Ginger-sourced drug-loaded microparticles for targeting colorectal cancer and application thereof

Abstract

The invention belongs to the technical field of preparation of anti-tumor drugs, and particularly relates to ginger-sourced drug-loaded microparticles for targeting colorectal cancer and application of the ginger-sourced drug-loaded microparticles. The ginger-sourced drug-loaded microparticles for targeting colorectal cancer provided by the invention comprise ginger-sourced microparticles and drugs which are wrapped by the ginger-sourced microparticles and can inhibit colorectal cancer, and the surfaces of the ginger-sourced microparticles are also modified with transferrin binding ligands. The ginger-sourced drug-loaded microparticles can target colorectal tumor tissues and penetrate into the deep part of the tumor through oral delivery, so that the ginger-sourced drug-loaded microparticles are efficiently taken by colorectal cancer tumor cells and tumor stem cells. In addition, the ginger-sourced drug-loaded microparticles can also cooperate with drugs to inversely polarize tumor-associated macrophages into M1 type, improve the tumor microenvironment, enhance the drug sensitivity of colorectal cancer tumor cells, effectively kill colorectal cancer tumor cells and tumor stem cells, and achieve efficient inhibition of colorectal cancer.

Description

Technical Field

[0001] The present application belongs to the technical field of anti-tumor drug preparation, and more specifically, to ginger-derived drug-loaded microparticles and applications for targeting colorectal cancer. Background Art

[0002] Existing colorectal cancer treatment drugs are mainly administered intravenously, which has a series of problems such as poor patient adaptability (inconvenient to use), large amounts of drugs enriched in the liver leading to hepatotoxicity, and poor tumor targeting. Oral administration has good adaptability, but oral free chemotherapy drugs have problems such as irregular oral absorption and severe gastrointestinal reactions. Using nanocarriers to load free chemotherapy drugs can reduce gastrointestinal adverse reactions and improve drug absorption efficiency, but the drugs are usually absorbed in the small intestine and then enter the blood, resulting in drug enrichment in liver tissue, and low efficiency in colorectal targeting. Loading transferrin on the surface of nanocarriers can achieve colorectal targeting and improve the absorption of chemotherapy drugs by intestinal epithelial cells, but transferrin has poor oral stability and is easily degraded by gastric juice and intestinal juice, making it difficult to achieve drug colorectal targeting.

[0003] Therefore, it is of great significance to develop a drug that can be orally delivered, has colorectal targeting, and can be efficiently taken up by colorectal cancer tumor cells. Summary of the invention

[0004] In view of the defects of the prior art, the purpose of this application is to provide ginger-derived drug-loaded microparticles and applications for targeting colorectal cancer, aiming to solve the problems that existing colorectal cancer drugs are difficult to deliver orally, have weak colorectal targeting after oral administration, have low drug absorption rate in intestinal epithelial cells, and the therapeutic effect of colorectal cancer drugs needs to be improved.

[0005] To achieve the above objectives, the present application provides ginger-derived drug-loaded microparticles for targeting colorectal cancer, including ginger-derived microparticles, and drugs capable of inhibiting colorectal cancer encapsulated in the ginger-derived microparticles; the surface of the ginger-derived microparticles is also modified with a transferrin-binding ligand.

[0006] Preferably, the mass ratio of the ginger-derived microparticles, the transferrin-binding ligand and the drug capable of inhibiting colorectal cancer is 100:(0.5-20):(0.5-20).

[0007] Preferably, the transferrin binding ligand comprises a transferrin binding polypeptide and a conjugate, the transferrin binding polypeptide is conjugated to the conjugate, and the conjugate can be connected to the surface of the ginger-derived microparticles by phospholipid exchange, electrostatic adsorption or covalent coupling.

[0008] Preferably, the conjugate is selected from at least one of DSPE-PEG-Mal, DSPE-PEG-Man, NH2-PEG-DSPE, N3-PEG-DSPE, GA and PEI.

[0009] Preferably, the transferrin binding polypeptide is a cyclic polypeptide (cp), and its amino acid sequence is at least one of CGGHc(KYLRW), CGGGc(KRIFMV) and c(GGGPFWWW)P.

[0010] Preferably, the transferrin binding ligand is selected from at least one of DSPE-PEG-cp, NH2-PEG-cp, N3-PEG-cp, GA-cp and PEI-cp.

[0011] Preferably, the above-mentioned drug capable of inhibiting colorectal cancer may be one or more of, but not limited to, irinotecan, doxorubicin, 5-fluorouracil, oxaliplatin and folinic acid.

[0012] Preferably, the average particle size of the ginger-derived drug-loaded microparticles is 100 nm to 1000 nm.

[0013] The present application also provides a method for preparing the ginger-derived drug-loaded microparticles, comprising the following steps:

[0014] S1. performing differential centrifugation on ginger juice to obtain ginger-derived microparticles;

[0015] S2, incubating the ginger-derived microparticles and the transferrin-binding ligand, modifying the transferrin-binding ligand on the surface of the ginger-derived microparticles by phospholipid exchange, electrostatic adsorption or covalent coupling, and collecting the ginger-derived microparticles modified with the transferrin-binding ligand;

[0016] S3. Mix and incubate the above-mentioned transferrin-binding ligand-modified ginger-derived microparticles and the above-mentioned drug capable of inhibiting colorectal cancer, and collect the ginger-derived drug-loaded microparticles for targeting colorectal cancer.

[0017] Preferably, in step S1, the differential centrifugation is performed as follows:

[0018] The ginger juice is centrifuged at a centrifugal force of 2,000g to 4,000g for 10min to 30min, the supernatant is centrifuged at a centrifugal force of 10,000g to 12,000g for 0.5h to 1h, and then the supernatant is centrifuged at a centrifugal force of 150,000g to 200,000g for 1h to 1.5h, and the precipitate is collected to obtain ginger-derived microparticles.

[0019] Preferably, in step S2 and step S3, the collection conditions are: collection at 4°C and a centrifugal force of 100,000 g to 200,000 g.

[0020] On the other hand, the present application also provides a drug for treating colorectal cancer, which includes the above-mentioned ginger-derived drug-loaded microparticles.

[0021] On the other hand, the present application also provides a drug capable of reverse polarizing M2 macrophages to M1 macrophages, which comprises the above-mentioned ginger-derived drug-loaded microparticles.

[0022] In general, the above technical solutions conceived by this application have the following technical advantages compared with the prior art:

[0023] (1) The ginger-derived drug-loaded microparticles provided in the present application use ginger-derived microparticles modified with transferrin-binding ligands as drug delivery carriers, and can be taken orally to allow drugs that inhibit colorectal cancer to accumulate at the site of colorectal cancer tumors, thereby reducing the toxic and side effects of drugs on normal tissues. At the same time, they also have strong transcytosis ability, can penetrate intestinal epithelial cells and tumor tissues, and improve the efficiency of drug uptake by colorectal inflammatory tissues or colorectal cancer tumor tissues, thereby enhancing the deep penetration ability of primary colorectal cancer tumor tissues and achieving efficient inhibition of colorectal cancer.

[0024] (2) The present application discovered through experiments that the ginger-derived drug-loaded microparticles can reverse polarize tumor-associated macrophages to the M1 type, significantly enhancing the efficacy of the drug while improving the tumor microenvironment, inhibiting the proliferation of colorectal cancer tumor cells, reducing the number of colorectal cancer tumor nodules, reducing tumor resistance, and enhancing the killing effect on colorectal cancer tumor cells and tumor stem cells.

[0025] (3) In a preferred embodiment, compared with oral administration of CPT, GEVs, lp-GEVs, cp-GEVs, CPT@GEVs, and CPT@lp-GEVs alone, the ginger-derived drug-loaded microparticles CPT@cp-GEVs provided in the present application showed a synergistic effect in the targeted treatment of colorectal cancer, and its therapeutic effect was significantly better than that of other treatment groups, and even better than the simple sum of the technical effects of a single CPT and a single cp-GEVs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the mass ratio range of transferrin binding ligand and microparticles in the embodiment;

[0027] Figure 2 is the particle size of ginger-derived drug-loaded microparticles modified with transferrin-binding ligand in the embodiment; wherein content (a) is ginger-derived microparticles modified with cyclic peptide cp, and content (b) is ginger-derived drug-loaded microparticles;

[0028] Figure 3 The results of uptake and efflux of ginger-derived microparticles modified with cyclic peptide cp in Example 2; wherein content (a) is the uptake result of microparticles by caco-2 cells, and content (b) is the efflux result of microparticles by caco-2 cells;

[0029] Figure 4 is the transcytosis capacity of the ginger-derived microparticles modified with the cyclic peptide cp in Example 3;

[0030] Figure 5 The targeting and safety of the ginger-derived microparticles modified with the cyclic peptide cp in Example 4; wherein content (a) is that the microparticles are enriched in the intestinal muscle layer, content (b) is that the microparticles are enriched in the colorectal part, content (c) is that the microparticles target MC38 colon cancer cells, content (d) is that the microparticles are enriched in the intestinal part of mice with primary colorectal cancer, and content (e) is the content of the microparticles in the intestine of healthy mice;

[0031] Figure 6 The deep penetration ability of ginger-derived microparticles modified with cyclic peptide cp in Example 5 on tumor spheres and colorectal cancer tumors; wherein content (a) is CT26 tumor stem cell spheres, and content (b) is colorectal cancer tumors of primary colorectal cancer mice;

[0032] Figure 7 The effects of the ginger-derived microparticles modified with the cyclic peptide cp in Example 6 on macrophage reverse polarization, tumor cell proliferation and drug efficacy; wherein content (a) is the expression level of macrophage CD80, content (b) is the expression level of M1 macrophage CD86, content (c) is the activity of MC38 colon cancer tumor cells, and content (d) is the killing of MC38 colon cancer tumor cells by the drug;

[0033] Figure 8 The killing effect of ginger-derived drug-loaded microparticles modified with cyclic peptide cp in Example 7 on MC38 colon cancer cells and CT26 tumor stem cells; wherein content (a) is the result of killing MC38 colon cancer cells, and content (b) is the result of killing CT26 tumor stem cells;

[0034] Fig. 9 The inhibition of tumor nodules in primary colorectal cancer by ginger-derived drug-loaded microparticles modified with cyclic peptide cp in Example 8;

[0035] Fig.10 The effect of ginger-derived drug-loaded microparticles modified with cyclic peptide cp on the size of tumor nodules in primary colorectal cancer in Example 8;

[0036] Fig.11The reverse polarization effect of ginger-derived drug-loaded microparticles modified with cyclic peptide cp on tumor-associated macrophages in Example 9; wherein content (a) is CD80 + The proportion of macrophages, content (b) is CD86 + The proportion of macrophages;

[0037] Fig.12 The cytotoxic effect of ginger-derived drug-loaded microparticles modified with cyclic peptide cp on tumor stem cells in colorectal cancer in Example 10; wherein content (a) is the number of tumor stem cells SP, and content (b) is the number of tumor stem cells CD133. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0039] The term "effective dose" refers to an amount that, when administered to a subject, shows the effect of improving, treating, detecting and diagnosing cancer, or inhibiting or slowing down the progression of cancer. The term "subject" can be an animal, preferably a mammal, especially an animal including a human being, or a cell, tissue, or organ from an animal. The subject can be a patient who needs these effects.

[0040] The document (J.Agric.Food Chem.2022,70,4725-4734) reports that ginger exosomes can be specifically internalized by intestinal cells through endocytosis and microendocytosis mediated by macrophages and vesicle proteins, which can inhibit the expression of inflammatory factors (TNF-α, IL-6, IL-1β) in LPS-induced macrophages, thereby alleviating colorectal inflammation. The inventors of the present application have experimentally found that ginger-derived drug-loaded microparticles modified with transferrin-binding polypeptides can reverse polarize tumor-associated macrophages to M1 type, thereby improving the tumor microenvironment, and the drugs that can inhibit colorectal cancer that are synergistically encapsulated enhance the inhibitory effect on colorectal cancer tumors, and the therapeutic effect is significant. Specifically, the ginger-derived drug-loaded microparticles for targeted treatment of colorectal cancer provided by the present application include ginger-derived microparticles, and drugs that can inhibit the proliferation of colorectal cancer cells encapsulated by the ginger-derived microparticles; the surface of the above-mentioned ginger-derived microparticles is also modified with transferrin-binding ligands.

[0041] In some embodiments, the mass ratio of the ginger-derived microparticles, the transferrin-binding ligand, and the drug capable of inhibiting colorectal cancer is 100:(0.5-20):(0.5-20).

[0042] In a preferred embodiment, the mass ratio of the ginger-derived microparticles, the transferrin-binding ligand and the drug capable of inhibiting colorectal cancer is 100:(1-20):(0.5-20).

[0043] In a more preferred embodiment, the mass ratio of the ginger-derived microparticles, the transferrin-binding ligand and the drug capable of inhibiting colorectal cancer is 100:(1-10):(0.5-20).

[0044] In the present application, the above-mentioned drug capable of inhibiting colorectal cancer can be a medically recognized drug capable of inhibiting the proliferation of colorectal cancer cells. In some embodiments, the above-mentioned drug capable of inhibiting colorectal cancer includes but is not limited to one or more of irinotecan, doxorubicin, 5-fluorouracil, oxaliplatin and folinic acid.

[0045] In some embodiments, the transferrin binding ligand includes a transferrin binding polypeptide and a conjugate, the transferrin binding polypeptide is coupled to the conjugate, and the conjugate can be connected to the surface of the ginger-derived microparticles by phospholipid exchange, electrostatic adsorption or covalent coupling. In some embodiments, the conjugate includes but is not limited to at least one of DSPE-PEG-Mal, DSPE-PEG-Man, NH2-PEG-DSPE, N3-PEG-DSPE, GA and PEI. When the conjugate is DSPE-PEG-Mal, DSPE-PEG-Man, NH2-PEG-DSPE or N3-PEG-DSPE, it can be connected to the surface of the ginger-derived microparticles by phospholipid exchange; when the conjugate is GA (glutaraldehyde) or PEI (polyethyleneimine), it can be connected to the surface of the ginger-derived microparticles by electrostatic adsorption.

[0046] In some embodiments, the transferrin binding polypeptide is a cyclic polypeptide (cp), and its amino acid sequence is at least one of CGGHc(KYLRW), CGGGc(KRIFMV) and c(GGGPFWWW)P.

[0047] In some embodiments, the transferrin binding ligand is selected from at least one of DSPE-PEG-cp, NH2-PEG-cp, N3-PEG-cp, GA-cp and PEI-cp.

[0048] In some embodiments, the average particle size of the ginger-derived drug-loaded microparticles is 100 nm to 1000 nm.

[0049] On the other hand, the present application also provides a method for preparing the ginger-derived drug-loaded microparticles, comprising the following steps:

[0050] S1. Incubating the ginger-derived microparticles with the transferrin-binding ligand, modifying the transferrin-binding ligand on the surface of the ginger-derived microparticles by phospholipid exchange, electrostatic adsorption or covalent coupling, and collecting the ginger-derived microparticles modified with the transferrin-binding ligand;

[0051] S2. Mix and incubate the above-mentioned transferrin-binding ligand-modified ginger-derived microparticles and the above-mentioned drug capable of inhibiting colorectal cancer, and collect the ginger-derived drug-loaded microparticles for targeting colorectal cancer.

[0052] As an optional manner, in the above method for preparing ginger-derived drug-loaded microparticles, step S1 specifically comprises the following steps:

[0053] S1-1. Preparing ginger-derived microparticles: performing differential centrifugation on ginger juice, collecting the supernatant, and obtaining ginger-derived microparticles;

[0054] S1-2, preparing a transferrin binding ligand: dissolving the above conjugate in an organic solvent, mixing the above transferrin binding polypeptide and the above conjugate, stirring to carry out a coupling reaction, and then dialyzing and freeze-drying to obtain a transferrin binding ligand;

[0055] S1-3. Preparation of ginger-derived microparticles modified with transferrin-binding ligands: incubate the ginger-derived microparticles and the transferrin-binding ligands, modify the transferrin-binding ligands onto the surface of the ginger-derived microparticles by phospholipid exchange, electrostatic adsorption or covalent coupling, and collect the ginger-derived microparticles modified with transferrin-binding ligands.

[0056] As an optional method, in step S1-1, the differential centrifugation specifically includes the following steps:

[0057] The ginger juice is centrifuged at a centrifugal force of 2,000g to 4,000g for 10min to 30min to remove plant residues, and then the supernatant is centrifuged at a centrifugal force of 10,000g to 12,000g for 0.5h to 1h to remove cell debris protein precipitate, and then the supernatant is centrifuged at a centrifugal force of 150,000g to 200,000g for 1h to 1.5h, and the precipitate is collected to obtain ginger-derived microparticles.

[0058] In some embodiments, the ginger juice is obtained by squeezing ginger juice and filtering through a 200-mesh sieve.

[0059] In some embodiments, in step S1-2, the coupling reaction is carried out under stirring at room temperature for 3 h to 5 h.

[0060] In some embodiments, in step S1-3, the mass ratio of the ginger-derived microparticles to the transferrin-binding ligand is 100:(0.5-20).

[0061] In a preferred embodiment, in step S1-3, the mass ratio of the ginger-derived microparticles to the transferrin-binding ligand is 100:(1-20).

[0062] In some embodiments, in step S1-3, the incubation time is 6 hours to 12 hours.

[0063] In some embodiments, in steps S2 and S3, the collection conditions are: collection at a centrifugal force of 100,000 g to 200,000 g at 4°C.

[0064] The ginger-derived drug-loaded microparticles for targeting colorectal cancer provided in the present application include ginger-derived microparticles, and transferrin targeting molecules modified on the surface of the ginger-derived microparticles, and the ginger-derived microparticles also encapsulate drugs that can inhibit colorectal cancer. The ginger-derived drug-loaded microparticles can target colorectal tumor tissues and penetrate deep into the tumor through oral delivery, and then be efficiently taken up by colorectal cancer tumor cells and tumor stem cells. In addition, the ginger-derived drug-loaded microparticles can also cooperate with drugs to reverse polarize tumor-associated macrophages to M1 type, improve the tumor microenvironment, enhance the drug sensitivity of colorectal cancer tumor cells, effectively kill colorectal cancer tumor cells and tumor stem cells, and achieve efficient inhibition of colorectal cancer.

[0065] On the other hand, the present application also provides a drug for treating colorectal cancer, which includes the above-mentioned ginger-derived drug-loaded microparticles.

[0066] In some embodiments, the above-mentioned colorectal cancer includes at least one of colorectal cancer and rectal cancer.

[0067] On the other hand, the present application also provides a drug capable of reverse polarizing tumor-associated macrophages into M1 macrophages, which comprises the above-mentioned ginger-derived drug-loaded microparticles.

[0068] It is understood that the ginger-derived drug-loaded microparticles can be included in a drug for treating colorectal cancer and a drug for reverse polarizing tumor-associated macrophages to M1 macrophages together with a pharmaceutically acceptable carrier, excipient and / or diluent. The ginger-derived drug-loaded microparticles can be provided in the form of a single drug or a kit.

[0069] The above-mentioned drug for treating colorectal cancer and the drug for reverse polarizing tumor-associated macrophages into M1 macrophages provided in the present application can be administered to mammals including humans by any method. For example, by oral or parenteral administration. Based on this, the above-mentioned drug can be formulated into a preparation for oral administration or parenteral administration according to the administration route.

[0070] The total effective dose of the drug for treating colorectal cancer and the drug for reverse polarizing tumor-associated macrophages to M1 macrophages provided in the present application can be administered to the patient in a single dose, or in a long-term multiple dose according to a fractionated treatment scheme. Those skilled in the art can determine the appropriate effective dose of the drug based on the patient's age, weight, gender, severity of the disease, diet, route of administration, number of treatments, etc., and therefore the total dose of the drug is not limited.

[0071] The present application also provides a method for targeted treatment of colorectal cancer, comprising administering an effective dose of a drug for treating colorectal cancer to a subject in need thereof, wherein the drug for treating colorectal cancer comprises the ginger-derived drug-loaded microparticles as an active ingredient.

[0072] It should be understood that materials with the same or similar types, models, qualities, properties or functions as the reagents and instruments used in the following examples can be used to implement the present application. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0073] The following are examples:

[0074] Various cells, reagents and experimental animals used in the following examples:

[0075] Caco-2 cells, MC38 colon cancer cells, CT26 tumor cells, and mouse macrophage cell line RAW264.7 cells were purchased from the Center for Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China); C57 mice were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.; transferrin was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; DSPE-PEG-Man was purchased from Shanghai Pengshuo Biotechnology Co., Ltd.; cyclic peptides (cp: CGGHc(KYLRW)) and linear peptides (lp: CGGGHKYLRW) were purchased from Hefei Guopi Biotechnology Co., Ltd.; transferrin-binding ligands DSPE-PEG-cp and DSPE-PEG-lp were constructed by conventional molecular biology methods; irinotecan hydrochloride (CPT) was purchased from Dalian Meilun Biotechnology Co., Ltd.; anti-CD45 antibody, CD133 antibody, CD11b antibody, F4 / 80 antibody, CD80 antibody, and CD86 antibody were purchased from biolegend.

[0076] Example 1 Transferrin Binding Ligand Modified Ginger-Derived Drug-Loaded Microparticles

[0077] (1) Ginger juice was squeezed and passed through a 200-mesh sieve. The filtered ginger juice was centrifuged at 2,000 g for 20 min to remove plant residues. The supernatant was centrifuged at 10,000 g for 40 min to remove cell debris and protein precipitate. The supernatant was centrifuged at 150,000 g for 1.5 h to collect the precipitate, i.e., ginger-derived microparticles (abbreviated as GEVs).

[0078] (2) The transferrin binding polypeptide (cyclic polypeptide cp: CGGHc(KYLRW), linear polypeptide lp: CGGGHKYLRW) and the conjugate (DSPE-PEG-Man) were placed in a PBS system and rotated and stirred for 4 h, and then freeze-dried to obtain transferrin binding ligands (DSPE-PEG-cp, DSPE-PEG-lp). The affinity of the cyclic polypeptide and the linear polypeptide was analyzed by TRA. The results are shown in Table 1.

[0079] Table 1 Affinity of cyclic peptide cp and linear peptide lp

[0080] Peptides <![CDATA[Binding rate constant (M -1 s -1 )]]> <![CDATA[Dissociation rate constant (s -1 )]]> Dissociation equilibrium constant (M) cp <![CDATA[5.27×10 4 ]]> <![CDATA[2.48×10 -4 ]]> <![CDATA[4.71×10 -9 ]]> lp <![CDATA[5.92×10 4 ]]> <![CDATA[7.01×10 -4 ]]> <![CDATA[1.18×10 -8 ]]>

[0081] In Table 1, the dissociation rate of cyclic peptides and proteins is lower than that of linear peptides, and the dissociation equilibrium constant is significantly smaller than that of linear peptides, indicating that the affinity of cyclic peptides and proteins is significantly higher than that of linear peptides.

[0082] (3) GEVs were labeled with the fluorescent dye CFSE, and then GEVs and transferrin-binding ligands were incubated at 4°C for 12 hours. The mass ratio of transferrin-binding ligands to microparticles was adjusted to 1:10, 1:50, and 1:100. The transferrin-binding ligands were modified onto ginger microparticles by phospholipid exchange, and the ginger-derived microparticles modified with transferrin-binding ligands were obtained by centrifugation at 150,000 g for 90 minutes (ginger-derived microparticles modified with cyclic peptides, ginger-derived microparticles modified with linear peptides, lp-GEVs). The ginger-derived microparticles modified with transferrin-binding ligands and MC38 tumor cells were incubated at 37°C and 5% carbon dioxide incubator for 4 hours, and the intracellular fluorescence was detected. The results are shown in Table 2. Figure 1 .

[0083] Depend on Figure 1 It can be seen that at the same modification ratio, cp-GEVs mediated the most MC38 tumor cell uptake compared to GEVs and lp-GEVs. Among different modification ratios, the mass ratio of transferrin binding ligand to microparticles was 1:50 and 1:10, which mediated more MC38 tumor cell uptake. The mass ratio of transferrin binding ligand to microparticles was 1:10 for subsequent experiments.

[0084] (4) Mix equal volumes of 1 mg / mL (protein amount) of cp-GEVs and 500 μg / mL of irinotecan hydrochloride (CPT), incubate at 37°C on a shaker for 2 hours to allow the drug to be encapsulated by the ginger-derived microparticles, and then centrifuge at 150,000 g for 90 minutes to obtain ginger-derived drug-loaded microparticles modified with transferrin-binding ligands (abbreviated as CPT@cp-GEVs). According to the above treatment method, ginger-derived drug-loaded microparticles encapsulating only the drug (abbreviated as CPT@GEVs) and ginger-derived drug-loaded microparticles modified with linear polypeptides (abbreviated as CPT@lp-GEVs) were prepared. The particle size of each treatment group was detected using a dynamic light scattering (DLS) particle size analyzer. The results are shown in Table 1. Figure 2 .

[0085] Figure 2 The average particle size of each treatment group was 200nm~1000nm, indicating that DSPE-PEG-cp modification and drug loading of ginger-derived microparticles did not basically change the particle size of the microparticles.

[0086] Example 2 Cyclic peptide CP-modified ginger-derived microparticles are taken up and excreted by Caco-2 cells

[0087] The CFSE fluorescent dye-labeled GEVs, lp-GEVs, and cp-GEVs prepared in Example 1 were mixed with transferrin at a molar ratio of 1:1 and incubated at 37°C for 1 h. The microparticles with a final concentration of 25 μg / mL (protein amount) after incubation were added to caco-2 cells cultured in serum-free medium and cultured for 4 h. The fluorescence intensity in the caco-2 cells was detected to observe the uptake of the microparticles by the caco-2 cells. The results are shown in Figure 3 Content (a). Then continue to culture for 0h, 1h, 2h, and 4h, use flow cytometry to detect the fluorescence intensity of Caco-2 cells, and observe the efflux of microparticles by Caco-2 cells. The results are shown in Figure 3 Content (b).

[0088] like Figure 3 As shown in content (a), compared with GEVs and lp-GEVs, the fluorescence intensity of cp-GEVs in caco-2 cells is stronger, indicating that cp-GEVs can significantly promote the uptake of ginger microparticles by caco-2 cells. Figure 3 As shown in content (b), the fluorescence intensity of cp-GEVs in caco-2 cells decreased significantly with the increase of culture time, indicating that cp-GEVs have the highest exocytosis efficiency and higher intestinal epithelial transport efficiency, which promotes the excretion of cp-GEVs by caco-2 cells.

[0089] Example 3 Ginger-derived microparticles modified with cyclic peptide cp have strong transcytosis ability

[0090] MC38 colon cancer cells were spread on a glass slide, GEVs dye, lp-GEVs and cp-GEVs labeled with CFSE dye prepared in 2.1 were mixed with transferrin at a molar ratio of 1:1 and incubated at 37°C for 1 hour, then the incubated microparticles were added to MC38 colon cancer cells and cultured in a 37°C, 5% carbon dioxide incubator for 4 hours, and then the microparticles adsorbed on the cell surface were washed off with PBS to obtain a P1 glass slide, and the fluorescence intensity of the cells on the P1 glass slide was detected by flow cytometry. Another P1 glass slide and another glass slide covered with MC38 colon cancer cells were placed in a new culture dish, cultured in a 37°C, 5% carbon dioxide incubator for 12 hours, and then the surface microparticles were washed off with PBS to obtain a P2 glass slide, and the fluorescence intensity of the cells on the P2 glass slide was detected by flow cytometry. Take another new P2 slide and another slide covered with MC38 colon cancer cells and place them in a new culture dish. After culturing in a 37°C, 5% carbon dioxide incubator for 12 hours, wash the surface microparticles with PBS to obtain a P3 slide. Flow cytometry was used to detect the fluorescence intensity of the cells on the P3 slide. The results are shown in Figure 4 .

[0091] like Figure 4 As shown, the fluorescence intensity of cp-GEVs in each group of slides was significantly higher than that of GEVs and lp-GEVs, and the ratio of the fluorescence intensity of cp-GEVs to GEVs increased, indicating that cp-GEVs has the strongest tumor cell transcytosis ability.

[0092] Example 4 Targeting and safety of ginger-derived microparticles modified with cyclic peptide cp

[0093] (1) GEVs, lp-GEVs, and cp-GEVs labeled with the fluorescent dye IR780 were prepared according to the method of Example 1, and then the mice were anesthetized, the abdominal cavity was opened to expose the colorectal area, and the intestinal segment of about 1 cm was ligated up and down with cotton thread. IR780 dye, IR780 dye-labeled GEVs, lp-GEVs, and cp-GEVs were injected into the intestinal segment, respectively. After 1 hour, the mice were killed and the intestines were removed. After paraffin embedding and sectioning, the fluorescence intensity of the intestinal muscle side was detected using a confocal microscope. The results are shown in FIG. Figure 5 Content(a).

[0094] Depend on Figure 5 Content (a) shows that compared with IR780, GEVs, and lp-GEVs, cp-GEVs are significantly enriched in the intestinal muscle layer, indicating that cp-GEVs have the strongest ability to penetrate the intestinal epithelium and can improve the absorption efficiency of cp-GEVs by the intestinal muscle layer.

[0095] (2) Eight-week-old C57 male mice were selected, and their drinking water was replaced with 2.5% dextran sulfate sodium solution (DSS) filtered through 0.2 μm. The solution was replaced every 2 days. After 7 days, IR780 fluorescent dye was dispersed in 100 μL PBS as the IR780 treatment group. GEVs, lp-GEVs and cp-GEVs labeled with IR780 dye were dispersed in 100 μL PBS according to the protein quantification of 10 mg / kg, and they were respectively used as the GEVs treatment group, lp-GEVs treatment group and cp-GEVs treatment group. The amount of fluorescent dye used in each treatment group was the same. Then, each treatment group was gavaged with mice to construct a colorectal inflammation model mouse. After 12 hours, the mice were killed, and the heart, liver, spleen, lung, kidney and intestine were dissected. The intestinal fluorescent image was obtained by small animal live imaging. The distribution ratio of each treatment group in each tissue was statistically analyzed. The results are shown in Figure 5 Content (b).

[0096] Depend on Figure 5 Content (b) shows that compared with other tissues and other drug treatment groups, cp-GEVs are significantly enriched in the colorectal area. The reason for this may be that cp-GEVs can achieve colorectal targeting by binding to transferrin in the inflammatory area of ​​colorectal inflammation. In addition, liver tissue also has a certain fluorescence intensity. The reason for this may be that cp-GEVs are efficiently absorbed by colorectal cells after binding to transferrin. In the absence of tumors (colorectal inflammation model), cp-GEVs reach the liver through blood circulation.

[0097] (3) GEVs, lp-GEVs, and cp-GEVs labeled with CFSE fluorescent dye were mixed with transferrin at a molar ratio of 1:1 and incubated at 37°C for 1 h. The incubated microparticles were added to MC38 colon cancer cells at protein concentrations of 12.5 μg / mL, 25 μg / mL, and 50 μg / mL and cultured in a 37°C, 5% carbon dioxide incubator for 4 h. The fluorescence intensity of MC38 colon cancer cells was detected by flow cytometry. The results are shown in Table 2. Figure 5 Content (c).

[0098] Depend on Figure 5 Content (c) shows that at different final concentrations, cp-GEVs have the strongest colorectal cancer cell targeting compared with GEVs and lp-GEVs.

[0099] (4) Eight-week-old C57 male mice were intraperitoneally injected with 10 mg / kg (drug / body weight) of azomethane (AOM). Three days later, the drinking water was replaced with a 2.5% by mass dextran sodium sulfate solution, and then replaced every two days. After seven days of feeding, the drinking water was replaced again for 14 days. The above operation was repeated three times as one cycle to construct a primary colorectal cancer mouse model. IR780 dye was dispersed in 100 μL PBS as the IR780 treatment group. GEVs, lp-GEVs and cp-GEVs labeled with IR780 dye were dispersed in 100 μL PBS according to the protein quantification of 10 mg / kg, and they were respectively used as the GEVs treatment group, lp-GEVs treatment group and cp-GEVs treatment group. The amount of fluorescent dye used in each treatment group was the same. Then, each treatment group was gavaged with primary colorectal cancer mice, and the mice were killed 12 hours later. The complete intestine (from stomach to anus) was taken out, and the intestinal fluorescence was detected by small animal live imaging and the fluorescence intensity of the colorectal segment was counted. The results are shown in Figure 5 Content (d).

[0100] Depend on Figure 5 Content (d) shows that the fluorescence intensity of cp-GEVs in the intestine of mice with primary colorectal cancer is significantly higher than that of GEVs and lp-GEVs. The reason may be that the intestine of mice with primary colorectal cancer contains transferrin, and the ginger microparticles modified with cyclic peptides can adhere to the inflammatory intestinal wall more and be absorbed by the intestine, thereby being more accurately retained in the colorectal area and achieving tumor targeting of colorectal cancer.

[0101] (5) Eight-week-old C57 male mice were taken, and IR780 fluorescent dye was dispersed in 100 μL PBS as the IR780 treatment group. GEVs, lp-GEVs and cp-GEVs labeled with IR780 dye were dispersed in 100 μL PBS according to the protein quantification of 10 mg / kg, and they were respectively used as the GEVs treatment group, lp-GEVs treatment group and cp-GEVs treatment group. The amount of fluorescent dye in each treatment group was the same. Then, each treatment group was gavaged with healthy mice, and the mice were killed 12 hours later. The complete intestine (from stomach to anus) was removed, and the intestinal fluorescence was detected by small animal live imaging and the fluorescence intensity of the colorectal segment was counted. The results are shown in Figure 5 Content(e).

[0102] Depend on Figure 5 Content (e) shows that the fluorescence intensity of cp-GEVs in the intestine of healthy mice is the lowest. The reason may be that the intestine of healthy mice does not contain transferrin, and the ginger microparticles modified with cyclic peptides are more easily excreted from the intestine, thereby reducing the toxic side effects on the healthy intestine.

[0103] Example 5 Deep penetration ability of ginger-derived microparticles modified with cyclic peptide cp on tumor spheres and colorectal cancer tumors

[0104] (1) Add 5 mL of a 1% low-density agarose solution sterilized at high temperature to a cell culture dish, and after cooling, obtain a low-adsorption plate. Then, add 4 million CT26 tumor cells to the above culture dish, and add 10 mL of tumor stem cell culture medium (based on DMEM / F-12 serum-free culture medium, add 2% B27, 20 ng / mL mouse epidermal growth factor and 2 mM glutamine). The culture medium is replaced every two days to remove dead cells. After culturing for 5 days, CT26 tumor stem cell spheres with strong stemness are obtained. The CFSE dye-labeled GEVs, lp-GEVs, and cp-GEVs prepared in Example 1 were mixed with transferrin at a molar ratio of 1:1 and incubated at 37°C for 1 h. The microparticles with a final concentration of 25 μg / mL (protein amount) after incubation were added to a 6-well plate containing tumor stem cell spheres and cultured for 4 h. The microparticles were centrifuged at 1200 rpm and washed with PBS to remove the residual plant exosomes on the surface of the tumor cell spheres. After that, 4% paraformaldehyde was added to fix at room temperature for 10 min. Then, the microparticles were placed on a confocal dish and scanned on the Z axis using a confocal microscope to observe the ability of GEVs, lp-GEVs, and Cp-GEVs to infiltrate the tumor stem cell spheres at different depths. The results are shown in Figure 6 Content(a).

[0105] Depend on Figure 6 Content (a) shows that the penetration rate of Cp-GEVs into tumor spheroids at different depths is significantly higher than that of GEVs and lp-GEVs. In addition, green fluorescence is widely distributed in tumor spheroids treated with cp-GEVs, while the fluorescence of GEVs and lp-GEVs is mainly scattered at the edge of the tumor spheroids. The reason for this may be that after cp-GEVs bind to transferrin, the transcytosis ability of tumor cells is enhanced, thereby promoting the deep penetration of cp-GEVs into tumor spheroids.

[0106] (2) The colorectal cancer tumor nodules of the primary colorectal cancer mice in Example 4 were removed, embedded in paraffin and sectioned, and the infiltration of the primary colorectal cancer tumors by each treatment group was observed using a confocal microscope. The fluorescence intensity at the depth of each section of 50 μm from the edge of the tumor to the depth was counted. The results are shown in Table 1. Figure 6 Content (b).

[0107] Depend on Figure 6 As can be seen from content (b), compared with IR780, GEVs, and lp-GEVs, cp-GEVs can effectively infiltrate deep into the tumor, indicating that cp-GEVs have a stronger ability to penetrate deep into the tumor.

[0108] Example 6 Ginger microparticles modified with cyclic peptide cp can reverse polarization of macrophages to M1 phenotype, promote tumor cell proliferation, and enhance the efficacy of irinotecan

[0109] (1) PBS, CFSE fluorescent dye-labeled GEVs, lp-GEVs, cp-GEVs and transferrin were mixed at a molar ratio of 1:1 and incubated at 37°C for 1 h. The incubated microparticles were then added to Raw264.7 cells and cultured in a 37°C, 5% carbon dioxide incubator for 24 h. The microparticles adsorbed on the cell surface were washed off with PBS to obtain single cells. CD80 antibodies and CD86 antibodies were then added, and the protein expression levels of each treatment group were detected by flow cytometry. The results are shown in Figure 7 .

[0110] Depend on Figure 7 Content (a), by Figure 7 Content (b) shows that compared with PBS, GEVs, and lp-GEV, the expression levels of macrophage CD80 and CD86 in the cp-GEVs treatment group were the highest, indicating that cp-GEVs have the strongest ability to upregulate CD80 and CD86 and can reverse polarize macrophages to the M1 phenotype.

[0111] (2) CFSE dye-labeled GEVs, lp-GEVs, and cp-GEVs were mixed with transferrin at a molar ratio of 1:1 and incubated at 37°C for 1 hour. The incubated microparticles were added to Raw264.7 macrophages cultured in serum-free medium and incubated for 24 hours. The supernatant was centrifuged at 2,000g and filtered through a 0.2μm filter membrane to remove impurities, and the supernatant of macrophage culture medium treated with GEVs, lp-GEVs, and cp-GEVs was obtained. The above supernatant was mixed with complete culture medium in an equal volume of 1:1 and then cultured in MC38 colon cancer tumor cells (culture environment was CM-GEVs, CM-lp-GEVs, CM-cp-GEVs). The culture environment NM was a mixture of serum-free medium and complete culture medium in an equal volume of 1:1; the culture environment CM was a mixture of supernatant of macrophage culture medium not treated with microparticles and complete culture medium in an equal volume of 1:1. CCK8 was used to detect the proliferation of colorectal cancer cells in different culture environments at 0h, 6h, 12h, 24h, 36h, 48h, and 72h. The results are shown in Figure 7 Content (c).

[0112] Depend on Figure 7Content (c) shows that compared with other culture environments, the culture environment CM-cp-GEVs treatment group can effectively inhibit the proliferation of MC38 colon cancer tumor cells. The reason may be that the tumor-associated macrophages in the supernatant of the macrophage culture medium treated with cp-GEVs are reverse polarized to M1 type, which improves the tumor microenvironment. When the supernatant is mixed with complete culture medium and used for MC38 colon cancer tumor cells, it can inhibit the proliferation of MC38 colon cancer tumor cells.

[0113] (3) 10 μg / mL irinotecan hydrochloride was added to the different culture environments in (2) above, and then MC38 colon cancer tumor cells were cultured. CCK8 was used to detect the killing effect of the drug on MC38 colon cancer tumor cells when the MC38 colon cancer tumor cells were cultured in the culture environment with the drug for 0 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h. The results are shown in Figure 7 Content (d).

[0114] Depend on Figure 7 Content (d) shows that the culture environment CM-cp-GEVs with added drugs has the strongest killing effect on MC38 colon cancer tumor cells. The reason may be that the macrophages in the supernatant of the macrophage culture medium treated with cp-GEVs are reverse polarized to M1 type, which improves the tumor microenvironment and significantly increases the efficacy of irinotecan through the synergistic effect between components.

[0115] Example 7 Cyclic peptide cp-modified ginger-derived drug-loaded microparticles kill MC38 colon cancer cells and CT26 tumor stem cells

[0116] (1) PBS was used as the G1 group (100 μL); free CPT was used as the G2 group (0.375 μg / mL); GEVs, CPT@GEVs, CPT@lp-GEVs and CPT@cp-GEVs were used as the G3 group, G4 group, G5 group and G6 group, respectively. The final concentrations of the G3 to G6 groups were all 25 μg / mL (protein amount). Then the last treatment groups were added to the MC38 tumor cells and cultured in a 37°C, 5% carbon dioxide incubator for 24 hours, and then CCK8 reagent was added. The microplate reader was used to detect the killing of MC38 colon cancer cells by the G1 to G6 treatment groups. The results are shown in Figure 8 Content(a).

[0117] like Figure 8 As shown in content (a), compared with PBS, GEVs, and CPT, CPT@GEVs, CPT@lp-GEVs, and CPT@cp-GEVs can all kill MC38 colon cancer cells, among which CPT@cp-GEVs (G6 group) has the strongest effect in killing MC38 colon cancer cells.

[0118] (2) The CT26 tumor stem cell spheres prepared in Example 5 were added to a 96-well plate at a concentration of 100 per well, and then PBS (100 μL, G1 group), free CPT (0.375 μg / mL, G2 group), vesicle group GEVs (G3 group), CPT@GEVs (G4 group), CPT@lp-GEVs (G5 group) and CPT@cp-GEVs (G6 group) were added. The final concentration of groups G3 to G6 was 25 μg / mL (protein amount). After culturing in a 37°C, 5% carbon dioxide incubator for 24 h, 10 μL of CCK8 reagent was added to each well. The killing of CT26 tumor stem cells by each treatment group was detected using an ELISA instrument. The results are shown in Table 2. Figure 8 Content (b).

[0119] like Figure 8 As shown in content (b), CPT@cp-GEVs has the strongest effect in killing CT26 tumor stem cells.

[0120] Example 8 Effect of ginger-derived drug-loaded microparticles modified with cyclic peptide cp on inhibiting tumor nodules in primary colorectal cancer

[0121] A primary colorectal cancer mouse model was constructed according to the method of Example 4, wherein in the third cycle, the drinking water was replaced with a 2.5% sodium dextran sulfate solution by mass, and PBS (100 μL, G1 group); free CPT (150 μg / kg dispersed in 100 μL PBS, G2 group); vesicle group GEVs (G3 group), lp-GEVs (G4 group), cp-GEVs (G5 group), CPT@GEVs (G6 group), CPT@lp-GEVs (G7 group) and CPT@cp-GEVs (G8 group) were dispersed in 100 μL PBS according to the protein quantitative 10 mg / kg, and the mice were gavaged, and the drug was administered once every 2 days, for a total of 8 times. Five days after the end of the drug administration, the mice were killed, the intestines from the cecum to the anus were removed, and the number of tumor nodules in the intestinal cavity was counted after longitudinal dissection with scissors. The results are shown in Fig. 9 The intestinal tract from the cecum to the anus was embedded in paraffin, cut longitudinally from the anus, and stained with HE. The pathological structure was observed under a microscope. The results are shown in Fig.10 .

[0122] Depend on Fig. 9 It can be seen that compared with other treatment groups, the administration of CPT@GEVs, CPT@lp-GEVs, and CPT@cp-GEVs can significantly inhibit the occurrence of tumor nodules in mice with primary colorectal cancer, and the number of tumor nodules in the CPT@cp-GEVs treatment group was the lowest, indicating that oral administration of CPT@cp-GEVs has a better therapeutic effect. Fig.10In the experiment, the size of colorectal cancer tumors in mice with primary colorectal cancer that were intragastrically administered with CPT@cp-GEVs was smaller than that in the other treatment groups. In summary, the synergistic effect of the three components in CPT@cp-GEVs was confirmed.

[0123] Example 9: Effect of cyclic peptide cp-modified ginger-derived drug-loaded microparticles on the reverse polarization of tumor-associated macrophages

[0124] The colorectal cancer tumor nodules of the primary colorectal cancer mice in Example 8 were taken out to prepare single cells, and then anti-CD45 antibody, CD11b antibody, F4 / 80 antibody and CD80 antibody were added, and CD80 was detected by flow cytometry. + Macrophages (CD45 + CD11b + F4 / 80 + CD80 + cells), see Fig.11 Content (a); Add anti-CD45 antibody, CD11b antibody, F4 / 80 antibody and CD86 antibody, and use flow cytometry to detect CD86 + Macrophages (CD45 + CD11b + F4 / 80 + CD86 + The results are shown in Fig.11 Content (b).

[0125] like Fig.11 Content (a), Fig.11 As shown in (b), CD80 in the CPT@cp-GEVs treatment group + Macrophages, CD86 + The proportion of macrophages in the two groups was better than that in the other treatment groups, indicating that oral administration of CPT@cp-GEVs in primary colorectal cancer can significantly upregulate the expression of CD80 in colorectal cancer tumor tissues. + Macrophages, CD86 + The ratio of macrophages can effectively reverse the polarization of tumor-associated macrophages to M1 type.

[0126] Example 10 Cyclic peptide cp-modified ginger-derived drug-loaded microparticles kill tumor stem cells in colorectal cancer

[0127] The primary colorectal cancer mouse tumor nodules in Example 8 were taken out to prepare single cells, and then anti-CD45 antibody and Hochest dye were added to count SP cells (CD45 - Hochest - The results are shown in Fig.12Content (a); Add anti-CD45 antibody and CD133 antibody to count CD133 cells (CD45 - CD133 + The results are shown in Fig.12 Content (b).

[0128] Depend on Fig.12 Content (a), Fig.12 Content (b) shows that the number of tumor stem cells (including SP cells and CD133 cells) in primary colorectal cancer mice gavaged with CPT@cp-GEVs was significantly reduced, and its ability to kill tumor stem cells was the strongest. Its effect of killing tumor stem cells was significantly better than that of other treatment groups, confirming the synergistic effect of the three components in the ginger-derived drug-loaded microparticles (CPT@cp-GEVs) modified with cyclic peptide cp.

[0129] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. Ginger-derived drug-loaded microparticles for targeting colorectal cancer, characterized in that: It comprises ginger-derived microparticles and drugs capable of inhibiting colorectal cancer which are wrapped in the ginger-derived microparticles; the surfaces of the ginger-derived microparticles are also modified with transferrin-binding ligands.

2. The ginger-derived drug-loaded microparticles according to claim 1, characterized in that: The mass ratio of the ginger-derived microparticles, the transferrin-binding ligand and the drug capable of inhibiting colorectal cancer is 100:(0.5-20):(0.5-20).

3. The ginger-derived drug-loaded microparticles according to claim 1, characterized in that: The transferrin binding ligand comprises a transferrin binding polypeptide and a conjugate, wherein the transferrin binding polypeptide is conjugated to the conjugate, and the conjugate can be connected to the surface of the ginger-derived microparticles by phospholipid exchange, electrostatic adsorption or covalent coupling; The transferrin binding polypeptide is a cyclic polypeptide, and its amino acid sequence is at least one of CGGHc(KYLRW), CGGGc(KRIFMV) and c(GGGPFWWW)P.

4. The ginger-derived drug-loaded microparticles according to claim 1, characterized in that: The drug capable of inhibiting colorectal cancer is selected from one or more of irinotecan, doxorubicin, 5-fluorouracil, oxaliplatin and folinate.

5. The ginger-derived drug-loaded microparticles according to claim 1, characterized in that: The average particle size of the ginger-derived drug-loaded microparticles is 100nm to 1000nm.

6. A method for preparing ginger-derived drug-loaded microparticles according to any one of claims 1 to 5, characterized in that: The steps include: S1. performing differential centrifugation on ginger juice to obtain ginger-derived microparticles; S2, incubating the ginger-derived microparticles and the transferrin-binding ligand, modifying the transferrin-binding ligand on the surface of the ginger-derived microparticles by phospholipid exchange, electrostatic adsorption or covalent coupling, and collecting the ginger-derived microparticles modified with the transferrin-binding ligand; S3. Mix and incubate the transferrin-binding ligand-modified ginger-derived microparticles and the drug capable of inhibiting colorectal cancer, and collect the ginger-derived drug-loaded microparticles for targeting colorectal cancer.

7. The preparation method according to claim 6, characterized in that: In step S1, the differential centrifugation is performed as follows: The ginger juice is centrifuged at a centrifugal force of 2,000g to 4,000g for 10min to 30min, the supernatant is centrifuged at a centrifugal force of 10,000g to 12,000g for 0.5h to 1h, and then the supernatant is centrifuged at a centrifugal force of 150,000g to 200,000g for 1h to 1.5h, and the precipitate is collected to obtain ginger-derived microparticles.

8. The preparation method according to claim 6, characterized in that: In step S2 and step S3, the collection conditions are: collecting at 4°C and a centrifugal force of 100,000g to 200,000g.

9. A drug for treating colorectal cancer, characterized in that: The drug comprises the ginger-derived drug-loaded microparticles as described in any one of claims 1 to 5.

10. A drug capable of reverse polarizing tumor-associated macrophages to M1 macrophages, characterized in that: The drug comprises the ginger-derived drug-loaded microparticles as described in any one of claims 1 to 5.

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