Preparation of glycyrrhetinic acid-quaternary ammonium salt chitosan / carboxymethyl chitosan nanoparticles

Abstract

The invention discloses a preparation method of glycyrrhetinic acid at quaternary ammonium salt chitosan / carboxymethyl chitosan nanoparticles, and belongs to the technical field of medicines. Quaternary ammonium salt chitosan is used as a nano-drug delivery system carrier, carboxymethyl chitosan is used as a cross-linking agent, glycyrrhetinic acid and adriamycin are modified on the surface through ion exchange to serve as model drugs, and glycyrrhetinic acid and quaternary ammonium salt chitosan / carboxymethyl chitosan nano-particles are prepared. The nanoparticle provided by the invention can control the release of the antitumor drug in the inner cavity aiming at the microenvironment of tumor cells, can be used as a targeted transportation tool of the antitumor drug, and has a great application prospect in the field of targeted therapy of tumors.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, and in particular to the preparation of glycyrrhetinic acid@quaternary ammonium salt chitosan / carboxymethyl chitosan nanoparticles. Background Art

[0002] Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver. In China, liver cancer ranks as the fifth most common malignant tumor and the second leading cause of cancer-related death. A statistical analysis of 17 studies on the survival rate of Chinese cancer patients published in The Lancet Global Health showed that the 5-year survival rate of Chinese liver cancer patients was only 12.1%. Treatment options for HCC include: local ablation therapy, resection and liver transplantation. Advanced patients can only rely on systemic chemotherapy and transarterial chemoembolization (TACE) for treatment. However, systemic chemotherapy often has multidrug resistance and severe side effects, making it difficult to produce sustainable therapeutic effects and placing a huge burden on the patient's body, ultimately leading to unsatisfactory treatment effects and low survival rates for HCC.

[0003] This therapy, which uses tumor-targeting ligands and nanotechnology to encapsulate drugs to extend the plasma half-life and reduce the toxic side effects of systemic chemotherapy, is called targeted nano drug delivery system (TNDDS).

[0004] TNDDS usually modifies receptor-specific ligands on the surface of nanocarriers, such as peptides, carbohydrates, proteins and antibodies, and achieves targeted drug delivery by specifically binding the modified ligands to receptors overexpressed on tumor cells. At the same time, TNDDS can be specifically stable and circulate for a long time in the body due to its small particle size, large specific surface area and high surface energy, which significantly improves the bioavailability of drugs, improves the stability of drugs and achieves sustained release of drugs.

[0005] Studies have found that receptors highly expressed on the surface of liver cancer cells include glycyrrhetinic acid receptor (GA-R) and folic acid receptor (FA-R). They have high affinity with receptors overexpressed on liver cells or HCC cells, and modified nanoparticles can be internalized by malignant cells through receptor-mediated endocytosis, leading to intracellular drug release and greater cytotoxicity. However, in patients with liver disease, FA-functionalized nanocarriers such as liposomes enter the blood and bind to natural immunoglobulins in large quantities, depriving FA of receptor recognition and accelerating complement activation in the body, resulting in reduced targeting efficiency. Glycyrrhetinic acid (GA) is one of the main active ingredients of traditional Chinese medicine licorice, and its chemical structure belongs to pentacyclic triterpenoid compounds. The expression of GA receptor (GA-R) in liver cancer cells is significantly higher than that in normal liver cells. The binding of GA to GA-R is highly specific and saturated, and the internalization of GA is mainly mediated by ligand-receptor interaction, which ensures the high efficiency of GA absorption by cancer cells. In the experiment of Tian et al., the uptake rate of GA-modified wogonin liposomes in HepG2 cells was 2.5 times that of L-02 cells, indicating that there are more GA-specific binding sites on HepG2 cells compared with L-02 cells. This result also suggests that GA modification can distinguish liver cancer cells from normal liver cells. Based on previous studies, GA-R has significant advantages in achieving efficient targeting of liver cancer. A large number of research reports have shown that GA-modified nanodrug delivery systems (GA-TNDDS) have excellent liver cancer targeting. Summary of the invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to provide a preparation method for glycyrrhetinic acid @ quaternary ammonium salt chitosan / carboxymethyl chitosan nanoparticles. The present invention uses quaternary ammonium salt chitosan as a carrier of a nano drug delivery system, carboxymethyl chitosan as a cross-linking agent, uses ion exchange to modify glycyrrhetinic acid on the surface, and uses doxorubicin as a model drug to prepare glycyrrhetinic acid @ quaternary ammonium salt chitosan / carboxymethyl chitosan nanoparticles.

[0007] 1. Selection of carriers for nanodrug delivery systems.

[0008] Quaternary ammonium chitosan is a product obtained by chemical modification of chitosan. It has many useful properties of chitosan and has a positive charge.

[0009] Due to its cationic nature, quaternary ammonium chitosan can effectively fight negatively charged bacteria. This makes it show great potential in the field of antibacterial. In addition, since quaternary ammonium chitosan has adjustable solubility and biocompatibility, it can be used to prepare a variety of drug delivery systems and biomedical devices. It is reported that quaternary ammonium chitosan has good adaptability to various organs or muscles, skin, and teeth of the body, and has good biocompatibility and no antigenicity. In the body, chitosan can be absorbed and degraded into small molecular active substances such as glucosamine, which can finally be absorbed by the body.

[0010] At the same time, many studies have shown that chitosan has a certain inhibitory effect on tumor cells. Common theories include charge inhibition and acid-base inhibition. It causes charge imbalance on the cell surface, reduces cell adhesion, relies on amino groups to regulate the acidic environment inside cancer cells, and increases lymphocyte activity, ultimately achieving growth inhibition of cancer cells.

[0011] 2. Selection of cross-linking agent.

[0012] Carboxymethyl chitosan is a water-soluble chitosan derivative and an amphoteric polyelectrolyte. It is formed by introducing a hydrophilic group -CH2COOH into the chitosan polymer chain. After carboxymethylation, chitosan has improved water solubility, especially in neutral and alkaline solutions. It has film-forming, thickening, moisturizing, chelating and other properties. Therefore, it is widely used in agriculture, medicine, health products, cosmetics, preservation, environmental protection and other aspects.

[0013] In view of the acidic environment within tumor cells, based on the pH sensitivity and high solubility of carboxymethyl chitosan, in order to obtain a pH-sensitive nanodrug carrier, carboxymethyl chitosan was used as a cross-linking agent for quaternary ammonium chitosan in this experiment.

[0014] 3. Selection of liver cancer cell surface targets.

[0015] Glycyrrhetinic acid (GA) is one of the main active ingredients of traditional Chinese medicine licorice, and its chemical structure belongs to pentacyclic triterpenoid compounds. The expression of GA receptor (GA-R) in liver cancer cells is significantly higher than that in normal liver cells. The binding of GA to GA-R is highly specific and saturated, and the internalization of GA is mainly mediated by ligand-receptor interaction, which ensures the high efficiency of GA absorption by cancer cells. In the experiment of Tian et al., the uptake rate of GA-modified wogonin liposomes in HepG2 cells was 2.5 times that of L-02 cells, indicating that there are more GA-specific binding sites on HepG2 cells compared with L-02 cells. This result also suggests that GA modification can distinguish liver cancer cells from normal liver cells. Based on previous studies, GA-R has significant advantages in achieving efficient targeting of liver cancer. A large number of research reports have shown that GA-modified nanodrug delivery system (GA-TNDDS) has excellent liver cancer targeting. Due to the different H configurations at the C-18 position, GA has two isomers, 18α-GA and 18β-GA. In current research, 18β-GA is often used as a liver cancer targeting ligand, so the discussion of this project only involves 18β-GA (hereinafter referred to as GA).

[0016] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0017] The invention discloses a drug-loaded microsphere, which is characterized in that the components include quaternary ammonium salt chitosan and carboxymethyl chitosan.

[0018] The present invention also discloses a pharmaceutical composition, which comprises the drug-loaded microspheres as described above, glycyrrhetinic acid and doxorubicin.

[0019] The present invention also discloses the application of the drug-loaded microspheres and the drug composition in the preparation of tumor-targeting drugs.

[0020] The present invention also discloses a method for preparing the drug-loaded microspheres described above, which is characterized by comprising the following steps: Step 1: Preparation of carboxymethyl chitosan; Step 2: Preparation of quaternary ammonium salt chitosan; Step 3: Preparation of quaternary ammonium salt-carboxymethyl chitosan microspheres.

[0021] Furthermore, the specific steps of step 1 are: dispersing 1.61g of chitosan in 16mL of propanol, adding 5mL of sodium hydroxide solution (40%, w / v) dropwise, and stirring at 50°C for 1h in a round-bottom flask equipped with a mechanical stirrer; then adding 23mL of chloroacetic acid solution (10%, w / v) to the above solution, and then grouping and stirring at 70°C for 4h; thereafter precipitating with anhydrous ethanol, washing with 90% ethanol, and filtering the obtained solid and freeze-drying it to constant weight to obtain carboxymethyl chitosan that meets the requirements.

[0022] Furthermore, the specific steps of step 2 are as follows: weigh 1.61g chitosan and disperse it in 60mL isopropanol, and swell it at room temperature for 4h; slowly add 12.5mL 2,3-epoxypropyltrimethylammonium chloride aqueous solution (65%, w / v) under reflux conditions, and divide it into several experimental groups and continue to react for 10h at 60°C. After the reaction, adjust the pH of the reaction system to neutral with 10% hydrochloric acid and then filter it, soak and wash the obtained solid with 90% ethanol, precipitate it with anhydrous ethanol, and dry the final product in an electric constant temperature forced air drying oven at 55°C to constant weight to obtain quaternary ammonium chitosan that meets the requirements.

[0023] Furthermore, the specific steps of step 3 are: dissolving the quaternary ammonium salt chitosan obtained in step 2 in an appropriate amount of deionized water, filtering with a micron filter to obtain a filtrate with a particle size of less than 0.3 microns; adding the carboxymethyl chitosan solution obtained in step 1 dropwise to the above filtrate; stirring at room temperature at a speed of 1000 rpm for half an hour; after the reaction is completed, centrifuging the milky white reaction system at high speed for 10 minutes, filtering with suction, and freeze-drying to obtain quaternary ammonium salt-carboxymethyl chitosan drug-loaded microspheres.

[0024] The present invention also discloses the use of quaternary ammonium salt-carboxymethyl chitosan drug-loaded microspheres prepared by any of the above-mentioned preparation methods in the preparation of tumor-targeting drugs.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0026] (1) The present invention uses quaternary ammonium salt chitosan: chitosan derivatives and chitosan have the characteristics of low toxicity and high biocompatibility. Among them, quaternary ammonium salt chitosan can carry out ion exchange with the help of the cations it carries, providing sites for the successful modification of glycyrrhetinic acid.

[0027] (2) The crosslinking agent of the present invention uses carboxymethyl chitosan: carboxymethyl chitosan has good pH sensitivity, which helps to achieve efficient drug release in the acidic liver cancer environment and reduce loss in the blood. At the same time, compared with the traditional anionic crosslinking agent sodium tripolyphosphate, carboxymethyl chitosan is easy to metabolize and is not easy to cause enteritis.

[0028] (3) Biological target selection: Glycyrrhetinic acid receptor: Membrane proteins and receptors highly expressed on the surface of liver cancer cells include asialoglycoprotein receptor (ASGP-R), glycyrrhetinic acid receptor (GA-R), transferrin receptor (TF-R), folate receptor (FA-R), epidermal growth factor receptor (EGF-R), etc. They have high affinity with receptors overexpressed on hepatocytes or HCC cells. The modified nanoparticles can be internalized by malignant cells through receptor-mediated endocytosis, leading to intracellular drug release and greater cytotoxicity. Glycyrrhetinic acid is one of the main active ingredients of traditional Chinese medicine licorice, and its chemical structure belongs to pentacyclic triterpenoid compounds. In the experiment of Tian et al., the uptake rate of GA-modified wogonin liposomes in HepG2 cells was 2.5 times that of L-02 cells, indicating that there are more GA-specific binding sites on HepG2 cells compared with L-02 cells. This result also suggests that GA modification can distinguish liver cancer cells from normal liver cells. Based on previous studies, GA-R has significant advantages in achieving efficient targeting of liver cancer. And a large number of research reports show that GA-modified nanodrug delivery system (GA-TNDDS) has excellent liver cancer targeting. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The results of functional group modification were determined by infrared spectroscopy.

[0030] Figure 2 1 The H NMR spectrum further confirmed the structure of the chitosan derivatives.

[0031] Figure 3 Particle size of quaternary ammonium chitosan.

[0032] Figure 4 Carboxymethyl chitosan particle size.

[0033] Figure 5 Particle size of quaternary ammonium salt-carboxymethyl chitosan combined microspheres. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0035] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0036] Example, preparation of glycyrrhetinic acid@quaternary ammonium salt chitosan / carboxymethyl chitosan nanoparticles of the present invention.

[0037] 1. Synthesis of cross-linking agent carboxymethyl chitosan.

[0038] Carboxymethyl chitosan is a water-soluble chitosan derivative with pH sensitivity and high solubility. The internal environment of tumor cells is acidic. In order to obtain a pH-sensitive nano drug carrier, the present invention selects carboxymethyl chitosan as a crosslinking agent for quaternary ammonium salt chitosan.

[0039] 2. Exploration of optimal reaction conditions and temperature.

[0040] ① Preparation of carboxymethyl chitosan.

[0041] 1.61g (10mmol) of chitosan was dispersed in 16mL of propanol, and 5mL of sodium hydroxide solution (40%, w / v) was added dropwise, and stirred at 50℃ for 1h in a round-bottom flask equipped with a mechanical stirrer. Then 23mL of chloroacetic acid solution (10%, w / v) was added to the above solution, and then divided into groups and stirred at 70℃ for 1h, 2h, and 4h respectively. After precipitation with anhydrous ethanol and washing with 90% ethanol, the solid obtained by suction filtration was freeze-dried to constant weight to obtain carboxymethyl chitosan that met the requirements. The results of the physical and chemical property determination showed that the physical and chemical properties of the carboxymethyl chitosan obtained when stirred for 4h were better.

[0042] ② Preparation of quaternary ammonium salt chitosan.

[0043] 1.61g (10 mmol) chitosan was weighed and dispersed in 60 mL isopropanol and swollen at room temperature for 4h. 12.5mL 2,3-epoxypropyltrimethylammonium chloride aqueous solution (65%, w / v) was slowly added dropwise under reflux conditions, and the mixture was divided into several experimental groups and continued to react for 10h at 60℃, 65℃, 70℃, and 75℃. After the reaction, the pH of the reaction system was adjusted to neutral with 10% hydrochloric acid and then filtered. The obtained solid was soaked and washed with 90% ethanol and precipitated with anhydrous ethanol. The final product was dried in an electric constant temperature blast drying oven at 55℃ to constant weight, and then the physical and chemical properties were determined. The results show that the quaternary ammonium salt chitosan prepared at 60℃ has excellent physical and chemical properties.

[0044] Product recovery rate: Quaternary ammonium salt chitosan 2.9882g8.127g+1.61g =9.737g 30.69% Carboxymethyl chitosan 2.0584g1.61g+2.3g=3.91g 52.64%.

[0045] 3. Preparation of quaternary ammonium salt chitosan nanoparticles.

[0046] Quaternary ammonium chitosan nanoparticles were prepared using an ion gel method.

[0047] Quaternary ammonium salt chitosan is dissolved in an amount of deionized water, and micron-grade filter is used to filter to obtain a filtrate having a particle diameter lower than 0.3 micron. Prepare carboxymethyl chitosan solution, and add dropwise in the above-mentioned filtrate. In room temperature, stir half an hour with the speed of 1000rpm. After reaction finishes, the milky white reaction system is subjected to high-speed centrifugation for 10 minutes, and suction filtration, freeze-drying, obtain quaternary ammonium salt chitosan nano particles.

[0048] Detection of physical and chemical properties of quaternary ammonium chitosan and carboxymethyl chitosan nanoparticles (infrared, Zeta potential, scanning electron microscopy, etc.).

[0049] .

[0050] The changes in particle size and potential demonstrated the success of the synthesized microspheres.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A drug-loaded microsphere, characterized in that: Components include: Quaternary ammonium chitosan and carboxymethyl chitosan.

2. A pharmaceutical composition comprising the drug-loaded microspheres according to claim 1, glycyrrhetinic acid and doxorubicin.

3. Use of the drug-loaded microspheres according to claim 1 and the pharmaceutical composition according to claim 2 in the preparation of tumor-targeting drugs.

4. A method for preparing the drug-loaded microspheres according to claim 1, characterized in that: The following steps are involved: Step 1: Preparation of carboxymethyl chitosan; Step 2: Preparation of quaternary ammonium salt chitosan; Step 3: Preparation of quaternary ammonium salt-carboxymethyl chitosan microspheres.

5. The preparation method according to claim 4, characterized in that: The specific steps of step 1 are: dispersing 1.61 g of chitosan in 16 mL of propanol, dropping 5 mL of sodium hydroxide solution (40%, w / v), stirring at 50° C. for 1 h in a round-bottom flask equipped with a mechanical stirrer; then adding 23 mL of chloroacetic acid solution (10%, w / v) to the above solution, and then grouping and stirring at 70° C. for 4 h; thereafter precipitating with anhydrous ethanol, washing with 90% ethanol, and filtering the obtained solid and freeze-drying it to constant weight to obtain carboxymethyl chitosan that meets the requirements.

6. The preparation method according to claim 4, characterized in that: The specific steps of step 2 are: weighing 1.61g chitosan and dispersing it in 60mL isopropanol, swelling it at room temperature for 4h; slowly dropping 12.5mL 2,3-epoxypropyltrimethylammonium chloride aqueous solution (65%, w / v) under reflux conditions, dividing it into several experimental groups and continuing the reaction for 10h at 60°C, and after the reaction, adjusting the pH of the reaction system to neutral with 10% hydrochloric acid and then filtering it, soaking and washing the obtained solid with 90% ethanol, precipitating it with anhydrous ethanol, and finally drying the product in an electric constant temperature forced air drying oven at 55°C to constant weight to obtain quaternary ammonium salt chitosan that meets the requirements.

7. The preparation method according to claim 4, characterized in that: The specific steps of step 3 are: dissolving the quaternary ammonium salt chitosan obtained in step 2 in an appropriate amount of deionized water, filtering with a micron filter to obtain a filtrate with a particle size of less than 0.3 microns; adding the carboxymethyl chitosan solution obtained in step 1 dropwise to the above filtrate; stirring at room temperature at a speed of 1000 rpm for half an hour; after the reaction is completed, centrifuging the milky white reaction system at high speed for 10 minutes, filtering with suction, and freeze-drying to obtain quaternary ammonium salt-carboxymethyl chitosan drug-loaded microspheres.

8. Use of quaternary ammonium salt-carboxymethyl chitosan drug-loaded microspheres prepared by the preparation method according to claims 4 to 7 in the preparation of tumor targeting drugs.

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