WO2017223462A1 - Composition and method for adhering biomaterials to a target surface - Google Patents

Abstract

Described herein are compositions and methods for bonding biomaterials to tissues and inorganic surfaces.

Description

COMPOSITION AND METHOD FOR ADHERING BIOMATERIALS TO A TARGET

SURFACE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/354,352 filed June 24, 2016, the content of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention is directed to compositions and method for bonding biomatenals formed from carbohydrates and proteins to organic and inorganic surfaces. More specifically, the invention relates to compositions comprising transglutaminase (TG) and glutamic acid rich polypeptides, such as casein, and their use for bonding biomaterials, such as chitosan and collagen biomaterials, to surfaces of organic and inorganic substrates. The invention also relates to compositions and methods for wound healing.

BACKGROUND OF THE INVENTION

[0003] Natural biomaterials, such as chitosan and collagen, are useful for biomedical applications because they are biocompatible, mechanically robust and biodegradable, but it is difficult to rapidly and tightly bond them to living tissues. Thus, there is need in the art for compositions and methods that can rapidly and tightly bond biomaterials to surface of organic (such as living tissue) and inorganic substrates (such as materials used in medical products). The present invention partly addresses this need.

SUMMARY OF THE INVENTION

[0004] Embodiments of various aspects provided herein relate to compositions and methods for bonding of biomaterials to tissues and inorganic surfaces. The inventors have discovered inter alia that casein enhances bonding of different material by transglutaminase. Thus, in one aspect, the invention provides a method for bonding or adhering a biomaterial to a target surface. Generally, the method comprises applying an effective amount of a transglutaminase and a glutamic acid rich polypeptide to a target surface and contacting a biomaterial to the target surface where the transglutaminase and the glutamic acid rich polypeptide have been applied.

[0005] In another aspect, the invention provides a method for promoting wound healing. Generally, the method comprises applying a transglutaminase and a glutamic acid rich polypeptide to a surface of a wound.

[0006] In yet another aspect, the invention provides a method for forming a coating layer on a target surface. The method comprises applying a polymer, a transglutaminase and a glutamic acid rich polypeptide to the same portion of a target surface.

[0007] In some embodiments, the coating layer can act as an adhesive, e.g., as a dressing on a wound surface, or as a glue to bond two surfaces together. In some embodiments, the adhesive coating layer is selective for adhesion to the target surface.

[0008] In some embodiments, the coating layer can act as a sealant or provide a physical barrier, e.g., to reduce or stop a fluid leakage or permeation such as bleeding, or to protect a substrate or surface being coated from an external disturbance (e.g., bacteria, light, moisture, or any combinations thereof).

[0009] In some embodiments, the coating layer can form a casing, e.g., a "skin" to enclose a material therein.

[0010] In some embodiments, the coating layer can seal lung punctures.

[0011] In still another aspect, the invention provides a three component composition. The composition comprises as a first component a polymer in a flowable form, wherein the polymer comprises an amino group; as a separate second component a transglutaminase in a flowable form; and as a separate third component a glutamic acid rich polypeptide in a flowable form.

[0012] In embodiments of the various aspects of the invention, the glutamic acid rich polypeptide is casein.

[0013] In various embodiments of the aspects of the invention, the transglutaminase is a mammalian or microbial transglutaminase.

BRIEF DESCRIPTION OF THE FIGURES

[0014] FIGS. la-Id show the microbial transglutaminase (mTG) reaction and mTG- mediated bonding of biomaterial films. FIG. la shows mTG-catalyzed reactions with relevance for biomaterial bonding. The upper reaction is a deamination that occurs in the absence of amine substrates and the presence of water. mTG catalyzes the hydrolysis of the glutaminyl residue, resulting in loss of the amine group. In the lower reaction, the presence of a primary amine group results in formation of a covalent bond between both molecules. The involved atoms are O (red), N (blue) and H (white). FIG. lb is a bar graph showing bonding strengths produced by commercial mTG preparations containing mTG and maltodextrin (mTG+Ma) or both

components plus casein (mTG+Ca). FIG. lc is a graph showing kinetics of the mTG bonding reaction measured at the macroscale as the adhesive force between surfaces of two muscle tissues. FIG. Id is a graph showing more detailed examination of the first 15 minutes of the reaction dynamics shown in FIG. lc.

[0015] FIGS. 2a-2b show mTG-mediated bonding of biomaterials to an inorganic PDMS substrate. FIG. 2a shows the chemistry that underlies APTES functionalization of the PDMS surface. The highly reactive silane group in APTES silanizes the surface by forming covalent bonds with surface atoms. FIG. 2b is a bar graph showing bonding strength produced between contacting chitosan or collagen films and untreated (PDMS) or APTES-modified PDMS

(PDMS+Aptes) substrates.

[0016] FIGS. 3a-3e show the applicability of mTG bonding of biomaterial films and foams to native tissues. FIG. 3a shows the diagram of the ASTM F2392 standard protocol to measure burst strength of surgical sealants that are used to fill a 3 mm diameter hole on a collagen surface. FIG. 3b is a bar graph showing performance of different sealing materials tested in the ASTM F2392 assay presented as average maximum and ultimate pressures required to burst the seals. FIG. 3c is a bar graph showing bonding strength of a chitosan film bonded to heart and liver surface using mTG and casein (mTG+Ca) preparation. FIG. 3d is a bar graph showing bonding strength of a chitosan film adhered to skin epidermis or dermis (after epidermis is removed); control indicates bonding of chitosan to dermis, however no adhesion is observed to undamaged (fully cornified) epidermis. FIG. 3e shows a photomicrograph of a chitosan foam bonded to the surfaces of a 1 cm irregularly shaped defect in an explanted pig muscle using mTG+Ca (note the seamless nature of the adhesion).

[0017] FIGS. 4a-4e show use of mTG as a sealant for tissue punctures. FIG. 4a is a graph showing the burst test performed on a 1 cm diameter hole in the wall of a small intestine on an explanted pig, repaired with a chitosan film patch bonded to the tissue with mTG+Ca. The patch resisted a linear increment of hydraulic pressure applied through the intestinal lumen for several minutes, reaching the equivalent to the maximum physiological pressures in the human body before bursting. FIG. 4b shows images of the chitosan patch before (top) and after (bottom) on a region of the native intestine on the opposite side from where the patch burst at high pressure, corresponding to point C on the graph shown in FIG. 4a. (data not shown). FIG. 4c shows image and digital rendering of the double-canister spray device designed to form a conformal coating of chitosan with mTG+Ca with adhesive properties. Each component is loaded into separate barrels, and pushed through the nozzles using the same pressure source. The

exchangeable nozzles (blue and red in the image) have different output diameters, enabling control of the ratio between different components in the mix. FIG. 4d shows photographs of a 3 cm deep puncture in an explanted pig lung before (top) and after (bottom) it was sprayed for 10 min while cyclically inflating and deflating the lungs using the double-canister spray method (data not shown). FIG. 4e shows a scanning electron microscopic image of the

chitosan+mTG+Ca film produced at the surface of the lung using the spray method, which demonstrates its highly porous nature.

DETAILED DESCRIPTION OF THE INVENTION

[0018] Embodiments of the various aspects disclosed herein include a transglutaminase.

[0019] Embodiments of the various aspect disclosed herein include applying different components, such as one or more of a biomaterial, a transglutaminase, a glutamic acid rich polypeptide and a polymer to a target surface. The various components that are applied to the target surface can be formulated in a solution, an emulsion, an aerosol, a foam, an ointment, a paste, a lotion, a powder, a gel, a hydrogel, a hydrocolloid, a microparticle, a nanoparticle, or a cream.

[0020] Without limitations, the components can be formulated in separate compositions, all together in one composition, or some together in one composition and others in separate compositions. In various embodiments, the transglutaminase and the glutamic acid rich polypeptide are formulated in one composition. In some embodiments, the biomaterial or the polymer is formulated in a separate composition and the transglutaminase and the glutamic acid rich polypeptide are formulated in one composition.

[0021] In various embodiments, at least one of the biomaterial, transglutaminase and glutamic acid rich polypeptide or at least one of the polymer, transglutaminase and the glutamic acid rich polyopeptide can be provided in a flowable form, e.g., a state of a material that is capable of flowing. Examples of a flowable form include, but are not limited to, a liquid, a fluid, a solution, powder, particles (e.g., nanoparticles, or microparticles), or fibers (e.g., nanofibers or microfibers), a suspension, a colloid, a gel, an emulsion (e.g., oil-in-water or water-in-oil), an aerosol, a foam, an ointment, a paste, or any combinations thereof. In some embodiments, at least two of the biomaterial, transglutaminase, glutamic acid rich polypeptide and polymer are in liquid form. In some embodiments, at least two of the biomaterial, transglutaminase, glutamic acid rich polypeptide and polymer are in powder form.

[0022] Selection of an appropriate flowable form for the biomaterial, transglutaminase, glutamic acid rich polypeptide and polymer can be determined based on a number of factors, e.g., but not limited to, viscosity and/or solubility of the biomaterial, transglutaminase, glutamic acid rich polypeptide and/or polymer, reaction rate (and reaction time) of crosslinking upon contact, and any combinations thereof.

[0023] By way of example only, when the biomaterial or polymer used in the compositions and methods described herein comprises chitosan, it is more desirable to provide chitosan in a liquid than in powder (e.g., pre-dissolution of chitosan in a solvent vs. chitosan powder), partly because chitosan generally requires a specific pH condition for dissolution, and its dissolution rate is much slower than a crosslinking reaction rate with the transglutaminase (e.g., on a time scale of less than a second). Thus, if chitosan were provided in powder for use in the methods described herein, contacting chitosan powder in a transglutaminase solution could potentially result in formation of a shell of cross-linked chitosan encapsulating unreacted chitosan. Accordingly, pre-dissolution of chitosan in an acidic solvent can be more beneficial for formation of a more homogenous coating layer. Similarly, in some embodiments, it is also more desirable to provide a solution of transglutaminase so that it can react with a chitosan solution to form a more homogenous coating layer upon contact on a target surface.

[0024] The biomaterial, transglutaminase, glutamic acid rich polypeptide and polymer in a flowable form can be applied to a target surface by any methods known in the art to deliver a flowable material, including, but not limited to, extrusion, atomization, spraying, pumping, and any combinations thereof. For example, in one embodiment, application to a target surface is by a delivery method comprising atomization of the component to be applied. Exemplary atomization methods can include, but are not limited to, syringe extrusion, coaxial air flow method, mechanical disturbance method, electrostatic force method, electrostatic bead generator method, spraying, atomization using a rotary or centrifugal atomizer, air atomization (e.g., using a spray gun and air pressure), pressure atomization, vacuum atomization (e.g., by spraying from high pressure into low pressure zone), ultrasonic atomization, and any combinations thereof.

[0025] In air driven atomization, solutions can be broken into fine droplets with the aid of air flow pressure. In some embodiments, the air flow pattern can be altered to form coaxial pattern for formation of uniform droplets or particles. Coaxial air flow technique generally uses concentric streams of air which shear the liquid droplets released from one or more needles. Alternatives to the air driven mechanism include electrostatic field, mechanical disturbance and electrostatic force. Electrostatic mechanism generally utilizes a potential difference between a capillary tip such as a nozzle and a flat counter electrode to reduce the diameter of the droplets by applying an additional force (i.e., electric force) in the direction of gravitational force in order to overcome the upward capillary force of liquid. In mechanical disturbance method, liquid droplets can be broken into fine droplets using a mechanical disturbance. Typically, vibrations including ultrasonic atomization can be as a mechanical disturbance to produce fine droplets. In electrostatic force method, electrostatic forces can destabilize a viscous jet, where the electrostatic force can be used to disrupt the liquid surface instead of a mechanical disturbance.

[0026] Depending on various atomization methods, each atomization condition can be independently controlled to provide a desired atomized droplet size. For example, an aerosol of a solution can be controlled by changing instrumental/process, and/or material parameters. Exemplary instrumental/process parameters that can be varied include, but are not limited to, air pressure of a spray, nozzle size (e.g., nozzle diameter), atomization power output, flow rate of a spray, height of a nozzle head (e.g., distance of the nozzle head from a target surface), atomization duration, and material parameters that can be varied include, but are not limited to, concentration and/or viscosity of the solution, and/or concentration of a plasticizer, if any.

[0027] In one embodiment, biomaterial, transglutaminase, glutamic acid rich polypeptide or the polymer, transglutaminase and the glutamic acid rich polypeptide can be sprayed to a target surface, e.g., forming an aerosol or a mist of fine particles. In some embodiments, the delivery method can further comprise mixing the amino-polymer and transglutaminase. For example, when the amino-polymer and the transglutaminase are to be sprayed to a target surface, each component can be sprayed at an angle such that both components contact with each other in air and a mixing of the components occurs during the flight in air. [0028] The biomaterial or polymer, transglutaminase and glutamic acid rich polypeptide can be applied to the same portion of a target surface in a sequential or concurrent manner. In specific embodiments, the components can be applied concurrently or simultaneously to the same portion of a target surface. In such embodiments, the components can be individually applied to a target surface at the same time such that both components can contact and mix with each other before the mixture deposits on the target surface. For example, the components can be sprayed as separate entities concurrently or simultaneously to the same portion of a target surface such that the sprayed components contact and mix with each other during flight in air prior to depositing on the target surface.

[0029] Alternatively, the components can be applied as a single mixture to a target surface, provided that no significant pre-crosslinking in the mixture of the components occurs prior to its application to the target surface. For example, since the crosslinking reaction is generally rapid (e.g., on a time scale of less than a second), it is desirable to apply the mixture to a target surface immediately once the different components are in contact with each other.

[0030] The pre-determined volume ratio of different components applied to a target substrate or surface can vary with a number of factors, including, but not limited to, selection of an appropriate biomaterial or polymer, viscosity of the different compositions to be used, desired properties of a resulting material. In some embodiments, the pre-determined volume ratio of two components to be applied can range from about 10: 1 to about 1 : 10, or from about 5: 1 to about 1 :5, or from about 3 : 1 to about 1 :3. In some embodiments, the pre-determined volume ratio can range from about 1 : 1 to about 1 :5, or from about 1 : 1 to about 1 : 3.

[0031] In some embodiments, the method can further comprise applying an additive, in addition to the biomaterial or polymer and the transglutaminase and glutamic acid rich polypeptide, to a target surface. The additive can be mixed with one of the other prior to applying to a target surface. Alternatively, the additive can be independently applied to a target surface in concurrent with the application of the other components to the target surface. An additive can be any molecule, compound, or agent that can produce an effect (e.g., a beneficial effect) on and/or in close proximity to the target surface, and/or that can confer a new property/feature to the target surface. Examples of an additive that can be employed can include, without limitations, a living cell (e.g., a stem cell), a therapeutic agent, a hemostatic agent, an antiseptic agent (e.g., an antibiotic), a wound healing agent, a cross-linking agent, flavorings, colorings, nutraceuticals, a cell growth factor, a peptide, a peptidomimetic, an antibody or a portion thereof, an antibody-like molecule, nucleic acid, a plasticizer (e.g., glycerol), a nanoparticle or microparticle, a nanofiber, and any combinations thereof.

[0032] Without wishing to be limited, the methods disclosed herein can be performed on a target substrate or surface more than once (e.g., twice, three times, four times or more). For example, upon formation of a coating layer on a target substrate or surface by applying a first polymer composition and a first transglutaminase composition thereto, a second polymer composition and a second transglutaminase composition can be applied on the first coating layer, thereby forming a second coating layer on top of the coating layer. The applied second amino- polymer composition (including, e.g., types and/or concentrations of the polymer(s), and optionally additive(s)) can be the same as or different from the first polymer composition. Similarly, the second transglutaminase composition (including, e.g., sources and/or origins of transglutaminases) can be the same as or different from the first transglutaminase composition. Accordingly, a method for providing a multi-layer coating layer is also provided herein.

[0033] In various embodiments, the biomaterial, transglutaminase, glutamic acid rich polypeptide and polymer can be provided in an aerosol. For example, at least one (e.g., one, two, three or all four) of the biomaterial, transglutaminase, glutamic acid rich polypeptide and polymer is an aerosol.

[0034] Without limitations, a target surface can be any surface to which a biomaterial or a polymer is to be bonded or adhered. In some embodiments, a target surface can be surface of an organic or inorganic substrate. For example, the target surface can be surface of a tissue or organ. In some embodiments, the target surface can include surfaces of hepatic, cardiac and dermal tissues.

[0035] In one embodiment, target surface includes a wound. As used herein, the term "wound" refers to physical disruption of the continuity or integrity of tissue structure caused by a physical (e.g., mechanical) force, a biological (e.g., thermic or actinic force), or a chemical means. In particular, the term "wound" encompasses wounds of the skin. The term "wound" also encompasses contused wounds, as well as incised, stab, lacerated, open, penetrating, puncture, abrasions, grazes, burns, frostbites, corrosions, wounds caused by ripping, scratching, pressure, and biting, and other types of wounds. In particular, the term encompasses ulcerations (i.e., ulcers), preferably ulcers of the skin. The term "wound" also includes surgical wounds. [0036] The wound can be acute or chronic. As used herein, the term "chronic wound" refers to a wound that does not fully heal even after a prolonged period of time (e.g., 2 to 3 months or longer). Chronic wounds, including pressure sores, venous leg ulcers and diabetic foot ulcers, can simply be described as wounds that fail to heal. Whilst the exact molecular pathogenesis of chronic wounds is not fully understood, it is acknowledged to be multi-factorial. As the normal responses of resident and migratory cells during acute injury become impaired, these wounds are characterized by a prolonged inflammatory response, defective wound extracellular matrix (ECM) remodelling and a failure of re-epithelialisation.

[0037] The wound can be an internal wound, e.g. where the external structural integrity of the skin is maintained, such as in bruising or internal ulceration, or external wounds, particularly cutaneous wounds, and consequently the tissue can be any internal or external bodily tissue. In some embodiment the tissue is skin (such as human skin), i.e. the wound is a cutaneous wound, such as a dermal or epidermal wound.

[0038] Wounds can be classified in one of two general categories, partial thickness wounds or full thickness wounds. A partial thickness wound is limited to the epidermis and superficial dermis with no damage to the dermal blood vessels. A full thickness wound involves disruption of the dermis and extends to deeper tissue layers, involving disruption of the dermal blood vessels. The healing of the partial thickness wound occurs by simple regeneration of epithelial tissue. Wound healing in full thickness wounds is more complex.

[0039] In some embodiments, the wound is selected from the group consisting of cuts and lacerations, surgical incisions or wounds, punctures, grazes, scratches, compression wounds, abrasions, friction wounds (e.g. nappy rash, friction blisters), decubitus ulcers (e.g. pressure or bed sores); thermal effect wounds (burns from cold and heat sources, either directly or through conduction, convection, or radiation, and electrical sources), chemical wounds (e.g. acid or alkali burns) or pathogenic infections (e.g. viral, bacterial or fungal) including open or intact boils, skin eruptions, blemishes and acne, ulcers, chronic wounds, (including diabetic-associated wounds such as lower leg and foot ulcers, venous leg ulcers and pressure sores), skin graft/transplant donor and recipient sites, immune response conditions, e.g. psoriasis and eczema, stomach or intestinal ulcers, oral wounds, including a ulcers of the mouth, damaged cartilage or bone, amputation wounds, corneal lesions, and any combinations thereof.

[0040] In some embodiments, the wound can be selected from the group consisting of cuts and lacerations, surgical incisions, punctures, grazes, scratches, compression wounds, abrasions, friction wounds, chronic wounds, ulcers, thermal effect wounds, chemical wounds, wounds resulting from pathogenic infections, skin graft/transplant donor and recipient sites, immune response conditions, oral wounds, stomach or intestinal wounds, damaged cartilage or bone, amputation sites, corneal lesions and lung punctures.

[0041] The compositions and methods disclosed herein can be used for wound healing. For example, compositions and methods described herein can be used for suture-less closure of wounds. As used herein, the term "wound healing" refers to a regenerative process with the induction of an exact temporal and spatial healing program comprising wound closure and the processes involved in wound closure. The term "wound healing" encompasses but is not limited to the processes of granulation, neovascularization, fibroblast, endothelial and epithelial cell migration, extracellular matrix deposition, reepithelialization, and remodeling. The term "wound healing" includes the restoration of tissue integrity. It will be understood that this can refer to a partial or a full restoration of tissue integrity. Treatment of a wound thus refers to the promotion, improvement, progression, acceleration, or otherwise advancement of one or more stages or processes associated with the wound healing process.

[0042] As used herein, the term "wound closure" refers to the healing of a wound wherein sides of the wound are rejoined to form a continuous barrier (e.g., intact skin). As used herein, the term "granulation" refers to the process whereby small, red, grain-like prominences form on a raw surface (that of wounds) as healing agents. As used herein, the term "neovascularization" refers to the new growth of blood vessels with the result that the oxygen and nutrient supply is improved. Similarly, the term "angiogenesis" refers to the vascularization process involving the development of new capillary blood vessels. As used herein, the term "cell migration" refers to the movement of cells (e.g., fibroblast, endothelial, epithelial, etc.) to the wound site.

[0043] As used herein, the term "extracellular matrix deposition" refers to the secretion by cells of fibrous elements (e.g., collagen, elastin, reticulin), link proteins (e.g., fibronectin, laminin), and space filling molecules (e.g., glycosaminoglycans). As used herein, the term "reepithelialization" refers to the reformation of epithelium over a denuded surface (e.g., wound). As used herein the term "remodeling" refers to the replacement of and/or devascularization of granulation tissue.

[0044] In embodiments of the various aspects disclosed herein, a wound healing agent can be applied to the wound. As used herein, a "wound healing agent" is a compound or composition that actively promotes wound healing process. Exemplary wound healing agents include, but are not limited to dexpanthenol; growth factors; enzymes, hormones; povidon-iodide; fatty acids; anti-inflammatory agents; antibiotics; antimicrobials; antiseptics; cytokines; thrombin; angalgesics; opioids; aminoxyls; furoxans; nitrosothiols; nitrates and anthocyanins; nucleosides, such as adenosine; and nucleotides, such as adenosine diphosphate (ADP) and adenosine triphosphate (ATP); neutotransmitter/neuromodulators, such as acetylcholine and 5- hydroxytryptamine (serotonin/5 -HT); histamine and catecholamines, such as adrenalin and noradrenalin; lipid molecules, such as sphingosine-1 -phosphate and lysophosphatidic acid; amino acids, such as arginine and lysine; peptides such as the bradykinins, substance P and calcium gene-related peptide (CGRP); nitric oxide; and any combinations thereof.

[0045] Exemplary growth factors include, but are not limited to, fibroblast growth factor (FGF), FGF-1, FGF-2, FGF-4, FGF-a, FGF-β, plateletderived growth factor (PDGF), insulin- binding growth factor (IGF), IGF-1, IGF-2, heparin-binding growth factor- 1, heparin-binding growth factor-2, epidermal growth factor (EGF), transforming growth factor (TGF), TGF-a, TGF-β , cartilage inducing factors-A and -B, osteoid-inducing factors, osteogenin, vascular endothelial growth factor, bone growth factors, collagen growth factors, insulin-like growth factors, and their biologically active derivatives.

[0046] Embodiments of the various aspects disclosed herein include a transglutaminase. As used herein, by "transglutaminase" is meant a member of the group of enzymes identified by Enzyme Commission System of Classification No. 2.3.2.13 (EC 2.3.2.13). Skilled artisan is well aware that transglutaminases are enzymes that catalyze an acyl transfer reaction of a γ- carboxamide group of a glutamine residue in a peptide chain. Transglutaminases form 8-(y-Glu)- Lys crosslinks in and between protein molecules when an ε-amino group of a lysine residue in a protein acts as an acyl receptor. Transglutaminases can also deaminate a glutamine residue into a glutamic acid residue when water acts as an acyl receptor.

[0047] Such transglutaminases include calcium-independent transglutaminases and calcium- dependent transglutaminases. The former include an enzyme derived from microorganisms (see, for example, JP-A-1-27471, content of which is incorporated herein by reference in its entirety), and the latter include an enzyme derived from the guinea pig's liver (see, JP-B-150382, content of which is incorporated herein by reference in its entirety), an enzyme derived from fish (see, for example, Seki Nobuo et al. Nihon Suisan Gakkaishi, vol. 56, No. 1, p. 125 (1990), content of which is incorporated herein in its entirety), and the like. Further, it includes enzymes produced by gene recombination. See JP-A-1-300889, JP-A-5-199883, and JP-A-6-225775, contents of all which are incorporated herein by reference in their entireties).

[0048] The transglutaminase for use in the methods of the present invention can be from a natural or a synthetic source, e.g., recombinant. Thus, transglutaminase prepared (i.e. extracted) from mammalian tissue samples, as well as mammalian transglutaminases expressed by recombinant means are included herein. Furthermore, variants of naturally-occurring mammalian transglutaminases are also included.

[0049] In some embodiments, the transglutaminase is a mammalian transglutaminase. Mammalian transglutaminases can be obtained from animal cells and tissues and cellular products.

[0050] In some embodiments, transglutaminase is a tissue transglutaminase (tTgase).

[0051] In some embodiments, transglutaminase is a microbial transglutaminase. A microbial transglutaminase can be isolated from one or more of a Streptomyces hygroscopicus strain, Streptoverticillium Baldaccii, Streptoverticillium mobaraense, or Escherichia Coli. See for example, Cui L et al., Bioresource Technology (2008) 99(9): 3794-3800, content of which is incorporated herein by reference in its entirety).

[0052] In some embodiments, transglutaminase is a human transglutaminase. Human transglutaminase can be prepared from human tissue or cells. For example, a human transglutaminase can be extracted from human tissue sources such as lung, liver, spleen, kidney, heart muscle, skeletal muscle, eye lens, endothelial cells, erythrocytes, smooth muscle cells, bone and macrophages.

[0053] Alternatively, human transglutaminase can be obtained from a culture of human cells that express a mammalian transglutaminase, using cell culture methodology well known in the art. Preferred cell line sources of such transglutaminases include, but are not limited to, human endothelial cell line ECV304 (for tissue transglutaminase) and human osteosarcoma cell line MG63.

[0054] In some embodiments, transglutaminase is a calcium-independent transglutaminase.

In some other embodiments, transglutaminase is a calcium-dependent transglutaminase.

[0055] Some exemplary transglutaminases include, but are not limited to, Factor XIII A (fibrin stabilizing factor), Type 1 transglutaminase (keratinocyte transglutaminase,), Type 2 transglutaminase (tissue transglutaminase), Type 3 transglutaminase (epidermal transglutaminase), Type 4 transglutaminase (prostate transglutaminase), Type 5 transglutaminase (Transglutaminase X), Type 6 transglutaminase (Transglutaminase Y), and Type 7 transglutaminase (Transglutaminase Z).

[0056] It will be appreciated by those skilled in the art that the source of the transglutaminase can be selected according to the particular use (e.g. site of implantation) of the medical implant material. For example, if the medical implant material is to be used as artificial bone, it can be beneficial for the material to comprise a bone-derived transglutaminase. Thus, in the method of the present invention, any of transglutaminases can be used, and the origin and the production process thereof are not limited.

[0057] The transglutaminase can be derived from mammals or microorganisms. Moreover, humanized recombinant transglutaminase can also be used.

[0058] Some commercially available transglutaminases derived from mammals, such as guinea pig liver-derived transglutaminase, goat-derived transglutaminase and rabbit-derived transglutaminase, are available from Oriental Yeast Co., Ltd., Upstate USA Inc. and Biodesign International. Other non-limiting examples of commercially available transglutaminase products include those produced by Ajinomoto Co. (Kawasaki, Japan), such as Activa TG-TI, Activa TG- FP, Activa TG-GS, Activa TG-RM, and Activa MP; and those produced by Yiming Biological Products Co. (Jiangsu, China), such as TG-B and TG-A.

[0059] The transglutaminase can be applied in any suitable form including, but not limited to, solutions, emulsions (oil-in-water, water-in-oil), aerosols, foams, ointments, pastes, lotions, powders, gels, hydrogels, hydrocolloids, creams, and any combinations thereof. In one embodiment, the transglutaminase is applied in the form of a powder.

[0060] The transglutaminase can optionally be in a pharmaceutical acceptable composition. A pharmaceutically acceptable composition comprises a transglutaminase formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. In some embodiments, the pharmaceutically acceptable composition can also include any co-factors, such as salts, for the transglutaminase.

[0061] Generally, the transglutaminase activity is enhanced at elevated temperature. Thus, the bonding can be performed at any temperature. In some embodiments, the bonding is performed at an elevated temperature e.g., 30°C or higher, 35°C or higher, 40°C or higher, 45°C or higher, 50°C or higher, 55°C or higher, or 60°C or higher. In some embodiments, bonding is performed at room temperature, e.g., from about 15°C to about 25°C. In some embodiments, bonding is performed at a temperature of about 20°C to about 30°C.

[0062] As used herein, an "effective amount of a transglutaminase" means the amount of transglutaminase which is effective to for adhering together two surfaces. Accordingly, in some embodiments, effective amount of a transglutaminase is from about ^g to about lOOmg per cm² of contact surface area. In some embodiments, effective amount of a transglutaminase is selected from the group consisting of from about lmg to about 50mg per cm², from about 5mg to about 40mg per cm², from about lOmg to about 30mg per cm², from about 15mg to about 25mg per cm², or about 20mg per cm².

[0063] As used herein, in context of applying a transglutaminase, the term "applying" refers to increasing the amount or activity of a transglutaminase at the desired site. Accordingly, the term "applying" embraces topical applications of transglutaminase at the desired site, increasing the expression of a host transglutaminase at the desired site, and/or increasing the activity of a transglutaminase at the desired site. Without wishing to be bound by a theory, one can increase the expression or activity of a host transglutaminase by applying a composition that increases the expression and/or activity of the transglutaminase. For example, Davies et al. (J. Biol. Chem. (1985) 260:5166-5174) describe inducing transglutaminase expression using retinoic acid. Exemplary transglutaminase activators include, but are not limited to, thrombin, TIG3 protein, calcium chloride, and sphingosylphosphorylcholine. Additionally, Sigma-Aldrich sells the Transglutaminase Assay Kit which can be used for screening activators of transglutaminase.

[0064] Embodiments of the various aspects disclosed herein include a glutamic acid rich polypeptide. As used herein, a "glutamic acid rich polypeptide" refers to polypeptides that have a plurality of glutamic acids. For example, at least 5% of the amino acids in the polypeptide are glutamic acid. In some embodiments, the glutamic acid rich polypeptide is casein.

[0065] As used herein, an "effective amount of a glutamic acid rich polypeptide" means the amount of glutamic acid rich polypeptide which is effective to enhance bonding of two components with a transglutaminase. In some embodiments, effective amount of a glutamic acid rich polypeptide is from about ^g to about lOOmg per cm² of contact surface area. In some embodiments, effective amount of a t glutamic acid rich polypeptide is selected from the group consisting of from about lmg to about 50mg per cm², from about 5mg to about 40mg per cm², from about lOmg to about 30mg per cm², from about 15mg to about 25mg per cm², or about 20mg per cm².

[0066] Embodiments of the various aspects disclosed herein include a biomaterial. As used herein, the term "biomaterial" refers to any material that is biocompatible. As used herein, the term "biocompatible material" refers to any polymeric material that does not deteriorate appreciably and does not induce a significant immune response or deleterious tissue reaction, e.g., toxic reaction or significant irritation, over time when implanted into or placed adjacent to the biological tissue of a subject, or induce blood clotting or coagulation when it comes in contact with blood. Suitable biocompatible materials include derivatives and copolymers of a polyimides, poly(ethylene glycol), polyvinyl alcohol, polyethyleneimine, and polyvinylamine, polyacrylates, polyamides, polyesters, polycarbonates, and polystyrenes.

[0067] In some embodiments, the biomaterial comprises a carbohydrate-based polymer, wherein the polymer comprises at least one amino group. In some embodiments, the biomaterial comprises collagen or gelatin.

[0068] In some embodiments, the biomaterial comprises a composite laminate material. In various embodiments, at least one layer of the composite laminate material comprises a protein and at least one layer of the composite laminate material comprises a carbohydrate. Exemplary composite laminate materials amenable to the present invention are described for example in US Patent Application No. 13/819,391, content of which is incorporated herein by reference in its entirety.

[0069] In some embodiments, the biomaterial is in form of a medical implant device. As used herein, the term "medical implant device" refers to devices for implementation into a subject's body. Exemplary medical implant devices include, but are not limited to, artificial tissues, artificial organs, prosthetic devices, drug delivery devices, wound dressings, fibers, nanoparticles, microparticles, foams, and sponges. Without limitations, a medical implant device can be in any form including, but not limited to 3-D scaffolds, fibers, foams, sponges, films, and any combinations thereof. A medical implant device can be used for permanent substitution of an organ (function).

[0070] In some embodiments, the medical implant device is a foam or sponge.

[0071] In some embodiments, the medical implant device comprises a nanoparticle or a microparticle.

[0072] In one embodiment, the medical implant device is a wound dressing. Exemplary wound dressings include, but are not limited to bandages, gauzes, tapes, meshes, nets, adhesive plasters, films, membranes, and patches. In some embodiments, a wound dressing can comprise a composite material described herein.

[0073] In some embodiments, the medical implant device is associated with a protein which is cross-linkable by a transglutaminase. As used in context of a medical implant device, the term "associated with" refers to a medical implant device which is coated with, includes, or comprises a transglutaminase linkable protein. In some embodiments, the medical implant device is coated with the transglutaminase linkable protein. By "coated" is meant that the transglutaminase linkable protein is applied to the surface of the medical implant device. Thus, the medical implant device can be painted or sprayed with a solution comprising a transglutaminase linkable protein. Alternatively, the medical implant device can be dipped in a solution of transglutaminase linkable protein solution.

[0074] The transglutaminase linkable protein can be covalently or non-covalently associated with the medical implant device, e.g. at the external surface of the medical implant device. Once associated with the medical implant device, the transglutaminase linkable protein provides means of attaching the medical implant device to a tissue or organ.

[0075] As used herein, the term "cross-linkable by a transglutaminase" refers to a protein or polypeptide which serves as a substrate for a transglutaminase. Accordingly, a transglutaminase cross-linkable protein is or comprises a transglutaminase substrate. As used herein, the term "transglutaminase substrate" refers to a peptide or polypeptide sequence with an appropriate transglutaminase target for cross-linking. Without limitations, a transglutaminase linkable protein is or comprises a transglutaminase substrate selected from the group consisting of aldolase A, glyceraldehyde-3 -phosphate dehydrogenase, phosphorylase kinase, crystalline, glutathione S-transferase, actin, myosin, troponin, β-tublin, tau, rho, histone, a-oxoglutarate dehydrogenase, β-lactoglobulin, cytochromes, erythrocyte band III, CD38, acetylcholine esterase, collagen, entactin, fibronectin, fibrin, silk, fibroin, fibrinogen, vitronectin, osteopontin, nidogen, laminin, LTBP-1, osteonectin, osteopontin, osteocalcin, thrombospondin, substance P, phospholipases A₂, midkine, wheat gelatin, whey proteins, casein, soy proteins, pea legumin, Candida albicans surface proteins, HIV envelop glycoproteins gpl20 and gp41, HIV aspartyl proteinase, hepatitis C virus core protein, fragments thereof that are capable of binding to a transglutaminase, and combinations thereof. In some embodiments, the transglutaminase linkable protein is silk fibroin or a fragment thereof that is capable of being cross-linked by a transglutaminase.

[0076] Peptide and polypeptide sequences with an appropriate transglutaminase target for cross-linking are known in the art. Non-limiting examples of such peptides are described, for example in U.S. Pat. No. 5,428,014; No. 5,939,385; and No. 7,208,171, content of all of which is incorporated herein by reference. U.S. Pat. No. 5,428,014 describes biocompatible, bioadhesive, transglutaminase cross-linkable polypeptides wherein transglutaminase is known to catalyze an acyl-transfer reaction between the γ-carboxamide group of protein-bound glutaminyl residues and the ε-amino group of Lys residues, resulting in the formation of 8-(y-glutamyplysine isopeptide bonds. U.S. Pat. No. 5,939,385 describes biocompatible, bioadhesive transglutaminase cross-linkable polypeptides.

[0077] U.S. Pat. No. 7,208,171, describes the rational design of transglutaminase substrate peptides. The design strategy was based on maximizing the number of available acyl acceptor lysine-peptide substrates and acyl donor glutaminyl-peptide substrates available for transglutaminase cross-linking. Beyond this, the Lys and Glu substrate peptides were designed to possess basic features of known biomacromolecular and synthetic peptide substrates of transglutaminase. For example, the Glu substrate peptides contained 2-5 contiguous Glu residues, based on evidence that peptides become better transglutaminase substrates with increasing length of Glu repeats and that proteins containing two or more adjacent Glu residues are known to be good substrates. A Leu residue was placed adjacent to the Glu near the C- terminus in several peptides, because this has been shown to result in a significant increase in Glu specificity. Regarding the Lys substrate peptides, it has been shown that the composition and sequence of the amino acids adjacent to lysine residues in peptide and protein substrates can have an effect on the amine specificity. Finally, in all peptides a Gly residue was added on the C-terminal side to act as a spacer between the peptide and the polymer in the peptide-polymer conjugates, so that the peptide in the conjugate may be more accessible to enzyme.

[0078] A medical implant device can be fabricated from any biocompatible material. As used herein, the term "biocompatible material" refers to any polymeric material that does not deteriorate appreciably and does not induce a significant immune response or deleterious tissue reaction, e.g., toxic reaction or significant irritation, over time when implanted into or placed adjacent to the biological tissue of a subject, or induce blood clotting or coagulation when it comes in contact with blood. Suitable biocompatible materials include derivatives and copolymers of a polyimides, poly(ethylene glycol), polyvinyl alcohol, polyethyleneimine, and polyvinylamine, polyacrylates, polyamides, polyesters, polycarbonates, and polystyrenes.

[0079] In some embodiments, the medical implant device is fabricated from a material selected from the group consisting of carbohydrate polymers, proteins, silk fibroin, polydimethylsiloxane, polyimide, polyethylene terephthalate, polymethylmethacrylate, polyurethane, polyvinylchloride, polystyrene polysulfone, polycarbonate, polymethylpentene, polypropylene, a polyvinylidine fluoride, polysilicon, polytetrafluoroethylene, polysulfone, acrylonitrile butadiene styrene, polyacrylonitrile, polybutadiene, poly(butylene terephthalate), poly(ether sulfone), poly(ether ether ketones), poly(ethylene glycol), styrene-acrylonitrile resin, poly(trimethylene terephthalate), polyvinyl butyral, polyvinylidenedifluoride, poly(vinyl pyrrolidone), and any combination thereof.

[0080] A medical implant device can be fabricated from a biodegradable material, e.g., a biodegradable polymer. As used herein, the term "biodegradable" describes a material which can decompose under physiological conditions into breakdown products. Such physiological conditions include, for example, hydrolysis (decomposition via hydrolytic cleavage), enzymatic catalysis (enzymatic degradation), and mechanical interactions. As used herein, the term "biodegradable" also encompasses the term "bioresorbable", which describes a substance that decomposes under physiological conditions to break down to products that undergo bioresorption into the host-organism, namely, become metabolites of the biochemical systems of the host organism.

[0081] The term "biodegradable polymer", as used herein, refers to a polymer that at least a portion thereof decomposes under physiological conditions. The polymer can thus be partially decomposed or fully decomposed under physiological conditions.

[0082] Exemplary biodegradable polymers include, but are not limited to, polyanhydrides, polyhydroxybutyric acid, polyorthoesters, polysiloxanes, polycaprolactone, poly(lactic-co- glycolic acid), poly(lactic acid), poly(glycolic acid), and copolymers prepared from the monomers of these polymers.

[0083] In some embodiments, the medical implant device is fabricated from a biocompatible, biodegradable material.

[0084] Suitable polymers which can be used for fabricating a medical implant device include, but are not limited to, one or a mixture of polymers selected from the group consisting of carbohydrate polymers; silk; glycosaminoglycan; fibrin; poly-ethyleneglycol (PEG); C2 to C4 polyalkylene glycols (e.g., propylene glycol); polyhydroxy ethyl methacrylate; polyvinyl alcohol; polyacrylamide; poly (N-vinyl pyrolidone); poly glycolic acid (PGA); poly lactic-co- glycolic acid (PLGA); poly e-carpolactone (PCL); polyethylene oxide; poly propylene fumarate (PPF); poly acrylic acid (PAA); hydrolysed polyacrylonitrile; polymethacrylic acid; polyethylene amine; polyanhydrides; polyhydroxybutyric acid; polyorthoesters; polysiloxanes; polycaprolactone; poly(lactic-co-glycolic acid); poly(lactic acid); poly(glycolic acid); alginic acid; esters of alginic acid; pectinic acid; esters of pectinic acid; carboxy methyl cellulose; hyaluronic acid; esters of hyaluronic acid; heparin; heparin sulfate; chitosan; carboxymethyl chitosan; chitin; pullulan; gellan; xanthan; collagen; carboxymethyl starch; carboxymethyl dextran; chondroitin sulfate; cationic guar; cationic starch as well as salts and esters thereof.

[0085] In some embodiments, the medical implant device is fabricated from a carbohydrate- based polymer. In one embodiment, the carbohydrate polymer is chitin, chitosan or a derivative thereof.

[0086] In some embodiments, the medical implant device is fabricated from a transglutaminase linkable protein.

[0087] In some embodiments, the medical implant device is fabricated from a composite material described herein.

[0088] Embodiments of the various aspects disclosed herein include a polymer. In various embodiments, the polymer comprises at least one group linkable by a transglutaminase. An exemplary group that is linkable by a transglutaminase is an amino group.

[0089] In some embodiments, the polymer comprises a protein, e.g., a transglutaminase linkable protein. In some embodiments, the polymer comprises a carbohydrate-based polymer. Exemplary carbohydrate-based polymers include, but are not limited to, chitin and chitosan.

[0090] In some embodiments, the polymer comprising at least one amino group includes a carbohydrate-based amino-polymer. As used herein, the term "carbohydrate-based amino- polymer," includes, but is not limited to, oligomers or polymers that contain monomers having the formula C_m(H₂0)_n and at least one amino group, wherein m and n are > 3 and wherein m and n can be same or different. In some embodiments, m and n are independently 3, 4, 5, 6, or 7. Carbohydrate-based amino-polymers include, but are not limited to, compounds such as oligosaccharides, polysaccharides, glycoproteins, glycolipids, and any combinations thereof. Any carbohydrate-based polymer comprising an amino group that can crosslink with another reactive group of another molecule (e.g., carboxamide group of a molecule or a polymer) in the presence of a transglutaminase can be used in any embodiments of the methods and devices described herein.

[0091] In some embodiments of this and other aspects described herein, the carbohydrate- based amino-polymer can comprise at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 or sugar monomers.

[0092] Without limitation, the carbohydrate-based amino-polymer can comprise sugar monomers independently selected from the group consisting of erythrose, threose, ribose, arabinose, xylose, lyxose, ribulose, xylulose, allose, altrose, glucose, mannose, gulose, idose, galactose, galactosamine, N-acetylgalactose, glucosamine, N-acetylglucosamine, sialic acid, talose, psicose, fructose, sorbose, tagatose, fucose, fuculose, rhamonse, sedoheptulose, octose, sulfoquinovose, glycosaminoglycan and nonose (neuraminic acid), wherein the sugar may be optionally substituted. Without limitation, each sugar can independently have the L- or the D- conformation.

[0093] The linkage between two sugar monomers can independently have a a- or β- configuration. Furthermore, the linkage between the two sugars can be 1— »3, 1— »4, 1— »5, or 1→6.

[0094] In some embodiments, at least one (e.g., 1, 2, 3, or 4) hydroxyl group of the sugar monomer can be replaced by an amino group. In some embodiments, the hydroxyl group at position 2 of the sugar monomer can be replaced by an amino group. The amino group can be optionally substituted with a C1-C6 alkyl or an acyl group. In some embodiments, C1-C6 alkyl groups include methyl, ethyl, propyl, butyl, and t-butyl. In one embodiment, the acyl group comprises acetyl.

[0095] In some embodiments of this and other aspects described herein, the carbohydrate amino-polymer can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) disaccharide, trisaccharide or tetrasaccharide monomers independently selected from the group consisting of sucrose, lactulose, lactose, maltose, trehalose, cellobiose, kojibiose, nigerose, isomaltose, β,β-Trehalose, α,β-Trehalose, sophorose, laminaribiose, gentibiose, turanose, maltulose, palatinose, gentibiulose, mannobiose, melibiose, rutinose, rutinulose, xylobiose, raffinose, melezitose, acarbose and stachyose.

[0096] As used herein, the term "oligosaccharide" refers without limitation to several (e.g., five to ten) covalently linked monosaccharide units. As used herein, the term "polysaccharide" refers without limitation to many (e.g., eleven or more) covalently linked sugar units. Polysaccharides can have molecular masses up to millions of Daltons. Exemplary oligosaccharides and polysaccharides include, but are not limited to, fructooligosaccharide, galactooligosaccharides, mannanoligosaccharides, glycogen, starch (amylase, amylopectin), glycosaminoglycans (e.g., hyaluronic acid, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratin sulfate, and heparin), cellulose, beta-glucan (e.g., zymosan, lentinan, sizofiran), maltodextrin, inulin, levan beta (2— »6), chitin, and chitosan.

[0097] In some embodiments of this and other aspects described herein, the carbohydrate- based amino-polymer can comprise chitin or a derivative thereof. In one embodiment, chitin derivative comprises chitosan (a-(l-4) 2-amino-2-deoxy-P-D-glucan) or a derivative thereof. It will be understood by those skilled in the art that chitosan can also include all derivatives of chitin, or poly-N-acetyl-D-glucosamine (including all polyglucosamine and oligomers of glucosamine materials of different molecular weights), in which at least about 20% of the acetyl groups (e.g., at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more, including 100% of the acetyl groups) have been removed through hydrolysis. Generally, chitosans are a family of cationic, binary heteropoly saccharides composed of (1— »4)-linked 2-acetamido-2-deoxy-(3-D-glucose (GlcNAc, A-unit) and 2-amino-2-deoxyP-D-glucose, (GlcN; D-unit) (Varum K. M. et al., Carbohydr. Res., 1991, 217: 19-27; Sannan T. et al., Macromol. Chem., 1776, 177: 3589-3600). Chitosan, chitosan derivatives or salts (e.g., but not limited to, nitrate, phosphate, sulphate, hydrochloride, glutamate, lactate or acetate salts) of chitosan can be used in any embodiments of the methods and devices described herein. Any art-recognized chitosan and derivatives thereof, e.g., the ones described in the U.S. Patent No. 7,125,967 and 7,288,532, the content of which are incorporated herein by reference, can be provided as the polymer comprising an amino group used in some embodiments of the methods and devices described herein.

[0098] As used herein, the term "chitosan derivatives" are intended to include ester, ether or other derivatives formed by bonding of acyl and/or alkyl groups with OH groups, but not the H₂ groups, of chitosan. Examples are O-alkyl ethers of chitosan and O-acyl esters of chitosan. Additional exemplary derivatives of chitosan include, but are not limited to, N-(aminoalkyl) chitosans, succinyl chitosans, quteraminated chitosans, N-acylated chitosans (e.g., caproyl chitosan, octanoyl chitosan, myristoyl chitosan, and palmitoyl chitosan), N-methylene phosphonic chitosans, N-lauryl -N-methylene phosphonic chitosans, N-lauryl-carboxymethyl chitosans, N-alkyl-O-sulfated chitosans, thiolated chitosans (e.g., chitosan-2-iminthiolane, chitosan-4-thiobutylamidine, and chitosan-thioglycolic acid), trimethylchitosan, and phosphorylated chitosans).

[0099] Modified chitosans, e.g., those chitosans conjugated to polyethylene glycol, can also be utilized in any embodiments of the methods and devices described herein.

[00100] Chitosans of various viscosity (for example CL113, G210 and CLIO) can be obtained from various sources, e.g., chitosan produced by deacetylation of chitin, which, for example, can be obtained from the structural element in the exoskeleton of crustaceans (such as crabs and shrimp) and cell walls of fungi, or from commercial sources including, but not limited to, PRONOVA Bioamino-polymer, Ltd. (UK); SEIGAGAKU America Inc. (Maryland, USA); MERON (India) Pvt, Ltd. (India); VANSON Ltd. (Virginia, USA); and AMS Biotechnology Ltd. (UK).

[00101] Any carbohydrate-based polymer comprising at least one amino group can be used in any embodiments of the compositions and methods described herein. One of skill in the art is well aware of synthetic methods which can be used for the synthesis of carbohydrate-based amino-polymers. See for example, Stick, R.V., Carbohydrates: The Sweet Molecules of Life.; Academic Press, pp 113-177 (2002); Crich, D. & Dudkin V., J. Am. Chem. Soc, 123 :6819-6825 (2001); Garegg, P. J., Chemtracts-Org. Chem., 5:389 (1992); Mayer, T. G, Kratzer, B. & Schmidt, R. R. Synthesis of a GPI anchor of the yeast Saccharomyces cerevisiae. Angew. Chem. Int. Ed. Engl. 33, 2177-2181 (1994); Seifert, J., Lergenmuller, M. & Ito, Y. Synthesis of an a- (2,3)-sialylated complex-type undecasaccharide. Angew. Chem. Int. Ed. 39, 531-534 (2000); Wang, Z.-G. et al. Toward fully synthetic homogeneous glycoproteins: a high mannose core containing glycopeptide containing carrying full H-type 2 human blood group specificity. Angew. Chem. Int. Ed. 40, 1728-1732 (2001); Caruthers, M. H. Gene synthesis machines: DNA chemistry and its uses. Science 230, 281-285 (1985); Sears, P. & Wong, C.-H. Toward automated synthesis of oligosaccharides and glycoproteins. Science 291, 2344-2350 (2001); Zhang, Z. et al. Programmable one-pot oligosaccharide synthesis. J. Am. Chem. Soc. 121, 734- 753 (1999; Nishimura, S. Automated glycosynthesizer 'Golgi' by mimicking biosynthetic process. Tanpakushitsu Kakusan Koso 48, 1220-1225 (2003); Plante, O. J., Palmacci, E. R. & Seeberger, P. H. Automated solid-phase synthesis of oligosaccharides. Science 291, 1523-1527 (2001); Andrade, R. B., Plante, O. J., Melean, L. G. & Seeberger, P. H. Solid-phase oligosaccharide synthesis: preparation of complex structures using a novel linker and different glycosylating agents. Org. Lett. 1, 1811-1814 (1999); Love, K. R. & Seeberger, P. H. Automated solid-phase synthesis of protected tumor-associated antigen and blood group determinant oligosaccharides. Angew. Chem. Int. Ed. 43, 602-605 (2004); and Seeberger, P.H. & Werz, D.B. Synthesis and medical applications of oligosaccharides. Nature 446, 1046-1051 (2007), content of all of which is herein incorporated by reference.

[00102] Embodiments of the various aspects disclosed herein include a protein. Without limitations, the protein can be selected from the group consisting of collagen, gelatin, perculin, abductin, fibrin, fibroin, elastin, resilin, fibronectin, fibrinogen, keratin, titin, actin, Arp2/3, coronin, dystrophin, FtsZ, myosin, spectrin, Tau (protein), tubulin, F-spondin, Pikachurin, protein fragments, synthetic peptides, genetically expressed portions of proteins, fragments thereof, and any combinations thereof. In some embodiments, the protein is collagen or gelatin.

[00103] In one aspect, the invention provides a three component composition. The three components are in flowable form. Generally, the first component is a polymer comprising an amino group, i.e., an amino polymer; the second component is a transglutaminase; and the third component is a glutamic acid rich polypeptide. The three components can be in separate formulations, in one formulation or two in one formulation and the other in a separate formulation. In some embodiments, the amino polymer is in a separate formulation than the transglutaminase and the glutamic acid rich polypeptide. In one embodiment the transglutaminase and the glutamic acid rich polypeptide are in one formulation.

[00104] Embodiments of the various aspects disclosed herein can be described by one or more of the following numbered paragraphs:

1. A method for bonding or adhering a biomaterial to a target surface, the method

comprising applying an effective amount of a transglutaminase and a glutamic acid rich polypeptide to a target surface and contacting a biomaterial to the target surface where the transglutaminase and the glutamic acid rich polypeptide have been applied.

The method of paragraph 1, wherein the glutamic acid rich polypeptide is casein.

The method of paragraph 1 or 2, wherein the biomaterial comprises a protein or a carbohydrate-based polymer comprising at least one amino group.

The method of paragraph 3, wherein the carbohydrate-based polymer comprises chitin or chitosan.

The method of paragraph 3, wherein the protein is collagen or gelatin.

The method of any of paragraphs 1-5, wherein the biomaterial comprises a composite laminate material comprising a layer of carbohydrate-based polymer and a layer of protein.

The method of paragraph 6, wherein the carbohydrate-based polymer of the composite laminate material comprises chitin or chitosan.

The method of paragraph 6 or 7, wherein the protein of the composite laminate material comprises collagen, gelatin or silk fibroin.

The method of any of paragraphs 1-8, wherein the biomaterial is in form of a medical implant device.

The method of paragraph 9, wherein the medical implant device is selected from the group consisting of artificial tissues, artificial organs, prosthetic devices, drug delivery devices, wound dressings, films, foams, sponges, scaffolds, meshes, hemostatic materials, and any combinations thereof.

The method of paragraph 9 or 10, wherein the medical implant device is a wound dressing selected from the group consisting of bandages, gauzes, tapes, meshes, nets, adhesive plasters, films, membranes, patches, microparticles, nanoparticles, and any combinations thereof.

The method of any of paragraphs 1-11, wherein at least one of the biomaterial, the transglutaminase and the glutamic acid rich polypeptide is formulated in a solution, an emulsion, an aerosol, a foam, an ointment, a paste, a lotion, a powder, a gel, a hydrogel, a hydrocolloid, a microparticle, a nanoparticle, or a cream.

The method of any of paragraphs 1-12, wherein the biomaterial, the transglutaminase and the glutamic acid rich polypeptide are formulated in separate compositions. The method of any of paragraphs 1-12, wherein at least two of the biomaterial, the transglutaminase and the glutamic acid rich polypeptide are formulated in one

composition.

The method of paragraph 14, wherein all three of the biomaterial, the transglutaminase and the glutamic acid rich polypeptide are formulated in one composition.

The method of any of paragraphs 1-15, wherein the target surface is a surface of a tissue or organ.

The method of paragraph 16, wherein the target surface is a surface of a hepatic, cardiac, intestinal, pulmonary or dermal tissue.

The method of any of paragraph 1-17, wherein the target surface is a wound.

The method of paragraph 18, wherein the wound is selected from the group consisting of cuts and lacerations, surgical incisions, punctures, grazes, scratches, compression wounds, abrasions, friction wounds, chronic wounds, ulcers, thermal effect wounds, chemical wounds, wounds resulting from pathogenic infections, skin graft/transplant donor and recipient sites, immune response conditions, oral wounds, stomach or intestinal wounds, damaged cartilage or bone, amputation sites, corneal lesions and lung punctures. The method of any of paragraphs 1-19, wherein the transglutaminase is a mammalian or microbial transglutaminase.

A method for promoting wound healing, the method comprising applying a

transglutaminase and a glutamic acid rich polypeptide to a surface of a wound.

The method of paragraph 21, wherein the glutamic acid rich polypeptide is casein.

The method of paragraph 21 or 22, wherein at least one of the transglutaminase and the glutamic acid rich polypeptide is formulated in a solution, an emulsion, an aerosol, a foam, an ointment, a paste, a lotion, a powder, a gel, a hydrogel, a hydrocolloid, a microparticle, a nanoparticle, or a cream.

The method of any of paragraphs 21-23, wherein the transglutaminase and the glutamic acid rich polypeptide are formulated in separate compositions.

The method of any of paragraphs 21-23, wherein the transglutaminase and the glutamic acid rich polypeptide are formulated in one composition.

The method of any of paragraphs 21-25, further comprising applying a polymer to the wound, wherein the polymer comprises an amino group. The method of paragraph 26, wherein the polymer is a carbohydrate-based polymer. The method of paragraph 27, wherein the carbohydrate-based polymer comprises chitin or chitosan.

The method of any of paragraphs 26-29, wherein the polymer is in form of a solution, an emulsion, an aerosol, a foam, an ointment, a paste, a lotion, a powder, a gel, a hydrogel, a bandage, a gauze, a tape, a mesh, a net, an adhesive plaster, a film, a membrane, a patch, a microparticle, a nanoparticle or any combinations thereof.

The method of any of paragraphs 26-30, wherein the polymer is formulated in a composition with at least one of the transglutaminase and the glutamic acid rich polypeptide.

The method of paragraph 30, wherein the polymer and the transglutaminase are formulated together in one composition.

The method of paragraph 30, wherein all three of the polymer, the transglutaminase and the glutamic acid rich polypeptide are formulated in one composition.

The method of any of paragraphs 21-32, wherein the wound is selected from the group consisting of cuts and lacerations, surgical incisions, punctures, grazes, scratches, compression wounds, abrasions, friction wounds, chronic wounds, ulcers, thermal effect wounds, chemical wounds, wounds resulting from pathogenic infections, skin graft/transplant donor and recipient sites, immune response conditions, oral wounds, stomach or intestinal wounds, damaged cartilage or bone, amputation sites, corneal lesions and lung punctures.

The method of any of paragraphs 21-33, further comprising applying a wound healing agent to the wound.

The method of any of paragraphs 21-34, wherein the transglutaminase is a mammalian or microbial transglutaminase.

A method for forming a coating layer on a target surface, the method comprising:

applying a polymer, a transglutaminase and a glutamic acid rich polypeptide to the same portion of a target surface.

The method of paragraph 36, wherein the glutamic acid rich polypeptide is casein.

The method of paragraph 36 or 37, wherein the polymer is a protein or a carbohydrate- based polymer comprising at least one amino group. The method of paragraph 38, wherein the carbohydrate-based polymer comprises chitin or chitosan.

The method of paragraph 38, wherein the protein is collagen or gelatin.

The method of any of paragraphs 36-40, wherein at least one of the polymer, the transglutaminase and the glutamic acid rich polypeptide is formulated in a solution, an emulsion, an aerosol, a foam, an ointment, a paste, a lotion, a powder, a gel, a hydrogel, a hydrocolloid, a microparticle, a nanoparticle, or a cream.

The method of any of paragraphs 36-41, wherein the polymer, the transglutaminase and the glutamic acid rich polypeptide are formulated in separate compositions.

The method of any of paragraphs 36-41, wherein at least two of the polymer, the transglutaminase and the glutamic acid rich polypeptide are formulated in one

composition.

The method of paragraph 43, wherein the transglutaminase and the glutamic acid rich polypeptide are formulated in one composition.

The method of paragraph 44, wherein all three of the polymer, the transglutaminase and the glutamic acid rich polypeptide are formulated in one composition.

The method of any of paragraphs 36-45, wherein the target surface is a surface of a tissue or organ.

The method of paragraph 46, wherein the target surface is a surface of a hepatic, cardiac, intestinal, pulmonary or dermal tissue.

The method of any of paragraph 36-47, wherein the target surface is a wound.

The method of paragraph 48, wherein the wound is selected from the group consisting of cuts and lacerations, surgical incisions, punctures, grazes, scratches, compression wounds, abrasions, friction wounds, chronic wounds, ulcers, thermal effect wounds, chemical wounds, wounds resulting from pathogenic infections, skin graft/transplant donor and recipient sites, immune response conditions, oral wounds, stomach or intestinal wounds, damaged cartilage or bone, amputation sites, corneal lesions and lung punctures. The method of any of paragraphs 36-49, wherein the transglutaminase is a mammalian or microbial transglutaminase.

A three component composition comprising as a first component a polymer in a flowable form, wherein the polymer comprises an amino group; as a separate second component a transglutaminase in a flowable form; and as a separate third component a glutamic acid rich polypeptide in a flowable form.

The composition of paragraph 51, wherein at least two of the first, second and the third component are formulated together.

The composition of paragraph 51 or 52, wherein the second and the third component are formulated together.

The composition of any of paragraphs 51-53, wherein the flowable form includes a liquid, powder, or a combination thereof. The composition of any of paragraphs 51-54, wherein at least one of the first, the second and third component is in a liquid form. The composition of any of paragraphs 51-55, wherein at least one of the first, the second and third component is in a powder form. The composition of any of paragraphs 51-56, wherein the second and the third component are provided in a powder form. The composition of any of paragraphs 51-56, wherein the second and the third component are provided in a liquid form. The composition of paragraph 57 or 58, wherein the first component is in a liquid form. The composition of paragraph 57 or 58, wherein the first component is in form of a liquid. The composition of any of paragraphs 51-54, wherein at least one of the first, the second and third component is in form of an aerosol. The composition of paragraph 61, wherein the first component is in form of an aerosol. The composition of paragraph 61 or 62, wherein the second component is in form of an aerosol. The composition of any of paragraphs 61-63, wherein the third component is in form of an aerosol. The composition of any of paragraphs 51-64, wherein the glutamic acid rich polypeptide is casein. 66. The composition of any of paragraphs 51-65, wherein the polymer is a protein or a carbohydrate-based polymer comprising at least one amino group.

67. The composition of paragraph 66, wherein the carbohydrate-based polymer comprises chitin or chitosan.

68. The composition of paragraph 66, wherein the protein is collagen or gelatin.

69. The method of any of paragraphs 51-68, wherein the polymer, transglutaminase and

glutamic acid rich polypeptide are applied concurrently to the target surface.

70. The composition of any of paragraphs 51-69, wherein the transglutaminase is a

mammalian or microbial transglutaminase.

Definitions

[00105] Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[00106] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not. The terms "comprising" and "comprises" include the terms "consisting of and "consisting essentially of."

[00107] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[00108] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[00109] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages can mean ±1%.

[00110] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise.

[00111] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[00112] The terms "decrease", "reduced", "reduction", "decrease" or "inhibit" are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, "reduced", "reduction" or "decrease" or "inhibit" means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%), or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g. absent level as compared to a reference sample), or any decrease between 10-100%) as compared to a reference level.

[00113] The terms "increased", "increase" or "enhance" or "activate" are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms "increased", "increase" or "enhance" or "activate" means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%), or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100%) as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[00114] The term "statistically significant" or "significantly" refers to statistical significance and generally means a two standard deviation (2SD) below normal, or lower, concentration of the marker. The term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p-value.

[00115] As used herein, the terms "effective" and "effectiveness" includes both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of the treatment to result in a desired biological effect in the patient. Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ and/or organism level (often referred to as side-effects) resulting from administration of the treatment. "Less effective" means that the treatment results in a therapeutically significant lower level of pharmacological effectiveness and/or a therapeutically greater level of adverse physiological effects.

[00116] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate, e.g., a human. The terms, "patient" and "subject" are used interchangeably herein. The terms, "patient" and "subject" are used interchangeably herein. A subject can be male or female.

[00117] Preferably, the subject is a mammal. The mammal can be a human, non -human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of disorders associated with autoimmune disease or inflammation. In addition, the methods and compositions described herein can be used to treat domesticated animals and/or pets.

[00118] By "contact surface area" is meant the surface area of the target surface needed for bonding. For wounds, the contact surface area can mean the size of the wound and/or the total exposed area of the wound.

[00119] As used here, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[00120] As used herein, the term "pharmaceutically-acceptable carrier" means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C2-C12 alchols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein.

[00121] To the extent not already indicated, it will be understood by those of ordinary skill in the art that any one of the various embodiments herein described and illustrated can be further modified to incorporate features shown in any of the other embodiments disclosed herein. Thus, other embodiments are within the scope and spirit of the invention. Further, while the description above refers to the invention, the description may include more than one invention.

EXAMPLES

[00122] The invention now being generally described, it will be more readily understood by reference to the following example, which IS included merely for purposes of illustration of certain aspects and embodiments of the present invention, and is not intended to limit the invention.

Example 1: Direct bonding of chitosan biomaterials to tissue using transglutaminase for surgical repair or device implantation.

[00123] Natural biomaterials, such as chitosan and collagen, are useful for biomedical applications because they are biocompatible, mechanically robust and biodegradable, but it is difficult to rapidly and tightly bond them to living tissues. In the discussed experiment, the microbial enzyme transglutaminase (mTG) was used to rapidly (< 5 min) bond chitosan and collagen biomaterials to the surfaces of hepatic, cardiac and dermal tissues, as well as to functionalized polydimethylsiloxane (PDMS) materials that are used in medical products. The mTG-bonded Shrilk patches composed of a chitosan-fibroin laminate effectively sealed intestinal perforations, and a newly developed two-component mTG- bonded chitosan spray effectively repaired ruptures in a breathing lung when tested ex vivo. The mechanical strength of mTG- catalyzed chitosan adhesive bonds were comparable to those generated by commonly used surgical glues. These results suggest that mTG preparations may be broadly employed to bond various types of organic materials, including polysaccharides, proteins and functionalized inorganic polymers to living tissues, which may open new avenues for biomedical engineering, medical device integration and tissue repair.

[00124] In the discussed experiment, a method was developed to test the binding of chitosan biomaterials to biological tissues in order to open up new paths for biomedical engineering and medical device development. Use of transglutaminase (TG) enzyme was focused on due to its rapid and highly efficient bonding to primary amine groups, which are found at high density in chitosan and other biological molecules, such as collagen, and are relevant to tissue engineering and regenerative medicine. Tissue-derived TG has been employed to bond pieces of cartilage [see for example, Jiirgensen, K., et al., A New Biological Glue for Cartilage-Cartilage Interfaces: Tissue Transglutaminase 79, 85-193 (1997)] and microbial TG (mTG) has been used to crosslink protein gels, including fibrin and gelatin gels, which were shown to exhibit improved cell attachment and resistance to protease degradation. See for example, Chau, D.Y. S., et al., The cellular response to transglutaminase-cross-linked collagen. Biomaterials, 26(33), 6518-6529 (2005). TGs also have been investigated for the crosslinking of proteins to non-proteinaceous molecules that require functional groups, such as hyaluronic acid [see for example, Picard, J., S. Giraudier, and Larreta-Garde, V. Controlled remodeling of a protein-poly saccharide mixed gel: examples of gelatin-hyaluronic acid mixtures. Soft Matter, 5(21), 4198-4205 (2009)] and peptide-modified polyethyleneglycol (PEG). See for example, Sperinde, J.J. and Griffith, L. G. Control and Prediction of Gelation Kinetics in Enzymatically Cross-Linked Poly(ethylene glycol) Hydrogels. Macromolecules, 33(15), 5476-5480 (2000). mTG was chosen rather than tissue-derived TG in the discussed experiment because it can be produced much more efficiently at lower cost, and it does not require the presence of calcium ions to be activated, which enables many applications not supported by tissue- derived TG. The results show that mTG can be used to bond chitosan and collagen materials to organic substrates including different tissue types as well as inorganic substrates modified to contain sterically available amine groups. However, this process is only generalizable when the mTG preparation contains casein which is rich in glutamic acid residues. The results also show that mTG-mediated bonding of films made of chitosan strengthened with fibroin can be used to effectively seal intestinal perforations and a specifically designed mTG-based chitosan spray can be used to repair an injured breathing lung using ex vivo models.

Results and Discussion

mTG preparations with casein efficiently bond chitosan to organic surfaces

[00125] When glutaminic residues are exposed to mTG in the absence of a contacting substrate containing primary amines, the enzyme catalyzes the hydrolysis of the residues, resulting in the loss of the material's amine groups (FIG. la). However, when another structure is present containing primary amines (e.g. chitosan), mTG catalyzes an acyl transfer reaction between a glutaminyl residue (acyl donor) and a primary amine (acyl receptor) [see for example, Yokoyama, K., N. Nio, and Kikuchi, Y. Properties and applications of microbial transglutaminase. Applied Microbiology and Biotechnology, 64(4), 447-454 (2004)], thereby covalently linking to the structure. This process is extremely fast and gives rise to strong covalent bonds with high resistance to degradation. See for example, Lorand, L. and Graham, R. M. Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat. Rev. Mol. Cell Biol. 4(2), 140-156 (2003).

[00126] In common mTG preparations, carbohydrates (e.g., maltodextrin, saccharose, or mannose) are added to increase the stability of the enzyme against thermal degradation [see for example, Cui, L., et al., Stabilization of a new microbial transglutaminase from Streptomyces hygroscopicus WSH03-13 by spray drying. Process Biochemistry, 41(6), 1427-1431 (2006)], and proteins, such as casein, are added to protect mTG against degradation by extracellular proteolytic enzymes. See for example, Junqua, M., et al., Optimization of microbial transglutaminase production using experimental designs. Applied Microbiology and Biotechnology, 48(6), 730-734 (1997). Thus, to investigate mTG as a bonding reagent for chitosan materials, the ability of two different commercial mTG preparations to bind chitosan and collagen films were compared: one preparation contained only maltodextrin (mTG+Ma) and another contained both this carbohydrate and casein (mTG+Ca). The mTG preparation was applied as a powder to one of the surfaces before the two materials were brought together without applying pressure. Since both surfaces are hydrated and hydrophilic, capillary forces held the films in close contact. 10 minutes after mTG application, the adhesion force required to separate two mTG-bonded films was quantified using a standard t-peel test protocol (ASTM D1876). The results show that while the mTG+Ma preparation effectively bonded collagen substrates together, it was unable to bond chitosan substrates to each other or to collagen surfaces. On the other hand, the preparation containing casein (mTG+Ca) bonded collagen to both itself and to chitosan substrates, although it still did not mediate chitosan-chitosan bonding (FIG. lb). Thus, the presence of casein (which is rich in glutamic acid residues) in the preparation appears to be necessary for mTG to be able to rapidly and tightly bind chitosan to other organic surfaces, such as collagen, however is not necessary to mediate the collagen- collagen bonding, as the collagen protein already contains many internal glutamic acid residues. This finding that different mTG preparations produce different bonding results also might explain contradictions in past rheological studies of biopolymeric gels formed by mTG crosslinking, where identities of mTG stabilizers were not reported. See for example, Schorsch, C, et al., Cross-linking casein micelles by a microbial transglutaminase conditions for formation of transglutaminase-induced gels. International Dairy Journal, 10(8), 519-528 (2000); Chen, T., et al, Enzyme-catalyzed gel formation of gelatin and chitosan: potential for in situ applications. Biomaterials, 24(17), 2831-2841 (2003); Benjakul, S., et al, Effect of chitin and chitosan on gelling properties of surimi from barred garfish (Hemiramphus far). Journal of the Science of Food and Agriculture, 81(1), 102-108 (2001).

[00127] Importantly, while the optimal reaction temperature for material bonding by mTG has been reported to be around 50°C, rapid bonding of two organic (i.e. collagen) films was observed even at room temperature using mTG+Ca preparation (FIG. lc). Moreover, the maximum load necessary to separate these films was reached within 15 minutes at room temperature (FIGS, lc, Id), with more than 50% of the maximum resistance to separation already being reached within 5 minutes after the reaction was initiated (FIG. Id). Thus, the mTG preparation with casein enables rapid and strong bonding of chitosan and collagen materials to organic surfaces, indicating its suitability for use under physiological conditions in medically relevant tissue microenvironments. in T -mediated bonding of chitosan and collagen to inorganic materials

[00128] Implanted medical devices, electrochemical sensors, and actuators require strong and preferentially seamless interfaces between inorganic materials and living tissues, as do recently developed microfluidic Organ-on-a-chip' culture devices. See for example, Huh, D., et al, Reconstituting Organ-Level Lung Functions on a Chip. Science, 328(5986), 1662-1668 (2010); Bhatia, S.N. and Ingber, D. E. Microfluidic organs-on-chips. Nat. Biotech, 32(8), 760-772 (2014). Polydimethylsiloxane (PDMS) is a silicone polymer broadly used in medical devices, as well as microfluidic organ-on-a-chip devices, which does not normally serve as a substrate for mTG-mediated adhesion because it lacks endogenous reactive amine groups. Thus, mTG catalyzed adhesion of engineered organic biofilms to include bonding of these biofilms to PDMS was explored by first functionalizing its surface with amine groups using 3- Triethoxysilylpropylamine (APTES) before addition of mTG (FIG. 2a). Similar bonding studies were performed using collagen or chitosan films with mTG+Ma versus mTG+Ca, and adhesion strength was measured. Both collagen and chitosan films show limited adhesion to unfunctionalized PDMS, without any significant differences between the material or preparation used (p values ranged between 0.2 and 0.9) (FIG. 2b). In contrast, pretreatment with APTES resulted in a significant increase of bonding strength between films of chitosan or collagen and PDMS surfaces using mTG+Ca (p = 0.007 and p = 0.04, respectively), with the chitosan-PDMS bond displaying greater strength of adhesion (FIG. 2b). Interestingly, there was no detectable difference in the strength of bonding between collagen films and functionalized PDMS surfaces using different mTG preparations (i.e., mTG+Ma vs. mTG+Ca, p = 0.8). This is in direct contrast to the results with chitosan films where only the casein-containing mTG preparation was able to effectively bond the chitosan to the functionalized PDMS (p = 0.006) and there was no significant effect on bonding when mTG+Ma was used with functionalized or unfunctionalized PDMS (p > 0.051).

[00129] Together, these findings suggest that mTG preparations containing casein can mediate bonding of chitosan and collagen to inorganic surfaces, such as PDMS, which are integral parts of various types of biomedical implants and microfluidic devices. This approach could facilitate develop of medical technologies with synthetic elements, such as sensors, actuators or structural components, and allow them to leverage the biocompatibility and absorbability of these natural biomaterials.

Comparison of mTG-mediated bonding versus surgical glues

[00130] To investigate the potential of mTG as a medical bonding reagent for natural biomaterials, the mechanical strength of a chitosan patch bonded to a collagen film was compared using the mTG+Ca preparation versus the bonding strength produced using two common commercial surgical glues, Evicel and Progel. Evicel (Johnson & Johnson, USA) is fibrin-based and the most broadly employed sealant for surgical applications. Progel (C.R. Bard, USA) is an example of a newer generation of surgical glues specifically used for the treatment of pleural air leaks that are based on the use of a functional polymer and a bonding reagent (in the case of Progel, PEG functionalized with succinate groups and human serum albumin, respectively). The three materials were assayed with the "Standard Test Method for Burst Strength of Surgical Sealants" (ASTM F2392), a standard industrial protocol that measures the ultimate pressure (pressure necessary to break the sealing) of a surgical patch covering a 3 mm circular perforation on standard tissue mimic surface (e.g., collagen film #320; Nippi, Inc., Japan) (FIG. 3a). As per the ASTM protocol, the ultimate pressure is reported as the average pressure (n=5) necessary to break the sealing. However, due to variability introduced during the application, maximum pressure reached among all the samples is also reported, as it is indicative of bonding strength produced by each adhesion method independently of the user's skills.

[00131] These studies revealed that although both glues produced clinically relevant bonding strengths, the Progel glue provided much stronger adhesive properties than the Evicel glue (FIG. 3b). However, the Progel material was less flexible and conformal than the Evicel glue, which produced clumps rather than conformal coatings (data not shown). Importantly, although the mechanical adhesive strength of the mTG-mediated bond between the chitosan and collagen films was not as strong as that of the Progel, it was significantly greater than that of the Evicel (p = 0.02) and thus, the mTG bioglue produced a material bond that is well in the range of medically useful sealants. These findings suggest that mTG-mediated bonding of chitosan materials may be well-suited for a spectrum of biomedical applications where rapid and strong bonding of biomaterials are required. mTG-mediated chitosan bonding to whole tissues

[00132] To investigate the medical potential of mTG-mediated bonding of chitosan to living tissues, mTG+Ca method was used to attach chitosan films to whole porcine liver, heart and skin explants. Chitosan films were covered with the mTG powder and placed on the surface of the tissue for 10 min at room temperature. Again no pressure was applied during the reaction time, and instead the film conformed to the shape and held itself in place because of the small amounts of water it holds on its surface and the action of resultant capillary forces.

[00133] These studies revealed that addition of mTG resulted in a 3- to 6-fold increase in the strength of bonding between chitosan films and liver (p=0.03) or heart (p=0.002) tissues, respectively (FIG. 3c). In contrast, when mTG was used to adhere chitosan to the epidermal surface of the skin explant, it did not produce any detectable (p=0.7) bonding of the film to the tissue (FIG. 3d). This is to be expected given that the stratum corneum of the epidermis is rich in endogenous tTG, which results in cross- linking of virtually all available amines within the epidermal cell layer [see for example, Eckhart, L., et al., Cell death by cornification. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1833(12), 3471-3480 (2013)], and apparently, none are available to link to chitosan using exogenous mTG. This possibility was supported by the finding that mechanical removal of the epidermis and exposure of the underlying dermis resulted in extraordinarily strong bonding to chitosan films using mTG (p=0.02), even surpassing that observed for other organ surfaces (FIG. 3d). In addition, it was explored whether mTG can also be used to bond chitosan foams to tissues to help fill larger tissue defects. It was confirmed that chitosan foams can be similarly bonded to tissues by using mTG+Ca to adhere chitosan foams to an irregularly shaped 1 cm defect in an explanted latissimus dorsi muscle from a domestic pig. Again, these foams exhibited firm attachment to the tissue boundaries within 10 minutes after application, and the adhesion between the foam and tissue was virtually seamless with the border being difficult to detect when analyzed with photomicroscopy (FIG. 3e). mTG-bonded chitosan films as surgical sealants

[00134] To further explore the potential biomedical uses of mTG-bonded chitosan materials, mTG-mediated bonding of chitosan films was examined on a punctured tissue. Gastrointestinal perforations occur in a variety of illnesses including Crohn's disease, appendicitis and ulcers, and if not treated in time, they lead to life threatening abdominal infections and sepsis. Except for very mild cases, their repair requires a surgical procedure that reestablishes a tight barrier separating the gastrointestinal lumen and the abdominal space. An ex vivo intestinal perforation model was developed by surgically incising a 1 cm slit hole in the wall of an explanted pig small intestine. To repair the perforation, mTG+Ca was used to bond a 3 x 3 cm² chitosan-fibroin laminate film, known as Shrilk [see for example, Fernandez, J.G. and Ingber, D. E., Unexpected Strength and Toughness in Chitosan-Fibroin Laminates Inspired by Insect Cuticle. Advanced Materials, 24(4), 480-484 (2012)], over the hole by placing the mTG powder-coated film in contact with the inner surface of the intestinal wall surrounding the damaged area (e.g., mimicking placement via endoscopic surgery). A burst test was performed 10 minutes later by clamping both ends of the intestinal segment onto the end of a hollow tube and flowing saline into the lumen to increase intraluminal hydraulic pressure. Impressively, the chitosan-fibroin composite film seal systematically remained intact even when the pressure was raised to more than 125 kPa (FIG. 4a); however, the experiment ended when the intestinal wall opposite to the site of the sealed perforation burst (FIG. 4b; data not shown). The intestine burst at pressure values twenty times higher than the physiological pressure present in the intestine [see for example, Tasaka, K. and Farrar, J. T., Intraluminal pressure of the small intestine of the unanesthetized dog. Pflugers Arch, 364(1), 35-44 (1976)]; this pressure is even higher than levels in the circulatory system experienced during a hypertensive crisis, which is the highest physiological pressure generated within the human body that has been found in the literature. Thus, chitosan patches sealed in place using mTG could potentially be used for surgical repair for various tissues.

A sprayable mTG-bonded method for repair of lung punctures

[00135] Although chitosan films were used to seal punctures in the intestine, the organ was explanted and static in that study. It is more difficult to use a film of fixed shape and size to seal punctures and tears in living organs that experience dynamic movements, such as in the lung that undergoes cyclic breathing motions, or in organs with irregular shaped defects. Thus, it was explored whether a sprayable form of chitosan and mTG sealant can be developed. One major challenge to explore this possibility was that chitosan and casein exhibit opposite behaviors in basic and acidic environments: chitosan dissolves in acid and precipitates in alkaline solutions, while the opposite is true for casein. Thus, to create a sprayable form of chitosan, it must be dissolved in a diluted acid, to prevent the combination of both components in a single spraying solution.

[00136] To overcome the different pH dependencies of chitosan and casein, a double-canister spray system was developed (FIG. 4c) in which one canister contained chitosan in an acidic solution and the other contained mTG with casein in a lightly basic solution. Both solutions are sprayed simultaneously onto the same surface. When both components interact at the tissue surface, the pH of the combined solution neutralizes and gives rise to an even and homogenous chitosan-mTG-casein precipitate with adhesive properties. The resulting properties of the conformal coating produced by the two-component spray differed from those of pure chitosan films that were used to test bonding strength in that they are much more elastic, but less tough.

[00137] The double-spray system's ability to seal a punctured lung was investigated next. A 3x1 cm deep incision was made on the surface of one lobe of an explanted pig lung while the damaged lung was cyclically inflated and deflated to reproduce the breathing cycle using an external air source connected to the trachea (data not shown). By using the double-canister spray system to apply chitosan and mTG+Ca directly to the injured surface of the breathing lung, the puncture and the surrounding area was covered with a conformal elastic film that effectively sealed the leak within a few minutes (FIG. 4d). Scanning electron microscopic analysis of the sealed tissue surface revealed a seamless bond, as well as the porous nature of the sprayed chitosan+mTG+Ca sealant (FIG. 4e). The speed and easy applicability of the double- spray chitosan-mTG sealant method, combined with the biocompatibility and antimicrobial properties of chitosan, should make it useful for treatment of internal and external wounds of variable sizes, including exposed dermal surfaces of burn injuries. Interestingly, based on the current finding that mTG does not bond chitosan to epidermis, this method potentially can be used to create a sprayable skin for burn injuries that could serve as a protective cuticle that spontaneously detaches over time as the epidermal cells migrate into the healing wound.

[00138] Although these bonded materials were not studied in vivo, past studies have shown that chitosan implants only induce minimal chronic inflammation and foreign body reaction in vivo. See for example, Bavariya, A.J., et al., Evaluation of biocompatibility and degradation of chitosan nanofiber membrane crosslinked with genipin. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 102(5), 1084-1092 (2014). Together with their favorable mechanical adhesion properties, this makes mTG-mediated bonding of chitosan materials an ideal method to stably bond functional chitosan implants in different tissue contexts. Bonding of porous chitosan foams to dermis, for example, might be useful for mechanical treatment strategies used for large non-healing wounds, such as Vacuum Assisted Closure (VAC) therapy, which would benefit from use of bioabsorbable porous scaffolds that provide both good physical properties and strong attachment to the wound site. See for example, Nather, A., et al., Effectiveness of vacuum-assisted closure (VAC) therapy in the healing of chronic diabetic foot ulcers. Ann Acad Med Singapore, 39(5), 353-358 (2010).

[00139] In conclusion, the discussed experiment demonstrates how mTG preparations containing casein can be used to bond chitosan to materials composed of proteins, such as collagen and living tissues, as well as to inorganic surfaces of polymers that are used in medical and microfluidic devices, such as PDMS. The rapid action and the versatility of the bonding process make it suitable for a broad number of applications, ranging from surgical sealants to coatings for implantable medical devices or sensor/actuators to better integrate biomaterials and living tissues within microfluidic devices or BioMEMS. In short, the ability of mTG to rapidly and strongly bond chitosan and other biomaterials to both living tissues and inorganic surfaces offers a new way to seamlessly integrate living and non-living materials.

Materials and Methods Transglutaminase preparations

[00140] Two microbial transglutaminase (mTG) preparations containing Streptoverticillium calcium-independent TG were obtained from Ajinomoto Food Ingredients LLC (Chicago, USA). One preparation contained approximately 1% (w/w) enzyme stabilized in maltodextrin, whereas the other was stabilized using maltodextrin (0.39 w/w) and casein (0.6 w/w). Enzymatic preparations were stored in a powder form to facilitate transport of mTG at room and higher temperatures, and because this formulation could enable its use across a broad spectrum of applications in the future (e.g., first aid and battlefield assistance). In the case of films and foams, the enzymatic preparations were directly used in their powder form, sprinkling the surface of the material with an even thin coating prior to apposing it to another surface for attachment. In the case of the double-canister spray system, mTG preparations were used as a 3% (w/v) solution in 4% (w/v) NaOH to maintain their activity. See for example, Yokoyama, K., N. Nio, and Kikuchi, Y. Properties and applications of microbial transglutaminase. Applied Microbiology and Biotechnology, 64(4), 447-454 (2004). Although higher mTG concentrations enhanced the film- forming capabilities of the spray components, they were not useful for spraying due to micelle formation in the solution.

Chitosan and collagen materials

[00141] Chitosan films and foams were produced as reported before. See for example, Fernandez, J.G. and Ingber, D.E. Bioinspired Chitinous Material Solutions for Environmental Sustainability and Medicine. Advanced Functional Materials, 23(36), 4454-4466 (2013); Fernandez, J.G. and Ingber, D. E., Unexpected Strength and Toughness in Chitosan-Fibroin Laminates Inspired by Insect Cuticle. Advanced Materials, 24(4), 480-484 (2012). Chitosan films were used to investigate bonding strength using standardized industrial methods and ASTM protocols; chitosan foams were used to investigate tissue defect filling. Chitosan films were produced by solvent evaporation casting of a 2% (w/v) solution of 80% deacetylated chitosan in a 1% (v/v) acetic acid solution. Films were neutralized with a 4% NaOH solution (w/v). The method for producing Shrilk films composed of a laminate of layers of chitosan and fibroin has been previously described in detail. See for example, Fernandez, J.G. and Ingber, D. E., Unexpected Strength and Toughness in Chitosan-Fibroin Laminates Inspired by Insect Cuticle. Advanced Materials, 24(4), 480-484 (2012). Chitosan foams were made by freeze drying a 1% (w/v) solution of chitosan in a 0.5% (v/v) acetic acid solution, neutralizing it in 4% (w/v) NaOH, and intensely washing in double ionized water. Due to the randomness of the foam structure, this configuration is not suitable for a standardized measurement of bonding strength, and studies with living tissues were carried out instead, as described in the results. Collagen films were produced from the Collagen casing #320 (Nippi, Inc., Japan) standard for ASTM protocols (e.g. ASTM F2392). Chitosan films strengthened with fibroin (i.e. Shrilk) were produced by sequential deposition and neutralization of the components, as described previously. See for example, Fernandez, J.G. and Ingber, D. E., Unexpected Strength and Toughness in Chitosan-Fibroin Laminates Inspired by Insect Cuticle. Advanced Materials, 24(4), 480-484 (2012).

Mechanical adhesion testing

[00142] Adhesion measurements were performed using two ASTM methods. ASTM D1876 (t-peel test) experiments were performed with an Instron 3342 instrument (500N, Instron, USA) to measure the strength of adhesion of films bonded to flat surfaces. Studies were carried out on biopolymer films, collagen substrates, and tissue s(i.e. skin, lung, heart, and muscle) shaped in rectangular (1 cm wide x 6 cm long) strips. Both surfaces of the adhesion test were fixed to an aluminum support (200 μπι thick) to avoid the effect of the film and substrate stretching when measuring surface adhesion; pulling speed was 10 mm/min. The treated side of the films was placed in direct contact, but they were not subjected to pressure during the reaction time, and instead were held in place by capillary forces.

[00143] To measure burst pressures, the ASTM F2392 standard protocol was used, which was developed to measure burst strength of surgical sealants. A 4 mm diameter patch of chitosan and mTG+Ca powder was used to cover a 3 mm diameter hole punched in the center of a collagen film (Collagen casing #320, Nippi, Inc., Japan) prepared as per the ASTM protocol. Assays were performed 5 min after application of the adhesive (mTG or surgical sealant) in all studies. The system was filled with water on top of the patch and air was delivered with a syringe pump (World Precision Instruments Inc., Florida, USA) at a rate of 2 ml/min from at the bottom. Pressure was measured using a differential pressure sensor (Pasco, California, USA). Burst pressure was observed as a drop in pressure while air bubbles were observed passing through the broken seal of the patch. In all the mechanical tests, the sample size and statistical significance were chosen assuming a normal distribution [see for example, Hess, P.E., et al., Uncertainties in Material and Geometric Strength and Load Variables. Naval Engineers Journal, 114(2), 139-166 (2002)], and all error bars indicate standard deviation.

[00144] Lung tests with the double spray were performed using explanted lungs of domestic pig (sus domesticus) obtained from a local slaughterhouse. A deep hole (3 x 1.5 cm) was formed on the surface of one lung, and the trachea was connected to an external air pump. Continuous cyclic airflow to fully inflate and deflate the lungs was provided to mimic breathing motions of the lung. Tests were carried out twice on the same pair of lungs. Intestinal tests were performed by attaching both ends of a 15 cm section of explanted small intestine from a domestic pig to 3 cm diameter tube. A 1 cm hole was incised in the intestine wall and subsequently repaired using mTG in powder form to attach a 3 cm patch of Shrilk (chitosan-fibroin laminate film; see for example, Fernandez, J.G. and Ingber, D. E., Unexpected Strength and Toughness in Chitosan- Fibroin Laminates Inspired by Insect Cuticle. Advanced Materials, 24(4), 480-484 (2012)) from inside the lumen of the intestine. The system was filled with pressurized water containing blue dye (to enhance contrast) and pressure was measured using a differential pressure sensor (Pasco, California, USA).

[00145] All patents and other publications identified herein are expressly incorporated herein by reference for all purposes. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

Claims

CLAIMS What is claimed is:

1. A method for bonding or adhering a biomaterial to a target surface, the method

comprising applying an effective amount of a transglutaminase and a glutamic acid rich polypeptide to a target surface and contacting a biomaterial to the target surface where the transglutaminase and the glutamic acid rich polypeptide have been applied.

2. The method of claim 1, wherein the glutamic acid rich polypeptide is casein.

3. The method of claim 1, wherein the biomaterial comprises a protein or a carbohydrate- based polymer comprising at least one amino group.

4. The method of claim 3, wherein the carbohydrate-based polymer comprises chitin or chitosan.

5. The method of claim 3, wherein the protein is collagen or gelatin.

6. The method of claim 1, wherein the biomaterial comprises a composite laminate material comprising a layer of carbohydrate-based polymer and a layer of protein.

7. The method of claim 6, wherein the carbohydrate-based polymer of the composite

laminate material comprises chitin or chitosan.

8. The method of claim 6, wherein the protein of the composite laminate material comprises collagen, gelatin or silk fibroin.

9. The method of claim 1, wherein the biomaterial is in the form of a medical implant

device.

10. The method of claim 9, wherein the medical implant device is selected from the group consisting of artificial tissues, artificial organs, prosthetic devices, drug delivery devices, wound dressings, films, foams, sponges, scaffolds, meshes, hemostatic materials, and any combinations thereof.

11. The method of claim 9, wherein the medical implant device is a wound dressing selected from the group consisting of bandages, gauzes, tapes, meshes, nets, adhesive plasters, films, membranes, patches, microparticles, nanoparticles, and any combinations thereof.

12. The method of claim 1, wherein at least one of the biomaterial, the transglutaminase and the glutamic acid rich polypeptide is formulated in a solution, an emulsion, an aerosol, a foam, an ointment, a paste, a lotion, a powder, a gel, a hydrogel, a hydrocolloid, a microparticle, a nanoparticle, or a cream.

13. The method of claim 1, wherein the biomaterial, the transglutaminase and the glutamic acid rich polypeptide are formulated in separate compositions.

14. The method of claim 1, wherein at least two of the biomaterial, the transglutaminase and the glutamic acid rich polypeptide are formulated in one composition.

15. The method of claim 14, wherein all three of the biomaterial, the transglutaminase and the glutamic acid rich polypeptide are formulated in one composition.

16. The method of claim 1, wherein the target surface is a surface of a tissue or organ.

17. The method of claim 16, wherein the target surface is a surface of a hepatic, cardiac, intestinal, pulmonary or dermal tissue.

18. The method of claim 1, wherein the target surface is a wound.

19. The method of claim 18, wherein the wound is selected from the group consisting of cuts and lacerations, surgical incisions, punctures, grazes, scratches, compression wounds, abrasions, friction wounds, chronic wounds, ulcers, thermal effect wounds, chemical wounds, wounds resulting from pathogenic infections, skin graft/transplant donor and recipient sites, immune response conditions, oral wounds, stomach or intestinal wounds, damaged cartilage or bone, amputation sites, corneal lesions and lung punctures.

20. The method of claim 1, wherein the transglutaminase is a mammalian or microbial

transglutaminase.

21. A method for promoting wound healing, the method comprising applying a

transglutaminase and a glutamic acid rich polypeptide to a surface of a wound.

22. The method of claim 21, wherein the glutamic acid rich polypeptide is casein.

23. The method of claim 21, wherein at least one of the transglutaminase and the glutamic acid rich polypeptide is formulated in a solution, an emulsion, an aerosol, a foam, an ointment, a paste, a lotion, a powder, a gel, a hydrogel, a hydrocolloid, a microparticle, a nanoparticle, or a cream.

24. The method of claim 21, wherein the transglutaminase and the glutamic acid rich

polypeptide are formulated in separate compositions.

25. The method of claim 21, wherein the transglutaminase and the glutamic acid rich

polypeptide are formulated in one composition.

26. The method of claim 21, further comprising applying a polymer to the wound, wherein the polymer comprises an amino group.

27. The method of claim 26, wherein the polymer is a carbohydrate-based polymer.

28. The method of claim 27, wherein the carbohydrate-based polymer comprises chitin or chitosan.

29. The method of claim 26, wherein the polymer is in form of a solution, an emulsion, an aerosol, a foam, an ointment, a paste, a lotion, a powder, a gel, a hydrogel, a bandage, a gauze, a tape, a mesh, a net, an adhesive plaster, a film, a membrane, a patch, a microparticle, a nanoparticle or any combinations thereof.

30. The method of claim 26, wherein the polymer is formulated in a composition with at least one of the transglutaminase and the glutamic acid rich polypeptide.

31. The method of claim 30, wherein the polymer and the transglutaminase are formulated together in one composition.

32. The method of claim 30, wherein all three of the polymer, the transglutaminase and the glutamic acid rich polypeptide are formulated in one composition.

33. The method of claim 21, wherein the wound is selected from the group consisting of cuts and lacerations, surgical incisions, punctures, grazes, scratches, compression wounds, abrasions, friction wounds, chronic wounds, ulcers, thermal effect wounds, chemical wounds, wounds resulting from pathogenic infections, skin graft/transplant donor and recipient sites, immune response conditions, oral wounds, stomach or intestinal wounds, damaged cartilage or bone, amputation sites, corneal lesions and lung punctures.

34. The method of claim 21, further comprising applying a wound healing agent to the

wound.

35. The method of claim 21, wherein the transglutaminase is a mammalian or microbial transglutaminase.

36. A method for forming a coating layer on a target surface, the method comprising:

applying a polymer, a transglutaminase and a glutamic acid rich polypeptide to the same portion of a target surface.

37. The method of claim 36, wherein the glutamic acid rich polypeptide is casein.

38. The method of claim 36, wherein the polymer is a protein or a carbohydrate-based

polymer comprising at least one amino group.

39. The method of claim 38, wherein the carbohydrate-based polymer comprises chitin or chitosan.

40. The method of claim 38, wherein the protein is collagen or gelatin.

41. The method of claim 36, wherein at least one of the polymer, the transglutaminase and the glutamic acid rich polypeptide is formulated in a solution, an emulsion, an aerosol, a foam, an ointment, a paste, a lotion, a powder, a gel, a hydrogel, a hydrocolloid, a microparticle, a nanoparticle, or a cream.

42. The method of claim 36, wherein the polymer, the transglutaminase and the glutamic acid rich polypeptide are formulated in separate compositions.

43. The method of claim 36, wherein at least two of the polymer, the transglutaminase and the glutamic acid rich polypeptide are formulated in one composition.

44. The method of claim 43, wherein the transglutaminase and the glutamic acid rich

polypeptide are formulated in one composition.

45. The method of claim 44, wherein all three of the polymer, the transglutaminase and the glutamic acid rich polypeptide are formulated in one composition.

46. The method of claim 36, wherein the target surface is a surface of a tissue or organ.

47. The method of claim 46, wherein the target surface is a surface of a hepatic, cardiac, intestinal, pulmonary or dermal tissue.

48. The method of any of claims 36-47, wherein the target surface is a wound.

49. The method of claim 48, wherein the wound is selected from the group consisting of cuts and lacerations, surgical incisions, punctures, grazes, scratches, compression wounds, abrasions, friction wounds, chronic wounds, ulcers, thermal effect wounds, chemical wounds, wounds resulting from pathogenic infections, skin graft/transplant donor and recipient sites, immune response conditions, oral wounds, stomach or intestinal wounds, damaged cartilage or bone, amputation sites, corneal lesions and lung punctures.

50. The method of claim 36, wherein the transglutaminase is a mammalian or microbial transglutaminase.

51. A three component composition comprising as a first component a polymer in a flowable form, wherein the polymer comprises an amino group; as a separate second component a transglutaminase in a flowable form; and as a separate third component a glutamic acid rich polypeptide in a flowable form.

52. The composition of claim 51, wherein at least two of the first, second and the third

component are formulated together.

53. The composition of claim 51, wherein the second and the third component are formulated together.

54. The composition of claim 51, wherein the flowable form includes a liquid, powder, or a combination thereof.

55. The composition of claim 51, wherein at least one of the first, the second and third

component is in a liquid form.

56. The composition of claim 51, wherein at least one of the first, the second and third

component is in a powder form.

57. The composition of claim 51, wherein the second and the third component are provided in a powder form.

58. The composition of claim 51, wherein the second and the third component are provided in a liquid form.

59. The composition of claim 57, wherein the first component is in a liquid form.

60. The composition of claim 57, wherein the first component is in form of a liquid.

61. The composition of claim 51, wherein at least one of the first, the second and third

component is in form of an aerosol.

62. The composition of claim 61, wherein the first component is in form of an aerosol.

63. The composition of claim 61, wherein the second component is in form of an aerosol.

64. The composition of claim 61, wherein the third component is in form of an aerosol.

65. The composition of claim 51, wherein the glutamic acid rich polypeptide is casein.

66. The composition of claim 51, wherein the polymer is a protein or a carbohydrate-based polymer comprising at least one amino group.

67. The composition of claim 66, wherein the carbohydrate-based polymer comprises chitin or chitosan.

68. The composition of claim 66, wherein the protein is collagen or gelatin.

69. The method of claim 51, wherein the polymer, transglutaminase and glutamic acid rich polypeptide are applied concurrently to the target surface. The composition of claim 51, wherein the transglutaminase is a mammalian or microbial transglutaminase.