CN114618015B

CN114618015B - PHA microsphere containing collagen and preparation method and application thereof

Info

Publication number: CN114618015B
Application number: CN202210385770.7A
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: 2022-11-25
Anticipated expiration: 2042-04-13
Also published as: CN114618015A

Abstract

The invention discloses a PHA microsphere containing collagen, a preparation method and application thereof. The method of the present invention comprises preparing a first liquid phase by adding type I collagen to water, the concentration of type I collagen in the first liquid phase being controlled to be 0.1-95% 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 0.1-100 mg/mL; mixing the first liquid phase and the second liquid phase in a volume ratio of 3-15. The PHA microsphere containing the collagen solves the problems of low cell adhesion, easy agglomeration, adhesion, insufficient histocompatibility and the like of the microsphere, and has wide application prospect in the fields of medical and cosmetic filling, cell culture and implantation medical appliances.

Description

PHA microsphere containing collagen and preparation method and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to PHA microspheres containing collagen, a preparation method thereof 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 remain in the body for 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, biodegradability.

The collagen is natural protein of the body, has larger affinity, weaker antigenicity, good biocompatibility and biodegradation safety to protein molecules on the surface of the skin, can be degraded and absorbed, and has good adhesion. The collagen molecule peptide chain has various reaction groups, such as hydroxyl, carboxyl, amino and the like, is easy to absorb and combine various enzymes and cells to realize immobilization, and has the characteristics of good affinity and strong adaptability to the enzymes and the cells. In addition, collagen is easy to process and shape, so the purified collagen can be made into many different forms of materials, such as films, tapes, sheets, sponges, beads, etc., but the application in the form of films is the most reported.

However, the pure collagen has crisp texture after being dried, weak film forming capability, low film ductility, easy drying crack, poor water resistance, easy swelling when meeting water and easy degradation in vivo. Therefore, in practical application, collagen is often modified by a certain method, so that the tensile strength and the anti-degradation capability of the collagen are improved, the expansion rate is reduced, and the mechanical property and the water resistance of the collagen are improved.

In the prior art, when PHA material is prepared into 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 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 microspheres containing collagen, and the microspheres are not easy to agglomerate and adhere and have higher cell adhesion and histocompatibility. In addition, the preparation method of the invention does not involve copolymer synthesis, so that the problem of solvent residue is hardly existed, and the safety of the preparation is greatly improved. Meanwhile, the microsphere prepared by the method has uniform appearance and good sphericity, and is more favorable for filling in shape. Specifically, the present invention includes the following.

In one aspect of the present invention, there is provided a method for preparing PHA microspheres containing collagen, comprising the steps of:

(1) Adding type I collagen to water to prepare a first liquid phase, wherein the concentration of type I collagen in the first liquid phase is controlled to be 0.1-95% by 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 0.1-100mg/mL, and the liposoluble polymer comprises PHA; and

(3) Mixing the first liquid phase and the second liquid phase in a volume ratio of 3-15.

According to the method for preparing PHA microspheres containing collagen of the present invention, preferably, the weight average molecular weight of PHA is 2-100 ten thousand daltons.

According to the preparation method of the collagen-containing PHA microspheres of the present invention, preferably, 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 microspheres containing collagen of the present invention, preferably, the concentration of PHA in the PHA organic solution is 10-30mg/mL.

According to the method for preparing PHA microspheres containing collagen of the present invention, preferably, the organic solvent in step (2) is one or more selected from N-methylpyrrolidone, dichloromethane, chloroform and acetonitrile.

According to the method for preparing PHA microspheres comprising collagen of the present invention, preferably, the first liquid phase is formulated in the range of 3-39 ℃ in the step (1).

According to the method for preparing PHA microspheres containing collagen of the present invention, preferably, the molecular weight of the type I collagen is 2K-300K daltons.

In a second aspect of the present invention, there is provided PHA microspheres comprising collagen, produced by the method of the first aspect.

The PHA microspheres containing collagen according to the present invention preferably have a particle size of 20-60 μm.

In a third aspect of the invention, there is provided the use of PHA microspheres comprising collagen according to the second aspect in the preparation of a tissue bulking agent.

The PHA microspheres containing collagen have high clinical value, and the problems of stiff touch and strong foreign body sensation of PHA microspheres used for tissue filling are solved. The PHA microsphere prepared by the invention has the surface layer adhered with collagen, the collagen is not easy to crack and fall off by the preparation process, and PHA and the collagen are well combined and swell when meeting tissue fluid to absorb water to form the PHA microsphere with soft outside and hard inside. The PHA microsphere is used for skin filling and tissue filling, the touch feeling and the comfort are enhanced, the biocompatibility of the PHA microsphere is enhanced at multiple angles, and the tissue filling is more fit with a human body in use.

The method controls the raw materials and the process, so that the grain diameter of the prepared PHA microspheres can be controlled to be 20-60 mu m, the PHA microspheres have excellent cell adhesion, the microspheres have good dispersibility, no adhesion among the microspheres, and uniform and controllable shapes. In addition, the adhered collagen does not crack or peel, so that the collagen can be widely applied to preparation of tissue fillers and medical and cosmetic products.

Drawings

FIG. 1 is a schematic flow diagram of the preparation process of the present invention.

FIG. 2 is a scanning electron micrograph of microspheres prepared according to example 1 of the present invention.

FIG. 3 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. 4 is a scanning electron micrograph of microspheres prepared according to example 2 of the present invention.

FIG. 5 shows the results of particle size and span measurements using a laser particle sizer for microspheres prepared according to example 2 of the present invention.

FIG. 6 is an electron micrograph of microspheres obtained in comparative example 1 of the present invention.

FIG. 7 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 that 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 microspheres containing collagen ]

In one aspect of the invention, there is provided PHA microspheres comprising collagen, which may sometimes be referred to herein simply as "microspheres of the invention". In the microsphere, PHA is well combined with collagen, and the PHA microsphere with soft outer part and hard inner part is formed when the PHA and the collagen absorb water and swell when meeting tissue fluid.

Type I collagen is used in the aqueous phase preparation of the present invention. Through a great deal of research, the inventor finds that specific collagen has influence on the shape of the final microsphere, particularly holes appear on the surface of the microsphere, so that the microsphere is not beneficial to tissue filling. In some embodiments, the use of type I collagen compared to type II collagen greatly improves the morphology uniformity of the microspheres, ensuring good sphericity.

The molecular weight of the type I collagen of the present invention is not particularly limited, and may be controlled generally between 2K to 300K daltons, preferably between 3K to 200K, more preferably between 5K to 150K, for example 10K to 100K daltons.

The type I collagen in the present invention is uniformly distributed to PHA microspheres without 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 (PHBHHx), 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 in 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, 15 million, 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 25-56 μm, and more preferably 35-56 μm. A particle size of 20 to 60 μm is necessary 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, even causing skin breakdown. 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.

The compression elasticity of the PHA microsphere containing the collagen is 60-65%, and the excellent compression elasticity ensures that the PHA microsphere can be used for filling skin and tissues, enhances the comfortable feeling, and has more deep and wide application in the field of tissue filling. The method of measuring the compression elasticity is known in the art, and can be measured, for example, by a physical property analyzer using a compression induction force of 10g, an induction method of automation, a plunger lowering speed of 1mm/s, and a holding time of 10 seconds.

[ production method ]

In another aspect of the present invention, there is provided a method for preparing PHA microspheres containing collagen, 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 the type I collagen 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 type I collagen in the first liquid phase of the present invention is generally controlled to be 0.1 to 95%, preferably 1 to 10%, more preferably 1 to 8%, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, etc. on a weight basis.

Preferably, the first liquid phase is formulated in a thermostatic water bath in the range of 3-39 ℃, preferably also 20-35 ℃, more preferably 20-30 ℃.

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 fat-soluble 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 0.1-100mg/mL, preferably 5-60mg/mL, and more preferably 10-50mg/mL.

Examples of the organic solvent include N-methylpyrrolidone, dichloromethane, chloroform, and acetonitrile. One or more of the above solvents may be used in the present invention. 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 PHA in methylene chloride, 40mg/mL of PHA/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 that of the second liquid phase, and the volume ratio of the two is generally in the range of (3-15): 1, such as 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10. The first liquid phase is either too small or too large in volume to facilitate microsphere formation. For example, if the first liquid phase and the second liquid phase are equal in volume, or smaller than the second liquid phase, the collagen-containing PHA microspheres of the present invention will 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 0.08MPA, preferably between 0.01 and 0.06MPA, and further preferably between 0.02 and 0.06 MPA. The mixing emulsification time is generally between 20 and 500 minutes, preferably between 50 and 150 minutes, more preferably between 80 and 130 minutes. The pore diameter of the membrane tube is 0.8-50 μm, preferably 5-30 μm. The stirring speed during mixing and emulsification can be controlled within 150-1000r/min, preferably 150-400r/min, more preferably 300-350r/min. After emulsification, water is added for solidification, and the stirring speed after water addition is 50-500r/min, preferably 50-200r/min. The curing time is from 1 to 48 hours, preferably from 12 to 24 hours.

In an exemplary mixed emulsification scheme, the volume ratio of the second liquid phase to the first liquid phase is controlled to be 1/5, the emulsification pressure is 0.04MPA, the length of the membrane tube is 15 μm, and the emulsification time is 120min; the rotation speed of the emulsification and stirring is 350r/min. And after emulsification is finished, starting curing, wherein an overhead stirrer and a 10cm stirring paddle are used for curing, 280mL of water is additionally added, the stirring speed is 120r/min, and the curing time is 24h, so that the injectable PHA microspheres are obtained.

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 microspheres comprising collagen, preferably for the preparation of tissue bulking agents. 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.

The PHA microspheres containing the collagen provided by the invention have good dispersibility, no adhesion among the PHA microspheres, uniform shape and spherical shape. The adhered collagen does not crack or peel. Combines the advantages complementation of collagen and PHA in the biomedical application field, and the excellent biocompatibility of the collagen is complemented with the excellent mechanical property of the PHA. The functional microspheres absorb water and swell when meeting tissue fluid to form PHA microspheres with soft outside and hard inside. The skin-tissue filling material is used for filling skin and tissue, the touch comfort is enhanced, and the application of the skin-tissue filling material in the field of tissue filling is deeper and wider.

Example 1

The preparation process of the PHA microspheres of this example is shown in FIG. 1, and specifically follows:

s1, preparing an oil phase: dissolving 0.8g of P34HB in 20mL of dichloromethane to prepare 40mg/mL of PHA/organic solvent solution, wherein the weight average molecular weight of P34HB is 15 ten thousand;

s2, preparing a water phase: carrying out water bath at 25 ℃, and preparing a type I collagen water solution with the mass fraction of 3%;

s3, emulsifying the oil phase into a rotating water phase through a membrane emulsifier, controlling the volume ratio of the oil phase to the water phase to be 1/5, the emulsifying pressure to be 0.04MPA, the length of a membrane tube to be 15 mu m, and the emulsifying time to be 120min; the length of the magneton is 5cm, and the emulsifying and stirring speed is 350r/min. And after emulsification is finished, starting curing, wherein an overhead stirrer and a 10cm stirring paddle are used for curing, 280mL of water is additionally added, the stirring speed is 120r/min, and the curing time is 24h, so that the injectable PHA microspheres are obtained.

Example 2

The PHA microspheres of this example were prepared as follows:

s2, preparing a water phase: carrying out water bath at 25 ℃, and preparing a type I collagen water solution with the mass fraction of 5%;

s3, emulsifying the oil phase into a rotating water phase through a membrane emulsifier, controlling the volume ratio of the oil phase to the water phase to be 1/5, the emulsifying pressure to be 0.04MPA, the length of a membrane tube to be 15 mu m, and the emulsifying time to be 120min; the length of the magneton is 5cm, and the emulsifying and stirring speed is 350r/min. And after the emulsification is finished, starting curing, wherein an overhead stirrer and a 10cm stirring paddle are used for curing, 280mL of water is additionally added, the stirring speed is 120r/min, and the curing time is 24h, so that the injectable PHA microspheres are obtained.

Example 3

s1, preparing an oil phase: dissolving 0.8g of P34HB & collagen composite material in 20mL of dichloromethane to prepare 40mg/mL of P34HB & collagen composite material/organic solvent solution, wherein the P34HB in the P34HB & collagen composite material accounts for 70% of the mass fraction of the composite material, and the weight average molecular weight of the P34HB is 15 ten thousand;

Comparative example 1

The preparation of PHA microspheres of this comparative example was as follows:

s2, preparing a water phase: carrying out water bath at 25 ℃, and preparing a type II collagen aqueous solution with the mass fraction of 3%;

The experimental result is shown in fig. 6, which is an electron micrograph of the prepared PHA microspheres, and it can be seen that the microspheres have holes, which are not beneficial for tissue filling.

Comparative example 2

The preparation of PHA microspheres of this comparative example was as follows:

s2, preparing a water phase: carrying out water bath at 25 ℃, and preparing a type III collagen water solution with the mass fraction of 3%;

The experimental results are shown in fig. 7, which is an electron micrograph of the prepared PHA microspheres, and it can be seen that the microspheres have holes, which are not beneficial for tissue filling.

Test example 1

Scanning electron micrographs of PHA microspheres prepared in example 1 are shown in figure 2. As can be seen from FIG. 2, 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. The adhered collagen can be seen from the electron microscope picture, and does not crack or peel.

FIG. 3 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. 3, the average particle size of the microspheres obtained in example 1 is 56 μm, and table 1 shows the span value (the span value is the degree of dispersion of particle size distribution) of example 1 measured by a laser particle sizer, and the measurement result shows that the span value is 0.680, which can indicate that the injectable PHA microspheres of this example have uniform particle size.

TABLE 1

Test example 2

The scanning electron micrograph of the PHA microspheres prepared in example 2 is shown in fig. 4. As can be seen from FIG. 4, the PHA microspheres obtained in example 2 have good dispersibility, no adhesion between them, uniform morphology and spherical shape as seen in an electron micrograph. The adhered collagen can be seen from the electron microscope picture, and does not crack or peel.

FIG. 5 shows the average particle size and span (degree of dispersion of particle size distribution) of PHA microspheres analyzed by laser particle sizer. As can be seen from fig. 5, the average particle size of the microspheres obtained in example 2 is 35 μm, and table 2 shows the span value (the span value is the degree of dispersion of particle size distribution) of example 2 measured by a laser particle sizer, and the measurement result shows that the span value is 0.670, which indicates that the injectable PHA microspheres of this example have uniform particle size.

TABLE 2

Test example 3

The microspheres prepared in examples 1 to 3 and comparative examples 1 to 2 were subjected to measurement of compression elasticity, and a test apparatus: a physical property analyzer; setting parameters: compression mode, compressive induction force 10g, induction mode: the compression head was automatically lowered at a rate of 1mm/s for a duration of 10 seconds, the displacement of the compression head downward was measured, and the compression elasticity of the microspheres was calculated from the displacement/microsphere diameter 100%, and generally, the compression elasticity was 40% or more, which was acceptable, and the results are shown in table 3.

TABLE 3

Group of	Elasticity under compression
		Comparative example 1	25％
Comparative example 2	20％
		Example 1	60％
Example 2	65％
		Example 3	68％

The results show that the microspheres prepared in examples 1-3 of the invention have good compression elasticity, so that the microspheres can be used for filling skin and tissues, have enhanced tactile comfort and can be applied more deeply and widely in the field of tissue filling.

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. A method for preparing PHA microspheres containing collagen is characterized by comprising the following steps:

(1) Adding type I collagen into water to prepare a first liquid phase, wherein the concentration of the type I collagen in the first liquid phase is controlled to be 0.1-95% by weight, and the molecular weight of the type I collagen is 2K-300K daltons;

2. The method for the preparation of PHA microspheres containing collagen according to claim 1, wherein the weight average molecular weight of said PHA is comprised between 2 and 100 kilodaltons.

3. The method for preparing PHA microspheres containing collagen according to claim 1, wherein said PHA is selected from one or more of poly- β -hydroxybutyrate (PHB), copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV), copolyester of 3-hydroxybutyrate with 3-hydroxyhexanoate (PHBHHx) and poly (3-hydroxybutyrate-co-4-hydroxybutyrate) (P34 HB).

4. The method for preparing PHA microspheres containing collagen according to claim 3, wherein the concentration of PHA in said PHA organic solution is 5-90mg/mL.

5. The method for preparing PHA microspheres containing collagen according to claim 1, wherein the organic solvent in step (2) is one or more selected from the group consisting of N-methylpyrrolidone, dichloromethane, chloroform and acetonitrile.

6. The method for the preparation of PHA microspheres containing collagen according to claim 1, wherein said first liquid phase is formulated in said step (1) at a temperature ranging from 3 to 39 ℃.

7. PHA microspheres containing collagen, produced by the method according to any one of claims 1-6.

8. The collagen-containing PHA microspheres of claim 7, wherein said microspheres have a particle size of 20-60 μm.

9. Use of the PHA microspheres containing collagen according to claim 7 in the preparation of tissue bulking agents.