CN102188391B - Method for preparing granulocyte-macrophage colony stimulating factor microsphere - Google Patents

Abstract

A method for preparing granulocyte-macrophage colony-stimulating factor microspheres in the field of nanomedicine technology, by dispersing granulocyte-macrophage colony-stimulating factor dextran particles in an organic solution with slow-release or controlled-release functional materials Then add the suspension to the oil phase (O) to form microspheres by stirring or swirling, and transfer the microspheres to the glycerin-modified water phase (W) to form a double emulsion, and finally the double emulsion Granulocyte-macrophage colony-stimulating factor microspheres were obtained after solidification treatment. The microspheres prepared by the invention are regular and without adhesion; the encapsulation rate is high, the burst release is small, and the drug loading capacity is high.

Description

Method for preparing granulocyte-macrophage colony-stimulating factor microspheres

technical field

The invention relates to a method in the technical field of nanometer medicine, in particular to a method for preparing granulocyte-macrophage colony-stimulating factor (GM-CSF) microspheres by a glycerol method.

Background technique

In the pharmaceutical industry, from drug discovery to clinical application, the last link is drug preparation. Some of the drugs need long-term administration to be cured; others need targeted and other local administration. To achieve these goals, raw materials must be prepared into corresponding dosage forms. For example, drugs that require long-term administration but have a short half-life in the body should be prepared as sustained-release dosage forms; for the treatment of some tumors, some drugs need to be targeted to the disease, such as embolization microsphere preparations that target tumor blood vessels; Since recombinant technology has been used in the expression and production of therapeutic proteins for more than 20 years, so far, more than 30 protein drug products have been put into clinical use, and nearly 200 are in the process of approval and development. ), Gene Technology (Genentech) and a number of new large pharmaceutical companies. Compared with the rapid development of protein macromolecular drugs, their dosage form technology progresses slowly. On the one hand, protein macromolecular drugs are not absorbed orally and have a short half-life in the body, so they need to be administered by injection; on the other hand, many hormone and cytokine protein drugs have a long treatment cycle, and long-term and frequent injections are necessary, which also affects patients. The main reason for compliance. The research and development of dosage forms of sustained-release protein drugs, such as the W/O/W method, the S/O/W method with low encapsulation efficiency, and the S/O/O method that is easy to release due to the loss of activity during the preparation of microparticles, etc. . It is imperative to develop a method for preparing protein microspheres with active protection and improving encapsulation efficiency and burst release. So far, there is no report on the preparation of granulocyte-macrophage colony-stimulating factor microspheres by the glycerol method.

After searching the prior art documents, [Lee E.S., Kwon M.J., Lee H., and Kim J.J., Stabilization of protein encapsulated in poly(lactide-co-glycolide) microspheres by novelviscous S/W/O/W method, International Journal of Pharmaceutics 331(2007) 27-37l, (LeeE.S., Kwon M.J., LeeH., and KimJ.J., reported the stable encapsulation of protein in PLGA microspheres, International Journal of Pharmaceutical Sciences, 2007, 331: 27-37). Lee E.S. et al. reported in the literature that microspheres were prepared using the S/W/O/W method. This document utilizes protein cyclodextrin to be freeze-dried and then ground, and the ground protein cyclodextrin is added to a high-viscosity polysaccharide solution and a dichloromethane solution of PLGA to emulsify, and finally to the water phase to harden the microspheres and only target proteins Drugs, no other drugs have been reported. However, protein cyclodextrin particles are inevitably insoluble in contact with polysaccharide aqueous solution, and the dissolved protein aqueous solution is easy to contact with PLGA dichloromethane, and there is an oil-water interface, which leads to aggregation. Also cause encapsulation efficiency is not high, there is incomplete release. [Morita T., Sakamura Y., Horikiri Y., Suzuki T., Yoshino H., Protein encapsulation into biodegradable microspheres by a novel S/O/W emulsion method using poly(ethylene glycol) as a protein micronization adjuvant of enzyme Control, 69 (2000) 435-444] (Morita T. Sakamura Y., Horikiri Y., Suzuki T., Yoshino H., Preparation of protein-loaded microspheres by S/O/W emulsification method using PEG as an adjuvant for protein micronization, "Journal of Controlled Release", 2000, 69: 435-444) Morita T et al reported in this document that protein-loaded microspheres were prepared using a new S/O/W emulsification method. It is just that the surfactant has been changed. The previous report used more PVA, but in this document, PEG was used instead. However, the low encapsulation efficiency and the shortcoming of burst release and incomplete release still cannot be overcome. So far, there has been no report on the preparation of granulocyte-macrophage colony-stimulating factor microspheres by the glycerol method.

Contents of the invention

The present invention aims at the above-mentioned deficiencies in the prior art, and provides a method for preparing granulocyte-macrophage colony-stimulating factor microspheres. The prepared microspheres are regular and non-adhesive; the encapsulation rate is high, the burst release is small, and the drug loading capacity is high. high.

The present invention is achieved through the following technical solutions. The present invention forms a suspension by dispersing granulocyte-macrophage colony-stimulating factor dextran particles in an organic solution with slow-release or controlled-release functional materials, and then The suspension is added to the oil phase (O) and stirred or vortexed to form microspheres, and the microspheres are transferred to the glycerol-modified water phase (W) to form a double emulsion, and finally the double emulsion is solidified to obtain granulocyte-macrophage Colony-stimulating factor microspheres.

The granulocyte-macrophage colony-stimulating factor dextran particle is composed of 1-50 wt% of granulocyte-macrophage colony-stimulating factor and 1-50 wt% of dextran, and its particle size is 0.1-10 μm; The granulocyte-macrophage colony-stimulating factor dextran particles are formed by water-water emulsion method, low-temperature spray drying method, phase separation method, supercritical method or metal ion and granulocyte-macrophage colony-stimulating factor. Composite method was prepared.

The oil phase (O) refers to: an organic solution containing a slow-release or controlled-release material, the oil phase (O) is prepared by dissolving the controlled-release or slow-release functional material in an organic solvent, and its weight percent concentration is 1 -30% (w/w).

The described functional material with sustained release or controlled release adopts polylactic acid (PLA), polylactic acid-polyglycolic acid (PLGA), a combination of PLA and PLGA, polyethylene glycol-polylactic acid (PLA-PEG), polyethylene Glycol-polyglycolic acid (PLGA-PEG), polyglycolic acid-polyethylene glycol-polyglycolic acid (PLGA-PEG-PLGA), polylactic acid-polyethylene glycol-polylactic acid (PLA-PEG-PLA), Combinations of polylactic acid and polylactic acid-polyglycolic acid, polyethylene glycol-polycaprolactone (PEG-PCL), polycaprolactone poly-ethylene glycol-polycaprolactone (PCL-PEG-PCL) or poly Caprolactone (PCL);

Described organic solution adopts dichloromethane, ethyl acetate, acetonitrile or acetone organic solution.

The weight percent concentration of the organic solution of the functional material with sustained release or controlled release is: 1-30% (w/w).

The glycerol-modified aqueous phase (W) refers to: a combination containing surfactant, glycerin, water and NaCl, by adding a weight percent concentration of 0.5-10% (w/w) surfactant, 1.5-100 % (w/w) glycerin, 0-10% (w/w) NaCl and 0-98% (wt) water are mixed.

Described surfactant adopts polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), poloxamer (poloxmer), Tween, Polysorbate or ethyl cellulose;

The stirring refers to mechanical stirring for 0.1-5 minutes to form microspheres of 10-500 μm;

The solidification treatment refers to: add double emulsion dropwise to 1000mL sodium chloride solution containing 1-10% (w/w), and stir for 1-4 hours, collect microspheres by centrifugation and wash 3-5 times, then freeze Dry microspheres.

The invention relates to the microsphere prepared by the above method, the composition and mass percentage of which are: dextran particle 1-20%, functional material 80-99%, and the particle diameter of the microsphere is 1-500μm.

The invention has the advantages of: overcoming the shortcomings of low encapsulation rate and serious burst release of granulocyte-macrophage colony-stimulating factor with good water solubility; The interface, high shear force, interface, and cross-linking agent, etc., can maintain the activity of biologically active substances for a long time under mild conditions. The storage cost is greatly reduced and the curative effect is improved, and the encapsulation rate is higher than 40% and the activity is higher than that of the control group, and the release curve in vitro is better than that of the control group.

The present invention selects suitable oil phase (O) and glycerin-modified water phase (W) and suitable controlled-release or slow-release materials, so that water-soluble drug particles or oil-soluble drugs are prepared into oil-insoluble Particles, avoiding the disadvantages of conventional W/O, W/O/W, S/O/W with low encapsulation efficiency, severe burst release of S/O/O, and environmental pollution; adopt this method Prepare microspheres, whose particle size can be controlled according to different needs, without polluting the environment; it can avoid the effect on the treatment of drugs, especially those drugs with unstable physical and chemical properties and sensitive to oil-water interface, such as particles Cell-macrophage colony-stimulating factor drug. The surface of the microparticles is smooth and round, the particles are regular and non-adhesive, and the particle size can be adjusted from 1 μm to 500 μm according to the needs. The lyophilized powder is white and fine, loose, does not collapse, does not adhere, and has good redispersibility. It can be applied to the preparation of various drug sustained-release microspheres and vaccine adjuvants. The biological cell activity of the prepared granulocyte-macrophage colony-stimulating factor sustained-release microspheres is 20% higher than that of the control group; the release in vitro is also better than that of the control group.

Description of drawings

Fig. 1 is microsphere micrograph prepared by glycerol method;

In the figure: (A) m5-S/O/W: 100% (w/w) glycerol (B) m4-S/O/W: 80% (w/w) glycerol, (C) m3-S/O /W: 60% (w/w) glycerol, (D)m2-S/O/W: 40% (w/w) glycerol, (E)m1-S/O/W: 20% (w/w) Glycerol, (F) W/O/W control.

Fig. 2 is the scanning electron micrograph of microsphere prepared by glycerol method;

In the figure: (A) m5-S/O/W: 100% (w/w) glycerol (B) m4-S/O/W: 80% (w/w) glycerol, (C) m3-S/O /W: 60% (w/w) glycerol, (D)m2-S/O/W: 40% (w/w) glycerol, (E)m1-S/O/W: 20% (w/w) Glycerol, (F) W/O/W control.

Fig. 3 is a schematic diagram of in vitro release curve;

In the figure: ◇: m4-S/O/W: 80% (w/w) glycerol, ▲: m3-S/O/W: 60% (w/w) glycerol; ●: m2-S/O/W : 40% (w/w) glycerol; Δ: m1-S/O/W: 20% (w/w) glycerol; W/O/W control group.

Detailed ways

The embodiments of the present invention are described in detail below. This embodiment is implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operating procedures are provided, but the protection scope of the present invention is not limited to the following implementation example.

Example 1: A method for preparing granulocyte-macrophage colony-stimulating factor (GM-CSF) microspheres by the glycerol method

(1) Take 10 mg of granulocyte-macrophage colony-stimulating factor dextran particles of 5 μm and a dichloromethane solution of polylactic acid-glycolic acid (50:50, PLGA) with a molecular weight of 47,000 at a concentration of 20% by weight Stir, vortex or sonicate 1mL for 1-5 minutes to form a uniform suspension, that is, solid-in-oil (S/O) emulsion;

(2) Add the emulsion obtained in step (1) dropwise to the glycerin-modified aqueous phase (W) (W) containing 20% by weight and stir, vortex or sonicate for 0.1-5 minutes to form double emulsion;

(3) The double emulsion of step (2) is added dropwise in the 1000mL sodium chloride solution that the weight percent concentration is 5% (w/w) and stirs 2-4 hour;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

(5) Then store it in an environment with room temperature and humidity of 30% for two years, take it out and measure its biological activity with cells;

The above-mentioned microspheres passed through a microscope (as shown in Figure 1A) and a scanning electron microscope (as shown in Figure 2A) showed that the particle size was about 40 μm-80 μm; the encapsulation rate was 90%, while the control group was only 50% and the cell activity was 49% higher than that of the control group % is only 11% less active than the bulk drug used for preparation (newly purchased bulk drug). The in vitro release profile is shown in Figure 3, and the burst release was lower than that of the control group.

Embodiment 2: A kind of method for preparing granulocyte-macrophage colony-stimulating factor (GM-CSF) microspheres by glycerol method

(1) Take 50 mg of 10 μm granulocyte-macrophage colony-stimulating factor dextran particles and 1 mL of a dichloromethane solution of 20% polylactic acid-glycolic acid (50:50, PLGA) with a molecular weight of 47,000 and stir , Vortex or ultrasonic for 1-5 minutes to form a uniform suspension, that is, solid-in-oil (S/O) emulsion;

(2) Add the emulsion obtained in step (1) dropwise to the glycerin-modified aqueous phase (W) (W) containing a concentration of 40% by weight and stir, vortex or sonicate for 0.1-5 minutes to form a double emulsion;

(3) The double emulsion of step (2) is added dropwise in the 1000mL sodium chloride solution that the weight percent concentration is 10% (w/w) and stirs 2-4 hour;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

(5) Then store it in an environment with room temperature and humidity of 30% for two years, take it out and measure its biological activity with cells;

The above-mentioned microspheres are passed through a microscope (as shown in Figure 1B) and a scanning electron microscope (as shown in Figure 1B) to show that the particle size is about 40 μm-80 μm; the encapsulation rate is 90%, while the control group is only 50% and the cell activity is 48.7% higher than that of the control group % is only 11% less active than the bulk drug used for preparation (newly purchased bulk drug). The in vitro release profile is shown in Figure 3, and the burst release was lower than that of the control group.

Embodiment three: a kind of method for preparing granulocyte-macrophage colony-stimulating factor (GM-CSF) microspheres by glycerol method

(1) Take 5 mg of granulocyte-macrophage colony-stimulating factor dextran particles of 5 μm and 1 mL of a dichloromethane solution of polylactic acid (PLA) with a molecular weight of 83,000 at a concentration of 20% and stir, vortex or sonicate for 1-5 A uniform suspension is formed within minutes, that is, a solid-in-oil (S/O) emulsion;

(2) Add the emulsion obtained in step (1) dropwise to the glycerin-modified aqueous phase (W) (W) containing 60% by weight and stir, vortex or sonicate for 0.1-5 minutes to form double emulsion;

(3) The double emulsion of step (2) is added dropwise in the 1000mL sodium chloride solution that the weight percent concentration is 1% (w/w) and stirs 2-4 hour;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

(5) Then store it in an environment with room temperature and humidity of 30% for two years, take it out and measure its biological activity with cells;

The above-mentioned microspheres were passed through a microscope (as shown in Figure 1C) and a scanning electron microscope (as shown in Figure 1C) to show that the particle size was about 40 μm-80 μm; the encapsulation rate was 91%, while the control group was only 50% and the cell activity was 49% higher than that of the control group % is only 11% less active than the bulk drug used for preparation (newly purchased bulk drug). The in vitro release profile is shown in Figure 3, and the burst release was lower than that of the control group.

Embodiment four: A kind of method for preparing granulocyte-macrophage colony-stimulating factor (GM-CSF) microspheres by glycerol method

(1) Take 5 mg of granulocyte-macrophage colony-stimulating factor dextran particles of 1 μm and 1 mL of a dichloromethane solution of 20% polylactic acid (PLA) with a molecular weight of 83,000, stir, vortex or sonicate for 1-5 A uniform suspension is formed within minutes, that is, a solid-in-oil (S/O) emulsion;

(2) Add the emulsion obtained in step (1) dropwise to the glycerin-modified aqueous phase (W) (W) containing 80% by weight and stir, vortex or sonicate for 0.1-5 minutes to form double emulsion;

(3) The double emulsion of step (2) is added dropwise in the 1000mL sodium chloride solution that percentage concentration is 5% (w/w) and stirs 2-4 hour;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

(5) Then put it in an environment with room temperature and humidity of 30% for two years, take it out and measure its biological activity with cells;

The above-mentioned microspheres passed through a microscope (as shown in Figure 1D) and a scanning electron microscope (as shown in Figure 1D) showed that the particle size was about 40 μm-80 μm; the encapsulation rate was 91%, while the control group was only 50% and the cell activity was 49% higher than that of the control group % is only 11% less active than the bulk drug used for preparation (newly purchased bulk drug). The in vitro release profile is shown in Figure 3, and the burst release was lower than that of the control group.

Embodiment five: A kind of method for preparing granulocyte-macrophage colony-stimulating factor (GM-CSF) microspheres by glycerol method

(1) Take 5 mg of granulocyte-macrophage colony-stimulating factor dextran particles of 1 μm and 1 mL of a dichloromethane solution of 20% polylactic acid (PLA) with a molecular weight of 83,000, stir, vortex or sonicate for 1-5 A uniform suspension is formed within minutes, that is, a solid-in-oil (S/O) emulsion;

(2) Add the emulsion obtained in step (1) dropwise to the glycerin-modified aqueous phase (W) (W) containing 100% by weight and stir, vortex or sonicate for 0.1-5 minutes to form double emulsion;

(3) The double emulsion of step (2) is added dropwise in the 1000mL sodium chloride solution that the weight percent concentration is 8% (w/w) and stirs 2-4 hour;

(4) Collect the microspheres obtained in step (3) by centrifugation, wash with water for 3-5 times, and freeze-dry to obtain the microspheres.

(5) Then store it in an environment with room temperature and humidity of 30% for two years, take it out and measure its biological activity with cells;

The above-mentioned microspheres are passed through a microscope (as shown in Figure 1E) and a scanning electron microscope (as shown in Figure 1E) to show that the particle size is about 20 μm-80 μm; the encapsulation rate is 92%, while the control group is only 50% and the cell activity is 50% higher than that of the control group % is only 10% less active than the bulk drug used for preparation (newly purchased bulk drug). The in vitro release profile is shown in Figure 3, and the burst release was lower than that of the control group.

Claims (4)

1. A granulocyte-macrophage colony-stimulating factor microsphere, characterized in that its components and mass percentages are: granulocyte-macrophage colony-stimulating factor dextran particles 1-20%, slow-release or controlled 80-99% of the released functional material, the particle size of the microsphere is 1-500μm; By dispersing granulocyte-macrophage colony-stimulating factor dextran particles in an organic solution with slow-release or controlled-release functional materials to form a suspension, that is, a solid emulsion in oil, and transfer the emulsion to a glycerol-modified A double emulsion is formed in the water phase (W), and finally the double emulsion is solidified to obtain the granulocyte-macrophage colony-stimulating factor microspheres; The granulocyte-macrophage colony-stimulating factor dextran particle is composed of 1-50wt% granulocyte-macrophage colony-stimulating factor and 1-50wt% dextran, and its particle size is 0.1-10 μm. The granulocyte-macrophage colony-stimulating factor dextran particles are formed by water-water emulsion method, low-temperature spray drying method, phase separation method, supercritical method or metal ion and granulocyte-macrophage colony-stimulating factor. Composite method is prepared; The functional material for sustained release or controlled release uses polylactic acid, polylactic acid-polyglycolic acid, a combination of PLA and PLGA, polyethylene glycol-polylactic acid, polyethylene glycol-polyglycolic acid, polyglycolic acid-poly Ethylene glycol-polyglycolic acid, polylactic acid-polyethylene glycol-polylactic acid, combinations of polylactic acid and polylactic acid-polyglycolic acid, polyethylene glycol-polycaprolactone, polycaprolactone-polyethylene glycol - polycaprolactone or polycaprolactone; The glycerol-modified aqueous phase (W) refers to: a combination containing surfactant, glycerin, water and NaCl, by adding a weight percent concentration of 0.5-10% (w/w) surfactant, 1.5-100 % (w/w) glycerin, 0-10% (w/w) NaCl and 0-98% (wt) water are mixed. 2. The granulocyte-macrophage colony-stimulating factor microsphere according to claim 1, characterized in that, the organic solution with slow-release or controlled-release functional material is obtained by dissolving the slow-release or controlled-release functional material. It is prepared in an organic solvent, and its weight percent concentration is 1-30% (w/w). 3. the granulocyte-macrophage colony-stimulating factor microsphere according to claim 1, is characterized in that, described tensio-active agent adopts polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, porolidone Sham, Tween, polysorbate, or ethylcellulose. 4. The granulocyte-macrophage colony-stimulating factor microsphere according to claim 1, characterized in that, the solidification treatment refers to: the double emulsion is added dropwise to 1000mL containing 1-10% (w/w) sodium chloride solution, and stirred for 1-4 hours, centrifuged to collect microspheres and washed 3-5 times, and then freeze-dried to obtain microspheres.

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