Extracellular matrix-based adhesive composition comprising methacryloyl-substituted gelatin

Abstract

The present invention relates to an extracellular matrix-based adhesive composition comprising methacryloyl-substituted gelatin and specifically, comprises an extracellular matrix-containing hydrogel, methacryloyl-substituted gelatin, and a gelatin curing agent, wherein the methacryloyl-substituted gelatin (GelMA) has a molecular weight in the range of 50-200 kDa, and optionally, has a degree of methacryloylation (DOM) of 40-80%, and thus exhibits excellent effects in terms of mechanical properties, adhesive strength, elasticity, and the like.

Description

Extracellular matrix-based adhesive composition containing methacryloyl-substituted gelatin

[0001] The present invention relates to an extracellular matrix-based adhesive composition, and more specifically, to a bioadhesive based on an extracellular matrix and comprising methacryloyl-substituted gelatin, having excellent mechanical properties, adhesive strength, elasticity, etc., a method for manufacturing the same, and uses thereof.

[0002] Bioadhesives are a general term for adhesive materials used for bonding between biological tissues, such as skin, blood vessels, organs, and bones, or for bonding artificial materials with biological tissues. They primarily utilize substances that possess adhesive properties to various biological samples, such as biological cells and proteins. Bioadhesives can be applied in various clinical fields, including tissue adhesives, hemostatic agents, scaffolds for tissue engineering, hydrogels for drug delivery, tissue fillers, and wound healing. Bioadhesives require strong adhesive and cross-linking capabilities and must maintain their function within the body for an extended period. Furthermore, they must adhere instantaneously within the body, where tissue fluid is present, without generating heat or harmful substances, and without causing rejection reactions.

[0003] Currently commercialized or practical bioadhesives include cyanoacrylate instant adhesives, fibrin glue, gelatin glue, and polyurethane-based adhesives. However, bioadhesives utilizing synthetic polymers exhibit very weak strength in the presence of aqueous solutions within the body. Furthermore, cyanoacrylate, gelatin, and polyurethane-based bioadhesives have been reported to have in vivo toxicity, and causing side effects such as immune reactions in the human body is pointed out as a major limitation. Additionally, fibrin-based bioadhesives currently used in actual patients have limited applications due to their very low adhesive capacity, despite having few side effects. Moreover, most currently commercialized or practical bioadhesives have low bioactivity, which actually hinders self-healing of lesion sites and inhibits tissue regeneration. To overcome these problems, it is necessary to develop an ideal type of bioadhesive that possesses strong adhesive and cross-linking capabilities, exhibits minimal side effects within the body, and supports tissue regeneration.

[0004] Meanwhile, the cornea is a transparent, avascular tissue located on the anterior surface of the eyeball that protects the eye from the outside. As the first passage of light in the eye, it plays a crucial role in light refraction and transmission; however, because the cornea is constantly exposed to the external environment, it is easily damaged and susceptible to various diseases. According to the World Health Organization, approximately 285 million people suffer from visual impairments, mostly due to corneal diseases, and there are various corneal conditions and their complications, such as dry eye syndrome, Sjögren's syndrome, and bacterial keratitis. Chronic corneal diseases cause corneal opacity, necessitating corneal transplants, and more than 1.5 million new cases of corneal blindness are reported annually. Of these, less than 5% receive transplant treatment due to a shortage of donor tissue and high surgical costs. Corneal damage and infection cause scarring and thinning of the corneal stroma, which can lead to vision loss.

[0005] Corneal transplantation is essential in cases of corneal damage caused by severe corneal diseases or trauma. However, globally, there is an absolute shortage of corneal donors compared to the number of patients awaiting transplantation, resulting in an average waiting period of approximately eight years. Consequently, various approaches are being employed for conservative treatment, such as using tissue adhesives like cyanoacrylate adhesives and fibrin glue, or transplanting amniotic membranes to protect the wound and reduce inflammation. Nevertheless, there are currently no therapeutic agents available worldwide to induce healing and regeneration of the corneal epithelial tissue or stroma in cases of fatal corneal damage. To overcome these limitations, there is an urgent need for the development of biocompatible tissue repair biomaterials that are highly transparent, facilitate engraftment, and enable regeneration and repair. In particular, biocompositions with high adhesive properties that adhere well to tissues even in humid environments, such as the surface of the eye, are emerging as a new technology that can enhance the convenience of medical procedures.

[0006] In this regard, the inventors have developed an extracellular matrix-based bioadhesive through Korean Published Patent No. 10-2023-0050638 (Title of Invention: Extracellular Matrix-based Bioadhesive), which is an adhesive in the form of a composition comprising an extracellular matrix-containing hydrogel and a gelatin curing agent, wherein the extracellular matrix-containing hydrogel is gelatinized. However, the above-mentioned bioadhesive also has the disadvantage of having somewhat low adhesive strength, as well as lacking mechanical properties and elasticity against compressive stimulation.

[0007] Accordingly, there is always a need to develop bioadhesives that possess excellent adhesive strength while being suitable for various uses, such as corneal tissue repair, as their mechanical properties and elasticity against compressive stress are similar to those of biological corneal tissue.

[0008] [Prior Art Literature]

[0009] [Patent Document] Republic of Korea Published Patent No. 10-2023-0050638 (April 17, 2023)

[0010] The present invention aims to solve the aforementioned problems and provides an adhesive composition having excellent viscosity and mechanical strength.

[0011] Furthermore, the present invention aims to provide an adhesive composition that exhibits physical properties similar to human corneal tissue, including excellent adhesion, elasticity, and elasticity against compressive stimulation, as well as burst pressure and transparency.

[0012] Furthermore, the present invention aims to provide an adhesive composition that can be used for various purposes and applications by possessing high mechanical properties, adhesive strength, and elasticity exceeding the level of commercial tissue adhesives.

[0013] In addition, the present invention is intended to provide a useful application as an adhesive biomaterial for the healing and / or regeneration of corneal defects and / or damages, such as dry eye or corneal ulcers.

[0014] The extracellular matrix-based adhesive composition according to the present invention for achieving the above-mentioned purpose comprises an extracellular matrix-containing hydrogel, a gelatin curing agent, and a methacryloyl-substituted gelatin, wherein the methacryloyl-substituted gelatin has a molecular weight in the range of 50 kDa to 200 kDa.

[0015] Here, the methacryloyl-substituted gelatin may be GelMA (gelatin-methacryloyl).

[0016] In addition, the methacryloyl-substituted gelatin may have a molecular weight in the range of 90 kDa to 200 kDa.

[0017] In addition, the methacryloyl-substituted gelatin can have a molecular weight in the range of 120 kDa to 180 kDa.

[0018] In addition, the methacryloyl-substituted gelatin may have a molecular weight in the range of 150 kDa to 170 kDa.

[0019] In addition, the above methacryloyl-substituted gelatin may be included at a concentration of 5 to 15% (w / v).

[0020] In addition, the above methacryloyl-substituted gelatin may have a degree of methacryloyl substitution (DOM) of 40 to 80 percent.

[0021] In addition, the above methacryloyl-substituted gelatin can have a degree of methacrylolylation (DOM) of 50 to 70%.

[0022] In addition, the above-mentioned extracellular matrix-containing hydrogel may be a gelatinized decellularized extracellular matrix derived from the corneal stroma.

[0023] In addition, the above gelatin curing agent may be a photoinitiator composed of ruthenium and sulfuric acid.

[0024] In addition, the extracellular matrix-based adhesive composition according to the present invention can be used for corneal tissue repair.

[0025]

[0026] Specific details of other embodiments are included in the detailed description and drawings.

[0027] The present invention is characterized by comprising a gelatin curing agent and methacryloyl-substituted gelatin in a hydrogel containing an extracellular matrix, wherein the methacryloyl-substituted gelatin has a molecular weight within a specific range, thereby providing an adhesive composition having excellent mechanical properties.

[0028] In addition, the present invention has the effect of providing an adhesive composition in which the methacryloyl-substituted gelatin has a degree of methacrylolylation (DOM) within a specific range, thereby exhibiting excellent adhesion, elasticity, and elasticity against compressive stimulation, as well as physical properties such as burst pressure and transparency similar to human corneal tissue.

[0029] Since the adhesive composition according to the present invention possesses high mechanical properties, adhesive strength, and elasticity exceeding the level of commercial tissue adhesives, it can be used for various purposes and applications.

[0030] In particular, since the composition according to the present invention has transparency, burst pressure, and transparency at the level of human corneal tissue, it adheres well to the lesion site and can provide a useful application as an adhesive biomaterial for the healing and regeneration of corneal defects or damage.

[0031] Figure 1 is a schematic diagram showing the characteristics of methacryloyl-substituted gelatin according to one example of the present invention, based on molecular weight and degree of methacryloylation (DoM).

[0032] Figure 2 is the result of analyzing the gelation kinetics according to visible light irradiation time and the molecular weight of methacryloyl-substituted gelatin in a bioadhesive composition according to an example of the present invention.

[0033] Figure 3 is the result of analyzing the storage modulus of a bioadhesive in which cross-linking is induced by light, according to the molecular weight of methacryloyl-substituted gelatin in a bioadhesive composition according to an example of the present invention.

[0034] Figure 4 is the result of analyzing the gelation kinetics according to visible light irradiation time for each methacryloyl substitution rate of methacryloyl-substituted gelatin in a bioadhesive composition according to an example of the present invention.

[0035] Figure 5 is the result of analyzing the storage modulus of a bioadhesive in which cross-linking is induced by light, according to the methacryloyl substitution rate of methacryloyl-substituted gelatin in a bioadhesive composition according to an example of the present invention.

[0036] Figure 6 is the result of analyzing the adhesive strength (lap shear strength) according to the methacryloyl substitution rate of methacryloyl-substituted gelatin in a bioadhesive composition according to an example of the present invention.

[0037] Figure 7 is the result of analyzing the elasticity against compressive stimulation according to the methacryloyl substitution of methacryloyl-substituted gelatin in a bioadhesive composition according to an example of the present invention.

[0038] Figure 8 is the result of analyzing the pressure that the bioadhesive withstands until it ruptures when gas pressure is applied at a constant rate, according to the methacryloyl substitution of the methacryloyl-substituted gelatin in the bioadhesive composition according to one example of the present invention.

[0039] Figure 9 is the result of analyzing light transmittance according to the methacryloyl substitution rate of methacryloyl-substituted gelatin in a bioadhesive composition according to an example of the present invention.

[0040] Figure 10 is the result of analyzing the average transmittance in the visible light region according to the methacryloyl substitution rate of methacryloyl-substituted gelatin in a bioadhesive composition according to an example of the present invention.

[0041] The present invention is capable of various modifications and may have various embodiments; specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and it should be understood that it includes all modifications, equivalents, and substitutions that fall within the spirit and scope of the present invention. In describing the present invention, detailed descriptions of related prior art are omitted if it is determined that such detailed descriptions may obscure the essence of the present invention.

[0042] The terms used in this application are used merely to describe specific embodiments and are not intended to limit the invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, terms such as "comprising" or "having" are intended to specify the presence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0043] Terms such as "first," "second," etc., may be used to describe various components, but said components should not be limited by said terms. These terms are used solely for the purpose of distinguishing one component from another.

[0044] The extracellular matrix-based adhesive composition according to the present invention basically comprises an extracellular matrix-containing hydrogel, a gelatin curing agent, and methacryloyl-substituted gelatin.

[0045] The above-mentioned hydrogel containing extracellular matrix may be a hydrogel derived from or based on the extracellular matrix. The term "extracellular matrix (ECM)" as used in the present invention refers to the extracellular portion of animal tissue that typically provides structural support to animal cells while simultaneously performing various other important functions. The extracellular matrix is ​​a defining characteristic of connective tissue in animals and consists of various forms of proteins, including collagen and glycosaminoglycans (GAGs). This extracellular matrix may be tissue from animals such as pigs and cattle, and can be extracted from various organs.

[0046] In the present invention, it is preferable that the extracellular matrix be a decellularized extracellular matrix when considering in vivo applications. Decellularization is a technique for removing all xenogeneic cells that may induce an immune response in biological tissues and isolating the extracellular matrix. The present invention has developed an adhesive tissue repair biomaterial with a decellularized extracellular matrix (dECM) as the main component. In particular, the decellularized extracellular matrix derived from corneal tissue contains numerous proteins that maintain the differentiation, activity, and homeostasis of corneal tissue cells, thereby promoting the regeneration of the corneal epithelium and stroma, and securing the high transparency and mechanical properties characteristic of the cornea. The decellularized extracellular matrix has the effect of minimizing the immune response during allograft or xenograft by removing cells that can act as antigens inducing an immune response. Because the types and number of cells and the physical properties of the tissue itself differ depending on the tissue, decellularization is performed using various chemical substances such as acids, bases, hypotonic solutions, hypertonic solutions, and detergents. Furthermore, the aforementioned cellular matrix may be used while maintaining the tissue's own structure after undergoing only the decellularization process, but it may also be used by dissolving it in an acidic solution after freeze-drying and grinding processes, or by converting it into a hydrogel form after undergoing a neutralization process. Additionally, considering the primary application, it is more preferable for the aforementioned extracellular matrix to be a corneal-derived decellularized extracellular matrix. The corneal-derived decellularized extracellular matrix is ​​derived from corneal stromal tissue and contains proteins that aid in cell adhesion or proteins that facilitate cell growth and the expression of function, in addition to the physical structure surrounding the cells. The corneal-derived decellularized extracellular matrix preferably contains collagen fibers from which telopeptides have been removed.

[0047] In the present invention, the hydrogel containing the extracellular matrix can be gelatinized. The hydrogel containing the extracellular matrix can preferably be gelatinized by thermal denaturation of collagen, a component of the extracellular matrix. The term "gelatinized" used in the present invention refers to a state in which the hydrogel containing the extracellular matrix is ​​denatured by heat and possesses rheological properties identical or similar to those of gelatin. Gelatin is a type of derivative protein obtained by treating collagen with hot water; while it only swells in cold water, it dissolves in hot water to become a sol and acquires flowability. As the collagen is a major component of the extracellular matrix, when heated with water, it denatures, dissolves, and elutes in a colloidal state, converting into gelatin. Since collagen, a major component of the extracellular matrix, exists in a solidified state at body temperature (approx. 37°C) where the bioadhesive is applied, it is difficult to apply a non-gelatinized hydrogel containing the extracellular matrix evenly and easily to the lesion site, resulting in inconvenience in use. In addition, hydrogels containing non-gelatinized extracellular matrix do not harden easily even when a gelatinizing agent is added, and their adhesion is significantly reduced when applied to lesions.

[0048] In addition, the above-mentioned hydrogel containing extracellular matrix may be a decellularized extracellular matrix derived from the corneal matrix that has been gelatinized. When the adhesive tissue repair biomaterial composition according to the present invention is based on an extracellular matrix derived from corneal tissue, it includes not only collagen constituting the corneal tissue but also naturally derived ECMs constituting the corneal tissue related to ocular development, wound healing, and tissue reconstruction, and in particular, it includes major ECMs related to maintaining corneal homeostasis such as keratokan, lumican, and decorin, thereby exhibiting excellent effects in regenerating damaged corneal tissue into clear and transparent original corneal tissue.

[0049] In addition, in the extracellular matrix-based adhesive composition according to the present invention, the content of the extracellular matrix-containing hydrogel is not significantly limited, and considering the hydrogel-forming ability, smooth thermal denaturation, uniform mixing with gelatin curing agent, or the convenience of use or adhesive strength of the bioadhesive, it is possible to have 1 to 5% (w / v), it is preferable to have 2 to 4% (w / v), and it is more preferable to have 1.5 to 3% (w / v).

[0050] The adhesive composition according to the present invention, which includes such an extracellular matrix-containing hydrogel, not only has rheological properties identical or similar to gelatin, but also has flowability at temperatures above 30°C and can be applied evenly and easily to damaged lesion sites on the corneal surface, and can rapidly induce cross-linking using visible light.

[0051] The above gelatin curing agent is a substance that cures gelatin in the adhesive composition according to the present invention.

[0052] The term "gelatin curing agent" as used in the present invention refers to a substance or a combination of substances that converts gelatin into a solid state by cross-linking it through simple addition, heat treatment, or light irradiation. Various types of gelatin curing agents may be used, such as inorganic compounds containing polyvalent metal ions, or organic compounds including aldehydes and quinones. Generally, photoinitiators used as gelatin curing agents include Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) and Irgacure 2959. Specific examples of gelatin curing agents in the present invention include a combination of ruthenium (Ru) and sodium persulfate (SPS) that induces the curing of gelatin by irradiation with visible light (particularly blue light); and riboflavin that induces the curing of gelatin by irradiation with ultraviolet light. Or, there are combinations of EDC / NHS [(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride / Nhydroxysuccinimide] that induce hardening of gelatin by simple addition or a predetermined heat treatment, grape seed extract, dialdehyde starch, glutaraldehyde, etc.

[0053] In an extracellular matrix-based bioadhesive according to one example of the present invention, the gelatin curing agent may be a photoinitiator composed of ruthenium and sulfuric acid, considering biosafety, ease of use, and adhesive ability. For example, it may be selected from a combination of ruthenium and sodium persulfate or riboflavin, and more preferably from a combination of ruthenium and sodium persulfate. It is known that when irradiated with visible light (particularly blue light), the ruthenium and sodium persulfate oxidize aromatic residues containing tyrosine, and the oxidized aromatic residues are converted into free radicals and form covalent bonds with each other, for example, di-tyrosine covalent bonds, thereby inducing a cross-linking reaction.

[0054] After incorporating ruthenium and sodium persulfate as photoinitiators into a bio-derived extracellular matrix hydrogel, when exposed to visible light, Ru in the presence of the electron acceptor SPS 2+ is Ru 3+ It is photodegraded into Ru 3+ The presence of [the substance] in turn oxidizes aromatic residues containing tyrosine, which are abundant in the extracellular matrix derived from living organisms, and the oxidized tyrosine groups are further converted into tyrosyl free radicals and stabilized by forming covalent dityrosine bonds with nearby tyrosine groups.

[0055] The Ru / SPS crosslinking system can induce crosslinking very rapidly due to the high absorbance of Ru in visible light and its chemical stability in the excited state. Since the bio-derived extracellular matrix is ​​rich in tyrosine transport proteins, this Ru / SPS system can promote the crosslinking of hydrogels more rapidly in a cell-friendly manner. In particular, tyrosine oxidized by Ru reacts with hydroxide ions (HO-) to become L-3,4-dihydroxyphenylalanine (L-Dopa), and since this Dopa possesses naturally occurring adhesive properties, it can increase the adhesion of bio-derived extracellular matrix hydrogels.

[0056] In addition, when the gelatin curing agent is selected from a combination of ruthenium and sodium persulfate, considering biosafety and adhesive ability, it is preferable that the concentration of ruthenium in the bioadhesive be 0.1 to 2 mM and the concentration of sodium persulfate be 1 to 20 mM, and it is more preferable that the concentration of ruthenium be 0.2 to 1.5 mM and the concentration of sodium persulfate be 2 to 15 mM. The inventors prepared an extracellular matrix-based adhesive composition by adding ruthenium and sodium persulfate, which are one of the visible light-activated photoinitiators, in a ratio of 1:10 with final concentrations of 0.5-1 mM and 5-10 mM, respectively, and stirring. Through this, it was possible to effectively induce crosslinking by ionizing using visible light and through the methacrylate polymerization reaction of methacryloyl-substituted gelatin (GelMA).

[0057] The above methacryloyl-substituted gelatin may be methacrylated gelatin.

[0058] That is, methacryloyl-substituted gelatin can be a methacryloyl based on gelatin, which is a type of hydrogel. For example, it can be a material that enables crosslinking by chemically introducing methacrylate functional groups into gelatin.

[0059] Such methacryloyl-substituted gelatin can be GelMA (Gelatin-MethAcryloyl), which is a typical semi-synthetic hydrogel and is one of the versatile hydrogels capable of controlling mechanical properties while being biologically applicable like gelatin molecules. Unlike ordinary gelatin, gelatin with synthesized methacryloyl groups can be crosslinked by forming rapid covalent bonds in the presence of ultraviolet light and a photoinitiator.

[0060] The inventors focused on the molecular weight and the degree of methacryloylation (DoM; Degree of methacryloylation or DOF; Degree of functionalization) of such methacryloyl-substituted gelatin.

[0061] Figure 1 is a schematic diagram showing the characteristics according to the molecular weight and methacryloyl substitution rate of methacryloyl-substituted gelatin used according to an example of the present invention.

[0062] In other words, mechanical properties can vary depending on the molecular weight of methacryloyl-substituted gelatin. As the molecular weight of the gelatin decreases, the structure of the gelatin becomes relatively shorter, so the mechanical properties after crosslinking are softer and have lower stiffness compared to gelatin with a high molecular weight. Additionally, since the crosslinking density can be adjusted by controlling the methacryloyl substitution rate, it is also possible to finely adjust the mechanical properties of the gelatin by modifying the substitution rate (Fig. 1).

[0063] After conducting repeated research to enhance the adhesive strength of a bioadhesive comprising an extracellular matrix-containing hydrogel and a gelatin curing agent, the inventors completed the present invention by confirming that the mechanical properties of the material can be finely adjusted according to the molecular weight and level of methacryloyl substitution of the methacryloyl substituent gelatin, as can be seen in the examples and experimental examples described below.

[0064] The inventors confirmed that the physical properties of extracellular matrix-based bioadhesives differ depending on the type of GelMA, a methacryloyl-substituted gelatin, specifically the gelatin molecular weight and the level of methacryloyl substitution. Accordingly, they prepared various methacryloyl-substituted gelatins in extracellular matrix adhesive compositions with different gelatin molecular weights and levels of methacryloyl substitution, observed changes in physical properties as bioadhesives, and modified and optimized the material properties as an adhesive biomaterial for repairing damaged corneas.

[0065] According to the present invention, the methacryloyl-substituted gelatin may have a molecular weight in the range of 50 kDa to 200 kDa, a molecular weight in the range of 90 kDa to 200 kDa, a molecular weight in the range of 120 kDa to 180 kDa, or a molecular weight in the range of 150 kDa to 170 kDa. If the molecular weight is lower than the above range, it has a relatively short structural shape, so it has the disadvantage of exhibiting somewhat softer physical properties when gelled by inducing cross-linking covalent bonds, and if the molecular weight is higher than the above range, it has the disadvantage of increased stiffness and reduced adhesive strength.

[0066] As can be seen in the examples and experimental examples described below, the gelation kinetics according to visible light irradiation time were analyzed for methacryloyl-substituted gelatin according to the molecular weight of the bioadhesive composition according to one example of the present invention. As a result, the mechanical strength of the bioadhesives to which gelatin methacryloyl with molecular weights of 90 kDa and 160 kDa, respectively, was added increased rapidly upon irradiation with blue light (400-500 nm), thereby confirming that crosslinking can be induced by blue light for both types of gelatin methacryloyl with molecular weights (see Fig. 2). Furthermore, when comparing the mechanical strength or elasticity (storage modulus) of the bioadhesive composition according to one example of the present invention, it was confirmed that the bioadhesive containing 160 kDa gelatin had more than twice the crosslinking strength of the bioadhesive containing 90 kDa gelatin (see Fig. 3).

[0067] In addition, as an example of the present invention, the methacryloyl-substituted gelatin may have a methacryloyl substitution rate (DOM) of 40 to 80%, and it is also possible to have a DOM of 50 to 70%. Since the methacryloyl-substituted gelatin forms covalent bonds through the methacryloyl group when crosslinking is induced, if the methacryloyl substitution rate is higher than the above range, the crosslinking of the gelatin is induced more densely, which increases strength but has the disadvantage of potentially decreasing transparency. In addition, if the methacryloyl substitution rate is lower than the above range, the pore size increases, which may increase transparency but has the disadvantage of significantly decreasing mechanical strength.

[0068] As can be confirmed in the examples and experimental examples described below, when the gelation kinetics according to visible light irradiation time were analyzed for different substitution rates (40%, 60%, 80%) of methacryloyl-substituted gelatin in the bioadhesive composition according to one example of the present invention, the mechanical strength increased rapidly in all types of gelatin, and in particular, the highest mechanical strength and crosslinking strength were exhibited immediately after blue light irradiation at a substitution rate of 60% (see Fig. 4). Furthermore, according to the results of the analysis of the storage modulus, the strongest storage modulus and crosslinking strength were exhibited at a substitution rate of 60% (see Fig. 5), and according to the results of the analysis of lap shear strength, adhesive strength similar to or greater than that of commercial bioadhesives was exhibited at all substitution rates, with the strongest adhesive strength being exhibited at a substitution rate of 60% (see Fig. 6). In addition, the analysis of elasticity under compressive stimulation showed that there was no significant difference in compressive elasticity according to the substitution rate; however, among them, the elasticity under compressive stimulation was best when the methacryloyl substitution rate was 60% (see Fig. 7). Furthermore, the analysis of burst pressure indicated that bioadhesives at all substitution rates could be safely applied to the human eye within the normal range, and among them, the substitution rate of 60% showed the best burst pressure (see Fig. 8). Additionally, the analysis of visible light transmittance showed transmittance levels exceeding those of the human cornea at all substitution rates (see Figs. 9 and 10).

[0069] In addition, as another example of the present invention, the methacryloyl-substituted gelatin may be included at a concentration of 5 to 15% (w / v). When the methacryloyl-substituted gelatin is included in the above range, not only is the viscosity and mechanical strength excellent, but the adhesion and elasticity are also the best. That is, if the content of the methacryloyl-substituted gelatin is less than the above range, there is a disadvantage that the adhesion, viscosity, mechanical strength, adhesion, and elasticity are all insufficient, and if it exceeds the above range, there is a disadvantage that the adhesion is lower and the elasticity becomes too high.

[0070] The present invention is characterized by comprising a gelatin curing agent and methacryloyl-substituted gelatin in a hydrogel containing an extracellular matrix, wherein the methacryloyl-substituted gelatin has a molecular weight within a specific range, thereby providing an adhesive composition having excellent mechanical properties.

[0071] In addition, the present invention has the effect of providing an adhesive composition in which the methacryloyl-substituted gelatin has a degree of methacrylolylation (DOM) within a specific range, thereby exhibiting excellent adhesion, elasticity, and elasticity against compressive stimulation, as well as physical properties such as burst pressure and transparency similar to human corneal tissue.

[0072] As described above, the present invention has elasticity similar to that of biological corneal tissue, so it is suitable for use in corneal tissue repair and can provide useful applications as an adhesive biomaterial for healing and / or regeneration of corneal defects and / or damages such as dry eye or corneal ulcers.

[0073] Accordingly, the extracellular matrix-based adhesive composition according to the present invention can be used for corneal tissue repair. The composition of the present invention is an adhesive tissue repair material, and in particular, when composed of an extracellular matrix derived from the corneal matrix, it is clear and transparent and exhibits a light transmittance of 90% or more. In addition, since it exhibits adhesive properties, it shows high adhesion even on a moist ocular surface, has high elasticity against shear stimuli such as rubbing or blinking the eyes, and can be composed of an elastic biopolymer that has elasticity similar to biological tissue against vertical pressing stimuli.

[0074] Furthermore, the adhesive composition according to the present invention can be used as a corneal repair biomaterial useful for healing and inducing regeneration and reconstructing tissue in corneal damage and deficiency caused by various causes such as Sjögren's syndrome, neurotrophic keratitis, keratoconus, and corneal ulcers. This corneal tissue repair composition is biocompatible with corneal tissue, is biodegradable and remains in the corneal tissue for more than 30 days after implantation, and can be structurally replaced by regenerated autologous corneal tissue.

[0075] A method for using an extracellular matrix-based adhesive composition according to an example of the present invention may include the step of applying the adhesive composition to a lesion site and applying heat or irradiating light. For example, if the extracellular matrix-based adhesive composition comprises a combination of ruthenium and sodium persulfate as a gelatin curing agent, applying the adhesive composition to a lesion site and irradiating visible light, specifically blue light with a wavelength of 350 to 500 nm (preferably 400 to 450 nm) for a period of 20 to 120 seconds (preferably 30 to 100 seconds), can form a bioadhesive film with excellent adhesive strength and elasticity.

[0076] In fact, if the adhesive composition according to the present invention is applied to the surface of the eye and blue light is irradiated for 30 seconds to 2 minutes, an adhesive strength (adhesive strength), which is the strength to withstand shear stress, can be secured up to 36 to 42 N.

[0077] In addition, when the adhesive composition according to the present invention is subjected to a load that acts substantially perpendicularly to the surface of the eye and is pressed down to a depth of 100 μm, the compressive stress, which is the resistance force generated in correspondence with that magnitude, was found to be approximately 9,500 to 11,000 μN, thereby imparting characteristics similar to those of biological corneal tissue.

[0078] The present invention may be better understood by the following examples, which are for illustrative purposes only and are not intended to limit the scope of protection defined by the appended claims.

[0079] Preparation Example: Preparation of corneal-derived decellularized extracellular matrix (Co-dECM) hydrogel

[0080] A decellularized extracellular matrix (Co-dECM) hydrogel derived from the cornea was prepared as follows. First, the cornea was isolated from a porcine eye, and the epithelium and endothelium were removed from the corneal tissue to obtain a pure corneal stromal layer. Then, it was washed with a PBS buffer solution containing 100 units / mL penicillin and 0.1 mg / mL streptomycin. Subsequently, the stromal tissue was placed in a 20 mM ammonium hydroxide solution (NH4OH; 4.98 N aqueous solution) containing 0.5% Triton X-100 and stirred for approximately 4 hours. Afterward, the stromal tissue was washed with distilled water and stirred with a Tris-HCl (hypotonic Tris hydrochloride; pH 7.4) buffer solution for approximately 24 hours. Subsequently, the stromal tissue was placed in a 10 mM Tris-HCl solution containing 1% (v / v) Triton X-100 and stirred at 37°C for about 24 hours to obtain corneal-derived decellularized extracellular matrix (Co-dECM) tissue. Afterward, the corneal-derived decellularized extracellular matrix (Co-dECM) tissue was sterilized by treating it with a 1% peracetic acid solution in 50% ethanol for about 10 hours. After completing the decellularization process, the corneal-derived decellularized extracellular matrix (Co-dECM) was freeze-dried overnight. 0.2 g of corneal-derived decellularized extracellular matrix was added to 10 mL of acetic acid solution (0.5 M) supplemented with 0.02 g of pepsin and stirred for 3 days to obtain a homogeneous corneal-derived decellularized extracellular matrix hydrogel with a concentration of 2% (w / v). The above hydrogel was filtered through a 40 μm mesh, adjusted to pH 7.0 - 7.3 using 10N sodium hydroxide, stored at 4 ℃, and used in subsequent experiments.

[0081] Example: Preparation of an extracellular matrix-based adhesive composition

[0082] A gelatinized extracellular matrix-based hydrogel was obtained by heat-denaturing the 2% (w / v) extracellular matrix-based hydrogel prepared in the above preparation example by raising the temperature to 50–56°C and maintaining it for 20–40 minutes. Subsequently, a biomaterial composition was prepared by adding 10% (w / v) GelMA to the gelatinized hydrogel. The GelMA used had molecular weights of 90 kDa and 160 kDa, respectively, and methacryloyl substitution rates of 40%, 60%, and 80%, respectively. Then, ruthenium and sodium persulfate, which are gelatin curing agents, were added at final concentrations of 0.5 mM and 5 mM, respectively, and stirred to prepare the final adhesive biomaterial composition. At this time, care was taken to prevent the ruthenium and the hydrogel mixed with ruthenium from being exposed to light. The solvent for both the ruthenium solution and the sodium persulfate solution above was DPBS (Dulbecco's phosphate-buffered saline).

[0083] Subsequently, when used in experiments, the adhesive biomaterial composition prepared above was irradiated with blue light of a wavelength of about 400 to 500 nm, and tyrosine residues present inside the gelatinized extracellular matrix-based hydrogel were oxidized and converted into tyrosine free radicals, and formed di-tyrosine covalent bonds with nearby tyrosine residues to promote curing, thereby inducing adhesion and crosslinking.

[0084] Experimental Example 1: Comparison of Gelation Kinetics and Crosslinking Strength According to Gelatin Molecular Weight

[0085] Bioadhesives prepared according to the above example, containing gelatin methacryloyl with molecular weights of 90 kDa and 160 kDa respectively and having the same methacryloyl substitution rate (60% substitution), were applied to a rheometer, and the gelation kinetics over time were analyzed.

[0086] Figure 2 is the result of analyzing the gelation kinetics according to visible light irradiation time and the molecular weight of methacryloyl-substituted gelatin in a bioadhesive composition according to an example of the present invention.

[0087] As shown in Figure 2, the mechanical strength of the bioadhesives containing gelatin of two different molecular weights increased rapidly upon irradiation with blue light (400-500 nm), confirming that crosslinking can be induced in both gelatin methacryloyl of two different molecular weights by blue light.

[0088] Figure 3 is the result of analyzing the storage modulus of a bioadhesive in which cross-linking is induced by light, according to the molecular weight of methacryloyl-substituted gelatin in a bioadhesive composition according to an example of the present invention.

[0089] As shown in Figure 3, when comparing the mechanical strength or storage modulus of the bioadhesives with cross-linking induced immediately after blue light irradiation, the adhesive mixed with gelatin with a molecular weight of 90 kDa showed 1074.5±120.6 Pa, while the adhesive mixed with gelatin with a molecular weight of 160 kDa showed 2652.3±116.2 Pa. It was confirmed that the bioadhesive containing 160 kDa gelatin had more than twice the cross-linking strength of the bioadhesive containing 90 kDa gelatin, and a very strong statistically significant difference was observed (90 kDa vs. 160 kDa, p=0.00008).

[0090] Since the crosslinking strength and elasticity of such bioadhesives are very important physical properties as tissue adhesives, the subsequent mechanical properties were observed based on a molecular weight of 160 kDa and changes in the mechanical properties of the bioadhesive according to methacryloyl substitution rates of 40%, 60%, and 80%.

[0091] Experimental Example 2: Comparison of Gelation Kinetics and Crosslinking Strength According to Gelatin Methacryloyl Substitution Rate

[0092] For bioadhesives prepared according to the above example and having the same molecular weight of 160 kDa and methacryloyl substitution rates of 40%, 60%, and 80%, respectively, the gelation kinetics over time were analyzed.

[0093] Figure 4 is the result of analyzing the gelation kinetics according to visible light irradiation time for each methacryloyl substitution rate of methacryloyl-substituted gelatin in a bioadhesive composition according to an example of the present invention.

[0094] As shown in Figure 4, mechanical strength increased rapidly in all types of gelatin immediately after blue light irradiation, confirming that crosslinking was induced in all substitution rate groups. In particular, the highest mechanical strength and crosslinking power were exhibited immediately after blue light irradiation at a substitution rate of 60%. This was maintained for 10 minutes after blue light irradiation.

[0095] Figure 5 is the result of analyzing the storage modulus of a bioadhesive in which cross-linking is induced by light, according to the methacryloyl substitution rate of methacryloyl-substituted gelatin in a bioadhesive composition according to an example of the present invention.

[0096] As shown in Figure 5, when comparing the mechanical strength or elasticity of each bioadhesive observed immediately after blue light irradiation, the bioadhesive mixed with gelatin methacryloyl at a substitution rate of 60% exhibited the strongest elasticity and crosslinking strength. Next, the substitution rate of 40% showed high elasticity, and the substitution rate of 80% showed the lowest elasticity, but there was no statistical significance between the substitution rates of 40% and 80% (40%, 1461.8±279.2 Pa; 60%, 2652.3±116.2 Pa; 80%, 1289.8±155.9 Pa; 40% vs. 60%, p=0.00242; 40% vs. 80%, p=0.404; 60% vs. 80%, p=0.00026).

[0097] Experimental Example 3: Comparison of Adhesive Strength According to Gelatin Methacryloyl Substitution Rate

[0098] The adhesive strength was analyzed for bioadhesives prepared according to the above example, which have the same molecular weight of 160 kDa and have methacryloyl substitution rates of 40%, 60%, and 80%, respectively.

[0099] Figure 6 is the result of analyzing the adhesive strength (lap shear strength) according to the methacryloyl substitution rate of methacryloyl-substituted gelatin in a bioadhesive composition according to an example of the present invention.

[0100] As shown in Figure 6, the adhesive strength was found to be 19.8±10.0 kPa at a substitution rate of 40%, 66.6±23.1 kPa at a substitution rate of 60%, and 43.8±19.6 kPa at a substitution rate of 80%. Since commercial bioadhesives such as fibrin glue are known to typically have an adhesive strength of around 20 kPa, it was confirmed that adhesive strengths of similar or greater levels were exhibited at all substitution rates in comparison. Among them, the strongest adhesive strength was observed at a substitution rate of 60%, followed by 80% and 40% (40% vs. 60%, p=0.0011; 40% vs. 80%, p=0.0235; 60% vs. 80%, p=0.0961).

[0101] From a clinical perspective, since shear stimuli such as rubbing the eyes or blinking the eyelids are routinely applied to the cornea, a bioadhesive with a 60% replacement rate that exhibits high adhesive strength appears to have excellent performance for clinical application.

[0102] Experimental Example 4: Comparison of Elasticity under Compression Stimulation According to Gelatin Methacryloyl Substitution Rate

[0103] For bioadhesives prepared according to the above example and having the same molecular weight of 160 kDa and methacryloyl substitution rates of 40%, 60%, and 80%, respectively, the elasticity under compressive stimulation was analyzed.

[0104] That is, the elasticity of each cross-linked bioadhesive composition against compressive stimulation was confirmed by applying the bioadhesive according to one embodiment of the present invention to CellScale’s MicroTester G2 instrument and measuring the compressive modulus until shear occurs.

[0105] Figure 7 is the result of analyzing the elasticity against compressive stimulation according to the methacryloyl substitution of methacryloyl-substituted gelatin in a bioadhesive composition according to an example of the present invention.

[0106] As shown in Figure 7, there was no significant difference in compressive elasticity according to the substitution rate (40%, 8.36±1.43 kPa; 60%, 11.15±5.47 kPa; 80%, 10.16±0.71 kPa), but among them, the elasticity against compressive stimulation was best when the methacryloyl substitution rate was 60%, and compared to the commercial bioadhesive fibrin glue having a compressive strength of about 3-10 kPa or less, it was confirmed that all substitution rate groups had a similar level of compressive elasticity.

[0107] Experimental Example 5: Comparison of Burst Pressure According to Gelatin Methacryloyl Substitution Rate

[0108] For bioadhesives prepared according to the above example and having the same molecular weight of 160 kDa and methacryloyl substitution rates of 40%, 60%, and 80%, respectively, the burst pressure was compared and analyzed.

[0109] That is, the rupture pressure was measured by applying the bioadhesive according to one embodiment of the present invention to a burst pressure measuring device manufactured in accordance with ASTM standard F2392-04. This is an evaluation to verify whether the bioadhesive can withstand intraocular pressure; based on human standards, normal intraocular pressure is considered to be 10–20 mmHg, and intraocular pressure elevated due to glaucoma, etc., is considered to be 30 mmHg or higher. A pig cornea prepared to a certain size was placed on the measuring part of the device, and a penetrating wound of 2 mm was made in the center of the cornea to prepare for measurement. The penetrating site was covered with each bioadhesive according to the present invention, and the pressure the adhesive withstood until rupture was measured when gas pressure was applied to the cornea at the same flow rate (2 mL / min).

[0110] Figure 8 is the result of analyzing the pressure that the bioadhesive withstands until it ruptures when gas pressure is applied at a constant rate, according to the methacryloyl substitution of the methacryloyl-substituted gelatin in the bioadhesive composition according to one example of the present invention.

[0111] As shown in Figure 8, the intrarupture pressure was observed to be 22.75±4.19 mmHg for a substitution rate of 40%, 129.14±64.30 mmHg for a substitution rate of 60%, and 31.50±14.61 mmHg for an substitution rate of 80%. Since normal intraocular pressure is 10–20 mmHg, it is expected that bioadhesives of all substitution rates can be safely applied to human eyes within the normal range. In the case of fibrin glue, the intrarupture pressure has been observed to be approximately 100–150 mmHg. When compared to the above results, it was confirmed that the bioadhesive composed of a 60% substitution rate exhibits an intrarupture pressure at the level of fibrin glue, a currently commercialized bioadhesive.

[0112] Experimental Example 6: Comparison of Visible Light Transmittance According to Gelatin Methacryloyl Substitution Rate

[0113] For bioadhesives prepared according to the above example and having the same molecular weight of 160 kDa and methacryloyl substitution rates of 40%, 60%, and 80%, respectively, light transmittance was measured and comparatively analyzed.

[0114] The most important histological characteristic of the cornea is high permeability, and in the development of biomaterials for corneal repair, the ability to transmit light as is and deliver visual information directly to the optic nerve is important. The transparency of the human cornea is approximately 87%, and the transmittance of contact lenses is known to be around 95%. Using an absorbance measuring device, the light transmittance (Fig. 9) and the average transmittance in the visible light region of 380–700 nm wavelength range (Fig. 10) of the bioadhesive according to one embodiment of the present invention were measured.

[0115] Figure 9 is the result of analyzing light transmittance according to the methacryloyl substitution rate of methacryloyl-substituted gelatin in a bioadhesive composition according to an example of the present invention.

[0116] Figure 10 is the result of analyzing the average transmittance in the visible light region according to the methacryloyl substitution rate of methacryloyl-substituted gelatin in a bioadhesive composition according to an example of the present invention.

[0117] As shown in Fig. 10, the average transmittance in the visible light region (wavelengths of 380–700 nm) was 96.01±1.87% for the adhesive with a substitution rate of 40%, 92.89±8.90% for 60%, and 92.45±6.00% for 80%. Light transmittance showed a decreasing trend in the order of 40%, 60%, and 80% substitution rates, which is expected to be due to the increase in cross-linking density and decrease in pore size as the methacryloyl substitution rate increases. However, transmittances exceeding those of the human cornea were observed at all substitution rates.

[0118] Although the present invention has been illustrated and described above in relation to specific preferred embodiments, it is obvious to those skilled in the art that the present invention may be modified and varied without departing from the technical features or scope of the invention as defined by the following claims.

Claims

1. A hydrogel containing extracellular matrix, a gelatin hardener, and methacryloyl-substituted gelatin, comprising The above methacryloyl-substituted gelatin has a molecular weight in the range of 50 kDa to 200 kDa, Extracellular matrix-based adhesive composition.

2. In Paragraph 1, An extracellular matrix-based adhesive composition characterized in that the above-mentioned methacryloyl-substituted gelatin is GelMA (gelatin-methacryloyl).

3. In Paragraph 2, An extracellular matrix-based adhesive composition characterized in that the above methacryloyl-substituted gelatin has a molecular weight in the range of 90 kDa to 200 kDa.

4. In Paragraph 2, An extracellular matrix-based adhesive composition characterized in that the above methacryloyl-substituted gelatin has a molecular weight in the range of 120 kDa to 180 kDa.

5. In Paragraph 2, An extracellular matrix-based adhesive composition characterized in that the above methacryloyl-substituted gelatin has a molecular weight in the range of 150 kDa to 170 kDa.

6. In Paragraph 4, An extracellular matrix-based adhesive composition characterized by containing the above methacryloyl-substituted gelatin at a concentration of 5 to 15% (w / v).

7. In Paragraph 4, An extracellular matrix-based adhesive composition characterized in that the above methacryloyl-substituted gelatin has a degree of methacrylolylation (DOM) of 40 to 80 percent.

8. In Paragraph 4, An extracellular matrix-based adhesive composition characterized in that the above methacryloyl-substituted gelatin has a degree of methacrylolylation (DOM) of 50 to 70 percent.

9. In Paragraph 4, The above-mentioned extracellular matrix-containing hydrogel is an extracellular matrix-based adhesive composition characterized by decellularized extracellular matrix derived from the corneal stroma being gelatinized.

10. In Paragraph 4, An extracellular matrix-based adhesive composition characterized in that the gelatin curing agent is a photoinitiator combined with ruthenium and sulfuric acid.

11. In any one of paragraphs 1 through 10, An extracellular matrix-based adhesive composition characterized by being for corneal tissue repair.

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