined storage time.
22. The solid state composition of the immediately preceding claim, wherein the predetermined storage time is 1 month or more.
23. The solid state composition of the immediately preceding claim, wherein the predetermined storage time is 1 year or more.
24. The solid state composition of claim 19, wherein the aqueous solvent is one of fresh water or sea water.
25. The solid state composition of any one of the preceding claims, further comprising an additive.
26. The solid state composition of the immediately preceding claim, wherein the additive is at least one of calcium chloride, a metal ion, a nanoparticle, iron oxide, or iron chloride.
27. The solid state composition of any one of the preceding claims, wherein the freeze-dried silk fibroin is dopamine-substituted.
28. A method of making a solid state composition, the method comprising: a) grinding together a freeze-dried silk fibroin and tannic acid in a ratio by weight of silk fibroin protein to tannic acid of between 1 :10 and 100:1, thereby forming a solid-state coacervate.
29. The method of claim 28, further comprising, contacting the solid-state coacervate with an aqueous solvent to obtain the coacervate-based adhesive.
30. The method of claim 28, wherein the ratio is substantially 1:1.
31. The solid state composition or the method of any one of the preceding claims, wherein the solid state composition is pre-heated prior to its application.
32. The solid state composition or the method of any one of the preceding claims, wherein the solid state composition has a dry adhesive strength that is greater than a first comparison dry adhesive strength, wherein the first comparison dry adhesive strength is from a first comparison solid state composition that is not pre-heated and is otherwise identical to the solid state composition, wherein the dry adhesive strength is optionally 5% greater, 10% greater, 25% greater, 50% greater, 75% greater, or 100% greater than the first comparison dry adhesive strength, wherein the dry adhesive strength is optionally at least 500 kPa, at least 750 kPa, at least 1 MPa, at least 2 MPa, at least 3 MPa, or at least 4 MPa.
33. The solid state composition or the method of any one of the preceding claims, wherein the solid state composition has a wet adhesive strength that is greater than a first comparison wet adhesive strength, wherein the first comparison wet adhesive strength is from a first comparison solid state composition that is not pre-heated and is otherwise identical to the solid state composition, wherein the wet adhesive strength is optionally 5% greater, 10% greater, 25% greater, 50% greater, 75% greater, or 100% greater than the first comparison wet adhesive strength, wherein the wet adhesive strength is optionally at least 200 kPa, at least 500 kPa, at least 750 kPa, at least 1 MPa, or at least 2 MPa.
34. The solid state composition or the method of any one of the preceding claims, wherein the solid state composition has a wet adhesive strength that is greater than a second comparison wet adhesive strength, wherein the second comparison wet adhesive strength is from a first comparison solid state composition that has not been immersed in an aqueous environment for at least 18 hours and is otherwise identical to the solid state composition, wherein the wet adhesive strength is at least 100 times greater, at least 75 times greater, at least 50 times greater, at least 20 times greater, or at least 10 times greater than the second comparison wet adhesive strength.
35. The solid state composition or the method of any one of the preceding claims, wherein the solid state composition includes dopamine and has a wet adhesive duration that is longer than a second comparison wet adhesive duration, wherein the second comparison wet adhesive duration is from a first comparison solid state composition that does not include dopamine and is otherwise identical to the solid state composition, wherein the wet adhesive duration is at least 10 times longer, at least 7 times longer, at least 5 times longer, at least 3 times longer, or at least 2 times longer than the second comparison wet adhesive strength.
36. The solid state composition or the method of any one of claim 18 to the preceding claims, wherein the freeze-dried silk fibroin is a dopamine-substituted silk fibroin, and the dopaminesubstituted silk fibroin has a degree of polydopamine-substitution of between 5% and 50%, between 10% and 40%, or between 25% and 35%, wherein the degree of polydopamine-substitution is measured as a percentage by weight of polydopamine substituents related to the weight of a silk fibroin backbone.
37. The solid state composition or the method of the immediately preceding claim, wherein the polydopamine-substituted silk fibroin is polydopamine-substituted at one or more target amino acids, wherein the one or more target amino acids are selected from the group consisting of cysteine, tyrosine, arginine, lysine, histidine, phenylalanine, proline, and combinations thereof.
38. The solid state composition or the method of claims 36 to 37, wherein the polydopamine- substituted silk fibroin is polydopamine-substituted at one or more tyrosines.
39. The solid state composition or the method of claims 36 to 38, wherein the polydopamine- substituted silk fibroin is substituted with polydopamine having 15, 13, 12, 10, 9, or 8 polymeric units or less.
40. The solid state composition or the method of any one of claims 36 to 39, wherein the polydopamine-substituted silk fibroin is made by mixing silk fibroin with soluble dopamine.
41. The solid state composition or the method of claims 36 to 40, wherein the polydopamine- substituted silk fibroin has a structure that is indistinguishable from a comparison structure that is made by mixing comparison silk fibroin with a comparison soluble dopamine.
42. The solid state composition or the method of claim 40 or 41 , wherein the soluble dopamine and/or the comparison soluble dopamine is dopamine hydrochloride.
43. The solid state composition or the method of any one of claims 40 to 42, wherein mixing silk fibroin with soluble dopamine uses a ratio by weight of silk fibroin to soluble polydopamine of between 1 :2 and 10:1, between 1:1 and 5:1, or between 1.25:1 and 3: 1, including but not limited to, at least 1 :2, at least 1.5:1, at least 1:1, at least 1.1 : 1, at least 1.2:1 , at least 1.25: 1, at least 1.3:1, at least 1.4:1, at least 1.5:1, a least 1.75:1, at least 2:1, at least 3:1, or at least 4:1 and at most 10:1, at most 9:1, at most 8:1, at most 7:1, at most 6:1, at most 5: 1, at most 4:1, at most 3:1, at most 2: 1, or at most 1 : 1.
44. The solid state composition or the method of any one of claims 40 to 43, wherein the mixing comparison silk fibroin with comparison soluble polydopamine in a comparison aqueous solution uses a ratio by weight of comparison silk fibroin to comparison soluble polydopamine of between 1:2 and 10:1, between 1 :1 and 5:1, or between 1.25:1 and 3:1, including but not limited to, at least 1:2, at least 1.5:1, at least 1:1, at least 1.1 :1, at least 1.2:1, at least 1.25:1, at least 1.3:1, at least 1.4:1, at least 1.5:1, a least 1.75: 1, at least 2:1, at least 3:1, or at least 4: 1 and at most 10: 1, at most 9:1, at most 8: 1, at most 7:1, at most 6: 1, at most 5: 1, at most 4:1, at most 3: 1, at most 2:1, or at most 1 : 1.
45. The solid state composition or the method of any one of the preceding claims, wherein applying the solid state composition underwater provides instantaneous adhesion without a need for curing.
46. The solid state composition or the method of any one of the preceding claims, wherein the solid state composition provides adhesion between two articles when submerged in simulated sea water.
47. The solid state composition or the method of any one of the preceding claims, wherein the solid state composition provides adhesion between two articles when submerged in distilled water.
48. The solid state composition or the method of any one of the preceding claims, wherein the solid state composition provides adhesion between two articles when submerged in either distilled water or simulated sea water.
49. The solid state composition or the method of any one of the preceding claims, wherein the silk fibroin has a silk fibroin backbone with a weight average or number average molecular weight of between 25 kDa and 150 kDa.
50. The solid state composition or the method of claim 49, wherein the silk fibroin backbone has a weight average or number average molecular weight of between 30 kDa and 125 kDa.
51. The solid state composition or the method of claim 49, wherein the silk fibroin backbone has a weight average or number average molecular weight of between 35 kDa and 100 kDa.
52. The solid state composition or the method of claim 49, wherein the silk fibroin backbone has a weight average or number average molecular weight of between 25 kDa and 75 kDa.
53. The solid state composition or the method of claim 49, wherein the silk fibroin backbone has a weight average or number average molecular weight of between 100 kDa and 150 kDa.
54. The solid state composition or the method of claim 49, wherein the silk fibroin backbone has a weight average or number average molecular weight of between 50 kDa and 125 kDa.
55. The solid state composition or the method of claim 49, wherein the silk fibroin backbone has a weight average or number average molecular weight of between 40 kDa and 90 kDa.
56. The solid state composition or the method of claim 49, wherein the silk fibroin backbone has a weight average or number average molecular weight of between 55 kDa and 105 kDa.
57. The solid state composition or the method of claim 49, wherein the silk fibroin backbone has a weight average or number average molecular weight of between 45 kDa and 65 kDa.
58. The solid state composition or the method of any one of the preceding claims, wherein the solid state composition provides adhesion between surfaces without addition of metal cations.
59. The solid state composition or the method of claim 58, wherein the solid state composition provides stronger adhesion properties between surfaces upon addition of the metal cations.
60. The solid state composition or the method of any one of the preceding claims, wherein the solid state composition further comprises a therapeutic agent, wherein the therapeutic agent is optionally an antibiotic.
61. A method of using the solid state composition or the solid state composition made by the method of any one of the preceding claims, the method comprising: contacting a first article and a second article together with an adhesive amount of the solid state composition, thereby adhering the first and second articles to one another.
62. The method of claim 61, wherein the first article is a marine animal.
63. The method of claim 61 or 62, wherein the second article is a water-permeable tag.
64. The method of any one of claims 61 to 63, wherein the second article includes an electronic component.
65. A delivery device or syringe comprising the solid state composition of or made by the method of any of claims 1 to 13, 18 to 60, or 68 to 72.
66. A method of using the solid state composition of or made by the method of any one of the preceding claims, the method comprising: contacting the solid state composition with an aqueous solvent within a delivery device or syringe to obtain a coacervate-based adhesive; and dispensing the coacervate-based adhesive from the delivery device or syringe onto at least one surface of a first article or a second article and contacting the first and second articles together with the coacervate-based adhesive on the at least one surface, thereby adhering the first and second articles to one another.
67. The method of claim 66, wherein the contacting can be completed in under 5 seconds.
68. The solid state composition of or made by the method of any one of claims 1 to 13 or 18 to
60, wherein the solid state composition further comprises an additive.
69. The solid state composition of the immediately preceding claim, wherein the additive mass is between 10% and 80% of the mass of the solid state composition, including but not limited to at least 10%, at least 30%, at least 50%, or at least 70%, and at most 80%, at most 60%, at most 40%, or at most 20%.
70. The solid state composition of either one of the two immediately preceding claims, wherein the additive comprises at least one of a gas-evolving molecule (e.g., succinic acid or sodium carbonate) or a salt that produces an exothermic reaction with water (e.g., magnesium chloride).
71. The solid state composition of or made by the method of any one of claims 1 to 13, 18 to 60, or 68 to 70, wherein the solid state composition is compressed into a solid state tablet.
72. A method of making a solid state composition tablet, the method comprising compressing the solid state composition of or made by the method of any one of claims 1 to 13, 18 to 60, or 68 to 71 to form a solid state tablet, wherein the compression optionally comprises applying a pressure between 0.001 MPa and 5 MPa, including but not limited to, at least 0.001 MP, at least 0.01 MPa, 0.1 MPa, or at least 1 MPa, and at most 5 MPa, at most 2 MPa, at most 0.5 MPa, at most 0.05 MPa, or at most 0.005 MPa.
PCT/US2025/027523 2024-05-02 2025-05-02 Solid-state coacervate adhesives and methods of making and using the same Pending WO2025231379A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463641806P 2024-05-02 2024-05-02
US63/641,806 2024-05-02

Publications (1)

Publication Number Publication Date
WO2025231379A1 true WO2025231379A1 (en) 2025-11-06

Family

ID=97562375

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/027523 Pending WO2025231379A1 (en) 2024-05-02 2025-05-02 Solid-state coacervate adhesives and methods of making and using the same

Country Status (1)

Country Link
WO (1) WO2025231379A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024006607A2 (en) * 2022-06-03 2024-01-04 Trustees Of Tufts College Underwater animal tagging adhesives and methods of making and using the same
US20240082405A1 (en) * 2013-03-15 2024-03-14 Trustees Of Tufts College Low Molecular Weight Silk Compositions and Stabilizing Silk Compositions

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20240082405A1 (en) * 2013-03-15 2024-03-14 Trustees Of Tufts College Low Molecular Weight Silk Compositions and Stabilizing Silk Compositions
WO2024006607A2 (en) * 2022-06-03 2024-01-04 Trustees Of Tufts College Underwater animal tagging adhesives and methods of making and using the same

Similar Documents

Publication Publication Date Title
US10731046B2 (en) Biopolymer-based inks and use thereof
Zhou et al. Bioglass activated albumin hydrogels for wound healing
JP6006214B2 (en) Adhesive containing tannin, polyethylene glycol and water, lower alcohol or mixtures thereof
EP2970411B1 (en) Low molecular weight silk compositions and stabilizing silk compositions
Jeon et al. Body temperature-activated protein-based injectable adhesive hydrogel incorporated with decellularized adipose extracellular matrix for tissue-specific regenerative stem cell therapy
US20210155812A1 (en) Silk ink compositions and methods of making and using the same
US20170218228A1 (en) Three Dimensional Printing of Bio-Ink Compositions
WO2018071639A1 (en) Three-dimensional (3-d) printing inks made from natural extracellular matrix molecules
Lee et al. Bioinspired nanoparticulate medical glues for minimally invasive tissue repair
Wu et al. Formation and degradation tracking of a composite hydrogel based on UCNPs@ PDA
Fiejdasz et al. Biopolymer-based hydrogels as injectable materials for tissue repair scaffolds
CN102178978B (en) Medical adhesive and preparation method of medical adhesive
WO2025231379A1 (en) Solid-state coacervate adhesives and methods of making and using the same
Shkand et al. Assessment of alginate hydrogel degradation in biological tissue using viscosity-sensitive fluorescent dyes
WO2024145272A1 (en) Modular bone scaffold
Kiryukhin et al. Arrays of Biocompatible and Mechanically Robust Microchambers Made of Protein–Polyphenol–Clay Multilayer Films
WO2024158919A2 (en) Silk bioplastics and method of making same
WO2025054426A1 (en) Edible transferable holographic image films and methods
Jawadi et al. Bio-Inspired Muco-Adhesive Polymers for Drug Delivery Applications. Polymers 2022, 14, 5459
Ghavamzadeh et al. Bioadhesion and biocompatibility evaluations of gelatin and polyacrylic acid as a crosslinked hydrogel in vitro
WO2025144993A1 (en) Transdermal optical silk microneedle sensors for continuous monitoring for physiological analytes in interstitial fluid
WO2025193717A1 (en) Sound mediated assembly of biopolymers
WO2024259420A2 (en) Systems and methods of dry bioprinting with biopolymers
WO2025155859A1 (en) Dual crosslinked silk-based materials for in situ deposition or printing
US20250325495A1 (en) Silk nanoparticle synthesis: tuning size, dispersity, and surface chemistry for drug delivery

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25798816

Country of ref document: EP

Kind code of ref document: A1