阅读说明：本技术 一种磁响应前列地尔微球的制备方法 (Preparation method of magnetic response alprostadil microspheres ) 是由 李强 曾菲 陈涛 郑传东 于 2021-09-10 设计创作，主要内容包括：本发明公开了一种磁响应前列地尔微球的制备方法,通过双乳化-溶剂挥发法制备出聚乳酸-羟基乙酸共聚物(PLGA)-前列地尔微球,之后使用静电吸附自组装法,制备得到磁响应前列地尔微球,其磁响应前列地尔微球载药量达到3.2wt.％,包覆率达到70％以上,释药率达80％以上。(The invention discloses a preparation method of a magnetic response alprostadil microsphere, which is characterized in that a polylactic acid-glycolic acid copolymer (PLGA) -alprostadil microsphere is prepared by a double emulsification-solvent volatilization method, and then the magnetic response alprostadil microsphere is prepared by an electrostatic adsorption self-assembly method, wherein the drug loading rate of the magnetic response alprostadil microsphere reaches 3.2 wt.%, the coating rate reaches more than 70%, and the drug release rate reaches more than 80%.)

1. A preparation method of a magnetic response alprostadil microsphere mainly comprises the following steps of preparing a polylactic acid-glycolic acid copolymer (PLGA) -alprostadil microsphere by a double emulsification-solvent volatilization method, and preparing the magnetic response alprostadil microsphere by using an electrostatic adsorption self-assembly method, wherein the preparation method comprises the following steps:

(1) weighing polylactic acid-glycolic acid copolymer (PLGA) and putting the PLGA into dichloromethane solution, and oscillating to fully dissolve the PLGA;

(2) adding the alprostadil solution in the step (1), and emulsifying by using an ultrasonic probe to form a primary emulsion (W)₁/O)；

(3) Mixing W formed in step (2)₁Adding the/O solution into polyvinyl alcohol (PVA) solution, homogenizing with high speed homogenizer to form secondary emulsion (W)₁/O/W₂)；

(4) Mixing the secondary emulsion (W) obtained in step (3)₁/O/W₂) The solution is placed in a stirrer for overnight stirring, so that the surface of the microsphere is solidified, and dichloromethane is volatilized as much as possible;

(5) subpackaging the solution stirred in the step (4) into centrifuge tubes, centrifuging in a high-speed refrigerated centrifuge, taking out test tubes from the centrifuge, discarding supernatant, adding a proper amount of double distilled water for washing, fully and uniformly mixing by using a vortex mixer, centrifuging again, washing again, and carrying out centrifugal washing for three times;

(6) adding the PLGA-alprostadil microsphere suspension obtained in the step (5) into a Polyethyleneimine (PEI) solution, oscillating, fully mixing uniformly, performing shaking adsorption, centrifuging, removing supernatant, adding deionized water, performing vortex oscillation dispersion again, performing centrifugal washing twice, and removing Polyethyleneimine (PEI) which is not adsorbed;

(7) adding gamma-Fe in the step (6)₂O₃And @ DMSA, performing co-incubation adsorption, performing magnetic separation and washing three times under the same condition, and re-dispersing in deionized water to obtain the magnetic response alprostadil microspheres.

2. The method for preparing a magnetic-response alprostadil microsphere according to claim 1, wherein the dosage of the polylactic-co-glycolic acid (PLGA) in the step (1) is 500mg, and the dichloromethane solution is 20 ml.

3. The method for preparing a magnetic-response alprostadil microsphere according to claim 1, wherein the amount of the alprostadil solution used in the step (2) is 5ml, the solubility is 0.5-3ug/ml, and the emulsifying time of the primary emulsion is 50-150 s.

4. The preparation method of the magnetic response alprostadil microsphere according to claim 1, characterized in that the amount of the polyvinyl alcohol solution used in the step (3) is 100ml, the concentration is 1% -5%, and the homogenization time of the high-speed dispersion homogenizer is 5-15 min.

5. The method for preparing a magnetically responsive alprostadil microsphere as claimed in claim 1, wherein the amount of Polyethyleneimine (PEI) used in step (6) is 100ml, the solution concentration is 1% -5%, the adsorption time is 10-30min, the centrifugation conditions are 1500-3500rpm, the time is 5-10min, and the temperature is 10-20 ℃.

6. The method for preparing a magnetic-response alprostadil microsphere according to claim 1, wherein the adsorption time of the co-incubation in the step (7) is 20-40 min.

Technical Field

The invention relates to the field of magnetic targeted therapy, in particular to a preparation method of a magnetic response alprostadil microsphere.

Background

Alprostadil, also known as prostaglandin E, is a natural Prostaglandin (PG) substance, is an oxidation product of polyunsaturated fatty acid dihomor-linoleic acid, and has similar properties and characteristics of endogenous prostaglandin E. Endogenous prostaglandin E is a prostaglandin substance widely distributed in normal organisms, is relatively active in chemical nature, and is usually inactivated rapidly after local action. The alprostadil is used as a prostaglandin substance with high biological activity, has various biological effects, can obviously inhibit the contraction of capillary smooth muscle, reduce platelet aggregation, inhibit the generation of thromboxane A2, increase the deformability of red blood cells and reduce the aggregation of the red blood cells, thereby improving microcirculation, and simultaneously can reduce the secretion of endothelin by acting on vascular endothelium to ensure that the activity and the expression of endothelial nitric oxide synthase are increased, increase the content of serum nitric oxide, play various roles of inhibiting nerve injury, preventing and reversing the formation of atheromatous lipid plaques and the like. Under the action of an external magnetic field, the magnetic alprostadil microspheres can realize the targeted delivery of the alprostadil to the pathological change part, reduce the drug concentration of other tissues of the whole body and realize the purpose of targeted therapy.

The magnetic fluid is used as a magnetic drug-carrying particle, and is a stable and functional colloidal solution or suspension with magnetism formed by highly and uniformly dispersing superfine ferromagnetic or superparamagnetic particles which are treated by a surfactant and have a nanometer grade (the particle size is between a few nanometers and a few hundred nanometers) into a certain base liquid (carrier liquid). Therefore, the magnetic fluid has the strong magnetism of a solid material and the rheological property of a liquid material, is a common magnetic particle, can be combined with a high molecular substance to prepare and form a magnetic high molecular substance, the magnetic high molecular substance has good surface effect, when the particle size reaches the nanometer level, the amount of the medicine carried by the formed magnetic nanometer high molecular substance can be increased rapidly, and the medicine can move rapidly and be separated rapidly under the action of an external magnetic field, so that the curative effect of the medicine can be improved obviously. Has been widely applied to the fields of magnetofluid thermal therapy, magnetic embolism sealing therapy, magnetic medicine targeted therapy, imaging examination, cell separation and the like. In the magnetic drug targeting treatment, the magnetic fluid can be used as magnetic carrier particles of drugs, carries specific drugs to perform targeted movement and location accumulation in vivo under the action of an external magnetic field, improves the concentration of the drugs at a target position, and reduces the toxicity and adverse reactions of the drugs to other normal tissues of the whole body. In recent years, scholars at home and abroad carry out a series of basic experiments, animal experiments and clinical experiments on the targeted therapy of the magnetic medicament, and the results show that the scheme of the magnetic targeted therapy is safe and effective. At present, the research in the field mainly focuses on the aspect of tumor treatment, but no report is found in the field of prevention and treatment of organ ischemia-reperfusion injury, particularly the field of spinal ischemia-reperfusion injury.

Disclosure of Invention

The invention aims to provide a preparation method of a magnetic response alprostadil microsphere, which is suitable for the field of spinal ischemia reperfusion injury.

The technical scheme of the invention is as follows:

the invention provides a preparation method of a magnetic response alprostadil microsphere, which mainly prepares a polylactic acid-glycolic acid copolymer (PLGA) -alprostadil microsphere by a double emulsification-solvent volatilization method, and then prepares the magnetic response alprostadil microsphere by using an electrostatic adsorption self-assembly method, and specifically comprises the following steps:

(1) weighing polylactic acid-glycolic acid copolymer (PLGA) and putting the PLGA into dichloromethane solution, and oscillating to fully dissolve the PLGA;

(2) adding the alprostadil solution in the step (1), and emulsifying by using an ultrasonic probe to form a primary emulsion (W)₁/O)；

(3) Mixing W formed in step (2)₁Adding the/O solution into polyvinyl alcohol (PVA) solution, homogenizing with high speed homogenizer to form secondary emulsion (W)₁/O/W₂)；

(4) Mixing the secondary emulsion (W) obtained in step (3)₁/O/W₂) The solution is placed in a stirrer for overnight stirring, so that the surface of the microsphere is solidified, and dichloromethane is volatilized as much as possible;

(5) subpackaging the solution stirred in the step (4) into centrifuge tubes, centrifuging in a high-speed refrigerated centrifuge, taking out test tubes from the centrifuge, discarding supernatant, adding a proper amount of double distilled water for washing, fully and uniformly mixing by using a vortex mixer, centrifuging again, washing again, and carrying out centrifugal washing for three times;

(6) adding the PLGA-alprostadil microsphere suspension obtained in the step (5) into a Polyethyleneimine (PEI) solution, oscillating, fully mixing uniformly, performing shaking adsorption, centrifuging, removing supernatant, adding deionized water, performing vortex oscillation dispersion again, performing centrifugal washing twice, and removing Polyethyleneimine (PEI) which is not adsorbed;

(7) adding gamma-Fe in the step (6)₂O₃And @ DMSA, performing co-incubation adsorption, performing magnetic separation and washing three times under the same condition, and re-dispersing in deionized water to obtain the magnetic response alprostadil microspheres.

In step (1), the amount of polylactic-co-glycolic acid (PLGA) was 500mg and the methylene chloride solution was 20 ml.

In the step (2), the dosage of the alprostadil solution is 5ml, the solubility is 0.5-3ug/ml, and the emulsifying time of the primary emulsion is 50-150 s.

In the step (3), the dosage of the polyvinyl alcohol solution is 100ml, the concentration is 1% -5%, and the homogenizing time of the high-speed dispersion homogenizer is 5-15 min.

In the step (6), the dosage of the Polyethyleneimine (PEI) is 100ml, the solution concentration is 1% -5%, the adsorption time is 10-30min, the centrifugation condition is that the rotation speed is 1500-.

In step (7), the co-incubation adsorption time is 20-40 min.

The invention prepares the magnetic response alprostadil microspheres by a double emulsification-solvent volatilization method and an electrostatic adsorption self-assembly method, and the medicine with magnetic response prepared by wrapping the medicine and the magnetic material in the macromolecular coupling agent by a physical and chemical method is gathered at a target position, thereby improving the concentration of the medicine at the target position, improving the treatment effect of diseases and reducing the adverse reaction of the whole body.

Drawings

FIG. 1 is a flow chart of preparation of alprostadil PLGA microspheres;

FIG. 2 is a flow chart of the preparation of the magnetic response alprostadil microspheres;

FIG. 3 is an Atomic Force Microscope (AFM) image of a magnetic response alprostadil microsphere;

FIG. 4 is a Scanning Electron Microscope (SEM) image of a magnetic response alprostadil microsphere;

FIG. 5 is a magnetic hysteresis loop plot of a magnetically responsive alprostadil microsphere;

fig. 6 is a graph showing the cumulative release of magnetically responsive alprostadil microspheres in phosphate buffered saline.

Detailed Description

The present invention is further illustrated by the following specific examples, but it should be understood by those skilled in the art that the specific examples of the present invention are not intended to limit the present invention in any way, and any equivalents based on the present invention are within the scope of the present invention.

Examples

1. Preparation method of magnetic response alprostadil microspheres

(1) 500mg of polylactic-co-glycolic acid (PLGA) was weighed into 20ml of a methylene chloride solution and sufficiently dissolved by shaking.

(2) Adding 5ml alprostadil (1ug/ml) solution in the step (1), emulsifying for 100s with ultrasonic probe to form primary emulsion (W)₁/O)。

(3) Mixing W formed in step (2)₁Adding the/O solution into 100ml of 3% polyvinyl alcohol (PVA) solution, homogenizing with high speed homogenizer for 10min to obtain secondary emulsion (W)₁/O/W₂)。

(4) Mixing the secondary emulsion (W) obtained in step (3)₁/O/W₂) The solution was stirred overnight in a stirrer to solidify the microsphere surface and evaporate the dichloromethane as much as possible.

(5) And (3) respectively filling the solution stirred in the step (4) into 50ml centrifuge tubes, centrifuging in a high-speed refrigerated centrifuge, taking out the test tubes from the centrifuge, discarding the supernatant, adding a proper amount of double distilled water for washing, fully and uniformly mixing by using a vortex mixer, centrifuging again, washing again, performing centrifugal washing for three times, collecting, and storing in a refrigerator at 4 ℃ for later use.

(6) And (3) adding 10ml of the alprostadil PLGA microsphere suspension obtained in the step (5) into 25ml of 3% polyethylene imine (PEI) solution, oscillating, fully mixing, shaking for adsorption for 15min, centrifuging (3000rpm, 5min and 15 ℃), removing supernatant, adding deionized water, again performing vortex oscillation and dispersion, centrifuging and washing twice, and removing polyethylene imine (PEI) which is not adsorbed.

(7) Adding 5ml of gamma-Fe in the step (6)₂O₃@ DMSA, performing magnetic separation and washing for three times under the same condition, re-dispersing in deionized water to obtain magnetic response alprostadil microspheres, sealing the finally collected sample with a sealing film, and storing in a refrigerator at 4 ℃ for later use.

2. Performance characterization result of magnetic response alprostadil microspheres

2.1 magnetic response Atomic Force Microscope (AFM) picture of alprostadil microsphere

As shown in figure 3, the magnetic response alprostadil microspheres prepared by the double emulsification-solvent volatilization method have micro spherical structures after being naturally dried, the dry particle size is below 100nm, but the two have a certain agglomeration phenomenon.

2.2 Scanning Electron Microscope (SEM) image of magnetic response alprostadil microsphere

From fig. 4, it can be known that the magnetic response alprostadil microspheres prepared by the double emulsification-solvent volatilization method are all spherical, have good monodispersity, have microsphere diameter of about 20nm, and have a small amount of agglomeration phenomenon.

2.3 magnetic response Vibration Sample Magnetometer (VSM) hysteresis loop diagram of alprostadil microsphere

As shown in FIG. 5, the specific saturation magnetization of the magnetic response alprostadil microspheres is 82.4065 emu/g.

2.4 drug loading and drug loading conditions of magnetic response alprostadil microspheres

The drug loading rate of the magnetic response alprostadil microspheres can be 3.2 wt% according to a standard working curve, and the coating rate reaches 77.9%.

As can be seen from FIG. 6, the release rate of the magnetic PLGA microspheres in phosphate buffered saline solution within 1h reaches 15.0%. The drug release rate after 120h is 72.4%, and the drug release rate after 240h is stabilized at 84.4%.