CN101040849A - Nanometer micro-capsules having the same size for carrying medicine and the method of preparing the same - Google Patents

Abstract

The invention relates to a nano drug-loaded microcapsule product with uniform size and a preparation method thereof. A degradable polymer is used to embed a hydrophilic drug, and the drug embedding rate is high and the activity is maintained well. controllable; the method is carried out through the following steps: (1) dissolving the degradable polymer in at least one organic solvent; (2) adding the aqueous drug solution to the organic solution obtained in step (1), and homogenizing Emulsification or ultrasonic dispersion to form W ₁ /O emulsion; (3) Pour the emulsion obtained in step (2) into double emulsion, and form W ₁ /O/W ₂ pre-double emulsion through homogeneous emulsification or mechanical stirring; (4) The pre-double emulsion obtained in step (3) is obtained by pressure through a microporous membrane to obtain a uniform double emulsion; (5) the organic solvent in the double emulsion obtained in step (4) is removed; (6) centrifugal washing step (5 ) obtained solidified solution, and collect nano drug-loaded microcapsules with uniform size; the preparation method has simple process, mild operating conditions, good repeatability, easy amplification, and short time-consuming.

Description

Nano drug-loaded microcapsule product with uniform size and preparation method thereof

technical field

The invention relates to a preparation method of nanometer microcapsules in the fields of medicine, biochemical industry and the like, in particular to a preparation method of nanometer drug-loaded microcapsules with uniform size.

technical background

In the late 1970s, Narty and others first proposed the concept of nano-microcapsules. They found that nano-microcapsules have many unique properties, which made them widely used in many fields. For example: nano-microcapsules as drug carriers have good targeting and sustained release. When drug particles exist in the form of nanocapsules, injecting them into veins will not cause capillary blockage, because the smallest capillary in the human body is 4 μm in diameter, and nanocapsules can easily pass through. The drug nano-microcapsule can be used as a slow-release drug preparation for injection. When injected into a specific part of the human body, the drug can be concentrated in this part to exert its drug effect, and it has the advantages of protecting the drug, reducing the release concentration of the drug, and being easy to control. People's research has also found that the carcinogenic effect of certain substances is not only determined by the nature of the substance itself, but also related to the particle size of these substances when they enter the human body or animal body. Generally, the smaller the particle, the smaller the carcinogenic effect, so the use of drug nanocapsules can reduce the adverse side effects of the drug on the human body. Due to the above-mentioned advantages of nano drug-loaded microcapsules, more and more scientific researchers are devoting themselves to studying the preparation methods of nano microcapsules.

At present, the most commonly used preparation method of nanocapsules for embedding hydrophilic drugs is the double emulsion-solidification method, but the traditional double emulsion-solidification method has long used mechanical stirring, homogeneous emulsification or ultrasonic and other vigorous methods for the double emulsion. Preparation, such as: the preparation method in the patent "polymer nanoparticles and drug capsules and preparation" (publication number CN 1733310), this method greatly increases the possibility of drug inactivation, and the energy consumption is too high. At the same time, because the mechanical emulsification method is not easy to quantitatively control, the prepared carrier microcapsules often have uneven particle size, low drug embedding rate, and poor amplification repeatability. The inhomogeneous particle size of the drug carrier will also lead to the existence of the following problems: (1) Due to the different absorption sites and absorption mechanisms in the body of the drug carrier with different particle sizes, if the particle size is not uniform, the particle size and the absorption mechanism of the drug carrier cannot be adjusted. (2) When conducting in vivo and in vitro pharmacological experiments, since the particle size of the drug carrier directly affects the release rate of the encapsulated drug, if the particle size of the drug carrier is not uniform, it will This leads to poor repeatability of drug release, which directly affects the sustained and controlled release effect of the drug carrier.

In addition, the preparation methods of nano drug-loaded microcapsules also include emulsion polymerization, polymer precipitation and emulsification diffusion. In the above three methods, hemoglobin nanocapsules with a particle size of 0.05 μm-1 μm are prepared by using polyisobutylacrylocyanate or polylactic acid or polylactic acid-polyethylene glycol copolymer as a membrane component to embed hemoglobin. However, all three methods use a large amount of organic solvents, which can easily denature the drug. Although the particle size of the microcapsules formed by the emulsification diffusion method is small, about 0.1 μm, it cannot effectively embed hemoglobin (the embedment rate is too low, and the embedment rate has not been reported). Generally speaking, these three methods have disadvantages such as drug variability, low embedding rate, and the preparation process requires a large amount of organic solvents, which is not easy to scale up.

In 1996, Suzuki K. proposed rapid membrane emulsification technology (pre-mix membrane emulsification) (Suzuki, K., Shuto, I., Hagura, Y., Food Sci.Technol.Int.Tokyo, 2 (1), 43- 47, 1996). Although this method has the advantages of uniform emulsion droplet size, mild operating conditions, short time consumption, and easy industrial scale-up, at present, only emulsions can be prepared using this technology, which is difficult to store stably for a long time and is not suitable for embedding. And store drugs such as proteins that are easily inactivated, so it is difficult to apply them in real life.

In view of the above problems, the present invention improves the rapid membrane emulsification technology, so as to prepare solid nano drug-loaded microcapsules with uniform size.

Contents of the invention

The purpose of the present invention is to overcome the many shortcomings of the above-mentioned nano drug-loaded microcapsules, and provide a nano drug-loaded microcapsule product with uniform size;

Another object of the present invention is to provide a method for preparing the nano drug-loaded microcapsule with uniform size.

Embodiments of the present invention are as follows:

The nano drug-loaded microcapsule product with uniform size provided by the present invention is characterized in that the size of the nano drug-loaded microcapsule is uniform and controllable, and the diameter distribution coefficient (CV value) is in the range of 1%-20%; The average particle size range of drug-loaded microcapsules is 50-1000nm; the drugs are hydrophilic drugs, including proteins, polypeptides, enzymes, vaccines, genes, hormones, antibiotics, anticancer agents, Chinese herbal medicines and Its mixture; the entrapment rate of the drug reaches above 80%, and reaches above 90% under optimized conditions.

The method for preparing nanometer drug-loaded microcapsules with uniform size provided by the invention is characterized in that the steps are as follows:

(1) dissolving the degradable polymer in at least one organic solvent;

(2) adding the aqueous drug solution to the organic solution obtained in step (1), and forming a W ₁ /O emulsion through homogeneous emulsification or ultrasonic dispersion;

(3) Pour the emulsion obtained in step (2) into double emulsion W ₂ , and form W ₁ /O/W ₂ pre-double emulsion through homogeneous emulsification or mechanical stirring;

(4) the obtained pre-multi-emulsion of step (3) is obtained by pressure through the microporous membrane to obtain the multi-emulsion of uniform size;

(5) The double emulsion obtained in step (4) is evaporated under reduced pressure, stirred and volatilized at normal temperature and pressure, cross-flow diffusion dialysis or solvent extraction to remove the organic solvent therein;

(6) The solidified solution obtained in the step (5) is centrifuged and washed to obtain the nano drug-loaded microcapsules of the present invention with uniform size, freeze-dried, and collected.

The degradable polymer includes one or more polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer, polylactic acid-polyethylene glycol copolymer, polycaprolactone, polyorthoester, polyanhydride, Polyphosphazene and its copolymers with other polymers; the hydrophilic drugs include proteins, polypeptides, enzymes, vaccines, genes, hormones, antibiotics, anticancer agents, Chinese herbal medicines and mixtures thereof; Described organic solvent is any one or more mixed organic solvents in methylene chloride, ethyl acetate, ethyl propionate, propyl acetate and acetone; Described pressure is 300-2000kPa; Described double emulsion _W2 is a mixture obtained by dissolving a water-soluble emulsifier and/or an additive in water.

The particle size of the nano drug-loaded microcapsules prepared in the present invention can be well controlled by the pore size of the microporous membrane, so the particle size of the nano drug-loaded microcapsules is uniform and controllable, and the diameter distribution coefficient (CV value) is between 1% and 20 % range, the particle size range is 50-1000nm. The repeatability of the experiment is good, the activity of the embedded drug is well maintained, and the embedding rate is high, which can well exert the targeting and sustained release effect of the nano drug-loaded microcapsule.

The method for preparing nano drug-loaded microcapsules with uniform size provided by the invention has simple process, short time consumption, and is easy to scale up for large-scale production.

The drugs in the present invention are hydrophilic drugs, including proteins, polypeptides, enzymes, vaccines, genes, hormones, antibiotics, anticancer agents, Chinese herbal medicines and mixtures thereof, most of which are easily inactivated and denatured. However, the preparation method of nano-microcapsules embedding hydrophilic drugs in the present invention mainly relies on passing the pre-complex emulsion with a larger particle size through the microporous membrane under pressure conditions to obtain nano-scale microcapsules, and the emulsion is evenly stressed. . Although homogeneous emulsification or ultrasound is also used in the operation, these steps are only for the preparation of pre-double emulsion with larger particle size, so the selected emulsification shear force is small and the emulsification time is short. Therefore, compared with the traditional double emulsion-solidification method, the preparation conditions of the nano drug-loaded microcapsules in the present invention are milder. At the same time, in the present invention, the contact time between the hydrophilic drug and the organic solvent is short, and the amount of the organic solvent is small. Moreover, since the preparation method of the present invention has mild operating conditions and no local excessive shear force, the embedding rate of the hydrophilic drug can reach more than 80%, and it can reach more than 90% under optimal conditions.

The polymer material selected in the present invention is one or more polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer, polylactic acid-polyethylene glycol copolymer, polycaprolactone, polyorthoester, polyanhydride , Polyphosphazene and its copolymers with other polymers. They are all degradable polymer materials, have good biocompatibility, and have no toxic side effects to the human body.

The organic solvent used in the present invention is a mixed organic solvent of any one or more of dichloromethane, ethyl acetate, ethyl propionate, propyl acetate and acetone, and the specific type or volume depends on the film material used.

The pressure used in the present invention is 300-2000kPa, and the specific value depends on the required particle size and experimental system.

Description of drawings

Fig. 1 is the scanning electron micrograph of the nanometer microcapsule of embedding hydrophilic medicine that embodiment 1 gains;

Fig. 2 is the particle size distribution curve of the nano-microcapsule of embedding hydrophilic drug obtained in Example 1;

FIG. 3 is a confocal photo of the nanocapsules embedded with hydrophilic drugs obtained in Example 1. FIG.

Detailed ways

The technical scheme of the present invention will be further introduced below by specific embodiments:

The invention provides a preparation method for nano-microcapsules embedding hydrophilic drugs, comprising:

(1) dissolving the degradable polymer in at least one organic solvent;

(2) adding the aqueous drug solution to the organic solution obtained in step (1), and forming a W ₁ /O emulsion through homogeneous emulsification or ultrasonic dispersion;

(3) Pour the emulsion obtained in step (2) into double emulsion W ₂ , and form W ₁ /O/W ₂ pre-double emulsion through homogeneous emulsification or mechanical stirring;

(4) the obtained pre-multi-emulsion of step (3) is obtained by pressure through the microporous membrane to obtain the multi-emulsion of uniform size;

(5) The double emulsion obtained in step (4) is evaporated under reduced pressure, stirred and volatilized at normal temperature and pressure, cross-flow diffusion dialysis or solvent extraction to remove the organic solvent therein;

(6) The solidified solution obtained in the step (5) is centrifuged and washed to obtain the nano drug-loaded microcapsules of the present invention with uniform size, freeze-dried, and collected.

Example 1.

Pour 0.5 mL of 100 mg/mL hemoglobin aqueous solution (inner water phase) into 4 mL of ethyl acetate solution (oil phase) in which 50 mg of polylactic acid-polyethylene glycol copolymer was dissolved, and homogeneously emulsify at 6000 rpm for 30 s to obtain W ₁ /O colostrum. Then pour the primary emulsion into 30 mL of 1.0 wt % polyvinyl alcohol (PVA) in 0.9 wt % sodium chloride (NaCl) solution (external aqueous phase), and homogeneously emulsify at 3000 rpm for 60 s to obtain pre-double emulsion. The pre-double emulsion was pressed through a microporous membrane device (with a membrane pore size of 1.4 μm) under a nitrogen pressure of 800 kPa to obtain a double emulsion. The obtained solution was then poured into 200 mL of solidification liquid, and stirred at 200 rpm for 10 min. The solidified solution is centrifuged to obtain nano drug-loaded microcapsules, which are freeze-dried and collected. The surface morphology of the microcapsules was observed using a field emission scanning electron microscope (JEOL SEM Company, Japan) (as shown in Figure 1). The volume-average particle diameter and distribution of the microcapsules were measured with a laser particle size analyzer (Malvern Company, USA) (as shown in Figure 2), and it was detected that the volume-average particle diameter of the microcapsules was 376nm, and the CV value of the particle size distribution coefficient was 19.13%. The activity of the hemoglobin samples in the nanocapsules was measured by an oxygen analyzer (TCS Company, USA). The specific steps for the determination of the entrapment rate of hemoglobin are as follows: Accurately weigh 10 mg of freeze-dried microcapsules, add 4.0 mL of 0.1 M NaOH solution containing 2.0 wt % SDS, shake at room temperature for 24 hours, after the microcapsules are completely dissolved (hydrolyzed), add 0.4 M was neutralized with hydrochloric acid, and the protein content was determined by the Peterson-Lowry method. According to the embedding rate formula:

Protein embedment rate (EE) = (measured protein loading rate/theoretical protein loading rate) × 100%

The calculated embedding rate was 90.15%. It was further found through confocal detection that the hemoglobin labeled with FITC could be evenly distributed in the microcapsules (as shown in Figure 3).

Example 2.

Pour 0.5 mL of 10 mg/mL DNA aqueous solution (inner water phase) into 4 mL of dichloromethane/ethyl acetate solution (oil phase) dissolved with 100 mg of lactic acid-glycolic acid copolymer (PLGA), and emulsify homogeneously at 5000 rpm 30s, get W ₁ /O colostrum. Then pour the primary emulsion into 30mL of 2.0wt% PVA solution (outer water phase), and homogeneously emulsify at 3000rpm for 60s to obtain pre-double emulsion. The pre-double emulsion was pressed through a microporous membrane device (with a membrane pore size of 5.2 μm) under a nitrogen pressure of 700 kPa to obtain a double emulsion. The obtained solution was then poured into 50 mL of solidified liquid, and stirred for 3 mm at 200 rpm to remove ethyl acetate. Then the obtained solution was stirred and volatilized at normal temperature and pressure for 2 h, and the stirring speed was 200 rpm to remove dichloromethane. The solidified solution is centrifuged to obtain nano drug-loaded microcapsules, which are freeze-dried and collected. The volume-average particle size and distribution of the microcapsules were measured with a laser particle size analyzer (Malvern Company, USA). It was detected that the volume average particle diameter of the microcapsules was 547nm, the CV value of the particle size distribution coefficient was 16.21%, and the embedding rate was 91.09%.

Example 3.

Pour 1 mL of 10 mg/mL HBsAg solution (inner water phase) into 5 mL of methylene chloride solution (oil phase) in which 150 mg of polylactic acid was dissolved, and homogeneously emulsify at 5000 rpm for 30 s to obtain W ₁ /O colostrum. Then pour the primary emulsion into 50 mL of 1.0 wt% PVA aqueous solution (outer water phase), and homogeneously emulsify at 3000 rpm for 60 seconds to obtain pre-double emulsion. The pre-double emulsion was pressed through a microporous membrane device (with a membrane pore size of 9.0 μm) under a nitrogen pressure of 400 kPa to obtain a double emulsion. Then, the obtained solution was stirred and volatilized at normal temperature and pressure for 2 h at a stirring speed of 200 rpm to remove dichloromethane. The solidified solution is centrifuged to obtain nano drug-loaded microcapsules, which are freeze-dried and collected. The volume-average particle size and distribution of the microcapsules were measured with a laser particle size analyzer (Malvern Company, USA). It was detected that the volume average particle diameter of the microcapsules was 836nm, the CV value of the particle size distribution coefficient was 18.26%, and the embedding rate was 85.32%.

Example 4.

Pour 1 mL of 100 mg/mL insulin aqueous solution (inner water phase) into 5 mL of ethyl acetate solution (oil phase) in which 150 mg of polylactic acid-monomethoxypolyethylene glycol was dissolved, and homogeneously emulsify at 5000 rpm for 30 s to obtain W ₁ /O colostrum. Then pour the primary emulsion into 50mL 0.5wt% PVA aqueous solution (outer water phase), and homogeneously emulsify at 3000rpm for 60s to obtain pre-double emulsion. The pre-double emulsion was pressed through a microporous membrane device (with a membrane pore size of 1.4 μm) under a nitrogen pressure of 500 kPa to obtain a double emulsion. The obtained solution was then poured into 50 mL of solidified liquid, stirred at 200 rpm for 5 min, and ethyl acetate was removed. The solidified solution is centrifuged to obtain nano drug-loaded microcapsules, which are freeze-dried and collected. The volume average particle size and distribution of the microcapsules were measured by a laser particle size analyzer (Malvern Company, USA). It was detected that the volume average particle diameter of the microcapsules was 428nm, the CV value of the particle size distribution coefficient was 17.89%, and the embedding rate was 92.60%.

Claims (10)

1. A nano drug-loaded microcapsule product with uniform size, characterized in that the nano drug-loaded microcapsule has a uniform and controllable size, and the diameter distribution coefficient (CV value) defined by the following formula is in the range of 1%-20% , Coefficient of Variation (CV) $\mathrm{<span\; class="notranslate">\; cv</span>}<span\; class="notranslate">\; =</span>{<span\; class="notranslate">\; (</span>\underset{\mathrm{<span\; class="notranslate">\; i</span>}<span\; class="notranslate">\; =</span>\mathrm{<span\; class="notranslate">\; 1</span>}}{\overset{\mathrm{<span\; class="notranslate">\; no</span>}}{\mathrm{<span\; class="notranslate">\; \&Sigma;</span>}}}\frac{{<span\; class="notranslate">\; (</span>{\mathrm{<span\; class="notranslate">\; d</span>}}_{\mathrm{<span\; class="notranslate">\; i</span>}}<span\; class="notranslate">\; -</span>\stackrel{<span\; class="notranslate">\; \&OverBar;</span>}{\mathrm{<span\; class="notranslate">\; d</span>}}<span\; class="notranslate">\; )</span>}^{\mathrm{<span\; class="notranslate">\; 2</span>}}}{\mathrm{<span\; class="notranslate">\; N</span>}}<span\; class="notranslate">\; )</span>}^{\frac{\mathrm{<span\; class="notranslate">\; 1</span>}}{\mathrm{<span\; class="notranslate">\; 2</span>}}}<span\; class="notranslate">\; /</span>\stackrel{<span\; class="notranslate">\; \&OverBar;</span>}{\mathrm{<span\; class="notranslate">\; d</span>}}<span\; class="notranslate">\; \&times;</span>\mathrm{<span\; class="notranslate">\; 100</span>}<span\; class="notranslate">\; \%</span>$ In the above formula, d _i is the diameter of each microcapsule, d is the number average diameter, N is the number of microcapsules used to calculate the diameter, and N>300. 2. The nano drug-loaded microcapsule product with uniform size according to claim 1, characterized in that the average particle diameter of the nano drug-loaded microcapsules is in the range of 50-1000 nm. 3. The nano drug-loaded microcapsule product with uniform size according to claim 1, characterized in that the drug is a hydrophilic drug, including proteins, polypeptides, enzymes, vaccines, genes, hormones with medicinal value , antibiotics, anticancer agents, Chinese herbal medicines and mixtures thereof. 4. The drug-carrying nano-capsule product with uniform size according to claim 1, characterized in that the embedding rate of the drug is over 80%, and under optimized conditions it is over 90%. 5. A method for preparing the nano drug-loaded microcapsule with uniform size according to claim 1, characterized in that the steps are as follows: (1) dissolving the degradable polymer in at least one organic solvent; (2) adding the aqueous drug solution to the organic solution obtained in step (1), and forming a W ₁ /O emulsion through homogeneous emulsification or ultrasonic dispersion; (3) Pour the emulsion obtained in step (2) into double emulsion W ₂ , and form W ₁ /O/W ₂ pre-double emulsion through homogeneous emulsification or mechanical stirring; (4) the obtained pre-multi-emulsion of step (3) is obtained by pressure through the microporous membrane to obtain the multi-emulsion of uniform size; (5) The double emulsion obtained in step (4) is evaporated under reduced pressure, stirred and volatilized at normal temperature and pressure, cross-flow diffusion dialysis or solvent extraction to remove the organic solvent therein; (6) The solidified solution obtained in the step (5) is centrifuged and washed to obtain the nano drug-loaded microcapsules of the present invention with uniform size, freeze-dried, and collected. 6. The preparation method of nano drug-loaded microcapsules with uniform size according to claim 5, characterized in that, the degradable polymer is one or more polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer substances, polylactic acid-polyethylene glycol copolymer, polycaprolactone, polyorthoester, polyanhydride, polyphosphazene and its copolymers with other polymers. 7. The preparation method of nanometer drug-loaded microcapsules with uniform size according to claim 5, characterized in that, the drug is a hydrophilic drug, including proteins, polypeptides, enzymes, vaccines, genes, etc. , hormones, antibiotics, anticancer agents, Chinese herbal medicines and their mixtures. 8. The method for preparing the nano-sized drug-loaded microcapsule with uniform size according to claim 5, wherein the organic solvent is dichloromethane, ethyl acetate, ethyl propionate, propyl acetate and acetone. Any one or more mixed organic solvents. 9. The preparation method of nano drug-loaded microcapsules with uniform size according to claim 5, characterized in that the pressure is 300-2000 kPa. 10. The preparation method of nano drug-loaded microcapsules with uniform size according to claim 5, characterized in that said double emulsion _W2 is a mixture of water-soluble emulsifier and/or additives dissolved in water.

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