A gamma-polyglutamic acid grafted cholesterol amphiphilic polymer and its preparation method and application

Abstract

The present invention belongs to the field of drug-loaded nanomicelles and relates to a γ-polyglutamic acid grafted cholesterol amphiphilic polymer and its preparation method and application. γ-polyglutamic acid is activated by 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and then subjected to an esterification reaction with cholesterol to obtain a γ-polyglutamic acid grafted cholesterol amphiphilic polymer, namely γ-PGA-g-CHOL. The γ-polyglutamic acid grafted cholesterol amphiphilic polymer is used to prepare cannabidiol-loaded nanomicelles by dialysis. The CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles prepared by the present invention significantly improve the solubility and bioavailability of CBD in water, and improve its transport efficiency and anti-inflammatory effect in the intestine.

Description

Technical Field

[0001] The invention belongs to the field of drug-loaded nano micelles and relates to a gamma-polyglutamic acid grafted cholesterol amphiphilic polymer and a preparation method and application thereof. Background Art

[0002] Cannabidiol (CBD), an extract from hemp, has multiple pharmacological effects, including anti-inflammatory, anti-epileptic, anti-cancer, and anti-coronavirus activity. [1-3] Compared with the homologous addictive ingredient Δ9-tetrahydrocannabinol, CBD has the advantages of being non-addictive and non-hallucinogenic. As the medical value of CBD is gradually discovered, as of January 2019, 41 countries in the world have declared medical marijuana legal, and more than 50 countries have declared CBD legal. my country also allowed the legal cultivation of industrial hemp in Yunnan and Heilongjiang provinces in 2010 and 2018 respectively. The relaxation of policies has brought opportunities for the development of medicinal marijuana. [4-6] From 2018 to 2020, the United States, the European Union, and Australia successively approved the high-purity oral liquid cannabidiol preparation developed by the British GW Pharmaceuticals. Listing [7] , which has set off a research boom on cannabidiol in many fields.

[0003] However, CBD is a highly lipophilic compound with extremely low water solubility. Its oral bioavailability is only 6%, which is affected by its poor solubility and large first-pass effect. This limits the clinical application of CBD. [8-10] In the existing literature reports at home and abroad, there are few studies on increasing the water solubility of CBD and improving oral bioavailability. Koch et al.

[11] The prepared CBD amorphous solid preparations have problems such as poor stability and low drug loading; Knaub et al.

[12] A CBD self-emulsifying drug delivery system (SEDDS-CBD) was developed. Although it improved the oral bioavailability of CBD, it has large individual absorption differences, complex preparations, and low safety.

[0004] Amphiphilic nanomicelles are a novel form of drug encapsulation. Their carrier material is an amphiphilic block copolymer with good biocompatibility and biodegradability. By encapsulating insoluble drugs through physical encapsulation and other methods, they can significantly improve the water solubility of drugs, have high safety, and have small particle size, strong tissue permeability and cell penetration, and also have the advantages of sustained and controlled release. [13-15] , which can be used as an excellent choice for CBD preparation research. The development of high-efficiency and low-toxic amphiphilic polymers has become the primary goal of constructing CBD nanomicelle drug delivery systems.

[0005] Natural polyamino acid materials have better tissue affinity, non-immunogenicity and biodegradability, and are more advantageous for use as drug carriers. [16-18]As one of the three natural polyamino acids discovered so far, γ-polyglutamic acid is a biopolymer material that can be synthesized by microbial fermentation. Its main chain is composed of D, L-glutamic acid monomers connected by γ-amide bonds to form a Nylon-4 structure. It can be degraded, absorbed, metabolized and excreted in the body and is not prone to accumulation and toxic side effects. [19-21] The free carboxyl groups on its side chains give it water solubility and chemical modification properties that are superior to those of natural polysaccharides. Preliminary studies have shown the application potential of γ-PGA in the field of drug delivery. In particular, the use of amphiphilic block grafted γ-PGA derivatives and the self-assembly of drug-loaded nanomicelles with a hydrophobic core and hydrophilic shell structure is a current research hotspot.

[0006] CBD is effectively loaded into amphiphilic nanomicelles and then orally administered to prepare the drug-loaded nanomicelles. After entering the body, it is mainly absorbed in the small intestine.

[22] . Caco-2 cells are derived from human colon adenocarcinoma cells, have a microvilli structure, and can produce enzymes related to the brush border epithelium of the small intestine. After cell culture and maturity, they can form a complete cell monolayer membrane. The structure and function are similar to those of small intestinal epithelial cells. It is internationally recognized as one of the best models for studying the absorption and transport functions of small intestinal epithelial cells. It has good stability and high reproducibility in in vitro culture. Compared with animal experiments, it is easy to operate, takes less time, and requires a small amount of sample. It is widely used in rapid screening of drug activity in vitro and scientific research on intestinal related issues. Therefore, it can be used as an in vitro model for the study of oral absorption of CBD amphiphilic nanomicelles.

[0007] Whether a biologically active compound can exert its effect in vivo depends largely on the drug concentration and bioavailability in plasma, and the route of administration has a significant impact on the drug's pharmacokinetic parameters. Studies have shown that oral administration greatly limits the oral bioavailability of CBD due to the first-pass effect, while sublingual, intravenous, and inhalation administration methods reduce the absorption of CBD in the gastrointestinal tract, thereby having a significant impact on the pharmacokinetic parameters of CBD.

[23] Therefore, studying the pharmacokinetic process of CBD and two drug-loaded nanomicelles in rats after oral administration can preliminarily evaluate the difference in oral bioavailability of CBD and CBD-loaded nanomicelles, and provide a theoretical basis for the clinical application of CBD preparations.

[0008] Therefore, it is urgent to develop water-soluble CBD amphiphilic nanomicelles, improve the bioavailability of CBD, enhance its intestinal transport efficiency and anti-inflammatory effect, and explore the absorption and anti-inflammatory mechanism of CBD and its amphiphilic nanomicelles. This study will provide strong theoretical support for the huge development of CBD in the medical field.

[0009] References

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[21] Ye Ran. Development and anti-tumor treatment of bee venom / polyglutamic acid liposome preparations[D]. Shanghai Jiao Tong University, 2019.

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[22] Franco V, Gershkovich P, Perucca E, et al. The interplay between liver first-pass effect and lymphaticabsorption of cannabidiol and its implications for cannabidiol oral formulations[J]. Clinical Pharmacokinetics, 2020, 59(12): 1493-1500.

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[23] Taylor L, Gidal B, Blakey G, et al. A phase I, randomized, double-blind, placebo-controlled, single ascending dose, multipledose, and food effecttrial of the safety, tolerability and pharmacokinetics of highly purified cannabidiol in healthy subjects [J]. CNS drugs, 2018, 32(11): 1053-1067. Summary of the Invention

[0033] The technical problem to be solved by the present invention is to provide a gamma-polyglutamic acid grafted cholesterol amphiphilic polymer and a preparation method and application thereof in view of the deficiencies in the prior art.

[0034] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0035] The present invention discloses a method for preparing a γ-polyglutamic acid grafted cholesterol amphiphilic polymer, comprising the following steps:

[0036] (1) uniformly mixing γ-polyglutamic acid and phosphate buffer to obtain a first mixed solution; adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the first mixed solution, and performing an activation reaction at room temperature for 40 to 60 minutes to obtain an activation solution;

[0037] (2) Cholesterol and dioxane are mixed uniformly to obtain a second mixed solution; the second mixed solution is added to the activation solution obtained in step (1), and an esterification reaction is carried out at room temperature in the dark for 16 to 32 hours; after the reaction is completed, the reaction solution is dialyzed, the dialyzate obtained is centrifuged, and the supernatant is freeze-dried to obtain a γ-polyglutamic acid grafted cholesterol amphiphilic polymer, namely γ-PGA-g-CHOL.

[0038] In some embodiments, the phosphate buffer has a pH of 5.4; the concentration of carboxyl groups in the first mixed solution is 0.15 to 0.30 mol·L -1 ; The molar ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the carboxyl group in the γ-polyglutamic acid is 1 to 2.5:1; the activation reaction has a reaction time of 50 minutes.

[0039] In some embodiments, preferably, the phosphate buffer has a pH of 5.4; the concentration of carboxyl groups in the first mixed solution is 0.25 mol·L -1 ; The molar ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the carboxyl group in the γ-polyglutamic acid is 2:1; the activation reaction has a reaction time of 50 minutes.

[0040] In some embodiments, the concentration of cholesterol in the second mixed solution is 0.1 to 0.6 mmol·mL -1 ; The molar ratio of the hydroxyl group in cholesterol in the second mixed solution to the carboxyl group in γ-polyglutamic acid in the first mixed solution is 2:1 to 1:1.5; the reaction time of the esterification reaction is 24h.

[0041] In some embodiments, preferably, the concentration of cholesterol in the second mixed solution is 0.15 mmol·mL -1 ; The molar ratio of hydroxyl groups in cholesterol in the second mixed solution to carboxyl groups in γ-polyglutamic acid in the first mixed solution is 1.5:1; the reaction time of the esterification reaction is 24h.

[0042] In some embodiments, the specific operation of the dialysis is: placing the reaction solution in a dialysis bag with a molecular weight cutoff of 10,000, dialyzing with deionized water for 36 hours, and changing the water every 2 hours; the specific operation of the dialysate centrifugation is: centrifugation at 5,000 rpm for 20 minutes.

[0043] The γ-polyglutamic acid grafted cholesterol amphiphilic polymer prepared by the above preparation method is also within the protection scope of the present invention.

[0044] The use of the above-mentioned γ-polyglutamic acid grafted cholesterol amphiphilic polymer in the preparation of cannabidiol-loaded drug-loaded nanomicelles is also within the scope of protection of the present invention.

[0045] Specifically, the use of a γ-polyglutamic acid grafted cholesterol amphiphilic polymer in the preparation of cannabidiol-loaded drug-loaded nanomicelles comprises the following steps:

[0046] (i) mixing the aforementioned γ-polyglutamic acid grafted cholesterol amphiphilic polymer with deionized water to obtain an amphiphilic polymer aqueous solution; oscillating the amphiphilic polymer aqueous solution at a constant temperature for 16 to 32 hours in an ice bath, and then subjecting the solution to ultrasonic treatment with a probe in an ice bath; centrifuging after the ultrasonic treatment, and filtering the supernatant with a filter membrane to obtain an amphiphilic polymer blank micelle solution; and freeze-drying the amphiphilic polymer blank micelle solution to obtain amphiphilic polymer blank micelles;

[0047] (ii) mixing cannabidiol with tetrahydrofuran to obtain a cannabidiol solution; mixing the amphiphilic polymer blank micelles obtained in step (i) with deionized water and stirring to obtain an amphiphilic polymer blank micelle aqueous solution; adding the cannabidiol solution to the amphiphilic polymer blank micelle aqueous solution, stirring at 300-400 rpm at room temperature for 5-10 hours, placing the reaction solution in a dialysis bag for dialysis after the stirring is completed, filtering the dialyzate, and freeze-drying to obtain cannabidiol-loaded drug nanomicelles.

[0048] Specifically, in step (i), the concentration of the γ-polyglutamic acid grafted cholesterol amphiphilic polymer in the amphiphilic polymer aqueous solution is 0.0141 mg·mL -1 ; The probe ultrasonic treatment has an ultrasonic power of 100 to 250 W and an ultrasonic time of 5 to 9 minutes; the centrifugal operation is centrifugation at 4000 rpm for 20 minutes; the supernatant membrane filtration is filtering the supernatant through a 0.45 μm microporous membrane.

[0049] Specifically, preferably, in step (i), the probe is ultrasonically treated with an ultrasonic power of 150 W and an ultrasonic time of 5 min.

[0050] Wherein, in step (1), preferably, the amphiphilic polymer aqueous solution is oscillated at a constant temperature of 4° C. for 24 hours.

[0051] Specifically, in step (i), the average particle size of the blank amphiphilic polymer micelles is 117.3±1.2 nm, and the Zeta potential is -20.1±1.5 mV.

[0052] Specifically, in step (ii), the concentration of cannabidiol in the cannabidiol solution is 1 to 3 mg·mL -1 The concentration of the amphiphilic polymer blank micelles in the aqueous solution is 3 to 5 mg mL -1 ; The volume ratio of tetrahydrofuran in the cannabidiol solution to deionized water in the amphiphilic polymer blank micelle aqueous solution is 1:5~7.

[0053] Specifically, preferably, in step (ii), the concentration of cannabidiol in the cannabidiol solution is 1 mg·mL -1 The concentration of the amphiphilic polymer blank micelles in the aqueous solution of the amphiphilic polymer blank micelles is 5 mg·mL -1 ; The volume ratio of tetrahydrofuran in the cannabidiol solution to deionized water in the amphiphilic polymer blank micelle aqueous solution is 1:5.

[0054] Wherein, preferably, the cannabidiol solution is added to the aqueous solution of blank micelles of the amphiphilic polymer and stirred at 400 rpm at 25° C. for 6 h.

[0055] Specifically, in step (ii), the cannabidiol-loaded nanomicelles had an encapsulation efficiency of 84.46% ± 0.35%, a drug loading of 8.78% ± 0.28%, an average particle size of 163.1 ± 2.3 nm, a polydispersity index PDI of 0.205 ± 0.048, and a Zeta potential of -16.5 ± 1.7 mV.

[0056] After stirring, the reaction solution was placed in a dialysis bag for dialysis. The specific operation of the dialysis was to place the reaction solution in a dialysis bag and dialyze it with deionized water for 24 hours, changing the water every 2 hours.

[0057] The dialysis bag used in the dialysis process had a MWCO of 3500Da and a batch number of 409C026, and was purchased from Jinkelong (Beijing) Biotechnology Co., Ltd.

[0058] Wherein, the dialysate filtration is filtering the dialysate through a 0.45 μm organic filter membrane.

[0059] Beneficial effects:

[0060] (1) The present invention successfully prepared the amphiphilic polymer γ-PGA-g-CHOL by grafting the side chain carboxyl group of γ-polyglutamic acid with the hydroxyl group in the cholesterol molecule. Combined with ultrasound-assisted self-assembly technology, the response surface method was used to optimize the preparation process of blank nanomicelles with particle size as an indicator. Then, the CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles were prepared by dialysis. The results of dynamic light scattering and scanning electron microscopy confirmed that the obtained blank nanomicelles and drug-loaded nanomicelles had appropriate particle sizes and concentrated distribution, stable structures, and spherical morphology.

[0061] (2) The CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles prepared by the present invention significantly improved the solubility of CBD in water; in vitro and in vivo experiments demonstrated that the preparation of CBD / (γ-PGA-g-CHOL) improved the bioavailability of CBD, its transport efficiency in the intestine, and its anti-inflammatory effect.

[0062] (3) The present invention found that the uptake and transport modes of CBD and CBD / (γ-PGA-g-CHOL) in Caco-2 cells include caveolin-dependent and clathrin-dependent endocytosis, passive diffusion, and active transport mediated by efflux proteins. The transport of CBD is affected by the efflux proteins BCRP and MRP2, while the transport of CBD / (γ-PGA-g-CHOL) is not affected by the efflux protein MRP2, but is affected by the efflux proteins BCRP and P-gp. In addition, CBD / (γ-PGA-g-CHOL) can significantly improve the LPS-induced inflammatory damage response of Caco-2 cells by inhibiting IL-8, IL-1β, and TNF-α cellular inflammatory factors, and can regulate cell viability and TEER values ​​to protect the intestinal mucosal barrier. The present invention provides a strong theoretical basis for the great development of CBD in the field of medicine.

[0063] (4) The present study found that the uptake and transport modes of CBD and CBD / (γ-PGA-g-CHOL) in Caco-2 cells include caveolin-dependent and clathrin-dependent endocytosis, passive diffusion, and efflux protein-mediated active transport. In addition, CBD / (γ-PGA-g-CHOL) can significantly improve inflammatory damage responses by inhibiting cellular inflammatory factors and protect the intestinal mucosal barrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more apparent.

[0065] Figure 1 The synthetic route of γ-PGA-g-CHOL.

[0066] Figure 2 Infrared spectra of γ-PGA, CHOL and γ-PGA-g-CHOL.

[0067] Figure 3 These are the hydrogen nuclear magnetic resonance spectra of γ-PGA and γ-PGA-g-CHOL.

[0068] Figure 4 Figure 2 is the fluorescence emission spectra of pyrene in γ-PGA-g-CHOL nanomicelle solutions with different mass concentrations.

[0069] Figure 5 For I 375 / I 385 and the logarithmic relationship between the mass concentration of γ-PGA-g-CHOL nanomicelles.

[0070] Figure 6 The effect of alcohol-acid ratio on the particle size and PDI of blank nanomicelles.

[0071] Figure 7 The effect of carboxyl concentration in γ-PGA on the particle size and PDI of blank nanomicelles.

[0072] Figure 8 This is the effect of activation reaction time on the particle size and PDI of blank nanomicelles.

[0073] Figure 9 This is the effect of ultrasonic power on the particle size and PDI of blank nanomicelles.

[0074] Figure 10 This is the effect of ultrasound time on the particle size and PDI of blank nanomicelles.

[0075] Figure 11 This is the multiple regression Pareto chart.

[0076] Figure 12 This is the particle size distribution diagram of γ-PGA-g-CHOL blank nanomicelles.

[0077] Figure 13 This is the potential diagram of γ-PGA-g-CHOL blank nanomicelles.

[0078] Figure 14 Scanning electron micrograph of γ-PGA-g-CHOL blank nanomicelles.

[0079] Figure 15 This is a standard curve chart of cannabidiol CBD.

[0080] Figure 16 This is the particle size distribution diagram of CBD / (γ-PGA-g-CHOL) nanomicelles.

[0081] Figure 17 This is the potential diagram of CBD / (γ-PGA-g-CHOL) nanomicelles.

[0082] Figure 18 This is a scanning electron micrograph of CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles.

[0083] Figure 19 The stability of CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles at room temperature or 37°C.

[0084] Figure 20 This is the in vitro release curve of CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles.

[0085] Figure 21 This is the effect of γ-PGA-g-CHOL blank nanomicelles, CBD and CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles on cell survival rate.

[0086] Figure 22 This is the effect of γ-PGA-g-CHOL blank nanomicelles, CBD and CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles on cell membrane integrity.

[0087] Figure 23 Diagram of the Caco-2 cell status at different growth stages in the Transwell chamber.

[0088] Figure 24 This is a detection diagram for measuring the transmembrane electrical resistance (TEER) of Caco-2 cells.

[0089] Figure 25 ALP activity was measured in Caco-2 cell monolayers.

[0090] Figure 26 This is a diagram showing the leakage of a marker (sodium fluorescein).

[0091] Figure 27 Figure 2 shows the effects of time, concentration, temperature and endocytosis inhibitors on the uptake characteristics of Caco-2 cells; Figure A shows time, Figure B shows concentration, Figure C shows temperature, and Figure D shows endocytosis inhibitors.

[0092] Figure 28 The results of in vivo pharmacokinetic experiments of CBD and CBD / (γ-PGA-g-CHOL); Figure A is the blood drug concentration-time curve, and Figure B is the pharmacokinetic parameters.

[0093] Figure 29 The inflammatory response of Caco-2 cells stimulated by different concentrations of LPS; A is the cell survival rate, B is the LDH activity, and C is the level of cellular inflammatory factors.

[0094] Figure 30 Effects of CBD and CBD / (γ-PGA-g-CHOL) on LPS-induced inflammatory response of Caco-2 cells; A is cell survival rate, B is LDH activity; *P<0.05, **P<0.01.

[0095] Figure 31 The effects of CBD and CBD / (γ-PGA-g-CHOL) on the TEER value of LPS-induced inflammatory response in Caco-2 cells.

[0096] Figure 32 The effects of CBD and CBD / (γ-PGA-g-CHOL) on the secretion levels of cellular inflammatory factors; A is IL-8, B is IL-1β, and C is TNF-α; among them, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0097] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0098] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0099] 1. Experimental materials:

[0100] γ-Polyglutamic acid (γ-PGA, MW = 20000-30000, analytical grade, batch number: P20211209016, Shanghai Linghan Scientific Instrument Co., Ltd.); cholesterol (CHOL, MW = 386.6535, analytical grade, batch number: C14217584, Shanghai MacLean Biochemical Technology Co., Ltd.); 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 98.5%, MW = 191.7, batch number: C14321643, Shanghai MacLean Biochemical Technology Co., Ltd.); 1, 4-Dioxane (analytical grade 99%, batch number: C13799035, Shanghai McLean Biochemical Technology Co., Ltd.); disodium hydrogen phosphate (analytical grade, batch number: 20210123, Tianjin Komiou Chemical Reagent Co., Ltd.); sodium dihydrogen phosphate (analytical grade, batch number: 20210123, Tianjin Beichen Fangzheng Chemical Reagent Factory); potassium bromide (spectrally pure, batch number: C14089606, Shanghai McLean Biochemical Technology Co., Ltd.); pyrene (97%, batch number: C12790936, Shanghai McLean Biochemical Technology Co., Ltd.); dialysis bag (MWCO 8000-14000Da, batch number: 409C021, Jinkelong (Beijing) Biotechnology Co., Ltd.); cannabidiol (batch number: 7003, Shanghai Shidande Standard Technology Service Co., Ltd.); potassium dihydrogen phosphate (analytical grade, batch number: 20070403, Xianshuigu Industrial Park, Jinnan District, Tianjin); dialysis bag (MWCO3500Da, batch number: 409C026, Jinkelong (Beijing) Biotechnology Co., Ltd.).

[0101] 2. Preparation of the phosphate buffer solution used in the embodiments of the present invention:

[0102] 0.2 mol·L -1 Sodium hydrogen phosphate solution and 0.2 mol·L -1 Preparation of sodium dihydrogen phosphate solution: Accurately weigh 3.1202 g of NaH2PO4·2H2O solid powder and 7.1628 g of Na2HPO4·12H2O solid powder into two 100 mL brown volumetric flasks, make up to volume, shake thoroughly and set aside.

[0103] Preparation of phosphate buffer (pH = 5.40): pipette appropriate amount of 0.2 mol·L -1 Sodium hydrogen phosphate solution and 0.2 mol·L -1 After the sodium dihydrogen phosphate solution is fully stirred, the pH value is measured by a pH meter at 5.40.

[0104] 3. Determination of drug loading and encapsulation efficiency of nanomicelles

[0105] Accurately weigh an appropriate amount of drug-loaded nanomicelles and dissolve them in 1 mL of ultrapure water. Add 9 mL of acetonitrile and mix thoroughly. Ultrasonicate for 30 min to disaggregate the drug-loaded nanomicelles and completely dissolve cannabidiol. Filter through a 0.22 μm microporous filter membrane and inject the sample according to the chromatographic conditions. Record the peak area and calculate the CBD concentration according to the CBD standard curve equation. Calculate the encapsulation efficiency and drug loading according to the following formula.

[0106]

[0107] The chromatographic conditions were as follows: column: Agilent Eclipse XDB-C18 (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile: 0.1 wt% phosphoric acid = 80:20; detection wavelength: 210 nm; column temperature: 30.0° C.; flow rate: 1.0 mL min -1 , injection volume: 20 μL, detection time: 20 min, isocratic elution.

[0108] The CBD standard curve was established by accurately weighing 25.00 mg of CBD standard substance in a 25 mL volumetric flask, adding acetonitrile to the scale line, and shaking well to obtain 1 g·L -1 Mother solution. Transfer appropriate amount of mother solution to 10mL volumetric flask, dilute to the mark with acetonitrile, and prepare 2μg·mL -1 , 5μg·mL -1 , 10 μg·mL -1 , 20 μg·mL -1 , 40 μg·mL -1 , 60 μg·mL -1 , 80 μg·mL -1 , 100 μg·mL -1 , 150 μg·mL -1 The acetonitrile solution of CBD was filtered through a 0.22μm organic microporous filter membrane and tested according to the chromatographic conditions. The linear regression equation of CBD was obtained by taking the different concentrations of the drug as the horizontal axis and the chromatographic peak area as the vertical axis. The CBD standard curve is shown in the figure below. Figure 15 As shown, the results showed that CBD was in the range of 2 to 200 μg·mL -1The linear relationship is good within the concentration range, and the CBD linear regression equation is: Y = 145610X - 47161, R 2 =0.9999.

[0109] Example 1: Synthesis of γ-polyglutamic acid grafted cholesterol amphiphilic polymer (γ-PGA-g-CHOL)

[0110] (1) Accurately weigh γ-PGA (MW = 20,000-30,000, 0.292 g, containing 2 mmol of carboxyl groups) in a 100 mL beaker, add 20 mL of phosphate buffer (pH = 5.40), and stir at room temperature for 30 min to obtain a first mixed solution. EDC·HCl (0.766 g, 4 mmol) was weighed and added to the first mixed solution. An activation reaction was carried out at room temperature for 40 min to obtain an activated solution.

[0111] (2) Cholesterol (1.159 g, 3 mmol) was fully dissolved in 20 mL of dioxane solution to obtain a second mixed solution; the second mixed solution was slowly added dropwise to the activation solution obtained in step (1), and the esterification reaction was carried out at room temperature in the dark with stirring for 24 h; after the reaction, the reaction solution was placed in a dialysis bag (molecular weight cut-off of 10,000) and dialyzed with deionized water for 36 h, with the water changed every 2 h to remove the reaction by-products, and the obtained dialyzate was centrifuged at 5,000 rpm for 20 min. The supernatant was freeze-dried to obtain a γ-polyglutamic acid grafted cholesterol amphiphilic polymer, i.e., γ-PGA-g-CHOL.

[0112] The synthetic route of γ-PGA-g-CHOL is as follows Figure 1 As shown, the free carboxyl groups of the γ-PGA side chain reacted with the hydroxyl groups of cholesterol after activation by EDC·HCl to form the amphiphilic copolymer γ-PGA-g-CHOL.

[0113] Example 2: Verification of γ-polyglutamic acid grafted cholesterol amphiphilic polymer (γ-PGA-g-CHOL)

[0114] (1) Infrared measurement: 2 mg each of γ-PGA (MW = 20,000-30,000), cholesterol, and γ-PGA-g-CHOL (prepared in Example 1) were weighed and mixed with potassium bromide and pressed into tablets. Infrared spectroscopy was performed using a NICOLET IS10 Fourier transform spectrometer to obtain absorption spectra.

[0115] Figure 2 The infrared spectra of γ-PGA, CHOL and γ-PGA-g-CHOL are shown in Figure 2. The peaks in the γ-PGA spectrum are assigned as follows: 3434 cm -1 (OH and NH symmetric stretching vibration superposition peak), 1610 cm -1Near (the superposition peak of the stretching vibration peak of -C=O in the amide group and the in-plane bending peak of NH), 1366cm -1 (CN stretching vibration peak), 1084 cm -1 (CO stretching vibration peak); the peaks in the cholesterol spectrum are assigned as follows: 3404cm -1 (OH stretching vibration peak), 2932~2866cm -1 (CH stretching vibration peak), 1466~1376cm -1 (CH bending vibration peak), 1056~1022cm -1 (CO stretching vibration peak); the peaks in the γ-PGA-g-CHOL polymer spectrum are assigned as follows: 1656~1588cm -1 (superimposed peak of amide absorption band I and amide absorption band II), 1171 cm -1 (CO stretching vibration peak) and 2970 cm -1 (CH stretching vibration peak); Among them, the infrared spectrum of γ-PGA-g-CHOL is 1656~1588cm compared with γ-PGA. -1 The superposition peak of amide absorption band I and amide absorption band II is enhanced, 1171 cm -1 The CO stretching vibration peak at position 3 was significantly enhanced and red-shifted, which proved that the carboxyl group of the γ-PGA side chain was successfully grafted onto the hydroxyl group at position 3 of cholesterol.

[0116] (2) H-NMR spectrum determination: 5 mg of γ-PGA (MW = 20000-30000) and 5 mg of γ-PGA-g-CHOL (prepared in Example 1) were weighed and dissolved in 0.5 mL of deuterated water (D2O). Each sample was transferred to a nuclear magnetic resonance tube and tested in a nuclear magnetic resonance spectrometer (operating frequency of 600 MHz) to obtain the corresponding material. 1 H-NMR spectra were analyzed using Mestrenova software.

[0117] Figure 3 The H NMR spectra of γ-PGA and γ-PGA-g-CHOL are shown. The peak at 4.79 ppm corresponds to the D2O solvent peak. The peak positions for γ-PGA are assigned as follows: 1.83-2.18 ppm (β, 2H), 2.22-2.44 ppm (γ, 2H), and 3.98-4.20 ppm (α, 1H). The area integrals of the H proton peaks at the α, β, and γ positions of the monomer are approximately 1:2:2, consistent with the γ-PGA structure. After grafting the cholesterol group, the methyl peak at 0.88 ppm is significantly enhanced, and the H at the 3-position of the cholesterol group appears in the product, with a downfield chemical shift to 4.57 ppm, confirming the successful synthesis of γ-PGA-CHOL.

[0118] Example 3: Preparation and process optimization of amphiphilic polymer blank micelles

[0119] (1) The amphiphilic polymer γ-PGA-g-CHOL used in this example was prepared according to the preparation method of Example 1, except that the molar ratio of hydroxyl groups in cholesterol to carboxyl groups in γ-PGA (hereinafter referred to as the alcohol-acid ratio) was changed to 2.5:1, 2:1, 1.5:1, 1:1, and 1:1.5, and the carboxyl group concentration of γ-polyglutamic acid in the first mixed solution was 0.15 mol·L -1 , 0.20mol·L -1 , 0.25 mol·L -1 , 0.30 mol·L -1 , 0.35 mol·L -1 The activation reaction time was 30 min, 40 min, 50 min, 60 min, and 70 min to obtain different batches of γ-PGA-g-CHOL, which were then used to prepare amphiphilic polymer blank micelles.

[0120] (2) Determination of critical micelle concentration of amphiphilic polymer: The critical micelle concentration of γ-PGA-g-CHOL was determined by steady-state fluorescence spectroscopy. Pyrene was used as a fluorescent probe and 6×10 -5 mol·L -1 100 μL of pyrene acetone solution was placed in a 15 mL stoppered test tube and the acetone was blown dry with nitrogen. -4 ~1mg·mL -1 Eight groups of gradient concentrations of amphiphilic polymer aqueous solutions (γ-PGA-g-CHOL prepared in Example 1) were added to stoppered test tubes to make the final concentration of pyrene 6.0×10 - 7 mol·L -1 After mixing evenly, ultrasonicate for 40 minutes and place at room temperature in the dark overnight. The fluorescence emission spectra of pyrene at different concentrations were measured using a F97PRO fluorescence spectrophotometer. The excitation wavelength was 334 nm, the excitation bandwidth and emission bandwidth were 10 nm and 5 nm respectively, and the fluorescence emission spectrum scanning range was 360-480 nm. The fluorescence intensity of pyrene I 375 / I 385 Plot the logarithm of the concentration to determine the critical micelle concentration.

[0121] like Figure 4 As shown in the figure, the first emission peak and the third emission peak of pyrene in the γ-PGA-g-CHOL solution are located at 375 nm and 385 nm, respectively, and the fluorescence intensity of pyrene increases with the increase of micelle concentration in the γ-PGA-g-CHOL micelle solution, indicating that the fluorescence quantum yield of pyrene increases with the increase of concentration.

[0122] like Figure 5 As shown, the mass concentration of γ-PGA-g-CHOL micelle solution increased from 0.0001 mg·mL -1 Increase to 0.05 mg mL -1 When I 375 / I 385 The decrease from about 1.78 to 1.51 indicates that pyrene is transferred from the polar aqueous solution to the hydrophobic core of the micelle with lower polarity. At this time, the critical micelle concentration (CMC) is 0.0141 mg·mL -1 , indicating that lower concentrations of γ-PGA-g-CHOL in aqueous solution can self-assemble to form thermodynamically stable micelles for drug encapsulation and release; Note: The critical micelle concentration is often used to evaluate the thermodynamic stability of micelles. The smaller the CMC, the easier it is to form stable micelles.

[0123] (3) Preparation of amphiphilic polymer blank micelles by ultrasonic probe-assisted self-assembly method and single factor investigation: 0.141 mg of γ-PGA-g-CHOL (γ-PGA-g-CHOL of different batches prepared in this example) was mixed with 10 mL of deionized water to obtain an amphiphilic polymer aqueous solution; the amphiphilic polymer aqueous solution was oscillated at a constant temperature at 4°C for 24 h, and then subjected to probe ultrasonic treatment under ice bath conditions (to prevent the temperature from being too high during the ultrasonic process, the ultrasonic conditions were set to on for 5 s and off for 5 s); after the ultrasonic treatment, the mixture was centrifuged at 4000 rpm for 20 min, and the supernatant was filtered through a 0.45 μm microporous membrane to obtain an amphiphilic polymer blank micelle solution; the amphiphilic polymer blank micelle solution was freeze-dried to obtain amphiphilic polymer blank micelles.

[0124] Among them, a single factor investigation was conducted: the molar ratio of hydroxyl groups in cholesterol to carboxyl groups in γ-PGA (hereinafter referred to as the alcohol-acid ratio) in the preparation process of γ-PGA-g-CHOL was 2.5:1, 2:1, 1.5:1, 1:1, and 1:1.5, and the carboxyl concentration of γ-polyglutamic acid in the first mixed solution was 0.15 mol·L -1 , 0.20mol·L -1 , 0.25 mol·L -1 , 0.30 mol·L -1 , 0.35 mol·L -1 The activation reaction time was 30min, 40min, 50min, 60min and 70min; the probe ultrasonic power (50W, 100W, 150W, 200W, 250W) and probe ultrasonic time (1min, 3min, 5min, 7min, 9min) during the preparation of amphiphilic polymer blank micelles were investigated, and the blank nanomicelle samples prepared under different conditions were obtained. The surface Zeta potential, particle size and polydispersity index PDI were determined using Zetasizer Nano ZS90.

[0125] (4) Single factor experimental results:

[0126] Effect of alcohol-acid ratio on particle size and PDI of blank nanomicelles: Figure 6 As shown, as the molar ratio of cholesterol decreases, the PDI value and particle size first decrease and then increase. This is because when the molar ratio of cholesterol is too high, the solution viscosity coefficient is large, the diffusion coefficient is extremely small, and the grafting reaction is poor, resulting in large particle size and PDI values. At a low molar ratio of cholesterol, the forward driving force of the reaction is insufficient, resulting in a low grafting rate, large particle size and PDI values. At an alcohol-to-acid ratio of 1.5:1, the PDI value and particle size both reach their minimum values, at 0.181±0.005 and 172.1±2.4 nm, respectively. Therefore, an alcohol-to-acid ratio of 1.5:1 is selected.

[0127] Effect of carboxyl concentration in γ-PGA in the first mixed solution on the particle size and PDI of blank nanomicelles: Figure 7 The concentration of carboxyl groups in γ-PGA is less than 0.25 mol·L -1 When the concentration of carboxyl groups in γ-PGA in the first mixed solution increases, the PDI value and particle size decrease. This is because as the concentration of carboxyl groups in γ-PGA increases, more side chain carboxyl groups in the reaction system are activated, which is conducive to the forward reaction. When the concentration of carboxyl groups in γ-PGA is greater than 0.25 mol·L -1 When the particle distance in the system is small, the solution viscosity coefficient is large, the solute molecular diffusion coefficient is reduced, the grafting effect of the product is reduced, and the particle size value is affected. Therefore, the concentration of carboxyl groups in γ-PGA is selected to be 0.25 mol·L -1 At this time, the particle size of the blank nanomicelles was 149.3±1.8nm, and the PDI was 0.161±0.006.

[0128] Effect of activation reaction time on blank nanomicelle particle size and PDI: Figure 8 As shown, activation reaction times between 30 and 70 minutes have little effect on particle size, with PDI values ​​remaining stable between 0.10 and 0.25. When activation times are below 50 minutes, fewer γ-PGA side chain carboxyl groups are activated. However, when activation times are above 50 minutes, the grafting rate decreases due to the decomposition of the activated intermediates, resulting in increased blank nanomicelle size and PDI values. EDC·HCl has the best activation effect on γ-PGA when the activation time is 50 minutes, so an activation time of 50 minutes was selected, resulting in a particle size of 136.5±1.3 nm and an optimal PDI value of 0.153±0.006.

[0129] Effect of ultrasonic power on the particle size and PDI of blank nanomicelles: During the ultrasonic self-assembly process of nanomicelles, the mechanical wave of the ultrasonic probe only plays an initiating auxiliary role, such as Figure 9 As shown in the figure, increasing the ultrasonic power enhances the self-assembly-assisted effect; however, excessive ultrasonic power destroys the nanomicelle structure in the system, leading to an increase in particle size and PDI. At an ultrasonic power of 150 W, the particle size was 132.7 ± 1.8 nm and the PDI was 0.155 ± 0.005, so 150 W was the selected ultrasonic power.

[0130] Effect of ultrasonic time on the particle size and PDI of blank nanomicelles: Figure 10 As shown, longer sonication times lead to a more robust self-assembly effect and smaller particle size. The particle size and PDI values ​​reach their lowest values ​​at 5 minutes. Further increases in sonication time increase these values, likely due to the partial destruction of the blank nanomicelle structure when sonication is prolonged. Therefore, a 5-minute sonication time was selected, achieving an optimal particle size of 120.2 ± 1.6 nm and a PDI of 0.140 ± 0.007.

[0131] (5) Plackett-Burman experiment and results

[0132] Based on the results of single-factor experiments, with the particle size of the blank γ-PGA-g-CHOL nanomicelles as the response value, five influencing factors were selected: the alcohol-acid ratio, the concentration of carboxyl groups in the first mixed solution of γ-polyglutamic acid, the activation reaction time, the ultrasonic power, and the ultrasonic time. Using the Plackett-Burman test, the key factors that significantly affected the particle size and PDI of the blank nanomicelles were screened. Each factor was set at either high (+1) or low (-1) levels, and 12 sets of experiments were conducted. The experimental factors and their levels are shown in Table 1.

[0133] Table 1 Factors and levels of the Plackett-Burman experiment

[0134]

[0135] The results of the Plackett-Burman test and the evaluation of the effects of various factors are shown in Tables 2 and 3. Table 3 shows that the significant factors affecting the particle size and PDI of the γ-PGA-g-CHOL blank nanomicelles are the carboxyl group concentration of the γ-PGA in the first mixed solution, the ultrasonication time, and the alcohol-to-acid ratio. Based on the P values, the order of importance is: carboxyl group concentration of the γ-PGA in the first mixed solution (P2) > ultrasonication time (P5) > alcohol-to-acid ratio (P1). The carboxyl group concentration of the γ-PGA in the first mixed solution has a highly significant effect on the particle size and PDI of the γ-PGA-g-CHOL blank nanomicelles (P < 0.01). Activation time and ultrasonic power are non-significant factors. Considering preparation efficiency and cost control, the ultrasonic power and activation time were fixed at 150 W and 50 min, respectively, in subsequent experiments.

[0136] Table 2 Plackett-Burman experimental design and results

[0137]

[0138] Table 3 Evaluation of the effects of each factor in the Plackett-Burman experiment

[0139]

[0140]

[0141] Note: * indicates significant difference (P<0.05), ** indicates extremely significant difference (P<0.01).

[0142] The results of the Plackett-Burman experiment were analyzed according to the Pareto law, and the multivariate regression Pareto arrangement chart was generated using Minitab 2018 software. Figure 11 The carboxyl concentration of γ-PGA, ultrasonic time and alcohol-acid ratio in the first mixed solution have a great influence on the particle size and PDI of γ-PGA-g-CHOL blank nanomicelles. Therefore, these three factors were selected as significant factors for the steepest climbing test and Box-Behnken experiment.

[0143] (6) Steepest climbing test

[0144] Regression analysis of the data in Table 2 yielded the fitting equation: Y1 = 159.67 - 6.28P1 - 11.11P2 + 2.73P3 + 1.08P4 - 7.28P5. The direction of the steepest ramp experiment was designed based on the sign of the coefficients of each factor in the fitting equation, and the step size was determined based on the results of the single-factor experiment. Table 4 shows the steepest ramp experiment design and results. Experiment 3 yielded the smallest blank micelle size, so experimental condition 3 was selected as the zero level for the Box-Behnken experiment.

[0145] Table 4 Steepest climbing experiment design and results

[0146]

[0147] (7) Response surface optimization experiment

[0148] Based on the results of the Plackett-Burman and steepest-hill-crossing experiments, a three-factor, three-level Box-Behnken experiment was conducted to optimize the preparation process of blank γ-PGA-g-CHOL nanomicelles using particle size as the response value (Y) for the three selected factors: the alcohol-to-acid ratio, the carboxyl group concentration of γ-PGA in the first mixed solution, and the ultrasonication time. The experimental factors and levels are shown in Table 5.

[0149] Table 5 Factors and levels of Box-Behnken design

[0150]

[0151] Establishment of regression model and analysis of variance:

[0152] Based on the results of the Plackett-Burman and steepest-hill-scale experiments, a total of 17 experiments were designed using the Box-Behnken principle, with three factors and three levels, at fixed ultrasonic power of 150 W and activation time of 50 min. The alcohol-to-acid ratio (P1), the carboxyl group concentration of γ-PGA in the first mixed solution (P2), and the ultrasonic time (P5) were selected as response factors, and the particle size of the blank nanomicelles (Y) was used as the response value. The response surface optimization experimental design and response value results are shown in Table 6.

[0153] The experimental data in Table 6 were subjected to regression analysis using Design-Expert 13.0.1.0 software, and the fitting equation was obtained as follows: Y = 116.16 - 1.95P1 - 2.68P2 + 2.05P5 - 2.00P1P2 - 2.85P1P5 + 0.25P2P5 + 12.00P12 + 18.34P22 + 9.84P52.

[0154] As shown in Table 7, the model F value is 63.04, P < 0.0001, indicating that the model is extremely significant. The model lack of fit P = 0.9178 (P > 0.05), the lack of fit term is not significant, indicating that the model has a good fit and can predict the particle size of γ-PGA-g-CHOL blank nanomicelles within the range of experimental variables. The model determination coefficient R 2 =0.9878, the adjusted determination coefficient R 2 Adj =0.9721, indicating that the model can explain 97.21% of the variation in the particle size of blank nanomicelles. The model can be used to analyze and predict the optimal preparation process of blank nanomicelles. It can also be seen from Table 7 that the linear terms P1, P2, P5, the interaction terms P1P5 and the quadratic term P1 2 、P2 2 、P5 2 The effect on the particle size of blank nanomicelles was significant (P<0.05). According to the F value, the three single factors that significantly affected the particle size of γ-PGA-g-CHOL blank nanomicelles were ranked from largest to smallest as follows: P2 (carboxyl group concentration of γ-PGA in the first mixed solution) > P5 (ultrasound time) > P1 (alcohol-acid ratio).

[0155] Table 6 Response surface optimization experimental design and experimental results

[0156]

[0157] Table 7 Response surface variance analysis table

[0158]

[0159]

[0160] Note: * indicates significant difference (P<0.05), ** indicates extremely significant difference (P<0.01).

[0161] Optimization process verification experiment:

[0162] The optimal preparation process conditions were predicted by the experimental results processed by Design-Expert 13.0.1.0 software: the ultrasonic power and activation reaction time were fixed at 150 W and 50 min, respectively, the alcohol-acid ratio was 1.538:1, and the concentration of carboxyl groups in γ-PGA in the first mixed solution was 0.254 mol·L -1 , ultrasonic time was 4.811 min, and the optimal particle size of γ-PGA-g-CHOL blank nanomicelles was predicted to be 115.8 nm.

[0163] Based on the feasibility of actual experimental operation, the above predicted process parameters were adjusted as follows: the alcohol-acid ratio was 1.5:1, the concentration of carboxyl groups in γ-PGA in the first mixed solution was 0.25 mol·L -1 , ultrasonic time 5min. In order to verify the optimized process conditions, three parallel experiments were carried out, and the average particle size of γ-PGA-g-CHOL blank nanomicelles was measured to be 117.3±1.2nm, which was close to the predicted value of 115.8nm, indicating that the model is accurate and the process conditions are highly practical.

[0164] (8) Particle size distribution and Zeta potential determination of blank nanomicelles

[0165] Preparation of γ-PGA-g-CHOL blank nanomicelle sample: The preparation method of γ-PGA-g-CHOL was the same as that of Example 1, wherein the alcohol-acid ratio was 1.5:1, and the concentration of carboxyl groups in γ-PGA in the first mixed solution was 0.25 mol·L -1 , the activation reaction time is 50 min; the preparation of γ-PGA-g-CHOL blank nanomicelles is the same as that in Example 3 "(3) Preparation of amphiphilic polymer blank micelles by ultrasonic probe-assisted self-assembly method", wherein the ultrasonic power is 150 W and the ultrasonic time is 5 min (i.e., the optimal process conditions).

[0166] Take an appropriate amount of γ-PGA-g-CHOL blank nanomicelle sample and dissolve it in deionized water. Use Zetasizer NanoZS90 to measure the surface Zeta potential, particle size and polydispersity index (PDI) of the blank nanomicelle. Figure 12 、 Figure 13 As shown, the particle size of the optimal γ-PGA-g-CHOL blank nanomicelles showed a normal distribution, mainly concentrated between 40 and 300 nm, with an average particle size of 117.3 ± 1.2 nm and a potential of -20.1 ± 1.5 mV, proving that the nanomicelles had good stability.

[0167] (9) Scanning electron microscopy (SEM) surface morphology observation of blank nanomicelles

[0168] Take an appropriate amount of γ-PGA-g-CHOL blank nano-micelle freeze-dried powder (prepared under "(8)" in Example 3) and stick it on the conductive carbon film double-sided tape, put it on the sample stage of the ion sputtering instrument and spray it with gold for 30 seconds, observe it under a scanning electron microscope and take pictures. Figure 14 As shown, the freeze-dried sample powder of the γ-PGA-g-CHOL blank nanomicelles is in a spherical state.

[0169] Example 4: Preparation, Optimization and Characterization of CBD / (γ-PGA-g-CHOL) Drug-Loaded Nanomicelles

[0170] (1) Preparation of CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles

[0171] The preparation method of γ-PGA-g-CHOL used in this example is the same as that in Example 1, except that the alcohol-acid ratio is 1.5:1, and the concentration of carboxyl groups in γ-PGA in the first mixed solution is 0.25 mol·L -1 , the activation reaction time is 50min.

[0172] (i) 0.141 mg of γ-PGA-g-CHOL (prepared in this example) was mixed with 10 mL of deionized water to obtain an amphiphilic polymer aqueous solution; the amphiphilic polymer aqueous solution was thermostatted at 4° C. for 24 h, and then subjected to probe ultrasonic treatment in an ice bath (to prevent the temperature from rising too high during the ultrasonic treatment, the ultrasonic condition was set to 5 s on and 5 s off) with an ultrasonic power of 150 W and an ultrasonic time of 5 min; after the ultrasonic treatment, the mixture was centrifuged at 4000 rpm for 20 min, and the supernatant was filtered through a 0.45 μm microporous membrane to obtain an amphiphilic polymer blank micelle solution; the amphiphilic polymer blank micelle solution was freeze-dried to obtain amphiphilic polymer blank micelles.

[0173] (ii) mixing cannabidiol with an organic solvent to obtain a cannabidiol solution; mixing the amphiphilic polymer blank micelles obtained in step (i) with 10 mL of deionized water, stirring at 25° C. for 30 min to obtain an amphiphilic polymer blank micelle aqueous solution; adding the cannabidiol solution dropwise to the amphiphilic polymer blank micelle aqueous solution, stirring at 25° C., and after stirring, placing the reaction solution in a dialysis bag (MWCO 3500Da, batch number: 409C026, Jinkelong (Beijing) Biotechnology Co., Ltd.) and dialyzing it with deionized water for 24 h, changing the water every 2 h, and filtering the dialyzate through a 0.45 μm organic filter membrane. After freeze-drying, the cannabidiol-loaded nanomicelles, i.e., CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles, were obtained.

[0174] (2) Single factor experiment of preparation process

[0175] The preparation method in Example 4 (1) was subjected to a single-factor experiment, with the following factors being the choice of organic solvent [CBD / (γ-PGA-g-CHOL) group: DMSO, acetone, THF, ethanol, DMF], the dosage of CBD [CBD / (γ-PGA-g-CHOL) group: 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg], the concentration of amphiphilic polymer blank micelles in the aqueous solution [CBD / (γ-PGA-g-CHOL) group: 3 mg mL -1 , 4mg·mL -1 , 5mg·mL -1 , 6mg·mL-1 , 7mg·mL -1 ], organic phase / aqueous phase volume ratio [CBD / (γ-PGA-g-CHOL) group: 1:3, 1:4, 1:5, 1:6, 1:7], stirring time [CBD / (γ-PGA-g-CHOL) group: 2h, 4h, 6h, 8h, 10h], and stirring speed [CBD / (γ-PGA-g-CHOL) group: 200rpm / min, 300rpm / min, 400rpm / min, 500rpm / min, 600rpm / min] were used as influencing factors to explore their effects on the particle size, PDI, encapsulation efficiency, and drug loading of drug-loaded nanomicelles, and to obtain the optimal preparation process; Note: The organic phase / aqueous phase volume ratio here refers to the organic solvent in the cannabidiol solution / deionized water in the amphiphilic polymer blank micelle aqueous solution; the stirring time and stirring speed here refer to the stirring time and stirring speed after the cannabidiol solution is dropwise added to the amphiphilic polymer blank micelle aqueous solution.

[0176] (3) Single-factor experimental results

[0177] Selection of organic solvent: CBD / (γ-PGA-g-CHOL) nanomicelles were prepared by dialysis method. While keeping other factors in the formulation unchanged (dosage was 2 mg, the concentration of amphiphilic polymer blank nanomicelles was 4 mg mL -1 The organic phase was varied to include DMSO, acetone, THF, ethanol, and DMF. The effect of the organic phase selection on the particle size, polydispersity coefficient, encapsulation efficiency, and drug loading of CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles was investigated. The results were published in the journal Nature Communications.

[0178] As shown in Table 8, when THF was used as the solvent, the encapsulation efficiency and drug loading reached a maximum of 58.69% and 6.81%, and the particle size and PDI reached a minimum of 138.3 ± 1.8 nm and 0.214 ± 0.017, respectively. The encapsulation efficiency and drug loading of the other organic solvents were relatively low, and the particle size and PDI were relatively large. This may be due to the presence of cholesteryl groups in the γ-PGA-g-CHOL blank nanomicelles, which makes them more compatible with organic solvents with lower polarity, such as THF. Furthermore, CBD is easily soluble in THF, and the increase in the contact area of ​​the reactants makes the mixed system more uniform and stable, which is conducive to the effective loading of CBD during dialysis. Therefore, THF was finally selected as the organic reagent for preparing CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles.

[0179] Table 8 Effect of organic solvent selection on CBD / (γ-PGA-g-CHOL)

[0180]

[0181] Dosage of cannabidiol: When the CBD content of the blank nanomicelles increases, the particle size of the drug-loaded nanomicelles will tend to increase, and the smaller the PDI, the more concentrated the particle size distribution of the drug-loaded nanomicelles. As shown in Table 9, under the premise that other factors in the prescription remain unchanged, with the increase of the dosage, the encapsulation efficiency and drug loading rate both show a trend of first increasing and then decreasing. This is because when the concentration of the blank nanomicelles is constant, with the increase of the dosage, more CBD is loaded, the particle size increases, and the encapsulation efficiency and drug loading rate both increase; when the dosage is too high, the hydrophobic core of the blank nanomicelles is overloaded with CBD, and the encapsulation efficiency and drug loading rate decrease significantly; when the dosage is 1.5 mg, the encapsulation efficiency and drug loading rate reach peak values, which are 63.24% and 7.54%, respectively, and compared with other dosage groups, the particle size is larger and the distribution is concentrated. Therefore, the optimal dosage is selected as 1.5 mg.

[0182] Table 9 Effect of dosage on CBD / (γ-PGA-g-CHOL)

[0183]

[0184]

[0185] Concentration of amphiphilic polymer blank nanomicelles: As shown in Table 10, as the concentration of amphiphilic polymer blank nanomicelles increases, the encapsulation efficiency of drug-loaded nanomicelles gradually increases. However, when the concentration of blank nanomicelles is greater than 5 mg mL -1 When the encapsulation effect of CBD reaches saturation, the encapsulation rate does not increase significantly, and the drug loading decreases continuously; at the same time, as the concentration of blank nanomicelles increases, the particle size of the drug-loaded nanomicelles first increases and then tends to be stable. This may be because the particle size of the drug-loaded nanomicelles increases with the increase of CBD loading in the early stage, and then the CBD loading tends to saturation, and the particle size of the drug-loaded nanomicelles does not change significantly; the PDI values ​​of each drug-loaded nanomicelle are within 0.3, and the particle size dispersion is excellent. Taking all factors into consideration, the optimal blank nanomicelle concentration is set to 5 mg mL -1 .

[0186] Table 10 Effect of blank nanomicelle concentration on CBD / (γ-PGA-g-CHOL)

[0187]

[0188] Organic / aqueous phase volume ratio: The organic / aqueous phase volume ratio is crucial for nanomicelle formation and directly affects its formation and drug loading capacity. During dialysis, the amphiphilic polymer self-assembles into nanomicelles. As shown in Table 11, the encapsulation efficiency and drug loading gradually increase at organic / aqueous phase ratios of 1:3 and 1:4. At a ratio of 1:5, the encapsulation efficiency and drug loading reach their maximum values ​​of 82.17% and 8.13%, respectively. At ratios of 1:6 and 1:7, the encapsulation efficiency and drug loading decrease. Therefore, an organic / aqueous phase ratio of 1:5 was ultimately selected.

[0189] Table 11 Effect of organic phase / aqueous phase volume ratio on CBD / (γ-PGA-g-CHOL)

[0190]

[0191] Stirring Time: During the dialysis pretreatment process, prolonged stirring time promotes a more homogeneous and stable system of the organic and aqueous phases, which is crucial for maximizing CBD loading into the blank nanomicelles during the subsequent dialysis process. However, excessive stirring time can interfere with the formation of the steady-state structure of the drug-loaded nanomicelles during dialysis, leading to partial release of the loaded CBD and reduced encapsulation efficiency and drug loading. As shown in Table 12, with increasing stirring time, both encapsulation efficiency and drug loading initially increase and then decrease. After stirring for 4 hours, the encapsulation efficiency and drug loading reached 82.28% and 8.22%, respectively, indicating a relatively successful preparation. Therefore, a stirring time of 4 hours was selected.

[0192] Table 12 Effect of stirring time on CBD / (γ-PGA-g-CHOL)

[0193]

[0194] Stirring speed: As shown in Table 13, when the stirring speed is in the range of 200-600 rpm, the encapsulation efficiency and drug loading first increase and then decrease. When the stirring speed is 400 rpm, the encapsulation efficiency and drug loading reach the maximum value of 82.50% and 8.25%. When the stirring speed is lower than 400 rpm, the system cannot reach a stable equilibrium state within the same stirring time; when the stirring speed is higher than 400 rpm, the reaction contact area between the CBD molecules and the blank nanomicelles may be relatively reduced due to the strong stirring force, and the steady state of the drug-loaded nanomicelles may be destroyed, resulting in poor CBD loading effect. Therefore, the final stirring speed was selected to be 400 rpm.

[0195] Table 13 Effect of stirring speed on CBD / (γ-PGA-g-CHOL)

[0196]

[0197] (4) Orthogonal experiment and results

[0198] Orthogonal experiment: Based on the results of single factor experiment, the CBD / (γ-PGA-g-CHOL) group selected four factors: dosage, concentration of blank amphiphilic polymer nanomicelles, volume ratio of organic phase to aqueous phase, and stirring time for L9(3 4 ) Orthogonal experiments were designed using an orthogonal table. Encapsulation efficiency and drug loading were used as evaluation indicators to determine the optimal combination. The factor level table is shown in Table 14.

[0199] Table 14 Orthogonal factor level design table (CBD / (γ-PGA-g-CHOL) group)

[0200]

[0201] Results of orthogonal experiment of CBD / (γ-PGA-g-CHOL): The results of orthogonal experiment of CBD / (γ-PGA-g-CHOL) are shown in Table 15. As shown in Table 16 and Table 17, with encapsulation efficiency and drug loading as the evaluation indicators, according to the R value, the influencing factors are X2>X 3> X1>X4, that is, the concentration of amphiphilic blank nanomicelles> organic phase / aqueous phase volume ratio> dosage> stirring time. The optimized condition combination for the best process is X13X22X32X43. That is, the best preparation process is: under the premise of fixed organic reagent THF, stirring speed 400rpm, dosage of 2mg, and blank nanomicelle concentration of 5mg·mL -1 , the volume ratio of organic phase to aqueous phase was 1:5, and the stirring time was 6 h.

[0202] Table 15 Orthogonal experiment results (CBD / (γ-PGA-g-CHOL) group)

[0203]

[0204] Table 16 Encapsulation efficiency range analysis (CBD / (γ-PGA-g-CHOL) group)

[0205]

[0206] Table 17 Analysis of drug loading range (CBD / (γ-PGA-g-CHOL) group)

[0207]

[0208] (5) Verification experiment

[0209] According to the optimal formulation and process described above (the optimal parameters obtained in "(4) Orthogonal Experiment" in Example 4, and the method for preparing "(1) Preparation of CBD / (γ-PGA-g-CHOL) Drug-Loaded Nanomicelles" in Example 4), three batches of CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles were prepared. Their morphologies were observed, and their encapsulation efficiencies and drug loadings were measured, respectively. The encapsulation efficiencies of the CBD / (γ-PGA-g-CHOL) group were 84.46% ± 0.35%, and the drug loading was 8.78% ± 0.28%.

[0210] (6) Determination of water solubility of cannabidiol and its results

[0211] An excess CBD control sample and CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles (prepared according to the optimal parameters obtained in "(4) Orthogonal Experiment" in Example 4 and the method of "(1) Preparation of CBD / (γ-PGA-g-CHOL) Drug-Loaded Nanomicelles" in Example 4) were weighed and placed in 100 mL conical flasks. 50 mL of deionized water was added to each flask and stirred at 200 rpm for 48 h at 37°C. The suspension was centrifuged at 5000 rpm for 20 min, and 1 mL of the supernatant was added to 9 mL of acetonitrile. The mixture was mixed and sonicated for 5 min. The suspension was filtered through a 0.22 μm organic microporous membrane and the CBD content was determined according to the chromatographic conditions described above.

[0212] The results showed that the water solubility of raw CBD was 0.124 μg·mL -1 , while the solubility of CBD in CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles was 52.73 μg·mL -1 , that is, the water solubility of CBD in CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles increased by 425 times.

[0213] (7) Determination of particle size distribution and zeta potential of CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles

[0214] Take an appropriate amount of CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelle powder and prepare 2.0 mg·mL with deionized water. -1 The concentration of CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles was determined, and the surface Zeta potential, particle size and PDI were determined.

[0215] Three batches of CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles were prepared using the optimal process obtained by orthogonal optimization (the preparation method was based on the optimal parameters obtained in "(4) Orthogonal Experiment" in Example 4 and the method of "(1) Preparation of CBD / (γ-PGA-g-CHOL) Drug-Loaded Nanomicelles" in Example 4). The average particle size, PDI, and Zeta potential were measured to be 163.1±2.3 nm, 0.205±0.048, and -16.5±1.7 mV, respectively. The particle size and potential measurement results are shown in FIG. Figure 16 、 Figure 17 .

[0216] (8) Scanning electron microscopy (SEM) surface morphology observation of drug-loaded nanomicelles

[0217] An appropriate amount of CBD / (γ-PGA-g-CHOL) nanomicelle freeze-dried powder (prepared according to the optimal parameters obtained in "(4) Orthogonal Experiment" in Example 4 and the method of "(1) Preparation of CBD / (γ-PGA-g-CHOL) Drug-Loaded Nanomicelles" in Example 4) was placed on the conductive carbon film double-sided tape, placed on the sample stage of the ion sputtering instrument, sprayed with gold for 30 seconds, and observed and photographed under a scanning electron microscope.

[0218] from Figure 18 It can be seen that the CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles have regular micromorphology, are approximately spherical, and have a smooth surface. The particle size of the drug-loaded nanomicelles is significantly larger than that of the γ-PGA-g-CHOL blank nanomicelles.

[0219] (9) Study on the stability of drug-loaded nanomicelles

[0220] The CBD / (γ-PGA-g-CHOL) polymer micelles prepared by the optimal process (prepared according to the optimal parameters obtained in "(4) Orthogonal Experiment" in Example 4 and the method of "(1) Preparation of CBD / (γ-PGA-g-CHOL) Drug-Loaded Nanomicelles" in Example 4) were placed in a vial and placed at room temperature and 37°C. The particle size changes of the two groups of drug-loaded micelles were measured at 24 h, 48 h, 72 h, 120 h and 168 h, respectively, to observe the stability of the drug-loaded nanomicelles.

[0221] Depend on Figure 19 As shown in the figure, the particle size of CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles did not change significantly at room temperature and 37°C, indicating that both drug-loaded nanomicelles exhibited good storage stability during storage for up to 7 days.

[0222] (10) In vitro drug release behavior of drug-loaded nanomicelles

[0223] An appropriate amount of CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelle freeze-dried powder (prepared according to the optimal parameters obtained in "(4) Orthogonal Experiment" in Example 4 and the method of "(1) Preparation of CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles" in Example 4) was weighed and dissolved in 1 mL of deionized water, respectively, encapsulated in a dialysis bag (molecular weight cutoff 3500 Da), and placed in 20 mL of PBS solution containing 0.5% Tween 80 (w / v) at pH = 5.5 and pH = 7.4, respectively, and placed in a constant temperature shaker at 37°C and an oscillation rate of 100 rpm to simulate in vitro drug release. 1 mL samples were collected at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, and 144 h, respectively. Each sample was tested in parallel three times, and the release medium of each sample was replaced with an equal amount of fresh medium. The amount of CBD released from the CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles was detected according to the chromatographic conditions described above. The cumulative release rate of CBD at different time points was calculated, and the release curve was plotted with the cumulative release rate of CBD as the ordinate and the in vitro release time as the abscissa. The cumulative release rate of CBD was calculated using the following formula.

[0224]

[0225] Wherein, Er: cumulative release of CBD; Ve: volume of PBS buffer replaced; V0: total volume of release medium; C i : The concentration of the sample at the i-th sampling, i = 1, ..., n-1; m CBD : CBD mass in drug-loaded nanomicelles; n: number of times of buffer replacement; C n : The concentration of the sample at the nth sampling time.

[0226] The results are as follows Figure 20 As shown in the figure, the 144h cumulative release rates of CBD / (γ-PGA-g-CHOL) were 25.92% (pH=7.4) and 47.34% (pH=5.5), respectively. In the PBS medium with pH=5.5, the release rate of CBD was relatively fast, mainly because in the slightly acidic medium, a large number of free carboxyl groups on the hydrophilic side chains of γ-PGA-g-CHOL tended to be protonated, intramolecular and intermolecular hydrogen bonds were strengthened, and the secondary structure was mainly α-helix. Its molecular flexibility was poor, and its self-assembly performance and drug loading capacity were weakened. Under neutral pH conditions, γ-PGA-g-CHOL had good molecular flexibility, and the surface potential of the self-assembled nanomicelles formed was low, which was conducive to CBD loading and the formation of a stable drug-loading structure. In addition, since the burst release of drugs is mainly related to the rapid release of drugs embedded or adsorbed on the surface or near the surface of the nanomicelles in a short period of time, Figure 20It shows that there is no obvious burst release of CBD after being loaded into γ-PGA-g-CHOL blank micelles, indicating that the drug is basically embedded in the nanomicelles and bound by hydrophobic forces. With the gradual dissolution and degradation of the carrier material, cannabidiol is continuously released at a relatively slow rate. Under different pH conditions, the drug release rate is different, indicating that the release of drugs in nanomicelles is pH-dependent.

[0227] Example 5: Study on oral absorption and anti-inflammatory mechanism of CBD / (γ-PGA-g-CHOL) nanomicelles

[0228] The CBD / (γ-PGA-g-CHOL) nanomicelles used in this example were prepared according to the optimal parameters obtained in "(4) Orthogonal Experiment" in Example 4 and the method of "(1) Preparation of CBD / (γ-PGA-g-CHOL) Drug-Loaded Nanomicelles" in Example 4.

[0229] The amphiphilic polymer blank micelles used in this example were prepared according to the preparation method of "Step (i)" under "(1) Preparation of CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles" in Example 4.

[0230] 1. Experimental methods

[0231] 1.1 Cell Culture

[0232] Caco-2 cells were cultured in MEM medium containing fetal bovine serum (FBS, 10%), penicillin-streptomycin double antibody solution (100 U·mL -1 )-Streptomysin(100μg·mL -1 ) and cultured in a 5v / v% CO2 constant temperature incubator at 37°C and 95% humidity. 2 ) culture flasks until the cells grow to a density of more than 90%, and then digest them with 0.25% trypsin-0.2% EDTA solution at a ratio of 1:2 or 1:3 before passage.

[0233] 1.2 Determination of safe drug concentration

[0234] 1.2.1 CCK-8 assay

[0235] The CCK-8 method was used to investigate the effects of γ-PGA-g-CHOL blank micelles, CBD and CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles on the survival rate of Caco-2 cells. 5Caco-2 cells were seeded in a 96-well plate at a density of 100 μL per well. 200 μL of PBS solution (pH = 7.2-7.4, 0.01 M) was seeded in the outer wells of the 96-well plate to provide a humid environment and cultured in a 37°C incubator. After the Caco-2 cells attached to the wall, 10 μL of drug-containing MEM was added to each well to make the final concentration of γ-PGA-g-CHOL blank micelles 0-1000 μg·mL -1 The final concentration of CBD in CBD or CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles was 2.5 μg mL -1 , 5μg·mL -1 , 7.5 μg·mL -1 , 10 μg·mL -1 , 12.5 μg·mL -1 , 15 μg·mL -1 The cells were grouped and treated with corresponding drugs. Each group had six replicate wells, and the control group had 6 wells (with cells, no drugs, and MEM culture medium). After incubation for 24 hours, 10 μL of CCK-8 solution was added to each well. After incubation at 37°C for 1 hour, the absorbance (OD) value at a wavelength of 450 nm was measured using a microplate reader and calculated according to the following formula: Cell survival rate (%) = OD 处理孔 / OD 对照孔 × 100%. 处理孔 Indicates the cell absorbance value after adding drugs, OD 对照孔 It represents the cell absorbance value of blank control.

[0236] Test results such as Figure 21 As shown, the experimental results show that: Figure 21 As shown in A, in order to verify the good biocompatibility of the polymer material, we used γ-PGA-g-CHOL blank micelles to treat Caco-2 cells. The results showed that in the range of 0 to 1000 μg mL -1 There was no significant change in cell viability at the concentration of Figure 21 As shown in B, 0-15 μg·mL -1 Within the experimental concentration range, the cell survival rates of CBD and CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles were greater than 90%, indicating that γ-PGA-g-CHOL blank micelles, CBD and CBD / (γ-PGA-g-CHOL) did not cause damage to the cells.

[0237] 1.2.2 LDH release detection

[0238] The lactate dehydrogenase (LDH) release assay was used to investigate the effects of γ-PGA-g-CHOL blank micelles, CBD, and CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles on the cell membrane integrity of Caco-2 cells. Caco-2 cell suspensions were cultured at a concentration of 1×10 5 The cells were seeded at a density of 100 μL per well in a 96-well plate. 200 μL of PBS solution (pH = 7.2-7.4, 0.01 M) was added to the outer wells of the 96-well plate to provide a humid environment. The cells were cultured in a 37°C incubator. After the Caco-2 cells attached to the wall, 10 μL of drug-containing MEM was added to each well to make the final concentration of γ-PGA-g-CHOL blank micelles 0-1000 μg·mL -1 The final concentration of CBD in CBD or CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles was 2.5 μg mL -1 , 5μg·mL -1 , 7.5 μg·mL -1 , 10 μg·mL -1 , 12.5 μg·mL -1 , 15 μg·mL -1 , with blank MEM as the control group (cells, no drug, MEM medium added). The cells were grouped and treated with the corresponding drug intervention, with six replicates in each group. After incubation for 24 hours, 20 μL of the supernatant from each well was removed and added to another new 96-well plate. The reagent solution was added according to the requirements of the LDH release kit, and then the absorbance (OD) value at a wavelength of 570 nm was measured using a microplate reader. The following formula was used to calculate: Cell membrane integrity (%) = OD 处理孔 / OD 对照孔 × 100%. 处理孔 Represents the absorbance value of the cell supernatant after drug treatment, OD 对照孔 It represents the absorbance value of the blank control cell supernatant.

[0239] Test results such as Figure 22 As shown, the experimental results show that: Figure 22 As shown, when the concentration of γ-PGA-g-CHOL blank micelles was 0~1000 μg·mL -1 , CBD and CBD / (γ-PGA-g-CHOL) drug-loaded nanomicelles in the range of 0-15 μg·mL -1 Within the experimental concentration range, the release amount of LDH did not change significantly, indicating that within the experimental concentration range, γ-PGA-g-CHOL blank micelles, CBD and CBD / (γ-PGA-g-CHOL) did not destroy the integrity of the cell membrane.

[0240] 1.3 Establishment and characterization of the Caco-2 cell monolayer model

[0241] 1.3.1 Establishment of Caco-2 cell monolayer model

[0242] Caco-2 cells were cultured at 1 × 10 cells per ml. 5 Cells were seeded into Transwell chambers at a density of 100 cells / mL. 0.5 mL of cell suspension was added to the apical side (AP) and 1.5 mL of complete culture medium (MEM medium prepared in "1.1, Cell Culture") was added to the basolateral side (BL). The culture medium was changed every two days for the first week after seeding, and daily thereafter. Culture was continued for 21 days.

[0243] 1.3.2 Characterization of the Caco-2 Cell Monolayer Model

[0244] 1.3.2.1 Microscopic observation

[0245] The status of Caco-2 cells at different growth stages in Transwell chambers was observed under a microscope. Figure 23 shown.

[0246] 1.3.2.2 Transephrine electrical resistance (TEER) detection

[0247] Caco-2 cells were inoculated into Transwell chambers according to the above method, and the TEER of cells at different growth times was measured using a resistance meter (ERS). Before the measurement, the electrodes were sterilized by soaking in 70% ethanol for 15-20 minutes, air-dried for 15 seconds, and then placed in a PBS solution (pH = 7.2-7.4, 0.01M) for 15 minutes. The electrodes were then inserted vertically into the culture wells, with the electrode ends not touching the bottom of the chamber. Three locations in each well were randomly selected for measurement three times. The measured values ​​of the experimental group inoculated with Caco-2 cells were recorded as R, and the measured values ​​of the blank group without Caco-2 cells were recorded as R o , calculate the actual resistance value of each well according to the formula: TEER=(R t -R o )×A(Ω·cm 2 ). Where R t is the measured value of the experimental group inoculated with Caco-2 cells, R o The values ​​are the values ​​of the blank group without Caco-2 cells inoculated, and A is the area of ​​the Transwell membrane.

[0248] The results are as follows Figure 24 As shown in the experimental results, the TEER of the Caco-2 cell model established in this experiment increased with the extension of culture time, indicating that the tightness gradually increased with the extension of culture time. The TEER value reached 800Ω·cm in the second week of culture. 2 By 21 days, the TEER value tended to be stable, indicating that a complete and tight cell monolayer was formed.

[0249] 1.3.2.3 Alkaline phosphatase activity assay

[0250] An ALP / AKP kit was used to measure ALP activity in Caco-2 cell monolayers and evaluate the growth and differentiation characteristics of Caco-2 cells. ALP decomposes disodium phenyl phosphate. The resulting free phenol reacts with 4-aminoantipyrine in an alkaline solution and is oxidized by potassium ferrocyanide to form a red inner derivative. The intensity of the red color can be used to determine enzyme activity. Culture fluid from culture dishes (n=3) was used as a sample, and ALP activity in the cell fluid was measured according to the instructions provided with the ALP / AKP kit.

[0251] The results are as follows Figure 25 As shown, the experimental results showed that: the results of ALP activity detection found that the ALP activity ratios of the AP side and the BL side on the 3rd, 7th, 14th and 21st days were 1.02±0.02, 1.78±0.12, 2.97±0.21 and 4.23±0.09, respectively, indicating that when the Caco-2 cells were cultured for 21 days, the distribution of alkaline phosphatase was very asymmetric, with most of the enzyme on the brush border side, and the Caco-2 cell model had polarity characteristics.

[0252] 1.3.2.4. Fluorescein sodium transmittance test

[0253] Prepare 100 μg·mL -1 The fluorescein sodium stock solution was diluted to the following concentrations: 0, 1, 5, 10, 20, and 100 μg mL -1 After the Transwell plate has been plated for 21 days, randomly select one experimental well on each plate as the sodium fluorescein transport well. The control group is a well without cells. Remove the culture medium in the upper and lower chambers of the wells, add an appropriate amount of sterile PBS solution (pH = 7.2-7.4, 0.01M) to rinse, remove the solution in the sodium fluorescein transport well, and add 0.5mL of 100μg·mL to the upper chamber of the well. -1 1.5 mL of fluorescein sodium solution was added to the lower chamber (pH = 7.2-7.4, 0.01 M), and the cells were placed in a 37°C constant temperature CO2 cell culture incubator for 0.5, 1, 1.5, 2, and 2.5 h. The absorbance of the samples was measured at 490 nm using a microplate reader, and a standard curve was drawn to calculate the transmittance of fluorescein sodium in each well.

[0254] Fluorescein sodium is highly water-soluble and can be transported from the intercellular space, so it can be used to detect the integrity of cell monolayers. Figure 26As shown, the permeability of sodium fluorescein gradually increased over time within 0 to 2.5 hours of exposure to sodium fluorescein in the Caco-2 cell model. Compared with the control group, the permeability of sodium fluorescein in the experimental group was significantly reduced and increased slowly. These results indicate that the compactness and integrity of the Caco-2 cell model are good, meeting the requirements of transport experiments.

[0255] 1.4. CBD Content Determination in Caco-2 Cells and Rat Plasma

[0256] 1.4.1 Sample processing method

[0257] Cell sample processing: Take 100 μL of cell sample, add 300 μL of methanol to precipitate protein, vortex and let stand for 30 minutes, centrifuge at 12000 rpm for 20 minutes, and take the supernatant for injection.

[0258] Plasma sample processing: Take 100 μL of plasma, add 5 μL of 70% methanol and internal standard working solution (10 ng mL -1 A solution of 5 μL of diphenhydramine (5 μL), 100 μL of acetonitrile, and 400 μL of methyl tert-butyl ether was added. The mixture was vortexed for 1 minute and centrifuged at 12,000 rpm for 10 minutes. The supernatant was blown dry with nitrogen. The residue was reconstituted with 100 μL of 70% methanol and centrifuged at 12,000 rpm for 10 minutes. The supernatant was sampled.

[0259] 1.4.2 Chromatographic conditions

[0260] CBD detection method in Caco-2 cells: Chromatographic conditions: chromatographic column: Agilent Eclipse XDB-C18 (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile: 0.1 wt% formic acid = 80:20; detection wavelength: 210 nm, column temperature: 30.0°C, flow rate: 1.0 mL min -1 , injection volume: 20 μL, detection time: 20 min, isocratic elution.

[0261] Method for detecting CBD in plasma: Chromatographic conditions are as follows: chromatographic column: CORTECS C18 Column (150 mm × 4.6 mm, 2.7 μm), mobile phase A: 0.1 wt% formic acid-water, mobile phase B: acetonitrile; gradient elution: 0-2 min, A:B = 30:70; 2-9 min, A:B = 10:90; 9.1-16 min, A:B = 30:70; column temperature: 30.0°C, flow rate: 0.3 mL / min, injection volume: 5 μL.

[0262] The mass spectrometry conditions used were: ESI source; full ion detection; and multiple reaction monitoring (MRM).

[0263] Mass spectrometry detection ions and parameters: cannabidiol, detection range: 315.2-316.2 m / z, declustering voltage: 97.6 V, collision voltage: 31.9 V; diphenhydramine, detection range: 255.2-257.3 m / z, declustering voltage: 97.6 V, collision voltage: 31.9 V.

[0264] 1.4.3. Standard curve drawing

[0265] Cells: Accurately weigh the CBD reference substance and dissolve the blank cell suspension to a concentration of 500 μg mL -1 The mother solution was diluted with PBS buffer (pH = 7.2-7.4, 0.01 M) in the concentration gradient of 0.5, 1, 3, 6, 15, 30, 50, and 100 μg mL -1 After processing according to the "Cell Sample Processing" method under "1.4.1. Sample Processing Method", the sample was injected according to the "CBD Detection Method in Caco-2 Cells" in "1.4.2. Chromatographic Conditions". The peak area of ​​the analyte was used as the ordinate and the concentration of the analyte was used as the abscissa. The regression equation was Y = 99884X - 15221, R 2 =0.9998, good linear relationship.

[0266] Plasma: Take 100 μL of blank mouse plasma and add 10 μL of different concentrations of CBD methanol solution to prepare CBD concentrations of 10, 20, 40, 100, 200 and 400 ng·mL -1 A series of solutions were treated according to the "Plasma Sample Treatment" method under "1.4.1. Sample Treatment Method" and then determined according to the "CBD Detection Method in Plasma" under "1.4.2. Chromatographic Conditions". A standard curve was drawn with the concentration of the CBD reference substance in plasma as the horizontal axis and the peak area ratio of CBD and diphenhydramine as the vertical axis. The regression equation was calculated as Y = 0.01812X + 0.44521, R 2 =0.9991, good linear relationship.

[0267] 1.5. Study on the uptake characteristics of CBD / (γ-PGA-g-CHOL) nanomicelles

[0268] 1.5.1 Effect of time on Caco-2 cell uptake characteristics

[0269] Caco-2 cells (cultured in Example 5 "1.1, Cell Culture") were cultured at 1×10 5 The cells were inoculated at a density of 100 μg·mL in 6-well plates, and the medium was changed every other day. After one week, the medium was changed every day. After culturing for 14 days, the cells were gently washed 3 times with PBS solution (pH = 7.2-7.4, 0.01M) and then discarded. 15 μg·mL -12 mL of a PBS solution (pH = 7.2-7.4, 0.01 M) containing CBD or CBD / (γ-PGA-g-CHOL) nanomicelles was incubated in a 37°C incubator for 0.5, 1.0, 1.5, 2, 2.5, and 3 hours, respectively. Three wells were set up at each time point. After incubation, the drug-containing buffer was discarded, and pre-chilled PBS was added to terminate cellular uptake. The cell monolayer was quickly rinsed three times. 200 μL of cell lysis buffer was added to each well and lysed on ice for 5 minutes. Cells were scraped with a cell scraper into an Eppendorf tube and disrupted by ultrasound. A portion of the sample was processed according to the "Cell Sample Processing" method under "1.4.1. Sample Processing Method" and the CBD content was determined by HPLC (see "CBD Detection Method in Caco-2 Cells" under "1.4.2. Chromatographic Conditions"). The intracellular CBD concentration in each well was divided by the normalized total protein value per well and expressed as μg / mg protein.

[0270] 1.5.2 Effect of concentration on Caco-2 cell uptake characteristics

[0271] The cell treatment was the same as in 1.5.1, and the concentrations of 2.5, 5, 7.5, 10, 12.5, and 15 μg mL -1 CBD or CBD / (γ-PGA-g-CHOL) nanomicelles in PBS (pH = 7.2-7.4, 0.01 M) were added to the cells, 2 mL per well. Three wells were prepared for each concentration and cultured in a 37°C incubator for 4 h. After incubation, the drug-containing buffer was discarded, pre-chilled PBS was added to terminate cellular uptake, and the cell monolayer was quickly rinsed three times. Other steps were the same as in 1.5.1.

[0272] 1.5.3 Effect of temperature on Caco-2 cell uptake characteristics

[0273] The cell treatment was the same as in 1.5.1, and 15 μg·mL -1 Prepare 2 mL of a PBS solution (pH 7.2-7.4, 0.01 M) containing CBD or CBD / (γ-PGA-g-CHOL) nanomicelles and incubate at 4°C and 37°C for 1.5 h, respectively. Set up three wells at each temperature. After incubation, discard the drug-containing buffer, add pre-chilled PBS to terminate cellular uptake, and quickly rinse the cell monolayer three times. Other steps are the same as in 1.5.1.

[0274] 1.5.4 Effects of endocytosis inhibitors on Caco-2 cell uptake characteristics

[0275] The cell treatment was the same as in 1.5.1, and 15 μg·mL -12 mL of PBS solution (pH = 7.2-7.4, 0.01 M) of CBD or CBD / (γ-PGA-g-CHOL) nanomicelles was added, and MEM (Control group), 10 mmol·L -1 of MβCD and 10 μg·mL -1 Incubate 3 mL of Chlorpromazine at 37°C for 1 hour, with three wells set up at each temperature. After incubation, discard the drug-containing buffer, add pre-chilled PBS to terminate cellular uptake, and quickly rinse the cell monolayer three times. Other steps are the same as in 1.5.1.

[0276] For detailed data, please see Figure 27 , the experimental results show that: Figure 27 As shown in A, the drug concentration is 15 μg·mL -1 During the experiment, the uptake of CBD and CBD / (γ-PGA-g-CHOL) in Caco-2 cells increased first and then reached saturation with the extension of time from 0 to 3 hours, and the uptake reached the maximum at 2 hours. The results showed that the maximum uptake of CBD and CBD / (γ-PGA-g-CHOL) in Caco-2 cells was at 2 hours, which will be the time point for subsequent uptake experiments. Figure 27 As shown in Figure 2, at 2 h, the uptake of CBD and CBD / (γ-PGA-g-CHOL) in Caco-2 cells increased with increasing concentration, and there was no saturation phenomenon; at the same concentration, the uptake of the CBD / (γ-PGA-g-CHOL) group was significantly higher than that of the CBD group, suggesting that the uptake of CBD and CBD / (γ-PGA-g-CHOL) may not require a carrier and is a concentration-dependent passive diffusion or endocytosis method. Figure 27 C shows that the uptake of CBD and CBD / (γ-PGA-g-CHOL) in Caco-2 cells increases with increasing temperature, and the relationship between the uptake of CBD group and temperature is particularly significant, indicating that the uptake of CBD and CBD / (γ-PGA-g-CHOL) in cells is energy-dependent, and may be an active uptake or endocytosis method. Figure 27 D shows that compared with the blank group (MEM added), the cellular uptake of CBD and CBD / (γ-PGA-g-CHOL) decreased after the addition of endocytosis inhibitor MβCD or Chlorpromazine, indicating that endocytosis is involved in the cellular uptake of CBD and CBD / (γ-PGA-g-CHOL).

[0277] 1.6 Bilateral transport experiments of CBD / (γ-PGA-g-CHOL) nanomicelles in Caco-2 cell monolayer model

[0278] 1.6.1. Study on the transport efficiency of CBD / (γ-PGA-g-CHOL) nanomicelles in a Caco-2 cell monolayer model

[0279] Select a Caco-2 cell monolayer model that meets the transport requirements. Replace the cell culture medium with a blank PBS solution (pH = 7.2-7.4, 0.01M) at 37°C, culture the cells in the Transwell plate for 30 minutes (the cells were cultured in Example 5 "1.3.1, Establishment of Caco-2 cell monolayer model"), and gently rinse the Transwell membrane with a pipette to remove impurities on the cell surface. Transport from AP to BL: Add 15μg·mL -1 0.5 mL of PBS solution of CBD or CBD / (γ-PGA-g-CHOL) nanomicelles was added to the BL side, and 1.5 mL of blank PBS solution was added to the BL side. The mixture was incubated in a constant temperature oscillator at 37°C. 100 μL of the lower layer was sampled at 30, 60, 90, and 120 min, and the same volume of blank PBS solution was added. Transport from BL to AP: 15 μg mL -1 1.5 mL of PBS solution of CBD or CBD / (γ-PGA-g-CHOL) nanomicelles was added, 0.5 mL of blank PBS solution was added to the AP side, and the cells were incubated in a constant temperature oscillator at 37 ° C. 100 μL of the upper layer of the side without CBD or CBD / (γ-PGA-g-CHOL) was sampled at 30, 60, 90, and 120 min, and the same volume of blank PBS solution was added. 300 μL of methanol was added to the removed sample, vortexed for 1 min, allowed to stand for 30 min, centrifuged at 15000 rpm for 15 min, and 20 μL was injected for measurement (according to the "CBD detection method in Caco-2 cells" in "1.4.2, Chromatographic conditions"). Papp (cm / s) and ER were calculated according to the following formula:

[0280]

[0281] Where A is the permeable membrane area, which is the bottom area of ​​the donor pool Transwel1 membrane here; C0 is the initial drug concentration; dQ / dt is the drug transport amount per unit time; P app(B→A) represents P transported from the BL side to the AP side of the Transwell membrane app value; P app(A→B) It represents the Papp value of transport from the AP side to the BL side of the Transwell membrane.

[0282] 1.6.2. Study on the transport mechanism of CBD / (γ-PGA-g-CHOL) nanomicelles in a Caco-2 cell monolayer model

[0283] The effects of efflux protein inhibitors on the transport of CBD and CBD / (γ-PGA-g-CHOL) nanomicelles were further investigated using P-glycoprotein (P-gp) inhibitor verapamil (Ver), multidrug resistance-associated protein 2 (MRP2) inhibitor MK-571, and breast cancer resistance-associated protein (BCRP) inhibitor KO-143.

[0284] The experimental steps are the same as those in 1.6.1, except that: AP→BL transport: 15 μg·mL -1 0.5 mL of PBS solution of CBD or CBD / CCMC, CBD or CBD / CCMC+45.5 μg / mL Ver, CBD or CBD / CCMC+26.9 μg / mL MK-571, CBD or CBD / CCMC+4.7 μg / mL KO-143 was added to the BL side, and 1.5 mL of blank PBS solution was added. Similarly, for BL→AP transport: 15 μg·mL -1 1.5 mL of PBS solution containing CBD or CBD / CCMC, CBD or CBD / CCMC + 45.5 μg / mL Ver, CBD or CBD / CCMC + 26.9 μg / mL MK-571, or CBD or CBD / CCMC + 4.7 μg / mL KO-143 was added to the AP side. 0.5 mL of blank PBS solution was added to the AP side. After incubation at 37°C for 2 hours, samples were collected from the untreated side. The apparent permeability coefficient (Papp) and efflux rate (ER) were calculated according to the formula to investigate the effects of efflux protein inhibitors on the transport of CBD and CBD / CCMC.

[0285] The experimental results are shown in Table 18. The experimental results show that compared with the CBD group, the Papp (A→B) 、Papp (B→A) There was no significant change in the ER and CBD, indicating that the transport of CBD may not be related to P-gp. Compared with the CBD group, the Papp (A→B) Significant increase, Papp (B→A) The results showed that CBD may be the substrate of efflux proteins MRP2 and BCRP, and the transport of CBD was affected by MRP2 and BCRP. Compared with the CBD-γ-PGA-g-CHOL group, the Papp (A→B) 、Papp (B→A) There was no significant change in ER and Papp after adding Ver and KO-143 (A→B) Increase, Papp (B→A)The results showed that the transport of CBD-γ-PGA-g-CHOL was affected by BCRP and P-gp. Compared with the CBD group, CBD-γ-PGA-g-CHOL was not affected by MRP2, which means that CBD-γ-PGA-g-CHOL may avoid the efflux protein MRP2 and thus increase the transport of drugs.

[0286] Table 18 Results of bilateral transport experiments of CBD / (γ-PGA-g-CHOL) nanomicelles in the Caco-2 cell monolayer model

[0287]

[0288]

[0289] “*” indicates that the results were significantly different from those in the CBD group, and “#” indicates that the results were significantly different from those in the CBD-γ-PGA-g-CHOL group.

[0290] 1.7 Pharmacokinetic Studies of CBD and CBD / (γ-PGA-g-CHOL) Nanomicelles

[0291] Twelve male SD rats were gavage-administered with CBD or CBD / (γ-PGA-g-CHOL) nanomicelles (100 mg / kg). Approximately 100 μL of blood was collected from the orbital cavity at 0.5, 1, 1.25, 1.5, 1.75, 2, 4, 8, 24, and 48 hours. The blood was placed in a heparinized centrifuge tube and centrifuged at 6000 rpm for 5 minutes. The sample was transferred to a polypropylene tube and stored at -80°C until testing. The obtained samples were processed according to the "Plasma Sample Processing" method under "1.4.1, Sample Processing Method" and then assayed according to the "CBD Detection Method in Plasma" under "1.4.2, Chromatographic Conditions."

[0292] The research results are as follows Figure 28 As shown in the figure, the experimental results show that compared with free CBD, the area under the drug-time curve of CBD / (γ-PGA-g-CHOL) nanomicelles increases, the average residence time increases, and the clearance rate decreases, indicating that CBD / (γ-PGA-g-CHOL) nanomicelles can prolong the in vivo circulation time of CBD and slow down the clearance rate.

[0293] 1.8. Study on the anti-inflammatory activity of CBD and CBD / (γ-PGA-g-CHOL) nanomicelles

[0294] 1.8.1 LPS concentration screening

[0295] According to the experimental design: divided into 0.1, 1, 10, 25, 50 μg·mL -1There were 5 groups of LPS. Caco-2 cells grown to the logarithmic phase (obtained in Example 5, "1.1, Cell Culture") were digested and centrifuged, and 1×10 5 Cells were seeded into 96-well plates at a cell density of 100 μL per well. After culturing for 24 h, 100 μL of LPS-containing culture medium was added to the five experimental groups. LPS was not added to the control group (Control group). The culture flasks were placed in an incubator and cultured for 24 h before CCK-8, LDH, and ELISA release detection was performed.

[0296] Test results are shown in Figure 29 The experimental results showed that: CCK-8 and LDH detection found 1μg·mL -1 LPS had no effect on cell viability. When the LPS concentration was ≥10 μg·mL -1 When the cell viability decreased significantly, the cell membrane damage became more serious. The secretion level of inflammatory factors was detected by ELISA method. The results showed that 0-10 μg·mL -1 After LPS intervention, inflammatory factors IL-8, IL-1β, and TNF-α increased significantly. -1 , the level of cellular inflammatory factors tends to be stable. After comprehensive consideration, 10μg·mL was selected -1 As the experimental concentration of LPS.

[0297] 1.8.2 Establishment of Cellular Inflammation Model and Experimental Grouping

[0298] This study was divided into normal control group, model group (LPS group), and experimental group (LPS+CBD group, LPS+CBD / (γ-PGA-g-CHOL) group). Based on the established Caco-2 cell transport model, the model group and the experimental group were cultured with MEM medium (10 μg mL - 1 The cells were treated with LPS for 24 h to prepare the cell inflammation model; the normal control group was cultured with normal cell culture medium. The experimental groups were added with 5, 10, and 15 μg mL -1 The model group and the control group were added with equal amounts of PBS solution.

[0299] 1.8.3. Anti-inflammatory Activity Detection of CBD / (γ-PGA-g-CHOL) Drug-Loaded Nanomicelles

[0300] The anti-inflammatory activity of CBD and CBD / (γ-PGA-g-CHOL)-loaded nanomicelles was compared by measuring cell viability and transmembrane electrical resistance. Cell viability was assessed using CCK-8 assays and LDH release assays, and transmembrane electrical resistance was measured using the same methods as described in "1.3.2.2, Transmembrane Electrical Resistance (TEER) Measurement" above.

[0301] Test results are shown in Figure 30 and Figure 31 The experimental results showed that LPS was used to induce inflammatory damage in Caco-2 cells. The results showed that after adding CBD and CBD / (γ-PGA-g-CHOL) for intervention, compared with the LPS stimulation group, both could significantly increase cell survival rate and reduce LDH activity, suggesting that CBD and CBD / (γ-PGA-g-CHOL) can inhibit LPS-induced inflammatory damage in Caco-2 cells and have good anti-inflammatory activity ( Figure 30 The results of transmembrane electrical resistance (TEER) measurements in the Caco-2 cell model showed that compared with the blank control group, the TEER value of the LPS-induced Caco-2 monolayer model in the model group decreased rapidly within 0 to 5 hours and tended to stabilize after 8 hours. Compared with the LPS group, the TEER values ​​of the Caco-2 cell model were significantly increased after 5 hours of treatment with CBD and CBD / (γ-PGA-g-CHOL) treatment, with the CBD / (γ-PGA-g-CHOL) group showing a more significant increase. This suggests that CBD and CBD / (γ-PGA-g-CHOL) can increase transmembrane electrical resistance, reduce intestinal permeability, and improve LPS-induced barrier function damage in the Caco-2 cell model. This ability is concentration-dependent, and the anti-inflammatory effect of the CBD / (γ-PGA-g-CHOL) group is superior to that of the CBD group.

[0302] 1.8.4. Study on the Anti-inflammatory Mechanism of CBD and CBD / (γ-PGA-g-CHOL) Nanomicelles

[0303] Detect the secretion and expression of inflammatory factors and tight junction proteins: After cells are plated and grouped according to the experimental grouping procedure in "1.8.2," collect the cell culture supernatant at 4°C. Take an appropriate amount of the culture supernatant and detect the secretion levels of inflammatory factors such as IL-1β, IL-8, and TNF-α according to the ELISA kit instructions.

[0304] Test results are shown in Figure 32 The experimental results showed that compared with the normal control group, the secretion of IL-8, IL-1β, and TNF-α in the LPS group was significantly increased, causing inflammatory damage; compared with the LPS group, different concentrations of CBD and CBD / (γ-PGA-g-CHOL) could reduce the secretion levels of IL-8, IL-1β, and TNF-α, and CBD / (γ-PGA-g-CHOL) had a more significant inhibitory effect on cellular inflammatory factors, suggesting that CBD and CBD / (γ-PGA-g-CHOL) can alleviate the inflammatory response by regulating the secretion levels of cellular inflammatory factors, and CBD / (γ-PGA-g-CHOL) has better anti-inflammatory activity.

[0305] The present invention provides a γ-polyglutamic acid-grafted cholesterol amphiphilic polymer, its preparation method, and its application. There are many methods and approaches for implementing this technical solution. The above is only a preferred embodiment of the present invention. It should be noted that those skilled in the art may make various improvements and modifications without departing from the principles of the present invention, and such improvements and modifications are also within the scope of protection of the present invention. Any components not specified in this embodiment may be implemented using existing technologies.

Claims

1. Use of a γ-polyglutamic acid grafted cholesterol amphiphilic polymer in the preparation of cannabidiol-loaded drug-loaded nanomicelles, characterized in that: The steps include: (i) mixing a γ-polyglutamic acid grafted cholesterol amphiphilic polymer with deionized water to obtain an amphiphilic polymer aqueous solution; oscillating the amphiphilic polymer aqueous solution at a constant temperature for 16 to 32 hours in an ice bath, and then subjecting the solution to probe ultrasonic treatment in an ice bath; centrifuging after the ultrasonic treatment, filtering the supernatant with a filter membrane to obtain an amphiphilic polymer blank micelle solution; and freeze-drying the amphiphilic polymer blank micelle solution to obtain amphiphilic polymer blank micelles; (ii) mixing cannabidiol with tetrahydrofuran to obtain a cannabidiol solution; mixing the amphiphilic polymer blank micelles obtained in step (i) with deionized water and stirring to obtain an amphiphilic polymer blank micelle aqueous solution; adding the cannabidiol solution to the amphiphilic polymer blank micelle aqueous solution, stirring at 300-400 rpm at room temperature for 5-10 hours, and dialyzing the reaction solution in a dialysis bag after the stirring is completed. The dialyzed solution is filtered and freeze-dried to obtain cannabidiol-loaded drug nanomicelles; The preparation method of the γ-polyglutamic acid grafted cholesterol amphiphilic polymer comprises the following steps: (1) Mixing γ-polyglutamic acid and phosphate buffer evenly to obtain a first mixed solution; adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the first mixed solution, and performing an activation reaction at room temperature for 40 to 60 minutes to obtain an activated solution; (2) Cholesterol and dioxane are mixed evenly to obtain a second mixed solution; the second mixed solution is added to the activation solution obtained in step (1), and the esterification reaction is carried out at room temperature in the dark for 16 to 32 hours; after the reaction is completed, the reaction solution is dialyzed, the dialyzate obtained is centrifuged, and the supernatant is freeze-dried to obtain a γ-polyglutamic acid grafted cholesterol amphiphilic polymer, namely γ-PGA-g-CHOL.

2. The use according to claim 1, characterized in that The phosphate buffer solution has a pH of 5.4; the concentration of carboxyl groups in the first mixed solution is 0.15-0.30 mol·L -1 The molar ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to the carboxyl group in the γ-polyglutamic acid is 1~2.5:1; the activation reaction has a reaction time of 50 min.

3. The use according to claim 1, characterized in that The concentration of cholesterol in the second mixed solution is 0.1-0.6 mmol·mL -1 The molar ratio of the hydroxyl group in the cholesterol in the second mixed solution to the carboxyl group in the γ-polyglutamic acid in the first mixed solution is 2:1~1:1.5; the reaction time of the esterification reaction is 24 h.

4. The use according to claim 1, characterized in that In step (i), the concentration of the γ-polyglutamic acid grafted cholesterol amphiphilic polymer in the amphiphilic polymer aqueous solution is 0.0141 mg·mL -1 ; The probe ultrasonic treatment has an ultrasonic power of 100~250 W and an ultrasonic time of 5~9 min; the centrifugal operation is centrifugation at 4000 rpm for 20 min; the supernatant membrane filtration is filtering the supernatant through a 0.45 µm microporous membrane.

5. The use according to claim 1, characterized in that In step (i), the average particle size of the blank amphiphilic polymer micelles is 117.3±1.2 nm, and the Zeta potential is -20.1±1.5 mV.

6. The use according to claim 1, characterized in that In step (ii), the concentration of cannabidiol in the cannabidiol solution is 1-3 mg·mL -1 The concentration of the amphiphilic polymer blank micelles in the aqueous solution is 3~5 mg•mL -1 ; The volume ratio of tetrahydrofuran in the cannabidiol solution to deionized water in the amphiphilic polymer blank micelle aqueous solution is 1:5~7.

7. The use according to claim 1, characterized in that In step (ii), the cannabidiol-loaded nanomicelles had an encapsulation efficiency of 84.46%±0.35%, a drug loading of 8.78%±0.28%, an average particle size of 163.1±2.3 nm, a polydispersity index PDI of 0.205±0.048, and a zeta potential of -16.5±1.7 mV.

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