Paclitaxel ganoderma lucidum spore oil self-nanoemulsion composite particle taking ganoderma lucidum spore as carrier as well as preparation method and application of paclitaxel ganoderma lucidum spore oil self-nanoemulsion composite particle

Abstract

The invention relates to paclitaxel ganoderma lucidum spore oil self-nanoemulsion composite particles with ganoderma lucidum spores as a carrier and a preparation method of the paclitaxel ganoderma lucidum spore oil self-nanoemulsion composite particles, and belongs to the technical field of medicines. Pretreated Ganoderma lucidum spores (GLS) are obtained through process screening and serve as a drug carrier, and paclitaxel-loaded Ganoderma lucidum spore oil self-nanoemulsion (PGS) is loaded into the Ganoderma lucidum spores (GLS) to obtain paclitaxel-loaded composite particles (PGS (at) GLS). An in-vitro release curve shows that the PGS-atGLS has the characteristic of slow release after reaching the intestinal tract; in-vivo pharmacodynamic experiments show that PGS-atGLS can significantly inhibit development and metastasis of mouse colorectal cancer; tumor tissue slice immunofluorescence analysis shows that PGS-atGLS can effectively play a role in treating colon cancer. The paclitaxel self-nanoemulsion-loaded ganoderma lucidum spore composite particles synergistically promote cancer immune circulation by inducing immunogen cell death and immunoregulation, significantly improve the treatment effect of colon cancer, and have good biological safety.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and in particular relates to a paclitaxel and ganoderma lucidum spore oil self-nanoemulsion composite particle using ganoderma lucidum spore as a carrier and a preparation method thereof. Background Art

[0002] Colorectal cancer (CRC) is a common digestive tract cancer originating from the large intestinal epithelial tissue. It mainly occurs in middle-aged and elderly people over 40 years old and has a high mortality rate. Lymph node metastasis is one of the most important reasons for the poor prognosis of colorectal cancer patients. However, conventional chemotherapy or other oral drugs have difficulty reaching the lymph nodes and cannot effectively inhibit the occurrence and development of micrometastatic lesions therein.

[0003] The occurrence of cancer is the result of gene mutation and changes in normal cell regulation processes. The body's autoimmunity will respond and a series of reactions will occur, including: tumor cells release tumor antigens, which are captured by dendritic cells and presented to T cells, initiating and activating the response of effector T cells to cancer-specific antigens. The activated effector T cells are transported and infiltrated into tumor lesions, specifically identifying and binding to cancer cells, and ultimately killing tumor cells; and the killed cancer cells will release more tumor-associated antigens; the above process is repeated, which is called the cancer immune cycle. However, without drug intervention, the tumor site may lack antigens and presentation, resulting in the inability to effectively start and operate the above process, which ultimately leads to the continuous development and metastasis of the tumor.

[0004] Paclitaxel (PTX) is a tetracyclic diterpenoid organic compound extracted and separated from the bark of natural Taxus brevifolia. It inhibits the proliferation of tumor cells by destroying the dynamic balance of continuous polymerization and depolymerization between tubulin and microtubules. It has good anti-tumor effects and is mostly used in the form of injections for the treatment of various cancers. However, injection administration brings great inconvenience to patients and causes hypersensitivity reactions and serious side effects in many patients. However, the poor water solubility and permeability of paclitaxel make the development of its oral preparations face many problems. In addition, drug resistance of paclitaxel is also one of the important factors affecting its effective anti-tumor effect. Therefore, it is crucial to improve the efficacy of the drug by combining it with other active ingredients and improve its solubility and permeability through preparations to effectively deliver it into the body.

[0005] Ganoderma lucidum spore oil (GLSO) is a light yellow to yellow clear oily liquid extracted from the reproductive cells of the traditional Chinese medicine Ganoderma lucidum, i.e., Ganoderma spores. Its main components include Ganoderma triterpenes and fatty acids. It has the effects of inhibiting tumor cell proliferation and regulating immunity. Its immunoregulatory mechanism is related to enhancing the phagocytic ability of macrophages, increasing the number of lymphocytes, improving the CD4+ / CD8+T cell ratio, promoting the maturation of dendritic cells and increasing the levels of cytokines such as IFN-γ, TNF-α, and IL-1 in the body. However, Ganoderma lucidum spore oil is not easy to preserve and is easily oxidized. Its effect is limited by its low water solubility. For some special populations, it is difficult to absorb due to insufficient bile secretion, thus affecting its full immunoregulatory effect. Therefore, improving its solubility and promoting its effective absorption through preparations are the prerequisites for fully exerting its therapeutic effect.

[0006] Ganoderma lucidum spore (GLS) is the reproductive cell of the traditional Chinese medicine Ganoderma lucidum. It has an oval cystic structure with a double-wall structure and a diameter of about 5μm. The cell wall of Ganoderma lucidum spores is rich in Ganoderma lucidum β-glucan, which cannot be digested in the stomach and small intestine and is degraded by bacterial fermentation in the colon. It is reported that β-glucan is selectively recognized by receptors such as Dectin-1, complement receptor 3 (CR3) or Toll-like receptors (TLRs) expressed on the surface of immune cells in the form of pathogen-associated molecular patterns, and specifically binds to immune cells, thereby stimulating the occurrence of adaptive immune responses and exerting anti-tumor effects.

[0007] Designing a composite microparticle drug delivery system that encapsulates multiple active ingredients and simultaneously delivers chemotherapy drugs and immunomodulatory active components is one of the effective strategies to promote cancer immune circulation and improve the therapeutic effect of drugs on tumors. Self-Nanoemulsifying Drug Delivery System (SNEDDS) is a homogeneous liquid composed of an oil phase, a drug, and a surfactant. It can be rapidly and spontaneously emulsified into a nanoemulsion when it comes into contact with water, and then undergoes lipolysis and transepithelial transport, and finally excretes cells in the form of chylomicrons, enters the lacteals and lymphatic system, thereby increasing the lymphatic transport of drugs and improving the oral bioavailability of poorly soluble drugs. SNEDDS is used to simultaneously deliver the poorly soluble chemotherapy drug paclitaxel and the immunomodulatory active component Ganoderma lucidum spore oil. Its key feature is that the component that exerts the drug effect is used as the oil phase required for the preparation, and the drug and adjuvant are combined while improving the solubility of the above two drug components, thereby improving the delivery efficiency. The modified Ganoderma lucidum spores are then used as a natural drug reservoir, and the drugs are encapsulated by vacuum adsorption, which improves the drug stability while effectively avoiding the premature emulsification of paclitaxel and Ganoderma lucidum spore oil in the stomach, increasing the entry of drugs into the lymphatic system through the intestine, thereby improving the therapeutic effect on colorectal cancer.

[0008] The delivery of paclitaxel preparations and their therapeutic effects on tumors in the existing technology also need further research and improvement. Summary of the invention

[0009] The purpose of the present invention is to provide a paclitaxel-loaded Ganoderma lucidum spore composite microparticle for treating colorectal cancer and a preparation method thereof. The paclitaxel-loaded Ganoderma lucidum spore composite microparticle prepared by the present invention can simultaneously deliver chemotherapeutic drugs and immunomodulatory active components, improve drug solubility, and promote drug absorption via oral lymphatic transport pathway.

[0010] The drugs and excipients involved include paclitaxel, Ganoderma lucidum spore oil, emulsifiers and co-emulsifiers.

[0011] Among them, paclitaxel is a clinically recognized chemotherapy drug that has the effect of causing immunogenic cell death of tumor cells; Ganoderma lucidum spore oil and Ganoderma lucidum spores have a regulatory effect on stimulating immunity; emulsifiers have gelling and emulsifying properties, which can cause self-nanoemulsions to undergo a gel state before emulsification. Combined with the porous load of Ganoderma lucidum spores, it can avoid premature emulsification and release of drugs in the stomach, so that more drugs can be absorbed into the body through the intestinal lymphatic pathway.

[0012] The applicant used a self-emulsifying drug delivery system to prepare paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion, and effectively improved the solubility of the two drugs by selecting suitable emulsifiers and co-emulsifiers. Then, hollow and porous blank Ganoderma lucidum spore microparticles that still retained their intact form were obtained through multiple acid and alkali treatments, and the drug-loaded self-nanoemulsion was loaded into the Ganoderma lucidum spore microparticles by negative pressure adsorption, thereby obtaining Ganoderma lucidum spore composite microparticles loaded with paclitaxel.

[0013] The present invention is achieved through the following technical solutions:

[0014] A paclitaxel-loaded Ganoderma lucidum spore composite microparticle, characterized in that it is composed of two major parts: paclitaxel Ganoderma lucidum spore oil self-nanoemulsion (PGS) and Ganoderma lucidum spore (GLS);

[0015] The mass ratio of the Ganoderma lucidum spores to the Paclitaxel Ganoderma lucidum spore oil self-nanoemulsion is 1:5-10;

[0016] The paclitaxel ganoderma spore oil self-nanoemulsion comprises the following components:

[0017] (a), paclitaxel (PTX);

[0018] (b), Ganoderma lucidum spore oil (GLSO);

[0019] (c), an emulsifier, wherein the emulsifier is selected from one of polyoxyethylene 40 hydrogenated castor oil (RH40), Tween 80, lauroyl polyoxyethylene-32 glyceride or caprylic / capric macrogol glyceride;

[0020] (d) an emulsifier, wherein the emulsifier is selected from propylene glycol monolaurate, polyethylene glycol 400, 1,2-propylene glycol, diethylene glycol monoethyl ether or propylene glycol monolaurate.

[0021] The paclitaxel ganoderma spore oil self-nanoemulsion has a PTX content of 0.08-0.64%, a GLSO content of 10-40%, an emulsifier content of 27-72%, an auxiliary emulsifier content of 14-60%, and a stirring speed of 200-600 rpm to obtain paclitaxel-ganoderma spore oil self-nanoemulsion (PTX-GLSO SMEDDS, PGS).

[0022] The average emulsified particle size of PGS is 26 nm, the polydispersity index (PDI) is about 0.08, the drug loading (calculated as paclitaxel) ranges from 0.15% to 1.41%, and the encapsulation efficiency can be as high as 90.53%.

[0023] The method for preparing the paclitaxel-loaded Ganoderma lucidum spore composite microparticles is characterized by comprising the following steps:

[0024] (a) Preparation of Paclitaxel Ganoderma Lucidum Spore Oil Nanoemulsion (PGS) Pre-Concentrate:

[0025] Weighing GLSO, an emulsifier and an auxiliary emulsifier in a prescribed proportion, stirring at room temperature to completely mix them, and obtaining a Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate containing 10-40% of GLSO by mass;

[0026] PTX in the prescribed proportion is added to the above pre-concentrated solution, stirred in the dark to completely dissolve and mix evenly to obtain PGS.

[0027] (b) Preparation of defatted Ganoderma lucidum spores:

[0028] The degreasing treatment method is selected from one of solvent extraction, ultrasound-assisted extraction, supercritical CO2 extraction, and gradient centrifugation. The following is a detailed description of the steps using solvent extraction as an example:

[0029] Disperse a certain mass of Ganoderma lucidum spores in a certain volume of acetone (control the ratio of acetone to spores to be 10-20:1 (V / W)), the unit is mL / g, stir and reflux for 8-24h, control the stirring speed to be 200-600rpm, and control the reflux temperature to be 58-65°C;

[0030] The organic solvent in the reflux mixture is removed by suction filtration and the mixture is naturally dried.

[0031] (c) Alkali-treated Ganoderma lucidum spore particles:

[0032] Dispersing the defatted and naturally dried Ganoderma lucidum spores in a potassium hydroxide solution of a certain concentration (the ratio of dry spores to alkaline solution is controlled to be 1:20-25 (W / V)), and the potassium hydroxide concentration range is 6-18%;

[0033] Reflux in a water bath with stirring for 2-6h, water bath temperature 70-80°C, stirring speed 200-600rpm;

[0034] Centrifuge at 3500-4500 rpm for 15-20 min, discard the supernatant, and wash with hot distilled water 3-6 times to remove alkali-soluble impurities.

[0035] (d) Acid-treated Ganoderma lucidum spore particles:

[0036] The alkaline treated Ganoderma lucidum spores are dispersed in phosphoric acid (the ratio of spores to phosphoric acid is controlled to be 1:15-1:25 (W / V)), the mass concentration of phosphoric acid is 80-85%, heated in a water bath with stirring for 2-6 hours, the water bath temperature is 40-50° C., and the stirring speed is 200-600 rpm.

[0037] (e) Washing the Ganoderma lucidum spore particles:

[0038] Disperse the above spore phosphoric acid suspension in hot distilled water, control the water temperature to 60-65°C, control the volume ratio of original spore mass to distilled water to be 1:100-1:120, and stir until completely mixed;

[0039] Centrifuge at 3500-4500 rpm for 15-20 min, discard the supernatant, and repeat the above operation 3-6 times to remove acid-soluble impurities and residual phosphoric acid until the pH of the supernatant is close to neutral (control pH 6.0-7.0).

[0040] (f) Alcohol-washed Ganoderma lucidum spore particles:

[0041] Disperse the washed Ganoderma lucidum spores in anhydrous ethanol (control the ratio of spores to anhydrous ethanol to be 1:10-1:20 (W / V)), and stir until the dispersion is uniform;

[0042] Centrifuge at 3500-4500 rpm for 15-20 min, discard the supernatant, and repeat the above operation 2-4 times.

[0043] (g) Dry Ganoderma lucidum spore particles:

[0044] The spores after alcohol washing were placed in a vacuum drying oven and dried under reduced pressure to a constant weight (temperature controlled at 60-65° C.), thereby obtaining a blank carrier of Ganoderma lucidum spores.

[0045] (h) Drug loading: Vacuum negative pressure drug loading:

[0046] The drug loading method is selected from one of vacuum decompression method, stirring method and centrifugation method;

[0047] Preferably, the drug loading method is a vacuum decompression method, which is simple and easy to operate and convenient for mass production. The following is a detailed description of the steps using the vacuum decompression method as an example;

[0048] Vortex mixing the paclitaxel Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate and the blank Ganoderma lucidum spore particles, and controlling the mass ratio of the two to be 5-10:1;

[0049] Place the above mixture in a vacuum drying oven, control the temperature at 28-32°C, control the pressure at -0.01-0.08MPa, and maintain for 2-4h;

[0050] The vacuum loaded self-nanoemulsion and spore mixture was taken out and centrifuged at 3500-4500rpm for 15-20min to collect the drug-loaded spores in the lower layer. The spore surface was rinsed with ethanol and dried naturally at room temperature to obtain Ganoderma lucidum spore composite particles (PGS@GLS) loaded with paclitaxel Ganoderma lucidum spore oil self-nanoemulsion.

[0051] The obtained PGS@GLS needs to be stored in a sealed state at ≤25°C.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] (1) The technical obstacle faced in loading liquid self-nanoemulsion into the porous hollow spore capsule of Ganoderma lucidum is the leakage of self-nanoemulsion. The self-nanoemulsion prepared by the present invention adopts a specific emulsification system. After the Ganoderma lucidum spores enter the body and encounter the water environment, they will be in a gel state, effectively preventing the drug from being released from the spores too early.

[0054] (2) Efficacy: The combination of paclitaxel and ganoderma spore oil and ganoderma spore can significantly increase the expression of calreticulin in tumor tissues, the infiltration of mature dendritic cells, and the ratio of CD8+T lymphocytes and mature dendritic cells in mesenteric lymph nodes. The above-mentioned calreticulin is a marker protein that triggers immunogenic cell death (the initial step of the cancer immune cycle), mature dendritic cells play an antigen presentation role (the second step of the cancer immune cycle), and CD8+T lymphocytes are important immune cells that eliminate tumor cells (the third and fourth steps of the cancer immune cycle). They work together to promote immunity and enhance anti-tumor effects.

[0055] (3) Based on the oral absorption characteristics of self-nanoemulsion, by encapsulating paclitaxel Ganoderma lucidum spore oil in Ganoderma lucidum spore microcapsules, the drug can be released more slowly and lastingly, specifically increasing the concentration of the drug in the mesenteric lymph nodes, thereby improving the therapeutic effect of the drug on intestinal tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Dried Ganoderma lucidum spores before (left) and after (right) treatment;

[0057] Figure 2 A is the morphology of Ganoderma lucidum spores before treatment under an inverted phase contrast microscope. Figure 2 B is the morphology of Ganoderma lucidum spores after treatment under an inverted phase contrast microscope;

[0058] Figure 3 A is a scanning electron microscope image of Ganoderma lucidum spores before treatment. Figure 3 B is the scanning electron micrograph of Ganoderma lucidum spores after treatment;

[0059] Figure 4 A is the transmission electron microscopy image of Ganoderma lucidum spores before treatment. Figure 4 B is the transmission electron microscopy image of Ganoderma lucidum spores after treatment;

[0060] Figure 5 The appearance of the nanoemulsion was taken by transmission electron microscopy;

[0061] Figure 6 In vitro cumulative release of paclitaxel from PGS@GLS and PTX@GLS (mean±SD, n=6);

[0062] Figure 7 A is a schematic diagram showing the changes of drug fluorescence distribution in mesenteric lymph nodes over time. Figure 7 B is the graph showing the changes in the mean fluorescence intensity of mesenteric lymph nodes over time in vivo;

[0063] Figure 8 A is a representative picture of the in vivo imaging results of each group of animals (n=6), Figure 8 B is the image of the average fluorescence intensity of the abdomen of mice in the model group changing over time. Figure 8 C is the image of the average fluorescence intensity of the abdomen of mice in the PTX iv group changing over time. Figure 8 D is the image of the average fluorescence intensity of the abdomen of mice in the GLS group changing over time. Figure 8 E is the image of the average fluorescence intensity of the abdomen of mice in the PGS group changing over time. Figure 8 F is the image of the average fluorescence intensity of the abdomen of mice in the PGS@GLS group changing over time;

[0064] Fig. 9 The weight changes of mice;

[0065] Fig.10 The results of the mouse body condition scoring are shown in Table 1 (mean ± SD, n = 6);

[0066] Fig.11 A is the anatomical diagram of the development of intestinal tumors in mice in each drug-treated group. Fig.11 B is the statistical chart of the number of intestinal tumors in each drug-treated group. Fig.11 C is the analysis of colorectal tumor mass of mice in each drug-treated group, mean±SD, n=3);

[0067] Fig.12 A is a schematic diagram of the flow cytometric analysis results of mature dendritic cells in the tumor tissues of mice in each drug-treated group; Fig.12 B is a schematic diagram of the flow cytometric analysis results of CD8+T cells in the tumor tissues of mice in each drug-treated group; Fig.12 C is the quantitative results of mature dendritic cells in the tumor tissues of mice in each drug-treated group; Fig.12 D is the quantitative results of mature dendritic cells in the mesenteric lymph nodes of mice in each drug-treated group; Fig.12 E is the quantitative results of CD8+T cells in the tumor tissues of mice in each drug-treated group; Fig.12 F is the quantitative results of CD8+T cells in the mesenteric lymph nodes of mice in each drug-treated group.

[0068] Fig.13 The immunofluorescence staining results of calreticulin and HMGB1 in tumor tissues of mice in each drug-treated group (scale bar = 50 μm);

[0069] Fig.14The results of immunofluorescence staining of tumor tissue cell proliferation and apoptosis in each drug-treated group (scale bar = 50 μm);

[0070] Fig.15 The infiltration of CD8+T lymphocytes in the tumor tissue of each drug-treated group;

[0071] Fig.16 The statistical graphs of the counts of white blood cells (WBC), lymphocytes (Lymph), platelets (PLT), red blood cells (RBC), and hemoglobin (HGB) in the blood of mice in each drug administration group (mean±SD, n=3, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001);

[0072] Fig.17 Results of H&E staining of the main organs and tumor tissue sections of mice in each drug-treated group;

[0073] Fig.18 Schematic diagram of PGS@GLS composite particles activating the cancer immune cycle and inhibiting the development of colorectal cancer. DETAILED DESCRIPTION

[0074] Example 1

[0075] 1) Accurately weigh 20 g of Ganoderma lucidum spores, place in 400 mL of acetone, stir and reflux at 65° C. for 12 h, filter, and dry naturally to obtain defatted Ganoderma lucidum spores;

[0076] 2) Weigh 16 g of the dried Ganoderma lucidum spores after acetone defatting, add them to 400 mL of 12% (W / V) potassium hydroxide solution, reflux and stir at 80° C. water bath for 2 h; centrifuge at 4200 rpm for 15 min, discard the supernatant, resuspend with 60° C. distilled water, stir and wash, centrifuge, repeat three times, and obtain the Ganoderma lucidum spores after alkali treatment;

[0077] 3) Redispersing in 320 mL of phosphoric acid, stirring in a water bath at 40° C. for 2 h, to obtain alkali- and acid-treated Ganoderma lucidum spores;

[0078] 4) Disperse the above spore phosphate suspension in 2000 mL hot distilled water (60° C.), stir for 0.5 h until completely mixed, centrifuge at 4200 rpm for 15 min, and discard the supernatant; repeat the above water washing operation 3 times, and the pH of the supernatant is close to neutral;

[0079] 5) Re-disperse the washed Ganoderma lucidum spores in 200 mL of anhydrous ethanol, stir until uniformly dispersed, centrifuge at 4200 rpm for 15 min, and discard the supernatant; repeat the above operation twice;

[0080] 6) The spore precipitate was dispersed in 50 mL of anhydrous ethanol, evaporated at room temperature until no obvious liquid was present, and then moved to a vacuum drying oven at 60°C and dried under reduced pressure to constant weight to obtain treated Ganoderma lucidum spore microparticles (GLS). The treated Ganoderma lucidum spores were in the form of loose dispersed microparticles with slightly darker color. Figure 1 .

[0081] The GLS in Example 1 was observed under an inverted microscope. It was found that compared with the untreated Ganoderma lucidum spores, the GLS was hollow, the double wall was thinner, and one end of the spore was open. Figure 2 .

[0082] Example 2

[0083] RH40 5150 mg, L90 3000 mg, and Ganoderma lucidum spore oil 1850 mg were accurately weighed, and magnetic stirring was performed at room temperature until they were completely mixed to obtain Ganoderma lucidum spore oil self-nanoemulsion pre-concentrated solution.

[0084] Example 3

[0085] 14.8 mg of paclitaxel was accurately weighed and placed in the pre-concentrated solution of Ganoderma lucidum spore oil from nanoemulsion obtained in Example 2, and magnetically stirred at room temperature in the dark until completely dissolved to obtain paclitaxel-Ganoderma lucidum spore oil from nanoemulsion pre-concentrated solution (PGS).

[0086] Example 4

[0087] 1) Accurately weigh 7.4 mg of paclitaxel, dissolve it in 1000 μL of anhydrous ethanol, and then mix it with 1000 mg of GLS in Example 1, and vortex until completely mixed;

[0088] 2) Place the above mixture in a vacuum drying oven at -0.01 MPa for 2 h;

[0089] 3) After taking out, centrifuge at 4200 rpm for 15 min, collect the drug-loaded spores in the lower layer, rinse with 95% ethanol, and finally dry naturally at room temperature to obtain Ganoderma lucidum spores loaded with the raw material drug paclitaxel (PTX@GLS).

[0090] Example 5

[0091] 1) 5000 mg of the Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate in Example 2 was taken, mixed with 1000 mg of the GLS in Example 1, and vortexed until completely mixed;

[0092] 2) Place the above mixture in a vacuum drying oven at -0.01 MPa for 2 h;

[0093] 3) After taking out, centrifuge at 4200 rpm for 15 min, collect the drug-loaded spores in the lower layer, rinse with 95% ethanol, and finally dry naturally at room temperature to obtain Ganoderma lucidum spore composite particles (GS@GLS) loaded with Ganoderma lucidum spore oil self-nanoemulsion.

[0094] Example 6

[0095] 1) 5000 mg of the paclitaxel Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate in Example 3 was taken, mixed with 1000 mg of the GLS in Example 1, and vortexed until completely mixed;

[0096] 2) Place the above mixture in a vacuum drying oven at -0.01 MPa for 2 h;

[0097] 3) After taking out, centrifuge at 4200 rpm for 15 min, collect the drug-loaded spores in the lower layer, rinse with 95% ethanol, and finally dry naturally at room temperature to obtain paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion-loaded Ganoderma lucidum spore composite particles (PGS@GLS).

[0098] Example 7

[0099] 4500 mg of Tween 80, 3000 mg of L90 and 2500 mg of Ganoderma lucidum spore oil were accurately weighed, and magnetic stirring was performed at room temperature until the mixture was completely mixed to obtain a Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate.

[0100] 20 mg of paclitaxel was accurately weighed and placed in the above-obtained pre-concentrated solution of Ganoderma lucidum spore oil from nanoemulsion, and magnetically stirred at room temperature in the dark until completely dissolved to obtain paclitaxel-Ganoderma lucidum spore oil from nanoemulsion pre-concentrated solution (PGS).

[0101] The method for preparing the Ganoderma lucidum spore composite microparticles loaded with paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion from the paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate is the same as that in Example 6.

[0102] Example 8

[0103] 5250 mg of lauroyl polyoxyethylene 32 glyceride, 3000 mg of L90, and 1750 mg of Ganoderma lucidum spore oil were accurately weighed, and magnetic stirring was performed at room temperature until the mixture was completely mixed to obtain a Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate.

[0104] 28 mg of paclitaxel was accurately weighed and placed in the above-obtained pre-concentrated solution of Ganoderma lucidum spore oil from nanoemulsion, and magnetically stirred at room temperature in the dark until completely dissolved to obtain paclitaxel-Ganoderma lucidum spore oil from nanoemulsion pre-concentrated solution (PGS).

[0105] The method for preparing the Ganoderma lucidum spore composite microparticles loaded with paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion from the paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate is the same as that in Example 6.

[0106] Example 9

[0107] 4700 mg of caprylic capric acid macrogol glyceride, 2300 mg of Span 80, and 3000 mg of Ganoderma lucidum spore oil were accurately weighed, and magnetic stirring was performed at room temperature until the mixture was completely mixed to obtain a Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate.

[0108] 48 mg of paclitaxel was accurately weighed and placed in the above-obtained pre-concentrated solution of Ganoderma lucidum spore oil from nanoemulsion, and magnetically stirred at room temperature in the dark until completely dissolved to obtain paclitaxel-Ganoderma lucidum spore oil from nanoemulsion pre-concentrated solution (PGS).

[0109] The method for preparing the Ganoderma lucidum spore composite microparticles loaded with paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion from the paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate is the same as that in Example 6.

[0110] Example 10

[0111] RH40 6430 mg, PEG400 2144 mg, and Ganoderma lucidum spore oil 1429 mg were accurately weighed, and magnetic stirring was performed at room temperature until they were completely mixed to obtain a Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate.

[0112] 30 mg of paclitaxel was accurately weighed and placed in the above-obtained pre-concentrated solution of Ganoderma lucidum spore oil from nanoemulsion, and magnetically stirred at room temperature in the dark until completely dissolved to obtain paclitaxel-Ganoderma lucidum spore oil from nanoemulsion pre-concentrated solution (PGS).

[0113] The method for preparing the Ganoderma lucidum spore composite microparticles loaded with paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion from the paclitaxel-Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate is the same as that in Example 6.

[0114] Experimental Example 1

[0115] Untreated Ganoderma lucidum spores and acid-base treated Ganoderma lucidum spores (GLS) samples were evenly dispersed with 95% ethanol, adhered to conductive glue and sprayed with gold. The morphology of Ganoderma lucidum spores before and after treatment was observed and compared using a scanning electron microscope. It can be observed that the untreated Ganoderma lucidum spores are oval in shape with a size of about 5×8μm, with a spore navel at one end, a relatively smooth surface, and a slight closed depression. After acid-base washing, the spore size is slightly reduced to about 4×7μm, the surface is rough, with a porous structure, and the spore navel is open and broken. The results are shown in Figure 3 .

[0116] Experimental Example 2

[0117] A small amount of untreated Ganoderma lucidum spores and PGS@GLS obtained in Example 6 were taken, and were evenly dispersed with 95% ethanol respectively. 10 μL was taken and dropped onto a copper mesh and dried. A transmission electron microscope was used to perform transmission imaging at a voltage of 80 kV to observe the morphology of the drug-loaded Ganoderma lucidum spore composite particles. It can be intuitively observed that the untreated Ganoderma lucidum spores are opaque due to the complete spore biological structure (including polysaccharides, proteins and other substances) inside. The transparency of the Ganoderma lucidum spore image after acid-base treatment increased significantly, and the outline morphology remained basically unchanged but there were multiple transparent holes in the middle, indicating that the acid-base water washing and other treatment operations can effectively remove the internal substances of the spores and form small holes through the spore cyst wall, but did not destroy the basic structure of the spore cyst shell. Figure 4 .

[0118] Experimental Example 3

[0119] The samples obtained in Example 3 were dispersed in deionized water, and their average particle size, PDI, drug loading and encapsulation efficiency were measured. The results showed that the average particle size of the prepared paclitaxel Ganoderma lucidum spore oil self-nanoemulsion was (26.160±0.734) nm, the PDI was 0.022±0.013, and the drug loading and encapsulation efficiency (calculated as paclitaxel) were 1.41% and 90.53%, respectively.

[0120] Experimental Example 4:

[0121] The emulsified sample obtained in Experimental Example 3 was dripped onto a transmission electron microscope copper grid, negatively stained with phosphotungstic acid, and imaged using a transmission electron microscope after drying. The results showed that the emulsified droplets were uniformly circular in shape after nanoemulsification, and the size was consistent with the result of the nanoparticle sizer. Figure 5 .

[0122] Experimental Example 5:

[0123] The drug-loaded spores in Example 4 and Example 6 were immersed in pH 1.2, pH 6.8 and pH 7.4 buffers respectively, and the temperature was set to 37°C. Samples were taken at 2h, 3h, 5h, 7h, 8h, 12h and 24h, and the cumulative release amount (%) of paclitaxel at different time points was measured. The results showed that the cumulative release amount of paclitaxel in Example 4 was less than 20% in the first 2h (pH 1.2), and then it was released slowly. The cumulative release amount exceeded 80% at 8h (pH 7.4), and the cumulative release amount exceeded 90% at 12h (pH 7.4). The cumulative release amount of the drug-loaded spores in Example 6 was less than 50% at 12h. Figure 6 , indicating that after the paclitaxel Ganoderma lucidum spore oil is encapsulated in the Ganoderma lucidum spore microcapsules, the drug can be slowly released, and more dissolved paclitaxel can be detected in the release medium after the paclitaxel is prepared into nanoemulsion, indicating that its dissolution ability is effectively improved.

[0124] Experimental Example 6

[0125] 1) Fifty-four healthy male Kunming mice aged 6-8 weeks with an average body weight of about 18-22 g were selected and adaptively raised for one week. Before the experiment, the animals were randomly divided into three groups, with 18 mice in each group, and weighed and marked;

[0126] 2) The mice in the three groups were gavaged with free DiR (Free DiR), DiR-labeled paclitaxel-loaded Ganoderma lucidum spore oil nanoemulsion (PGS), and DiR-labeled paclitaxel-loaded Ganoderma lucidum spore composite microparticles (PGS@GLS).

[0127] 3) The mesenteric lymph nodes of mice in each group were collected at 1, 2, 4, 6, 12, and 24 hours after administration, and the fluorescence intensity of the mesenteric lymph nodes of mice in different groups at different time points was detected by the small animal imaging system. Figure 7 The fluorescence intensity of the mesenteric lymph nodes of mice in the PGS group and PGS@GLS group at each time point was significantly stronger than that of the free DiR group. The fluorescence intensity of the PGS group was the highest at 2h, and the fluorescence intensity of the PGS@GLS group reached a peak at 6h, and remained at a high intensity 24h after administration. The increase in fluorescence intensity indicates that the self-nanoemulsion preparation is conducive to more absorption of drugs into the mesenteric lymph nodes; the difference in the change time of the peak fluorescence intensity reflects the difference in drug release and absorption in the body. After oral administration, the peak fluorescence distribution in the lymph nodes of the PGS@GLS group shifted backward compared with the PGS group, indicating that the self-nanoemulsion after GLS encapsulation will be released more slowly and persistently in the body.

[0128] Experimental Example 7

[0129] 1) 40 healthy male Balb / c mice aged 6-8 weeks with an average body weight of about 18-22 g were selected for adaptive feeding for one week. The mice were fasted but not watered for 12 hours the day before modeling, and then weighed and marked;

[0130] 2) After the animal was anesthetized, it was fixed in a supine position, the skin was prepared and disinfected, a small incision was made in the abdomen and the cecum was externalized, and a 30G needle was used to insert 2×10 7 luc-CT26 cells were injected into the cecal wall and sutured;

[0131] 3) The mice were randomly divided into 5 groups, including model group (C), commercial paclitaxel injection group (PTX), blank Ganoderma lucidum spore (GLS) group, paclitaxel Ganoderma lucidum spore oil nanoemulsion (PGS) group and paclitaxel Ganoderma lucidum spore composite microparticles (PGS@GLS) group, with 8 mice in each group. After the model was successfully established, it was marked as day 1. Except for the model group and the commercial paclitaxel injection group, the other 5 groups were orally gavaged with the corresponding preparations. The daily dose of PTX for colon cancer mice was 5 mg / kg, and it was administered continuously for 5 days, followed by 5 days of drug withdrawal, and one cycle was repeated. The commercial paclitaxel injection group was injected with paclitaxel injection by tail vein on day 1 and day 6, respectively.

[0132] 4) Perform in vivo imaging of small animals on days 1, 5, 10, 15, and 20 to dynamically monitor the tumor development in each mouse. The results are shown in Figure 8 AF, the average fluorescence intensity of the abdomen of mice in the PGS@GLS group increased the least over time, and the average fluorescence intensity was the lowest at the end of the experiment. On the 21st day, the abdominal tumors of mice in the control group increased significantly, and the average fluorescence intensity was 126.7 times that of the first day, while the growth of abdominal tumors in mice in the paclitaxel intravenous injection group, GLS group, PGS group and PGS@GLS group was inhibited to a certain extent, and the average fluorescence intensity was 44.3 times, 37.4 times, 17.0 times and 11.9 times that of the first day, respectively. The fluorescence intensity of the abdomen of mice in the PGS@GLS group was significantly lower than that of the other groups.

[0133] 5) After administration, the mice were weighed every other day to dynamically monitor their weight changes. The results are shown in Fig. 9 The body weight of mice in the PTX intravenous injection group decreased significantly on the 7th day, and showed an overall downward trend during the entire experiment, indicating that paclitaxel injection has a large toxic and side effect on the body; the body weight of mice in the PGS@GLS group was relatively stable, with no obvious increase or decrease.

[0134] 6) On the 20th day, the mice were observed and their body condition scores (BCS) were comprehensively evaluated according to Table 1. The results are shown in Fig.10 The intravenous injection of PTX alone caused the mice to become emaciated and in poor condition; the mice in the PGS@GLS group were in better condition with a high overall score. Fig. 9 The results of body weight changes showed that the mice in the PGS@GLS group did not experience significant weight loss or emaciation caused by tumor loading after administration;

[0135] Table 1 Scoring criteria for mouse physical condition

[0136]

[0137] 7) On day 21, mice were killed after monitoring fluorescence intensity by in vivo imaging, whole blood was collected, and the whole intestinal segment and attached tumor were removed after dissection to observe the difference in tumor growth between groups. The tumor on the intestinal segment was peeled off and weighed for analysis. The results are shown in Fig.11 , the average number of intestinal tumors in mice in the PGS@GLS administration group was the smallest, the volume was smaller, and the average total tumor weight was the smallest.

[0138] The mesenteric lymph nodes were dissected, and tumor samples were selected and digested to prepare single cell suspension samples. Fluorescent antibodies were added for incubation, and the proportions of mature dendritic cells (mDCs) and CD8+T cells in the mesenteric lymph nodes and tumor tissues of each group of mice were detected by flow cytometry. Fig.12 , the proportion of mDCs and CD8+T cells in the tumor tissues and mesenteric lymph nodes of mice in the PGS@GLS administration group increased significantly.

[0139] Tumor samples were selected from each group for fixation, paraffin embedding, and sectioning. Immunogenic cell death in tumor tissues was analyzed by CRT and HMGB1 immunofluorescence staining. The results are shown in Fig.13 The fluorescence intensity of CRT in mouse tumor tissue was significantly enhanced near the cell nucleus in the PGS@GLS-treated group, and the fluorescence intensity of HMGB1 was significantly weakened on the cell nucleus in the PGS@GLS-treated group, indicating that PGS@GLS caused CRT externalization in the cell nucleus and the release of HMGB1 in the nucleus, which could significantly induce immunogenic cell death in tumor cells.

[0140] 8) Tumor samples were fixed, paraffin-embedded, and sliced ​​in each group. Tumor cell proliferation and apoptosis were analyzed by Ki67 and Tunel immunofluorescence staining. The results are shown in Fig.14 The number of Ki67-positive cells in mouse tumor tissues was significantly reduced in the PGS@GLS-treated group, indicating that the proliferation rate of tumor cells in the PGS@GLS-treated group was relatively low. In addition, TUNEL staining showed that the tumor cells in the PGS@GLS-treated group had the strongest apoptosis signal, reflecting that PGS@GLS has the most significant effect on promoting tumor cell apoptosis.

[0141] 9) Tumor samples were selected from each group for fixation, paraffin embedding, and sectioning. Immunofluorescence staining was used to analyze the CD8+ T cell infiltration in the tumor tissue. The results are shown in Fig.15 , the CD8+T lymphocyte infiltration level of mice in the PGS@GLS administration group was the highest.

[0142] 10) Analyze the white blood cell (WBC) count, lymphocyte (Lymph) count, red blood cell (RBC) count, hemoglobin (HGB), and platelet (PLT) count in the mouse blood to evaluate the safety of the preparation. The results are shown in Fig.16,Compared with the control group, the counts of white blood cells, lymphocytes, platelets, red blood cells, and hemoglobin in the PGS@GLS-administered group fluctuated within the normal range, showing good biosafety.

[0143] Fig.16 There was no significant change in the blood indexes of mice in the PGS@GLS group compared with those of normal mice. The red blood cells and hemoglobin of mice in the PTX intravenous injection group were significantly decreased compared with those of normal mice.

[0144] 11) The heart, liver, spleen, lung, kidney and tumor tissues of mice were removed, fixed, paraffin-embedded and sectioned. The structure and physiological morphology of each tissue were observed by hematoxylin-eosin staining (H&E staining). The results are shown in Fig.17 The cell density of tumor tissue in the PGS@GLS-treated mice was significantly reduced, and other major organ tissues were not significantly damaged.

[0145] Fig.18 Schematic diagram of the PGS@GLS composite microparticles prepared in this application activating the cancer immune cycle and inhibiting the development of colorectal cancer.

Claims

1. A Ganoderma lucidum spore composite microparticles containing paclitaxel-loaded Ganoderma lucidum spore oil self-nanoemulsion, characterized in that: The invention is composed of the following components: ganoderma lucidum spores and paclitaxel ganoderma lucidum spore oil self-nanoemulsion; in terms of mass, the mass ratio of the ganoderma lucidum spores to the paclitaxel ganoderma lucidum spore oil self-nanoemulsion is 1:5-10.

2. The Ganoderma lucidum spore composite microparticles of paclitaxel-loaded Ganoderma lucidum spore oil self-nanoemulsion according to claim 1, characterized in that: The paclitaxel ganoderma spore oil self-nanoemulsion is composed of the following components: Paclitaxel, Ganoderma lucidum spore oil, emulsifier, co-emulsifier.

3. The Ganoderma lucidum spore composite microparticles of paclitaxel-loaded Ganoderma lucidum spore oil self-nanoemulsion according to claim 2, characterized in that: The emulsifier is selected from one of polyoxyethylene 40 hydrogenated castor oil, Tween 80, lauroyl polyoxyethylene 32 glyceride, and caprylic / capric macrogol glyceride.

4. The Ganoderma lucidum spore composite microparticles of paclitaxel-loaded Ganoderma lucidum spore oil self-nanoemulsion according to claim 2, characterized in that: The auxiliary emulsifier is selected from one of polyethylene glycol 400, 1,2-propylene glycol, diethylene glycol monoethyl ether, and propylene glycol monolaurate.

5. The Ganoderma lucidum spore composite microparticles of paclitaxel-loaded Ganoderma lucidum spore oil self-nanoemulsion according to claim 2, characterized in that: Calculated by mass percentage: paclitaxel 0.08-0.64%, ganoderma lucidum spore oil 10-40%, emulsifier 27-72%, and co-emulsifier 14-60%.

6. The method for preparing the Ganoderma lucidum spore composite microparticles of the Ganoderma lucidum spore oil self-nanoemulsion of paclitaxel according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1: mixing Ganoderma lucidum spore oil, an emulsifier, and an emulsifier co-to obtain a Ganoderma lucidum spore oil self-nanoemulsion pre-concentrate, and adding paclitaxel to the pre-concentrate to obtain paclitaxel Ganoderma lucidum spore oil self-nanoemulsion; S2: defatting the Ganoderma lucidum spores to obtain defatted Ganoderma lucidum spores; S3: dispersing the defatted Ganoderma lucidum spores in an alkaline solution and an acid solution successively, washing and drying, to obtain a Ganoderma lucidum spore blank carrier; S4: Loading the paclitaxel and Ganoderma lucidum spore oil self-nanoemulsion onto the Ganoderma lucidum spore blank carrier to obtain the Ganoderma lucidum spore composite microparticles loaded with the paclitaxel and Ganoderma lucidum spore oil self-nanoemulsion.

7. The preparation method according to claim 6, characterized in that: The S2 defatting treatment method is selected from one of solvent extraction, ultrasound-assisted extraction, supercritical CO2 extraction, and gradient centrifugation.

8. The preparation method according to claim 7, characterized in that: The ratio of the organic solvent to the ganoderma lucidum spores used in the solvent extraction method is 10-20:1 (V / W), and the unit is mL / g.

9. The preparation method according to any one of claims 6 to 8, characterized in that: The S4 load treatment method is selected from one of a vacuum decompression method, a stirring method and a centrifugation method.

10. Use of the Ganoderma lucidum spore composite microparticles in the self-nanoemulsion of paclitaxel-loaded Ganoderma lucidum spore oil according to any one of claims 1 to 5 in the preparation of a pharmaceutical preparation for preventing colon cancer or rectal cancer.

Images