Tissue regeneration-promoting drug delivery composition and drug delivery kit containing the same

Abstract

The present invention relates to a hydrogel-based drug delivery system that can deliver a required amount of drug to a desired location more efficiently over a long period of time. The system comprises an amphiphilic block copolymer comprising a mixture of a first poloxamer and a second poloxamer with different molecular weights, and a protein extract (VdECM) containing elastin and collagen. The tissue regeneration-promoting drug delivery composition of the present invention is mixed with a drug and administered to the body, thereby increasing the drug release time in the body. It has phase-change properties, being in a liquid sol phase at room temperature but becoming a gel at body temperature, making it easy to inject as an injection and applicable to various body sites. It also contains a protein extract (VdECM) containing elastin, which is involved in the regeneration process of damaged tissue, and is therefore effective in rapidly repairing damaged tissue.

Description

[Technical Field]

[0001] The present invention relates to a hydrogel-based drug delivery system that can more efficiently deliver a required amount of drug to a desired location over a long period of time, and to a composition for drug delivery with excellent sustained release properties that is mixed with a drug and administered into the body to increase the release time of the drug in the body, and a drug delivery kit containing the same.

[0002] The drug delivery composition according to the present invention is easy to handle in a sol state at room temperature, but undergoes a phase transition to a gel state at temperatures such as body temperature and has the property of gradually decomposing in the body. This allows the drug mixed with it to be released continuously over a long period of time, thereby improving the therapeutic effect of the drug.

[0003] The present invention also relates to a tissue regeneration-promoting drug delivery composition and a drug delivery kit comprising the same, which can promote tissue regeneration by further containing a protein extract (VdECM) containing elastin and collagen obtained through a multi-stage decellularization process. [Background technology]

[0004] As we move into an aging society, there is a demand for effective treatment methods for various diseases. As the aging population and the prevalence of chronic diseases increase, the market related to drug delivery, which can efficiently deliver the required amount of drug to the desired location while minimizing side effects, is growing rapidly.

[0005] In particular, various products are commercially available as drug delivery carriers or drug delivery kits that can supply local anesthetics into the body for the purpose of pain control. However, these products have the advantage of being absorbed into the body so quickly that they can provide a rapid effect, but at the same time, they have the disadvantage that, due to their extremely fast absorption rate, they are unlikely to provide a long-term, sustained pain-reducing effect.

[0006] To overcome the problems associated with local anesthetics for pain management, various types of drug delivery systems have been commercialized.

[0007] For example, after surgery, a catheter with multiple micropores is inserted into the surgical site or the surrounding nerve tissue, and an elastic pump is then connected to continuously administer a local anesthetic. However, since the drug injection catheter is inserted into the body, pain can occur, the catheter holes can become clogged, and the catheter must be removed after use. This method also has drawbacks such as restricting the patient's freedom of movement.

[0008] As an alternative to these mechanical devices, there is an implant method that has been developed in the form of a mixture of bioabsorbable collagen sponge and an amide local anesthetic (bupivacaine HCl) and is mainly used in inguinal hernia surgery. The use of a bioabsorbable material has the advantage of eliminating the need for a further removal process, but because it is a sponge-shaped product with a fixed size and shape, it is difficult to use over a wider area than the product's size allows, and it must be cut to fit the size of the application area. It also has the disadvantage of being difficult to insert into the body through narrow administration channels.

[0009] In recent years, a drug delivery system for pain control has been commercialized that encapsulates a local anesthetic (bupicacaine HCl) in liposomes and gradually releases the local anesthetic over approximately 72 hours. The liposomes used in this drug delivery system are small, spherical lipid structures that gradually decompose and are absorbed into the body while retaining the local anesthetic. However, they must be stored refrigerated (2-8°C), making them vulnerable to long-term storage. Furthermore, the stability of liposomes remains poor, making it difficult to achieve stable drug release in the body over long periods of time. Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention has been made to effectively solve the problems of the conventional techniques as described above, and an object of the present invention is to provide a novel drug delivery composition and a drug delivery kit containing the same, which can be easily supplied to the body in a liquid phase and then form a stable drug delivery structure through a phase change caused by body temperature, thereby efficiently releasing drug components in the body over a long period of time, and can be decomposed and absorbed in the body, thereby promoting tissue regeneration. [Means for solving the problem]

[0011] A tissue regeneration-promoting drug delivery composition according to one embodiment of the present invention comprises an amphiphilic block copolymer comprising a mixture of a first poloxamer and a second poloxamer having different molecular weights, and a protein extract (VdECM) containing elastin and collagen.

[0012] The amphiphilic block copolymer is in a sol phase at room temperature and can change into a gel phase at body temperature.

[0013] The tissue regeneration-promoting drug delivery composition can contain 0.05 to 10 wt%, preferably 0.05 to 6 wt%, of a protein extract (VdECM), 1 to 40 wt% of a first poloxamer, 1 to 40 wt% of a second poloxamer, and the remainder being ultrapure water.

[0014] The first poloxamer preferably has a polyethylene oxide (PEO)-polypropylene oxide (PPO)-polyethylene oxide (PEO) block copolymer structure and has a weight-average molecular weight of 5,000 to 15,000, and the second poloxamer more preferably has a polyethylene oxide (PEO)-polypropylene oxide (PPO)-polyethylene oxide (PEO) block copolymer structure and has a weight-average molecular weight of 8,000 to 20,000.

[0015] The protein extract (VdECM) contained in the tissue regeneration-promoting drug delivery composition according to one embodiment of the present invention can be produced or obtained through a pretreatment step of preparing and pretreating non-human mammalian tissue, a first inactivation step of inactivating viruses contained in the pretreated tissue using alcohol, a decellularization step of removing cells from the virus-inactivated tissue, and a second inactivation step of inactivating viruses contained in the decellularized tissue using acid.

[0016] It is preferable that the DNA content of the tissue that has undergone the second inactivation step is 50 ng / mg or less, and the reduction rate (L) of the elastin content of the tissue that has undergone the pretreatment step to the second inactivation step is 20% or less.

[0017] The decellularization step may include a primary decellularization step in which cells are removed from virus-inactivated tissue using an aqueous base solution, and a secondary decellularization step in which the primarily decellularized tissue is treated with an enzyme to remove cells. The primary decellularization step may include a first decellularization step performed using a mixture of n-PrOH and NaOH, and a second decellularization step performed using an aqueous sodium hydroxide solution of greater than 0.05 M and less than 0.2 M.

[0018] The non-human mammalian tissue used in the present invention is preferably any one or more of blood vessels, ligaments and tendons derived from mammals.

[0019] The secondary decellularization step is preferably carried out using DNase.

[0020] Another embodiment of the present invention is a drug delivery kit in the form of a syringe or injector filled with the tissue regeneration-promoting drug delivery composition described above.

[0021] Another embodiment of the present invention includes a tissue regeneration-promoting drug delivery carrier in which the tissue regeneration-promoting drug delivery composition according to one embodiment and a drug are mixed in a 1:1 weight ratio. [Effects of the Invention]

[0022] The tissue regeneration-promoting drug delivery composition according to the present invention comprises poloxamer, a temperature-sensitive material, which has phase-change properties such that it is in a liquid sol phase at room temperature but becomes a gel at body temperature. This makes it easy to inject as an injection and applicable to various body parts.

[0023] It also contains a protein extract (VdECM) containing elastin components, which are involved in the regeneration process of damaged wound tissue, and can contribute to the rapid recovery of damaged wound tissue.

[0024] Furthermore, the elastin is composed of at least 70% hydrophobic amino acids, and can be combined with poloxamer, an amphiphilic temperature-sensitive polymer with hydrophilic and hydrophobic properties, to improve its stability in the body. This improves tissue adhesion, and after injection into the body via syringe, it adheres stably to the injected tissue without flowing out, making it effective for drug delivery to localized sites.

[0025] In particular, the poloxamer and protein extract (VdECM) are bioabsorbed within 14 days, eliminating the need for a separate removal process. Furthermore, the use of amphiphilic poloxamer, which possesses both hydrophobic and hydrophilic properties, allows for the use of both hydrophilic and hydrophobic drugs. This allows for the use of various drugs, such as growth factors, steroids, antibiotics, analgesics, and topical anticancer drugs, and allows for uniform mixing with a variety of drugs. [Brief explanation of the drawings]

[0026] [Figure 1] 1(A) and 1(B) are graphs showing the experimental results of Experimental Example 1. [Figure 2] FIG. 2 is a graph showing the experimental results of Experimental Example 2. [Figure 3] 3(A) and 3(B) are graphs showing the experimental results of Experimental Example 3. [Figure 4]4(A) and 4(B) are graphs showing the experimental results of Experimental Example 4. [Figure 5] FIG. 5 is a table showing the experimental results of Experimental Example 5. [Figure 6] FIG. 6 shows the results of an experiment on cytotoxicity with changes in the concentration of a protein extract in the drug delivery composition of the present invention. [Figure 7] FIG. 7 shows the results of measuring the change in viscosity with changing the concentration of the protein extract in the drug delivery composition of the present invention. [Figure 8] FIG. 8 shows the results of measuring the change in viscosity with changing the concentration of the protein extract in the drug delivery composition of the present invention. [Figure 9] FIG. 9 shows the results of measuring the change in adhesive strength with the change in concentration of the protein extract in the drug delivery composition of the present invention. [Figure 10] FIG. 10 shows the results of measuring the stability of the gel over time as the concentration of the protein extract in the drug delivery composition of the present invention changes. [Figure 11] FIG. 11 shows the results of measuring the amount of drug released over time to confirm the drug delivery performance of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0027] Before proceeding to a detailed description of preferred embodiments of the present invention, it should be made clear that the terms and words used in this specification and claims should not be interpreted in a limited way to their ordinary or dictionary meanings, but should be interpreted in a way that is consistent with the technical ideas of the present invention.

[0028] Throughout the specification, when a part is said to "comprise" a certain element, this does not mean excluding other elements, but means further including other elements, unless specifically stated to the contrary.

[0029] Furthermore, throughout the specification, unless otherwise specified, "%" used to indicate the concentration of a particular substance means (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid.

[0030] The term "mammal" as used herein means mammals other than humans, and even if the term "mammal" is simply used without any reference to excluding humans, it should be understood to mean mammals other than humans, and the expression "cells are removed" should be understood in a comprehensive sense to include destruction or removal of DNA.

[0031] Additionally, unless the context clearly dictates a particular sequence, each step may be performed differently from the sequence specified, i.e., each step may be performed in the same sequence as the sequence specified, may be performed substantially simultaneously, or may be performed in reverse order.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and in the event of conflict, the description of the present invention, including definitions, shall prevail.

[0033] One embodiment of the present invention provides a tissue regeneration-promoting drug delivery composition comprising an amphiphilic block copolymer and a protein extract (VdECM) containing elastin and collagen. The amphiphilic block copolymer may comprise a mixture of a first poloxamer and a second poloxamer having different molecular weights. The protein extract (VdECM) is obtained by a multi-step decellularization process of non-human mammalian tissue, such as mammalian tissues such as blood vessels, ligaments, and tendons. The protein extract contains approximately 60% elastin and 40% collagen, as well as trace amounts of growth factors and bioactive substances.

[0034] The tissue regeneration-promoting drug delivery composition may contain 0.05 to 10 wt%, preferably 0.05 to 6 wt%, of the protein extract (VdECM), 1 to 40 wt% of the first poloxamer and 1 to 40 wt% of the second poloxamer, and the remainder preferably contains ultrapure water as a solvent.

[0035] It is more preferable to use an alkaline aqueous solution containing a basic substance at a concentration of 0.01 to 10 N in ultrapure water to dissolve the powdered protein extract (VdECM), and after the protein extract has dissolved, to use an ultrapure water-based neutral solvent that has been neutralized with an organic acid to a neutral range, preferably a pH value in the range of 7.0 to 7.5.

[0036] Examples of basic substances used in this process include sodium hydroxide, potassium hydroxide, ammonia, sodium carbonate, sodium bicarbonate, potassium carbonate, and potassium bicarbonate. Examples of organic acids used to neutralize the alkaline aqueous solution in which the protein extract has been dissolved include 0.01 to 10 N acetic acid, lactic acid, hydrochloric acid, citric acid, succinic acid, glycolic acid, perchloric acid, carboxylic acid, and sulfonic acid.

[0037] The protein extract powder is dissolved in an alkaline aqueous solution to form a solution, which is then neutralized to a neutral range using an organic acid and subjected to a primary filtration process, followed by dissolving the first and second poloxamers. After dissolving the first and second poloxamers, the solution is subjected to a degassing process to remove air bubbles from the composition through low-temperature, slow stirring, and / or reduced pressure treatment, followed by packaging and sterilization, thereby producing a tissue regeneration-promoting drug delivery composition according to the present invention.

[0038] The drug delivery composition can be packaged in a first syringe, which is a syringe-shaped container, but the drug to be mixed with the drug delivery composition can also be loaded into a separate second syringe, and the discharge ports of the first and second syringes can then be connected to each other using a connecting member to pre-mix the drug delivery composition and the drug, which can then be injected or applied to the affected area or application site.

[0039] Elastin maintains patterns and shapes in the extracellular matrix (ECM) and provides elasticity. It also binds to elastin-binding sites (elastin receptor complex (ERC), GAGs, and integrin αVβ3) present in the cell membrane, regulating intercellular signals.

[0040] The main roles of elastin include regulating cell signaling, migration, attachment, proliferation, survival, development, differentiation, phenotype, ECM production, and physiology. It also promotes re-epithelialization, promotes regeneration of injured tissue, promotes angiogenesis, and promotes MMP-1 expression. Thus, elastin plays a crucial role in regulating tissue structure and physiological activity in normal tissues. Furthermore, exogenous administration of elastin components to injured tissues can promote the production of elastic fiber tissue, restoring the tissue to a state similar to the original tissue.

[0041] Thus, elastin plays various roles in the repair or healing process of injured tissue. For example, during the process of hemostasis as injured blood vessels contract, elastin promotes the migration of inflammatory cells to the injured wound site and the release of related factors. During the process of removing necrotic tissue from the injured wound site, elastin promotes the migration, adhesion, proliferation, and differentiation of epithelial cells at the wound site, promotes the proliferation of cells and extracellular matrix at the injured wound site, and is involved in the synthesis of collagen and elastin, which form the basic framework for wound healing. It also promotes the angiogenesis process and promotes elastin expression, thereby restoring elasticity similar to that of existing tissue.

[0042] The amphiphilic block copolymer contained in the drug delivery composition is preferably a mixture of a first poloxamer and a second poloxamer with different molecular weights. The first poloxamer preferably has a weight-average molecular weight of approximately 5,000 to 15,000, and the second poloxamer preferably has a weight-average molecular weight in the range of approximately 8,000 to 20,000. The first poloxamer and the second poloxamer can each be contained in the drug delivery composition in an amount ranging from 1 to 40 wt %. The use of a mixture of two poloxamers with different molecular weights as an amphiphilic block copolymer offers the advantages of improved stability in the body and ease of use compared to the use of a single poloxamer.

[0043] That is, to create a dosage form that can be stored for more than about 7 days using a single poloxamer, a high concentration of poloxamer must be used. However, dosage forms designed for high phase stability have the problem of poor usability due to their high viscosity at room temperature, making them unsuitable for use as drug delivery compositions.

[0044] However, in the case of the present invention, the mixture of two different types of poloxamer and a protein extract (VdECM) exhibits a liquid phase at room temperature, making it easy to inject as an injection such as a syringe formulation.After being injected into the body, the phase changes to a hard gel as the temperature rises due to body temperature, and this has the advantage of being able to maintain high phase stability for a long period of time, such as approximately 7 days or more.

[0045] The amphiphilic block copolymer has the properties of both hydrophilic and hydrophobic polymers. Therefore, when a poorly soluble hydrophobic drug is applied after forming the hydrogel, the drug can be homogenized inside the hydrogel due to the interaction between the hydrophobic groups. This has the advantage that the hydrophilic drug can also be easily homogenized in the hydrogel.

[0046] Poloxamer and poloxamine, representative amphiphilic block copolymers, are composed of a hydrophilic PEO block and a hydrophobic PPO block. They are effective in carrying both hydrophobic and hydrophilic drugs and exhibit a reversible sol-gel phase transition at a specific temperature, making them easy to use. Furthermore, they are biocompatible and non-reactive as nonionic substances, making them suitable for use as drug delivery materials.

[0047] In contrast, in the case of a single material, an amphiphilic block copolymer, the gelation reaction occurs at a relatively high concentration, and it has the characteristic of being rapidly absorbed and excreted from the body within a few hours after administration, and there is a limit to its ability to remain in the body for a long period of time and perform the function of continuously releasing a drug at a constant concentration for a certain period of time.

[0048] The first poloxamer is a poloxamer having a polyethylene oxide (PEO)-polypropylene oxide (PPO)-polyethylene oxide (PEO) structure, and preferably has a weight-average molecular weight of 5,000 to 15,000. It is preferably an amphiphilic copolymer containing polypropylene oxide (PPO) with a weight-average molecular weight of 4,000 and approximately 50 to 75 wt% polyethylene oxide (PEO), and having an HLB value of 24 or less.

[0049] The second poloxamer also preferably has a polyethylene oxide (PEO)-polypropylene oxide (PPO)-polyethylene oxide (PEO) structure and has an average molecular weight of 8,000 to 20,000. An amphiphilic copolymer containing polypropylene oxide (PPO) with a weight-average molecular weight of 3,300 and approximately 55 to 85 wt% polyethylene oxide (PEO) and having an HLB value of 24 or higher can be used.

[0050] Next, the protein extract (VdECM) obtained by the multi-step decellularization process will be described in more detail. The protein extract (VdECM) contained in the tissue regeneration-promoting drug delivery composition according to the present invention is obtained through the following steps: a preparation step of preparing non-human mammalian tissue; a pretreatment step of pretreating the tissue; a first inactivation step of inactivating viruses contained in the pretreated tissue using alcohol; a primary decellularization step of removing cells from the virus-inactivated tissue using an aqueous base solution; a secondary decellularization step of treating the primary decellularized tissue with an enzyme to remove cells; and a second inactivation step of inactivating viruses contained in the decellularized tissue using an acid.

[0051] In this case, the DNA content of the tissue after the second inactivation step is 50 ng / mg or less, preferably 20 ng / mg or less, 10 ng / mg or less, and more preferably 5 ng / mg or less.Moreover, it is more preferable that the reduction rate (L) of the elastin content of the tissue after the pretreatment step to the second inactivation step is 20% or less.

[0052] Here, the reduction rate (L) is defined by the following formula 1, where L0 means the elastin content of the tissue at the preparation stage, and L f denotes the elastin content of the tissue after the second inactivation step.

[0053]

number

[0054] The preparation step is a step of preparing tissue from a non-human mammal, where the mammal may be a mammal other than a human, such as a pig, horse, cow, or sheep, and the tissue may be one or more of blood vessels, ligaments, and tendons derived from such a mammal.

[0055] To produce a multi-step decellularized protein extract (VdECM), tissue is first harvested from a mammal. The harvested mammalian tissue is prepared in a preparation step by removing any excess tissue and blood adhering to the mammalian tissue, washing, drying, and cutting. The prepared mammalian tissue can be stored frozen and thawed for use when needed. When using frozen tissue material, the preparation step can involve removing and preparing the frozen tissue material.

[0056] Next, in the preparation step, the prepared mammalian tissue is pretreated by a pretreatment step. This step is a step of washing the mammalian tissue. If the mammalian tissue is frozen, it can be thawed, washed, and cut in this step. In this step, distilled water, purified water, physiological saline, etc. can be used as washing water, and distilled water is preferably used. From this step until the second inactivation step, physical agitation can be performed.

[0057] The mammalian tissue prepared through this pretreatment step can then undergo a first inactivation step, a primary decellularization step, a secondary decellularization step, and a second inactivation step to remove cells, crude fat, viruses, and other foreign substances that may induce immune and foreign body reactions. Decellularization through this multi-step reaction significantly improves the decellularization efficiency and foreign body removal efficiency at each step, increasing the amount of tissue that can be processed at one time and shortening the processing time, resulting in improved productivity and yield of decellularized protein extract (VdECM).

[0058] First, the first inactivation step is a step of inactivating viruses contained in the tissue pretreated with alcohol.

[0059] This step is performed to first inactivate viruses within the tissue before decellularizing it, thereby more effectively removing cells, crude fat, and foreign matter in the first and second decellularization steps.

[0060] The alcohol used in this step may be n-propanol, and in order to obtain the effect of inactivating viruses, it is preferable to use an aqueous n-propanol solution with a concentration of 50 to 90%, and more preferably an aqueous n-propanol solution with a concentration of 65 to 80%.

[0061] In the present invention, n-propanol is used as the alcohol for virus inactivation in the first inactivation step. When n-propanol is used for virus inactivation, the tissue becomes softer. This softness allows the treatment solution to penetrate more easily into the tissue, resulting in uniform virus inactivation and decellularization. As a result, the efficiency of the inactivation and decellularization processes is improved, resulting in more uniform tissue, and the quality and reliability of the resulting decellularized protein extract (VdECM) are improved.

[0062] Generally, when ethanol is used for virus inactivation, it has the characteristic of fixing and hardening the mammalian tissue being decellularized. However, if ethanol is used instead of n-propanol at this stage of the present invention, the tissue will become hard, which is undesirable as it reduces the efficiency of the subsequent process of loosening the tissue to improve the penetration of the treatment substance.

[0063] Furthermore, since isopropanol has a low viral inactivation effect, using isopropanol in the first inactivation step will not result in sufficient viral inactivation, and the decellularization efficiency in the subsequent primary and secondary decellularization steps will decrease. Therefore, it is preferable to use n-propanol in the first inactivation step.

[0064] At this stage, the pretreated tissue can be treated with 10 to 25 parts by weight, preferably 20 to 25 parts by weight, of isopropanol aqueous solution per 100 parts by weight.

[0065] The primary decellularization step is a step of removing cells by treating the virus-inactivated tissue after the first inactivation step with a basic aqueous solution.

[0066] This step includes a first decellularization step in which the tissue is treated with a mixture of an aqueous base solution and an alcohol, and a second decellularization step in which the tissue is treated with an aqueous base solution, where the aqueous base solution is preferably an aqueous sodium hydroxide solution and the alcohol is preferably n-propanol, in which case the first decellularization step is performed by treating the tissue with a mixture of n-propanol and sodium hydroxide, and the second decellularization step is performed by treating the tissue with an aqueous sodium hydroxide solution.

[0067] When the decellularization process is carried out in two stages in this way, not only are decellularized tissue removed in the first decellularization stage, but foreign matter such as crude fat is also removed, and a more effective decellularization process is carried out in the second decellularization stage.

[0068] In each of the first and second decellularization stages, the tissue can be treated with 10 to 25 parts by weight, preferably 20 to 25 parts by weight, per 100 parts by weight of treatment solution, which is approximately twice the conventional treatment volume.The multi-stage method of the present invention enables efficient decellularization treatment, and thus has the effect of being able to sufficiently treat twice the volume of tissue compared to conventional methods.

[0069] Specifically, the first decellularization step involves treating the virus-inactivated tissue with a mixture of n-propanol and sodium hydroxide. During this step, the crude fat in the tissue undergoes a saponification reaction with the sodium hydroxide, resulting in the separation and removal of the crude fat from the tissue. The n-propanol promotes this saponification reaction and causes the products of the saponification reaction to aggregate together, allowing the crude fat to be separated and removed from the tissue more quickly and efficiently.

[0070] In this case, the treatment time for treating the tissue with a mixture of n-propanol and sodium hydroxide is 10 to 48 hours, preferably 15 to 40 hours, and more preferably 18 to 28 hours, and the treatment temperature may be 0 to 30° C., preferably 10 to 28° C., and more preferably 18 to 25° C. Treatment within this temperature range for the above time period can achieve sufficient crude fat removal efficiency without significant damage to the tissue structure or loss of elastin.

[0071] The mixture of n-propanol and sodium hydroxide may be an aqueous solution with an n-propanol concentration of 50 to 95%, preferably 60 to 90%, and more preferably 65 to 85%, and with a sodium hydroxide concentration of more than 0.05 M and less than 0.2 M. These concentration ranges are concentration ranges that can minimize tissue damage in the first decellularization step while effectively removing foreign matter such as crude fat. In particular, if the sodium hydroxide concentration is below this range, the processing efficiency of the first decellularization step decreases, and if it is outside this range, the tissue structure itself will collapse and dissolve, so it is preferable to use n-propanol and an aqueous sodium hydroxide solution within the concentration ranges described above.

[0072] The second decellularization step involves treating the tissue that has undergone the first decellularization step with a basic solution to soften the tissue, loosening its structure and allowing the decellularization reaction to occur. Crude fatty acids and various foreign substances contained in the tissue act as a physical and chemical barrier to the decellularization reaction, but because the tissue has already undergone the first decellularization step and crude fat and various foreign substances have been removed, the decellularization reaction can occur more effectively in the second decellularization step.

[0073] The treatment time for treating the tissue with the basic aqueous solution at this stage is 10 to 48 hours, preferably 15 to 40 hours, and more preferably 18 to 28 hours, and the treatment temperature may be 0 to 30°C, preferably 10 to 28°C, and more preferably 18 to 25°C. When treated within this temperature range for the above period of time, the tissue structure is sufficiently gently deformed without collapse of the tissue structure or loss of elastin, allowing for an appropriate decellularization reaction to occur.

[0074] In this case, the basic aqueous solution used as the treatment solution may be an aqueous sodium hydroxide solution, and the concentration of sodium hydroxide contained in the aqueous sodium hydroxide solution may be greater than 0.05 M and less than 0.2 M. If the sodium hydroxide concentration is 0.05 M or less, the tissue structure will not be sufficiently loosened, resulting in a problem of reduced decellularization efficiency in the second decellularization step described below. If the sodium hydroxide concentration is 0.2 M or more, some tissues will exceed the loosening level in this step and will disintegrate or dissolve, resulting in a significant decrease in the final yield and a risk of elastin loss. Therefore, it is preferable to use an aqueous sodium hydroxide solution of the aforementioned concentration.

[0075] The primary decellularization step is followed by a secondary enzymatic decellularization step, and neutralization and washing steps can be performed between these two steps.

[0076] The neutralization process is a step in which the tissue is treated with an acidic aqueous solution to neutralize the sodium hydroxide used in the primary decellularization step. The type of acidic aqueous solution used here can be, for example, an acidic aqueous solution containing at least one of hydrochloric acid, sulfuric acid, acetic acid, and peracetic acid, but is not limited to these, and the concentration can be adjusted appropriately depending on the working environment and conditions.

[0077] The washing step is performed to prevent a decrease in enzymatic reactivity due to residues during enzymatic treatment in the subsequent secondary decellularization step. A buffer solution may be used as the washing solution in the washing step, such as, but not limited to, PBS (Phosphate buffered saline) solution.

[0078] The secondary decellularization step is a step in which the primary decellularized tissue is treated with an enzyme to remove cells, and is a step in which the primary decellularized tissue is treated with a DNA degrading enzyme to remove DNA in the primary decellularized tissue.

[0079] At this stage, the tissue can be treated with 10 to 25 parts by weight, preferably 20 to 25 parts by weight, per 100 parts by weight of the treatment solution containing DNase. As mentioned above, this is nearly double the treatment amount compared to conventional treatments, and is an achievable treatment amount due to the increased treatment efficiency achieved by the multi-stage decellularization process of the present invention.

[0080] To obtain sufficient enzyme activity and DNA degradation efficiency at this stage, the treatment time for treating the tissue with DNase can be 10 to 35 hours, preferably 18 to 30 hours, and the treatment temperature can be 30 to 45°C, preferably 35 to 42°C.

[0081] In the present invention, the concentration of DNase contained in the treatment solution is 0.0001 to 0.005 wt%, which is several to several hundred times lower than the DNase concentrations typically used in similar conventional techniques. The primary decellularized tissue of the present invention undergoes the primary decellularization step, which removes foreign matter, including crude fat, from the tissue and loosens the tissue structure. When DNase is added in this state, it can penetrate very easily into the tissue structure, ensuring at least the same or higher DNA removal efficiency even when a low concentration of DNase is used. Therefore, such low concentrations of DNase can be used in the present invention.

[0082] However, if the concentration of the DNase is less than the above range, the efficiency of DNA degradation decreases, so it is preferable to use it at a concentration of 0.0001% or more. If the concentration exceeds the above range, the improvement in DNA degradation efficiency is extremely small compared to the amount of DNase added, making it uneconomical, so it is preferable to use the DNase in the concentration range described above.

[0083] The tissue decellularized through the secondary decellularization step undergoes a second inactivation step to further remove viruses, and a bleaching and / or delipidation step can be performed between these two steps. The bleaching step is a step to remove color from the tissue and can be performed, for example, by treating the tissue with hydrogen peroxide, and the delipidation step is a step to further remove lipids contained in the tissue and can be performed, for example, using a ketone solution, preferably an acetone solution.

[0084] Next, a second inactivation step is carried out to further inactivate viruses contained in the tissue that has undergone the secondary decellularization step. This step uses acid to inactivate viruses in the tissue, specifically, treating the tissue with a mixture of organic acid and alcohol.

[0085] The organic acid used in this step can be peracetic acid, which is an oxidizing agent that can inactivate viruses by inducing damage to non-specific free radicals in the virus. The peracetic acid used in this step can be used at a concentration of 0.05 to 1%.

[0086] The alcohol used in this step may be a lower alcohol having 1 to 4 carbon atoms, such as methanol, ethanol, or propanol, and is preferably ethanol. Ethanol is preferred because it inactivates viruses and fixes the tissue structure to make the tissue stronger.

[0087] Through these steps, the virus is finally inactivated, crude fat and DNA are removed, and a protein extract (VdECM) containing various extracellular matrix components including elastin can be obtained.

[0088] The DNA content of the resulting protein extract (VdECM) is 50 to 1000 kJ / mg or less, preferably 20 ng / mg or less, 10 ng / mg or less, and more preferably 5 ng / mg or less, which is an extremely low DNA content, thereby minimizing the induction of immune and various foreign body reactions.

[0089] Furthermore, the protein extract (VdECM) has an extremely low elastin content, which is reduced by the pretreatment and second inactivation steps, and therefore has the advantage of having effects such as elastin-mediated wound healing and scar prevention. Specifically, the reduction rate (L) of the elastin content of tissue that has undergone the pretreatment and second inactivation steps is 20% or less. This value is significantly lower than the very high reduction rate (L) of elastin content of 50% or more that is achieved when conventional decellularization methods are applied.

[0090] After the second inactivation step, additional steps such as washing, packaging, sterilization, processing, etc. may be performed. Washing may be performed using a buffer solution, distilled water, etc., and packaging may be performed to prevent contamination during storage and transportation of the protein extract (VdECM) and for easy handling, and the protein extract (VdECM) may be packaged in a preservative solution.

[0091] Sterilization is performed to remove any further contamination that may occur after the second inactivation step, and may be performed by, for example, but not limited to, electron beam irradiation or radiation.

[0092] Processing refers to processing the protein extract (VdECM) into a form that can be used, and methods such as drying, powdering, and solution formation can be applied. After processing in this manner, the protein extract (VdECM) can be used in the form of a bioink or an injection, and in the case of the tissue regeneration-promoting adhesion preventing coating composition according to the present invention, it can be prepared in the form of a liquid injection for ease of processing and use.

[0093] Meanwhile, another embodiment of the present invention relates to a method for producing a mammalian protein extract (VdECM), and some of the explanations overlapping with one embodiment of the present invention will be omitted.

[0094] Specifically, the manufacturing method is a method for manufacturing a decellularized protein extract (VdECM) and includes the following steps: a preparation step of preparing non-human mammalian tissue; a pretreatment step of pretreating the tissue; a first inactivation step of inactivating viruses contained in the pretreated tissue using alcohol; a primary decellularization step of removing cells from the virus-inactivated tissue using an aqueous base solution; a secondary decellularization step of enzymatically treating the primary decellularized tissue to remove cells; and a second inactivation step of inactivating viruses contained in the decellularized tissue using an acid.

[0095] In this case, the DNA content of the tissue that has undergone the second inactivation step may be 50 ng / mg or less, and the reduction rate (L) of the elastin content of the tissue that has undergone the pretreatment step to the second inactivation step may be 20% or less.

[0096] Here, the reduction rate (L) of the elastin content of the tissue is defined by the following formula 1, where L0 is the elastin content of the tissue in the preparation stage, and L f is the elastin content of the tissue after the second inactivation step.

[0097]

number

[0098] First, the preparation step involves preparing tissue from a non-human mammal, where the mammal is a mammal other than a human, such as a pig, horse, cow, or sheep, and the tissue may be one or more of blood vessels, ligaments, and tendons derived from such a mammal.

[0099] The pre-treatment step is a step of washing the mammalian tissue prepared in the preparation step, and if necessary, the tissue can be cut into appropriate sizes in this step, and if the tissue was prepared in a frozen state in advance, it can be thawed in this step.

[0100] The first inactivation step is a step of inactivating viruses contained in the pretreated tissue using alcohol, and although ethanol is generally used for virus inactivation, n-propanol is used in the present invention because ethanol stiffens the tissue, reducing the softening efficiency in subsequent steps, whereas n-propanol softens the tissue, improving the softening efficiency in subsequent steps.

[0101] The primary decellularization step is a step in which tissue from which viruses have been inactivated after the first inactivation step is treated with a basic aqueous solution to remove cells. This step includes a first decellularization step in which the tissue is treated with a mixture of a basic aqueous solution and alcohol, and a second decellularization step in which the tissue is treated with a basic aqueous solution.

[0102] Here, it is preferable that the aqueous base solution is an aqueous sodium hydroxide solution, and the alcohol is n-propanol.

[0103] Specifically, the first decellularization step involves treating the virus-inactivated tissue with a mixture of n-propanol and sodium hydroxide, which removes crude fat and foreign matter from the tissue and partially softens it. The second decellularization step involves treating the tissue with an aqueous sodium hydroxide solution, which softens the tissue and simultaneously causes a decellularization reaction. However, this effect can be further enhanced by removing foreign matter, including crude fat, in advance.

[0104] Sodium hydroxide in the first and second decellularization steps can be present at a concentration greater than 0.05 μM and less than 0.2 μM, which is the preferred concentration range for preventing tissue breakdown and dissolution while allowing sufficient saponification and softening reactions by sodium hydroxide to occur.

[0105] Next, after the primary decellularization step, a secondary decellularization step using an enzyme is performed, and a neutralization step and a washing step can be performed between these two steps. The neutralization step is a step of treating with an acidic aqueous solution to neutralize the sodium hydroxide used in the primary decellularization step, and the washing step is a step performed to prevent a decrease in enzyme reactivity due to residues when enzyme treatment is performed in the subsequent secondary decellularization step.

[0106] The secondary decellularization step is a step in which the primary decellularized tissue is treated with a DNase to remove DNA from the tissue. The primary decellularized tissue of the present invention undergoes the primary decellularization step, removing foreign matter, including crude fat, from the tissue and loosening the tissue structure. When a DNase is introduced in this state, it penetrates the tissue structure very easily, allowing for DNA removal efficiency at least equal to or greater than that achieved with a low concentration of DNase. Therefore, in the present invention, a low concentration of DNase of 0.0001 to 0.005 wt% is used.

[0107] If the concentration of the DNase is below the above range, the efficiency of DNA degradation will decrease, and if it exceeds the above range, the degree of improvement in DNA degradation efficiency will be extremely small compared to the amount of DNase added, making it uneconomical. Therefore, it is preferable to use a DNase within the above concentration range.

[0108] The tissue decellularized through the secondary decellularization step undergoes a second inactivation step to further remove viruses, and a decolorization step and / or delipidation step may be performed between these two steps.

[0109] The decolorization step is a step of removing color from the tissue, and can be performed, for example, by treating the tissue with hydrogen peroxide. The delipidation step is a step of further removing lipids contained in the tissue, and can be performed, for example, using a ketone solution, preferably an acetone solution.

[0110] Next, a second inactivation step is performed to further inactivate viruses contained in the tissue that has undergone the secondary decellularization step. This step involves treating the tissue with a mixture of an organic acid and an alcohol, where peracetic acid can be used as the organic acid and ethanol can be used as the alcohol. Treating the tissue with this mixed solution can effectively remove viruses from the tissue.

[0111] Through these steps, viruses are finally inactivated, crude fat and DNA are removed, and a protein extract (VdECM) containing various extracellular matrix components including elastin can be produced.

[0112] The DNA content of the protein extract (VdECM) thus obtained is 50 to 1000 kJ / mg or less, preferably 20 ng / mg or less, 10 ng / mg or less, and more preferably 5 ng / mg or less, which is an extremely low DNA content, thereby minimizing the induction of immune and various foreign body reactions.

[0113] Furthermore, the protein extract (VdECM) has the advantage of having a very low elastin content, which is reduced by the pretreatment and second inactivation steps, and thus has the effect of promoting wound healing and scarring through elastin. Specifically, the reduction rate (L) of the elastin content of tissue that has undergone the pretreatment and second inactivation steps is 20% or less. This value is significantly lower than the very high reduction rate (L) of elastin content of 50% or more that is observed when conventional decellularization methods are applied.

[0114] After the second inactivation step, further steps such as washing, packaging, sterilization, processing, etc. may be performed. Washing may be performed using a buffer solution, distilled water, etc., packaging is performed to prevent contamination during storage and transportation of the protein extract (VdECM) and for easy handling, and sterilization is performed to remove further contamination that may occur after the second inactivation step.

[0115] Specific examples of the present invention will be described below. However, the scope of the present invention is not limited to the following preferred examples, and those skilled in the art can implement various modifications of the content described in this specification without departing from the scope of the present invention.

[0116] [Manufacturing Example 1] 1. Preparation and pre-treatment stages First, the aorta connected to the pig heart was prepared, which had been washed, dried, cut, and frozen. After thawing, it was washed with distilled water and then cut into pieces of 50 x 50 mm to prepare raw tissue samples.

[0117] 2. First inactivation step Next, 250 g of the raw tissue sample was placed in 1 L of 70% n-propanol and stirred at 20°C and 150 rpm for 24 hours to carry out the first inactivation step.

[0118] 3-1. Primary decellularization stage (first decellularization stage) Next, a mixed solution containing distilled water, n-propanol at a concentration of 70%, and sodium hydroxide at a concentration of 0.1M was prepared, and the raw tissue sample was mixed so that it contained 23 parts by weight per 100 parts by weight of the mixed solution.The first decellularization step was then carried out by stirring at a temperature of 20°C and a stirring speed of 150 rpm for 24 hours.

[0119] 3-2. Primary decellularization stage (secondary decellularization stage) Next, the tissue sample that had undergone the first decellularization step was mixed with 100 parts by weight of a 0.1 M aqueous sodium hydroxide solution prepared in advance so that it contained 24 parts by weight, and then the mixture was stirred for 24 hours under the same conditions as the previous step to carry out the second decellularization step.

[0120] Subsequently, the tissue sample that had undergone the second decellularization step was immersed in an aqueous solution of acetic acid, stirred and neutralized, and then washed with a PBS solution.

[0121] 4. Second Decellularization Stage Next, the tissue sample was placed in a treatment solution containing DNase at a concentration of 0.00011% by weight and stirred at 150 rpm for 23 hours at 37°C to perform a secondary decellularization step. After the secondary decellularization step, the tissue sample was decolorized by treatment with 3% hydrogen peroxide solution at 20°C for 1 hour, and then degreased by treatment with 20% acetone solution at 20°C for 1 hour.

[0122] 5. Second Inactivation Step Next, the tissue samples were immersed in a mixed solution of 70% n-propanol and 0.2% peracetic acid in distilled water at 20°C for 4 hours and 30 minutes to carry out the second inactivation step.

[0123] Finally, the tissue sample that had undergone all of the above treatments was washed with PBS solution and distilled water to prepare a multi-stage decellularized protein extract (VdECM) according to one embodiment of the present invention.

[0124] [Experimental Example 1] A multi-step decellularized protein extract (VdECM) was prepared using the same method as in Preparation Example 1, and the first, second, and second decellularization steps were performed sequentially. Tissue samples were collected immediately after each step and the elastin content per mg of tissue sample was measured. The results are shown in Figure 1(A).

[0125] Furthermore, the elastin content of the original tissue was measured during the preparation stage, and the percentage of the elastin content at each stage relative to the elastin content of the original tissue is shown in Figure 1(B).

[0126] In Figures 1(A) and 1(B), the first step refers to the first decellularization stage, the second step refers to the second decellularization stage, and the third step refers to the secondary decellularization stage.

[0127] The elastin content was measured using the Fastin Elastin Assay kit F2000 (Biocolor) according to the kit's protocol, followed by analysis of elastin at an absorbance of 513 nm using the multimode plate reader Victor Nivo™ (Perkin Elmer), and the elastin content was measured by comparing with a standard substance.

[0128] Referring to the results in Figures 1(A) and 1(B), it can be seen that the elastin content decreases slightly as each process progresses, but at the completion of the final process, approximately 80% or more of the elastin remains.

[0129] [Experimental Example 2] A multi-stage decellularized protein extract (VdECM) was prepared using the same method as in Preparation Example 1, but one step each from the first, second, and secondary decellularization steps was omitted. Tissue samples that had been processed through the final process were collected and the elastin content per mg of tissue sample was measured. The elastin content of the original tissue in the preparation stage was measured using the same method, and the L value was calculated. The results are shown in Table 1.

[0130] The elastin content in the protein extract (VdECM) obtained by omitting each preparation step was calculated as a percentage of the elastin content in the original tissue, and the results are shown in Figure 2.

[0131] The elastin content was measured in the same manner as in Experimental Example 1. In Table 1 and Figure 2, Step 1 refers to the first decellularization stage, Step 2 refers to the second decellularization stage, and Step 3 refers to the secondary decellularization stage.

[0132] [Table 1]

[0133] First, Table 1 shows that the L value is 20% or less when all steps are performed and when one step is omitted. Furthermore, Figure 2 shows that omitting a specific step does not significantly affect the elastin content. Therefore, these experimental results confirm that omitting any one of the first decellularization step, the second decellularization step, and the secondary decellularization step in the process according to one embodiment of the present invention does not significantly affect the elastin content.

[0134] [Experimental Example 3] The experiment was conducted in the same manner as in Experimental Example 1, and after each step, DNA content was measured instead of elastin content. DNA content was measured using the Quant-iT™ PicoGreen™ dsDNA Assay kits and dsDNA Reagents (Invitrogen) according to the kit's protocol, and then the DNA amount was measured at an absorbance of 480-520 nm using a multimode plate reader Victor Nivo™ (Perkin Elmer).

[0135] The DNA content per mg of tissue after each step is shown in Figure 3(A), and the DNA content of the tissue after each step relative to the DNA content of the original tissue is shown as a percentage in Figure 3(B). In Figures 3(A) and 3(B), step 1 refers to the first decellularization step, step 2 refers to the second decellularization step, and step 3 refers to the secondary decellularization step.

[0136] Referring to the results of Figures 3(A) and 3(B), it can be seen that most of the DNA is removed after the first decellularization step, the second decellularization step, and the secondary decellularization step.

[0137] [Experimental Example 4] The same experiment as in Experimental Example 2 was performed, and the DNA content of the protein extract (VdECM) prepared by omitting one step each was measured in the same manner as in Experimental Example 3.

[0138] The DNA content per mg of protein extract (VdECM) obtained by omitting each step is shown in Figure 4(A), and the DNA content of the tissue obtained by omitting each step relative to the DNA content of the original tissue is shown as a percentage in Figure 4(B). In Figures 4(A) and 4(B), the first step refers to the first decellularization step, the second step refers to the second decellularization step, and the third step refers to the secondary decellularization step.

[0139] 4(A) and 4(B), it can be seen that DNA is reliably removed by going through all three of the first, second, and secondary decellularization steps. In particular, it was found that the efficiency of DNA removal was significantly reduced when the second or secondary decellularization steps were omitted. Therefore, this experiment showed that going through all of the steps sequentially is more effective in removing DNA.

[0140] [Experimental Example 5] A multi-stage decellularized protein extract (VdECM) was produced using a method similar to that used in Preparation Example 1, and tissue was treated in the first decellularization step while varying the concentration of sodium hydroxide contained in the treatment solution to 0.005M, 0.1M, 0.2M, and 0.3M.

[0141] Photographs of the tissue during and after the second decellularization step (Step 2) were taken during the experiment and are shown in Figure 5. In addition, the DNA content in 1 mg of the final protein extract (VdECM) was measured using the same method as in Experimental Example 3, and the content of the final protein extract (VdECM) relative to the amount of tissue initially added was expressed as a percentage. The yield of the protein extract (VdECM) was calculated, and H&E stained photographs were taken and are shown in Figure 5.

[0142] Referring to FIG. 5, when the NaOH concentration was 0.05M, the yield was good, but the DNA content was high and unremoved cell nuclei were present in the tissue, indicating low decellularization efficiency.

[0143] In contrast, when treated with 0.1M NaOH, the tissue structure was well maintained in the second decellularization step, and the DNA content after final treatment was very low at 0.6ng / mg, with a high yield of 20.8%.

[0144] When treated with 0.2 M NaOH, the DNA content was low, but some of the tissue disintegrated during the primary decellularization step, resulting in a final yield that was less than half that of treatment with 0.1 M NaOH. When treated with 0.3 M NaOH, most of the tissue was found to be dissolved, and it is believed that no tissue was actually obtained.

[0145] From these experimental results, it was confirmed that in order to obtain a protein extract (VdECM) in which tissue breakdown or dissolution does not occur in the primary decellularization step, the yield is excellent, and DNA in the tissue has been sufficiently removed, the concentration of NaOH contained in the processing solution in the primary decellularization step, particularly the second decellularization step, should be greater than 0.05M and less than 0.2M.

[0146] [Experimental Example 6] A multi-stage decellularized protein extract (VdECM) was produced using a method similar to that used in the manufacturing example. In the first decellularization stage, the sodium hydroxide concentration in the treatment solution was 0.005M and 0.1M, and the tissue was treated with Dnase at a concentration ranging from 0.0001 to 0.003 wt%. The DNA content ratio of each final protein extract (VdECM) and the yield of the protein extract (VdECM) were calculated, and the results are summarized in Table 2.

[0147] [Table 2]

[0148] The results in Table 2 show that, at a constant NaOH concentration, increasing the DNase concentration results in a lower DNA concentration in the final protein extract (VdECM). However, despite treatment with a DNase concentration several to several tens of times lower at a 0.1M NaOH concentration than at a 0.05M NaOH concentration, the DNA concentration in the final protein extract (VdECM) was actually lower, suggesting that the difference in yield is not significant. The results of this experiment show that DNA removal efficiency is significantly lower at a 0.05M NaOH concentration compared to a 0.1M NaOH concentration, indicating that a NaOH concentration above 0.05M is preferred in the treatment solution used in the primary decellularization step.

[0149] [Manufacturing Example 2] The protein extract (VdECM) prepared in Preparation Example 1 was dissolved in 0.1N aqueous sodium hydroxide solution to various concentrations, and then 0.1N aqueous acetic acid solution was added to adjust the pH to 7.4, which is in the neutral range, to prepare a solution. Ultrapure water was used as the solvent for the aqueous solution.

[0150] Drug delivery compositions containing various concentrations of protein extract (VdECM) were prepared, as shown in Table 3 below. The concentration of the amphiphilic copolymer mixture in Groups 1 to 6 of each drug delivery composition was kept constant at 40 wt%. The amphiphilic copolymer used was a 1:1 mixture of a first poloxamer and a second poloxamer. The first poloxamer was a PEO-PPO-PEO copolymer with a weight-average molecular weight of 12,600 g / mol, and the second poloxamer was a PEO-PPO-PEO copolymer with a weight-average molecular weight of 15,000 g / mol.

[0151] [Table 3]

[0152] [Experimental Example 7] First, a cytotoxicity test was conducted on the drug delivery composition using varying concentrations of protein extract. Cytotoxicity measurements were conducted in accordance with the Common Criteria and Test Methods for Biological Safety of Medical Devices (ISO 10993-5:2009), and sample dissolution was conducted in accordance with ISO 10993-12. Groups 1 to 6 in Table 3 were used as the target samples. After dissolution in cell culture medium at 37°C for 24 hours at 100 rpm according to the standard test method, the samples were cultured in 2 x 10 L929 cells. 5 After seeding in a 6-well plate, cells cultured for 24 hours were treated with 2 ml of the eluate. Morphological changes in the cells were measured for 2 days (qualitative evaluation) and the cell count was measured (quantitative evaluation). Based on this, the cell viability (RCC) was calculated using the following formula 2.

[0153]

number

[0154] As can be seen from Figure 6, the results of the cytotoxicity test of the drug delivery composition according to the present invention confirmed that there was no cytotoxicity even when the concentration of the extracted protein (VdECM) was increased up to 6 wt%, and the RCC value also tended to increase as the concentration of the protein extract increased up to 4 wt%.

[0155] However, cytotoxicity was observed when the protein extract concentration was high at 12 wt%, which is presumably due to the increased hydrophobicity, a characteristic of the protein extract, resulting in increased viscosity when the protein extract concentration was too high, resulting in cytotoxicity.

[0156] In Figure 6, "Media" is a blank test solution, a sample eluted under the same conditions and in the same manner as the test sample without containing the test sample. "Negative" is a sample treated with a substance that does not cause a cytotoxic reaction and was used to determine the basic degree of cell reaction. "Positive" is a sample treated with a substance that causes a reproducible cytotoxic reaction and was used to confirm that the cytotoxicity test procedure was performed without any problems.

[0157] With the exception of Group 6, which contained a protein extract (VdECM) at a concentration of 12 wt%, the RCC values ​​of all test groups were 70% or higher, confirming that the criterion for cytotoxicity (RCC 70% or higher) was met.

[0158] Figure 6 shows that a similar cytotoxicity experiment was conducted on samples obtained by mixing and diluting the samples from Groups 1 to 6 with equal amounts of distilled water (see Group 1 (mixed) to Group 6 (mixed) in Figure 6). The results were the same as those for the samples before dilution with distilled water.

[0159] [Experimental Example 8] Drug delivery compositions (Groups 1 to 6) containing various concentrations of protein mixture (VdECM) listed in Table 3 were mixed with an equal volume of ultrapure water (Groups 1 to 6 (mixed)), and the viscosity was measured with temperature. The results are shown in Figure 7. The samples in Groups 1 to 6 were diluted 1:1 with ultrapure water before viscosity measurement. Therefore, the concentration of the protein extract in the viscosity measurement samples was half that of Groups 1 to 6.

[0160] It was found that as the concentration of the protein extract increased, the temperature at which the sol-gel transition occurred decreased, and the maximum viscosity value increased in proportion to the concentration of the protein extract up to a concentration of 6 wt%.

[0161] However, in the case of Group 6, which contained 12 wt% protein extract, a decrease in viscosity (a decrease in the maximum viscosity value) was observed at body temperature, 37°C. In particular, in the case of Group 6, gelation began to occur at room temperature, 25°C, making it undesirable for use as a drug delivery composition. This can be clearly confirmed by comparing the viscosity values ​​of the drug delivery composition at room temperature (25°C) and body temperature (37°C) at a shear rate of 1 / s, as shown in Figure 8.

[0162] As is clear from the results shown in Figure 8, when the concentration of the undiluted protein extract solution is less than 12 wt%, more preferably 6 wt% or less, the sol-gel transition proceeds effectively as the temperature changes from room temperature to body temperature. However, when the concentration of the protein extract is excessively increased, the sol-gel transition does not proceed smoothly at room temperature and body temperature.

[0163] [Experimental Example 9] After injecting the drug delivery composition according to the present invention into the body, adhesive strength was measured to confirm whether it could be stably positioned in the injected tissue. For groups 1 to 5 (excluding group 6, which was found to be undesirable in Experimental Example 8), a probe was vertically pressed with a predetermined force, and then the probe was vertically lifted from each of groups 1 to 5 to debond them, and the change in the measured normal stress value was observed.

[0164] The samples for measuring adhesive strength were prepared by diluting each of the samples in Groups 1 to 5 with the same amount of ultrapure water at a 1:1 ratio, similar to the viscosity measurement process described above. Therefore, the concentration of the protein extract contained in the samples used in the actual adhesive strength measurement experiment was equivalent to half of the concentration listed in Table 3.

[0165] The adhesive strength measurement experiment was carried out at room temperature (25° C.) and body temperature (37° C.) in the same manner as the viscosity measurement in Example 8, and the results are shown in FIG.

[0166] As can be seen from the results in Figure 9, when the protein extract (VdECM) concentration was below 2 wt% (Groups 1 to 3), the adhesive strength at body temperature (37°C) decreased compared to room temperature (25°C), but when the concentration was between 4 and 6 wt% (Groups 4 and 5), the adhesive strength at body temperature increased compared to room temperature, confirming that Group 4, with a protein extract (VdECM) concentration of 4 wt% (the concentration of protein extract contained in the actual sample was approximately 2 wt%), had the highest adhesive strength at body temperature.

[0167] [Experimental Example 10] Next, we investigated the phase stability of the gel formulations produced when the drug delivery compositions of the present invention underwent a phase change to form gels. Five grams of drug delivery compositions containing protein extracts of various concentrations (Groups 1 to 5) shown in Table 3 were mixed with 5 grams of ultrapure water, placed in tubes, and allowed to gel for approximately 30 minutes at body temperature (37°C).

[0168] After that, 2.5 g of phosphate buffered saline (PBS) was added to the tube, and the tube was stored at 37°C with shaking at approximately 50 rpm. After 24 hours, the tube was turned upside down and the height of the remaining drug delivery carrier was measured. The tube was then replaced with fresh PBS and stored under the same conditions (37°C, 50 rpm).

[0169] This process was repeated every 24 hours, and the height of the gelled drug delivery system was repeatedly measured every 24 hours until the remaining gel, which was the drug delivery carrier, was completely decomposed. The results are summarized in Figure 10.

[0170] As can be seen from the results in Figure 10, the drug delivery composition gel (Group 1) not containing the protein extract (VdECM) was completely degraded in 7 days, while Group 2 was degraded in 11 days, Group 3 in 12 days, and Groups 3 and 4 in 14 days.

[0171] This means that by including a protein extract in the drug delivery composition within a specified concentration range, the gel stability after phase change can be increased, and when inserted into the body, the drug delivery carrier retains its gel form for a long period of time, thereby providing excellent sustained-release functionality by gradually releasing the drug contained in the drug delivery carrier over a long period of time.

[0172] [Experimental Example 11] To confirm the actual drug release behavior of the drug delivery compositions according to the present invention, the drug delivery compositions of Groups 1 to 5 were mixed with the drug ropivacaine HCl at a 1:1 ratio, and the amount of drug released every 24 hours was measured for 120 hours (5 days). The results are summarized in Figure 11.

[0173] As is clear from the results in FIG. 11, in the case of Group 1, which did not contain the protein extract, the drug ropivacaine HCl was completely released after 72 hours (drug release 100%).

[0174] In contrast, it was confirmed that groups 2 to 5, which contained protein extract, gradually released the drug over a longer period of time compared to group 1, which did not contain protein extract, and groups 4 and 5 were found to have the best sustained drug release properties.

[0175] In fact, the known in vivo half-life of the single drug ropivacaine HCl is 1.8±0.7 hours when injected intravenously and 4.2±1 hours when injected epidurally. Considering this, it was found that when the drug is administered using the drug delivery composition of the present invention, it can be supplied to the body stably for a longer period of time than when injected using existing injection methods. [Industrial Applicability]

[0176] The drug delivery composition of the present invention comprises an amphiphilic block copolymer comprising a mixture of a first poloxamer and a second poloxamer having different molecular weights, and a protein extract (VdECM) containing elastin and collagen. After being easily supplied to the body in a liquid form, the composition undergoes a phase change due to body temperature to form a stable drug delivery structure, which releases drug components over a long period of time in the body and can be decomposed and absorbed in the body, thereby promoting tissue regeneration and therefore having industrial applicability.

Claims

1. an amphiphilic block copolymer comprising a mixture of a first poloxamer and a second poloxamer having different molecular weights; A tissue regeneration-promoting drug delivery composition comprising: a protein extract (VdECM) containing elastin and collagen;

2. The tissue regeneration-promoting drug delivery composition according to claim 1, wherein the amphiphilic block copolymer is in a sol phase at room temperature and changes into a gel phase at body temperature.

3. The tissue regeneration-promoting drug delivery composition according to claim 1, characterized in that it comprises 0.05 to 10 wt% protein extract (VdECM), 1 to 40 wt% first poloxamer, 1 to 40 wt% second poloxamer, and the remainder ultrapure water.

4. The tissue regeneration-promoting drug delivery composition according to claim 1, characterized in that the first poloxamer has a polyethylene oxide (PEO)-polypropylene oxide (PPO)-polyethylene oxide (PEO) block copolymer structure and has a weight-average molecular weight of 5,000 to 15,000.

5. The tissue regeneration-promoting drug delivery composition according to claim 1, characterized in that the second poloxamer has a polyethylene oxide (PEO)-polypropylene oxide (PPO)-polyethylene oxide (PEO) block copolymer structure and a weight-average molecular weight of 8,000 to 20,000.

6. The protein extract (VdECM) a pre-treatment step of providing and pre-treating non-human mammalian tissue; a first inactivation step in which viruses contained in the pretreated tissue are inactivated using alcohol; a decellularization step to remove cells from the virus-inactivated tissue; a second inactivation step using acid to inactivate viruses contained in the decellularized tissue, the DNA content of the tissue after the second inactivation step is 50 ng / mg or less; The tissue regeneration-promoting drug delivery composition according to claim 1, characterized in that the reduction rate (L) of the elastin content of the tissue after the pretreatment step to the second inactivation step is 20% or less.

7. The decellularization step comprises: a primary decellularization step of removing cells from the virus-inactivated tissue using a basic aqueous solution; The tissue regeneration-promoting drug delivery composition according to claim 6, further comprising a secondary decellularization step of enzymatically treating the primarily decellularized tissue to remove cells.

8. The first decellularization step a first decellularization step carried out with a mixture of n-PrOH and NaOH; The tissue regeneration-promoting drug delivery composition according to claim 7, further comprising a second decellularization step carried out using an aqueous sodium hydroxide solution of greater than 0.05 M and less than 0.2 M.

9. The tissue regeneration-promoting drug delivery composition according to claim 6, wherein the non-human mammalian tissue is any one or more of blood vessels, ligaments, and tendons derived from a mammal.

10. The tissue regeneration-promoting drug delivery composition according to claim 7 , wherein the secondary decellularization step is performed using DNase.

11. A drug delivery kit filled with the tissue regeneration-promoting drug delivery composition according to any one of claims 1 to 10.

12. A tissue regeneration-promoting drug delivery carrier in which the tissue regeneration-promoting drug delivery composition according to any one of claims 1 to 10 and a drug are mixed in a weight ratio of 1:1.

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