Capsule for oral delivery of mRNA and preparation method and application thereof

Abstract

The invention relates to a capsule for oral delivery of mRNA as well as a preparation method and application of the capsule, and belongs to the technical field of biological medicines. The capsule comprises a capsule body and a capsule cap, a flexible film is arranged on the inner surface of the capsule body, a non-flexible film is arranged at an opening of the capsule body to cover the opening, the outer surface of the capsule body and the outer surface of the capsule cap are coated with pH sensitive layers, and the pH sensitive layers contain pH sensitive polymers. The capsule for oral delivery of mRNA can realize oral delivery of solid or liquid mRNA, has good stability, drug loading capacity and release kinetics, can maintain a complete form in the stomach, can be rapidly disintegrated in the intestinal tract, improves the stability of the mRNA active component in a complex gastrointestinal environment, and improves the bioavailability of mRNA. And a technical foundation is laid for developing non-intrusive and modularized administration dosage forms.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and relates to a capsule for oral delivery of mRNA, a preparation method thereof, and an application thereof. Background Art

[0002] mRNA, also known as messenger RNA, is responsible for the translation and synthesis of proteins in cells. mRNA technology imports in vitro artificially synthesized mRNA into cells to make the cells become factories for synthesizing proteins. The novel coronavirus vaccine based on mRNA technology has achieved breakthrough achievements in disease prevention and control. The technological progress in this field has promoted the extension of mRNA therapy technology from the prevention of infectious diseases to cancer immunotherapy, genetic disease replacement therapy, and regenerative medicine and other fields. However, there are certain clinical application limitations in the existing administration routes of mRNA preparations: Injection administration can quickly achieve systemic distribution, with accurate dosage and wide applicability. However, its intravenous / muscular injection mode that relies on professional medical personnel operation is difficult to meet the rapid immunization needs during the outbreak of sudden infectious disease pandemics, and the patient compliance is significantly reduced in the long-term treatment of chronic diseases with repeated administration. Although the novel nebulization administration method of mRNA preparations in existing research has the advantages of non-invasive, high local concentration, and avoidance of the first-pass effect of the liver through targeted delivery through the respiratory mucosa or lungs, it faces challenges such as complex nebulization devices, the need for patient cooperation in nebulization, single disease, and difficult control of drug stability.

[0003] The oral administration route occupies a dominant position in the field of drug treatment due to its non-invasiveness, convenience, and wide applicability, and is particularly suitable for chronic disease management and large-scale public health interventions. High patient compliance is one of its core advantages. In terms of economy and accessibility, the production and storage costs of oral drugs are relatively low. At the disease treatment level, oral administration can accurately target local lesions in the digestive tract, etc. Therefore, the development of orally delivered mRNA therapeutic drugs has gradually become a research hotspot. For example, CN115515559A discloses the rectal delivery of messenger RNA, and the mRNA is encapsulated in lipid nanoparticles, which contain one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids.

[0004] In summary, developing a novel oral delivery carrier for mRNA therapeutic drugs and expanding the oral delivery tools for mRNA therapeutic drugs are of great significance for the field of development of orally administered mRNA therapeutic drugs. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art and actual needs, the present invention provides a capsule for oral delivery of mRNA, a preparation method thereof, and an application thereof, which improves the stability of mRNA active ingredients in the complex gastrointestinal environment and lays a technical foundation for the development of non-invasive and modular dosage forms.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a capsule for oral delivery of mRNA, the capsule comprising a capsule body and a capsule cap, the inner surface of the capsule body being provided with a flexible film, the flexible film comprising at least one of a polyolefin composite film, a hydroxypropyl methylcellulose film, a silicone rubber film, a polyvinyl alcohol film, a pullulan film or a silicone elastomer film; an opening of the capsule body being covered with a non-flexible film, the non-flexible film comprising at least one of a bio-based film, a vinyl polymer film or a medical-grade polyester PET film; the outer surfaces of the capsule body and the capsule cap being coated with a pH-sensitive layer, the pH-sensitive layer containing a pH-sensitive polymer, the pH-sensitive polymer comprising at least one of an acrylic acid polymer and its derivatives, chitosan, hypromellose acetate succinate, hypromellose phthalate, poly(lactic-co-glycolic acid), dextran, pectin or sodium alginate.

[0008] The present invention designs a capsule for oral delivery of mRNA, designs a specific capsule structure, including designing the internal structure of the capsule body and the opening sealing method, and designing a pH-sensitive layer on the outer surface of the capsule, etc., which can realize oral delivery of mRNA in solid form or liquid form, has good stability, drug loading capacity and release kinetics, can maintain an intact form in the stomach, and rapidly disintegrate in the intestine, significantly improving the stability of the mRNA active ingredient in the complex gastrointestinal environment, and laying a technical foundation for the development of non-invasive and modular dosage forms.

[0009] Preferably, the outer surfaces of the capsule body and the capsule cap are further coated with the flexible film, and the flexible film is located between the outer surface and the pH-sensitive layer.

[0010] In the present invention, by coating the flexible film (which can be coated in one layer) on the outer surfaces of the capsule body and the capsule cap, a non-snap rigid capsule can be obtained, and this rigid capsule will not deform or squeeze to release its contents during intestinal peristalsis.

[0011] Preferably, the materials of the capsule body and the capsule cap are selected from at least one of gelatin, gum arabic, starch and its derivatives, alginate, mulberry silk gum, hydroxypropyl methylcellulose, pullulan, trehalose, hypromellose phthalate, polyvinylpyrrolidone copolymer or polyvinyl alcohol.

[0012] Preferably, the area of the non-flexible film is at least 10% larger than the area of the opening of the capsule body, and it can be selected to be 20%, 30%, etc. larger.

[0013] In the present invention, the molecular weight range of the methacrylic acid-ethyl acrylate copolymer is about 20 - 250 KDa, and the molar ratio of methacrylic acid to ethyl acrylate is 1:1 or 1:2. The molecular weight range of hypromellose acetate succinate: about 7 - 15 KDa, degree of substitution: acetyl group (0.5 - 1.0), succinyl group (0.2 - 0.5). The molecular weight range of hypromellose phthalate: about 4.5 - 13 KDa, degree of substitution: phthaloyl group about 15%. The molecular weight range of chitosan is about 5 - 20 KDa, degree of polymerization: 100–200. The molecular weight range of poly(lactic-co-glycolic acid) is about 5 - 200 KDa. The molecular weight range of dextran is about 10 - 100KDa, degree of polymerization is about 100 - 5000. The molecular weight range of pectin is about 100 - 1000 KDa, and the molecular weight range of sodium alginate is 30 - 250KDa. Such as Eudragit L100-55 (molecular weight 250 KDa, degree of substitution of carboxyl group 10%), etc.

[0014] In a second aspect, the present invention provides a method for preparing the capsule for oral delivery of mRNA described in the first aspect, and the preparation method includes:

[0015] Coating the inner surface of the capsule body with the flexible film, covering the opening of the capsule body with a non-flexible film, and coating the outer surfaces of the capsule body and the capsule cap with a pH-sensitive polymer solution to obtain the capsule for oral delivery of mRNA.

[0016] Preferably, the mass percentage of the pH-sensitive polymer in the pH-sensitive polymer solution is 1% - 60%, for example, it can be 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 56%, 57%, 58% or 59%, etc.

[0017] In a third aspect, the present invention provides the application of the capsule for oral delivery of mRNA described in the first aspect in the preparation of oral mRNA products.

[0018] In the present invention, the designed capsule for oral delivery of mRNA can orally deliver the mRNA therapeutic drug to the intestine, generate pressure release and contraction according to the intestinal pH value, get rid of the traditional invasive administration methods such as intramuscular, subcutaneous, intravenous, etc. for mRNA nucleic acid drugs, and can effectively develop oral mRNA products such as mRNA therapeutic drugs, etc.

[0019] In a fourth aspect, the present invention provides an mRNA oral delivery system, and the delivery system includes the capsule for oral delivery of mRNA described in the first aspect and mRNA, and the mRNA is encapsulated in the capsule body by the non-flexible film.

[0020] It is understandable that any mRNA can be delivered using the capsule, and the mRNA can be an mRNA drug, such as any mRNA sequence capable of producing anti-PD1, including but not limited to self-reporting gene anti-PD1 mRNA, self-amplifying anti-PD1 mRNA, and circular anti-PD1 mRNA. Such as interleukin 10 (IL10)-mRNA, etc. The capsule can remain intact in the stomach, disintegrate rapidly in the intestine, release the IL10-mRNA drug, cross the intestinal mucosal barrier to reach the intestinal tissue for effective in vivo transfection and IL10 protein translation, and can significantly reduce the levels of pro-inflammatory cytokines in peripheral blood and intestinal tissue, alleviating colitis. The mRNA can also be EGFP-mRNA, Fluci mRNA, etc.

[0021] Preferably, the mRNA is also loaded in a nano-delivery carrier.

[0022] In the present invention, the mRNA is loaded in a nano-delivery carrier and then orally delivered using the capsule.

[0023] Preferably, the nano-delivery carrier includes any one or a combination of at least two of cationic lipid complexes, lipid nanoparticles, polymer nanoparticles, or lipid-polymer hybrid nanoparticles.

[0024] Preferably, the lipid-polymer hybrid nanoparticles contain cationic molecules, polymers, and biocompatible modification molecules.

[0025] Preferably, the cationic molecules include any one or a combination of at least two of ammonia molecule derivatives, positively charged amphiphilic lipid compounds, ammonium salts and their derivatives, or polyamides and their derivatives; the polymers include any one or a combination of at least two of polylactic acid and its copolymers, carbon phospholipids and their derivatives, amino acids and their derivatives, polyols and their derivatives, or polyacrylic acid and its derivatives; the biocompatible modification molecules include any one or a combination of at least two of polyethylene glycol and its derivatives, mannitol, dextran, carboxy dextran, liposomes, albumin, tetraethyl orthosilicate, or polyglutamic acid.

[0026] Preferably, the morphology of the mRNA is in liquid form or nanoparticle form.

[0027] In the present invention, a lipid-polymer hybrid nano-delivery system (LPH.NPs) can be used to load mRNA to prepare liquid mRNA.

[0028] In a fifth aspect, the present invention provides a preparation method of the mRNA oral delivery system described in the fourth aspect, and the preparation method includes:

[0029] Take the capsule for oral delivery of mRNA described in the first aspect, place the mRNA in the capsule body, cover the opening of the capsule body with the non-flexible membrane, and assemble the capsule body and the capsule cap.

[0030] Sixth aspect, the present invention provides the application of the mRNA oral delivery system described in the fourth aspect in the preparation of drugs.

[0031] Preferably, the drug includes drugs for treating intestinal and other diseases, and the diseases include but are not limited to intestinal diseases such as colitis, intestinal polyps, colorectal cancer, pancreatic cancer, liver cancer, and other systemic diseases such as melanoma.

[0032] Compared with the prior art, the present invention has at least the following beneficial effects:

[0033] The present invention designs a capsule for oral delivery of mRNA, designs a specific capsule structure, constructs a new mRNA delivery platform, breaks through the technical bottleneck of relying on professional medical resources through a non-invasive self-administered drug mode, improves patient compliance and the emergency response rate of public health emergencies; breaks through the mucosal immunogenic characteristics that are difficult to achieve with existing injection administration, activates the systemic immune response and mucosal immune defense system simultaneously through the gut-associated lymphoid tissue (GALT), constructs a pathogen invasion barrier mainly composed of secretory IgA, and has unique value in the prevention of digestive tract infectious diseases; realizes specific gastrointestinal cell transfection through targeted delivery technology, forms an advantage in the local treatment dimension of digestive tract diseases such as inflammatory bowel disease and colorectal cancer that cannot be achieved by existing injection administration, significantly improves the stability of the mRNA active ingredient in the complex gastrointestinal environment, and lays a technical foundation for the development of non-invasive and modular dosage forms. Description of the Drawings

[0034] Figure 1 It is a schematic diagram of the RNAcap capsule structure.

[0035] Figure 2 It is a schematic diagram of the preparation of Capsule-(A)B and Capsule-(B)B.

[0036] Figure 3 It is a comparison result diagram of the loading capacity of Capsule-(A)B and Capsule-(B)B.

[0037] Figure 4 It is a schematic diagram for evaluating the rapid release ability after removing the capsule cap of Capsule-(A)B and Capsule-(B)B.

[0038] Figure 5 It is a comparison result diagram of the rapid release of the contents after removing the capsule cap of the aforementioned Capsule-(A)B and Capsule-(B)B in a neutral environment (pH 7.4).

[0039] Figure 6Release curves of capsule-(A)B and capsule-(A)A removing the capsule cap for RB-loaded.

[0040] Figure 7 Stability results of RNACap (capsule-(A)B) loaded with RB.

[0041] Figure 8 Visualization release results of pH-sensitive capsule L100-55-RNACap loaded with RB in aqueous buffers at pH 1.2 and pH 7.4.

[0042] Figure 9 Cumulative release curve results of pH-sensitive capsules L100-55-RNACap and L100-RNACap loaded with RB in aqueous buffers at pH 1.2 and pH 7.4, where Figure A is the cumulative release curve of capsule L100-55-RNACap, Figure B is the cumulative release curve of RNACap capsule, and Figure C is the cumulative release curve of capsule L100-RNACap.

[0043] Figure 10 Characterization results of liquid mRNA nanomedicine, where Figure A is the particle size result, Figure B is the polydispersity coefficient result, Figure C is the potential result, and Figure D is the electron microscopy result.

[0044] Figure 11 Ex vivo images of the gastrointestinal tract of rats excised at various time points from 15 min to 6 h after intragastric administration of L100-55-Cy5-mRNA-RNAcap.

[0045] Figure 12 Ex vivo images of other organs of rats except the gastrointestinal tract at various time points from 15 min to 6 h after intragastric administration of L100-55-Cy5-mRNA-RNAcap.

[0046] Figure 13 Ex vivo images of the gastrointestinal tract of rats excised 6 h after intragastric administration of L100-Cy5-mRNA-RNAcap.

[0047] Figure 14 Absorption results of Cy5-mRNA in the rat intestine.

[0048] Figure 15 Effect diagrams of non-snap rigid capsules and RNAcap for oral delivery of therapeutic drug IL10mRNA nanomedicine, where Figure A is a schematic diagram for the evaluation of the effect of orally delivered nanomedicine, and Figure B is the delivery effect diagram evaluated by Elisa for non-snap rigid capsules and RNAcap.

[0049] Figure 16It is the effect diagram of the RNAcap oral delivery of the therapeutic drug-loaded IL10mRNA-LPH.NP nanoparticles and the delivery of IL10mRNA-LNP nanoparticles.

[0050] Figure 17 It is the result diagram of the IL10 protein expression of the RNAcap oral delivery of the therapeutic drug IL10mRNA-LPH.NP nanoparticles at different intestinal sites.

[0051] Figure 18 It is the H&E section result diagram of the small intestine and colon of the RNAcap oral delivery of the therapeutic drug IL10mRNA-LPH.NP nanoparticles.

[0052] Figure 19A It is the result diagram of the in vivo safety evaluation of the RNAcap oral delivery of the therapeutic IL10mRNA-LPH.NP nanoparticles.

[0053] Figure 19B It is the result diagram of blood biochemical indexes such as AST, ALT, BUN, WBC, and NE.

[0054] Figure 19C It is the result diagram of blood routine indexes such as LY, RBC, and Hb.

[0055] Figure 19D It is the HE result diagram of organs such as the intestine, heart, liver, spleen, lung, and kidney.

[0056] Figure 20 It is the schematic diagram of the RNAcap oral delivery of IL10mRNA-LPH.NP nanoparticles for the treatment of rat colitis.

[0057] Figure 21 It is the result diagram of the RNAcap oral delivery of IL10mRNA-LPH.NP nanoparticles for the treatment of rat colitis. Among them, Figure A is the result of continuously monitoring the relative body weight of rats for 8 days, Figure B is the result of continuously monitoring the disease activity index of rats for 8 days, and Figure C is the result of measuring the colon length of rats at the end of the experiment.

[0058] Figure 22 It is the result diagram of the RNAcap oral delivery of IL10mRNA-LPH.NP nanoparticles for the treatment of rat colitis. Among them, Figure A is the detection of the intestinal tissue of the treated rats, Figure B is the level of IL10 in the intestinal tissue, Figure C is the level of TNFα in the intestinal tissue, and Figure D is the level of IL-1β in the intestinal tissue.

[0059] Figure 23The figure shows the test results of the intestinal tissues of rats after treatment with the RNAcap oral delivery of IL10mRNA-LPH.NP nanoparticles for colitis. Among them, Figure A shows the level of IL6 in the intestinal tissue, Figure B shows the level of IL17A in the intestinal tissue, and Figure C shows the level of MCP-1 in the intestinal tissue.

[0060] Figure 24 The figure shows the results after the RNAcap oral delivery of IL10mRNA-LPH.NP nanoparticles for the treatment of rat colitis. Among them, Figure A shows the detection of the serum of the treated rats, Figure B shows the level of IL10 in the serum, Figure C shows the level of TNFα in the serum, and Figure D shows the level of IL-1β in the serum.

[0061] Figure 25 The figure shows the test results of the serum of rats after treatment with the RNAcap oral delivery of IL10mRNA-LPH.NP nanoparticles for colitis. Among them, Figure A shows the level of IL6 in the serum, Figure B shows the level of IL17A in the serum, and Figure C shows the level of MCP-1 in the serum.

[0062] Figure 26 The figure shows the results of HE staining of the intestinal tissues of rats after treatment with the RNAcap oral delivery of IL10mRNA-LPH.NP nanoparticles for colitis. Detailed implementation mode

[0063] The technical solution of the present invention will be further described below in conjunction with the drawings and through specific implementation modes. However, the following examples are only simple examples of the present invention and do not represent or limit the scope of the protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0064] For those not specifying specific technologies or conditions in the examples, they shall be in accordance with the technologies or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0065] The present invention constructs a novel mRNA delivery platform by developing an oral delivery system, including designing a capsule for oral delivery of mRNA drugs (named RNAcap), and the structural schematic diagram is as Figure 1As shown, the capsule includes a capsule body and a capsule cap. The materials of the capsule body and the capsule cap are selected from materials that can be quickly dissolved in aqueous solutions, such as gelatin, gum arabic, starch and its derivatives, alginate, mulberry silk gum, hypromellose, pullulan, trehalose, hypromellose phthalate, polyvinylpyrrolidone copolymer, or polyvinyl alcohol, etc.; a flexible film is provided on the inner surface of the capsule body (selectable polyolefin composite film, hydroxypropyl methylcellulose film, silicone rubber film, polyvinyl alcohol film, pullulan polysaccharide film, or silicone elastomer film, etc., such as Parafilm M sealing film), and a non-flexible film (selectable bio-based film, vinyl polymer film, or medical-grade polyester PET film, etc., such as polyethylene food wrap) is provided at the opening of the capsule body to cover the opening. The outer surfaces of the capsule body and the capsule cap are coated with a pH-sensitive polymer (selectable methyl methacrylate-ethyl acrylate copolymer, chitosan, hypromellose acetate succinate, hypromellose phthalate, poly(lactic-co-glycolic) acid copolymer, [(2-vinylpyridine)-co-(butyl methacrylate)] copolymer, cellulose acetate phthalate, dextran, pectin, or sodium alginate, etc., such as Eudragit L100-55). Optionally, a non-snap rigid capsule can also be prepared, including first coating a flexible film on the outer surfaces of the capsule body and the capsule cap and then coating a pH-sensitive polymer.

[0066] Example 1

[0067] In this example, an RNACap capsule for oral delivery of liquid mRNA (LPH.NPs) drug was prepared.

[0068] The capsule oral delivery system for encapsulating liquid mRNA drug used an FDA-approved gelatin capsule (size 5 and size 9, product of Torpac company), which consisted of a capsule body and a capsule cap that could be quickly dissolved in aqueous solutions. The specific operation steps are as follows:

[0069] 1. First, a flexible film A (Parafilm M sealing film, Thermo Fisher Scientific) was coated on the inner surface of the capsule body. By applying pressure using a rod-shaped tool, it was ensured that the flexible film A was completely adhered to the inner surface of the capsule, and the excess film material was carefully removed to achieve the maximum loading capacity. Attention should be paid to avoiding the rupture of film A resulting in liquid leakage.

[0070] 2. After loading 100 μL of rhodamine B (RB) solution (Sigma-Aldrich) (for subsequent tests), the opening of the capsule body was covered with a non-flexible film B (polyethylene food wrap, Thermo Fisher Scientific). The diameter of film B was designed to be 30% larger than the diameter of the capsule to ensure complete coverage. When installing the capsule cap, pressure was applied to the non-flexible film B to effectively seal the capsule body and prevent leakage.

[0071] 3. Coat a layer of pH-sensitive polymer (Eudragit L100-55, a product of Evonik Industries AG, a copolymer of methacrylic acid and ethyl acrylate) and dry it in air to obtain the final L100-55-RNACap capsules loaded with RB.

[0072] Coat a layer of another pH-sensitive polymer, Eudragit L100, to obtain the final L100-RNACap capsules loaded with RB.

[0073] 4. Replace RB in the above capsules with the mRNA nanoplatform to obtain pH-sensitive RNACap loaded with the mRNA nanoplatform.

[0074] In addition, to optimize the membrane for coating the inner surface of the capsule, referring to the above method, flexible membrane A or non-flexible membrane B was coated on the inner surface of the capsule to prepare capsule-(A)B and capsule-(B)B respectively. The structural schematic diagrams are as Figure 2 shown. Subsequently, the RB solution was loaded, sealed with non-flexible membrane B and capped. The obtained capsules were imaged and their drug loading was determined.

[0075] To optimize the sealing membrane, capsule-(A)B and capsule-(A)A were prepared respectively. Referring to the above method, flexible membrane A was coated on the inner surface of the capsule. Subsequently, the RB solution was loaded and sealed with flexible membrane A and non-flexible membrane B respectively and capped.

[0076] First, remove the capsule cap, and then soak for 20 min before immersing the obtained capsules in phosphate buffer (1×, pH 7.4) in a 6-well plate. At the predetermined time points, the culture dish was imaged using a Syngene PXi imager (Syngene), and 100 μL of solution was collected from each well to determine the release kinetics of RB. The results were as follows: both capsule (A)B and capsule (B)B successfully encapsulated the RB solution without damaging the capsules ( Figure 3 ). In addition, capsule (A)B accommodated a higher content volume than capsule (B)B.

[0077] According to the design of the present invention ( Figure 1 ), the sealing mechanism relies on the force exerted by the capsule cap, which rapidly dissolves in the intestine, thereby releasing the pressure applied on the sealing membrane, causing the sealing membrane to quickly separate to release the contents. Capsule (A)B and capsule (A)A were placed in an aqueous solution at pH 7.4 after removing the capsule caps ( Figure 4 ). The sealing membrane of capsule (A)B immediately separated after 3.5 minutes of incubation and released 50% of RB into the solution. In contrast, the sealing membrane of capsule (A)A adhered tightly to the capsule body, and no obvious release of RB was observed even after 20 min of incubation ( Figure 5 , Figure 6 ).

[0078] Test for stability: RNACap was incubated at room temperature for 60 hours. At the predetermined time points, RNACap was imaged using a Syngene PXi imager (Syngene) to monitor the stability of the system. The result was that RNACap loaded with RB (capsule (A)B) remained intact at room temperature for at least 60 hours without any leakage ( Figure 7 ).

[0079] Test for pH-sensitive release effect: RNACap coated with L100-55 remained intact in acidic gastric pH (pH 1.2) solution for at least 2 hours ( Figure 8 , Figure 9 Figure A in). In contrast, 50% of RB was released after incubation in a solution with neutral intestinal pH (pH 7.4) for 49 min. Without an enteric coating membrane (RB-RNACap), RNACap rapidly dissolved and released the loaded material into the solution under both pH conditions ( Figure 9 Figure B in). Another copolymer, Eudragit L100, can also protect RNACaps in an acidic environment. However, compared with RNACap coated with L100-55, RNACap coated with L100 had a slightly slower release rate of the loaded material in a neutral pH environment (50% released in 66 min, Figure 9 Figure C in).

[0080] Example 2

[0081] In this example, lipid nanoparticles of anti-PD1 mRNA and EGFP mRNA were prepared.

[0082] I. Chemically modified Fluci mRNA, EGFP mRNA, and IL10 mRNA were synthesized in vitro.

[0083] Luciferase protein, enhanced green fluorescent protein (EGFP), and IL10 mRNA were synthesized using in vitro transcription technology (IVT). To avoid immune stimulation caused by mRNA, conventional uridine triphosphate was replaced with pseudouridine-5'-triphosphate (Pseudo-UTP). The specific process is as follows:

[0084] 1. Using plasmids carrying the open reading frames of Fluci, EGFP, and murine IL10 genes with T7 promoter and T7 terminator as templates for polymerase chain reaction (PCR) amplification (the plasmids were purchased from Tsingke Biotechnology and Miaoling Biotechnology respectively and amplified in DH5α), with the T7 promoter sequence as the forward primer and the T7 terminator sequence as the reverse primer for PCR amplification. The Novazyme PCR kit was used, and the reaction was carried out for a total of 34 cycles at 98°C for 10 s, 60°C for 5 s, and 72°C for 15 s to amplify the template. The amplified products were separated by 1% gel electrophoresis and purified using the Novazyme gel extraction kit to form linearized DNA as the template for in vitro transcription;

[0085] 2. For in vitro transcription (IVT), the Novazyme T7 transcription kit was used together with 1 - 2 μg of in vitro transcription template, guanosine triphosphate, 5-methyl-cytidine triphosphate, adenosine triphosphate, and pseudouridine-5'-triphosphate. The reaction was carried out at 37°C for 2 h, followed by deoxyribonuclease (DNase) treatment to obtain Fluci mRNA, EGFP mRNA, and IL10 mRNA;

[0086] 3. For the synthesis of Cy5-mRNA, the Novazyme T7 transcription kit was used together with 1 - 2 μg of in vitro transcription template, guanosine triphosphate, 5-methyl-cytidine triphosphate, adenosine triphosphate, Cy5-labeled pseudouridine-5'-triphosphate, and pseudouridine-5'-triphosphate. The reaction was carried out at 37°C for 2 h, followed by deoxyribonuclease (DNase) treatment to obtain Cy5-mRNA.

[0087] 4. The above-mentioned mRNA was purified by semi-preparative high performance liquid chromatography (HPLC) to obtain the total ion current chromatogram of the in vitro transcription products. The salts in the buffer were removed by centrifugation using an ultrafiltration device (EMD Millipore, MWCO 100 kDa). After washing 3 times with enzyme-free water, the above-mentioned mRNA was collected in enzyme-free water for further use or stored in an -80°C refrigerator; further purified Fluci mRNA, EGFP mRNA, and IL10 mRNA were obtained.

[0088] II. Preparation and Characterization of Nanopreparations:

[0089] Fluci mRNA, EGFP mRNA, and IL10 mRNA nanopreparations (lipid-polymer hybrid nanoparticles encapsulating mRNA, LPH.NPs) were prepared by a self-assembly method. The method specifically includes the following steps:

[0090] 1. Synthesis of cationic molecule G0-C14: A methanol solution (CAS No.: 155773-2-1) of 1 mmol of PAMAM dendrimer (ethylenediamine core, generation G0) and 7 mmol of 1,2-epoxytetradecane (CAS No.: 3234-28-4) were added to a 20 mL reaction flask to form a mixed reaction solution. Methanol solution was removed by vacuum distillation. Then, under argon protection, the reaction was carried out at 90 °C with a stirring speed of 800 rpm for 48 h. After the reaction, the reaction product was diluted with dichloromethane, and then separated and purified by silica gel column chromatography to obtain the cationic lipid G0-C14.

[0091] 2. Preparation of the stock solutions of key components: G0-C14, PLGA, and DSPE-PEG were fully dissolved in N,N-dimethylformamide (DMF) to obtain the corresponding stock solutions of components - G0-C14 (2.5 mg / mL), PLGA (5.0 mg / mL), and DSPE-PEG (20 mg / mL).

[0092] 3. Prepare 50 mM and 10 mM citrate buffer (pH = 4.0) and perform nuclease-free treatment with DEPC.

[0093] 4. Preparation of mRNA / G0-C14 complex: 10 μg of EGFP mRNA and anti PD1 mRNA (1 μg / μL) were taken respectively, and 2.5 μL of citrate buffer (50 mM) was added thereto. After mixing evenly, it was quickly added to 50 μL of G0-C14 DMF solution under vortexing, and vortexed for another 20 s to obtain the mRNA / G0-C14 complex.

[0094] 5. 50 μL of PLGA DMF solution and 25 μL of DSPE-PEG DMF solution were successively added to the above mRNA / G0-C14 complex and mixed evenly to obtain solution A.

[0095] 6. Under vortexing, solution A was quickly added to 625 μL of citrate buffer (10 mM), and vortexed for another 50 s.

[0096] 7. Under vortexing, 625 μL of 1×PBS was slowly added dropwise to the above mixed solution.

[0097] 8. The organic solvents and free compounds in the formed nanoparticle formulation dispersion were removed by centrifugation using an ultrafiltration device (EMD Millipore, MWCO 100 kDa); after washing three times with high-pressure water, it was made up to 500 μL with 1×PBS buffer at pH 7.4 to obtain mRNA / G0-C14 LPH.NPs (mRNA concentration: 20 ng / μL) for further use or stored in a refrigerator at 4°C. The prepared nanoparticles were characterized using dynamic light scattering (DLS) and transmission electron microscopy (TEM), and the results are shown in Figure 10 , where Figure A shows the particle size results, Figure B shows the polydispersity coefficient results, Figure C shows the potential results, and Figure D shows the electron microscopy results.

[0098] The LNPs liquid nanoparticle formulation includes: the ionizable lipid is heptan-9-yl 8-(2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)aminooctanoate (SM-102), the co-lipids are 1,2-distearoyl-sn-glycero-3-phosphate (dsc), cholesterol, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), with molar ratios of 50:10:38.5:1.5 respectively. After mixing evenly, it forms the lipid phase. The mRNA is mixed with citrate buffer to form the aqueous phase.

[0099] Under vortexing, the lipid phase was rapidly added to the aqueous phase and mixed evenly. The organic solvents and free compounds in the formed nanoparticle formulation dispersion were removed by centrifugation using an ultrafiltration device (EMD Millipore, MWCO 100 kDa); after washing three times with high-pressure water, it was made up to 500 μL with 1×PBS buffer at pH 7.4 to obtain mRNA / LNP for further use or stored in a refrigerator at 4°C.

[0100] Example 3

[0101] In this example, the preparation of RNACap for oral delivery of liquid mRNA (LPH.NPs) drugs to rats was carried out.

[0102] Gelatin capsule No. 9 approved by the FDA was selected for oral administration to rats. Referring to Example 1, first, a flexible film A was coated on the inner surface of the capsule body. After loading 10 μL of the mRNA nanoparticle solution (prepared in Example 2) respectively, the opening of the capsule body was covered with a non-flexible film B. The capsule was sealed by installing the capsule cap. Finally, a pH-sensitive polymer was coated and air-dried to obtain the final RNAcap capsule of liquid mRNA drug that can be orally administered to rats.

[0103] Example 4

[0104] This example involves the preparation of a non-snap rigid capsule (Rigid-Cap) for oral administration of liquid mRNA (LPH.NPs) to rats.

[0105] The non-snap rigid capsule (rigid cap) is prepared by adding a flexible film A to the outer surfaces of the capsule body and cap. These additional films prevent the capsule from softening and dissolving in the neutral pH environment of the intestine. The specific steps are as follows:

[0106] Both the inner and outer surfaces of a size 9 gelatin capsule are coated with a flexible film A. After separately loading 10 μL of the mRNA nanoparticle formulation solution (prepared in Example 2), the opening of the capsule body is covered with a non-flexible film B. Then, the capsule is sealed by attaching a capsule cap that has been pre-coated with flexible film A on its outer surface. Finally, the capsule is immersed in a polymer solution (Eudragit L100-55, Evonik Industries AG) for coating to obtain the final rigid capsule (Rigid-Cap).

[0107] Example 5

[0108] This example involves the in vivo distribution test of oral delivery of liquid mRNA (LPH.NPs) drugs using capsules.

[0109] Sprague-Dawley rats with an average body weight of 500 g were used. Before oral administration of Cy5-mRNA-RNACap (L100-55) to the rats, the rats were fasted for 12 - 16 h. Each RNACap contained 50 μg of Cy5-mRNA (a total of 6 RNACaps), and the drug was administered by oral gavage. At different time points after administration (15 min, 1 h, 2 h, 4 h, and 6 h), the rats were euthanized, and then the stomach, intestine, and other major organs (including the heart, lung, spleen, and kidney) were removed for ex vivo imaging using a small animal in vivo imaging system. Similarly, Cy5-mRNA-RNACap coated with L-100 was also administered to the rats orally, and the rats were euthanized and imaged 6 h after administration.

[0110] The results are as Figure 11As shown, the ex vivo images of the gastrointestinal tract excised 15 min after administration showed that all administered RNACaps remained in the stomach and all six RNACaps were intact, indicating that RNACaps can withstand the harsh gastric environment. All RNACaps were still in the stomach 1 h after administration, and some RNACaps entered the intestine 2 h after administration (h). The 2-h period clearly showed the different states of RNACaps after oral administration, including the intact state in the stomach, the partially dissolved state in the intestine, the extruded state in the intestine (with obvious shape change), and the completely dissolved state (a large amount of Cy5-mRNA NPs released into the intestine). At 4 h and 6 h after administration, no intact RNACaps were observed in the stomach and intestine, indicating that all RNACaps entered the intestine and were subsequently dissolved, releasing Cy5-mRNA ( Figure 11 ). In addition, no obvious fluorescence was detected in other major organs of the rats ( Figure 12 ). However, for the transport of L100-coated RNACaps in vivo, even after 6 h, undissolved RNACaps remained in the intestine ( Figure 13 ).

[0111] Example 6

[0112] In this example, in vivo absorption tests of capsule oral delivery of liquid mRNA (LPH.NPs) drugs were carried out.

[0113] Using SD rats with an average body weight of 500 g, after fasting for 12 - 16 h, 6 Cy5-mRNA (LPH.NPs)-RNACaps were orally administered, and each RNACap contained 50 μg of Cy5-mRNA. The rats were euthanized 4 h after oral administration, and then the intestinal tissues were collected and immediately frozen in O.C.T. embedding medium (Tissue-Tek) using liquid nitrogen. Intestinal tissue sections with a thickness of 12 μm were obtained using a Leica CM1900 cryostat. The obtained sections were stained with a DNA fluorescent dye (Hoechst 33342) for cell nuclei, and then washed with PBS. The samples were observed by a confocal laser scanning microscope (Olympus FV1000).

[0114] The results were as Figure 14 shown. The intestine of rats treated or untreated with Cy5-mRNA-RNAcap of the present invention was sectioned and imaged. The strong fluorescence signal of Cy5 in the sections of rats treated with Cy5-mRNA-RNAcap indicated that NPs were able to deliver Cy5-mRNA to the intestine.

[0115] Example 7

[0116] In this example, it was verified that RNACap capsules are vulnerable to the squeezing force of intestinal contractions, resulting in rapid release of the contents.

[0117] Compare the effects of intestinal peristalsis on the delivery efficiency of oral administration of therapeutic liquid IL10-mRNA drugs using non-snap rigid capsules and RNACap capsules.

[0118] As Figure 15 shown in Figure A, using SD rats with an average body weight of 500 g, after fasting for 12 - 16 h, 6 IL-10-mRNA (LPH.NPs)-RigidCap and IL-10-mRNA (LPH.NPs)-RNACap were respectively given by oral gavage, and each capsule contained 50 µg of IL-10-mRNA. Blood samples were collected at 24 h, 48 h, and 72 h after oral administration, and then the IL-10 level was measured by enzyme-linked immunosorbent assay (ELISA).

[0119] The results are as Figure 15 shown in Figure B. The present invention utilizes intestinal peristalsis to trigger the rapid release of substances in RNACap. To test whether peristalsis is indeed crucial for the delivery mechanism, the present invention fabricated a non-elastic rigid capsule (rigid cap) that does not deform due to intestinal peristalsis. Although RNACap softens at the neutral pH of the intestine, allowing intestinal contractions to squeeze and release the substances therein, due to the presence of an additional tough coating film on the non-elastic rigid capsule, even after removing the intestinal envelope, the non-snap rigid capsule remains rigid, making it resistant to the contraction force; therefore, contraction-driven release does not occur, and thus the blood IL-10 level at all time points is significantly lower than that of the RNACap group.

[0120] Example 8

[0121] In this example, the therapeutic liquid IL10-mRNA drug is orally delivered using RNACap capsules.

[0122] Compare the delivery efficiency of oral administration of lipid nanoparticle (LNPs) mRNA drugs using RNACap capsules with that of oral administration of liquid mRNA (LPH.NPs) drugs using RNACap capsules.

[0123] Using SD rats with an average body weight of 500 g, after fasting for 12 - 16 h, 6 IL-10-mRNA (LPH.NPs)-RNACap and IL-10-mRNA (LNPs)-RNACap were respectively given by oral gavage, and each capsule contained 50 µg of IL-10-mRNA. Blood samples were collected at 24 h, 48 h, and 72 h after oral administration, and then the IL-10 level was measured by enzyme-linked immunosorbent assay (ELISA).

[0124] The results are as Figure 16As shown, after oral administration of LNPs-RNACap, the level of IL-10 in the blood of rats at each time point was lower than that of rats given LPH.NPs-RNACap.

[0125] At 24 h after oral administration, the rats were euthanized, and then the intestines were collected for further analysis.

[0126] For enzyme-linked immunosorbent assay (ELISA), the intestines were divided into 9 parts and cut into small pieces. Then, 200 mg of tissue from each part was added to 4 mL of tissue protein extraction reagent (Thermo Fisher Scientific) containing a protease inhibitor (Thermo Fisher Scientific). The tissue was homogenized and incubated on ice for 30 min, followed by centrifugation (10,000 g, 4 °C, 10 min). The supernatant (protein) was collected, and the IL-10 level was analyzed by ELISA.

[0127] Oral administration of IL-10-mRNA (LPH.NPs)-RNACap could induce significant expression of IL-10 in the duodenum, jejunum, ileum, cecum, and colon ( Figure 17 ). As shown by H&E staining, no obvious toxicity or intestinal damage was observed after oral administration of IL-10-mRNA (LPH.NPs)-RNACap ( Figure 18 ).

[0128] Example 9

[0129] This example tested the in vivo safety of IL-10-mRNA (LPH.NPs)-RNACap.

[0130] Using SD rats with an average body weight of 500 g, on days 2, 5, and 8, after fasting for 12 - 16 h, they were orally administered 25 µg of IL-10-mRNA (LPH.NPs)-RNACap (3 RNACaps), and untreated rats served as the control group ( Figure 19A ). After the last administration, the rats were euthanized, and intestinal, other major organ, and blood samples were collected for analysis.

[0131] The results were as follows: compared with the untreated group, there were no significant differences in the plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), or blood urea nitrogen (BUN) levels in the IL-10-mRNA (LPH.NPs)-RNACap group, indicating that oral administration of RNACap did not cause liver or kidney toxicity ( Figure 19B). Additionally, compared with the untreated group, RNACap treatment did not cause significant changes in hematological parameters, including white blood cell count (WBC), neutrophils (NE), lymphocytes (LY), red blood cell count (RBC), platelets (PLT), hemoglobin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), or mean corpuscular hemoglobin concentration (MCHC) ( Figure 19B , Figure 19C ). Moreover, histopathological examination (H&E staining) showed that oral administration of RNACap did not cause any obvious damage to the intestine or other major organs ( Figure 19D ). Collectively, these results indicate that RNACap can serve as a non-toxic platform for oral liquid mRNA therapeutics.

[0132] Example 10

[0133] This example evaluated the therapeutic efficacy of orally delivered RNACap capsules containing therapeutic liquid mRNA (LPH.NPs) in rats.

[0134] The therapeutic effect of IL10-mRNA (LPH.NPs)-RNACap was evaluated in a dextran sulfate sodium (DSS)-induced rat colitis model. Acute colitis was induced by providing drinking water containing 6.0% DSS (Cayman Chemicals) for 8 consecutive days. Rats treated with only water or only DSS served as the control groups. The body weight, fecal characteristics, and bleeding of the rats were monitored daily. The disease activity index (DAI) of the rats was calculated based on inflammation-related parameters such as percentage of body weight loss, fecal characteristics, and bleeding. The specific scoring criteria were as follows: body weight loss (percentage relative to the initial weight, 0 indicating no loss; 1 indicating 1%-5%; 2 indicating 6%-10%; 3 indicating 11%-20%; 4 indicating >20%), fecal characteristics (0 indicating normal; 2 indicating loose feces; 4 indicating diarrhea), and bleeding (0 indicating no bleeding; 1 indicating occult blood positive; 2 indicating occult blood positive and visible fecal bleeding; 4 indicating obvious bleeding with bloodstains around the anus). After the treatment ended, blood samples were collected and the rats were euthanized. The colon length was measured and the colon was cut into small pieces. Subsequently, 200 mg of colon tissue was added to 4 mL of tissue protein extraction reagent (Thermo Fisher Scientific) containing a protease inhibitor (Thermo Fisher Scientific). The tissue was homogenized and incubated on ice for 30 min, followed by centrifugation (10,000 g, 4 °C, 10 min). The supernatant (protein) was collected. Then, the levels of IL-10, TNF-α, IL-1β, IL-6, IL-17A, and MCP-1 in the colon tissue and serum were analyzed by enzyme-linked immunosorbent assay (ELISA). The ELISA kits used were: rat TNF-α ELISA (Biolegend), rat IL-1β / IL-1F2 ELISA (R&D Systems), rat IL-6 ELISA (R&D Systems), rat IL-17A ELISA (Biolegend), rat JE / MCP-1 / CCL2 ELISA (R&D Systems).

[0135] DSS-induced colitis can cause obvious symptoms such as physical discomfort, body weight loss, shortening of the colon length, and an increase in the disease activity index (DAI), which is determined based on parameters such as body weight loss, diarrhea, and rectal bleeding. Specifically, acute colitis was induced by allowing rats to freely drink drinking water supplemented with 6.0% DSS for 8 consecutive days. After the onset of DSS-induced colitis, IL10-mRNA (LPH.NPs)-RNACap (25 μg mRNA in 3 RNACaps per rat) was orally administered on days 2, 5, and 8, respectively ( Figure 20). Compared with the DSS group, the DSS + IL10-mRNA(LPH.NPs)-RNACap group showed less weight loss ( Figure 21 Panel A in Figure 21 ), lower DAI increase ( Figure 21 Panel B in Figure 22 ), and longer colon length ( Figure 23 Panel C in Figure 24 ). Then, we detected the levels of inflammation-related cytokines and proteins in colon tissues and blood, including IL-10, TNF-α, IL-1β, IL-6, IL-17A, and monocyte chemoattractant protein-1 (MCP-1). In tissues, ELISA analysis showed that compared with the control group, the DSS group had lower levels of anti-inflammatory IL-10 and higher levels of pro-inflammatory IL-1β, IL-6, IL-17A, and MCP-1, indicating that DSS induced severe inflammation ( Figure 25 ). Compared with the DSS group, the DSS + IL10-mRNA(LPH.NPs)-RNACap group had less DSS-induced inflammatory response, increased IL-10 level, and decreased levels of IL-1β, IL-6, IL-17A, and MCP-1. In blood, the DSS + IL10-mRNA(LPH.NPs)-RNACap group had higher IL-10 level and lower levels of TNF-α, IL-1β, IL-17A, IL-6, and MCP-1 than the DSS group ( Figure 26 ). Finally, the severity of inflammation in each group was evaluated by H&E staining. Compared with the DSS group, the DSS + IL10-mRNA(LPH.NPs)-RNACap group had less inflammatory response (

[0136] In summary, the present invention designs a capsule for oral delivery of mRNA, designs a specific capsule structure, including the internal structure of the capsule body and the opening sealing method, and designs a pH-sensitive layer on the outer surface of the capsule, etc., which can achieve oral delivery of mRNA in solid or liquid form, has good stability, drug loading capacity, and release kinetics, can maintain an intact form in the stomach, and rapidly disintegrate in the intestine, significantly improving the stability of the mRNA active ingredient in the complex gastrointestinal environment, laying a technical foundation for the development of non-invasive and modular dosage forms.

[0137] The applicant declares that the above description is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A capsule for oral delivery of mRNA, characterized in that, The capsule includes a capsule body and a capsule cap. A flexible film is provided on the inner surface of the capsule body. The flexible film includes at least one of a polyolefin composite film, a hydroxypropyl methylcellulose film, a silicone rubber film, a polyvinyl alcohol film, a pullulan polysaccharide film, or a silicone elastomer film. An inflexible film covers the opening at the opening of the capsule body. The inflexible film includes at least one of a bio-based film, a vinyl polymer film, or a medical-grade polyester PET film. The outer surfaces of the capsule body and the capsule cap are coated with a pH-sensitive layer. The pH-sensitive layer contains a pH-sensitive polymer. The pH-sensitive polymer includes at least one of an acrylic polymer and its derivatives, chitosan, hypromellose acetate succinate, hypromellose phthalate, poly(lactic-co-glycolic acid), dextran, pectin, or sodium alginate.

2. The capsule for oral delivery of mRNA according to claim 1, characterized in that, The outer surfaces of the capsule body and the capsule cap are further coated with the flexible film. The flexible film is located between the outer surface and the pH-sensitive layer.

3. The capsule for oral delivery of mRNA according to claim 1, characterized in that, The materials of the capsule body and the capsule cap are selected from at least one of gelatin, gum arabic, starch and its derivatives, alginic acid, mulberry silk gum, hydroxypropyl methylcellulose, pullulan, trehalose, hypromellose phthalate, polyvinylpyrrolidone copolymer, or polyvinyl alcohol. The area of the inflexible film is at least 10% larger than the area of the opening of the capsule body.

4. A method for preparing the capsule for oral delivery of mRNA according to any one of claims 1 - 3, characterized in that, The preparation method includes: Coating the flexible film on the inner surface of the capsule body, covering the opening of the capsule body with the inflexible film, and coating a pH-sensitive polymer solution on the outer surfaces of the capsule body and the capsule cap to obtain the capsule for oral delivery of mRNA.

5. The method for preparing the capsule for oral delivery of mRNA according to claim 4, characterized in that, The mass percentage of the pH-sensitive polymer in the pH-sensitive polymer solution is 1% - 60%.

6. Use of the capsule for oral delivery of mRNA according to any one of claims 1 - 3 in the preparation of an oral mRNA product.

7. An mRNA oral delivery system, characterized in that, The delivery system includes the capsule for oral delivery of mRNA according to any one of claims 1-3 and mRNA. The mRNA is encapsulated in the capsule body by the inflexible film.

8. The mRNA oral delivery system according to claim 7, characterized in that, The mRNA is further loaded in a nano-delivery carrier. The nano-delivery carrier includes any one or a combination of at least two of cationic lipid complexes, lipid nanoparticles, polymer nanoparticles, or lipid-polymer hybrid nanoparticles. The lipid-polymer hybrid nanoparticles contain cationic molecules, polymers, and biocompatibility-modifying molecules. The cationic molecules include any one or a combination of at least two of ammonia molecule derivatives, positively charged amphiphilic lipid compounds, ammonium salts and their derivatives, or polyamides and their derivatives. The polymers include any one or a combination of at least two of polylactic acid and its copolymers, carbon phospholipids and their derivatives, amino acids and their derivatives, polyols and their derivatives, or polyacrylic acid and its derivatives. The biocompatibility-modifying molecules include any one or a combination of at least two of polyethylene glycol and its derivatives, mannitol, dextran, carboxy dextran, liposomes, albumin, tetraethyl orthosilicate, or polyglutamic acid. The morphology of the mRNA is in a liquid form or a solid form.

9. A method for preparing the mRNA oral delivery system according to claim 7 or 8, characterized in that, The preparation method includes: Take the capsule for oral delivery of mRNA according to any one of claims 1-3, place the mRNA in the capsule body, cover the opening of the capsule body with the non-flexible membrane, and assemble the capsule body and the capsule cap.

10. Use of the mRNA oral delivery system according to claim 7 or 8 in the preparation of a drug; The drug includes drugs for treating intestinal diseases or other systemic diseases; The intestinal diseases include at least one of colitis, intestinal polyps or colorectal cancer; The other systemic diseases include at least one of pancreatic cancer, liver cancer or melanoma.

Images