Elemane colon-targeting nano-capsule, preparation method and use thereof

Abstract

The application discloses a elemene colon-targeting nano-capsule and a preparation method and application thereof, and belongs to the technical field of pharmaceutical preparations, and comprises a core and a pH-sensitive enteric polymer coated on the surface of the core, wherein the core comprises elemene and a self-nanoemulsifying lipid material, and the self-nanoemulsifying lipid material comprises an oil phase, an emulsifier and a co-emulsifier. The nano-capsule has small particle size, uniform dispersion, a clear capsule structure, high drug loading and high encapsulation efficiency, can protect the drug from being released in advance in the stomach and small intestine, and can be effectively delivered to the colon. Animal experiments show that the nano-capsule releases the drug in the form of nanoemulsion in simulated colon fluid, can significantly inhibit the tumor growth of in-situ tumor-bearing mice, has good biological safety, and significantly improves the anti-colorectal cancer therapeutic effect of the drug.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, and specifically relates to an elemol colon-targeting nanocapsule, its preparation method, and its uses. Background Technology

[0002] Colorectal cancer (CRC) is a common malignant tumor of the digestive tract. It is a malignant tumor originating from the colorectal mucosal epithelium and glands, formed under the combined effects of multiple pathogenic factors. It has a high global incidence rate and is trending towards affecting younger people, seriously threatening life and health. Clinically, a comprehensive treatment approach is often used, including surgery and postoperative adjuvant radiotherapy and chemotherapy. However, insufficient accumulation of drug concentrations at the colorectal cancer tumor site often hinders the full realization of the anti-cancer efficacy of the drugs.

[0003] Elemene (EL) is an effective active monomer isolated and extracted from the rhizome of the traditional Chinese medicine Curcuma zedoaria. It is a Class II non-cytotoxic plant-derived antitumor drug with independent intellectual property rights approved in my country. Basic research suggests that it exerts its antitumor effects through multiple mechanisms, including inhibiting tumor cell proliferation, inducing tumor cell apoptosis, and inhibiting tumor tissue angiogenesis. Clinical studies have found that elemenene not only has definite therapeutic effects on various tumors but also possesses certain immunoprotective effects. It exhibits synergistic effects when used in combination with radiotherapy and chemotherapy, relieving cancer pain, increasing white blood cell count, and inhibiting platelet aggregation. It has advantages such as low toxicity, high efficacy, and broad spectrum, and is unlikely to induce drug resistance. Currently, elemenene is widely used in the clinical treatment of various cancers, such as lung cancer, gastric cancer, and colorectal cancer.

[0004] Oral colon-targeted drug delivery systems (OCDDS) are novel targeted drug delivery systems that utilize drug delivery technology to deliver drugs orally without release in the upper gastrointestinal tract, instead releasing them in the colon via the ileocecal junction to exert local or systemic therapeutic effects. OCDDS significantly enhances local efficacy, overcomes many shortcomings of traditional oral formulations, and shows broad application prospects in the treatment of various gastrointestinal diseases such as ulcerative colitis, Kernro disease, and colon cancer.

[0005] Nanocapsules (NCs) are nanoparticles with a vesicular structure. Their core acts as a liquid reservoir for drugs, primarily using a lipophilic solvent (usually oil); the outer shell is typically made of polymers, especially biodegradable polymers. Drug delivery systems employing this vesicular structure offer the following advantages: 1. High drug loading capacity: the lipid core optimizes drug solubility, allowing for greater concentrations of poorly soluble drugs within the nanoparticles; 2. Drug "protection" within the nanocapsule prevents degradation due to tissue irritation at the drug delivery site; 3. By screening different polymer shell materials, responsive release from nanocapsules at specific tissue sites can be achieved.

[0006] Therefore, based on the unique advantages of the nanocapsule structure, this invention provides an elemene colon-targeting nanocapsule, which solves the problems of insufficient effective concentration and poor bioavailability of existing elemene drugs at the tumor site of colorectal cancer. The elemene colon-targeting nanocapsule of this invention can protect the drug from premature release in the stomach and small intestine, and realize the rapid release of the drug in the form of nanoemulsion in the colon environment, which 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 objective of this invention is to provide an elemene colon-targeting nanocapsule, its preparation method, and its uses. This elemene colon-targeting nanocapsule protects the drug from premature release in the stomach and small intestine, enabling rapid drug release in the colonic environment in the form of a nanoemulsion. Its lipid core increases the solubility of poorly soluble drugs and enhances permeability in tumor tissue; the polymer shell can dissolve in the colon based on the pH environment of the digestive tract, allowing the drug to be retained in the digestive tract and released into the colonic lumen. The released drug accumulates and permeates in colorectal cancer tumor tissue in the form of a nanoemulsion, exhibiting good anti-tumor therapeutic effects.

[0008] Elecithin is an effective active monomer of traditional Chinese medicine with broad anti-tumor activity. This invention uses an emulsification-solvent diffusion method to prepare elecithin colon-targeting nanocapsules (EL@NCs). First, an elecithin self-emulsification method is used to prepare an elecithin self-nanoemulsion (EL@SNE) core to increase the solubility and stability of elecithin. Then, a multi-level dispersion technique is used to coat the core with a pH-sensitive polymer to obtain elecithin colon-targeting nanocapsules. The in vitro release, gastrointestinal retention, tumor targeting and in vivo pharmacodynamics are investigated.

[0009] To achieve the above objectives, the present invention provides an elemene colon-targeting nanocapsule comprising a core and a pH-sensitive enteric polymer coated on the surface of the core, wherein the core comprises elemene and a self-emulsifying lipid material, and the self-emulsifying lipid material comprises an oil phase, an emulsifier, and a co-emulsifier.

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

[0011] Further, the oil phase is a medium-chain fatty acid; the emulsifier is selected from one or more of polyoxyethylene hydrogenated castor oil, lauroyl polyoxy-32 glycerol ester, castor oil polyoxyethylene ether, or octanoic acid-capric acid-polyethylene glycol glycerol ester; the co-emulsifier is selected from one or more of diethylene glycol monoethyl ether, polyethylene glycol 400, or propylene glycol monooctanoate.

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

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

[0014] A pH-sensitive enteric polymer was coated onto the surface of the elemene self-nanoemulsion to obtain the elemene colon-targeting nanocapsule.

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

[0016] The preparation of elemene self-nanoemulsion includes: first stirring and mixing elemene and an oil phase to obtain an elemene solution; stirring and mixing an emulsifier and a co-emulsifier to obtain a self-nanoemulsion blank matrix; and adding the elemene solution dropwise into the self-nanoemulsion blank matrix and stirring and mixing it a second time to obtain the elemene self-nanoemulsion.

[0017] And / or,

[0018] The preparation of pH-sensitive enteric polymer solution includes: dissolving pH-sensitive enteric polymer in water-saturated ethyl acetate solution and adjusting the pH of the system to weakly alkaline to obtain pH-sensitive enteric polymer solution;

[0019] And / or,

[0020] The preparation of elemene colon-targeting nanocapsules includes: vortexing the elemene nanoemulsion with the pH-sensitive enteric polymer solution to obtain a first mixed emulsion; adding the first mixed emulsion dropwise to a saturated aqueous solution of ethyl acetate, followed by a third stirring and mixing process to emulsify and diffuse, resulting in a second mixed emulsion; mixing the second mixed emulsion with pure water followed by a fourth stirring process to allow solvent diffusion, forming an elemene colon-targeting nanocapsule solution; and sequentially concentrating, centrifuging, and dialyzing the elemene colon-targeting nanocapsule solution to obtain the elemene colon-targeting nanocapsules.

[0021] Further, 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 elemol solution is added at a rate of 0.1–1 mL / min, the amount of the ethyl acetate saturated aqueous solution is 3–6 times that of the first mixed emulsion, and the amount of pure water is 8–12 times that of the second mixed emulsion.

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

[0024] In another aspect, the present invention also provides the use of the aforementioned elemene colon-targeting nanocapsules in the preparation of an oral colon-targeting formulation for the treatment of colon cancer.

[0025] Compared with existing technologies, the present invention has the following beneficial effects: The elemol colon-targeting nanocapsules prepared by the present invention are an oral colon-targeting formulation. The nanocapsules have small particle size, uniform dispersion, and a well-defined capsule structure, resulting in superior drug loading and encapsulation efficiency. This protects the drug from premature release in the stomach and small intestine, allowing for rapid release at the colon in the form of nanoemulsions. Due to the presence of pH-sensitive enteric polymers, the surface maintains a negative charge under acidic conditions (pH 1.2–6.8), while under alkaline conditions (pH 7.4), the pH-sensitive enteric polymers dissolve, releasing the drug in the form of even smaller nanoemulsions. This achieves aggregation and penetration at the colon tumor site, demonstrating a certain degree of tumor targeting.

[0026] In vitro studies have shown that the elemene colon-targeting nanocapsules of the present invention rapidly release drugs in the form of nanoemulsions in simulated colonic fluid, and exhibit certain tumor targeting in a mouse model of orthotopic colorectal cancer. In vivo pharmacodynamic studies have shown that, compared with elemene raw material and marketed drugs (elemene oral emulsion), the elemene colon-targeting nanocapsules of the present invention can significantly inhibit tumor growth in orthotopic tumor-bearing mice and have good biosafety, indicating that this formulation can significantly improve the anti-colorectal cancer therapeutic effect of the drug. Attached Figure Description

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

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

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

[0030] Figure 4 The following are transmission electron microscopy (TEM) images of the in vitro release of the present invention: elemene colon-targeting nanocapsules (EL@NCs) were observed under TEM after being incubated in SCF for 0 h (left), 2 h (middle), and 6 h (right);

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

[0032] Figure 6 The FTIC analysis diagrams of the present invention are shown: elemene (EL); blank matrix (CON-SNE); elemene self-nanoemulsion (EL@SNE); Eudragit@S100; elemene colon-targeting nanocapsules (EL@NCs);

[0033] Figure 7 The following IVIS images (n=36) show the in vivo imaging (top) and gastrointestinal biodistribution (bottom) of DID, DID@SNE and DID@NCs in mice at 2, 4, 6 and 24 hours after administration of the present invention.

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

[0035] Figure 9 The following IVIS images (n=6) show the biodistribution of the tumor-bearing intestine in a fluorescent field (top) and a bright field (bottom) three days after administration to the orthotopic colon cancer model mice of the present invention.

[0036] Figure 10 The images show the biodistribution IVIS images (left) and relative tumor volume assessment (right) of mice in each experimental group of the orthotopic colon cancer model mouse of the present invention (n=30);

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

[0038] Figure 12 The process of preparing the elemene colon-targeting nanocapsules (EL@NCs) of the present invention and their colon-targeted release are illustrated in the diagram. Detailed Implementation

[0039] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included within this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range. The invention will now be described in detail with reference to embodiments.

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

[0041] To achieve the above objectives, a first aspect of the present invention provides an elemene colon-targeting nanocapsule comprising a core and a pH-sensitive enteric polymer coated on the surface of the core, wherein the core comprises elemene and a self-emulsifying lipid material, the self-emulsifying lipid material comprising an oil phase, an emulsifier, and a co-emulsifier.

[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, insoluble at pH values ​​below 7, and can be widely used as a coating material for oral colon-targeted drug delivery systems. Therefore, to protect the drug and nanocarrier from degradation in the acidic environment of the stomach and to achieve targeted and localized delivery to the colonic epithelium, this invention selects ES as the 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 nonionic emulsifier, such as, but not limited to, one or more of polyoxyethylene hydrogenated castor oil, lauroyl polyoxy-32 glycerol ester, castor oil polyoxyethylene ether, or PEG-32 glycerol octanoate; the co-emulsifier includes, 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. More preferably, the medium-chain fatty acid is caprylic / capric triglyceride (purchased from Gattefossé China; product name: Labrafac). @ WL1349).

[0045] By using the aforementioned self-nanoemulsion lipid material and elemene to form a self-nanoemulsion system, the solubility of elemene can be effectively improved, which is conducive to the absorption of elemene in vivo, and at the same time provides a certain degree of tumor targeting.

[0046] To further improve the solubility, permeability, and targeted effect of the drug, in a preferred embodiment of the present invention, the mass ratio of the elemol, 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 elemene colon-targeting nanocapsules described in this invention have an average particle size of 130–140 nm, a polydispersity index of 0.17–0.21, and a surface charge of -13.33–-12.61 mV; the drug loading and encapsulation efficiency of the elemene colon-targeting nanocapsules are 4.6–5.5% and 77–80.6%, respectively.

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

[0049] A mixture of elemene, oil phase, emulsifier and co-emulsifier is used to obtain elemene self-nanoemulsion;

[0050] The elemene colon-targeting nanocapsules were prepared by coating the surface of the elemene self-nanoemulsion with a pH-sensitive enteric polymer.

[0051] In a preferred embodiment of the present invention, a pH-sensitive enteric polymer is coated onto the surface of the elemene nanoemulsion using a graded diffusion technique to prepare the elemene colon-targeting nanocapsules. Graded diffusion refers to a technique that utilizes the different solubilities of a bridging agent (in this invention, a pH-sensitive enteric polymer) in different solvents to generate diffusion motion and prepare nanocapsules.

[0052] In a preferred embodiment of the present invention, the preparation method of the elemene colon-targeting nanocapsules includes the following steps:

[0053] Step S1, Preparation of elemene self-nanoemulsion: elemene and oil phase are first stirred and mixed to obtain elemene solution; emulsifier and co-emulsifier are stirred and mixed to obtain self-nanoemulsion blank matrix; the elemene solution is added dropwise to the self-nanoemulsion blank matrix and stirred and mixed a second time to obtain elemene self-nanoemulsion;

[0054] Step S2, Preparation of pH-sensitive enteric polymer solution: Dissolve the pH-sensitive enteric polymer in water-saturated ethyl acetate solution and adjust the pH of the system to weakly alkaline to obtain the pH-sensitive enteric polymer solution;

[0055] Step S3, Preparation of elemene colon-targeting nanocapsules: The elemene nanoemulsion is vortexed with the pH-sensitive enteric polymer solution to obtain a first mixed emulsion; the first mixed emulsion is added dropwise to an ethyl acetate saturated aqueous solution, and after a third stirring and mixing, emulsification and diffusion are carried out to obtain a second mixed emulsion; the second mixed emulsion is mixed with pure water after a fourth stirring and mixing, and solvent diffusion is carried out to form an elemene colon-targeting nanocapsule solution; the elemene colon-targeting nanocapsule solution is concentrated, centrifuged, and dialyzed sequentially to obtain the elemene colon-targeting nanocapsules.

[0056] To further improve the efficacy and targeted effect of elemene colon-targeting 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 nanoemulsion to the pH-sensitive enteric polymer is 1:1 to 4.

[0057] To further improve the uniformity of the elemene nanoemulsion and the solubility of the drug, in a preferred embodiment of the present invention, the first stirring and mixing speed is 200-500 rpm for 1-15 min; the vortex mixing time is 1-5 min; the elemene solution is added at a rate of 0.1-1 mL / min; and the second stirring and mixing speed is 200-500 rpm for 5-30 min.

[0058] 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 ethyl acetate saturated aqueous solution is 3 to 6 times the amount of the first mixed emulsion; the dropping rate of the first mixed emulsion is 0.1 to 1 mL / min; and 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-processing of the elemene colon-targeting nanocapsule solution includes the following steps: the elemene colon-targeting nanocapsule solution is concentrated, centrifuged, and dialyzed sequentially to obtain elemene colon-targeting nanocapsules.

[0060] Specifically, the process includes the following steps: concentrating the elemene colon-targeting 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 with a capacity of 8 kDa–14 kDa for 18–30 h to obtain elemene colon-targeting nanocapsules, freeze-drying the elemene colon-targeting nanocapsules, and storing them at 4°C for subsequent use.

[0061] The elemene described in this invention, after being reemulsified with 50 times the amount of water, has an average particle size of 40-45 nm, a polydispersity index of 0.10-0.12, and a surface charge of -6.07--5.73 mV.

[0062] This invention prepares elemene colon-targeting nanocapsules (EL@NCs) via an emulsification-solvent diffusion method. Using lipid self-nanoemulsion as the core, pH-sensitive enteric polymer is encapsulated using hierarchical diffusion technology. The process basically includes four steps: preparation of the lipid core elemene self-nanoemulsion (EL@SNE), preparation of the pH-sensitive enteric polymer solution for the capsule material (ES), hierarchical diffusion encapsulation, and post-treatment.

[0063] Among them, the lipid-core elemene self-nanoemulsion is prepared by mixing drugs, oil phase, emulsifiers, and co-emulsifiers to form a self-nanoemulsion system. It has advantages such as improving the solubility of poorly soluble drugs, encompassing drugs with different lipid solubilities, facilitating drug absorption in vivo, and also exhibiting certain tumor targeting properties. In the graded diffusion coating process, the mixed emulsion of elemene self-nanoemulsion and pH-sensitive enteric polymer solution is emulsified and solvent diffused in saturated aqueous solution of ethyl acetate and pure water, respectively. The pH-sensitive enteric polymer is utilized as a polymer material. The solubility of ethyl acetate in water-saturated water, water saturated with ethyl acetate, and pure water is different. Self-driven fractional diffusion preparation has the advantages of simple operation, low energy consumption, and removal of residual organic solvents, making it green and environmentally friendly.

[0064] The preparation of the lipid core elemene nanoemulsion and the formulation of the pH-sensitive enteric polymer solution for the encapsulation material determine the efficiency of pH-sensitive enteric polymer encapsulation during fractional diffusion. Simultaneously, by synergistically controlling process parameters such as drug addition rate, solvent ratio, stirring rate, and time during emulsification-solvent diffusion, elemene colon-targeting nanocapsules (EL@NCs) were successfully prepared. The method of this invention is relatively simple in design and safe and feasible to operate.

[0065] A third aspect of the present invention also provides the use of the aforementioned elemene colon-targeting nanocapsules in the preparation of an oral colon-targeting formulation for the treatment of colon cancer.

[0066] The present application will be further described in detail below with reference to specific embodiments, which 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 raw material (purchased from Dalian Jingang Pharmaceutical Co., Ltd., purity 99.9%) and dissolve it in 90 mg of Labrafac@WL1349 (caprylic / capric triglyceride, purchased from Gattefossé China). Stir magnetically at room temperature until completely dissolved (stirring speed 200 rpm, stirring time 5 min) to obtain a solution containing 50% EL drug.

[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 magnetically at 500 rpm at room temperature until completely dissolved to obtain a blank matrix;

[0070] 3) The EL drug solution was then added dropwise to the blank matrix at a rate of 0.5 mL / min, and the mixture was stirred for 10 min at a speed of 200 rpm to obtain EL@SNE. In the experiment, EL@SNE was diluted and re-emulsified with 50 times the amount of water. It can be observed that the prepared EL@SNE suspension is translucent with a light blue opalescence due to the Tyndall effect, and the experimental operation has good reproducibility and stability.

[0071] Example 2

[0072] 1) Put 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 an Eudradit@S100 solution (mass concentration of 0.05%).

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

[0074] 3) The above mixed emulsion was added dropwise to 25 mL of ethyl acetate-saturated aqueous solution at a dropping rate of 0.1 mL / min using a staged diffusion technique, and the mixture was magnetically stirred at 200 rpm for 10 min to achieve emulsification and diffusion.

[0075] 4) Pour the mixed emulsion treated in step 3) into 250mL of water at once, mix and equilibrate by magnetic stirring at 500rpm for 30min, and then replace the solvent;

[0076] 5) Take the mixed emulsion after step 4) and concentrate it to 5 mL in a rotary evaporator at 32 °C. Centrifuge at 2000 r / min for 20 min, collect the supernatant, and dialyze it in a dialysis bag (8 kDa ~ 14 kDa) for 24 hours to obtain elemene colon-targeting nanocapsules (EL@NCs). Freeze-dry the nano suspension for 72 h using a vacuum freeze dryer and store it at 4 °C for subsequent use.

[0077] Example 3

[0078] The EL@SNE and EL@NCs from Examples 1 and 2 were observed using transmission electron microscopy. Comparative observation revealed that the nanoparticles were all spherical, but with varying degrees of edge blurring, confirming that the EL@NCs possess a rigid nanocapsule structure. The results are shown in [Figure number missing]. Figure 1 .

[0079] Example 4

[0080] Three parallel preparations of EL@SNE and EL@NCs from Examples 1 and 2 were made. Their average particle size, PDI polydispersity index, and Zeta potential were measured using a dynamic light scattering instrument. The results showed that the average particle size of the re-emulsified EL@SNE was 43.08 nm ± 2.06 nm, the PDI polydispersity index was 0.11 ± 0.01, and the surface charge was -5.90 mV ± 0.17. The average particle size of the EL@NCs was 135.60 nm ± 4.18 nm, the PDI polydispersity index was 0.19 ± 0.02, and the surface charge was -12.97 mV ± 0.36. This further confirms that the coating of ES on the surface of EL@SNE forms EL@NCs, causing changes in particle size and potential. The results are shown in [Figure number missing]. Figure 2 .

[0081] Example 5

[0082] The EL@NCs from Example 2 were placed in a constant temperature water bath shaker, and their release was measured over 180 minutes in different simulated digestive fluids (simulated gastric juice SGF, simulated intestinal juice SIF, and simulated colonic juice SGF). The cumulative release (%) at different time points was further examined. The results showed that Example 2 resulted in less than 20% drug release in the first 5 hours (pH 1.2 and 6.8). When the pH was increased to 7.4, a cumulative release of 92.4% ± 3.6% of the EL was achieved. (See attached figures). Figure 3 .

[0083] Example 6

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

[0085] Example 7

[0086] The EL@SNE in Examples 1 and 2, DSC analysis was performed on physical mixtures of EL@SNE and Eudergit@S100, and EL@NCs. Specifically, 10 mg each of EL@SNE, Eudergit@S100, the physical mixture of EL@SNE and Eudergit@S100, and EL@NCs were weighed and filled into aluminum crucibles. The crucibles were then covered with lids (with a small hole punched in the lid) and pressed together using a press. A blank aluminum crucible was used as a reference. A nitrogen atmosphere was maintained at a flow rate of 20.0 mL / min. The temperature was increased within the range of 20–460 °C, and the scanning rate was 10.0 °C / min. Differential scanning calorimetry (DSC) curves were obtained, and the results are shown below. Figure 5 .

[0087] Example 8

[0088] Fourier transform infrared (FTIR) spectroscopy was performed on the EL, blank lipid matrix (CON-SNE), EL@SNE, Eudergit@S100, and EL@NCs from Examples 1 and 2 to detect any interactions between the components. Specifically, EL, CON-SNE, EL@SNE, Eudergit@S100, and EL@NCs samples were weighed. The sample (powder) was mixed with dry KBr at a 1:100 ratio, finely ground, and then compressed into a tablet. FTIR spectra were then analyzed at 500–4000 cm⁻¹. -1 The analysis scan was performed within the wavenumber range, and the background absorbance was subtracted to obtain the infrared spectrum of the sample. The results are shown in [Figure number missing]. Figure 6 .

[0089] Example 9

[0090] Hydrophobic near-infrared dye DID was used as a fluorescent probe to load onto the nanoparticle coating formulation instead of the core drug. DID@SNE and DID@NCs were prepared using the same DID concentration (1.5 mg DID / g, based on an average mouse weight of 30 g) and the same preparation process, with free DID as a control. The drug was administered by gavage at predetermined time points (2, 4, 6, and 24 h), and anesthesia was maintained by nasal inhalation of 2.0% isoflurane. Photoexcitation was performed at 644 nm, and emission wavelength was detected at 664 nm. Whole-body fluorescence images of mice were obtained using an IVISLumina LT imaging system. Mice were euthanized by cervical dislocation at each time point, and the entire gastrointestinal tract from stomach to rectum was immediately harvested for in vitro observation of fluorescence signal distribution in the mouse GIT. Results are shown below. Figure 7 .

[0091] Example 10

[0092] 1) Take 48 healthy male SPF-grade Balb / c mice with an average weight of about 20-30g, acclimatize them at room temperature for two weeks, fast them for 18 hours before modeling but allow them to drink water, and then inject them intraperitoneally with 20% urethane anesthetic saline (0.006mL / g), at a dose of 1.2mg / g.

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

[0094] 3) Whole-body fluorescence images were acquired using the IVIS LuminaLT imaging system on days 3 and 7 after modeling for modeling observation. See [link to image]. Figure 8 .

[0095] Example 11

[0096] Six tumor-bearing mice successfully modeled in Example 10 were administered DID@NCs orally for three consecutive days. Twenty-four hours after the last administration, the mice were euthanized by cervical dislocation. Whole intestinal tissue was harvested, and fluorescence localization of the entire intestinal tissue was obtained using the IVIS LuminaLT imaging system and compared with bright-field imaging. Figure 9 .

[0097] Example 12

[0098] 1) Forty-two tumor-bearing mice that were successfully modeled in Example 10 were taken, with an average weight of about 20-30g. The mice were randomly divided into 6 groups, including blank control group (PBS group), excipient matrix control group (Placebo group), elemene raw material group (Free EL group), commercially available elemene oral emulsion (Marketed drug group), elemene self-nanoemulsion (EL@SNE group) and elemene colon-targeting nanocapsules (EL@NCs group), with 7 mice in each group, and each group was housed in the same cage;

[0099] 2) Mice were administered the drug by gavage daily for two weeks. Tumor changes in mice were assessed using the IVIS LuminaLT imaging system on days 0, 7, 14, and 21. (See attached image.) Figure 10 Bioluminescence results and relative tumor volume analysis of different groups showed that, compared with the PBS group and the Placebo group, the Free EL group and the Marketed drug group could slow down the rapid growth of tumors, while the EL@SNE group and the EL@NCs group with nanostructures could significantly inhibit tumor growth, especially the elemene nanocapsules EL@NCs with colon localization function, with a relative tumor volume of 0.813±0.102 after 21 days.

[0100] Example 13

[0101] Twelve mice were administered the drug in the groups described in Example 12 for 14 days. Heart, liver, spleen, lung, and kidney tissues from each group were collected for hematoxylin-eosin (H&E) staining to assess the biosafety of the formulation. (See attached table). Figure 11 .

[0102] The process of preparing the elemene colon-targeting nanocapsules of the present invention and its colon-targeted release are illustrated in the following diagram. Figure 12As shown, in vitro studies have demonstrated that the elemene colon-targeting nanocapsules of the present invention rapidly release drugs in the form of nanoemulsions in simulated colonic fluid, exhibiting certain tumor targeting in a mouse model of orthotopic colorectal cancer. In vivo pharmacodynamic studies have shown that, compared with elemene raw material and marketed drugs (elemene oral emulsion), the elemene colon-targeting nanocapsules of the present invention can significantly inhibit tumor growth in orthotopic tumor-bearing mice and have good biosafety, indicating that this formulation can significantly improve the anti-colorectal cancer therapeutic effect of the drug.

[0103] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. The present invention can also be implemented in other specific ways or forms without departing from its spirit or essential characteristics. Therefore, the described embodiments should be considered illustrative rather than limiting in any respect. The scope of the present invention should be defined by the appended claims, and any variations equivalent to the intent and scope of the claims should also be included within the scope of the present invention.

Claims

1. A colon-targeting nanocapsule containing elemol, characterized in that, The product comprises a core and a pH-sensitive enteric polymer coating the surface of the core. The core includes elemol and a self-emulsifying lipid material, the self-emulsifying lipid material comprising an oil phase, an emulsifier, and a co-emulsifier. The pH-sensitive enteric polymer is Eudradit® S100, which is an anionic copolymer of methacrylic acid and methyl acrylate. The oil phase is a medium-chain fatty acid. The emulsifier is selected from one or more of polyoxyethylene hydrogenated castor oil and castor oil polyoxyethylene ether. The co-emulsifier is selected from one or more of diethylene glycol monoethyl ether and polyethylene glycol 400. The preparation method of the elemene colon-targeting nanocapsules includes the following steps: mixing elemene, an oil phase, an emulsifier, and a co-emulsifier to obtain elemene self-nanoemulsion; coating the surface of the elemene self-nanoemulsion with a pH-sensitive enteric polymer to obtain the elemene colon-targeting nanocapsules.

2. The elemene colon-targeting nanocapsule according to claim 1, characterized in that, The elemene colon-targeting nanocapsules have an average particle size of 130-140 nm, a polydispersity index of 0.17-0.21, and a surface charge of -13.33--12.61 mV; the drug loading and encapsulation efficiency of the elemene colon-targeting nanocapsules are 4.6-5.5% and 77-80.6%, respectively.

3. A method for preparing elemene colon-targeting nanocapsules as described in claim 1 or 2, characterized in that, Includes the following steps: A mixture of elemene, oil phase, emulsifier and co-emulsifier is used to obtain elemene self-nanoemulsion; A pH-sensitive enteric polymer was coated onto the surface of the elemene self-nanoemulsion to obtain the elemene colon-targeting nanocapsule.

4. The method for preparing elemene colon-targeting nanocapsules according to claim 3, characterized in that, Includes the following steps: The preparation of elemene self-nanoemulsion includes: first stirring and mixing elemene and an oil phase to obtain an elemene solution; stirring and mixing an emulsifier and a co-emulsifier to obtain a self-nanoemulsion blank matrix; and adding the elemene solution dropwise into the self-nanoemulsion blank matrix and stirring and mixing it a second time to obtain the elemene self-nanoemulsion. And / or, The preparation of pH-sensitive enteric polymer solution includes: dissolving pH-sensitive enteric polymer in water-saturated ethyl acetate solution and adjusting the pH of the system to weakly alkaline to obtain pH-sensitive enteric polymer solution; And / or, The preparation of elemene colon-targeting nanocapsules includes: vortexing the elemene nanoemulsion with the pH-sensitive enteric polymer solution to obtain a first mixed emulsion; The first mixed emulsion was added dropwise to a saturated aqueous solution of ethyl acetate, and then stirred and mixed in a third stage to emulsify and diffuse, thus obtaining a second mixed emulsion. The second mixed emulsion and pure water were mixed by a fourth stirring process, and the solvent diffused to form an elemol colon-targeting nanocapsule solution. The elemene colon-targeting nanocapsule solution was concentrated, centrifuged, and dialyzed sequentially to obtain the elemene colon-targeting nanocapsules.

5. The method for preparing elemene colon-targeting nanocapsules according to claim 4, 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%.

6. The method for preparing elemene colon-targeting nanocapsules according to claim 4, characterized in that, The elemene solution is added at a rate of 0.1-1 mL / min; the amount of the ethyl acetate saturated aqueous solution is 3-6 times that of the first mixed emulsion; the first mixed emulsion is added at a rate of 0.1-1 mL / min; and the amount of pure water is 8-12 times that of the second mixed emulsion.

7. The method for preparing elemene colon-targeting nanocapsules according to claim 4, characterized in that, The elemene nanoemulsion, after being reemulsified with 50 times the amount of water, has an average particle size of 40-45 nm, a polydispersity index of 0.10-0.12, and a surface charge of -6.07--5.73 mV.

8. The use of the elemene colon-targeting nanocapsules as described in claim 1 or 2, characterized in that, The use of the elemene colon-targeting nanocapsules in the preparation of oral colon-targeting formulations for the treatment of colon cancer.

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