CN100418537C - Amphotericin B slow-release microspheres and preparation method thereof - Google Patents

Abstract

The invention belongs to the technical field of nanomaterials and biomedicine, and in particular relates to an amphotericin B sustained-release microsphere and a preparation method thereof. The invention uses a degradable polymer as a drug carrier, uses amphotericin B as a drug load, and adopts a phase separation method to prepare a degradable slow-release microsphere. In the present invention, the oil phase is a degradable polymer and amphotericin B dissolved in acetone solution or a degradable polymer in a co-solvent; the water phase is one of distilled water, physiological saline or 5% glucose injection added with an emulsifier or Add emulsifier in distilled water, normal saline or 5% glucose injection to form a suspension formed by mixing amphotericin B, add the oil phase to the water phase, stir, naturally volatilize to remove acetone, and then dialyze it , remove the unencapsulated drug, and finally freeze-dry it into powder to obtain the required sustained-release microspheres. This product is biodegradable, with a smooth and round surface, good uniformity, regular particles, and a particle size of 120nm. Below, the encapsulation rate is 30-70%, and it has good sustained release performance.

Description

Amphotericin B slow-release microspheres and preparation method thereof

technical field

The invention belongs to the technical field of nanomaterials and biomedicine, and in particular relates to an amphotericin B sustained-release microsphere and a preparation method thereof.

Background technique

Amphotericin B (amphotericin B) is a macrocyclic polyene antibiotic that can selectively combine with ergosterol in the fungal cell membrane to form pores, thereby increasing the permeability of the membrane, leading to the leakage of important substances in the cell and causing death. Due to its high toxicity, intravenous administration can cause toxic side effects such as fever, chills, and rapid heart rate, and it is difficult to cross the blood-brain barrier.

A large number of data show that high encapsulation rate and small particle size are the necessary conditions to reduce toxicity and improve curative effect. The phase separation method is simple to operate, which is to disperse the drug to be encapsulated in the polymer solution or in the aqueous solution. The non-solvent method allows the polymer to coagulate and precipitate around the dispersed encapsulated particles to form microspheres. Although it consumes a large amount of organic solvent, it has good repeatability, large amount of drug coating and uniform particle size. At present, there are many studies on liposomes of amphotericin B at home and abroad, and some of them have been industrialized. Zhao Rongsheng from the School of Pharmacy of Peking University has prepared amphotericin B liposomes by thin film-ultrasonic method. (Zhao Rongsheng, Yan Baoxia, Hou Xinpu's development and quality evaluation of amphotericin B liposomes. Pharmaceutical Journal, 2000, 9 (35): 593-595) Rima Kassab etc. prepared PLA containing amphotericin B by solvent evaporation method, PLGA Microspheres, the particle size is 100-200μm. (Rima Kassab, Helene Parrot-Lopez, Molecular recognition by Kluyveromyces of amphotericin B-loaded, galactose-tagged, polylactic acid microspheres, Bioorganic and Medicinal Chemistry 10(2002) 1767ard-1775) (Gallis, H.A., Drew, Drew . W., mphotericin B: 30 years of clinical experience. v. Infect. Dis. 1990(12), 308-329)

In order to control the release rate of the drug, reduce the peak and valley of the blood drug level, and reduce the drug level in the whole body, biodegradable materials can be used as drug carriers. Considering that polylactic acid only depends on its molecular weight and molecular weight distribution to adjust its degradation rate, it has great limitations, and the lactone ring-opened homopolymer PLA is a hydrophobic substance, and its degradation cycle is also difficult to control. Only by changing the hydrophilicity, hydrophobicity and crystallinity of the material through copolymerization with other monomers can the degradation rate of the polymer be controlled according to the molecular weight of the copolymer and the type and ratio of the comonomer. Copolymers based on polylactic acid, especially copolymers of glycolic acid and lactic acid and copolymers of PLA and PEG, have excellent hemocompatibility and biocompatibility, hydrophilicity and softness, and have increasingly attracted people's attention. .

Contents of the invention

The object of the present invention is to propose a kind of amphotericin B slow release microsphere and preparation method thereof.

A kind of amphotericin B sustained-release microspheres proposed by the present invention, the medicine wrapped is amphotericin B, the carrier of the medicine is a biodegradable polymer, the particle diameter of the obtained slow-release microspheres is below 120nm, and the stable release is 80 hours or more.

In the present invention, the biodegradable polymer is polylactic acid, poly(lactic acid-ethylene glycol), poly(lactic acid-glycolic acid), poly(lactic acid-glycolic acid-ethylene glycol), polycaprolactone, Poly(lactic acid-caprolactone), one of polyhydroxyalkanoate and polyβ-hydroxybutyrate.

In the present invention, the weight average molecular weight of the biodegradable polymer is 5000-50000.

The preparation method of the amphotericin B sustained-release microspheres proposed by the present invention, the specific steps are as follows: the oil phase is a biodegradable polymer and the acetone solution in which amphotericin B is dissolved in a co-solvent, and the water phase is distilled water adding an emulsifier , normal saline or 5% glucose injection, the oil phase is added to the water phase, stirred, natural volatilization to remove acetone, and then dialyzed to remove unencapsulated drugs, and finally freeze-dried into powder, namely get the desired product;

The specific conditions are:

The co-solvent is one of dimethyl sulfoxide (DMSO) or methanol (MeOH), the concentration of amphotericin B in dimethyl sulfoxide is 0.2-20.0mg/ml, and the concentration of amphotericin B in methanol is 0.05-0.1mg/ml;

The concentration of the polymer in the acetone solution is 10-50mg/ml;

The concentration of amphotericin B in acetone solution is 0.1-10mg/ml;

The volume ratio of the oil phase to the water phase is 2:1-10:1;

The amount of the emulsifier added is 0.1-20 mg/ml of the water phase.

In the present invention, amphotericin B can be dissolved in the oil phase through a co-solvent, and can be suspended in the water phase, and the amphotericin B is suspended in the water phase. The specific steps of the preparation method of amphotericin B are as follows : the oil phase is the acetone solution of the biodegradable polymer, the water phase is the distilled water added with emulsifier, physiological saline or 5% glucose injection mixed with amphotericin B to form a suspension, the oil phase is added to In the water phase, stir, naturally volatilize to remove acetone, then dialyze it to remove unencapsulated drug, and finally freeze-dry it into powder to obtain the desired product;

The specific conditions are:

The concentration of the polymer in the acetone solution is 10-50mg/ml;

The concentration of amphotericin B in the aqueous phase is 0.01-0.5mg/ml;

The volume ratio of the oil phase to the water phase is 2:1-10:1;

The amount of the emulsifier added is 0.1-20 mg/ml of the water phase.

In the present invention, the amphotericin B is directly suspended in the water phase by mixing the amphotericin B with distilled water, physiological saline or 5% glucose injection, and ultrasonically vibrating for 15-30 minutes to form a uniform suspension.

In the present invention, the emulsifier is one of polyvinyl alcohol (PVA), Tween-80 or Span-80.

In the present invention, the molecular weight of polyvinyl alcohol (PVA) is 7000-14000.

Those skilled in the art can understand that in the process of adding the oil phase to the water phase, if the mixing of the oil phase and the water phase can be promoted by means such as stirring; in the process of adding the oil phase to the water phase, the oil phase can be quickly injected into the water phase Slowly drop into the water phase than the oil phase to make the two completely mixed.

The method of the present invention has the advantages of: the phase separation method is adopted, the preparation operation is simple, and the acetone/water is used as the oil/water two-phase system, stable drug sustained-release microspheres can be obtained without adhesion, the microspheres are smooth spherical, and the release is stable , The slow-release performance is adjustable, which can meet more application requirements. The particle size of the obtained sustained-release microspheres is below 120nm, the encapsulation rate is 20-60%, and can be released stably for more than 80 hours.

Detailed ways

Further detailed description will be given below in conjunction with the examples.

Example 1

The polymer adopts poly(lactic acid-ethylene glycol), wherein the molecular weight of polyethylene glycol is 8000, and the copolymerization ratio of poly(lactic acid-ethylene glycol) copolymer is 5/1, and the feeding amount is 0.4076g, 3mg amphotericin B Dissolve in 1ml of DMSO, put the two into 20ml of acetone to form an oil phase, add 0.12g of Tween-80 to 20ml of distilled water to form a water phase, stir at 1000 rpm, drop the oil phase into the water phase, and remove the acetone by natural volatilization. Then it is dialyzed to remove the unencapsulated drug, and finally it is freeze-dried into powder. The particle size of the obtained microsphere is 118±18nm, the encapsulation rate of amphotericin B is 42.7%, and it can be released stably within 75 hours.

Example 2

The polymer is polylactic acid PLA, the molecular weight is 5000, the dosage is 0.4352g, 10mg amphotericin B is dissolved in 1mlDMSO, the two are put into 20ml acetone to form an oil phase, 0.3g Tween-80 is added to 20ml distilled water to form water Phase, under stirring at 1000 rpm, the oil phase is dropped into the water phase, the acetone is naturally volatilized to remove the acetone, and then it is dialyzed to remove the unencapsulated drug, and finally it is freeze-dried into powder, and the particle size of the obtained microspheres is 97± 12nm, the encapsulation efficiency is 30.6%. Sustainable and stable release within 53 hours.

Example 3

The polymer is polycaprolactone, the molecular weight is 8000, the dosage is 0.8032g, 20mg amphotericin B is dissolved in 20mlMeOH, the two are put into 20ml acetone to form an oil phase, 1g Tween 80 is added to 20ml distilled water to form a water phase, Under the stirring state of 1000 rpm, the oil phase is dripped into the water phase, and the acetone is naturally volatilized to remove the acetone, and then it is dialyzed to remove the unencapsulated drug, and finally it is freeze-dried into powder, and the particle size of the obtained microspheres is 110±23nm. The encapsulation efficiency was 45.7%.

Example 4

The polymer adopts poly-β-hydroxybutyrate, the molecular weight of the polymer is 8000, 20 mg amphotericin B is dissolved in 10 ml MeOH, other conditions are the same as in Example 3, the obtained microsphere particle size is 99 ± 18 nm, and the encapsulation efficiency is 36.5% .

Example 5

Without adding co-solvent, put amphotericin B into the water phase, put 4.1mg amphotericin B and 0.6g Span 80 into 20ml distilled water to form a suspension, other conditions are the same as in Example 1, and the obtained microspheres The diameter is 101±16nm, the encapsulation rate of amphotericin B is 57.6%, and the stable release lasts for more than 80 hours.

Example 6

Put amphotericin B into the water phase without adding a co-solvent, and use poly(lactic acid-ethylene glycol) as the polymer, wherein the molecular weight of polyethylene glycol is 4000, and the copolymerization ratio of poly(lactic acid-ethylene glycol) copolymer is 5/1, the feeding amount is 0.4021g, other conditions are the same as in Example 2, the obtained microsphere particle diameter is 82.5±17nm, and the amphotericin B encapsulation efficiency is 61.3%.

Example 7

Put amphotericin B into the water phase without adding a co-solvent, and use poly(lactic acid-ethylene glycol) as the polymer, wherein the molecular weight of polyethylene glycol is 4000, and the copolymerization ratio of poly(lactic acid-ethylene glycol) copolymer is 2/1, the feeding amount is 0.4021g, other conditions are the same as in Example 3, the particle diameter of the obtained microspheres is 54.7±8nm, and the amphotericin B encapsulation efficiency is 53.5%.

Example 8

Without adding co-solvent, amphotericin B is put into the water phase, the polymer adopts poly(lactic acid-ethylene glycol), the molecular weight of polyethylene glycol is 6000, and the copolymerization ratio of poly(lactic acid-ethylene glycol) copolymer is 5/1, the feeding amount is 0.4034g, add 1% PVA aqueous solution, other conditions are the same as embodiment 5, the obtained microsphere particle size is 94.6 ± 28nm, the encapsulation efficiency of amphotericin B is 45.6%.

Example 9

Polymer adopts poly(lactic acid-glycolic acid), the concentration of amphotericin B in aqueous solution is 0.01mg/ml, and other conditions are with example 5, and gained microsphere particle diameter is 98.3 ± 21nm, and amphotericin B encapsulation efficiency is 23.1 %.

Example 10

Polymer adopts poly(lactic acid-caprolactone), and amphotericin B concentration in aqueous solution is 0.5mg/ml, and other conditions are with example 5, and gained microsphere particle diameter is 101.5 ± 15nm, and amphotericin B encapsulation efficiency 40.8%.

Claims (8)

1. A slow-release microsphere of amphotericin B, characterized in that: the medicine wrapped is amphotericin B, the carrier of the medicine is a biodegradable polymer, and the particle diameter of the slow-release microsphere obtained is below 120nm, stable Release for more than 80 hours; the oil phase is an acetone solution in which a biodegradable polymer and amphotericin B are dissolved in a co-solvent, and the water phase is one of distilled water with emulsifier added, normal saline or 5% glucose injection, Or the oil phase is the acetone solution of the biodegradable polymer, and the water phase is the suspension that is mixed with amphotericin B in one of emulsifying agent distilled water, physiological saline or 5% glucose injection; then the oil phase Add it into the water phase, stir, remove the acetone by natural volatilization, then dialyze it to remove the unencapsulated drug, and finally freeze-dry it into powder to obtain the required amphotericin B sustained-release microspheres; wherein, the The co-solvent is one of dimethyl sulfoxide or methanol. 2. amphotericin B sustained-release microspheres according to claim 1, is characterized in that described biodegradable polymer is polylactic acid, poly(lactic acid-ethylene glycol), poly(lactic acid-glycolic acid), One of poly(lactic acid-glycolic acid-ethylene glycol), polycaprolactone, poly(lactic acid-caprolactone), polyhydroxyalkanoate, and polyβ-hydroxybutyrate. 3. The amphotericin B sustained-release microspheres according to claim 1 and 2, characterized in that the weight average molecular weight of the biodegradable polymer is 5000-50000. 4. a preparation method of amphotericin B slow-release microspheres as claimed in claim 1, is characterized in that concrete steps are as follows: oily phase is the acetone that biodegradable polymer and amphotericin B are dissolved in co-solvent Solution, the water phase is one of distilled water, normal saline or 5% glucose injection with emulsifier added, the oil phase is added to the water phase, stirred, acetone is naturally volatilized, and then dialyzed to remove unencapsulated drugs , and finally freeze-dry it into powder to obtain the desired product; The specific conditions are: The co-solvent is one of dimethylsulfoxide or methanol, the concentration of amphotericin B in dimethylsulfoxide is 0.2-20.0mg/ml, and the concentration of amphotericin B in methanol is 0.05-0.1mg/ml ml; The concentration of the polymer in the acetone solution is 10-50mg/ml; The concentration of amphotericin B in acetone solution is 0.1-10mg/ml; The volume ratio of the oil phase to the water phase is 2:1-10:1; The amount of the emulsifier added is 0.1-20 mg/ml of the water phase. 5. a preparation method of amphotericin B slow-release microspheres as claimed in claim 1, is characterized in that concrete steps are as follows: oil phase is the acetone solution of biodegradable polymer, and water phase is the acetone solution that adds emulsifier Mix distilled water, normal saline or 5% glucose injection into a suspension formed by amphotericin B, add the oil phase to the water phase, stir, and naturally volatilize to remove acetone, then dialyze it to remove uncoated Seal the drug, and finally freeze-dry it into powder to obtain the desired product; The specific conditions are: The concentration of the polymer in the acetone solution is 10-50mg/ml; The concentration of amphotericin B in the aqueous phase is 0.01-0.5mg/ml; The volume ratio of the oil phase to the water phase is 2:1-10:1; The amount of the emulsifier added is 0.1-20 mg/ml of the water phase. 6. the preparation method of amphotericin B sustained-release microspheres according to claim 5 is characterized in that amphotericin B is directly suspended in aqueous phase by amphotericin B and distilled water, normal saline or 5 One of the % glucose injections was mixed and ultrasonically oscillated for 15-30 minutes to form a uniform suspension. 7. according to the preparation method of the described amphotericin B sustained release microsphere of claim 4 or 5, it is characterized in that emulsifier is one of polyvinyl alcohol, Tween 80 or Span 80. 8. the preparation method of amphotericin B sustained-release microspheres according to claim 7, is characterized in that polyvinyl alcohol molecular weight is 7000-14000.