Ginsenoside Rh2 pH sensitive liposome and preparation method thereof

Abstract

The invention belongs to the technical field of liposome drugs, and discloses a ginsenoside Rh2 pH sensitive liposome and a preparation method thereof, the ginsenoside Rh2 pH sensitive liposome comprises 1 part by weight of ginsenoside Rh2, 14 parts by weight of soybean lecithin, 6 parts by weight of cholesterol, and 5-7 parts by weight of cholesterol succinic acid monoester. The ginsenoside Rh2 pH-sensitive liposome provided by the invention is good in biocompatibility and high in stability, and due to the pH-sensitive characteristic of the ginsenoside Rh2 pH-sensitive liposome, a drug active component ginsenoside Rh2 wrapped in the ginsenoside Rh2 pH-sensitive liposome can be released in a targeted manner in an acidic tumor environment, so that the drug active component is gathered in tumor tissues, and the drug is prevented from losing action due to metabolism in a drug circulation process. Experiments in a mouse liver cancer model prove that the liposome has an anti-tumor effect of remarkably inhibiting tumor growth and metastasis in vivo.

Description

Technical Field

[0001] The invention belongs to the technical field of liposome medicines, and in particular relates to a ginsenoside Rh2 pH-sensitive liposome and a preparation method thereof. Background Art

[0002] Liver cancer is a major cancer worldwide, and its incidence rate is increasing year by year. Despite advances in surgery, interventional therapy, molecular targeted drugs, and immunotherapy, the prognosis of patients with advanced liver cancer remains dismal. Therefore, there is an urgent need to develop more effective therapeutic drugs to improve the prognosis of patients with liver cancer.

[0003] Ginsenoside Rh2 is a monomer compound extracted and separated from ginseng. It has strong anti-cancer activity, can inhibit tumor cell proliferation and induce apoptosis, reverse tumor cell resistance to anticancer drugs, improve the immunity of cancer patients, inhibit tumor invasion and metastasis, and has inhibitory effects on lung cancer, liver cancer, breast cancer, leukemia and other tumor cells. It has very low or even no toxicity to normal cells or tissues. However, the solubility of ginsenoside Rh2 monomer in water is low and its bioavailability is poor. How to improve its bioavailability has become an important topic for pharmaceutical researchers.

[0004] The main research direction at present is to prepare ginsenoside Rh2 into new dosage forms such as liposomes, microemulsions or submicroemulsions, inclusion compounds, and nanoemulsions by encapsulation and other methods. Liposomes are artificially prepared microscopic spherical carriers and are drug delivery systems that encapsulate drugs in bilayer vesicles similar to biological membrane structures. Liposomes are often used as universal carriers for water-soluble and insoluble drugs, diagnostics, and nutritional products.

[0005] As a widely studied and applied drug delivery system, liposome preparations have the advantages of high drug loading capacity, easy degradation and low toxicity compared with traditional drug preparations, and can effectively encapsulate various biomolecules such as vitamins, minerals, proteins and antioxidants. This type of preparation can effectively concentrate drugs at the lesion site and prevent them from losing their effect due to metabolism during drug circulation.

[0006] It is well known that the average pH value of the extracellular microenvironment of tumor cells is between 6.5 and 7.0, while the pH value of normal tissues and blood is about 7.4. How to design liposomes that can encapsulate ginsenoside Rh2 so that it can release anticancer drugs in an acidic tumor environment is the key to improving the bioavailability of ginsenoside Rh2.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and, on the one hand, provide a ginsenoside Rh2 pH-sensitive liposome having an anti-liver cancer effect and capable of targeted release of anticancer drugs in an acidic tumor environment.

[0009] Another aspect of the present invention provides a method for preparing the ginsenoside Rh2 pH-sensitive liposome.

[0010] In order to solve the above technical problems, the basic concept of the technical solution adopted by the present invention is: The first aspect of the present invention provides a ginsenoside Rh2 pH-sensitive liposome, comprising 1 part by weight of ginsenoside Rh2, 14 parts by weight of soybean lecithin, 6 parts by weight of cholesterol, and 5-7 parts by weight of cholesterol succinate.

[0011] A further embodiment includes 1 part by weight of ginsenoside Rh2, 14 parts by weight of soybean lecithin, 6 parts by weight of cholesterol, and 6 parts by weight of cholesterol succinate.

[0012] In the present invention, soybean lecithin (PC), cholesterol (CHOL) and cholesterol succinate monoester (CHEMS) are used as the membrane materials of liposomes, and the active drug ingredient ginsenoside Rh2 is encapsulated inside the membrane materials. The use of cholesterol succinate monoester gives the liposomes pH-sensitive properties. The liposomes have a higher encapsulation rate in an environment close to the pH value of human blood, while the encapsulation rate is reduced in an acidic environment, and can be used as a drug targeting anti-cancer ingredients.

[0013] Through experimental tests, the mass ratio of PC, CHOL and CHEMS in the membrane material is controlled at 14:6:5~7, and liposomes with pH-sensitive properties can be obtained. When the mass ratio is controlled at 14:6:6, the pH-sensitive properties of the liposomes are more significant, and the particle size is appropriate. It can be predicted that the targeted release of drugs in the treatment of liver cancer will be better.

[0014] The second aspect of the present invention provides a method for preparing the above-mentioned ginsenoside Rh2 pH-sensitive liposomes, comprising the following steps: (1) adding 14 parts by weight of soybean lecithin, 6 parts by weight of cholesterol, 5-7 parts by weight of cholesterol succinate and 1 part by weight of ginsenoside Rh2 into an organic solvent and mixing well; (2) Rotary evaporation to form a thin film, followed by vacuum drying; (3) Add PBS buffer solution for hydration; (4) Perform high pressure homogenization two to six times to obtain ginsenoside Rh2 pH-sensitive liposomes.

[0015] Herein, PBS buffer solution refers to phosphate buffered saline.

[0016] In a further embodiment, in step (1), 1 part by weight of ginsenoside Rh2 is dissolved in methanol, and then the methanol solution of ginsenoside Rh2 is uniformly mixed with other raw materials and an organic solvent; Preferably, the ratio of ginsenoside Rh2 to methanol is 1 mg: 0.3-0.4 ml.

[0017] In a further embodiment, in step (1), the ratio of ginsenoside Rh2 to organic solvent is 1 mg:1 ml; Preferably, the organic solvent is chloroform.

[0018] As a specific implementation method, in step (1), soybean lecithin, cholesterol and cholesterol succinate are accurately weighed according to the ratio, added into a round-bottom flask, and then an organic solvent is added; ginsenoside Rh2 is accurately weighed according to the ratio, dissolved in a certain amount of methanol, added into the above round-bottom flask, and mixed thoroughly.

[0019] In a further embodiment, in step (2), the conditions for rotary evaporation include: water bath temperature of 40° C. and rotation speed of 24 r / min.

[0020] In a further embodiment, in step (2), the vacuum drying temperature is 80° C. and the vacuum drying time is 1 h.

[0021] As a specific implementation method, a round-bottom flask is placed on a rotary evaporator, and the parameters of the rotary evaporator are set to a water bath temperature of 40°C and a rotation speed of 24r / min to evaporate the organic solvent and form a uniform yellow-white film at the bottom of the round-bottom flask; then, it is transferred to a vacuum drying oven and vacuum dried at 80°C for 1h.

[0022] In a further embodiment, in step (3), the hydration temperature is 60°C and the hydration time is 30-60 min; Preferably, the added PBS buffer solution is preheated to 60° C., and the pH value of the PBS buffer solution is 7.4; Preferably, hydration is performed using a benchtop thermostatic shaker for rotation.

[0023] In a further scheme, in step (3), PBS buffer solution is added at a ratio of 1 mg:4 ml of ginsenoside Rh2 to PBS buffer solution.

[0024] As a specific implementation method, a PBS buffer solution with a pH value of 7.4 is preheated to 60° C., added to a round-bottom flask with a yellow-white film formed on the bottom, and rotated and hydrated at 60° C. for 30 to 60 minutes using a desktop constant temperature oscillator to obtain a uniform yellow-white solution.

[0025] The “rotational hydration” mentioned herein refers to the oscillator adopting a cyclotronic oscillation mode.

[0026] In a further embodiment, in step (4), high pressure homogenization is performed four times; Preferably, the pressure of the high-pressure homogenization is 400 bar.

[0027] As a specific implementation method, the yellow-white solution obtained in step (3) is homogenized four times at a pressure of 400 bar using a high-pressure homogenizer.

[0028] In a further embodiment, in step (4), the ginsenoside Rh2 pH-sensitive liposomes obtained after homogenization are placed in a refrigerator and protected from light.

[0029] In a specific embodiment of the present invention, the method for preparing the ginsenoside Rh2 pH-sensitive liposomes comprises the following steps: (1) Accurately weigh 14 parts by weight of PC, 6 parts by weight of CHOL and 5-7 parts by weight of CHEMS, add them into a round-bottom flask, and then add a certain amount of organic solvent; accurately weigh 1 part by weight of ginsenoside Rh2, dissolve it in methanol according to the ratio of ginsenoside Rh2 to methanol of 1 mg: 0.3-0.4 ml, add it into the above round-bottom flask, and mix thoroughly; wherein the ratio of ginsenoside Rh2 to organic solvent is 1 mg: 1 ml; (2) placing the round-bottom flask on a rotary evaporator, setting the parameters of the rotary evaporator to a water bath temperature of 40°C and a rotation speed of 24 r / min, and removing the organic solvent by rotary evaporation until a uniform yellow-white film is formed at the bottom of the round-bottom flask; transferring the mixture to a vacuum drying oven, and vacuum drying the mixture at 80°C for 1 h; (3) Add a PBS buffer solution preheated to 60°C and having a pH value of 7.4 into a round-bottom flask, wherein the ratio of ginsenoside Rh2 to PBS buffer solution is 1 mg:4 ml. Use a desktop constant temperature oscillator to rotate and hydrate at 60°C for 30-60 min to obtain a uniform yellow-white solution. (4) The yellow-white solution is homogenized two to six times using a high-pressure homogenizer at a pressure of 400 bar to obtain ginsenoside Rh2 pH-sensitive liposomes, which are then stored in a refrigerator away from light.

[0030] After adopting the above technical scheme, the present invention has the following beneficial effects compared with the prior art.

[0031] The present invention helps to solve the problems of poor drug biocompatibility, low stability, and less distribution of active drug ingredients in tumor tissue cells in the prior art. The provided ginsenoside Rh2 pH-sensitive liposomes have the advantages of good biocompatibility and high stability, and thanks to its pH-sensitive properties, can target release of the active drug ingredient ginsenoside Rh2 encapsulated therein in an acidic tumor environment, so that ginsenoside Rh2 accumulates in tumor tissue. Through experimental verification in a mouse liver cancer model, the liposomes of the present invention have an anti-tumor effect of significantly inhibiting tumor growth and metastasis in vivo.

[0032] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings are part of the present invention and are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention, but do not constitute an improper limitation of the present invention. Obviously, the drawings described below are only some embodiments. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. In the accompanying drawings: Figure 1 is a transmission electron microscope (TEM) image of the ginsenoside Rh2 pH-sensitive liposome prepared in Example 1 of the present invention; Figure 2 The figure is a comparison of the morphologies of liposomes prepared at different CHEMS dosages in Experimental Example 1 of the present invention in different pH environments; Figure 3 is the particle size of the liposomes prepared at different CHEMS dosages in Experimental Example 1 of the present invention tested at different pH values; Figure 4 is the PDI of the liposomes prepared at different CHEMS dosages in Experimental Example 1 of the present invention tested at different pH values; Figure 5 This is a comparison of the morphologies of liposomes prepared at different homogenization times in Experimental Example 2 of the present invention in environments with different pH values; Figure 6 is the particle size of the liposomes prepared at different homogenization times in Experimental Example 2 of the present invention tested at different pH values; Figure 7 is the PDI of the liposomes prepared at different homogenization times in Experimental Example 2 of the present invention tested at different pH values; Figure 8 is the standard curve of ginsenoside Rh2 in Experimental Example 3 of the present invention; Fig. 9 is the cumulative release rate curve of ginsenoside Rh2 in Experimental Example 5 of the present invention; Fig.10is a graph showing changes in particle size and PDI of ginsenoside Rh2 pH-sensitive liposomes stored at 4°C for a certain period of time in Experimental Example 6 of the present invention; Fig.11 is the tumor volume growth curve of each group of nude mice in Experimental Example 7 of the present invention; Fig.12 is the tumor weight of each group of nude mice after treatment in Experimental Example 7 of the present invention; Fig.13 These are photos of tumors in each group of nude mice after treatment in Experimental Example 7 of the present invention; Fig.14 This is the body weight change curve of each group of nude mice during the treatment period in Experimental Example 7 of the present invention.

[0034] It should be noted that these drawings and textual descriptions are not intended to limit the conceptual scope of the present invention in any way, but are intended to illustrate the concept of the present invention for those skilled in the art by referring to specific embodiments. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. The following embodiments are used to illustrate the present invention but are not used to limit the scope of the present invention.

[0036] Example 1 This embodiment provides a method for preparing ginsenoside Rh pH-sensitive liposomes, which specifically comprises the following steps: (1) Accurately weigh 140 mg of PC, 60 mg of CHOL and 60 mg of CHEMS, add them into a 250 ml round-bottom flask, and then add 10 ml of chloroform; accurately weigh 10 mg of ginsenoside Rh2, dissolve it in 4 ml of methanol, add it to the above round-bottom flask, and mix thoroughly; (2) placing the round-bottom flask on a rotary evaporator, setting the parameters of the rotary evaporator to a water bath temperature of 40°C and a rotation speed of 24 r / min, and removing the organic solvent by rotary evaporation until a uniform yellow-white film is formed at the bottom of the round-bottom flask; transferring the mixture to a vacuum drying oven, and vacuum drying the mixture at 80°C for 1 h; (3) Add 40 ml of PBS buffer solution preheated to 60°C and pH 7.4 to the round-bottom flask and rotate and hydrate at 60°C for 30 min using a desktop constant temperature oscillator to obtain a uniform yellow-white solution. (4) The yellow-white solution is homogenized four times using a high-pressure homogenizer at a pressure of 400 bar to obtain ginsenoside Rh2 pH-sensitive liposomes, which are then stored in a refrigerator away from light for future use.

[0037] The ginsenoside Rh2 pH-sensitive liposomes prepared in this example were diluted 10 times with PBS buffer solution, dropped onto a copper grid, and negatively stained with 1% phosphotungstic acid. After drying, the morphology was observed under a transmission electron microscope. Figure 1 As shown in the TEM image, the prepared liposomes are spherical nanoparticles with a particle size of less than 200 nm.

[0038] Test Example 1 This test example refers to the preparation method provided in Example 1, and three groups of different blank liposome samples are prepared by adjusting the amount of CHEMS used therein for testing.

[0039] 1. Preparation of blank liposomes In this test example, the preparation method of blank liposomes includes the following steps: (1) Accurately weigh 140 mg of PC, 60 mg of CHOL and X mg of CHEMS, add them into a 250 ml round-bottom flask; then add 10 ml of chloroform and mix thoroughly; (2) Place the round-bottom flask on a rotary evaporator, set the parameters of the rotary evaporator to a water bath temperature of 40°C and a rotation speed of 24 r / min, and remove the organic solvent by rotary evaporation until a uniform yellow-white film is formed at the bottom of the round-bottom flask; transfer to a vacuum drying oven and vacuum dry at 80°C for 1 h.

[0040] (3) Add 40 ml of PBS buffer solution preheated to 60°C and pH 7.4 to the round-bottom flask and rotate and hydrate at 60°C for 30 min using a desktop constant temperature oscillator to obtain a uniform yellow-white solution. (4) The yellow-white solution was homogenized four times using a high-pressure homogenizer at a pressure of 400 bar to obtain pH-sensitive blank liposomes, which were then stored in a refrigerator away from light for future use.

[0041] In the preparation process of three different groups of samples, the amount of CHEMS Xmg used in step (1) is respectively: 50 mg for the first group, 60 mg for the second group, and 70 mg for the third group.

[0042] 2. Morphology comparison The pH values ​​of the three groups of samples prepared above were adjusted to 7.21, 5.28 and 4.54 respectively. The morphologies of the samples at different pH values ​​are shown in Fig. Figure 2 As shown, samples with different pH values ​​were adjusted with 1M HCl solution or NaOH solution.

[0043] exist Figure 2In the figure, the CHEMS dosage of the first, second and third groups from left to right is 50mg, 60mg and 70mg respectively, and the numbers 1, 2 and 3 of each group correspond to pH values ​​of 7.21, 5.28 and 4.54 respectively. As can be seen from the figure, as the pH value of the solution decreases, its color deepens successively, and the color change of the second group is particularly obvious, indicating that the pH sensitivity of the liposome is more significant when the CHEMS dosage is 60mg.

[0044] 3. Particle size and PDI determination The samples at different pH values ​​obtained above were measured for their particle size and PDI using Anton Paar particle size analyzer. The particle size measurement results are shown in Figure 3 The PDI determination results are shown in Figure 4 shown.

[0045] When the CHEMS dosage changes between 50 and 70 mg, Figure 3 It can be seen that the increase in the amount of CHEMS is accompanied by a decrease in the liposome particle size. When the amount of CHEMS is 60 mg, the particle size is appropriate and changes significantly with the pH value. Figure 4 It can be seen that when the CHEMS dosage is 60 mg, the PDI is smaller, indicating that the dispersion is more stable, and the dosage is less than when the CHEMS content is 70 mg, saving drugs.

[0046] Based on the above test results, in the preferred embodiment of the present invention, the mass ratio of PC, CHOL and CHEMS is controlled to be 14:6:6, and in subsequent test examples, a scheme with a CHEMS dosage of 60 mg is used for further testing.

[0047] Test Example 2 In this test example, three groups of different blank liposome samples were prepared by adjusting the number of high-pressure homogenizations with reference to the preparation method provided in Example 1 for testing.

[0048] 1. Preparation of blank liposomes In this test example, the preparation method of blank liposomes includes the following steps: (1) Accurately weigh 140 mg of PC, 60 mg of CHOL and 60 mg of CHEMS, add them into a 250 ml round-bottom flask; then add 10 ml of chloroform and mix thoroughly; (2) Place the round-bottom flask on a rotary evaporator, set the parameters of the rotary evaporator to a water bath temperature of 40°C and a rotation speed of 24 r / min, and remove the organic solvent by rotary evaporation until a uniform yellow-white film is formed at the bottom of the round-bottom flask; transfer to a vacuum drying oven and vacuum dry at 80°C for 1 h.

[0049] (3) Add 40 ml of PBS buffer solution preheated to 60°C and pH 7.4 to the round-bottom flask and rotate and hydrate at 60°C for 30 min using a desktop constant temperature oscillator to obtain a uniform yellow-white solution. (4) The yellow-white solution is homogenized Y times at a pressure of 400 bar using a high-pressure homogenizer to obtain pH-sensitive blank liposomes, which are then stored in a refrigerator away from light for future use.

[0050] In the preparation process of three groups of different samples, the number of high pressure homogenization in step (4) is Y times: twice for the first group, four times for the second group, and six times for the third group.

[0051] 2. Morphology comparison The pH values ​​of the three groups of samples prepared above were adjusted to 7.24, 5.38 and 4.50 respectively. The morphologies of the samples at different pH values ​​are shown in Figure 5 As shown, samples with different pH values ​​were adjusted with 1M HCl solution or NaOH solution.

[0052] exist Figure 5 In the figure, the high pressure homogenization times of the first, second and third groups from left to right are two, four and six times respectively, and the numbers 1, 2 and 3 of each group correspond to pH values ​​of 7.24, 5.38 and 4.50 respectively. As can be seen from the figure, as the pH value of the solution decreases, its color deepens successively, and, among the three groups of samples, the color change of the second group is particularly obvious, indicating that the pH sensitivity of the liposome is more obvious when the high pressure homogenization times is four times.

[0053] 3. Particle size and PDI determination The samples at different pH values ​​obtained above were measured for their particle size and PDI using Anton Paar particle size analyzer. The particle size measurement results are shown in Figure 6 The PDI determination results are shown in Figure 7 shown.

[0054] When the high pressure homogenization times are changed between two and six times, Figure 6 It can be seen that the increase in the number of high-pressure homogenization times will be accompanied by a decrease in the liposome particle size, but too many homogenization times will result in the liposome particle size not changing significantly with the pH value. If the homogenization times are too few, the liposome particle size will be too large. When the high-pressure homogenization times are four, the particle size is appropriate and changes significantly with the pH value. Figure 7 It can be seen that when the high-pressure homogenization number is four times, the PDI is small, indicating that the dispersion is more uniform than the other two groups. When the homogenization number is increased to six times, although the PDI is lower than 0.3, which is a relatively stable dispersion level, it is significantly higher than the other two groups of samples, indicating that the uniformity of the liposomes is worse in comparison.

[0055] Based on the above test results, the preferred scheme of the present invention performs four high-pressure homogenizations, and the subsequent test examples adopt the scheme of four high-pressure homogenizations for further testing.

[0056] Test Example 3 This test example is used to establish a standard curve for ginsenoside Rh2, and the specific method is as follows.

[0057] Ginsenoside Rh2 was accurately weighed, dissolved in 75% ethanol solution, transferred into a volumetric flask and fixed to volume to prepare a mother solution. The mother solution was then diluted in turn to prepare a standard series of solutions with final concentrations of 0.98, 0.49, 0.33, 0.26 and 0.19 mg / mL, respectively. The precise concentration test results of the standard series of solutions are shown in Table 1.

[0058] Using 75% ethanol solution as background, the absorbance of the above standard series solutions was measured at 208 nm using a UV-visible spectrophotometer. The results are shown in Table 1. Figure 8 As shown), the linear regression equation is Y=1.5072x+0.0067, and the concentration range is 0.10~1.00mg / ml.

[0059] Table 1 Test Example 4 In this test example, ginsenoside Rh2 pH-sensitive liposomes were prepared according to the preparation method provided in Example 1, and the particle size, PDI and potential, as well as the encapsulation efficiency and drug loading of the liposomes were measured under different pH values. Among them, samples with different pH values ​​were adjusted with 1M HCl solution or NaOH solution.

[0060] 1. Determination of particle size, PDI and Zeta potential of ginsenoside Rh2 pH-sensitive liposomes The sample was added to a cuvette and a potential sample cell, and the particle size, PDI, and Zeta potential of the sample were measured in sequence using a particle size analyzer. The results are shown in Table 2.

[0061] 2. Determination of encapsulation efficiency and drug loading Take 1 ml of sample solution in the inner tube of a refrigerated centrifuge tube, refrigerate and centrifuge for 1 hour at a temperature of 4 ° C and a speed of 6500 r / min. After the liquid in the inner tube is shaken clean, take 0.5 ml of the liquid in the outer tube, add 1.5 ml of anhydrous ethanol, put it into a cuvette, and use a UV-visible spectrophotometer to measure its absorbance at 208 nm, where the reference solution is an ethanol solution with a volume concentration of 75%. According to the absorbance results measured, the concentration of unencapsulated ginsenosides is calculated using the standard curve of ginsenosides established in Test Example 3, and the encapsulation rate is calculated using the following formula: .

[0062] Furthermore, the drug loading can be calculated according to the following formula: .

[0063] The results of encapsulation efficiency and drug loading are shown in Table 2.

[0064] Table 2 From the data in Table 2, it can be seen that the particle size, PDI and Zeta potential of the prepared liposomes at different pH values, as well as the encapsulation efficiency and drug loading, are significantly different, indicating that the prepared liposomes have pH-sensitive properties. At different pH values, the PDI is less than 0.3, and the absolute value of the Zeta potential is greater than 25 mV, indicating that the liposomes, as nanoparticles, can be stably dispersed in solutions of different pH values.

[0065] The test results of the encapsulation rate showed that the encapsulation rate showed a downward trend as the pH value decreased. In other words, the prepared liposomes had a high encapsulation rate in the normal pH environment of the human body and blood, but a relatively low encapsulation rate in the acidic environment near the tumor, thus achieving the purpose of concentrated release of ginsenoside Rh2 at the lesion site.

[0066] Test Example 5 This test example tests the sustained release performance of ginsenoside Rh2 pH-sensitive liposomes prepared by the preparation method provided in Example 1. The specific test method includes the following steps: A. Prepare ginsenoside Rh2 pH-sensitive liposomes according to the preparation method provided in Example 1, divide them into three groups, adjust the pH values ​​to 7.4, 5.2 and 4.6 respectively, and prepare three groups of PBS buffer solutions with pH values ​​of 7.4, 5.2 and 4.6 respectively, wherein samples with different pH values ​​are adjusted with 1M HCl solution or NaOH solution; B. Take 5 mL of ginsenoside Rh2 pH-sensitive liposome samples with different pH values ​​and add them to the pre-treated dialysis bags respectively. Then put the dialysis bags into light-proof brown bottles, add 50 mL of PBS buffer solution with corresponding pH value respectively, and keep them aside for later use. C. Place the three brown bottles in a tabletop constant temperature oscillator, adjust the temperature to 37°C (simulating human body temperature), and oscillate slowly for 48 hours. Start timing from the oscillation. Take 1 mL of dialysate at 30 minutes, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, and 48 hours respectively, and add 1 mL of PBS buffer solution of the corresponding pH value to make up the difference. D. Use an ultraviolet-visible spectrophotometer to measure the absorbance of all samples obtained in step C at a wavelength of 208 nm, and use the standard curve established in Test Example 3 to calculate the concentration of ginsenoside Rh2 therein, and further calculate to obtain the relationship curve between the cumulative release rate of ginsenoside Rh2 and time of each group of samples, as shown in FIG. Fig. 9 shown.

[0067] Depend on Fig. 9 It can be seen that under the same time, the cumulative release rate of ginsenoside Rh2 in samples with lower pH values ​​is higher, especially in samples with a pH value of 4.6, the cumulative release rate in 6 hours is close to 100%. This shows that the liposomes provided by the scheme of the present invention have pH-sensitive properties. In an environment where the pH value of normal human tissue and blood is about 7.4, the amount of ginsenoside Rh2 released is extremely low, which can prevent the drug from losing its effect during the circulation process with metabolism. In an acidic tumor environment, the ginsenoside Rh2 encapsulated therein can be targeted for release, causing ginsenoside Rh2 to aggregate in tumor tissues, thereby improving the bioavailability of ginsenoside Rh2.

[0068] Test Example 6 This test example tests the stability of the ginsenoside Rh2 pH-sensitive liposomes prepared by the preparation method provided in Example 1.

[0069] Specifically, ginsenoside Rh2 pH-sensitive liposomes were prepared according to the preparation method provided in Example 1, and the samples were stored at 4°C for 30 days. Samples were taken at 1, 15, and 30 days, and the particle size and PDI of the samples were measured at different pH values ​​(the pH adjustment method was the same as in Experimental Example 3). The results are as follows: Fig.10 shown.

[0070] As can be seen from the figure, after different storage times, the particle size and PDI of the samples measured at different pH values ​​showed obvious differences, indicating that as the storage time increases, the samples still maintain pH sensitivity. In addition, the PDI obtained from the above tests are all less than 0.3, indicating that after different storage times, in different pH environments, the liposomes can be stably dispersed in the solution.

[0071] Test Example 7 This test example investigated the in vivo anti-liver cancer activity of ginsenoside Rh2 pH-sensitive liposomes prepared according to the preparation method provided in Example 1. The specific method is as follows.

[0072] 1. Establishment of nude mouse subcutaneous tumor model (1) Preparation of cell suspension ① Huh7 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin in a cell culture incubator at 37°C and 5% CO2; ② Digest the cells that have grown to 80% to 90% of the culture dish with trypsin, centrifuge at 1000 rpm, remove the supernatant, add PBS to wash the cells, resuspend them in PBS again, count the cells, and adjust the number to 2.5×10 7 cells / ml; ③ According to the number of nude mice, prepare a sufficient amount of cell suspension for use in inoculating nude mice; store the prepared cell suspension in a 4°C refrigerator and inoculate nude mice as soon as possible.

[0073] (2) Establishment of nude mouse subcutaneous tumor model The injection site was uniformly set at 0.3 cm from the axilla of the right forelimb of nude mice (BALB / c-Nude (6-8W) (22±2g). After disinfection with iodine cotton balls, a 1 ml syringe was used to extract the mixed cell suspension (density 2.5×10 7 After the syringe needle penetrates the skin of the nude mouse, slightly lift the needle, move it to the injection site, and slowly inject the cell suspension. After the injection is completed, press the needle with a sterile cotton ball, and dispose of the experimental equipment and waste harmlessly. Put the newly modeled nude mouse into a cage with new bedding, and observe the nude mouse's mental state, activity and eating conditions the next day, and whether there is redness or swelling at the injection site.

[0074] 2. Dosage method The nude mice were randomly divided into 6 groups, 3 mice in each group, and the specific groups were as follows: (1) High concentration of ginsenoside Rh2 liposome group (25 mg / kg Rh2 NPs); (2) High concentration of ginsenoside Rh2 monomer group (25 mg / kg Rh2); (3) low concentration ginsenoside Rh2 liposome group (15 mg / kg Rh2 NPs); (4) Low concentration ginsenoside Rh2 monomer group (15 mg / kg Rh2); (5) empty liposome group (NPs); (6) Blank control group (Control).

[0075] When small nodules appeared at the inoculation site of nude mice (approximately the 6th day after inoculation), the tumor volume was measured and medication was started. The specific administration method was as follows: The high-concentration ginsenoside Rh2 liposome group was treated with ginsenoside Rh2 pH-sensitive liposomes injected into the tail vein at a dose of 25 mg / kg / time ginsenoside Rh2 pH-sensitive liposomes, once every 3 days for 4 weeks; The high-concentration ginsenoside Rh2 monomer group was treated with ginsenoside Rh2 monomer tail vein injection, with an injection volume of 25 mg / kg / time ginsenoside Rh2 monomer, once every 3 days for 4 weeks; The low-concentration ginsenoside Rh2 liposome group was treated with ginsenoside Rh2 pH-sensitive liposomes injected into the tail vein at a dose of 15 mg / kg / time ginsenoside Rh2 pH-sensitive liposomes, once every 3 days for 4 weeks; The low-concentration ginsenoside Rh2 monomer group was treated with ginsenoside Rh2 monomer tail vein injection, with an injection dose of 15 mg / kg / time ginsenoside Rh2 monomer, once every 3 days for 4 weeks; The empty liposome group was treated with tail vein injection of blank liposomes, with an injection volume of 0.2 ml / time of blank liposomes, once every 3 days for 4 weeks; The blank control group was treated with tail vein injection of normal saline, with an injection volume of 0.2 ml / time, once every 3 days for 4 weeks.

[0076] 3. Detection indicators (1) Animal observation: record the weight, mental state, excitement, biting, activity, etc. of the nude mice; (2) Tumor measurement: After the tumor is visible to the naked eye, the short diameter (W) and long diameter (L) of the transplanted tumor are measured with a vernier caliper and the formula V (cm 3 )=L×W 2 / 2 Calculate the volume of the tumor and measure the tumor size every six days; (3) Final sampling: 36 days after nude mice were implanted with tumors, tumor tissues were collected, and the volume and weight of the tumors were calculated. The tumors were then divided into two parts for storage: ① fixation with 4% paraformaldehyde, and ② cryopreservation at -80°C.

[0077] In this experimental example, the growth of tumor volume in each group of nude mice during the treatment process was recorded, and a tumor volume growth curve was drawn, such as Fig.11 As shown in the figure, it can be seen that the tumor growth curve of the 25 mg / kg ginsenoside Rh2 liposome group was the slowest, followed by the 25 mg / kg ginsenoside Rh2 monomer group, followed by the 15 mg / kg ginsenoside Rh2 liposome group, the 15 mg / kg ginsenoside Rh2 monomer group, the empty liposome group and the blank control group (*P < 0.05). Fig.12 The tumor weight of nude mice in each group after treatment (*P<0.05). Fig.13The pictures of tumors isolated from nude mice in each group after treatment are shown. By comparing with the blank control group or the empty liposome group, it can be seen that 15mg / kg and 25mg / kg ginsenoside Rh2 monomers, 15mg / kg and 25mg / kg ginsenoside Rh2 liposomes have obvious anti-liver cancer effects, and ginsenoside Rh2 liposomes have better anti-liver cancer effects than ginsenoside Rh2 monomers of the same concentration, indicating that the ginsenoside Rh2 pH-sensitive liposomes provided by the present invention have a more significant effect in inhibiting tumor growth than ginsenoside Rh2 monomers.

[0078] During the treatment, the body weight of nude mice in each group was measured every 6 days. Fig.14 As shown. It can be seen that during the experiment, the weight of nude mice in each group varied between 15 and 25 g, and the difference in weight change was small. Weight change is an important indicator of systemic toxicity. The above results show that the liposomes did not show severe systemic toxicity.

[0079] The above results show that the 25 mg / kg ginsenoside Rh2 liposome group has the best anti-liver cancer effect. The ginsenoside Rh2 pH-sensitive liposome provided by the present invention can improve the anti-liver cancer effect of ginsenoside Rh2 by encapsulating ginsenoside Rh2 in liposomes with pH-sensitive properties and releasing the drug in an acidic tumor environment.

[0080] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with the present application can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A ginsenoside Rh2 pH-sensitive liposome, characterized in that: The invention comprises 1 weight part of ginsenoside Rh2, 14 weight parts of soybean lecithin, 6 weight parts of cholesterol and 5-7 weight parts of cholesterol succinate.

2. The ginsenoside Rh2 pH-sensitive liposome according to claim 1, characterized in that: The invention comprises 1 part by weight of ginsenoside Rh2, 14 parts by weight of soybean lecithin, 6 parts by weight of cholesterol and 6 parts by weight of cholesterol succinate.

3. A method for preparing ginsenoside Rh2 pH-sensitive liposomes as claimed in claim 1 or 2, characterized in that: The following steps are involved: (1) adding 14 parts by weight of soybean lecithin, 6 parts by weight of cholesterol, 5-7 parts by weight of cholesterol succinate and 1 part by weight of ginsenoside Rh2 into an organic solvent and mixing well; (2) Rotary evaporation to form a thin film, followed by vacuum drying; (3) Add PBS buffer solution for hydration; (4) Perform high pressure homogenization two to six times to obtain ginsenoside Rh2 pH-sensitive liposomes.

4. The method for preparing ginsenoside Rh2 pH-sensitive liposomes according to claim 3, characterized in that: In step (1), 1 part by weight of ginsenoside Rh2 is dissolved in methanol, and then the methanol solution of ginsenoside Rh2 is evenly mixed with other raw materials and an organic solvent.

5. The method for preparing ginsenoside Rh2 pH-sensitive liposomes according to claim 3, characterized in that: In step (1), the ratio of ginsenoside Rh2 to organic solvent is 1 mg:1 ml.

6. The method for preparing ginsenoside Rh2 pH-sensitive liposomes according to any one of claims 3 to 5, characterized in that: In step (2), the conditions for rotary evaporation include: water bath temperature of 40° C. and rotation speed of 24 r / min.

7. The method for preparing ginsenoside Rh2 pH-sensitive liposomes according to any one of claims 3 to 5, characterized in that: In step (2), the vacuum drying temperature is 80° C. and the vacuum drying time is 1 h.

8. The method for preparing ginsenoside Rh2 pH-sensitive liposomes according to any one of claims 3 to 5, characterized in that: In step (3), the hydration temperature is 60°C and the hydration time is 30-60 min.

9. The method for preparing ginsenoside Rh2 pH-sensitive liposomes according to any one of claims 3 to 5, characterized in that: In step (3), PBS buffer solution is added according to the ratio of ginsenoside Rh2 to PBS buffer solution of 1 mg:4 ml.

10. The method for preparing ginsenoside Rh2 pH-sensitive liposomes according to any one of claims 3 to 5, characterized in that: In step (4), high-pressure homogenization is performed four times, and / or the pressure of high-pressure homogenization is 400 bar.

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