CN100457187C - VEGF slowly releasing injection microsphere support and its prepn and use - Google Patents

Abstract

The invention discloses a VEGF slow-release injection microsphere scaffold, which consists of a matrix, VEGF, a protective agent and ammonium bicarbonate, and is characterized in that the matrix is polylactic acid:glycolic acid=25:75-75:25. Lactic acid-glycolic acid copolymer; the protective agent includes zinc carbonate, serum albumin, trehalose and mannitol; the weight ratio of VEGF to polylactic acid-glycolic acid copolymer is 1:100000 to 1:10, and the microsphere particle diameter 50 μm to 1000 μm at room temperature. Its preparation adopts the following methods: w/o/w solvent volatilization method, or w/o/w solvent volatilization method in which medicine is added in the form of micropowder. The method of the invention is simple, easy to operate and has good reproducibility. The protein drug VEGF is released in vitro for more than 4 weeks in the microsphere scaffold, and its release approximately conforms to the zero-order kinetic model, which can be applied to the repair and treatment of various tissue vascular defects.

Description

VEGF slow-release injection microsphere scaffold and its preparation method and application

technical field

The invention relates to the technical field of medicine, and relates to a protein-loaded drug VEGF (vascular endothelial growth factor, VEGF) slow-release injection microsphere stent and a preparation method and application thereof.

Background technique

Vascular endothelial growth factor (vascular endothelial growth factor, VEGF) is a growth factor that specifically acts on vascular endothelial cells, can promote the proliferation, metastasis and differentiation of new blood vessels, and because VEGF can enhance vascular permeability , so it is also called vascular permeability factor. It was first purified from the conditioned medium of bovine pituitary follicular stellate cells by Ferrara et al. in 1989. Recombinant human vascular endothelial growth factor (rhVEGF) has entered the clinical trial stage from extensive preclinical research in the treatment of ischemic diseases and is expected to become a therapeutic drug for clinical application in the near future. (N. Ferrara, K. Alitalo. Clinical applications of angiogenic growth factors and their inhibitors, [J]. Nature Med. 1999, 5 (12): 1359). However, similar to other growth factors, VEGF is degraded too quickly in vivo due to its short half-life (Jeffrey L. Cleland Ph.D, Eillen T. Puenas et al. Cuthbertson Development of poly-(D, L-Lactide-co-glycolide) microspheres fermulation containing recombinant human vascular endothelial growth factor to promote local angiogenesis [J]. Controlled Release 2001, 72: 13), thus limiting its clinical application.

Contents of the invention

The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art, combine the advantages of microsphere preparation technology and tissue engineering scaffold technology to provide a slow-release injection microsphere scaffold of VEGF and its preparation method, the protein drug VEGF can be released continuously up to 4 Zhou, to increase the survival rate of cells and promote their growth, thereby increasing the formation of new blood vessels, for the treatment of local ischemic diseases.

In recent years, biodegradable polymer materials have been widely used in the preparation of sustained-release microspheres of protein and polypeptide drugs and tissue engineering technology, among which poly(lactide-co-glycolide), PLGA], due to its Good biocompatibility and biodegradability have been approved by the US FDA as a pharmaceutical polymer material. Using PLGA as the microsphere matrix, the degradation experiments in vivo and in vitro proved that during the drug release process, the entire polymer skeleton was degraded uniformly. With the decrease of molecular weight, the hydrophilicity of the skeleton material was enhanced, and the protein and polypeptide (Wang YM, Sato H, Horikoshi.In vitro and in vivo evaluation of taxol release fromploy(lactic-co-glycotic acid)microspheres containing isopropyl myristate and degradation of the microspheres.J Control Rel,1957,49:1 ). The present invention uses PLGA as a matrix to prepare VEGF slow-release microsphere scaffold. The present invention uses PLGA as the basic skeleton to prepare VEGF slow-release injection microsphere scaffolds, which are porous protein-loaded drug microspheres. On the one hand, the holes in the microspheres are planted by cell culture and corresponding cells are positioned to the tissue defect site through injection. On the other hand, VEGF in microspheres can release drugs at a constant release rate to stimulate cell growth and differentiation to form new tissues.

The VEGF sustained-release injection microsphere scaffold of the present invention comprises a matrix, VEGF, a protective agent and ammonium bicarbonate, and is characterized in that the matrix is polylactic acid-hydroxyl of polylactic acid:glycolic acid=25:75-75:25 Acetic acid copolymer; the protective agent includes zinc carbonate, serum albumin, trehalose and mannitol; the weight ratio of VEGF to polylactic acid-glycolic acid copolymer is 1: 100000 to 1: 10, and the particle size of microspheres is at room temperature 50μm to 1000μm.

There are two methods for preparing the VEGF slow-release injection microsphere scaffold of the present invention:

1.w/o/w solvent evaporation method: microspheres are prepared by w/o/w double emulsification-solvent evaporation method

Prepare the oil phase

The matrix material PLGA is dissolved in the organic solvent dichloromethane to make an oil phase with a concentration of 30mg-300mg/ml;

Preparation of the inner aqueous phase

Take an appropriate amount of VEGF, protective agent and ammonium bicarbonate dissolved in water to form an internal water phase, the concentration of ammonium bicarbonate is 2%-50%, the concentration of VEGF is 1μg-1mg/ml, and the protective agent is selected from zinc carbonate, serum albumin, seaweed Sugar and mannitol, etc., the content is 1-5%;

Preparation of microspheres

Add the inner water phase to the above-mentioned oil phase for homogenization (or ultrasonication) to form colostrum, quickly drop the colostrum into 0.01-6% polyvinyl alcohol (PVA) aqueous solution, and fully homogenize with mechanical stirring (stirring speed is 100 ~1800rpm), continue to stir at low speed for 4 hours at room temperature (stirring speed is 100~300rpm), wash, collect, and freeze-dry.

2. The w/o/w solvent evaporation method in which the drug is added in the form of micropowder:

Prepare the oil phase

The matrix material PLGA is dissolved in the organic solvent dichloromethane to make an oil phase with a concentration of 30mg-300mg/ml;

Preparation of the inner aqueous phase

Dissolve an appropriate amount of ammonium bicarbonate in water to form an internal water phase, and the concentration of ammonium bicarbonate is 2%-50%;

Preparation of VEGF micropowder

Disperse an appropriate amount of polyethylene glycol (PEG), VEGF and protective agent (the ratio is 4:1:2) in water, freeze-dry, wash with dichloromethane, centrifuge, remove PEG, obtain VEGF powder, protect Agent is selected from zinc carbonate, serum albumin, trehalose and mannitol etc.;

Preparation of microspheres

Add the VEGF micropowder and the inner water phase to the oil phase, homogenize, disperse, emulsify and mix to form colostrum, quickly drop the colostrum into 0.01-6% polyvinyl alcohol (PVA) aqueous solution, and fully homogenize with mechanical stirring (stirring speed is 100-1800rpm), continue to stir at low speed for 4 hours at room temperature (stirring speed is 100-300rpm), wash, collect, and freeze-dry.

The matrix material PLGA used in the present invention has a molecular weight of 3000-40000, a ratio of polylactic acid (PLA):glycolic acid (PGA) of 25:75-75:25, and a concentration of 30mg-300mg/ml. Homogenization conditions: 2000 ~ 10000rpm.

The microsphere scaffold in the present invention adopts the combination of the conventional preparation method of microspheres and the tissue engineering scaffold foaming method. The microsphere scaffold is composed of vascular endothelial cell growth factor, biodegradable matrix and additives, and is prepared into VEGF slow-release injection microspheres bracket, see Figure 1.

The VEGF sustained-release microspheres of the present invention have been released in vitro for more than 4 weeks, and the release conforms to an approximate zero-order model, and can be used for the treatment of local ischemic diseases.

Description of drawings

Figure 1 is a VEGF slow-release injection microsphere scaffold,

Fig. 2 is the in vitro cumulative release-time curve of the VEGF slow-release injection microspheres prepared by method 1,

Fig. 3 is the in vitro cumulative release-time curve of VEGF sustained-release injection microspheres prepared by method 2.

Detailed ways

Example 1: Preparation of VEGF slow-release microsphere scaffold by w/o/w solvent evaporation method

Dissolve 100 mg of PLGA (PLA:PGA=75:25, Mw=10,000) in 3.2 ml of dichloromethane to make an oil phase, dissolve 3 μg of VEGF and 100 mg of ammonium bicarbonate in 1 ml of redistilled water (containing 3% trehalose, 5 % mannitol) to form the inner water phase, which is added to the above-mentioned oil phase, homogenized to form w/o colostrum, 120ml of 0.1% PVA solution is placed in a stirring vessel, and the colostrum is stirred (450rpm) Quickly add it into the external water phase to fully homogenize. After five minutes, reduce the speed to 200rpm and add 30ml of distilled water to the external water phase. Stir at room temperature for 4 hours. After the microspheres are hardened, filter, wash, and freeze-dry. After sealing and dispensing, it can be irradiated and sterilized. The particle size is about 500 μm.

Embodiment 2: The w/o/w solvent volatilization method that medicine is added in the form of micropowder prepares VEGF slow-release microsphere stent

Disperse 1.2 mg of PEG (PEG6000), 300 μg of VEGF and protective agent (600 μg of zinc carbonate) in 1 ml of double-distilled water, vortex and mix for about 3 minutes, subpackage, freeze-dry, wash with dichloromethane, centrifuge, remove PEG, and obtain VEGF Micronized. Dissolve 100 mg of PLGA (PLA:PGA=75:25, Mw=10000) in 3.2 ml of dichloromethane to make an oil phase, and dissolve 100 mg of ammonium bicarbonate in 1 ml of redistilled water (containing 3% trehalose, 5% manna Alcohol) to form the inner water phase, add it to the above oil phase, homogenize for three minutes, add micronized medicine and homogenize at a low speed for one minute to form w/o colostrum, put 120ml of 0.1% PVA solution in In a stirring container, quickly add colostrum into the external water phase under stirring (450rpm) to fully homogenize. After five minutes, reduce the speed to 200rpm and add 30ml of distilled water to the external water phase, stir at room temperature for 4 hours, and the microspheres harden After that, it was filtered, washed, and freeze-dried. After sealing and dispensing, it can be irradiated and sterilized. The particle size is about 500 μm.

Embodiment 3: VEGF sustained-release microspheres are tested through in vitro release

Instruments: 0508-2 desktop low-speed centrifuge (Shanghai Medical Instrument Co., Ltd.); XW-80 vortex mixer (Shanghai First Medical College Instrument Factory); CARY 100 UV spectrophotometer (Varian, USA); CS501 super constant temperature Water bath (Shanghai Pudong Rongfeng Scientific Instrument Co., Ltd.), FA1004 1/10,000 electronic balance (Shanghai Balance Instrument Factory);

Method and operation: Accurately weigh about 20 mg of drug-containing microspheres or blank microspheres and place them in a 7ml centrifuge tube, add 1.5ml of 10mM pH 7.2 phosphate buffer (containing 0.02% sodium azide as a bacteriostatic agent, 0.02% F -68 as a wetting agent), placed in a constant temperature water bath shaker at 37°C, with an oscillation speed of 100rpm. Take out the centrifuge tubes on day 1 and day 3 respectively, place them in a centrifuge at 2000rpm for 5min, carefully suck out 2mL of the release medium and replace it with an equal amount of new release medium, then take samples every 3-4 days or more, and use blank microspheres The released supernatant was used as a blank control, and the sample was added according to the requirements of the BCA kit. After the reaction was developed, the absorbance was measured at 562nm, and it was substituted into the standard curve to calculate the content of BSA in the released solution.

(1) VEGF slow-release microsphere scaffold prepared by method 1

The results are shown in Figure 2. Experiments have shown that the release of protein drugs in microsphere scaffolds in vitro lasts for more than 4 weeks, and the release approximately conforms to the zero-order kinetic model.

(2) VEGF slow-release microsphere scaffold prepared by method 2

The results are shown in Figure 3. Experiments have shown that the release of protein drugs in microsphere scaffolds in vitro lasts for more than 4 weeks, and the release approximately conforms to the zero-order kinetic model.

The invention relates to the technical field of medicine and the technical field related to tissue engineering, and is an innovation in the preparation method of a polymer microsphere scaffold for tissue engineering and the application of the encapsulated protein drug. The novelty of the present invention lies in that it changes the traditional concept that the traditional stent needs to be surgically implanted in the body, and the application of the injection form reduces the inconvenience of the traditional stent requiring surgical implantation and increases the patient's compliance. Adding cell-growth-promoting growth factor protein drugs to the microsphere scaffold to make it slowly released after injection, so as to increase cell survival rate, promote cell growth, promote cell growth and reproduction, and form fresh tissue in tissue defect parts in the body ,. The method of the invention is simple, easy to operate and has good reproducibility. The release of protein drugs in microsphere scaffolds lasted more than 4 weeks in vitro, and the release approximated zero-order kinetics. The VEGF slow-release injection microsphere scaffold of the present invention can be applied to the repair and treatment of various tissue and blood vessel defects.

Claims (4)

1. VEGF slowly releasing injection microsphere support, its composition is substrate, VEGF, protective agent and ammonium bicarbonate, it is characterized in that described substrate is polylactic acid: hydroxyacetic acid=25: 75-75: 25 polylactic acid-glycolic guanidine-acetic acid copolymer; Described protective agent is selected from zinc carbonate, serum albumin, trehalose and mannitol; The part by weight of VEGF and polylactic acid-glycolic guanidine-acetic acid copolymer is 1: 100000 to 1: 10, and microspherulite diameter is 50 μ m to 1000 μ m when room temperature.

2. a kind of VEGF slowly releasing injection microsphere support according to claim 1 is characterized in that its preparation employing w/o/w solvent evaporation method, and concrete steps are as follows:

Substrate is dissolved in organic solvent makes oil phase, concentration is 30mg-300mg/ml; Get water in VEGF, protective agent and the formation soluble in water of foaming agent ammonium bicarbonate, wherein ammonium bicarbonate concentration is 2%-50%, and VEGF concentration is 1 μ g～1mg/ml, and protective agent content is 1～5%; Interior water is added above-mentioned oil phase, emulsifying forms colostrum, colostrum is dripped in the 0.01-6% polyvinyl alcohol water solution rapidly, mixing speed is the abundant homogenize of mechanical agitation under 100～1800rpm, mixing speed is that 100～300rpm stirred 4 hours under the room temperature, washing is collected, and lyophilization promptly gets the VEGF slowly releasing injection microsphere support through the preparation of W/O/W solvent evaporation method.

3. a kind of VEGF slowly releasing injection microsphere support according to claim 1 is characterized in that the w/o/w solvent evaporation method that its preparation adopts medicine to add with the micropowder form, and concrete steps are as follows:

Substrate is dissolved in organic solvent makes oil phase, concentration is 30mg-300mg/ml, gets water in the formation soluble in water of an amount of foaming agent ammonium bicarbonate, and ammonium bicarbonate concentration is 2%-50%; Polyethylene Glycol (PEG), VEGF and protective agent are scattered in the water in 4: 1: 2 ratios, after the lyophilization,, remove PEG, obtain the VEGF micropowder with washed with dichloromethane, centrifugal; Interior water is added oil phase, after water oil phase mixing homogenize a few minutes, add through lyophilization and handle the VEGF micropowder that obtains, homogenize dispersion and emulsion mixing forms colostrum, colostrum is dripped in 0.01-6% polyvinyl alcohol (PVA) aqueous solution rapidly, mixing speed is the abundant homogenize of 100～1800rpm mechanical agitation, mixing speed is that 100～300rpm stirred 4 hours under the room temperature, washing, collect, lyophilization promptly gets the VEGF slowly releasing injection microsphere support of medicine with the w/o/w solvent evaporation method preparation of micropowder form adding.

4. the application of the described a kind of VEGF slowly releasing injection microsphere support of claim 1 in the medicine for preparing damaged reparation of all kinds of tissue blood vessels and treatment.

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