Beta-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside ck, preparation method and application thereof

Abstract

The present application relates to the technical field of nanomedicine, in particular to a β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticle carrying ginsenoside CK, a preparation method and application. A first esterification reaction is performed between a β-cyclodextrin solution and a carboxyl-activated carboxymethyl chitosan solution to obtain a β-cyclodextrin-carboxymethyl chitosan polymer; a second esterification or amidation reaction is performed by adding carboxyl-activated deoxycholic acid to the β-cyclodextrin-carboxymethyl chitosan polymer to obtain a β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer; an ethanol solution of ginsenoside CK is mixed with a water solution of the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer to obtain a whole particle, and a freeze-drying protective agent is added for freeze-drying to obtain the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticle carrying ginsenoside CK. The present application focuses on the design of an intelligent carrier, realizes the synergistic optimization of high drug loading and intelligent delivery, and overcomes the defects of the prior art.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine technology, specifically to β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK, their preparation method, and applications. Background Technology

[0002] Ginsenoside CK is a compound with diverse pharmacological activities. It exhibits significant inhibitory effects on various tumors, such as lung cancer, liver cancer, and breast cancer, through mechanisms including inducing apoptosis, inhibiting cell proliferation, suppressing angiogenesis, and inhibiting cancer cell metastasis. These properties make it a promising candidate for clinical research and application. However, ginsenoside CK has poor water solubility and is unstable in the gastrointestinal fluid environment, which greatly limits its oral bioavailability.

[0003] In existing technologies, such as the patent CN201610109341.1, a method for preparing chitosan-based drug-loaded nanoparticles is disclosed. This method involves bonding a hydrophobic drug to a hydrophilic substrate to construct amphiphilic polymer molecules. The amphiphilic molecules are then mixed with the drug. During the self-assembly of the amphiphilic molecules to form nanomicelles, the free drug is encapsulated by intermolecular interactions between the bonded drug and the free drug, resulting in chitosan-based drug-loaded nanoparticles. Sufficiently high drug loading can reduce the amount of carrier material used, lowering toxicity risks and production costs. For example, liposomal chemotherapy drugs require a drug loading of ≥5% to meet clinical dosage requirements, such as doxorubicin liposomes. However, the chitosan-based drug-loaded nanoparticles prepared by this technique can only achieve a maximum drug loading of 20%, and there are still many shortcomings in terms of uniformity, stability, and bioavailability, limiting their clinical application.

[0004] For example, patent CN202211034090.7 discloses glycyrrhizin-loaded nanoparticles, with relative encapsulation efficacies of baicalin, paeoniflorin, and glycyrrhizin of 31.7%, 22.56%, and 23.8%, respectively. The drug loading and encapsulation efficacies are generally low, failing to meet the requirements for targeted delivery of higher drug concentrations. CN202111086004.2 discloses a method for preparing and applying drug-loaded polymer nanoparticles responsive to reactive oxygen species. The prepared nanoparticles have a particle size of 300nm~500nm, are regularly spherical, and have good dispersibility, but the drug loading rate is only 5%~10%, resulting in excessively large administration volumes. For example, intravenous injection requires a large amount of fluid, affecting patient compliance. CN201910537009.9 discloses a method for preparing a core-shell structured multifunctional mesoporous silica drug carrier. Although the prepared composite nanoparticles have strong drug loading capacity, the drug loading in various simulated drugs is only about 30%. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK, their preparation method, and applications. This invention uses carboxymethyl chitosan as a raw material, grafting hydrophobic cavities of β-cyclodextrin onto the carboxymethyl chitosan, followed by grafting deoxycholic acid to form a β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer. Simultaneously, the polymer is granulated via dialysis while undergoing hydrophobic self-assembly. The hydrophobic core of the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer captures the glycosyl groups of hydrophobic ginsenoside CK into the core, completing the encapsulation of ginsenoside CK and obtaining a drug-loaded nanoparticle suspension. Subsequently, the suspension is freeze-dried after adding a lyophilization protectant to obtain β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK. This invention focuses on intelligent carrier design to achieve synergistic optimization of high drug loading and intelligent delivery, while also possessing uniformity and stability, overcoming the shortcomings of existing technologies.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The first objective of this invention is to provide a method for preparing β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK, comprising the following steps:

[0008] S1. Using N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine as activators, the activators are mixed with an aqueous solution of carboxymethyl chitosan. The carboxyl groups on the carboxymethyl chitosan are activated by the activators to obtain a carboxyl-activated carboxymethyl chitosan solution.

[0009] S2. After dissolving β-cyclodextrin in a solvent, mix it with a carboxyl-activated carboxymethyl chitosan solution to carry out the first esterification reaction, and obtain β-cyclodextrin-carboxymethyl chitosan polymer.

[0010] S3. Add carboxyl-activated deoxycholic acid dropwise to the β-cyclodextrin-carboxymethyl chitosan polymer to carry out a second esterification or amidation reaction to obtain the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer.

[0011] The purpose of adding the dropwise particles is to improve the dispersibility of the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles and prevent them from agglomerating.

[0012] S4. An ethanol solution of ginsenoside CK was added dropwise to an aqueous solution of β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer. Self-assembly and granulation via hydrophobic drive and dialysis were then performed to obtain a drug-loaded nanoparticle suspension. Ethanol was chosen because ginsenoside CK is only soluble in alcohol solvents, and ethanol is used as a solvent in multiple esterification reactions. Among organic solvents, ethanol has the lowest toxicity.

[0013] The self-assembly here is a physical process rather than a chemical reaction, which does not require the breaking or formation of covalent bonds, and its driving force comes from the weak interactions between molecules.

[0014] S5. After mixing the drug-loaded nanoparticle suspension and the freeze-drying protectant, freeze-drying was performed to obtain β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK.

[0015] Preferably, during the esterification reaction, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine are used as activators to activate deoxycholic acid to obtain carboxyl-activated deoxycholic acid; wherein the mass-volume ratio of deoxycholic acid, N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and β-cyclodextrin-carboxymethyl chitosan polymer is 5mg~10mg:4mg:2.5mg:0.9mL.

[0016] Preferably, during the amidation reaction, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride are used as activators to activate deoxycholic acid to obtain carboxyl-activated deoxycholic acid; wherein the mass-volume ratio of deoxycholic acid, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and β-cyclodextrin-carboxymethyl chitosan polymer is 4 mg~6 mg:4 mg:5 mg:0.9 mL.

[0017] Preferably, the mass ratio of β-cyclodextrin to carboxyl-activated carboxymethyl chitosan is 5:1~3.

[0018] Preferably, the mass ratio of β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer to ginsenoside CK is 5:3~7.

[0019] Preferably, the freeze-drying protectant is selected from at least one of mannitol, sucrose, and trehalose.

[0020] Preferably, the mass-to-volume ratio of the lyophilization protectant to the drug-loaded nanoparticle suspension is 0.1g~1g:100mL.

[0021] Preferably, when preparing the β-cyclodextrin-carboxymethyl chitosan polymer, the conditions for the first esterification reaction are: stirring at room temperature for 12-24 hours.

[0022] Preferably, when preparing the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer, the conditions for the second esterification reaction are: stirring at room temperature for 12-24 hours.

[0023] Preferably, when preparing the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer, the amidation reaction conditions are: stirring at room temperature for 12-24 hours.

[0024] Preferably, the solvent for dissolving β-cyclodextrin is selected from dimethyl sulfoxide or a mixture of ethanol and water.

[0025] The second objective of this invention is to provide β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK prepared by the above-mentioned method, wherein the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK have a core-shell structure, with the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer as the shell and ginsenoside CK as the core.

[0026] Preferably, the drug loading of ginsenoside CK in the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles is 86.67%. Here, the drug loading of ginsenoside CK refers to the percentage by mass of the drug encapsulated in the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer within the ginsenoside CK-loaded β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles.

[0027] Preferably, the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK have an average particle size of 200 nm to 350 nm and are uniform in size.

[0028] A third objective of this invention is to provide the application of the above-mentioned β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK in the preparation of antitumor drugs.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] 1. This invention provides a method for preparing β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK. The method involves dissolving β-cyclodextrin in a solvent and mixing it with a carboxyl-activated carboxymethyl chitosan solution for a first esterification reaction to obtain a β-cyclodextrin-carboxymethyl chitosan polymer. Carboxyl-activated deoxycholic acid is then added dropwise to the β-cyclodextrin-carboxymethyl chitosan polymer for a second esterification or amidation reaction to obtain another β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer. An ethanol solution of ginsenoside CK is then added dropwise to an aqueous solution of the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer for self-assembly and simultaneous dialysis granulation to obtain a drug-loaded nanoparticle suspension. The drug-loaded nanoparticle suspension is then mixed with a lyophilization protectant and freeze-dried to obtain β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK. The β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK prepared by this invention not only effectively overcome the bottleneck of limited bioavailability of ginsenoside CK due to its low water solubility and sensitivity to the gastrointestinal environment, achieving a significant improvement in drug loading efficiency, but also solve the problem of traditional drug-loaded nanoparticles being unable to simultaneously achieve high drug loading capacity and intelligent delivery through the synergistic effect of hydrophobic cavity confinement effect and pH-responsive structure dynamic regulation.

[0031] 2. In the preparation of β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK, this invention innovatively introduces β-cyclodextrin with a hydrophobic cavity structure into carboxymethyl chitosan. Through the principle of like dissolves like, and the dialysis of the aqueous solution of the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer, the hydrophobic substance ginsenoside CK is forced to be captured into the hydrophobic core of the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer, which significantly improves the drug loading capacity and aqueous solution stability of ginsenoside CK.

[0032] 3. The β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK prepared in this invention innovatively incorporate freeze-drying protection technology. Specifically, the freeze-drying protectant trehalose forms a high-viscosity amorphous glassy state (i.e., non-crystalline) during freeze-drying, inhibiting ice crystal growth and reducing mechanical damage to the core-shell structure of the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK, thus significantly enhancing their stability and instantaneous resolubility.

[0033] 4. The β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK prepared in this invention, hereinafter referred to as nanoparticles, achieve pH-responsive intelligent delivery of ginsenoside CK through molecular structure design via a second esterification reaction and an amidation reaction. Specifically, the ester bonds formed by the reaction of carboxymethyl chitosan and β-cyclodextrin enable the nanoparticles to exhibit strong chemical bonding in the acidic environment of gastric acid at pH 1.2, promoting their contraction to form a dense structure and effectively protecting ginsenoside CK from acidic degradation. In the intestinal fluid environment at pH 7.4, the ester bonds weaken, causing the carboxymethyl chitosan shell network structure of the nanoparticles to become looser, achieving a sustained-release effect of ginsenoside CK, promoting gradient absorption and utilization by intestinal cells, and significantly improving the bioavailability of ginsenoside CK. In addition, some nanoparticles are absorbed whole by intestinal cells and transported to the tumor microenvironment at pH 5.8. The amide bonds in their molecular structure can break in response to the weakly acidic microenvironment, triggering the specific release of ginsenoside CK from the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles, forming a dual pH response mechanism. This structural characteristic not only effectively avoids the destruction of active ingredients by gastric acid, but also achieves precise targeted drug release through the response of the tumor microenvironment. Attached Figure Description

[0034] Figure 1 CD-g-CMDA of Example 1 E Transmission electron microscopy image and structural schematic diagram of the @CK-NPs suspension, where a represents CD-g-CMDA from Example 1. E Transmission electron microscopy image of the CK-NPs suspension, b is CD-g-CMDA from Example 1. E Schematic diagram of the structure of @CK-NPs suspension.

[0035] Figure 2 The ingredients are ginsenoside CK, β-cyclodextrin, carboxymethyl chitosan, deoxycholic acid, and CD-g-CMDA from Example 1. E Infrared spectrum of @CK-NPs suspension.

[0036] Figure 3 CD-g-CMDA of Example 1 E @CK-NPs suspension and CD-g-CMDA from Example 8 A Drug loading diagram of @CK-NPs suspension.

[0037] Figure 4 CD-g-CMDA of Example 1 E @CK-NPs suspension and CD-g-CMDA from Example 8 A Particle size distribution of @CK-NPs suspension.

[0038] Figure 5 For Comparative Example 1, CD-g-CMDA E @CK D -Particle size distribution diagram of NPs.

[0039] Figure 6 CD-g-CMDA in Example 1 under different pH conditions E Release characteristics diagram of @CK-NPs.

[0040] Figure 7 CD-g-CMDA of Examples 1 to 4 E @CK-NPs and CD-g-CMDA of Comparative Example 1 E @CK D - Particle size change diagram after instantaneous resolution of NPs.

[0041] Figure 8 CD-g-CMDA of Examples 1 and 4-7 E Particle size change diagram of @CK-NPs after instantaneous resolution.

[0042] Figure 9 For Comparative Example 2, CD-g-CMDA E and CD-g-CMDA E The in vitro biosafety evaluation results of -NPs are shown in the figure, where (a) is the comparison example 2 CD-g-CMDA. E The in vitro biosafety evaluation results are shown in Figure (b), which is the comparison example 2 CD-g-CMDA. E -Graph showing the in vitro biosafety evaluation results of NPs.

[0043] Figure 10 CD-g-CMDA of Example 1 E The inhibitory effects of CK-NPs and ginsenoside CK on the proliferation of HepG2 cells at different treatment times are shown in the figures. (a) represents a treatment time of 24 h, and (b) represents a treatment time of 48 h. Detailed Implementation

[0044] The technical solution of the present invention will be clearly and completely described below with reference to the data in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0045] It should be noted that the technical terms used in this invention are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of this invention. Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the following embodiments of this invention can be purchased commercially or prepared by existing methods. Among them, carboxymethyl chitosan is denoted as CMCS; N,N'-dicyclohexylcarbodiimide is denoted as DCC; 4-dimethylaminopyridine is denoted as DMAP; N-hydroxysuccinimide is denoted as NHS; and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is denoted as EDC-HCl.

[0046] In existing technologies, chitosan-based nanodelivery systems generally suffer from bottlenecks such as limited drug loading capacity, insufficient encapsulation efficiency, and limited environmental responsiveness. While chitosan-based nanoparticles, exemplified by CN201610109341.1, achieve drug bonding and encapsulation through amphiphilic polymer self-assembly, their maximum drug loading capacity is only 20%, and their particle uniformity and stability in the gastric acid environment are poor, making it difficult to ensure effective drug delivery in the complex digestive system. The glycyrrhizin nanoparticles developed in CN202211034090.7 have an encapsulation rate of less than 25% for polyglycosides, significantly affecting the establishment of drug concentration gradients. Although the reactive oxygen species-responsive nanosystem designed in CN202111086004.2 exhibits good dispersibility, its 5%–10% drug loading rate necessitates a doubling of the dosage, severely limiting its feasibility for clinical application. Although CN201910537009.9 uses a mesoporous silica carrier to increase drug loading to 30%, its rigid structure leads to a sluggish pH response, making it impossible to achieve precise drug release in the tumor microenvironment. Current technologies lack intelligent carriers that can simultaneously meet the requirements of high drug loading, multi-stage pH response, and cross-barrier delivery. This is especially true for active ingredients like ginsenoside CK, which possess both strong hydrophobicity and acid sensitivity; traditional delivery systems struggle to overcome the dual technical barriers of gastrointestinal degradation and targeted release.

[0047] To address the problems existing in the prior art, this invention provides a method for preparing β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK, comprising the following steps: dissolving β-cyclodextrin in a solvent and mixing it with a carboxyl-activated carboxymethyl chitosan solution to perform a first esterification reaction, obtaining a β-cyclodextrin-carboxymethyl chitosan polymer; adding carboxyl-activated deoxycholic acid dropwise to the β-cyclodextrin-carboxymethyl chitosan polymer and performing a second esterification reaction or amidation reaction, obtaining a β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer; adding an ethanol solution of ginsenoside CK dropwise to an aqueous solution of the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer, performing self-assembly and dialysis granulation to obtain a drug-loaded nanoparticle suspension; mixing the drug-loaded nanoparticle suspension with a lyophilization protectant and then freeze-drying to obtain β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK.

[0048] To address the limitation of drug loading in existing technologies, this invention constructs a ternary composite system of β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid. Utilizing the principle of similarity-miscibility between the hydrophobic cavities of β-cyclodextrin and deoxycholic acid, the drug loading efficiency of ginsenoside CK is enhanced, increasing it to 86.67%, significantly breaking through the 20%~30% drug loading limit of existing chitosan-based carriers.

[0049] To address the problem of insufficient responsiveness to multiple environments in existing technologies, this invention constructs a three-tiered delivery mechanism—gastric acid contraction protection, intestinal fluid sustained release, and tumor microacid targeted release—by leveraging the differentiated response characteristics of ester and amide bonds in the molecular structure, thereby achieving intelligent delivery across biological barriers.

[0050] To address the particle stability defects in existing technologies, this invention innovatively introduces a freeze-drying protectant to form a glassy protective layer. By inhibiting ice crystal growth, the integrity of the nanoparticle structure is maintained, resulting in a particle size retention rate of >95% after reconstitution.

[0051] To address the issue of low bioavailability in existing technologies, the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles of ginsenoside CK in this invention have a particle size of 200nm~350nm, which enhances the transmembrane absorption efficiency in the intestine. At the same time, the cholic acid transporter-mediated effect of deoxycholic acid promotes tumor-targeted accumulation, thereby improving the bioavailability of the drug compared to free ginsenoside CK.

[0052] To enable those skilled in the art to more clearly understand the technical solution of the present invention, the following will describe it in detail with reference to specific embodiments:

[0053] Example 1

[0054] A method for preparing β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK includes the following steps:

[0055] Preparation of S1, β-cyclodextrin-carboxymethyl chitosan polymer:

[0056] First, 1g of CMCS was weighed and dissolved in 100mL of deionized water to obtain an aqueous solution of carboxymethyl chitosan. 800mg of DCC and 500mg of DMAP were dissolved together in 20mL of dimethyl sulfoxide solution. After mixing, the mixture was added dropwise to the obtained aqueous solution of carboxymethyl chitosan. The mixture was stirred at 1000r / min for 2h at room temperature to obtain a dimethyl sulfoxide solution of carboxyl-activated carboxymethyl chitosan, denoted as CMCS solution.

[0057] Next, 5g of β-cyclodextrin was dissolved in 60mL of dimethyl sulfoxide solution to obtain a dimethyl sulfoxide solution of β-cyclodextrin.

[0058] Finally, the dimethyl sulfoxide solution of β-cyclodextrin was mixed with the CMCS solution, stirred at 800 r / min at room temperature, and esterified for 24 h to obtain the β-cyclodextrin-carboxymethyl chitosan polymer, denoted as β-CD-CMCS.

[0059] Preparation of S2, β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer:

[0060] 500 mg of deoxycholic acid, 400 mg of DCC, and 250 mg of DMAP were dissolved in 10 mL of dimethyl sulfoxide solution and stirred for 2 h to obtain a dimethyl sulfoxide solution of activated carboxy-deoxycholic acid. The activated carboxy-deoxycholic acid dimethyl sulfoxide solution was then added dropwise to 90 mL of β-CD-CMCS, and the mixture was stirred at 900 r / min for 24 h at room temperature to carry out an esterification reaction. This yielded a β-cyclodextrin-carboxymethyl chitosan polymer, i.e., an esterified β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer, denoted as CD-g-CMDA, resulting from the esterification of deoxycholic acid with the hydroxyl groups in carboxymethyl chitosan. E .

[0061] S3. Dissolve 5 mg of ginsenoside CK in 5 mL of ethanol solution to obtain the CK solution dissolved in ethanol; then take 5 mg of CD-g-CMDA E Dissolved in 5 mL of deionized water, CD-g-CMDA is obtained. E An aqueous solution of CK dissolved in ethanol was added dropwise to CD-g-CMDA. E After stirring in an aqueous solution for 2 hours, the solution was placed into a dialysis bag with a molecular weight of 7000 and dialyzed in 800 mL of pure aqueous solution for 24 hours to obtain a drug-loaded nanoparticle suspension, denoted as CD-g-CMDA.E @CK-NPs suspension.

[0062] S4. Place CD-g-CMDA E @CK-NPs suspension and trehalose were mixed at a mass ratio of 100:0.4. After mixing, the mixture was pre-cooled overnight at -20°C and then freeze-dried in a freeze dryer until completely freeze-dried to obtain β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK, denoted as CD-g-CMDA. E @CK-NPs.

[0063] Example 2

[0064] A method for preparing β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK is the same as the preparation steps in Example 1, except that trehalose in step S4 is replaced with sucrose, and includes the following steps:

[0065] Preparation of S1, β-cyclodextrin-carboxymethyl chitosan polymer:

[0066] First, 1g of CMCS was weighed and dissolved in 100mL of deionized water to obtain an aqueous solution of carboxymethyl chitosan. 800mg of DCC and 500mg of DMAP were dissolved together in 20mL of dimethyl sulfoxide solution. After mixing, the mixture was added dropwise to the obtained aqueous solution of carboxymethyl chitosan. The mixture was stirred at 1000r / min for 2h at room temperature to obtain a dimethyl sulfoxide solution of carboxyl-activated carboxymethyl chitosan, denoted as CMCS solution.

[0067] Next, 5g of β-cyclodextrin was dissolved in 60mL of dimethyl sulfoxide solution to obtain a dimethyl sulfoxide solution of β-cyclodextrin.

[0068] Finally, the dimethyl sulfoxide solution of β-cyclodextrin was mixed with the CMCS solution, stirred at 800 r / min at room temperature, and esterified for 24 h to obtain the β-cyclodextrin-carboxymethyl chitosan polymer, denoted as β-CD-CMCS.

[0069] Preparation of S2, β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer:

[0070] 500 mg of deoxycholic acid, 400 mg of DCC, and 250 mg of DMAP were dissolved in 10 mL of dimethyl sulfoxide solution and stirred for 2 h to obtain a dimethyl sulfoxide solution of activated carboxy-deoxycholic acid. The activated carboxy-deoxycholic acid dimethyl sulfoxide solution was then added dropwise to 90 mL of β-CD-CMCS, and the mixture was stirred at 900 r / min for 24 h at room temperature to carry out an esterification reaction. This yielded a β-cyclodextrin-carboxymethyl chitosan polymer, i.e., an esterified β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer, denoted as CD-g-CMDA, resulting from the esterification of deoxycholic acid with the hydroxyl groups in carboxymethyl chitosan. E .

[0071] S3. Dissolve 5 mg of ginsenoside CK in 5 mL of ethanol solution to obtain the CK solution dissolved in ethanol; then take 5 mg of CD-g-CMDA E Dissolved in 5 mL of deionized water, CD-g-CMDA is obtained. E An aqueous solution of CK dissolved in ethanol was added dropwise to CD-g-CMDA. E After stirring in an aqueous solution for 2 hours, the solution was placed into a dialysis bag with a molecular weight of 7000 and dialyzed in 800 mL of pure aqueous solution for 24 hours to obtain a drug-loaded nanoparticle suspension, denoted as CD-g-CMDA. E @CK-NPs suspension.

[0072] S4. Place CD-g-CMDA E @CK-NPs suspension and sucrose were mixed at a mass ratio of 100:0.4. After mixing, the mixture was pre-cooled overnight in a refrigerator at -20°C and then freeze-dried in a freeze dryer until completely freeze-dried to obtain β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK.

[0073] Example 3

[0074] A method for preparing β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK is the same as the preparation steps in Example 1, except that trehalose in step S4 is replaced with mannitol, and includes the following steps:

[0075] Preparation of S1, β-cyclodextrin-carboxymethyl chitosan polymer:

[0076] First, 1g of CMCS was weighed and dissolved in 100mL of deionized water to obtain an aqueous solution of carboxymethyl chitosan. 800mg of DCC and 500mg of DMAP were dissolved together in 20mL of dimethyl sulfoxide solution. After mixing, the mixture was added dropwise to the obtained aqueous solution of carboxymethyl chitosan. The mixture was stirred at 1000r / min for 2h at room temperature to obtain a dimethyl sulfoxide solution of carboxyl-activated carboxymethyl chitosan, denoted as CMCS solution.

[0077] Next, 5g of β-cyclodextrin was dissolved in 60mL of dimethyl sulfoxide solution to obtain a dimethyl sulfoxide solution of β-cyclodextrin.

[0078] Finally, the dimethyl sulfoxide solution of β-cyclodextrin was mixed with the CMCS solution, stirred at 800 r / min at room temperature, and esterified for 24 h to obtain the β-cyclodextrin-carboxymethyl chitosan polymer, denoted as β-CD-CMCS.

[0079] Preparation of S2, β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer:

[0080] 500 mg of deoxycholic acid, 400 mg of DCC, and 250 mg of DMAP were dissolved in 10 mL of dimethyl sulfoxide solution and stirred for 2 h to obtain a dimethyl sulfoxide solution of activated carboxy-deoxycholic acid. The activated carboxy-deoxycholic acid dimethyl sulfoxide solution was then added dropwise to 90 mL of β-CD-CMCS, and the mixture was stirred at 900 r / min for 24 h at room temperature to carry out an esterification reaction. This yielded a β-cyclodextrin-carboxymethyl chitosan polymer, i.e., an esterified β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer, denoted as CD-g-CMDA, resulting from the esterification of deoxycholic acid with the hydroxyl groups in carboxymethyl chitosan. E .

[0081] S3. Dissolve 5 mg of ginsenoside CK in 5 mL of ethanol solution to obtain the CK solution dissolved in ethanol; then take 5 mg of CD-g-CMDA E Dissolved in 5 mL of deionized water, CD-g-CMDA is obtained. E An aqueous solution of CK dissolved in ethanol was added dropwise to CD-g-CMDA. E After stirring in an aqueous solution for 2 hours, the solution was placed into a dialysis bag with a molecular weight of 7000 and dialyzed in 800 mL of pure aqueous solution for 24 hours to obtain a drug-loaded nanoparticle suspension, denoted as CD-g-CMDA. E @CK-NPs suspension.

[0082] S4. Place CD-g-CMDA E@CK-NPs suspension and mannitol were mixed at a mass ratio of 100:0.4. After mixing, the mixture was pre-cooled overnight in a refrigerator at -20°C and then freeze-dried in a freeze dryer until completely freeze-dried to obtain β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK.

[0083] Example 4

[0084] A method for preparing β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK is the same as the preparation steps in Example 1, except that the mass fraction of trehalose in step S4 is replaced from 0.4% to 1%, and includes the following steps:

[0085] Preparation of S1, β-cyclodextrin-carboxymethyl chitosan polymer:

[0086] First, 1g of CMCS was weighed and dissolved in 100mL of deionized water to obtain an aqueous solution of carboxymethyl chitosan. 800mg of DCC and 500mg of DMAP were dissolved together in 20mL of dimethyl sulfoxide solution. After mixing, the mixture was added dropwise to the obtained aqueous solution of carboxymethyl chitosan. The mixture was stirred at 1000r / min for 2h at room temperature to obtain a dimethyl sulfoxide solution of carboxyl-activated carboxymethyl chitosan, denoted as CMCS solution.

[0087] Next, 5g of β-cyclodextrin was dissolved in 60mL of dimethyl sulfoxide solution to obtain a dimethyl sulfoxide solution of β-cyclodextrin.

[0088] Finally, the dimethyl sulfoxide solution of β-cyclodextrin was mixed with the CMCS solution, stirred at 800 r / min at room temperature, and esterified for 24 h to obtain the β-cyclodextrin-carboxymethyl chitosan polymer, denoted as β-CD-CMCS.

[0089] Preparation of S2, β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer:

[0090] 500 mg of deoxycholic acid, 400 mg of DCC, and 250 mg of DMAP were dissolved in 10 mL of dimethyl sulfoxide solution and stirred for 2 h to obtain a dimethyl sulfoxide solution of activated carboxy-deoxycholic acid. The activated carboxy-deoxycholic acid dimethyl sulfoxide solution was then added dropwise to 90 mL of β-CD-CMCS, and the mixture was stirred at 900 r / min for 24 h at room temperature to carry out an esterification reaction. This yielded a β-cyclodextrin-carboxymethyl chitosan polymer, i.e., an esterified β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer, denoted as CD-g-CMDA, resulting from the esterification of deoxycholic acid with the hydroxyl groups in carboxymethyl chitosan. E .

[0091] S3. Dissolve 5 mg of ginsenoside CK in 5 mL of ethanol solution to obtain the CK solution dissolved in ethanol; then take 5 mg of CD-g-CMDA E Dissolved in 5 mL of deionized water, CD-g-CMDA is obtained. E An aqueous solution of CK dissolved in ethanol was added dropwise to CD-g-CMDA. E After stirring in an aqueous solution for 2 hours, the solution was placed into a dialysis bag with a molecular weight of 7000 and dialyzed in 800 mL of pure aqueous solution for 24 hours to obtain a drug-loaded nanoparticle suspension, denoted as CD-g-CMDA. E @CK-NPs suspension.

[0092] S4. Place CD-g-CMDA E @CK-NPs suspension and trehalose were mixed at a mass ratio of 100:1. After mixing, the mixture was pre-cooled overnight in a refrigerator at -20°C and then freeze-dried in a freeze dryer until completely freeze-dried to obtain β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK.

[0093] Example 5

[0094] A method for preparing β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK is the same as the preparation steps in Example 1, except that the mass fraction of trehalose in step S4 is replaced from 0.4% to 0.6%, and includes the following steps:

[0095] Preparation of S1, β-cyclodextrin-carboxymethyl chitosan polymer:

[0096] First, 1g of CMCS was weighed and dissolved in 100mL of deionized water to obtain an aqueous solution of carboxymethyl chitosan. 800mg of DCC and 500mg of DMAP were dissolved together in 20mL of dimethyl sulfoxide solution. After mixing, the mixture was added dropwise to the obtained aqueous solution of carboxymethyl chitosan. The mixture was stirred at 1000r / min for 2h at room temperature to obtain a dimethyl sulfoxide solution of carboxyl-activated carboxymethyl chitosan, denoted as CMCS solution.

[0097] Next, 5g of β-cyclodextrin was dissolved in 60mL of dimethyl sulfoxide solution to obtain a dimethyl sulfoxide solution of β-cyclodextrin.

[0098] Finally, the dimethyl sulfoxide solution of β-cyclodextrin was mixed with the CMCS solution, stirred at 800 r / min at room temperature, and esterified for 24 h to obtain the β-cyclodextrin-carboxymethyl chitosan polymer, denoted as β-CD-CMCS.

[0099] Preparation of S2, β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer:

[0100] 500 mg of deoxycholic acid, 400 mg of DCC, and 250 mg of DMAP were dissolved in 10 mL of dimethyl sulfoxide solution and stirred for 2 h to obtain a dimethyl sulfoxide solution of activated carboxy-deoxycholic acid. The activated carboxy-deoxycholic acid dimethyl sulfoxide solution was then added dropwise to 90 mL of β-CD-CMCS, and the mixture was stirred at 900 r / min for 24 h at room temperature to carry out an esterification reaction. This yielded a β-cyclodextrin-carboxymethyl chitosan polymer, i.e., an esterified β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer, denoted as CD-g-CMDA, resulting from the esterification of deoxycholic acid with the hydroxyl groups in carboxymethyl chitosan. E .

[0101] S3. Dissolve 5 mg of ginsenoside CK in 5 mL of ethanol solution to obtain the CK solution dissolved in ethanol; then take 5 mg of CD-g-CMDA E Dissolved in 5 mL of deionized water, CD-g-CMDA is obtained. E An aqueous solution of CK dissolved in ethanol was added dropwise to CD-g-CMDA. E After stirring in an aqueous solution for 2 hours, the solution was placed into a dialysis bag with a molecular weight of 7000 and dialyzed in 800 mL of pure aqueous solution for 24 hours to obtain a drug-loaded nanoparticle suspension, denoted as CD-g-CMDA. E @CK-NPs suspension.

[0102] S4. Place CD-g-CMDA E @CK-NPs suspension and trehalose were mixed at a mass ratio of 100:0.6. After mixing, the mixture was pre-cooled overnight in a refrigerator at -20°C and then freeze-dried in a freeze dryer until completely freeze-dried to obtain β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK.

[0103] Example 6

[0104] A method for preparing β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK is the same as the preparation steps in Example 1, except that the mass fraction of trehalose in step S4 is replaced from 0.4% to 0.8%, and includes the following steps:

[0105] Preparation of S1, β-cyclodextrin-carboxymethyl chitosan polymer:

[0106] First, 1g of CMCS was weighed and dissolved in 100mL of deionized water to obtain an aqueous solution of carboxymethyl chitosan. 800mg of DCC and 500mg of DMAP were dissolved together in 20mL of dimethyl sulfoxide solution. After mixing, the mixture was added dropwise to the obtained aqueous solution of carboxymethyl chitosan. The mixture was stirred at 1000r / min for 2h at room temperature to obtain a dimethyl sulfoxide solution of carboxyl-activated carboxymethyl chitosan, denoted as CMCS solution.

[0107] Next, 5g of β-cyclodextrin was dissolved in 60mL of dimethyl sulfoxide solution to obtain a dimethyl sulfoxide solution of β-cyclodextrin.

[0108] Finally, the dimethyl sulfoxide solution of β-cyclodextrin was mixed with the CMCS solution, stirred at 800 r / min at room temperature, and esterified for 24 h to obtain the β-cyclodextrin-carboxymethyl chitosan polymer, denoted as β-CD-CMCS.

[0109] Preparation of S2, β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer:

[0110] 500 mg of deoxycholic acid, 400 mg of DCC, and 250 mg of DMAP were dissolved in 10 mL of dimethyl sulfoxide solution and stirred for 2 h to obtain a dimethyl sulfoxide solution of activated carboxy-deoxycholic acid. The activated carboxy-deoxycholic acid dimethyl sulfoxide solution was then added dropwise to 90 mL of β-CD-CMCS, and the mixture was stirred at 900 r / min for 24 h at room temperature to carry out an esterification reaction. This yielded a β-cyclodextrin-carboxymethyl chitosan polymer, i.e., an esterified β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer, denoted as CD-g-CMDA, resulting from the esterification of deoxycholic acid with the hydroxyl groups in carboxymethyl chitosan. E .

[0111] S3. Dissolve 5 mg of ginsenoside CK in 5 mL of ethanol solution to obtain the CK solution dissolved in ethanol; then take 5 mg of CD-g-CMDA E Dissolved in 5 mL of deionized water, CD-g-CMDA is obtained. E An aqueous solution of CK dissolved in ethanol was added dropwise to CD-g-CMDA. E After stirring in an aqueous solution for 2 hours, the solution was placed into a dialysis bag with a molecular weight of 7000 and dialyzed in 800 mL of pure aqueous solution for 24 hours to obtain a drug-loaded nanoparticle suspension, denoted as CD-g-CMDA. E @CK-NPs suspension.

[0112] S4. Place CD-g-CMDA E@CK-NPs suspension and trehalose were mixed at a mass ratio of 100:0.8. After mixing, the mixture was pre-cooled overnight in a refrigerator at -20°C and then freeze-dried in a freeze dryer until completely freeze-dried to obtain β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK.

[0113] Example 7

[0114] A method for preparing β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK is the same as the preparation steps in Example 1, except that the mass fraction of trehalose in step S4 is replaced from 0.4% to 1.2%, and includes the following steps:

[0115] Preparation of S1, β-cyclodextrin-carboxymethyl chitosan polymer:

[0116] First, 1g of CMCS was weighed and dissolved in 100mL of deionized water to obtain an aqueous solution of carboxymethyl chitosan. 800mg of DCC and 500mg of DMAP were dissolved together in 20mL of dimethyl sulfoxide solution. After mixing, the mixture was added dropwise to the obtained aqueous solution of carboxymethyl chitosan. The mixture was stirred at 1000r / min for 2h at room temperature to obtain a dimethyl sulfoxide solution of carboxyl-activated carboxymethyl chitosan, denoted as CMCS solution.

[0117] Next, 5g of β-cyclodextrin was dissolved in 60mL of dimethyl sulfoxide solution to obtain a dimethyl sulfoxide solution of β-cyclodextrin.

[0118] Finally, the dimethyl sulfoxide solution of β-cyclodextrin was mixed with the CMCS solution, stirred at 800 r / min at room temperature, and esterified for 24 h to obtain the β-cyclodextrin-carboxymethyl chitosan polymer, denoted as β-CD-CMCS.

[0119] Preparation of S2, β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer:

[0120] 500 mg of deoxycholic acid, 400 mg of DCC, and 250 mg of DMAP were dissolved in 10 mL of dimethyl sulfoxide solution and stirred for 2 h to obtain a dimethyl sulfoxide solution of activated carboxy-deoxycholic acid. The activated carboxy-deoxycholic acid dimethyl sulfoxide solution was then added dropwise to 90 mL of β-CD-CMCS, and the mixture was stirred at 900 r / min for 24 h at room temperature to carry out an esterification reaction. This yielded a β-cyclodextrin-carboxymethyl chitosan polymer, i.e., an esterified β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer, denoted as CD-g-CMDA, resulting from the esterification of deoxycholic acid with the hydroxyl groups in carboxymethyl chitosan. E .

[0121] S3. Dissolve 5 mg of ginsenoside CK in 5 mL of ethanol solution to obtain the CK solution dissolved in ethanol; then take 5 mg of CD-g-CMDA E Dissolved in 5 mL of deionized water, CD-g-CMDA is obtained. E An aqueous solution of CK dissolved in ethanol was added dropwise to CD-g-CMDA. E After stirring in an aqueous solution for 2 hours, the solution was placed into a dialysis bag with a molecular weight of 7000 and dialyzed in 800 mL of pure aqueous solution for 24 hours to obtain a drug-loaded nanoparticle suspension, denoted as CD-g-CMDA. E @CK-NPs suspension.

[0122] S4. Place CD-g-CMDA E @CK-NPs suspension and trehalose were mixed at a mass ratio of 100:1.2. After mixing, the mixture was pre-cooled overnight in a refrigerator at -20°C and then freeze-dried in a freeze dryer until completely freeze-dried to obtain β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK.

[0123] Example 8

[0124] A method for preparing β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK includes the following steps:

[0125] Preparation of S1, β-cyclodextrin-carboxymethyl chitosan polymer:

[0126] First, 1g of CMCS was weighed and dissolved in 100mL of deionized water to obtain an aqueous solution of carboxymethyl chitosan. 800mg of DCC and 500mg of DMAP were dissolved together in 20mL of dimethyl sulfoxide solution. After mixing, the mixture was added dropwise to the obtained aqueous solution of carboxymethyl chitosan. The mixture was stirred at 1000r / min for 2h at room temperature to obtain a dimethyl sulfoxide solution of carboxyl-activated carboxymethyl chitosan, denoted as CMCS solution.

[0127] Next, 5g of β-cyclodextrin was dissolved in 60mL of dimethyl sulfoxide solution to obtain a dimethyl sulfoxide solution of β-cyclodextrin.

[0128] Finally, the dimethyl sulfoxide solution of β-cyclodextrin was mixed with the CMCS solution, stirred at 800 r / min at room temperature, and esterified for 24 h to obtain the β-cyclodextrin-carboxymethyl chitosan polymer, denoted as β-CD-CMCS.

[0129] S2. Dissolve 0.5 g of deoxycholic acid, 250 mg of NHS, and 0.5 g of EDC-HCl in 15 mL of anhydrous ethanol. Stir for 30 min under light-protected conditions, then add dropwise to 90 mL of β-CD-CMCS. Perform an amidation reaction at 900 r / min for 24 h at room temperature to obtain an amidated β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer, denoted as CD-g-CMDA. A .

[0130] S3. Dissolve 5 mg of ginsenoside CK in 5 mL of ethanol solution to obtain the CK solution dissolved in ethanol; then take 5 mg of CD-g-CMDA A Dissolved in 5 mL of deionized water, CD-g-CMDA is obtained. A An aqueous solution of CK dissolved in ethanol was added dropwise to CD-g-CMDA. A After stirring in an aqueous solution for 2 hours, the solution was placed into a dialysis bag with a molecular weight of 7000 and dialyzed in 800 mL of pure aqueous solution for 24 hours to obtain a drug-loaded nanoparticle suspension, denoted as CD-g-CMDA. A @CK-NPs suspension.

[0131] S4. Place CD-g-CMDA A @CK-NPs suspension and trehalose were mixed at a mass ratio of 100:0.4. After mixing, the mixture was pre-cooled overnight at -20°C and then freeze-dried in a freeze dryer until completely freeze-dried to obtain β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK, denoted as CD-g-CMDA. A @CK-NPs.

[0132] Comparative Example 1

[0133] A method for preparing β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK is the same as the preparation steps in Example 1, except that the amount of trehalose, the freeze-drying protectant in step S4, is replaced from 0.4% to 0, and includes the following steps:

[0134] Preparation of S1, β-cyclodextrin-carboxymethyl chitosan polymer:

[0135] First, 1g of CMCS was weighed and dissolved in 100mL of deionized water to obtain an aqueous solution of carboxymethyl chitosan. 800mg of DCC and 500mg of DMAP were dissolved together in 20mL of dimethyl sulfoxide solution. After mixing, the mixture was added dropwise to the obtained aqueous solution of carboxymethyl chitosan. The mixture was stirred at 1000r / min for 2h at room temperature to obtain a dimethyl sulfoxide solution of carboxyl-activated carboxymethyl chitosan, denoted as CMCS solution.

[0136] Next, 5g of β-cyclodextrin was dissolved in 60mL of dimethyl sulfoxide solution to obtain a dimethyl sulfoxide solution of β-cyclodextrin.

[0137] Finally, the dimethyl sulfoxide solution of β-cyclodextrin was mixed with the CMCS solution, stirred at 800 r / min at room temperature, and esterified for 24 h to obtain the β-cyclodextrin-carboxymethyl chitosan polymer, denoted as β-CD-CMCS.

[0138] Preparation of S2, β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer:

[0139] 500 mg of deoxycholic acid, 400 mg of DCC, and 250 mg of DMAP were dissolved in 10 mL of dimethyl sulfoxide solution and stirred for 2 h to obtain a dimethyl sulfoxide solution of activated carboxy-deoxycholic acid. The activated carboxy-deoxycholic acid dimethyl sulfoxide solution was then added dropwise to 90 mL of β-CD-CMCS, and the mixture was stirred at 900 r / min for 24 h at room temperature to carry out an esterification reaction. This yielded a β-cyclodextrin-carboxymethyl chitosan polymer, i.e., an esterified β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer, denoted as CD-g-CMDA, resulting from the esterification of deoxycholic acid with the hydroxyl groups in carboxymethyl chitosan. E .

[0140] S3. Dissolve 5 mg of ginsenoside CK in 5 mL of ethanol solution to obtain the CK solution dissolved in ethanol; then take 5 mg of CD-g-CMDA E Dissolved in 5 mL of deionized water, CD-g-CMDA is obtained. E An aqueous solution of CK dissolved in ethanol was added dropwise to CD-g-CMDA. E After stirring in an aqueous solution for 2 hours, the solution was placed into a dialysis bag with a molecular weight of 7000 and dialyzed in 800 mL of pure aqueous solution for 24 hours to obtain a drug-loaded nanoparticle suspension, denoted as CD-g-CMDA. E @CK-NPs suspension.

[0141] S4. Place CD-g-CMDA E The CK-NPs suspension was pre-cooled overnight at -20°C and then freeze-dried in a freeze dryer until completely freeze-dried to obtain β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK, denoted as CD-g-CMDA. A @CK D -NPs.

[0142] Comparative Example 2

[0143] A method for preparing β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles is the same as the preparation steps in Example 4, except that ginsenoside CK is not added in step S3, and includes the following steps:

[0144] Preparation of S1, β-cyclodextrin-carboxymethyl chitosan polymer:

[0145] First, 1g of CMCS was weighed and dissolved in 100mL of deionized water to obtain an aqueous solution of carboxymethyl chitosan. 800mg of DCC and 500mg of DMAP were dissolved together in 20mL of dimethyl sulfoxide solution. After mixing, the mixture was added dropwise to the obtained aqueous solution of carboxymethyl chitosan. The mixture was stirred at 1000r / min for 2h at room temperature to obtain a dimethyl sulfoxide solution of carboxyl-activated carboxymethyl chitosan, denoted as CMCS solution.

[0146] Next, 5g of β-cyclodextrin was dissolved in 60mL of dimethyl sulfoxide solution to obtain a dimethyl sulfoxide solution of β-cyclodextrin.

[0147] Finally, the dimethyl sulfoxide solution of β-cyclodextrin was mixed with the CMCS solution, stirred at 800 r / min at room temperature, and esterified for 24 h to obtain the β-cyclodextrin-carboxymethyl chitosan polymer, denoted as β-CD-CMCS.

[0148] Preparation of S2, β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer:

[0149] 500 mg of deoxycholic acid, 400 mg of DCC, and 250 mg of DMAP were dissolved in 10 mL of dimethyl sulfoxide solution and stirred for 2 h to obtain a dimethyl sulfoxide solution of activated carboxy-deoxycholic acid. The activated carboxy-deoxycholic acid dimethyl sulfoxide solution was then added dropwise to 90 mL of β-CD-CMCS, and the mixture was stirred at 900 r / min for 24 h at room temperature to carry out an esterification reaction. This yielded a β-cyclodextrin-carboxymethyl chitosan polymer, i.e., an esterified β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer, denoted as CD-g-CMDA, resulting from the esterification of deoxycholic acid with the hydroxyl groups in carboxymethyl chitosan. E .

[0150] S3. Take 5mg of CD-g-CMDA E Dissolved in 5 mL of deionized water, CD-g-CMDA is obtained. E The solution was then placed in an aqueous solution; subsequently, it was placed in a dialysis bag with a molecular weight of 7000 and dialyzed in 800 mL of pure aqueous solution for 24 h to obtain a nanoparticle suspension, denoted as CD-g-CMDA. E -NPs suspension.

[0151] S4. Place CD-g-CMDA E -NPs suspension and trehalose were mixed at a mass ratio of 100:1, pre-cooled overnight at -20°C, and then freeze-dried until completely dry to obtain β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles, denoted as CD-g-CMDA. E -NPs.

[0152] Transmission electron microscopy was used to examine CD-g-CMDA from Example 1. E Morphological characterization of the CK-NPs suspension was performed, and observations were made. Figure 1 It was found that CD-g-CMDA in Example 1... E The @CK-NPs suspension exhibits a regular spherical structure with a smooth and flat surface, excellent particle dispersion, and no obvious aggregation was observed. The particle size distribution is uniform, with an average particle size of approximately 300 nm according to measurements.

[0153] Characterization was performed using Fourier transform infrared absorption spectroscopy, and observations were made. Figure 2 It was found that, compared with ginsenoside CK, β-cyclodextrin, carboxymethyl chitosan, and deoxycholic acid, CD-g-CMDA in Example 1... E @CK-NPs suspension at 945cm -1 The characteristic peak generated by the α-pyranyl vibration of β-cyclodextrin appeared at 1626 cm⁻¹. -1 The appearance of a stretching vibration absorption peak at 1589 cm⁻¹ indicates successful esterification; the absorption peak at 1589 cm⁻¹ further confirms this. -1 A new peak appeared at 3000 cm⁻¹, generated by the vibration of primary amine groups. -1 ~3750cm -1 The grafting of cyclodextrin significantly enhanced the intensity of the hydroxyl absorption peak of carboxymethyl chitosan. This further demonstrates the successful grafting of β-cyclodextrin and carboxymethyl chitosan, and the successful preparation of β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK.

[0154] observe Figure 3 The drug loading of β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK prepared by dialysis was found to be higher than that of CD-g-CMDA in Example 8. A At that time, CD-g-CMDA A The drug loading of the CK-NPs suspension was 95.95%; when the shell was CD-g-CMDA from Example 1... E At that time, CD-g-CMDA E The drug loading of the @CK-NPs suspension was 83.3%.

[0155] observe Figure 4It was found that, compared to the CD-g-CMDA prepared by amidation in Example 8, A @CK-NPs suspension, CD-g-CMDA prepared by esterification reaction in Example 1. E @CK-NPs suspensions showed better performance in terms of both particle size and dispersion.

[0156] Depend on Figure 5 Therefore, the CD-g-CMDA of Embodiment 1 of the present invention is shown to be... E @CK-NPs have an average particle size of 291.05 nm and a PDI of 0.139, exhibiting a narrow and uniform particle size distribution.

[0157] observe Figure 6 The results showed that after 35 hours in an environment with a pH of 1.2, the cumulative release of ginsenoside CK was 13.88%, which effectively reduced drug release in the acidic gastric environment. At pH 7.4, which is considered a normal physiological environment, the cumulative release of ginsenoside CK after 35 hours was 31.1%. In an environment with a pH of 5.8, 40% of ginsenoside CK had already been released after 1 hour, mainly due to the release of ginsenoside CK contained in β-cyclodextrin. Between 1 and 3 hours, the release of ginsenoside CK was slow. The cumulative release rate decreased from 40.11% to 40.15%, showing almost no increase. Between 3 and 5 hours, the weakly acidic environment weakened the ester bond between the carboxyl group of carboxymethyl chitosan and the hydroxyl group of β-cyclodextrin in the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK. The shell network structure gradually became loose, and ginsenoside CK began to be released in large quantities, with a cumulative release rate as high as 83.27% after 5 hours. Between 5 and 7 hours, the cumulative release rate increased by about 10% per hour on average until ginsenoside CK was completely released.

[0158] Depend on Figure 7 and Figure 8 It was found that among the different types of freeze-drying protectants in Examples 1 to 3, trehalose had the best effect; in Examples 1 and 4 to 7, and when the amount of trehalose added accounted for CD-g-CMDA E When the @CK-NPs suspension was 1%, the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles carrying ginsenoside CK showed the smallest change in particle size before and after freeze-drying, indicating the best freeze-drying protection effect.

[0159] Examples 1 to 8 of this invention all yielded β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK, and the effects were parallel. The following uses ginsenoside CK, the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK from Example 1 of this invention, and the amidated β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer and β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles from Comparative Example 2 as examples to test the in vitro biosafety and proliferation inhibition effects on HepG2 liver cancer cells. HepG2 liver cancer cells are referred to as HepG2 cells. Specific application methods and results are as follows:

[0160] Application method:

[0161] a. The CD-g-CMDA of Comparative Example 2 was analyzed using the MTT assay. E and CD-g-CMDA E The in vitro biosafety of -NPs was assessed, and the specific procedures are as follows:

[0162] Collect HepG2 cells in the logarithmic growth phase, and seed 100 μL of each cell into 96-well plates using a pipette, ensuring approximately 8000 HepG2 cells per well. After 24 hours of adherent culture, discard the old culture medium. Add different volumes of CD-g-CMDA from Comparative Example 2 prepared with the corresponding culture medium. E and CD-g-CMDA E -NPs solution, i.e., 50 μL, 40 μL, 30 μL, 20 μL, and 10 μL, was added to each well and incubated for 24 h and 48 h respectively. Then, 20 μL of MTT solution (5 mg / mL) was added to each well, and incubation was continued for 4 h. The culture medium in each well was carefully aspirated, and 150 μL of dimethyl sulfoxide was added to each well. The mixture was shaken to fully dissolve the crystals, and the absorbance was measured at 490 nm using a microplate reader.

[0163] HepG2 cell viability was calculated using the following formula:

[0164]

[0165] The A490 sample group represents the absorbance value of the experimental group at a wavelength of 490 nm using an ELISA reader, and includes HepG2 cells, MTT solution, MEM culture medium, and CD-g-CMDA. A @CK-NPs solution; A490 blank group represents the absorbance value of the microplate reader at a wavelength of 490nm without the addition of any drug, including HepG2 cells, MEM culture medium, and MTT solution; A490 control group represents the absorbance value of the blank group with added free ginsenoside CK at a wavelength of 490nm, including MEM culture medium and MTT solution.

[0166] Depend on Figure 9 From (a) in Comparative Example 2, we find that CD-g-CMDA E At a concentration of 100 μg / mL, the cell safety index was slightly below 80% at the 48-hour stage. However, due to its high drug loading capacity, the required carrier concentration in practical applications can be significantly reduced to below 50 μg / mL. Under this concentration, the survival rate of HepG2 cells remained stable above 93% at both 24 and 48 hours, indicating good in vitro biosafety and potential as a drug carrier. Further combined... Figure 9 (b) shows that when CD-g-CMDA of Comparative Example 2 with a nanoparticle mass concentration of 200 μg / mL is subjected to... E Even after treatment with -NPs, the survival rate of HepG2 cells remained above 90%. This result further confirms the efficacy of CD-g-CMDA. E -NPs exhibit excellent biosafety in drug delivery applications, providing experimental evidence for their potential applications in the biomedical field.

[0167] b. The inhibitory effects of β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK and ginsenoside CK on the proliferation of HepG2 cells were tested using the MTT assay in Example 1. The specific operation method was the same as in step a, except that the amidated CD-g-CMDA and CD-g-CMDA-NPs solutions in Comparative Example 2 were replaced with β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK and ginsenoside CK in Example 1. The results are as follows:

[0168] observe Figure 10The results showed that the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK of Example 1 of this invention exhibited a significant dose-dependent inhibitory effect on HepG2 cells, and the inhibitory effect was more pronounced in the 48h treatment group than in the 24h treatment group. Specifically, as the concentration of ginsenoside CK in the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK increased from 0 μmol / L to 50 μmol / L, the survival rate of HepG2 cells in the 24h treatment group decreased from 99.42% to 75.43%, while the survival rate of HepG2 cells in the 48h treatment group decreased from 100% to 46.90%. This is because, with prolonged treatment time, the endocytosis of ginsenoside CK-loaded β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles by HepG2 cells increased, leading to the accumulation of these nanoparticles within the HepG2 cells. This, in turn, increased the release of ginsenoside CK from the HepG2 cell environment. As the active ingredient, the large-scale release of ginsenoside CK effectively induced apoptosis in HepG2 cells and inhibited their proliferation. This is consistent with the characteristic that the ginsenoside CK-loaded β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles at pH 5.8 and 34 hours showed almost complete drug release, further validating the effectiveness and controllability of nanoparticles as drug delivery carriers. In conclusion, the ginsenoside CK-loaded β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles prepared in this invention have a significant inhibitory effect on the proliferation of HepG2 cells.

[0169] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range, as well as any value between the two endpoints, can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

Claims

1. A method for preparing β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK, characterized in that, Includes the following steps: After dissolving β-cyclodextrin in a solvent, it was mixed with a carboxyl-activated carboxymethyl chitosan solution to carry out the first esterification reaction, thereby obtaining a β-cyclodextrin-carboxymethyl chitosan polymer. Carboxyl-activated deoxycholic acid is added dropwise to β-cyclodextrin-carboxymethyl chitosan polymer to carry out a second esterification or amidation reaction to obtain β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer. An ethanol solution of ginsenoside CK was added dropwise to an aqueous solution of β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer to perform self-assembly and granulation by dialysis, resulting in a drug-loaded nanoparticle suspension. After mixing the drug-loaded nanoparticle suspension with a freeze-drying protectant, freeze-drying was performed to obtain β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK.

2. The method for preparing β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK according to claim 1, characterized in that, During the second esterification reaction, N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine were used as activators to activate deoxycholic acid to obtain carboxyl-activated deoxycholic acid. The mass-to-volume ratio of deoxycholic acid, N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine, and β-cyclodextrin-carboxymethyl chitosan polymer is 5 mg~10 mg: 4 mg: 2.5 mg: 0.9 mL.

3. The method for preparing β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK according to claim 1, characterized in that, When the amidation reaction is carried out, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride are used as activators to activate deoxycholic acid to obtain carboxyl-activated deoxycholic acid. The mass-volume ratio of deoxycholic acid, N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to β-cyclodextrin-carboxymethyl chitosan polymer is 4 mg~6 mg:4 mg:5 mg:0.9 mL.

4. The method for preparing β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK according to claim 1, characterized in that, The mass ratio of β-cyclodextrin to carboxyl-activated carboxymethyl chitosan is 5:1~3.

5. The method for preparing β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK according to claim 1, characterized in that, The mass ratio of β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer to ginsenoside CK is 5:3~7.

6. The method for preparing β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK according to claim 1, characterized in that, The freeze-drying protectant is selected from at least one of mannitol, sucrose, and trehalose.

7. A type of β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticle loaded with ginsenoside CK, characterized in that, The nanoparticles containing ginsenoside CK are prepared by the preparation method according to any one of claims 1 to 6, and the nanoparticles are in the form of a core-shell structure, with the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid polymer as the shell and ginsenoside CK as the core.

8. The β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK according to claim 7, characterized in that, The average particle size of the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK is 200nm~350nm, and the particle size is uniform.

9. The use of the β-cyclodextrin-carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with ginsenoside CK as described in claim 7 in the preparation of antitumor drugs.

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