CN118021773A - Preparation method and application of gossypol acetate self-assembled nanocrystalline drug - Google Patents
Preparation method and application of gossypol acetate self-assembled nanocrystalline drug Download PDFInfo
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- CN118021773A CN118021773A CN202410193182.2A CN202410193182A CN118021773A CN 118021773 A CN118021773 A CN 118021773A CN 202410193182 A CN202410193182 A CN 202410193182A CN 118021773 A CN118021773 A CN 118021773A
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- gossypol acetate
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- nanocrystalline
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- NIOHNDKHQHVLKA-UHFFFAOYSA-N acetic acid;7-(8-formyl-1,6,7-trihydroxy-3-methyl-5-propan-2-ylnaphthalen-2-yl)-2,3,8-trihydroxy-6-methyl-4-propan-2-ylnaphthalene-1-carbaldehyde Chemical compound CC(O)=O.CC(C)C1=C(O)C(O)=C(C=O)C2=C(O)C(C=3C(O)=C4C(C=O)=C(O)C(O)=C(C4=CC=3C)C(C)C)=C(C)C=C21 NIOHNDKHQHVLKA-UHFFFAOYSA-N 0.000 title claims abstract description 254
- 239000003814 drug Substances 0.000 title claims abstract description 222
- 229940079593 drug Drugs 0.000 title claims abstract description 213
- 238000002360 preparation method Methods 0.000 title claims abstract description 30
- 239000003381 stabilizer Substances 0.000 claims abstract description 60
- 229920001661 Chitosan Polymers 0.000 claims description 45
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 42
- 239000000725 suspension Substances 0.000 claims description 39
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 39
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- 238000003760 magnetic stirring Methods 0.000 claims description 25
- 239000002904 solvent Substances 0.000 claims description 22
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 18
- 238000003756 stirring Methods 0.000 claims description 16
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
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- 238000002156 mixing Methods 0.000 claims description 7
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 6
- FKRCODPIKNYEAC-UHFFFAOYSA-N ethyl propionate Chemical compound CCOC(=O)CC FKRCODPIKNYEAC-UHFFFAOYSA-N 0.000 claims description 6
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 3
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- QBKSWRVVCFFDOT-UHFFFAOYSA-N gossypol Chemical compound CC(C)C1=C(O)C(O)=C(C=O)C2=C(O)C(C=3C(O)=C4C(C=O)=C(O)C(O)=C(C4=CC=3C)C(C)C)=C(C)C=C21 QBKSWRVVCFFDOT-UHFFFAOYSA-N 0.000 description 6
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- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 3
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- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
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- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
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- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
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- IYDGMDWEHDFVQI-UHFFFAOYSA-N phosphoric acid;trioxotungsten Chemical compound O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.O=[W](=O)=O.OP(O)(O)=O IYDGMDWEHDFVQI-UHFFFAOYSA-N 0.000 description 1
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- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/11—Aldehydes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Inorganic Chemistry (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Engineering & Computer Science (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention belongs to the technical field of medicines, and particularly relates to a preparation method and application of a gossypol acetate self-assembled nanocrystalline drug. According to the invention, the hydrophobic drug gossypol acetate is self-assembled into a nano structure, and then a small amount of stabilizer is used for improving the stability and solubility of the nano drug. Under the condition of avoiding introducing other nano carriers, the preparation method enables drug monomer molecules to form nano-sized particles through self-assembly, thereby avoiding biological immune response and toxicity problems possibly caused by carrier introduction, and simultaneously retaining the advantages of the nano-particles in overcoming drug resistance and improving anti-tumor effect of chemotherapeutic drugs. The preparation method is simple, the repeatability is good, the dosage of the obtained nano crystal medicine can be effectively reduced, and the nano crystal medicine has good safety and stability.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a preparation method and application of a gossypol acetate self-assembled nanocrystalline drug.
Background
Nanocarrier-assisted drug delivery systems have created a new approach to cancer treatment and have made significant progress in recent years. However, nanocarrier-assisted drug delivery systems still have their inevitable drawbacks. For example, the loading rate of chemotherapeutic drugs is not ideal, generally below 10% (W/W), resulting in the need to inject large amounts of nanocarriers to achieve therapeutic effects in order to achieve efficacy, which may reduce patient compliance. Furthermore, most vectors do not have a direct therapeutic effect themselves and may elicit additional long-or short-term toxic responses. Also, some carriers may interact with specific cell surface receptors, eliciting adverse immune responses, and thus affecting therapeutic efficacy.
In the field of antitumor, gossypol acetate shows inhibition effect on various tumor cells, but is poorly water-soluble and easily oxidized in the environment. At present, the patents related to gossypol and its derivative preparations are mainly concentrated in the fields of oral liquid, injection and the like. CN1144089a discloses water-soluble gossypol preparation and its preparation method, including oral liquid, injection, powder, tablet and capsule etc.; CN101810577B discloses a gossypol intravenous injection fat emulsion for treating tumor; CN102138916B discloses cyclodextrin inclusion compounds of l-gossypol or its acetate for treating tumors; however, these oral dosage forms have certain limitations in terms of antitumor therapy. The intravenous injection emulsion is unfavorable for the effect of the medicine on tumor tissue due to the large size of the carrier.
Disclosure of Invention
The invention aims to provide a preparation method and application of a gossypol acetate self-assembled nanocrystalline drug, which are used for solving the problems that aggregation, agglomeration, crystal growth or crystalline state change and low drug activity are easy to occur along with the reduction of the particle size of the drug in the existing preparation process of the nanocrystalline drug.
In order to solve the technical problems, the invention specifically provides the following technical scheme:
A preparation method of a gossypol acetate self-assembled nanocrystalline drug comprises the following steps: dissolving gossypol acetate in a good solvent to obtain a gossypol acetate drug solution, adding the gossypol acetate drug solution into a poor solvent, and stirring to obtain a gossypol acetate drug suspension; dissolving a stabilizer in an aqueous medium to obtain a stabilizer solution; mixing the gossypol acetate drug suspension with a stabilizer solution to obtain a gossypol acetate self-assembled nanocrystalline drug; the stabilizer is mainly divided into an ionic stabilizer and a nonionic stabilizer, wherein the ionic stabilizer is mainly used for keeping the stability of the gossypol acetate drug suspension nanosystem through electrostatic repulsion, and the nonionic stabilizer is mainly used for keeping the stability of the gossypol acetate drug suspension nanosystem through space obstruction; meanwhile, the stabilizer is loaded on the surface of the gossypol acetate to further improve the stability and solubility of the drug, so that nano particles with uniform particle size are formed, the bioavailability of the hydrophobic drug gossypol acetate is improved, the circulation time of the hydrophobic drug gossypol acetate in blood is prolonged, and the targeting effect is achieved, so that the targeting drug is particularly suitable for cancer treatment.
Preferably, the hydration particle size of the gossypol acetate self-assembled nanocrystalline drug is 50-1000 nm; the hydrated particle size of the gossypol acetate self-assembled nanocrystalline drug refers to the particle size of a hydrate formed by dispersing the gossypol acetate self-assembled nanoparticle in water after being treated by a surface modification stabilizer.
Preferably, the good solvent comprises dichloromethane, chloroform, ethyl acetate, ethyl propionate, propyl acetate, acetone, methanol, dimethyl sulfoxide, acetonitrile, and ethanol; the poor solvent includes water.
More preferably, the good solvent comprises dimethyl sulfoxide or ethanol.
Preferably, the concentration of the gossypol acetate drug solution is 1-100 mg/mL, the concentration is the total concentration of the gossypol acetate in the good solvent, and the too large or too small concentration can cause the particle size of the finally formed gossypol acetate self-assembled nanocrystalline drug.
Preferably, the stabilizer is any one of polyvinylpyrrolidone, polyethylene glycol, chitosan and hyaluronic acid.
Preferably, the aqueous medium comprises water or an acetic acid solution.
Preferably, the concentration of the stabilizer solution is 1-2 mg/mL, the concentration is the total concentration of the stabilizer in the aqueous medium, the concentration depends on the concentration of the above gossypol acetate drug solution, and too large or too small concentration can cause the complete adsorption of the gossypol acetate self-assembled nano particles and the stabilizer.
Preferably, the concentration of the gossypol acetate drug suspension is 5-20% (v/v); the volume ratio of the gossypol acetate drug suspension to the stabilizer solution is 1:1-10, and the stabilizer needs to be used for completely covering the surface of the crystal nucleus of the gossypol acetate self-assembled nanocrystalline drug to form more gossypol acetate self-assembled nanocrystalline drugs.
Preferably, the stirring is magnetic stirring, the magnetic stirring rotating speed is 50-2000 rpm, the magnetic stirring temperature is 4-50 ℃, and the magnetic stirring time is 0.1-5 h; mixing to ultrasonic treatment under ice bath or room temperature; the ultrasonic condition is water bath ultrasonic or probe ultrasonic, the ultrasonic power is 30-500W, and the ultrasonic condition is limited to ensure that the gossypol acetate and the stabilizer are fully adsorbed, so that the gossypol acetate self-assembled nanocrystalline drug with uniform particle size is formed.
More preferably, the magnetic stirring temperature is room temperature, ensuring that the stabilizer has a sufficiently high diffusion rate to rapidly cover the surface of the drug that forms the gossypol acetate self-assembled nanocrystals, so as to provide sufficient electron or space repulsive force between the particles, thereby forming the gossypol acetate self-assembled nanocrystals with uniform particle size.
The invention also provides a preparation method of the gossypol acetate self-assembled nanocrystalline drug, which specifically comprises the following steps:
Dissolving gossypol acetate in a good solvent to obtain a gossypol acetate drug solution, adding the gossypol acetate drug solution into a poor solvent, and stirring to obtain a gossypol acetate drug suspension; dissolving a stabilizer and an auxiliary agent in an aqueous medium to obtain a stabilizer solution; mixing the gossypol acetate drug suspension with a stabilizer solution to obtain a gossypol acetate self-assembled nanocrystalline drug; the auxiliary agent and the stabilizer produce synergistic effect, and the stability of the system in the crystallization induction stage is controlled, so that the crystal nucleus formed by the gossypol acetate in the crystal nucleus formation stage is increased, and the crystal grain size is reduced.
Preferably, the good solvent comprises dichloromethane, chloroform, ethyl acetate, ethyl propionate, propyl acetate, acetone, methanol, dimethyl sulfoxide, acetonitrile, and ethanol; the poor solvent includes water.
More preferably, the good solvent comprises dimethyl sulfoxide or ethanol.
Preferably, the concentration of the gossypol acetate drug solution is 1-100 mg/mL, and the concentration is the total concentration of the gossypol acetate in the good solvent.
Preferably, the stabilizer is any one of polyvinylpyrrolidone, polyethylene glycol, chitosan and hyaluronic acid; the auxiliary agent comprises erythritol.
Preferably, the aqueous medium comprises water or an acetic acid solution.
Preferably, the concentration of the stabilizer in the stabilizer solution is 1-2 mg/mL.
Preferably, the concentration of the adjuvant in the stabilizer solution is from 0.1 to 1 mg/mL.
Preferably, the volume ratio of the gossypol acetate drug suspension to the stabilizer solution is 1:1-10.
Preferably, the stirring is magnetic stirring, the magnetic stirring rotating speed is 50-2000 rpm, the magnetic stirring temperature is 4-50 ℃, and the magnetic stirring time is 0.1-5 h; mixing to ultrasonic treatment under ice bath or room temperature; the ultrasonic condition is water bath ultrasonic or probe ultrasonic, and the ultrasonic power is 30-500W.
More preferably, the stirring temperature is room temperature.
Preferably, the gossypol acetate self-assembled nanocrystalline drug prepared by any one of the above methods.
The invention also provides application of the gossypol acetate self-assembled nanocrystalline drug in an injected emulsion formulation.
In a preferred embodiment, the preparation method of the gossypol acetate self-assembled nanocrystalline drug specifically comprises the following steps:
Dissolving gossypol acetate in dimethyl sulfoxide to obtain a gossypol acetate medicinal solution; dripping the gossypol acetate drug solution into water, and stirring to obtain a gossypol acetate drug suspension; dissolving chitosan in acetic acid solution to obtain chitosan solution; adding the gossypol acetate drug suspension into chitosan solution, and performing ultrasonic treatment to obtain the gossypol acetate self-assembled nanocrystalline drug.
Preferably, the concentration of the gossypol acetate drug solution is 1-100 mg/mL, and the concentration is the total concentration of the gossypol acetate in the good solvent.
Preferably, the chitosan is a low molecular weight chitosan having a molecular weight in the range of 50000-190000 Da.
Preferably, the chitosan solution has a concentration of 1-2 mg/mL.
Preferably, the concentration of the acetic acid solution is 0.5-2% (w/w).
Preferably, the concentration of the gossypol acetate drug suspension is 5-20% (v/v).
Preferably, the volume ratio of the gossypol acetate drug suspension to the chitosan solution is 1:1-10.
Preferably, the stirring is magnetic stirring, the magnetic stirring rotating speed is 50-2000 rpm, the magnetic stirring temperature is 4-50 ℃, and the magnetic stirring time is 0.1-5 h; the ultrasonic condition is water bath ultrasonic or probe ultrasonic under ice bath or room temperature, and the ultrasonic power is 30-500W.
In a preferred embodiment, the preparation method of the gossypol acetate self-assembled nanocrystalline drug specifically comprises the following steps:
Dissolving gossypol acetate in ethanol to obtain a gossypol acetate drug solution; dripping the gossypol acetate drug solution into water, and stirring to obtain a gossypol acetate drug suspension; dissolving chitosan in acetic acid solution to obtain chitosan solution; adding the gossypol acetate drug suspension into chitosan solution, and performing ultrasonic treatment to obtain the gossypol acetate nanocrystalline drug.
Preferably, the concentration of the gossypol acetate drug solution is 1-100 mg/mL, and the concentration is the total concentration of the gossypol acetate in the good solvent.
Preferably, the chitosan is a low molecular weight chitosan having a molecular weight in the range of 50000-190000 Da.
Preferably, the chitosan solution has a concentration of 1-2 mg/mL.
Preferably, the concentration of the gossypol acetate drug suspension is 5-20% (v/v).
Preferably, the volume ratio of the gossypol acetate drug suspension to the chitosan solution is 1:1-10.
Preferably, the stirring is magnetic stirring, the magnetic stirring rotating speed is 50-2000 rpm, the magnetic stirring temperature is 4-50 ℃, and the magnetic stirring time is 0.1-5 h; the ultrasonic condition is water bath ultrasonic or probe ultrasonic under ice bath or room temperature, and the ultrasonic power is 30-500W.
In a preferred embodiment, the preparation method of the gossypol acetate self-assembled nanocrystalline drug specifically comprises the following steps:
Dissolving gossypol acetate in dimethyl sulfoxide to obtain a gossypol acetate medicinal solution; dripping the gossypol acetate drug solution into water, and stirring to obtain a gossypol acetate drug suspension; dissolving chitosan and erythritol into an acetic acid solution to obtain a stabilizer solution; adding the gossypol acetate drug suspension into the stabilizer solution, and performing ultrasonic treatment to obtain the gossypol acetate nanocrystalline drug.
Preferably, the concentration of the gossypol acetate drug solution is 1-100 mg/mL, and the concentration is the total concentration of the gossypol acetate in the good solvent.
Preferably, the chitosan is a low molecular weight chitosan having a molecular weight in the range of 50000-190000 Da.
Preferably, the aqueous medium comprises water or an acetic acid solution.
Preferably, the concentration of the low molecular weight chitosan in the stabilizer solution is 1-2 mg/mL.
Preferably, the concentration of erythritol in the stabilizer solution is in the range of 0.1-1 mg/mL.
Preferably, the concentration of the gossypol acetate drug suspension is 5-20% (v/v).
Preferably, the volume ratio of the gossypol acetate drug suspension to the stabilizer solution is 1:1-10.
Preferably, the stirring is magnetic stirring, the magnetic stirring rotating speed is 50-2000 rpm, the magnetic stirring temperature is 4-50 ℃, and the magnetic stirring time is 0.1-5 h; mixing to ultrasonic treatment under ice bath or room temperature; the ultrasonic condition is water bath ultrasonic or probe ultrasonic, and the ultrasonic power is 30-500W.
The invention provides a gossypol acetate self-assembled nanocrystalline drug, the preparation method adopts a simple and environment-friendly precipitation method, no toxic organic reagent and nano carrier are introduced, the possible biotoxicity and immunogenicity are avoided, and the invention has the characteristics of high yield and easy expansion production; the hydrophobic gossypol acetate is self-assembled into a nano structure, and then a stabilizer is used for loading on the surface of the gossypol acetate, so that the stability, the solubility and the dispersibility in aqueous solution of the gossypol acetate are further improved; the gossypol acetate and the stabilizer are fully mixed to form nano particles with uniform particle size, so that the bioavailability of the hydrophobic drug gossypol acetate is improved, the circulation time of the hydrophobic drug gossypol acetate in blood is prolonged, meanwhile, the nano-scale property enables the hydrophobic drug gossypol acetate to have good permeation enhancement effect (EPR effect) and vascular permeability enhancement effect (EPR effect) retention, and the targeting effect is achieved, so that the hydrophobic drug is particularly suitable for cancer treatment, and can achieve the same or even better tumor inhibition effect with lower drug concentration.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.
FIG. 1 is a Transmission Electron Microscope (TEM) photograph of the gossypol acetate self-assembled nanocrystalline drug prepared in example 1;
FIG. 2 is a graph showing the Zeta potential stability of the gossypol acetate self-assembled nanocrystalline drug in example 2;
FIG. 3 is a graph showing the results of the viability of A549 cells after treatment of the gossypol acetate self-assembled nanoparticles prepared in examples 1-5 and comparative example 1 with 48 h.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. The chitosan used in the examples of the present invention was Sigma (Sigma-Aldrich) low molecular weight chitosan with a molecular weight of 50,000-190,000 Da.
Example 1: preparation method of gossypol acetate self-assembled nanocrystalline drug
S1, dissolving 20 mg gossypol acetate in 1 mL dimethyl sulfoxide, and performing vortex dissolution to obtain a gossypol acetate drug solution;
S2, 200 mu L of the gossypol acetate drug solution is dripped into 800 mu L of water, and magnetically stirred for 2 hours at 25 ℃ and 350 rpm rotation speed to obtain a gossypol acetate drug suspension;
S3, dissolving 1 mg low molecular weight chitosan in 1mL of 1% (v/v) acetic acid solution, and performing vortex dissolution to obtain chitosan solution;
S4, adding 200 mu L of gossypol acetate drug suspension into the chitosan solution, and performing water bath ultrasonic treatment at room temperature of 300W to obtain gossypol acetate nanocrystalline drug;
s5, dialyzing the synthesized nanocrystalline medicine with deionized water, and removing free gossypol acetate and chitosan after dialysis.
Example 2: preparation method of gossypol acetate self-assembled nanocrystalline drug
S1, dissolving 20 mg gossypol acetate in 1mL ethanol, and performing vortex dissolution to obtain a gossypol acetate drug solution;
s2, dripping 100 mu L of the gossypol acetate drug solution into 900 mu L of water, and magnetically stirring for 2 hours at 25 ℃ and 350 rpm rotation speed to obtain a gossypol acetate drug suspension;
s3, dissolving 1 mg low molecular weight chitosan in 1 mL of 1% (v/v) acetic acid solution, and performing vortex dissolution to obtain chitosan solution;
S4, adding 200 mu L of gossypol acetate drug suspension into the chitosan solution, and performing water bath ultrasonic treatment at room temperature of 300W to obtain gossypol acetate nanocrystalline drug;
s5, dialyzing the synthesized nanocrystalline medicine with deionized water, and removing free gossypol acetate and chitosan after dialysis.
Example 3: preparation method of gossypol acetate self-assembled nanocrystalline drug
S1, dissolving 5 mg gossypol acetate in 1 mL ethanol, and performing vortex dissolution to obtain a gossypol acetate drug solution;
s2, dripping 100 mu L of the gossypol acetate drug solution into 900 mu L of water, and magnetically stirring for 2 hours at 25 ℃ and 350 rpm rotation speed to obtain a gossypol acetate drug suspension;
S3, dissolving 2 mg low molecular weight chitosan in 1mL of 1% (v/v) acetic acid solution, and performing vortex dissolution to obtain chitosan solution;
S4, adding 200 mu L of gossypol acetate drug suspension into the chitosan solution, and performing water bath ultrasonic treatment at room temperature of 300W to obtain gossypol acetate nanocrystalline drug;
s5, dialyzing the synthesized nanocrystalline medicine with deionized water, and removing free gossypol acetate and chitosan after dialysis.
Example 4: preparation method of gossypol acetate self-assembled nanocrystalline drug
S1, dissolving 1 mg gossypol acetate in 1 mL dimethyl sulfoxide, and performing vortex dissolution to obtain a gossypol acetate drug solution;
s2, dripping 80 mu L of the gossypol acetate drug solution into 920 mu L of water, and magnetically stirring for 2 hours at 25 ℃ and 350 rpm rotation speed to obtain a gossypol acetate drug suspension;
S3, dissolving 1 mg low molecular weight chitosan in 1mL of 1% (v/v) acetic acid solution, and performing vortex dissolution to obtain chitosan solution;
S4, adding 200 mu L of gossypol acetate drug suspension into the chitosan solution, and performing water bath ultrasonic treatment at room temperature of 300W to obtain gossypol acetate nanocrystalline drug;
s5, dialyzing the synthesized nanocrystalline medicine with deionized water, and removing free gossypol acetate and chitosan after dialysis.
Example 5: preparation method of gossypol acetate self-assembled nanocrystalline drug
S1, a preparation method of a gossypol acetate drug solution is the same as that of example 4;
s2, a preparation method of a gossypol acetate drug suspension is the same as that of example 4;
S3, dissolving 1 mg low molecular weight chitosan and 1 mg erythritol in 1 mL of 1% (v/v) acetic acid solution, and performing vortex dissolution to obtain a stabilizer solution;
S4, a preparation method of a gossypol acetate nanocrystalline drug is the same as that of example 4;
s5, dialyzing the synthesized nanocrystalline medicine with deionized water, and removing free gossypol acetate, chitosan and erythritol after dialysis.
Comparative example 1: preparation method of gossypol acetate self-assembled nanocrystalline drug
S1, dissolving 20 mg gossypol acetate in 1 mL dimethyl sulfoxide, and performing vortex dissolution to obtain a gossypol acetate drug solution;
S2, 200 mu L of the gossypol acetate drug solution is dripped into 800 mu L of water, and magnetically stirred for 2 hours at 25 ℃ and 350 rpm rotation speed to obtain a gossypol acetate drug suspension;
S3, dissolving 0.25 mg low molecular weight chitosan in 1mL of 1% (v/v) acetic acid, and performing vortex dissolution to obtain chitosan solution;
s4, adding 500 mu L of gossypol acetate drug suspension into the chitosan solution, and performing water bath ultrasonic treatment at room temperature of 300W to obtain gossypol acetate nanocrystalline drug;
s5, dialyzing the synthesized nanocrystalline medicine with deionized water, and removing free gossypol acetate and chitosan after dialysis.
Test example 1:
1. Transmission Electron Microscope (TEM) measurement
1.1 Sample
The gossypol acetate self-assembled nanocrystalline drug prepared in example 1.
1.2 Method
The gossypol acetate self-assembled nanocrystalline drug is properly diluted by double distilled water and then is adhered to a copper mesh with a supporting film, when the drops on the mesh are not dried, the solution is put into a phosphotungstic acid solution for negative dyeing, then the copper mesh is clamped by forceps, the excessive dye solution is sucked by filter paper, and the solution is dried at room temperature, so that the microsphere is in a examine and accept-grain form by a transmission electron microscope. A Transmission Electron Microscope (TEM) photograph of the gossypol acetate self-assembled nanocrystalline drug of example 1 is shown in FIG. 1.
Fig. 1 is a Transmission Electron Microscope (TEM) photograph of the self-assembled nano-crystalline drug of gossypol acetate prepared in example 1, and as shown in fig. 1, it can be seen that the self-assembled nano-crystalline drug of gossypol acetate prepared in example 1 has small and uniform particle size, which illustrates that the preparation method of the invention in example 1 can reduce agglomeration of the self-assembled nano-crystalline drug of gossypol acetate.
2. Hydrated particle size analysis
2.1 Sample
The gossypol acetate self-assembled nanocrystalline drugs prepared in examples 1-5 and comparative example 1.
2.2 Method
Taking the trace amount of the gossypol acetate self-assembled nanocrystalline medicines prepared in examples 1-5 and comparative example 1, diluting a sample with 10 mu mol/ml NaCl solution, placing the diluted sample into a sample cell of a granularity analyzer, setting operation parameters, repeating for 3 times, and storing data. The hydrated particle sizes of the gossypol acetate self-assembled nanocrystalline drugs of examples 1-5 and comparative example 1 are shown in Table 1.
TABLE 1 median value of hydrated particle size of gossypol acetate self-assembled nanocrystalline drug
Table 1 shows the median value of the hydration particle sizes of the gossypol acetate self-assembled nanocrystalline medicines prepared in examples 1-5 and comparative example 1, and it can be seen from Table 1 that the hydration particle sizes of the gossypol acetate self-assembled nanocrystalline medicines prepared in examples 1-5 reach nanoscale, and have good nano effect. The hydrated particle size of the gossypol acetate self-assembled nanocrystalline drug prepared in example 2 is smaller than that of the gossypol acetate self-assembled nanocrystalline drug prepared in example 1, which indicates that the use of ethanol as a good solvent to dissolve the gossypol acetate can reduce the particle size of the gossypol acetate self-assembled nanocrystalline drug. The hydrated particle size of the gossypol acetate self-assembled nanocrystalline drug prepared in example 3 is smaller than that of the gossypol acetate self-assembled nanocrystalline drug prepared in example 2, which indicates that increasing the concentration of the stabilizer solution can reduce the particle size of the gossypol acetate self-assembled nanocrystalline drug. The hydrated particle size of the gossypol acetate self-assembled nanocrystalline drug prepared in example 4 is smaller than that of the gossypol acetate self-assembled nanocrystalline drug prepared in example 3, which shows that the concentration of the stabilizer solution is increased, and the particle size of the gossypol acetate self-assembled nanocrystalline drug is further reduced. The hydration particle size of the gossypol acetate self-assembled nanocrystalline drug prepared in example 5 is smaller than that of the gossypol acetate self-assembled nanocrystalline drug prepared in example 4, which shows that the auxiliary agent and the stabilizer produce synergistic effect, and the stability of the gossypol acetate system in the crystallization induction stage is controlled, so that crystal nuclei generated by the gossypol acetate in the crystal nucleus formation stage are increased, and the crystal particle size is reduced. The hydration particle size of the gossypol acetate self-assembled nanocrystalline drug prepared in comparative example 1 is smaller than that of the gossypol acetate self-assembled nanocrystalline drug prepared in example 1, which shows that when the concentration of the stabilizer solution is 1-2 mg/mL and the volume ratio of the gossypol acetate drug suspension to the stabilizer solution is 1:1-10, the stabilizer can be used for completely covering the crystal nucleus surface of the gossypol acetate self-assembled nanocrystalline drug to form nano particles with uniform particle size, and the particle size of the gossypol acetate self-assembled nanocrystalline drug is reduced.
3. Hydrated particle size stability analysis
3.1 Sample
The gossypol acetate self-assembled nanocrystalline drugs prepared in examples 1-5 and comparative example 1.
3.2 Method
The gossypol acetate self-assembled nanocrystalline drugs prepared in examples 1 to 5 and comparative example 1 were placed in a refrigerator at 4℃and their particle size change was measured every 2 hours, while the drug concentration in the supernatant was measured to determine the drug leakage amount, and total observation was performed for 11 days. The results of the hydration particle size stability of the gossypol acetate self-assembled nanocrystalline drug of examples 1-5 are shown in Table 2.
Table 2 stability of hydrated particle size of gossypol acetate self-assembled nanocrystalline drug
Table 2 is a graph showing the stability of the hydrated particle size of the self-assembled nano-crystalline drug of gossypol acetate in example 3, and as shown in Table 2, it can be seen that the self-assembled nano-crystalline drug of gossypol acetate prepared in examples 1-5 after 11 days of standing still maintains stable hydrated particle size, demonstrating that the self-assembled nano-crystalline drug of gossypol acetate prepared by the preparation method of examples 1-5 of the present invention has good stability. The stability of the hydrated particle size of the gossypol acetate self-assembled nanocrystalline drug prepared in example 2 is superior to that of the gossypol acetate self-assembled nanocrystalline drug prepared in example 1, which indicates that the use of ethanol as a good solvent for dissolving the gossypol acetate can improve the stability of the hydrated particle size of the gossypol acetate self-assembled nanocrystalline drug. The stability of the hydrated particle size of the gossypol acetate self-assembled nanocrystalline drug prepared in example 3 is superior to that of the gossypol acetate self-assembled nanocrystalline drug prepared in example 2, which indicates that increasing the concentration of the stabilizer solution can increase the stability of the hydrated particle size of the gossypol acetate self-assembled nanocrystalline drug. The stability of the hydrated particle size of the gossypol acetate self-assembled nanocrystalline drug prepared in example 4 is superior to that of the gossypol acetate self-assembled nanocrystalline drug prepared in example 3, which indicates that the concentration of the stabilizer solution is increased, and the stability of the hydrated particle size of the gossypol acetate self-assembled nanocrystalline drug is further improved. The stability of the hydrated particle size of the gossypol acetate self-assembled nanocrystalline drug prepared in example 5 is superior to that of the gossypol acetate self-assembled nanocrystalline drug prepared in example 4, which shows that the auxiliary agent and the stabilizer produce synergistic effect, and the stability of the gossypol acetate system in the crystallization induction stage is controlled, so that the crystal nucleus formed by the gossypol acetate in the crystal nucleus formation stage is increased, the crystal particle size is reduced, and the stability of the hydrated particle size is improved. The stability of the hydration particle size of the gossypol acetate self-assembled nanocrystalline drug prepared in comparative example 1 is inferior to that of the gossypol acetate self-assembled nanocrystalline drug prepared in example 1, which indicates that when the concentration of the stabilizer solution is 1-2 mg/mL and the volume ratio of the gossypol acetate drug suspension to the stabilizer solution is 1:1-10, the stabilizer can be used for completely covering the surface of the crystal nucleus of the gossypol acetate self-assembled nanocrystalline drug to form nano particles with uniform particle size, and the stability of the hydration particle size of the gossypol acetate self-assembled nanocrystalline drug is increased.
4. Zeta potential detection
4.1 Sample
The gossypol acetate self-assembled nanocrystalline drug prepared in example 2.
4.2 Method
Under the condition of room temperature, the gossypol acetate self-assembled nanocrystalline drug prepared in the example 2 is diluted to a proper concentration by a proper amount of deionized water, and the Zeta potential of the gossypol acetate self-assembled nanocrystalline drug is measured by a Markov laser particle sizer. The Zeta potential of the gossypol acetate self-assembled nanocrystalline drug in example 2 is shown in fig. 2.
Fig. 2 is a graph showing the Zeta potential stability of the gossypol acetate self-assembled nanocrystalline drug in example 2, and as shown in fig. 2, it can be seen that the gossypol acetate self-assembled nanocrystalline drug prepared in example 2 after 11 days of placement still maintains stable Zeta potential, which indicates that the stability of the gossypol acetate self-assembled nanocrystalline drug prepared by the method of example 2 of the present invention is good.
5. In vitro tumor cell inhibition studies
5.1 Sample
The gossypol acetate self-assembled nanocrystalline drugs prepared in examples 1-5 and comparative example 1.
5.2 Method
Cell proliferation and toxicity were performed on a549 cells (CCK-8 method experiment, whereby the effect of cell viability was examined on the gossypol acetate self-assembled nanoparticles prepared in examples 1 to 5 and comparative example 1, as follows:
S1, cell culture
Human non-small cell lung cancer A549 cells were cultured in 1640 liquid medium containing 10% fetal bovine serum, and placed in an incubator at 37℃with 5% carbon dioxide.
S2, cell viability assay
Cells were seeded into 96-well plates at 2000 cells/well density. After the wall is attached to 24 h, the cells are divided into 3 groups, namely a blank group, a gossypol acetate group and a gossypol acetate nanoparticle group. The blank group is not treated, and the other components are respectively added with the gossypol acetate and the gossypol acetate nanocrystalline medicines with the same concentration, and the concentration of the gossypol acetate and the gossypol acetate nanocrystalline medicines is 8 uM. After 48 hours of drug treatment, the culture medium is removed; mu.L of a cell culture medium containing 10% (by volume) of CCK-8 was added to each well, and after incubation in an incubator for 2h hours, the absorbance at 450: 450nm was measured on a microplate reader. After subtracting the absorbance value of the blank solution from the absorbance value of each group, the absorbance value of each hole is divided by the absorbance value of the control group to obtain the cell viability. Each set was provided with 6 parallel wells.
S3, anti-tumor mechanism of gossypol acetate
At 48h of drug treatment time, the anti-tumor mechanism of gossypol acetate is to inhibit the growth of tumor cells by inhibiting ATP production in mitochondria. However, since the produced ATP has no effect, the cell viability is inhibited within 48 hours to a certain level. The cell viability results of the gossypol acetate self-assembled nanoparticles of examples 1-5 and comparative example 1 after 48h treatments are shown in FIG. 3.
FIG. 3 is a graph showing the results of the cell viability of A549 cells tested by CCK-8 method after treatment of 48h with the gossypol acetate self-assembled nanoparticles prepared in examples 1-5 and comparative example 1 at a concentration of 8 uM, as shown in FIG. 3, it can be seen that the gossypol acetate self-assembled nanoparticles prepared in examples 1-5 have a significant effect on the viability of A549 cells, and the inhibition rate of the gossypol acetate self-assembled nanoparticles on A549 cells is highest compared with that of the single gossypol acetate treatment and blank group, indicating that the gossypol acetate nanocrystals obtained by the preparation method of examples 1-5 of the present invention have high pharmaceutical activity. The inhibition effect of the gossypol acetate self-assembled nanocrystalline drug prepared in example 2 on tumor cells is superior to that of the gossypol acetate self-assembled nanocrystalline drug prepared in example 1, which shows that the use of ethanol as a good solvent for dissolving the gossypol acetate can improve the bioavailability of the gossypol acetate self-assembled nanocrystalline drug. The inhibition effect of the gossypol acetate self-assembled nanocrystalline drug prepared in example 3 on tumor cells is superior to that of the gossypol acetate self-assembled nanocrystalline drug prepared in example 2, which indicates that increasing the concentration of the stabilizer solution can increase the bioavailability of the gossypol acetate self-assembled nanocrystalline drug. The inhibition effect of the gossypol acetate self-assembled nanocrystalline drug prepared in example 4 on tumor cells is superior to that of the gossypol acetate self-assembled nanocrystalline drug prepared in example 3, which shows that the concentration of the stabilizer solution is increased, and the drug activity of the gossypol acetate self-assembled nanocrystalline drug is further improved. The inhibition effect of the gossypol acetate self-assembled nanocrystalline drug prepared in example 5 on tumor cells is superior to that of the gossypol acetate self-assembled nanocrystalline drug prepared in example 4, which shows that the auxiliary agent and the stabilizer produce synergistic effect, and the stability of the system of the gossypol acetate in the crystallization induction stage is controlled, so that the crystal nucleus formed by the gossypol acetate in the crystal nucleus formation stage is increased, the crystal grain size is reduced, the stability of the hydrated grain size is improved, and the bioavailability and the pharmaceutical activity are improved. The inhibition effect of the gossypol acetate self-assembled nanocrystalline drug prepared in comparative example 1 on tumor cells is worse than that of the gossypol acetate self-assembled nanocrystalline drug prepared in example 1, which shows that when the concentration of the stabilizer solution is 1-2 mg/mL and the volume ratio of the gossypol acetate drug suspension to the stabilizer solution is 1:1-10, the dosage of the stabilizer can be enough to completely cover the crystal nucleus surface of the gossypol acetate self-assembled nanocrystalline drug to form more nano particles with uniform particle size, and the drug activity is increased.
The above examples and/or embodiments are merely for illustrating the preferred embodiments and/or implementations of the present technology, and are not intended to limit the embodiments and implementations of the present technology in any way, and any person skilled in the art should be able to make some changes or modifications to the embodiments and/or implementations without departing from the scope of the technical means disclosed in the present disclosure, and it should be considered that the embodiments and implementations are substantially the same as the present technology.
Claims (10)
1. A preparation method of a gossypol acetate self-assembled nanocrystalline drug comprises the following steps: dissolving gossypol acetate in a good solvent to obtain a gossypol acetate drug solution, adding the gossypol acetate drug solution into the poor solvent, stirring to obtain a gossypol acetate drug suspension, and dissolving a stabilizer in an aqueous medium to obtain a stabilizer solution; mixing the gossypol acetate drug suspension with a stabilizer solution to obtain the gossypol acetate self-assembled nanocrystalline drug.
2. The method for preparing the gossypol acetate self-assembled nanocrystalline drug according to claim 1, wherein the good solvent comprises dichloromethane, chloroform, ethyl acetate, ethyl propionate, propyl acetate, acetone, methanol, dimethyl sulfoxide, acetonitrile, ethanol; the poor solvent includes water.
3. The method for preparing the gossypol acetate self-assembled nanocrystalline drug according to claim 1, wherein the concentration of the gossypol acetate drug solution is 1-100 mg/mL.
4. The method for preparing a gossypol acetate self-assembled nanocrystalline drug according to claim 1, wherein the stabilizer is any one of polyvinylpyrrolidone, polyethylene glycol, chitosan and hyaluronic acid.
5. The method for preparing a gossypol acetate self-assembled nanocrystalline drug according to claim 1, wherein the aqueous medium comprises water or acetic acid solution.
6. The method for preparing a gossypol acetate self-assembled nanocrystalline drug according to claim 1, wherein the concentration of the stabilizer solution is 1-2 mg/mL.
7. The method for preparing the gossypol acetate self-assembled nanocrystalline drug according to claim 1, wherein the concentration of the gossypol acetate drug suspension is 5-20% (v/v); the volume ratio of the gossypol acetate drug suspension to the stabilizer solution is 1:1-10.
8. The method for preparing the gossypol acetate self-assembled nanocrystalline drug according to claim 1, wherein the stirring is magnetic stirring, the magnetic stirring rotation speed is 50-2000 rpm, the magnetic stirring temperature is 4-50 ℃, and the magnetic stirring time is 0.1-5 h; the mixing is ultrasonic treatment under ice bath or room temperature; the ultrasonic condition is water bath ultrasonic or probe ultrasonic, and the ultrasonic power is 30-500W.
9. The gossypol acetate self-assembled nanocrystalline drug prepared by any one of the methods of claims 1-8.
10. Use of the gossypol acetate self-assembled nanocrystalline drug according to claim 9 in an injectable emulsion formulation.
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