WO2022124508A1 - Injectable composition for tissue repair and method for preparing same - Google Patents

Abstract

The present invention relates to an injectable composition for tissue repair and a method for preparing same, in which a short-term tissue repair effect and a long-term tissue repair effect by inducing collagen production can be provided, and skin health damaged by aging and irritation can be restored. The present invention also provides: an injectable composition for tissue repair, which is a ready-to-use injectable composition without the need for dilution before use, and thus can be immediately used, and is uniformly dispersed, thus exhibiting a uniform tissue repair effect; and a method for preparing same.

Description

Injection composition for tissue repair and manufacturing method thereof

The present invention relates to an injection composition for tissue repair and a method for preparing the same, and more particularly, to an injection composition for tissue repair and a method for preparing the same .

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

The hyaluronic acid, used as the injection composition for tissue repair, has a problem of insufficient long-term lasting effect because reabsorption in the body occurs very rapidly between 2 weeks and 2 months. Accordingly, as in Korean Patent Application Laid-Open No. 10-2004-0072008, products in which hyaluronic acid and a cross-linking material are cross-linked to each other to extend the resorption period are being sold. However, these cross-linked products have also been reported to have problems due to the toxicity of the cross-linking material.

In order to replace the injection composition for tissue repair containing hyaluronic acid, a number of tissue repair products using biodegradable polymers have also been developed. After processing into sized particles, it was developed and used as a formulation dispersed through media with viscosity.

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

The formulation using a polymer that is decomposed in vivo has problems in that the micro-particles are clogged with the needle during injection, and the particles are not uniformly dispersed, so that the 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 is insignificant.

Therefore, when the injection composition for tissue repair is administered by injection, it exhibits an immediate tissue repair effect and at the same time can exhibit a long-term continuous tissue repair effect, reduces discomfort in the procedure, and the particles are uniformly dispersed in a tissue that can be used There is an urgent need to prepare an injection composition for restoration.

[Prior art literature]

[Patent Literature]

(Patent Document 1) KR10-2004-0072008 A1

An object of the present invention is an injection for tissue repair that can be injected into the body to restore skin health damaged by aging and stimulation through improvement of the physiological environment, induce an immediate tissue repair effect and collagen production, and can continuously repair tissue for a long time To provide a composition and a method for preparing the same.

Another object of the present invention is an injection composition ready for use without the need to dilute before use when used as an injection, and there is no problem of dosing discomfort in which particles clog the injection needle during injection, and the composition is uniformly dispersed and uniform To provide an injection composition for tissue repair and a method for preparing the same, which exhibits a tissue repair effect.

In order to achieve the above object, an injection composition for tissue repair according to an embodiment of the present invention, wherein the composition for tissue repair injection comprises: microspheres comprising a biodegradable polymer; and hyaluronic acid (HA), wherein the composition has a value of 3,400 to 3,600 according to Formula 1:

[Equation 1]

G ^* /sinδ

here,

G ^* is the complex shear modulus,

δ is the phase angle.

The tissue repair injection composition may further include a polynucleotide (Polynucleotide, PN).

The ratio (elastic component/viscous component) of the composition to the elastic component and the viscous component is 6 to 7.

The microspheres have a spherical shape with a uniform surface, an average diameter of 35 to 55 μm, and a standard deviation of 3.0 to 5.5 with respect to the average diameter.

The specific surface area of the microspheres is 1.40 to 1.50 m ² /g.

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

The microspheres can be bioabsorbed within 1 to 3 years after injection into the body.

The injectable composition for tissue repair does not need to be diluted before use, and is ready to use.

A method for preparing an injection composition for tissue repair according to another embodiment of the present invention comprises the steps of 1) preparing microspheres comprising a biodegradable polymer; 2) preparing a buffer solution; 3) preparing a PN dilution solution by mixing polynucleotide (PN) with the buffer solution of step 2); 4) preparing a mixed dilution solution by mixing sodium hyaluronate gel with the PN dilution solution; 5) mixing and defoaming the microspheres of step 1) with the mixed diluent; and 6) injecting the diluent containing the microspheres of step 5) into the pre-filled syringe.

The buffer solution may include sodium chloride, di-sodium phosphate, sodium dihydrogen phosphate and water for injection.

Step 5) includes: 5-1) mixing the mixed dilution solution in which the microspheres are mixed at a speed ratio of revolution and rotation of 1:1 to 2:1 for 1 to 5 minutes; 5-2) waiting for 1 to 5 minutes after the completion of the mixing; 5-3) repeating steps 5-1) and 5-2) 2 to 4 times; and 5-4) degassing the mixed dilution solution in which the microspheres are mixed at a speed ratio of revolution and rotation of 2:1 to 5:1 for 5 to 20 minutes.

Step 6) is to be injected in a ready to use state without the need for dilution before use.

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

An injection composition for tissue repair according to an embodiment of the present invention comprises: microspheres comprising a biodegradable polymer; and hyaluronic acid (HA), wherein the composition has a value of 3,400 to 3,600 according to Formula 1 below:

[Equation 1]

G ^* /sinδ

here,

G ^* is the complex shear modulus,

δ is the phase angle.

The existing tissue repair injection composition contains hyaluronic acid as a main component, and the hyaluronic acid is present in joint fluid, cartilage, skin, and the like. When the hyaluronic acid is used as an injection composition for tissue repair and injected into the body, the hyaluronic acid attracts water molecules, thereby increasing moisture and skin elasticity in the body.

However, the injection composition for tissue repair containing such hyaluronic acid only has a maintenance period of 6 to 12 months, so there is an inconvenience of having to periodically inject the injection composition into the body.

Techniques using biodegradable polymer particles as an injection composition for tissue repair have been developed, and these injectable compositions do not only serve to fill volume in depressions in the body, but fundamentally induce collagen production, so that the tissue repair effect can be maintained for a long time. can

However, in the case of these biodegradable polymer particles, immediately after injection into the body, there was a problem that the immediate tissue repair effect did not appear.

Accordingly, in order to improve the problems of using conventional hyaluronic acid and biodegradable polymer particles as an injection composition for tissue repair, the injectable composition for tissue repair of the present invention comprises: microspheres comprising a biodegradable polymer; and hyaluronic acid (HA), wherein the composition has a value of 3,400 to 3,600 according to Formula 1 below:

[Equation 1]

G ^* /sinδ

here,

G ^* is the complex shear modulus,

δ is the phase angle.

That is, by combining the advantages of using hyaluronic acid and using biodegradable polymer particles, a short-term tissue repair effect can be exhibited, and natural skin luster can be expressed by adding moisture to the skin, and as biodegradable polymer particles are used, , by inducing collagen production, it is possible to improve the maintenance period of the tissue repair effect.

In addition, by further including a polynucleotide having a skin regeneration effect, it is possible to restore skin health damaged by aging and irritation through improvement of the physiological environment in the body.

The injectable composition for tissue repair of the present invention contains hyaluronic acid and microspheres, and is mainly characterized by exhibiting viscoelastic behavior characteristics, and due to the above characteristics, the uniformly mixed injectable composition is ready for use (Ready to use) can be provided.

For the injectable composition for tissue repair of the present invention, the viscous and elastic behavior characteristics can be analyzed by measuring the complex shear modulus (G ^* ) and the phase angle (δ).

A value that reflects the two characteristics of G ^* and δ is G ^* /sinδ, and G ^* /sinδ is a characteristic value indicating the viscoelastic properties of the composition. Generally, a composition with strong elasticity has a high G ^* /sinδ value and a strong binder. has a low value.

As in the present invention, in order to provide a uniformly mixed injection composition containing hyaluronic acid and microspheres in a ready to use form, the value according to Equation 1 should be 3,400 to 3,600.

That is, when the value falls within the above range, it may be provided not only as a uniform injectable composition, but also may be provided in a ready-to-use form, enabling direct administration without a dilution process prior to administration as an injection.

In general, the viscosity and elasticity of the injection composition are indicated by hyaluronic acid, and the properties may be affected depending on the content range of hyaluronic acid.

However, the injection composition of the present invention is characterized in that it further contains microspheres and polynucleotides (PN) containing a biodegradable polymer, thereby affecting the viscosity and elasticity of the injection composition.

As described above, the viscosity and elasticity of the injection composition itself are changed under the influence of additional mixing of microspheres and polynucleotides, and the value according to Equation 1 can be expressed by adjusting the content range of the injection composition.

As it satisfies the range of values according to Equation 1, it will be said that it is possible to provide an injection composition for tissue repair in a ready to use form without dilution before use as in the present invention.

Specifically, in the injection composition for tissue repair of the present invention, the injection composition of the present invention has a viscosity of 75 to 85 Pa, a measured elasticity of 500 to 550 Pa, and an elastic component and viscous The ratio (elastic component / viscous component) to the component) is characterized in that 6 to 7.

As it exhibits elasticity and viscous properties within the above range, it is possible to provide it as a uniformly mixed injectable composition, as well as provide it in a ready to use form.

In addition, when provided within the above range, it not only facilitates injection into the body, but also induces collagen production by including hyaluronic acid, microspheres and polynucleotides containing biodegradable polymers, and recovery of skin health damaged by aging and stimulation All effects can be increased.

That is, when the elastic and viscous properties of the composition are included within the scope of the present invention, it is possible to provide a uniform injectable composition, and the hyaluronic acid, microspheres and polynucleotides maintain a uniform state even within the skin tissue after administration. Therefore, there is no difference in effect according to the site.

Conventionally, when microparticles containing biodegradable polymers are used as an injection composition for tissue repair, there is a problem in that the particle diameter of the microparticles is not uniform, so that the tissue repair effect does not appear evenly when injected into the body.

1 relates to microspheres comprising a biodegradable polymer according to an embodiment of the present invention, and it can be seen that the surface exhibits a uniform spherical shape.

FIG. 2 is an SEM measurement photograph of biodegradable polymer particles used as a composition for tissue repair in other products, and it can be seen that they exhibit an irregular shape rather than a spherical shape.

3 is an SEM measurement photograph of biodegradable polymer particles used as a composition for tissue repair in other products. Although the spherical shape is the same as in the present invention, it can be confirmed that the surface of the particles is not uniform.

2 and 3 relate to biodegradable polymer particles used as an injection composition for conventional tissue repair. can

As described above, the conventional injectable composition for tissue repair has a problem in that the shape of the microspheres is not constant, the particle diameters are different from each other, or the surface is not uniform, so that the tissue repair effect does not appear evenly when injected into the body.

Specifically, if the shape of the microspheres does not show a constant size or if the surface is not uniform, there is a difference in the rate of decomposition when injected into the body, thereby causing a problem in that the tissue repair effect in the skin is different.

Specifically, the microspheres have an average diameter of 35 to 55 μm, a standard deviation of the average diameter of 3.0 to 5.5, and a specific surface area of 1.40 to 1.50 m ² /g.

As described above, the main feature is that the dispersion of the average diameter is small, which means that the diameter range of the microspheres included in the composition is very narrowly distributed, and the surface is uniformly formed to represent a large specific surface area value. .

The microspheres are distributed in a uniform size, exhibit a large specific surface area value, and can be absorbed into the body within 1 to 3 years after injection into the body.

Specifically, the composition for tissue repair of the present invention contains microspheres including hyaluronic acid and biodegradable polymer at the same time as described above, and can induce collagen production by hyaluronic acid and microspheres, resulting in a short-term tissue repair effect is shown by hyaluronic acid, and even if the tissue repair effect by hyaluronic acid is reduced, it can exhibit a tissue repair effect for a long time by the microspheres containing the biodegradable polymer.

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

A method for preparing an injection composition for tissue repair according to another embodiment of the present invention comprises the steps of 1) preparing microspheres comprising a biodegradable polymer; 2) preparing a buffer solution; 3) preparing a PN dilution solution by mixing polynucleotide (PN) with the buffer solution of step 2); 4) preparing a mixed dilution solution by mixing a sodium hyaluronate dilution solution with the PN dilution solution; 5) mixing and defoaming the microspheres of step 1) with the mixed diluent; and 6) injecting the diluent containing the microspheres of step 5) into the pre-filled syringe.

Step 1) is a step of preparing microspheres containing a biodegradable polymer.

Specifically, 1) step may include a) preparing a first mixture; b) preparing a second mixture; c) injecting the first mixture into the microchannel in a straight direction; d) injecting the second mixture into microchannels on both sides or on one side; e) collecting microspheres; f) agitating the collected microspheres; and g) washing and drying the microspheres.

Step a) is a step of preparing a first mixture, dissolving a biodegradable polymer in an organic solvent to prepare a first mixture, wherein the biodegradable polymer is polylactic acid, polylactide, polylactic-co-glycolic acid , polylactide-co-glycolide (PLGA), polyphosphazine, polyiminocarbonate, polyphosphoester, polyanhydride, polyorthoester, polycaprolactone, polyhydroxyvalate, polyhydroxybutyrate , polylactic acid, polyamino acids, and combinations thereof, preferably polylactic acid (PLLA), but is not limited to the above examples.

In addition, the organic solvent is immiscible with water, for example, any one or more selected from the group consisting of chloroform, chloroethane, dichloroethane, trichloroethane, and mixtures thereof, preferably dichloromethane, It is not limited to, an organic solvent capable of dissolving a biodegradable polymer, and is not limited to the above examples, and any organic solvent that can be easily selected by a person skilled in the art can be used.

More specifically, the biodegradable polymer in the first mixture contains 1 to 10% by weight, preferably 3 to 7% by weight, but is not limited to the above example.

Step b) is a step of preparing a second mixture, and a second mixture is prepared by dissolving a surfactant in water. The surfactant can be used without limitation as long as the biodegradable polymer solution can help form a stable emulsion. Specifically, it is at least one selected from the group consisting of nonionic surfactants, anionic surfactants, cationic surfactants, and mixtures thereof, and more specifically, methylcellulose, polyvinylpyrrolidone, lecithin, gelatin, polyvinyl alcohol , polyoxyethylene sorbitan fatty acid ester, polyoxyethylene castor oil derivative, sodium lauryl sulfate, sodium stearate, ester amine, linear diamine, pattiamine, and any one or more selected from the group consisting of mixtures thereof, preferably Although it is polyvinyl alcohol, it is not limited to an example.

More specifically, the second mixture is a surfactant dissolved in water, and the surfactant may be included in an amount of 0.1 to 5% by weight, preferably 0.1 to 0.5% by weight, but is not limited thereto.

Steps c) and d) are steps of injecting the first mixture and the second mixture into the microchannel formed on the wafer to flow.

More specifically, the microchannel may be formed on a material selected from the group consisting of a silicon wafer or a polymer film, but examples of the material are not limited to the above example, and any material capable of forming a microchannel may be used.

The polymer film is polyimide, polyethylene, fluorinated ethylene propylene, polypropylene, polyethylene terephthalate, polyethylene naphthalate, polysulfone ( Polysulfone) and mixtures thereof may be selected from the group consisting of, but not limited to the above examples.

As an example, aluminum is deposited on a silicon wafer using an e-beam evaporator, and photoresist is patterned on the aluminum using a photolithography technique. Thereafter, aluminum is etched using photoresist as a mask, silicon is etched by DRIE (deep ion reactive etching) using aluminum as a mask after photoresist is removed, and after aluminum is removed, glass is anodized and sealed on the wafer. Thus, the microchannel is prepared.

The microchannel has an average diameter of 80 to 120 μm, preferably 100 μm, but is not limited thereto.

However, the average diameter of the microchannel can be changed according to the range of the injection pressure. As an example, when the diameter of the channel is 100 μm, the second mixture may be injected at a pressure of 1,500 to 2,500 mbar, and the first mixture may be injected at a pressure of 300 to 700 mbar.

The average diameter of the microchannel is closely related to the average diameter of the particles, but is also closely related to the injection pressure of the first mixture and the second mixture, so it is not limited to the above example, and the average diameter of the particles to be prepared or when injected It can be changed according to the pressure conditions.

In step c), the first mixture is injected into the microchannel in the linear direction to flow, and in step d), the microchannel on both sides or one side is formed to form an intersection point with the microchannel in the linear direction. to flow by injecting

That is, the first mixture flows along the linear microchannel, and the second mixture flows along the microchannel forming an intersection with the linear microchannel on both sides or one side based on the linear microchannel, encounter with the flow of the first mixture.

At this time, when the first mixture is injected into the microchannel in a straight direction, it is injected under a constant pressure condition and flows at a constant flow rate, and the pressure condition at this time is 300 to 700 mbar, preferably 500 mbar, but it is not limited to examples.

In addition, when the second mixture is injected into the microchannels of both sides or one side, it is injected under a constant pressure condition and flows at a constant flow rate, and the pressure condition at this time is 1500 to 2500 mbar, preferably 2000 mbar, but not limited to examples does not

That is, in order to make the flow of the second mixture forming an intersection with the flow of the first mixture flow at a faster flow rate than the first mixture injected into the linear microchannel, the second mixture is flowed under a higher pressure condition.

As described above, by differentiating the flow rates of the first mixture and the second mixture, and making the flow rate of the second mixture faster than the flow rate of the first mixture, the flow of the first mixture and the flow of the second mixture are relatively more The second mixture having a high flow rate compresses the first mixture, and at this time, the biodegradable polymer in the first mixture produces spherical microspheres due to the repulsive force of the first mixture and the second mixture.

Step e) is a step of collecting microspheres, and the microspheres are collected in a water tank containing the second mixture to prevent aggregation between initially generated microspheres.

In step e), the second mixture prepared in step b), that is, a mixed solution of a surfactant and water, is used. After the second mixture is prepared in step b), a part is injected into the microchannel, and the other part is injected into the microchannel. is used to prevent agglomeration between the collected microspheres by moving them to the water bath of step e).

Step f) is a step of stirring the collected microspheres in a water bath, and the microspheres are stirred at a constant temperature condition and agitation speed, and the organic solvent present on the surface of the sustained-release particles is evaporated and removed. At this time, the stirring conditions include the steps of primary stirring at a speed of 150 to 650 rpm for 0.5 to 1.5 hours at 10 to 15 ℃; And after the first stirring step, it proceeds in the order of the second stirring step at a speed of 500 to 1,500 rpm for 2.0 to 4.0 hours at 50 to 70 ℃.

The stirring speed proceeds with a different stirring speed in the first and second stirring steps, and the stirring process is performed using a faster speed in the second stirring step than in the first stirring step.

In addition to the stirring speed, the temperature conditions are also characterized by stirring by increasing the temperature in the secondary stirring process compared to the primary stirring process, and as the temperature is increased stepwise, the organic solvent present on the surface of the microspheres The evaporation rate can be controlled. That is, by slowly evaporating the organic solvent present on the surface of the microsphere, microspheres having a smooth surface can be prepared.

The temperature at which the first mixture and the second mixture flow through the microchannel is also 15 to 20°C, preferably 15°C. That is, after flowing through the microchannel and forming an intersection to generate microspheres, the temperature is constantly maintained at 15 to 20° C. until the collected microspheres are first stirred. It is possible to manufacture and maintain spherical particles only by maintaining a low temperature in the manufacturing process of the microspheres. That is, when it is not a low-temperature condition, it is difficult to manufacture particles having a constant spherical shape.

Finally, step g) is a step of washing and drying the sustained-release particles, and the microspheres from which all organic solvents have been removed on the surface by stirring are washed several times with sterilized filtered purified water to remove the surfactant remaining in the microspheres, , then freeze-dried.

The freeze-drying conditions include: freezing at -45 to -40°C for 1 to 15 hours; After the freezing step, the first drying step for 1 to 15 hours by raising the temperature to -30 to -20 ℃ under 150 to 250 μbar conditions; After the first drying step, the second drying step for 1 to 15 hours by raising the temperature to 1 to 5 ℃; and raising the temperature to 20 to 40° C. after the second drying step and performing a third drying step for 1 to 25 hours.

As the freeze-drying process proceeds as described above, as the solvent remaining on the surface of the microspheres is sublimated and removed, surface damage can be prevented, and the formation of pores during the solvent removal process is prevented. It enables the production of particles with an even surface.

If the surface of the particles is not prepared evenly as shown in FIG. 2 by manufacturing differently from the above conditions, the decomposition rate of the microspheres increases, and thus a problem may occur in which the tissue repair effect cannot be exhibited for a long time as in the present invention.

Step 2) is a step of preparing a buffer solution. The buffer solution contains sodium chloride, dibasic sodium phosphate, sodium dihydrogen phosphate and water for injection.

Specifically, sodium chloride, dibasic sodium phosphate and sodium dihydrogen phosphate are dissolved in water for injection in a weight ratio of 1:0.1:0.03 to 1:0.2:0.1, and the pH of the prepared buffer is 6 to 8.

Step 3) is a step of preparing a PN dilution solution by mixing polynucleotide (PN) with the buffer solution of step 2), and 10 to 30 parts by weight of the polynucleotide is mixed with respect to 100 parts by weight of the buffer solution. The PN dilution solution is then stirred for 10 to 50 minutes at a speed of 50 to 150 rpm in order to be uniformly mixed.

Step 4) is to prepare a mixed dilution solution by mixing sodium hyaluronate gel with the PN dilution solution. After the mixing, the stirring process was performed for 5 to 20 minutes at a speed of 50 to 150 rpm.

5) Mixing and defoaming the microspheres of step 1) with the mixed dilution solution, specifically, 5-1) the mixing dilution solution in which the microspheres are mixed, and the speed ratio of revolution and rotation is 1:1 to 2:1 mixing for 1 to 5 minutes with a furnace; 5-2) waiting for 1 to 5 minutes after the completion of the mixing; 5-3) repeating steps 5-1) and 5-2) 2 to 4 times; and 5-4) degassing the mixed dilution solution in which the microspheres are mixed at a speed ratio of revolution and rotation of 2:1 to 5:1 for 5 to 20 minutes.

By proceeding with the steps of 5-1) to 5-3), the microspheres, polynucleotides and hyaluronic acid in the composition can be uniformly mixed, and then by adjusting the revolution and rotation rate ratio in step 5-4), It is possible to proceed with the optimal defoaming process.

As the mixing and defoaming process of step 5) is performed, the composition of the present invention can be provided as a ready-to-use formulation, and then, in step 6), injecting the diluent into the prefilled syringe proceed with

The present invention is put into the body, it can restore the skin health damaged by aging and stimulation through improvement of the physiological environment, induces an immediate tissue repair effect and collagen production, and enables continuous tissue repair for a long time.

In addition, when used as an injection, it is a ready-to-use injection composition that does not need to be diluted before use, so there is no problem of dosing discomfort due to particles clogging the injection needle during injection, and the composition is uniformly dispersed, resulting in a uniform tissue repair effect can indicate

1 is an SEM measurement photograph of microspheres according to an embodiment of the present invention.

Figure 2 is an SEM measurement of the biodegradable polymer particles according to an embodiment of the present invention.

3 is an SEM measurement photograph of biodegradable polymer particles according to an embodiment of the present invention.

4 is an experimental result regarding the tissue repair and tissue repair lasting effect of the composition according to an embodiment of the present invention.

The present invention provides an injection composition for tissue repair, the composition comprising: microspheres comprising a biodegradable polymer; And hyaluronic acid (Hyaluronic acid, HA), wherein the composition relates to an injection composition for tissue repair having a value of 3,400 to 3,600 according to Formula 1:

[Equation 1]

G ^* /sinδ

here,

G ^* is the complex shear modulus,

δ is the phase angle.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily carry out the present invention. However, the present invention may be embodied in several different forms and is not limited to the embodiments described herein.

Preparation Example 1

Preparation of microspheres

Polylactic acid (PLLA) was dissolved in dichloromethane to prepare a first mixture. In this case, polylactic acid (PLLA) in the first mixture was included in a proportion of 5% by weight.

Polyvinyl alcohol as a surfactant was mixed with water to prepare a second mixture containing 0.25 wt% of polyvinyl alcohol.

The first mixture and the second mixture were injected into microchannels formed on a silicon wafer to flow. At this time, in order to flow the first mixture and the second mixture at a constant flow rate, the first mixture was flowed under a pressure condition of 500 mbar, and the second mixture was flowed under a pressure condition of 2,000 mbar. The temperature condition was maintained at 15°C.

The microspheres formed at the intersection where the flow of the first mixture and the flow of the second mixture meet were collected in a water bath containing the second mixture.

The microspheres collected in the water bath were first stirred at 15° C. at a rate of 200 to 400 rpm for 1 hour, and then the temperature was raised to 60° C., followed by second stirring at a speed of 800 to 1,200 rpm for 3 hours.

After stirring, the microspheres were washed several times with sterile filtered purified water, and freeze-dried to prepare microspheres.

Preparation 2

Preparation of an injectable composition for tissue repair

Sodium chloride, dibasic sodium phosphate and sodium dihydrogen phosphate were added to 1000cc of water for injection in a weight ratio of 1:0.15:0.06 and mixed to prepare a buffer solution. The prepared buffer solution has a pH of 6 to 7.

A PN dilution solution was prepared by mixing 20 parts by weight of polynucleotide with respect to 150 parts by weight of the buffer solution and stirring at 100 rpm for 30 minutes. 800 parts by weight of sodium hyaluronate gel was mixed with 150 parts by weight of the PN diluted solution, and stirred at a speed of 100 rpm for 10 minutes.

Thereafter, the microspheres prepared in Preparation Example 1 were mixed, and the revolving and rotating speed ratio was 1.2:1, mixed for 1 to 5 minutes, and after waiting for 2 minutes, the mixing process was performed again. Thereafter, the defoaming process was performed for 10 minutes at a speed ratio of 3:1 for revolution and rotation.

The composition after the mixing and defoaming process was completed was prepared in a ready-to-use form by filling a syringe.

Experimental Example 1

Confirmation of surface shape by freeze-drying conditions of microspheres

When preparing the microspheres of Preparation Example 1, the freeze-drying conditions were changed as follows, and then, SEM images were measured to confirm whether the surface was uniform.

	process conditions
Preparation Example 1	Freezing at -40°C for 15 hours; After the freezing step, the first drying step for 15 hours by raising the temperature to -25 ℃ under 150 to 250 μbar conditions; After the first drying step, the second drying step for 15 hours by raising the temperature to 4 ℃; and raising the temperature to 25° C. after the second drying step and drying the third for 25 hours.
Comparative Example 1	After freeze-drying in Preparation Example 1, the temperature was increased to 25° C. and dried for 55 hours.
Comparative Example 2	In Preparation Example 1, the freeze-drying process was not performed, and drying was performed in the first to third drying steps.
Comparative Example 3	In Preparation Example 1, the third drying step was increased to 45° C. and dried.

When pores are formed on the surface, the surface is non-uniform and the decomposition rate is fast when injected into the body, which may cause a problem in that the tissue repair effect cannot be exhibited for a long period of time.

As a result of the SEM measurement, the case shown in FIG. 1 is denoted by O, and the case where it is formed non-uniformly as shown in FIG. 3 is denoted by X.

	Preparation Example 1	Comparative Example 1	Comparative Example 2	Comparative Example 3
surface shape	O	X	X	X

As shown in Table 2 above, when prepared under freeze-drying conditions in the present invention, microspheres with a uniform surface as shown in FIG. 1 can be prepared, but under other conditions, microspheres with a non-uniform surface are prepared as shown in FIG. 3 Confirmed.

Experimental Example 2

Evaluation of the physical properties of the injectable composition for tissue repair

The elasticity (Storage modulus, elastic modulus), viscosity (Loss modulus, viscous modulus), complex viscosity, and phase angle of the injection composition were measured.

First, the resistance to a given force was measured by injecting a sample between a balanced plate and a microscopically vibrating and rotating geometry. The shear strain was determined in the amplitude sweep evaluation, and the viscoelasticity characteristics were confirmed through the frequency sweep evaluation. Measurement conditions were shear strain: 0.15 %, temperature: 25 ℃, frequency gap: 0.5 mm, and the corresponding frequency (0.1 ~ 10 Hz) was measured and the measured value was confirmed at 1 Hz.

For the comparative experiment, the above physical property values were also measured for Rejuran S (Comparative Example 4) and The CHAEUM No. 4 (Comparative Example 5), which are commercially available filler products.

Through the measured values, the values of Equation 1 below were derived, and the ratios for elasticity and viscosity were calculated:

[Equation 1]

G ^* /sinδ

here,

G ^* is the complex shear modulus,

δ is the phase angle.

The measurement and calculation results are shown in Table 3 below.

	Frequency (Hz)	Shear modulus (complex component) (Pa)	Shear modulus (elastic component) (Pa)	Shear modulus (viscous component) (Pa)	phase angle (°)	Equation 1	ratio
Preparation 2	One	531.8	525.7	80.4	8.7	3515.782	6.54
hyaluronic acid	One	-	394.1	50.04	7.27	0	7.88
Comparative Example 4	One	-	5.031	1.746	19.13	0	2.88
Comparative Example 5	One	-	460.4	29.81	3.7	0	15.44

As shown in Table 3 above, the injection composition of the present invention can confirm that the value of Formula 1 is included within the scope of the present invention, measured only for hyaluronic acid, or for tissue repair injection compositions on the market When measured, it was confirmed that the shear modulus value was not measured, so that the value of Equation 1 as in the present invention was not derived.

It can be confirmed that the value of Formula 1 corresponds to a value measured only when provided as an injection composition for tissue repair such as the present invention.

Experimental Example 3

Tissue repair effect and tissue repair lasting effect

After anesthetizing a 6-week-old nude mouse to evaluate the tissue repair and lasting effect of the injection composition (Zoletil:rompun=3:1), the composition of Preparation Example 2 is on the left side, and the control example (the chaeum sub-) on the right side. Q) was injected subcutaneously.

A total of 60 mice were used, and 0.15 mL was constantly injected subcutaneously, and the tissue repair power and persistence of the administered samples were confirmed according to the lapse of 4 weeks, 8 weeks, 12 weeks, 16 weeks, 20 weeks, and 24 weeks immediately after injection. .

The experimental results are shown in FIG. 4 . Specifically, the composition of Preparation Example 2 maintained the injected form as compared to the Control Example, while the Control Example did not gradually maintain the shape during the test period and spread to the surrounding tissues.

Foreign body reaction was initially seen in all mice injected with the composition of Preparation Example 2, but showed a tendency to gradually decrease. On the other hand, in the control sample, there was no initial foreign body reaction after injection, but it gradually expanded during the test period.

According to the experimental results, it will be said that the composition of Preparation Example 1 has better stability against foreign body reactions and the effectiveness of maintaining the injected form compared to the control example.

Although preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention as defined in the following claims are also provided. is within the scope of the

The present invention relates to an injection composition for tissue repair and a method for preparing the same, and more particularly, to an injection composition for tissue repair and a method for preparing the same .

Claims (12)

1. As an injection composition for tissue repair,

   The tissue repair injection composition comprises: microspheres comprising a biodegradable polymer; and hyaluronic acid (HA),

   The composition has a value of 3,400 to 3,600 according to Formula 1 below

   Injectable composition for tissue repair:

   [Equation 1]

   G ^* /sinδ

   here,

   G ^* is the complex shear modulus,

   δ is the phase angle.
2. According to claim 1,

   The tissue repair injection composition further comprises a polynucleotide (Polynucleotide, PN)

   An injectable composition for tissue repair.
3. According to claim 1,

   The ratio (elastic component / viscous component) to the elastic (elastic component) and the viscous component of the composition is 6 to 7

   An injectable composition for tissue repair.
4. According to claim 1,

   The microspheres have a spherical shape with a uniform surface, an average diameter of 35 to 55 μm, and a standard deviation of 3.0 to 5.5 for the average diameter.

   An injectable composition for tissue repair.
5. According to claim 1,

   The specific surface area of the microspheres is 1.40 to 1.50 m ² /g

   An injectable composition for tissue repair.
6. According to claim 1,

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

   An injectable composition for tissue repair.
7. According to claim 1,

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

   An injectable composition for tissue repair.
8. According to claim 1,

   The tissue repair injection composition is ready to use, without the need for dilution before use (Ready to Use)

   An injectable composition for tissue repair.
9. 1) preparing microspheres comprising a biodegradable polymer;

   2) preparing a buffer solution;

   3) preparing a PN dilution solution by mixing polynucleotide (PN) with the buffer solution of step 2);

   4) preparing a mixed dilution solution by mixing sodium hyaluronate gel with the PN dilution solution;

   5) mixing and defoaming the microspheres of step 1) with the mixed diluent; and

   6) injecting the diluent containing the microspheres of step 5) into a pre-filled syringe

   A method for preparing an injectable composition for tissue repair.
10. 10. The method of claim 9,

    The buffer solution includes sodium chloride, di-sodium phosphate, sodium dihydrogen phosphate and water for injection

    A method for preparing an injectable composition for tissue repair.
11. 10. The method of claim 9,

    Step 5) is,

    5-1) mixing the mixed dilution solution in which the microspheres are mixed in a ratio of 1:1 to 2:1 for revolution and rotation for 1 to 5 minutes;

    5-2) waiting for 1 to 5 minutes after the mixing is completed;

    5-3) repeating steps 5-1) and 5-2) 2 to 4 times; and

    5-4) comprising the step of defoaming the mixed dilution solution in which the microspheres are mixed at a rate ratio of 2:1 to 5:1 for 5 to 20 minutes

    A method for preparing an injectable composition for tissue repair.
12. 10. The method of claim 9,

    In step 6), there is no need to dilute before use, and it is injected in a ready to use state.

    A method for preparing an injectable composition for tissue repair.

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