WO2021033810A1 - Injectable composition for tissue repair and preparation method therefor - Google Patents

Abstract

The present invention relates to an injectable composition for tissue repair. According to the present invention, an injectable composition for tissue repair and a method for preparing same can be provided, in which the injectable composition as a ready-to-use injectable composition without the need for dilution before use induces an immediate tissue repair effect and collagen production, thus exhibiting a long-lasting tissue repair effect, and further comprises a polynucleotide, and thus can restore skin health damaged by aging and stimuli, through improvement in physiological environments in the body. In addition, in the injectable composition for tissue repair according to the present invention, natural extracts are uniformly mixed in microparticles using a biodegradable polymer, and thus, when the microparticles are injected as an injection, the occurrence of side effects such as inflammatory responses, edema, and dermal necrosis can be prevented.

Description

Injectable composition for tissue repair and its manufacturing method

The present invention relates to an injectable composition for tissue repair and a method for manufacturing the same, and more particularly, to an injectable composition for tissue repair showing an effect of improving damaged skin, an immediate tissue repair effect, and a long-term tissue repair effect through collagen formation, and a manufacturing method thereof. .

Injectable compositions for tissue repair are generally for cosmetic purposes, for filling or replacing biological tissues (filling of wrinkles, remodeling of the face, increasing lip volume, etc.), and It has been used for therapeutic purposes to rehydrate the skin by mesotherapy.

Hyaluronic acid, which is used as an injection composition for tissue repair, has a problem of inadequate long-lasting effect because it is reabsorbed in vivo very quickly between 2 weeks and 2 months. Accordingly, products in which hyaluronic acid and a crosslinked material are crosslinked to each other to extend the reabsorption period, as in Korean Patent Laid-Open No. 10-2004-0072008, are on the market. However, these crosslinked products have also been reported to have problems due to toxicity of the crosslinking material.

In order to replace the above injection composition for tissue repair containing hyaluronic acid, a number of products for tissue repair using polymers that can be degraded in vivo have also been developed.It is a formulation using existing biocompatible polymers. After processing into sized particles, it was developed and used as a formulation dispersed through a viscous media.

Formulation in which polylactic acid (PLA) having a particle diameter of 20 to 50 μm is dispersed in an aqueous solution of carboxymethylcellulose (CMC) or polycaprolactone having a particle diameter of 20 to 50 μm (Polycaprolactone) (PCL) particles were dispersed in CMC and glycerin (Glycerin) aqueous solution was used.

The formulation using the above-described polymer that can be degraded in vivo has a problem in that the microparticles are clogged in the needle during injection, and there is a problem in that the particles are not uniformly dispersed, so that a uniform tissue repair effect cannot be obtained.

In addition, the polymer may be injected into the body to induce collagen formation, thereby exhibiting a tissue repair effect, but, like hyaluronic acid, the effect of immediate tissue repair was insufficient.

Accordingly, when an injection composition for tissue repair is administered by injection, it can exhibit an immediate tissue repair effect and a long-lasting tissue repair effect, reduce discomfort in the procedure, and the number of tissues that can be used with particles evenly dispersed. There is an urgent need to prepare an injectable composition for administration.

In addition, there is a need to develop a product that can prevent problems such as inflammatory reactions, swelling, and skin necrosis that occur as side effects by administration of an injection composition for tissue repair.

It is an object of the present invention to provide an injectable composition for tissue repair and a method for producing the same, capable of continuously repairing tissue for a long period of time by inducing an immediate tissue repair effect and collagen production.

Another object of the present invention is to provide an injection composition for tissue repair and a method of manufacturing the same, which is injected into the body and can restore the health of skin damaged by aging and irritation through improvement of the physiological environment.

Another object of the present invention is that natural extracts are uniformly mixed in microparticles using biodegradable polymers, so when the microparticles are injected as an injection, the natural extracts are introduced into the body as they are decomposed in the body, resulting in inflammatory reactions, swelling and skin. To provide an injection composition for tissue repair that can prevent the occurrence of side effects such as necrosis and a method of manufacturing the same.

Another object of the present invention is an injection composition that is ready for use without the need to dilute before use, when used as an injection, and can be used immediately, and an injection composition for tissue repair showing a uniform tissue repair effect by dispersing the injection composition uniformly And to provide a method of manufacturing the same.

In order to achieve the above object, the injection composition for tissue repair according to an embodiment of the present invention comprises microparticles comprising a biodegradable polymer; Hyaluronic acid; And a polynucleotide, wherein the microparticles are evenly distributed in a fine powder form of a natural extract, and the natural extract includes centella asiatica extract.

The injection composition for tissue repair is ready to use without needing to be diluted before use.

The natural extracts further include those selected from the group consisting of beetroot extract, aloe vera extract, ginkgo biloba extract, and mixtures thereof.

The biodegradable polymer is one or more selected from the group consisting of polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, and derivatives thereof.

The microparticles are bioabsorbed within 1 to 3 years after injection into the body.

The average particle diameter of the microparticles is 20 to 70㎛.

A method of preparing an injection composition for tissue repair according to another embodiment of the present invention includes: 1) preparing a suspension by mixing a natural extract in the form of fine particles in an organic solvent in which a biodegradable polymer is dissolved; 2) preparing a second mixture by dissolving a surfactant in water; 3) injecting the suspension of step 1) into a microchannel in a linear direction to flow; 4) Injecting the second mixture of step 2) into microchannels formed on both sides or one side so that the suspension of step 3) can form an intersection with the microchannels flowing in a linear direction, and flowing, the straight line of the suspension Preparing microparticles in a form in which natural extracts in the form of fine particles are evenly distributed in spherical biodegradable polymer particles by crossing the flow in the direction and the flow of the second mixture; 5) collecting the microparticles generated at the intersection of step 4); 6) stirring the microparticles collected in step 5) to evaporate and remove the organic solvent present in the microparticles; 7) washing and drying the microparticles of step 6), and 8) secondary drying the spherical microparticles of step 7) by vacuum or freeze drying; And 9) the secondary dried spherical microparticles of step 8). Hyaluronic acid; It includes preparing an injection composition for tissue repair by mixing polynucleotide and water for injection.

The natural extract in the form of fine particles in step 1) is prepared in the form of fine particles by pulverizing the natural extract after freeze drying, and the fine particles have a diameter of 10 to 5000 nm.

The natural extracts further include those selected from the group consisting of beetroot extract, aloe vera extract, ginkgo biloba extract, and mixtures thereof.

The biodegradable polymer is one or more selected from the group consisting of polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, and derivatives thereof.

Hereinafter, the present invention will be described in more detail.

Injectable composition for tissue repair according to an embodiment of the present invention comprises microparticles comprising a biodegradable polymer; Hyaluronic acid; And a polynucleotide, wherein the microparticles are evenly distributed in a fine powder form of a natural extract, and the natural extract includes centella asiatica extract.

In the present invention, tissue repair may mean temporarily or semi-permanently improving or repairing wrinkles on the face or body by injection of the composition, improving contours, forming a sense of volume of tissues, and regenerating tissues such as scar healing. The tissue may mean a face or a part of a body.

In the present invention, hyaluronic acid is used in the sense of including all of the hyaluronic acid itself as well as its salts and derivatives. Therefore, the hyaluronic acid aqueous solution used hereinafter is a concept including both an aqueous solution of hyaluronic acid, an aqueous solution of hyaluronic acid salt, an aqueous solution of hyaluronic acid derivative, and an aqueous mixture of these.

The hyaluronic acid is a linear polymer polysaccharide in which β-DN-acetylglucosamine and β-D-glucuronic acid are alternately linked, and is distributed in connective tissues such as subcutaneous tissues and cartilage tissues of mammals. It is present in the umbilical cord, etc., and is known to exist in the capsular membrane of streptococcus or bacillus. General methods for obtaining hyaluronic acid include extraction from chicken heads, umbilical cords, and the like, and methods of extracting and purifying streptococci and bacillus after culturing them.

Natural hyaluronic acid, which has excellent biocompatibility, does not have cross-species specificity, does not have tissue or organ specificity, improves skin moisturization, maintains skin elasticity, reduces damage to the lower layers of the skin in case of skin damage, and contains collagen, a major component of the skin. It also acts like a lubricant to smooth the movement through the silo.

When the hyaluronic acid is used as an injection composition for tissue repair and is injected into the body, hyaluronic acid attracts water molecules, thereby adding moisture and elasticity to the body.

However, since such an injection composition for tissue repair containing hyaluronic acid has a maintenance period of only 6 to 12 months, there is an inconvenience that the injection composition must be periodically injected into the body. In particular, when natural hyaluronic acid is used as it is, mechanical properties are not good, and because it is easily decomposed and removed by an enzyme called hyaluronidase present in the body, there are limitations in various applications.

Accordingly, technologies that use microparticles containing biodegradable polymers as an injection composition for tissue repair containing hyaluronic acid have been developed, not only filling the volume in the depression in the body, but inducing collagen production in the muscle, The tissue repair effect is maintained for a long time.

However, in the case of such microparticles, there arises a problem that an immediate tissue repair effect does not appear immediately after injection into the body.

Therefore, in order to improve the problems of using hyaluronic acid and microparticles as an injection composition for tissue repair, the injection composition for tissue repair according to an embodiment of the present invention includes microparticles containing a biodegradable polymer; Hyaluronic acid; And a polynucleotide, wherein the microparticles are evenly distributed in a natural extract in the form of a fine powder.

That is, by combining the advantages of using hyaluronic acid and the case of using microparticles containing biodegradable polymers, a short-term tissue repair effect can be obtained, and a natural skin gloss can be obtained by adding moisture to the skin, and the microparticles By evenly distributing the natural extract in the form of fine powder, it is possible to improve the maintenance period of the tissue repair effect by inducing collagen production.

In addition, as a polynucleotide, which has a skin regeneration effect, is additionally included, the health of the skin damaged by aging and irritation can be restored through improvement of the physiological environment in the body.

In addition, since the microparticles are evenly distributed in a fine powder form of natural extract, even when injected directly into the body, side effects such as inflammatory reaction, swelling, and skin necrosis can be prevented.

The natural extract is centella asiatica extract.

Centella asiatica extract is a plant that originates in the tropical regions of the Indian Ocean and grows ideally at an altitude of 600 meters above sea level in humid and shady areas, and is also called gotu kola, indian pennywork or tiger grass. These centella asiaticoside, madecasosside, asiatic acid and madecassic acid, called triterpenoids (saponins), are present in the body, and they regulate collagen synthesis in the human body. It is known to promote blood circulation by strengthening the vestibule, and to help improve memory.

The natural extracts further include those selected from the group consisting of beetroot extract, aloe vera extract, ginkgo biloba extract, and mixtures thereof.

Angelica dahurica BENTH (Angelica dahurica BENTH) is a biennial or triennial plant of the parsley family, a dicotyledonous plant, and has a thick rhizome, and the stem stands upright and grows to a height of 1.5m while pruning branches. The leaves are divided twice in the shape of a feather, and the broken pieces are oval or lanceolate, with a sharp tip, and the edges are unevenly split or serrated. It is distributed nationwide, and it grows in wet places such as mountain streams. It has the effect of pain relief and small bells, and the applied diseases include headache, migraine, various neuralgia, toothache, and abdominal pain.

The natural extract refers to an extract extracted by a general extraction method that can be extracted by an expert in the art, and the extract is used in the form of a powder.

Specifically, the Guritdae extract and Centella asiatica extract are fermented products of Guritdae and Centella asiatic fermented using black yeast, which are freeze-dried and used in powder form.

The black yeast (Aureobasidium pullulans) contains a large amount of beta glucan and is known to be excellent in moisturizing, elasticity, and atopic pruritus. Since beta-glocans constitute the cell wall of yeast, amino acids that act as natural moisturizing factors add elasticity as well as moisturization, and excellent skin affinity can increase immunity.

The fermented product of Guritdae and Centella asiatica is fermented using black yeast and uniformly mixed in microparticles using a biodegradable polymer included in the tissue repair injection composition of the present invention.

When fermentation of the beetroot and centella asiatica using black yeast, it is possible to increase the extraction rate of the content of activated active ingredients in the beetroot and centella asiatica, and exist as a more stable compound, so that immediate tissue repair when prepared as an injection composition for tissue repair It can increase the effect of restoring the health of damaged skin by collagen formation, aging and irritation.

Preferably, the microparticles of the tissue repair injection composition may include 30 to 50 parts by weight of fermented beetroot and 30 to 50 parts by weight of fermented centella asiatica per 100 parts by weight of the biodegradable polymer.

The black yeast fermented copper stalk fermented product and centella fermented product are uniformly mixed in the microparticles using the biodegradable polymer of the present invention, and when the microparticles are injected as an injection, they are introduced into the body as they are decomposed in the body, resulting in an inflammatory reaction. It is possible to provide an injection composition for tissue repair and a method of manufacturing the same, which can prevent the occurrence of side effects such as edema and skin necrosis.

In addition, compared to a composition for tissue repair using only microparticles using a biodegradable polymer, the tissue repair effect can be maintained for a long time, and when the natural extract is distributed in the microparticles in the form of fine powder, hyaluronic acid and polynucleotide ( Polynucleotide) and other constituents are mixed together to increase the effect of recovering the health of damaged skin due to collagen formation, aging and irritation.

Therefore, according to an embodiment of the present invention, the composition for tissue repair is uniformly distributed in microparticles containing a biodegradable polymer in a fine powder form of a fermented beetroot and a fermented centipede.

Preferably, the microparticles of the composition for tissue repair of the present invention may further include aloe vera fermented with black yeast and gingko leaves in addition to the fermented copper stalk fermented product and centella asiatic fermented product fermented with black yeast.

Aloe Vera (Barbados aloe) is native to the Mediterranean region and Africa. The gel-like sap contained in the leaves is known to be effective in treating burns and arthritis. It is a bushy perennial plant, and the stem is short with a height of 30-60cm and grows up to 100cm. From this stem, many long and hard leaves come out in a rosette shape. The leaves are 15-40cm long and 2-7cm wide, with white spots on a pale green background, thick, fleshy, and succulent. The bottom is wide and the top is narrowed, and the back is round and the front is slightly indented. There are small greyish thorns in the shape of teeth on the edge. There is a slight groove at the tip of the leaf and the tip is sharp. Small coffin-shaped flowers bloom in winter and spring. Flowers are bright yellow or orange in color, forming a racemic inflorescence and blooming downwards. It is easy to cultivate, is resistant to diseases and pests, and has excellent oxygen generation at night, making it a good indoor air purification plant. It is suitable for cultivation indoors such as bedrooms, living rooms, and offices, and is widely used as a medicinal plant. It is used for various purposes such as raw materials for cosmetics and pharmaceuticals, edible, and has been found to be effective in sterilization and neutralization of toxins, fatigue recovery and skin neutralization, antipyretic, constipation and ulcers. It contains about 300 different ingredients such as vitamins A, C, D, E, B12, fatty acids, amino acids, selenium, calcium and aloin, aloesin. India, North Africa, Manila, etc. are very wide.

Ginkgo biloba contains flavone glycosides such as quercetin, camperol, and isorhamnetin, so antioxidant and cytoprotective effects have been reported.Bilobadide, zincolide ( It contains terpenes (heterosides) such as ginkgolide) A, Zincolide B, Zincolide C, and Zincolide J. It is used to inhibit platelet aggregation by platelet activating factor (PAF) and ADP, and to relax blood vessels. It is reported to be dragon. In addition, it has corticoid-like, anti-ischemic properties, and is known as an antagonist of terminal benzodiapine receptors that induces anti-stress activity.

As aloe vera fermented with the black yeast and ginkgo biloba are additionally included, the effect of recovering damaged skin health due to collagen formation, aging, and irritation due to the mixing action between each component is increased.

Preferably, the microparticles of the tissue repair composition are based on 100 parts by weight of the biodegradable polymer, 30 to 50 parts by weight of fermented beetroot, 30 to 50 parts by weight of fermented centella, 30 to 50 parts by weight of fermented aloe vera and fermented ginkgo leaves It may contain from 10 to 20 water.

More preferably, according to an embodiment of the present invention, the composition for tissue repair is uniformly distributed in microparticles containing a biodegradable polymer in which a fermented beetroot fermented product, a centella fermented product, aloe vera fermented product, and a ginkgo leaf fermentation product in fine powder form are evenly distributed. will be.

The composition for tissue repair of the present invention and a method for preparing the same include distributing a natural extract in the form of fine powder evenly on microparticles containing a biodegradable polymer.

The biodegradable polymer is one or more selected from the group consisting of polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, and derivatives thereof.

Specifically, the biodegradable polymer refers to a property that medical materials are harmless and easy to adapt to the living body. Since the target organism is a wide range of various tissues and organs, including blood, it is subdivided into blood compatibility (antithrombogenicity) and tissue compatibility, but the biodegradable polymer refers to a polymer that can be widely applied.

The microparticles are bioabsorbed within 1 to 3 years after injection into the body.

More specifically, the tissue repair injection composition is bioabsorbed within 1 to 3 years after injection into the body, and has a tissue repair effect for a short period of time, that is, 6 to 12 months by hyaluronic acid, and thereafter, tissue repair by microparticles The effect appears.

The average particle diameter of the microparticles is 20 to 70㎛.

When microparticles containing a conventional biodegradable polymer are used as an injection composition for tissue repair, there is a problem in that the particle diameter of the microparticles is uneven, and the early repair effect does not appear evenly when injected into the body.

Accordingly, the microparticles contained in the tissue repair injection composition of the present invention have an average diameter of 20 to 70 μm, and can be injected into the body as microparticles having a certain size, and may have a uniform tissue repair effect.

Preferably, in the case of the particle size, the injection composition is smoothly injected into the body, and it can exhibit a uniform tissue repair effect, which can induce an immediate tissue repair effect and collagen production, and damaged by aging and irritation through improvement of the physiological environment. It can be provided as an injection composition for tissue repair that can restore skin health.

Conventional tissue repair compositions are used in the form of diluting in water for injection before use for injection into the body and injecting them.

That is, the prepared composition for tissue repair is sold without being mixed with water for injection, and is mixed with water for injection before injection by injection in a hospital, and then injected into the body.

In the case of such injection, it is unclear whether it is possible to uniformly mix according to the amount of water for injection or whether it is mixed with appropriate water for injection, and it is inevitable to depend on the skill level of the user.

The composition for tissue repair is uniformly mixed in water for injection as well as the components constituting it to minimize pain to the patient at the time of injection, and as a uniformly mixed injection agent is injected, it provides an even tissue repair effect when injected into the body. Can be indicated.

That is, when a non-uniform injection is injected, not only a foreign body sensation and pain can be felt when the injection is administered to a patient, but also a difference can be exhibited in the tissue repair effect due to the non-uniformity of the injection in the body.

Accordingly, in the present invention, the composition for tissue repair is provided as an injection composition, which is provided in a ready-to-use form, so that the user can omit the step of mixing in water for injection before injection and provide it in a form that can be injected immediately. do.

The tissue repair injection composition includes microparticles comprising a biodegradable polymer; Hyaluronic acid; And a polynucleotide, and in actual use, aggregation between microparticles occurs due to attractive force between microparticles. Therefore, a cumbersome problem arises in that the process of suspending and shaking must be repeated.

Accordingly, the injection composition for tissue repair according to an embodiment of the present invention does not need to be diluted before use, is ready to use, and can be directly injected into the body, and the problem of aggregation between microparticles in the injection composition Does not occur, so the injection composition can be uniformly dispersed.

In order to provide the tissue repair injection composition in a ready-to-use form, a syringe made of a polyolefin resin, a polypropylene resin, and a mixture thereof and a rubber stopper is filled.

At this time, the tissue repair composition to be mixed with the tissue repair injection composition includes 30 to 50 parts by weight of hyaluronic acid and 30 to 50 parts by weight of polynucleotide based on 100 parts by weight of spherical microparticles.

It exhibits excellent effects on the initial tissue repair effect and organ tissue repair effect within the above range, and can be used in an optimal range capable of recovering skin health damaged by collagen formation, aging, and stimulation by polynucleotides.

In order to be prepared in the ready-to-use form, an injection solution for tissue repair is prepared using 2.0 to 5.0 ml of a suspension solution for 1 vial (84.0 mg) of the tissue repair composition.

Thereafter, it is mixed with 100 ml of 0.9% physiological saline for injection to prepare an injection composition for tissue repair that is ready for use.

The suspension solvent is prepared by mixing D-mannitol, sodium carboxymethylcellulose, polysorbate 80, and water for injection.

A method of preparing an injection composition for tissue repair according to another embodiment of the present invention includes: 1) preparing a suspension by mixing a natural extract in the form of fine particles in an organic solvent in which a biodegradable polymer is dissolved; 2) preparing a second mixture by dissolving a surfactant in water; 3) injecting the suspension of step 1) into a microchannel in a linear direction to flow; 4) Injecting the second mixture of step 2) into microchannels formed on both sides or one side so that the suspension of step 3) can form an intersection with the microchannels flowing in a linear direction, and flowing, the straight line of the suspension Preparing microparticles in a form in which natural extracts in the form of fine particles are evenly distributed in spherical biodegradable polymer particles by crossing the flow in the direction and the flow of the second mixture; 5) collecting the microparticles generated at the intersection of step 4); 6) stirring the microparticles collected in step 5) to evaporate and remove the organic solvent present in the microparticles; 7) washing and drying the microparticles of step 6), and 8) secondary drying the spherical microparticles of step 7) by vacuum or freeze drying; And 9) the secondary dried spherical microparticles of step 8). Hyaluronic acid; It includes preparing an injection composition for tissue repair by mixing polynucleotide and water for injection.

The organic solvent in step 1) is one or more selected from the group consisting of dichloromethane, chloroform, chloroethane, dichloroethane, trichloroethane, and mixtures thereof.

The natural extract in the form of fine particles in step 1) is prepared in the form of fine particles by pulverizing the natural extract after freeze drying, and the fine particles have a diameter of 10 to 5000 nm.

When the natural extract is prepared in the form of fine particles with a diameter of 10 to 5000 nm, it can be uniformly mixed in microparticles using a biodegradable polymer, and when injected as an injection, the natural extract is evenly introduced into the body and decomposed, resulting in inflammation. It is possible to provide an injection composition for tissue repair and a method of manufacturing the same that can prevent the occurrence of side effects such as reaction, swelling and skin necrosis.

The natural extracts further include those selected from the group consisting of beetroot extract, aloe vera extract, ginkgo biloba extract, and mixtures thereof.

The biodegradable polymer is one or more selected from the group consisting of polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, and derivatives thereof.

The method for preparing an injection composition for tissue repair according to another embodiment of the present invention is not particularly limited in use, and may be used as a skin cosmetic or medical filler requiring resorption in vivo, and in particular, subcutaneous or It can be used as an injectable filler in the skin, but is not limited to the above example.

According to the injection composition for tissue repair of the present invention, as it induces an immediate tissue repair effect and collagen production, it shows a tissue repair effect continuously for a long time, and as it further contains a polynucleotide, aging and aging through improvement of the physiological environment in the body It is possible to provide an injection composition for tissue repair and a method of manufacturing the same that can restore the health of skin damaged by irritation.

In addition, the tissue repair injection composition of the present invention is uniformly mixed with natural extracts in microparticles using biodegradable polymers, so when the microparticles are injected as an injection, side effects such as inflammatory reaction, swelling and skin necrosis are prevented. It can be prevented, and it is provided as a ready-to-use injection composition without the need to dilute before use.

1 is an experimental result of suppression of nitric oxide (NO) production of an injection composition for tissue repair according to an embodiment of the present invention.

2 is an experimental result of inhibition of PGE production of an injection composition for tissue repair according to an embodiment of the present invention.

3 is an experimental result of the inhibition of TNF-α production of an injection composition for tissue repair according to an embodiment of the present invention.

4 is an experimental result of inhibition of IL-1β production of an injection composition for tissue repair according to an embodiment of the present invention.

The present invention is a microparticle containing a biodegradable polymer; Hyaluronic acid; And a polynucleotide, wherein the microparticles are evenly distributed in a fine powder form of a natural extract, and the natural extract includes a centella asiatica extract.

1. Preparation of natural extract

Preparation of beetroot powder

After washing and removing moisture, hot water was extracted for 4 hours at a temperature of 130°C in an extractor, and then dried at -40 to -44°C. The freeze-dried copper plate was pulverized into a powder of 300 mesh or more to prepare 84.6 nm copper plate powder (CP).

Preparation of other natural extracts

Using the same method as the method for preparing the beetroot powder, 84.6 nm centella powder (AP), aloe vera powder (RP), and ginkgo leaf powder (GP) were prepared.

Preparation of fermented beetroot powder

After washing and removing moisture, the beetroot was fermented at low temperature for 48 hours in a fermentor mixed with black yeast. Thereafter, hot water extraction was performed at a temperature of 130° C. in an extractor for 4 hours, and then dried at -40 to -44° C. temperature condition. The freeze-dried copper plate was pulverized into a powder of 300 mesh or more to prepare 84.6 nm fermented copper plate powder (BCP).

Preparation of other fermented natural powders

Using the same method as the method for preparing the beetroot powder, 84.6 nm fermented centella powder (BAP), fermented aloe vera powder (BRP), and fermented ginkgo leaf powder (BGP) were prepared.

2. Preparation of microparticles containing biodegradable polymers

A suspension was prepared by mixing the 84.6 nm copper stalk powder, centella asiatica powder, aloe vera powder and ginkgo leaf powder in a solvent in which polycaprolactone (PCL) having a molecular weight (Mn) of 45,000 was dissolved in dichloromethane (boiling point: 39.6°C). .

As a surfactant, polyvinyl alcohol (PVA) having a molecular weight of 85,000 to 124,000 was dissolved in water at 0.25% by weight to prepare 250 mL of a second mixture.

The suspension was injected into a microchannel to flow at 100 μl/min, and the second mixture was injected to flow in the first flow and the second flow with 90° on both sides of the suspension flow, and then at 1000 μl/min. Let it flow.

A dispersed phase in the form of droplets was formed at the intersection of the suspension and the second mixture and in a single flow after the intersection. The dispersed phase in the form of droplets was collected in 100 mL of a receiver solution by dissolving polyvinyl alcohol (PVA) at 0.25% by weight in purified water, and allowed to stand at room temperature (25°C) for about 24 hours to extract a dichloromethane solvent. After the second mixture containing microparticles is stirred, the microparticles are washed and the residual polyvinyl alcohol and dichloromethane solution are evaporated to remove them. Thereafter, microparticles in which natural extracts in the form of fine particles are evenly distributed in spherical biodegradable polymer particles through washing and drying processes of the microparticles were finally mixed in a composition ratio as shown in Table 1 below.

	MX1	MX2	MX3	MX4	MX5	MX6	MX7	MX8	MX9	MX10	MX11	MX12	MX13
PCL	100	100	100	100	100	100	100	100	100	100	100	100	100
BCP	-	20	30	40	50	60	-	-	-	-	-	-	-
BAP	-	20	30	40	50	60	50	50	-	-	-	-	-
BRP	-	20	30	40	50	60	-	50	-	-	-	-	-
BGP	-	-	5	10	20	30	20	20	-	-	-	-	-
CP	-	-	-	-	-	-	-	-	20	30	40	50	60
AP	-	-	-	-	-	-	-	-	20	30	40	50	60
RP	-	-	-	-	-	-	-	-	20	30	40	50	60
GP	-	-	-	-	-	-	-	-	-	5	10	20	30

(Unit: parts by weight)

3. Preparation of injection composition for tissue repair

For each 100 parts by weight of each of the freeze-dried spherical microparticles MX1 to MX13, 40 parts by weight of hyaluronic acid (manufacturer: SK Bioland, Korea) and polynucleotides (NEB (New England Biolabs, USA) (Purchased from) 40 parts by weight and uniformly suspended in 2.0 mL of a suspension solution to prepare injectable compositions TL1 to TL13 for tissue repair.

The suspension solvent was prepared in the composition shown in Table 2 below.

Content standard	Purpose of mixing	Ingredient name	amount	unit
2.0 mL	Isotonic topic	D-Mannitol	100.0	mg
	Suspending agent	Soduim Carboxymethylcellulose	10.0	mg
	Suspending agent		Polysorbate 80	10.0	mg
	solvent	Injection water	Remainder

The suspended tissue repair injection compositions TL1 to TL13 were mixed in 100 ml of 0.9% physiological saline for injection, and filled in a syringe made of a cyclic polyolefin-based resin and a syringe made of a rubber stopper, and prepared in a ready-to-use form.

[Experimental Example 1: Toxicity Test]

In order to evaluate the toxicity of the prepared tissue repair injection compositions TL1 to TL13, differences in toxicity and side effects expressions were confirmed when the administration of the tissue repair injection compositions TL1 to TL13 in a rat repeated dose toxicity test model.

Six-week-old male and female SD rats were administered orally once per group at the same morning time each day after administration of the tissue repair injection composition for 13 weeks. One dose was administered in an amount of 3.75 mg/kg to 5 mg/kg. Thereafter, mortality, general symptoms, weight change, feed and water intake were observed.

As a result, no dead individuals occurred in the group administered with the tissue repair injections TL1 to TL13 within the experimental period, and in view of the above experimental results, it was confirmed that the toxicity of the injection composition for tissue repair of the present invention was not a problem.

[Experimental Example 2: Evaluation of homogeneous mixing]

It was evaluated visually whether or not homogeneous mixing according to the manufacturing method of the injection composition for tissue repair according to an embodiment of the present invention. After 10 days of the prepared tissue repair injection compositions TL1 to TL13, it was evaluated whether or not precipitation occurred.

As a result of observation, it was confirmed that the injection compositions TL1 to TL13 for tissue repair according to the present invention did not cause precipitation even after 10 days elapsed.

It was confirmed that the microparticles according to an embodiment of the present invention do not cause agglomeration problem, so that they can be directly injected into the body without repeating the suspension and shaking process before use.

[Experimental Example 3: Anti-inflammatory Effect Test of Injectable Composition for Tissue Repair]

In order to investigate the excellent anti-inflammatory effects of the prepared tissue repair injection compositions TL1 to TL13 _{, inflammatory cytokines such as Prostaglandin E 2} (PGE ₂ ), Interleukin (IL)-1β, and Tumor Necrosis Factor-α (TNF-α) are produced. The effect on it was verified.

First, RAW 264.7 cells, a mouse macrophage cell line, were pre-sale from American type culture collections (ATCC). , Incubated in 5% CO _{2 conditions.}

DMEM medium and FBS were purchased from Invitrogen-Gibco (Grand Island, NY), and _{ELISA kits for quantification of TNF-α, IL-1β, IL-6 and PGE 2} were R&D system, Inc. (St. Louis, MO). And BD biosciences (San Diego, CA).

After culturing the cells under the same conditions as above, the supernatant of the group treated with nothing (control) and the group treated with the tissue repair injection compositions TL1 to TL13 were collected. Thereafter, the content of PGE2, IL-1β, and TNF-α (Enzo LifeScience) was quantified using an ELISA kit.

The results are shown in Figs. 1 to 4, and as can be seen in Figs. 1 to 4, the tissue repair injection compositions TL1 to TL13 significantly inhibit NO, PGE2, TNF-α and IL-1β. Confirmed.

In particular, TL2 to TL13 containing natural extracts showed high anti-inflammatory effects, and it was confirmed that the most excellent anti-inflammatory effects were exhibited in the case of TL4 and TL5 using fermented natural extracts further containing fermented Goridae extract.

Therefore, it will be said that the injection composition for tissue repair in the mouse macrophage line RAW 264.7 exhibits more excellent anti-inflammatory effects when TL4 and TL5 containing fermented natural extracts are used.

[Experimental Example 4: Evaluation of tissue repair ability of an injection composition for tissue repair]

In order to confirm the tissue repair effect of the tissue repair injection composition, the composition was injected into SD rats, and tissue repair power and collagen content were evaluated after 0, 4, 8, and 12 weeks.

Specifically, the injection volume that can be injected into the mouse was set to 0.5 mL through a preliminary experiment and injected intradermally. Immediately after the injection, the tissue into which the composition was injected was photographed to measure the volume, and after sacrifice, collagen expression was confirmed through immunohistochemistry. Even after 4, 8, and 12 weeks of injection, the tissue volume and collagen expression were also confirmed in Table 2 below.

	Tissue repair power (%)				Collagen content (Score 0-10)
	0 weeks	4 weeks	8 weeks	12 weeks	0 weeks	4 weeks	8 weeks	12 weeks
TL1	100	45	21	13	0	One	One	One
TL2	100	61	53	40	0	3	3	4
TL3	100	69	59	49	0	3	4	4
TL4	100	76	67	58	0	6	7	8
TL5	100	80	70	62	0	7	7	8
TL6	100	68	59	47	0	5	6	6
TL7	100	70	61	51	0	5	7	7
TL8	100	75	67	54	0	6	6	7
TL9	100	51	42	32	0	One	One	One
TL10	100	59	48	37	0	2	2	3
TL11	100	59	50	41	0	2	2	3
TL12	100	65	55	44	0	3	3	3
TL13	100	58	48	36	0	2	3	3
Jubiderm (HA filler)	100	48	10	8	0	One	One	One
Scultra (PLA filler)	100	21	10	15	0	3	5	3

As shown in Table 3, it was confirmed that the tissue repair injection compositions TL1 to TL13 showed superior tissue repair power and retention period compared to Scultra and Juvidum, which are products of a biodegradable polymer or crosslinked hyaluronic acid alone.

In particular, it was confirmed that the immediate tissue repair effect and collagen formation were increased in TL2 to TL13 containing natural extracts, and in the case of TL4 and TL5 containing fermented natural extracts further comprising fermented beetroot extract, better tissue repair It was found to increase the effect and collagen formation.

The present invention relates to an injectable composition for tissue repair and a method for manufacturing the same, and more particularly, to an injectable composition for tissue repair showing an effect of improving damaged skin, an immediate tissue repair effect, and a long-term tissue repair effect through collagen formation, and a manufacturing method thereof. .

Claims (10)

1. Microparticles containing biodegradable polymers;

   Hyaluronic acid; And

   Contains polynucleotide,

   The microparticles are evenly distributed in a fine powder form of natural extract,

   The natural extract containing centella asiatica extract

   Injectable composition for tissue repair.
2. The method of claim 1,

   The tissue repair injection composition does not need to be diluted before use, and is ready to use (Ready to Use)

   Injectable composition for tissue repair.
3. The method of claim 1,

   The natural extract further comprises one selected from the group consisting of beetroot extract, aloe vera extract, ginkgo biloba extract, and mixtures thereof

   Injectable composition for tissue repair.
4. The method of claim 1,

   The biodegradable polymer is any one or more selected from the group consisting of polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, and derivatives thereof.

   Injectable composition for tissue repair.
5. The method of claim 1,

   The microparticles are bioabsorbed within 1 to 3 years after injection into the body.

   Injectable composition for tissue repair.
6. The method of claim 1,

   The average particle diameter of the microparticles is 20 to 70㎛

   Injectable composition for tissue repair.
7. 1) preparing a suspension by mixing a natural extract in the form of fine particles in an organic solvent in which a biodegradable polymer is dissolved;

   2) preparing a second mixture by dissolving a surfactant in water;

   3) injecting the suspension of step 1) into a microchannel in a linear direction to flow;

   4) injecting the second mixture of step 2) into microchannels formed on both sides or one side so that the suspension of step 3) forms an intersection with the microchannels flowing in a linear direction to flow,

   Preparing microparticles in a form in which the flow of the suspension in the linear direction and the flow of the second mixture cross, and the natural extract in the form of fine particles is evenly distributed in the spherical biodegradable polymer particles;

   5) collecting the microparticles generated at the intersection of step 4);

   6) stirring the microparticles collected in step 5) to evaporate and remove the organic solvent present in the microparticles;

   7) washing and drying the microparticles of step 6),

   8) secondary drying the spherical microparticles of step 7) by vacuum or freeze drying; And

   9) the secondary dried spherical microparticles of step 8); Hyaluronic acid; Containing the step of preparing an injection composition for tissue repair by mixing polynucleotide and water for injection

   Method for producing an injection composition for tissue repair.
8. The method of claim 7,

   The natural extract in the form of fine particles of step 1) is prepared in the form of fine particles by pulverizing the natural extract after freeze drying,

   The fine particles have a diameter of 10 to 5000 nm

   A method for producing an injection composition for tissue repair.
9. The method of claim 7,

   The natural extract further comprises one selected from the group consisting of beetroot extract, aloe vera extract, ginkgo biloba extract, and mixtures thereof

   Method for producing an injection composition for tissue repair.
10. The method of claim 7,

    The biodegradable polymer is any one or more selected from the group consisting of polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, and derivatives thereof.

    Method for producing an injection composition for tissue repair.

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