Highly porous spider silk fiber

Abstract

At least 100m 2 A composition comprising porous major ampullate spidroin protein (MaSp)-based fibers characterized by a BET surface area of ​​1000 nm / g is disclosed. Additionally, articles comprising the composition and methods for making the same are disclosed.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to PCT International Patent Application No. PCT / IL2020 / 050752, filed on 5 July 2020, and U.S. Provisional Patent Application No. 63 / 134,343, filed on 6 January 2021. The entire contents of the above documents are incorporated by reference as if they were fully described herein.

[0002] In some embodiments, the present invention relates to compositions comprising MaSp (large bottle-shaped glandular spirulin) protein fibers, the preparation thereof, and methods for using the same. [Background technology]

[0003] Dragline spider silk is known in the art as the silk used by orbweb-spinning spiders to construct the frame and radius of their webs, as well as to create lifelines for falling or escaping danger. To enable these tasks, dragline fibers exhibit remarkably high toughness due to a combination of high elasticity and strength, and are therefore considered the strongest fibers, whether natural or artificial. For example, dragline is six times stronger than high-tensile steel in its diameter and three times stronger than Kevlar, one of the strongest synthetic fibers ever created. Dragline silk consists of two main polypeptides, usually called large vial-shaped glandular spidolin (MaSp) 1 and 2, and ADF-3 and ADF-4 in the garden spider (Araneus diadematus). These proteins have apparent molecular weights ranging from 200 to 720 kDa, depending on the sample age and analytical conditions. Known dragline silk spiroins consist of highly repeating blocks alternating between alanine-rich segments that form crystalline β-sheets within the fiber and glycine-rich segments that are more flexible and largely lack ordered structure. The C-terminal region is non-repeating and highly conserved across species, forming an α-helix structure. The N-terminal region of dragline silk proteins has also been found to be highly conserved among different spiroins and across different spider species. Numerous attempts have been made to synthesize spider silk using bacterial, yeast, plant, and mammalian cells in tissue culture, and further, genetic engineering using transgenic goats. The need for improved compositions and methods to produce fibers with mechanical properties similar to natural spider silk remains unaddressed. [Overview of the project]

[0004] According to one aspect, the present invention provides a minimum of 100m 2 The present invention provides a composition comprising porous, large bottle-shaped glandular spidoin protein (MaSp) fibers characterized by a BET surface area of ​​1 / g and a residual amount of organic solvent.

[0005] In some embodiments, the MaSp fibers are in the form of particles having a size in the range of 0.5 μm to 2 μm.

[0006] In some embodiments, the organic solvent can form an azeotropic mixture with water.

[0007] In some embodiments, the organic solvent includes t-butanol.

[0008] In some embodiments, the composition further comprises additional agents.

[0009] In some embodiments, the w / w concentration of further agents in the composition is 1 to 80%.

[0010] In some embodiments, the w / w ratio of the additional agent to the porous MaSp fiber is 100:1 to 1:10.

[0011] In some embodiments, the rate of further drug release from the composition is reduced by at least 10% compared to the control.

[0012] In some embodiments, the composition further comprises a thermoplastic polymer selected from polyester, polyamide, polyol, polyurethane, polyethylene, nylon, polyolefin, polyacrylate, polycarbonate, polylactic acid (PLA) or copolymers thereof, polycaprolactone (PCL), rubber, cellulose, or any combination thereof.

[0013] In some embodiments, the weight / weight (w / w) ratio of the MaSp fiber to the polymer is 0.01:1 to 1:1.

[0014] In some embodiments, the composition contains 0.01% to 50% (w / w) of MaSp fibers.

[0015] In some embodiments, the tensile strength of the composition is increased by at least 20% compared to the control.

[0016] In another embodiment, a method for obtaining dried large vial-shaped glandular spidoin protein (MaSp) fibers comprises (a) mixing MaSp fibers with a liquid containing an organic solvent to obtain a mixture, and (b) providing the mixture under conditions suitable for substantially removing the liquid from the mixture, thereby obtaining at least 100 m 2 A method is provided for obtaining dried MaSp fibers characterized by a BET surface area of ​​1 / g and a residual amount of organic solvent.

[0017] In some embodiments, the liquid optionally includes an aqueous solvent.

[0018] In some embodiments, the organic solvent can form an azeotropic mixture with water.

[0019] In some embodiments, the organic solvent includes t-butanol.

[0020] In another embodiment, dried porous bottle-shaped glandular spidoin protein (MaSp) fibers obtained by the method of the present invention are provided.

[0021] In some embodiments, the dried MaSp fibers are the MaSp fibers of the composition of the present invention.

[0022] In some embodiments, the dried MaSp fibers feature either (i) an improved loading capacity for further agents, or (ii) a sustained release profile.

[0023] In some embodiments, the improved load capacity includes a w / w ratio of additional agents to porous MaSp fibers of 100:1 to 1:10.

[0024] In another embodiment, an article comprising the composition of the present invention or the dried MaSp-based fibers of the present invention is provided.

[0025] In some embodiments, the article may be in the form of reinforced plastic, a bottle, a container, a package, a cable, a tube, a film, a rope, a thread, or a fabric.

[0026] In some embodiments, the article features at least one improved mechanical property compared to the properties of a control article, where the property is selected from the group consisting of Young's modulus, tensile strength, fracture strain, yield point, toughness, work of fracture, impact strength, tear strength, flexural modulus, flexural strain and stress at a particular elongation, wear, UV resistance, and gas permeability.

[0027] In some embodiments, the article further includes a carrier.

[0028] In another embodiment, a method is provided for adding further agents to a subject, comprising administering an article of the present invention to the subject, thereby adding further agents to the subject.

[0029] Unless otherwise specified, all technical and / or scientific terms used herein have the same meaning as commonly understood by those skilled in the art in the industry to which the present invention pertains. Similar or equivalent methods and materials may be used in the implementation or testing of embodiments of the present invention, but exemplary methods and / or materials are described below. In case of any conflict, the present specification, including definitions, shall prevail. Furthermore, materials, methods, and examples are illustrative and not necessarily limiting.

[0030] Further embodiments and the full scope of the present invention will become apparent from the detailed description given below. However, it should be understood that the detailed description and specific examples illustrating preferred embodiments of the present invention are given merely as examples, as various variations and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawing]

[0031] [Figure 1]Figures 1A to 1E are SEM images of lyophilized porous MaSp fibers from t-butanol suspensions (Figures 1A, 1C, and 1E) and H2O suspensions (Figures 1B and 1D) at different magnifications. [Figure 2] This graph shows the stress-strain curves of polyurethane (PU) (E394POTA) reinforced with t-butanol and freeze-dried 15% SVX-E in water, compared to the control (original polyurethane (PU) (E394POTA)). [Figure 3] Figures 3A and 3B are graphs showing the release profiles of hyaluronic acid (HA) from SVX-E freeze-dried with t-butanol (Figure 3A) and SVX-E freeze-dried with water (Figure 3B). [Figure 4] Figures 4A and 4B are graphs showing the release profiles of glycolic acid (GA) from SVX-E freeze-dried with t-butanol (Figure 4A) and SVX-E freeze-dried with water (Figure 4B). [Figure 5] The differential scanning calorimetry (DSC) curve of spider silk protein (SVX-E) expressed in bacteria is shown. [Figure 6] The differential scanning calorimetry (DSC) curves of the SVX-E are shown during temperature increases from 25°C to 280°C (Curve 1), cooling to 50°C (Curve 2), and further temperature increases to 350°C (Curve 3). [Figure 7] Figures 7A and 7B show the thermogravimetric analysis (TGA) curves of spider silk protein (SVX-E) expressed in bacteria. [Modes for carrying out the invention]

[0032] According to some embodiments, the present invention provides highly porous, large bottle-shaped glandular spidoin protein (MaSp) fiber.

[0033] According to some embodiments, the present invention provides at least 100m 2The present invention provides a composition comprising porous, large bottle-shaped glandular spidoin protein (MaSp) fibers characterized by a BET surface area of ​​1 / g and a residual amount of organic solvent.

[0034] The present invention is partly based on the remarkable finding that the tensile strength of a composition comprising porous MaSp fibers lyophilized in an organic solvent and reinforced with a thermoplastic polymer is at least 20% higher than that of a control. The present invention is partly based on the remarkable finding that the tensile strength of a composition comprising porous MaSp fibers lyophilized in t-butanol and reinforced with a thermoplastic polymer is at least 20% higher than that of MaSp fibers lyophilized in water.

[0035] The present invention is partly based on the surprising discovery that a composition containing porous MaSp fibers freeze-dried with an organic solvent features a larger BET surface area compared to porous MaSp fibers freeze-dried with water. The present invention is partly based on the surprising discovery that a composition containing porous MaSp fibers freeze-dried with t-butanol features a larger BET surface area compared to porous MaSp fibers freeze-dried with water.

[0036] The present invention is based in part on the surprising discovery that a composition comprising porous MaSp-based fibers that are dried from an organic solvent and fortified with a further agent exhibits a slower release rate of the further agent as compared to a control. The present invention is based in part on the surprising discovery that a composition comprising porous MaSp-based fibers that are dried from t-butanol and fortified with a further agent (such as hyaluronic acid or glycolic acid) exhibits a slower release rate of the further agent as compared to MaSp-based fibers that are dried from water and fortified with a further agent. The present invention is based in part on the surprising discovery that a composition comprising porous MaSp-based fibers that are lyophilized from an organic solvent and fortified with a further agent exhibits a slower release rate of the further agent as compared to a control. The present invention is based in part on the surprising discovery that a composition comprising porous MaSp-based fibers that are lyophilized from t-butanol and fortified with a further agent (such as hyaluronic acid or glycolic acid) exhibits a slower release rate of the further agent as compared to MaSp-based fibers that are lyophilized from water and fortified with a further agent (illustrated in FIGS. 3A-3B and FIGS. 4-4B).

[0037] Composition According to some embodiments, a composition comprising porous major ampullate spidroin (MaSp)-based fibers is provided. In some embodiments, the fibers are characterized by a BET surface area of at least 100 m 2 / g. In some embodiments, the composition comprises a residual amount of an organic solvent. In some embodiments, the fibers of the present invention are characterized by a BET surface area of at least 100 m 2 / g or about 180 m 2 / g or more and a residual amount of an organic solvent.

[0038] According to some embodiments, (i) a residual amount of an organic solvent, and (ii) at least 100 m 2 / g, at least 150 m 2 / g, at least 1 eighty m 2 / g, at least 200 m 2 / g, at least 210 m 2 / g, at least 250 m2 / g, at least 300m 2 / g, at least 350m 2 / g, at least 400m 2 / g, at least 450m 2 / g, at least 500m 2 / g, at least 800m 2 / g, at least 1000m 2 / g, at least 1500m 2 / g, at least 2000m 2 / g, at least 2500m 2 / g or at least 5000m 2 A porous MaSp fiber is provided, characterized by a BET surface area of ​​ / g (including any value in between). Each possibility represents a distinct embodiment of the present invention.

[0039] In some embodiments, 100-5500m 2 / g, 100-5000m 2 / g, 100~2500m 2 / g, 100-2000m 2 / g, 100~1000m 2 / g, 100-500m 2 / g, 100-250m 2 / g, 100-200m 2 / g, 120~5500m 2 / g, 120~5000m 2 / g, 120~2500m 2 / g, 120-2000m 2 / g, 120~1000m 2 / g, 120~500m 2 / g, 120-250m 2 / g, 120-200m 2 / g, 150~5500m 2 / g, 150~5000m 2 / g, 150-2500m 2 / g, 150-2000m 2 / g, 150~1000m 2 / g, 150~500m 2 / g, 150-250m 2 / g or 150-200m 2Porous MaSp fibers are provided, characterized by a BET surface area of ​​ / g (including any range in between). Each possibility represents a distinct embodiment of the present invention. In some embodiments, the porous MaSp fibers are substantially devoid of organic solvents and / or water (e.g., devoid of trace amounts of organic solvents and / or water).

[0040] In one aspect of the present invention, (i) at least 100m 2 / g, at least 150m 2 / g, at least 180m 2 / g, at least 200m 2 / g, at least 210m 2 / g, at least 250m 2 / g, at least 300m 2 / g, at least 350m 2 / g, at least 400m 2 / g, at least 450m 2 / g, at least 500m 2 / g, at least 800m 2 / g, at least 1000m 2 / g, at least 1500m 2 / g, at least 2000m 2 / g, at least 2500m 2 / g or at least 5000m 2 A porous MaSp fiber is provided, characterized by (ii) a BET surface area of ​​1 / g (including any value in between), (ii) a residual amount of organic solvent as specified herein (e.g., t-butanol), and / or a water content as specified herein (e.g., less than 100 ppm).

[0041] In one aspect of the present invention, (i) at least 100m 2 / g, at least 150m 2 / g, at least 180m 2 / g, at least 200m 2 / g, at least 210m 2 / g, at least 250m 2 / g, at least 300m 2 / g, at least 350m 2 / g, at least 400m2 / g, at least 450m 2 / g, at least 500m 2 / g, at least 800m 2 / g, at least 1000m 2 / g, at least 1500m 2 / g, at least 2000m 2 / g, at least 2500m 2 / g or at least 5000m 2 (ii) BET surface area in g / g (including any value in between), (ii) DSC pattern (and / or T) as described herein. d (iii) a porous MaSp fiber is provided, characterized by (iii) a residual amount of organic solvent (e.g., tert-butanol) and / or a water content (e.g., less than 100 ppm) as specified herein. In some embodiments, the porous MaSp fiber comprises a plurality of MaSp polymers as specified herein. In some embodiments, the porous MaSp fiber is in the form of particles. In some embodiments, the porous MaSp fiber is in the form of particles in the composition of the present invention.

[0042] In some embodiments, porous MaSp fibers and / or compositions of the present invention comprising them are further characterized by improved properties compared to a control, where the improved properties are one or more of the following: additional drug loading capacity, extended release time of additional drugs encapsulated therein, and / or improved mechanical strength (e.g., tensile strength).

[0043] According to several embodiments, compositions comprising porous MaSp fibers in the form of particles are provided. In some embodiments, these particles are referred to herein as “porous particles”. In some embodiments, the particles have sizes (average particle diameters) in the range of 0.5 μm to 2 μm, 0.7 μm to 1.5 μm, 0.8 μm to 1.5 μm, 0.9 μm to 1.5 μm, 0.5 μm to 1 μm, 0.7 μm to 1 μm, 0.8 μm to 1 μm, 0.9 μm to 1 μm, 0.5 μm to 1.3 μm, 1.0 μm to 2 μm, 1.0 μm to 2 μm, 1.0 μm to 2 μm, 0.5 μm to 1.2 μm, 0.7 μm to 1.3 μm, 0.7 μm to 1.2 μm, or 0.9 μm to 1.2 μm (including any range in between). Each possibility represents a distinct embodiment of the present invention. In some embodiments, particle size refers to the average particle size in an aqueous solvent (e.g., an aqueous dispersion) as measured by laser diffraction (see the section on examples). In some embodiments, particle size refers to the number-average particle size. Those skilled in the art will know that the average particle size (e.g., number-average particle size) can be measured by laser diffraction or using SEM imaging. The number-average particle size can be calculated according to a well-known equation. In some embodiments, particle size refers to the dry particle size (e.g., the size of a particle substantially lacking an outer shell containing, for example, water molecules, as measured by SEM).

[0044] In some embodiments, the particles are spherical, elliptical, and / or cylindrical. In some embodiments, the particles have a length dimension (for example, along the longitudinal axis of the particle) in the range of 0.5 μm to 2 μm, 0.7 μm to 1.5 μm, 0.8 μm to 1.5 μm, 0.9 μm to 1.5 μm, 0.5 μm to 1 μm, 0.7 μm to 1 μm, 0.8 μm to 1 μm, 0.9 μm to 1 μm, 0.5 μm to 1.3 μm, 0.5 μm to 1.2 μm, 0.7 μm to 1.3 μm, 0.7 μm to 1.2 μm, or 0.9 μm to 1.2 μm (including any range in between). In some embodiments, the particles have a width dimension (for example, perpendicular to the longitudinal axis of the particle) in the range of 0.01 μm to 0.5 μm, 0.01 μm to 0.05 μm, 0.05 μm to 0.1 μm, 0.1 μm to 0.2 μm, 0.2 μm to 0.3 μm, 0.3 μm to 0.4 μm, and 0.4 μm to 0.5 μm (including any range in between).

[0045] In some embodiments, the porous MaSp fibers are in the form of multiple aggregated particles in the composition of the present invention. In some embodiments, the porous MaSp fibers are in the form of multiple distinct particles in the composition of the present invention. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, and at least 99.9% by weight of porous MaSp fibers (including any range between them) are in the form of multiple particles, where such particles are as described herein. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, and at least 99.9% by weight of the protein content of the composition are in the form of multiple particles.

[0046] In some embodiments, the MaSp fiber comprises or consists of an insoluble MaSp polymer. In some embodiments, the insoluble MaSp polymer is in the form of particles. In some embodiments, the insoluble MaSp polymer is insoluble in organic solvents. In some embodiments, the insoluble MaSp polymer is insoluble in aqueous solutions. The terms “MaSp polymer” and “MaSp fiber” as used herein are interchangeable herein.

[0047] As used herein, the term “insoluble” refers to a material that does not dissolve when exposed to an excess of solvent but can be dispersed to varying degrees. In some embodiments, “insoluble” refers to a material that is soluble in the solvent at concentrations of less than 10%, less than 5%, less than 2%, or less than 1%. In some embodiments, “insoluble” refers to a material that can only be partially dissolved in the solvent at a concentration of less than 0.01% by weight. The solvents according to the present invention include organic solvents and aqueous solutions. In some embodiments, the solvent includes an aqueous solution of a surfactant. In some embodiments, the solvent includes an aqueous solution of urea.

[0048] In some embodiments, the MaSp fiber is characterized by a specified differential scanning calorimetry (DSC) pattern. In some embodiments, “DSC pattern” is intended to refer to the location of the peak. In some embodiments, “peak” is intended to refer to the exothermic peak. Throughout this specification, “location of the peak” or “peak position” refers to the peak along the temperature axis in the thermogram pattern, and in some embodiments, it may refer to the location of the peak at any peak intensity. Those skilled in the art will understand that the data obtained in a DSC measurement is determined in part by the equipment used and the environmental conditions (e.g., humidity) at the time the measurement is performed.

[0049] In some embodiments, the MaSp fibers are characterized by a DSC pattern showing an endothermic peak in the range of 200°C to 280°C. In some embodiments, the disclosed compositions are characterized by a DSC pattern showing an endothermic peak in the range of 200°C to 270°C, 200°C to 260°C, 200°C to 250°C, 210°C to 280°C, 212°C to 280°C, 215°C to 280°C, 216°C to 280°C, 220°C to 280°C, 210°C to 250°C, 212°C to 250°C, 215°C to 250°C, 216°C to 250°C, 220°C to 250°C, 210°C to 245°C, 210°C to 242°C, or 215°C to 245°C (including any range in between). Each possibility represents a distinct embodiment of the present invention.

[0050] In some embodiments, the MaSp fibers are further characterized by DSC patterns exhibiting endothermic peaks at least 280°C–350°C, 290°C–350°C, 300°C–350°C, 310°C–350°C, 280°C–330°C, 290°C–330°C, 300°C–330°C, 310°C–330°C, or 320°C–330°C (including any range in between). Each possibility represents a distinct embodiment of the present invention.

[0051] In some embodiments, MaSp fibers have a glass transition temperature (T) of 200°C-250°C, 210°C-250°C, 220°C-250°C, 230°C-250°C, 200°C-240°C, 210°C-240°C, 220°C-240°C, 230°C-240°C, 200°C-230°C, or 210°C-230°C (including any range in between) as determined by DSC. g ) is characterized by each possibility representing a distinct embodiment of the present invention.

[0052] In some embodiments, MaSp fibers are T determined by DSC to be 260°C-320°C, 270°C-320°C, 280°C-320°C, 290°C-320°C, 260°C-310°C, 270°C-310°C, 280°C-310°C, or 290°C-310°C (including any range in between). g This is characterized by the fact that each possibility represents a distinct embodiment of the present invention.

[0053] In some embodiments, the MaSp fibers are characterized by a DSC pattern exhibiting an endothermic peak at least 5°C to 100°C, at least 10°C to 100°C, at least 15°C to 100°C, at least 12°C to 100°C, at least 25°C to 100°C, at least 5°C to 80°C, at least 10°C to 80°C, at least 15°C to 80°C, at least 12°C to 80°C, at least 25°C to 80°C, at least 5°C to 50°C, at least 10°C to 50°C, at least 15°C to 50°C, at least 12°C to 50°C, or at least 25°C to 50°C lower than the DSC pattern of the corresponding composition containing the (MaSp) fibers. Each possibility represents a distinct embodiment of the present invention.

[0054] In some embodiments, MaSp fibers lack DSC peaks in the range of approximately -100°C to approximately 190°C. In some embodiments, the disclosed compounds lack DSC peaks in the range of approximately -100°C to approximately 25°C. In some embodiments, the disclosed compositions are characterized by a DSC pattern showing that they lack exothermic peaks in the range of at least 40°C to 70°C.

[0055] In some embodiments, MaSp fibers lack a DSC peak in the range of approximately -100°C to approximately -50°C. In some embodiments, the disclosed compound lacks a DSC peak in the range of approximately -50°C to approximately 0°C. In some embodiments, the disclosed compound lacks a DSC peak in the range of approximately -0°C to approximately -25°C.

[0056] The term "decomposition temperature (T)" as used in this specification. d The term "thermal decomposition" refers to the temperature at which decomposition occurs. Thermal decomposition is a process of broad chemical species changes caused by heat.

[0057] The term "glass transition temperature (T)" as used herein refers to the glass transition temperature (T) used in this specification. g The term ) refers to a material that is an elastic and viscous amorphous liquid (T>T g ) from brittle, glassy amorphous solid (T <T grefers to the temperature at which it transitions to (1). This liquid - glass transition (or simply glass transition) is a reversible transition. The glass transition temperature (T g ), when it exists, is generally lower than the melting temperature (T m ) of the crystalline state of the material.

[0058] In some embodiments, the MaSp - based fibers are characterized by having an amide peak in the range of 1615 cm -1 to 1635 cm -1 as measured by FTIR analysis. In some embodiments, the disclosed compositions are characterized by having an amide peak in the range of 1620 cm -1 to 1635 cm -1 , 1620 cm -1 to 1630 cm -1 , 1621 cm -1 to 1630 cm -1 or 1620 cm -1 to 1625 cm -1 (including any range therebetween), each possibility representing a distinct embodiment of the invention.

[0059] In some embodiments, the MaSp - based fibers lack a peak in the range of 1700 cm -1 to 1800 cm -1 as measured by FTIR analysis.

[0060] In one embodiment, the MaSp - based polymer of the present invention aggregates by self - assembly. "Self - assembly" means that the monomers of the present invention, i.e., the synthetic spider silk protein, naturally bind to each other in an energetically favorable way under normal physiological conditions or at room temperature to create a macromolecular structure having the properties described herein. Furthermore, the MaSp - based fibers of the present invention are extremely elastic and, once aggregated, can withstand extreme chemical attacks such as solubilization in a 10% surfactant solution and boiling for at least 1 hour.

[0061] According to some embodiments, compositions are provided that comprise porous large bottle-shaped glandular spidoin protein (MaSp) fibers and a residual amount of organic solvent as described herein.

[0062] In some embodiments, the organic solvents disclosed herein can form an azeotrope with water.

[0063] As used herein, the terms “azeotropic mixture” or “azeotropic mixture” refer to a system of two or more components whose liquid and vapor compositions are equal at a given pressure and temperature. In practice, this means that the components of an azeotropic mixture have a constant boiling point or are essentially constant boiling points and are generally thermodynamically inseparable during phase transitions. The vapor composition formed by boiling or evaporation of an azeotropic mixture is identical or substantially identical to the original liquid composition. Solvents that can form azeotropic mixtures with water are well known, documented in the art, and will be apparent to those skilled in the art.

[0064] In some embodiments, the organic solvent is selected from the group consisting of ethanol, isopropyl alcohol, t-butanol, 2-butanol, n-butanol, acetonitrile, ethyl acetate, DMF, DMSO, THF, TFA, toluene, hexane, heptane, methyl ethyl ketone, diethyl ketone, methyl isobutyl ketone, dioxane, ethers (e.g., diethyl ether) and any combination thereof.

[0065] In some embodiments, the organic solvent is a Class 3 solvent. In some embodiments, the Class 3 solvent is selected from the group consisting of acetic acid, acetone, anisole, 1-butanol, 2-butanol, butyl acetate, tert-butyl methyl ether, cumene, dimethyl sulfoxide, ethanol, ethyl acetate, ethyl ether, ethyl formate, formic acid, heptane, isobutyl acetate, isopropyl acetate, methyl acetate, 3-methyl-1-butanol, methyl ethyl ketone, methyl isobutyl ketone, 2-methyl-1-propanol, pentane, 1-pentanol, 1-propanol, 2-propanol, propyl acetate, and tetrahydrofuran.

[0066] In some embodiments, the compositions and / or MaSp fibers of the present invention contain residual amounts of organic solvents within a pharmaceutically acceptable or cosmetically acceptable range. Accordingly, the residual amounts of organic solvents disclosed herein are pharmaceutically acceptable or, in some embodiments, cosmetically acceptable amounts in the compositions and / or fibers of the present invention.

[0067] In some embodiments, the compositions and / or MaSp fibers of the present invention contain an organic solvent in concentrations of 0.1 ppm to 100 ppm, 0.5 ppm to 100 ppm, 0.9 ppm to 100 ppm, 1 ppm to 100 ppm, 5 ppm to 100 ppm, 10 ppm to 100 ppm, 20 ppm to 100 ppm, 0.1 ppm to 50 ppm, 0.5 ppm to 50 ppm, 0.9 ppm to 50 ppm, 1 ppm to 50 ppm, 5 ppm to 50 ppm, 10 ppm to 50 ppm, 20 ppm to 50 ppm, 0.1 ppm to 20 ppm, 0.5 ppm to 20 ppm, 0.9 ppm to 20 ppm, 1 ppm to 20 ppm, 5 ppm to 20 ppm, or 10 ppm to 20 ppm (including any range in between). Each possibility represents a distinct embodiment of the present invention.

[0068] In some embodiments, the composition is substantially water-free. In some embodiments, the composition is characterized by a water content of less than 100 ppm, less than 70 ppm, less than 50 ppm, less than 40 ppm, less than 20 ppm, less than 10 ppm, less than 10 ppm, less than 5 ppm, less than 1 ppm, less than 0.5 ppm, or less than 0.1 ppm (including any value in between). Each possibility represents a separate embodiment of the present invention.

[0069] In some embodiments, the composition further comprises additional agents.

[0070] In some embodiments, further agents are selected from biological agents, pharmaceuticals, nutrients, and dietary supplements.

[0071] As used herein, the term “biological agent” (also known as “biomaterial”) refers to any substance or material of biological origin. For example, the term “biological agent” includes cells (including stem cells), proteins, peptides, or nucleic acids (including nucleic acid analogs). As used herein, the term “pharmaceutical product” (also known as “pharmaceutical compound”) refers to any biological or chemical substance that can be used to treat, cure, prevent, prophylactic or diagnose a pathological condition, such as a disease or disorder, or otherwise to improve physical, mental or spiritual health. Accordingly, as envisioned in the context of this invention, the term “pharmaceutical product” includes any agent having a therapeutic, diagnostic or preventive effect, i.e., any therapeutic, diagnostic or prophylactic agent.

[0072] Pharmaceuticals may be drugs that affect or are involved in tissue growth, cell growth, or cell differentiation; drugs that can induce biological effects such as immune responses; or drugs that can play any other role in one or more biological processes.

[0073] Non-exclusive examples of pharmaceuticals include, but are not limited to, antimicrobial agents such as antibacterial agents (e.g., antibiotics), antiviral or antifungal agents, immunosuppressants, anti-inflammatory agents, anti-allergic agents, anticoagulants, antirheumatic agents, antipsoriatic agents, sedatives, muscle relaxants, anti-migraine drugs, antidepressants, insecticides, growth factors, hormones, hormone antagonists, antibodies, adjuvants in combination with immunoactive compounds such as antibodies, antioxidants, proteins such as glycoproteins, lipoproteins or enzymes (e.g., hyaluronidase), polysaccharides, free radical scavengers, radiotherapeutic agents, photodynamic therapy agents, dyes (e.g., fluorescent dyes), contrast agents, disinfectants, preservatives, or any combination thereof.

[0074] The drug may also be a small molecule compound. The term "small molecule compound" refers to a molecule that can act on or influence biological processes. Small molecules can include any number of therapeutic agents currently known and in use, or they may be small molecules synthesized in a library of such molecules for screening of biological functions. Small molecule compounds typically have a molecular weight of less than about 5,000 daltons (Da), preferably less than about 2,500 Da, more preferably less than 1,000 Da, and most preferably less than about 500 Da.

[0075] As used herein, “nutrients” are substances used in the metabolism of organisms that must be taken in from the environment or from the chemicals necessary for an organism to live and grow. Organic nutrients include carbohydrates, fats, proteins (amino acids), and vitamins. Inorganic nutrients are food minerals, water, and oxygen. Preferred nutrients are macronutrients such as carbohydrates, amino acids, or proteins, and micronutrients such as vitamins.

[0076] Non-limiting examples of carbohydrates include, but are not limited to, monosaccharides or stereoisomers thereof such as glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, growth, idose, galactose, talose, dihydroxyacetone, erythrolose, ribulose, xylulose, psicose, fructose, sorbose, and tagatose; amino sugars such as galactosamine, glucosamine, sialic acid, and N-acetylglucosamine; sulfosaccharides such as sulfoquinovose; disaccharides such as sucrose, lactulose, lactose, maltose, trehalose, or maltobiose; and oligosaccharides such as fructooligosaccharides (FOS), galactooligosaccharides (GOS), or mannanoligosaccharides (MOS).

[0077] As used herein, the term “dietary supplement” (also known as “food supplement” or “nutritional supplement”) refers to a preparation intended to provide nutrients such as vitamins, minerals, fiber, fatty acids, or amino acids that are lacking or not consumed in sufficient quantities in a person’s diet.

[0078] Non-exclusive examples of dietary supplements include, but are not limited to, steroids or their derivatives such as dehydroepiandrosterone (DHEA) and pregnenolone, hormones such as melatonin, and hydrazine sulfate, caffeine, catechins, soy isoflavones, glucosamine, and coenzyme Q10. 10 Other substances include ephedrine alkaloids such as ephedrine, synephrine, norephedrine, or pseudoephedrine.

[0079] Further agents may be positively or negatively charged. Alternatively, further agents may be electrically neutral. Preferably, further agents are positively or negatively charged. The terms "positive charge" and "cation," and "negative charge" and "anion" can be used synonymously.

[0080] In some embodiments, MaSp polymer particles contain multiple pores (i.e., spaces or lumens) formed by the twisted polymer chains of the MaSp polymer. In some embodiments, the entangled MaSp polymers form a matrix. In some embodiments, further agents fill the matrix or at least some of the pores within the particles. In some embodiments, further agents are encapsulated in the multiple pores.

[0081] In some embodiments, the physical interaction is referred to as the encapsulation (i.e., capture) of further drugs within a matrix formed by the MaSp polymer. In some embodiments, the matrix is ​​bound to or in contact with the further drugs. In some embodiments, the (MaSp) fibers are bound to the further drugs.

[0082] In some embodiments, additional agents are stably encapsulated within multiple pores of the particles. In some embodiments, the stably encapsulated additional agents feature a sustained-release profile (e.g., on the surface of the application site or in a solution). In some embodiments, the particles encapsulating the additional agents substantially prevent the rapid release of the additional agents from there.

[0083] In some embodiments, the stably encapsulated additional agent features an extended release time compared to a control, which is as described herein.

[0084] As used herein, the term “stablely encapsulated” refers to the ability of this composition to substantially prevent further rapid release of the drug from there.

[0085] In some embodiments, the w / w concentration of further agents in the composition is 1%-80%, 3%-80%, 5%-80%, 10%-80%, 20%-80%, 30%-80%, 50%-80%, 1%-60%, 3%-60%, 5%-60%, 10%-60%, 20%-60%, 30%-60%, 50%-60%, 1%-50%, 3%-50%, 5%-50%, 10%-50%, 20%-50%, 30%-80%, or 50-99.9% (including any range in between). Each possibility represents a distinct embodiment of the present invention.

[0086] In some embodiments, the mixture substantially contains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, and at least 99% by weight of further agents. In some embodiments, the mixture substantially contains at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, and at least 99% by weight of further agents, which are bonded to the MaSp fibers by non-covalent bonds, physical interactions, or both. Each possibility represents a distinct embodiment of the present invention. Non-covalent bonds are well known in the art and include, in particular, hydrogen bonds, pp stacking, van der Waals interactions, and the like.

[0087] In some embodiments, the physical interaction is referred to as the encapsulation (i.e., capture) of further drugs within a matrix formed by the MaSp polymer. In some embodiments, the matrix is ​​bound to or in contact with the further drugs.

[0088] In some embodiments, the further agent is in contact with or bound to the MaSp fiber. In some embodiments, the polymer reinforced with the further agent is physically bound to the MaSp fiber. In some embodiments, the further agent fills at least some of the pores on the surface or within the MaSp fiber. In some embodiments, the further agent is encapsulated by the MaSp fiber. In some embodiments, the further agent is in contact with or bound to the fibrils. In some embodiments, the further agent is encapsulated by entangled particles (also used herein as the “matrix”). In some embodiments, the further agent is encapsulated by the particles. In some embodiments, the further agent is incorporated into the MaSp fiber. In some embodiments, the further agent is embedded within the MaSp fiber. In some embodiments, the further agent is embedded within the matrix. In some embodiments, the matrix is ​​doped with the further agent. In some embodiments, the further agent is located within multiple pores. In some embodiments, the further agent is located between fibrils. In some embodiments, the further agent is located within a lumen, which is defined by the entangled fibers of the matrix. In some embodiments, additional drugs are encapsulated within the fibrils. In some embodiments, additional drugs are encapsulated within each lumen (or cavity) of the fibrils.

[0089] In some embodiments, the coupling is via non-covalent bonding, physical interaction, or both.

[0090] In some embodiments, the additional agent fills 20% to 100% of the pore volume. In some embodiments, the additional agent fills 55% to 100%, 60% to 100%, 55% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 50% to 99%, 50% to 98%, 50% to 97%, 50% to 95%, 50% to 90%, 70% to 90%, or 70% to 95% (including any range in between). Each possibility represents a distinct embodiment of the present invention.

[0091] In some embodiments, the additional agent fills 20% to 100% of the volume (e.g., lumen) of the particle. In some embodiments, the additional agent fills 55% to 100%, 60% to 100%, 55% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 50% to 99%, 50% to 98%, 50% to 97%, 50% to 95%, 50% to 90%, 70% to 90%, or 70% to 95% (including any range in between) of the volume of the particle. Each possibility represents a separate embodiment of the present invention.

[0092] In some embodiments, the w / w ratio of the further agent in the composition of the present invention to the MaSp fiber is 100:1~1:10, 100:1~8:1, 100:1~6:1, 100:1~4:1, 100:1~1:1, 100:1~10:1, 90:1~1:10, 90:1~8:1, 90:1~6:1, 90:1~4:1, 90:1~1:1, 90:1~10:1, 50:1~1:10, 50:1~8:1, 50:1~6:1, 50 The ranges are 1-4:1, 50:1-1:1, 50:1-10:1, 20:1-1:10, 20:1-8:1, 20:1-6:1, 20:1-4:1, 20:1-1:1, 20:1-10:1, 10:1-1:10, 10:1-8:1, 8:1-6:1, 6:1-4:1, 4:1-3:1, 3:1-2:1, 2:1-1:1, 1:1-1:2, 1:2-1:3, 1:3-1:5, 1:5-1:10 (including any range in between). Each possibility represents a distinct embodiment of the present invention.

[0093] In some embodiments, the w / w ratio of further agents in the composition to MaSp fibers is at most 100:1, at most 80:1, at most 40:1, at most 20:1, at most 10:1, at most 6:1, at most 5:1, at most 4:1, at most 3:1, at most 2:1, and at most 1:1 (including any range in between). Each possibility represents a separate embodiment of the present invention.

[0094] In some embodiments, the rate of release of further agents from the composition is reduced by at least 10% compared to the control. In some embodiments, the rate of release of further agents from the composition is reduced by at least 10%, at least 20%, at least 30%, at least 50%, at least 70%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 400%, at least 450%, at least 500%, at least 600%, at least 700%, at least 800%, and at least 1000% (including any range or value in between). Each possibility represents a distinct embodiment of the present invention.

[0095] In some embodiments, the control is a MaSp-based fiber freeze-dried from water. As illustrated below (Figures 3 and 4), the compositions of the present invention feature an improved release profile (i.e., a reduced release rate) of further agents compared to a control (e.g., a MaSp-based fiber freeze-dried from water).

[0096] In some embodiments, the release period is extended by at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, and at least 1000% (including any range or value in between) compared to a control. Each possibility represents a distinct embodiment of the present invention.

[0097] In some embodiments, the composition further comprises a thermoplastic polymer. As used herein, “thermoplastic” refers to a material that becomes softer when heated and harder when cooled. Thermoplastic materials can be cooled and heated several times without any change in their chemical or mechanical properties.

[0098] In some embodiments, the thermoplastic polymer is selected from polyester, polyamide, polyol, polyurethane, polyethylene, nylon, polyolefin, polyacrylate, polycarbonate, polylactic acid (PLA) or copolymers thereof, polycaprolactone (PCL), rubber, cellulose, or any combination thereof.

[0099] In some embodiments, the weight / weight (w / w) ratio of MaSp fibers to high melt temperature polymers is 0.01:1 to 1:1, 0.02:1 to 1:1, 0.05:1 to 1:1, 0.09:1 to 1:1, 0.1:1 to 1:1, 0.5:1 to 1:1, or 0.9:1 to 1:1 (including any range in between). Each possibility represents a distinct embodiment of the present invention.

[0100] In some embodiments, this method is suitable for concentrations of 0.01%~90% (w / w), 0.05%~90% (w / w), 0.09%~90% (w / w), 0.1%~90% (w / w), 0.5%~90% (w / w), 0.9%~90% (w / w), 1%~90% (w / w), 5%~90% (w / w), 10%~90% (w / w), 15%~90% (w / w), 20%~90% (w / w), 30%~90% (w / w), and 0.01%. ~80%(w / w), 0.05%~80%(w / w), 0.09%~80%(w / w), 0.1%~80%(w / w), 0.5%~80%(w / w), 0.9%~80%(w / w), 1%~80%(w / w) , 5%~80%(w / w), 10%~80%(w / w), 15%~80%(w / w), 20%~80%(w / w), 30%~80%(w / w), 0.01%~50%(w / w), 0.05%~50%(w / w) ), 0.09%~50%(w / w), 0.1%~50%(w / w), 0.5%~50%(w / w), 0.9%~50%(w / w), 1%~50%(w / w), 5%~50%(w / w), 10%~50%(w / w), 15%~50%(w / w), 20%~50%(w / w), 30%~50%(w / w), 0.01%~20%(w / w), 0.05%~20%(w / w), 0.09%~20%(w / w), 0.1%~2 The MaSp fibers include 0% (w / w), 0.5%~20% (w / w), 0.9%~20% (w / w), 1%~20% (w / w), 5%~20% (w / w), 0.01%~10% (w / w), 0.05%~10% (w / w), 0.09%~10% (w / w), 0.1%~10% (w / w), 0.5%~10% (w / w), 0.9%~10% (w / w), or 1%~10% (w / w) (including any range in between). Each possibility represents a distinct embodiment of the present invention.

[0101] In some embodiments, one or more properties of the composition, selected from, for example, thermal stability, Young's modulus, tensile strength, yield point, abrasion resistance, and tensile stress, are increased by at least 1%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 50%, at least 100%, at least 200%, or at least 500%. Each possibility represents a distinct embodiment of the present invention.

[0102] In some embodiments, the tensile strength of the composition is increased by at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 50%, at least 100%, at least 200%, or at least 500% (including any value in between) compared to the control. Each possibility represents a distinct embodiment of the present invention.

[0103] In some embodiments, the composition is characterized by a Young's modulus in the range of 50 MPa to 170 MPa, 52 MPa to 170 MPa, 60 MPa to 170 MPa, 68 MPa to 170 MPa, 90 MPa to 170 MPa, 100 MPa to 170 MPa, 101 MPa to 170 MPa, 105 MPa to 170 MPa, 101 MPa to 160 MPa, or 105 MPa to 160 MPa (including any range in between). Each possibility represents a separate embodiment of the present invention.

[0104] "Tenacity" or "tensile strength" refers to the weight of a filament that can withstand before fracture. The maximum specific stress produced by a tensile test to fracture a material is typically that of a filament, yarn, or fabric. According to specific embodiments, the MaSp polymer of the present invention has a tensile strength of approximately 100-3000 MPa (MPa = N / mm²). 2 It has a tensile strength of approximately 300-3000 MPa, approximately 500-2700 MPa, approximately 700-2500 MPa, approximately 900-2300 MPa, approximately 1100-2000 MPa, approximately 1200-1800 MPa, approximately 1300-1700 MPa, or approximately 1400-1600 MPa. More specifically, it is approximately 1500 MPa.

[0105] "Toughness" refers to the energy required to break a MaSp-based polymer. This is the area under the stress-strain curve, and is sometimes called "breaking energy" or "work of fracture." According to certain embodiments, the MaSp-based polymer of the present invention has a toughness of approximately 20-1000 MJ / m². 3 , about 50~950MJ / m 3 , about 100~900MJ / m 3 , about 120~850MJ / m 3 , about 150~800MJ / m 3 , about 180~700MJ / m 3 , about 180~750MJ / m 3 , about 250~700MJ / m 3 , about 280~600MJ / m 3 , about 300~580MJ / m 3 , about 310~560MJ / m 3 , about 320~540MJ / m 3 Or approximately 350-520 MJ / m 3 Most notably, approximately 350-520 MJ / m³ 3 It possesses toughness.

[0106] "Elasticity" refers to the property of a material that tends to recover its original size and shape after deformation. Plasticity, or deformation without recovery, is the opposite of elasticity. In the molecular arrangement of MaSp polymers, recoverable deformation, i.e., elastic deformation, is made possible by the elongation (reorientation) of interatomic and intermolecular structural bonds. Conversely, the breakdown of intermolecular bonds and their reformation to new stable positions causes irreversible deformation, i.e., plastic deformation.

[0107] "Elongation" refers to an increase in length expressed as a percentage or fraction of the initial length.

[0108] "Fine diameter" refers to the average diameter of a MaSp-based polymer or filament (e.g., biofilament), which is usually expressed in microns (μm).

[0109] While not bound by any particular theory, we assume that the high porosity of the MaSp fibers disclosed herein predetermines several advantageous properties of the fibers, such as release profile, tensile strength, and optionally load capacity.

[0110] In some embodiments, the MaSp proteins or MaSp polymers used interchangeably herein are in the form of fibers. As used herein, “fiber” means a thin string of fibrous material consisting of two or more filaments twisted together. “Filament” means an elongated, thread-like object or structure of indeterminate length ranging from microscopic to more than one mile. Specifically, synthetic spider silk filaments are microscopic and proteinaceous. “Biofilament” means a filament made from a protein containing recombinant spider silk protein. In some embodiments, the term “fiber” does not include unstructured aggregates or precipitates.

[0111] In some embodiments, the MaSp fiber comprises multiple MaSp polymers. In some embodiments, the multiple MaSp polymers comprise polymers having different chemical compositions and / or different molecular weights (MW). In some embodiments, the multiple MaSp polymers comprise polymers having different numbers of repeating regions.

[0112] In some embodiments, the composition of the present invention substantially comprises a single MaSp polymer or MaSp fiber as described herein. In some embodiments, the composition of the present invention substantially comprises a single further agent or a plurality of further agents (e.g., 2, 3, 4, 5, 10, etc.). In some embodiments, the MaSp polymer or MaSp fiber substantially lacks further non-MaSp proteins. In one embodiment, the MaSp polymer or MaSp fiber substantially lacks further polymers (e.g., synthetic polymers, non-MaSp peptides, non-MaSp proteins).

[0113] In some embodiments, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, at least 99.5%, at least 99.9%, or 80-99%, 90-99.9% (including any range in between) by weight of the composition of the present invention consists of the MaSp fiber of the present invention (e.g., a single MaSp fiber species) and optionally one or more further agents described herein. In some embodiments, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, at least 99.5%, at least 99.9% (including any range in between) by weight of the protein content in the composition of the present invention consists of the MaSp fiber of the present invention. In some embodiments, at least 80%, at least 90%, at least 95%, at least 97%, at least 99%, at least 99.5%, at least 99.9% (including any range in between) by weight of the polymer content in the composition of the present invention consists of the MaSp fiber of the present invention.

[0114] In some embodiments, at least 90%, at least 95%, at least 97%, at least 99%, at least 99.5%, at least 99.9%, at least 99.99%, or 80-99%, 90-99.9% (including any range in between) by weight of the MaSp-based fiber of the present invention consists of the MaSp-based polymer of the present invention (e.g., a single amino acid sequence or multiple amino acid sequences as described herein).

[0115] In some embodiments, the protein fiber is characterized by a size of at least one dimension (e.g., diameter, length). For example, but not limited to, the diameter of the fiber may be 10 nm to 1 μm, 20 to 100 nm, or 10 to 50 nm.

[0116] In one embodiment, the MaSp fiber consists of multiple MaSp polymers (for example, each of the MaSp polymers is selected from peptides, polyamino acids, or polypeptides having the same or different amino acid sequences). In one embodiment, the multiple MaSp polymers have the same amino acid sequence selected from the sequence numbers described herein. In one embodiment, the multiple MaSp polymers are arranged within nanofibrils. In one embodiment, the multiple nanofibrils are arranged within or constitute the fiber. In one embodiment, the monomers or nanofibrils in the MaSp fiber have a diameter of 4 to 16 nm. In one embodiment, the monomers or nanofibrils in the MaSp fiber have a diameter of 6 to 14 nm. In one embodiment, the monomers or nanofibrils in the MaSp fiber have a diameter of 8 to 12 nm. In some embodiments, the MaSp fiber contains multiple fibrils (e.g., nanofibrils). In some embodiments, the fiber having a mutated amino acid sequence substantially lacks nanofibrils. In some embodiments, the fiber having a mutated amino acid sequence is in the form of non-porous particles.

[0117] In some embodiments, the nanofibrils have a diameter of, for example, 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, about 20 nm, about 21 nm, about 22 nm, about 23 nm, about 24 nm, about 25 nm, about 26 nm, about 27 nm, about 28 nm, about 29 nm, about 30 nm, about 31 nm, about 32 nm, about 33 nm, about 34 nm, about 35 nm, about 36 nm, about 37 nm, about 38 nm, about 40 nm, about 42 nm, about 44 nm, about 46 nm, about 48 nm or about 50 nm (including any value or range in between). In one embodiment, the nanofibril has a diameter of 3 to 7 nm. In another embodiment, the nanofibril has a diameter of 4 to 6 nm.

[0118] In one embodiment, the MaSp fiber has a diameter of 70 to 450 nm. In one embodiment, the MaSp fiber has a diameter of 80 to 350 nm. In one embodiment, the MaSp fiber has a diameter of 80 to 300 nm. In one embodiment, the MaSp fiber has a diameter of 150 to 250 nm. In one embodiment, the MaSp fiber or MaSp polymer is arranged as a coil. In one embodiment, a single fiber, i.e., one MaSp polymer, is arranged as a coil. In one embodiment, the coil has a diameter of 5 to 800 μm. In one embodiment, the coil has a diameter of 5 to 500 μm. In one embodiment, the coil has a diameter of 5 to 30 μm. In one embodiment, the coil has a diameter of 5 to 20 μm. In one embodiment, the MaSp fiber or MaSp polymer has a length of 5 to 800 μm. In one embodiment, the MaSp fiber or MaSp polymer has a length of 30 to 300 μm. In some embodiments, the length of the MaSp fiber is 1–200 μm, 10–100 μm, 100–500 μm, or 200–500 μm (including any range in between).

[0119] In some embodiments, the MaSp fibers contain multiple pores. In some embodiments, the MaSp fibers are in the form of particles described herein. In some embodiments, the composition contains multiple MaSp fibers. In some embodiments, the multiple MaSp fibers contain fibers having different chemical compositions. In some embodiments, the multiple MaSp fibers are in the form of particles having different sizes and / or different structures. In some embodiments, the multiple MaSp fibers are in the form of particles having different porosity (expressed as BET surface area). Porosity may be measured using BET surface analysis as described, for example, S. Brunauer, P. Hemmett and E. Teller, J. Am. Chem. Soc., 1938, 60, 309, the entire disclosure of which is incorporated herein by reference.

[0120] In some embodiments, the MaSp fibers of the present invention are characterized by a porous structure. In some embodiments, the MaSp fibers are porous fibers. In some embodiments, the MaSp fibers of the present invention are in the form of porous particles having a mesh-like or spongy structure, where the particle size is as described herein. In some embodiments, the porous particles are in the form of a matrix as described herein (e.g., comprising a plurality of mesh-like intertwined MaSp polymers defining a plurality of cavities or lumens). In some embodiments, the MaSp fibers of the present invention comprise a plurality of distinct porous particles, where the porous particles are as described herein (e.g., having a median diameter in the range of 0.5 μm to 1.5 μm). On the other hand, the control MaSp fibers (e.g., dried from an aqueous solution) are in the form of substantially less porous fibers having a size greater than 2 μm and are substantially uniform filamentous fibers lacking, for example, distinct porous particles.

[0121] Figure 1 shows an exemplary structure of the porous fiber of the present invention compared to a similar non-porous MaSp-based fiber.

[0122] In some embodiments, the MaSp fibers of the present invention are characterized by pore diameters of at least 10 nm, at least 20 nm, at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 80 nm, and at least 100 nm (including any range in between). In some embodiments, the MaSp fibers of the present invention are characterized by pore diameters of 20 to 80 nm and 20 to 60 nm (including any range in between). In some embodiments, the pore diameter values ​​described herein are median values. In some embodiments, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, and at least 95% of the pores are characterized by the pore diameters described herein.

[0123] While not bound by any particular theory or mechanism, we assume that the control MaSp fibers (e.g., dried from an aqueous solution) have pore sizes greater than 500 nm, greater than 1 μm, or even larger.

[0124] In some embodiments, the porous structure or porous MaSp fiber is characterized by at least 30% (e.g., 30-99%) porosity. In some embodiments, the porous structure is characterized by at least 50% (e.g., 50-99%) porosity. In some embodiments, the porous structure is characterized by at least 60% (e.g., 60-99%) porosity. In some embodiments, the porous structure is characterized by at least 70% (e.g., 70-99%) porosity. In some embodiments, the porous structure is characterized by at least 80% (e.g., 80-99%) porosity. In some embodiments, the porous structure is characterized by at least 90% (e.g., 90-99%) porosity. In some embodiments, the porous structure is characterized by approximately 90% porosity.

[0125] In this specification, the term "porosity" refers to the proportion of volume of a material consisting of voids (e.g., a "sponge-like" material). In another embodiment, porosity is measured by dividing the voids within the surface area by the total surface area (porous and non-porous).

[0126] In some embodiments, the porous structure of the disclosed MaSp fibers allows for the efficient absorption of further agents or polymers into the multiple pores of the MaSp fibers of the present invention. The improved porosity of the MaSp fibers is assumed to contribute to increased retention of further agents within them compared to controls (e.g., similar fibers dried from aqueous solutions) and to have substantially lower void and / or BET values ​​(as illustrated in Figure 1). That is, without being bound by any particular theory, this remarkable finding can be explained in terms of the structure and porosity of the disclosed fibers, which are strictly distinguishable from naturally occurring spider silk or similar MaSp fibers (e.g., having the same amino acid sequence) that are dried from aqueous solutions, feature a "collapsed" structure, and have a substantially less porous structure compared to the fibers of the present invention. As illustrated in Figure 1, the fibers freeze-dried from aqueous solutions are substantially in the form of a film.

[0127] In some embodiments, the MaSp fibers of the present invention have a BET value of at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, or at least 2000% or more compared to a control, where the control is as described herein.

[0128] In some embodiments, the porous MaSp fibers are in the form of particles described herein. In some embodiments, the particles are porous particles. In some embodiments, the particles are substantially non-aggregated particles. In some embodiments, the particles contain a plurality of pores (i.e., spaces or lumens) formed by twisted polymer chains of the MaSp polymer. In some embodiments, the entangled MaSp polymers form a matrix. In some embodiments, further agents fill at least some of the pores in the matrix or particles. In some embodiments, further agents are encapsulated by the plurality of pores.

[0129] Goods In another aspect of the present invention, an article comprising the composition of the present invention or the dried MaSp-based fibers of the present invention is provided.

[0130] In another aspect of the present invention, an article comprising the composition of the present invention or freeze-dried MaSp fibers of the present invention is provided.

[0131] In some embodiments, the article may be in the form of reinforced plastic, a bottle, a container, a package, a cable, a tube, a film, a rope, a thread, or a fabric.

[0132] In some embodiments, the article is characterized by at least one improved mechanical property compared to the properties of an article without the composition, wherein the property is selected from the group consisting of Young's modulus, tensile strength, fracture strain, yield point, toughness, work of fracture, impact strength, tear strength, flexural modulus, flexural strain and stress at a particular elongation, wear, UV resistance, and gas permeability.

[0133] In some embodiments, the article further includes a carrier.

[0134] In some embodiments, the article is a cosmetic (e.g., color cosmetics, powders, face cleansers). The cosmetic may be one of those described elsewhere in this specification or known to those skilled in the art. Non-limiting examples of products include moisturizers, creams, lotions, emollients, foundations, night creams, lipsticks, cleansers, toners, sunscreens, masks, anti-aging products, deodorants, antiperspirants, perfumes, and colognes.

[0135] In some embodiments, the carrier is a physiologically suitable carrier. Exemplary physiologically suitable carriers are listed below, and further physiologically suitable carriers are well known in the art.

[0136] In some embodiments, the carrier includes an emulsifier. The emulsifier can reduce interphase tension and improve the formulation and stability of the emulsion. The emulsifier may be nonionic, cationic, anionic, or zwitterionic (see McCutcheon's (1986); U.S. Patents 5,011,681, 4,421,769, and 3,755,560).

[0137] Non-limiting examples of emulsifiers include glycerin esters, propylene glycol esters, polyethylene glycol fatty acid esters, polypropylene glycol fatty acid esters, sorbitol esters, sorbitan anhydride esters, carboxylic acid copolymers, glucose esters and ethers, ethoxylated ethers, ethoxylated alcohols, alkyl phosphates, polyoxyethylene aliphatic ether phosphates, fatty acid amides, acyl lactylates, soaps, TEA stearate, DEA oleth-3 phosphate, polyethylene glycol 20 sorbitan monolaurate (polysorbate 20), polyethylene glycol 5 soybean sterols, steareth-2, steareth-20, steareth-21, ceteareth-20, PPG-2 methyl glucose ether distearate, ceteth-10, polysorbate 80, cetyl phosphate, potassium cetyl phosphate, diethanolamine cetyl phosphate, polysorbate 60, glyceryl stearate, PEG-100 stearate, or any combination thereof.

[0138] method In another aspect of the present invention, a method for obtaining dried large vial-shaped glandular spidolin protein (MaSp) fibers comprises the steps of (a) mixing MaSp fibers with a liquid containing an organic solvent to obtain a mixture, and (b) providing the mixture under conditions suitable for substantially removing the liquid from the mixture, thereby obtaining at least 100 m 2A method is provided for obtaining dried MaSp fibers characterized by a BET surface area of ​​1 / g and optionally a residual amount of organic solvent. In some embodiments, steps (a) and (b) are performed sequentially or simultaneously. In some embodiments, step (a) is performed before step (b).

[0139] In some embodiments, the dried MaSp fibers are the MaSp fibers of the present invention as described herein. In some embodiments, the MaSp fibers of the present invention are dried by the method disclosed herein.

[0140] Another aspect of the present invention provides a method for obtaining freeze-dried large vial-shaped glandular spidoin protein (MaSp) fibers, comprising the steps of (a) mixing MaSp fibers with a liquid to obtain a mixture, and (b) subjecting the mixture to conditions suitable for freeze-drying, thereby providing a method for obtaining freeze-dried MaSp fibers.

[0141] In some embodiments, the method is for obtaining dried large vial-shaped glandular spidoin protein (MaSp) fiber, and the method comprises (a) mixing wet MaSp fiber with a liquid containing an organic solvent to obtain a mixture, and (b) providing the mixture under conditions suitable for substantially removing the liquid from the mixture, thereby obtaining dried MaSp fiber. In some embodiments, the method comprises a step of preparing wet MaSp fiber, which is performed before carrying out either step (a) or (b). In some embodiments, the wet MaSp fiber is obtained (e.g., extracted) from one of the expression systems described herein. In some embodiments, the wet MaSp fiber is isolated from host cells or host organisms (also referred to as “expression system”).

[0142] In some embodiments, the method includes the steps of mixing MaSp fibers (e.g., obtained from an expression system and containing water) with a liquid containing an organic solvent to obtain a mixture, and subjecting the mixture to conditions suitable for drying the MaSp fibers. In some embodiments, the mixing step includes adding the liquid to wet MaSp fibers or vice versa. In some embodiments, the addition is done by pouring the liquid onto the wet MaSp fibers. In some embodiments, the mixing step includes adding the wet MaSp fibers to the liquid.

[0143] In some embodiments, suitable drying conditions include conditions sufficient to remove at least 99%, at least 99.9%, at least 99.99%, at least 99.999%, or more of the solvent (e.g., the liquid and water content of the wet fibers) from the mixture, thereby obtaining dried MaSp-based fibers. In some embodiments, suitable drying conditions include exposure to heat and / or IR or MW radiation with an intensity sufficient to remove at least 99%, at least 99.9%, at least 99.99%, at least 99.999%, or more of the solvent (e.g., the liquid and water content of the wet fibers) from the mixture. In some embodiments, suitable drying conditions include exposure to heat and / or IR or MW radiation for a period of time sufficient to remove at least 99%, at least 99.9%, at least 99.99%, at least 99.999%, or more of the solvent (e.g., the liquid and water content of the wet fibers) from the mixture.

[0144] In some embodiments, suitable drying conditions include vacuuming the mixture for a period of time sufficient to remove at least 99%, at least 99.9%, at least 99.99%, at least 99.999%, or more of the solvent (e.g., the liquid and water content of the wet fibers) from the mixture. In some embodiments, suitable drying conditions include conditions suitable for freeze-drying the mixture.

[0145] In some embodiments, suitable drying conditions include (i) exposing the mixture to conditions suitable for freezing the mixture to obtain a frozen mixture, and (ii) exposing the frozen mixture to a vacuum for a time sufficient to substantially remove the solvent (e.g., the liquid and water content of the wet fibers). In some embodiments, suitable drying conditions include (i) subjecting the mixture to a temperature below the freezing point of the mixture to obtain a frozen mixture, and (ii) applying a sufficient vacuum to the frozen mixture for a time sufficient to remove at least 99%, at least 99.9%, at least 99.99%, at least 99.999% or more of the solvent (e.g., the liquid and water content of the wet fibers) from the mixture. In some embodiments, steps (i) and (ii) are performed sequentially or simultaneously. In some embodiments, suitable drying conditions are for obtaining the MaSp fibers of the present invention characterized by the BET and optionally the residual water content and / or residual amount of solvent as specified herein.

[0146] In some embodiments, the liquid optionally includes an aqueous solvent. In some embodiments, the liquid includes an organic solvent (e.g., t-butanol) in 50-100% w / w, 50-70% w / w, 70-80% w / w, 80-100% w / w, 80-90% w / w, 90-95% w / w, 95-99% w / w, 99-100% w / w (including any range in between), and optionally includes water or aqueous solution in 30-0.1% w / w, optionally 30-20% w / w, 20-0.1% w / w, 20-10% w / w, 10-0.1% w / w (including any range in between).

[0147] In some embodiments, the organic solvent can form an azeotropic mixture with water. In some embodiments, the organic solvent is in a w / w ratio to moist MaSp fibers sufficient to obtain the dry MaSp fibers of the present invention. In some embodiments, the organic solvent is in a w / w ratio to moist MaSp fibers sufficient to form an azeotropic mixture with the water content of the moist MaSp fibers. In some embodiments, step (a) of the method includes contacting or mixing moist MaSp fibers (e.g., obtained from an expression system and containing water) with a sufficient amount of liquid (here the liquid is as described herein). In some embodiments, step (a) of the method includes contacting or mixing moist MaSp fibers (e.g., obtained from an expression system and containing water) with a liquid containing an organic solvent to obtain a mixture, where the w / w ratio of the liquid to moist MaSp fibers is sufficient to form an azeotropic mixture. In some embodiments, step (b) of the method includes subjecting the mixture to conditions sufficient to evaporate or remove the azeotropic mixture. Those skilled in the art will be able to adjust the liquid / fiber weight ratio to obtain optimal drying or freeze-drying of MaSp fibers. Optimal drying conditions and the optimal liquid / fiber weight ratio can be evaluated by calculating the porosity of dried (e.g., freeze-dried) MaSp fibers as disclosed herein.

[0148] In some embodiments, the organic solvent is selected from the group consisting of ethanol, isopropyl alcohol, t-butanol, 2-butanol, n-butanol, acetonitrile, dichloromethane, benzene, ethyl acetate, DMF, DMSO, THF, TFA, toluene, hexane, heptane, methyl ethyl ketone, diethyl ketone, methyl isobutyl ketone, dioxane, and any combination thereof. In some embodiments, the solvent is t-butanol. In some embodiments, the organic solvent is as described above herein (e.g., a Class 3 solvent).

[0149] In some embodiments, suitable conditions for substantially removing liquid from the mixture include centrifugation, filtration, sedimentation, freeze-drying, or any combination thereof.

[0150] Another aspect of the present invention provides a method for obtaining freeze-dried large vial-shaped glandular spidoin protein (MaSp) fibers, comprising the steps of (a) contacting the MaSp fibers with an organic solvent for a period of time, and (b) separating the MaSp fibers from the solvent, thereby obtaining the MaSp fibers.

[0151] Another aspect of the present invention provides a method for purifying large vial-shaped glandular spidoin protein (MaSp) fibers, comprising the steps of (a) contacting the MaSp fibers with an organic solvent for a period of time, and (b) separating the MaSp fibers from the solvent to purify the MaSp fibers.

[0152] In some embodiments, the method includes repeating step (a) one to five times, two to five times, or three to five times (including any range in between). Each possibility represents a separate embodiment of the present invention.

[0153] In some embodiments, the period is between 10 seconds (s) and 10 minutes (min).

[0154] In some embodiments, the contact step includes mixing, high-shear mixing, overhead stirring, homogenization, or a combination thereof.

[0155] In some embodiments, the separation step includes centrifugation, filtration, sedimentation, freeze-drying, or any combination thereof.

[0156] In another aspect of the present invention, dried porous large bottle-shaped glandular spidoin protein (MaSp) fibers obtained by the method described herein are provided.

[0157] In another aspect of the present invention, lyophilized porous bottle-shaped glandular spidoin protein (MaSp) fibers obtained by the methods described herein are provided.

[0158] In some embodiments, dried MaSp fibers are characterized by an improved loading capacity for further agents. In some embodiments, MaSp fibers dried from an organic solvent are characterized by an improved loading capacity for further agents compared to MaSp fibers dried from water. In some embodiments, MaSp fibers dried from an organic solvent are characterized by an improved loading capacity for further agents compared to a control. In some embodiments, the control is MaSp fibers dried from water. As used herein, “loading capacity” refers to the amount of further agent loaded per unit weight of dried MaSp fibers in the composite of the present invention. Loading capacity can be calculated by dividing the total weight of dried MaSp fibers by the total amount of further agent trapped in the composite of the present invention.

[0159] In some embodiments, the improved load capacity includes a w / w ratio of 100:1 to 1:10 of additional agents to porous MaSp fibers in the composite material of the present invention. In some embodiments, the w / w ratio of additional agents to MaSp fibers is 100:1 to 1:10, 100:1 to 8:1, 100:1 to 6:1, 100:1 to 4:1, 100:1 to 1:1, 100:1 to 10:1, 90:1 to 1:10, 90:1 to 8:1, 90:1 to 6:1, 90:1 to 4:1, 90:1 to 1:1, 90:1 to 10:1, 50:1 to 1:10, 50:1 to 8:1, 50:1 to 6:1, 50:1 to 4: These ranges are 1, 50:1~1:1, 50:1~10:1, 20:1~1:10, 20:1~8:1, 20:1~6:1, 20:1~4:1, 20:1~1:1, 20:1~10:1, 10:1~1:10, 10:1~8:1, 8:1~6:1, 6:1~4:1, 4:1~3:1, 3:1~2:1, 2:1~1:1, 1:1~1:2, 1:2~1:3, 1:3~1:5, 1:5~1:10 (including any range in between). In some embodiments, improvements are made compared to the comparison as described herein. Each possibility represents a distinct embodiment of the present invention.

[0160] In some embodiments, the w / w ratio of further agents in the composite material of the present invention to MaSp-based fibers is at most 100:1, at most 80:1, at most 40:1, at most 20:1, at most 10:1, at most 6:1, at most 5:1, at most 4:1, at most 3:1, at most 2:1, and at most 1:1 (including any range in between). Each possibility represents a distinct embodiment of the present invention.

[0161] In some embodiments, dried MaSp fibers are characterized by improved retention of further agents. In some embodiments, MaSp fibers dried from organic solvents are characterized by improved retention of further agents compared to MaSp fibers dried from water.

[0162] In another aspect of the present invention, a composite material comprising dried MaSp fibers and further agents is provided. In some embodiments, the composite material comprises MaSp fibers bound to the further agents (e.g., by physical adsorption and / or non-covalent bonding or interaction). In some embodiments, a composition comprising MaSp fibers and further agents is referred to herein as the “composite material”. In some embodiments, the composite material comprises further agents homogeneously dispersed in a matrix of the MaSp fibers of the present invention. In some embodiments, the composite material comprises further agents encapsulated, adsorbed, or embedded in the MaSp fibers of the present invention (e.g., in lumens or cavities as described herein). In some embodiments, the composite material of the present invention is substantially homogeneous or uniform (e.g., substantially lacking aggregates and / or phase separation).

[0163] As used herein, the term “composite material” refers to a material made from two or more constituent materials having dissimilar chemical or physical properties, which, when combined, create a substance that has different properties from the individual elements.

[0164] In some embodiments, the compositions and / or composites of the present invention are substantially stable. In some embodiments, a composite comprising MaSp fibers (also used herein as “MaSp fibers” or “MaSp fibers of the present invention”) dried from an organic solvent and further agents has improved stability compared to a composite comprising MaSp fibers dried from water and further agents. In some embodiments, the further agents are stably encapsulated within the multiple pores of the particles. In some embodiments, the terms “improved stability” and “stably encapsulated” refer to the ability of the composite to substantially prevent the release of further agents from there. In some embodiments, the term “improved stability” refers to the ability of the composite to substantially retain its physical properties and / or chemical composition. In some embodiments, the term “improved stability” refers to the ability of the composite to substantially lack aggregates and / or phase separation and / or leakage of further agents under suitable storage conditions, such as in a formulation (e.g., medicated cosmetic and / or pharmaceutical composition). In some embodiments, the compositions and / or composites are said to be stable if they substantially lack phase separation over the period disclosed herein. As used herein, the term “stable” refers to the chemical and / or physical stability of the compositions and / or composites of the present invention.

[0165] In some embodiments, the compositions and / or composites of the present invention are stable if the concentration of further agents in the compositions and / or composites of the present invention does not decrease by more than 1%, 5%, or 10% over a period of six months at a temperature of 20°C or lower. In some embodiments, the compositions and / or composites are stable if the concentration of further agents in the compositions and / or composites does not decrease by more than 1%, 5%, or 10% (including any range in between) over a period of six months under the appropriate storage conditions described herein.

[0166] In some embodiments, suitable storage conditions include storage temperatures of 1–60°C, 1–10°C, 10–30°C, 30–40°C, 40–50°C, and 50–60°C (including any range in between). In some embodiments, suitable storage conditions include ambient atmosphere. In some embodiments, suitable storage conditions include the storage temperatures described herein and storage times of at least 1 month (m), at least 2 m, at least 3 m, at least 4 m, at least 5 m, at least 6 m, at least 7 m, at least 8 m, at least 10 m, at least 12 m, and at least 2 years (including any range or value in between). In some embodiments, the term “stable” refers to the storage stability of the composition and / or composite, where storage stability includes stability under the suitable storage conditions described herein.

[0167] In some embodiments, dried MaSp fibers feature a sustained-release profile compared to controls. As used herein, “sustained-release” refers to the ability to gradually and slowly release additional agents over a long period of time, enabling a sustained effect (e.g., compared to controls).

[0168] In some embodiments, the encapsulated additional drug features a sustained-release profile (e.g., at the application site or in solution). In some embodiments, the particles encapsulating the additional drug substantially prevent the rapid release of the additional drug from there.

[0169] In some embodiments, the release rate of further agents from the composite material containing MaSp fibers and further agents dried from an organic solvent is reduced by at least 10% compared to the composite material containing MaSp fibers and further agents dried from water. In some embodiments, the release rate of further agents from the composite material containing MaSp fibers and further agents dried from an organic solvent is reduced by at least 10%, at least 20%, at least 30%, at least 50%, at least 70%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 400%, at least 450%, at least 500%, at least 600%, at least 700%, at least 800%, or at least 1000% (including any range or value in between) compared to the composite material containing MaSp fibers and further agents dried from water. Each possibility represents a distinct embodiment of the present invention.

[0170] In some embodiments, the release rate of additional agents from the composite material containing MaSp fibers and further agents dried from the organic solvent is reduced by at least 10% compared to the control.

[0171] In some embodiments, the control is a MaSp-based fiber freeze-dried from water. As illustrated below (Figures 3 and 4), the composites of the present invention feature an improved release profile (i.e., a reduced release rate) of further agents compared to a control (e.g., a composite containing a MaSp-based fiber freeze-dried from water).

[0172] In some embodiments, the release period is extended by at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, and at least 1000% (including any range or value in between) compared to a control. Each possibility represents a distinct embodiment of the present invention.

[0173] In another embodiment, a method is provided for adding further agents to a subject, comprising administering a composition or article of the present invention to the subject, thereby adding further agents to the subject. In some embodiments, the subject is selected from human subjects or animal subjects.

[0174] extruded product According to several embodiments, the present invention provides extruded articles and / or compositions containing the MaSp fibers of the present invention, and high melt-temperature polymers. In some embodiments, the MaSp fibers and / or extruded articles containing the MaSp fibers of the present invention are extrudeable or moldable (e.g., characterized by sufficient physical and / or chemical stability during processing by extrusion and / or molding). In some embodiments, molding is selected from the group consisting of melt extrusion, injection molding, spinning, melt blowing and thermoforming or any combination thereof. In some embodiments, the extruded article is in the form of a composite material described herein. In some embodiments, the extruded article is in the form of a high melt-temperature polymer reinforced with the MaSp fibers of the present invention.

[0175] In some embodiments, MaSp fibers and / or extruded articles containing them are stable when exposed to conditions suitable for molding or extrusion. In some embodiments, conditions suitable for molding include exposing the MaSp fibers and / or extruded articles to temperatures around their softening point. In some embodiments, conditions suitable for molding include exposing the MaSp fibers and / or extruded articles to temperatures around their Tm. In some embodiments, conditions suitable for molding include exposure to heat in the range of 100 to 300°C, thereby melting or softening the extruded articles. In some embodiments, conditions suitable for molding or extrusion include subjecting the extruded articles to conditions suitable for their melting or softening.

[0176] In some embodiments, the weight / weight (w / w) ratio of MaSp fibers to high melt temperature polymers is 0.01:1 to 1:1, 0.02:1 to 1:1, 0.05:1 to 1:1, 0.09:1 to 1:1, 0.1:1 to 1:1, 0.5:1 to 1:1, or 0.9:1 to 1:1 (including any range in between). Each possibility represents a distinct embodiment of the present invention.

[0177] In some embodiments, the extruded product has a viscosity of 0.01%~50% (w / w), 0.05%~50% (w / w), 0.09%~50% (w / w), 0.1%~50% (w / w), 0.5%~50% (w / w), 0.9%~50% (w / w), 1%~50% (w / w), 5%~50% (w / w), 10%~50% (w / w), 15%~50% (w / w), 20%~50% (w / w), 30%~50% (w / w), 0.01%~20% (w / w), and 0.05%~20% (w / w). This includes MaSp fibers in concentrations of 0.09%~20%(w / w), 0.1%~20%(w / w), 0.5%~20%(w / w), 0.9%~20%(w / w), 1%~20%(w / w), 5%~20%(w / w), 0.01%~10%(w / w), 0.05%~10%(w / w), 0.09%~10%(w / w), 0.1%~10%(w / w), 0.5%~10%(w / w), 0.9%~10%(w / w), or 1%~10%(w / w) (including any range in between). Each possibility represents a distinct embodiment of the present invention.

[0178] In some embodiments, the high melting temperature polymer is bonded to the MaSp-based fiber by non-covalent bonds, physical interactions, or both.

[0179] In some embodiments, the high melting temperature polymers are 200°C to 400°C, 210°C to 400°C, 220°C to 400°C, 250°C to 400°C, 270°C to 400°C, 280°C to 400°C, 200°C to 390°C, 210°C to 390°C, 220°C to 390°C, 250°C to 390°C, 270°C to 390°C, 280°C to 390°C, 200°C to 380°C, 210°C to 380°C, 220°C to 380°C, 250°C to 380°C, 270°C to 380°C, 280°C to 380°C, Melting temperature (T) of 200℃~350℃, 210℃~350℃, 220℃~350℃, 250℃~350℃, 270℃~350℃, 280℃~350℃, 200℃~310℃, 210℃~310℃, 220℃~310℃, 250℃~310℃, 270℃~310℃, 280℃~310℃, 200℃~300℃, 210℃~300℃, 220℃~300℃, 250℃~300℃, 270℃~300℃ or 280℃~380℃ (including any range in between) m ) is characterized by each possibility representing a distinct embodiment of the present invention.

[0180] As used herein, the term “high melting point polymer” refers to a polymer characterized by a continuous use temperature (CUT) or relative temperature index (RTI) above 150°C. These temperatures are considered the maximum effective use temperature of a material above which its critical properties are not unacceptably impaired by thermal decomposition. The maximum continuous use temperature is the maximum allowable temperature above which, if exceeded, the mechanical properties (tensile strength, impact strength) or electrical properties (dielectric strength related to insulation) of the material deteriorate significantly during the reasonable lifespan of the product under test.

[0181] In some embodiments, the high melting point polymer is a thermoplastic polymer. As used herein, "thermoplastic" refers to a material that becomes softer when heated and harder when cooled. Thermoplastic materials can be cooled and heated several times without any change in their chemical or mechanical properties.

[0182] In some embodiments, high-temperature thermoplastics are also referred to as “high-performance plastics” or “high-performance polymers.” As used herein, the term “high-performance polymer” refers to a group of polymer materials known to retain their desirable mechanical, thermal, and chemical properties when subjected to harsh environmental conditions such as high temperature, high pressure, and corrosive chemicals.

[0183] In some embodiments, the high melting temperature polymers are poly(vinyl chloride) (PVC), poly(6-aminocaproic acid), poly(caprolactam), poly(hexamethylene sevacamide) (nylon 6,10), poly(vinyl alcohol), poly(decamethylene adipamide) (nylon 10,6), poly(hexamethylene sbellamide) (nylon 6,8), poly(styrene) (PS), poly(4-methylpentene) (PMP), and poly(ethylene terephthalate). The material is selected from the group consisting of (PET), poly(hexamethylene adipamide)(nylon 6,6), poly(acrylonitrile)(PAN), poly(tetrafluoroethylene)(PTFE), polyarylate, ethylene vinyl acetate (EVA), polybutylene terephthalate (PBT), poly(1,4-cyclohexanedimethylene terephthalate)(PCT), polyether ether ketone (PEEK), and any copolymers and combinations thereof.

[0184] In some embodiments, the MaSp fibers are characterized by thermal stability at temperatures of 200°C to 300°C for up to 5 hours. In some embodiments, the extruded product is characterized by thermal stability at temperatures of 200°C to 300°C, 210°C to 300°C, 220°C to 300°C, 250°C to 300°C, 270°C to 300°C or 280°C to 380°C (including any range in between) for up to 5 hours. Each possibility represents a distinct embodiment of the present invention. In some embodiments, the extruded product is characterized by thermal stability at temperatures of 200°C to 300°C for up to 5 hours, up to 2 hours, up to 1 hour, up to 40 minutes, up to 20 minutes or up to 10 minutes (including any range in between). Each possibility represents a distinct embodiment of the present invention.

[0185] In some embodiments, the extruded product is characterized by thermal stability at temperatures of 200°C to 300°C over periods ranging from 1 minute to 5 hours, 2 minutes to 5 hours, 5 minutes to 5 hours, 10 minutes to 5 hours, 20 minutes to 5 hours, 30 minutes to 5 hours, 1 minute to 2 hours, 2 minutes to 2 hours, 5 minutes to 2 hours, 10 minutes to 2 hours, 20 minutes to 2 hours, 30 minutes to 2 hours, 1 minute to 1 hour, 2 minutes to 1 hour, 5 minutes to 1 hour, 10 minutes to 1 hour, 20 minutes to 1 hour, 30 minutes to 1 hour, 1 minute to 40 minutes, 2 minutes to 40 minutes, 5 minutes to 40 minutes, 10 minutes to 40 minutes, 20 minutes to 40 minutes, 1 minute to 20 minutes, 2 minutes to 20 minutes, 5 minutes to 20 minutes, or 10 minutes to 20 minutes (including any range in between). Each possibility represents a separate embodiment of the present invention.

[0186] In some embodiments, MaSp fibers are characterized by thermal stability at temperatures of 200°C–300°C, 210°C–300°C, 220°C–300°C, 250°C–300°C, 270°C–300°C, or 280°C–380°C (including any range in between) for up to 5 hours. Each possibility represents a distinct embodiment of the present invention.

[0187] In some embodiments, MaSp fibers are characterized by thermal stability at temperatures between 200°C and 300°C for up to 5 hours, up to 2 hours, up to 1 hour, up to 40 minutes, up to 20 minutes, or up to 10 minutes (including any range in between). Each possibility represents a distinct embodiment of the present invention.

[0188] In some embodiments, MaSp fibers are characterized by thermal stability at temperatures of 200°C to 300°C over periods ranging from 1 minute to 5 hours, 2 minutes to 5 hours, 5 minutes to 5 hours, 10 minutes to 5 hours, 20 minutes to 5 hours, 30 minutes to 5 hours, 1 minute to 2 hours, 2 minutes to 2 hours, 5 minutes to 2 hours, 10 minutes to 2 hours, 20 minutes to 2 hours, 30 minutes to 2 hours, 1 minute to 1 hour, 2 minutes to 1 hour, 5 minutes to 1 hour, 10 minutes to 1 hour, 20 minutes to 1 hour, 30 minutes to 1 hour, 1 minute to 40 minutes, 2 minutes to 40 minutes, 5 minutes to 40 minutes, 10 minutes to 40 minutes, 20 minutes to 40 minutes, 1 minute to 20 minutes, 2 minutes to 20 minutes, 5 minutes to 20 minutes, or 10 minutes to 20 minutes (including any range in between). Each possibility represents a separate embodiment of the present invention.

[0189] In some embodiments, MaSp fibers are available in concentrations of 10,000 psi to 24,000 psi, 12,000 psi to 24,000 psi, 15,000 psi to 24,000 psi, 18,000 psi to 24,000 psi, 10,000 psi to 21,000 psi, 12,000 psi to 21,000 psi, 15,000 psi to 21,000 psi, and 18,000 psi to 21,000 psi. The compression strength is characterized in the range of psi, 20000psi~21000psi, 10000psi~20000psi, 12000psi~20000psi, 15000psi~20000psi, 18000psi~20000psi, 10000psi~17000psi, 12000psi~17000psi, or 15000psi~17000psi (including any range in between). Each possibility represents a distinct embodiment of the present invention.

[0190] In some embodiments, MaSp fibers are characterized by flexural strengths in the ranges of 12,000 psi to 25,000 psi, 12,000 psi to 21,000 psi, 15,000 psi to 21,000 psi, 18,000 psi to 21,000 psi, 20,000 psi to 21,000 psi, 12,000 psi to 20,000 psi, 15,000 psi to 20,000 psi, 18,000 psi to 20,000 psi, 12,000 psi to 17,000 psi, or 15,000 psi to 17,000 psi (including any range in between). Each possibility represents a distinct embodiment of the present invention.

[0191] The term "extruded product" refers to a product in which the composition is heated and / or compressed until it is molten (or softened) and then extruded. As used herein, the term "extrudeable composition" refers to the ability of the composition to be extruded. As used herein, "extruded product" and "extrudeable composition" refer not only to compositions containing thermoplastic polymers, but also to polymers that are readily extruded by known techniques or otherwise behave similarly to thermoplastic polymers with respect to the extrusion process.

[0192] In some embodiments, the extruded product is characterized by improved thermal stability compared to the thermal stability of high-melting-temperature polymers that do not contain MaSp fibers.

[0193] In some embodiments, the extruded product is characterized by at least one improved mechanical property compared to the properties of high melting temperature polymers that do not contain MaSp fibers, where the property is selected from the group consisting of Young's modulus, storage modulus, loss modulus, tensile strength, fracture strain, yield point, toughness, work of fracture, impact strength, tear strength, flexural modulus, flexural strain and stress at a particular elongation, and wear.

[0194] According to some embodiments, the present invention provides articles comprising the extruded articles described herein.

[0195] In some embodiments, the article may be in the form of reinforced plastic, a bottle, a container, a package, a cable, a tube, a film, a rope, a thread, or a fabric.

[0196] In some embodiments, the article is characterized by at least one improved mechanical property compared to the properties of an article without the composition, wherein the property is selected from the group consisting of Young's modulus, tensile strength, fracture strain, yield point, toughness, work of fracture, impact strength, tear strength, flexural modulus, flexural strain and stress at a particular elongation, wear, UV resistance, and gas permeability.

[0197] In some embodiments, the present invention provides abrasion-resistant extruded articles. In some embodiments, the present invention provides extruded articles having improved abrasion resistance. As used herein, the term “abrasion resistance” refers to the ability of a material to stop displacement when exposed to the relative movement of hard particles or protrusions. Abrasion resistance can be measured by various tests known in the art, such as abrasion (Taber) tests, Gardner scrubber tests, and sand-dropping tests.

[0198] In some embodiments, one or more properties selected from thermal stability, Young's modulus, tensile strength, yield point, abrasion resistance, and tensile stress are increased by, for example, at least 1%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 50%, at least 100%, at least 200%, or at least 500%. Each possibility represents a distinct embodiment of the present invention.

[0199] In some embodiments, one or more properties selected from thermal stability, Young's modulus, tensile strength, yield point, abrasion resistance, and tensile stress are increased by, for example, at least 100%, at least 150%, at least 250%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 1000%, at least 1500%, at least 2000%, at least 2500%, or at least 3000%. Each possibility represents a distinct embodiment of the present invention.

[0200] In some embodiments, the composition is characterized by a Young's modulus in the range of 50 MPa to 170 MPa, 52 MPa to 170 MPa, 60 MPa to 170 MPa, 68 MPa to 170 MPa, 90 MPa to 170 MPa, 100 MPa to 170 MPa, 101 MPa to 170 MPa, 105 MPa to 170 MPa, 101 MPa to 160 MPa, or 105 MPa to 160 MPa (including any range in between). Each possibility represents a separate embodiment of the present invention.

[0201] In some embodiments, at least two properties selected from thermal stability, Young's modulus, tensile strength, yield point, abrasion resistance, and tensile stress are increased by, for example, at least 1%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 50%, at least 100%, at least 200%, or at least 500%. Each possibility represents a distinct embodiment of the present invention.

[0202] In some embodiments, at least three properties selected from thermal stability, Young's modulus, tensile strength, yield point, abrasion resistance, and tensile stress are increased by, for example, at least 1%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 50%, at least 100%, at least 200%, or at least 500%. Each possibility represents a distinct embodiment of the present invention.

[0203] In some embodiments, thermal stability is increased by, for example, at least 1%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 50%, at least 100%, at least 200%, or at least 500%. Each possibility represents a distinct embodiment of the present invention.

[0204] In some embodiments, the Young's modulus is increased by, for example, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 50%, at least 100%, at least 200%, or at least 500%. Each possibility represents a distinct embodiment of the present invention.

[0205] In some embodiments, the tensile strength is increased by, for example, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, or at least 50%. Each possibility represents a distinct embodiment of the present invention.

[0206] In some embodiments, the yield point is raised by, for example, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, or at least 50%. Each possibility represents a distinct embodiment of the present invention.

[0207] In some embodiments, the abrasion resistance is increased by, for example, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, or at least 50%. Each possibility represents a distinct embodiment of the present invention.

[0208] In some embodiments, the extruded product is characterized by structural strength derived from MaSp fibers, with more than 20% of the structural strength incorporated. In some embodiments, the composite material is characterized by structural strength derived from MaSp fibers, with more than 30% of the structural strength incorporated.

[0209] In some embodiments, the extruded product is characterized by structural strength derived from MaSp fibers incorporating more than 1% of the tensile strength. In some embodiments, the extruded product is characterized by structural strength derived from MaSp fibers incorporating more than 5% of the tensile strength. In some embodiments, the extruded product is characterized by structural strength derived from MaSp fibers incorporating more than 10% of the tensile strength. In some embodiments, the extruded product is characterized by structural strength derived from MaSp fibers incorporating more than 20% of the tensile strength. In some embodiments, the extruded product is characterized by tensile strength obtained from MaSp fibers incorporating more than 30% of the structural strength.

[0210] MaSp fiber The terms “large bottle gland spidoin protein” and “spirulina protein” are used synonymously throughout this specification and encompass all known large bottle gland spidoin proteins, which are typically abbreviated as “MaSp” or, in the case of the garden spider, “ADF.” These large bottle gland spidoin proteins are generally of two types, 1 and 2. These terms further include non-natural proteins disclosed herein that have a high degree of identity and / or similarity to at least the repeating regions of known large bottle gland spidoin proteins. Further preferred spider silk proteins include MaSp2, MiSp, MiSp2, AcSp, FLYS, FLAS, and flagellar gland-derived proteins.

[0211] As used herein, the terms “repeating region,” “repeating sequence,” or “repeat” refer to recombinant protein sequences derived from repeat units that occur multiple times naturally in spider silk amino acid sequences (e.g., in the MaSp-1 protein). Those skilled in the art will know that the primary structure of spider silk proteins is thought to consist primarily of a series of small variations of unit repeats. These unit repeats in naturally occurring proteins are often different from one another; that is, there is little or no exact overlap of unit repeats along the length of the protein. In some embodiments, synthetic spider silk of the present invention is prepared such that the primary structure of the protein contains many exact repeats of a single unit repeat. In further embodiments, the synthetic spider silk of the present invention contains many repeats of one unit repeat along with many repeats of a second unit repeat. Such structures are similar to typical block copolymers. Alternatively, unit repeats of several different sequences may be combined to obtain synthetic spider silk proteins with properties suitable for specific applications. As used herein, the term “direct repeat” refers to tandem repeats (head-tail arrangement) having similar repeats. In another embodiment, the repeats used to form the synthetic spider silk of the present invention are direct repeats. In some embodiments, such repeats do not exist in nature (i.e., they are not naturally occurring amino acid sequences).

[0212] An exemplary sequence containing a repeating sequence is ADF-4:(SEQ ID NO: 1). In some embodiments, the synthetic repeating sequence of the present invention is based on one or more repeating sequences derived from ADF-4(SEQ ID NO: 1) (e.g., having a high degree of identity as defined herein). As used herein, the term “based on” refers to a sequence having a high degree of homology to the repeating sequence.

[0213] In some embodiments, each repeat sequence includes up to 60 amino acids, up to 55 amino acids, up to 50 amino acids, up to 49 amino acids, up to 48 amino acids, up to 47 amino acids, up to 46 amino acids, up to 45 amino acids, up to 44 amino acids, up to 43 amino acids, up to 42 amino acids, up to 41 amino acids, up to 40 amino acids, up to 39 amino acids, up to 38 amino acids, up to 37 amino acids, up to 36 amino acids, or up to 35 amino acids, where each possibility represents a separate embodiment of the present invention. In some embodiments, each repeat sequence includes 5 to 60 amino acids, 10 to 55 amino acids, 15 to 50 amino acids, 20 to 45 amino acids, 25 to 40 amino acids, 25 to 39 amino acids, or 28 to 36 amino acids, where each possibility represents a separate embodiment of the present invention. In some embodiments, each repeat sequence comprises 30–40 amino acids, 31–39 amino acids, 32–38 amino acids, 33–37 amino acids, and 34–36 amino acids, where each possibility represents a distinct embodiment of the present invention. In further embodiments, each repeat sequence comprises 35 amino acids.

[0214] In some embodiments, the repeating region is independently the amino acid sequence (X1) shown in Formula 1. Z X2GPGGYGPX3X4X5GPX6GX7GGX8GPGGPGX9X 10 The formula includes such that X1 is independently A or G in each case.

[0215] In some embodiments, (X1) Z At least 50% of X is A, Z is an integer between 5 and 30, X2 is S or G, X3 is G or E, X4 is G, S or N, X5 is Q or Y, X6 is G or S, X7 is P or R, X8 is Y or Q, X9 is G or S, X 10 It is either S or G.

[0216] In another embodiment, the repeat region of the MaSP1 protein includes the amino acid sequence shown in SEQ ID NO: 2 (SGPGGYGPGSQGPSGPGGYGPGGPGSS). In yet another embodiment, the repeat region of the MaSP1 protein includes the amino acid sequence shown in SEQ ID NO: 3 (AAAAAAAASGPGGYGPGSQGPSGPGGYGPGGPGSS).

[0217] In another embodiment, homologs of repeat regions of the MaSP1 protein are provided that share at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with SEQ ID NO: 1. Each possibility represents a distinct embodiment of the present invention.

[0218] In another embodiment, the homologue shares at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with SEQ ID NO: 2. Each possibility represents a distinct embodiment of the present invention.

[0219] In another embodiment, the repeat region of the MaSP1 protein has the amino acid sequence shown in SEQ ID NO: 1.

[0220] In another embodiment, the MaSP1 protein includes a single N-terminal region selected from the group consisting of SEQ ID NO: 4 (MSYYHHHHHHDYDIPTTENLYFQGAMDPEFKGLRRRAQLV), SEQ ID NO: 5 (MSYYHHHHHHDYDIPTTENLYFQGAMDPEFKGLRRRAQLVRPLSNLDNAP), SEQ ID NO: 6 (MSYYHHHHHHDYDIPTTENLYFQGAMDPEFKGLRRRAQLVDPPGCRNSARAGSS), or any functional homologue, variant, derivative, or fragment thereof. In another embodiment, the C-terminal region homologue shares at least 70% homology with any one of SEQ ID NOs: 4-6.

[0221] In another embodiment, the MaSP1 protein further comprises a single C-terminal region selected from the group consisting of SEQ ID NO: 7 (VAASRLSSPAASSRVSSAVSSLVSSGPTNGAAVSGALNSLVSQISASNPGLSGCDALVQALLELVSALVAILSSASIGQVNVSSVSQSTQMISQALS), SEQ ID NO: 8 (GPSGPGAYGPSPSASASVAASRLSSPAASSRVSSAVSSLVSSGPTNGAAVSGALNSLVSQISASNPGLSGCDALVQALLELVSALVAILSSASIGQVNVSSVSQSTQMISQALS), or any functional homologue, variant, derivative, fragment, or mutant thereof. In another embodiment, the N-terminal region homologue shares at least 70% homology with SEQ ID NOs: 7-8.

[0222] In some embodiments, the MaSp-based fibers comprise a mixture of proteins disclosed in International Publication No. 2017025964, the entirety of which is incorporated herein by reference.

[0223] In some embodiments, the MaSP1 protein further comprises at least one tag sequence. Non-limiting examples of tags that can be used in the present invention include His tags, HA tags, and T7 tags. Those skilled in the art are well aware of other suitable tags or other fusion partners.

[0224] As used herein, “amino acids” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimes that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. “Amino acid analogs” refer to compounds that have the same basic chemical structure as naturally occurring amino acids, namely hydrogen, a carboxyl group, an amino group, and an alpha carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have a modified R group or a modified peptide skeleton but retain the same basic chemical structure as naturally occurring amino acids. “Amino acid mimes” refer to compounds that have a structure different from the general chemical structure of amino acids but function similarly to naturally occurring amino acids. Amino acids may be referred herein by either their commonly known three-letter or one-letter symbols as recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0225] An "amino acid sequence" or "peptide sequence" is the order in which amino acid residues linked by peptide bonds are located within a chain of peptides and proteins. This sequence is generally reported from the N-terminus, which contains a free amino group, to the C-terminus, which contains a free carboxyl group. While amino acid sequences are often called peptides, or protein sequences, when they represent the primary structure of a protein, proteins are defined as amino acid sequences folded into a specific three-dimensional structure and typically undergo post-translational modifications such as phosphorylation, acetylation, glycosylation, sulfhydryl bond formation, and cleavage. Therefore, the terms "amino acid sequence" or "peptide sequence" must be distinguished from the term "protein."

[0226] As used herein, “isolated” or “substantially purified” means, in the context of the synthetic spider silk amino acid sequences or nucleic acid molecules encoding them as illustrated by the present invention, that the amino acid sequences or polynucleotides have been removed from their natural environment or altered from their natural state. Thus, “isolated” does not necessarily reflect the degree to which the amino acid sequences or nucleic acid molecules are purified. However, it will be understood that such molecules that are purified to some extent are “isolated.” If a molecule does not exist in its natural environment, i.e., does not exist naturally, then the molecule is “isolated,” regardless of where it exists. For example, an amino acid sequence or polynucleotide that does not exist naturally in humans is “isolated,” even if it is present in humans.

[0227] The terms “isolated” or “substantially purified,” when applied to amino acid sequences or nucleic acids, mean that the amino acid sequence or nucleic acid essentially does not contain other cellular components to which it is naturally associated. It may exist in a homogeneous state, or in either dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The amino acid sequence or nucleic acid, which is the main species present in the formulation, is substantially purified.

[0228] In some embodiments, the repeat is a homolog, variant, or derivative of the repeat region of the MaSp1 protein or a fragment thereof. In some embodiments, the repeat is a homolog, variant, or derivative of the repeat region of the ADF-4 protein or a fragment thereof.

[0229] As used herein in relation to "functional homologues, variants, derivatives, or fragments," the term "functional" refers to an amino acid sequence having a biological function or activity identified by a specified functional assay. More specifically, the specified functional assay is the formation of self-assembling fibers in cells expressing the functional homologue, variant, derivative, or fragment.

[0230] An amino acid sequence or nucleic acid sequence is a homolog of the corresponding amino acid sequence or nucleic acid if it is determined that the homology is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%.

[0231] The terms “identical,” “substantial identity,” “substantial homology,” or “identical percentage” refer to two or more sequences or subsequences that, in the context of two or more amino acid or nucleic acid sequences, are the same amino acid residue or nucleotide, or have a specified percentage of the same amino acid residue or nucleotide (i.e., approximately 60% identity across a specified region (e.g., SEQ ID NO: 2 or 3 of the amino acid sequence) when compared and aligned for maximum match across a comparison window or specified region, or at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identity) when compared and aligned for maximum match across a comparison window or specified region. Such sequences are then said to be “substantial identity.” This definition may also refer to or apply to complements of test sequences. This definition includes sequences with deletions and / or additions, as well as sequences with substitutions. Preferred algorithms can account for gaps, etc.

[0232] Typically, one sequence acts as a reference sequence for sequence comparison, and the test sequence is compared to it. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Preferably, default program parameters can be used, or other parameters can be specified. The sequence comparison algorithm then calculates the degree of sequence identity of the test sequence to the reference sequence based on the program parameters.

[0233] Naturally, the present invention further encompasses amino acid sequences comprising n repeats of any one variant of SEQ ID NOs: 1, 2, or 3. As used herein, the terms “variant” or “substantially similar” include amino acid or nucleotide sequences that differ from a specifically identified sequence in which one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 25) amino acid residues or nucleotides are deleted, substituted, or added. Variants may be naturally occurring allele variants or non-natural variants. Variants or substantially similar sequences refer to amino acid sequences or nucleic acid fragments that may be characterized by a degree of identity between those amino acid or nucleotide sequences and the amino acid or nucleotide sequences described herein, as determined by common algorithms used in state-of-the-art art. Preferred fragments of amino acids or nucleic acids have an amino acid or nucleotide sequence having at least about 40 or 45% sequence identity, preferred about 50 or 55% sequence identity, more preferably about 60 or 65% sequence identity, more preferably about 70 or 75% sequence identity, more preferably about 80 or 85% sequence identity, and even more preferably about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity when compared to a reference sequence. Each possibility represents a distinct embodiment of the present invention.

[0234] In one embodiment, the MaSp polymer is a fiber.

[0235] In one embodiment, the MaSp polymer consists of monomers. In one embodiment, multiple monomers are arranged within nanofibrils. In one embodiment, multiple nanofibrils are arranged within or constitute a fiber. In one embodiment, the monomers or nanofibrils within the MaSp polymer or fiber have a diameter of 4 to 16 nm. In one embodiment, the monomers or nanofibrils within the MaSp polymer or fiber have a diameter of 6 to 14 nm. In one embodiment, the monomers or nanofibrils within the MaSp polymer or fiber have a diameter of 8 to 12 nm. In one embodiment, the fiber or MaSp polymer has a diameter of 70 to 450 nm. In one embodiment, the protein fiber or MaSp polymer has a diameter of 80 to 350 nm. In one embodiment, the fiber or MaSp polymer has a diameter of 80 to 300 nm. In one embodiment, the fiber or MaSp polymer has a diameter of 150 to 250 nm. In one embodiment, the fiber or MaSp polymer is arranged as a coil. In one embodiment, a single fiber, i.e., one MaSp-based polymer, is arranged as a coil. In one embodiment, the coil has a diameter of 5 to 800 μm. In one embodiment, the coil has a diameter of 5 to 500 μm. In one embodiment, the coil has a diameter of 5 to 30 μm. In one embodiment, the coil has a diameter of 5 to 20 μm. In one embodiment, the fiber or MaSp-based polymer has a length of 5 to 800 μm. In one embodiment, the fiber or MaSp-based polymer has a length of 30 to 300 μm.

[0236] In one embodiment, the fiber or MaSp polymer is branched. In one embodiment, the fiber or MaSp polymer contains 1 to 10 branches. In one embodiment, the fiber or MaSp polymer does not contain carbohydrates. In one embodiment, the fiber or MaSp polymer is not glycosylated. In one embodiment, the fiber or MaSp polymer does not contain fats or fatty acids. In one embodiment, the fiber or MaSp polymer does not contain phosphorus. In one embodiment, the fiber or MaSp polymer does not contain further non-MaSp proteins. In one embodiment, the fiber or MaSp polymer does not contain further polymers (e.g., synthetic polymers, non-MaSp peptides, non-MaSp proteins). In one embodiment, the fiber or MaSp polymer substantially does not contain further polymers. In one embodiment, "does not contain" means "lacks" or essentially "lacks."

[0237] In one embodiment, the length-to-diameter aspect ratio of the fiber or MaSp polymer is at least 1:10. In one embodiment, the length-to-diameter aspect ratio of the fiber or MaSp polymer is at least 1:10 to 1:1500. In one embodiment, the length-to-diameter aspect ratio of the fiber or MaSp polymer is at least 1:50 to 1:1000. In one embodiment, the length-to-diameter aspect ratio of the fiber or MaSp polymer is at least 1:100 to 1:1200. In one embodiment, the length-to-diameter aspect ratio of the fiber or MaSp polymer is at least 1:100 to 1:1000. In one embodiment, the length-to-diameter aspect ratio of the fiber or MaSp polymer is at least 1:500 to 1:1000.

[0238] As used herein, the terms derivative and functional derivative mean the amino acid sequences of the present invention having any insertions, deletions, substitutions, and modifications.

[0239] Naturally, as used herein, the term “insertion” means the addition of any number of amino acid residues to the sequence of the present invention, specifically 1 to 50 amino acid residues, more specifically 20 to 1 amino acid residues, and more specifically 1 to 10 amino acid residues. Most specifically, these are 1, 2, 3, 4, 5, 6, 7, 8, 9 to 10 amino acid residues. Furthermore, the amino acid sequence of the present invention may be extended at its N-terminus and / or C-terminus by a variety of identical or different amino acid residues.

[0240] An amino acid "substitution" is the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties; that is, a conservative amino acid substitution. Amino acid substitutions may be made based on the similarity of the residues involved in terms of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar, neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0241] In another embodiment, the repeat sequence of the present invention has 17 or fewer, 16 or fewer, 15 or fewer, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, or 7 or fewer amino acid substitutions in any one of sequence numbers 2 or 3. In one embodiment, the repeat sequence of the present invention has at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 amino acid substitutions in any one of sequence numbers 1, 2 or 3.

[0242] Those skilled in the art with respect to amino acid sequences will recognize that individual substitutions, deletions, or additions to an amino acid, nucleic acid, peptide, polypeptide, or protein sequence that alter, add, or delete a single amino acid or a small proportion of amino acids in the encoded sequence are “conservatively modified variants” in which the alteration results in the substitution of an amino acid with a chemically similar amino acid. Tables of conservative substitutions that provide functionally similar amino acids are well known in the art. Such conservatively modified variants also include, but are not excluded from, the polymorphic variants, interspecific homologs, and alleles of the present invention.

[0243] For example, substitutions may be made in which an aliphatic amino acid (G, A, I, L, or V) is replaced with another member of the following groups, i.e., substitutions in which one polar residue is replaced with another, such as replacing arginine with lysine, glutamic acid with aspartic acid, or glutamine with asparagine. Each of the following eight groups includes other exemplary amino acids in which substitutions are conserved: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M).

[0244] A conservative nucleic acid substitution is a nucleic acid substitution that yields the conservative amino acid substitution defined above.

[0245] The amino acid sequence variant of the present invention may have at least 80% sequence similarity, at least 85% sequence similarity, 90% sequence similarity, or at least 95%, 96%, 97%, 98%, or 99% sequence similarity at the amino acid level with respect to the repeat unit represented by one of SEQ ID NOs: 2 or 3.

[0246] The amino acid sequence of the present invention may contain 2 to 70 repeats of SEQ ID NO: 1 or 3 or any fragment thereof. A "fragment" constitutes a fraction of an amino acid or DNA sequence of a specific region. A fragment of a peptide sequence is at least one amino acid shorter than a specific region, and a fragment of a DNA sequence is at least one base pair shorter than a specific region. The fragment may be cleaved at the C-terminus or N-terminus side or both. The amino acid fragment may contain at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 24, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33 or at least 34 amino acids of SEQ ID NO: 1 or 3. Each possibility represents a separate embodiment of the present invention.

[0247] Mutants of the amino acid sequence of the present invention are characterized by one or more (point mutants) of that amino acid for one or more of another amino acid and about 10 or fewer exchanges. They are the result of corresponding mutations at the DNA level that result in different codons.

[0248] Furthermore, the present invention relates to derivatives of the amino acid sequences of the present invention. The derivatives of the amino acid sequences of the present invention have functional groups such as amino, hydroxyl, mercapto or carboxyl groups derivatized, for example, glycosylated, acylated, amidated or esterified, respectively. In glycosylated derivatives, the oligosaccharide is usually linked to asparagine, serine, threonine and / or lysine. The acylated derivatives are acylated particularly by naturally occurring organic or inorganic acids such as acetic acid, phosphoric acid or sulfuric acid, which usually occur particularly at the N-terminal amino group or the hydroxy group of tyrosine or serine, respectively. The ester is an ester of a naturally occurring alcohol such as methanol or ethanol. Further derivatives are salts, particularly pharmaceutically acceptable salts, such as metal salts such as alkali metal and alkaline earth metal salts, such as sodium, potassium, magnesium, calcium or zinc salts, or ammonium salts formed with ammonia or a suitable organic amine, such as a lower alkylamine such as triethylamine, a hydroxy-lower alkylamine such as 2-hydroxyethylamine, etc.

[0249] In some embodiments, the silk protein of the present invention lacks post-translational modifications.

[0250] In some embodiments, the silk protein of the present invention is biodegradable. This property can always be important, for example, in the field of medicine, when the silk protein is for in vivo use where biodegradation is desired. This property may be applied to suture materials as well as wound closure and coating systems.

[0251] According to some aspects, the MaSp-based fibers of the present invention are produced using an expression vector containing a suitable nucleic acid sequence, where the nucleic acid sequence is under the expression control of a promoter and optionally regulatory sequences operably linked. Exemplary expression systems such as the expression system disclosed in PCT / IL2020 / 050752 are known in the art.

[0252] In some embodiments, MaSp proteins self-assemble to form a predetermined structure. In some embodiments, MaSp proteins exist as a network. In some embodiments, MaSp proteins exist as a complex. In some embodiments, MaSp proteins induce predetermined secondary structures, such as β-turns, γ-turns, β-sheets, and α-helix structures.

[0253] In some embodiments, the MaSp proteins and / or MaSp polymers used interchangeably herein are in the form of fibers, and therefore, in some embodiments of the present invention, the terms MaSp polymer and MaSp fiber are used interchangeably herein. As used herein, “fiber” means a thin string of fibrous material consisting of two or more filaments twisted together. “Filament” means an elongated, thread-like object or structure of indeterminate length ranging from microscopic to more than one mile. Specifically, synthetic spider silk filaments are microscopic and proteinaceous. “Biofilament” means a filament made from a protein containing recombinant spider silk protein. In some embodiments, the term “fiber” does not include unstructured aggregates or precipitates.

[0254] In some embodiments, the protein fiber is characterized by a size of at least one dimension (e.g., diameter, length). For example, but not limited to, the diameter of the fiber may be 10 nm to 1 μm, 20 to 100 nm, or 10 to 50 nm.

[0255] In some embodiments, the fiber consists of nanofibrils. In some embodiments, the nanofibrils have a diameter of, for example, 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, about 20 nm, about 21 nm, about 22 nm, about 23 nm, about 24 nm, about 25 nm, about 26 nm, about 27 nm, about 28 nm, about 29 nm, about 30 nm, about 31 nm, about 32 nm, about 33 nm, about 34 nm, about 35 nm, about 36 nm, about 37 nm, about 38 nm, about 40 nm, about 42 nm, about 44 nm, about 46 nm, about 48 nm or about 50 nm (including any value or range in between). Each possibility represents a distinct embodiment of the present invention. In one embodiment, the nanofibril has a diameter of 3 to 7 nm. In another embodiment, the nanofibril has a diameter of 4 to 6 nm.

[0256] In some embodiments, the disclosed fiber lengths are 1–200 μm, 10–100 μm, 100–500 μm, or 200–500 μm.

[0257] In some embodiments of any one of the embodiments described herein, the disclosed fibers (e.g., particles) are characterized by a porous structure. In some embodiments, the porous structure is characterized by at least 30% (e.g., 30-99%) porosity. In some embodiments, the porous structure is characterized by at least 50% (e.g., 50-99%) porosity. In some embodiments, the porous structure is characterized by at least 60% (e.g., 60-99%) porosity. In some embodiments, the porous structure is characterized by at least 70% (e.g., 70-99%) porosity. In some embodiments, the porous structure is characterized by at least 80% (e.g., 80-99%) porosity. In some embodiments, the porous structure is characterized by at least 90% (e.g., 90-99%) porosity. In some embodiments, the porous structure is characterized by about 90% porosity.

[0258] In this specification, the term "porosity" refers to the percentage of volume of a material consisting of voids (e.g., a "sponge-like" material). In another embodiment, porosity is measured by dividing the voids or lumens within the surface area by the total surface area (porous and non-porous).

[0259] In some embodiments, the porous structure of the disclosed fibers allows for efficient absorption of water onto the fiber surface. That is, although not bound by any particular theory, this remarkable finding can be explained in terms of the structure of the disclosed fibers and their porosity, which are strictly distinct from natural spider silk found in nature.

[0260] In some of the embodiments described herein, the disclosed fibers are characterized by having an average diameter of nanoscale.

[0261] In some embodiments, the disclosed fibers are characterized by having an average diameter in the range of 1 to 50 nm. In some such embodiments, the average diameter is in the range of 3 to 50 nm. In some such embodiments, the average diameter is in the range of 5 to 50 nm. In some such embodiments, the average diameter is in the range of 1 to 40 nm. In some such embodiments, the average diameter is in the range of 1 to 30 nm. In some such embodiments, the average diameter is in the range of 5 to 40 nm.

[0262] In some embodiments, the MaSp fibers contain multiple pores. In some embodiments, the porous MaSp fibers contain multiple fibrils (e.g., nanofibrils). In some embodiments, the MaSp fibers are in the form of particles as described below in this specification. In some embodiments, the MaSp fibers are as described below in this specification. In some embodiments, the composition contains multiple MaSp fibers. In some embodiments, the multiple MaSp fibers contain fibers having different chemical compositions and / or different molecular weights (MW).

[0263] As further illustrated in the following sections on embodiments, in some embodiments, the multiple disclosed fibers may be in the form of a self-assembling structure or matrix. In some embodiments, this matrix can be made suitable for biomaterial applications.

[0264] In some embodiments, the matrix is ​​suitable for cell proliferation and for maintaining or promoting cell activity, as further demonstrated herein.

[0265] In some embodiments, the term “self-assembly” refers to a structure resulting from a self-assembly process (e.g., a spontaneous self-assembly process) based on a series of associative chemical reactions between at least two domains of the fiber, which occurs when associating groups on one domain are sufficiently close together and oriented to allow constitutive association with another domain. In other words, associative interaction means proximity that causes the attachment of one or more domains of a fiber to each other. In some embodiments, the attached domains are not parallel to each other. Configurations are also conceivable in which there are three or more domains of the self-assembly structure, each engaged in a different plane.

[0266] In some embodiments, the density of the self-assembled fibers (e.g., about 80% voids) is 0.1 g / cm³. 3 ~0.4g / cm 3 Or 0.2 g / cm³ 3 ~0.3g / cm 3 It is worth noting that the range is within this range. In exemplary embodiments, the density of the self-assembling fibers is approximately 0.26 g / cm³. 3 That is the case.

[0267] General matters As used herein, the term "approximately" refers to a range of ±10%.

[0268] The terms "comprises", "comprising", "includes", "including", "having" and their conjugations mean "including, but not limited to".

[0269] The term "consisting of" means "including and limited to those".

[0270] The term "consisting essentially of" means that the present composition, method or structure may include additional components, steps and / or parts, but only if the additional components, steps and / or parts do not substantially change the basic and novel characteristics of the composition, method or structure according to the claim.

[0271] As used herein, the word "exemplary" is used to mean "serving as an example, instance or illustration". Any embodiment described as "exemplary" should not necessarily be construed as being more preferred or advantageous than other embodiments and / or as excluding the incorporation of features from other embodiments.

[0272] As used herein, the word "optionally" is used to mean "provided in some embodiments and not provided in other embodiments". Any particular embodiment of the present invention may include a plurality of "optional" features as long as such features are not contradictory.

[0273] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly indicates otherwise. For example, the terms "compound" or "at least one compound" may include a plurality of compounds including mixtures thereof.

[0274] Throughout this application, various embodiments of the invention may be presented in range form. Naturally, range form is merely for convenience and brevity and should not be interpreted as an unchangeable limitation on the scope of the invention. Therefore, range descriptions should be interpreted to include all possible sub-ranges specifically disclosed, as well as the individual numbers within those ranges. For example, a range description such as 1-6 should be interpreted to include specifically disclosed sub-ranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, as well as the individual numbers within those ranges, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0275] Wherever a range of numbers is indicated herein, it is intended to include any enumerated numbers (fractions or integers) within that range. The phrases “the range between the first and second indicators” and “the range between the first and second indicators” are used interchangeably herein and are intended to include the first and second indicators and all fractions and integers between them.

[0276] As used herein, the term “method” refers to a manner, means, techniques and procedures for accomplishing a given task, including, but not limited to, manners, means, techniques and procedures that are known or readily developed from manners, means, techniques and procedures known to practitioners of the fields of chemistry, pharmacology, biology, biochemistry and medicine.

[0277] As used herein, the term “treat” includes inhibiting, substantially blocking, slowing or reversing the progression of a disease, substantially improving the clinical or aesthetic symptoms of a disease, or substantially preventing the appearance of the clinical or aesthetic symptoms of a disease.

[0278] For clarity, it should be understood that certain features of the Invention described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the Invention described in the context of a single embodiment may also be provided separately or as any preferred partial or combination in any other described embodiment of the Invention. Certain features described in the context of different embodiments should not be considered essential features of those embodiments unless the embodiment cannot be carried out without those elements.

[0279] Various embodiments and aspects of the present invention, detailed herein and claimed in the following claims, are experimentally supported in the following examples. [Examples]

[0280] Next, the following embodiments illustrate some embodiments of the present invention in conjunction with the above description, in a non-limiting manner.

[0281] Experimental Procedure Bacterial growth Bacteria expressing pET24R (produced according to the procedure disclosed in PCT / IL2020 / 050752) were seeded in 3 mL of LB medium containing chloramphenicol and kanamycin as a starter culture, grown until the OD reached approximately 0.6 (measured in 100 μL in a 96-well plate), and then seeded into growth medium.

[0282] This bacterium was grown at 37°C with shaking, and after about 20 hours, OD 600 And β-sheet specific staining was performed. Significant β-sheet staining appeared after 24 hours. Further details are disclosed in PCT / IL2020 / 050752, which is incorporated herein by reference.

[0283] General protocol for reinforcing polymers with spider silk fibers The spider silk fiber suspension was centrifuged and resuspended in a sufficient amount of solvent. This suspension was poured into the polymer solution. The polymer and spider silk polymer suspensions were thoroughly mixed until homogeneity was achieved. The solution was then dried.

[0284] Example 1: Purification of SVX fibers produced by bacterial cells Bacteria (e.g., bacteria expressing pET24R) were centrifuged and resuspended in deionized water. A surfactant solution was then added, and the resulting suspension was shaken overnight at 37°C. After centrifugation, the pellet (particularly containing isolated MaSp fibers) was resuspended in 6M urea. After centrifugation, the pellet was resuspended and washed several times with the surfactant solution.

[0285] The resulting pellets (isolated MaSp fibers) were then washed with t-butanol, and this washing was repeated 2-3 times. Then, t-butanol was added to the MaSp fibers until a homogeneous suspension was obtained. The suspension was stirred to prevent the formation of aggregates. After centrifugation, the MaSp fibers were dried by freeze-drying, which included freezing the suspension (e.g., in liquid nitrogen) until the solvent was completely evaporated, and then applying a vacuum to the frozen suspension. The inventors further obtained freeze-dried MaSp fibers as described herein by successfully mixing t-butanol with up to 20% w / w water to obtain a suspension and freeze-drying this mixture. While the exact freeze-drying conditions may vary, those skilled in the art will be able to adjust the freeze-drying conditions (e.g., vacuum, freeze-drying time, freezing temperature, etc.) to obtain freeze-dried MaSp fibers as disclosed herein.

[0286] Figures 1A, 1C, and 1E show the results for fibers obtained after purification and freeze-drying with t-butanol, compared to similar fibers freeze-dried from water.

[0287] Example 2: Mechanical properties In an exemplary experiment, polyurethane (PU) (E394POTA) was reinforced with 15% SVX-E lyophilized from t-butanol or water. The stress-strain curves of the PU reinforced with t-butanol and 15% SVX-E lyophilized from water, compared to the control (unreinforced PU), are shown in Figure 2.

[0288] Example 3: Loading and release of hyaluronic acid To evaluate the release of hyaluronic acid (HA) from SVX-E lyophilized with t-butanol and SVX-E lyophilized with water, formulations were prepared using different SVX-E and HA. Hyaluronic acid (1:1 w / w) was added to SVX-E lyophilized with t-butanol and SVX-E lyophilized with water.

[0289] The release of HA was evaluated using water washing, and the ratio of HA in the SVX-HA complex was measured using Fourier transform infrared spectroscopy (FTIR).

[0290] Graphs showing the HA release profiles from SVX-E freeze-dried with t-butanol and SVX-E freeze-dried with water are shown in Figures 3A and 3B.

[0291] SVX-E lyophilized with t-butanol and fortified with HA exhibits a slower HA release rate compared to SVX-E lyophilized with water and fortified with HA.

[0292] Example 4: Loading and release of glycolic acid To evaluate the release of glycolic acid (GA) from SVX-E lyophilized with t-butanol and SVX-E lyophilized with water, formulations were prepared using different SVX-E and HA. GA (1:1 w / w) was added to SVX-E lyophilized with t-butanol and SVX-E lyophilized with water.

[0293] The release of GA was evaluated using water washing, and the ratio of GA in the SVX-GA complex was measured using FTIR.

[0294] Graphs showing the GA release profiles from SVX-E freeze-dried with t-butanol and SVX-E freeze-dried with water are shown in Figures 4A and 4B.

[0295] It can be observed that SVX-E lyophilized with t-butanol and enhanced with GA exhibits a slower release rate of GA compared to SVX-E lyophilized with water and enhanced with GA.

[0296] Example 5: BET surface area The surface area was determined from nitrogen adsorption data using the BET (Brunauer, Emmett, Teller) method described in S. Brunauer, P. Emmett, E. Teller, J. Am. Chem. Soc. 1938, 60, 309-331. BET surface area measurements were performed using Nova Station A and Quantachrome NovaWin (Quantachrome Instruments) version 11.02.

[0297] Sample preparation A 0.0168 g powdered SVX sample was heated at 120°C for 3 hours to remove gas. Analysis was performed using nitrogen at a bath temperature of 273°K for 308 minutes.

[0298] The surface area of ​​SVX dried from t-butanol and SXV dried from water was measured. The calculated surface area of ​​SVX dried from t-butanol was approximately 180 m². 2 The value was / g. The calculated surface area of ​​SVX dried from water was considerably lower than that of SVX dried from t-butanol, with the calculated value being approximately 85m². 2 It was / g.

[0299] Example 6: Thermal stability of large bottle-shaped glandular spidoin protein (MaSp) polymers Analysis of differential scanning calorimetry (DSC) curves of the provided spider silk polymer (SVX-E) expressed in bacteria revealed that SVX-E did not exhibit a melting peak. Instead, it showed small glass transition temperature (Tg) regions at approximately 220°C and 280°C. A decomposition peak was also observed at approximately 330°C (Figure 5).

[0300] Figure 6 shows the DSC curves of the SVX-E as the temperature rises from 25°C to 280°C (curve 1), cools to 50°C (curve 2), and rises again to 350°C (curve 3).

[0301] The thermogravimetric analysis (TGA) curves of SVX-E (Figures 7A-7B) show that at a heating rate of 10°C / min, the weight loss at temperatures below 100°C was only about 5% of the absorbed water. A significant weight loss (over 1% / hour) can be observed starting above 230°C.

[0302] While the present invention has been described along with its specific embodiments, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Therefore, the appended claims are intended to encompass the spirit of the invention and all such alternatives, modifications, and variations that are broadly included.

[0303] All publications, patents, and patent applications referenced herein are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated herein by reference. Furthermore, any citation or specification of references in this application should not be construed as an admission that such references are available to the invention as prior art. Section headings, insofar as they are used, should not necessarily be construed as restrictive.

Claims

1. At least 100m ２ A composition comprising a porous large bottle-shaped glandular spidoin protein (MaSp) polymer characterized by a BET surface area of ​​0.1 ppm to 100 ppm and a residual amount of t-butanol, wherein the MaSp polymer is in the form of particles having an average size in the range of 0.5 μm to 2 μm, the particles being spherical or elliptical, and the MaSp polymer is a protein comprising a single N-terminal region, a single C-terminal region, and a repeating region having the amino acid sequence shown in formula 1: (X 1) Z X 2 GPGGYGPX 3 X 4 X 5 GPX 6 GX 7 GGX 8 GPGGGPGX 9 X 10, where X 1 is independently A or G in each case, Z is an integer from 5 to 30, X 2 is S or G, X 3 is G or E, X 4 A composition in which X5 is G, S, or N, X6 is Q or Y, X7 is P or R, X8 is Y or Q, X9 is G or S, and X10 is S or G.

2. The BET surface area is 100 to 5500 m². ２ The composition according to claim 1, wherein the amount is / g.

3. The composition according to claim 1, wherein the repeating region comprises SEQ ID NO: 2 (SGPGGGYGPGSQGPGPGGGYGPGGGPGSS) or SEQ ID NO: 3 (AAAAAAASGPGGGYGPGSQGPGPGGGYGPGGGPGSS), or a homolog having at least 90% homology to SEQ ID NO:

2.

4. The composition according to claim 1, wherein the single N-terminal region consists of Sequence ID No. 4 (MSYYHHHHHHDYDIPTTENLYFQGAMDPEFKGLRRRAQLV) and the single C-terminal region consists of Sequence ID No. 7 (VAASRLSSPAASSRVSSAVSSLVVSGPTNGAAVSGALNSLVVSQISASNPGLSGCDALVQALLELVSSALVALVAILSSASIGQVNVSSVSQSTQMISQALS).

5. The composition according to claim 1, further comprising further agents.

6. The composition according to claim 5, wherein the w / w concentration of the further agent in the composition is 1 to 80%.

7. The composition according to claim 5, wherein the w / w ratio of the further agent to the porous MaSp polymer is 100:1 to 1:

10.

8. The composition according to claim 1, wherein the release rate of the further agent from the composition is reduced by at least 10% compared to a control in which the MaSp polymer is freeze-dried from water.

9. The composition according to claim 1, further comprising a thermoplastic polymer selected from polyester, polyamide, polyol, polyurethane, polyethylene, nylon, polyolefin, polyacrylate, polycarbonate, polylactic acid (PLA) or copolymers thereof, polycaprolactone (PCL), rubber, cellulose or any combination thereof.

10. The composition according to claim 9, wherein the weight / weight (w / w) ratio of the MaSp-based polymer to the polymer is 0.01:1 to 1:

1.

11. The composition according to claim 1, comprising 0.01% to 50% (w / w) of the MaSp-based polymer.

12. The composition according to claim 1, wherein the tensile strength of the composition is increased by at least 20% compared to the control.

13. A method for obtaining dried large bottle-shaped glandular spidoin protein (MaSp) polymers, (a) A step of mixing a MaSp polymer as defined in claim 1 with a liquid containing t-butanol to obtain a mixture, and (b) A step of subjecting the mixture to conditions suitable for substantially removing the liquid from the mixture. This includes, thereby, at least 100 m ２ A method for obtaining the dried MaSp polymer characterized by a BET surface area of ​​1 / g and a residual amount of t-butanol ranging from 0.1 ppm to 100 ppm.

14. The method according to claim 13, wherein the liquid further comprises water.

15. The method according to claim 13, wherein step (b) is carried out by freeze-drying.

16. A composition comprising a porous large bottle-shaped glandular spidoin protein (MaSp) polymer according to claim 1, and a carrier selected from a pharmaceutically acceptable carrier and a pharmaceutically acceptable carrier.

17. An article comprising the composition described in claim 1, which is in the form of reinforced plastic, a container, packaging material, a cable, a tube, a film, a rope, a thread, or a fabric.

18. An article according to claim 17, characterized by at least one improved mechanical property compared to the properties of the article not comprising the composition, wherein the property is selected from the group consisting of Young's modulus, tensile strength, fracture strain, yield point, toughness, work of fracture, impact strength, tear strength, flexural modulus, flexural strain and stress at a particular elongation, wear, UV resistance, and gas permeability.

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