Composition and adhesive

Abstract

To provide a composition with superior biocompatibility and tissue adhesion.SOLUTION: A composition contains gelatin, alcohol, and a solvent containing water. In a matrix containing the gelatin, droplets containing the alcohol and the solvent are dispersed.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a composition and an adhesive.

Background Art

[0002] In Japan, which has entered a super-aged society, the development of minimally invasive medicine is required, and the development of various biocompatible biomaterials and medical materials is progressing. For example, a tissue adhesive is a medical material that can quickly close and repair defects and wounds by adhering tissues to each other with an adhesive, and has various advantages such as shortening the operation time and promoting tissue regeneration.

[0003] Currently, fibrin glue is cited as a tissue adhesive used clinically, but problems include low adhesive strength and the risk of virus infection. Cyanoacrylate-based adhesives exhibit high adhesive ability, but the problem is their strong toxicity. In addition, adhesives using polymers having reactive functional groups exhibit high biocompatibility, but there is concern that an inflammatory reaction may be caused by a chemical reaction, and since it is necessary to mix two liquid components, handling is difficult.

[0004] Under such circumstances, the development of a tissue adhesive composed of a single liquid component and excellent in biocompatibility and tissue adhesiveness is required.

[0005] For example, Non-Patent Document 1 describes a temperature-responsive hydrogel that is liquid at low temperatures and forms a gel at high temperatures. In addition, Non-Patent Document 2 describes a temperature-responsive adhesive using a recombinant protein and a surfactant.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

[0007] However, the hydrogel described in Non-Patent Document 1, while having excellent handling properties due to being a one-component type, suffers from low gel strength and poor biocompatibility with biological tissues because it is a synthetic polymer. Furthermore, the hydrogel described in Non-Patent Document 2, while having excellent handling properties and adhesive strength, raises concerns about the toxicity of the surfactant.

[0008] The present invention has been made in view of the above circumstances, and provides a composition that can be used as a tissue adhesive and has excellent biocompatibility and tissue adhesion properties. [Means for solving the problem]

[0009] The inventors, after diligently studying to achieve the above objectives, found that the objectives could be achieved with the following configuration.

[0010] [1] A composition, Gelatin and, Alcohol and, A solvent containing water, A composition in which droplets containing the alcohol and the solvent are dispersed in a matrix containing the gelatin. [2] The composition according to [1], wherein the sol-gel transition temperature of the composition is 38°C to 50°C. [3] The composition according to [1] or [2], wherein the shear modulus at 37°C is 1000 Pa or more. [4] The composition according to any one of [1] to [3], wherein the shear modulus at 50°C is 200 Pa or less. [5] The composition according to any one of [1] to [4], wherein the concentration of gelatin in the composition is 5% to 20% by mass, and the concentration of alcohol in the composition is 2.5% to 10% by mass. [6] The composition according to [5], wherein the concentration of the alcohol in the composition is 5% by mass to 10% by mass. [7] The composition according to [5], wherein the concentration of gelatin in the composition is 10% to 20% by mass, and the concentration of alcohol in the composition is 5% to 10% by mass. [8] The composition according to any one of [1] to [7], wherein the ratio (A / G) of the concentration of alcohol in the composition to the concentration of gelatin in the composition (G) is 1 / 10 to 20 / 10. [9] The composition according to any one of [1] to [8], wherein the gelatin is derived from pig tendons.

[10] The composition according to any one of [1] to [9], wherein the weight-average molecular weight of the gelatin is 200,000 to 500,000.

[11] The composition according to any one of [1] to

[10] , wherein the alcohol is a polyalkylene glycol.

[12] The composition according to

[11] , wherein the polyalkylene glycol is polyethylene glycol.

[13] The composition according to

[11] or

[12] , wherein the weight-average molecular weight of the polyalkylene glycol is 6000 or more.

[14] The composition according to

[13] , wherein the weight-average molecular weight of the polyalkylene glycol is 6,000 to 40,000.

[15] The composition according to any one of [1] to

[14] , wherein the solvent is a buffer solution.

[16] A composition according to any one of [1] to

[15] , which is a hydrogel having a coacervate structure.

[17] An adhesive comprising any of the compositions described in [1] to

[16] . [Effects of the Invention]

[0011] The present invention provides a composition with excellent biocompatibility and tissue adhesion properties. This composition can be used, for example, as an adhesive (tissue adhesive). [Brief explanation of the drawing]

[0012] [Figure 1] This is a phase-contrast microscope image of the coacervate hydrogel (sample 1 prepared in the example). [Figure 2] These are confocal laser scanning microscope images of hydrogels (samples 2-1 to 2-12 prepared in the example) prepared by varying the concentrations of fluorescently modified porcine tendon-derived gelatin (TG) and polyethylene glycol (PEG). [Figure 3A] This figure shows the relationship between temperature and shear modulus in hydrogels with varying PEG concentrations (samples 3-1 to 3-5 prepared in the examples). [Figure 3B] This figure shows the sol-gel transition temperatures (gelation temperatures) of hydrogels with varying PEG concentrations (samples 3-1 to 3-5 prepared in the examples). [Figure 4A]Confocal laser scanning microscope photographs of hydrogels (samples 4-1 to 4-8 prepared in the examples) prepared using fluorescently modified TG and PEGs with different molecular weights. [Figure 4B] It is a diagram showing the relationship between the molecular weight of PEG and the shear storage modulus in the hydrogel (samples 4-1 to 4-8 prepared in the examples). [Figure 5] It is a diagram showing the evaluation results of the gelation rate of the coacervate hydrogel (sample 1) prepared in the example and TG without PEG addition (sample 3-4). [Figure 6] It is a diagram showing the evaluation results of the thixotropic properties of the coacervate hydrogel (sample 1) prepared in the example and TG without PEG addition (sample 3-4). [Figure 7] It is a diagram showing the results of the injectability test of the coacervate hydrogel (sample 1) prepared in the example. [Figure 8] It is a diagram showing the results of the tensile test of the coacervate hydrogel (sample 1) prepared in the example and TG without PEG addition (sample 3-4). [Figure 9] It is a diagram showing the results of the in-water stability test of the coacervate hydrogel (sample 1) prepared in the example, the coacervate hydrogel added with a degrading enzyme, and TG without PEG addition (sample 3-4). [Figure 10A] It is a diagram showing the results of the adhesion test at 50 °C of the coacervate hydrogel (sample 1) prepared in the example and TG without PEG addition (sample 3-4). [Figure 10B] It is a diagram showing the results of the adhesion test at 37 °C of the coacervate hydrogel (sample 1) prepared in the example and TG without PEG addition (sample 3-4). [Figure 11] It is a diagram showing the results of the cytotoxicity test of the coacervate hydrogel (sample 1) prepared in the example and TG without PEG addition (sample 3-4). [Figure 12] It is a diagram showing the biocompatibility and biodegradability of the coacervate hydrogel (sample 1) prepared in the example and TG without PEG addition (sample 3-4). [Figure 13] This figure shows the evaluation results of the adhesion prevention ability of the coacervate hydrogel (sample 1) prepared in the example. [Modes for carrying out the invention]

[0013] The present invention will be described in detail below. The following description of the constituent elements may be based on typical embodiments of the present invention, but the present invention is not limited to such embodiments.

[0014] In this specification, a numerical range represented by "~" means a range that includes the numbers written before and after "~" as the lower and upper limits, respectively.

[0015] Furthermore, in the notation of groups (atomic groups) in this specification, the notation that does not specify substituted or unsubstituted includes both substituted and unsubstituted groups, to the extent that it does not impair the effects of the present invention. For example, "alkyl group" includes not only unsubstituted alkyl groups but also substituted alkyl groups. This is also true for each compound.

[0016] [Composition] The composition of this embodiment comprises gelatin, alcohol, and a solvent containing water (hereinafter referred to as "aqueous solvent" as appropriate). The composition of this embodiment forms a non-fluid hydrogel at around human body temperature (around 37°C). More specifically, the composition of this embodiment is a coacervate hydrogel having a structure in which droplets containing alcohol and an aqueous solvent are dispersed in a gelatin matrix (a so-called coacervate structure).

[0017] The gelatin used in this embodiment is not particularly limited, but from the viewpoint of forming a stable coacervate hydrogel at around human body temperature (around 37°C), gelatin derived from animals such as cattle or pigs, which has a relatively high sol-gel transition temperature, is preferred, gelatin derived from pig skin or pig tendons is more preferred, and gelatin derived from pig tendons is particularly preferred. By using gelatin, a polymer of biological origin, as the main component, the composition of this embodiment can be made more biocompatible.

[0018] The gelation temperature (sol-gel transition temperature) of gelatin is preferably, for example, 33°C or higher, 37°C or higher, or 38°C or higher. The composition of this embodiment has a coacervate structure, which allows its gelation temperature to be higher than that of the raw material gelatin. If the gelation temperature of the raw material gelatin is above the above temperature, the composition of this embodiment can obtain a sufficiently high gelation temperature, and as a result, a more stable and stronger coacervate hydrogel can be formed at around human body temperature. The upper limit of the gelation temperature of gelatin is not particularly limited, but for example, it is 50°C or lower.

[0019] The molecular weight of gelatin is not particularly limited, but for example, the weight-average molecular weight may be 100,000 to 500,000, 200,000 to 500,000, or 250,000 to 400,000. If the weight-average molecular weight of gelatin is within the above range, the composition of this embodiment can form a more stable and stronger coacervate hydrogel.

[0020] The alcohol used in this embodiment is not particularly limited and can be appropriately selected within the range that achieves the effects of this embodiment. When the composition of this embodiment is used in a living organism, it is preferable that the alcohol is non-toxic or has extremely low toxicity. Furthermore, from the viewpoint of forming a more stable and strong coacervate hydrogel, it is preferable that it is soluble in an aqueous solvent (water-soluble).

[0021] Examples of alcohols used in this embodiment include polyalkylene glycol (linear, branched, and functionally modified (amino, thiol, and carboxyl groups)), polyglycidol, and ethanol. From the viewpoint of forming a coacervate hydrogel that is less toxic to living organisms and is more stable and stronger, polyalkylene glycol (with, for example, 2 to 4 carbon atoms in the alkylene group in the repeating unit) is preferred as the alcohol used in this embodiment, and polyethylene glycol is more preferred.

[0022] The molecular weight of the polyalkylene glycol is not particularly limited, but it is preferable that the weight-average molecular weight is 6,000 or more, or 6,000 to 40,000. When the molecular weight of the polyalkylene glycol is within the above range, the composition of this example can form a more stable and stronger coacervate hydrogel.

[0023] The alcohol in this embodiment may be a single type of alcohol or a mixture of multiple types of alcohols.

[0024] The aqueous solvent used in this embodiment is not particularly limited as long as its main component is water, but for example, ultrapure water, physiological saline, boric acid, phosphoric acid, carbonic acid, and various inorganic salt buffer solutions (buffer solutions) or mixtures thereof can be used.

[0025] The concentrations of each component in the composition of this embodiment are not particularly limited, as long as the composition can form a coacervate hydrogel at around human body temperature (around 37°C). For example, the gelatin concentration in the composition may be 5% to 20% by mass, and the alcohol concentration may be 2.0% to 10% by mass, 2.5% to 10% by mass, or 5% to 10% by mass. Alternatively, the gelatin concentration in the composition may be 10% to 20% by mass, and the alcohol concentration may be 5% to 10% by mass. If the gelatin concentration and / or alcohol concentration in the composition are within the above ranges, the composition is more likely to form a more stable and stronger coacervate hydrogel.

[0026] Furthermore, while the ratio of gelatin to alcohol in the composition is not particularly limited, from the viewpoint of forming a more stable and stronger coacervate hydrogel, the ratio of the alcohol concentration (A) to the gelatin concentration (G) in the composition (A / G) may be 1 / 10 to 20 / 10, 2 / 10 to 20 / 10, or 2 / 10 to 10 / 10.

[0027] The amount of water in the composition is not particularly limited. Depending on the application, it may be appropriately adjusted to a range of, for example, 80 to 99% by mass of the composition.

[0028] The composition of this embodiment may consist only of gelatin, alcohol, and an aqueous solvent, or it may contain general-purpose additives to the extent that it achieves the effects of this embodiment. When the composition of this embodiment is used in a living organism, it may contain pharmaceutically acceptable known additives. Examples of such additives include, for example, blood coagulation factor XIII, trypsin inhibitors (such as aprotinin), albumin, collagen, polyglycolic acid (PGA), isoleucine, glycine, arginine, glutamic acid, surfactants, pH adjusters, sodium chloride, calcium chloride, sugar alcohols (such as glycerol and mannitol), and sodium citrate. The concentration of additives in the composition may be, for example, 5% by mass or less, 1% by mass or less, or 0% by mass.

[0029] The gelation temperature (sol-gel transition temperature) of the composition of this embodiment is preferably, for example, 38°C to 50°C, or 44°C to 48°C. Furthermore, the shear modulus (storage modulus G') of the composition at 37°C is preferably, for example, 1000 Pa or higher. Because the composition of this embodiment has a coacervate structure, its gelation temperature is higher than that of raw material gelatin, and it can become a more stable and stronger gel at around human body temperature. This property makes it excellent as a bioadhesive, and the composition of this embodiment can be applied as a tissue adhesive. The upper limit of the shear modulus (storage modulus G') of the composition at 37°C is not particularly limited, but is, for example, 20,000 Pa or less.

[0030] Furthermore, the shear modulus (storage modulus G') of the composition of this embodiment at 50°C is preferably, for example, 200 Pa or less. If the composition of this embodiment is in a sol state at temperatures higher than human body temperature and has a sufficiently low shear modulus, it can be used as a temperature-responsive tissue adhesive that is applied to a living body in a sol state above body temperature and then gelled at body temperature after application. The lower limit of the shear modulus (storage modulus G') of the composition at 50°C is not particularly limited, but for example, it is 2 Pa or more.

[0031] The composition of this embodiment can be produced by mixing gelatin, alcohol, an aqueous solvent (a solvent containing water), and additives as needed, using a general method.

[0032] As described above, the inventors have found that the composition of this embodiment becomes a coacervate hydrogel at around human body temperature (e.g., 37°C), and that this coacervate structure increases the gel's strength and its adhesive strength to tissues. Furthermore, the inventors have found that coacervation improves the gelation rate, mechanical strength, and water stability of the composition of this embodiment, as well as enabling injection with less force (thixotropic properties) and improving adhesion prevention when used in vivo. The composition of this embodiment, possessing these properties, can be used in various applications described below, such as adhesives (tissue adhesives for biological use).

[0033] [Uses of the composition] <Tissue adhesive> The tissue adhesive of this embodiment comprises the composition of this embodiment described above. The tissue adhesive of this embodiment is temperature-responsive and has excellent handling properties, biocompatibility, and tissue adhesion.

[0034] The tissue adhesive of this embodiment is in a sol state in the range of approximately 40°C to 60°C, and becomes a gel state when left to stand for 5 to 30 minutes at 37°C or below (e.g., 10°C to 37°C). In other words, the tissue adhesive of this embodiment is temperature-responsive. Therefore, the tissue adhesive of this embodiment can be applied to tissue defect sites using a spray device or syringe, for example, by heating it to a sol state at 40°C to 60°C. Furthermore, after application of the tissue adhesive of the invention, a hydrogel is formed by contact with body temperature of approximately 37°C, thereby physically filling the defect in the affected area and promoting tissue regeneration. Other applications (functions) of the tissue adhesive include hemostasis, wound closure, adhesion prevention, pancreatic juice leakage prevention, pressure ulcer treatment, muscle tissue regeneration, and wound healing in the gastrointestinal mucosa. The means for heating the tissue adhesive are not particularly limited, and any known means can be suitably employed.

[0035] Thus, the tissue adhesive of this embodiment can exhibit good viscosity and tissue adhesion by adjusting the temperature, and since it can be used as a one-component type, it has excellent handling properties.

[0036] Furthermore, the adhesive of this embodiment has thixotropic properties. Thixotropic properties are characteristics in which the viscoelasticity of a material decreases when stress is applied, and gradually recovers when the application is stopped. Due to the thixotropic properties, the adhesive of this embodiment can be easily injected with a syringe even in a gel state, and can be applied to tissue while still in a gel state, that is, without heating it to a sol state.

[0037] Furthermore, the tissue adhesive of this embodiment can also be compounded with a decellularized matrix, which is the matrix structure of biological tissue. By compounding with the extracellular matrix components that remain after removing cellular components from the organs and tissues of animals such as pigs and cows, it is possible to impart biological function to the tissue adhesive. As the decellularized matrix, decellularized matrices prepared from organs such as the bladder, heart, liver, pancreas, and small intestine can be used.

[0038] Furthermore, a bio-tissue adhesive kit is also provided, which includes the tissue adhesive of this embodiment as described above. In addition to the tissue adhesive of this embodiment, the bio-tissue adhesive kit may also include injection devices such as spray devices and syringes, packaging containers, and the like.

[0039] <Other uses> The composition of this embodiment can be used to create a drug delivery carrier (such as a local delivery carrier or a sustained-release delivery carrier) by incorporating various drugs to be delivered (including proteins, etc.). In other words, the drug delivery carrier of this embodiment comprises a hydrogel (the composition described above) and various desired drugs.

[0040] The drugs are not particularly limited, but examples include anticancer drugs, anti-inflammatory drugs, antithrombotic drugs, antibiotics, biological agents, or growth factors such as fibroblast growth factor, vascular endothelial growth factor, and hepatocyte growth factor. Furthermore, the drug delivery carrier can also be used as a vaccine carrier by carrying viral or cancer antigen proteins.

[0041] Furthermore, in the field of regenerative medicine, the composition of this embodiment, when heated to 37 degrees Celsius, can be mixed with a cell suspension and used as a scaffold material for cell transplantation. [Examples]

[0042] The present invention will be described in more detail below based on examples. The materials, amounts used, proportions, processing content, and processing procedures shown in the following examples can be modified as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be interpreted as being limited by the examples shown below.

[0043] 1. Microscopic observation of coacervate hydrogels [Sample 1] Gelatin derived from pig tendons (TG: Tendon gelatin, purchased from Nitta Gelatin) was dissolved in phosphate buffer (PBS: Phosphate buffered saline, pH=7.4) at 50°C. A predetermined amount of polyethylene glycol (PEG: Polyethylene glycol, weight-average molecular weight: 10,000) was added to the resulting aqueous TG solution and mixed, and the mixture was allowed to stand at 50°C for 5 to 30 minutes (final concentration TG: 20% by mass, PEG: 5% by mass). Subsequently, the mixture was allowed to stand at 25 to 37°C for 30 minutes to obtain a hydrogel (composition).

[0044] <Rating> The obtained sample 1 was observed using a phase-contrast microscope. As shown in Figure 1, a coacervate structure was confirmed in which droplets containing PEG and solvent (phosphate buffer) were dispersed in a gelatin-containing matrix. In sample 1, it is presumed that the addition of PEG to the TG aqueous solution dehydrated the TG, inducing liquid-liquid phase separation.

[0045] 2. Effects of TG and PEG concentrations in coacervation Samples (hydrogels) were prepared by varying the concentrations of PEG and fluorescently modified TG, and then observed under a microscope.

[0046] [Sample 2-1] TG was dissolved in dimethyl sulfoxide, and fluorescein isothiocyanate (FITC) was added and the reaction was carried out. After reprecipitation with ethanol, it was dissolved again in water at 10 mg / mL and dialyzed using a dialysis membrane (molecular weight fraction: 10 kDa). Subsequently, FITC-modified TG was obtained by lyophilization. FITC-modified TG was dissolved in PBS (pH=7.4) at 50°C. A predetermined amount of PEG (weight-average molecular weight: 10,000) was added to the solution and mixed, and the mixture was allowed to stand at 50°C for 5 to 30 minutes (final concentration FITC-modified TG: 2.5% by mass, PEG: 2.5% by mass). Subsequently, sample 2-1 (hydrogel) was obtained by allowing the mixture to stand at 25 to 37°C for 30 minutes.

[0047] [Samples 2-2 to 2-4] Each sample was prepared in the same manner as sample 2-1, except that the final concentration of PEG was set to 2.5% by mass, and the final concentrations of FITC-modified TG were set to 5%, 10%, and 20% by mass, respectively.

[0048] [Samples 2-5 to 2-8] Each sample was prepared in the same manner as Sample 2-1, except that the final concentration of PEG was set to 5% by mass, and the final concentrations of FITC-modified TG were set to 2.5%, 5%, 10%, and 20% by mass, respectively. The composition of Sample 2-8 is the same as that of Sample 1 described above.

[0049] [Samples 2-9 to 2-12] Each sample was prepared in the same manner as sample 2-1, except that the final concentration of PEG was set to 10% by mass, and the final concentrations of FITC-modified TG were set to 2.5%, 5%, 10%, and 20% by mass, respectively.

[0050] <Rating> The obtained samples 2-1 to 2-12 were observed using a confocal laser scanning microscope for fluorescence. Figure 2 shows the images in grayscale. Regions containing fluorescently modified TG are shown in lighter colors (gray), and regions without TG are shown in darker colors (black). In samples 2-3 to 2-4, 2-6 to 2-8, and 2-10 to 2-12, a coacervate structure was confirmed in which droplets containing PEG and solvent (phosphate buffer) were dispersed within the gelatin matrix.

[0051] On the other hand, in samples 2-1 to 2-2, no liquid-liquid phase separation occurred, and a coacervate structure could not be confirmed. In addition, in samples 2-5 and 2-9, coacervates in which gelatin-containing droplets were dispersed in PEG and solvent were confirmed.

[0052] From the above results, the following can be inferred: A stable coacervate hydrogel is easily formed when the gelatin concentration in the composition is 5% to 20% by mass and the alcohol concentration is 2.5% to 10% by mass. Furthermore, a more stable coacervate hydrogel is easily formed when the gelatin concentration in the composition is 5% to 20% by mass and the alcohol concentration is 5% to 10% by mass, and even more so when the gelatin concentration in the composition is 10% to 20% by mass and the alcohol concentration is 5% to 10% by mass.

[0053] 3. Viscoelasticity Measurement The viscoelasticity of the sample (hydrogel) was measured by varying the concentration of PEG.

[0054] [Samples 3-1 to 3-3] Samples 3-1 to 3-3 were prepared in the same manner as Sample 1, except that the final concentration of TG was set to 20% by mass, and the final concentrations of PEG were set to 2%, 5%, and 10% by mass, respectively. The composition of Sample 3-2 is the same as that of Sample 1 described above.

[0055] [Sample 3-4] Samples 3 and 4 were prepared in the same manner as Sample 1, except that the final concentration of TG was set to 20% by mass and PEG was not added (i.e., the final concentration of PEG was 0% by mass).

[0056] [Samples 3-5] Samples 3-5 were prepared in the same manner as Sample 1, except that gelatin derived from pig skin (SG: Skin gelatin, purchased from Nitta Gelatin) was used instead of gelatin derived from pig tendons, and PEG was not added (i.e., final concentration of PEG: 0% by mass).

[0057] <Rating> The following evaluations were performed on each of the prepared samples 3-1 to 3-5. A sample (100 μL) heated to 50°C was placed on the stage of a rheometer (viscoelasticity measuring device, Anton Paar, Rheoplus®) and clamped with a 10 mm diameter jig. During the measurement, the temperature of the stage was changed from 20°C to 50°C, and the relationship between temperature and shear modulus (storage modulus G' and loss modulus G") was evaluated. Figure 3A shows the relationship between temperature and shear modulus (storage modulus G') for each sample, and Figure 3B shows the gelation temperature (sol-gel transition temperature) for each sample. The gelation temperature shown in Figure 3B is defined as the temperature at which the relative magnitudes of the storage modulus G' and the loss modulus G'' are reversed for each sample (the intersection point of the storage modulus G' curve and the loss modulus G'' curve on a graph with temperature on the x-axis and shear modulus on the y-axis).

[0058] As shown in Figure 3A, the shear modulus of all samples decreased with increasing temperature. When comparing porcine tendon gelatin (TG, sample 4) and porcine skin gelatin (SG, sample 5) at around human body temperature (37°C), the shear modulus of porcine tendon gelatin (TG) was considerably higher than that of porcine skin gelatin (SG). This is because, as shown in Figure 3B, the gelation temperature of porcine tendon gelatin (TG) is high, above 38°C, and it is in a gel state at around 37°C, while the gelation temperature of porcine skin gelatin (SG) is low, at 32°C, and it is in a sol state at around 37°C.

[0059] In samples 3-1 to 3-3, coacervates prepared by adding PEG to porcine tendon gelatin (TG) showed higher gelation temperatures and higher shear moduli than porcine tendon gelatin (SG). Furthermore, the higher the PEG concentration, the higher the shear moduli and gelation temperature tended to be. In samples 3-2 to 3-3 (PEG: 5-10 mass%), the shear moduli at 37°C was over 1000 Pa, and the gelation temperature was 44-48°C.

[0060] Thus, it was found that adding alcohol such as PEG to gelatin to form a coacervate structure increases the gelation temperature, resulting in a highly stable gel state at around human body temperature. Based on these results, although experimental data are not shown here, it is presumed that if gelatin derived from pig skin is used as the gelatin, the gelation temperature will also increase through coacervation, and a composition that forms a stable coacervate gel at 37°C can be obtained.

[0061] Furthermore, at 50°C, the shear modulus of the samples with PEG concentrations of 2-10% by mass (samples 3-1 to 3-3) was 200 Pa or less, indicating a sol state. From these results, it was confirmed that samples 3-1 to 3-3 can be used as temperature-responsive tissue adhesives, applied to living tissue in a sol state above body temperature and then gelling at body temperature after application.

[0062] 4. The effect of PEG molecular weight on coacervation Samples (hydrogels) were prepared using fluorescently modified TG (FITC-modified TG) and PEG with different molecular weights, and then observed under a microscope.

[0063] [Sample 4-1] Fluorescently modified TG (FITC-modified TG) was synthesized using the same method as described for sample 2-1 above, and the synthesized FITC-modified TG was dissolved in PBS (pH=7.4) at 50°C. A predetermined amount of PEG with a weight-average molecular weight of 400 was added to the solution and mixed, and the mixture was allowed to stand at 50°C for 5 to 30 minutes (final concentration TG: 20% by mass, PEG: 5% by mass). Subsequently, a hydrogel was obtained by allowing the mixture to stand at 25 to 37°C for 30 minutes.

[0064] [Samples 4-2 to 4-7] Samples 4-2 to 4-7 were prepared in the same manner as sample 4-1, except that PEG with weight-average molecular weights of 1,500, 6,000, 10,000, 40,000, 100,000, and 300,000 was used instead of PEG with a weight-average molecular weight of 400.

[0065] [Samples 4-8] Sample 4-8 was prepared in the same manner as Sample 4-1, except that PEG was not added (final concentration of PEG: 0% by mass). Sample 4-8 is shown as Control in Figures 4A and 4B.

[0066] <Rating> The obtained samples 4-1 to 4-8 were observed using a confocal laser scanning microscope for fluorescence. Figure 4A shows a grayscale representation of the results. In Figure 4A, regions containing fluorescently modified TG are shown in lighter colors (gray), and regions without TG are shown in darker colors (black). As shown in Figure 4A, in samples with a weight-average molecular weight of 6,000 or more (samples 4-3 to 4-7), a coacervate structure was confirmed in which droplets containing PEG and a solvent (phosphate buffer) were dispersed in the gelatin matrix.

[0067] On the other hand, in samples with a weight-average molecular weight of 1,500 or less, and in the Control samples (samples 4-1 to 4-2 and 4-8) that did not contain PEG, liquid-liquid phase separation did not occur, and the coacervate structure could not be confirmed.

[0068] Furthermore, the following evaluations were performed on samples 4-1 to 4-8. A sample (100 μL) heated to 50°C was placed on the stage of a rheometer (viscoelasticity measuring device, Anton Paar, Rheoplus®) and clamped with a 10 mm diameter jig. The stage temperature was switched to 37°C, and the shear modulus (storage modulus G') at 37°C was measured. The results are shown in Figure 4B. Samples with a weight-average molecular weight of 6,000 or more in which a coacervate structure was confirmed to have a higher shear storage modulus and be stronger, more stable gels than the Control (TG without PEG). In particular, higher shear storage moduli were observed in the weight-average molecular weight range of 6,000 to 40,000, and the maximum value was shown when using PEG with a weight-average molecular weight of 10,000, with a shear storage modulus of 1.7 kPa. Furthermore, when using PEG with a weight-average molecular weight exceeding 10,000, the slight decrease in shear storage modulus is presumed to be due to a decrease in the solubility of PEG, which causes some aggregation.

[0069] 5. Gelation rate of coacervate hydrogels The gelation rates of Sample 1 (final concentration TG: 20% by mass, weight-average molecular weight: 10,000 PEG: 5% by mass) and Samples 3-4 (TG without added PEG) were evaluated using the method described below.

[0070] Each sample (100 μL) heated to 50°C was placed on the stage of a rheometer (viscoelasticity measuring device, Anton Paar, Rheoplus®) and clamped with a 10 mm diameter jig. First, measurement of each sample was started at 50°C, and 3 seconds after the start of measurement, the temperature was switched to 37°C and the measurement continued. The relationship between the measurement time and the shear modulus (storage modulus G' and loss modulus G") was evaluated. The results are shown in Figure 5. On the horizontal axis of Figure 5, "0 minutes" represents the time when the measurement temperature was switched to 37°C.

[0071] Sample 1 (coacervate hydrogel), heated to 50°C immediately after the start of measurement, had a G' value lower than G'' and was in a sol state. However, as soon as the temperature was switched to 37°C, G' increased, exceeding G'' after 2 minutes, indicating gelation. On the other hand, samples 3-4 (TG without PEG) required 20 minutes to gel. From these results, it is presumed that in coacervate hydrogel, the gelation rate improved because TG was dehydrated by PEG during coacervation and concentrated in the TG matrix.

[0072] 6. Thixotropic properties The thixotropic properties of Sample 1 (final concentration TG: 20% by mass, weight-average molecular weight: 10,000 PEG: 5% by mass) and Samples 3-4 (TG without added PEG) were evaluated using the method described below.

[0073] A sample (100 μL) heated to 50°C was placed on the stage of a rheometer (viscoelasticity measuring device, Anton Paar, Rheoplus®) and clamped with a 10 mm diameter jig. Under measurement conditions of 37°C, the shear modulus (storage modulus G' and loss modulus G") was measured while changing the strain to 1% and 300% every 5 minutes. The results are shown in Figure 6.

[0074] As shown in Figure 6, the gel was initially destroyed by a 300% strain, causing a decrease in shear modulus. However, when the strain was reduced to 1%, the shear modulus recovered. Thus, it was confirmed that both samples 1 and 3-4 exhibited thixotropic properties. In each sample, the gel was formed using hydrogen bonding, a reversible physical interaction, as the driving force. Therefore, it is presumed that the shear modulus recovered because the broken bonds were reformed.

[0075] Furthermore, compared to samples 3-4 (TG without PEG), sample 1, which is a coacervate hydrogel, exhibits a lower shear modulus when strain is applied, which is expected to make syringe injection easier.

[0076] 7. Injectability Test The injectability of Sample 1 (final concentration TG: 20% by mass, weight-average molecular weight: 10,000 PEG: 5% by mass) was evaluated by the method described below. Three types of syringes equipped with subcutaneous injection needles of needle gauges 23G, 25G, and 27G were prepared, and their weights were measured in advance. 1 mL of Sample 1 (gel) was filled into each syringe, the weight was measured again, and the syringes were left standing at 45°C for 1 hour. After that, the syringes were fixed to a measuring stand, and the gel was injected by compressing the syringes using a tensile testing machine (Shimadzu Corporation) (maximum stress setting: 50N, 100 mm / min). The weight of the syringes after measurement was measured, and the weight of the injected gel was calculated. The results are shown in Figure 7.

[0077] As shown in Figure 7, Sample 1 exhibited a high injectability of 80% with syringes of all needle gauges, and the stress required for injection was 20N. A stress of around 20N is sufficient for injection by hand. This indicates that the gel can be injected with a syringe by heating and can be applied as a tissue adhesive.

[0078] 8. Measurement of mechanical strength by tensile testing Sample 1 (final concentration TG: 20% by mass, weight-average molecular weight: 10,000 PEG: 5% by mass) and Samples 3-4 (TG without added PEG) were heated to 50°C and poured into ISO 37-2 size silicone molds, respectively, and gelled at 25°C for 60 minutes. Subsequently, tensile tests of the gels were performed at 25°C using a texture analyzer. The results are shown in Figure 8.

[0079] As shown in Figure 8, the coacervate hydrogel (TG-PEG, sample 1) showed a higher elongation rate compared to TG (samples 3-4) (TG: 224%, TG-PEG: 353%). Furthermore, the coacervate hydrogel (TG-PEG) showed a higher breaking strength compared to TG (samples 3-4) (TG: 0.15 MPa, TG-PEG: 0.40 MPa), indicating that the mechanical strength of the gel was improved by intermolecular hydrogen bonding.

[0080] 9. Underwater stability 100 μL of sample 1 (final concentration TG: 20% by mass, weight-average molecular weight: 10,000 PEG: 5% by mass) and samples 3-4 (TG without added PEG) were each injected into 2 mL tubes and gelled at 25 °C for 60 minutes. 1 mL of PBS was then added to the tubes and incubated at 37 °C for a specified time. The supernatant was then removed, and the remaining gel was freeze-dried and its weight measured. The results are shown in Figure 9.

[0081] As shown in Figure 9, the coacervate hydrogel (TG-PEG, sample 1) exhibited a slower rate of weight loss and higher water stability under physiological conditions compared to TG (samples 3-4).

[0082] Furthermore, the same test was performed by adding 1 mL of 1 mg / mL collagenase solution to the gel of sample 1 instead of PBS. The combined results are shown in Figure 9. Sample 1 (TG-PEG collagenase) with added collagenase solution gradually decreased in weight and almost completely disappeared after 24 hours. From these results, it was confirmed that the coacervate hydrogel of sample 1 is a biodegradable material that is broken down by enzymes in tissues in vivo.

[0083] 10. Adhesion Test Adhesion tests were conducted using pig large intestines (Shibaura Organs). The test method followed the standard of the American Society for Testing and Materials (ASTM F-2258-05). First, the pig large intestine was opened and washed with physiological saline. The obtained tissue was cut into 2.5 cm square tissue pieces and fixed to the upper and lower fixtures of the test apparatus using cyanoacrylate adhesive. At this time, the inner side of the colon tissue was adhered to the fixture, and the outer side of the tissue was placed in contact with the other side. The temperature of the pig large intestine tissue during measurement was maintained at 37°C using a hot plate.

[0084] Next, 300 μL of sample 1 (final concentration TG: 20% by mass, weight-average molecular weight: 10,000 PEG: 5% by mass) and samples 3-4 (TG without added PEG), heated to 50°C, were applied to the tissue, respectively. Immediately afterward, the samples were pressed together at 2N using the upper jig, and after being pressed for 3 minutes, the adhesive strength was measured by pulling upwards. The results are shown in Figure 10A. The same test was also performed on tissue without any gel or other additives, and this was used as the control. As shown in Figure 10A, samples 1 and 3-4 showed higher adhesive strength compared to the control.

[0085] Next, a similar experiment was performed by varying the temperature of the adhesive. First, the gel, heated to 50°C, was applied to the tissue, and then the gel temperature was lowered to 37°C by incubation at 37°C for 10 minutes. Then, the tissue was pressed with a 2N pressure using the upper jig, and after being pressed for 3 minutes, the adhesive strength was measured by pulling it upwards. The results are shown in Figure 10B. Samples 3-4 (TG) without PEG added showed relatively high tissue adhesion strength, while sample 1 (TG-PEG) showed low adhesion strength. This is thought to be because a stable gel layer was formed at 37°C in sample 1, which has a higher gelation temperature.

[0086] The results shown in Figures 10A and 10B clearly demonstrate that sample 1 (TG-PEG) exhibits high tissue adhesion at high temperatures (50°C), but once the temperature drops to near body temperature (37°C) and a gel forms, it does not adhere to other tissues. From this, it was confirmed that the coacervate hydrogel of sample 1 can be used as a temperature-responsive tissue adhesive with adhesion prevention capabilities.

[0087] 11. Cytotoxicity Test 100 μL of sample 1 (final concentration TG: 20% by mass, weight-average molecular weight: 10,000 PEG: 5% by mass) and samples 3-4 (TG without PEG) were warmed to 50°C and injected into 2 mL tubes, then gelled at 25°C for 60 minutes. 1 mL of RPMI1640 medium (10% fetal bovine serum, 1% penicillin streptomycin) was added, and after incubation at 37°C for a specified time, the supernatant was collected. Toxicity was evaluated using mouse fibroblasts (L929 cells) as evaluation cells. L929 cells were cultured in RPMI1640 medium (10% fetal bovine serum, 1% penicillin streptomycin) at 37°C in a 5% CO2 incubator. 2 x 10 4L929 cells were seeded in a 96-well plate and pre-cultured for 24 hours. The collected supernatant was added to each well and cultured for another 24 hours. After the culture was complete, the cell count was quantified using a cell counting kit (WST-8, DOJINDO). The results (cell viability) are shown in Figure 11. Both samples 3-4 and sample 1 showed high cell viability. These results confirmed that the coacervate hydrogel (sample 1) exhibited high cell compatibility.

[0088] 12. Biocompatibility and biodegradability testing The biocompatibility and biodegradability of the sample (hydrogel) were evaluated by implanting it into C57BL / 6J mice (6-8 weeks old, female).

[0089] First, 100 μL of sample 1 (final concentration TG: 20% by mass, weight-average molecular weight: 10,000 PEG: 5% by mass) and samples 3-4 (TG without added PEG) were heated to 50°C and placed in 1 mm thick silicone molds, respectively, and gelled at 25°C for 60 minutes. The resulting gels were sterilized by UV irradiation. Under isoflurane inhalation anesthesia, the hair on the backs of mice was shaved, disinfected with 70% ethanol, and then the skin was incised with a scalpel and each sample was implanted subcutaneously. The mice were euthanized after 1, 3, and 7 days, the materials and tissues were excised, and tissue sections were observed.

[0090] As shown in Figure 12, hematoxylin-eosin observation revealed that no gel residue was observed in samples 3-4 (TG) after 1 day, indicating degradation. On the other hand, sample 1 (TG-PEG) remained subcutaneously even after 1 day, and was found to have degraded and been absorbed after 3 days. These results confirm that sample 1's stability in the biological environment is improved and it is compatible with the body through coacervation. Furthermore, it was confirmed that sample 1 is biodegradable and ultimately degraded and absorbed.

[0091] 13. Evaluation of adhesion prevention ability using a rat peritoneal adhesion model. To verify the adhesion prevention ability, a rat peritoneal adhesion model was used. The abdominal hair of Sprague-Dawley rats (SD rats) was shaved, disinfected with 70% ethanol, and then a 5 cm laparotomy was performed. The cecum was exposed, an abrasion was created with gauze, and it was returned to its original position. The surface tissue of the peritoneum of the abdominal wall adjacent to the cecum (1 cm x 2 cm) was dissected and excised with a scalpel. 500 μL of sample 1 (final concentration TG: 20% by mass, weight-average molecular weight: 10,000 PEG: 5% by mass), warmed to 45°C, was applied to the peritoneal tissue and the surface of the cecum. After standing for about 5 minutes to confirm gelation, the cecum was returned to the abdominal cavity, and an antibiotic (amikamicin, 1 mg / kg) was added. The muscle layer was sutured, the skin wound was closed with an autoclip, and disinfection was performed.

[0092] On day 14, the abdomen was opened, and the presence or absence of adhesions was visually checked and scored according to the following criteria. A rat peritoneal adhesion model in which no treatment was given to abrasions was also tested and observed in the same manner (Control). The results are shown in Figure 13.

[0093] <Criteria for evaluating collusion> 0: No collusion 1: Weak adhesion (bonding between tissues at one point) 2: Moderate adhesion (adhesion at multiple points between tissues) 3: Widespread adhesion (partial adhesion between tissue surfaces) 4. Serious collusion (complete collusion between organizations)

[0094] As shown in Figure 13, strong adhesions were observed between the peritoneum and cecum in untreated rats (Control). On the other hand, no adhesions were observed in rats treated with sample 1, and re-epithelialization of mesothelial cells occurred on the surface of the damaged peritoneal and cecal tissues, confirming tissue regeneration. These results confirm that sample 1 has an adhesion-preventing function. [Industrial applicability]

[0095] The compositions of the present invention are useful as biomaterials such as tissue adhesives and medical materials. In particular, the tissue adhesives exhibit excellent handling properties, biocompatibility, and tissue adhesion.

Claims

1. A composition, It contains gelatin, alcohol, and an aqueous solvent. A droplet containing the alcohol and the aqueous solvent is dispersed in the gelatin matrix. The gelatin has a sol-gel transition temperature of 33°C to 50°C and a weight-average molecular weight of 200,000 to 500,000. The alcohol is polyethylene glycol having a weight-average molecular weight of 6,000 to 40,000. A composition wherein the concentration of gelatin in the composition is 5% to 20% by mass, and the concentration of alcohol is 2.5% to 10% by mass.

2. The composition according to claim 1, wherein the sol-gel transition temperature of the composition is 38°C to 50°C.

3. The composition according to claim 1, wherein the shear modulus at 37°C is 1000 Pa or more.

4. The composition according to claim 1, wherein the shear modulus at 50°C is 200 Pa or less.

5. The composition according to claim 1, wherein the weight-average molecular weight of the polyethylene glycol is 6,000 to 10,000.

6. The composition according to claim 1, wherein the concentration of the alcohol in the composition is 5% by mass to 10% by mass.

7. The concentration of gelatin in the composition is 10% by mass to 20% by mass. The composition according to claim 1, wherein the concentration of the alcohol in the composition is 5% by mass to 10% by mass.

8. The composition according to claim 1, wherein the ratio (A / G) of the concentration of alcohol (A) in the composition to the concentration of gelatin (G) in the composition is 2 / 10 to 20 / 10.

9. The composition according to claim 1, wherein the gelatin is derived from pig tendons.

10. The composition according to claim 1, wherein the aqueous solvent is at least one selected from the group consisting of water, physiological saline, and buffer solution.

11. The composition according to claim 1, wherein the aqueous solvent is a buffer solution.

12. The composition according to claim 1, which is a hydrogel having a coacervate structure.

13. An adhesive comprising the composition according to any one of claims 1 to 12.

Images