CaHA / PLGA composite microsphere, preparation method and application of CaHA / PLGA composite microsphere in soft tissue filling agent

Abstract

The invention discloses CaHA / PLGA (poly (lactic-co-glycolic acid)) composite microspheres, a preparation method and application of the CaHA / PLGA composite microspheres in a soft tissue filler. The preparation method of the composite microspheres comprises the following steps: dissolving PLGA in dichloromethane to obtain a first solution, dissolving polyvinyl alcohol in water to obtain a second solution, mixing the two solutions, continuously stirring until dichloromethane is completely volatilized, and freeze-drying to obtain PLGA microspheres; the preparation method comprises the following steps: adding calcium salt and phosphate into water, dissolving, regulating the pH until the solution is clear, adding urea and a template agent, dissolving, and carrying out hydrothermal reaction to obtain CaHA microspheres; carrying out alkali treatment on the PLGA microspheres to obtain activated PLGA microspheres, dispersing CaHA microspheres in a dispersing agent, adding the activated PLGA microspheres into the dispersion liquid, oscillating, and freeze-drying to obtain the product. The soft tissue filling agent is prepared from CaHA / PLGA (poly (lactic-co-glycolic acid)) composite microspheres and gel. The mechanism of the CaHA / PLGA composite microspheres for stimulating collagen regeneration is milder, the inflammation risk is lower, and the defects of a traditional soft tissue filling agent can be overcome.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and specifically to a CaHA / PLGA composite microsphere, a preparation method thereof, and an application thereof in a soft tissue filler. Background Art

[0002] At present, soft tissue fillers are widely used in the field of medical aesthetics. Their core properties include immediate filling effect, biocompatibility, tissue repair ability, and controllability of the degradation process. However, the existing mainstream microsphere materials on the market, PLLA (poly-L-lactic acid) and PCL (polycaprolactone), still have certain limitations. For example, during the degradation process of PLLA, local acidification may be caused by the accumulation of lactic acid, leading to an inflammatory reaction, and at the same time, its ability to induce tissue collagen regeneration is limited; the degradation rate of PCL is relatively slow, which may have an adverse impact on the long-term residence and biological safety of the patient's body. In addition, there is still an imbalance between the degradation cycle of these fillers and the tissue regeneration function: some fillers can achieve a longer filling maintenance time, but their biological activity is low and they cannot effectively promote tissue repair and collagen regeneration.

[0003] PLGA (poly(lactic-co-glycolic acid)) is a relatively excellent biomaterial. However, it has limitations similar to those of the aforementioned materials, such as lack of biological activity, generation of lactic acid during the degradation process, which may cause inflammation, and it has a faster degradation rate. Therefore, PLGA is usually used in hard tissue fields such as drug carriers and tissue engineering scaffolds. In order to reduce the inflammation caused by PLGA during decomposition, those skilled in the prior art usually use it in combination with CaHA (calcium hydroxyapatite), but it is still mainly used for hard tissue repair, and there is a large gap in the research in the field of soft tissue filling. Summary of the Invention

[0004] To solve at least one of the above problems, the present invention provides a CaHA / PLGA composite microsphere, a preparation method thereof, and an application thereof in a soft tissue filler, which can be preferably applied to the filling of soft tissues and promote tissue repair and collagen regeneration.

[0005] The technical solution of the present invention is as follows: A preparation method of a CaHA / PLGA composite microsphere, comprising the following steps:

[0006] Dissolve PLGA in dichloromethane to obtain a first solution, dissolve polyvinyl alcohol in water to obtain a second solution, mix the above two solutions and continuously stir until dichloromethane completely volatilizes, and then obtain PLGA microspheres after freeze-drying; the concentration of PLGA in the first solution is 5-10%, the concentration of polyvinyl alcohol in the second solution is 0.1-1%, and the volume ratio of the first solution to the second solution is 1:10-20;

[0007] Calcium salts and phosphates are taken, added to water and dissolved. Subsequently, the pH is adjusted until the solution is clear. Then, urea and a template agent are added, and after their dissolution, a hydrothermal reaction is carried out, followed by separation and purification to obtain CaHA microspheres.

[0008] The PLGA microspheres are subjected to alkali treatment to obtain activated PLGA microspheres. CaHA microspheres are taken and dispersed in a dispersant, and the activated PLGA microspheres are added to the aforementioned dispersion. After constant-temperature oscillation, it is freeze-dried to obtain CaHA / PLGA composite microspheres.

[0009] One embodiment of the present invention is that the molecular weight of PLGA is 30 - 100 KDa, and the molar ratio of LA (lactic acid) to GA (glycolic acid) in its raw materials is 15:85 - 85:15. For the molar ratio of LA to GA in PLGA, the higher the proportion of GA, the faster the degradation rate of the product of the present invention. Therefore, in the actual production and use process, we can select PLGA with a suitable ratio according to the actual situation.

[0010] One embodiment of the present invention is that during the preparation of PLGA microspheres, the stirring speed is 300 - 800 rpm.

[0011] One embodiment of the present invention is that during the preparation of CaHA microspheres, the molar ratio of calcium salts to phosphates is 1.67, the molar ratio of urea to the calcium salts is 0.56:1, and the molar ratio of the template agent to the calcium salts is 1:20 - 200; the calcium salt is one of calcium nitrate and calcium chloride, the phosphate is one of disodium hydrogen phosphate, sodium dihydrogen phosphate, and ammonium phosphate, and the template agent is cyclohexanehexacarboxylic acid.

[0012] One embodiment of the present invention is that during the preparation of CaHA microspheres, the hydrothermal reaction temperature is 120 - 180 °C, and the reaction time is 3 - 12 h; the separation and purification steps are as follows: after removing the solvent, it is washed several times with deionized water and ethanol in sequence, and then dried to obtain.

[0013] One embodiment of the present invention is that during the preparation of CaHA / PLGA composite microspheres, the alkali solution for alkali treatment is a 0.1 - 1 mol / L sodium hydroxide or potassium hydroxide solution, and the alkali treatment method is that the alkali solution covers the PLGA microspheres and soaks for 30 - 180 s; the dispersant is ethanol.

[0014] One embodiment of the present invention is that during the preparation of CaHA / PLGA composite microspheres, the mass ratio of CaHA microspheres to PLGA microspheres is 1:1 - 20.

[0015] Another object of the present invention is to disclose a CaHA / PLGA composite microsphere, which is prepared by any of the above methods.

[0016] Another object of the present invention is to disclose the application of the above-mentioned CaHA / PLGA composite microspheres in soft tissue fillers, including the following steps: mixing and compounding the above-mentioned CaHA / PLGA composite microspheres with a gel to obtain a soft tissue filler. Of course, as is well known to those skilled in the art, for materials in medical filling, before use, they all need to be disinfected and sterilized. Among them, the disinfection and sterilization methods can adopt conventional methods in the art such as ultraviolet sterilization and ethylene oxide sterilization. Those skilled in the art can select appropriate disinfection and sterilization methods according to the actual situation. At the same time, the soft tissue filler of the present invention can be applied to the middle and lower layer soft tissue filling: when filling in the middle layer, it can promote the collagen deposition of fibroblasts; when approaching the periosteum layer deeply, it can also promote periosteal osteogenesis, so as to achieve bone shaping.

[0017] One embodiment of the present invention is that the mass-volume fraction of the CaHA / PLGA composite microspheres is 1-5%; in the gel, the gel material is one of carboxymethyl cellulose and hyaluronic acid, and the mass-volume fraction of the gel material is 1-5%, which is the same as that of a conventional gel, and the remaining material is water.

[0018] Beneficial effects:

[0019] (1) The CaHA / PLGA composite microspheres prepared by the present invention have CaHA uniformly loaded on the surface of the PLGA microspheres, and their particle sizes are distributed in the range of 20-60 μm. And different component and performance composite microspheres can be obtained by adjusting the ratio of CaHA to PLGA. PLGA provides the structural support and controllable degradation characteristics of the microspheres. The surface-modified CaHA can effectively reduce the adverse effects of local acidic degradation products on tissues, and at the same time regulate the microenvironment through the slow release of calcium ions to promote tissue regeneration. The presence of surface CaHA can enhance the adhesion and activation of fibroblasts, thus significantly increasing the collagen secretion level. At the same time, the PLGA microspheres are solid spheres. As the PLGA material degrades, the microspheres form cavities while maintaining the volume unchanged, providing good conditions for long-term tissue support and ingrowth.

[0020] (2) The CaHA / PLGA composite microspheres prepared by the present invention are compounded with a gel to obtain a soft tissue filler. Among them, the gel component provides stability for the filler and plays a role in moisturizing and nourishing during early injection. The CaHA / PLGA composite microspheres provide a long-term filling effect and stimulate collagen regeneration. Different component and performance soft tissue fillers can be obtained by adjusting the ratio of CaHA, which can be applied to the facial soft tissue filling of different layers.

[0021] (3)In summary, compared with the commonly used synthetic polymer-filled microspheres such as PLLA and PCL at present, the CaHA / PLGA composite microspheres have a milder mechanism of stimulating collagen regeneration and a lower risk of inflammation; their degradation period is moderate, and they have better tissue repair activity, which can achieve better filling effects; moreover, the design of the soft tissue filler of the present invention can not only make up for the deficiencies of traditional soft tissue fillers, but also further promote the development of multifunctional and high-performance medical beauty materials. Description of the Drawings

[0022] Figure 1 Scanning electron micrograph and particle size distribution diagram of the CaHA / PLGA composite microspheres prepared in Example 1;

[0023] Figure 2 Scanning electron micrograph and particle size distribution diagram of the CaHA / PLGA composite microspheres prepared in Example 2;

[0024] Figure 3 Scanning electron micrograph for evaluating the in vitro degradation performance of the CaHA / PLGA composite microspheres prepared in Example 1;

[0025] Figure 4 Static diagram and shear / viscosity curve of the soft tissue filler prepared in Example 3;

[0026] Figure 5 Optical density value and Sirius red staining of the co-culture of the CaHA / PLGA composite microspheres prepared in Example 1 with fibroblasts;

[0027] Figure 6 Section staining diagram of the soft tissue filler prepared in Example 4 after being injected into rats. Detailed Description of the Invention

[0028] The following will clearly and completely describe the specific implementation manners of the present invention in conjunction with examples and drawings. Obviously, the described examples are only a part of the embodiments of the present invention, rather than all embodiments.

[0029] In the following examples, unless otherwise specified, the medicaments used are all conventional commercial products.

[0030] In the following examples, unless otherwise specified, the methods used are all conventional operations in the art.

[0031] Example 1: A preparation method of CaHA / PLGA composite microspheres, comprising the following steps:

[0032] Take 250 mg of PLGA (molar ratio of LA to GA is 85:15, 100 KDa) and dissolve it in 4 mL of dichloromethane to obtain a first solution. Take polyvinyl alcohol and dissolve it in 50 mL of water to obtain a second solution with a volume-mass fraction of 1%. Mix the above two solutions and continuously stir under the condition of 800 rpm until dichloromethane completely volatilizes to obtain PLGA microspheres;

[0033] Weigh 2.36 g of calcium nitrate and 2.15 g of disodium hydrogen phosphate respectively, add them to 50 mL of deionized water and dissolve. Then adjust the pH with nitric acid until the solution is clear. After that, add 1.68 g of urea and 5 mmol / L of cyclohexanehexacarboxylic acid. After its dissolution, carry out a hydrothermal reaction at 150 °C for 3 h. Take the solid phase and wash it several times with deionized water and absolute ethanol successively, and dry it at 60 °C to obtain CaHA microspheres;

[0034] Treat the PLGA microspheres with 0.1 M sodium hydroxide solution to obtain activated PLGA microspheres. Disperse the CaHA microspheres in absolute ethanol, and add the activated PLGA microspheres to the above dispersion liquid according to the mass ratio of PLGA microspheres to CaHA microspheres of 1:2. After constant temperature oscillation at 37 °C, freeze-dry to obtain CaHA / PLGA composite microspheres.

[0035] Example 2: A preparation method of CaHA / PLGA composite microspheres, comprising the following steps:

[0036] Take 200 mg of PLGA (molar ratio of LA to GA is 50:50, 30 KDa) and dissolve it in 4 mL of dichloromethane to obtain a first solution. Take polyvinyl alcohol and dissolve it in 50 mL of water to obtain a second solution with a volume-mass fraction of 1%. Mix the above two solutions and continuously stir under the condition of 600 rpm until dichloromethane completely volatilizes to obtain PLGA microspheres;

[0037] Weigh 2.36 g of calcium nitrate and 2.15 g of disodium hydrogen phosphate respectively, add them to 50 mL of deionized water and dissolve. Then adjust the pH with nitric acid until the solution is clear. After that, add 1.68 g of urea and 1 mmol / L of cyclohexanehexacarboxylic acid. After its dissolution, carry out a hydrothermal reaction at 150 °C for 5 h. Then wash and dry it successively with deionized water and absolute ethanol to obtain CaHA microspheres;

[0038] Treat the PLGA microspheres with 1 M sodium hydroxide solution to obtain activated PLGA microspheres. Disperse the CaHA microspheres in absolute ethanol, and add the activated PLGA microspheres to the above dispersion liquid according to the mass ratio of PLGA microspheres to CaHA microspheres of 1:4. After constant temperature oscillation at 37 °C, freeze-dry to obtain CaHA / PLGA composite microspheres.

[0039] Example 3: A soft tissue filler, comprising 200 mg of the CaHA / PLGA composite microspheres prepared in Example 1, 200 mg of carboxymethyl cellulose, and 20 mL of deionized water. The preparation method of the soft tissue filler is as follows:

[0040] Weigh carboxymethyl cellulose and dissolve it in deionized water to obtain a gel solution. Take the CaHA / PLGA composite microspheres and mix them evenly with the gel solution to obtain a soft tissue filler.

[0041] Example 4: A soft tissue filler, comprising 200 mg of the CaHA / PLGA composite microspheres prepared in Example 1, 200 mg of hyaluronic acid, and 20 mL of deionized water. The preparation method of the soft tissue filler is as follows:

[0042] Weigh hyaluronic acid and dissolve it in deionized water to obtain a gel solution. Take the CaHA / PLGA composite microspheres and mix them evenly with the gel solution to obtain a soft tissue filler.

[0043] To further illustrate the effects of the CaHA / PLGA composite microspheres and the soft tissue filler prepared in the embodiments of the present invention, specific test examples are given below.

[0044] 1. Microscopic morphology

[0045] Take the CaHA / PLGA composite microspheres prepared in Example 1 and Example 2, observe their microscopic morphology by scanning electron microscopy, and count their particle size distribution. The final results are as Figure 1 and Figure 2 shown.

[0046] Among them, Figure 1 is the scanning electron micrograph and particle size distribution diagram of the CaHA / PLGA composite microspheres of Example 1, Figure 2 is the scanning electron micrograph and particle size distribution diagram of the CaHA / PLGA composite microspheres of Example 2.

[0047] Take the CaHA / PLGA composite microspheres prepared in Example 1, soak them in a phosphate buffer solution with a pH of 7.4, shake them at a constant temperature of 37 °C for 3 months, freeze-dry them, and observe their microscopic morphology by scanning electron microscopy. The final results are as Figure 3 shown.

[0048] From Figure 1 (a), it can be seen that for the CaHA / PLGA composite microspheres prepared in Example 1, more CaHA is loaded on their surface and the loading is relatively uniform; from Figure 1 (b), it can be seen that for the CaHA / PLGA composite microspheres prepared in Example 1, the particle size distribution range is 30 - 60 μm, and the average particle size is 44.19 ± 4.84 μm.

[0049] From Figure 2 (a), it can be seen that the CaHA / PLGA composite microspheres prepared in Example 2 have relatively less CaHA loaded on their surfaces; from Figure 2 (b), it can be seen that the CaHA / PLGA composite microspheres prepared in Example 2 have a particle size distribution range of 20 - 60 μm and an average particle size of 45.99 ± 7.76 μm.

[0050] Comparing Example 1 and Example 2, CaHA / PLGA composite microspheres with a particle size distribution in the range of 20 - 60 μm can be obtained within the parameter range, and by adjusting the ratio of CaHA to PLGA, the loading amount of CaHA on their surfaces can be regulated.

[0051] From Figure 3 it can be seen that the CaHA / PLGA composite microspheres prepared in Example 1 have started to degrade after 3 months of in vitro degradation test, and a large number of pores have formed on their surfaces and inside, but the volume change of the microspheres is relatively small, which provides favorable conditions for the long-term support of soft tissue filling and the ingrowth of collagen fibers.

[0052] 2. Stability and viscosity

[0053] Take the soft tissue filler prepared in Example 3 and let it stand at room temperature for 1 hour. The photo is as shown in Figure 4 (a); at the same time, take the soft tissue filler prepared in Example 3 and test its viscosity under different shear conditions. The final result is as shown in Figure 4 (b).

[0054] From Figure 4 (a), it can be seen that the soft tissue filler prepared in Example 3 can maintain a steady state within 1 h without stratification or sedimentation; from Figure 4 (b), it can be seen that the soft tissue filler prepared in Example 3 shows the characteristics of shear thinning and its viscosity is generally low, so it has good injectability.

[0055] 3. Optical density value and Sirius red staining

[0056] Seed fibroblasts at a density of 1×10 5 cells / well in a 24-well plate. Take 1 mg of the CaHA / PLGA composite microspheres prepared by the preparation method of Example 1 and co-culture them with fibroblasts. On the 1st, 2nd, and 3rd days, the cell optical density (OD) value is detected by the CCK-8 method, and on the 14th day, Sirius red staining is performed to observe the collagen deposition situation. The final result is as shown in Figure 5 .

[0057] From Figure 5(a) It can be seen that the CaHA / PLGA composite microspheres significantly promote the proliferation of fibroblasts and have no negative impact on their proliferation status. From Figure 5 (b) It can be seen that on the 14th day, a large amount of collagen deposition (red) is formed by fibroblasts, and the staining is deeper around the CaHA / PLGA composite microspheres.

[0058] 4. Section staining map after injection into rats.

[0059] Take the soft tissue filler of Example 4 and perform hematoxylin-eosin (H&E) staining and Masson staining on the tissue sections of rats subcutaneously injected for 4 weeks. The results are as Figure 6 shown.

[0060] From Figure 6 (a), it can be seen that no inflammatory tissue is formed; from Figure 6 (b), it can be seen that collagen fibers (blue) are formed around the injection site. It shows that the soft tissue filler of Example 4 not only does not cause inflammation, but also can effectively promote the regeneration and repair of soft tissues.

[0061] The above are only the preferred embodiments of the present invention and do not impose any form of limitation on the present invention. Although the present invention is disclosed as above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to equivalent embodiments by using the above-disclosed technical content within the scope of the technical solution of the present invention. However, as long as it does not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A preparation method of CaHA / PLGA composite microspheres, characterized in that, It includes the following steps: Dissolve PLGA in dichloromethane to obtain a first solution, dissolve polyvinyl alcohol in water to obtain a second solution, mix the above two solutions and continuously stir until dichloromethane completely volatilizes, and obtain PLGA microspheres after freeze-drying; the concentration of PLGA in the first solution is 5-10%, the concentration of polyvinyl alcohol in the second solution is 0.1-1%, and the volume ratio of the first solution to the second solution is 1:10-20; Take calcium salt and phosphate, add them to water and dissolve, then adjust the pH until the solution is clear, then add urea and a template agent, and carry out a hydrothermal reaction after its dissolution and separate and purify to obtain CaHA microspheres; Carry out alkali treatment on the PLGA microspheres to obtain activated PLGA microspheres, disperse the CaHA microspheres in a dispersant, add the activated PLGA microspheres to the above-mentioned dispersion liquid, and freeze-dry after constant temperature oscillation to obtain CaHA / PLGA composite microspheres.

2. The method according to claim 1, wherein The molecular weight of the PLGA is 10-500KDa, and the molar ratio of LA and GA in its raw materials is 15:85-85:

15.

3. The method according to claim 1, characterized in that, During the preparation process of the PLGA microspheres, the stirring speed is 300-800 rpm.

4. The method according to claim 1, characterized in that During the preparation process of the CaHA microspheres, the molar ratio of calcium salt and phosphate is 1.67, the molar ratio of urea to the calcium salt is 0.56:1, and the molar ratio of the template agent to the calcium salt is 1:20-200; the calcium salt is one of calcium nitrate and calcium chloride, the phosphate is one of disodium hydrogen phosphate, sodium dihydrogen phosphate, and ammonium phosphate, and the template agent is cyclohexane hexacarboxylic acid.

5. The method according to claim 1, wherein During the preparation process of the CaHA microspheres, the hydrothermal reaction temperature is 120-180°C, and the reaction time is 3-12 h; the separation and purification steps are: after removing the solvent, wash with deionized water and ethanol several times in sequence, and then dry to obtain.

6. The method according to claim 1, wherein During the preparation process of the CaHA / PLGA composite microspheres, the alkali solution for alkali treatment is 0.1-1 mol / L sodium hydroxide or potassium hydroxide solution, and the alkali treatment method is that the alkali solution covers the PLGA microspheres and soaks for 30-180 s; the dispersant is anhydrous ethanol.

7. The method according to claim 1, wherein During the preparation process of the CaHA / PLGA composite microspheres, the mass ratio of CaHA microspheres to PLGA microspheres is 1:1-20.

8. A CaHA / PLGA composite microsphere prepared by the method according to any one of claims 1-7.

9. Use of the CaHA / PLGA composite microspheres according to claim 8 in a soft tissue filler, characterized in that, Mix and compound the CaHA / PLGA composite microsphere of claim 8 with a gel to obtain a soft tissue filler.

10. The application according to claim 9, characterized in that, The mass-volume fraction of the CaHA / PLGA composite microsphere is 1-5%; in the gel, the gel material is one of carboxymethyl cellulose and hyaluronic acid, and the mass-volume fraction of the gel material is 1-5%, and the rest is sterile water.

Images