CN114588310A

CN114588310A - PHA (polyhydroxyalkanoate) composite hyaluronic acid microspheres and preparation method and application thereof

Info

Publication number: CN114588310A
Application number: CN202210384597.9A
Authority: CN
Inventors: 郭建俊; 宋春艳; 吕金艳; 余柳松; 司徒卫
Original assignee: Zhuhai Medfa Biotechnology Co ltd
Current assignee: Zhuhai Medfa Biotechnology Co ltd
Priority date: 2022-04-13
Filing date: 2022-04-13
Publication date: 2022-06-07
Anticipated expiration: 2042-04-13
Also published as: CN114588310B

Abstract

The invention discloses PHA composite hyaluronic acid microspheres and a preparation method and application thereof. The method of the present invention comprises preparing a first liquid phase by adding polyvinyl alcohol to water, the concentration of polyvinyl alcohol in the first liquid phase being controlled to be 0.1-5% on a weight basis; adding the liposoluble polymer into an organic solvent to prepare a second liquid phase, wherein the concentration of the liposoluble polymer in the second liquid phase is controlled to be 5-100 mg/mL; mixing the first liquid phase and the second liquid phase at a volume ratio of 3-15:1 to prepare the microspheres. The PHA composite hyaluronic acid microsphere solves the problems of low cell adhesion, easy agglomeration, adhesion, insufficient histocompatibility and the like, and has wide application prospect in the fields of medical beauty filling, cell culture and implanted medical instruments.

Description

PHA (polyhydroxyalkanoate) composite hyaluronic acid microspheres and preparation method and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to PHA composite hyaluronic acid microspheres and a preparation method and application thereof in the fields of tissue filling and the like.

Background

As people age or are affected by certain diseases, muscle and collagen tissues in human bodies can generate functional degeneration with different degrees, which causes problems of skin depression, gastric reflux and the like, so that people invent various fillers to fill the depressed skin or stimulate the regrowth of muscle and collagen through foreign matters, such as hyaluronic acid, bovine collagen and the like. However, these substances as fillers have a short retention time of the filling effect, and thus the filling effect thereof needs to be maintained by frequent injection.

In order to achieve the long-term filling effect, people try to use biodegradable materials to prepare microspheres as fillers, such as polyvinyl alcohol (PVA), polymethyl methacrylate (PMMA) and the like. Although the filling effect of these fillers is maintained for a significantly longer time, these materials are left in the body too long, releasing harmful substances and thus causing a series of side reactions that are harmful to human health.

In recent years, biodegradable high polymer materials come into the visual field of people, and the material is non-toxic and non-rejection to human bodies and can be gradually degraded along with the metabolism of the human bodies and then discharged out of the human bodies. Polyhydroxyalkanoates, which is called polyhydroxyakanoates in English, called PHA for short, is a natural high-molecular biological material, and is an intracellular polyester synthesized by microorganisms. PHA is one of the most desirable biomedical materials due to its good biocompatibility and biodegradability.

In the prior art, when the PHA material is used for preparing injectable microspheres, the prepared microspheres are easy to agglomerate and adhere to form large massive substances, which brings inconvenience to subsequent injection, and the prepared microspheres have weak capability of adhering cells and can not be well compatible with tissues, thereby greatly hindering the application of the biodegradable material microspheres as a filling agent. Therefore, a novel microsphere preparation scheme is urgently needed to solve the problems of low cell adhesion, easy agglomeration, adhesion, insufficient tissue compatibility and the like of the microsphere.

The information in this background is only for the purpose of illustrating the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art that is known to a person skilled in the art.

Disclosure of Invention

In order to solve at least part of technical problems in the prior art, the invention provides PHA composite hyaluronic acid microspheres, which are not easy to agglomerate and adhere under the same conditions and have higher cell adhesion and histocompatibility. In addition, the preparation method of the invention overcomes the technical problem of uniformly distributing the hyaluronic acid to the PHA microspheres and prepares the PHA composite hyaluronic acid microspheres with good performance. Specifically, the present invention includes the following.

In one aspect of the present invention, a method for preparing PHA composite hyaluronic acid microspheres is provided, which comprises the following steps:

(1) adding polyvinyl alcohol into water to prepare a first liquid phase, wherein the concentration of the polyvinyl alcohol in the first liquid phase is controlled to be 0.1-5% on the basis of weight;

(2) adding a liposoluble polymer into an organic solvent to prepare a second liquid phase, wherein the concentration of the liposoluble polymer in the second liquid phase is controlled to be 5-100mg/mL, and the liposoluble polymer is a composite material of PHA and cross-linked hyaluronic acid; and

(3) mixing the first liquid phase and the second liquid phase in a volume ratio of 3-15:1 to prepare microspheres.

According to the method for preparing PHA composite hyaluronic acid microspheres, the crosslinking degree of the crosslinked hyaluronic acid is preferably 10% -40%.

According to the method for preparing PHA composite hyaluronic acid microspheres, hyaluronic acid is preferably added into PHA organic solution, the mixture is dissolved by stirring, and the mixture is evaporated and dried to obtain the PHA composite hyaluronic acid microspheres, wherein the weight ratio of PHA to hyaluronic acid is 5-20: 1.

According to the method for preparing PHA composite hyaluronic acid microspheres, the concentration of PHA in the PHA organic solution is preferably 10-30 mg/mL.

According to the preparation method of the PHA composite hyaluronic acid microspheres, preferably, the weight average molecular weight of the PHA is 2-100 ten thousand daltons, and the PHA is selected from one or more of poly-beta-hydroxybutyrate (PHB), copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV), copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx), and poly (3-hydroxybutyrate-co-4-hydroxybutyrate) (P34 HB).

According to the method for preparing PHA composite hyaluronic acid microspheres, the organic solvent in step (2) is preferably one or more selected from N-methylpyrrolidone, dichloromethane, chloroform and acetonitrile.

In a second aspect of the present invention, there is provided PHA composite hyaluronic acid microspheres prepared by the method of the first aspect.

The PHA composite hyaluronic acid microspheres provided by the invention have the particle size of 20-60 μm.

The PHA composite hyaluronic acid microspheres provided by the invention have preferably an adsorption concentration of more than 4000 μ g/g for bovine serum albumin.

In a third aspect of the invention, there is provided a use of the PHA composite hyaluronic acid microspheres according to the second aspect in the preparation of a tissue filler.

The PHA composite hyaluronic acid microspheres have high clinical value, the physical and chemical properties of the crosslinked hyaluronic acid are obviously improved, and the biocompatibility and biodegradability of the crosslinked hyaluronic acid are still unchanged. The hyaluronic acid molecules are prolonged by the crosslinking reaction, and the solubility of the hyaluronic acid molecules is changed, so that the mechanical strength is improved, and the degradation of organisms is resisted.

The PHA composite material is prepared by modifying PHA through polymer hyaluronic acid, and then is emulsified with water phase by taking the PHA composite material as an oil phase to prepare PHA composite microspheres. By controlling the raw materials and the process, the grain diameter of the prepared PHA composite microspheres can be controlled to be 20-60 mu m, the static water contact angle is 60-90 degrees, the adsorption concentration of the PHA composite microspheres to bovine serum albumin is more than 4000 mu g/g, and the PHA composite microspheres have good hydrophilicity and hydrophobicity, excellent cell adhesion and uniform and controllable grain diameter, so that the PHA composite microspheres can be widely applied to preparation of tissue fillers and medical and aesthetic products. In addition, the preparation method provided by the invention is simple, has few operation steps, and can obtain uniform PHA composite microspheres without complex screening.

Drawings

FIG. 1 is a scanning electron micrograph of microspheres prepared according to example 1 of the present invention.

FIG. 2 shows the results of particle size and span measurements using a laser particle sizer for microspheres made according to example 1 of the present invention.

FIG. 3 is a hyaluronic acid release profile of microspheres prepared in example 1 of the present invention.

FIG. 4 is a photograph of a composite material of microspheres obtained in comparative example 1 of the present invention.

FIG. 5 is an electron micrograph of microspheres obtained in comparative example 2 of the present invention.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.

[ PHA complex hyaluronic acid microspheres ]

In one aspect of the invention, there is provided PHA complexing hyaluronic acid microspheres, which may sometimes be referred to herein simply as "microspheres of the invention". The microsphere is not easy to agglomerate and adhere, has higher cell adhesion and histocompatibility, and is prepared by the aqueous phase of polyvinyl alcohol and the organic phase of a composite material.

The composite material of the present invention uses cross-linked hyaluronic acid instead of uncrosslinked hyaluronic acid. The hyaluronic acid molecules are more agglomerated through crosslinking, the supporting force of the hyaluronic acid is enhanced, and the hyaluronic acid becomes a good filling material.

The degree of crosslinking of the crosslinked hyaluronic acid in the present invention is not particularly limited, but is generally controlled to 10 to 40%, preferably 10 to 30%, and further preferably 20 to 40%. If the degree of crosslinking is outside the above range, it is disadvantageous for the effect of the present invention. On the other hand, when the crosslinking degree becomes low, for example, less than 10%, the microspheres are strongly adhered, do not disperse, and even cannot be prepared. On the other hand, when the degree of crosslinking becomes high, for example, higher than 40%, the produced microspheres tend to be unstable, and chipping or even cracking occurs during the production. The degree of crosslinking can be controlled by, for example, the amount of the crosslinking agent used and the reaction time. Further achieving controllable degradation time.

The molecular weight of the crosslinked hyaluronic acid in the present invention is not particularly limited, and may be controlled generally between 0.9 million and 2 million daltons, preferably between 1 million and 1000 million daltons, more preferably between 5 million and 500 million daltons, for example, between 10 million and 100 million daltons. The larger the molecular weight, the less susceptible to degradation, but if the molecular weight is too large, the cell adhesion becomes poor.

The crosslinked hyaluronic acid in the present invention forms composite microspheres by uniformly distributing hyaluronic acid to a PHA composite material without a chemical reaction with PHA, the type of PHA is not particularly limited, and examples thereof include, but are not limited to, poly- β -hydroxybutyrate (PHB), copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV), copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate (phbhxx), and poly (3-hydroxybutyrate-co-4-hydroxybutyrate) (P34 HB). The present invention may employ one or more of the above ingredients. When a plurality of components are used, the amount ratio of the components is not particularly limited, and can be freely mixed by those skilled in the art as needed.

The molecular weight of the PHA of the present invention is not particularly limited, but is generally controlled to be between 2 and 100 million daltons, preferably between 5 and 90 million daltons, more preferably between 10 and 80 million daltons, for example, 20 million, 30 million, 40 million, 50 million, 60 million, 70 million, and the like.

The particle size of the microspheres in the invention is controlled to be 20-60 μm, preferably 25-50 μm, and more preferably 30-40 μm. A particle size of 20-60 μm is essential for the purposes of the present invention. If the diameter of the microspheres is less than 20 microns, the microspheres may be phagocytosed by human cells. On the other hand, if the microspheres are too large, injection is not facilitated, and in severe cases, the needle may be blocked, or even skin may be cracked. Although the particle size of the microspheres may vary within the above ranges, the microspheres of the present invention have a more uniform particle size distribution.

[ production method ]

In another aspect of the present invention, there is provided a method for preparing PHA composite hyaluronic acid microspheres, which includes, but is not limited to, the following steps:

In the present invention, the step (1) is a step of preparing a first liquid phase, which is an aqueous phase. The step (1) comprises dissolving or dispersing polyvinyl alcohol in an aqueous solvent. Wherein the aqueous solvent includes water and mixtures thereof with other aqueous solvents. Water, such as purified or distilled water, is preferably used in the present invention. The dissolution or dispersion can be accelerated by, for example, stirring. The concentration of the polyvinyl alcohol in the first liquid phase of the present invention is generally controlled to be 0.1 to 8%, preferably 0.5 to 4%, more preferably 1 to 3%, such as 1%, 1.5%, 2%, 2.5%, 3%, etc. on a weight basis.

In the present invention, the step (2) is a step of preparing a second liquid phase, which is an oil phase. And the step (2) comprises adding the liposoluble polymer into an organic solvent to prepare a solution with a certain concentration. Wherein the concentration of the fat-soluble polymer is controlled to be 5-100mg/mL, preferably 5-60mg/mL, and more preferably 10-50 mg/mL.

In the present invention, the liposoluble polymer is a composite of PHA and crosslinked hyaluronic acid, which is a mixture of the two materials, and is not a reactant. The weight ratio of PHA to hyaluronic acid in the composite material is generally 5-20:1, preferably 6-15:1, more preferably 10-15. The composite material may be prepared by known methods, and in exemplary embodiments, the composite material is prepared as follows: firstly, adding an organic solvent into PHA to prepare a PHA solution with the concentration of 5-40mg/mL, then adding crosslinked hyaluronic acid into 400mL of PHA solution, stirring for dissolving, performing rotary evaporation at 20-100 ℃, and performing freeze drying to obtain the composite material. The concentration of the PHA solution is preferably 10-40mg/mL, and still more preferably 15-30 mg/mL. The amount of the crosslinked hyaluronic acid added is 0.1 to 10g, preferably 0.8 to 8 g. The rotary evaporation temperature is preferably 30 to 60 ℃ and further preferably 35 to 50 ℃, and rotary evaporation is not particularly limited as long as removal of the organic solvent can be achieved. In an exemplary preparation method, the spin-steaming is performed at a fixed oil bath temperature using a spin-steaming bottle.

Examples of the organic solvent include N-methylpyrrolidone, dichloromethane, chloroform, and acetonitrile. The present invention may use one or more of the above solvents. When a plurality of solvents are used, the amount ratio of each solvent is not particularly limited, and can be freely set by a skilled person as needed. In an exemplary step (2) comprising dissolving the composite in methylene chloride, a 30mg/mL PHA composite/organic solvent solution was formulated.

In the present invention, the step (3) is a step of preparing microspheres by mixing two phases. The volume of the first liquid phase is larger than the volume of the second liquid phase, and the volume ratio of the two phases is generally in the range of (5-10):1, for example 6:1, 7:1, 9: 1. The first liquid phase is either too small or too large in volume to facilitate microsphere formation. For example, if the volumes of the first liquid phase and the second liquid phase are equal, or smaller than the second liquid phase, the hyaluronic acid microspheres of the present invention may not be formed.

In the present invention, the mixing of the first liquid phase and the second liquid phase may be performed by a membrane emulsifier. When the mixing is carried out using a membrane emulsifier, the emulsifying pressure is controlled to be between 0.005 and 0.05MPA, preferably between 0.05 and 0.03 MPA. The mixing emulsification time is generally between 40 and 240 minutes, preferably between 50 and 150 minutes, more preferably between 60 and 70 minutes. The stirring speed during mixing and emulsification can be controlled at 100-500r/min, preferably 150-400r/min, and more preferably 200-300 r/min. After emulsification, the organic solvent can be further stirred and volatilized, and the microspheres can be prepared by freeze-drying.

In an exemplary mixed emulsification scheme, the volume ratio of the second liquid phase to the first liquid phase was controlled to 1/7, the emulsification pressure was 0.015MPA, the membrane tube length was 6.1 μm, and the emulsification time period was 70 min; the emulsifying and stirring speed is 220 r/min. After the emulsification is finished, the organic solvent is volatilized and freeze-dried, water can be added when the organic solvent is volatilized, and the mixture is stirred for 20-50h at the speed of 100-150 r/min. Finally, freeze-drying for 20-40h to obtain the microspheres of the invention, wherein the freeze-drying equipment is not particularly limited, and a freeze-drying apparatus known in the art can be used.

It will be understood by those skilled in the art that the order of steps (1) and (2) is not particularly limited as long as the object of the present invention can be achieved. Further, both steps may be performed simultaneously. In addition, it will be understood by those skilled in the art that other steps or operations may be included before, after, or between any of the above steps (1) - (3), such as to further optimize and/or improve the methods of the present invention.

[ use ]

The invention also provides the use of PHA composite hyaluronic acid microspheres, preferably for preparing tissue fillers. The microsphere has good biocompatibility, biodegradability and uniform particle size, has good cell compatibility with cells in vivo, and can allow the cells to grow on the microsphere well.

Meanwhile, the microspheres can be well dispersed in water for injection, and particularly, the crosslinked hyaluronic acid is uniformly dispersed in the PHA composite material, can continuously play a role in the degradation process and has long action time.

Example 1

The PHA composite microspheres of this example were prepared as follows:

firstly, preparing a composite material:

the preparation method comprises the steps of taking methylene dichloride as a solvent, taking P34HB as PHA (3-hydroxybutyrate-co-4-hydroxybutyrate) as poly (3-hydroxybutyrate-co-4-hydroxybutyrate with the weight-average molecular weight of 3-6W, preparing a solution with the concentration of 20mg/mL, taking 400mL of P34HB solution, adding 8g of hyaluronic acid (the crosslinking degree is 20%), stirring for dissolution, putting the solution into a 1L rotary evaporation bottle, carrying out rotary evaporation at the oil bath temperature of 45 ℃ for 20 hours, recovering the solvent by using condensed water at the temperature of-10 ℃, and carrying out freeze drying on the paste after rotary evaporation at the temperature of-40 ℃ to obtain the PHA composite material (hyaluronic acid & P34 HB).

Secondly, preparing the microspheres:

(1) adopting dichloromethane as a solvent, and preparing the PHA composite material prepared by the method into PHA composite material solution with the mass concentration of 30mg/mL to obtain an oil phase;

(2) dissolving polyvinyl alcohol (PVA) in 280mL of water to prepare a water phase with the PVA mass concentration of 1.5%; mixing 40mL of the oil phase prepared in the step (1) with the water phase to obtain a mixed solution;

(3) adding the mixed solution into a conventional membrane emulsifier of Zhongkesen glow, setting the buffer pressure to be 0.015MPa, and checking the air tightness: 0.03MPa, 6.1 mu m and no air bubble; then carrying and using a 6.1um membrane tube, adjusting the emulsifying pressure to be 0.015MPa, and emulsifying and stirring the mixture at the rotating speed of 220r/min for emulsifying for 70 min; after the emulsification is finished, adding 280mL of water, stirring for 40h at the rotating speed of 150r/min, and naturally volatilizing the organic solvent; and finally, freeze-drying for 31h to prepare the PHA microspheres.

Example 2

Firstly, preparing a composite material:

preparing a PHA (P34HB) solution with the concentration of 20mg/mL by taking dichloromethane as a solvent; then taking 400mL of P34HB solution, adding 0.89g of hyaluronic acid (the degree of crosslinking is 30%), stirring for dissolution, putting into a 1L rotary evaporation bottle, carrying out rotary evaporation for 20h at the temperature of 45 ℃ oil bath, and recovering the solvent by adopting condensed water at the temperature of-10 ℃; and freeze-drying the paste after rotary evaporation at-40 ℃ to obtain the PHA composite material (hyaluronic acid & P34 HB).

Secondly, preparing the microspheres:

(3) adding the mixed solution into a conventional membrane emulsifier of Zhongkesen glow, setting the buffer pressure to be 0.015MPa, and checking the air tightness: 0.03MPa, 6.1 mu m and no bubbles; then carrying and using a 6.1um membrane tube, adjusting the emulsifying pressure to be 0.012MPa, and emulsifying and stirring the mixture at the rotating speed of 220r/min for emulsifying for 60 min; after the emulsification is finished, adding 280mL of water, stirring for 40h at the rotating speed of 120r/min, and naturally volatilizing the organic solvent; and finally, freeze-drying for 31h to prepare the PHA microspheres.

Comparative example 1

Firstly, preparing a PHA composite material:

preparing a PHA (P34HB) solution with the concentration of 20mg/mL by taking dichloromethane as a solvent; then taking 400mL of P34HB solution, adding 0.89g of hyaluronic acid (the degree of crosslinking is 1.5%), stirring for dissolution, putting into a 1L rotary evaporation bottle, carrying out rotary evaporation for 20h at the temperature of 45 ℃ oil bath, and recovering the solvent by adopting condensed water at the temperature of-10 ℃; and freeze-drying the paste after rotary evaporation at-40 ℃ to obtain the PHA composite material (hyaluronic acid & P34 HB).

Secondly, a preparation method of PHA microspheres:

(1) adopting dichloromethane as a solvent, and preparing the PHA composite material prepared by the method into a PHA composite material solution with the mass concentration of 30mg/mL to obtain an oil phase;

(3) adding the mixed solution into a conventional membrane emulsifier of Zhongkesen glow, setting the buffer pressure to be 0.015MPa, and checking the air tightness: 0.03MPa, 6.1 mu m and no air bubble; then carrying and using a 6.1um membrane tube, adjusting the emulsifying pressure to be 0.012MPa, and emulsifying and stirring the mixture at the rotating speed of 220r/min for emulsifying for 60 min; after the emulsification is finished, adding 280mL of water, stirring for 40h at the rotating speed of 120r/min, and naturally volatilizing the organic solvent; and finally, freeze-drying for 31h to prepare the PHA microspheres.

The experimental results are shown in fig. 4, which is a photograph of the composite material prepared. At normal temperature, it is a material similar to that between liquid and solid states. Microspheres made using this material adhere strongly and do not disperse.

Comparative example 2

Firstly, preparing the PHA composite material:

preparing a PHA (P34HB) solution with the concentration of 20mg/mL by taking dichloromethane as a solvent; then taking 400mL of P34HB solution, adding 0.89g of hyaluronic acid (the crosslinking degree is 55%), stirring for dissolution, putting into a 1L rotary evaporation bottle, carrying out rotary evaporation for 20h at the oil bath temperature of 45 ℃, and recovering the solvent by adopting condensed water at the temperature of-10 ℃; and freeze-drying the paste after rotary evaporation at-40 ℃ to obtain the PHA composite material (hyaluronic acid & P34 HB).

Secondly, a preparation method of PHA microspheres:

The experimental results are as follows: comparative example 2 photographs of the microspheres are shown in figure 5. During the formation of the microspheres, debris may occur and the microspheres may break.

Test example 1

The scanning electron micrographs of the PHA microspheres prepared in example 1 are shown in figure 1. As can be seen from FIG. 1, the PHA microspheres obtained in example 1 have good dispersibility, no adhesion between them, uniform morphology and spherical shape as seen in an electron micrograph.

FIG. 2 is a graph showing the average particle size and span (degree of dispersion of particle size distribution) of PHA microspheres analyzed by a laser particle sizer. As can be seen from fig. 2, the microspheres had uniform particle size and good sphericity.

Test example 2

To further examine the protein adsorption of PHA microspheres, the following experiments were performed:

(1) drawing a protein growth curve: BSA was used to prepare a 3mg/ml solution, which was scanned at full wavelength using a spectrophotometer to detect the maximum absorption wavelength of BSA albumin at 278.58 nm. Then, a series of concentration gradient solutions of 0.5, 1, 1.5, 3, 4 and 5mg/ml are prepared, the absorbance value of each concentration solution is detected at the maximum absorption wavelength, and a standard curve is drawn.

(2) Preparing 5mg/ml BSA solution, and respectively incubating the prepared PHA microspheres, wherein the specific incubation process is as follows: taking 10mL of bovine serum albumin solution, respectively adding 0.03g of the prepared PHA microspheres, incubating at 25 ℃ for 5 hours, detecting the light absorption value of the incubated bovine serum albumin solution (supernatant solution), and calculating the specific adsorption concentration according to a standard curve.

When the biological material is in contact with a physiological environment, the biological material is not rejected in vivo, has no serious side effect related to filler, and needs good cell adhesion, and a very important point in the cell adhesion is that a large amount of protein is adhered to the cell surface, and the protein adsorption is an important cell adhesion driving force. The CT1 charge ball is a commonly used cell culture microcarrier, and the higher the value, the better the protein adsorption. The value of the charge ball adsorption of bovine serum albumin by the CT1 test is 600 mug/g. The adsorption concentration of the PHA microspheres prepared in example 1 to bovine serum albumin is 4000 mug/g; through comparative analysis, the PHA microsphere prepared by the invention has excellent protein adsorption, and the adsorption concentration is 7-8 times of that of CT1 charge spheres, thereby showing that the PHA microsphere has excellent cell adhesion and can be used for medical beauty filling, cell culture, implantation of medical instruments and the like. The BSA adsorption results of examples 1-2 and comparative examples 1-2 are shown in Table one.

TABLE 1

Group of	BSA adsorption (μ g/g)
		Example 1	4000
Example 2	3200
		Comparative example 1	300
Comparative example 2	450

Test example 3

1g of the microspheres prepared in example 1 was placed in 100ml of pbs buffer solution, the temperature was maintained at 37 ℃, the content of hyaluronic acid in the solution was measured by high performance liquid chromatography every 10 days, and a hyaluronic acid release curve was plotted as shown in fig. 3. The sustained release of the hyaluronic acid in the release process and the continuous increase of the concentration of the hyaluronic acid in the solution are shown, and the PHA composite hyaluronic acid microspheres prepared by the invention can continuously play a role in the degradation process and have long action time.

While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Many modifications and variations may be made to the exemplary embodiments of the present description without departing from the scope or spirit of the present invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.

Claims

1. The preparation method of the PHA composite hyaluronic acid microspheres is characterized by comprising the following steps:

(3) mixing the first liquid phase and the second liquid phase at a volume ratio of 3-15:1 to form microspheres.

2. The method for preparing PHA microspheres of claim 1, wherein the degree of crosslinking of the crosslinked hyaluronic acid is 10% to 40%.

3. The method of claim 1, wherein the PHA microspheres are prepared by adding hyaluronic acid to PHA solution, dissolving the mixture under stirring, evaporating the solution, and drying the solution, wherein the weight ratio of PHA to hyaluronic acid is 5-20: 1.

4. The method for preparing PHA microspheres of hyaluronic acid compounded according to claim 3, wherein the concentration of PHA in the PHA organic solution is 10-30 mg/mL.

5. The method for preparing PHA complex hyaluronic acid microspheres of claim 1, wherein the PHA has a weight-average molecular weight of 2-100 ten thousand daltons and is selected from one or more of poly- β -hydroxybutyrate (PHB), copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV), copolyester of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx), and poly (3-hydroxybutyrate-co-4-hydroxybutyrate) (P34 HB).

6. The method for preparing PHA microspheres of hyaluronic acid complex according to claim 1, wherein the organic solvent in step (2) is one or more selected from N-methylpyrrolidone, dichloromethane, chloroform and acetonitrile.

7. PHA-complexed hyaluronic acid microspheres, obtainable by the process according to any one of claims 1-6.

8. The PHA composite hyaluronic acid microspheres according to claim 7, wherein the particle size of the microspheres is 20-60 μm.

9. The PHA composite hyaluronic acid microspheres according to claim 8, wherein the adsorption concentration of the microspheres to bovine serum albumin is more than 4000 μ g/g.

10. Use of PHA in combination with hyaluronic acid microspheres according to any of claims 7-9 for the preparation of a tissue filler.