Poly-L-lactic acid microspheres for injection and preparation method thereof

Abstract

The invention relates to the technical field of compounds, in particular to poly-L-lactic acid microspheres for injection and a preparation method of the poly-L-lactic acid microspheres. The microspheres prepared under the condition of high-speed shearing dispersion or mechanical stirring are uniform in particle size, narrow in particle size range and good in reproducibility. A polylactic acid raw material with proper molecular weight is screened, microspheres are manufactured through a membrane emulsification method, then the microspheres with the required particle size are screened through a screening mechanism, and the surfaces of the microspheres are smooth.

Description

Technical Field

[0001] The invention relates to the technical field of compounds, in particular to poly-L-lactic acid microspheres for injection and a preparation method thereof. Background Art

[0002] At present, injectable beauty refers to injecting injectable materials directly into a local or specific part of the human body to improve the appearance or shape of the human body. The most common injectable products on the market are hyaluronic acid products, which will shift after injection. Multiple supplementary injections may cause cross-linking agent residues or excessive exposure, causing delayed allergic reactions. The new injectable beauty filler poly-L-lactic acid is derived from a biodegradable synthetic polymer in the α-hydroxy acid family. It has good biocompatibility and has been widely used in dissolvable sutures, bone screws and facial implants.

[0003] The world's first "baby face injection" was invented by the Dermik laboratory of Sanofi-Aventis in Europe. It was approved by the FDA in 2009 as an injectable cosmetic agent for the treatment of wrinkles. Its molecular weight is 120,000-170,000 Da, and its intrinsic viscosity is 3-4 dL / g. Its degradation period in the body is 18-24 months. 【1】 , its slow degradation kinetics are attributed to the morphology of the prepared microspheres / microparticles, the size of the microspheres / microparticles, the intrinsic viscosity and molecular weight of polylactic acid. The retention time of the filler in the body, excluding individual differences, is mostly related to the control of the above-mentioned physical and chemical indicators. First, select the raw materials with suitable molecular weight. The diameter of the prepared microspheres should be greater than 5μm, preferably greater than 20μm, to avoid being phagocytosed by macrophages. Their diameter should be less than 150μm, preferably less than 63μm, to ensure that the size of the microspheres is large enough to avoid being phagocytosed by dermal macrophages or passing through the capillary wall, and to ensure good needle permeability; secondly, the particle morphology is smooth. Smooth microspheres are relatively sharp and irregular particles, which will greatly reduce the inflammatory response; finally, the molecular weight of the raw materials selected for the preparation of microspheres, according to the Mark-Houwink equation, [η]=KM^α, α=0.73, K (dl / g)=5.45×10 -4 , calculate the relationship between the intrinsic viscosity of the polymer and the viscosity-average molecular weight.

[0004] When poly-L-lactic acid fillers are injected in vivo, they will cause clinical foreign body inflammatory reactions: including I) host protein absorption, II) cell aggregation, and III) fibrotic encapsulation. Protein adsorption occurs immediately after injection, causing local mast cells to release histamine and proinflammatory cytokines, triggering neutrophils and monocytes to migrate to the implant material, and monocytes to differentiate into macrophages. When macrophages cannot phagocytize foreign bodies, macrophages aggregate and fuse to foreign body giant cells (FBGCs), and the resulting FBGCs form a continuous layer of encapsulated materials to protect the body. Macrophages release transforming growth factors, aggregate and stimulate fibroblast proliferation. Macrophages and fibroblasts are in autocrine and paracrine communication, followed by a large amount of collagen fiber distribution and the resulting type I collagen deposition in the extracellular matrix. Fibroblasts further differentiate into myofibroblasts and synthesize more type III collagen than type I. Subsequently, PLLA degrades, and collagen accumulates over time. The above chronic inflammatory process is also key to clinical efficacy.

[0005] The Chinese invention patent with publication number CN109010910B uses the oil-water emulsification method and the volatility of organic solvents to prepare porous polylactic acid microspheres. The microspheres are uneven in size. The process is optimized and a screening process is added on this basis to prepare microspheres that meet the requirements. Summary of the invention

[0006] The present invention provides a poly-L-lactic acid microsphere for injection and a preparation method thereof. The microspheres prepared under high-speed shear dispersion or mechanical stirring conditions have uniform particle size, narrow particle size range and good reproducibility. Poly-lactic acid materials with suitable molecular weight are screened, microspheres are manufactured by membrane emulsification, and then microspheres with the required particle size are screened by a screening mechanism, and the surface of the microspheres is smooth.

[0007] The technical solution of the present invention is as follows: A method for preparing poly-L-lactic acid microspheres for injection comprises the following steps: Poly (L-lactic acid) microspheres were prepared by membrane emulsification technology. The preparation method was as follows: PLLA was dissolved in dichloromethane or chloroform as an organic phase, and a polyvinyl alcohol (PVA) solution containing 0.5% to 2.5% by mass and / or sodium dodecyl sulfate aqueous solution or / and Tween80 was used as an aqueous phase.

[0008] The method also includes the following steps: loading the organic phase into an organic phase tank, loading the aqueous phase into an aqueous phase stirring tank, and rapidly squeezing the organic phase through a membrane emulsifier into the aqueous phase under the pressure of liquid nitrogen, with a stirring speed of 100 rpm to 400 rpm, an emulsification pressure of 0.5 bar to 5 bar, and a frequency of an aqueous phase circulation pump of 10% to 60%, to form an emulsion with uniform particle size, and removing the organic solvent to obtain solidified microspheres, which are then washed, centrifuged, and freeze-dried.

[0009] Or it also includes the following steps: quickly adding the organic phase to the aqueous phase, high-speed shear emulsification at 6000r· / min~10000r· / min to form O / W type colostrum, quickly passing the colostrum through a membrane emulsifier under the pressure of liquid nitrogen to form an emulsion with uniform particle size, adding the emulsion to a 0.5%~2.5% PVA aqueous solution, stirring at a speed of 100rpm~400rpm, an emulsification pressure of 0.5bar~5bar, a water phase circulation pump frequency of 10%~60%, removing the organic solvent, and obtaining solidified microspheres, which are then washed, centrifuged, and freeze-dried.

[0010] The method also includes the following steps: cutting a 1 cm section of conductive tape and sticking it on a sample table, evenly spreading the injectable poly-L-lactic acid microsphere sample on the conductive tape, inverting the sample table, gently tapping it to make the unadhered particles fall off, then turning the sample table face up, using an ear bulb to blow away the unadhered powder on the sample table to obtain a uniform powder layer; irradiating the sample table under an infrared baking lamp for 15 to 30 minutes to keep the sample surface dry, then spraying gold on the sample for 50 to 80 seconds, and observing the sample morphology using a Phenom scanning electron microscope.

[0011] Poly-L-lactic acid microspheres for injection obtained according to the preparation method.

[0012] The beneficial effects of the present invention are as follows: the present invention selects polylactic acid materials with suitable molecular weight, firstly manufactures microspheres by membrane emulsification, and then selects microspheres with required particle size by screening process, and the surface of the microspheres is smooth.

[0013] The filler prepared by the polylactic acid microspheres for injection prepared by the present invention is supplemented with sodium carboxymethyl cellulose and mannitol. After injection into an animal body, a large amount of collagen fibers can be seen distributed at the implantation point, and the arrangement is relatively neat. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1-1 This is a schematic diagram of the morphology of the poly (L-lactic acid) microspheres for injection of the present invention, with a scale bar of 50 µm.

[0015] Figure 1-2 This is a schematic diagram of the morphology of the poly-L-lactic acid microspheres for injection of the present invention, with a scale bar of 30 µm.

[0016] Figure 2 This is a schematic diagram of the number of poly-L-lactic acid microspheres for injection of the present invention.

[0017] Figure 3 Schematic diagram of particle size and distribution of poly (L-lactic acid) microspheres for injection of the present invention.

[0018] Figure 4 The figure is a schematic diagram of HE staining and immunofluorescence staining of the test sample group and the marketed control group 78 weeks after implantation of the present invention. DETAILED DESCRIPTION

[0019] The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. Example 1

[0020] Poly (L-lactic acid) microspheres were prepared by membrane emulsification technology. The preparation method was as follows: PLLA was dissolved in dichloromethane as the organic phase, and an aqueous solution containing 1.5% PVA by mass and 0.6% Tween80 by mass was used as the aqueous phase. The organic phase was loaded into an organic phase tank, and the aqueous phase was loaded into an aqueous phase stirring tank. The organic phase was rapidly squeezed through a membrane emulsifier under the pressure of liquid nitrogen, with a stirring speed of 300 rpm, an emulsification pressure of 1 bar, and a frequency of a water phase circulation pump of 20% to form an emulsion with uniform particle size. The solidified microspheres were washed, centrifuged, and freeze-dried.

[0021] Cut a 1cm section of conductive tape and stick it on the sample table. Evenly spread the injectable poly-L-lactic acid microsphere sample on the conductive tape. Invert the sample table and tap it gently to make the unadhered particles fall off. Then turn the sample table upside down and use an ear-cleaning bulb to blow away the unadhered powder on the sample table to obtain a uniform powder layer. Irradiate the sample table under an infrared baking lamp for 20 min to keep the sample surface dry. Then spray the sample with gold for 70 s and observe the sample morphology using a Phenom scanning electron microscope. D50=48.4μm, D90=58μm, span: 0.63. Example 2

[0022] Poly (L-lactic acid) microspheres were prepared by membrane emulsification technology. The preparation method was as follows: PLLA was dissolved in dichloromethane as the organic phase, and an aqueous solution containing 1.5% PVA by mass and 0.6% Tween80 by mass was used as the aqueous phase. The organic phase was quickly added to the aqueous phase, and high-speed shear emulsification was performed at 8,000 r / min to form an O / W type colostrum. The colostrum was quickly passed through a membrane emulsifier under the pressure of liquid nitrogen to form an emulsion with uniform particle size. The emulsion was added to a 1% PVA aqueous solution with a stirring speed of 300 rpm, an emulsification pressure of 0.8 bar, and a frequency of an aqueous phase circulation pump of 15%. The organic solvent was removed to obtain solidified microspheres, which were then washed, centrifuged, and freeze-dried. Example 3

[0023] The poly-L-lactic acid microspheres for injection prepared in Example 1 were mixed with sodium carboxymethyl cellulose and mannitol and then freeze-dried to prepare a white dry powder solid. The solid was sterilized and packaged in vials, halogenated butyl rubber stoppers and aluminum-plastic combination caps. It was a sterile product for single use. Dissolved in sterile water for injection at a ratio of 367.5 mg / 5 ml, allowed to stand for more than 6 hours after dissolution, and injected into the rabbit subcutaneously with a disposable sterile syringe. 0.1 ml was tested according to the subcutaneous implantation test method in 16886.6-2015.

[0024] Statistical analysis of SEM images was performed using ImagePro software, as above. Figure 1-1 (Scale bar is 50µm) As shown in the figure, when the magnification is 1200 times, it can be observed that the poly-L-lactic acid microspheres for injection are spherical, and the particle size is between 40-63µm. Figure 1-2 (The scale bar is 30µm). When the magnification is 2100 times, it can be observed that the surface of the microspheres is smooth, which is conducive to injection and effectively reduces the production of inflammatory reactions. The particle size range of poly-L-lactic acid microspheres is 40-63μm, which is conducive to injection and is not easily captured and cleared by capillary walls or immune cells (such as macrophages). It is also conducive to needle passage and suitable for subsequent in vivo implantation applications.

[0025] pass Figure 2 From the analysis, it can be observed that the particle size of the injectable poly-L-lactic acid microspheres is mainly concentrated in the range of 40~63μm.

[0026] pass Figure 3 From the analysis, it can be observed that the D50 of the injectable poly-L-lactic acid microspheres is 45.15 μm, and the spheres ranging from 40 to 63 μm account for 72.38%, with a span of 0.444.

[0027] pass Figure 4 The tissue structure of the subcutaneous implantation site of the test sample and the negative control was observed with the naked eye. Histopathological examination showed that there was no irritation reaction in the test sample and the negative control at 78 weeks. Hematoxylin-eosin staining showed that most of the test sample had been absorbed under the microscope, and a very small number of scattered test sample particles were surrounded by collagen tissue and adipose tissue. Masson's trichrome staining showed that a large number of collagen fibers were distributed around the implantation site of the test sample, and the arrangement was relatively neat. Immunofluorescence staining showed that the expression of matrix metalloproteinase 9 in the test sample was significantly reduced.

[0028] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only preferred examples of the invention and are not intended to limit the present invention. Without departing from the novel spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing poly (L-lactic acid) microspheres for injection, characterized in that The following steps are involved: Poly (L-lactic acid) microspheres were prepared by membrane emulsification technology. The preparation method was as follows: PLLA was dissolved in dichloromethane or chloroform as an organic phase, and an aqueous solution containing 0.5% to 2.5% by mass of PVA or sodium dodecyl sulfate or 0.1% to 1% by mass of Tween80 was used as an aqueous phase.

2. The method for preparing poly-L-lactic acid microspheres for injection according to claim 1, characterized in that The method also includes the following steps: loading the organic phase into an organic phase tank, loading the aqueous phase into an aqueous phase stirring tank, and rapidly squeezing the organic phase through a membrane emulsifier under the pressure of liquid nitrogen, with a stirring speed of 100 rpm to 400 rpm, an emulsification pressure of 0.5 bar to 5 bar, and a frequency of an aqueous phase circulation pump of 10% to 60%, to form an emulsion with uniform particle size, and removing the organic solvent to obtain solidified microspheres, which are then washed, centrifuged, and freeze-dried.

3. The method for preparing poly-L-lactic acid microspheres for injection according to claim 1, characterized in that The method also includes the following steps: rapidly adding the organic phase to the aqueous phase, performing high-speed shear emulsification at 6000 r· / min~10000 r· / min to form O / W colostrum, rapidly passing the colostrum through a membrane emulsifier under the pressure of liquid nitrogen to form an emulsion with uniform particle size, adding the emulsion to a 0.5%~2.5% PVA aqueous solution, stirring at a speed of 100rpm~400rpm, an emulsification pressure of 0.5bar~5bar, a water phase circulation pump frequency of 10%~60%, removing the organic solvent, and obtaining solidified microspheres which are then washed, centrifuged, and freeze-dried.

4. The method for preparing poly (L-lactic acid) microspheres for injection according to claim 2 or 3, wherein the sieving is performed using a stainless steel sieve.

5. The method for preparing poly-L-lactic acid microspheres for injection according to claim 2 or 3, characterized in that The method also includes the following steps: cutting a 1 cm section of conductive tape and sticking it on a sample table, evenly spreading the injectable poly-L-lactic acid microsphere sample on the conductive tape, inverting the sample table, gently tapping it to make the unadhered particles fall off, then turning the sample table face up, using an ear bulb to blow away the unadhered powder on the sample table to obtain a uniform powder layer; irradiating the sample table under an infrared baking lamp for 15 to 30 minutes to keep the sample surface dry, then spraying gold on the sample for 50 to 80 seconds, and observing the sample morphology using a Phenom scanning electron microscope.

6. Poly (L-lactic acid) microspheres for injection obtained according to the preparation method according to any one of claims 1 to 5.

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