KR20230059749A - Hydrophilic injection type skin filling composition, preparation method and application thereof - Google Patents

Abstract

The present invention relates to the field of medical technology, and specifically discloses a hydrophilic injectable dermal filling composition and a manufacturing method and application thereof. The composition of the present invention includes polymeric microspheres, polyethylene glycol succinimide glutarate, and a dispersion, wherein the polymeric microspheres are a polylactic acid-polycaprolactone-polyethylene glycol copolymer, and the dispersion contains cross-linked sodium hyaluronate and protein. And, the protein is mussel protein or silk fibroin. The composition can achieve accurate targeting and position-occupying charging effect at the action site without the risk of position shift, and causes the targeting and immediate charging effect. It can reduce the reaction, and the clinical application is safe and effective.

Description

Hydrophilic injection type skin filling composition and preparation method and application thereof {Hydrophilic injection type skin filling composition, preparation method and application thereof}

The present invention relates to the field of pharmaceutical technology, and specifically to a hydrophilic injectable dermal filling composition and a manufacturing method and application thereof.

In recent years, with the expansion of the medical beauty industry, medical beauty injection fillers are developing rapidly, people's interest in aesthetic level and safety and health is gradually increasing, and safe and effective injection products are receiving widespread attention. However, currently marketed microspheres and sodium hyaluronate filled products used for injection filling have poor affinity with the human skin, the injection site is easily moved, and the dispersibility of microspheres is low, resulting in easy precipitation and aggregation. Due to this, it is difficult to ensure a stable effect in the long term after injection.

Artificially synthesized polymeric microspheres reduce the risk of infection compared to animal-derived fillers, are relatively safe, and have a long duration, but lack relatively good biocompatibility. Sodium hyaluronate is a filling material with good biocompatibility and a wide range of applications, but the problem of shifting the position of sodium hyaluronate filling is often pointed out by people, and the shifting of the injection site greatly reduces the filling effect. That is, although sodium hyaluronate has relatively excellent biocompatibility, it is not the most ideal filler for injection because of its relatively poor adhesive effect with human cells.

Chinese Patent No. 201980018465.0 discloses a polycaprolactone microsphere filler containing collagen peptides and a method for manufacturing the same, but polycaprolactone is easily lumped together to reduce the stimulation effect of microspheres on collagen production and cause side effects such as nodules, This is also not an ideal injectable filling material.

In view of the above problems, it is still necessary to develop new injectable dermal filling compositions.

Regarding the problems of the prior art, an object of the present invention is to have good biocompatibility, reduce the incidence of side effects such as subcutaneous nodules and inflammation after transplantation, have a good immediate filling effect, and have a hydrophilic injectable skin filling that does not easily move the position. It is to provide a composition and its manufacturing method and application.

In order to achieve the above object, the technical solution of the present invention is as follows:

A composition comprising polymeric microspheres, polyethylene glycol succinimide glutarate (PEG-SG) and a dispersion, wherein the polymeric microspheres are polylactic acid-polycaprolactone-polyethylene glycol copolymer, and the dispersion is cross-linked sodium hyaluronate and protein A composition comprising a, wherein the protein is mussel protein or silk fibroin.

In the present invention, a dispersion containing specific amphiphilic polymer microspheres and specific components is blended and used, and protein, PEG-SG and cross-linked sodium hyaluronate are mixed together to form a composition system in a liquid state to form a specific solid-liquid interpenetrating gel bionic It is rapidly formed into an extracellular matrix structure, so that the polymer microspheres of the present invention can stably exist in the composition system in a specific dispersed form, and the obtained composition is not easy to move, has a low swelling rate, and can be filled immediately after targeting effectiveness and good biocompatibility.

Preferably, the protein is a mussel protein to achieve extensive adhesion localization of cells, local antioxidant, anti-inflammatory, site-occupying filling and repair effect on needle holes.

In the present invention, the mass ratio of the protein and the cross-linked sodium hyaluronate is 1: (15 to 25), preferably 1:20; and/or

The mass ratio of the cross-linked sodium hyaluronate and the polyethylene glycol succinimide glutarate is (80 to 120): 1, preferably 100: 1.

In the present invention, the mass ratio of the sum of the masses of the polyethylene glycol succinimide glutarate and the dispersion and the mass of the polymeric microspheres is (6 to 9): (1 to 4), and better targeting and immediate filling effect and proliferation In order to achieve a sustained stimulating effect, it is preferably 7.5 : 2.5.

Under the above-mentioned preferred material ratio of the present invention, it has better uniform dispersibility of microspheres and immediate filling effect.

In the present invention, the dispersion further contains a phosphate buffer and glycerin, and has an osmotic pressure of 250 to 350 mOsm/L; The concentration of the cross-linked sodium hyaluronate in the dispersion is 15 to 30 mg/ml, preferably 24 mg/ml to obtain a better filling effect with low swelling; and/or

The mass fraction of the glycerin in the dispersion is 0.48 to 2%, and is preferably 1% in order to consider fluidity and moisturizing properties together.

The phosphate buffer in the dispersion of the present invention enables the composition of the present invention to achieve isotonicity with the human body. Its pH is preferably 7.3.

In the present invention, the intrinsic viscosity of the polymer microspheres is 0.15 to 1.5 dl/g (prepared with a 0.5% chloroform solution at 25 ° C, referring to the viscosity measurement method of Pharmacopoeia 20200663), and the proportion of PCL in the polymer is 40 to 70 wt%. In order to consider the effect of stimulating collagen growth and the hydrophilic effect together, preferably, the intrinsic viscosity of the polymeric microspheres is 1.2dl/g, and the proportion of PCL in the polymer is 50wt%; and/or

The polyethylene glycol succinimide glutarate is 4-armed and has a molecular weight of 5000 to 40000, preferably 10000; and/or

The molecular weight of the cross-linked sodium hyaluronate is 1000000 to 2500000, preferably 1500000.

In the present invention, the crosslinking agent in the crosslinked sodium hyaluronate is divinylsulfone, and the specific crosslinking method is: Dissolve 1g of sodium hyaluronate in a sodium hydroxide solution having a mass fraction of 10%, add 5mg of divinylsulfone and stir to crosslink. Sodium hyaluronate is obtained, and at the end, 1 mol/L hydrochloric acid solution is added to adjust the pH to 7.0 to 7.4.

In the present invention, according to the use site of the polymer microspheres and the recovery effect demand, the molecular weight, viscosity and blending ratio of the main raw materials are adjusted to achieve the purpose of different decomposition time (the decomposition period can be 1 to 5 years).

Dispersibility, injectability, degradability, and localization of the composition may be considered together within the range of the viscosity of the polymer microspheres, the molecular weight of protein and cross-linked sodium hyaluronate, and the mixing ratio of each material.

Preferably, the particle diameter range of the polymer microspheres of the present invention is 35 to 45 μm.

In addition, the present invention provides a method for preparing the above-described composition comprising the step of preparing polymer microspheres through a shear emulsification method, a membrane emulsification method or a spray drying method;

In order to achieve a better target particle size distribution and yield of microspheres, a preferred method for producing polymer microspheres is membrane emulsification.

Specifically, it includes mixing an organic solvent and a polylactic acid-polycaprolactone-polyethylene glycol copolymer to obtain an oil phase; The organic solvent is at least one of dichloromethane, tetrahydrofuran and chloroform, and is preferably a mixed solvent composed of dichloromethane or tetrahydrofuran and dichloromethane in a volume ratio of (1 to 2) : (8 to 10).

In the present invention, when preparing by applying shear emulsification or membrane emulsification, the step of mixing an emulsifier and water to obtain an aqueous phase is further included; The emulsifier is at least one of polyvinyl alcohol, Span, Tween, and carboxymethyl cellulose, and preferably polyvinyl alcohol or polyvinyl alcohol (viscosity 5 to 50 mPa s) and Tween-80 (9 to 10) : It is a mixed solvent composed of a mass ratio of 1; and/or

The volume ratio of the oil phase and the aqueous phase is 1: (4 to 10).

In the present invention, the mass fraction of the polylactic acid-polycaprolactone-polyethylene glycol copolymer in the oil phase is 2 to 15%, and the mass fraction of the emulsifier in the aqueous phase is 0.1 to 3%.

In the present invention, when preparing polymer microspheres, it is possible to ensure that the microspheres obtained after emulsification have a better physical shape through a specific oil phase concentration and aqueous phase composition and concentration. Specifically, the sphericity of the particle size of the microspheres obtained is good, the particle size distribution is narrow, the surface is smooth, and the yield can be ideal.

In a preferred form, when prepared by applying a membrane emulsification method, the organic solvent is tetrahydrofuran and dichloromethane in a volume ratio of 1:9; The mass fraction of the polylactic acid-polycaprolactone-polyethylene glycol copolymer in the oil phase is 8%, and the mass fraction of the emulsifier in the aqueous phase is 1%; Considering realizing a better target microsphere yield, the volume ratio of the oil phase and the aqueous phase is 1:6.

When produced by applying the membrane emulsification method, the oil phase is passed through a membrane under a pressure of 0.1 to 0.4 kPa, stirred and mixed with the water phase at 4 to 8 ° C, the stirring speed is 200 to 500 r / min, and the membrane The pore size is 10 to 20 μm, and after the passage of the oil phase through the membrane is completed, the oil phase is continuously stirred (200 to 500 r/min) for 10 to 60 min to emulsify, and then the oil phase is continuously stirred (200 to 500 r/min) at 15 to 25 ° C. volatilize the solvent.

Preferably, the prepared microspheres are washed several times until there is no residual aqueous phase solvent (washed 3 to 5 times with clean water), and after screening, microspheres having a target particle size are obtained.

After the passage of the oil phase through the membrane is completed, it is emulsified by continuous stirring (at 4 to 8 ° C) and continuously stirred at 15 to 25 ° C to volatilize the oil phase solvent and the speed used when mixing the oil phase and water phase is the same do.

Preferably, when the oil phase passes through the membrane and is stirred and mixed with the water phase at 4 to 8 ° C., when the mixture is emulsified by continuous stirring, and when the oil phase solvent is volatilized by continuous stirring at 15 to 25 ° C., the speed is 250 r. /min, the oil phase extrusion pressure is 0.2 kPa, the membrane has a pore diameter of 20 μm, and the oil phase solvent is volatilized by continuously stirring at 15 to 25° C. for 4 hours.

When preparing by applying the shear emulsification method, the oil phase is mixed with the aqueous phase drop by drop while stirring at 4 to 8 ° C., the stirring speed is 800 to 1000 r / min, the time is 20 to 40 min, and then 15 to 25 stirring continuously at 400 to 500 r/min for 3 to 5 h; Preferably, when mixing, the stirring speed is 1000 r/min, the time is 30 min, and then the stirring is continued at 15 to 25° C. at a rate of 500 r/min for 5 h;

When manufacturing by applying the spray drying method, the oil phase is spray dried, the inlet temperature of the spray drying process is 60 to 85 ° C, the outlet temperature is 25 to 50 ° C, the feed rate of the raw material is 10 to 25 ml / min, and the high-pressure air flow rate is 400 to 600 L/h; Preferably, the inlet temperature is 85°C, the outlet temperature is 25°C, the feed rate of the raw material is 25ml/min, and the high-pressure air flow rate is 600L/h.

In the method for producing polymer microspheres of the present invention, mixing and using each condition can effectively mix the characteristics of raw materials for microsphere production, and can realize an ideal production effect.

In the present invention, the above-described composition can be obtained by uniformly mixing the obtained polymer microspheres with the dispersion and PEG-SG according to the ratio after primary screening.

In addition, the present invention provides an application of the above-described composition or the composition obtained by the above-described method in the manufacture of an injectable dermal filling product.

The composition obtained from the present invention can be used for subcutaneous filling injection, not for the purpose of diagnosis or treatment of a disease, and can stimulate collagen production in the deep layer of the skin itself, thereby forming a sustained recovery effect for a relatively long time. Specifically, it is possible to realize a cosmetic effect by applying to the face and neck.

The beneficial effects of the present invention are at least as follows:

The composition of the present invention has good re-solubility, high biocompatibility, can reduce the inflammatory response after material implantation, has antioxidant action and recovery effect for injection needle holes, and has protein for cells in the specific gel system of the present invention. Accuracy and fixation of the injection position can be realized through the adhesive action of the injection, and the risk of filler position shift can be solved, and the specific interpenetrating network structure of the composition of the present invention can maintain the effect of suspending and dispersing the microspheres, and has an immediate filling effect. to provide.

In addition, the polymer microspheres prepared by the method of the present invention have a narrow particle size distribution, high sphericity, and a smooth surface, and the incidence of side effects such as uneven decomposition after implantation, subcutaneous nodules, and redness can be more effectively reduced.

Hereinafter, preferred embodiments of the present invention will be described in detail by combining examples. It should be understood that the following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the gist and spirit of the present invention.

Experimental methods used in the following examples are all conventional methods unless otherwise specified. Materials, reagents, etc. used in the following examples can all be obtained through commercial routes unless otherwise specified.

Example 1

This experimental example provides a method for preparing the composition of the present invention, specifically including the following steps:

Step 1: Preparation of microspheres

Mixed reforming of polycaprolactone: ε-caprolactone, lactide, polyethylene glycol (molecular weight 2000) and toluene were added to the reaction vessel in a mass ratio of 2: 1: 1: 2, and Stannous octoate was used as a catalyst was added dropwise, reduced pressure repeatedly at 125 ° C., nitrogen was passed through to discharge oxygen, sealed, and reacted at that temperature for 48 h to obtain a product. The product was dissolved in chloroform, washed again and purified, After vacuum drying, a polylactic acid-polycaprolactone-polyethylene glycol copolymer was obtained.

The polylactic acid-polycaprolactone-polyethylene glycol copolymer prepared above using tetrahydrofuran and dichloromethane as solvents (the volume ratio of the two solvents is 1:9) (PCL occupies 50wt%, viscosity 1.2dl/ g) to prepare an oil phase, wherein the mass fraction of the polylactic acid-polycaprolactone-polyethylene glycol copolymer is 8%.

Polyvinyl alcohol (viscosity: 25 mPa·s) and Tween-80 were used as solutes (mass ratio of the two solutes was 10:1), and purified water was added to prepare an aqueous phase, where the mass fraction of the solute was 1%.

The volume ratio of the oil phase and the aqueous phase is 1:6.

Microspheres are prepared by membrane emulsification: the oil phase is extruded under a pressure of 0.2 kPa, passed through a membrane with a pore diameter of 20 μm, and mixed with the aqueous phase (ice bath conditions of 4 to 8 ° C.) under stirring, and the stirring speed is 250 r/ min, and after completion of the passage of the oil phase through the membrane, the mixture was emulsified by stirring at the same speed for 30 min, then the ice bath was removed, and then the oil phase solvent was volatilized by stirring at 21 ° C. for 4 h at 250 r/min. The prepared microspheres are washed 3 to 5 times with clean water. The particle diameter range of the obtained microspheres is 30 to 50 μm.

The microspheres obtained above were screened to obtain microspheres having a particle diameter of 35 to 45 μm (75.6% by mass in total microspheres).

Step 2: Preparation of Dispersion

Take 240 mg of cross-linked sodium hyaluronate (molecular weight 1500000), 12 mg of mussel protein, and 100 mg of glycerin, add a phosphate buffer (prepared with reference to Pharmacopoeia 20208002, pH 7.3), dissolve, and use 10 ml. Gel dispersion A was obtained by adjusting the osmotic pressure to 300 mOsm/L with sodium chloride.

Step 3: Preparation of Composition

After adding water to 2.4 mg of PEG-SG (4-arm, molecular weight 10000) to prepare a solution with a mass fraction of 15%, lyophilized powder was obtained by lyophilization (pre-freezing at -40 ° C. for 3 hours). Then, by operating a vacuum of 0.2 mbar, drying at a temperature gradient of -28 ℃, -18 ℃, 10 ℃, and 30 ℃ for 20 h, 8 h, 2 h and 2 h, respectively, to prepare a freeze-dried powder), the freeze-drying The powder was introduced into the gel dispersion A obtained in Step 2 together with the microspheres screened in Step 1 and mixed uniformly to obtain a uniform composition. The mass ratio of the total mass of PEG-SG and gel dispersion A to the mass of microspheres was 7.5:2.5.

Example 2

This example provides a method for preparing the composition of the present invention, specifically comprising the following steps:

Step 1: Preparation of microspheres

Mixed reforming of polycaprolactone: ε-caprolactone, lactide, polyethylene glycol (molecular weight 2000) and toluene were added to the reaction vessel in a mass ratio of 4: 3: 3: 10, and stannous octoate was added dropwise as a catalyst. . A lactic acid-polycaprolactone-polyethylene glycol copolymer was obtained.

The polylactic acid-polycaprolactone-polyethylene glycol copolymer prepared above using tetrahydrofuran and dichloromethane as solvents (the volume ratio of the two solvents is 2:8) (PCL occupies 40 wt%, viscosity 1.5 dl/ g) to prepare an oil phase, wherein the mass fraction of the polylactic acid-polycaprolactone-polyethylene glycol copolymer is 15%.

Polyvinyl alcohol (viscosity: 5 mPa·s) and Tween-80 were used as solutes (mass ratio of the two solutes was 9:1), and purified water was added to prepare an aqueous phase, where the mass fraction of the solute was 3%.

The volume ratio of the oil phase and the aqueous phase is 1:10.

Microspheres are prepared by the membrane emulsification method: the oil phase is extruded under a pressure of 0.4 kPa, passed through a membrane having a pore diameter of 10 μm, and mixed with the aqueous phase (ice bath conditions of 4 to 8 ° C.) under stirring, and the stirring speed is 200 r/ min, and after the passage of the oil phase through the membrane was completed, the mixture was emulsified by stirring at the same speed for 10 min, then the ice bath was removed, and then the oil phase solvent was volatilized by stirring at 22° C. for 4 h at 200 r/min. The prepared microspheres are washed 3 to 5 times with clean water. The particle diameter range of the obtained microspheres is 20 to 60 μm.

The microspheres obtained above were screened to obtain microspheres having a particle diameter of 35 to 45 μm (49.6% by mass in total microspheres).

Step 2: Preparation of Dispersion

Take 240 mg of cross-linked sodium hyaluronate (molecular weight 1500000), 12 mg of mussel protein, and 100 mg of glycerin, add a phosphate buffer (prepared with reference to Pharmacopoeia 20208002, pH 7.3), dissolve, and use 10 ml. Gel dispersion A was obtained by adjusting the osmotic pressure to 310 mOsm/L with sodium chloride.

Step 3: Preparation of Composition

2.4 mg of lyophilized powder of PEG-SG (4-arm, molecular weight 10000) (preparation method is the same as in Example 1) was added to the gel dispersion A obtained in Step 2 together with the microspheres screened in Step 1, By mixing uniformly, a uniform composition was obtained. The total mass of PEG-SG and gel dispersion A and the mass ratio of microspheres were 7.5:2.5.

Example 3

Step 1: Preparation of microspheres

Mixed reforming of polycaprolactone: ε-caprolactone, lactide, polyethylene glycol (molecular weight 2000) and toluene were added to the reaction vessel in a mass ratio of 2: 1: 1: 2, and stannous octoate was added dropwise as a catalyst. . A lactic acid-polycaprolactone-polyethylene glycol copolymer was obtained.

Using pure dichloromethane as a solvent, it was mixed with the polylactic acid-polycaprolactone-polyethylene glycol copolymer (PCL occupied by 50 wt%, viscosity 1.2 dl/g) obtained above to prepare an oil phase, where poly The mass fraction of the lactic acid-polycaprolactone-polyethylene glycol copolymer is 2%.

Polyvinyl alcohol (viscosity: 50 mPa·s) as a solute was put into purified water to prepare an aqueous phase, where the mass fraction of the solute was 0.1%.

The volume ratio of the oil phase and the aqueous phase is 1:4.

Microspheres are prepared by membrane emulsification: the oil phase is extruded under a pressure of 0.1 kPa, passed through a membrane with a pore diameter of 20 μm, and mixed with the aqueous phase (ice bath conditions of 4 to 8 ° C.) in a stirring state, and the stirring speed is 500 r/ min, and after completion of passing the oil phase through the membrane, the mixture was emulsified by stirring at 500 r/min for 60 min, then the ice bath was removed, and then stirred at 18° C. at 500 r/min for 4.5 h to volatilize the oil phase solvent. The prepared microspheres are washed 3 to 5 times with clean water. The particle diameter range of the microspheres obtained is 25 to 60 μm.

The microspheres obtained above were screened to obtain microspheres having a particle diameter of 35 to 45 µm (61.7% by mass in the total microspheres).

Step 2: Preparation of Dispersion

Take 240 mg of cross-linked sodium hyaluronate (molecular weight 1500000), 12 mg of mussel protein, and 100 mg of glycerin, add a phosphate buffer (prepared with reference to Pharmacopoeia 20208002, pH 7.3), dissolve, and use 10 ml. Gel dispersion A was obtained by adjusting the osmotic pressure to 280 mOsm/L with sodium chloride.

Step 3: Preparation of Composition

2.4 mg of lyophilized powder of PEG-SG (4-arm, molecular weight 10000) (preparation method is the same as in Example 1) was added to the gel dispersion A obtained in Step 2 together with the microspheres screened in Step 1, By mixing uniformly, a uniform composition was obtained. The total mass of PEG-SG and gel dispersion A and the mass ratio of microspheres were 7.5:2.5.

Example 4

This example provides a method for preparing the composition of the present invention, specifically comprising the following steps:

Step 1: Preparation of microspheres

Mixed reforming of polycaprolactone: ε-caprolactone, lactide, polyethylene glycol (molecular weight 2000) and toluene were added to the reaction vessel in a mass ratio of 2: 1: 1: 2, and stannous octoate was added dropwise as a catalyst. . A lactic acid-polycaprolactone-polyethylene glycol copolymer was obtained.

The polylactic acid-polycaprolactone-polyethylene glycol copolymer prepared above using tetrahydrofuran and dichloromethane as solvents (the volume ratio of the two solvents is 1:9) (PCL occupies 50wt%, viscosity 1.2dl/ g) to prepare an oil phase, wherein the mass fraction of the polylactic acid-polycaprolactone-polyethylene glycol copolymer is 8%.

Polyvinyl alcohol (viscosity: 25 mPa·s) and Tween-80 were used as solutes (mass ratio of the two solutes was 10:1), and purified water was added to prepare an aqueous phase, where the mass fraction of the solute was 1%.

The volume ratio of the oil phase and the aqueous phase is 1:6.

Microspheres are prepared by membrane emulsification: the oil phase is extruded under a pressure of 0.2 kPa, passed through a membrane with a pore diameter of 20 μm, and mixed with the aqueous phase (ice bath conditions of 4 to 8 ° C.) under stirring, and the stirring speed is 250 r/ min, and after completion of the passage of the oil phase through the membrane, the mixture was emulsified by stirring at 250 r/min for 30 min, then the ice bath was removed, and then stirred at 16° C. at 250 r/min for 5 h to volatilize the oil phase solvent. The prepared microspheres are washed 3 to 5 times with clean water. The particle diameter range of the microspheres obtained is 30 to 50 μm.

The microspheres obtained above were screened to obtain microspheres having a particle size of 35 to 45 µm (72.4% by mass in the total microspheres).

Step 2: Preparation of Dispersion

Take 300 mg of cross-linked sodium hyaluronate (molecular weight 1000000), 12 mg of mussel protein, and 50 mg of glycerin, add phosphate buffer (prepared with reference to Pharmacopoeia 20208002, pH 7.3), dissolve them, and use 10 ml regularly. Gel dispersion A was obtained by adjusting the osmotic pressure to 320 mOsm/L with sodium chloride.

Step 3: Preparation of Composition

2.5 mg of lyophilized powder of PEG-SG (4-arm, molecular weight 5000) (preparation method is the same as in Example 1) was added to the gel dispersion A obtained in Step 2 together with the microspheres screened in Step 1, By mixing uniformly, a uniform composition was obtained. The mass ratio of the total mass of PEG-SG and gel dispersion A to the mass of microspheres is 6:4.

Example 5

This example provides a method for preparing the composition of the present invention, specifically comprising the following steps:

Step 1: Preparation of microspheres

Mixed reforming of polycaprolactone: ε-caprolactone, lactide, polyethylene glycol (molecular weight 2000) and toluene were added to the reaction vessel in a mass ratio of 2: 1: 1: 2, and stannous octoate was added dropwise as a catalyst. . A lactic acid-polycaprolactone-polyethylene glycol copolymer was obtained.

The polylactic acid-polycaprolactone-polyethylene glycol copolymer prepared above using tetrahydrofuran and dichloromethane as solvents (the volume ratio of the two solvents is 1:9) (PCL occupies 50wt%, viscosity 1.2dl/g) ) to prepare an oil phase, wherein the mass fraction of the polylactic acid-polycaprolactone-polyethylene glycol copolymer is 8%.

Polyvinyl alcohol (viscosity: 25 mPa·s) and Tween-80 were used as solutes (mass ratio of the two solutes was 10:1), and purified water was added to prepare an aqueous phase, where the mass fraction of the solute was 1%.

The volume ratio of the oil phase and the aqueous phase is 1:6.

Microspheres are prepared by membrane emulsification: the oil phase is extruded under a pressure of 0.2 kPa, passed through a membrane with a pore diameter of 20 μm, and mixed with the aqueous phase (ice bath conditions of 4 to 8 ° C.) under stirring, and the stirring speed is 250 r/ min, and after the completion of passing the oil phase through the membrane, the mixture was emulsified by stirring at 250 r/min for 30 min, then the ice bath was removed, and then stirred at 19° C. at 250 r/min for 4 h to volatilize the oil phase solvent. The prepared microspheres are washed 3 to 5 times with clean water. The particle diameter range of the obtained microspheres is 30 to 50 μm.

The microspheres obtained above were screened to obtain microspheres having a particle diameter of 35 to 45 µm (74.3% by mass in total microspheres).

Step 2: Preparation of Dispersion

Take 150 mg of cross-linked sodium hyaluronate (molecular weight 2500000), 10 mg of mussel protein, and 200 mg of glycerin, add a phosphate buffer (prepared with reference to Pharmacopoeia 20208002, pH 7.3), dissolve, and use 10 ml. Gel dispersion A was obtained by adjusting the osmotic pressure to 300 mOsm/L with sodium chloride.

Step 3: Preparation of Composition

1.88 mg of lyophilized powder of PEG-SG (4-arm, molecular weight 40000) (preparation method is the same as in Example 1) was added to the gel dispersion A obtained in Step 2 together with microspheres screened in Step 1, By mixing uniformly, a uniform composition was obtained. The mass ratio of the total mass of PEG-SG and gel dispersion A to the mass of microspheres is 9:1.

Example 6

This experimental example provides a method for preparing the composition of the present invention, specifically including the following steps:

Step 1: Preparation of microspheres

Mixed reforming of polycaprolactone: ε-caprolactone, lactide, polyethylene glycol (molecular weight 2000) and toluene were added to the reaction vessel in a mass ratio of 4: 1: 1: 4, and stannous octoate was added dropwise as a catalyst. , The pressure was repeatedly reduced at 125 ° C. to discharge oxygen by passing nitrogen, sealed, and reacted at that temperature for 12 h to obtain a product. The product was dissolved in chloroform, washed again and purified, and dried in vacuum, A polylactic acid-polycaprolactone-polyethylene glycol copolymer was obtained.

The polylactic acid-polycaprolactone-polyethylene glycol copolymer prepared above using tetrahydrofuran and dichloromethane as solvents (the volume ratio of the two solvents is 1:9) (PCL occupies 70wt%, viscosity 0.15dl/g). ) to prepare an oil phase, wherein the mass fraction of the polylactic acid-polycaprolactone-polyethylene glycol copolymer is 8%.

Polyvinyl alcohol (viscosity: 25 mPa·s) and Tween-80 were used as solutes (mass ratio of the two solutes was 10:1), and purified water was added to prepare an aqueous phase, where the mass fraction of the solute was 1%.

The volume ratio of the oil phase and the aqueous phase is 1:6.

Microspheres are prepared by membrane emulsification: the oil phase is extruded under a pressure of 0.2 kPa, passed through a membrane with a pore diameter of 20 μm, and mixed with the aqueous phase (ice bath conditions of 4 to 8 ° C.) under stirring, and the stirring speed is 250 r/ min, and after the completion of passing the oil phase through the membrane, the mixture was emulsified by stirring at 250 r/min for 30 min, then the ice bath was removed, and then stirred at 20° C. for 4 h at 250 r/min to volatilize the oil phase solvent. The prepared microspheres are washed 3 to 5 times with clean water. The particle diameter range of the obtained microspheres is 20 to 60 μm.

The microspheres obtained above were screened to obtain microspheres having a particle diameter of 35 to 45 μm (53.5% by mass in the total microspheres).

Step 2: Preparation of Dispersion

Take 240 mg of cross-linked sodium hyaluronate (molecular weight 1500000), 12 mg of silk fibroin, and 100 mg of glycerin, add a phosphate buffer (prepared with reference to Pharmacopoeia 20208002, pH 7.3), dissolve them, and adjust the volume to 10 ml. Gel dispersion A was obtained by adjusting the osmotic pressure to 300 mOsm/L with sodium chloride.

Step 3: Preparation of Composition

2.4 mg of lyophilized powder of PEG-SG (4-arm, molecular weight: 40000) (preparation method is the same as in Example 1) was added to the gel dispersion A obtained in Step 2 together with the microspheres screened in Step 1 and uniformly mixed to obtain a uniform composition. The total mass of PEG-SG and gel dispersion A and the mass ratio of microspheres were 7.5:2.5.

Example 7

This example provides a method for preparing the composition, and the specific method for preparing the composition is the same as in Example 1. Distinctive points are: the manufacturing method of the microspheres is changed from the membrane emulsification method to the shear emulsification method.

Microspheres are prepared by shear emulsification method: The specific parameter of mechanical agitation is 1000 r/min, and the oil phase is added dropwise to the water phase under the conditions of a 5 ° C ice bath and stirring is activated, stirred for 30 min to emulsify, and then placed in an ice bath is removed, and the solvent is volatilized by stirring at a speed of 500 r/min for 5 h by reducing the rotation speed at 22 ° C., and the microspheres obtained are washed 3 to 5 times with clean water. The particle diameter range of the microspheres obtained is 10 to 80 μm.

The microspheres obtained above were screened to obtain microspheres having a particle size of 35 to 45 μm (36.6% by mass in total microspheres).

Example 8

This example provides a method for preparing the composition, and the specific method for preparing the composition is the same as in Example 1. Distinctive points are: the manufacturing method of the microspheres is changed from the membrane emulsification method to the spray drying method.

Preparation of microspheres by spray drying method: An oil phase having a mass fraction of a polylactic acid-polycaprolactone-polyethylene glycol copolymer at a concentration of 15% is prepared. The oil phase is put into the spray drying device, and the parameters specifically set are: the inlet temperature is 85 ° C, the outlet temperature is 25 ° C, the feed rate of the raw material is 25 ml / min, and the high-pressure air flow rate is 600 L / h. The particle diameter range of the microspheres obtained is 5 to 60 μm.

The microspheres obtained above were screened to obtain microspheres having a particle diameter of 35 to 45 μm (38.2% by mass in the total microspheres).

Comparative Example 1

This comparative example provides a preparation method of the composition, and the specific preparation method is the same as in Example 1. Distinguishing points: Polycaprolactone (molecular weight 8000) was used instead of polylactic acid-polycaprolactone-polyethylene glycol copolymer as a raw material for producing polymer microspheres. Specifically, the particle diameter range of the microspheres obtained is 5 to 65 μm. The microspheres obtained above were screened to obtain microspheres having a particle size of 35 to 45 μm (38.7% by mass in total microspheres).

Comparative Example 2

This comparative example provides a preparation method of the composition, and the specific preparation method is the same as in Example 1. Distinctive point: The composition was prepared using lyophilized powder of polyethylene glycol-propionaldehyde (4-arm, molecular weight: 10000) instead of lyophilized powder of polyethylene glycol succinimide glutarate.

Comparative Example 3

This comparative example provides a preparation method of the composition, and the specific preparation method is the same as in Example 1. Distinctive point: CMC-Na (molecular weight 700000) was used instead of cross-linked sodium hyaluronate in Example 1 as a component of gel dispersion A.

Comparative Example 4

This comparative example provides a preparation method of the composition, and the specific preparation method is the same as in Example 1. The difference is: the mass ratio of the total mass of PEG-SG and gel dispersion A to the mass of the microspheres is 10:1.

Comparative Example 5

This comparative example provides a preparation method of the composition, and the specific preparation method is the same as in Example 1. Distinguishing points are: the amount of cross-linked sodium hyaluronate (molecular weight 1500000) used is 100 mg, the mass ratio between cross-linked sodium hyaluronate and mussel protein is 30:1, the mass of mussel protein is 3.3 mg, and the mass of polyethylene glycol succinimide glutarate is It is 1 mg.

Comparative Example 6

This comparative example provides a preparation method of the composition, and the specific preparation method is the same as in Example 1. Distinctive points are: the mass fraction of the polylactic acid-polycaprolactone-polyethylene glycol copolymer in the oil phase is 1%, and the mass fraction of the emulsifier in the aqueous phase is 4%. Other steps are the same, and the obtained microspheres have a particle size range of 1 to 45 μm. The microspheres obtained above were screened to obtain microspheres having a particle diameter of 35 to 45 μm (34.8% by mass in total microspheres).

Experimental example 1

In this experimental example, the performance of the composition prepared and obtained in the above-described Examples and Comparative Examples was tested. Specific methods and results are as follows.

Observations on microsphere morphology:

A small amount of the microspheres after washing and natural drying were taken, and the appearance of the microspheres was observed under a scanning electron microscope at 200x and 500x magnification.

Swelling rate test:

After preparing the composition in the form of a module, the composition is cut into a fixed form, the mass M1 is precisely measured, transferred to a ground joint type Erlenmeyer flask, and put into a phosphate buffer having a pH of 7.2 to 7.4 preheated to 37 ° C. , the amount of solution used is at least 10 times the mass of the test sample, take out the sample after 24 hours, absorb and remove surface moisture with filter paper, and accurately measure the mass M2 of the gel, mass swelling ratio = (M2-M1)/M1Х100%.

Digestion cycle investigation:

The sample was sealed and placed in a constant temperature shaking water bath at 37° C., and a sample was taken every month to observe the degradation of microspheres.

Hydrophilic experiment:

The microspheres in the composition are separated, dissolved in dichloromethane, naturally volatilized on a slide to form a polymer film, and the slide is left with the film attached to a contact angle meter to measure the contact angle of the polymer film.

Stability Sample Neglect Investigation:

The sample is sealed and placed in a syringe, left upright at room temperature in a light-shielded environment, and the precipitation and aggregation of microspheres in the sample are observed at intervals of one day.

Cytocompatibility test:

Smooth muscle cells (purchased from Suzhou Beina Chuanglian Biotechnology Co., Ltd.) were resuscitated and cultured and passaged at 80% confluence, inoculated with the same weight of the composition obtained in different Examples and Comparative Examples, and then smooth muscle cells after inoculation. After digestion, the cell suspension was prepared, the suspension was added at a cell density of 5Х10 ³ cells/well, 100 μl of the smooth muscle cell culture medium was added, and then placed in a cell incubator at 37 °C, 5% CO ₂ and cultured for 7 d. , There are 4 duplicate wells in each group of samples, the growth state of cells is observed with a scanning electron microscope, the number of live/dead cells is counted through fluorescence staining, and the 450nm wavelength is used through the CCK8 method. By measuring the absorbance value at , the state of cell proliferation on the material is determined. Cell relative proliferation rate = live cells/dead cells.

In vivo animal testing:

(1) 48 purebred New Zealand white rabbits, 4 to 6 months old and weighing 210 to 215 g, were used as experimental animals, randomly divided into 8 groups of 6 rabbits in each group. The first group tests the composition of Example 1, the second group tests the composition of Example 4, the third group tests the composition of Example 5, and the fourth group tests the composition of Example 6. The fifth group tests the composition of Comparative Example 3, the sixth group tests the composition of Comparative Example 4, the seventh group tests the composition of Comparative Example 5, and the eighth group tests the composition of Comparative Example 6. to test

(2) On the day of transplantation, the rabbit's back was dehaired, disinfected with iodine tincture and ethanol normally, and a total of 10 points (0.5mL/point) were injected subcutaneously at 2cm intervals on both sides of the spine, and the injection site is marked as Coomassie Brilliant Blue. Color supplementation is applied to the injection site every 2 weeks to prevent discoloration due to metabolism.

(3) All animals were observed at 3 time points (1 week, 1 month, 3 months) after transplantation, the experimental animals were sacrificed at 3 months, and the subcutaneous tissue including the transplant material was cut to obtain a volume fraction of 10%. Put in phosphorus formaldehyde solution, fix for 48h, then carry out conventional dehydration, clearing, waxing, embedding, and conventional sectioning, section thickness is 3 μm; Immediately and 1 week after implantation, the diameter (horizontal and longitudinal radii, two oblique radii, and central thickness are measured, and the average value is taken as the hemisphere radius r), volume (volume = 2/3πr ³ ), As a result of the volume test immediately after injection, there is no significant difference in the volume of all Examples and Comparative Examples, and they are all 0.48 to 0.5 cm ³ ; At 1 month after transplantation, local appearance reactions and inflammatory reactions at the injection site were observed, and the animals were not sacrificed. The experimental results are shown in Table 1 and Table 2.

Sample Observation of microsphere appearance hydrophilic experiment Investigation of stability sample neglect Decomposition cycle investigation cytocompatibility
Experiment Swelling rate, % Example 1 The microspheres are complete and the surface is smooth. less than 90°
have hydrophilicity Dispersion is uniform within one year, finely divided layer Decomposition begins in year 1, complete decomposition in year 3 Cell relative proliferation rate 92% 15 Example 2 microspheres are complete,
smooth surface less than 90°
have hydrophilicity Dispersion is uniform within one year, finely divided layer Decomposition begins in year 1, complete decomposition in year 5 Cell relative proliferation rate 89% 16 Example 3 microspheres are complete,
smooth surface less than 90°
have hydrophilicity Dispersion is uniform within one year, finely divided layer Decomposition begins in year 1, complete decomposition in year 3 Cell relative proliferation rate 90% 15 Example 4 The microspheres are complete and the surface is smooth. less than 90°
have hydrophilicity Dispersion is uniform within one year, finely divided layer Decomposition begins in year 1, complete decomposition in year 3 Cell relative proliferation rate 88% 17 Example 5 The microspheres are complete and the surface is smooth. less than 90°
have hydrophilicity Dispersion is uniform within one year, finely divided layer Decomposition begins in year 1, complete decomposition in year 3 Cell relative proliferation rate 83% 18 Example 6 The microspheres are complete and the surface is smooth. less than 90°
have hydrophilicity Dispersion is uniform within one year, finely divided layer Decomposition begins at 9 months, complete decomposition by 1 year Cell relative proliferation rate 90% 16 Comparative Example 1 The microspheres are complete and the surface is smooth. greater than 90°
have hydrophobicity Dispersion is uniform within one year, finely divided layer Decomposition starts from year 1, complete decomposition in year 3 Cell relative proliferation rate 78% 16 Comparative Example 2 The microspheres are complete and the surface is smooth. less than 90°
have hydrophilicity Microspheres sedimented at 1 month Decomposition begins at 6 months, complete decomposition by 3 years Cell relative proliferation rate 82% 22 Comparative Example 3 microspheres are complete,
smooth surface less than 90°
have hydrophilicity Microspheres sedimented at 6 months Decomposition begins in year 1, complete decomposition in year 3 Cell relative proliferation rate 85% 23 Comparative Example 4 microspheres are complete,
smooth surface less than 90°
have hydrophilicity Dispersion is uniform within one year, finely divided layer Decomposition begins in year 1, complete decomposition in year 3 Cell relative proliferation rate 69% 19 Comparative Example 5 The microspheres are complete and the surface is smooth. Have less than 90° hydrophilicity Microspheres sedimented at 10 months Decomposition begins at 9 months, complete decomposition by 3 years Cell relative proliferation rate 88% 20 Comparative Example 6 Microspheres are incomplete and a large number of cracks exist. Have less than 90° hydrophilicity Dispersion is uniform within one year, finely divided layer Decomposition begins in year 1, complete decomposition in year 3 Cell relative proliferation rate 79% 19

Sample Animal test results local appearance reaction
(observe without sacrificing) Inflammatory reaction at the injection site
(observe without sacrificing) Changes in the fibrous layer at the injection site
(Observation after sacrificing) Histological observation of the injection site
(Observation after sacrificing) Volume 1 week after injection, cm ³ Example 1 There were no redness, ulcers, or nodules, and there was no abnormal feeling when touching the injection site with the hand, and the surface temperature was normal. No clear inflammatory response At the 3rd month, a collagen film is formed at all injection sites, and the subcutaneous position of the injection area is not clearly shifted. Significant proliferation of collagen fibers between cells at 3 months 0.53 Example 4 There were no redness, ulcers, or nodules, and there was no abnormal feeling when touching the injection site with the hand, and the surface temperature was normal. No clear inflammatory response At the 3rd month, a collagen film is formed at all injection sites, and the subcutaneous position of the injection area is not clearly shifted. Significant proliferation of collagen fibers between cells at 3 months 0.57 Example 5 There were no redness, ulcers, or nodules, and there was no abnormal feeling when touching the injection site with the hand, and the surface temperature was normal. No clear inflammatory response At the 3rd month, a collagen film is formed at all injection sites, and the subcutaneous position of the injection area is not clearly shifted. Significant proliferation of collagen fibers between cells at 3 months 0.58 Example 6 There were no redness, ulcers, or nodules, and there was no abnormal feeling when touching the injection site with the hand, and the surface temperature was normal. No clear inflammatory response At the 3rd month, a collagen film is formed at all injection sites, and the subcutaneous position of the injection area is not clearly shifted. Significant proliferation of collagen fibers between cells at 3 months 0.56 Comparative Example 3 There were no redness, ulcers, or nodules, and there was no abnormal feeling when touching the injection site with the hand, and the surface temperature was normal. No clear inflammatory response On the 7th day, the subcutaneous position of the injection area was relatively clear, and at the 3rd month, collagen films were formed at 3 to 4 injection sites per animal. A small amount of collagen fibers proliferated between cells at 3 months 0.68 Comparative Example 4 There were no redness, ulcers, or nodules, and there was no abnormal feeling when touching the injection site with the hand, and the surface temperature was normal. No clear inflammatory response At 3 months, collagen films are formed at 7 to 8 injection sites per animal, and the subcutaneous location of the injection area is not clearly shifted. A small amount of collagen fibers proliferated between cells at 3 months 0.63 Comparative Example 5 There were no redness, ulcers, or nodules, and there was no abnormal feeling when touching the injection site with the hand, and the surface temperature was normal. No clear inflammatory response At the 1st month, the subcutaneous location of the injection area was moved finely, and at the 3rd month, collagen films were formed at 5 to 6 injection sites per animal. A small amount of collagen fibers proliferated between cells at 3 months 0.65 Comparative Example 6 There were no redness, ulcers, or nodules, and there was no abnormal feeling when touching the injection site with the hand, and the surface temperature was normal. A mild inflammatory response is seen At 3 months, collagen films are formed at 7 to 8 injection sites per animal, and the subcutaneous location of the injection area is not clearly shifted. Significant proliferation of collagen fibers between cells at 3 months 0.61

Although the present invention has been described in detail with typical descriptions and embodiments as described above, some modifications or improvements are possible on the basis of the present invention, which are obvious to those skilled in the art. Therefore, all such modifications or improvements made on the basis of not departing from the spirit of the present invention fall within the scope of the claims of the present invention.

Claims (10)

A composition comprising polymeric microspheres, polyethylene glycol succinimide glutarate, and a dispersion, wherein the polymeric microspheres are polylactic acid-polycaprolactone-polyethylene glycol copolymer, and the dispersion contains cross-linked sodium hyaluronate and protein, wherein the The composition, characterized in that the protein is mussel protein or silk fibroin. According to claim 1, the mass ratio of the protein and the cross-linked sodium hyaluronate is 1: (15 to 25), preferably 1: 20; and/or
The composition, characterized in that the mass ratio of the cross-linked sodium hyaluronate and the polyethylene glycol succinimide glutarate is (80 to 120): 1, preferably 100: 1. The method of claim 2, wherein the weight ratio of the sum of the masses of the polyethylene glycol succinimide glutarate and the dispersion to the polymer microspheres is (6 to 9): (1 to 4), preferably 7.5 to 2.5. Characterized composition. The method according to any one of claims 1 to 3, wherein the dispersion further comprises a phosphate buffer and glycerin, and has an osmotic pressure of 250 to 350 mOsm/L; The concentration of the cross-linked sodium hyaluronate in the dispersion is 15 to 30 mg/ml, preferably 24 mg/ml; and/or
The composition, characterized in that the mass fraction of the glycerin in the dispersion is 0.48 to 2%, preferably 1%. The method according to any one of claims 1 to 4, wherein the polymer microspheres have an intrinsic viscosity of 0.15 to 1.5 dl/g, and a proportion of PCL in the polymer is 40 to 70 wt%. Preferably, the polymer microspheres The intrinsic viscosity is 1.2 dl/g, and the proportion of PCL in the polymer is 50 wt %; and/or
The polyethylene glycol succinimide glutarate is 4-armed and has a molecular weight of 5000 to 40000, preferably 10000; and/or
A composition, characterized in that the molecular weight of the cross-linked sodium hyaluronate is 1000000 to 2500000, preferably 1500000. As a method for producing the composition of any one of claims 1 to 5,
It includes a process of preparing polymer microspheres through shear emulsification, membrane emulsification or spray drying;
Specifically, it includes mixing an organic solvent and a polylactic acid-polycaprolactone-polyethylene glycol copolymer to obtain an oil phase; The organic solvent is at least one of dichloromethane, tetrahydrofuran and chloroform, preferably a mixed solvent composed of dichloromethane or tetrahydrofuran and dichloromethane in a volume ratio of (1 to 2): (8 to 10) How to characterize. The method according to claim 6, further comprising the step of obtaining an aqueous phase by mixing an emulsifier and water when preparing by applying a shear emulsification method or a membrane emulsification method; The emulsifier is at least one of polyvinyl alcohol, Span, Tween, and carboxymethyl cellulose, and is preferably a mixed solvent composed of polyvinyl alcohol or polyvinyl alcohol and Tween-80 in a mass ratio of (9 to 10): 1; and/or
The volume ratio of the oil phase and the aqueous phase is 1: (4 to 10). The method of claim 7, wherein the mass fraction of the polylactic acid-polycaprolactone-polyethylene glycol copolymer in the oil phase is 2 to 15%, and the mass fraction of the emulsifier in the aqueous phase is 0.1 to 3%. The method of claim 7 or 8, when the membrane emulsification method is applied, the oil phase is passed through a membrane under a pressure of 0.1 to 0.4 kPa, and mixed with the aqueous phase at 4 to 8 ° C., and the stirring speed is 200 to 500 r/min, the pore diameter of the membrane is 10 to 20 μm, and after the passage of the oil phase is completed, the oil phase is continuously stirred for emulsification for 10 to 60 minutes, and then the oil phase solvent is volatilized by continuing stirring at 15 to 25 ° C. make; Preferably, the pressure passing through the membrane is 0.2 kPa, the pore diameter of the membrane is 20 μm, and when the oil phase and the water phase are mixed and emulsified by continuous stirring, the stirring speed is 250 r/min, continuously at 15 to 25 ° C. The speed at which the oil phase solvent is volatilized by stirring is 250 r/min;
When preparing by applying the shear emulsification method, the oil phase is mixed with the aqueous phase drop by drop while stirring at 4 to 8 ° C., the stirring speed is 800 to 1000 r / min, the time is 20 to 40 min, and then 15 to 25 stirring continuously at 400 to 500 r/min for 3 to 5 h; Preferably, when mixing, the stirring speed is 1000 r/min, the time is 30 min, and then the stirring is continued at 15 to 25° C. at a rate of 500 r/min for 5 h;
When manufacturing by applying the spray drying method, the oil phase is spray dried, the inlet temperature of the spray drying process is 60 to 85 ° C, the outlet temperature is 25 to 50 ° C, the feed rate of the raw material is 10 to 25 ml / min, and the high-pressure air flow rate is 400 to 600 L/h; Preferably, the inlet temperature is 85 ° C, the outlet temperature is 25 ° C, the feed rate of the raw material is 25 ml / min, and the high-pressure air flow rate is 600 L / h. Application of the composition of any one of claims 1 to 5 or the composition prepared by the method of any one of claims 6 to 9 in the manufacture of an injectable dermal filling product.