Elemene colon-targeted nanocapsule as well as preparation method and application thereof

Abstract

The invention discloses an elemene colon-targeted nanocapsule and a preparation method and application thereof, and belongs to the technical field of pharmaceutical preparations, the elemene colon-targeted nanocapsule comprises an inner core and a pH-sensitive enteric polymer coating the surface of the inner core, the inner core comprises elemene and a self-nano-milk lipid material, and the self-nano-milk lipid material comprises an oil phase, an emulsifier and a co-emulsifier. The nanocapsule disclosed by the invention is relatively small in particle size and uniform in dispersion, has a capsule structure with a clear edge, is relatively excellent in drug loading capacity and encapsulation efficiency, and can be used for protecting drugs from being released in advance in the stomach and the small intestine and effectively conveying the drugs to the colon part. Animal experiments show that the nanocapsule disclosed by the invention can quickly release drugs in a simulated colon fluid in the form of nanoemulsion, and compared with an elemene raw material drug and an elemene marketing drug, the nanocapsule disclosed by the invention can remarkably inhibit tumor growth of in-situ tumor-bearing mice, has relatively good biological safety, and can be used for preparing a medicine for treating in-situ tumor-bearing mice. The anti-colorectal cancer treatment effect of the medicine is obviously improved.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to an elemene colon-targeted nanocapsule and a preparation method and use thereof. Background Art

[0002] Colorectal cancer (CRC) is a common digestive tract malignancy. It is a malignant tumor that occurs in the colorectal mucosal epithelium and glandular part under the combined action of multiple pathogenic factors. The incidence of cancer is high worldwide, and it tends to be younger, which seriously endangers life and health. Clinically, a combination of surgery and postoperative adjuvant radiotherapy and chemotherapy is often used, but the accumulation of drug concentration in the colorectal cancer tumor site is often insufficient, which hinders the full play of the anti-cancer effect of the drug.

[0003] Elemene (EL) is an effective active monomer separated and extracted from the rhizome of the traditional Chinese medicine Curcuma zedoaria. It is a Class II non-cytotoxic plant-derived anti-tumor drug approved by my country with independent intellectual property rights. Basic research suggests that it can exert anti-tumor effects through multiple mechanisms such as inhibiting tumor cell proliferation, inducing tumor cell apoptosis, and inhibiting tumor tissue angiogenesis. Clinical studies have found that elemene not only has a definite therapeutic effect on a variety of tumors, but also has a certain immune protection effect. It has a synergistic effect when used in combination with radiotherapy and chemotherapy, can relieve cancer pain, increase white blood cells and inhibit platelet aggregation. It has the advantages of low toxicity, high efficiency and broad spectrum, and is not easy to develop drug resistance. At present, elemene has been widely used in the clinical treatment of various tumors, such as lung cancer, gastric cancer, colorectal cancer and other cancers.

[0004] Oral colon-targeted drug delivery system (OCDDS) is a new type of targeted drug delivery system that uses drug delivery technology to ensure that the drug is not released in the upper digestive tract after oral administration, but is transported to the ileocecal region and then released in the colon to exert local or systemic therapeutic effects. OCDDS can significantly enhance local therapeutic effects, making up for the shortcomings of many traditional oral preparations, and has broad application prospects in the treatment of many gastrointestinal diseases such as ulcerative colitis, Kernrow's disease, colon cancer, etc.

[0005] Nanocapsules (NCs) are nanoparticles with a cystic structure. The core of the nanocapsule is a liquid reservoir for drugs, mainly a lipophilic solvent (usually oil); the shell is usually made of polymers, especially biodegradable polymers. The drug delivery system using this cystic structure has the following advantages: 1. It has a higher drug loading capacity. Since the lipid core optimizes the solubility of the drug, the concentration of poorly soluble drugs in the nanoparticles can be increased to a greater extent; 2. Since the drug is "protected" in the nanocapsule, it can prevent the degradation of the drug itself caused by tissue irritation at the administration site; 3. By screening different polymer shell materials, the responsive release of nanocapsules at specific tissue sites can be achieved.

[0006] Therefore, based on the special advantages of the nanocapsule structure, the present invention provides an elemene colon-targeted nanocapsule to solve the problems of insufficient effective concentration and poor bioavailability of existing elemene drugs at colorectal cancer tumor sites. The elemene colon-targeted nanocapsule of the present invention can protect the drug from being released prematurely in the stomach and small intestine, and realize the rapid release of the drug in the form of nanoemulsion in the colon environment, has a certain tumor targeting effect, and enhances the efficacy of the drug in the treatment of colorectal cancer. Summary of the invention

[0007] The main purpose of the present invention is to provide an elemene colon-targeted nanocapsule and a preparation method and use thereof. The elemene colon-targeted nanocapsule of the present invention can protect drugs from being released prematurely in the stomach and small intestine, and realize rapid release of drugs in the form of nanoemulsion in the colon environment. Its lipid core can increase the solubility of poorly soluble drugs and enhance the permeability in tumor tissues; the polymer capsule shell can dissolve the colon-soluble polymer material triggered by the pH environment of the digestive tract, so that the drug can be retained in the digestive tract and released in the colon cavity. The released drug accumulates and penetrates in the colorectal cancer tumor tissue in the form of nanoemulsion, which can exert a good anti-tumor therapeutic effect.

[0008] Elemene is an effective active monomer of traditional Chinese medicine with broad anti-tumor activity. The present invention prepares elemene colon-targeted nanocapsules (EL@NCs) by using an emulsification-solvent diffusion method. First, an elemene self-nanoemulsion (EL@SNE) inner core is prepared by a self-emulsification method to increase the solubility and stability of elemene. Then, a pH-sensitive polymer is coated on the inner core by a multi-stage dispersion technology to obtain elemene colon-targeted nanocapsules, and their in vitro release, digestive tract retention, tumor targeting and in vivo pharmacodynamics are investigated.

[0009] In order to achieve the above-mentioned object, the present invention provides an elemene colon-targeted nanocapsule, comprising an inner core and a pH-sensitive enteric polymer coated on the surface of the inner core, wherein the inner core comprises elemene and a self-nanoemulsifying lipid material, and the self-nanoemulsifying lipid material comprises an oil phase, an emulsifier and an emulsifier aid.

[0010] Furthermore, the pH-sensitive enteric polymer is an anionic copolymer of methacrylic acid and methyl acrylate.

[0011] Furthermore, the oil phase is a medium-chain fatty acid; the emulsifier is selected from one or more of polyoxyethylene hydrogenated castor oil, lauroyl polyoxyl-32 glyceride, castor oil polyoxyethylene ether or caprylic capric acid polyethylene glycol glyceride; the co-emulsifier is selected from one or more of diethylene glycol monoethyl ether, polyethylene glycol 400 or propylene glycol monocaprylate.

[0012] Furthermore, the average particle size of the elemene colon-targeted nanocapsule is 130-140 nm, the polydispersity index is 0.17-0.21, and the surface charge is -13.33--12.61 mV; the drug loading and encapsulation efficiency of the elemene colon-targeted nanocapsule are 4.6-5.5% and 77-80.6%, respectively.

[0013] Another aspect of the present invention provides a method for preparing the aforementioned elemene colon-targeted nanocapsules, comprising the following steps: mixing elemene, an oil phase, an emulsifier and an auxiliary emulsifier to obtain an elemene self-nanoemulsion;

[0014] The pH-sensitive enteric polymer is coated on the surface of the elemene self-nanoemulsion to obtain the elemene colon-targeted nanocapsule.

[0015] Furthermore, the preparation method of the elemene colon targeting nanocapsules comprises the following steps:

[0016] The preparation of elemene self-nanoemulsion comprises: first stirring and mixing elemene and an oil phase to obtain an elemene solution; stirring and mixing an emulsifier and an auxiliary emulsifier to obtain a self-nanoemulsion blank matrix; dropping the elemene solution into the self-nanoemulsion blank matrix and stirring and mixing for a second time to obtain the elemene self-nanoemulsion;

[0017] and / or,

[0018] The preparation of the pH-sensitive enteric polymer solution comprises: dissolving the pH-sensitive enteric polymer in a water-saturated ethyl acetate solution, and adjusting the pH of the system to a weak alkaline state to obtain the pH-sensitive enteric polymer solution;

[0019] and / or,

[0020] The preparation of elemene colon-targeted nanocapsules comprises: vortex-mixing the elemene self-nanoemulsion and the pH-sensitive enteric polymer solution to obtain a first mixed emulsion; dropping the first mixed emulsion into a saturated aqueous solution of ethyl acetate, performing a third stirring and mixing, emulsifying and diffusing, to obtain a second mixed emulsion; performing a fourth stirring and mixing of the second mixed emulsion and pure water, performing solvent diffusion, to form an elemene colon-targeted nanocapsule solution; and sequentially concentrating, centrifuging, and dialyzing the elemene colon-targeted nanocapsule solution to obtain the elemene colon-targeted nanocapsule.

[0021] Furthermore, the concentration of elemene in the elemene solution is 0.4 mg / mL to 0.6 mg / mL; and the mass concentration of the pH-sensitive enteric polymer in the pH-sensitive enteric polymer solution is 0.03 to 0.1%.

[0022] Furthermore, the dripping speed of the elemene solution is 0.1-1 mL / min, the amount of the saturated aqueous solution of ethyl acetate is 3-6 times the amount of the first mixed emulsion; and the amount of the pure water is 8-12 times the amount of the second mixed emulsion.

[0023] Furthermore, the average particle size of the elemene self-nanoemulsion after re-emulsification with 50 times the amount of water is 40-45 nm, the polydispersity index is 0.10-0.12, and the surface charge is -6.07--5.73 mV.

[0024] Another aspect of the present invention provides a use of the aforementioned elemene colon-targeted nanocapsule, which is use of the elemene colon-targeted nanocapsule in preparing an oral colon-targeted preparation for treating colon cancer.

[0025] Compared with the prior art, the present invention has the following beneficial effects: the prepared elegans colon-targeted nanocapsules are an oral colon-targeted preparation, the nanocapsules have a small particle size, are evenly dispersed, have a capsule structure with clear edges, have a good drug loading and encapsulation rate, can protect the drug from being released prematurely in the stomach and small intestine, and release it quickly in the form of nanoemulsion at the colon. Due to the presence of the pH-sensitive enteric polymer, the surface negative charge is always maintained under acidic conditions (pH 1.2-6.8), and under alkaline conditions (pH 7.4), the pH-sensitive enteric polymer is dissolved, and the drug is released in the form of nanoemulsion with a smaller particle size, achieving aggregation and penetration at the colon tumor site, and having certain tumor targeting.

[0026] In vitro experimental studies have shown that the elemene colon-targeted nanocapsules of the present invention can rapidly release drugs in the form of nanoemulsions in simulated colon fluid, and can exhibit certain tumor targeting properties in a mouse model of colorectal cancer orthotopic tumor-bearing mice; in vivo efficacy studies have shown that, compared with elemene raw materials and marketed drugs (elemene oral emulsion), the elemene colon-targeted nanocapsules of the present invention can significantly inhibit tumor growth in orthotopic tumor-bearing mice and have good biosafety, indicating that the preparation can significantly improve the anti-colorectal cancer therapeutic effect of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Transmission electron microscopy images of the elemene self-nanoemulsion (EL@SNE) of Example 1 of the present invention (left) and the elemene colon-targeted nanocapsules (EL@NCs) of Example 2 (right) are shown;

[0028] Figure 2 The particle size (left) and zeta potential diagram (right) of the elemene self-nanoemulsion (EL@SNE) of Example 1 and the elemene colon-targeted nanocapsules (EL@NCs) of Example 2 of the present invention are shown;

[0029] Figure 3 The in vitro cumulative release graphs (mean±SD, n=6) of elemene colon-targeted nanocapsules (EL@NCs) of Example 2 of the present invention in different simulated digestive fluids (left) and at different time points (right) are shown;

[0030] Figure 4 The in vitro release transmission electron microscopy analysis diagram of the present invention is shown: transmission electron microscopy observation of elemene colon-targeted nanocapsules (EL@NCs) after incubation in SCF for 0 h (left), 2 h (middle), and 6 h (right);

[0031] Figure 5 The DSC analysis diagrams of the present invention are shown: elemene self-nanoemulsion (EL@SNE); Eudrgit@S100; physical mixture of EL@SNE and Eudrgit@S100; elemene colon-targeted nanocapsules (EL@NCs);

[0032] Figure 6 The FTIC analysis diagram of the present invention is shown: elemene (EL); blank matrix (CON-SNE); elemene self-nanoemulsion (EL@SNE); Eudrgit@S100; elemene colon-targeted nanocapsules (EL@NCs);

[0033] Figure 7 The IVIS images of DID, DID@SNE and DID@NCs in vivo (upper) and gastrointestinal biodistribution (lower) at 2, 4, 6 and 24 hours after administration to mice of the present invention are shown (n=36);

[0034] Figure 8 The construction of the mouse orthotopic colon cancer model of the present invention is shown: 0 days after surgery (modeling) (left), 3 days after surgery (middle), and 7 days after surgery (right);

[0035] Fig. 9 The IVIS images of the biodistribution of the tumor-bearing intestine in the fluorescent field (upper) and bright field (lower) are shown three days after administration of the orthotopic colon cancer model mice of the present invention (n=6);

[0036] Fig.10 The IVIS images of biodistribution (left) and relative tumor volume evaluation (right) of each experimental group of mice in the orthotopic colon cancer model of the present invention are shown (n=30);

[0037] Fig.11 The H&E staining images of the main organ tissues of the orthotopic colon cancer model mice of the present invention are shown (n=12);

[0038] Fig.12 The schematic diagram shows the preparation process of elemene colon-targeted nanocapsules (EL@NCs) of the present invention and their colon-targeted release. DETAILED DESCRIPTION

[0039] It should be noted that, in the case of no conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or the intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope. The present invention will be described in detail below in conjunction with the embodiments.

[0040] In the process of preparing elemene colon-targeted nanocapsules of the present invention, elemene can be derived from elemene raw materials, elemene extracts and other substances.

[0041] In order to achieve the above-mentioned purpose, a first aspect of an embodiment of the present invention provides an elemene colon-targeted nanocapsule, comprising an inner core and a pH-sensitive enteric polymer coated on the surface of the inner core, wherein the inner core comprises elemene and a self-nanoemulsion lipid material, and the self-nanoemulsion lipid material comprises an oil phase, an emulsifier and an emulsifier aid.

[0042] In a preferred embodiment of the present invention, the pH-sensitive enteric polymer is an anionic copolymer of methacrylic acid and methyl acrylate, for example (Purchased from Evonik Industries AG).

[0043] S100 (ES) is an anionic copolymer based on methacrylic acid and methyl acrylate. It is a pH-sensitive enteric polymer that is insoluble when the pH value is lower than 7 and can be widely used as a coating material for oral colon-targeted drug delivery systems. Therefore, in order to protect drugs and nanocarriers from degradation in the acidic environment of the stomach and to deliver them in a targeted and localized manner to the colon epithelium, the present invention selects ES as an enteric polymer on the drug carrier to achieve colon targeting.

[0044] In a preferred embodiment of the present invention, the oil phase is a medium-chain fatty acid; the emulsifier is a non-ionic emulsifier, for example, it may include but is not limited to one or more of polyoxyethylene hydrogenated castor oil, lauroyl polyoxyl-32 glyceride, castor oil polyoxyethylene ether or caprylic capric acid polyethylene glycol glyceride; the co-emulsifier is but is not limited to one or more of diethylene glycol monoethyl ether, polyethylene glycol 400 or propylene glycol monocaprylate. Medium-chain fatty acids are saturated fatty acids composed of 8 to 10 carbon atoms. It is further preferred that the medium-chain fatty acid is caprylic capric triglyceride (purchased from Gattefossé China; model number is Labrafac @ WL1349).

[0045] The self-nanoemulsion system composed of the self-nanoemulsion lipid material and elemene can effectively improve the solubility of elemene, facilitate the absorption of elemene in the body, and provide certain tumor targeting.

[0046] In order to further improve the solubility and permeability of the drug and the directional targeting effect, in a preferred embodiment of the present invention, the mass ratio of the elemene, the oil phase, the emulsifier, the co-emulsifier and the pH-sensitive enteric polymer is 1:(0.8-1.2):(4.5-5):(4-4.5):(10-45).

[0047] The average particle size of the elemene colon targeting nanocapsule of the present invention is 130-140nm, the polydispersity index is 0.17-0.21, and the surface charge is -13.33--12.61mV; the drug loading and encapsulation efficiency of the elemene colon targeting nanocapsule are 4.6-5.5% and 77-80.6% respectively.

[0048] A second aspect of an embodiment of the present invention provides a method for preparing elemene colon-targeted nanocapsules, comprising the following steps:

[0049] Mixing elemene, an oil phase, an emulsifier and a co-emulsifier to obtain elemene self-nanoemulsion;

[0050] The pH-sensitive enteric polymer is coated on the surface of the elemene self-nanoemulsion to prepare the elemene colon-targeted nanocapsule.

[0051] In a preferred embodiment of the present invention, the pH-sensitive enteric polymer is coated on the surface of the elemene self-nanoemulsion by graded diffusion technology to prepare the elemene colon-targeted nanocapsules. Graded diffusion technology refers to the technology of preparing nanocapsules by utilizing the different solubility of the bridging agent (the pH-sensitive enteric polymer in the present invention) in different solvents to generate diffusion movement.

[0052] In a preferred embodiment of the present invention, the method for preparing the elemene colon targeting nanocapsules comprises the following steps:

[0053] Step S1, preparation of elemene self-nanoemulsion: elemene and an oil phase are first stirred and mixed to obtain an elemene solution; an emulsifier and a co-emulsifier are stirred and mixed to obtain a self-nanoemulsion blank matrix; the elemene solution is dropped into the self-nanoemulsion blank matrix and then stirred and mixed for a second time to obtain the elemene self-nanoemulsion;

[0054] Step S2, preparation of a pH-sensitive enteric polymer solution: dissolving the pH-sensitive enteric polymer in a water-saturated ethyl acetate solution, and adjusting the pH of the system to a weakly alkaline state to obtain a pH-sensitive enteric polymer solution;

[0055] Step S3, preparation of elemene colon-targeted nanocapsules: vortex-mixing the elemene self-nanoemulsion and the pH-sensitive enteric polymer solution to obtain a first mixed emulsion; dropping the first mixed emulsion into a saturated aqueous solution of ethyl acetate, performing a third stirring and mixing, emulsification and diffusion, to obtain a second mixed emulsion; performing a fourth stirring and mixing of the second mixed emulsion and pure water, solvent diffusion, to form an elemene colon-targeted nanocapsule solution; and sequentially concentrating, centrifuging, and dialyzing the elemene colon-targeted nanocapsule solution to obtain the elemene colon-targeted nanocapsule.

[0056] In order to further improve the efficacy and directional targeting effect of elemene colon-targeted nanocapsules, in a preferred embodiment of the present invention, the concentration of elemene in the elemene solution is 0.4 mg / mL to 0.6 mg / mL; the mass concentration of the pH-sensitive enteric polymer in the pH-sensitive enteric polymer solution is 0.03 to 0.1%; and the mass ratio of the elemene self-nanoemulsion to the pH-sensitive enteric polymer is 1:1 to 4.

[0057] In order to further improve the uniformity of elemene self-nanoemulsion and the solubility of the drug, in a preferred embodiment of the present invention, the rotation speed of the first stirring and mixing is 200-500 rpm, and the time is 1-15 min; the time of the vortex mixing is 1-5 min; the speed of dropping the elemene solution is 0.1-1 mL / min, and the rotation speed of the second stirring and mixing is 200-500 rpm, and the time is 5-30 min.

[0058] In order to further improve the efficiency and uniformity of pH-sensitive enteric polymer coating, in some preferred embodiments of the present invention, the vortex mixing time is 1 to 5 minutes; the amount of the saturated aqueous solution of ethyl acetate is 3 to 6 times the amount of the first mixed emulsion; the dripping speed of the first mixed emulsion is 0.1 to 1 mL / min; the amount of pure water is 8 to 12 times the amount of the second mixed emulsion.

[0059] In a preferred embodiment of the present invention, the post-treatment process of the elemene colon-targeted nanocapsule solution comprises the following steps: sequentially concentrating, centrifuging, and dialyzing the elemene colon-targeted nanocapsule solution to obtain elemene colon-targeted nanocapsules.

[0060] Specifically, the method comprises the following steps: concentrating the elemene colon-targeted nanocapsule solution in a rotary evaporator at 30-35° C., centrifuging the concentrate at 1500-2500 r / min for 10-30 min, collecting the supernatant, dialyzing the supernatant in a dialysis bag of 8kda-14kda for 18-30 h to obtain the elemene colon-targeted nanocapsule, and freeze-drying the elemene colon-targeted nanocapsule and storing it at 4° C. for subsequent use.

[0061] The average particle size of the elemene self-nanoemulsion after re-emulsification with 50 times the amount of water is 40-45 nm, the polydispersity index is 0.10-0.12, and the surface charge is -6.07--5.73 mV.

[0062] The present invention prepares elemene colon targeting nanocapsules (EL@NCs) by an emulsification-solvent diffusion method, takes lipid self-nanoemulsion as the core, and utilizes graded diffusion technology to achieve pH-sensitive enteric polymer coating, which basically includes four steps: preparation of lipid core elemene self-nanoemulsion (EL@SNE), preparation of capsule material pH-sensitive enteric polymer solution (ES), graded diffusion coating and post-treatment.

[0063] Among them, the lipid core elemene self-nanoemulsion is prepared by mixing drugs, oil phase, emulsifier, and co-emulsifier to form a self-nanoemulsion system. It has the advantages of improving the solubility of poorly soluble drugs and accommodating drugs with different lipid solubilities, which is beneficial to the absorption of drugs in the body and has certain tumor targeting. In the graded diffusion coating, the mixed emulsion of elemene self-nanoemulsion and pH-sensitive enteric polymer solution is emulsified-solvent diffused in ethyl acetate saturated aqueous solution and pure water respectively. The solubility in water-saturated ethyl acetate, ethyl acetate-saturated water and pure water is different. The self-driven graded diffusion preparation has the advantages of simple operation, low energy consumption, and can remove residual organic solvents, thus having the advantages of being green and environmentally friendly.

[0064] The preparation of lipid core elemene self-nanoemulsion and the preparation of pH-sensitive enteric polymer solution of capsule material determine the efficiency of pH-sensitive enteric polymer coating during graded diffusion, and the process parameters such as drug addition rate, solvent ratio, stirring mixing rate, time, etc. during emulsification-solvent diffusion are synergistically controlled to successfully prepare elemene colon-targeted nanocapsules (EL@NCs). The method of the present invention is relatively simple in design and safe and feasible in operation.

[0065] The third aspect of the embodiments of the present invention further provides a use of the aforementioned elemene colon-targeted nanocapsule, which is use of the elemene colon-targeted nanocapsule in preparing an oral colon-targeted preparation for treating colon cancer.

[0066] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0067] Example 1

[0068] 1) Accurately weigh 90 mg of EL API (purchased from Dalian Jingang Pharmaceutical Co., Ltd., purity 99.9%) and dissolve in 90 mg of Labrafac@WL1349 (caprylic capric triglyceride, purchased from Gattefossé China), and stir magnetically at room temperature until completely dissolved (stirring speed 200 rpm, stirring time 5 min) to obtain a 50% EL drug solution;

[0069] 2) Weigh 440 mg of polyoxyethylene hydrogenated castor oil (RH40) and 380 mg of diethylene glycol monoethyl ether (purchased from Gattefossé China, model Transcutol@HP) and stir them magnetically at 500 rpm at room temperature until they are completely dissolved to obtain a blank matrix;

[0070] 3) The EL drug solution was then added dropwise to the blank matrix at a dropping speed of 0.5 mL / min, and stirring was continued for 10 min at a stirring speed of 200 rpm to obtain EL@SNE. In the experiment, EL@SNE was diluted with 50 times water for re-emulsification. It can be observed that the prepared EL@SNE suspension was translucent with light blue opalescence due to the Tyndall phenomenon, and the experimental operation was reproducible and stable.

[0071] Example 2

[0072] 1) Eudradit@S100 (i.e. S100, an anionic copolymer of methacrylic acid and methyl acrylate, purchased from Evonik Industries AG) was dissolved in a water-saturated ethyl acetate solution, and the pH of the system was adjusted to 7.4 with N,N-dimethylacetamide to obtain Eudradit@S100 solution (mass concentration of 0.05%);

[0073] 2) 125 mg of EL@SNE in Example 1 was dissolved in 5 mL of Eudradit@S100 solution and vortexed for 2 min to obtain a mixed emulsion; wherein the mass ratio of elemene self-nanoemulsion (EL@SNE) to the anionic copolymer of methacrylic acid and methyl acrylate (Eudradit@S100) was 3:1; and the mass ratio of elemene, oil phase, emulsifier, co-emulsifier and pH-sensitive enteric polymer was 1:1:4.9:4.2:33.3.

[0074] 3) using a graded diffusion technique, the mixed emulsion was added dropwise to 25 mL of an aqueous solution saturated with ethyl acetate at a dropping speed of 0.1 mL / min, and mixed with a magnetic stirrer at 200 rpm for 10 min to emulsify and diffuse;

[0075] 4) Pour the mixed emulsion treated in step 3) into 250 mL of water at once, stir with a magnetic stirrer at 500 rpm for 30 min, and replace the solvent;

[0076] 5) The mixed emulsion treated in step 4) was concentrated to 5 mL in a rotary evaporator at 32° C., centrifuged at 2000 r / min for 20 min, the supernatant was collected, and dialyzed in a dialysis bag (8 kDa to 14 kDa) for 24 h to obtain elemene colon-targeted nanocapsules (EL@NCs). The nanosuspension was freeze-dried in a vacuum freeze dryer for 72 h and stored at 4° C. for subsequent use.

[0077] Example 3

[0078] The EL@SNE and EL@NCs in Example 1 and Example 2 were observed using a transmission electron microscope. Comparative observation revealed that the nanoparticles were all spherical, but with different degrees of edge blur, confirming that EL@NCs had a rigid nanocapsule structure. Figure 1 .

[0079] Example 4

[0080] The EL@SNE and EL@NCs in Example 1 and Example 2 were prepared in parallel for 3 times, and their average particle size, PDI polydispersity index and Zeta potential were measured by dynamic light scattering. The results showed that the average particle size of the prepared EL@SNE after re-emulsification was 43.08nm±2.06, the PDI polydispersity index was 0.11±0.01, and the surface charge was -5.90mV±0.17; the average particle size of EL@NCs was 135.60nm±4.18, the PDI polydispersity index was 0.19±0.02, and the surface charge was -12.97mV±0.36, which further confirmed that ES was coated on the surface of EL@SNE to form EL@NCs, causing changes in particle size and potential. The results are shown in Figure 2 .

[0081] Example 5

[0082] The EL@NCs of Example 2 were placed in a constant temperature water bath shaker, and the release in different simulated digestive fluids (simulated gastric fluid SGF, simulated intestinal fluid SIF, simulated colonic fluid SGF) within 180 minutes was measured. The cumulative release amount (%) of the drug at different time points was further investigated. The results showed that Example 2 could make the drug release amount less than 20% in the first 5 hours (pH 1.2 and 6.8). When the pH value was increased to 7.4, 92.4% ± 3.6% of EL was cumulatively released. The results are shown in Table 1. Figure 3 .

[0083] Example 6

[0084] After the EL@NCs of Example 5 were incubated in the simulated colon fluid SGF for 0 h, 2 h and 6 h, 1 mL of the solution was centrifuged at 6000 rpm for 10 min, and the supernatant was taken to observe the morphological changes of the nanoparticles using a transmission electron microscope. The results are shown in FIG. Figure 4 .

[0085] Example 7

[0086] The EL@SNE in Example 1 and Example 2, The physical mixture of EL@SNE and Eudrgit@S100, and EL@NCs were subjected to DSC analysis. That is, 10 mg of each sample of EL@SNE, Eudrgit@S100, the physical mixture of EL@SNE and Eudrgit@S100, and EL@NCs were weighed and filled into an aluminum crucible, and then the crucible cover (a small hole was pierced on the crucible cover) was added and placed on a press to press the crucibles together. A blank aluminum crucible was used as a reference, the flow rate of the atmospheric nitrogen was 20.0 mL / min, and the temperature was scanned in the range of 20 to 460°C at a scanning speed of 10.0°C / min to obtain a differential scanning calorimetry curve. The results are shown in Figure 5 .

[0087] Example 8

[0088] Fourier transform infrared spectroscopy was performed on EL, blank lipid matrix (CON-SNE), EL@SNE, Eudrgit@S100, and EL@NCs in Example 1 and Example 2 to detect whether there is any interaction between the components. That is, EL, CON-SNE, EL@SNE, Eudrgit@S100, and EL@NCs samples were weighed. The sample to be tested (powder) was mixed with dry KBr at a ratio of 1:100, and then pressed into tablets. The infrared spectrometer was used at 500-4000cm -1 The infrared spectrum of the sample was obtained by analyzing and scanning within the wave number range and subtracting the background absorption value. The results are shown in Figure 6 .

[0089] Example 9

[0090] The hydrophobic near-infrared dye DID was used as a fluorescent probe instead of the core drug to load into the nanoparticle coating formula. DID@SNE and DID@NCs loaded with DID dye were prepared with the same DID concentration (1.5 mg DID / g, based on an average weight of 30 g for mice) and the same preparation process, with free DID as the control. The drug was administered orally at the predetermined time points (2, 4, 6, 24 h), and anesthesia was maintained by inhaling 2.0% isoflurane through a nose cone. The light was excited at 644 nm, and the emission wavelength was detected at 664 nm. The whole-body fluorescence images of the mice were obtained by the IVISLumina LT imaging system. The mice were killed by cervical dislocation at each time point, and the entire gastrointestinal tract tissue from the stomach to the rectum was immediately observed in vitro for the distribution of fluorescence signals in the mouse GIT. The results are shown in Figure 7 .

[0091] Example 10

[0092] 1) 48 healthy male SPF Balb / c mice with an average body weight of about 20-30 g were selected and fed at room temperature for two weeks. The mice were fasted but not watered for 18 h before modeling and then intraperitoneally injected with 20% urethane anesthesia saline (0.006 mL / g) at a dose of 1.2 mg / g;

[0093] 2) Place the animal in supine position, clean the skin surface with iodine, and then make a longitudinal incision along the right abdominal line and appendix area. Use a 30g needle to insert a 2.5×10 7 CT-26WT-luc luciferase-labeled colorectal cancer cells were injected into the cecal wall. The abdominal cavity was sutured, and 20,000 units of penicillin potassium were injected intramuscularly after disinfection.

[0094] 3) On the 3rd and 7th days after modeling, whole-body fluorescence images were obtained using the IVIS LuminaLT imaging system for modeling observation. Figure 8 .

[0095] Embodiment 11

[0096] Six tumor-bearing mice successfully modeled in Example 10 were orally gavaged with DID@NCs for three consecutive days. The mice were killed by cervical dislocation 24 hours after the last administration. The whole intestinal tissue of the mice was obtained, and the fluorescence localization of the whole intestinal tissue was obtained using the IVIS LuminaLT imaging system, and compared with the bright field observation. Fig. 9 .

[0097] Example 12

[0098] 1) 42 tumor-bearing mice successfully modeled in Example 10, with an average body weight of about 20-30 g, were randomly divided into 6 groups, including a blank control group (PBS group), an excipient matrix control group (Placebo group), an elemene bulk drug group (Free EL group), a commercially available elemene oral emulsion (Marketed drug group), an elemene self-nanoemulsion (EL@SNE group) and an elemene colon-targeted nanocapsule (EL@NCs group), with 7 mice in each group, and each group was housed in the same mouse cage;

[0099] 2) Daily oral administration for 2 weeks, using IVIS LuminaLT imaging system to detect the changes in mouse tumors on days 0, 7, 14 and 21. Fig.10 The bioluminescence results and relative tumor volume analysis of different groups showed that compared with the PBS group and Placebo group, the Free EL group and the Marketed drug group could slow down the rapid growth of the tumor, while the EL@SNE group and the EL@NCs group with nanostructures could significantly inhibit the growth of the tumor, especially the EL@NCs with colon localization function, with the relative tumor volumes of 0.813±0.102 on day 21, respectively.

[0100] Embodiment 13

[0101] Twelve mice were taken from each group after 14 days of administration in Example 12, and the heart, liver, spleen, lung, and kidney tissues of each group of mice were stained with hematoxylin-eosin (H&E) to evaluate the biological safety of the preparation. Fig.11 .

[0102] The schematic diagram of the preparation process of the elemene colon-targeted nanocapsules and the colon-targeted release thereof is shown in FIG. Fig.12As shown, in vitro experimental studies have shown that the elemene colon-targeted nanocapsules of the present invention can rapidly release drugs in the form of nanoemulsions in simulated colon fluid, and can exhibit certain tumor targeting in a colorectal cancer orthotopic tumor-bearing mouse model; in vivo efficacy studies have shown that, compared with elemene raw materials and marketed drugs (elemene oral emulsion), the elemene colon-targeted nanocapsules of the present invention can significantly inhibit tumor growth in orthotopic tumor-bearing mice and have good biosafety, indicating that the preparation can significantly improve the anti-colorectal cancer therapeutic effect of the drug.

[0103] The above-described embodiments only represent the embodiments of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. The present invention can also be implemented in other specific ways or other specific forms without departing from the gist or essential features of the present invention. Therefore, the described embodiments should be regarded as illustrative rather than restrictive in any respect. The scope of the present invention should be described by the attached claims, and any changes equivalent to the intention and scope of the claims should also be included in the scope of the present invention.

Claims

1. An elemene colon-targeted nanocapsule, characterized in that: The invention comprises an inner core and a pH-sensitive enteric polymer coated on the inner core surface. The inner core comprises elemene and a self-nanoemulsifying lipid material. The self-nanoemulsifying lipid material comprises an oil phase, an emulsifier and an auxiliary emulsifier.

2. The elemene colon-targeted nanocapsule according to claim 1, characterized in that: The pH-sensitive enteric polymer is an anionic copolymer of methacrylic acid and methyl acrylate.

3. The elemene colon-targeted nanocapsule according to claim 1, characterized in that: The oil phase is a medium-chain fatty acid; the emulsifier is selected from one or more of polyoxyethylene hydrogenated castor oil, lauroyl polyoxyl-32 glyceride, castor oil polyoxyethylene ether or caprylic capric acid polyethylene glycol glyceride; the co-emulsifier is selected from one or more of diethylene glycol monoethyl ether, polyethylene glycol 400 or propylene glycol monocaprylate.

4. The elemene colon-targeted nanocapsule according to any one of claims 1 to 3, characterized in that: The average particle size of the elemene colon-targeted nanocapsules is 130 to 140 nm, the polydispersity index is 0.17 to 0.21, and the surface charge is -13.33 to -12.61 mV; The drug loading amount and encapsulation rate of the elemene colon-targeted nanocapsule are 4.6-5.5% and 77-80.6% respectively.

5. A method for preparing elemene colon-targeting nanocapsules according to any one of claims 1 to 4, characterized in that: The following steps are involved: Mixing elemene, an oil phase, an emulsifier and a co-emulsifier to obtain elemene self-nanoemulsion; The pH-sensitive enteric polymer is coated on the surface of the elemene self-nanoemulsion to obtain the elemene colon-targeted nanocapsule.

6. The method for preparing elemene colon targeting nanocapsules according to claim 5, characterized in that: The following steps are involved: The preparation of elemene self-nanoemulsion comprises: first stirring and mixing elemene and an oil phase to obtain an elemene solution; stirring and mixing an emulsifier and an auxiliary emulsifier to obtain a self-nanoemulsion blank matrix; dropping the elemene solution into the self-nanoemulsion blank matrix and stirring and mixing for a second time to obtain the elemene self-nanoemulsion; and / or, The preparation of the pH-sensitive enteric polymer solution comprises: dissolving the pH-sensitive enteric polymer in a water-saturated ethyl acetate solution, and adjusting the pH of the system to a weak alkaline state to obtain the pH-sensitive enteric polymer solution; and / or, The preparation of elemene colon-targeted nanocapsules comprises: vortex-mixing the elemene self-nanoemulsion and the pH-sensitive enteric polymer solution to obtain a first mixed emulsion; The first mixed emulsion is added dropwise to a saturated aqueous solution of ethyl acetate, and the mixture is stirred and mixed for emulsification and diffusion to obtain a second mixed emulsion; The second mixed emulsion and pure water are mixed by a fourth stirring process, and the solvent is diffused to form an elemene colon targeting nanocapsule solution; The elemene colon-targeted nanocapsule solution is sequentially concentrated, centrifuged, and dialyzed to obtain the elemene colon-targeted nanocapsule.

7. The method for preparing elemene colon targeting nanocapsules according to claim 6, characterized in that: The concentration of elemene in the elemene solution is 0.4 mg / mL to 0.6 mg / mL; the mass concentration of the pH-sensitive enteric polymer in the pH-sensitive enteric polymer solution is 0.03 to 0.1%.

8. The method for preparing elemene colon targeting nanocapsules according to claim 6, characterized in that: The dripping speed of the elemene solution is 0.1 to 1 mL / min; the amount of the saturated aqueous solution of ethyl acetate is 3 to 6 times the amount of the first mixed emulsion; the dripping speed of the first mixed emulsion is 0.1 to 1 mL / min; the amount of pure water is 8 to 12 times the amount of the second mixed emulsion.

9. The method for preparing elemene colon targeting nanocapsules according to claim 6, characterized in that: The average particle size of the elemene self-nanoemulsion after re-emulsification with 50 times the amount of water is 40-45 nm, the polydispersity index is 0.10-0.12, and the surface charge is -6.07--5.73 mV.

10. A use of the elemene colon targeting nanocapsule according to any one of claims 1 to 4, characterized in that: Use of the elemene colon-targeted nanocapsule in preparing an oral colon-targeted preparation for treating colon cancer.

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