CN116271225A

CN116271225A - Medical sterile collagen product loaded with polylactic acid-glycolic acid copolymer microspheres and preparation method thereof

Info

Publication number: CN116271225A
Application number: CN202310151752.7A
Authority: CN
Inventors: 初丽艳
Original assignee: Zhuhai Fuyuan Biotechnology Co ltd
Current assignee: Zhuhai Fuyuan Biotechnology Co ltd
Priority date: 2023-02-22
Filing date: 2023-02-22
Publication date: 2023-06-23

Abstract

The invention relates to the technical field of medical preparations and medical cosmetology, in particular to a medical sterile collagen product loaded with polylactic acid-glycolic acid copolymer microspheres, which comprises sterile polylactic acid-glycolic acid copolymer microspheres and recombinant human III type collagen, wherein the sterile polylactic acid-glycolic acid copolymer microspheres and the recombinant human III type collagen are subjected to radiation sterilization treatment.

Description

Medical sterile collagen product loaded with polylactic acid-glycolic acid copolymer microspheres and preparation method thereof

Technical Field

The invention relates to the technical field of medical preparations and medical cosmetology, in particular to a medical sterile collagen product loaded with polylactic acid-glycolic acid copolymer microspheres and a preparation method thereof.

Background

Polylactic acid-glycolic acid copolymer (PLGA, CAS number: 34346-01-5) is polymerized by two monomers, namely lactic acid and glycolic acid, is a degradable functional polymer organic compound, and different types of PLGA can be prepared by different monomer proportions. The external appearance of the water-soluble organic solvent is white particles, is nontoxic, insoluble in water and easily soluble in various polar organic solvents. The polylactic acid-glycolic acid copolymer has good biocompatibility, good organic high polymer compatibility and good biodegradability. It has longer half-life, adjustable and controllable retention time in human body, long curative effect retention time and 18-24 months effective time. PLGA is a degradable high molecular organic compound, and the degradation products are polylactic acid and polyglycolic acid, are byproducts of human metabolic pathways, have good biocompatibility, are widely applied to the fields of pharmacy and medical engineering materials, and have no toxic or side effect. Therefore, the product of the invention has no immune reaction and no adverse reactions such as red swelling and the like during injection. Is suitable for soft tissue filling in cosmetic and plastic surgery, such as facial wrinkle removing and dent filling, and can eliminate forehead transverse lines, fish tail lines, inter-eyebrow lines, nose lip lines, mouth Zhou Zhouwen and scar. The gel based on polylactic acid-glycolic acid copolymer containing recombinant type III human collagen can provide immediate and continuous plumping effect after injection.

Polylactic acid-glycolic acid copolymer as a filler raw material is generally formed into microspheres, and the size, shape, smoothness thereof have been proved to have a great influence on tissue reaction, and the extent of the reaction can be determined. The polylactic acid-glycolic acid copolymer microsphere with smooth surface and regular sphere shape can reduce inflammation to the greatest extent; the smoother the sphere of the polylactic acid-glycolic acid copolymer microsphere is, the more favorable the subcutaneous injection is, and the polylactic acid-glycolic acid copolymer microsphere with high smoothness can not only stimulate the fibroblast to wrap one by one under the wrinkles, but also reduce the occurrence of foreign body reaction.

The recombinant III type human collagen is full-length or partial amino acid sequence segment coded by human collagen specific type gene prepared by DNA recombination technology, or contains the combination of human collagen functional segment. The repeated unit of the amino acid sequence is the same as the specific functional area of the amino acid sequence of the human collagen protein, and the biocompatibility is good; and the 164.88 DEG flexible triple helix structure with special high cell adhesion can form a reticular fiber structure and has good repairing property.

The recombinant III type humanized collagen serving as a repairing filling raw material can directly play a role after being injected into a body, so that wrinkles are eliminated.

After 1-3 months, when the implanted collagen is gradually absorbed by a human body, the polylactic acid-glycolic acid copolymer microsphere also starts to play a role, stimulates the regeneration of the collagen in the human body, replaces the absorbed implanted collagen, and continuously maintains the wrinkle removing filling effect, so that the skin keeps the effect when the skin is just applied for a long time.

Since cosmetic products are generally administered by direct injection into subcutaneous tissue of the human body, if pathogens carried on the surface or inside of the product can cause infection of the patient and cause serious adverse reactions, biomedical fillers for human body implantation need sterilization. Sterilization not only needs to kill or remove microbial propagules and spores, improves the safety and effectiveness of injection to the greatest extent, but also needs to ensure the shape, the character, the stability and the clinical curative effect of the injection cosmetic product, so that the selection of a proper sterilization method has important significance for ensuring the quality of the cosmetic product. Various sterilization methods have been employed in the fields of medical device products and medical injection products for the past several decades, including autoclaving, ethylene oxide sterilization, plasma sterilization, and beta radiation, gamma radiation, or X-ray radiation. The proper sterilization method should be selected according to the characteristics of the implant material so as to avoid ineffectiveness of the sterilization process and reduce damage to the product.

Polylactic acid-glycolic acid copolymers are temperature sensitive and have a low glass transition temperature, so that a wet heat sterilization (e.g., autoclaving) method cannot be adopted. Co irradiation sterilization has been used for sterilization of microsphere formulations more and more, because Co irradiation sterilization has a strong penetrating power without increasing the temperature. However, since high-energy rays affect the physicochemical properties of the polymer by interacting with the polymer, the determination of irradiation dose is very important. The invention selects 10kGy, 15kGy and 25kGy irradiation doses, has good sterilization effect, has no obvious influence on the average particle size and span of the microspheres, and shows that the high-energy rays do not change the property of PLGA when the sterilization dose is less than or equal to 25kGy. The invention selects the highest dose of 25kGy to ensure the sterility of the final product and meet the hygienic standard of medicines.

In addition, polylactic acid-glycolic acid copolymer microspheres having regular spherical shapes and high surface smoothness can improve the fluidity of gel and prevent adverse foreign body reaction at the same time, because the overall biocompatibility of dermal fillers is determined by the regular shapes and smoothness of filled microparticles. If the surface of the filling particles is smooth, the collagen growth is stimulated; if the shape is irregular and the surface is rough, the surface is surrounded by macrophages and multinuclear giant cells, which leads to envelope fibrosis, inhibits the generation of collagen, and may lead to adverse foreign body reaction.

In addition, the gel containing the recombinant type III human collagen in the injectable medical sterile collagen product containing the polylactic acid-glycolic acid copolymer microspheres also needs to be sterilized. Because the gel contains recombinant III type human collagen, the gel is not suitable for a high-pressure steam sterilization method and other damp heat sterilization methods, and the method can greatly destroy the activity of the collagen. Therefore, gamma irradiation or beta irradiation sterilization is required.

Syringe materials such as polypropylene materials are known to discolor and become brittle when subjected to radiation sterilization and are therefore unsuitable for sterilization by gamma irradiation or beta irradiation. If the syringe is filled with the gel injection, a gas such as ethylene oxide cannot pass through the syringe. Therefore, the assembled medical sterile collagen product for injection can not be sterilized by using ethylene oxide. Therefore, the polylactic acid-glycolic acid copolymer microsphere can be effectively sterilized, and the sphere shape and the surface smoothness of the microsphere can be maintained. And how to sterilize the gel containing the recombinant III type humanized collagen, and simultaneously maintain the basic properties of viscosity, fluidity and the like of the gel, and ensure the activity of the collagen to the greatest extent not to be destroyed, and the preparation of the medical sterile collagen product containing the sterile polylactic acid-glycolic acid copolymer microsphere is a problem to be solved in the medical cosmetic field.

In summary, the invention solves the problems by designing a medical sterile collagen product loaded with polylactic acid-glycolic acid copolymer microspheres and a preparation method thereof.

Disclosure of Invention

The invention aims to provide a medical sterile collagen product loaded with polylactic acid-glycolic acid copolymer microspheres and a preparation method thereof, so as to solve the problems in the background technology.

In order to achieve the above purpose, the present invention provides the following technical solutions:

the medical sterile collagen product loaded with the polylactic acid-glycolic acid copolymer microsphere is characterized by comprising the sterile polylactic acid-glycolic acid copolymer microsphere and recombinant human III type collagen, wherein the sterile polylactic acid-glycolic acid copolymer microsphere and the recombinant human III type collagen are subjected to irradiation sterilization treatment, the surface of the sterile polylactic acid-glycolic acid copolymer microsphere is smooth, the average particle diameter range is 25-45 mu m, and the roundness is more than or equal to 0.95; the content of the recombinant human III type collagen is more than or equal to 0.2 percent.

As a preferable scheme of the invention, the sterile polylactic acid-glycolic acid copolymer microsphere is obtained by adopting gamma ray or beta ray irradiation sterilization treatment, wherein the irradiation dose is 15kGy, 20kGy or 25kGy.

As a preferable scheme of the invention, the proportion of the microsphere with the particle size of 25-50 μm in the sterile polylactic acid-glycolic acid copolymer microsphere is more than or equal to 70%, and the proportion of the microsphere with the particle size of less than 25 μm is less than or equal to 20%.

As a preferable scheme of the invention, the roundness change rate of the polylactic acid-glycolic acid copolymer microsphere before and after irradiation is less than or equal to 4.0%, and the recombinant type III human collagen is contained in CMC-Na gel or crosslinked sodium hyaluronate gel.

As a preferable scheme of the invention, CMC-Na gel or sodium hyaluronate gel of the recombinant III type human collagen is obtained by adopting gamma ray or beta ray irradiation sterilization treatment, wherein the irradiation dose is 6kGy, 9kGy or 12kGy.

As a preferable scheme of the invention, the activity change rate of the recombinant type III human collagen before and after irradiation is less than or equal to 6.0 percent, and the concentration change rate of the recombinant type III human collagen before and after irradiation is less than or equal to 10.0 percent.

As a preferred embodiment of the present invention, a sterile-processed syringe is also included.

The preparation method of the medical sterile collagen product loaded with the polylactic acid-glycolic acid copolymer microspheres comprises the following specific steps:

step 1: sterilizing by irradiation to obtain sterile polylactic acid-glycolic acid copolymer microspheres; sterilizing by beta rays to obtain CMC-Na gel or crosslinked sodium hyaluronate gel containing recombinant III type human collagen; sterilizing the syringe by using ethylene oxide;

step 2: adding the sterilized sterile polylactic acid-glycolic acid copolymer microspheres into a gel containing recombinant III-type humanized collagen, and uniformly mixing;

step 3: packaging the uniformly mixed gel loaded with the sterile polylactic acid-glycolic acid copolymer microsphere and containing the recombinant III-type humanized collagen into the injector;

wherein the steps of obtaining the aseptic polylactic acid-glycolic acid copolymer microsphere through irradiation sterilization treatment are specifically as follows:

step a: weighing a proper amount of polylactic acid-glycolic acid copolymer microspheres, placing the microspheres in a centrifuge tube, and marking the irradiation dose and the types of the microspheres on the centrifuge tube;

step b: sealing the centrifuge tube by using a sealing film in order to ensure the sterile state and no leakage in detection;

step c: filling the centrifuge tube into a packaging box, labeling the outside of the packaging box with a label, and marking sterilization dose information;

step d: receiving gamma ray irradiation;

as a preferred embodiment of the present invention, the following steps are performed for the recombinant type III humanized collagen-containing sterile CMC-Na gel:

step a: adding glycerol and propylene glycol into the gel, and stirring until the mixture is uniform;

step b: weighing the recombinant type III human collagen powder according to claim 1, dissolving the recombinant type III human collagen powder by using a small amount of water, adding the dissolved recombinant type III human collagen powder into the mixed gel in the step (1), and stirring the mixed gel until the mixed gel is uniformly mixed.

Step c: weighing a proper amount of CMC-Na gel containing recombinant III type humanized collagen, placing the CMC-Na gel in an ep tube, and marking the irradiation dose and the gel type on the ep tube;

step d: sealing the ep tube by using a sealing film in order to ensure the sterile state and no leakage in detection;

step e: filling the ep tube into a packaging box, labeling the outside of the box, and marking sterilization dosage information;

step f: receiving beta-ray irradiation;

wherein the step of mixing is carried out under vacuum condition.

10. The method for preparing a medical sterile collagen product loaded with polylactic acid-glycolic acid copolymer microspheres according to claim 8, wherein the preparation method comprises the following specific steps of:

step a: weighing the recombinant type III human collagen powder according to claim 1, dissolving the recombinant type III human collagen powder with a small amount of water, adding the dissolved recombinant type III human collagen powder into the crosslinked sodium hyaluronate gel, and stirring the mixture until the mixture is uniform.

Step b: weighing a proper amount of crosslinked sodium hyaluronate gel containing recombinant III-type humanized collagen, placing the crosslinked sodium hyaluronate gel in an ep tube, and marking the irradiation dose and the gel type on the ep tube;

step c: sealing the ep tube by using a sealing film in order to ensure the sterile state and no leakage in detection;

step d: filling the ep tube into a packaging box, labeling the outside of the box, and marking sterilization dosage information;

step e: receiving beta radiation.

Compared with the prior art, the invention has the beneficial effects that:

according to the invention, the sterile polylactic acid-glycolic acid copolymer microsphere obtained by adopting a certain dose of gamma or beta rays for irradiation has a colony number detection value reaching an undetected level, and the sterile polylactic acid-glycolic acid copolymer microsphere obtained by irradiation sterilization and the recombinant III-containing humanized collagen gel obtained by irradiation sterilization are uniformly mixed and then filled into an injector sterilized by ethylene oxide to form a sterile medical product, so that the sterile injection requirement can be met, the product quality can be better ensured, and the generation of side effects such as inflammatory reaction and the like is reduced, and the preparation method comprises the following steps:

(1) The medical sterile collagen product loaded with the polylactic acid-glycolic acid copolymer microspheres provided by the invention acts through the recombinant III type human collagen in the early stage of injection use, and long-acting shaping is realized through the growth stimulation of the polylactic acid-glycolic acid copolymer microspheres in the later stage, the effect of maintaining the skin activity is realized, and finally, the long-acting sustainable effect is achieved.

(2) The CMC-Na gel part of the recombinant III type human collagen contained in the product of the invention is added with the recombinant III type human collagen as a main active ingredient, and glycerol for injection and 1, 3-propanediol as an auxiliary humectant and a vitamin stabilizer, thereby further increasing the moisturizing effect of the product and reducing the irritation of the product to skin.

(3) The medical sterile collagen product loaded with the polylactic acid-glycolic acid copolymer microsphere provided by the invention has the advantages that the polylactic acid-glycolic acid copolymer microsphere, the gel containing the recombinant III type human collagen and the loaded syringe are sterilized by adopting different sterilization modes, and the biological safety of the product is high.

(4) The polylactic acid-glycolic acid copolymer microsphere related by the invention is uniformly dispersed in the gel containing the recombinant III type humanized collagen, so that side reaction caused by microsphere aggregation is greatly reduced, and the biological safety of the product is high.

Detailed Description

In the following, the technical solutions of the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and all other embodiments obtained by those skilled in the art without making creative efforts based on the embodiments of the present invention are included in the protection scope of the present invention.

The invention provides a technical scheme that:

the present invention is a medical sterile collagen product comprising sterile polylactic acid-glycolic acid copolymer microspheres, which is required to be injected into the facial skin of a human body, thus requiring strict sterilization of the product itself. To polylactic acid-glycolic acid copolymer microspheres contained therein. By researching the influence of gamma-ray and beta-ray irradiation sterilization on polylactic acid-glycolic acid copolymer microspheres, the sterilization effect of gamma-ray is found to be better than that of beta-ray. In addition, it has been found that both high doses of gamma rays and high doses of beta rays adversely affect the polylactic acid-glycolic acid copolymer microspheres themselves, mainly by varying the free radical content and surface smoothness of the polylactic acid-glycolic acid copolymer microspheres. Therefore, in sterilizing polylactic acid-glycolic acid copolymer microspheres, the irradiation mode and the irradiation dose will be very important. Regarding the gel containing the recombinant III type human collagen contained in the product, the influence of gamma-ray and beta-ray irradiation sterilization on the gel property and the collagen activity is researched, and the beta-ray sterilization effect is better than that of gamma-ray, and meanwhile, both the high-dose gamma-ray and the high-dose beta-ray can damage the gel property and the collagen activity. Thus, the manner of irradiation and the dose of irradiation will be important when sterilizing gels containing recombinant type III human collagen.

In the invention, CMC-Na gel contains recombinant III type humanized collagen, and glycerol and 1, 3-propylene glycol for injection are also added.

As described above, the glycerol content in the glycerol for injection used in the invention is more than or equal to 99.5%, and the safety is higher than that of common glycerol. As a humectant in the product, the glycerin for injection can obviously improve the water content of the skin horny layer, increase the skin elasticity, protect the skin from external stimulus, and enable the skin to be healthy, fine, smooth and full of vigor.

The 1, 3-propanediol is used in the invention as the organic polyol humectant, which is beneficial to the penetration of the active ingredient (recombinant III type human collagen) into the skin, and has higher moisturizing ability than that of the 1, 2-propanediol, lower uncomfortable reactions such as skin irritation, burning sensation and the like, and even reaches the level of no uncomfortable reaction.

The crosslinked sodium hyaluronate gel of the invention contains recombinant III type humanized collagen and is added with a crosslinking agent of divinyl sulfone. The divinyl sulfone can obviously improve the stability of the hyaluronic acid, and obviously enhance the resistance to hyaluronidase and free radicals, so that the retention time of the hyaluronic acid in the body is obviously prolonged.

The invention relates to a medical sterile collagen product containing sterile polylactic acid-glycolic acid copolymer microspheres, which comprises the sterile polylactic acid-glycolic acid copolymer microspheres, wherein the polylactic acid-glycolic acid copolymer microspheres are subjected to irradiation sterilization treatment, the surfaces of the polylactic acid-glycolic acid copolymer microspheres are smooth and spherical, the average particle size range is 25-45 mu m, and the roundness is more than or equal to 0.95, preferably more than 0.96, more than 0.97, more than 0.98 and more than 0.99.

As described above for the medical sterile collagen product, the roundness change rate of the polylactic acid-glycolic acid copolymer microsphere before and after irradiation is less than or equal to 4.0%, preferably less than or equal to 3.5%, 3.0%, 2.5% or 2%.

The medical sterile collagen product also comprises gel containing recombinant type III human collagen, which is obtained by beta-ray irradiation sterilization treatment, wherein the concentration change rate of the recombinant type III human collagen before and after irradiation is less than or equal to 10.0%, preferably less than or equal to 9.0%, 8.0%, 7.0%, 6.0%, 5.0%, 4.0%, 3.0% or 2.0%.

The medical sterile collagen product as described above, further comprising a sterile-processed syringe.

A method for preparing the sterilized polylactic acid-glycolic acid copolymer microsphere in claim 1, which comprises the following steps of:

step 1: weighing a proper amount of polylactic acid-glycolic acid copolymer microspheres, placing the microspheres in a centrifuge tube, and marking the irradiation dose and the types of the microspheres on the centrifuge tube;

step 2: sealing the centrifuge tube by using a sealing film in order to ensure the sterile state and no leakage in detection;

step 3: filling the centrifuge tube into a packaging box, labeling the outside of the packaging box with a label, and marking sterilization dose information;

step 4: receiving gamma-ray or beta-ray radiation.

A method as hereinbefore described wherein the irradiation dose is 10kGy, 15kGy or 25kGy.

A method for preparing a medical sterile collagen product containing sterile polylactic acid-glycolic acid copolymer microspheres as described above, comprising the step of uniformly mixing the sterile polylactic acid-glycolic acid copolymer microspheres with CMC-Na gel or crosslinked sodium hyaluronate gel containing recombinant III-type human collagen obtained after sterilization by beta-ray irradiation.

The preparation method comprises the following steps of: mixing under vacuum.

The preparation method adopts a vacuum stirring deaeration machine in the mixing process.

A method for preparing CMC-Na gel containing recombinant type III human collagen, which comprises the following specific steps:

step 1: adding glycerol and propylene glycol into the gel, and stirring until the mixture is uniform;

step 2: weighing the recombinant type III human collagen powder according to claim 1, dissolving the recombinant type III human collagen powder by using a small amount of water, adding the dissolved recombinant type III human collagen powder into the mixed gel in the step (1), and stirring the mixed gel until the mixed gel is uniformly mixed.

Step 3: weighing a proper amount of CMC-Na gel containing recombinant III type humanized collagen, placing the CMC-Na gel in an ep tube, and marking the irradiation dose and the gel type on the ep tube;

step 4: sealing the ep tube by using a sealing film in order to ensure the sterile state and no leakage in detection;

step 5: filling the ep tube into a packaging box, labeling the outside of the box, and marking sterilization dosage information;

step 6: receiving beta radiation.

A method as hereinbefore described wherein the irradiation dose is 5kGy, 8kGy or 10kGy.

A method for preparing a crosslinked sodium hyaluronate gel containing recombinant type iii human collagen as described above, comprising the following steps:

step 1: weighing the recombinant type III human collagen powder according to claim 1, dissolving the recombinant type III human collagen powder with a small amount of water, adding the dissolved recombinant type III human collagen powder into the crosslinked sodium hyaluronate gel, and stirring the mixture until the mixture is uniform.

Step 2: weighing a proper amount of crosslinked sodium hyaluronate gel containing recombinant III-type humanized collagen, placing the crosslinked sodium hyaluronate gel in an ep tube, and marking the irradiation dose and the gel type on the ep tube;

step 3: sealing the ep tube by using a sealing film in order to ensure the sterile state and no leakage in detection;

step 4: filling the ep tube into a packaging box, labeling the outside of the box, and marking sterilization dosage information;

step 5: receiving beta radiation.

A method for preparing a medical sterile collagen product as described above, comprising the following steps:

step 1: sterilizing by irradiation to obtain sterile polylactic acid-glycolic acid copolymer microspheres; the gel containing the recombinant III type humanized collagen is obtained through irradiation sterilization; sterilizing the syringe by using ethylene oxide;

step 3: and packaging the uniformly mixed gel containing the recombinant type III human collagen and loaded with the sterile polylactic acid-glycolic acid copolymer microspheres into the syringe.

The present invention is further illustrated by the following examples, but is not limited thereto.

Example 1: preparation of CMC-Na gel containing recombinant III type human collagen

And step 1, pouring a certain amount of PBS buffer solution with pH of 6.5-8 prepared by using dihydrogen phosphate and sodium hydroxide into a beaker, and stirring.

And step 2, slowly pouring a certain amount of CMC-Na powder (the weight ratio of the CMC-Na powder to the final product filling is 2-3 wt%) into the gel, fully contacting the powder with the buffer solution, and stirring for 2-5h until no caking, namely unstirred powder, is seen in the gel.

And 3, adding a certain amount of glycerin (the content of the glycerin in the final product filler is 0.9-1.1 wt.%) into the gel, and fully stirring for about 5min.

And step 4, weighing 0.02g of recombinant III type human collagen powder, dissolving the powder by using a small amount of water, adding the dissolved powder into the gel in the step 3, and magnetically stirring the dissolved powder until the dissolved powder is uniformly mixed to obtain CMC-Na gel containing the recombinant III type human collagen.

Example 2: the preparation of the sodium hyaluronate gel containing the recombinant III type human collagen is prepared by fully mixing the following components through high-speed stirring or grinding: 10% of cross-linked hyaluronic acid gel, 3% of recombinant III-type humanized collagen and the balance of water.

The preparation method of the recombinant III type human collagen sodium hyaluronate gel comprises the following steps:

step 1, sodium hyaluronate (mw=150ten thousand) was dissolved in a 2mol/L sodium hydroxide solution, and the mass concentration of sodium hyaluronate in the solution was 20%. After stirring until fully dissolved, divinyl sulfone was added. The mass concentration of the divinyl sulfone in the solution is 5%.

Step 2, the temperature is raised to 60 ℃ and kept mechanically stirred for 14 hours.

And 3, stopping heating, placing the obtained crosslinked hyaluronic acid gel in 0.05mo1/L hydrochloric acid solution, wherein the addition volume of the hydrochloric acid solution is 5 times of the volume of the crosslinked hyaluronic acid gel, stirring at room temperature, and reacting for 12 hours.

And 4, filtering out the hydrochloric acid solution by suction, adding 0.05mol/L of phosphate buffer solution with the pH value of 7.0 for dissolution, wherein the adding volume of the buffer solution is 50 times of the volume of the crosslinked hyaluronic acid gel.

And 5, dissolving the recombinant III type human collagen powder by using a small amount of water, adding the dissolved recombinant III type human collagen powder into the gel in the step 4, and stirring the mixture until the mixture is uniformly mixed to obtain the sodium hyaluronate gel containing the recombinant III type human collagen.

Example 3: the method for preparing gel by mixing crosslinked recombinant type III human collagen and crosslinked sodium hyaluronate comprises the following steps:

step 1, mixing recombinant III type human collagen with alginic acid aldehyde in NaOH solution, wherein the weight ratio of the recombinant III type human collagen to the first crosslinking agent is 35:1, preparing the crosslinked recombinant III-type humanized collagen through a heating crosslinking reaction, wherein the process condition of the heating reaction is 30 ℃, the reaction time is 8 hours, and the heating is performed by water bath.

Step 2, mixing sodium hyaluronate with 1, 4-butanediol diglycidyl ether in NaOH solution, wherein the weight ratio of the sodium hyaluronate to the second crosslinking agent is 25:1, heating to prepare sodium hyaluronate gel, wherein the process condition of the heating reaction is 40 ℃, and the reaction time is 10 hours.

And step 3, cutting the sodium hyaluronate gel into blocks, wherein the cutting mode is to cut the gel into blocks transversely and longitudinally by adopting a metal wire mesh, cut the gel into blocks transversely and longitudinally by adopting the metal wire mesh, and then mix the gel with the crosslinked recombinant human collagen to obtain the mixed gel.

And 4, neutralizing the mixed gel acid, flushing the mixed gel with a buffer solution, homogenizing, wherein the acid is lactic acid, neutralizing the mixed gel acid until the pH value of the system is 6-7, and the buffer solution is phosphate solution to obtain a gel finished product.

Example 4: preparation method of gel containing recombinant III type humanized collagen

Step 1, dissolving recombinant III type human collagen powder in sodium chloride solution with the mass concentration of 0.9% to prepare recombinant III type human collagen suspension;

and 2, regulating the pH value of the recombinant type III human collagen suspension to 7.5 by adopting a sodium hydroxide solution with the concentration of 0.15 mol/L. Hyaluronic acid is added, and the mass ratio of the recombinant III type humanized collagen to HA is 5:1, heating to 45 ℃ and stirring, and carrying out crosslinking reaction under the action of ultrasonic waves to obtain the gel. The power of the ultrasonic wave was 400W and the crosslinking reaction time was 13h.

And 3, washing gel to be neutral by using 0.75mol/L hydrochloric acid, and washing by using a sodium chloride solution with the mass concentration of 0.9% to obtain the crosslinked recombinant type III human collagen gel.

Example 5: the present example provides a CMC-Na gel containing recombinant humanized collagen, which is prepared from the following components: the recombinant type III human collagen with the molecular weight of 55KD, the recombinant type IV human collagen with the molecular weight of 165KD, the recombinant type IV human collagen with the molecular weight of 0.4%, sodium carboxymethylcellulose, the glycerol of 4%, the disodium edetate of 0.2%, the medical preservative methylparaben of 0.3% and the balance of purified water. The specific preparation process comprises the following steps:

step 1, adding 90.7mL of purified water into a stirring beaker, heating to 90 ℃ in a water bath, adding 0.3g of methylparaben, stirring and dissolving.

And 2, cooling to 50 ℃, adding 4g of CMC-Na, stirring and dissolving.

And 3, cooling to 30 ℃, adding 4g of glycerol, and stirring until the glycerol is completely dissolved.

And step 4, adding 0.2g of disodium ethylenediamine tetraacetate and stirring until the disodium ethylenediamine tetraacetate is completely dissolved.

And 5, adding 0.4g of recombinant type III human collagen and 0.4g of recombinant type IV human collagen, and stirring until the recombinant type III human collagen and the recombinant type IV human collagen are completely dissolved.

And step 6, slowly stirring, standing and swelling at room temperature until no obvious bubbles exist when the temperature is reduced to below 30 ℃, and obtaining the CMC-Na gel containing the recombinant humanized collagen.

Example 6: mixing the sterilized polylactic acid-glycolic acid copolymer microsphere and the sterilized sodium hyaluronate gel to form a sterilized medical product

And step 1, adding the microspheres into the gel. The mass of the microsphere is 19% of the sum of the mass of the sodium hyaluronate and the recombinant type III human collagen.

And 2, performing preliminary stirring by using a glass rod or a fine scraper. And then stirring and removing bubbles by using a vacuum stirring deaeration machine.

Example 7: injecting the medical sterile collagen product loaded with polylactic acid-glycolic acid copolymer microspheres into a sterilized syringe to form a sterile injection medical product

The syringe and needle cap enter the LAF Laminar Air Flow device (Laminar Air Flow) in a sterile manner. The cosmetic product is filled into the syringe by pouring to the correct volume and the end cap is attached to form a sterile barrier. All mixing and filling steps were performed aseptically in an ISO 5 LAF device, labeled, resulting in an aseptically injectable cosmetic product, which was then stored.

The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.

Claims

1. The medical sterile collagen product loaded with the polylactic acid-glycolic acid copolymer microsphere is characterized by comprising the sterile polylactic acid-glycolic acid copolymer microsphere and recombinant human III type collagen, wherein the sterile polylactic acid-glycolic acid copolymer microsphere and the recombinant human III type collagen are subjected to irradiation sterilization treatment, the surface of the sterile polylactic acid-glycolic acid copolymer microsphere is smooth, the average particle diameter range is 25-45 mu m, and the roundness is more than or equal to 0.95; the content of the recombinant human III type collagen is more than or equal to 0.2 percent.

2. The medical sterile collagen product loaded with polylactic acid-glycolic acid copolymer microspheres according to claim 1, wherein the sterile polylactic acid-glycolic acid copolymer microspheres are obtained by gamma-ray or beta-ray irradiation sterilization treatment, and the irradiation dose is 15kGy, 20kGy or 25kGy.

3. The medical sterile collagen product loaded with polylactic acid-glycolic acid copolymer microspheres according to claim 1, wherein the ratio of the microspheres with the particle size of 25-50 μm in the sterile polylactic acid-glycolic acid copolymer microspheres is more than or equal to 70%, and the ratio of the microspheres with the particle size of less than 25 μm is less than or equal to 20%.

4. The medical sterile collagen product loaded with polylactic acid-glycolic acid copolymer microspheres according to claim 1, wherein the roundness rate of the polylactic acid-glycolic acid copolymer microspheres before and after irradiation is less than or equal to 4.0%, and the recombinant type iii human collagen is contained in CMC-Na gel or crosslinked sodium hyaluronate gel.

5. The medical sterile collagen product loaded with polylactic acid-glycolic acid copolymer microspheres according to claim 4, wherein the CMC-Na gel or the sodium hyaluronate gel of the recombinant type III human collagen is obtained by adopting gamma ray or beta ray irradiation sterilization treatment, wherein the irradiation dose is 6kGy, 9kGy or 12kGy.

6. The medical sterile collagen product loaded with polylactic acid-glycolic acid copolymer microspheres according to claim 1, wherein the activity change rate of the recombinant type III human collagen before and after irradiation is less than or equal to 6.0%, and the concentration change rate of the recombinant type III human collagen before and after irradiation is less than or equal to 10.0%.

7. The medical sterile collagen product loaded with polylactic acid-glycolic acid copolymer microspheres and the preparation method thereof according to claim 1, further comprising a syringe subjected to sterile treatment.

8. The preparation method of the medical sterile collagen product loaded with polylactic acid-glycolic acid copolymer microspheres according to claim 7, comprising the following specific steps:

step d: receiving gamma radiation.

9. The method for preparing a medical sterile collagen product loaded with polylactic acid-glycolic acid copolymer microspheres according to claim 8, wherein the specific steps of the sterile CMC-Na gel containing recombinant type iii human collagen are as follows:

step f: receiving beta-ray irradiation;

wherein the step of mixing is carried out under vacuum condition.

step e: receiving beta radiation.