Bionic dextran drug carrier and its preparation method and application

Abstract

The present invention relates to the field of biomedicine, and more specifically to a biomimetic glucan drug carrier, its preparation method, and its application. In the present invention, cationic liposomes are used to encapsulate drugs, and yeast cell wall cystic structures are used to encapsulate the cationic liposomes, forming a two-stage encapsulation structure. This promotes intestinal absorption of the biomimetic glucan drug carrier, thereby improving drug delivery. The biomimetic glucan drug carrier of the present invention is particularly suitable for delivering ginsenosides.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a biomimetic glucan drug carrier and a preparation method and application thereof. Background Art

[0002] Primary hepatocellular carcinoma (PHC) poses a serious threat to public health and imposes a significant financial burden on patients. Common treatments for HCC include surgical resection, transcatheter arterial chemoembolization (TACE), and chemotherapy. Although chemotherapeutic drugs, such as 5-fluorouracil (5-FU), doxorubicin (ADM), and platinum-based drugs, play an important role in the treatment of HCC, drug sensitivity decreases with prolonged treatment, potentially due to drug resistance of tumor-associated targets. Currently, many researchers are focusing on the adjunctive treatment of HCC with medicinal and edible plants and their active ingredients. Various medicinal and edible plants have been reported to inhibit tumor growth and metastasis, improve the tumor microenvironment, and regulate pyroptosis and autophagy through various mechanisms. For patients who cannot tolerate surgery but have developed drug resistance, adjunctive therapy with medicinal and edible plants or their active ingredients can reverse HCC drug resistance, reduce tumor burden, and prolong patient survival.

[0003] Ginseng, the dried rhizome of the Araliaceae plant Panax ginseng, is a precious traditional Chinese medicinal material used in my country for over 2,000 years. Modern pharmacological studies have shown that ginseng possesses multiple physiological activities, including anti-tumor, cardiovascular protection, anti-inflammatory, and immunomodulatory. In particular, its main active ingredient, ginsenosides, has been reported to exhibit a wide range of anti-tumor effects. Studies have shown that the anti-liver cancer mechanism of ginsenosides may involve inhibiting liver cancer cell proliferation, inducing liver cancer cell differentiation, promoting liver cancer cell apoptosis, inhibiting liver cancer cell invasion and metastasis, reducing liver cancer cell drug resistance, and enhancing the body's immunity. However, the low water solubility, poor membrane permeability, and P-gp-mediated efflux of ginsenosides result in poor absorption after oral administration, resulting in low absolute bioavailability in vivo. Furthermore, oral administration of ginsenosides alone lacks targeting and cannot fully exert their maximum therapeutic effect. Currently, no technology addresses these two key application limitations of ginsenosides. Summary of the Invention

[0004] Based on this, the present invention provides a biomimetic glucan drug carrier and a preparation method and application thereof, which at least solves one problem in the prior art.

[0005] In a first aspect, the present invention provides a biomimetic glucan drug carrier, which comprises a cationic liposome and a yeast cell wall cystic structure encapsulating the cationic liposome, wherein the yeast cell wall cystic structure is a hollow yeast cell wall, and the cationic liposome is used to encapsulate the drug.

[0006] In the present invention, cationic liposomes are used to encapsulate drugs, and yeast cell wall cystic structures are used to encapsulate cationic liposomes, forming a two-stage encapsulation structure, which can promote the absorption of biomimetic dextran drug carriers by the intestine, thereby improving the drug loading effect.

[0007] In a second aspect, the present invention provides a method for preparing the biomimetic glucan drug carrier, comprising the following steps:

[0008] providing a cationic liposome solution;

[0009] Yeast powder and sodium hydroxide solution are mixed, stirred and centrifuged, the precipitate is collected and resuspended with water, the pH is adjusted to 4-5, incubated and centrifuged, and the insoluble particles are collected to obtain the yeast cell wall cystic structure;

[0010] The cationic liposome solution and the yeast cell wall cystic structure are mixed, ultrasonically treated, stirred and then centrifuged to collect the precipitate to obtain the biomimetic glucan drug carrier.

[0011] In a third aspect, the present invention provides use of the biomimetic glucan drug carrier in the preparation of drugs containing ginsenosides.

[0012] In a fourth aspect, the present invention provides a drug containing ginsenosides, comprising the biomimetic glucan drug carrier and ginsenosides, wherein the ginsenosides are encapsulated by the cationic liposomes in the biomimetic glucan drug carrier.

[0013] Due to the adoption of the above technical solution, the embodiments of the present invention have at least the following beneficial effects:

[0014] (1) The mechanism of action of the biomimetic glucan drug carrier is to simulate fungal invasion, target M cells and internal macrophages in the intestine, rapidly transport through the lymphatic circulation, and accumulate in inflammatory or tumor tissues under the stimulation of chemokines, thereby targeting the lesion area, solving the problem of poor absorption of ginsenosides;

[0015] (2) Using yeast cell wall cystic structures as a medium, an oral drug delivery vehicle was constructed to efficiently target macrophages and deliver ginsenosides, thereby achieving ginsenoside targeting to the tumor microenvironment;

[0016] (3) Yeast cell wall cystic structures, as pathogen-associated molecular patterns, can simulate the recognition of pathogens by pathogen recognition receptors on host immune cells, such as C-type lectin receptor dectin-1, complement receptor 3 (CR3) and Toll-like receptors (TLRs), thereby exerting immunomodulatory effects; both ginsenosides and yeast cell wall cystic structures have the effect of reversing the phenotype of tumor-associated macrophages (TAMs). The combination of ginsenosides and yeast cell wall cystic structures synergistically induces TAMs to repolarize from the M2 phenotype to the M1 phenotype. The biomimetic dextran drug carrier enhances the efficacy of ginsenosides in adjuvant therapy of tumors;

[0017] (4) The preparation method of biomimetic glucan drug carrier is simple and reproducible, which is suitable for the encapsulation of amphiphilic ginsenosides, and the encapsulation efficiency of cationic liposomes for ginsenosides is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a scanning electron microscope image of Rh2-lips in Example 3 of the present invention.

[0019] Figure 2 This is a scanning electron microscope image of the yeast glucan particles in Example 6 of the present invention.

[0020] Figure 3 This is a scanning electron microscope image of Rh2-lips GPs in Example 6 of the present invention.

[0021] Figure 4 This is a confocal image of Rh2-lips GPs in Example 6 of the present invention.

[0022] Figure 5 This is a graph showing the retention rate of Rh2 in the carrier of Rh2-lips in Example 3 and Rh2-lips GPs in Example 6 during simulated in vitro digestion.

[0023] Figure 6 This is a confocal image of the morphological changes of Rh2-lips during in vitro digestion in Example 3 of the present invention.

[0024] Figure 7 This is a confocal image of the morphological changes of Rh2-lips GPs during in vitro digestion in Example 6 of the present invention.

[0025] Figure 8 Graph showing the experimental results of macrophage uptake of Rh2-lips in Example 3 and Rh2-lips GPs in Example 6 of the present invention.

[0026] Figure 9 The figure shows the tumor conditions of mice in the animal experiment in the examples of the present invention.

[0027] Figure 10This is a dynamic monitoring chart of tumor volume changes in tumor-bearing mice in the animal experiment in the examples of the present invention.

[0028] Figure 11 These are in vivo imaging images of Rh2-lips in Example 3 and Rh2-lips GPs in Example 6 that were labeled with ICG (indocyanine green) by oral gavage in BALB / c tumor-bearing mice.

[0029] Figure 12 These are in vitro images of major organs and tumors in BALB / c tumor-bearing mice 24 hours after oral administration of ICG-labeled Rh2-lips in Example 3 and Rh2-lipsGPs in Example 6. DETAILED DESCRIPTION

[0030] The following is a clear and complete description of the concept of the present invention and the technical effects produced, so as to fully explain the purpose, scheme and effects of the present invention.

[0031] Ginsenosides or similar drugs have some shortcomings when used to treat diseases, such as low absolute bioavailability in the body and poor targeting. The present invention adopts the following technical solutions to improve these shortcomings.

[0032] In a first aspect, the present invention provides a biomimetic glucan drug carrier, which comprises a cationic liposome and a yeast cell wall cystic structure encapsulating the cationic liposome, wherein the yeast cell wall cystic structure is a hollow yeast cell wall, and the cationic liposome is used to encapsulate the drug.

[0033] In some preferred embodiments, the drug is ginsenoside.

[0034] In some preferred embodiments, the drug is ginsenoside Rh2, such as 20(S)-ginsenoside Rh2 (English name: 20(S)-Ginsenoside Rh2) or 20(R)-ginsenoside Rh2 (English name: 20(R)-GinsenosideRh2).

[0035] In some preferred embodiments, the cationic liposome comprises the following raw material components in parts by weight: 30 parts of hydrogenated soybean lecithin, 1-10 parts of cholesterol and 1.5-5 parts of octadecylamine.

[0036] In some preferred embodiments, the cationic liposome comprises the following raw material components in parts by weight: 30 parts of hydrogenated soybean lecithin, 6 parts of cholesterol and 6 parts of octadecylamine.

[0037] In a second aspect, the present invention provides a method for preparing the biomimetic glucan drug carrier, comprising the following steps:

[0038] providing a cationic liposome solution;

[0039] Yeast powder and sodium hydroxide solution are mixed, stirred and centrifuged, the precipitate is collected and resuspended with water, the pH is adjusted to 4-5, incubated and centrifuged, and the insoluble particles are collected to obtain the yeast cell wall cystic structure;

[0040] The cationic liposome solution and the yeast cell wall cystic structure are mixed, ultrasonically treated, stirred and then centrifuged to collect the precipitate to obtain the biomimetic glucan drug carrier.

[0041] In some preferred embodiments, the method for preparing the cationic liposome solution comprises the following steps:

[0042] Mixing hydrogenated soybean lecithin, cholesterol, octadecylamine and an organic solvent, and performing rotary evaporation to form a uniform lipid film;

[0043] Mixing the uniform lipid film with pure water at a temperature of 55-65° C. to perform a hydration reaction to form a liposome suspension;

[0044] The liposome suspension is ultrasonically treated in an ice-water bath to obtain the cationic liposome solution.

[0045] In some preferred embodiments, the organic solvent is anhydrous ethanol.

[0046] In a third aspect, the present invention provides use of the biomimetic glucan drug carrier in the preparation of drugs containing ginsenosides.

[0047] In a fourth aspect, the present invention provides a drug containing ginsenosides, comprising the biomimetic glucan drug carrier and ginsenosides, wherein the ginsenosides are encapsulated by the cationic liposomes in the biomimetic glucan drug carrier.

[0048] In some preferred embodiments, the drug containing ginsenoside is an anti-tumor drug containing ginsenoside.

[0049] In some preferred embodiments, the tumor is liver cancer.

[0050] Some typical embodiments are described below.

[0051] Example 1

[0052] Prepare ginsenoside Rh2 cationic liposomes according to the following steps:

[0053] Accurately weigh 15 mg of hydrogenated soy lecithin, 3 mg of cholesterol, 5 mg of octadecylamine, and 3 mg of ginsenoside, add 10 ml of anhydrous ethanol, and sonicate in a water bath to completely dissolve the raw materials;

[0054] Ethanol was removed by rotary evaporation at 40 rpm at 35°C for 45 min to form a uniform lipid film;

[0055] Add 60°C pure water for 30 minutes to form a liposome suspension;

[0056] The liposome suspension was sonicated in an ice-water bath (200w, 15min, 3s on 3s off) to reduce the particle size.

[0057] Example 2

[0058] Prepare ginsenoside Rh2 cationic liposomes according to the following steps:

[0059] Accurately weigh 15 mg of hydrogenated soy lecithin, 5 mg of cholesterol, 5 mg of octadecylamine, and 1 mg of ginsenoside, add 10 ml of anhydrous ethanol, and sonicate in a water bath to completely dissolve the raw materials;

[0060] The ethanol was removed by rotary evaporation at 40 rpm at 35°C for 45 minutes to form a uniform lipid film.

[0061] Add 60°C pure water for 30 minutes to form a liposome suspension;

[0062] The liposome suspension was sonicated in an ice-water bath (200w, 15min, 3s on 3s off) to reduce the particle size.

[0063] Example 3

[0064] Ginsenoside Rh2 cationic liposomes (abbreviated as Rh2-lips) were prepared according to the following steps:

[0065] Accurately weigh 15 mg of hydrogenated soy lecithin, 3 mg of cholesterol, 3 mg of octadecylamine, and 1 mg of ginsenoside, add 10 ml of anhydrous ethanol, and sonicate in a water bath to completely dissolve the raw materials;

[0066] Ethanol was removed by rotary evaporation at 40 rpm at 35°C for 45 min to form a uniform lipid film;

[0067] Add 60°C pure water for 30 minutes to form a liposome suspension;

[0068] The liposome suspension was sonicated in an ice-water bath (200w, 15min, 3s on 3s off) to reduce the particle size.

[0069] Example 4

[0070] Prepare blank cationic liposomes as follows:

[0071] Accurately weigh 15 mg of hydrogenated soy lecithin, 3 mg of cholesterol, and 3 mg of octadecylamine, add 10 ml of anhydrous ethanol, and sonicate in a water bath to completely dissolve the raw materials;

[0072] Ethanol was removed by rotary evaporation at 40 rpm at 35°C for 45 min to form a uniform lipid film;

[0073] Add 60°C pure water for 30 minutes to form a liposome suspension;

[0074] The liposome suspension was sonicated in an ice-water bath (200w, 15min, 3s on 3s off) to reduce the particle size.

[0075] Example 5

[0076] Blank biomimetic glucan drug carriers (abbreviated as lips GPs) were prepared according to the following steps:

[0077] Dissolve 20g of sodium hydroxide powder in 500ml of distilled water to obtain a 1M sodium hydroxide solution;

[0078] Weigh 20g of yeast powder, dissolve it in sodium hydroxide solution, place it in a shaker at 80℃ for one hour, centrifuge (3000rpm, 10min), and remove the supernatant;

[0079] Add 300 ml of distilled water and wash (stir evenly with a glass rod, centrifuge to remove the supernatant, and wash twice);

[0080] Add 300 ml of distilled water, adjust the pH to 4-5 with 2.5 M hydrochloric acid, place in a shaker at 60°C for one hour, centrifuge (3000 rpm, 10 min), and remove the supernatant;

[0081] Add 300 ml of distilled water and wash twice (stir evenly with a glass rod and centrifuge to remove the supernatant);

[0082] After washing four times with 100 mL of isopropanol, the mixture was washed twice with 100 mL of acetone, centrifuged and the supernatant was discarded. Yeast glucan particles (GPs) were obtained after drying.

[0083] 2.3 mg of yeast glucan particles were added to the blank cationic liposome solution prepared in Example 4. The mixture was stirred and incubated at room temperature for 1 hour, then centrifuged (3000 rpm, 10 min) to remove the supernatant. The precipitate was washed three times with water and placed in a -20°C environment for 1 day.

[0084] After two repeated freeze-thaw cycles, the mixture was freeze-dried to obtain lips GPs freeze-dried powder.

[0085] Example 6

[0086] The biomimetic glucan drug carrier containing ginsenoside Rh2 (abbreviated as Rh2-lipsGPs) was prepared according to the following steps:

[0087] Dissolve 20g of sodium hydroxide powder in 500ml of distilled water to obtain a 1M sodium hydroxide solution;

[0088] Weigh 20g of yeast powder, dissolve it in sodium hydroxide solution, place it in a shaker at 80℃ for one hour, centrifuge (3000rpm, 10min), and remove the supernatant;

[0089] Add 300 ml of distilled water and wash (stir evenly with a glass rod, centrifuge to remove the supernatant, and wash twice);

[0090] Add 300 ml of distilled water, adjust the pH to 4-5 with 2.5 M hydrochloric acid, place in a shaker at 60°C for one hour, centrifuge (3000 rpm, 10 min), and remove the supernatant;

[0091] Add 300 ml of distilled water and wash twice (stir evenly with a glass rod and centrifuge to remove the supernatant);

[0092] After washing 4 times with 100 mL of isopropanol, washing twice with 100 mL of acetone, centrifuging and discarding the supernatant, yeast glucan particles were obtained after drying.

[0093] 2.3 mg of yeast glucan particles were added to the ginsenoside Rh2 cationic liposome solution prepared in Example 3. The mixture was stirred and incubated at room temperature for 1 hour, then centrifuged (3000 rpm, 10 min) to remove the supernatant. The precipitate was washed three times with water and placed in a -20°C environment for 1 day.

[0094] After two repeated freeze-thaw cycles and freeze-drying, Rh2-lips GPs freeze-dried powder (a drug containing ginsenoside Rh2) was obtained.

[0095] The encapsulation efficiency of the cationic liposomes prepared in Example 1-3 was measured by liquid chromatography. Wherein, the liquid phase conditions are: the mobile phase consists of acetonitrile / water (60:40, v / v), the flow rate is 1.0 mL / min, and the injection volume is 10 μl. The DAD detector is set at 203 nm. Take an appropriate amount of cationic liposomes, add methanol to dilute five times to break the emulsion, and the concentration measured by liquid chromatography is C0; the cationic liposomes are first passed through a 0.45 μm microporous filter membrane to remove insoluble ginsenoside Rh2, and the filtrate is diluted five times with methanol to break the emulsion. The concentration measured by liquid chromatography is C1, and the filtrate is then ultrafiltered and centrifuged using a 50KD ultrafiltration tube (12000 rpm, 30 min), and the concentration of the outer tube solution is measured as C2; the encapsulation efficiency is calculated according to the following formula:

[0096] EE1(%)=(C1- C2) / C0 × 100%.

[0097] The encapsulation efficiency of the Rh2-lips GPs prepared in Example 6 was measured under the same conditions. Dextran powder and ginsenoside Rh2 cationic liposome solution were mixed and incubated for 1 hour, followed by centrifugation (3000 rpm, 10 min). The supernatant was collected and diluted five-fold with methanol to break the emulsion. The concentration was measured by liquid chromatography as C3. The encapsulation efficiency was calculated according to the following formula:

[0098] EE1(%)=(C1- C2-C3) / C0 × 100%.

[0099] The hydrated particle size, polydispersity index (PDI), and potential of the cationic liposomes prepared in Examples 1-4 were measured using a Malvern nanoparticle sizer. The cationic liposomes were dispersed in ultrapure water for analysis. All measurements were performed at 25°C, a scattering angle of 90°, and a laser wavelength of 659 nm.

[0100] Table 1 Characterization of cationic liposomes and biomimetic dextran drug carriers

[0101]

[0102] In the table, the results are expressed as mean ± standard deviation, and the number of measurements is n = 3; different letters in the same column indicate significant differences (P < 0.05).

[0103] The morphology of the product of Example 3 was observed by cold field emission scanning electron microscopy (SEM). An appropriate amount of the sample suspension was evenly applied on a silicon wafer, dried in a vacuum, and the surface morphology of the sample was observed after gold spraying. The results are shown in FIG. Figure 1 As shown, evenly dispersed spherical liposomes are clearly visible.

[0104] The morphology of the yeast glucan particles and the product of Example 6 was observed by scanning electron microscopy. Figure 2-Figure 4 As shown, yeast glucan particles (GPs) are extracted from yeast by an acid-base method. They are hollow porous cell wall microcapsules with β-1,3-D-glucan as the main component, and the particle size is about 2-4 μm. Compared with blank yeast glucan particles (GPs), the surface of the product of Example 6 is smooth without rough protrusions and pores.

[0105] In vitro digestion experiments were performed on the products of Example 3 and Example 6. The Rh2-lips suspension prepared in Example 3 and the Rh2-lips GPs suspension prepared in Example 6 were added to simulated gastric fluid (SGF: 3.2 mg / mL pepsin and 2.0 mg / mL sodium chloride), and the mixture was digested at 37.0°C and pH 1.2; samples were collected at 0, 30, 60, and 120 minutes; after 120 minutes of simulated gastric digestion, the pH value was adjusted to 7.5 using 0.1 mol / L sodium hydroxide solution to terminate gastric digestion; the remaining mixture was mixed with an equal volume of simulated intestinal fluid (SIF: 2.0 mg / mL pancreatin, 12.0 mg / mL bile salts, 6.8 mg / mL potassium dihydrogen phosphate, and 8.8 mg / mL sodium chloride), and samples were taken after 15, 30, 60, and 120 minutes, respectively.

[0106] The amount of ginsenoside Rh2 remaining in the carrier at different digestion times was measured by liquid chromatography. Figure 5-Figure 7 As shown in the figure, G0, G30, G60, and G120 represent digestion in simulated gastric fluid for 0, 30, 60, and 120 minutes, respectively, and I15, I30, I60, and I120 represent digestion in simulated intestinal fluid for 15, 30, 60, and 120 minutes, respectively. The products of Examples 3 and 6 remained stable during simulated gastric digestion, but during simulated intestinal digestion, the product of Example 3 was rapidly destroyed, releasing a large amount of ginsenoside Rh2, indicating that the liposomes were easily destroyed in intestinal fluid. The product of Example 6 showed no significant change in the content of ginsenoside Rh2 in the carrier in the intestine, indicating that the yeast glucan particles of Example 6 can protect ginsenoside Rh2 from degradation in the gastrointestinal tract.

[0107] The phagocytic effect of macrophages on the Rh2-lips prepared in Example 3 and the Rh2-lips GPs prepared in Example 6 was tested. Coumarin 6 (C6) was used to label the Rh2-lips prepared in Example 3 and the Rh2-lips GPs prepared in Example 6. RAW.264.7 cells were cultured at a density of 1×10 5 Cells were seeded at a density of 100 cells / well in confocal culture dishes. After 12 hours of culture, free coumarin 6 (control group), coumarin 6-labeled Rh2-lips (Rh2-lips group), and Rh2-lips GPs (Rh2-lipsGPs group) were added and incubated at 37°C for 4 hours. The cells were fixed with 4% paraformaldehyde and washed three times. Macrophages were stained with 4',6-diamidino-2-phenylindole (DAPI) and washed three times with PBS. The intensity of fluorescence colocalization between different treatment groups was observed under an inverted fluorescence microscope, and the phagocytosis of Rh2-lips and Rh2-lips GPs by RAW264.7 cells was analyzed.

[0108] To further investigate the targeting mechanism, an additional group of laminarin plus Rh2-lips GPs prepared in Example 6 was added, and RAW.264.7 cells were treated with laminarin (a dectin-1 inhibitor) 15 minutes before drug addition.

[0109] The results are as follows Figure 8 As shown in the figure, compared with the control group, the co-localization fluorescence of the Rh2-lips group was stronger, and the Rh2-lips GPs group showed the strongest fluorescence co-localization intensity; after adding laminarin, the dectin-1 receptor on the surface of macrophages was inhibited, and the co-localization fluorescence intensity was significantly reduced, indicating that Rh2-lips GPs enhanced the uptake of ginsenoside Rh2 by macrophages RAW.264.7, and it was mainly mediated by the dectin-1 receptor.

[0110] Animal experiments were conducted using 80 SPF female C57BL / 6J mice (4-6 weeks old). The mice were adaptively fed for 7 days. Resuscitated H22 hepatocellular carcinoma ascites cells were injected into the mouse peritoneal cavity. After culturing the ascites for 5-7 days, the ascites was removed and three volumes of red blood cell lysis buffer were added. The cells were reacted at 4°C for 2 minutes, centrifuged (1000 rpm, 4°C, 5 minutes), and the supernatant was removed. The cells were washed once with saline and resuspended. The H22 hepatocellular carcinoma cells were stained with trypan blue to detect viability. When the viability reached above 95%, the cells were diluted to a density of 1×10 cells. 7 / ml, subcutaneously injected into the right armpit of the mouse to be modeled, 0.2ml / mouse. Mice with successful modeling and similar tumor size were included in the experiment, and the experimental mice were divided into model group and sample group according to the random number method. Experimental sample groups: normal saline (model group), free ginsenoside Rh2, Rh2-lips prepared in Example 3, lips GPs prepared in Example 5, Rh2-lips GPs prepared in Example 6. During the experiment, all mice had free food and water, and the tumor volume and body weight of the mice were measured every three days. Gavage once a day for 12 consecutive days, the dosage was based on the concentration of ginsenoside Rh2 (12 mg / kg), and the Example 5 group was based on the GPs concentration in the Example 6 group. Detect or calculate the following indicators:

[0111] (1) Sample collection: At the end of the experiment, mice were fasted but not deprived of water for 12 h, and blood was collected from the eyeballs after isoflurane inhalation anesthesia. After the mice were killed, they were placed supine on the animal testing table, their limbs were fixed, and the hair was removed. The chest and abdomen were exposed, and the organs and tumor tissues were removed and weighed.

[0112] (2) Tumor inhibition rate TGI = 100% × (Tvol Control -Tvol Treated ) / Tvol Control, Tvol=final tumorvolume-initial tumor volume, where, Tvol Control Indicates the change in tumor volume during the model group experiment, Tvol Treated represents the change in tumor volume during the experiment of the sample group, final tumor represents the tumor volume at the end of the experiment, and initial tumor volume represents the tumor volume at the time of grouping;

[0113] (3) Serological index detection: The collected blood was placed at 4°C for 2 h, centrifuged at 3000 rpm for 15 min, and the serum was aspirated and divided into 1.5 mL centrifuge tubes. A portion of the serum was taken and the contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (BUN), and creatinine (CRE) were detected using an automatic biochemical analyzer;

[0114] (4) Immune factor detection: ELISA was used to determine the levels of tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), and interleukin-10 (IL-10) in serum samples, and immunoblotting was used to detect the levels of tumor necrosis factor-α (TNF-α), interleukin-2 (IL-2), and interleukin-10 (IL-10) in tumor tissues;

[0115] (5) Macrophage phenotype: On the day of sample collection, appropriate amount of tumor tissue was taken, 2 ml of collagenase was added, and the mixture was reacted at 4 °C for 1 hour. The mixture was then ground and 2 ml of fresh culture medium was added intermittently. The mixture was passed through a 200-mesh cell sieve to prepare a single-cell suspension. After centrifugation (2000 rpm, 5 min, 4 °C), the supernatant was removed and a 3% BSA PBS solution containing antibody fluorescence (where each ml of PBS contains 0.5 μl CD206 and 0.5 μl CD86). After staining for half an hour, the mixture was centrifuged, washed twice with PBS, and resuspended with sheath fluid. Macrophage phenotype analysis was performed by upflow cytometry.

[0116] The experimental data were statistically analyzed using SPSS 19.0 software, and the t-test was used to analyze the significance of the difference between the two groups. P < 0.05 indicated a significant difference.

[0117] like Figure 9 and Figure 10 As shown, compared with the other groups, the tumor in the Rh2-lips GPs experimental group prepared in Example 6 grew slowly and the tumor volume was significantly reduced (P < 0.05), indicating that the Rh2-lips GPs prepared in Example 6 had a good tumor inhibitory effect on mice.

[0118] Table 2 shows the ratio of M2 macrophages to M1 macrophages in the tumor tissues of each group of animals. It can be seen that the Rh2-lips GPs prepared in Example 6 significantly reduced the M2 / M1 value in the tumor tissues, indicating that the Rh2-lips GPs prepared in Example 6 enhanced the efficacy of ginsenoside Rh2 in reversing the macrophage phenotype.

[0119] Table 2 Ratio of M2 macrophages to M1 macrophages in tumor tissues of animals in each group

[0120]

[0121] In the table, the results are expressed as mean ± standard deviation, and the number of measurements is n = 3; different letters in the same column indicate significant differences (P < 0.05).

[0122] In addition, animal experiments were conducted to study the targeting of drugs to tumor tissues. The animals used were SPF female BALB / c mice (4-6 weeks old). The mice were adaptively fed for 7 days, and the resuscitated H22 liver cancer ascites cells were injected into the mouse peritoneal cavity. After the ascites was cultured for 5-7 days, the ascites was removed and 3 times the volume of red blood cell lysis buffer was added. The cells were reacted at 4°C for 2 minutes and centrifuged (1000 rpm, 4°C, 5 minutes). The supernatant was removed, the cells were washed once with physiological saline and resuspended. The activity of the H22 liver cancer cells was detected by trypan blue staining. When the cell density reached more than 95%, the cell density was diluted to 1×10 7 0.2 ml / mouse was injected subcutaneously into the right armpit of the mouse to be modeled. The tumor grew to about 400 mm. 3 The mice were then randomly divided into an ICG control group (indocyanine green), an ICG-lips group (Rh2-lips from Example 3 labeled with indocyanine green), and an ICG-lips GPs group (Rh2-lips GPs from Example 6 labeled with indocyanine green), with three mice in each group. They were administered ICG at a concentration of 50 μg / ml by gavage. In vivo imaging of the animals was performed 4, 8, and 24 hours after administration. After 24 hours, the mice were sacrificed by cervical dislocation, and the tumors, hearts, livers, spleens, lungs, and kidneys were removed for ex vivo imaging.

[0123] like Figure 11 and Figure 12 As shown, at the same time point after oral gavage, the fluorescence signal in the ICG-lips group was significantly stronger than that in the ICG control group, with the ICG-lips GPs group exhibiting the strongest fluorescence signal. Fluorescence peaked in all groups 4 hours after oral gavage, then decreased over time. 24-hour ex vivo imaging of major organs and tumors also revealed the strongest fluorescence signal in the tumors of the ICG-lips GPs group, followed by the ICG-lips group. These results demonstrate that the constructed carrier (lips GP) enhances drug targeting to tumor tissue.

[0124] While the present invention is illustrated by the above-described embodiments, the present invention is not limited to these embodiments, nor does it necessarily rely on these embodiments for implementation. Those skilled in the art will appreciate that any improvements to the present invention, equivalent replacements for raw materials in the products of the present invention, additions of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

Claims

1. A biomimetic glucan drug carrier for use in the preparation of a drug containing ginsenosides, characterized in that: The biomimetic glucan drug carrier comprises a cationic liposome and a yeast cell wall cystic structure encapsulating the cationic liposome, wherein the yeast cell wall cystic structure is a hollow yeast cell wall, and the cationic liposome is used to encapsulate the ginsenoside; the ginsenoside is ginsenoside Rh2; The combination of ginsenosides and yeast cell wall cystic structures can synergistically induce TAMs to repolarize from the M2 phenotype to the M1 phenotype; The preparation method of the cationic liposome solution comprises the following steps: Mixing hydrogenated soybean lecithin, cholesterol, octadecylamine and an organic solvent, and performing rotary evaporation to form a uniform lipid film; wherein the cationic liposome comprises the following raw material components in parts by weight: 30 parts hydrogenated soybean lecithin, 6 parts cholesterol and 6 parts octadecylamine; Mixing the uniform lipid film with pure water at a temperature of 55-65° C. to perform a hydration reaction to form a liposome suspension; The liposome suspension is ultrasonically treated in an ice-water bath to obtain the cationic liposome solution.

2. A medicament containing ginsenosides, characterized in that: The invention comprises a biomimetic glucan drug carrier and ginsenoside, wherein the biomimetic glucan drug carrier comprises a cationic liposome and a yeast cell wall cystic structure encapsulating the cationic liposome, and the ginsenoside is encapsulated by the cationic liposome in the biomimetic glucan drug carrier; wherein the yeast cell wall cystic structure is a hollow yeast cell wall, and the cationic liposome is used to encapsulate the ginsenoside; and the ginsenoside is ginsenoside Rh2; The combination of ginsenosides and yeast cell wall cystic structures can synergistically induce TAMs to repolarize from the M2 phenotype to the M1 phenotype; The preparation method of the cationic liposome solution comprises the following steps: Mixing hydrogenated soybean lecithin, cholesterol, octadecylamine and an organic solvent, and performing rotary evaporation to form a uniform lipid film; wherein the cationic liposome comprises the following raw material components in parts by weight: 30 parts hydrogenated soybean lecithin, 6 parts cholesterol and 6 parts octadecylamine; Mixing the uniform lipid film with pure water at a temperature of 55-65° C. to perform a hydration reaction to form a liposome suspension; The liposome suspension is ultrasonically treated in an ice-water bath to obtain the cationic liposome solution.

3. The medicament containing ginsenosides according to claim 2, characterized in that The medicine containing ginsenoside is an anti-liver cancer medicine containing ginsenoside.

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