PLGA-hydroxyapatite composite porous microsphere as well as preparation method and application thereof

Abstract

The invention provides a PLGA (poly (lactic-co-glycolic acid))-hydroxyapatite composite porous microsphere as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) adding 1-100 mL of a span80 solution and 0.25-25 g of PLGA (poly (lactic-co-glycolic acid)) into 10-1000 mL of ethyl acetate, stirring and dissolving to obtain a mixed solution; (2) continuously stirring the mixed solution, adding an ammonium bicarbonate solution containing hydroxyapatite, and emulsifying to obtain w / o emulsion; (3) uniformly mixing 100 mL to 10000 mL of a PVA solution and 10 mL to 1000 mL of a Tween 80 solution, continuously stirring, adding the w / o emulsion, and emulsifying to obtain a w / o / w emulsion; and (4) stirring until the organic solvent is volatilized, sieving, washing with water, centrifuging, and freeze-drying the obtained precipitate to obtain the composite porous microspheres. The preparation method is simple and easy to implement and simple to operate.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a PLGA-hydroxyapatite composite porous microsphere and a preparation method and application thereof. Background Art

[0002] The International Organization for Standardization (ISO) defines biomaterials as inanimate materials that are used for medical purposes in contact with living tissue to form functions, including biocompatible or biodegradable materials.

[0003] Regenerative medicine is the research focus, and bioregenerative materials, as an important branch of regenerative medicine, are receiving unprecedented attention. Orthopedics, trauma repair, and medical aesthetics are the main clinical application scenarios of bioregenerative materials.

[0004] Medical beauty regenerative injection products usually use biomedical polymer materials as the main ingredients. They are usually injected into the dermis and / or subcutaneous tissue in the form of microspheres, which stimulate the regeneration of the body's own collagen to achieve the effects of remodeling, firming, beauty and anti-aging. Common medical beauty injection regenerative materials are synthetic polymers. The main ingredients of polymer microspheres currently approved for marketing in China can be divided into polylactic acid (PLA) and polycaprolactone (PCL).

[0005] Hydroxyapatite CaHA is a bioceramic with extremely high biocompatibility. After injection, CaHA can promote the regeneration of collagen I and collagen III, elastin and proteoglycans, and induce tissue regeneration. Compared with other treatments such as PLLA, CaHA has two relatively visible advantages: thermal stability and hydrophilicity. However, there are also some disadvantages and limitations: high brittleness, low toughness, and long degradation time that cannot match the degradation speed with the protein regeneration speed.

[0006] PLGA is an FDA-approved biodegradable medical polymer material. When used alone, it lacks inductive properties and has an unsatisfactory repair effect. After being compounded with hydroxyapatite, it retains both the biological properties of hydroxyapatite and the mechanical properties of PLGA, while the degradation rate is controllable. However, the preparation process needs to be further improved. Summary of the invention

[0007] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. The present invention provides a PLGA-hydroxyapatite composite porous microsphere and a preparation method thereof, which can solve the problems of the microsphere preparation method being complicated, being difficult to scale up in batches, and reagents in the prior art. The provided method adopts an emulsified solvent volatilization method to form a uniform suspension by emulsification, and the suspension is a w / o emulsion; the w / o emulsion is added to the aqueous phase and stirred and emulsified again to form a w / o / w emulsion, and stirring is continued after emulsification until the volatile organic solvent is completely volatilized, and the desired PLGA-hydroxyapatite composite porous microsphere is obtained after post-treatment. This method has the advantages of being easy to operate and not requiring special equipment.

[0008] Specifically, the present invention provides the following technical solutions:

[0009] In a first aspect of the present invention, a method for preparing PLGA-hydroxyapatite composite porous microspheres is provided, comprising:

[0010] (1) adding 1-100 mL of span80 solution and 0.25-25 g of PLGA into 10-1000 mL of ethyl acetate and stirring to dissolve to obtain a mixed solution;

[0011] (2) continuously stirring the mixed solution at a first predetermined speed, adding an ammonium bicarbonate solution containing hydroxyapatite, and performing a first emulsification to obtain a w / o emulsion;

[0012] (3) 100 mL to 10,000 mL of the PVA solution and 10 mL to 1,000 mL of the Tween 80 solution are mixed, and the mixture is continuously stirred at a second predetermined speed, and the w / o emulsion is added to perform a second emulsification to obtain a w / o / w emulsion;

[0013] (4) The w / o / w emulsion is stirred until the organic solvent evaporates, then sieved, washed with water, and centrifuged. The resulting precipitate is freeze-dried to obtain PLGA-hydroxyapatite composite porous microspheres.

[0014] The present invention prepares PLGA-hydroxyapatite composite porous microspheres by an emulsified solvent volatilization method. In the preparation process, an organic solvent is introduced to construct an emulsion, PLGA is dissolved in a volatile organic solvent, and hydroxyapatite is added to the organic solution to form a w / o emulsion; the w / o emulsion is added to an aqueous phase to form a w / o / w emulsion, the organic solvent is volatilized, and the desired PLGA-hydroxyapatite composite porous microspheres are obtained after post-treatment.

[0015] According to some embodiments of the present invention, the span80 solution in step (1) is an ethyl acetate solution of span80, and the concentration of the ethyl acetate solution of span80 is 0.1wt% to 10wt%. According to a preferred embodiment, the concentration of the ethyl acetate solution of span80 is 0.5wt% to 2wt%, more preferably 1wt%.

[0016] According to some embodiments of the present invention, the first predetermined rotational speed and the second predetermined rotational speed are each independently 1000-10000 rpm. According to a preferred embodiment, the first predetermined rotational speed and the second predetermined rotational speed are each independently 5000-10000 pm. The time for the first emulsification and the second emulsification are each independently 1 min-30 min. According to a preferred embodiment, the time for the first emulsification and the second emulsification are each independently 5-30 min.

[0017] According to some embodiments of the present invention, the mass fraction of hydroxyapatite in the ammonium bicarbonate solution is 0.1% to 10%; the PVA solution is a PVA aqueous solution, and the concentration of the PVA aqueous solution is 0.1wt% to 10wt%; the Tween 80 solution is an aqueous solution of Tween 80, and the concentration of the Tween 80 aqueous solution is 0.5wt% to 50wt%.

[0018] According to some embodiments of the present invention, the stirring in step (4) is to stir the w / o / w emulsion under vacuum conditions until the organic solvent evaporates, and the stirring time is 1 to 5 hours.

[0019] According to some embodiments of the present invention, the average particle size of the PLGA-hydroxyapatite composite porous microspheres is 1-200 μm, preferably 10-60 μm, and more preferably 20-50 μm.

[0020] The second aspect of the present invention provides a method for preparing PLGA-hydroxyapatite composite porous microspheres, comprising:

[0021] (1) adding 1-100 mL of ethyl acetate solution of span80 and 0.25-25 g of PLGA into 10-1000 mL of ethyl acetate and stirring to dissolve to obtain a mixed solution;

[0022] (2) placing the mixed solution under a homogenizer, stirring at a speed of 5000 to 10000 rpm, adding an ammonium bicarbonate solution containing hydroxyapatite, and emulsifying for 5 to 30 minutes to obtain a w / o emulsion;

[0023] (3) 100 mL to 10,000 mL of the PVA aqueous solution and 10 mL to 1,000 mL of the Tween 80 solution are mixed and placed in a homogenizer, stirred at a speed of 5,000 to 10,000 rpm, and the w / o emulsion is added, and emulsified for 1 to 30 minutes to obtain a w / o / w emulsion;

[0024] (4) stirring the w / o / w emulsion until the organic solvent evaporates, sieving, washing with water, and centrifuging, and freeze-drying the resulting precipitate to obtain PLGA-hydroxyapatite composite porous microspheres;

[0025] The average particle size of the PLGA-hydroxyapatite composite porous microspheres is 10-60 μm.

[0026] The third aspect of the present invention provides a PLGA-hydroxyapatite composite porous microsphere, which is obtained according to the preparation method of the PLGA-hydroxyapatite composite porous microsphere according to any one of the first aspect or the second aspect.

[0027] In some embodiments of the present invention, the average particle size of the PLGA-hydroxyapatite composite porous microspheres is 1-200 μm, and the surface pore size is 0.1-5 μm.

[0028] The fourth aspect of the present invention provides use of the PLGA-hydroxyapatite composite porous microspheres described in the second aspect in the field of preparing biomedical materials.

[0029] The beneficial effects achieved by the present invention are at least:

[0030] The method provided by the present invention is easy to operate and does not require special equipment. The prepared PLGA-hydroxyapatite composite porous microspheres retain both the biological properties of hydroxyapatite and the mechanical properties of PLGA, while the degradation rate is controllable. Moreover, its surface is porous and has a sponge-like porous microsphere structure, with a larger volume at the same mass. When applied to the field of biological application materials, such as regenerative medical beauty injections, the immediate shaping effect is more obvious, and collagen fibers can be promoted to grow into the microspheres along the porous microsphere structure, thereby more effectively promoting the regeneration of collagen. The swelling, light transmittance and stiffness after multiple injections after injection will be smaller, and the effect will be relatively more natural. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is an electron microscopic image of PLGA-hydroxyapatite composite porous microspheres provided in Example 1 of the present invention.

[0032] Figure 2 It is an electron microscope image of PLGA-hydroxyapatite composite microspheres provided in Comparative Example 1 of the present invention.

[0033] Figure 3It is an electron microscope image of PLGA-hydroxyapatite composite porous microspheres provided in Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0034] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0035] The present invention provides a method for preparing PLGA-hydroxyapatite composite porous microspheres, comprising:

[0036] (1) adding 1-100 mL of span80 solution and 0.25-25 g of PLGA into 10-1000 mL of ethyl acetate and stirring to dissolve to obtain a mixed solution;

[0037] (2) placing the mixed solution under a homogenizer, stirring at a first predetermined speed, adding an ammonium bicarbonate solution containing hydroxyapatite, and performing a first emulsification to obtain a w / o emulsion;

[0038] (3) 100 mL to 10000 mL of the PVA solution and 10 mL to 1000 mL of the Tween 80 solution are mixed and placed under a homogenizer, stirred at a second predetermined speed, and the w / o emulsion obtained in step (2) is added to perform a second emulsification to obtain a w / o / w emulsion;

[0039] (4) The w / o / w emulsion in step (3) is stirred until the organic solvent evaporates, and then sieved, washed with water, and centrifuged to precipitate. The precipitate is freeze-dried to obtain PLGA-hydroxyapatite composite porous microspheres.

[0040] The present invention introduces an organic solvent to construct an emulsion through an emulsification solvent volatilization method. That is, PLGA is dissolved in a volatile organic solvent, hydroxyapatite is dissolved in an ammonium bicarbonate solution and added to the organic solution to form a w / o emulsion; an aqueous phase is prepared, the w / o emulsion is added to the aqueous phase to form a w / o / w emulsion, the organic solvent is volatilized, and the required PLGA-hydroxyapatite composite porous microspheres are obtained after post-treatment. Hydroxyapatite is pre-dissolved in an ammonium bicarbonate solution to form microspheres with a hollow structure. Ammonium bicarbonate can decompose to produce carbon dioxide gas during the treatment process, and this other expansion effect can form a cavity inside the microsphere. The use of ammonium bicarbonate not only helps to control the microstructure of the microsphere, but also can effectively realize the preparation of hollow microspheres.

[0041] The span80 solution is an ethyl acetate solution of span80, and its concentration is 0.1wt% to 10wt%. Although span80 can be dissolved in a variety of organic solvents (such as petroleum ether, propylene glycol, liquid paraffin, n-heptane, etc.), it was found during the research process that only span80 is soluble in ethyl acetate, and the prepared composite microspheres have better performance and more uniform surface porous structure. According to a preferred embodiment, the concentration of the ethyl acetate solution of span80 is 1 to 5wt%.

[0042] The first predetermined rotation speed and the second predetermined rotation speed are each independently 1000-10000 rpm (for example, 5000-10000 rpm); the time of the first emulsification and the second emulsification are each independently 1 min-30 min (for example, 5-20 min).

[0043] The mass fraction of hydroxyapatite in the ammonium bicarbonate solution is 0.1% to 10%; the PVA solution is an aqueous solution of PVA, and its concentration is 0.1wt% to 10wt% (for example, 1wt% to 10wt%); the Tween 80 solution is an aqueous solution of Tween 80, and its concentration is 0.5wt% to 50wt% (for example, 10wt% to 20wt%).

[0044] The stirring in step (4) is to continue vacuum stirring after emulsification until the volatile organic solvent is completely volatilized, and the stirring time is 1 to 5 hours.

[0045] According to some embodiments of the present invention, the average particle size of the PLGA-hydroxyapatite composite porous microspheres is 1-200 μm, such as 10-60 μm, more preferably 20-50 μm (such as 30-40 μm).

[0046] In some embodiments of the present invention, the average particle size of the PLGA-hydroxyapatite composite porous microspheres is 1-200 μm, and the surface pore size is 0.1-5 μm.

[0047] According to a preferred embodiment, the preparation method provided comprises:

[0048] (1) adding 1-100 mL of 1 wt%-10 wt% span80 ethyl acetate solution and 0.25-25 g of PLGA into 10-1000 mL of ethyl acetate and stirring to dissolve to obtain a mixed solution;

[0049] (2) placing the mixed solution under a homogenizer, stirring at a speed of 5000 to 10000 rpm, adding an ammonium bicarbonate solution containing hydroxyapatite, and emulsifying for 5 to 30 minutes to obtain a w / o emulsion;

[0050] (3) 100 mL to 10000 mL of 1 wt% to 10 wt% PVA aqueous solution and 10 mL to 1000 mL of 10 wt% to 20 wt% Tween 80 solution are mixed and placed under a homogenizer, stirred at a speed of 5000 to 10000 rpm, and the w / o emulsion is added, and emulsified for 5 to 30 minutes to obtain a w / o / w emulsion;

[0051] (4) stirring the w / o / w emulsion until the organic solvent evaporates, sieving, washing with water, and centrifuging, and freeze-drying the resulting precipitate to obtain PLGA-hydroxyapatite composite porous microspheres;

[0052] The average particle size of the PLGA-hydroxyapatite composite porous microspheres is 30-40 μm.

[0053] The technical scheme of the present invention is described below by specific examples. It should be noted that these examples are only used to facilitate the understanding of those skilled in the art and should not be regarded as limiting the scope of protection of the present invention. Unless otherwise specified, the reagents used in the examples can be obtained commercially.

[0054] Example 1

[0055] Example 1 PLGA-hydroxyapatite composite porous microspheres were prepared by the following method, including:

[0056] Step 1: add 1 mL of span80 ethyl acetate solution (concentration is 1 wt%) and 0.25 g of PLGA into 10 mL of ethyl acetate and stir to dissolve.

[0057] Step 2, placing the solution in step 1 under a homogenizer with a stable rotation speed of 5000 rpm, then adding the ammonium bicarbonate solution containing hydroxyapatite thereto, and fully emulsifying for 5 minutes to obtain a w / o emulsion.

[0058] Step 3, mix 100 mL of PVA aqueous solution (1 wt%) and 10 mL of Tween 80 aqueous solution (concentration 10 wt%) and put them under a homogenizer with a stable speed of 5000 rpm, then add the w / o emulsion in step 2 and fully emulsify for 5 minutes to obtain a w / o / w emulsion.

[0059] Step 4, stirring the w / o / w emulsion in step 3, stirring and vacuuming until the organic solvent evaporates (about 1 hour), then sieving, washing with water, centrifuging and precipitating, and then freeze-drying the precipitate to obtain composite microspheres.

[0060] The average particle size of the obtained composite microspheres is 40 μm, and the surface has micropores with a pore size of about 0.1-5 μm. The electron microscope image is as follows Figure 1 shown.

[0061] Example 2

[0062] Example 2 PLGA-hydroxyapatite composite porous microspheres were prepared by the following method, including:

[0063] Step 1: add 100 mL of span80 ethyl acetate solution (concentration is 1 wt%) and 20 g of PLGA into 800 mL of ethyl acetate and stir to dissolve.

[0064] Step 2, placing the solution in step 1 under a homogenizer with a stable rotation speed of 10000 rpm, then adding the ammonium bicarbonate solution containing hydroxyapatite thereto, and fully emulsifying for 30 minutes to obtain a w / o emulsion.

[0065] Step 3, 9000 mL of PVA aqueous solution (10 wt%) and 1000 mL of Tween 80 aqueous solution (concentration of 20 wt%) were mixed and placed under a homogenizer with a stable speed of 10000 rpm, and then the w / o emulsion in step 2 was added thereto and fully emulsified for 30 min to obtain a w / o / w emulsion.

[0066] Step 4, stirring the w / o / w emulsion in step 3, stirring and vacuuming until the organic solvent evaporates (about 5 hours), then sieving, washing with water, centrifuging and precipitating, and then freeze-drying the precipitate to obtain composite microspheres.

[0067] The obtained composite microspheres have an average particle size of 30 μm and have micropores on the surface, with the pore size of the micropores being about 0.1-5 μm.

[0068] Comparative Example 1

[0069] The difference between Comparative Example 1 and Example 1 is that in step 2, only hydroxyapatite is added for direct emulsification, and no ammonium bicarbonate solution containing hydroxyapatite is added.

[0070] The results showed that the obtained composite microspheres were solid and non-porous microspheres. Figure 2 .

[0071] Comparative Example 2

[0072] Comparative Example 2 is different from Example 1 in that the rotation speed and emulsification time are adjusted, specifically including:

[0073] Step 1: add 1 mL of span80 solution (concentration of 1 wt%) and 0.25 g of PLGA into 10 mL of ethyl acetate and stir to dissolve.

[0074] Step 2, placing the solution in step 1 under a homogenizer with a stable rotation speed of 500 rpm, then adding the ammonium bicarbonate solution containing hydroxyapatite thereto, and emulsifying for 1 minute to obtain a w / o emulsion.

[0075] Step 3, mix 80 mL PVA solution (0.1 wt%) and 8 mL Tween 80 solution (concentration 0.5 wt%) and put them under a homogenizer with a stable speed of 500 rpm, add the above w / o emulsion, and emulsify for 1 min to obtain a w / o / w emulsion.

[0076] Step 4, stirring the w / o / w emulsion, stirring and vacuuming until the organic solvent evaporates (about 1 hour), then sieving, washing with water, centrifuging and precipitating, and then freeze-drying the precipitate to obtain composite microspheres.

[0077] The results showed that the prepared composite microspheres were concave and could not be used. Figure 3 .

[0078] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "specific implementations", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example.

[0079] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for preparing PLGA-hydroxyapatite composite porous microspheres, characterized in that: include: (1) adding 1-100 mL of span80 solution and 0.25-25 g of PLGA into 10-1000 mL of ethyl acetate and stirring to dissolve to obtain a mixed solution; (2) continuously stirring the mixed solution at a first predetermined speed, adding an ammonium bicarbonate solution containing hydroxyapatite, and performing a first emulsification to obtain a w / o emulsion; (3) 100 mL to 10000 mL of the PVA solution and 10 mL to 1000 mL of the Tween 80 solution are mixed, and the mixture is continuously stirred at a second predetermined speed, and the w / o emulsion is added to perform a second emulsification to obtain a w / o / w emulsion; (4) The w / o / w emulsion is stirred until the organic solvent evaporates, and then sieved, washed with water, and centrifuged. The obtained precipitate is freeze-dried to obtain PLGA-hydroxyapatite composite porous microspheres.

2. The method for preparing PLGA-hydroxyapatite composite porous microspheres according to claim 1, characterized in that: The span80 solution in step (1) is an ethyl acetate solution of span80, and the concentration of the ethyl acetate solution of span80 is 0.1wt% to 10wt%, more preferably 0.5wt% to 2wt%.

3. The method for preparing PLGA-hydroxyapatite composite porous microspheres according to claim 1, characterized in that: The first predetermined rotation speed and the second predetermined rotation speed are independently 1000-10000 rpm; The time of the first emulsification and the second emulsification is independently 1-30 minutes.

4. The method for preparing PLGA-hydroxyapatite composite porous microspheres according to claim 1, characterized in that: The mass fraction of hydroxyapatite in the ammonium bicarbonate solution is 0.1% to 10%; The PVA solution is a PVA aqueous solution, and the concentration of the PVA aqueous solution is 0.1wt% to 10wt%; The concentration of the Tween 80 solution is 0.5 wt % to 50 wt %.

5. The method for preparing PLGA-hydroxyapatite composite porous microspheres according to claim 1, characterized in that: The stirring in step (4) is to stir the w / o / w emulsion under vacuum conditions until the organic solvent evaporates, and the stirring time is 1 to 5 hours.

6. The method for preparing PLGA-hydroxyapatite composite porous microspheres according to claim 1, characterized in that: The average particle size of the PLGA-hydroxyapatite composite porous microspheres is 1-200 μm, preferably 10-60 μm, and more preferably 20-50 μm.

7. A method for preparing PLGA-hydroxyapatite composite porous microspheres, characterized in that: include: (1) adding 1-100 mL of ethyl acetate solution of span80 and 0.25-25 g of PLGA into 10-1000 mL of ethyl acetate and stirring to dissolve to obtain a mixed solution; (2) placing the mixed solution under a homogenizer, stirring at a speed of 5000 to 10000 rpm, adding an ammonium bicarbonate solution containing hydroxyapatite, and emulsifying for 5 to 30 minutes to obtain a w / o emulsion; (3) 100 mL to 10,000 mL of the PVA aqueous solution and 10 mL to 1,000 mL of the Tween 80 solution are mixed and placed in a homogenizer, stirred at a speed of 5,000 to 10,000 rpm, and the w / o emulsion is added, and emulsified for 5 to 30 minutes to obtain a w / o / w emulsion; (4) stirring the w / o / w emulsion until the organic solvent evaporates, sieving, washing with water, and centrifuging, and freeze-drying the resulting precipitate to obtain PLGA-hydroxyapatite composite porous microspheres; The average particle size of the PLGA-hydroxyapatite composite porous microspheres is 10-60 μm.

8. A PLGA-hydroxyapatite composite porous microsphere, characterized in that: Obtained according to the preparation method of PLGA-hydroxyapatite composite porous microspheres according to any one of claims 1 to 7.

9. The PLGA-hydroxyapatite composite porous microspheres according to claim 8, characterized in that: The average particle size of the PLGA-hydroxyapatite composite porous microspheres is 1-200 μm, and the surface pore size is 0.1-5 μm.

10. Use of the PLGA-hydroxyapatite composite porous microspheres according to claim 8 or 9 in the field of preparing biomedical materials.

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