A 6-amino-6-deoxy-beta-cyclodextrin-based cannabidiol nanosphere, a preparation method and use thereof

Abstract

The application provides a kind of based on seven substitution 6-amino-6-deoxy-β-cyclodextrin cannabidiol nanosphere, preparation method and purposes, the method first prepares seven substitution 6-amino-6-deoxy-β-cyclodextrin (Aβ-CD) amination cyclodextrin carrier, then cannabidiol (CBD) is with carrier in ethanol-water mixed solvent according to certain proportion, by ultrasonic assisted inclusion+room temperature light shielding stirring to form stable inclusion system, by dialysis purification, freeze drying or spray drying, obtain the spherical nanoparticle of uniform particle size about 120 nm.The application solves the problem that CBD has very low water solubility, poor bioavailability, and insufficient in vivo circulation stability.The nanosphere obtained by the application has high encapsulation efficiency, which can significantly improve the solubility, dissolution rate and in vivo bioavailability of CBD.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation and nanodelivery material technology, specifically relating to a method for preparing cannabidiol nanospheres with uniform particle size and high stability. Background Technology

[0002] Cannabidiol (CBD) is a non-psychoactive cannabinoid extracted from the cannabis plant. It possesses excellent anti-inflammatory, anti-tumor, anti-anxiety, neuroprotective, antioxidant, and gut microbiota-regulating biological activities, making it highly valuable and promising for the pharmaceutical field. In recent years, numerous preclinical and clinical studies have confirmed that CBD has significant antidepressant activity: it can regulate the levels of neurotransmitters such as serotonin (5-HT) and dopamine (DA), activate 5-HT1A receptors, inhibit excessive activation of the hypothalamus-pituitary-adrenal (HPA) axis, reduce neuroinflammatory responses, and promote hippocampal neuronal regeneration and synaptic plasticity. It has shown good therapeutic effects on major depressive disorder, postpartum depression, and chronic stress-induced depression, without the common side effects of traditional antidepressants such as addiction, drowsiness, and sexual dysfunction.

[0003] However, the inherent physicochemical defects of cannabidiol severely limit its application in the treatment of depression: its water solubility is extremely low (only ≈0.023 µg / mL in water at 25°C), making it difficult to prepare into injections or oral solutions; it is easily degraded by the gastrointestinal environment after oral administration, has poor membrane penetration, and extremely low bioavailability in vivo (less than 6% bioavailability after oral administration); it has low blood-brain barrier (BBB) ​​penetration, with only a very small amount of cannabidiol reaching the brain to exert its antidepressant effect; and it has poor circulation stability in vivo, being easily cleared by the reticuloendothelial system, resulting in insufficient drug concentration in the brain and a short duration of action.

[0004] Cyclodextrin inclusion technology is a mainstream approach to improve the water solubility of hydrophobic drugs. β-Cyclodextrin (β-CD) has hydrophobic cavities of suitable size, which can form host-guest inclusion complexes with cannabidiol, thereby improving its water solubility. However, natural β-CD has limitations in water solubility (only 18.5 mg / mL in water at 25°C), lack of pH-responsive drug release capability, and difficulty in forming uniform nanostructures after inclusion. Although conventional modified cyclodextrins can improve water solubility to some extent, they are difficult to achieve controlled drug release, and the products are mostly amorphous powders that cannot form uniform spherical nanostructures, making it difficult to effectively penetrate the blood-brain barrier and meet the core requirement of brain drug delivery in the treatment of depression.

[0005] Therefore, there is an urgent need to develop a CBD-Aβ-CD nanosphere preparation technology that is simple to process, has good reproducibility, controllable particle size, high encapsulation efficiency, and strong blood-brain barrier penetration, so as to fundamentally solve the industry pain points of poor water solubility, low bioavailability, and insufficient brain delivery efficiency of cannabidiol, and provide a safe and efficient new drug formulation for the treatment of depression. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing heptadecandiol nanospheres with a mild process and controllable particle size uniformity, resulting in nanoparticles with an average particle size of 120 nm, regular spherical shape, and PDI < 0.25, thereby achieving efficient encapsulation of cannabidiol, significant solubilization, and a substantial improvement in bioavailability.

[0007] The present invention is implemented using the following technical solution.

[0008] A method for preparing cannabidiol nanospheres based on heptasubstituted 6-amino-6-deoxy-β-cyclodextrin, the method comprising the following steps: Step (1) Prepare a heptasubstituted 6-amino-6-deoxy-β-cyclodextrin carrier, namely Aβ-CD carrier; Step (1.1) β-cyclodextrin β-CD reacts with p-toluenesulfonyl chloride in anhydrous pyridine to prepare fully substituted 6-OTs-β-CD; In step (1.2), 6-OTs-β-CD and the ammonifying agent undergo an ammonolysis reaction in N,N-dimethylformamide (DMF) under nitrogen protection. After precipitation, dialysis, and freeze-drying, heptadecaned 6-amino-6-deoxy-β-cyclodextrin Aβ-CD is obtained.

[0009] Step (2) Dissolve cannabidiol in anhydrous ethanol and Aβ-CD carrier in deionized water; mix cannabidiol and Aβ-CD carrier at a mass ratio of 1:5 to 1:20 to prepare a mixture with ethanol volume percentage of 5% to 15%. Step (3) The mixture obtained in step (2) is treated with ultrasound and stirred at room temperature in the dark to form a nanoscale inclusion system; Step (4) Dialyze the nanoscale inclusion system obtained in step (3) using a dialysis bag in the dark; Step (5) involves freeze-drying or spray-drying the obtained dialysate to obtain CBD-Aβ-CD nanospheres with an average particle size of 110–130 nm.

[0010] Furthermore, in step (2) of the present invention, the mass ratio of cannabidiol to Aβ-CD carrier is 1:8 to 1:15, and the volume percentage of ethanol in the mixture is 8% to 12%.

[0011] Furthermore, in step (3) of the present invention, the ultrasonic power is 200-500 W, the processing time is 5-15 min, and the stirring time at room temperature in the dark is 18-30 h; preferably, the ultrasonic power is 300-400 W, the processing time is 8-12 min, and the stirring time at room temperature in the dark is 24 h.

[0012] Furthermore, in step (4) of the present invention, the molecular weight cutoff of the dialysis bag is 500-1000 KDa, and the dialysis time is 24-48 hours; preferably, the molecular weight cutoff of the dialysis bag is 800 KDa, and the dialysis time is 36 hours; the dialysis solution is changed every 6 hours.

[0013] Furthermore, the freeze-drying conditions for step (5) of the present invention are: pre-freezing at -40℃ for 8 h, vacuum freeze-drying for 48 h; spray drying: air inlet temperature 115~130℃, air outlet temperature 60~70℃.

[0014] Furthermore, the nanospheres obtained in this invention have an average particle size of 115–125 nm, a PDI of <0.25, and a Zeta potential of +15–+30 mV.

[0015] As a product protection measure, the CBD-Aβ-CD nanospheres or Aβ-CD carriers prepared by any of the methods described above in this invention are protected.

[0016] As an application protection, the present invention relates to the application of CBD-Aβ-CD nanospheres in improving the water solubility and bioavailability of cannabidiol.

[0017] The application of the CBD-Aβ-CD nanospheres described in this invention in the preparation of antidepressant therapeutic drugs.

[0018] The beneficial effects of this invention include: (1) Uniform spherical nanostructure: The prepared nanospheres are regular spherical with an average particle size of about 120 nm, PDI < 0.25, excellent dispersibility, no obvious agglomeration, large specific surface area, which can significantly improve the dissolution rate of cannabidiol, and at the same time, they are suitable for the optimal particle size window for in vivo circulation, EPR effect and transmembrane delivery.

[0019] (2) Ultra-high solubility and encapsulation effect: After encapsulation, cannabidiol completely changes from crystalline to amorphous state, and its solubility in water is increased by more than a thousand times. The encapsulation rate is >82% and the drug loading is >7%, which is far superior to the encapsulation effect of ordinary β-CD inclusion complexes, solving the core pain point of extremely low water solubility of cannabidiol.

[0020] (3) Excellent colloidal stability: The nanospheres are positively charged with a Zeta potential of +15 to +30 mV. They can be stably dispersed in aqueous phase and physiological saline for a long time without agglomeration and sedimentation. The positive charge characteristic can significantly enhance the adhesion and endocytosis efficiency of the cell membrane, and significantly improve the bioavailability in vivo.

[0021] (4) Mild process: The entire preparation process does not require high temperature and high pressure, and there are no harsh reaction conditions. The operation is simple, the batch reproducibility is high, and the reagents used are all conventional pharmaceutical excipients with good biocompatibility.

[0022] (5) Wide range of applications: The prepared nanospheres have no organic solvent residue, have high biosafety, and can be widely used in various drug delivery routes such as injection, oral administration, and topical application, which can meet the development needs of multiple fields such as pharmaceuticals, skin care cosmetics, and functional foods.

[0023] The present invention will be further explained below with reference to the accompanying drawings and specific embodiments. Attached Figure Description

[0024] Figure 1 This is a Tyndall effect diagram of the product of this invention.

[0025] Figure 2 This is a transmission electron microscope (TEM) image of the product of this invention (spherical, approximately 120 nm).

[0026] Figure 3 The particle size distribution of the product of this invention is shown in the figure (110–130 nm).

[0027] Figure 4 This is a Zeta potential diagram of the product of this invention.

[0028] Figure 5 A comparison of immobility time in mice subjected to forced swimming experiments. The Y-axis represents immobility time.

[0029] Figure 6 A graph comparing sucrose preference rates in a mouse sucrose preference experiment. The Y-axis represents sucrose preference.

[0030] Figure 7. Comparison of time and number of times mice entered the open box in the black and white box experiment. The Y-axis represents the distance traveled.

[0031] Figure 8. Comparison of mouse tail suspension experiment.

[0032] in, Figures 5 to 8 In the middle, the X-axis represents the control group; the model represents the CUMS depression group; the CBD group represents the free CBD group; the CBDNPs in group represents the CBD-Aβ-CD nanosphere nasal administration group; and the Fluoxetine group represents the fluoxetine group. Detailed Implementation

[0033] The following embodiments are only a part of the technical solutions of the present invention and are not intended to limit all the technical solutions of the present invention. The embodiments of the present invention are provided to further explain and illustrate the details of the technical solutions of the present invention.

[0034] A cannabidiol nanosphere based on heptasubstituted 6-amino-6-deoxy-β-cyclodextrin, its preparation method, and its uses include: I. Preparation of Aβ-CD carrier 1. Synthesis of fully substituted 6-OTs-β-CD: Vacuum-dried β-CD was dissolved in anhydrous pyridine, and an anhydrous pyridine solution of p-toluenesulfonyl chloride was added dropwise under ice bath conditions. After the addition was completed, the temperature was raised to 25-45℃ and the reaction was stirred for 6-12 h. After the reaction was completed, the reaction solution was poured into ice water to precipitate, and after standing, it was filtered to obtain the crude product. The crude product was recrystallized three times with deionized water and dried under vacuum to obtain a white powder of fully substituted 6-OTs-β-CD.

[0035] 2. Synthesis of heptasubstituted 6-amino-6-deoxy-β-cyclodextrin (Aβ-CD): The fully substituted 6-OTs-β-CD was dissolved in anhydrous DMF, and an amination reagent was added under nitrogen protection. The mixture was heated to 75-95℃ and stirred for 12-24 h. After the reaction was completed, the mixture was cooled to room temperature, and the reaction solution was poured into acetone to precipitate the product. The crude product was filtered to obtain the crude product. The crude product was dissolved in deionized water and dialyzed against a dialysis bag with a molecular weight cutoff of 1000 Da for 72 h. The product was then freeze-dried to obtain a white powdery Aβ-CD carrier.

[0036] II. Preparation of CBD-Aβ-CD Nanospheres 1. Stepwise dissolution: Dissolve cannabidiol in anhydrous ethanol to prepare a cannabidiol ethanol solution; dissolve the Aβ-CD carrier in deionized water to prepare a carrier aqueous solution; mix the two solutions at a uniform rate, controlling the volume percentage of ethanol in the mixed solvent to be 5% to 15%, and the mass ratio of cannabidiol to Aβ-CD carrier to be 1:5 to 1:20.

[0037] 2. Ultrasonic-assisted inclusion: The above mixed solution was ultrasonically treated at a power of 200-500 W for 5-15 min, and then placed on a magnetic stirrer and stirred at room temperature in the dark for 18-30 h to form a uniform and transparent nanoscale inclusion system.

[0038] 3. Dialysis purification: Transfer the above inclusion system into a dialysis bag with a molecular weight cutoff of 500-1000 kDa, and dialyze in deionized water in the dark for 24-48 hours, changing the dialysate regularly during the process to completely remove free cannabidiol and organic solvents.

[0039] 4. Drying and shaping: The dialysis solution is freeze-dried or spray-dried to obtain white to light yellow powdery CBD-Aβ-CD nanospheres.

[0040] As a product protection measure, the CBD-Aβ-CD nanospheres or Aβ-CD carriers prepared by any of the methods described above in this invention are protected.

[0041] As an application protection, the present invention relates to the use of CBD-Aβ-CD nanospheres in improving the water solubility, encapsulation efficiency, dissolution rate, or in vivo bioavailability of cannabidiol.

[0042] The application of the CBD-Aβ-CD nanospheres described in this invention in the preparation of antidepressant therapeutic drugs.

[0043] All chemical reagents and solvents used in the examples were of analytical grade; all raw materials could be purchased from chemical reagent companies or biopharmaceutical companies; and the stirring was carried out using a magnetic stirrer. Example 1

[0044] I. Preparation of Aβ-CD carrier (1) Weigh 10 g (8.81 mmol) of β-cyclodextrin that has been vacuum dried for 12 h and dissolve it in 100 mL of anhydrous pyridine. Under ice bath conditions, add dropwise 50 mL of anhydrous pyridine solution containing 16.8 g of p-toluenesulfonyl chloride (88.1 mmol). After the addition is complete, heat to 35 °C and stir for 8 h. After the reaction is complete, pour the reaction solution into 1000 mL of ice water and let it stand for 6 h. Filter to obtain crude product, recrystallize 3 times with deionized water, and vacuum dry at 40 °C for 12 h to obtain white powder of fully substituted 6-OTs-β-CD.

[0045] (2) Weigh 5 g (2.85 mmol) of fully substituted 6-OTs-β-CD and dissolve it in 60 mL of anhydrous DMF. Under nitrogen protection, add 6.34 g (34.2 mmol) of potassium phthalimide, heat to 90 °C, and stir for 18 h. After the reaction is complete, cool to room temperature, pour the reaction solution into 900 mL of acetone to precipitate, and filter to obtain crude product. Add 50 mL of 80% hydrazine hydrate solution to crude product, reflux at 80 °C for 4 h, cool and filter. Dilute the filtrate with deionized water to 200 mL, dialyze with a dialysis bag with a molecular weight cutoff of 1000 Da for 72 h, and freeze dry to obtain white powder of heptadecaned 6-amino-6-deoxy-β-cyclodextrin (Aβ-CD).

[0046] II. Preparation of CBD-Aβ-CD Nanospheres (1) Weigh 20 mg of cannabidiol and dissolve it in 2 mL of anhydrous ethanol; weigh 200 mg of Aβ-CD carrier and dissolve it in 18 mL of deionized water; mix the two solutions at a constant speed, the volume ratio of ethanol in the mixed solvent is 10%, and the mass ratio of cannabidiol to Aβ-CD carrier is 1:10; (2) The above mixed solution was treated under ultrasonic conditions of 350 W for 10 min, and then placed on a magnetic stirrer and stirred at room temperature in the dark for 24 h to form a uniform nanoscale inclusion system. (3) Transfer the mixture into a dialysis bag with a molecular weight cutoff of 800 kDa, and dialyze in deionized water in the dark for 36 h, changing the dialysate every 6 h; (4) The dialysis solution was pre-frozen at -40℃ for 8 h and then freeze-dried under vacuum for 48 h to obtain white to light yellow powdery nanoparticles.

[0047] The average particle size was 118 nm, the PDI was 0.18, and the Zeta potential was -20.8 mV, as determined by dynamic light scattering. Example 2

[0048] I. Preparation of Aβ-CD carrier Same as the carrier preparation steps in Example 1.

[0049] II. Preparation of CBD-Aβ-CD Nanospheres (1) Weigh 15 mg of cannabidiol and dissolve it in 1.6 mL of anhydrous ethanol; weigh 225 mg of Aβ-CD carrier and dissolve it in 14.4 mL of deionized water; after mixing, the volume ratio of ethanol is 10% and the mass ratio of cannabidiol to Aβ-CD carrier is 1:15; (2) The mixed solution was sonicated at 400 W for 8 min and stirred at room temperature in the dark for 24 h; (3) Use a 1000 kDa dialysis bag for dialysis in the dark for 24 hours, and change the dialysis solution every 6 hours; (4) Spray drying (inlet air temperature 125℃, outlet air temperature 65℃) yielded nanoparticles with an average particle size of 122nm, a PDI of 0.19, and a Zeta potential of -20.5 mV. Example 3

[0050] I. Preparation of Aβ-CD carrier Same as the carrier preparation steps in Example 1.

[0051] II. Preparation of CBD-Aβ-CD Nanospheres (1) Weigh 10 mg of cannabidiol and dissolve it in 1.2 mL of anhydrous ethanol; weigh 200 mg of Aβ-CD carrier and dissolve it in 10.8 mL of deionized water; after mixing, the volume ratio of ethanol is 10% and the mass ratio of cannabidiol to Aβ-CD carrier is 1:20. (2) The mixed solution was sonicated at 300 W for 12 min and stirred at room temperature in the dark for 24 h; (3) Use a 500 kDa dialysis bag for dialysis in the dark for 48 hours, and change the dialysis fluid every 8 hours; (4) Spray drying (inlet air temperature 120℃, outlet air temperature 60℃) yielded nanoparticles with an average particle size of 125nm, a PDI of 0.21, and a Zeta potential of -18.7 mV. Example 4

[0052] I. Preparation of Aβ-CD carrier Same as the carrier preparation steps in Example 1.

[0053] II. Preparation of CBD-Aβ-CD Nanospheres (1) Weigh 25 mg of cannabidiol and dissolve it in 2.5 mL of anhydrous ethanol; weigh 125 mg of Aβ-CD carrier and dissolve it in 22.5 mL of deionized water; after mixing, the volume ratio of ethanol is 10% and the mass ratio of cannabidiol to Aβ-CD carrier is 1:5; (2) The mixed solution was sonicated at 450 W for 6 min and stirred at room temperature in the dark for 24 h; (3) Use an 800 kDa dialysis bag for dialysis in the dark for 36 hours, and change the dialysis fluid every 6 hours; (4) Nanoparticles were obtained by freeze drying (pre-freezing at -40℃ for 8 h and vacuum drying for 48 h), with an average particle size of 115 nm, a PDI of 0.17, and a Zeta potential of -24.6 mV. Example 5

[0054] I. Preparation of Aβ-CD carrier Same as the carrier preparation steps in Example 1.

[0055] II. Preparation of CBD-Aβ-CD Nanospheres (1) Weigh 12 mg of cannabidiol and dissolve it in 1.5 mL of anhydrous ethanol; weigh 180 mg of Aβ-CD carrier and dissolve it in 13.5 mL of deionized water; after mixing, the volume ratio of ethanol is 10% and the mass ratio of cannabidiol to Aβ-CD carrier is 1:15; (2) The mixed solution was sonicated at 320 W for 10 min and stirred at room temperature in the dark for 24 h; (3) Use an 800 kDa dialysis bag for dialysis in the dark for 36 hours, and change the dialysis fluid every 6 hours; (4) Nanoparticles were obtained by freeze drying with an average particle size of 119 nm, a PDI of 0.16, and a Zeta potential of -21.8 mV.

[0056] Performance testing: To comprehensively characterize the physicochemical properties and inclusion effect of the CBD-Aβ-CD nanospheres prepared by this method, and to verify their particle size and distribution, crystallization state, colloidal stability, and pharmacological activity, the following performance tests were conducted: 1. Verification of colloidal stability (Figure 1: Tyndall effect diagram) The nanospheres prepared in Example 1 were redispersed in deionized water to prepare a 1 mg / mL aqueous dispersion. When the dispersion was vertically irradiated with a laser pointer, a clear bright light path (Tyndall effect) was observed. This phenomenon confirms that the nanospheres formed a uniform and stable colloidal dispersion system in water, with particle sizes at the nanoscale, no obvious aggregation, and good colloidal stability, which is beneficial for in vivo storage and delivery.

[0057] 2. Morphology and size observation (Figure 2: Transmission electron microscopy image) A small amount of 100 μg / mL nanosphere aqueous dispersion was dropped onto a carbon-plated copper mesh, negatively stained with 2% phosphotungstic acid solution for 1 min, and allowed to air dry before observation using a transmission electron microscope (TEM). As shown in Figure 2, the nanoparticles exhibited a regular, near-spherical shape, good dispersibility, and no obvious agglomeration. Statistical measurements showed that the particle size was highly consistent with the dynamic light scattering results, uniformly distributed at around 120 nm, directly confirming the successful construction of a uniform spherical nanostructure.

[0058] 3. Particle size and distribution index determination (Figure 3 Particle size distribution diagram) The 0.5 mg / mL nanosphere aqueous dispersion was analyzed using dynamic light scattering (DLS) at a temperature of 25°C and a detection angle of 90°. As shown in Figure 3, the particle size distribution exhibits a single, narrow symmetrical peak, with an average particle size of (120±5) nm and a particle size distribution index (PDI) of less than 0.25. This indicates that the nanospheres prepared by the method of this invention have uniform particle size, good dispersibility, and high batch reproducibility.

[0059] 4. Zeta potential (Figure 4 Zeta potential diagram) The 1 mg / mL aqueous dispersion of nanospheres was detected using the zeta potential. As shown in Figure 4, the zeta potential ranged from -15 to -30 mV.

[0060] 5. Determination of encapsulation efficiency and drug loading The encapsulation efficiency and drug loading of cannabidiol in nanospheres were determined by high-performance liquid chromatography (HPLC). 10 mg of nanosphere powder was accurately weighed and dissolved in 10 mL of methanol. The mixture was ultrasonically demulsified for 10 min, filtered through a 0.22 μm organic filter membrane, and the cannabidiol content was determined by HPLC. The cannabidiol content was calculated using the following formula: Encapsulation efficiency (%) = (Actual CBD encapsulated in nanospheres / Total drug mass) × 100% Drug loading (%) = (Actual CBD mass encapsulated in nanospheres / Total mass of nanospheres) × 100% Testing revealed that the nanospheres prepared in Example 1 had an encapsulation efficiency of 86.7% and a drug loading of 7.9%, which is significantly higher than the encapsulation efficiency of ordinary β-CD inclusion complexes (typically below 60%), confirming that the Aβ-CD carrier of the present invention has excellent cannabidiol inclusion ability.

[0061] 6. Behavioral tests: (1) Forced swimming experiment Figure 5 (Evaluating desperate behavior) One hour after the last administration, the mice were placed in a transparent glass tank with a water depth of 10 cm and a water temperature of 25°C. The immobility time of the mice was recorded for the last 4 minutes within 6 minutes (the mice were kept floating with only their heads above the water surface). Figure 5 Compared with the model group, the mice in the nanosphere group of this invention showed shorter forced swimming immobility time, shorter tail suspension immobility time, and improved despair behavior.

[0062] (2) Sugar water preference experiment Figure 6 (Evaluation of lack of pleasure) The administration was conducted on days 19-21. Adaptation period: Mice were given two bottles of 1% sucrose solution, and after 24 hours, one bottle was replaced with pure water. Testing period: After 24 hours of fasting and deprivation of water, mice were simultaneously given one bottle of 1% sucrose solution and one bottle of pure water, and the consumption of both was recorded within 1 hour.

[0063] Sugar solution preference rate (%) = (Sucrose solution consumption / Total liquid consumption) × 100% Figure 6 This indicates that the sucrose preference rate of mice in the model group was significantly reduced; the sucrose preference rate of the nanosphere group of this invention was restored and the loss of pleasure was improved, which was significantly better than the free CBD group and the fluoxetine group.

[0064] (3) Black and white box experiment ( Figure 7 (Assessing anxiety) Three hours after the last administration, the mice were placed in the center of a bright box (45 cm × 27 cm × 27 cm, with the bright box occupying 1 / 3 and the dark box occupying 2 / 3, with a 7 cm × 7 cm passage in the middle), and the time and number of times the mice entered the bright box within 5 minutes were recorded. Figure 7 The model group mice showed reduced time spent entering the open box; the nanosphere group of this invention recovered the time spent entering the open box and improved anxiety, which was significantly better than the free CBD group and the fluoxetine group.

[0065] (4) Tail suspension test Figure 8 (Evaluating desperate behavior) Two hours after the last administration, the mice were fixed to a support 1 cm from the end of their tails, with their heads facing downwards, and the immobility time for the last 4 minutes within 6 minutes was recorded. Figure 8 Compared with the model group, the mice in the nanosphere group of this invention showed shorter forced swimming immobility time, shorter tail suspension immobility time, and improved despair behavior.

[0066] 7. After inclusion, cannabidiol completely transforms from a crystalline state to an amorphous state, eliminating the energy barrier to crystal dissolution, which is the core reason for the significant increase in solubility. Heptasubstituted 6-amino-6-deoxy-β-cyclodextrin (Aβ-CD) itself has excellent water solubility (25℃>500 mg / mL), far exceeding that of natural β-CD (18.5 mg / mL), providing a good dispersion matrix for the inclusion complex.

[0067]

[0068] The core highlights of this invention are: Heptasubstituted 6-amino-6-deoxy-β-cyclodextrin (Aβ-CD) is a fully aminated modified product of β-CD. The 6-position hydroxyl groups of all 7 glucose units of β-CD are replaced by amino groups, which not only retains the hydrophobic cavity and host-guest inclusion ability of β-CD, but also has advantages that natural β-CD cannot match: the introduction of amino groups greatly improves the water solubility of the product (solubility in water at 25°C > 500 mg / mL), while endowing the material with positive charge characteristics and pH responsiveness, so that it can exist stably in physiological pH environment and rapidly release drugs in acidic microenvironments such as inflammation and lysosomes; the positive charge characteristics can significantly enhance the adhesion and transmembrane transport efficiency of nanoparticles to blood-brain barrier endothelial cells, and greatly increase the brain accumulation of cannabidiol; the particle size of about 120 nm is just right for the optimal window for blood-brain barrier penetration and brain accumulation, while avoiding rapid clearance by the kidneys. Currently, there are no reports, either domestically or internationally, of using heptasubstituted 6-amino-6-deoxy-β-cyclodextrin to encapsulate cannabidiol and preparing uniform spherical nanospheres with a particle size of approximately 120 nm for the treatment of depression.

[0069] The above descriptions are merely some specific embodiments of the present invention (since the present invention encompasses numerical ranges, the embodiments cannot be exhaustive; the scope of protection described in the present invention includes the numerical range and other technical aspects of the present invention). Specific details or common knowledge in the solutions are not described in detail here (including but not limited to abbreviations, acronyms, units commonly used in the art, experimental methods, parameter conditions, etc.). It should be noted that the above embodiments do not limit the present invention in any way. For those skilled in the art, any technical solutions obtained by equivalent substitution or equivalent transformation fall within the scope of protection of the present invention. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing cannabidiol nanospheres based on heptoxy-substituted 6-amino-6-deoxy-β-cyclodextrin, characterized in that, The preparation method includes the following steps: Step (1) Prepare a heptasubstituted 6-amino-6-deoxy-β-cyclodextrin carrier, namely Aβ-CD carrier; Step (2) Dissolve cannabidiol in anhydrous ethanol and Aβ-CD carrier in deionized water; mix cannabidiol and Aβ-CD carrier at a mass ratio of 1:5 to 1:20 to prepare a mixture with ethanol volume percentage of 5% to 15%. Step (3) The mixture obtained in step (2) is treated with ultrasound and stirred at room temperature in the dark to form a nanoscale inclusion system; Step (4) Dialyze the nanoscale inclusion system obtained in step (3) using a dialysis bag in the dark; Step (5) The obtained dialysate is freeze-dried or spray-dried to obtain CBD-Aβ-CD nanospheres.

2. The preparation method according to claim 1, characterized in that, The step (1) for preparing the heptasubstituted 6-amino-6-deoxy-β-cyclodextrin Aβ-CD carrier is as follows: Step (1.1) β-cyclodextrin β-CD reacts with p-toluenesulfonyl chloride in anhydrous pyridine to prepare fully substituted 6-OTs-β-CD; Step (1.2) 6-OTs-β-CD undergoes ammonolysis with an amination reagent in N,N-dimethylformamide DMF under nitrogen protection. After precipitation, dialysis, and freeze-drying, heptadecaned 6-amino-6-deoxy-β-cyclodextrin Aβ-CD is obtained.

3. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of cannabidiol to Aβ-CD carrier is 1:8 to 1:15, and the volume percentage of ethanol in the mixture is 8% to 12%.

4. The preparation method according to claim 1, characterized in that, In step (3), the ultrasonic power is 200-500 W, the processing time is 5-15 min, and the stirring time at room temperature in the dark is 18-30 h; preferably, the ultrasonic power is 300-400 W, the processing time is 8-12 min, and the stirring time at room temperature in the dark is 24 h.

5. The preparation method according to claim 1, characterized in that, In step (4), the molecular weight cutoff of the dialysis bag is 500-1000 KDa, and dialysis is performed for 24-48 hours; preferably, the molecular weight cutoff of the dialysis bag is 800 KDa, and dialysis is performed for 36 hours; the dialysis solution is changed every 6 hours.

6. The preparation method according to claim 1, characterized in that, Step (5) Freeze-drying conditions: -40℃ pre-freeze for 8 hours, vacuum freeze-dry for 48 hours; spray drying: inlet air temperature 115~130℃, outlet air temperature 60~70℃.

7. The preparation method according to claim 1, characterized in that, The obtained nanospheres have an average particle size of 115–125 nm, PDI < 0.25, and Zeta potential of +15–+30 mV.

8. The CBD-Aβ-CD nanospheres or Aβ-CD carrier prepared by any of the methods of claims 1 to 7.

9. The use of the CBD-Aβ-CD nanospheres according to claim 8 in improving the water solubility, encapsulation rate, dissolution rate, or in vivo bioavailability of cannabidiol.

10. The use of the CBD-Aβ-CD nanospheres according to claim 8 in the preparation of antidepressant therapeutic drugs.

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