CN105997941B

CN105997941B - Amphiphilic micelle nanocapsule and preparation method and application thereof

Info

Publication number: CN105997941B
Application number: CN201610320365.1A
Authority: CN
Inventors: 门毅; 徐文慧; 张文生
Original assignee: Beijing Normal University
Current assignee: Beijing Normal University
Priority date: 2016-05-14
Filing date: 2016-05-14
Publication date: 2020-01-14
Anticipated expiration: 2036-05-14
Also published as: CN105997941A

Abstract

The invention provides an amphiphilic micelle nanocapsule with a structure shown in a formula I, and a preparation method of the amphiphilic micelle nanocapsule. Meanwhile, the invention also provides application of the amphiphilic micelle nanocapsule in preparation of a drug-loaded micelle. The amphiphilic micelle nanocapsule provided by the invention has a core-shell structure with 'outer hydrophilic and inner hydrophobic', can effectively coat medicines, and has high encapsulation efficiency; the drug-loaded micelle can effectively improve the drug loading rate and the drug delivery rate of the drug, control the slow release of the drug, maintain the drug effect for a long time and improve the bioavailability of the drug in the drug delivery process.

Description

Amphiphilic micelle nanocapsule and preparation method and application thereof

Technical Field

The invention relates to the technical field of biomedical materials, in particular to an amphiphilic micelle nano-capsule and a preparation method and application thereof.

Background

The traditional Chinese medicine is a huge treasury, the curative effect of many traditional Chinese medicines not only withstands the inspection of long-term medical practice, but also is proved by modern scientific research institute, but the characteristics of some traditional Chinese medicines greatly limit the clinical application thereof. For example, panax notoginseng is a rare medicinal material in China, and the main active ingredient of the panax notoginseng is panax notoginseng saponins, so that the panax notoginseng saponins have the effects of expanding blood vessels, reducing myocardial oxygen consumption, prolonging blood coagulation time, regulating immunity, resisting cancer and the like; but it has the disadvantages of strong irritation, slightly bitter taste, easy moisture absorption, poor solubility and low bioavailability. The traditional oral preparations comprise panax notoginseng saponins tablets, panax notoginseng saponins granules and the like by taking the traditional oral preparation as a raw material, but the pharmacokinetics and in vivo metabolism studies show that: after the treatment by the traditional methods, the absorption rate of the panax notoginseng saponins is poor, and the elimination half-life period is longer.

Nanotechnology is continuously promoting technological progress, and thus has been widely used in the fields of information industry, aerospace, military, chemical industry, metallurgy, environmental protection, life science, and the like, due to the attention of more and more researchers. The nano drug delivery system is an application result of nano technology in the medical field, and refers to a drug delivery system with the particle diameter of 10-1000 nm, and the drug delivery system can enhance drug effect, reduce drug toxicity and change the in-vivo process of the drug, so that the specific targeting property and the release controllability of the drug are realized.

Aiming at the problems of poor absorption rate and long elimination half-life existing in the clinical application of traditional Chinese medicines, researchers try to use beta-cyclodextrin or polyethylene glycol-polylactic acid block copolymer, such as (PEG)₃PLA block copolymer as drug carrier, using nanometer drug delivery system to realize drug delivery in vivo. Compared with the traditional mode, the drug delivery mode has the advantages of improving the stability of the drug, and the slow release and controlled release of the drug, but still has the problems of low encapsulation efficiency and low drug-loading rate.

Disclosure of Invention

The invention aims to provide an amphiphilic micelle nanocapsule, a preparation method and application thereof, the amphiphilic micelle nanocapsule provided by the invention can effectively coat a medicament, and the encapsulation rate is high; the drug-loaded micelle prepared by taking the amphiphilic micelle nanocapsule as the carrier can effectively improve the drug-loaded capacity and the drug delivery rate of the drug in the drug delivery process, can control the slow release of the drug, maintains the drug effect for a long time and improves the bioavailability of the drug.

The technical scheme of the invention is as follows:

the invention provides an amphiphilic micelle nanocapsule, which has a structure shown in a formula I:

wherein x is 1-1000, y is 1-1000, and z is 1-1000.

Preferably, x is 2 to 700, y is 2 to 700, and z is 2 to 700.

The invention provides a preparation method of the amphiphilic micelle nanocapsule, which comprises the following steps:

(1) carrying out amination reaction on the beta-cyclodextrin to obtain aminated beta-cyclodextrin;

performing carboxylation reaction on methoxy polyethylene glycol-b-polylactide-glycolide to obtain carboxylated methoxy polyethylene glycol-b-polylactide-glycolide;

(2) and (2) reacting the carboxylated methoxy polyethylene glycol-b-polylactide-glycolide and the aminated beta-cyclodextrin obtained in the step (1) with N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in a protective atmosphere and a buffer solution to obtain the amphiphilic micelle nano-capsule with the structure shown in the formula I.

Preferably, the amination of the beta-cyclodextrin in the step (1) specifically includes the following steps:

mixing beta-cyclodextrin, paratoluensulfonyl chloride and a solvent I for reaction to obtain 6-paratoluensulfonyl-beta-cyclodextrin;

carrying out amination reaction on the 6-p-toluenesulfonyl-beta-cyclodextrin and an amination reagent to obtain aminated beta-cyclodextrin;

wherein the solvent I comprises sodium hydroxide solution or pyridine, and the amination reagent comprises a polyamine compound.

Preferably, the carboxylation reaction of the methoxy polyethylene glycol-b-polylactide-glycolide in the step (1) specifically comprises the following steps:

in a protective atmosphere, performing carboxylation reaction on methoxy polyethylene glycol-b-polylactide-glycolide, succinic anhydride, 4-dimethylamino pyridine and dichloromethane to obtain carboxylated methoxy polyethylene glycol-b-polylactide-glycolide.

Preferably, the mass ratio of the carboxylated methoxy polyethylene glycol-b-polylactide-glycolide, the aminated beta-cyclodextrin, the N-hydroxysuccinimide and the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the step (2) is 1: (1.5-3.5): (0.5-2.0): (3.0-7.0).

Preferably, the step (2) is specifically:

mixing the carboxylated methoxy polyethylene glycol-b-polylactide-glycolide, N-hydroxysuccinimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and a buffer solution, and carrying out an activation reaction in a protective atmosphere;

and adjusting the pH value of the activation reaction product to 6.7-7.3, mixing the activation reaction product with aminated beta-cyclodextrin, and reacting in a protective atmosphere to obtain the amphiphilic micelle nano-capsule with the structure shown in the formula I.

Preferably, the buffer solution in step (2) comprises a phosphate buffer solution or a tris hydrochloride buffer solution.

The invention provides an application of the amphiphilic micelle nanocapsule in the technical scheme or the amphiphilic micelle nanocapsule prepared by the method in the technical scheme in preparation of a drug-loaded micelle, wherein the drug-loaded micelle comprises an amphiphilic micelle nanocapsule carrier and a drug coated by the carrier.

Preferably, the medicament comprises notoginsenoside R1, notoginsenoside Rg1, adriamycin or andrographolide.

The invention provides an amphiphilic micelle nanocapsule with a structure shown in a formula I, and a preparation method of the amphiphilic micelle nanocapsule. The invention also provides application of the amphiphilic micelle nanocapsule in preparation of a drug-loaded micelle. The amphiphilic micelle nanocapsule provided by the invention has a core-shell structure with 'outer hydrophilic and inner hydrophobic', can effectively coat medicines, and has an encapsulation rate of 41.6%. The drug-carrying micelle provided by the invention has the drug-carrying capacity of 8.6%, and can effectively improve the drug delivery rate in the drug-carrying micelle administration process; meanwhile, the drug-loaded micelle provided by the invention can control the slow release of the drug, the time for maintaining the drug effect is as long as 48h, and the bioavailability of the drug can be effectively improved; in addition, the preparation method of the amphiphilic micelle nanocapsule is simple and convenient to operate, and the yield is up to 79.73%.

Detailed Description

wherein x is 1-1000, y is 1-1000, and z is 1-1000.

In the invention, x is preferably 2-700, more preferably 3-400, and most preferably 4-100; the y is preferably 2-700, more preferably 3-400, and most preferably 4-100: the z is preferably 2 to 700, more preferably 3 to 400, and most preferably 4 to 100.

The invention carries out amination reaction on beta-cyclodextrin (beta-CD) to obtain aminated beta-cyclodextrin (6-NH)₂- β -CD); methoxy polyethylene glycol diAnd (3) carrying out carboxylation reaction on alcohol-b-polylactide-glycolide (mPEG-PLGA) to obtain carboxylated methoxy polyethylene glycol-b-polylactide-glycolide (mPEG-PLGA-COOH).

In the present invention, the amination of β -cyclodextrin preferably specifically comprises the following steps:

mixing beta-cyclodextrin, p-toluenesulfonyl chloride (TsCl) and a solvent I for reaction to obtain 6-p-toluenesulfonyl-beta-cyclodextrin (6-OTs-beta-CD);

carrying out amination reaction on the 6-OTs-beta-CD and an amination reagent to obtain 6-NH₂-β-CD；

The invention mixes beta-CD, TsCl and solvent I to react to obtain 6-OTs-beta-CD. In the present invention, the mass ratio of β -CD, TsCl and solvent I is preferably 1: (4-12): (100-200), more preferably 1: (6-10): (120-140).

In the present invention, the solvent I preferably comprises sodium hydroxide solution or pyridine; the molar concentration of the sodium hydroxide solution is preferably 0.1-0.8 mol/L.

In the invention, the mixed reaction of the beta-CD, the TsCl and the solvent I is preferably specifically as follows:

dissolving beta-CD in a solvent I, stirring for 2-20 min to dissolve, adding part of TsCl into the obtained mixture, stirring to react for 1-3 h, adding the rest of TsCl, and continuously stirring to react for 2-8 h to obtain the 6-OTs-beta-CD.

The stirring mode adopted by the invention is not particularly limited, the technical scheme of stirring known to those skilled in the art can be adopted, and the magnetic stirring mode is preferably adopted for stirring in the invention. In the present invention, the rotation speed of the stirring is preferably 20 to 50rpm, and more preferably 30 to 40 rpm.

In the present invention, the portion of TsCl is preferably 40% to 60% by mass of TsCl.

According to the invention, after the reaction is completed, the obtained product is preferably subjected to post-treatment to obtain 6-OTs-beta-CD. In the present invention, the post-treatment preferably comprises the steps of:

after the reaction is finished, filtering, placing the obtained filtrate in an ice water bath, adjusting the pH value of the filtrate to be 5.8-6.2, stirring until the filtrate is turbid, refrigerating in a refrigerator at the temperature of 1-3 ℃ for 12-24 hours, and filtering again to obtain a 6-OTs-beta-CD crude product;

and mixing the crude 6-OTs-beta-CD product with secondary water, filtering while the crude product is hot after dissolution, refrigerating the obtained filtrate in a refrigerator at the temperature of 1-3 ℃ for 12-24 h for recrystallization, repeating the operation for 2-3 times, and vacuum-drying the recrystallized product at the temperature of 25-60 ℃ for 12-24 h to obtain the 6-OTs-beta-CD.

In the invention, the pH value of the filtrate is adjusted to 5.8-6.2 during the post-treatment. The method or the reagent for adjusting the pH value of the filtrate to 5.8-6.2 is not particularly limited, and the technical scheme for adjusting the pH value, which is well known to a person skilled in the art, can be adopted. According to the invention, the pH value of the filtrate is preferably adjusted to 5.8-6.2 by adopting 10-15% hydrochloric acid solution.

In the invention, after the pH value of the filtrate is adjusted to 5.8-6.2, stirring is carried out until the filtrate is turbid. The stirring mode adopted by the invention is not particularly limited, the technical scheme of stirring known to those skilled in the art can be adopted, and the magnetic stirring mode is preferably adopted for stirring in the invention. In the invention, the rotation speed of the stirring is preferably 20-50 rpm, and more preferably 30-40 rpm; the stirring time is preferably 4-6 h.

In the invention, the mixture is stirred until the mixture is turbid, and then refrigerated in a refrigerator at the temperature of 1-3 ℃ for 12-24 hours. In the invention, the refrigeration time is preferably 15-20 h.

In the present invention, after the crude 6-OTs-beta-CD is obtained, the crude 6-OTs-beta-CD is mixed with secondary water. In the invention, the mass ratio of the 6-OTs-beta-CD crude product to the secondary water is preferably 1: 90-110.

In the invention, the crude 6-OTs-beta-CD product is mixed with secondary water, dissolved and filtered while hot. In the present invention, the temperature of the dissolution is preferably 45 to 55 ℃.

In the invention, after the hot filtration is finished, the obtained filtrate is refrigerated in a refrigerator at the temperature of 1-3 ℃ for 12-24 h for recrystallization. In the present invention, the refrigerating time for recrystallization is preferably 15 to 20 hours.

In the invention, the product obtained by recrystallization is dried in vacuum for 12-24 h at 25-60 ℃ to obtain 6-OTs-beta-CD. In the invention, the temperature of the vacuum drying is preferably 35-45 ℃; the vacuum drying time is preferably 15-20 h; the vacuum degree of the vacuum drying is preferably 8-12 Pa.

After 6-OTs-beta-CD is obtained, the invention preferably carries out amination reaction on the 6-OTs-beta-CD and an amination reagent to obtain 6-NH₂- β -CD. In the present invention, the mass ratio of the 6-OTs- β -CD to the amination reagent is preferably 1: (50 to 150), more preferably 1: (70-120).

In the present invention, the amination reagent preferably includes a polyamine compound. In the present invention, the polyamine compound preferably includes ethylenediamine or triethylamine.

In the present invention, the amination reaction is preferably embodied as follows:

mixing the 6-OTs-beta-CD with an amination reagent, and carrying out amination reaction for 12-36 h at 50-85 ℃ under the stirring condition to obtain 6-NH₂-β-CD。

In the invention, the temperature of the amination reaction is preferably 60-70 ℃; the time of the amination reaction is preferably 18-28 h.

According to the invention, preferably, after the amination reaction is finished, the obtained product is subjected to post-treatment to obtain 6-NH₂- β -CD. In the present invention, the amination reaction post-treatment preferably comprises the steps of:

the amination reaction product is cooled to room temperature and then at 55And (4) at the temperature of 65 ℃ below zero, and performing rotary evaporation for 25-35 min. Standing, cooling to room temperature, adding precooled anhydrous ethanol into the obtained material, standing in ice water bath to obtain white precipitate, and filtering to obtain 6-NH₂-a crude beta-CD product;

reacting the 6-NH₂Dissolving the beta-CD crude product in ultrapure water, adding precooled absolute ethyl alcohol to obtain a white precipitate, filtering, repeating the operation for 2-3 times, and then drying in vacuum at the temperature of 20-50 ℃ for 2-3 days to obtain the 6-NH₂-β-CD。

Wherein, the 6-NH₂The mass ratio of the beta-CD crude product to the ultrapure water to the precooled absolute ethyl alcohol is preferably (1.8-2.2): (0.8-1.2): (180-220). In the invention, when the post-treatment of the amination reaction is carried out, the amination reaction product is steamed in a rotating way and stands still, the material obtained after cooling to room temperature is added with precooled absolute ethyl alcohol, the mixture stands still in ice water bath, white precipitate appears, and the 6-NH is obtained by filtering₂-beta-CD crude product. In the invention, the mass ratio of the material obtained after cooling to room temperature to the pre-cooled absolute ethyl alcohol is preferably 1: 90-110.

In the invention, the temperature of the precooled absolute ethyl alcohol is preferably 1-2 ℃.

In the invention, the standing time in the ice-water bath is preferably 2.5-3.5 h.

In the present invention, 6-NH is obtained₂After the crude product of-beta-CD, the 6-NH is added₂Dissolving the beta-CD crude product in ultrapure water, adding precooled absolute ethyl alcohol to obtain a white precipitate, filtering, repeating the operation for 2-3 times, and then drying in vacuum at the temperature of 20-50 ℃ for 2-3 days to obtain the 6-NH₂- β -CD. In the present invention, the 6-NH₂The mass ratio of the beta-CD crude product to the ultrapure water to the precooled absolute ethyl alcohol is preferably (1.8-2.2): (0.8-1.2): (180-220).

In the invention, the temperature of the vacuum drying is preferably 28-40 ℃; the vacuum degree of the vacuum drying is preferably 8-12 Pa.

In the present invention, the carboxylation reaction of methoxy polyethylene glycol-b-polylactide-glycolide preferably specifically comprises the following steps:

in a protective atmosphere, performing carboxylation reaction on methoxy polyethylene glycol-b-polylactide-glycolide (mPEG-PLGA), succinic anhydride, 4-dimethylaminopyridine and dichloromethane to obtain carboxylated methoxy polyethylene glycol-b-polylactide-glycolide (mPEG-PLGA-COOH).

In the present invention, the mass ratio of mPEG-PLGA, succinic anhydride, 4-dimethylaminopyridine and methylene chloride is preferably 1: (4.5-5.5): (0.25-0.5): (0.075-0.08).

According to the invention, before the mPEG-PLGA is subjected to a carboxylation reaction, succinic anhydride is preferably subjected to an activation treatment. In the present invention, the method for activating succinic anhydride preferably comprises the following steps:

succinic anhydride, 4-dimethylamino pyridine and dichloromethane are subjected to activation reaction in a protective atmosphere.

The type of the protective gas used in the protective atmosphere is not particularly limited in the present invention, and any gas known to those skilled in the art for use in protective atmospheres can be used. Nitrogen is preferably used as the shielding gas in the present invention. In the present invention, the activation reaction is preferably performed at room temperature, i.e., without heating or cooling; the time of the activation reaction is preferably 1-5 hours, and more preferably 2-4 hours.

After the succinic anhydride is activated, the activated reaction product and mPEG-PLGA preferably react in a protective atmosphere to obtain mPEG-PLGA-COOH.

The type of the protective gas used in the protective atmosphere is not particularly limited in the present invention, and any gas known to those skilled in the art for use in protective atmospheres can be used. Nitrogen is preferably used as the shielding gas in the present invention.

In the present invention, the reaction of the activated product with mPEG-PLGA is preferably carried out in an ice-water bath under stirring conditions. The stirring mode adopted by the invention is not particularly limited, the technical scheme of stirring known to those skilled in the art can be adopted, and the magnetic stirring mode is preferably adopted for stirring in the invention. In the present invention, the rotation speed of the stirring is preferably 20 to 50rpm, and more preferably 30 to 40 rpm.

In the invention, the reaction time of the activated product and mPEG-PLGA is preferably 12-48 h, and more preferably 20-35 h.

According to the invention, after the carboxylation reaction is completed, the obtained product is subjected to post-treatment to obtain mPEG-PLGA-COOH. In the present invention, the post-treatment of the carboxylation reaction preferably comprises the steps of:

centrifuging a product obtained after the carboxylation reaction is finished to obtain a supernatant;

washing and layering the supernatant for 2-3 times by using dilute hydrochloric acid and saturated sodium chloride solution respectively in sequence;

drying the lower layer liquid obtained by layering;

and filtering the dried product, mixing the obtained filtrate with pre-cooled anhydrous ether, sealing, refrigerating overnight in a refrigerator at 1-3 ℃, filtering again, and drying the filter cake in vacuum to obtain mPEG-PLGA-COOH.

When the post-treatment of the carboxylation reaction is carried out, the product obtained after the carboxylation reaction is finished is centrifuged to obtain supernatant. In the invention, the rotating speed of the centrifugation is preferably 8000-10000 rpm, more preferably 8500-9500 rpm; the time for centrifugation is preferably 5-10 min.

In the invention, after the supernatant is obtained, the supernatant is sequentially washed and layered for 2-3 times by using dilute hydrochloric acid and saturated sodium chloride solution respectively. In the invention, the pH value of the dilute hydrochloric acid is preferably 1.5-2.5. The present invention preferably performs the layering using a separatory funnel to yield the lower liquid layer.

In the present invention, after the layering is completed, the lower layer liquid obtained by the layering is dried. In the present invention, the drying is preferably carried out overnight using anhydrous sodium sulfate; wherein the anhydrous sodium sulfate is preferably added to the non-caking position.

In the invention, after the lower layer liquid obtained by layering is dried, the dried product is filtered, the obtained filtrate is mixed with precooled anhydrous ether, the mixture is refrigerated overnight at the temperature of-16 to-20 ℃ in a refrigerator after being sealed, and the filter cake is dried in vacuum after being filtered again, thus obtaining mPEG-PLGA-COOH. In the present invention, the volume ratio of the filtrate to the precooled anhydrous ether is preferably 1: (3-8).

In the present invention, the temperature of the pre-cooled dehydrated ether is preferably-16 to-20 ℃.

In the invention, the temperature of the vacuum drying is preferably 23-27 ℃; the vacuum drying time is preferably 20-28 h; the vacuum degree of the vacuum drying is preferably 8-12 Pa.

To obtain the 6-NH₂After the beta-CD and the mPEG-PLGA-COOH, the invention adds the mPEG-PLGA-COOH and 6-NH in a protective atmosphere and a buffer solution₂And (3) reacting the-beta-CD with N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to obtain the amphiphilic micelle nano-capsule (mPEG-PLGA-beta-CD) with the structure shown in the formula I.

In the invention, the mPEG-PLGA-COOH and 6-NH are₂The mass ratio of the-beta-CD, the N-hydroxysuccinimide and the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is preferably 1: (1.5-3.5): (0.5-2.0): (3.0 to 7.0), more preferably 1: (2.0-2.5): (1.0-1.5): (4.0-5.5).

In the present invention, the buffer solution preferably includes a phosphate buffer solution or a tris hydrochloride buffer solution. In the invention, the pH value of the phosphate buffer solution is preferably 5.8-6.2. In the invention, the pH value of the tris hydrochloride buffer solution is preferably 6.7-7.3.

In the present invention, the mPEG-PLGA-COOH and 6-NH are₂The reaction of beta-CD with N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is particularly preferably:

mixing the mPEG-PLGA-COOH, N-hydroxysuccinimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and a buffer solution, and carrying out an activation reaction in a protective atmosphere;

and adjusting the pH value of the activation reaction product to 6.7-7.3, then with 6-NH₂Mixing the beta-CD, and reacting in a protective atmosphere to obtain mPEG-PLGA-beta-CD.

The mPEG-PLGA-COOH, N-hydroxysuccinimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and a buffer solution are mixed and subjected to an activation reaction in a protective atmosphere. The type of the protective gas used in the protective atmosphere is not particularly limited in the present invention, and any gas known to those skilled in the art for use in protective atmospheres can be used. Nitrogen is preferably used as the shielding gas in the present invention.

In the invention, the time of the activation reaction is preferably 0.5-3.5 hours, and more preferably 1.0-2.5 hours.

In the present invention, the activation reaction is preferably carried out at room temperature with stirring. The stirring mode adopted by the invention is not particularly limited, the technical scheme of stirring known to those skilled in the art can be adopted, and the magnetic stirring mode is preferably adopted for stirring in the invention. In the present invention, the rotation speed of the stirring is preferably 20 to 50rpm, and more preferably 30 to 40 rpm.

After the activation reaction is finished, the pH value of a product after the activation reaction is adjusted to 6.7-7.3, and then the product is reacted with 6-NH₂Mixing the beta-CD, and reacting in a protective atmosphere to obtain mPEG-PLGA-beta-CD. According to the invention, a pH value regulator is preferably adopted to regulate the pH value of the product after the activation reaction is finished to be 6.7-7.3. In the present invention, the kind of the pH adjuster is not particularly limited, and a pH adjuster known to those skilled in the art may be used. The invention preferably uses saturated sodium hydroxide solution as the pH regulator.

In the present invention, the product after the activation reaction is reacted with 6-NH₂The reaction time of the beta-CD is preferably 3-9 h, and more preferably 4-7 h.

In the present invention, the product after the activation reaction is reacted with 6-NH₂The reaction of the-. beta. -CD is preferably carried out at room temperature with stirring. The stirring mode adopted by the invention is not particularly limited, the technical scheme of stirring known to those skilled in the art can be adopted, and the magnetic stirring mode is preferably adopted for stirring in the invention. In the present invention, the rotation speed of the stirring is preferably 20 to 50rpm, and more preferably 30 to 40 rpm.

The invention preferably involves reacting the product with 6-NH after the activation reaction₂And after the reaction of the-beta-CD is finished, carrying out post-treatment on the obtained product to obtain mPEG-PLGA-beta-CD. In the present invention, the product after the activation reaction is reacted with 6-NH₂The work-up of the-CD reaction preferably comprises the following steps:

dialyzing a product obtained after the reaction by adopting ultrapure water;

and (3) carrying out freeze drying on the dialyzate obtained by dialysis to obtain mPEG-PLGA-beta-CD.

In the present invention, the product after the activation reaction is reacted with 6-NH₂In the post-treatment of the-CD reaction, the product obtained after the reaction is dialyzed with ultrapure water. According to the invention, the reaction product is preferably placed in a dialysis bag for dialysis, and the dialysis time is preferably 12-48 h, and more preferably 20-35 h. In the present invention, the cut-off molecular weight of the dialysis bag is preferably 3500 Da. In the invention, before use, the dialysis bag is preferably boiled in boiling water for 20-40 min, and more preferably for 25-35 min; in the dialysis process, water is preferably replaced every 5-7 hours.

After the dialysis is completed, the dialysate obtained by the dialysis is subjected to freeze drying to obtain the mPEG-PLGA-beta-CD. In the present invention, the temperature of the freeze-drying is preferably-45 to-55 ℃; the vacuum degree of the freeze drying is preferably 8.0-10.0 Pa.

In the present invention, the mass ratio of the drug to the amphiphilic micelle nanocapsule is preferably 1: (10-15). In the present invention, the drug preferably comprises notoginsenoside R1, notoginsenoside Rg1, adriamycin or andrographolide.

The preparation method of the drug-loaded micelle is not specially limited, and the technical scheme for preparing the drug-loaded micelle known by the technical personnel in the field is adopted. The invention preferably adopts a dialysis method to prepare the drug-loaded micelle, and comprises the following steps:

dissolving the medicine and the amphiphilic micelle nanocapsule in methanol, dripping ultrapure water under the ultrasonic condition, and then carrying out ultrasonic treatment for 10-30 min; putting the obtained solution into a dialysis bag, and dialyzing for 12-48 h at room temperature; and freeze-drying the dialyzed solution to obtain the drug-loaded micelle.

In the present invention, the ratio of the mass of the drug to the volume of methanol and ultrapure water is preferably 10 mg: (0.5-2.0) mL: (2.0-4.0) mL.

In the invention, the speed of dripping the ultrapure water is preferably 0.25-0.35 mL/min.

In the invention, the power of the ultrasonic wave is preferably 280-320W.

In the present invention, the cut-off molecular weight of the dialysis bag is preferably 3500 Da. In the invention, before use, the dialysis bag is preferably boiled in boiling water for 20-40 min, and more preferably for 25-35 min; in the dialysis process, water is preferably replaced every 5-7 hours.

Preferably, water is replaced every 5-7 hours in the dialysis process.

In the present invention, the temperature of the freeze-drying is preferably-45 to-55 ℃; the vacuum degree of the freeze drying is preferably 8.0-10.0 Pa.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1:

drug-loaded micelle mPEG₂₀₀₀-PLGA₁₀₀₀Preparation of-beta-CD/R1 (x-45, y-7, z-7)

(1)6-NH₂Preparation of-beta-CD

Preparation of 6-OTs-beta-CD:

① preparation of crude product, in ice water bath, 1mmol of beta-CD is dissolved in 20mL of 0.2mol/L sodium hydroxide solution, after magnetic stirring at 35rpm for 5min, 2mmol of TsCl is added, after reaction for 2h, 2mmol of TsCl is added, and the reaction is continued for 3 h.

② separating the crude product, namely, the pH value of the solution after reaction is 13.26, filtering unreacted TsCl, putting the filtrate in an ice water bath, adjusting the pH value to 6.0, magnetically stirring at 35rpm until the solution is turbid, refrigerating at 2 ℃ for 24h in a refrigerator, and filtering to obtain the crude product of 6-OTs-beta-CD.

③ purifying the crude product by mixing the crude product of 6-OTs-beta-CD with secondary water, heating to dissolve, filtering while hot, refrigerating in a refrigerator at 2 deg.C for 12h to recrystallize, repeating the operation for 2 times, and vacuum drying the recrystallized product at 25 deg.C for 12h to obtain white powder of 6-OTs-beta-CD.

The resulting dry, clean sample, 6-OTs- β -CD, was weighed at 38.3% yield.

6-NH₂-preparation of β -CD:

① crude product was prepared by mixing 1.0mmol of 6-OTs-. beta. -CD with 60mmol of ethylenediamine and reacting at 60 ℃ with magnetic stirring for 24 h.

② separating crude product, cooling the product to room temperature, rotary steaming at 50 deg.C for 25min, standing, cooling to room temperature, adding 20mL precooled anhydrous ethanol into the obtained material, getting turbid system, standing in ice water bath, generating white precipitate, filtering to obtain 6-NH₂-beta-CD crude product.

③ purification of the crude product as 6-NH₂The mass ratio of the-beta-CD crude product to the ultrapure water to the precooled absolute ethyl alcohol is 2:1:200, and the obtained 6-NH₂Dissolving the crude product of the beta-CD in ultrapure water, adding precooled absolute ethyl alcohol to obtain white precipitate, filtering, repeating the operation for 2 times, and thenVacuum drying at 25 deg.C for 2 days to obtain light yellow powder 6-NH₂-β-CD。

Weighing the resulting dry neat sample 6-NH₂- β -CD, yield 80.16%.

(2) Carboxylated mPEG₂₀₀₀-PLGA₁₀₀₀(mPEG₂₀₀₀-PLGA₁₀₀₀Preparation of-COOH)

① activation of succinic anhydride 3.0mmol of succinic anhydride and 0.015g of 4-dimethylaminopyridine were dissolved in 5.0mL of dichloromethane and reacted at room temperature for 2h under nitrogen.

② preparation of crude product by mixing the activated material with 1.0mmol mPEG₂₀₀₀-PLGA₁₀₀₀OH (available from Jinan Dai Ting & Ting Bio-engineering Ltd.) was mixed and magnetically stirred for 24h at 35rpm in an ice water bath under nitrogen.

③ separating and purifying crude product, transferring all the products obtained after reaction into a centrifuge tube, centrifuging at 8000rpm for 10min, placing the supernatant into a separating funnel, washing with 15mL of dilute hydrochloric acid with pH 2 and 15mL of saturated sodium chloride solution for 2 times respectively, discharging the lower layer liquid after washing, adding anhydrous sodium sulfate to the position where no agglomeration occurs, drying overnight, filtering, adding 5 times volume of precooled anhydrous ether into the obtained filtrate, sealing, refrigerating at 2 deg.C overnight in a refrigerator, filtering, and vacuum drying the filter cake to obtain mPEG₂₀₀₀-PLGA₁₀₀₀-COOH。

The resulting dry, pure product mPEG was weighed₂₀₀₀-PLGA₁₀₀₀-COOH, yield 75.3%.

(3) Amphiphilic micelle nano-encapsulated mPEG₂₀₀₀-PLGA₁₀₀₀Preparation of-beta-CD

①mPEG₂₀₀₀-PLGA₁₀₀₀Activation of COOH: 1.0mmol mPEG₂₀₀₀-PLGA₁₀₀₀-COOH, 1.0mmol N-hydroxysuccinimide, 5.0mmol 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 6.0mL phosphate buffer pH 6.0 were mixed and the reaction magnetically stirred at room temperature 35rpm for 1h under nitrogen protection.

② preparation of crude product, which is obtained by regulating activation reaction with saturated sodium hydroxide solutionAfter the pH value of the reaction system of (1) was adjusted to 7.0, 2.5mmol of 6-NH was added₂beta-CD, magnetically stirring at 35rpm at room temperature under nitrogen protection for 5 h.

③ separating and purifying crude product, placing the product in dialysis bag boiled in boiling water for 30min, dialyzing with ultrapure water for 24h, changing water every 6h, freeze drying the obtained dialysate for 12h to obtain mPEG₂₀₀₀-PLGA₁₀₀₀-β-CD。

The resulting dry, pure product mPEG was weighed₂₀₀₀-PLGA₁₀₀₀beta-CD, yield 81.35%.

Subjecting the obtained product to¹HNMR analysis, the analysis result is: (H-1, 3.38, 3H), (H-2, H-3, 3.65, 4), (H-4, 4.82, 2H), (H-5, 5.20, 2H), (H-6, 1.55, 3H), (H-7, 2.81, 2H), (H-8, 2.84, 2H), (H-9, 2.00, 2H), (H-10, 2.31, 2H), the product was proved to be mPEG₂₀₀₀-PLGA₁₀₀₀-β-CD。

(4) Drug-loaded micelle mPEG₂₀₀₀-PLGA₁₀₀₀Preparation of-. beta. -CD/R1

Mixing 10mg of notoginsenoside R1 and 150mg of mPEG₂₀₀₀-PLGA₁₀₀₀Dissolving beta-CD in 1.0mL of methanol, dropwise adding 3mL of ultrapure water at the speed of 0.25mL/min under the ultrasonic condition of 300W, and further performing ultrasonic treatment for 20 min. The obtained solution is placed in a dialysis bag, and is dialyzed for 24 hours at room temperature, and water is replaced every 6 hours. Finally, freeze-drying the dialyzed solution at-50 ℃ and the vacuum degree of 9.0Pa to obtain the drug-loaded micelle mPEG₂₀₀₀-PLGA₁₀₀₀-β-CD/R1。

Weighing the obtained drug-loaded micelle mPEG₂₀₀₀-PLGA₁₀₀₀- β -CD/R1, yield 57.12%.

High performance liquid chromatography is adopted to determine the medicine carrying micelle mPEG₂₀₀₀-PLGA₁₀₀₀-drug loading and encapsulation efficiency of β -CD/R1, wherein the mobile phase: the organic phase is chromatographic pure acetonitrile, and the flow rate of the organic phase is 0.32 mL/min; the water phase is phosphoric acid water solution with the mass fraction of 0.05 percent, and the flow rate of the water phase is 1.28 mL/min. The drug-loaded micelle mPEG is calculated by using an external standard one-point method₂₀₀₀-PLGA₁₀₀₀Drug loading of beta-CD/R16.52 percent and the encapsulation efficiency is 36.2 percent.

The drug-loaded micelle mPEG prepared in example 1₂₀₀₀-PLGA₁₀₀₀- β -CD/R1 performing an in vitro release experiment comprising the steps of:

50mg of micelle mPEG for drug loading₂₀₀₀-PLGA₁₀₀₀Dissolving beta-CD/R1 in 1mL of phosphate buffer solution (pH 7.4), transferring the obtained drug-loaded micelle solution into a dialysis bag with the molecular weight cutoff of 1000Da, placing the dialysis bag into the phosphate buffer solution (pH 7.4), and dialyzing in a constant-temperature (T36.5 ℃) water bath shaking table.

At 0min, 5min, 15min, 30min, 45min, 1h, 2h, 4h, 6h, 8h, 10h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h and 108h, 1mL of dialysate was taken, and 1mL of newly prepared phosphate buffer (pH 7.4) was supplemented. Freeze-drying the dialysate, and testing the content of the drug by high performance liquid chromatography.

The test results show that the drug is still detectable at 48h release. The slow release of the drug can be realized by the drug-loaded micelle, and the drug effect can be maintained for a long time.

Example 2:

drug-loaded micelle mPEG₂₀₀₀-PLGA₂₀₀₀Preparation of-beta-CD/R1 (x-45, y-14, z-14)

(1)6-NH₂Preparation of-beta-CD

Preparation of 6-OTs-beta-CD:

① preparation of crude product, in ice water bath, 1mmol of beta-CD is dissolved in 20mL of 0.35mol/L sodium hydroxide solution, after magnetic stirring at 35rpm for 10min, 3mmol of TsCl is added, after reaction for 1.5h, 3mmol of TsCl is added, and the reaction is continued for 2.5 h.

② separating crude product, wherein the pH value of the solution after reaction is 12.69, filtering out unreacted TsCl, putting the filtrate in an ice water bath, adjusting the pH value to 6.0, magnetically stirring at 35rpm until the solution is turbid, refrigerating at 3 ℃ for 20h in a refrigerator, and filtering to obtain the crude product of 6-OTs-beta-CD.

③ purifying the crude product by mixing the crude product of 6-OTs-beta-CD with secondary water, heating to dissolve, filtering while hot, refrigerating in a refrigerator at 3 deg.C for 12h to recrystallize, repeating the operation for 2 times, and vacuum drying the recrystallized product at 25 deg.C for 12h to obtain white powder of 6-OTs-beta-CD.

The resulting dry, clean sample, 6-OTs- β -CD, was weighed to give a yield of 40.5%.

6-NH₂-preparation of β -CD:

① crude product was prepared by mixing 1.0mmol of 6-OTs-. beta. -CD with 65mmol of ethylenediamine and reacting at 55 ℃ with magnetic stirring for 20 h.

② separating crude product, cooling the product to room temperature, rotary steaming at 60 deg.C for 30min, standing, cooling to room temperature, adding 20mL precooled anhydrous ethanol into the obtained material, getting turbid system, standing in ice water bath, generating white precipitate, filtering to obtain 6-NH₂-beta-CD crude product.

③ purification of the crude product as 6-NH₂The mass ratio of the beta-CD crude product to the ultrapure water to the precooled absolute ethanol is 1.8: 1: 180, 6-NH to be obtained₂Dissolving the crude product of the beta-CD in ultrapure water, adding precooled absolute ethyl alcohol to obtain white precipitate, filtering, repeating the operation for 2 times, and then drying in vacuum at 35 ℃ for 1 day to obtain light yellow powder 6-NH₂-β-CD。

Weighing the resulting dry neat sample 6-NH₂- β -CD in 83.70% yield.

(2) Carboxylated mPEG₂₀₀₀-PLGA₂₀₀₀(mPEG₂₀₀₀-PLGA₂₀₀₀Preparation of-COOH)

① activation of succinic anhydride 2.5mmol of succinic anhydride and 0.014g of 4-dimethylaminopyridine were dissolved in 4.5mL of dichloromethane and reacted at room temperature for 3h under nitrogen.

② preparation of crude product by mixing the activated material with 1.0mmol mPEG₂₀₀₀-PLGA₂₀₀₀OH (available from Jinan Dai Ting & Ting Bio-engineering Ltd.) was mixed and magnetically stirred for 24h at 35rpm in an ice water bath under nitrogen.

③ separating and purifying the crude product by transferring all the products into centrifuge tube, centrifuging at 8000rpm for 10min, placing the supernatant into separating funnelEach of the two solutions was washed 2 times with 15mL of dilute hydrochloric acid having a pH of 2 and 15mL of saturated sodium chloride solution. After the washing was completed, the lower layer liquid was discharged, and anhydrous sodium sulfate was added to the non-blocking position and dried overnight. Filtering, adding 5 times volume of precooled anhydrous ether into the obtained filtrate, sealing, refrigerating at 3 deg.C overnight in a refrigerator, filtering, and vacuum drying the filter cake to obtain mPEG₂₀₀₀-PLGA₂₀₀₀-COOH。

The resulting dry, pure product mPEG was weighed₂₀₀₀-PLGA₂₀₀₀-COOH, yield 75.3%.

(3) Amphiphilic micelle nano-encapsulated mPEG₂₀₀₀-PLGA₂₀₀₀Preparation of-beta-CD

①mPEG₂₀₀₀-PLGA₂₀₀₀Activation of COOH: 1.0mmol mPEG₂₀₀₀-PLGA₂₀₀₀-COOH, 1.5mmol N-hydroxysuccinimide, 4.5mmol 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 5.5mL phosphate buffer pH 6.0 were mixed and the reaction magnetically stirred at room temperature 35rpm for 0.5h under nitrogen protection.

② preparation of crude product by adjusting pH of reaction system obtained after activation reaction to 7.0 with saturated sodium hydroxide solution, and adding 2.0mmol 6-NH₂beta-CD, magnetically stirring at 35rpm at room temperature under nitrogen protection for 6 h.

③ separating and purifying crude product, placing the product in dialysis bag boiled in boiling water for 30min, dialyzing with ultrapure water for 20h, changing water every 6h, freeze drying the obtained dialysate for 18h to obtain mPEG₂₀₀₀-PLGA₂₀₀₀-β-CD。

The resulting dry, pure product mPEG was weighed₂₀₀₀-PLGA₂₀₀₀- β -CD in 76.31% yield.

(4) Drug-loaded micelle mPEG₂₀₀₀-PLGA₂₀₀₀Preparation of-. beta. -CD/R1

Mixing 10mg notoginsenoside R1 and 100mg mPEG₂₀₀₀-PLGA₂₀₀₀Dissolving beta-CD in 1.5mL of methanol, dropwise adding 2.5mL of ultrapure water at the speed of 0.3mL/min under the ultrasonic condition of 300w, and further performing ultrasonic treatment for 10 min. Placing the obtained solution in dialysis bag, and thoroughly dissolving at room temperatureAnd (4) separating for 18h, and replacing water every 6 h. Finally, freeze-drying the dialyzed solution at-55 ℃ and the vacuum degree of 8.0Pa to obtain the drug-loaded micelle mPEG₂₀₀₀-PLGA₂₀₀₀-β-CD/R1。

Drug-loaded micelle mPEG₂₀₀₀-PLGA₂₀₀₀The yield of-beta-CD/R1 was 63.7%, the drug loading was 8.6%, and the encapsulation efficiency was 41.1%.

Example 3:

drug-loaded micelle mPEG₂₀₀₀-PLGA₁₀₀₀Preparation of-beta-CD/Rg 1 (x-45, y-7, z-7)

(1)6-NH₂Preparation of-beta-CD

Preparation of 6-OTs-beta-CD:

① preparation of crude product, in ice water bath, 1mmol of beta-CD is dissolved in 30mL of 0.4mol/L sodium hydroxide solution, after magnetic stirring at 35rpm for 5min, 2mmol of TsCl is added, after reaction for 1.5h, 3mmol of TsCl is added, and the reaction is continued for 3 h.

② separating crude product, wherein the pH value of the solution after reaction is 13.29, filtering out unreacted TsCl, putting the filtrate in an ice water bath, adjusting the pH value to 6.0, magnetically stirring at 35rpm until the solution is turbid, refrigerating at 3 ℃ for 24h in a refrigerator, and filtering to obtain the crude product of 6-OTs-beta-CD.

③ purifying the crude product by mixing the crude product of 6-OTs-beta-CD with secondary water, heating to dissolve, filtering while hot, refrigerating in a refrigerator at 3 deg.C for 24h to recrystallize, repeating the operation for 2 times, and vacuum drying the recrystallized product at 50 deg.C for 18h to obtain white powder 6-OTs-beta-CD.

The resulting dry, clean sample, 6-OTs- β -CD, was weighed at 45.4% yield.

6-NH₂-preparation of β -CD:

① crude product was prepared by mixing 1.0mmol of 6-OTs-. beta. -CD with 80mmol of ethylenediamine and reacting at 65 ℃ with magnetic stirring for 24 h.

② separating crude product, cooling the product to room temperature, rotary steaming at 65 deg.C for 35min, standing, cooling to room temperature, adding 30mL precooled anhydrous ethanol into the obtained material, getting turbid, standing in ice water bath, and getting the productWhite precipitate is filtered to obtain 6-NH₂-beta-CD crude product.

③ purification of the crude product as 6-NH₂The mass ratio of the beta-CD crude product to the ultrapure water to the precooled absolute ethanol is 2.2: 1: 220, 6-NH to be obtained₂Dissolving the crude product of the beta-CD in ultrapure water, adding precooled absolute ethyl alcohol, and obtaining 6-NH₂Dissolving the crude product of the beta-CD in ultrapure water, adding precooled absolute ethyl alcohol to obtain white precipitate, filtering, repeating the operation for 2 times, and then drying in vacuum at 50 ℃ for 1 day to obtain light yellow powder 6-NH₂-β-CD。

Weighing the resulting dry neat sample 6-NH₂- β -CD, yield 84.3%.

① activation of succinic anhydride 3.0mmol of succinic anhydride and 0.022g of 4-dimethylaminopyridine were dissolved in 5.0mL of dichloromethane and reacted at room temperature for 2h under nitrogen.

② preparation of crude product by mixing the activated material with 1.0mmol mPEG₂₀₀₀-PLGA₁₀₀₀OH (available from Jinan Dai Ting & Ting Bio-engineering Ltd.) was mixed and magnetically stirred for 20h at 35rpm in an ice water bath under nitrogen.

③ separating and purifying crude product, transferring all the products obtained after reaction into a centrifuge tube, centrifuging at 8000rpm for 10min, placing the supernatant into a separating funnel, washing with 15mL of dilute hydrochloric acid with pH 2 and 15mL of saturated sodium chloride solution for 2 times respectively, discharging the lower layer liquid after washing, adding anhydrous sodium sulfate to the position where no agglomeration occurs, drying overnight, filtering, adding 6 times volume of precooled anhydrous ether into the obtained filtrate, sealing, refrigerating at-18 deg.C overnight, filtering, and vacuum drying the filter cake to obtain mPEG₂₀₀₀-PLGA₁₀₀₀-COOH。

The resulting dry, pure product mPEG was weighed₂₀₀₀-PLGA₁₀₀₀-COOH, yield 70.6%.

①mPEG₂₀₀₀-PLGA₁₀₀₀Activation of COOH: 1.0mmol mPEG₂₀₀₀-PLGA₁₀₀₀-COOH, 2.0mmol N-hydroxysuccinimide, 5.0mmol 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 5.5mL phosphate buffer pH 6.0 were mixed and the reaction magnetically stirred at room temperature 35rpm for 0.5h under nitrogen protection.

③ separating and purifying crude product, placing the product in dialysis bag boiled in boiling water for 30min, dialyzing with ultrapure water for 24h, changing water every 6h, freeze drying the obtained dialysate for 18h to obtain mPEG₂₀₀₀-PLGA₁₀₀₀-β-CD。

The resulting dry, pure product mPEG was weighed₂₀₀₀-PLGA₁₀₀₀beta-CD, yield 76.3%.

(4) Drug-loaded micelle mPEG₂₀₀₀-PLGA₁₀₀₀Preparation of-beta-CD/Rg 1

Mixing 10mg of notoginsenoside Rg1 and 100mg of mPEG₂₀₀₀-PLGA₁₀₀₀Dissolving beta-CD in 1.0mL of methanol, dropwise adding 3mL of ultrapure water at the speed of 0.3mL/min under the ultrasonic condition of 300w, and further performing ultrasonic treatment for 15 min. The obtained solution is placed in a dialysis bag, and is dialyzed for 24 hours at room temperature, and water is replaced every 6 hours. Finally, freeze-drying the dialyzed solution at-50 ℃ and the vacuum degree of 8.0Pa to obtain the drug-loaded micelle mPEG₂₀₀₀-PLGA₁₀₀₀-β-CD/Rg1。

Drug-loaded micelle mPEG₂₀₀₀-PLGA₁₀₀₀The yield of the-beta-CD/Rg 1 was 59.7%, the drug loading was 8.3%, and the encapsulation efficiency was 41.2%.

Example 4:

drug-loaded micelle mPEG₂₀₀₀-PLGA₂₀₀₀Preparation of-beta-CD/Rg 1 (x-45, y-14, z-14)

(1)6-NH₂Preparation of-beta-CD

Preparation of 6-OTs-beta-CD:

① preparation of crude product, in ice water bath, 1mmol of beta-CD is dissolved in 35mL of 0.45mol/L sodium hydroxide solution, after magnetic stirring at 35rpm for 5min, 3mmol of TsCl is added, after 2h reaction, 3mmol of TsCl is added, and the reaction is continued for 3 h.

② separating the crude product, namely, the pH value of the solution after reaction is 13.26, filtering unreacted TsCl, putting the filtrate in an ice water bath, adjusting the pH value to 6.0, magnetically stirring at 35rpm until the solution is turbid, refrigerating at 3 ℃ for 24h in a refrigerator, and filtering to obtain the crude product of 6-OTs-beta-CD.

③ purifying the crude product by mixing the crude product of 6-OTs-beta-CD with secondary water, heating to dissolve, filtering while hot, refrigerating in a refrigerator at 3 deg.C for 24h to recrystallize, repeating the operation for 2 times, and vacuum drying the recrystallized product at 55 deg.C for 12h to obtain white powder 6-OTs-beta-CD.

The resulting dry, clean sample, 6-OTs- β -CD, was weighed to give a yield of 46.69%.

6-NH₂-preparation of β -CD:

① crude product was prepared by mixing 1.0mmol of 6-OTs-. beta. -CD with 100mmol of ethylenediamine and reacting at 65 ℃ with magnetic stirring for 24 h.

② separating crude product, cooling the product to room temperature, rotary steaming at 65 deg.C for 30min, standing, cooling to room temperature, adding 30mL precooled anhydrous ethanol into the obtained material, getting turbid system, standing in ice water bath, generating white precipitate, filtering to obtain 6-NH₂-beta-CD crude product.

③ purification of the crude product as 6-NH₂-the mass ratio of the beta-CD crude product, the ultrapure water and the precooled anhydrous ethanol is 2: 1.8: 200, the obtained 6-NH₂Dissolving the crude product of the beta-CD in ultrapure water, adding precooled absolute ethyl alcohol, and obtaining 6-NH₂Dissolving the crude product of the beta-CD in ultrapure water, adding precooled absolute ethyl alcohol to obtain white precipitate, filtering, repeating the operation for 2 times, and then drying in vacuum at 35 ℃ for 2 days to obtain light yellow powder 6-NH₂-β-CD。

The resulting dry clean sample 6 was weighed-NH₂- β -CD, yield 85.8%.

① activation of succinic anhydride 3.0mmol of succinic anhydride and 0.017g of 4-dimethylaminopyridine were dissolved in 5.0mL of dichloromethane and reacted at room temperature for 3h under nitrogen protection.

② preparation of crude product by mixing the activated material with 1.0mmol mPEG₂₀₀₀-PLGA₂₀₀₀OH (available from Jinan Dai Ting & Ting Bio-engineering Ltd.) was mixed and magnetically stirred for 20h at 35rpm in an ice water bath under nitrogen.

③ separating and purifying crude product, transferring all the products obtained after reaction into a centrifuge tube, centrifuging at 8000rpm for 10min, placing the supernatant into a separating funnel, washing with 15mL of dilute hydrochloric acid with pH 2 and 15mL of saturated sodium chloride solution for 2 times respectively, discharging the lower layer liquid after washing, adding anhydrous sodium sulfate to the position where no agglomeration occurs, drying overnight, filtering, adding 5 times volume of precooled anhydrous ether into the obtained filtrate, sealing, refrigerating at-18 deg.C overnight, filtering, and vacuum drying the filter cake to obtain mPEG₂₀₀₀-PLGA₂₀₀₀-COOH。

①mPEG₂₀₀₀-PLGA₂₀₀₀Activation of COOH: 1.0mmol mPEG₂₀₀₀-PLGA₂₀₀₀-COOH, 1.0mmol N-hydroxysuccinimide, 3.0mmol 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 5.5mL phosphate buffer pH 6.0 were mixed and the reaction magnetically stirred at room temperature 35rpm under nitrogen protection for 1.0 h.

② preparation of crude product by adjusting pH of reaction system obtained after activation reaction to 7.0 with saturated sodium hydroxide solution, and adding 2.0mmol 6-NH₂beta-CD, magnetically stirring at 30rpm at room temperature under nitrogen protection for 6 h.

③ separating and purifying crude product, placing the product in dialysis bag boiled in boiling water for 30min, dialyzing with ultrapure water for 20h, changing water every 6h, freeze drying the obtained dialysate for 24h to obtain mPEG₂₀₀₀-PLGA₂₀₀₀-β-CD。

The resulting dry, pure product mPEG was weighed₂₀₀₀-PLGA₂₀₀₀- β -CD in 78.91% yield.

(4) Drug-loaded micelle mPEG₂₀₀₀-PLGA₂₀₀₀Preparation of-beta-CD/Rg 1

Mixing 10mg of notoginsenoside Rg1 and 100mg of mPEG₂₀₀₀-PLGA₂₀₀₀Dissolving beta-CD in 1.0mL of methanol, dropwise adding 3mL of ultrapure water at the speed of 0.3mL/min under the ultrasonic condition of 300w, and further performing ultrasonic treatment for 20 min. The obtained solution is placed in a dialysis bag, and is dialyzed for 24 hours at room temperature, and water is replaced every 6 hours. Finally, freeze-drying the dialyzed solution at-50 ℃ and the vacuum degree of 8.0Pa to obtain the drug-loaded micelle mPEG₂₀₀₀-PLGA₂₀₀₀-β-CD/Rg1。

Drug-loaded micelle mPEG₂₀₀₀-PLGA₂₀₀₀The yield of the-beta-CD/Rg 1 was 68.7%, the drug loading was 8.1%, and the encapsulation efficiency was 41.6%.

Example 5:

drug-loaded micelle mPEG₅₀₀-PLGA₁₀₀₀₀Preparation of-beta-CD/R1 (x-10, y-69, z-86)

(1)6-NH₂Preparation of-beta-CD

Preparation of 6-OTs-beta-CD:

The resulting dry, clean sample, 6-OTs- β -CD, was weighed to give a yield of 40.51%.

6-NH₂-preparation of β -CD:

③ purification of the crude product as 6-NH₂-the mass ratio of the beta-CD crude product, the ultrapure water and the precooled anhydrous ethanol is 2:1:200, the obtained 6-NH₂Dissolving the crude product of the beta-CD in ultrapure water, adding precooled absolute ethyl alcohol to obtain white precipitate, filtering, repeating the operation for 2 times, and then drying in vacuum at 35 ℃ for 1 day to obtain light yellow powder 6-NH₂-β-CD。

Weighing the resulting dry neat sample 6-NH₂- β -CD in 83.70% yield.

(2) Carboxylated mPEG₅₀₀-PLGA₁₀₀₀₀(mPEG₅₀₀-PLGA₁₀₀₀₀Preparation of-COOH)

② preparation of crude product by mixing the activated material with 1.0mmol mPEG₅₀₀-PLGA₁₀₀₀₀OH (available from Jinan Dai Ting & Ting Bio-engineering Ltd.) was mixed and magnetically stirred for 24h at 35rpm in an ice water bath under nitrogen.

③ separation and purification of crude product by reacting the obtained productAll were transferred to a centrifuge tube, centrifuged at 8000rpm for 10min, the supernatant was placed in a separatory funnel, and washed 2 times with 15mL of dilute hydrochloric acid having a pH of 2 and 15mL of saturated sodium chloride solution, respectively. After the washing was completed, the lower layer liquid was discharged, and anhydrous sodium sulfate was added to the non-blocking position and dried overnight. Filtering, adding 5 times volume of precooled anhydrous ether into the obtained filtrate, sealing, refrigerating at 3 deg.C overnight in a refrigerator, filtering, and vacuum drying the filter cake to obtain mPEG₅₀₀-PLGA₁₀₀₀₀-COOH。

The resulting dry, pure product mPEG was weighed₅₀₀-PLGA₁₀₀₀₀-COOH, yield 75.83%.

(3) Amphiphilic micelle nano-encapsulated mPEG₅₀₀-PLGA₁₀₀₀₀Preparation of-beta-CD

①mPEG₅₀₀-PLGA₁₀₀₀₀Activation of COOH: 1.0mmol mPEG₅₀₀-PLGA₁₀₀₀₀-COOH, 1.5mmol N-hydroxysuccinimide, 4.5mmol 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 5.5mL phosphate buffer pH 6.0 were mixed and the reaction magnetically stirred at room temperature 35rpm for 0.5h under nitrogen protection.

③ separating and purifying crude product, placing the product in dialysis bag boiled in boiling water for 30min, dialyzing with ultrapure water for 20h, changing water every 6h, freeze drying the obtained dialysate for 18h to obtain mPEG₅₀₀-PLGA₁₀₀₀₀-β-CD。

The resulting dry, pure product mPEG was weighed₅₀₀-PLGA₁₀₀₀₀- β -CD in 76.31% yield.

(4) Drug-loaded micelle mPEG₅₀₀-PLGA₁₀₀₀₀Preparation of-. beta. -CD/R1

Mixing 10mg notoginsenoside R1 and 100mg mPEG₅₀₀-PLGA₁₀₀₀₀Dissolving beta-CD in 1.5mL of methanol, and dropwise adding 2.5 at a speed of 0.3mL/min under 300w of ultrasonic conditionsAnd 4, carrying out ultrasonic treatment for 10min by using mL of ultrapure water. The obtained solution is placed in a dialysis bag, and dialyzed for 18h at room temperature, and water is changed every 6 h. Finally, freeze-drying the dialyzed solution at-55 ℃ and the vacuum degree of 8.0Pa to obtain the drug-loaded micelle mPEG₅₀₀-PLGA₁₀₀₀₀-β-CD/R1。

Drug-loaded micelle mPEG₅₀₀-PLGA₁₀₀₀₀The yield of beta-CD/R1 was 63.74%, the drug loading was 8.6%, and the encapsulation efficiency was 41.1%.

Example 6:

drug-loaded micelle mPEG₁₀₀₀-PLGA₁₅₀₀₀Preparation of-beta-CD/R1 (x-22, y-104, z-129)

(1)6-NH₂Preparation of-beta-CD

Preparation of 6-OTs-beta-CD:

The resulting dry, clean sample, 6-OTs- β -CD, was weighed to give a yield of 68.55%.

6-NH₂-preparation of β -CD:

② separating crude product, cooling the product to room temperature, rotary steaming at 60 deg.C for 30min, standing, cooling to room temperature,adding 20mL of precooled absolute ethyl alcohol into the obtained material, enabling the system to become turbid, standing in ice water bath, generating white precipitate, and filtering to obtain 6-NH₂-beta-CD crude product.

③ purification of the crude product as 6-NH₂-the mass ratio of the beta-CD crude product, the ultrapure water and the precooled anhydrous ethanol is 2: 1.2: 200, the obtained 6-NH₂Dissolving the crude product of the beta-CD in ultrapure water, adding precooled absolute ethyl alcohol to obtain white precipitate, filtering, repeating the operation for 2 times, and then drying in vacuum at 35 ℃ for 1 day to obtain light yellow powder 6-NH₂-β-CD。

Weighing the resulting dry neat sample 6-NH₂- β -CD, yield 53.58%.

(2) Carboxylated mPEG₁₀₀₀-PLGA₁₅₀₀₀(mPEG₁₀₀₀-PLGA₁₅₀₀₀Preparation of-COOH)

② preparation of crude product by mixing the activated material with 1.0mmol mPEG₁₀₀₀-PLGA₁₅₀₀₀OH (available from Jinan Dai Ting & Ting Bio-engineering Ltd.) was mixed and magnetically stirred for 24h at 35rpm in an ice water bath under nitrogen.

③ separating and purifying crude product, transferring all the products obtained after reaction into a centrifuge tube, centrifuging at 8000rpm for 10min, placing the supernatant into a separating funnel, washing with 15mL of dilute hydrochloric acid with pH 2 and 15mL of saturated sodium chloride solution for 2 times respectively, discharging the lower layer liquid after washing, adding anhydrous sodium sulfate to the position where no agglomeration occurs, drying overnight, filtering, adding 5 times volume of precooled anhydrous ether into the obtained filtrate, sealing, refrigerating at 3 deg.C overnight, filtering, and vacuum drying the filter cake to obtain mPEG₁₀₀₀-PLGA₁₅₀₀₀-COOH。

The resulting dry, pure product mPEG was weighed₁₀₀₀-PLGA₁₅₀₀₀-COOH, yield 65.72%.

(3) Amphiphilic micelle nano-encapsulated mPEG₁₀₀₀-PLGA₁₅₀₀₀-β-Preparation of CD

①mPEG₁₀₀₀-PLGA₁₅₀₀₀Activation of COOH: 1.0mmol mPEG₁₀₀₀-PLGA₁₅₀₀₀-COOH, 1.5mmol n-hydroxysuccinimide, 4.5mmol 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 5.5mL phosphate buffer pH 6.0 were mixed and the reaction magnetically stirred at 35rpm for 0.5h at room temperature under nitrogen protection.

③ separating and purifying crude product, placing the product in dialysis bag boiled in boiling water for 30min, dialyzing with ultrapure water for 20h, changing water every 6h, freeze drying the obtained dialysate for 18h to obtain mPEG₁₀₀₀-PLGA₁₅₀₀₀-β-CD。

The resulting dry, pure product mPEG was weighed₁₀₀₀-PLGA₁₅₀₀₀- β -CD in 66.87% yield.

(4) Drug-loaded micelle mPEG₁₀₀₀-PLGA₁₅₀₀₀Preparation of-. beta. -CD/R1

Mixing 10mg notoginsenoside R1 and 100mg mPEG₁₀₀₀-PLGA₁₅₀₀₀Dissolving beta-CD in 1.5mL of methanol, dropwise adding 2.5mL of ultrapure water at the speed of 0.3mL/min under the ultrasonic condition of 300w, and further performing ultrasonic treatment for 10 min. The obtained solution is placed in a dialysis bag, and dialyzed for 18h at room temperature, and water is changed every 6 h. Finally, freeze-drying the dialyzed solution at-55 ℃ and the vacuum degree of 8.0Pa to obtain the drug-loaded micelle mPEG₁₀₀₀-PLGA₁₅₀₀₀-β-CD/R1。

Drug-loaded micelle mPEG₁₀₀₀-PLGA₁₅₀₀₀The yield of-beta-CD/R1 was 79.73%, the drug loading was 8.6%, and the encapsulation efficiency was 41.1%.

Example 7:

drug-loaded micelle mPEG₂₀₀₀-PLGA₂₀₀₀₀Preparation of-beta-CD/R1 (x-45, y-139, z-172)

(1)6-NH₂Preparation of-beta-CD

Preparation of 6-OTs-beta-CD:

The resulting dry, clean sample, 6-OTs- β -CD, was weighed at 75.15% yield.

6-NH₂-preparation of β -CD:

③ purification of the crude product as 6-NH₂-the mass ratio of the beta-CD crude product, the ultrapure water and the precooled anhydrous ethanol is 2:1: 190, reacting the obtained 6-NH₂Dissolving the crude product of the beta-CD in ultrapure water, adding precooled absolute ethyl alcohol to obtain white precipitate, filtering, repeating the operation for 2 times, and then drying in vacuum at 35 ℃ for 1 day to obtain light yellow powder 6-NH₂-β-CD。

Weighing the resulting dry neat sample 6-NH₂- β -CD in 63.48% yield.

(2) Carboxylated mPEG₂₀₀₀-PLGA₂₀₀₀₀(mPEG₂₀₀₀-PLGA₂₀₀₀₀Preparation of-COOH)

② preparation of crude product by mixing the activated material with 1.0mmol mPEG₂₀₀₀-PLGA₂₀₀₀₀OH (available from Jinan Dai Ting & Ting Bio-engineering Ltd.) was mixed and magnetically stirred for 24h at 35rpm in an ice water bath under nitrogen.

③ separating and purifying crude product, transferring all the products obtained after reaction into a centrifuge tube, centrifuging at 8000rpm for 10min, placing the supernatant into a separating funnel, washing with 15mL of dilute hydrochloric acid with pH 2 and 15mL of saturated sodium chloride solution for 2 times respectively, discharging the lower layer liquid after washing, adding anhydrous sodium sulfate to the position where no agglomeration occurs, drying overnight, filtering, adding 5 times volume of precooled anhydrous ether into the obtained filtrate, sealing, refrigerating at 3 deg.C overnight, filtering, and vacuum drying the filter cake to obtain mPEG₂₀₀₀-PLGA₂₀₀₀₀-COOH。

The resulting dry, pure product mPEG was weighed₂₀₀₀-PLGA₂₀₀₀₀-COOH, yield 70.16%.

(3) Amphiphilic micelle nano-encapsulated mPEG₂₀₀₀-PLGA₂₀₀₀₀Preparation of-beta-CD

①mPEG₂₀₀₀-PLGA₂₀₀₀₀Activation of COOH: 1.0mmol mPEG₂₀₀₀-PLGA₂₀₀₀₀-COOH, 1.5mmol n-hydroxysuccinimide, 4.5mmol 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 5.5mL phosphate buffer pH 6.0 were mixed and the reaction magnetically stirred at 35rpm for 0.5h at room temperature under nitrogen protection.

③ separation and purification of crude product:putting the product obtained after the reaction into a dialysis bag boiled by boiling water for 30min, dialyzing with ultrapure water for 20h, and replacing water every 6 h; freeze-drying the obtained dialysate for 18h to obtain mPEG₂₀₀₀-PLGA₂₀₀₀₀-β-CD。

The resulting dry, pure product mPEG was weighed₂₀₀₀-PLGA₂₀₀₀₀- β -CD, yield 67.56%.

(4) Drug-loaded micelle mPEG₂₀₀₀-PLGA₂₀₀₀₀Preparation of-. beta. -CD/R1

Mixing 10mg notoginsenoside R1 and 100mg mPEG₂₀₀₀-PLGA₂₀₀₀₀Dissolving beta-CD in 1.5mL of methanol, dropwise adding 2.5mL of ultrapure water at the speed of 0.3mL/min under the ultrasonic condition of 300w, and further performing ultrasonic treatment for 10 min. The obtained solution is placed in a dialysis bag, and dialyzed for 18h at room temperature, and water is changed every 6 h. Finally, freeze-drying the dialyzed solution at-55 ℃ and the vacuum degree of 8.0Pa to obtain the drug-loaded micelle mPEG₂₀₀₀-PLGA₂₀₀₀₀-β-CD/R1。

Drug-loaded micelle mPEG₂₀₀₀-PLGA₂₀₀₀₀The yield of-beta-CD/R1 was 63.25%, the drug loading was 8.6%, and the encapsulation efficiency was 41.1%.

Example 8:

drug-loaded micelle mPEG₃₀₀₀-PLGA₁₈₀₀₀Preparation of-beta-CD/R1 (x-68, y-125, z-155)

(1)6-NH₂Preparation of-beta-CD

Preparation of 6-OTs-beta-CD:

The resulting dry, clean sample, 6-OTs- β -CD, was weighed to give a yield of 55.32%.

6-NH₂-preparation of β -CD:

③ purification of the crude product as 6-NH₂The mass ratio of the beta-CD crude product to the ultrapure water to the precooled absolute ethanol is 1.9: 1:200, the obtained 6-NH₂Dissolving the crude product of the beta-CD in ultrapure water, adding precooled absolute ethyl alcohol to obtain white precipitate, filtering, repeating the operation for 2 times, and then drying in vacuum at 35 ℃ for 1 day to obtain light yellow powder 6-NH₂-β-CD。

Weighing the resulting dry neat sample 6-NH₂- β -CD, yield 58.25%.

(2) Carboxylated mPEG₃₀₀₀-PLGA₁₈₀₀₀(mPEG₃₀₀₀-PLGA₁₈₀₀₀Preparation of-COOH)

② preparation of crude product by mixing the activated material with 1.0mmol mPEG₃₀₀₀-PLGA₁₈₀₀₀OH (available from Jinan Dai Ting & Ting Bio-engineering Ltd.) was mixed and magnetically stirred for 24h at 35rpm in an ice water bath under nitrogen.

③ separating and purifying the crude product by transferring all the products into a centrifuge tube and separating at 8000rpmAfter 10min, the supernatant was placed in a separatory funnel and washed 2 times with 15mL of dilute hydrochloric acid having a pH of 2 and 15mL of saturated sodium chloride solution, respectively. After the washing was completed, the lower layer liquid was discharged, and anhydrous sodium sulfate was added to the non-blocking position and dried overnight. Filtering, adding 5 times volume of precooled anhydrous ether into the obtained filtrate, sealing, refrigerating at 3 deg.C overnight in a refrigerator, filtering, and vacuum drying the filter cake to obtain mPEG₃₀₀₀-PLGA₁₈₀₀₀-COOH。

The resulting dry, pure product mPEG was weighed₃₀₀₀-PLGA₁₈₀₀₀-COOH, yield 66.16%.

(3) Amphiphilic micelle nano-encapsulated mPEG₃₀₀₀-PLGA₁₈₀₀₀Preparation of-beta-CD

①mPEG₃₀₀₀-PLGA₁₈₀₀₀Activation of COOH: 1.0mmol mPEG₃₀₀₀-PLGA₁₈₀₀₀-COOH, 1.5mmol n-hydroxysuccinimide, 4.5mmol 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 5.5mL phosphate buffer pH 6.0 were mixed and the reaction magnetically stirred at 35rpm for 0.5h at room temperature under nitrogen protection.

③ separating and purifying crude product, placing the product in dialysis bag boiled in boiling water for 30min, dialyzing with ultrapure water for 20h, changing water every 6h, freeze drying the obtained dialysate for 18h to obtain mPEG₃₀₀₀-PLGA₁₈₀₀₀-β-CD。

The resulting dry, pure product mPEG was weighed₃₀₀₀-PLGA₁₈₀₀₀- β -CD in 57.56% yield.

(4) Drug-loaded micelle mPEG₃₀₀₀-PLGA₁₈₀₀₀Preparation of-. beta. -CD/R1

Mixing 10mg notoginsenoside R1 and 100mg mPEG₃₀₀₀-PLGA₁₈₀₀₀Dissolving beta-CD in 1.5mL of methanol, dropwise adding 2.5mL of ultrapure water at the speed of 0.3mL/min under the ultrasonic condition of 300w, and further performing ultrasonic treatment for 10 min. Will getThe obtained solution is placed in a dialysis bag, and is dialyzed for 18h at room temperature, and water is replaced every 6 h. Finally, freeze-drying the dialyzed solution at-55 ℃ and the vacuum degree of 8.0Pa to obtain the drug-loaded micelle mPEG₃₀₀₀-PLGA₁₈₀₀₀-β-CD/R1。

Drug-loaded micelle mPEG₃₀₀₀-PLGA₁₈₀₀₀The yield of-beta-CD/R1 was 68.74%, the drug loading was 8.6%, and the encapsulation efficiency was 41.1%.

Example 9:

drug-loaded micelle mPEG₅₀₀₀-PLGA₂₁₀₀₀Preparation of-beta-CD/R1 (x 113, y 146, z 181)

(1)6-NH₂Preparation of-beta-CD

Preparation of 6-OTs-beta-CD:

The resulting dry, clean sample, 6-OTs- β -CD, was weighed to give a yield of 60.32%.

6-NH₂-preparation of β -CD:

② separating crude product by cooling the product to room temperature, rotary steaming at 60 deg.C for 30min, standing, cooling to room temperature, adding 20mL of the obtained materialCold absolute ethanol to make the system turbid, standing in ice water bath to obtain white precipitate, and filtering to obtain 6-NH₂-beta-CD crude product.

Weighing the resulting dry neat sample 6-NH₂- β -CD, yield 51.98%.

(2) Carboxylated mPEG₅₀₀₀-PLGA₂₁₀₀₀(mPEG₅₀₀₀-PLGA₂₁₀₀₀Preparation of-COOH)

② preparation of crude product by mixing the activated material with 1.0mmol mPEG₅₀₀₀-PLGA₂₁₀₀₀OH (available from Jinan Dai Ting & Ting Bio-engineering Ltd.) was mixed and magnetically stirred for 24h at 35rpm in an ice water bath under nitrogen.

③ separating and purifying crude product, transferring all the products obtained after reaction into a centrifuge tube, centrifuging at 8000rpm for 10min, placing the supernatant into a separating funnel, washing with 15mL of dilute hydrochloric acid with pH 2 and 15mL of saturated sodium chloride solution for 2 times respectively, discharging the lower layer liquid after washing, adding anhydrous sodium sulfate to the position where no agglomeration occurs, drying overnight, filtering, adding 5 times volume of precooled anhydrous ether into the obtained filtrate, sealing, refrigerating at 3 deg.C overnight, filtering, and vacuum drying the filter cake to obtain mPEG₅₀₀₀-PLGA₂₁₀₀₀-COOH。

The resulting dry, pure product mPEG was weighed₅₀₀₀-PLGA₂₁₀₀₀-COOH, yield 58.63%.

(3) Amphiphilic micelle nano-encapsulated mPEG₅₀₀₀-PLGA₂₁₀₀₀Preparation of-beta-CD

①mPEG₅₀₀₀-PLGA₂₁₀₀₀Activation of COOH: 1.0mmol mPEG₅₀₀₀-PLGA₂₁₀₀₀-COOH, 1.5mmol n-hydroxysuccinimide, 4.5mmol 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 5.5mL phosphate buffer pH 6.0 were mixed and the reaction magnetically stirred at 35rpm for 0.5h at room temperature under nitrogen protection.

③ separating and purifying crude product, placing the product in dialysis bag boiled in boiling water for 30min, dialyzing with ultrapure water for 20h, changing water every 6h, freeze drying the obtained dialysate for 18h to obtain mPEG₅₀₀₀-PLGA₂₁₀₀₀-β-CD。

The resulting dry, pure product mPEG was weighed₅₀₀₀-PLGA₂₁₀₀₀- β -CD in 59.63% yield.

(4) Drug-loaded micelle mPEG₅₀₀₀-PLGA₂₁₀₀₀Preparation of-. beta. -CD/R1

Mixing 10mg notoginsenoside R1 and 100mg mPEG₅₀₀₀-PLGA₂₁₀₀₀Dissolving beta-CD in 1.5mL of methanol, dropwise adding 2.5mL of ultrapure water at the speed of 0.3mL/min under the ultrasonic condition of 300w, and further performing ultrasonic treatment for 10 min. The obtained solution is placed in a dialysis bag, and dialyzed for 18h at room temperature, and water is changed every 6 h. Finally, freeze-drying the dialyzed solution at-55 ℃ and the vacuum degree of 8.0Pa to obtain the drug-loaded micelle mPEG₅₀₀₀-PLGA₂₁₀₀₀-β-CD/R1。

Drug-loaded micelle mPEG₅₀₀₀-PLGA₂₁₀₀₀The yield of-beta-CD/R1 was 45.33%, the drug loading was 8.6%, and the encapsulation efficiency was 41.1%.

The embodiment shows that the amphiphilic micelle nanocapsule with the core-shell structure with the outer hydrophilic and inner hydrophobic properties can effectively coat the drug, and the encapsulation rate is up to 41.6%. The drug-carrying micelle provided by the invention has the drug-carrying capacity of 8.6%, and can effectively improve the drug delivery rate in the drug-carrying micelle administration process; meanwhile, the drug-loaded micelle provided by the invention can control the slow release of the drug, the time for maintaining the drug effect is as long as 48h, and the bioavailability of the drug can be effectively improved; in addition, the preparation method of the amphiphilic micelle nanocapsule is simple and convenient to operate, and the yield is up to 79.73%.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. Drug-loaded micelle mPEG₂₀₀₀-PLGA₁₀₀₀The preparation method of the-beta-CD/notoginsenoside R1 comprises the following steps:

(1)6-NH₂preparation of-beta-CD

Preparation of 6-OTs-beta-CD:

① preparation of crude product, in ice water bath, dissolving 1mmol of beta-CD in 20mL of 0.2mol/L sodium hydroxide solution, magnetically stirring at 35rpm for 5min, adding 2mmol of TsCl, reacting for 2h, adding 2mmol of TsCl, and continuing to react for 3 h;

② separating the crude product, namely, the pH value of the solution after reaction is 13.26, filtering unreacted TsCl, putting the filtrate in an ice water bath, adjusting the pH value to 6.0, magnetically stirring at 35rpm until the solution is turbid, refrigerating at 2 ℃ for 24h in a refrigerator, and filtering to obtain the crude product of 6-OTs-beta-CD;

③ purifying the crude product by mixing the crude product of 6-OTs-beta-CD with secondary water, heating to dissolve, filtering while hot, refrigerating in a refrigerator at 2 deg.C for 12h for recrystallization, repeating the operation for 2 times, and vacuum drying the recrystallized product at 25 deg.C for 12h to obtain white powder of 6-OTs-beta-CD;

6-NH₂-preparation of β -CD:

① preparation of crude product, mixing 1.0mmol of 6-OTs-beta-CD with 60mmol of ethylenediamine, and reacting for 24h under magnetic stirring at 60 ℃;

② separation of the crude product by cooling the product obtained after the reaction to room temperatureHeating, and rotary steaming at 50 deg.C for 25 min; standing, cooling to room temperature, adding 20mL precooled anhydrous ethanol into the obtained material, making the system turbid, standing in ice water bath, generating white precipitate, filtering to obtain 6-NH₂-a crude beta-CD product;

③ purification of the crude product as 6-NH₂The mass ratio of the-beta-CD crude product to the ultrapure water to the precooled absolute ethyl alcohol is 2:1:200, and the obtained 6-NH₂Dissolving the crude product of the beta-CD in ultrapure water, adding precooled absolute ethyl alcohol to obtain white precipitate, filtering, repeating the operation for 2 times, and then drying in vacuum at 25 ℃ for 2 days to obtain light yellow powder 6-NH₂-β-CD；

① activation of succinic anhydride, 3.0mmol of succinic anhydride and 0.015g of 4-dimethylamino pyridine are dissolved in 5.0mL of dichloromethane and reacted for 2h at room temperature under the protection of nitrogen;

② preparation of crude product by mixing the activated material with 1.0mmol mPEG₂₀₀₀-PLGA₁₀₀₀OH, mixing, and magnetically stirring for 24 hours at 35rpm in an ice water bath under the protection of nitrogen;

③ the crude product is separated and purified by transferring all the products obtained after the reaction into a centrifuge tube, centrifuging at 8000rpm for 10min, placing the supernatant into a separating funnel, washing respectively with 15mL of dilute hydrochloric acid with pH 2 and 15mL of saturated sodium chloride solution for 2 times, discharging the lower layer liquid after washing, adding anhydrous sodium sulfate until no agglomeration, drying overnight, filtering, adding 5 times volume of precooled anhydrous ether into the filtrate, sealing, refrigerating at 2 ℃ overnight in a refrigerator, filtering, and vacuum drying the filter cake to obtain mPEG₂₀₀₀-PLGA₁₀₀₀-COOH；

①mPEG₂₀₀₀-PLGA₁₀₀₀Activation of COOH: 1.0mmol mPEG₂₀₀₀-PLGA₁₀₀₀-COOH, 1.0mmol of N-hydroxysuccinimide, 5.0mmol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide saltMixing the acid salt with 6.0ml phosphate buffer solution with pH value of 6.0, and magnetically stirring at room temperature of 35rpm under the condition of nitrogen protection for reaction for 1 h;

② preparation of crude product by adjusting pH of reaction system obtained after activation reaction to 7.0 with saturated sodium hydroxide solution, and adding 2.5mmol 6-NH₂beta-CD, under the protection of nitrogen, magnetically stirring at 35rpm at room temperature for further reaction for 5 hours;

③ separating and purifying crude product, placing the product in dialysis bag boiled in boiling water for 30min, dialyzing with ultrapure water for 24h, changing water every 6h, freeze drying the obtained dialysate for 12h to obtain mPEG₂₀₀₀-PLGA₁₀₀₀-β-CD；

(4) Drug-loaded micelle mPEG₂₀₀₀-PLGA₁₀₀₀Preparation of-beta-CD/notoginsenoside R1

Mixing 10mg of notoginsenoside R1 and 150mg of mPEG₂₀₀₀-PLGA₁₀₀₀Dissolving beta-CD in 1.0mL of methanol, dripping 3mL of ultrapure water at the speed of 0.25mL/min under the ultrasonic condition of 300W, and then performing ultrasonic treatment for 20 min; putting the obtained solution into a dialysis bag, dialyzing for 24h at room temperature, and changing water every 6 h; finally, freeze-drying the dialyzed solution at-50 ℃ and the vacuum degree of 9.0Pa to obtain the drug-loaded micelle mPEG₂₀₀₀-PLGA₁₀₀₀-beta-CD/notoginsenoside R1.