Drug-loaded nano particle loaded with active ingredient of liquorice and preparation method of drug-loaded nano particle

Abstract

The invention relates to a drug-loaded nanoparticle loaded with an active ingredient of liquorice and a preparation method of the drug-loaded nanoparticle. The invention relates to a preparation method of drug-loaded nanoparticles loaded with licorice active ingredients. The preparation method comprises the following steps: (1) preparing mesoporous silica microspheres; (2) uniformly mixing the mesoporous silica microspheres and glycyrrhizic acid or 18beta glycyrrhetinic acid in water, stirring and reacting in a dark place, centrifugally washing, and drying in vacuum to obtain mesoporous silica microspheres loaded with active ingredients of liquorice; and (3) uniformly mixing the mesoporous silica microspheres loaded with the licorice active component, 3-sulfopropyl hexadecyl dimethyl betaine, methanol and tetrahydrofuran, stirring and reacting in a dark place under the protection of nitrogen, heating and concentrating, adding acetonitrile, centrifugally washing, and drying in vacuum to obtain the drug-loaded nanoparticles loaded with the licorice active component. According to the drug-loaded nano particles loaded with the active ingredients of liquorice and the preparation method of the drug-loaded nano particles, a nano drug delivery system with multi-responsiveness and good biocompatibility is prepared.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical nanomaterials, and particularly relates to drug-loaded nanoparticles loaded with licorice active ingredients and a preparation method thereof. Background Art

[0002] Globally, cancer is one of the most serious causes of death, among which lung cancer is one of the most common cancers, and about 25% of cancer deaths are related to lung cancer. Lung cancer is mainly divided into non-small cell lung cancer and small cell lung cancer. About one-third of lung cancer patients in the world live in China. Since the country started lung cancer screening in 2002, the mortality rate of lung cancer in men has gradually decreased, but the mortality rate in women has not decreased significantly. In both men and women, the incidence of lung cancer peaks between the ages of 75 and 80, and tumor metastasis occurs in most lung cancer patients. Malignant tumors often have poor prognosis and poor clinical outcomes, mainly because lung cancer is often diagnosed in the late stage.

[0003] For the treatment of lung cancer, modern medicine and traditional Chinese medicine have some corresponding methods. Chemotherapy, radiotherapy, and targeted therapy are typical modern medical treatments. Traditional Chinese medicine treatments for lung cancer include Chinese medicine, diet therapy, acupuncture, physical exercise and other methods. Both of these two types of treatments have their inevitable defects. For example, chemotherapy and radiotherapy will have great side effects on the patient's body and cause great harm; and Chinese medicine has the problem of complex ingredients and unclear mechanisms. The auxiliary treatment of modern medicine by Chinese medicine can enhance the efficacy and reduce side effects. For the problems of Chinese medicine, nanomaterials can greatly improve its shortcomings. In recent years, drug carrier treatment methods have been widely studied and applied. There are many types of nanocarriers, and the research is very extensive, such as liposomes, mesoporous silica, nanoemulsions, micelles, etc. Although a large number of studies are underway, their effects are still limited. Therefore, how to improve the therapeutic effect of nanocarriers has become a problem that must be solved. There are two main directions for research on this issue. One is to hide oneself as perfectly as possible, avoid tracking and clearance by the immune system, and achieve a long circulation effect in the body as much as possible; the other is to strengthen its targeting ability so that the carrier can load drugs as much as possible to target the tumor for treatment.

[0004] Licorice is one of the commonly used Chinese medicines in clinical practice. It has the functions of tonifying the spleen and replenishing qi, clearing away heat and detoxifying, removing phlegm and relieving cough, relieving acute pain and relieving pain, and harmonizing various medicines. The active ingredients glycyrrhizic acid and glycyrrhetinic acid have a good inhibitory effect on liver cancer, lung cancer, breast cancer and other cancers, and can also be used in combination with chemotherapy drugs to increase the efficacy. However, it also has problems such as low water solubility, low bioavailability, and high toxic side effects.

[0005] Compared with traditional dosage forms, Chinese medicine can be transformed into nanomedicine through nanotechnology, which has the advantages of high bioavailability, targeted distribution of drugs in the body, sustained and controlled release, enhanced efficacy, reduced toxicity, etc., and may solve the problem of difficulty in systemic administration of anticancer Chinese medicine and difficulty in achieving effective anticancer concentration in the local tumor. Nano-Chinese medicine has shown good development momentum and application prospects in improving bioavailability, enhancing efficacy, optimizing traditional dosage forms, and expanding the scope of application of Chinese medicine.

[0006] The main characteristics of mesoporous silica (MSN) are regular porous structure, uniform pore size arrangement, and independent pore sizes. It also has high specific surface area and pore volume as well as high thermal stability. Its pore size can also be controlled from a few nanometers to more than ten nanometers. In addition, mesoporous silica materials contain many silanol groups, so different functional groups can be modified on its surface and mesoporous channels to expand its application range. MSN has many applications in the medical field, including improving drug efficacy, overcoming resistance, gene delivery, photodynamic therapy, targeted delivery system, tumor immunotherapy, auxiliary imaging technology and other medical applications.

[0007] In view of this, the present invention proposes a new MSN nano drug delivery system loaded with licorice active ingredients and a preparation method thereof, which has multi-responsiveness and good biocompatibility and can be used for the adjuvant treatment of lung cancer. Summary of the invention

[0008] The invention aims to provide a method for preparing drug-loaded nanoparticles loaded with licorice active ingredients. The preparation method uses mesoporous silica microspheres as carriers, loads glycyrrhizic acid and 18β-glycyrrhetinic acid on the carriers respectively, and uses 3-sulfopropyl hexadecyl dimethyl betaine as a surface modification to prepare nano Chinese medicine by layer-by-layer structural modification.

[0009] In order to achieve the above purpose, the technical solution adopted is:

[0010] A method for preparing drug-loaded nanoparticles loaded with licorice active ingredients comprises the following steps:

[0011] (1) Preparation of mesoporous silica microspheres;

[0012] (2) Loading the active ingredient of licorice: After the mesoporous silica microspheres and glycyrrhizic acid or 18β-glycyrrhetinic acid are uniformly mixed in water, stirred in the dark for 22-26 hours, solid-liquid separation, centrifugal washing, and vacuum drying are performed to obtain mesoporous silica microspheres loaded with the active ingredient of licorice;

[0013] (3) After the mesoporous silica microspheres loaded with the active ingredient of licorice, 3-sulfopropyl hexadecyl dimethyl betaine, methanol and tetrahydrofuran are uniformly mixed, the mixture is stirred and reacted for 22-26 hours at 45-55° C. in the dark under nitrogen protection, and then heated and concentrated. After acetonitrile is added to promote precipitation, the mixture is solid-liquid separated, centrifuged and washed, and vacuum dried to obtain the drug-loaded nanoparticles loaded with the active ingredient of licorice.

[0014] Furthermore, the process of preparing mesoporous silica microspheres in step (1) is as follows: at a temperature of 75-85° C., triethylamine is added dropwise to water and stirred evenly, CTAB and salicylic acid are added and the stirring reaction is continued for 0.8-1.2 hours, and then tetraethyl silicate is added to react for 1.5-2.5 hours, cooled to room temperature, solid-liquid separation, centrifugal washing, collecting precipitates, vacuum drying, calcining, and collecting products.

[0015] Furthermore, in the step (2), the mass ratio of the mesoporous silica microsphere solution to glycyrrhizic acid is 2:1.3-1.7;

[0016] The mass ratio of the mesoporous silica microsphere solution to 18β-glycyrrhetinic acid is 2:1.3-1.7.

[0017] Furthermore, in the step (2), the mass ratio of the mesoporous silica microsphere solution to glycyrrhizic acid is 2:1.4;

[0018] The mass ratio of the mesoporous silica microsphere solution to 18β-glycyrrhetinic acid is 2:1.3.

[0019] Furthermore, in the step (3), the mass ratio of the mesoporous silica microspheres loaded with the licorice active ingredient to 3-sulfopropyl hexadecyl dimethyl betaine is 1:0.8-1.2;

[0020] The volume ratio of methanol to tetrahydrofuran is 1:1.5-2.5.

[0021] Furthermore, in the step (3), the mass ratio of the mesoporous silica microspheres loaded with licorice active ingredients to 3-sulfopropyl hexadecyl dimethyl betaine is 1:1;

[0022] The volume ratio of methanol to tetrahydrofuran is 1:2.

[0023] Furthermore, in the step (2), water and anhydrous ethanol are used as detergents in the centrifugal washing;

[0024] In the step (3), methanol is used as a washing agent in the centrifugal washing.

[0025] Furthermore, in the step (3), the solution is concentrated to one tenth of the original volume.

[0026] Another object of the present invention is to provide a drug-loaded nanoparticle loaded with licorice active ingredients, which is prepared by the above-mentioned preparation method, has multi-responsiveness and good biocompatibility, and can be used for the adjuvant treatment of lung cancer.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention uses typical nano-carrier mesoporous silica as a carrier, loads glycyrrhizic acid and 18β-glycyrrhetinic acid, which are active molecules of traditional Chinese medicine extracted from licorice and have anti-tumor, anti-inflammatory and analgesic effects, and finally uses zwitterionic 3-sulfopropyl hexadecyl dimethyl betaine, which performs well in invisible nanoparticles for drug delivery, as a surface modification material to prepare nano drug delivery systems SB@MSN-GA and SB@MSN-18β-Gly with multi-responsiveness and good biocompatibility for adjuvant treatment of lung cancer. The technical solution of the present invention has the following advantages:

[0029] 1. In the technical scheme of the present invention, licorice is a typical Chinese herbal medicine that can enter the lungs. Its main active ingredients are glycyrrhizic acid and 18β-glycyrrhetinic acid. Many studies before the present invention have confirmed that glycyrrhizic acid and 18β-glycyrrhetinic acid have good effects on anti-tumor, and have anti-inflammatory and antibacterial effects. The ingredients of Chinese herbal medicine are complex, and it is difficult to aggregate the drugs in a specific position, resulting in poor therapeutic effects. In the technical scheme of the present invention, glycyrrhizic acid molecules and 18β-glycyrrhetinic acid are loaded onto mesoporous silica nanocarriers, and modified on their surfaces so that they can aggregate and release the drugs according to the specific acidic environment of the tumor. In addition, most of the previous studies used a single component combined with nanotechnology for treatment, but in the present invention, it is not a single Chinese medicine active molecule that is nanosized for treatment, but the two main active ingredients in licorice are used in combination.

[0030] 2. In the technical solution of the present invention, the zwitterionic 3-sulfopropyl hexadecyl dimethyl betaine (SB) polymer selected as the outer layer modification material performs excellently in constructing stealth nanoparticles for drug delivery, has a certain sensitivity to pH and temperature, has low cytotoxicity, can effectively prolong the circulation time in the body, and is beneficial to the long circulation and targeted aggregation of nanocarriers in the body.

[0031] 3. In the technical solution of the present invention, mesoporous silica is used as a carrier. Mesoporous silica can be excreted from the body through the kidneys or liver within 4 weeks. Glycyrrhizic acid molecules and 18β-glycyrrhetinic acid have anti-inflammatory, antibacterial, and anti-tumor effects. 3-sulfopropylhexadecyl dimethyl betaine is stable to acid and alkali, non-toxic, and has a certain bactericidal property, so that the prepared drug-loaded hybrid nanoparticles have the characteristics of good biocompatibility, high safety, and no toxic side effects.

[0032] 4. The technical solution of the present invention has a simple and rapid preparation process, is highly universal, and is easy to scale up for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 SEM images of MSN, SB@MSN-GA, and SB@MSN-18β-Gly;

[0034] Figure 2 TEM images of MSN, SB@MSN-GA, and SB@MSN-18β-Gly;

[0035] Figure 3 BET and BJH diagrams of MSN, SB@MSN-GA, and SB@MSN-18β-Gly;

[0036] Figure 4 Particle size and Zeta potential diagram of MSN, SB@MSN-GA, and SB@MSN-18β-Gly of the present invention;

[0037] Figure 5 Thermogravimetric analysis diagrams of MSN, MSN-GA, MSN-18β-Gly, SB@MSN-GA, and SB@MSN-18β-Gly;

[0038] Figure 6 a, b are infrared spectra of MSN, GA, SB, MSN-GA, SB@MSN-GA, 18β-Gly, MSN-18β-Gly, and SB@MSN-18β-Gly, respectively;

[0039] Figure 7 a, b are the UV spectra and standard curves of GA of the present invention; c, d are the UV spectra and standard curves of 18β-Gly of the present invention;

[0040] Figure 8 The drug loading diagram of SB@MSN-GA and SB@MSN-18β-Gly of the present invention;

[0041] Fig. 9 a shows the drug release of SB@MSN-GA at different temperatures and pH values, and b shows the drug release of SB@MSN-18β-Gly at different temperatures and pH values;

[0042] Fig.10 It is the hemolytic performance diagram of MSN, MSN-GA, MSN-18β-Gly, SB@MSN-GA, and SB@MSN-18β-Gly;

[0043] Fig.11a and b are the cytotoxicity graphs of MSN, SB@MSN-GA, SB@MSN-18β-Gly, and SB@MSN-GA+SB@MSN-18β-Gly. DETAILED DESCRIPTION

[0044] In order to further explain the drug-loaded nanoparticles of a kind of loaded licorice active ingredient of the present invention and the preparation method thereof, and to achieve the expected invention purpose, the following is combined with the preferred embodiments, and the drug-loaded nanoparticles of a kind of loaded licorice active ingredient and the preparation method thereof, its specific implementation mode, structure, characteristics and efficacy proposed according to the present invention are described in detail as follows. In the following description, different "one embodiment" or "embodiment" do not necessarily refer to the same embodiment. In addition, the specific features, structures or characteristics in one or more embodiments can be combined in any suitable form.

[0045] The following will further introduce in detail the drug-loaded nanoparticles loaded with licorice active ingredients and the preparation method thereof in conjunction with specific embodiments of the present invention:

[0046] Conventional surgical methods have a great impact on the body. Chemotherapy and radiotherapy drugs not only have great toxic side effects but also cannot prolong survival or improve the quality of life. Most targeted drugs do not have satisfactory clinical effects and may even be off-target. Traditional Chinese medicine has limited therapeutic effects on cancer, and has complex ingredients and unclear mechanisms of action. In response to the above problems, the present invention provides a drug-loaded nanoparticle loaded with licorice active ingredients and a method for preparing the same. It is a nano-traditional Chinese medicine loaded with a nanocarrier and modified with an active ingredient of a traditional Chinese medicine and a method for preparing the same, which can effectively solve the problems of poor targeting ability of existing traditional Chinese medicines, complex ingredients, and great toxic side effects of conventional methods. The technical solution of the present invention is:

[0047] A method for preparing drug-loaded nanoparticles loaded with licorice active ingredients comprises the following steps:

[0048] (1) Preparation of mesoporous silica microspheres;

[0049] (2) Loading the active ingredient of licorice: After the mesoporous silica microspheres and glycyrrhizic acid or 18β-glycyrrhetinic acid are uniformly mixed in water, stirred in the dark for 22-26 hours, solid-liquid separation, centrifugal washing, and vacuum drying are performed to obtain mesoporous silica microspheres loaded with the active ingredient of licorice;

[0050] (3) After the mesoporous silica microspheres loaded with the active ingredient of licorice, 3-sulfopropyl hexadecyl dimethyl betaine, methanol and tetrahydrofuran are uniformly mixed, the mixture is stirred and reacted for 22-26 hours at 45-55° C. in the dark under nitrogen protection, and then heated and concentrated. After acetonitrile is added to promote precipitation, the mixture is solid-liquid separated, centrifuged and washed, and vacuum dried to obtain the drug-loaded nanoparticles loaded with the active ingredient of licorice.

[0051] In the above technical scheme, the nano drug delivery system of the present invention is a nano drug delivery system using mesoporous silica microspheres as carriers, glycyrrhizic acid and 18β-glycyrrhetinic acid are loaded on the carriers respectively, and the surface is modified with 3-sulfopropyl hexadecyl dimethyl betaine.

[0052] Glycyrrhizic acid and glycyrrhetinic acid exert their anti-tumor effects mainly through regulating cell cycle and apoptosis signals, and inhibit the proliferation and invasion of lung cancer cells by downregulating the activity of thromboxane synthase pathway. Zwitterionic 3-sulfopropyl hexadecyl dimethyl betaine (SB) polymer performs well in constructing invisible nanoparticles for drug delivery and has low cytotoxicity. It can not only effectively prolong the circulation time in the body, but also release more drugs in an acidic environment.

[0053] Preferably, the process of preparing mesoporous silica microspheres in step (1) is as follows: at a temperature of 75-85° C., triethylamine is added dropwise to water and stirred evenly, CTAB and salicylic acid are added and the stirring reaction is continued for 0.8-1.2 h, tetraethyl silicate is added and the reaction is continued for 1.5-2.5 h, the mixture is cooled to room temperature, solid-liquid separation is performed, centrifugal washing is performed, the precipitate is collected, vacuum drying is performed, calcination is performed, and the product is collected.

[0054] Preferably, in the step (2), the mass ratio of the mesoporous silica microsphere solution to glycyrrhizic acid is 2:1.3-1.7;

[0055] The mass ratio of the mesoporous silica microsphere solution to 18β-glycyrrhetinic acid is 2:1.3-1.7.

[0056] Further preferably, in the step (2), the mass ratio of the mesoporous silica microsphere solution to glycyrrhizic acid is 2:1.4;

[0057] The mass ratio of the mesoporous silica microsphere solution to 18β-glycyrrhetinic acid is 2:1.3.

[0058] Preferably, in the step (3), the mass ratio of the mesoporous silica microspheres loaded with licorice active ingredients to 3-sulfopropyl hexadecyl dimethyl betaine is 1:0.8-1.2;

[0059] The volume ratio of methanol to tetrahydrofuran is 1:1.5-2.5.

[0060] Further preferably, in the step (3), the mass ratio of the mesoporous silica microspheres loaded with licorice active ingredients to 3-sulfopropyl hexadecyl dimethyl betaine is 1:1;

[0061] The volume ratio of methanol to tetrahydrofuran is 1:2.

[0062] Preferably, in the step (2), water and anhydrous ethanol are used as detergents for centrifugal washing;

[0063] In the step (3), methanol is used as a washing agent in the centrifugal washing.

[0064] Preferably, in step (3), the solution is concentrated to one tenth of the original volume.

[0065] Example 1.

[0066] The specific steps are as follows:

[0067] (1) Preparation of mesoporous silica microspheres

[0068] Heat 50 mL of ultrapure water to 80° C. in an oil bath, slowly drop 0.068 g of triethylamine, and continue stirring for 30 min. Weigh 760 mg of CTAB and 336 mg of sodium salicylate and continue stirring at 80° C. for 1 h.

[0069] Then, 8 mL of tetraethyl silicate was slowly added dropwise to the resulting mixed solution and the reaction was continued for 1.5 h.

[0070] The reaction mixture was cooled to room temperature and centrifuged at 6000 r / min for 15 min to obtain a precipitate. The precipitate was washed by centrifugation, and washed three times with ultrapure water and anhydrous ethanol, each time at 10000 r / min for 10 min. The washed precipitate was collected and vacuum dried at 50°C overnight.

[0071] The dried product was placed in a muffle furnace for calcination. The settings of the muffle furnace were as follows: a starting temperature of 30°C, an end temperature of 550°C, a heating rate of 2°C / min, a heating time of 260min, a holding temperature of 550°C, a holding time of 240min, and cooled to room temperature to obtain the product MSN.

[0072] (2) Loading of glycyrrhizic acid and 18β-glycyrrhetinic acid

[0073] Glycyrrhizic acid and 18β-glycyrrhetinic acid were respectively prepared into 0.7 mg / mL and 0.65 mg / mL solutions with ultrapure water, and mesoporous silica microspheres were prepared into 1 mg / mL solution with ultrapure water, and ultrasonicated for 20 minutes to make them dispersed evenly, and then the mesoporous silica microsphere solution was mixed with glycyrrhizic acid solution and 18β-glycyrrhetinic acid solution, and the volume ratio of the mesoporous silica microsphere solution to the glycyrrhizic acid solution and the 18β-glycyrrhetinic acid solution was 1:1, and they were mixed and prepared into two mixed solutions. The obtained mixed solution was ultrasonicated for 20 minutes to make it evenly mixed, stirred at room temperature at a speed of 700 r / min in the dark for 24 hours, and centrifuged at 10000 r / min for 10 minutes to obtain a precipitate. The precipitate was washed by centrifugation, and washed twice with ultrapure water and anhydrous ethanol, each time at a speed of 10000r / min for 10min. The washed precipitate was collected and vacuum dried at 40°C overnight to obtain the products MSN-GA and MSN-18β-Gly.

[0074] (3) Coated with zwitterionic membrane

[0075] Weigh 15 mg of MSN-GA and 15 mg of MSN-18β-Gly into two round-bottom flasks, add 15 mg of 3-sulfopropyl hexadecyl dimethyl betaine to each flask, add 25 mL of methanol and 50 mL of tetrahydrofuran as solvents to each flask, introduce nitrogen to exhaust the air, and then ultrasonicate for 20 min to make the dispersion uniform. Stir in a 50°C oil bath at 700 r / min under nitrogen protection for 24 h in the dark.

[0076] Then, the solution was concentrated to one tenth of its original volume by rotary evaporation.

[0077] 20 mL of acetonitrile was added to promote precipitation, and the mixture was centrifuged at 10,000 r / min for 12 min to obtain a precipitate. The precipitate was centrifuged and washed four times with methanol, each time at 10,000 r / min for 12 min. The washed precipitate was collected and dried overnight at 40 °C under vacuum conditions to obtain the products SB@MSN-GA and SB@MSN-18β-Gly, respectively.

[0078] Example 2.

[0079] The specific steps are as follows:

[0080] (1) Preparation of mesoporous silica microspheres

[0081] Heat 50 mL of ultrapure water to 75° C. in an oil bath, slowly drop 0.068 g of triethylamine, and continue stirring for 30 min. Weigh 760 mg of CTAB and 336 mg of sodium salicylate and continue stirring at 75° C. for 1.2 h.

[0082] Then, 8 mL of tetraethyl silicate was slowly added dropwise to the resulting mixed solution and the reaction was continued for 2 h.

[0083] The reaction mixture was cooled to room temperature and centrifuged at 6000 r / min for 20 min to obtain a precipitate. The precipitate was washed by centrifugation, and washed three times with ultrapure water and anhydrous ethanol, each time at 10000 r / min for 15 min. The washed precipitate was collected and vacuum dried at 50°C overnight.

[0084] The dried product was placed in a muffle furnace for calcination. The settings of the muffle furnace were as follows: a starting temperature of 30°C, an end temperature of 550°C, a heating rate of 2°C / min, a heating time of 260min, a holding temperature of 550°C, a holding time of 240min, and cooled to room temperature to obtain the product MSN.

[0085] (2) Loading of glycyrrhizic acid and 18β-glycyrrhetinic acid

[0086] The mesoporous silica microspheres prepared in step (1) were prepared into two 2 mg / mL solutions with ultrapure water, and glycyrrhizic acid and 18β-glycyrrhetinic acid were added respectively. The mass ratios of mesoporous silica microspheres to glycyrrhizic acid and 18β-glycyrrhetinic acid were 2:1.7 and 2:1.7 respectively. Ultrasonication was performed for 40 minutes to make the mixture uniform.

[0087] Then, the mixture was stirred at 800 r / min in the dark for 22 h at room temperature, and centrifuged at 10,000 r / min for 12 min to obtain a precipitate. The precipitate was washed by centrifugation, and washed twice with ultrapure water and anhydrous ethanol, each time at 10,000 r / min for 12 min. The washed precipitate was collected and vacuum dried at 40°C overnight to obtain the products MSN-GA and MSN-18β-Gly.

[0088] (3) Coated with zwitterionic membrane

[0089] Weigh 15 mg of MSN-GA and 15 mg of MSN-18β-Gly into two round-bottom flasks, add 12 mg of 3-sulfopropyl hexadecyl dimethyl betaine to each flask, add 30 mL of methanol and 45 mL of tetrahydrofuran as solvents to each flask, introduce nitrogen to exhaust the air, and then ultrasonicate for 20 min to make the dispersion uniform. Stir in a 45°C oil bath at 700 r / min under nitrogen protection for 26 h in the dark.

[0090] Then, the solution was concentrated to one tenth of its original volume by rotary evaporation.

[0091] 20 mL of acetonitrile was added to promote precipitation, and the mixture was centrifuged at 10,000 r / min for 15 min to obtain a precipitate. The precipitate was washed by centrifugation and washed four times with methanol, each time at 10,000 r / min for 15 min. The washed precipitate was collected and dried overnight under vacuum at 40°C to obtain the products SB@MSN-GA and SB@MSN-18β-Gly, respectively.

[0092] Example 3.

[0093] The specific steps are as follows:

[0094] (1) Preparation of mesoporous silica microspheres

[0095] Heat 50 mL of ultrapure water to 85° C. in an oil bath, slowly drop 0.068 g of triethylamine, and continue stirring for 30 min. Weigh 760 mg of CTAB and 336 mg of sodium salicylate, and continue stirring at 85° C. for 0.8 h.

[0096] Then, 8 mL of tetraethyl silicate was slowly added dropwise to the resulting mixed solution and the reaction was continued for 2.5 h.

[0097] The reaction mixture was cooled to room temperature and centrifuged at 6000 r / min for 15 min to obtain a precipitate. The precipitate was washed by centrifugation, and washed 4 times with ultrapure water and anhydrous ethanol, each time at 10000 r / min for 15 min. The washed precipitate was collected and vacuum dried at 50°C overnight.

[0098] The dried product was placed in a muffle furnace for calcination. The settings of the muffle furnace were as follows: a starting temperature of 30°C, an end temperature of 550°C, a heating rate of 2°C / min, a heating time of 260min, a holding temperature of 550°C, a holding time of 240min, and cooled to room temperature to obtain the product MSN.

[0099] (2) Loading of glycyrrhizic acid and 18β-glycyrrhetinic acid

[0100] The mesoporous silica microspheres prepared in step (1) were prepared into two 2 mg / mL solutions with ultrapure water, and glycyrrhizic acid and 18β-glycyrrhetinic acid were added respectively. The mass ratios of mesoporous silica microspheres to glycyrrhizic acid and 18β-glycyrrhetinic acid were 2:1.3 and 2:1.3 respectively. Ultrasonication was performed for 40 minutes to make the mixture evenly mixed.

[0101] Then, the mixture was stirred at 600 r / min in the dark for 26 h at room temperature, and centrifuged at 10,000 r / min for 15 min to obtain a precipitate. The precipitate was washed by centrifugation, and washed twice with ultrapure water and anhydrous ethanol, each time at 10,000 r / min for 14 min. The washed precipitate was collected and vacuum dried at 40°C overnight to obtain the products MSN-GA and MSN-18β-Gly.

[0102] (3) Coated with zwitterionic membrane

[0103] Weigh 15 mg of MSN-GA and 15 mg of MSN-18β-Gly into two round-bottom flasks, add 18 mg of 3-sulfopropyl hexadecyl dimethyl betaine to each flask, add 30 mL of methanol and 75 mL of tetrahydrofuran as solvents to each flask, introduce nitrogen to exhaust the air, and then ultrasonicate for 20 min to make the dispersion uniform. Stir in a 55°C oil bath at 700 r / min under nitrogen protection for 22 h in the dark.

[0104] Then, the solution was concentrated to one tenth of its original volume by rotary evaporation.

[0105] 25 mL of acetonitrile was added to promote precipitation, and the mixture was centrifuged at 10,000 r / min for 15 min to obtain a precipitate. The precipitate was washed by centrifugation and washed with methanol 4 times, each time at 10,000 r / min for 15 min. The washed precipitate was collected and dried overnight under vacuum at 40°C to obtain the products SB@MSN-GA and SB@MSN-18β-Gly, respectively.

[0106] Example 4: SEM test

[0107] Method: Take a small amount of MSN, SB@MSN-GA, and SB@MSN-18β-Gly prepared in Example 1, dissolve them in ethanol and disperse them ultrasonically, drop the samples on the silicon wafer with a 200μl pipette, attach the silicon wafers to the conductive glue to prepare samples and mark them, where sample No. 1 is MSN, sample No. 2 is SB@MSN-GA, and sample No. 3 is SB@MSN-18β-Gly. The conductivity of the material is enhanced by 30s gold plating to facilitate obtaining better morphological features in SEM, and then put it into the SEM observation room for observation. After finding the sample at low magnification, the appropriate area can be selected for observation and photography at a magnification.

[0108] SEM test results: The final results are as follows Figure 1 shown. Figure 1a is a scanning electron microscope (SEM) image of MSN 1 μm, in which MSN presents a spherical structure; 1b is a scanning electron microscope (SEM) image of SB@MSN-GA 1 μm, and 1c is a scanning electron microscope (SEM) image of SB@MSN-18β-Gly 1 μm. The surface pores are covered, but the spherical structure is still maintained.

[0109] Example 5: TEM test

[0110] Method: Take a small amount of MSN, SB@MSN-GA, and SB@MSN-18β-Gly prepared in Example 1, dissolve them in ethanol and ultrasonically disperse them, and drop the samples on the copper mesh with a 10μl pipette, where sample No. 1 is MSN, sample No. 2 is SB@MSN-GA, and sample No. 3 is SB@MSN-18β-Gly. Use a TEM transmission electron microscope for scanning observation, find a suitable sample at a low magnification, and observe and take pictures at a high magnification.

[0111] TEM test results: The final results are as follows Figure 2 shown. Figure 2 a is the transmission electron microscope (TEM) image of MSN 100nm, in which its dendritic structure can be clearly seen; 2b is the transmission electron microscope (TEM) image of SB@MSN-GA 100nm, and 2c is the transmission electron microscope (TEM) image of SB@MSN-18β-Gly 100nm. The dendritic channels are covered, indicating that SB is successfully coated on the surface of the silicon sphere.

[0112] Example 6: Pore Size Analysis

[0113] Method: The pore size analysis was performed on the MSN, SB@MSN-GA and SB@MSN-18β-Gly prepared in Example 1.

[0114] The nitrogen adsorption-desorption isotherms were measured by an A500 instrument (Mike, USA), and the specific surface area was calculated from the adsorption data in the low pressure range using the Brunauer-Emmett-Teller (BET) model, and the pore size was determined according to the Barrett Joyner-Halenda (BJH) method.

[0115] Results: Figure 3 As shown in Figures a, b, and c, N 2 The adsorption-desorption curves (the inset is the corresponding pore size distribution curve) show that both before and after modification present a typical Langmuir IV type hysteresis loop, indicating that the material has a mesoporous structure. The changes in pore size, specific surface area, and pore volume before and after modification also indicate that SB@MSN-GA and SB@MSN-18β-Gly were successfully prepared.

[0116] Example 7: Particle size and Zeta potential analysis

[0117] Methods: The particle size and Zeta potential of MSN, SB@MSN-GA and SB@MSN-18β-Gly prepared in Example 1 were measured by a British Zetasizer Nano ZS90 instrument (Malvern, UK).

[0118] Results: Figure 4 As shown, the absolute values ​​of particle size and potential after modification increased, the stability of nanoparticles increased, and the changes in particle size and Zeta potential indirectly reflected the successful preparation of the nano-drug delivery system.

[0119] Example 8: Thermogravimetric curve drawing

[0120] Methods: The MSN, MSN-GA, MSN-18β-Gly, SB@MSN-GA and SB@MSN-18β-Gly prepared in Example 1 were heated in the range of 30-800°C under N 2 Thermogravimetric analysis (TGA) was performed at a rate of 10 K / min under atmosphere.

[0121] Results: Figure 5 As shown, the weight loss rate increased significantly after modification, proving the successful loading of the drug and the successful coating of the zwitterionic membrane.

[0122] Example 8: Infrared spectrum analysis

[0123] Methods: The MSN, GA, SB, MSN-GA, SB@MSN-GA, 18β-Gly, MSN-18β-Gly, and SB@MSN-18β-Gly in Example 1 were tested for Fourier transform infrared spectroscopy (FT-IR) on a Nicolet iS50 spectrophotometer (ThermoFisherScientific, USA).

[0124] Results: Figure 6 As shown, the infrared spectrum shows the interconnection between the functional groups in each modification process of MSN, proving that the desired drug carrier is synthesized.

[0125] Example 9: Preparation of GA and 18β-Gly standard curves

[0126] (1) Method: 10 mg of GA was accurately weighed and prepared into a 0.1 mg / mL stock solution for later use. The solution was then diluted into a series of solutions with a concentration gradient: 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, and 0.06 mg / mL. The absorbance of different drug concentrations at 252 nm was measured by UV-Vis, and a linear fit was performed to determine the GA standard curve equation.

[0127] Results: Figure 7 As shown in a, GA has the maximum absorbance at an absorption wavelength of 252 nm. Figure 7 As shown in b, the equation of the GA standard curve is C = 8.6521A + 0.0066 R 2 =0.9994.

[0128] (2) Method: 100 mg of 18β-Gly was accurately weighed and prepared into a 1 mg / mL stock solution for later use, which was then diluted into a series of solutions with a concentration gradient: 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, and 0.8 mg / mL. The absorbance of different drug concentrations at 250 nm was measured by UV-Vis, and a linear fit was performed to determine the equation of the 18β-Gly standard curve.

[0129] Results: Figure 7 As shown in Figure c, 18β-Gly has the maximum absorbance at an absorption wavelength of 250nm. Figure 7 d, the standard curve equation of 18β-Gly is C = 1.7097A-0.0021R 2 =0.9996.

[0130] Example 10: Analysis of drug loading performance of MSN

[0131] Methods: The following GA solutions with different concentration gradients were prepared: 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.55 mg / mL, 0.6 mg / mL, 0.65 mg / mL, 0.7 mg / mL, 0.75 mg / mL, 0.8 mg / mL, and 0.85 mg / mL. Then 13 portions of 1 mg / mL mesoporous silica microsphere carriers were prepared and added to the above solution. The solution was stirred in the dark for 24 h. The hydroxyl groups on GA and the hydroxyl groups on MSN were connected by hydrogen bonds, thereby loading the drug onto the carrier. The solution was centrifuged at 10,000 rpm for 10 min to achieve solid-liquid separation, and a precipitate and a supernatant were obtained.

[0132] The obtained precipitate was washed by centrifugation, and ultrapure water and anhydrous ethanol were used as detergents, and each was washed 3 times, and each centrifugation was centrifuged at a speed of 10000r / min for 10min. Ultrapure water and anhydrous ethanol were used to wash away free drugs and surface adsorbed drugs to obtain drug-loaded nanoparticles MSN@GA. The supernatant was collected and the drug absorbance at 252nm was measured. The drug loading rate was then calculated according to the formula:

[0133]

[0134] Where W z is the total mass of GA added; W z is the mass of GA in the supernatant; W t is the total mass of the carrier and the drug after drug loading.

[0135] The performance of MSN loaded with 18β-Gly was determined using the same method as above.

[0136] Results: Figure 8 As shown, the drug loading rate was the highest when the GA concentration was 0.7 mg / mL, which was 43.61%. The drug loading rate was the highest when the 18β-Gly concentration was 0.65 mg / mL, which was 36.56%.

[0137] Example 11: Analysis of drug release properties of SB@MSN-GA and SB@MSN-18β-Gly

[0138] Method: The drug carrier SB@MSN-GA prepared in Example 1 was dispersed in 2mL PBS solution (pH=7.4), added to a dialysis bag after ultrasonic treatment, and then transferred to 40mL PBS buffer solution containing different components. The pH variables were controlled at 7.4, 6.5 and 5.0 to study the effect of pH on the sustained release effect of the drug, and the temperature variables were controlled at 25℃, 37℃ and 43℃ to study the effect of temperature on the sustained release effect of the drug. It was placed in a constant temperature shaking incubator and shaken at a rate of 100rpm. 3mL of the release solution was taken at 0.5h, 1h, 2h, 3h, 4h, 6h, 8h, 10h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, and 96h, and an equal volume and concentration of sustained release solution was added. The absorbance was measured by UV-Vis, and the cumulative drug release rate of the drug carrier was calculated according to the following formula:

[0139] Cumulative drug release (%) = (drug mass outside the dialysis bag / drug mass in the carrier) × 100

[0140] The obtained data was imported into Origin for processing to obtain the results.

[0141] The drug release performance analysis of SB@MSN-18β-Gly was the same as the above method.

[0142] Results: The drug release performance of SB@MSN-GA was as follows Fig. 9 As shown in a, with the decrease of pH and the increase of temperature, the drug release rate gradually increased from the initial 7.15% to the final 21.24%.

[0143] The drug release performance of SB@MSN-18β-Gly is as follows Fig. 9 As shown in (b), with the decrease of pH and the increase of temperature, the drug release rate gradually increased from the initial 26.01% to the final 75.03%.

[0144] Example 12: Hemolytic Test of Nanoparticles

[0145] Methods: Hemolysis experiments were performed on the synthesized nanoparticles (MSN, MSN-GA, MSN-18β-Gly, SB@MSN-GA, SB@MSN-18β-Gly) using defibrinated sheep blood to evaluate the biocompatibility of the nanoparticles.

[0146] Results: Fig.10 As shown, the hemolysis rate of the modified mesoporous silica is less than 5%, which has good biocompatibility.

[0147] Example 13: Cytotoxicity test of nanocarriers

[0148] Methods: A549 cells were seeded in a 96-well plate at 5000 cells / well and incubated with different concentrations of MSN, SB@MSN-GA, SB@MSN-18β-Gly and SB@MSN-GA+SB@MSN-18β-Gly prepared in Example 1 for 24 h. Cell viability was detected by CCK-8 method. The toxicity of the material to the cells after treatment was determined by comparing the cell viability to determine the optimal drug carrier concentration, and the cell survival rate was calculated according to the formula:

[0149] Cell survival rate (%) = [(OD 1 -OD 0 ) / (OD 2 -OD 0 )]×100

[0150] Where: OD 1 is the absorbance of the experimental group; OD 2 is the absorbance of the control group; OD 0 is the absorbance of the blank group.

[0151] Drug group: cells + nanoparticles + culture medium + CCK-8

[0152] Control group: cells + culture medium + CCK-8

[0153] Blank group: culture medium + CCK-8

[0154] Results: Fig.11 As shown in Figure a, MSN exhibits good biocompatibility. In Figure b, SB@MSN-GA+SB@MSN-18β-Gly has a stronger killing effect on A549 cells than SB@MSN-GA and SB@MSN-18β-Gly, showing good tumor killing effect.

[0155] In combination with the embodiments, it can be seen that in the process of loading glycyrrhizic acid and 18β-glycyrrhetinic acid by the nanoparticles prepared by the present invention, the abundant silicon hydroxyl groups on the surface of MSN directly form intermolecular hydrogen bonds with the hydroxyl and carboxyl groups on the drug molecules, which reduces the steps of MSN group modification, greatly reduces the reaction cost, shortens the preparation cycle, simplifies the preparation process, and can reach 43.61% and 36.56% loading rates for glycyrrhizic acid and 18β-glycyrrhetinic acid, respectively. It can be seen from TEM and SEM that the prepared nanomaterial has strong encapsulation. Under the conditions of pH = 6.5 and 42°C, the release amount of 18β-Gly can reach more than 75%, which is higher than that of traditional nanocarriers. It releases more drugs under specific conditions, is more suitable for playing a role in the tumor microenvironment, and avoids premature leakage in normal tissues to cause damage. In the present invention, the entire preparation process is simple and fast, with high universality, and is easy to industrially scale up production.

[0156] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers used in the present embodiment to express the dosage, percentage, and other numerical values ​​should be understood as modified by the word "about" in all cases. Therefore, unless otherwise specified, the numerical parameters listed in the specification and the attached claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained. Each numerical parameter should at least be regarded as obtained according to the reported significant figures and by conventional rounding methods.

[0157] The above is only a preferred embodiment of the embodiment of the present invention, and does not impose any form of limitation on the embodiment of the present invention. Any simple modification, equivalent changes and modifications made to the above embodiment based on the technical essence of the embodiment of the present invention are still within the scope of the technical solution of the embodiment of the present invention.

Claims

1. A method for preparing drug-loaded nanoparticles loaded with licorice active ingredients, characterized in that: The following steps are involved: (1) Preparation of mesoporous silica microspheres: (2) Loading the active ingredient of licorice: After the mesoporous silica microspheres and glycyrrhizic acid or 18β-glycyrrhetinic acid are uniformly mixed in water, stirred in the dark for 22-26 hours, solid-liquid separation, centrifugal washing, and vacuum drying are performed to obtain mesoporous silica microspheres loaded with the active ingredient of licorice; (3) After the mesoporous silica microspheres loaded with the active ingredient of licorice, 3-sulfopropyl hexadecyl dimethyl betaine, methanol and tetrahydrofuran are uniformly mixed, the mixture is stirred and reacted for 22-26 hours at 45-55° C. in the dark under nitrogen protection, and then heated and concentrated. After acetonitrile is added to promote precipitation, the mixture is solid-liquid separated, centrifuged and washed, and vacuum dried to obtain the drug-loaded nanoparticles loaded with the active ingredient of licorice.

2. The preparation method according to claim 1, characterized in that: The process of preparing mesoporous silica microspheres in step (1) is as follows: at a temperature of 75-85° C., triethylamine is added dropwise to water and stirred evenly, CTAB and salicylic acid are added and the stirring reaction is continued for 0.8-1.2 hours, tetraethyl silicate is added and the reaction is continued for 1.5-2.5 hours, the mixture is cooled to room temperature, solid-liquid separation is performed, centrifugal washing is performed, the precipitate is collected, vacuum drying is performed, calcination is performed, and the product is collected.

3. The preparation method according to claim 1, characterized in that: In the step (2), the mass ratio of the mesoporous silica microsphere solution to glycyrrhizic acid is 2:1.3-1.7; The mass ratio of the mesoporous silica microsphere solution to 18β-glycyrrhetinic acid is 2:1.3-1.

7.

4. The preparation method according to claim 3, characterized in that: In the step (2), the mass ratio of the mesoporous silica microsphere solution to glycyrrhizic acid is 2:1.4; The mass ratio of the mesoporous silica microsphere solution to 18β-glycyrrhetinic acid is 2:1.

3.

5. The preparation method according to claim 1, characterized in that: In the step (3), the mass ratio of the mesoporous silica microspheres loaded with licorice active ingredients to 3-sulfopropyl hexadecyl dimethyl betaine is 1:0.8-1.2; The volume ratio of methanol to tetrahydrofuran is 1:1.5-2.

5.

6. The preparation method according to claim 5, characterized in that: In the step (3), the mass ratio of the mesoporous silica microspheres loaded with licorice active ingredients to 3-sulfopropyl hexadecyl dimethyl betaine is 1:1; The volume ratio of methanol to tetrahydrofuran is 1:

2.

7. The preparation method according to claim 1, characterized in that: In the step (2), water and anhydrous ethanol are used as detergents for centrifugal washing; In the step (3), methanol is used as a washing agent in the centrifugal washing.

8. The preparation method according to claim 1, characterized in that: In the step (3), the solution is concentrated to one tenth of the original volume.

9. A drug-loaded nanoparticle loaded with licorice active ingredients, characterized in that: The preparation method is described in any one of claims 1 to 8.

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