Composite composition for improving skin condition or treating bone-related inflammatory diseases, and method for preparing same

Abstract

The present invention relates to a composite composition for improving skin condition or treating bone-related inflammatory diseases, and a method for preparing same. The composite composition comprises a mixed composition in which a microparticle composition containing at least one of PDO, PLGA, or PLLA and an excipient composition containing at least one of HA or a nucleic acid molecule are mixed. The composition increases cell adhesiveness by stably inducing cell adhesion, has a retention period of 1 year or more, and has the effect of regenerating chondrocytes through the promotion of chondrogenic differentiation potential and collagen activity. Thus, the composition can be advantageously used as a filler or an intra-articular injection composition.

Description

Composite composition for improving skin condition or treating bone-related inflammatory diseases and method for preparing the same

[0001] The present invention relates to a composite composition for improving skin conditions or treating bone-related inflammatory diseases and a method for preparing the same.

[0002] Recently, due to the improvement in living standards and the aging of the population, interest in skin care, skin beauty, and arthritis treatment is increasing. To meet the diverse demands of consumers, various human-derived cells are being utilized, such as human umbilical cord blood cell culture fluid and exosomes, for use in improving skin wrinkles or treating arthritis.

[0003] PDRN (Polydeoxyribnucleotide) is a low molecular weight DNA fragment of about 50-1500 kDa, and PN (Polynucleotide) is a mixture of high molecular weight DNA and RNA of 1500-3000 kDa, and its absorption rate is relatively slower than that of PDRN.

[0004] Meanwhile, microparticles made of hydrophobic biodegradable polymers have the characteristic of being completely decomposed by hydrolysis in the body and excreted in urine, and have the advantage of being adjustable in duration from as long as 4 years to as short as 6 months depending on the type and molecular weight of the polymer.

[0005] Although methods for preparing compositions composed of hydrophobic biodegradable polymers such as Poly-L-Lactic acid (PLLA), Poly-ε-caprolactone (PCL), Poly-Lactic-co-glycolic acid (PLGA), and polydioxanone (PDO) have been proposed, PDO has the disadvantage of being difficult to maintain in the body for a long period due to its short degradation period, while PLGA, PLLA, and PCL are maintained in the body for a long period of more than one year due to their high hydrophobic properties, and side effects such as granuloma are known.

[0006] In particular, after microparticles of hydrophobic biodegradable polymers are injected into the human body, they can have the effect of regenerating collagen through a foreign body reaction; however, there is a disadvantage in that collagen regeneration is slow or ineffective because cell adsorption to the surface of the hydrophobic biodegradable polymer microparticles is difficult during this process.

[0007] Against this background, the inventors conducted research to develop a composition that is advantageous for cell adsorption and has a maintenance period of more than one year in the body. As a result, they provide a composite composition comprising a microparticle composition containing at least one of PDO, PLLA, and PLGA, and an excipient composition containing at least one of HA and nucleic acid molecules as an excipient. The present invention was completed by confirming that the composite composition increases cell adsorption, has a maintenance period of more than one year, and promotes chondrocyte differentiation ability and collagen activity.

[0008] The object of the present invention is to provide a method for preparing a composite composition comprising the steps of: i) preparing a composite microparticle composition comprising at least one of PDO microparticles, PLGA microparticles, and PLLA microparticles; ii) preparing an excipient composition comprising at least one of HA and nucleic acid molecules; iii) preparing a mixed composition provided by mixing the microparticle composition and the excipient composition; iv) preparing a dispersion by dispersing the mixed composition in a solvent in which a dispersant is dissolved; and v) preparing a freeze-dried body by freeze-drying the dispersion.

[0009] Another objective of the present invention includes providing a filler composition having improved cell adsorption, cartilage cell differentiation ability, and collagen activity by utilizing a composite composition prepared by the above-described manufacturing method.

[0010] Another objective of the present invention is to provide an intra-articular injection composition that utilizes the composite composition prepared by the above-described manufacturing method to increase cell adsorption and promote chondrocyte differentiation ability and collagen activity.

[0011] However, these tasks are exemplary and do not limit the scope of the invention.

[0012] The present invention provides a manufacturing method comprising: i) a step of preparing a composite microparticle composition comprising at least one of PDO microparticles, PLGA microparticles, and PLLA microparticles; ii) a step of preparing an excipient composition comprising at least one of HA and nucleic acid molecules; iii) a step of preparing a mixed composition by mixing the microparticle composition and the excipient composition; iv) a step of preparing a dispersion by dispersing the mixed composition in a solvent in which a dispersant is dissolved; and v) a step of preparing a freeze-dried body by freeze-drying the dispersion.

[0013] The PDO of the present invention is a polymer of several repeating ether-ester units and is a biodegradable and biocompatible polymer. The monomer is obtained by the ring-opening polymerization of p-dioxanone. The PDO is non-toxic, metabolizable and excretable, and has excellent physical properties, so it can be used as a surgical suture, drug delivery device, cardiovascular device, etc.

[0014] The PLGA of the present invention is a poly(lactic-co-glycolic acid) copolymer, which is a biodegradable and biocompatible polymer. Depending on the ratio of lactide to glycolide, different types of polymers can be obtained from the PLGA, and depending on the ratio, the crystallinity, glass transition temperature, hydrolysis time, etc. of the PLGA may vary. The PLGA is non-toxic, metabolizable and excretable, and has excellent physical properties, so it can be used as a therapeutic device such as an implant or surgical device.

[0015] The PLLA of the present invention is a copolymer of poly-L-lactide (Poly-L-Lactic acid (PLLA)), which is a biodegradable and biocompatible polymer. It was developed to treat lipoatrophy and is FDA-approved and effective for wrinkle improvement.

[0016] In one embodiment, the composite microparticle composition of step i) may include one, two, or all three of PDO microparticles, PLGA microparticles, and PLLA microparticles. The composite microparticle composition of one embodiment may include PDO and one or more of PLGA and PLLA. When the composite microparticle composition of one embodiment includes two or more of PDO, PLGA, and PLLA, it is defined as including two or more microparticles containing each individual polymer.

[0017] The HA of the present invention is hyaluronic acid, a polymer compound defined as a glycosaminoglycan having an unbranched polysaccharide chain produced by the condensation of thousands of disaccharide units, and exhibits high solubility in water. By forming cross-linked hyaluronic acid through a specific cross-linking agent and appropriate operating conditions (temperature, pH, etc.), a molecular framework can be created to maintain the shape and tone of the epidermal tissue.

[0018] The nucleic acid molecule of one embodiment may include, for example, PDRN and / or PN. The PDRN (Polydeoxyribonucleotide) of the present invention is a low-molecular-weight DNA fragment of about 50-1500 kDa that can aid in cell absorption, regeneration, and skin healing. The PN (Polynucleotide) is a mixture of high-molecular-weight DNA and RNA of 1500-3000 kDa that has a relatively slower absorption rate than PDRN, allowing it to persist in the body for a long period and exhibit effects on cell regeneration and skin healing. PDRN and PN are compounds containing DNA complexes, i.e., deoxyribonucleotide polymers, which can be used as activators necessary for cell regeneration without side effects and can promote cell differentiation.

[0019] In the method for preparing the composite composition of the present invention, the weight ratio of the fine particle composition (fine particles provided as PDO, PLGA, PLLA alone or mixed with two or more types) may be 60 to 99 weight% based on the total weight of the composite composition. For example, the weight ratio of the fine particles may be 50 to 95 weight%, 55 to 95 weight%, 60 to 95 weight%, 60 to 90 weight%, 65 to 90 weight%, 65 to 85 weight%, or 70 to 99.99 weight%, but is not limited thereto.

[0020] Based on the total weight of the composite composition, the weight ratio of the PDO microparticles may be 55 to 95 wt%, 55 to 90 wt%, 55 to 80 wt%, 60 to 80 wt%, or 65 to 75 wt%, but is not limited thereto. Based on the total weight of the composite composition, the weight ratio of the PLLA microparticles and the PLGA microparticles may each be independently 0 to 10 wt%, 0 to 8 wt%, 2.5 to 8 wt%, or 4 to 6 wt%, but is not limited thereto. Based on the total weight of the composite composition, the weight ratio of the excipient composition containing at least one of HA and nucleic acid molecules may be 0.01 to 40 wt%. For example, the weight ratio of the excipient composition may be 1 to 40 weight%, 5 to 40 weight%, 10 to 40 weight%, 15 to 40 weight%, 20 to 40 weight%, 25 to 35 weight%, or 1 to 5%, but is not limited thereto.

[0021] In the total weight of the above-mentioned injectable composition, the microparticle composition may be provided in an amount of 70 to 99.99 weight%, and the microparticle composition may consist of PDO alone.

[0022] In the case where the composition of one embodiment further includes PLLA and / or PLGA, it is more stably adsorbed to cells, which enhances collagen production and extends the collagen maintenance period, thereby providing a long-term tissue repair effect. For example, if a microparticle composition in which a small amount of PLLA and PLGA are provided alone or both mixed with the PDO-only microparticle composition is provided at 75% by weight of the total weight of the injectable composition, and an excipient composition containing at least one of HA and nucleic acid molecules is contained at 25% by weight, the excipient composition may be sufficiently present around the PDO microparticles containing a small amount of PLLA and PLGA.

[0023] The dispersant of the present invention is used to disperse physiologically active substances, and may be a low molecular weight dispersant or a high molecular weight dispersant polymer. The low molecular weight dispersant refers to a compound with a weight-average molecular weight of less than 15,000 MW, and the high molecular weight dispersant polymer refers to a compound that includes repeating covalent bonds between one or more monomers and has a weight-average molecular weight of 15,000 MW or more. As for the low molecular weight dispersant, it is not particularly limited as long as it is permitted as a pharmaceutical compound, a functional food compound, a functional cosmetic compound, etc. Specifically, it may be sugars, cyclodextrins, amino acids, organic acids, or other components, and one of these may be used alone or two or more may be used in combination.

[0024] The dispersant of the present invention may be selected from the group consisting of cells, growth factors, peptides, amino acids, proteins, sugars, natural polymers, and combinations thereof, but is not limited thereto.

[0025] In one embodiment, the dispersant may be dissolved in a solvent and then the mixed composition may be added to disperse the mixed composition, and the solvent may be water for injection, but is not limited thereto.

[0026] The cells and growth factors of the present invention may be one or more cells and growth factors selected from the group consisting of mesenchymal stem cells, induced pluripotent stem cells (iPSCs), fibroblast growth factor (FGF), epidermal growth factor (EGF), keratinocyte growth factor (KGF), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), granulocyte stimulating factor (GCSF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor-BB (PDGF-BB), brain-derived neurotrophic factor (BDNF), and glial-derived neurotrophic factor (GDNF), but are not limited thereto.

[0027] The peptides, amino acids, and proteins of the present invention may be alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or combinations thereof, but are not limited thereto.

[0028] The sugars of the present invention are glucose, mannose, idose, galactose, fucose, ribose, xylose, lactose, sucrose, maltose, trehalose, turanose, raffinose, maltotriose, acarbose, water-soluble cellulose, synthetic cellulose, sugar alcohol, glycerin, sorbitol, lactitol, mannitol, mannitol, xylitol, erythritol, polyol, derivatives thereof, or combinations thereof. It is possible, but is not limited to this.

[0029] The excipient composition of the present invention may be one or more selected from the group consisting of hyaluronic acid, polydeoxyribonucleotide, polynucleotide, chitosan, alginate, and combinations thereof, but is not limited thereto.

[0030] The manufacturing method of one embodiment may further include a step of modifying the surface of each microparticle prepared in step i) by hydrolyzing the microparticle at 100 to 140 ℃ for 30 to 100 minutes. The hydrolysis may be performed at 100 to 140 ℃ for 30 to 100 minutes, and more specifically, may be performed at 100 to 130 ℃ for 50 to 100 minutes or 50 to 90 minutes, but is not limited thereto.

[0031] In step v) above, freeze-drying may be performed at a temperature of -90 to -60 ℃ for 10 to 30 hours, and more specifically at a temperature of -80 to -70 ℃ for 10 to 20 hours, but is not limited thereto.

[0032] In a manufacturing method of one embodiment, the step of preparing a microparticle composition in step i) may include: ii) a step of preparing a first-1 solution by dissolving PDO, PLGA, and PLLA, respectively, in a solvent; i-ii) a step of preparing a first-2 solution by dissolving a surfactant in distilled water; i-iii) a step of preparing a first-3 solution by mixing and stirring the first-1 solution and the first-2 solution, and then removing the solvent; and i-iv) a step of obtaining biodegradable polymer microparticles by precipitating the biodegradable polymer microparticles generated in the first-3 solution and then removing the supernatant. In a manufacturing method of one embodiment, for example, when the microparticles contain only PDO, in step ii), only PDO is dissolved in a solvent to prepare the first-1 solution, and when they contain PDO and PLGA, PDO and PLGA, respectively, are dissolved in a solvent to prepare the first-1 solution.

[0033] In step ii) above, the solvent may be one or more selected from the group consisting of HFIP (1,1,1,3,3,3-Hexafluoro-2-propanol), chloroform, acetone, acetonitrile, dioxane, methoxybenzene, dichloromethane, dimethylformamide, dimethylsulfoxide and ethyl acetate, hexafluoroisopropanol, and anisole, but is not limited thereto.

[0034] In steps i-ii) above, the surfactant may be PVA (Polyvinyl alcohol), but is not limited thereto.

[0035] After steps i-iv) above, step iv) of washing the biodegradable polymer microparticles may be further included.

[0036] The average size of the biodegradable polymer microparticles of the present invention may be 20 to 100 μm, for example, 10 to 120 μm, 15 to 100 μm, 20 to 90 μm, or 20 to 70 μm, but is not limited thereto.

[0037] The microparticle composition of the present invention can be prepared by mixing PLGA microparticles, PLLA microparticles, each or both, with PDO microparticles.

[0038] The present invention may include the step of preparing a mixed composition by mixing the microparticle composition and the excipient composition, and preparing a dispersion by dissolving the mixture in a solvent such as water for injection.

[0039] In addition, the present invention provides a composite composition that can increase cell adsorption capacity and improve chondrocyte differentiation ability and collagen activity by the above-described manufacturing method.

[0040] The composite composition of the present invention can increase the secretion of growth factors VEGF and FGF-2, and can increase collagen activity by increasing the expression of Collagen type I and Collagen type III.

[0041] As described above, the above composite composition has excellent cell adsorption properties and excellent chondrocyte differentiation ability, and can improve skin condition or treat bone-related inflammatory diseases by increasing collagen activity through increased expression of growth factors and Collagen type I and Collagen type III.

[0042] That is, the above composite composition may be a composition for improving skin condition or treating bone-related inflammatory diseases.

[0043] The above skin condition improvement may include one or more selected from skin regeneration, wrinkle improvement, and tissue repair. The above bone-related inflammatory disease is included without limitation as long as it is an inflammatory disease occurring in the bone and its adnexal organs. For example, bone-related inflammatory disease may include arthritis, osteitis, osteochondritis, osteomyelitis, or osteonecrosis. In one embodiment, arthritis may include rheumatoid arthritis, degenerative arthritis, ankylosing spondylitis, intra-articular inflammation, septic arthritis, gouty arthritis, chondritis, and / or psoriatic arthritis. Osteitis may include tuberculous osteitis, and osteochondritis may include osteochondritis dissecans. In one embodiment, osteonecrosis may include avascular necrosis of the femoral head, Kienbeck's disease, avascular necrosis of the foot, and / or drug-induced osteonecrosis. In one embodiment, osteomyelitis may include acute osteomyelitis and / or chronic osteomyelitis.

[0044] In one embodiment, the nucleic acid molecule may include at least one of PN and PDRN. For example, the nucleic acid molecule of one embodiment may include PN.

[0045] According to one embodiment of the present invention, a composite composition for improving skin condition or treating bone-related inflammatory diseases may be provided. The composite composition of one embodiment may be prepared by the manufacturing method described above. Hereinafter, the same content as the manufacturing method described above may be applied to the composite composition to be described below, unless there is a contradiction.

[0046] A composite composition for improving skin conditions or treating bone-related inflammatory diseases according to one embodiment may include microparticles comprising at least one of polydioxanone (PDO), poly-L-lactic acid (PLLA), and poly(lactic-co-glycolic acid, PLGA), and an excipient composition comprising at least one of HA and nucleic acid molecules. In one embodiment, the microparticles may be dispersed and arranged among the excipient compositions.

[0047] The above microparticles may have been hydrolyzed and have their surfaces modified.

[0048] The above composite composition can increase the secretion of one or more of the growth factors VEGF (vascular endothelial growth factor) and FGF-2 (fibroblast growth factor-2).

[0049] The above composite composition can increase the expression of Collagen type I and Collagen type III.

[0050] The above composite composition can treat or prevent bone-related inflammatory diseases by alleviating inflammation.

[0051] In addition, the present invention may provide an injectable agent for improving skin conditions or treating bone-related inflammatory diseases, comprising the above-mentioned composite composition.

[0052] The injectable agent of the present invention can be used in a form administered to the skin area of ​​a subject using an injection device having a fine needle or canola shape.

[0053] The filler composition of one embodiment may be provided in the form contained in a vial or in the form of a pre-filled syringe. The pre-filled syringe or vial may comprise glass or plastic.

[0054] The filler composition of one embodiment may be provided in the form of a pre-filled syringe. The filler composition of one embodiment may be provided and distributed sealed in the form of a pre-filled syringe and may be reconstituted with water for injection (e.g., sterile water or physiological saline) before being administered to a subject requiring subsequent administration.

[0055] In one embodiment, the subject may be a mammal, including a human.

[0056] According to one embodiment of the present invention, a vial or injectable comprising the aforementioned filler composition; and a package comprising a dosing instruction sheet may be provided.

[0057] Depending on the use, the injectable of the present invention may additionally include a physiologically active substance, a local anesthetic, water for injection, sterile water, or distilled water.

[0058] In one embodiment, the composite composition may be administered by injection, for example, into the body of a subject (e.g., subcutaneous, intradermal, dermal, subcutaneous fat layer, epidermal layer and / or joint cavity).

[0059] The composition of one embodiment has a lower risk of side effects and higher stability than an injectable composition consisting of PLGA microparticles or PLLA microparticles alone, increases cell adsorption, has a maintenance period of more than one year, and can promote collagen activation and cell adsorption.

[0060] In addition, the present invention provides a composite composition that increases cell adsorption and promotes chondrocyte differentiation ability and collagen activity.

[0061] The above composite composition comprises a microparticle composition containing at least one of the PDO, PLGA, and PLLA of the present invention, and an excipient composition containing at least one of HA and nucleic acid molecules, and may additionally include a biocompatible carrier. The biocompatible carrier may include one or more selected from the group consisting of alginic acid and its salt, carboxymethyl cellulose and its salt, dextran and its salt, collagen, gelatin, and elastin, but is not limited thereto.

[0062] The composite composition of the present invention may additionally include a physiologically active substance, a local anesthetic, water for injection, sterile water, or distilled water depending on the use.

[0063] The present invention may provide a composite composition comprising a microparticle composition comprising one or more of PDO, PLGA, and PLLA, and an excipient composition comprising one or more of HA and nucleic acid molecules, and a method for preparing the same. The composite composition of one embodiment stably induces cell adsorption while increasing collagen production, has a maintenance period of one year or more, and can be usefully used as a filler or an intra-articular injection composition.

[0064] Of course, the scope of the present invention is not limited by these effects.

[0065] Figure 1 is a flowchart illustrating a method for preparing a composite composition for cell regeneration and collagen activation.

[0066] Figure 2 is a scanning electron microscope (SEM) image of PDO, PLGA, and PLLA microparticles constituting the microparticle composition.

[0067] Figure 3 is a scanning electron microscope (SEM) image of a mixture of HA and PN constituting an excipient composition.

[0068] Figure 4 is a scanning electron microscope (SEM) image of the present composition in which a microparticle composition containing a small amount of PLGA and PLLA microparticles is dispersed among excipients containing HA and nucleic acid molecules.

[0069] Figure 5 shows the results of confirming the degree of Collagen I and III production (12 weeks) using a rabbit arthritis model to evaluate the in vivo efficacy of a mixed composition of a microparticle composition containing small amounts of PLGA and PLLA microparticles in PDO microparticles and a bronchodilator composition.

[0070] Figures 6 and 7 show the results obtained by using a rabbit arthritis model to evaluate the in vivo efficacy of a mixed composition of a microparticle composition containing small amounts of PLGA and PLLA microparticles in PDO microparticles and an additive composition, using MMP-3, MMP-9, and MMP-13 mRNA and Collagen II and Agrrecan protein expression levels (12 weeks).

[0071]

[0072] The advantages and features of the present invention and the methods for achieving them will become clear by referring to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below but may be implemented in various different forms. These embodiments are provided merely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the present invention, and the present invention is defined only by the scope of the claims.

[0073] The terms used in this specification are for describing embodiments and are not intended to limit the invention. In this specification, the singular form includes the plural form unless specifically stated otherwise in the text. The terms "comprises" and / or "comprising" used in this specification do not exclude the presence or addition of one or more other components in addition to the components mentioned. Throughout the specification, the same reference numerals refer to the same components, and "and / or" includes each of the mentioned components and all combinations of one or more. Although terms such as "first," "second," etc., are used to describe various components, these components are not limited by these terms. These terms are used merely to distinguish one component from another. Therefore, the first component mentioned below may be the second component within the technical scope of the invention.

[0074] Unless otherwise defined, all terms used herein (including technical and scientific terms) may be used in a meaning commonly understood by those skilled in the art to which the present invention pertains. Additionally, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise.

[0075] In the following embodiments, singular expressions include plural expressions unless the context clearly indicates otherwise. In the drawings of the present disclosure, the size of components may be exaggerated or reduced for convenience of explanation. For example, the size and thickness of each component shown in the drawings are depicted arbitrarily for convenience of explanation, and therefore the present disclosure is not necessarily limited to what is depicted.

[0076] Where any embodiment of the present disclosure can be implemented differently, a specific order of operations may be performed differently from the order described. For example, two steps described consecutively may be performed substantially simultaneously or proceed in the reverse order of the order described.

[0077] In this disclosure, the word "exemplary" is used to mean "used as an example or illustration." Any embodiment described as "exemplary" in this disclosure should not be interpreted as being preferred or having an advantage over other embodiments.

[0078] Figure 1 is a flowchart illustrating the method for preparing a composite composition for cell regeneration and collagen activation. Figure 2 is a scanning electron microscope (SEM) image of PDO, PLGA, and PLLA microparticles constituting the microparticle composition. Figure 3 is a scanning electron microscope (SEM) image of a mixture of HA and PN constituting the excipient composition. Figure 4 is a scanning electron microscope (SEM) image of the composition of the present invention, in which a microparticle composition containing a small amount of PLGA and PLLA microparticles within PDO microparticles is dispersed among excipients containing HA and nucleic acid molecules. Figure 5 is a figure showing the results of confirming the degree of Collagen I and III production (12 weeks) using a rabbit arthritis model to evaluate the in vivo efficacy of a mixed composition of a microparticle composition containing a small amount of PLGA and PLLA microparticles within PDO microparticles and an excipient composition. Figures 6 and 7 show the results obtained by using a rabbit arthritis model to evaluate the in vivo efficacy of a mixed composition of a microparticle composition containing small amounts of PLGA and PLLA microparticles in PDO microparticles and an additive composition, using MMP-3, MMP-9, and MMP-13 mRNA and Collagen II and Agrrecan protein expression levels (12 weeks).

[0079] Hereinafter, with reference to FIGS. 1 to 7, the contents of the present invention will be explained in more detail through the following embodiments and experimental examples. However, the scope of the present invention is not limited only to the following embodiments and experimental examples, but includes variations of technical concepts equivalent thereto.

[0080] Example 1. Preparation of PDO microparticles

[0081] Solution 1-1 was prepared by dissolving 2g of PDO in 50mL of HFIP (Hexafluoroisopropanol). Solution 1-2 was prepared by dissolving 2g of PVA as a surfactant in 50mL of distilled water. Solution 1-1 and Solution 1-2 were mixed, and the prepared mixture was stirred at 400rpm for 3 days to remove the HFIP, thereby simultaneously obtaining Solution 1-3 containing PDO microparticles.

[0082] After stirring was finished, the polymer microparticles were allowed to settle by leaving the mixture for at least 24 hours, then the supernatant was removed and the PDO microparticles were separated. Distilled water was added to the separated PDO microparticles, and they were washed by stirring again. This washing step was performed a total of three times, and PDO microparticles were prepared (see Fig. 2a).

[0083] Example 2. Preparation of PLGA and PLLA microparticles

[0084] Solution 1-1 was prepared by dissolving 2g each of PLGA and PLLA in 50mL of chloroform. Solution 1-2 was prepared by dissolving 2g of PVA as a surfactant in 50mL of distilled water. Solution 1-1 and Solution 1-2 were mixed, and the chloroform was removed by stirring the prepared mixture at 400rpm for 3 days, thereby obtaining Solution 1-3 containing fine particles of PLGA and PLLA, respectively.

[0085] After stirring was finished, the polymer microparticles were allowed to settle by leaving the mixture for at least 24 hours, then the supernatant was removed and the PLGA and PLLA microparticles were separated. Distilled water was added to the separated PLGA and PLLA microparticles, and they were washed by stirring again. This washing step was performed a total of three times, and PLGA (see Fig. 2b) and PLLA (see Fig. 2c) microparticles were prepared (Fig. 2).

[0086] Example 3. Mixture of PDO, microparticles containing small amounts of PLLA and PLGA, and HA and PN excipients

[0087] A microparticle composition was prepared according to Table 1 below, and a total of 5 types of microparticle compositions totaling 10g were prepared.

[0088]

[0089] Each was added to 100 mL of sterile water for injection and stirred for 2 hours to prepare a completely dispersed solution. The dispersion was uniformly poured into a hemispherical mold with a diameter of 1 cm or into a cylindrical container and placed in an ultra-low temperature freezer at -75°C to be completely frozen for 12 hours.

[0090] Subsequently, a completely dried injectable composition was prepared by freeze-drying. The final composite, which was completed by mixing PDO+PLGA+PLLA : HA+PN in a ratio of 70 : 30 among the injectable compositions, is shown in Fig. 4.

[0091] Example 4. High-temperature, high-pressure hydrolysis treatment

[0092] By surface treating the microparticle compositions presented in Examples 1 and 2 above through a high-pressure steam sterilizer (120°C, 50 minutes), the cell engraftment rate was improved by hydrophilic properties and hydrolysis through surface modification of the microparticles.

[0093] Experimental Example 1. In vivo efficacy evaluation experiment

[0094] To evaluate the in vivo efficacy of a mixed composition of a microparticle composition containing PDO microparticles and an excipient composition, a rabbit arthritis model was utilized, and the degree of Collagen I and III formation was evaluated histologically through IF staining.

[0095] In addition, the expression levels of MMP-3, MMP-9, and MMP-13 mRNA, as well as Collagen II and Agrrecan proteins, were evaluated using qRT-PCR and Western blot with the same test model.

[0096] As shown in Figure 5, the test results observed the expression levels of Collagen I and III in cross-sections of rabbit joint tissue using a fluorescence microscope. At 12 weeks after administration of the test substance, the expression levels of Collagen I in the test group (product of the present invention) significantly increased (improved) by 268.71% and Collagen III by 498.83% compared to the negative control group (PBS). The expression level of Collagen I increased significantly by more than 13% when the composition of the present invention was administered compared to the administration of PN alone, and the expression level of Collagen III increased significantly by approximately 20% when the composition of the present invention was administered compared to the administration of HA alone (see Figure 5).

[0097] In the case of qRT-PCR for evaluating MMP-3, MMP-9, and MMP-13 mRNA expression levels, analysis was performed based on the MMP-3, MMP-9, and MMP-13 mRNA expression levels of the negative control (PBS) in rabbit joint tissue. As a result, the expression levels of MMP-3, MMP-9, and MMP-13 mRNA in the test group (product of the present invention) were significantly reduced (improved) by 85.52%, 87.12%, and 94.62%, respectively, compared to the negative control (PBS). A comparative test was also conducted with PN alone and HA alone regarding MMP-13 mRNA expression levels, and it was confirmed that MMP-13 mRNA expression levels were significantly reduced compared to the comparative example. (See Fig. 6) In addition, regarding the Western blot to evaluate Collagen II protein expression, analysis of the Collagen II protein expression in rabbit joint tissue based on the negative control (PBS) showed that at 12 weeks after administration of the test substance, the test group (product of the present invention) showed a tendency for Collagen II protein expression to increase (improve) by 39.64% compared to the negative control (PBS) (See Fig. 7).

[0098] This suggests that the mixed composition of the microparticle composition and the excipient composition of the present invention is effective in improving arthritis by inhibiting the secretion of Collagen I and III, MMP-3, MMP-9, and MMP-13, and enhancing the expression of Collagen II protein.

[0099]

[0100] <Reference Material 1>

[0101] Additional Example 1: In vitro cytotoxicity test (MTT assay) of the substance in Experimental Example 1 of the present invention

[0102]

[0103] An in vitro cytotoxicity test was conducted using the same composition as the mixture of PDO composite microparticles and HA and PN microparticles used in Experimental Example 1 of the present specification, and the results are as follows.

[0104] In this embodiment, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) analysis was performed to confirm human safety by comparing and evaluating the effects on cell viability of the substance of Experimental Example 1 of the present invention (hereinafter 'test substance') containing PDO, PLLA, PLGA, HA, and nucleic acid molecules, and the substance containing PLGA, HA, and PDRN (hereinafter 'comparative substance 1').

[0105]

[0106] 1. Treatment of test substance

[0107] Human articular chondrocyte (HC-a) cells were treated with the 'test substance' and 'comparative substance 1' of the present invention, respectively, to the final concentrations as follows.

[0108] Control: Group not treated with the test substance (treated with the same volume of medium containing water for injection)

[0109] Test Group 1: Treatment with test substance (microparticles of Experimental Example 1 of the present invention) at 10, 100 μg / mL

[0110] Comparison group: Treatment with 100 μg / mL of Comparison Substance 1 (microparticles containing PLGA, HA, and PDRN).

[0111]

[0112] 2. Measurement of cell viability via MTT assay

[0113] After culturing human articular chondrocytes (HC-a) cells, once the cell density reached 80%, the cells were transferred to 96-well plates for subculture. Subsequently, cell viability was measured using MTT reagent. In summary, HC-a cells were seeded into 96-well plates and cultured. The test substance and Comparison 1 were prepared and applied to each 96-well plate. After stimulating for 24 hours, 10 μl of MTT solution was added to each well, and the plates were incubated at room temperature for 4 hours. DMSO, a solubilizing solution, was added to dissolve the formed formazan crystals. The absorbance of the solution was measured at a wavelength of 570 nm using a microplate reader. Cell viability for each condition was calculated based on the absorbance values.

[0114]

[0115] 3. Result Analysis

[0116] Cell viability was calculated according to the following mathematical formula 1, with the absorbance value of the control group not treated with the test substance set to 100%.

[0117] [Mathematical Formula 1]

[0118] Cell viability (%) = (Absorbance of control group / Absorbance of test substance treated group) × 100

[0119]

[0120] The results are shown in Table 2 and Figure 8 below.

[0121]

[0122]

[0123]

[0124] As a result, the substance used in Experimental Example 1 of the present invention showed a significantly higher cell viability compared to Comparative Substance 1 (Cited Invention 1) treated at the same concentration (100 μg / mL). This strongly suggests that the substance of the present invention has significantly lower cytotoxicity compared to the comparative substance, thereby possessing excellent safety for in vivo application.

[0125]

[0126] <Reference Material 2>

[0127] Experimental Objective: Intentional inflammation was induced in chondrocytes using the same composition as the mixture of PDO composite microparticles and HA and PN microparticles used in Experimental Example 1 of the present specification, and the degree of inflammation alleviation was confirmed when samples were subsequently treated (nitrogen oxide (NO) quantification test).

[0128] Experimental Method:

[0129] 1) Dissolve the inflammatory cytokine (IL-1β) in PBS dissolved in 0.1% BSA to obtain a solution with a final concentration of 50 μg / ml.

[0130] 2) Chondrocyte cells were seeded into a 96-well plate, and each type of sample was added and cultured for 24 hours.

[0131] 3) Each experimental group was measured three times, and statistical significance between the two groups was verified using an independent sample t-test (Welch's T-test) assuming heteroscedasticity.

[0132] The closer the NO concentration is to the Control value, the greater the anti-inflammatory effect.

[0133]

[0134] Classification NO Concentration, uM123AvgControl 3.4652 3.5496 3.2601 3.424967 IL-1 β 4.5136 4.5065 4.6295 4.549867 IL-1 β + Comparative Substance 2 (Citation 2) Composition (PDO + PLLA + PCL) 3.8066 3.953 3.8427 3.867 433 IL-1 β + Present Composition (PDO + PLLA + PLGA + HA + PN) 3.389 3.3382 3.4307 3.385967

[0135] Referring to Table 3 and Figure 9, the average measured NO concentrations show that when the composition of the present invention is treated with IL-1 β, the values ​​are similar to those of the control group (untreated group) (average 3.3860 uM). However, when the composition of Comparative Substance 2 (Citation 2) is treated with IL-1 β, the NO values ​​are approximately 13% higher than those of the control group (untreated group) (average 3.8674 uM), indicating that the composition of the present invention significantly inhibited NO production more than the composition of Comparative Substance 2 (Citation 2) (p < 0.01). Through this, it was confirmed that the composition of the present invention has an excellent anti-inflammatory effect, showing a statistically significant difference compared to other samples.

[0136] Therefore, it is confirmed that the composition of the present invention exhibits an unpredictable and significant effect compared to the composition of comparative substance 2 (Citation 2).

[0137]

[0138] Although embodiments of the present invention have been described above, the present invention is not limited to the above embodiments and can be manufactured in various different forms, and those skilled in the art will understand that the present invention can be implemented in other specific forms without changing the technical concept or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0139]

Claims

1. i) A step of preparing a composite microparticle composition comprising polydioxanone (PDO) microparticles, poly-L-lactic acid (PLLA) microparticles, and poly(lactic-co-glycolic acid (PLGA) microparticles; ii) a step of preparing an excipient composition comprising HA and nucleic acid molecules; iii) a step of preparing a mixed composition by mixing the above-mentioned microparticle composition and excipient composition; iv) a step of preparing a dispersion by dispersing the above mixed composition in a solvent in which a dispersant is dissolved; and v) A step of freeze-drying the above dispersion to produce a freeze-dried body, and A method for preparing a composite composition, further comprising the step of modifying the surface of each microparticle prepared in step i) by hydrolyzing said microparticle at 100 to 140 ℃ for 30 to 100 minutes.

2. In Paragraph 1, A method for manufacturing a composite composition, characterized in that the weight ratio of the fine particle composition is 60 to 99.99 weight% based on the total weight of the composite composition.

3. In Paragraph 1, A method for preparing a composite composition, characterized in that the weight ratio of the excipient composition is 0.01 to 40 weight% based on the total weight of the composite composition.

4. In Paragraph 1, A method for preparing a composite composition, characterized in that the above-mentioned dispersant is one or more selected from the group consisting of cells, growth factors, peptides, amino acids, proteins, sugars, and natural polymers.

5. In Paragraph 4, A method for preparing a composite composition, characterized in that the above natural polymer is one or more selected from the group consisting of chitosan and alginate.

6. In Paragraph 1, A method for preparing a composite composition, characterized in that in step v) above, the freeze-drying is performed at a temperature of -90 to -60 ℃ for 10 to 20 hours.

7. In Paragraph 1, A method for preparing a composite composition, characterized in that the microparticles in step i) are provided from the following steps: ii) preparing a first-1 solution by dissolving each of PDO, PLGA, and PLLA in a solvent; i-ii) preparing a first-2 solution by dissolving a surfactant in distilled water; i-iii) preparing a first-3 solution by mixing and stirring each of the first-1 solution and the first-2 solution, and then removing the solvent; and i-iv) obtaining microparticles by precipitating each of the microparticles in the first-3 solution and then removing the supernatant.

8. In Paragraph 7, A method for preparing a composite composition, characterized in that, in step ii) above, the solvent is one or more selected from the group consisting of chloroform, acetone, acetonitrile, dioxane, methoxybenzene, dichloromethane, dimethylformamide, dimethylsulfoxide, ethyl acetate, hexafluoroisopropanol, and anisole.

9. In Paragraph 7, A method for preparing a composite composition, characterized in that, in step i-ii) above, the surfactant is PVA (Polyvinyl alcohol).

10. In Paragraph 1, The above composite composition is a composition for treating arthritis, and a method for preparing a composite composition.

11. In Paragraph 1, A method for preparing a composite composition, wherein the above composite composition is a composition for improving skin condition.

12. In Paragraph 1, A method for preparing a composite composition in which the nucleic acid molecule comprises at least one of PN and PDRN.

13. A composition comprising microparticles comprising polydioxanone (PDO), poly-L-lactic acid (PLLA), and poly(lactic-co-glycolic acid, PLGA), and an excipient composition comprising HA and nucleic acid molecules, and A composite composition in which the above-mentioned microparticles have been hydrolyzed and their surfaces modified.

14. In Paragraph 13, The above composite composition is characterized by increasing the secretion amount of one or more of the growth factors VEGF (vascular endothelial growth factor) and FGF-2 (fibroblast growth factor-2).

15. In Paragraph 13, The above composite composition is characterized by increasing the expression of Collagen type I and Collagen type III.

Images