Carboxymethyl chitosan nano freeze-dried powder as well as preparation method and application thereof

Abstract

The invention discloses carboxymethyl chitosan nano freeze-dried powder as well as a preparation method and application thereof, and belongs to the technical field of nano medicines. The preparation method of the carboxymethyl chitosan nano freeze-dried powder comprises the following steps: dissolving a carboxymethyl chitosan deoxycholic acid polymer with an alcohol solvent, adding the dissolved carboxymethyl chitosan deoxycholic acid polymer into water to prepare a turbid liquid, and carrying out first dialysis to obtain a carboxymethyl chitosan deoxycholic acid nano particle solution; adding ginsenoside CK powder into the solution, stirring at room temperature to enable ginsenoside CK to be loaded on the nanoparticles, and performing second dialysis with water after stirring to obtain a ginsenoside CK carboxymethyl chitosan-deoxycholic acid nanoparticle suspension; and then freeze-drying to obtain the carboxymethyl chitosan nano freeze-dried powder. The prepared carboxymethyl chitosan nano freeze-dried powder has controllable particle size and high drug loading capacity, can specifically release drugs in a tumor slightly acidic environment, remarkably improves the liver cancer treatment effect and biological safety, and has great market application potential.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nanomedicine, and more particularly to a carboxymethyl chitosan nanofreeze-dried powder, a preparation method and application thereof. BACKGROUND

[0002] Among all malignant tumors, the mortality rate of liver cancer is relatively high. At present, the traditional drugs for anti-liver cancer have the characteristics of hydrophobicity, low bioavailability and poor stability. The rare natural active product ginsenoside Compound K can effectively inhibit the proliferation of tumor cells by inducing the apoptosis and cycle arrest of various tumor cells. Compared with other anticancer drugs, it has the advantages of high biological activity and minimal side effects, but it also has the problems of poor stability during storage and application, and easy reduction of activity in the gastric acid environment.

[0003] The development of nanocarrier systems provides a new strategy for stable delivery of antitumor drugs. Carboxymethyl chitosan is widely used in drug delivery systems due to its good biodegradability, non-toxicity and easy chemical modification. Deoxycholic acid is a secondary bile acid that can improve the oral bioavailability of hydrophobic drugs by promoting intestinal epithelial absorption. For example, the patent CN108553647A discloses a preparation method of ginsenoside CK-chitosan micelle nanoparticles. This kind of patent uses deoxycholic acid-O-carboxymethyl chitosan as a carrier to coat ginsenoside CK to obtain chitosan micelle nanoparticles by ultrasonic self-assembly method, but its drug loading capacity is low, the dosage of the carrier during treatment is still relatively large, and the patient compliance is poor.

[0004] Particle size is a very critical parameter for the application of nanocarrier systems, which has a significant influence on the physicochemical properties, stability, drug loading capacity, and drug administration method of drug-loaded nanoparticles. It is particularly important to develop a new type of size-adjustable nanoparticle preparation process that can adapt to diversified market demand. For example, the patent CN110183613A discloses a preparation method of nanomicelle system. The drug-loaded micelles prepared by this method are filtered through a 0.45 μm filter to obtain uniform particles. However, the particle size of the particles prepared by this method is uncontrollable, and large particles are filtered out, resulting in low yield and serious waste of rare drugs.

[0005] In addition, the stability maintenance during nanoparticle storage and application directly affects the efficacy and safety of nanodrugs, and is also a quality evaluation index focused by regulatory departments in the drug approval process. CN109908105A discloses a preparation method of a deoxycholic acid modified nanocomposite. The deoxycholic acid modified low molecular weight chitosan / carboxymethyl chitosan nanocomposite is prepared by ion crosslinking method, and the oral delivery of rhein is effectively realized. However, the data of the patent shows that the particle stability is not high. As described in patent CN113461968B, carboxymethyl chitosan is used as a single emulsion stabilizer, and a high internal phase emulsion with an oil phase volume fraction of 90% can be prepared by shear mixing. The emulsion does not delaminate or oxidize during 8 months of storage, but the oil phase described in the preparation process of the emulsion includes vegetable oil, n-hexane and n-dodecane, etc. These organic reagents have potential risks to human safety as auxiliary materials. SUMMARY

[0006] In view of the above problems, the present application provides a carboxymethyl chitosan nanofreeze-dried powder and a preparation method and application thereof. The carboxymethyl chitosan nanofreeze-dried powder prepared by the present application has the advantages of stable and controllable particle size and high drug loading capacity. In addition, the carboxymethyl chitosan nanofreeze-dried powder can specifically release drugs in the acidic environment of tumors, significantly improve the treatment effect and biological safety of liver cancer, and has great market application potential.

[0007] The first object of the present application is to provide a preparation method of a carboxymethyl chitosan nanofreeze-dried powder, comprising the following steps: After the carboxymethyl chitosan deoxycholic acid polymer is dissolved in an alcohol solvent, it is added to water. During the addition process, the carboxymethyl chitosan deoxycholic acid polymer self-assembles to form nanoparticles, obtaining a suspension. The first dialysis is performed to remove free polymers or other small molecular impurities, obtaining a carboxymethyl chitosan deoxycholic acid nanoparticle solution.

[0008] The ginsenoside CK powder is added to the carboxymethyl chitosan deoxycholic acid nanoparticle solution. The ginsenoside CK is loaded onto the nanoparticles under room temperature and stirring. After the stirring is completed, the ginsenoside CK is loaded onto the carboxymethyl chitosan deoxycholic acid nanoparticles. After the stirring is completed, a dialysis bag with a molecular weight cut-off of 3500-14000 is used for the second dialysis to remove unencapsulated ginsenoside CK and regulate the particle size of the product, obtaining a ginsenoside CK carboxymethyl chitosan-deoxycholic acid nanoparticle suspension.

[0009] The ginsenoside CK carboxymethyl chitosan-deoxycholic acid nanoparticle suspension is freeze-dried to obtain a carboxymethyl chitosan nanofreeze-dried powder.

[0010] In a preferred embodiment of the present application, the molecular weight cut-off of the dialysis bag used in the first dialysis is 7000-14000, and the dialysis time is 12-24 hours. The main purpose of the first dialysis is to remove free polymers or other small molecular impurities, and it has an important influence on the particle size and drug loading of the nanoparticles. The molecular weight cut-off range can remove small molecular carboxymethyl chitosan and deoxycholic acid that do not form polymers, and can also enable the carboxymethyl chitosan deoxycholic acid polymer to self-assemble into nanoparticles with a target particle size. The dialysis time affects the stability of the nanoparticles, and the dialysis time is determined by monitoring the drug concentration in the dialysate.

[0011] In a preferred embodiment of the present application, the molecular weight cut-off of the dialysis bag used in the second dialysis is 3500-14000, and the dialysis time is 1-24 hours. The second dialysis is used to remove unencapsulated ginsenoside CK, and the particle size is controlled by the size of the molecular weight cut-off. The dialysis time affects the stability of the nanoparticles, and the dialysis time is determined by monitoring the drug concentration in the dialysate.

[0012] In a preferred embodiment of the present application, the ratio of carboxymethyl chitosan deoxycholic acid polymer to water is 1 mg-1.5 mg: 1 mL.

[0013] In a preferred embodiment of the present application, the molecular weight cut-off of the dialysis bag used in the first dialysis is 7000.

[0014] In a preferred embodiment of the present application, the ratio of carboxymethyl chitosan deoxycholic acid nanoparticle solution to ginsenoside CK powder is 20 ml: 1 mg-5 mg.

[0015] In a preferred embodiment of the present application, the ratio of carboxymethyl chitosan deoxycholic acid nanoparticle solution to ginsenoside CK powder is 20 ml: 3 mg.

[0016] In a preferred embodiment of the present application, a freeze-drying protective agent is added to the ginsenoside CK carboxymethyl chitosan-deoxycholic acid nanoparticle suspension before freeze-drying.

[0017] The freeze-drying protective agent is at least one of trehalose, sucrose, mannitol, and cyclodextrin.

[0018] In a preferred embodiment of the present application, the volume fraction of the freeze-drying protective agent in the ginsenoside CK carboxymethyl chitosan-deoxycholic acid nanoparticle suspension is 0.4%.

[0019] The second object of the present application is to provide a carboxymethyl chitosan nanopowder prepared by the above preparation method.

[0020] The third object of the present application is to provide the use of the above carboxymethyl chitosan nanopowder in the preparation of an antitumor drug.

[0021] Compared with the prior art, the present application has the following beneficial effects: 1. The present application provides a preparation method of a high drug loading, size adjustable carboxymethyl chitosan nanofreeze-dried powder, carboxymethyl chitosan deoxycholic acid polymer is dissolved with methanol and added dropwise into water to prepare a suspension, which is then added into a dialysis bag and dialyzed with water to obtain carboxymethyl chitosan deoxycholic acid nanoparticles; the prepared carboxymethyl chitosan deoxycholic acid nanoparticle solution is added into ginsenoside CK powder, and stirring is performed to load ginsenoside CK onto the nanoparticles; after dialysis of the ginsenoside CK-loaded nanoparticle mixture with water, a ginsenoside CK carboxymethyl chitosan-deoxycholic acid nanoparticle suspension is obtained; and the suspension is freeze-dried to obtain ginsenoside CK-loaded carboxymethyl chitosan nanofreeze-dried powder. The nanofreeze-dried powder prepared by the present application can effectively control the particle size, adapt to diversified market demands, and has a high drug loading capacity, can release a large amount of drugs in a tumor micro-acid environment, and release very little drugs in gastric acid and body fluid, which greatly improves the liver cancer treatment effect and biological safety, and has great market application potential.

[0022] 2. In the preparation process of the carboxymethyl chitosan nanofreeze-dried powder, the molecular cut-off amount of the dialysis bag in the second dialysis process is regulated to accurately control the particle size of the nanoparticles. The method is simple and repeatable, and can prepare nanoparticles with a specific particle size in the range of 50nm to 400nm according to different clinical application requirements, significantly improving the customizability of the product and better meeting the differentiated needs of drug delivery systems for nanoparticle size.

[0023] 3. In the preparation process of the carboxymethyl chitosan nanofreeze-dried powder, the molecular cut-off amount of the dialysis bag in the second dialysis step is innovatively regulated to accurately control the particle size of the nanoparticles. In the preparation process of the carboxymethyl chitosan nanofreeze-dried powder, deoxycholic acid is introduced, and the hydrophobic property of deoxycholic acid has strong affinity for hydrophobic drugs such as ginsenoside CK, which can capture more ginsenoside CK drugs into the nanoparticles, thereby significantly improving the drug loading capacity of the nanoparticles.

[0024] 4. The deoxycholic acid introduced in the preparation process of the carboxymethyl chitosan nanofreeze-dried powder can also promote the penetration of the nanoparticles into cell membranes, especially tumor cells, improve drug uptake, and the deoxycholic acid modified nanocarrier is more easily depolymerized in a tumor micro-acid environment with a pH of 6.5-7.0, realizes targeted drug release, and reduces the risk of normal tissue toxicity and side effects.

[0025] 5. The high-load, size-adjustable carboxymethyl chitosan nano freeze-dried powder prepared by the present invention innovatively improves the freeze-drying protection process during production. The innovative introduction of a freeze-drying protectant prevents water molecules from forming an amorphous glassy state during the freeze-drying process of the nanoparticles, reduces mechanical stress, and prevents ice crystals formed by water molecules during the freeze-thaw process from causing nanoparticle aggregation or collapse. This effectively maintains the three-dimensional skeleton structure of the nanoparticles, avoids skeleton damage and drug leakage caused by ice crystal growth, and significantly improves the stability of the nanoparticles. Simultaneously, it achieves an instantaneous reconstitution effect, improving the portability of the application.

[0026] 6. The carboxymethyl chitosan nano-freeze-dried powder prepared by the present invention has a particle size distribution ranging from 90 nm to 400 nm, a drug loading of 52.56%, and excellent stability during storage. After freeze-drying and reconstitution, turbidity is evident, with almost no visible particles, and the particle size variation does not exceed 10%. When used to deliver ginsenoside CK to human liver cancer cells, the cell survival rate was approximately 46.04% when CMDA@CK-NPs were added at a concentration of 50 μM. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a scanning electron microscope image of the drug-loaded nanoparticle powder prepared in Example 1.

[0028] Figure 2 This is the Fourier infrared spectrum of the drug-loaded nanoparticle powder prepared in Example 1.

[0029] Figure 3 This is the X-ray diffraction pattern of the drug-loaded nanoparticle powder prepared in Example 1.

[0030] Figure 4 The effect of different drug-to-polymer carrier ratios on the drug-loaded nanoparticle powder prepared in Example 1.

[0031] Figure 5 The release curves of the drug-loaded nanoparticle powder prepared in Example 1 under different pH environments are shown.

[0032] Figure 6 3. The effect of adding lyoprotectant on the instantaneous re-dissolution effect of CMDA@CK-NPs, wherein (a) is Example 1, (b) is Comparative Example 1, (c) is Example 8, and (d) is Comparative Example 2.

[0033] Figure 7 The effect of trehalose addition on the storage stability of CMDA@CK-NPs freeze-dried powder at 4°C, where (a) is Example 1 without trehalose, and (b) is Example 8 with 0.4% trehalose added.

[0034] Figure 8The in vitro safety of the empty carboxymethyl chitosan-deoxycholic acid nanoparticle lyophilized powder of Comparative Example 1 was evaluated at 24 h and 48 h.

[0035] Figure 9 The inhibition effect of the drug-loaded nanoparticle powder prepared in Example 1 on liver cancer cells HepG2, wherein (a) is 24 h, and (b) is 48 h. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present application will be clearly and completely described in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0037] In the present application, N-hydroxysuccinimide is denoted as NHS, and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is denoted as EDC•HCl.

[0038] Example 1 S1, Preparation of carboxymethyl chitosan-deoxycholic acid polymer: The synthesis method of carboxymethyl chitosan-deoxycholic acid polymer in CN116831992A was referred to. Specifically, 1.0 g of carboxymethyl chitosan was dissolved in 70 mL of aqueous solution, and stirred until dissolved to obtain a carboxymethyl chitosan aqueous solution. 0.8 g of deoxycholic acid, 0.5 g of NHS, and 1 g of EDC•HCl were dissolved in 30 mL of ethanol, and after stirring at room temperature for 30 min, they were added dropwise to the carboxymethyl chitosan aqueous solution at a uniform speed to obtain a solution of 100 mL of reaction system, which was stirred at 600 rpm on a magnetic stirrer for 24 h, and then dialyzed with water for 24 h, centrifuged at 12000 rpm for 10 min, the supernatant was discarded, and the precipitate was freeze-dried to obtain carboxymethyl chitosan-deoxycholic acid polymer, denoted as CMCS-DA.

[0039] S2, 20 mg of CMCS-DA was weighed, dissolved in 2 mL of methanol, and added dropwise to 20 mL of stirred ultrapure water at a uniform speed. During the dropwise addition process, the microparticles were self-assembled into a microparticle solution, and after the dropwise addition was completed, the microparticle solution was added to a dialysis bag with a molecular weight cut-off of 14000, and then dialyzed with water for 24 h to obtain a carboxymethyl chitosan-deoxycholic acid nanoparticle suspension, denoted as CMDA-NPs.

[0040] S3, 20 mL of the prepared CMDA-NPs solution was taken out after ultrasonic treatment for 5 min, 3 mg of ginsenoside CK was added to the above nanoparticle suspension, and the mixture was stirred at room temperature for 3 h to load ginsenoside CK on the nanoparticles. Then the CK-loaded nanoparticle mixture was transferred to a dialysis bag with a molecular weight cut-off of 14000, and dialyzed against water for 24 h to drive ginsenoside CK into the hydrophobic core of the nanoparticles, thereby obtaining carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with high amounts of ginsenoside CK, denoted as CMDA@CK3-NPs.

[0041] S4, 2 mL of the CMDA@CK3-NPs suspension was mixed with trehalose at a mass ratio of 100:0, and the mixture was uniformly mixed and then pre-cooled at -20°C overnight. Then the mixture was freeze-dried in a freeze dryer until completely dried, thereby obtaining the finished product of high ginsenoside CK-loaded carboxymethyl chitosan-deoxycholic acid nanoparticles in the form of a freeze-dried powder.

[0042] Example 2 S1, the preparation of carboxymethyl chitosan-deoxycholic acid polymer was carried out according to the synthesis method of carboxymethyl chitosan-deoxycholic acid polymer in CN116831992A. Specifically, 1.0 g of carboxymethyl chitosan was dissolved in 70 mL of aqueous solution, and stirred until dissolved to obtain a carboxymethyl chitosan aqueous solution. 0.8 g of deoxycholic acid, 0.5 g of NHS, and 1 g of EDC•HCl were dissolved in 30 mL of ethanol, and the mixture was stirred at room temperature for 30 min before being added dropwise to the carboxymethyl chitosan aqueous solution at a uniform speed to obtain a 100 mL reaction system solution. The solution was placed on a magnetic stirrer and stirred at 600 rpm for 24 h. The solution was dialyzed against water for 24 h, centrifuged at 12000 rpm for 10 min, and the supernatant was discarded. The precipitate was freeze-dried to obtain carboxymethyl chitosan-deoxycholic acid polymer, denoted as CMCS-DA.

[0043] S2, 20 mg of CMCS-DA was weighed and dissolved in 2 mL of methanol, and then added dropwise to 20 mL of stirred ultrapure water at a uniform speed. During the dropwise addition process, the solution self-assembled into microparticles. After the dropwise addition was completed, the solution was added to a dialysis bag with a molecular weight cut-off of 14000, and dialyzed against water for 24 h to obtain a carboxymethyl chitosan-deoxycholic acid nanoparticle suspension, denoted as CMDA-NPs.

[0044] S3, 20 mL of the prepared CMDA-NPs solution was taken out after ultrasonic treatment for 5 min, 1 mg of ginsenoside CK was added to the above nanoparticle suspension, and the mixture was stirred at room temperature for 3 h to load ginsenoside CK on the nanoparticles. Then the CK-loaded nanoparticle mixture was transferred to a dialysis bag with a molecular weight cut-off of 14000, and dialyzed against water for 24 h to drive ginsenoside CK into the hydrophobic core of the nanoparticles, thereby obtaining carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with high amounts of ginsenoside CK, denoted as CMDA@CK1-NPs.

[0045] S4, 2 mL of the CMDA@CK1-NPs suspension was mixed with trehalose at a mass ratio of 100:0, and the mixture was uniformly mixed and then pre-cooled at -20°C overnight. Then the mixture was freeze-dried in a freeze dryer until completely dried, thereby obtaining the finished product of high ginsenoside CK-loaded carboxymethyl chitosan-deoxycholic acid nanoparticles in the form of a freeze-dried powder.

[0046] Example 3 S1, the preparation of carboxymethyl chitosan-deoxycholic acid polymer was carried out according to the synthesis method of carboxymethyl chitosan-deoxycholic acid polymer in CN116831992A. Specifically, 1.0 g of carboxymethyl chitosan was dissolved in 70 mL of aqueous solution, and stirred until dissolved to obtain a carboxymethyl chitosan aqueous solution. 0.8 g of deoxycholic acid, 0.5 g of NHS, and 1 g of EDC•HCl were dissolved in 30 mL of ethanol, and the mixture was stirred at room temperature for 30 min before being added dropwise to the carboxymethyl chitosan aqueous solution at a uniform speed to obtain a 100 mL reaction system solution. The solution was placed on a magnetic stirrer and stirred at 600 rpm for 24 h, and then dialyzed against water for 24 h. The solution was centrifuged at 12000 rpm for 10 min, the supernatant was discarded, and the precipitate was freeze-dried to obtain carboxymethyl chitosan-deoxycholic acid polymer, denoted as CMCS-DA.

[0047] S2, 20 mg of CMCS-DA was weighed and dissolved in 2 mL of methanol, and then added dropwise to 20 mL of stirred ultrapure water at a uniform speed. During the dropwise addition process, the solution self-assembled into microparticles. After the dropwise addition was completed, the solution was added to a dialysis bag with a molecular weight cut-off of 14000, and dialyzed against water for 24 h to obtain a carboxymethyl chitosan-deoxycholic acid nanoparticle suspension, denoted as CMDA-NPs.

[0048] S3, 20 mL of the prepared CMDA-NPs solution was taken out after ultrasonic treatment for 5 min, and 2 mg of ginsenoside CK was added to the above nanoparticle suspension. The mixture was stirred at room temperature for 3 h to load ginsenoside CK onto the nanoparticles. Then the CK-loaded nanoparticle mixture was transferred to a dialysis bag with a molecular weight cut-off of 14000. The dialysis bag was dialyzed against water for 24 h to drive ginsenoside CK into the hydrophobic core of the nanoparticles, thereby obtaining carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with high amounts of ginsenoside CK, denoted as CMDA@CK2-NPs.

[0049] S4, 2 mL of the CMDA@CK2-NPs suspension was mixed with trehalose at a mass ratio of 100:0, and the mixture was pre-cooled at -20°C overnight. Then the mixture was freeze-dried in a freeze dryer until completely dried, thereby obtaining the finished product of carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with high amounts of ginsenoside CK in the form of a freeze-dried powder.

[0050] Example 4 S1, the preparation of carboxymethyl chitosan-deoxycholic acid polymer was carried out according to the synthesis method of carboxymethyl chitosan-deoxycholic acid polymer in CN116831992A. Specifically, 1.0 g of carboxymethyl chitosan was dissolved in 70 mL of aqueous solution, and stirred until dissolved to obtain a carboxymethyl chitosan aqueous solution. 0.8 g of deoxycholic acid, 0.5 g of NHS, and 1 g of EDC•HCl were dissolved in 30 mL of ethanol. After stirring at room temperature for 30 min, the solution was added to the carboxymethyl chitosan aqueous solution at a constant rate to obtain a 100 mL reaction system. The reaction was carried out on a magnetic stirrer at 600 rpm for 24 h, and then dialyzed against water for 24 h. The mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was discarded. The precipitate was freeze-dried to obtain carboxymethyl chitosan-deoxycholic acid polymer, denoted as CMCS-DA.

[0051] S2, 20 mg of CMCS-DA was weighed and dissolved in 2 mL of methanol, and then added to 20 mL of stirred ultrapure water at a constant rate. During the addition process, the solution self-assembled into microparticles. After the addition was completed, the solution was added to a dialysis bag with a molecular weight cut-off of 14000. The dialysis bag was dialyzed against water for 24 h to obtain a carboxymethyl chitosan-deoxycholic acid nanoparticle suspension, denoted as CMDA-NPs.

[0052] S3, 20 mL of the prepared CMDA-NPs solution was taken out after ultrasonic treatment for 5 min, and 4 mg of ginsenoside CK was added to the above nanoparticle suspension. The mixture was stirred at room temperature for 3 h to load ginsenoside CK onto the nanoparticles. Then the CK-loaded nanoparticle mixture was transferred to a dialysis bag with a molecular weight cut-off of 14000. The dialysis bag was dialyzed against water for 24 h to drive ginsenoside CK into the hydrophobic core of the nanoparticles, thereby obtaining carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with high amounts of ginsenoside CK, denoted as CMDA@CK4-NPs.

[0053] S4, 2 mL of the CMDA@CK4-NPs suspension was mixed with trehalose at a mass ratio of 100:0, and the mixture was pre-cooled at -20°C overnight. Then the mixture was freeze-dried in a freeze dryer until completely dried, thereby obtaining the finished high-ginsenoside-CK-loaded carboxymethyl chitosan-deoxycholic acid nanoparticle lyophilized powder.

[0054] Example 5 S1, the preparation of carboxymethyl chitosan-deoxycholic acid polymer was carried out according to the synthesis method of carboxymethyl chitosan-deoxycholic acid polymer in CN116831992A. Specifically, 1.0 g of carboxymethyl chitosan was dissolved in 70 mL of water solution, and stirred until dissolved to obtain a carboxymethyl chitosan aqueous solution. 0.8 g of deoxycholic acid, 0.5 g of NHS, and 1 g of EDC•HCl were dissolved in 30 mL of ethanol, and the solution was added to the carboxymethyl chitosan aqueous solution at a constant speed after stirring at room temperature for 30 min, to obtain a 100 mL reaction system solution. The solution was stirred at 600 rpm on a magnetic stirrer for 24 h, and then dialyzed against water for 24 h. The solution was centrifuged at 12000 rpm for 10 min, and the supernatant was discarded. The precipitate was freeze-dried to obtain carboxymethyl chitosan-deoxycholic acid polymer, denoted as CMCS-DA.

[0055] S2, 20 mg of CMCS-DA was weighed and dissolved in 2 mL of methanol, and then added to 20 mL of stirred ultrapure water at a constant speed. The solution self-assembled into microparticles during the addition process. After the addition was completed, the solution was added to a dialysis bag with a molecular weight cut-off of 14000. The dialysis bag was dialyzed against water for 24 h to obtain a carboxymethyl chitosan-deoxycholic acid nanoparticle suspension, denoted as CMDA-NPs.

[0056] S3, 20 mL of the prepared CMDA-NPs solution was taken out after ultrasonic treatment for 5 min, and 5 mg of ginsenoside CK was added to the above nanoparticle suspension. The mixture was stirred at room temperature for 3 h to load ginsenoside CK onto the nanoparticles. Then the CK-loaded nanoparticle mixture was transferred to a dialysis bag with a molecular weight cut-off of 14000. The dialysis bag was dialyzed against water for 24 h to drive ginsenoside CK into the hydrophobic core of the nanoparticles, thereby obtaining carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with high amounts of ginsenoside CK, denoted as CMDA@CK5-NPs.

[0057] S4, 2 mL of the CMDA@CK5-NPs suspension was mixed with trehalose at a mass ratio of 100:0, and the mixture was pre-cooled at -20°C overnight. Then the mixture was freeze-dried in a freeze dryer until completely dried, thereby obtaining the finished product of high ginsenoside CK-loaded carboxymethyl chitosan-deoxycholic acid nanoparticles in the form of a freeze-dried powder.

[0058] Example 6 S1, the preparation of carboxymethyl chitosan-deoxycholic acid polymer was carried out according to the synthesis method of carboxymethyl chitosan-deoxycholic acid polymer in CN116831992A. Specifically, 1.0 g of carboxymethyl chitosan was dissolved in 70 mL of aqueous solution, and stirred until dissolved to obtain a carboxymethyl chitosan aqueous solution. 0.8 g of deoxycholic acid, 0.5 g of NHS, and 1 g of EDC•HCl were dissolved in 30 mL of ethanol, and the mixture was stirred at room temperature for 30 min before being added dropwise to the carboxymethyl chitosan aqueous solution at a constant speed to obtain a 100 mL reaction system solution. The solution was placed on a magnetic stirrer and stirred at 600 rpm for 24 h. The solution was dialyzed against water for 24 h, centrifuged at 12000 rpm for 10 min, and the supernatant was discarded. The precipitate was freeze-dried to obtain carboxymethyl chitosan-deoxycholic acid polymer, denoted as CMCS-DA.

[0059] S2, 20 mg of CMCS-DA was weighed and dissolved in 2 mL of methanol, and then added dropwise to 20 mL of stirred ultrapure water at a constant speed. During the dropwise addition process, the solution self-assembled into microparticles. After the dropwise addition was completed, the solution was added to a dialysis bag with a molecular weight cut-off of 14000. The dialysis bag was dialyzed against water for 24 h to obtain a carboxymethyl chitosan-deoxycholic acid nanoparticle suspension, denoted as CMDA-NPs.

[0060] S3, 20 mL of the prepared CMDA-NPs solution was taken out after ultrasonic treatment for 5 min, 3 mg of ginsenoside CK was added to the above nanoparticle suspension, and the mixture was stirred at room temperature for 3 h to load ginsenoside CK on the nanoparticles. Then the CK-loaded nanoparticle mixture was transferred to a dialysis bag with a molecular weight cut-off of 7000, and dialyzed against water for 24 h to drive ginsenoside CK into the hydrophobic core of the nanoparticles, thereby obtaining carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with high amounts of ginsenoside CK, denoted as CMDA@CK3-NPs.

[0061] S4, 2 mL of the CMDA@CK3-NPs suspension was mixed with trehalose at a mass ratio of 100:0, and the mixture was uniformly mixed and then pre-cooled at -20°C overnight. Then the mixture was freeze-dried in a freeze dryer until completely dried, thereby obtaining the finished product of carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with high amounts of ginsenoside CK in the form of a freeze-dried powder.

[0062] Example 7 S1, the preparation of carboxymethyl chitosan-deoxycholic acid polymer was carried out according to the synthesis method of carboxymethyl chitosan-deoxycholic acid polymer in CN116831992A. Specifically, 1.0 g of carboxymethyl chitosan was dissolved in 70 mL of aqueous solution, and stirred until dissolved to obtain a carboxymethyl chitosan aqueous solution. 0.8 g of deoxycholic acid, 0.5 g of NHS, and 1 g of EDC•HCl were dissolved in 30 mL of ethanol, and the mixture was stirred at room temperature for 30 min before being added dropwise to the carboxymethyl chitosan aqueous solution at a uniform speed to obtain a solution of 100 mL of reaction system. The solution was placed on a magnetic stirrer and stirred at 600 rpm for 24 h. The solution was dialyzed against water for 24 h, centrifuged at 12000 rpm for 10 min, and the supernatant was discarded. The precipitate was freeze-dried to obtain carboxymethyl chitosan-deoxycholic acid polymer, denoted as CMCS-DA.

[0063] S2, 20 mg of CMCS-DA was weighed and dissolved in 2 mL of methanol, and then added dropwise to 20 mL of stirred ultrapure water at a uniform speed. During the dropwise addition process, the microparticles self-assembled into a solution. After the dropwise addition was completed, the solution was added to a dialysis bag with a molecular weight cut-off of 14000, and dialyzed against water for 24 h to obtain a carboxymethyl chitosan-deoxycholic acid nanoparticle suspension, denoted as CMDA-NPs.

[0064] S3, 20 mL of the prepared CMDA-NPs solution was taken out after ultrasonic treatment for 5 min, and 3 mg of ginsenoside CK was added to the above nanoparticle suspension. The mixture was stirred at room temperature for 3 h to load ginsenoside CK onto the nanoparticles. Then the CK-loaded nanoparticle mixture was transferred to a dialysis bag with a molecular weight cut-off of 3500. The dialysis bag was dialyzed against water for 24 h to drive ginsenoside CK into the hydrophobic core of the nanoparticles, thereby obtaining carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with high amounts of ginsenoside CK, denoted as CMDA@CK3-NPs.

[0065] S4, 2 mL of the CMDA@CK3-NPs suspension was mixed with trehalose at a mass ratio of 100:0, and the mixture was pre-cooled at -20°C overnight. Then the mixture was freeze-dried in a freeze dryer until completely dried, thereby obtaining the finished product of carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with high amounts of ginsenoside CK in the form of a freeze-dried powder.

[0066] The effect of the freeze-drying protectant on the nanoparticle freeze-dried powder was then studied.

[0067] Example 8 S1, the preparation of carboxymethyl chitosan-deoxycholic acid polymer was performed according to the synthesis method of carboxymethyl chitosan-deoxycholic acid polymer in CN116831992A. Specifically, 1.0 g of carboxymethyl chitosan was dissolved in 70 mL of an aqueous solution, and stirred until dissolved, thereby obtaining a carboxymethyl chitosan aqueous solution. 0.8 g of deoxycholic acid, 0.5 g of NHS, and 1 g of EDC•HCl were dissolved in 30 mL of ethanol, and the mixture was stirred at room temperature for 30 min before being added dropwise to the carboxymethyl chitosan aqueous solution at a constant speed, thereby obtaining a solution of the 100 mL reaction system. The solution was stirred at 600 rpm on a magnetic stirrer for 24 h, and then dialyzed against water for 24 h. The solution was centrifuged at 12000 rpm for 10 min, and the supernatant was discarded. The precipitate was freeze-dried, thereby obtaining carboxymethyl chitosan-deoxycholic acid polymer, denoted as CMCS-DA.

[0068] S2, 20 mg of CMCS-DA was weighed and dissolved in 2 mL of methanol, and then added dropwise to 20 mL of stirred ultrapure water at a constant speed. During the dropwise addition process, the solution self-assembled into microparticles. After the dropwise addition was completed, the solution was added to a dialysis bag with a molecular weight cut-off of 14000. The dialysis bag was dialyzed against water for 24 h, thereby obtaining a carboxymethyl chitosan-deoxycholic acid nanoparticle suspension, denoted as CMDA-NPs.

[0069] S3, 20 mL of the prepared CMDA-NPs solution was taken out after ultrasonic treatment for 5 min, and 3 mg of ginsenoside CK was added to the above nanoparticle suspension. The mixture was stirred at room temperature for 3 h to load ginsenoside CK onto the nanoparticles. Then the CK-loaded nanoparticle mixture was transferred to a dialysis bag with a molecular weight cut-off of 7000. The dialysis bag was dialyzed against water for 24 h to drive ginsenoside CK into the hydrophobic core of the nanoparticles, thereby obtaining carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with high amounts of ginsenoside CK, denoted as CMDA@CK3-NPs.

[0070] S4, 2 mL of the CMDA@CK3-NPs suspension was mixed with trehalose at a mass ratio of 100:0.4, and the mixture was uniformly mixed and then pre-cooled at -20°C overnight. Then the mixture was freeze-dried in a freeze dryer until completely dried, thereby obtaining the finished product of high ginsenoside CK-loaded carboxymethyl chitosan-deoxycholic acid nanoparticles in the form of a freeze-dried powder.

[0071] Example 9 S1, the preparation of carboxymethyl chitosan-deoxycholic acid polymer was performed according to the synthesis method of carboxymethyl chitosan-deoxycholic acid polymer in CN116831992A. Specifically, 1.0 g of carboxymethyl chitosan was dissolved in 70 mL of aqueous solution, and stirred until dissolved to obtain a carboxymethyl chitosan aqueous solution. 0.8 g of deoxycholic acid, 0.5 g of NHS, and 1 g of EDC•HCl were dissolved in 30 mL of ethanol, and the mixture was stirred at room temperature for 30 min before being added dropwise to the carboxymethyl chitosan aqueous solution at a uniform speed to obtain a solution of the 100 mL reaction system. The solution was stirred at 600 rpm on a magnetic stirrer for 24 h, and then dialyzed against water for 24 h. The solution was centrifuged at 12000 rpm for 10 min, and the supernatant was discarded. The precipitate was freeze-dried to obtain carboxymethyl chitosan-deoxycholic acid polymer, denoted as CMCS-DA.

[0072] S2, 20 mg of CMCS-DA was weighed and dissolved in 2 mL of methanol, and then added dropwise to 20 mL of stirred ultrapure water at a uniform speed. During the dropwise addition process, the solution self-assembled into microparticles. After the dropwise addition was completed, the solution was added to a dialysis bag with a molecular weight cut-off of 14000. The dialysis bag was dialyzed against water for 24 h to obtain a carboxymethyl chitosan-deoxycholic acid nanoparticle suspension, denoted as CMDA-NPs.

[0073] S3, 20 mL of the prepared CMDA-NPs solution was taken out after ultrasonic treatment for 5 min, and 3 mg of ginsenoside CK was added to the above nanoparticle suspension. The mixture was stirred at room temperature for 3 h to load ginsenoside CK onto the nanoparticles. Then the CK-loaded nanoparticle mixture was transferred to a dialysis bag with a molecular weight cut-off of 7000. The dialysis bag was dialyzed against water for 24 h to drive ginsenoside CK into the hydrophobic core of the nanoparticles, thereby obtaining carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with high amounts of ginsenoside CK, denoted as CMDA@CK3-NPs.

[0074] S4, 2 mL of the CMDA@CK3-NPs suspension was mixed with mannitol at a mass ratio of 100:0.4, and the mixture was pre-cooled at -20°C overnight. Then the mixture was freeze-dried in a freeze dryer until completely dried, thereby obtaining the finished high-ginsenoside-CK-loaded carboxymethyl chitosan-deoxycholic acid nanoparticle lyophilized powder.

[0075] Example 10 S1, the preparation of carboxymethyl chitosan-deoxycholic acid polymer was carried out according to the synthesis method of carboxymethyl chitosan-deoxycholic acid polymer in CN116831992A. Specifically, 1.0 g of carboxymethyl chitosan was dissolved in 70 mL of water solution, and stirred until dissolved to obtain a carboxymethyl chitosan aqueous solution. 0.8 g of deoxycholic acid, 0.5 g of NHS, and 1 g of EDC•HCl were dissolved in 30 mL of ethanol, and after stirring at room temperature for 30 min, the solution was added to the carboxymethyl chitosan aqueous solution at a constant speed, to obtain a 100 mL reaction system solution. The solution was stirred at 600 rpm on a magnetic stirrer for 24 h, and then dialyzed against water for 24 h. The solution was centrifuged at 12000 rpm for 10 min, and the supernatant was discarded. The precipitate was freeze-dried to obtain carboxymethyl chitosan-deoxycholic acid polymer, denoted as CMCS-DA.

[0076] S2, 20 mg of CMCS-DA was weighed and dissolved in 2 mL of methanol, and then added to 20 mL of stirred ultrapure water at a constant speed. During the addition process, the solution self-assembled into microparticles. After the addition was completed, the solution was added to a dialysis bag with a molecular weight cut-off of 14000. The dialysis bag was dialyzed against water for 24 h to obtain a carboxymethyl chitosan-deoxycholic acid nanoparticle suspension, denoted as CMDA-NPs.

[0077] S3, 20 mL of the prepared CMDA-NPs solution was taken out after ultrasonic treatment for 5 min, 3 mg of ginsenoside CK was added to the above nanoparticle suspension, and the mixture was stirred at room temperature for 3 h to load ginsenoside CK on the nanoparticles. Then the CK-loaded nanoparticle mixture was transferred to a dialysis bag with a molecular weight cut-off of 7000, and dialyzed against water for 24 h to drive ginsenoside CK into the hydrophobic core of the nanoparticles, thereby obtaining carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with high amounts of ginsenoside CK, denoted as CMDA@CK3-NPs.

[0078] S4, 2 mL of the CMDA@CK3-NPs suspension was mixed with sucrose at a mass ratio of 100:0.4, and the mixture was uniformly mixed and then pre-cooled at -20°C overnight. Then the mixture was freeze-dried in a freeze dryer until completely dried, thereby obtaining the finished product of high ginsenoside CK-loaded carboxymethyl chitosan-deoxycholic acid nanoparticle lyophilized powder.

[0079] Example 11 S1, the preparation of carboxymethyl chitosan-deoxycholic acid polymer was carried out according to the synthesis method of carboxymethyl chitosan-deoxycholic acid polymer in CN116831992A. Specifically, 1.0 g of carboxymethyl chitosan was dissolved in 70 mL of aqueous solution, and stirred until dissolved to obtain a carboxymethyl chitosan aqueous solution. 0.8 g of deoxycholic acid, 0.5 g of NHS, and 1 g of EDC•HCl were dissolved in 30 mL of ethanol, and the mixture was stirred at room temperature for 30 min, and then added to the carboxymethyl chitosan aqueous solution at a constant speed to obtain a 100 mL reaction system solution, which was stirred at 600 rpm on a magnetic stirrer for 24 h, dialyzed against water for 24 h, centrifuged at 12000 rpm for 10 min, the supernatant was discarded, and the precipitate was freeze-dried to obtain carboxymethyl chitosan-deoxycholic acid polymer, denoted as CMCS-DA.

[0080] S2, 20 mg of CMCS-DA was weighed and dissolved in 2 mL of methanol, and then added to 20 mL of stirred ultrapure water at a constant speed. During the addition process, the microparticles self-assembled into a solution. After the addition was completed, the solution was added to a dialysis bag with a molecular weight cut-off of 14000, and dialyzed against water for 24 h to obtain a carboxymethyl chitosan-deoxycholic acid nanoparticle suspension, denoted as CMDA-NPs.

[0081] S3, 20 mL of the prepared CMDA-NPs solution was taken out after ultrasonic treatment for 5 min, and 3 mg of ginsenoside CK was added to the above nanoparticle suspension. The mixture was stirred at room temperature for 3 h to load ginsenoside CK onto the nanoparticles. Then the CK-loaded nanoparticle mixture was transferred to a dialysis bag with a molecular weight cut-off of 7000. The dialysis bag was dialyzed against water for 24 h to drive ginsenoside CK into the hydrophobic core of the nanoparticles, thereby obtaining carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with high amounts of ginsenoside CK, denoted as CMDA@CK3-NPs.

[0082] S4, 2 mL of the CMDA@CK3-NPs suspension was mixed with trehalose at a mass ratio of 100:0.2, and the mixture was pre-cooled at -20°C overnight. Then the mixture was freeze-dried in a freeze dryer until completely dried, thereby obtaining the finished product of carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with high amounts of ginsenoside CK in the form of a freeze-dried powder.

[0083] Example 12 S1, the preparation of carboxymethyl chitosan-deoxycholic acid polymer was carried out according to the synthesis method of carboxymethyl chitosan-deoxycholic acid polymer in CN116831992A. Specifically, 1.0 g of carboxymethyl chitosan was dissolved in 70 mL of aqueous solution, and stirred until dissolved to obtain a carboxymethyl chitosan aqueous solution. 0.8 g of deoxycholic acid, 0.5 g of NHS, and 1 g of EDC•HCl were dissolved in 30 mL of ethanol, and the mixture was stirred at room temperature for 30 min before being added dropwise to the carboxymethyl chitosan aqueous solution at a constant speed to obtain a 100 mL reaction system solution. The solution was stirred at 600 rpm on a magnetic stirrer for 24 h, and then dialyzed against water for 24 h. The solution was centrifuged at 12000 rpm for 10 min, and the supernatant was discarded. The precipitate was freeze-dried to obtain carboxymethyl chitosan-deoxycholic acid polymer, denoted as CMCS-DA.

[0084] S2, 20 mg of CMCS-DA was weighed and dissolved in 2 mL of methanol, and then added dropwise to 20 mL of stirred ultrapure water at a constant speed. During the dropwise addition process, the microparticles self-assembled into a solution. After the dropwise addition was completed, the solution was added to a dialysis bag with a molecular weight cut-off of 14000. The dialysis bag was dialyzed against water for 24 h to obtain a carboxymethyl chitosan-deoxycholic acid nanoparticle suspension, denoted as CMDA-NPs.

[0085] S3, 20 mL of the prepared CMDA-NPs solution was taken out after ultrasonic treatment for 5 min, and 3 mg of ginsenoside CK was added to the above nanoparticle suspension. The mixture was stirred at room temperature for 3 h to load ginsenoside CK onto the nanoparticles. Then the mixture loaded with CK was transferred to a dialysis bag with a molecular weight cut-off of 7000. The dialysis bag was dialyzed against water for 24 h to drive ginsenoside CK into the hydrophobic core of the nanoparticles, thereby obtaining carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with high amounts of ginsenoside CK, denoted as CMDA@CK3-NPs.

[0086] S4, 2 mL of the CMDA@CK3-NPs suspension was mixed with trehalose at a mass ratio of 100:0.6, and the mixture was pre-cooled at -20°C overnight. Then the mixture was freeze-dried in a freeze dryer until completely dried, thereby obtaining the finished product of carboxymethyl chitosan-deoxycholic acid nanoparticles loaded with high amounts of ginsenoside CK in the form of a freeze-dried powder.

[0087] When the amount of trehalose added was changed, the viscosity of the carboxymethyl chitosan-deoxycholic acid nanoparticle freeze-dried powder system loaded with high amounts of ginsenoside CK increased slightly as the amount of trehalose added increased. Excessive sugar residues can change the surface properties (such as charge and hydrophilicity) of the nanoparticles, affecting subsequent drug release or cell uptake. The optimal mass ratio was 100:0.6.

[0088] Comparative Example 1 S1, the preparation of carboxymethyl chitosan-deoxycholic acid polymer was carried out according to the synthesis method of carboxymethyl chitosan-deoxycholic acid polymer in CN116831992A. Specifically, 1.0 g of carboxymethyl chitosan was dissolved in 70 mL of aqueous solution, and stirred until dissolved to obtain a carboxymethyl chitosan aqueous solution. 0.8 g of deoxycholic acid, 0.5 g of NHS, and 1 g of EDC•HCl were dissolved in 30 mL of ethanol, and after stirring at room temperature for 30 min, the solution was added to the carboxymethyl chitosan aqueous solution at a constant speed, obtaining a 100 mL reaction system solution. The solution was placed on a magnetic stirrer and stirred at 600 rpm for 24 h. The solution was dialyzed against water for 24 h, centrifuged at 12000 rpm for 10 min, the supernatant was discarded, and the precipitate was freeze-dried to obtain carboxymethyl chitosan-deoxycholic acid polymer, denoted as CMCS-DA.

[0089] S2, 20 mg of CMCS-DA was weighed and dissolved in 2 mL of methanol, and then added to 20 mL of stirred ultrapure water at a constant speed. During the addition process, the microparticles self-assembled into a particle solution. After the addition was completed, the solution was added to a dialysis bag with a molecular weight cut-off of 14000. The dialysis bag was dialyzed against water for 24 h to obtain a carboxymethyl chitosan-deoxycholic acid nanoparticle suspension, denoted as CMDA-NPs.

[0090] S3, 20 mL of the prepared CMDA-NPs solution was taken out after ultrasonic treatment for 5 min, transferred to a dialysis bag with a molecular weight cut-off of 7000, and dialyzed against water for 24 h to obtain unloaded carboxymethyl chitosan-deoxycholic acid nanoparticles, denoted as CMDA@-NPs.

[0091] S4, 2 mL of the CMDA@-NPs suspension was mixed with trehalose at a mass ratio of 100:0.4, and the mixture was uniformly mixed and pre-cooled at -20°C overnight. Then the mixture was freeze-dried in a freeze dryer until complete freeze-drying to obtain the finished product of unloaded carboxymethyl chitosan-deoxycholic acid nanoparticle freeze-dried powder.

[0092] The micro-morphology of the high-loading ginsenoside CK carboxymethyl chitosan-deoxycholic acid nanoparticles prepared in Example 1 was characterized by scanning electron microscopy, and it was observed that Figure 1 the nanoparticles were round, smooth in surface, and well dispersed. Statistical measurement showed that the average particle size was about 300 nm.

[0093] The high-loading ginsenoside CK carboxymethyl chitosan-deoxycholic acid nanoparticles prepared in Example 1 were characterized by Fourier transform infrared absorption spectrometer, and the results are shown in Figure 2 It was observed that Figure 2 compared with the CMDA-NPs, the high-loading CK nanoparticles had a unique α-glycosidic bond at 1080 cm -1 , indicating that the CMDA-NPs successfully loaded CK.

[0094] The high-loading CK nanoparticles prepared in Example 1 were characterized by X-ray diffraction, and the results are shown in Figure 3 It was observed that Figure 3 the diffraction peaks of the high-loading ginsenoside CK carboxymethyl chitosan-deoxycholic acid nanoparticles were similar to those of the CMDA-NPs. The diffraction peaks of ginsenoside CK did not appear in the X-ray diffraction pattern of the high-loading CK nanoparticles, indicating that ginsenoside CK was successfully loaded.

[0095] It should be noted that Figures 1-3 the characterization results of Example 1, so CMDA@CK-NPs refers to the product prepared in Example 1.

[0096] Examples 1-5 changed the amount of ginsenoside CK added, and the encapsulation efficiency and drug loading of the high-loading ginsenoside CK carboxymethyl chitosan-deoxycholic acid nanoparticles prepared in Examples 1 and 5 are shown in Figure 4 It was observed that Figure 4The results showed that the entrapment efficiency of CMDA@CK-NPs initially increased with the addition of CK and then decreased. This may be due to the fact that excess unentrapped CK was dialyzed out during the dialysis process, resulting in a decrease in the entrapment efficiency. The drug loading of CMDA@CK-NPs initially increased and then leveled off. This may be because as the CK addition ratio increased from 1:10 to 3:10, the CK loading on the CMDA-NPs gradually reached saturation. At this point, the drug loading of CMDA@CK-NPs was 64.4±0.964%. Considering the two key parameters of drug loading and entrapment efficiency, the drug loading and entrapment efficiency of CMDA@CK-NPs were optimal at a drug-to-polymer ratio of 3:10, reaching 64.4% and 79.4%, respectively. It should be noted that the CK:CMDA ratio increased from 1:10 to 5:10, respectively, and the CK addition amount in the examples ranged from 1 mg to 5 mg.

[0097] Table 1 Particle size of nanoparticles prepared in Example 1 and Examples 6-7 In Examples 1, 6, and 7, the molecular weight cutoff of the dialysis bag in step S3 was varied. The particle sizes of the nanoparticles prepared in Examples 1, 6, and 7 are shown in Table 1. When the molecular weight cutoff of the dialysis bag was 14,000, the particle size distribution was broad, with an average particle size of 195.1 nm and a drug loading of 44.1%. When the molecular weight cutoff of the dialysis bag was 3,500, the distribution was narrower, with an average particle size of 89.27 nm, and the drug loading remained relatively low at 29.1%. When the molecular weight cutoff of the dialysis bag was 7,000, the CMDA@CK-NPs nanoparticles exhibited a more concentrated particle size distribution, with an average particle size of 216.7 nm, ranging from 90 nm to 400 nm, and a drug loading of 52.56%.

[0098] The release curves of CMDA@CK3-NPs prepared in Example 1 under different pH environments are shown in the following table: Figure 5 As shown, observe Figure 5 The results showed that at pH 1.2 and 7.4, CK from the nanoparticles was virtually not released, effectively preventing drug release in normal physiological environments and reducing potential systemic and toxic side effects. Under weakly acidic conditions of pH 5.8, CK exhibited a significant pH-responsive release, with a cumulative release of 92.17% within 35 hours. This release characteristic of the particles facilitates selective, high-volume release of the drug at the tumor site, thereby enhancing drug targeting and therapeutic efficacy while minimizing damage to normal tissues.

[0099] Table 2 Effects of lyoprotectants on freeze-dried nanoparticle powders The high-loading nanoparticle lyophilized powder prepared in Example 1, Example 8 to Example 10, the type of lyoprotectant is changed, and the instant reconstitution is as follows Figure 6 and Table 2, it is observed that Figure 6 It is concluded that the high-loading CK nanoparticle lyophilized powder containing 0.4% trehalose has almost no visible particles, obvious turbidity phenomenon, and good effect. It is observed that Figure 7 It is concluded that the high-loading CMDA@CK3-NPs nanoparticle lyophilized powder containing 0.4% trehalose has good storage condition, compared with Figure 7 (a) high-loading CK nanoparticle lyophilized powder without 0.4% trehalose in Example 1, and Figure 7 (b) it can be seen that the high-loading CK nanoparticle lyophilized powder containing 0.4% trehalose has good storage stability within 11 days. It is observed that Figure 7 It is concluded that the average particle size of the CMDA@CK-NPs lyophilized powder containing 0.4% trehalose increases from 205.5 nm to 451.9 nm, and the polydispersity index increases from 0.172 to 0.281, which may be due to the shrinkage of the core of the nanoparticles at a higher room temperature during storage, resulting in an increase in particle size. This storage temperature is not conducive to prolonging the storage period of CMDA@CK-NPs. Therefore, the best storage method for CMDA@CK-NPs is to use 0.4% trehalose as a lyoprotectant during lyophilization to ensure the structural integrity, and the best storage condition is 4°C cold storage, and the storage container is an EP tube.

[0100] Example 13 This example is the in vitro biological safety test of the empty nanoparticle lyophilized powder prepared in Comparative Example 1 on hepatoma cells HepG2 cells, which will be referred to as HepG2 cells hereinafter. The specific application method and results are as follows:

[0101] Application method: a. The in vitro biological safety of the empty nanoparticle lyophilized powder of Comparative Example 1 is evaluated by MTT method, and the specific operation is as follows: HepG2 cells in the logarithmic growth phase were collected and 100 μL was taken each time with a pipette and inoculated into a 96-well plate so that the number of HepG2 cells in each well was about 8000. After culturing for 24 hours, the old culture medium was discarded. 100 μL of culture medium prepared with the empty nano-lyophilized powder of Example 1 was added to maintain the final concentration of the nanoparticles at 12.5, 25, 150, 200 and 400 μg / mL, and the culture was continued for 24 hours and 48 hours. Subsequently, 20 μL of MTT solution with a mass concentration of 5 mg / mL was added to each well and incubated for 4 hours. The culture medium in the well was carefully aspirated, and 150 μL of dimethyl sulfoxide was added to each well. The crystals were shaken to fully dissolve, and then the absorbance was measured at 490 nm using a microplate reader.

[0102] HepG2 cell viability was calculated using the following formula: Among them, A 490 The experimental group represents the absorbance of the experimental group at 490 nm, which includes cells, MTT, empty nanoparticles, and HepG2 cell culture medium; A 490 The zero-adjusted well indicates the absorbance at 490 nm, and the zero-adjusted well indicates MTT and HepG2 cell culture medium; A 490 The control group represents the absorbance of the control group at 490 nm, and the control group contains cells, MTT, and HepG2 cell-specific culture medium.

[0103] Depend on Figure 8 The in vitro biosafety evaluation results of empty CMDA-NPs at different concentrations are shown in Figures 4-7 As shown, after 48 hours of incubation with cancer cells, the cell viability of unloaded nanoparticles at concentrations ranging from 12.5 μg / mL to 400 μg / mL was slightly lower at 48 hours than at 24 hours. This is likely due to increased uptake of CMDA-NPs by HepG2 cells with prolonged incubation time. Furthermore, at concentrations as high as 150 μg / mL, HepG2 cell viability after treatment with lyophilized CMDA-NPs remained above 92.5%. The concentration of CMDA@CK-NPs used in subsequent experiments was far below 150 μg / mL. Therefore, CMDA-NPs possess excellent material safety and can be used as a carrier for drug delivery.

[0104] Example 14 This example tests the proliferation inhibition effect of the CMDA@CK3-NPs nanoparticle freeze-dried powder prepared in Example 1 on liver cancer cells HepG2.

[0105] HepG2 cells in the logarithmic growth phase were harvested and the cell concentration was adjusted. 100 μL of culture medium was pipetted into a 96-well plate, allowing approximately 6,000 to 8,000 cells per well. After attachment for 18 to 24 hours, the old culture medium was discarded and ginsenoside CK or CMDA@CK-NPs freeze-dried powder solutions at varying concentrations (12.5 μg / mL to 400 μg / mL) prepared in MEM medium were added. Cultures were continued for 24 and 48 hours. Subsequently, 20 μL of a 5 mg / mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution was added to each well. The cells were incubated for 4 hours. The culture medium was carefully aspirated and 150 μL of dimethyl sulfoxide was added to each well. After shaking to fully dissolve the crystals, the absorbance was measured at 490 nm using a microplate reader. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was designated as MTT.

[0106] Calculate cell viability using the following formula: Among them, A 490 The experimental group refers to the absorbance value of the experimental group at a wavelength of 490 nm on a microplate reader. The experimental group includes HepG2 cells, MEM culture medium, MTT solution and drug solution. 490 The blank group refers to the absorbance value of the blank group at a wavelength of 490 nm on the microplate reader. The blank group contains MEM culture medium and MTT solution. 490 The control group represents the absorbance value of the control group at a wavelength of 490 nm on a microplate reader, and the control group comprises HepG2 cells, MEM culture medium, and MTT solution.

[0107] Depend on Figure 9 As shown, CMDA@CK-NPs exhibited a more pronounced inhibitory effect on human HepG2 cell proliferation than free CK. This may be due to CMDA@CK-NPs being internalized into cells via receptor-mediated endocytosis. During this process, CK is not recognized by surface P-glycoprotein, thus preventing CK from being excreted, ensuring its accumulation within tumor cells and thus exerting its therapeutic effect. Furthermore, the inhibitory effect of CMDA@CK-NPs on HepG2 cells was greater at 48 hours than at 24 hours. This may be due to the fact that over time, cells internalized more CMDA@CK-NPs and released a large amount of CK into the slightly acidic microenvironment within the tumor, consistent with the drug release behavior of CMDA@CK-NPs at pH 5.8 from 22 to 34 hours. Furthermore, the inhibitory effect of CMDA@CK-NPs on HepG2 cells at 48 hours was dose-dependent, with a cell viability of approximately 46.04% when CMDA@CK-NPs were added at a concentration of 50 μM.

[0108] While the preferred embodiments of the application have been described, additional variations and modifications can be made to these embodiments by those skilled in the art once they have the benefit of the present disclosure without departing from the spirit and scope of the application. Accordingly, it is intended that the appended claims include all such modifications and variations as fall within the scope of the present application.

[0109] It is apparent that those skilled in the art can make various changes and modifications to the application without departing from the spirit and scope of the application. It is therefore intended that the present application cover all such changes and modifications that are within its scope.

Claims

1. A method for preparing carboxymethyl chitosan nano freeze-dried powder, characterized in that: The following steps are involved: Dissolving a carboxymethyl chitosan deoxycholic acid polymer in an alcohol solvent and adding the resulting solution to water, wherein the carboxymethyl chitosan deoxycholic acid polymer self-assembles into nanoparticles during the addition process to obtain a suspension, and performing a first dialysis to remove free polymer or other small molecular impurities to obtain a carboxymethyl chitosan deoxycholic acid nanoparticle solution; Ginsenoside CK powder was added to the carboxymethyl chitosan deoxycholic acid nanoparticle solution and stirred at room temperature to load ginsenoside CK onto the carboxymethyl chitosan deoxycholic acid nanoparticles. After stirring, a second dialysis was performed using a dialysis bag with a molecular weight cut-off of 3500 to 14000 to remove unencapsulated ginsenoside CK and adjust the product particle size to obtain a ginsenoside CK carboxymethyl chitosan deoxycholic acid nanoparticle suspension. The ginsenoside CK carboxymethyl chitosan-deoxycholic acid nanoparticle suspension is freeze-dried to obtain carboxymethyl chitosan nano freeze-dried powder.

2. The method for preparing a carboxymethyl chitosan nano freeze-dried powder according to claim 1, wherein: During the second dialysis, the dialysis time is 1h~24h.

3. The method for preparing a carboxymethyl chitosan nano freeze-dried powder according to claim 1, wherein: During the first dialysis, the molecular weight cutoff of the dialysis bag used is 7000~14000, and the dialysis time is 12h~24h.

4. The method for preparing a carboxymethyl chitosan nano freeze-dried powder according to claim 1, wherein: The ratio of carboxymethyl chitosan deoxycholic acid polymer and water is 1mg~1.5mg:1mL.

5. The method for preparing a carboxymethyl chitosan nano freeze-dried powder according to claim 1, wherein: The ratio of carboxymethyl chitosan deoxycholic acid nanoparticle solution and ginsenoside CK powder is 20 mL: 1 mg~5 mg.

6. The method for preparing a carboxymethyl chitosan nano freeze-dried powder according to claim 5, characterized in that: The ratio of carboxymethyl chitosan deoxycholic acid nanoparticle solution and ginsenoside CK powder was 20 mL:3 mg.

7. The method for preparing a carboxymethyl chitosan nano freeze-dried powder according to claim 1, characterized in that: Before freeze-drying, a freeze-drying protective agent was added to the suspension of ginsenoside CK carboxymethyl chitosan-deoxycholic acid nanoparticles; The freeze-drying protective agent is at least one of trehalose, sucrose, mannitol and cyclodextrin.

8. The method for preparing a carboxymethyl chitosan nano freeze-dried powder according to claim 1, characterized in that: The mass volume fraction of the lyoprotectant in the ginsenoside CK carboxymethyl chitosan-deoxycholic acid nanoparticle suspension is 0.2%~0.6%.

9. A carboxymethyl chitosan nano freeze-dried powder prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the carboxymethyl chitosan nano freeze-dried powder according to claim 9 in the preparation of anti-tumor drugs.

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