An oral drug delivery system targeting mesenteric lymph nodes and a method of preparing the same

Abstract

The application discloses an oral drug delivery system targeting mesenteric lymph nodes, prepares a novel LNP oral delivery carrier simulating drug release of chylomicron, and explores a pathway and method of intestinal chylomicron lymphatic vessel targeting by modifying a carrier material with a targeting group. Compared with the prior art, the application can significantly improve the ability of nano-drugs to penetrate the intestinal epithelial barrier, enhance the targeting of nano-drugs, and has significant advantages in improving vaccine stability, enhancing immune response and reducing side effects through calcium alginate hydrogel wrapping.

Description

Technical Field

[0001] This invention relates to an oral drug delivery system targeting mesenteric lymph nodes, its preparation method, and its application, belonging to the field of biopharmaceutical technology. Background Technology

[0002] Fatty acid transporter 4 (FATP4) on the surface of intestinal mucosal cells transports long-chain fatty acids (LCFAs) from the intestinal lumen into the cells and then releases them from the basal side of the intestinal mucosal epithelium into the lymphatic system via the endoplasmic reticulum, thus achieving specific absorption of LCFAs. These LCFAs then enter the bloodstream via the lymphatic system. The absorption rate of LCFAs from our daily diet exceeds 95%, demonstrating the high efficiency of this transport pathway. Among intestinal transporters, FATP4 has the highest expression level. Therefore, the FATP4 transporter is an ideal and reasonable target for exploring the absorption of active substances in the intestinal epithelium.

[0003] Currently, most cancer vaccines in clinical trials are administered via intramuscular or subcutaneous injection. Compared to injection, oral administration is generally considered to have better safety, better patient compliance, and lower medical costs. The gut is the largest immune organ, containing approximately 70% of the body's immune cells, and oral cancer vaccines can elicit a strong anti-tumor immune response, making them a promising alternative. However, oral cancer vaccines are limited by various physical and biological barriers in the gastrointestinal tract, including the harsh acidic environment of gastric juice, multiple digestive enzymes, a thick mucosal layer, and the challenges posed by tightly packed intestinal epithelial cells and the intestinal epithelial barrier. Therefore, how to make oral cancer vaccines tolerate the gastrointestinal environment, overcome the intestinal epithelial barrier, and interact with the abundant immune cells in the mucosa is an urgent problem to be solved. Summary of the Invention

[0004] The purpose of this invention is to provide a preparation strategy for an oral drug delivery system targeting mesenteric lymph nodes.

[0005] The technical solution adopted in this invention is as follows:

[0006] A compound called DMG-PEG1000-N3 has the following structural formula:

[0007]

[0008] A compound, DBCO-PEG1000-LCFA, has the following structural formula:

[0009]

[0010] The aforementioned compound DMG-PEG1000-N3, in combination with compound DBCO-PEG1000-LCFA, is used in the preparation of oral drug carriers targeting mesenteric lymph nodes.

[0011] The present invention also discloses an oral drug delivery system targeting mesenteric lymph nodes, comprising a bioactive agent and lipid nanoparticles (LNPs). The bioactive agent is encapsulated by the lipid nanoparticles (LNPs). The PEG end of the lipid nanoparticles (LNPs) is connected to a long-chain fatty acid group (LCFA), which is a saturated fatty acid or an unsaturated fatty acid with a carbon content of 16 ≤ C ≤ 24.

[0012] Preferably, the PEG-LCFA structure of the LNP is as follows:

[0013]

[0014] Preferably, the oral drug delivery system is further encased in a hydrogel shell, specifically sodium alginate, chitosan, microalgae, or polysaccharides.

[0015] Preferably, the bioactive agent refers to mRNA, siRNA, ASO, miRNA, or Aptamer.

[0016] The present invention also discloses a method for preparing the above-mentioned oral drug delivery system, the steps of which include: preparing a lipid-ethanol solution and a bioactive agent-citric acid buffer solution, preparing lipid nanoparticles encapsulating the bioactive agent by microfluidic mixing, and obtaining bioactive agent-LNP lipid nanoparticles.

[0017] Long-chain fatty acid groups were modified onto bioactive agent-LNP particles to obtain bioactive agent-LNP-LCFA lipid nanoparticles.

[0018] Bioactive agent-LNP-LCFA lipid nanoparticles were mixed with sodium alginate solution, and droplet microfluidics were used to utilize the reaction of alginate with CaCl2 at pH 6-8. 2+ The cross-linking properties of the reaction result in a layer of calcium alginate hydrogel shell encapsulating the bioactive agent-LNP-LCFA lipid nanoparticles.

[0019] Preferably, the molar percentage of the lipid-ethanol solution is SM102:DSPC:DMG-PEG1000-N3:Cholesterol = 46.3:9.4:1.6:42.7, and the flow rate ratio of the lipid-ethanol solution to the bioactive agent-citric acid buffer is 1:3.

[0020] Preferably, modifying the bioactive agent-LNP particles with long-chain fatty acid groups refers to reacting the bioactive agent-LNP particles with DBCO-PEG1000-LCFA, thereby modifying the LNP surface of the bioactive agent-LNP particles with LCFA-containing groups.

[0021] The present invention also discloses the application of the above-described oral drug delivery system in the preparation of drugs targeting intestinal cells or tissues.

[0022] Preferably, the cells are antigen-presenting dendritic cells (DCs), and the tissue is intestinal lymphoid tissue.

[0023] Beneficial effects:

[0024] Improving drug bioavailability: The carrier prepared in this invention, by modifying LNPs with LCFA groups, can mimic the natural behavior of chylomicrons, effectively transporting drugs from the intestine to the mesenteric lymphatic system, thereby bypassing first-pass metabolism in the liver and improving drug bioavailability. Enhancing drug targeting: Modifying the carrier material with targeting groups facilitates drug penetration of the intestinal barrier, allowing the drug to reach lymphocytes more precisely, reducing drug distribution in non-target areas, thereby reducing side effects and improving therapeutic efficacy. Promoting drug accumulation in the lymphatic system: Mimicking the drug release mechanism of chylomicrons helps drug accumulation in the mesenteric lymphatic system. LCFA-modified LNP nanoparticles more easily cross the intestinal epithelial barrier, thereby improving drug delivery efficacy.

[0025] Encapsulating the bioactive agent LNP-LCFA with a calcium alginate hydrogel shell using a droplet microfluidic chip can improve vaccine stability and bioavailability. The calcium alginate hydrogel shell protects vaccine components from gastric acid degradation, thereby enhancing vaccine stability and bioavailability in the gastrointestinal tract. It also enhances the immune response: by increasing intestinal permeability and macrophage uptake of the antigen, the calcium alginate hydrogel-encapsulated vaccine can improve the systemic immune response, triggering a stronger immune reaction. Furthermore, the slow release of the vaccine in the intestine may reduce local irritation or side effects caused by rapid, large-volume antigen release. Attached Figure Description

[0026] Figure 1 This study explores the modification efficiency of long-chain fatty acid groups (LCFA) on the surface of lipid nanoparticles (LNP).

[0027] Figure 2 The image shows the morphology of calcium alginate microspheres prepared by droplet microfluidic control (A) and the particle size distribution of the calcium alginate microspheres (B).

[0028] Figure 3 The image shows the drug release detection results of calcium alginate microspheres.

[0029] Figure 4 The image shows the in vivo biodistribution of orally administered calcium alginate microspheres.

[0030] Figure 5The image shows LCFA-modified lipid nanoparticles penetrating the intestinal epithelial barrier.

[0031] Figure 6 The image shows a translocation diagram of the intestinal lymphatic system for oral vaccines.

[0032] Figure 7 The image shows the anti-tumor effect of an oral vaccine.

[0033] Figure 8 The diagram shows the structure of mRNA-LNP-LCFA. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this application clearer, the present invention will be further described in detail below. However, it should be understood that the description herein is merely illustrative and not intended to limit the scope of the invention.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in the specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. All reagents and instruments used herein are commercially available, and the characterization methods involved are described in relevant prior art and will not be repeated here.

[0036] To further understand the present invention, the present invention will be described in detail below with reference to the preferred embodiments.

[0037] Example 1: Preparation of mRNA-LNP lipid nanoparticles.

[0038] The conventional LNP delivery carrier consists of four components: ionizable lipid SM-102 (CAS:2089251-47-6), auxiliary lipid DS PC (CAS:816-94-4), cholesterol Cholesterol (CAS:57-88-5), and polyethylene glycol-modified lipid DMG-PEG2000 (CAS:160743-62-4). The DMG-PEG2000 component is replaced with "DMG-PEG 1000-N3", while the other three components remain unchanged. First, a lipid-ethanol solution (SM102:DSPC:DMG-PEG1000-N3:Cholesterol = 46.3:9.4:1.6:42.7, molar percentage) and pH 4.0 citrate buffer were prepared to contain 0.125 mg / mL OVA mRNA (Yeasen Biotechnology (Shanghai) Co., Ltd.; 17103ES80), with a nitrogen-to-phosphorus ratio of 6:1. Lipid nanoparticles encapsulating the mRNA were prepared using microfluidic mixing. A suitable syringe sleeve was assembled, and the prepared lipid and nucleic acid phases (0.125 mg / mL OVA mRNA in citrate buffer) were drawn into the syringe, air bubbles were removed, and the syringe was connected to the chip. A flow rate of 1:3 was used to synthesize 1 mL of the final product and 0.2 mL of pre-product waste (i.e., 1.2 mL of total product). The syringe was assembled, and the syringe brand and parameters were modified or confirmed in the software. The synthesis parameters were then set. Flow rate ratio 1:3; total flow rate 12 mL / min, initial waste liquid 0.2 mL, subsequent waste liquid 0 mL.

[0039] The synthesis route of DMG-PEG1000-N3 is as follows:

[0040]

[0041] Where n is 22.

[0042] Step 1:

[0043] The raw material was dehydrated by adding TOL at 120°C, cooled to room temperature, dissolved by adding 10V DCM, and 3.5 eq of TEA was added. The temperature was lowered to 0°C, and 3 eq of MsCl was added dropwise. The mixture was then heated to room temperature and stirred overnight. After the reaction was complete, the temperature was lowered to 0°C, and the reaction was quenched by adding 10 times the volume of EtOH with MsCl. The mixture was concentrated to dryness at 35°C, dissolved by adding 10V water, back-extracted twice by 5V EA, dissolved by adding 15% NaCl to the aqueous phase, extracted three times by 5V DCM, dried the DCM phase, filtered, concentrated to dryness at 35°C, dissolved by adding 30V IPA at 50°C, crystallized by a high-low temperature cycling bath at -10°C, filtered, and vacuum dried to obtain the product.

[0044] Step 2:

[0045] The raw material was dissolved in 5V MeOH, and an equal amount of Pd / C was added. Hydrogen gas was introduced, and the mixture was stirred and aerated three times. The mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was filtered through diatomaceous earth, taking care not to dry it to prevent fire. The product was then concentrated at 45°C.

[0046] Step 3:

[0047] The raw material was dissolved in 10V THF, and 3 eq of TBAF and 3 eq of TMS-N3 were added sequentially. The mixture was heated to 65°C and stirred overnight. After the reaction was complete, the mixture was concentrated to dryness at 40°C, dissolved in 10V water, back-extracted three times with 5V EA, dissolved in 15% NaCl in the aqueous phase, extracted twice with 5V DCM, dried the DCM phase, filtered, and concentrated to dryness at 35°C to obtain the product containing TBAF.

[0048] Step 4:

[0049] The raw material was dissolved in 10V THF, and 10 eq of NaOH and 30 eq of epichlorohydrin were added sequentially. A small amount of BHT was used for protection, and the mixture was refluxed at 70°C overnight. After the reaction was complete, the mixture was cooled to room temperature, filtered, and diluted with 10V water and 10% NaH2PO4 to adjust the pH to approximately 7. The THF was concentrated at 40°C, and back-extracted four times with 5V EA. The aqueous phase was dissolved by stirring with 15% NaCl, and extracted twice with 5V DCM. The DCM phase was dried, filtered, and concentrated at 35°C to obtain the product.

[0050] Step 5:

[0051] The raw material was dissolved in a 10V 2mol / L KOH aqueous solution and stirred overnight at room temperature. After the reaction was complete, 15% NaCl was added to the reaction solution and stirred to dissolve. The mixture was extracted three times with DCM at 5V. The DCM phase was dried, filtered, and concentrated at 35℃ to obtain the product.

[0052] Step 6:

[0053] The raw material was dissolved in 10V DCM, and 2.8 eq of tetradecanoic acid and 0.4 eq of DMAP were added sequentially. The mixture was cooled to 0°C by venting, and 2.8 eq of DCC (dissolved in 1V DCM) was added dropwise. After the addition was complete, the mixture was brought to room temperature and stirred overnight. After the reaction was complete, the reaction solution was passed through diatomaceous earth and concentrated at 35°C to obtain the crude product.

[0054] Example 2 describes the modification of CPG-Cy3-LNP lipid nanoparticles with long-chain fatty acid groups (LCFA).

[0055] First, following the method in Example 1, a CPG-Cy3-LNP lipid nanoparticle solution (concentration: 100 ng / µL) was prepared by replacing the mRNA-citrate buffer with CPG-Cy3-citrate buffer. Specifically, a lipid-ethanol solution (SM102:DSPC:DMG-PEG1000-N3:Cholesterol = 46.3:9.4:1.6:42.7, molar percentage) and a pH 4.0 citrate buffer were used to prepare a 0.125 mg / mL CPG-Cy3 nucleic acid phase. Then, at room temperature, a DBCO-PEG1000-LCFA solution was slowly added dropwise to the CPG-Cy3-LNP lipid nanoparticle solution with azide groups on its surface. After gentle shaking, the LCFA groups were modified onto the LNP surface of the lipid nanoparticles via a click chemistry reaction. Flow cytometry was used to explore the changes in the modification ratio of LNPs obtained by adding different concentrations of elements. The results are as follows: Figure 1 As shown, DMG-PEG1000-N3 and DBCO-PEG1000-FITC were added at a molar ratio of 1:0.001. With increasing concentration of the added element, the modification ratio of the element to LNPs also increased. When the molar ratio of DMG-PEG1000-N3 to DBCO-PEG1000-FITC was 1:1.5, the modification ratio reached 98.7%. Further increases in concentration...

[0056] The amount of DBCO-PEG1000-FITC did not significantly increase the modification ratio of LNPs by the element, indicating that the element modification ratio of LNPs approached its maximum when the molar ratio of DMG-PEG1000-N3 to DBCO-PEG1000-FITC was 1:1.5. Therefore, in subsequent experiments, a molar ratio of DMG-PEG1000-N3 to DBCO-PEG1000-LCFA of 1:1.5 was selected as the element addition concentration for the preparation of LNP-LCFA.

[0057] The synthetic route for DBCO-PEG1k-LCFA is as follows:

[0058]

[0059] Where n is 22.

[0060] Step 1: Dissolve NH2-PEG1000-OH in dichloromethane, add triethylamine and (BOC)2O, react at room temperature for 12 hours, concentrate to remove dichloromethane, add tap water and stir to dissolve, wash twice with ethyl acetate. Add 15% w / w sodium chloride to the water tank and stir to dissolve. Extract twice with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, concentrate, add isopropanol and heat to dissolve, cool to crystallize, filter, and dry to obtain TBoc-PEG1000-OH.

[0061] Step 2: Dissolve TBoc-PEG1000-OH in dichloromethane, add DCC and DMAP, and react with hexadecanoic acid for 16 hours under ice bath conditions. Concentrate to remove dichloromethane, add tap water and stir to dissolve, then wash twice with ethyl acetate. Add 15% w / w sodium chloride to the water tank and stir to dissolve. Extract twice with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, concentrate, add isopropanol and ethyl acetate, heat to dissolve, cool to crystallize, filter, and dry to obtain...

[0062] TBoc-PEG1000-LCFA.

[0063] Step 3: Dissolve TBoc-PEG1000-HDA in dichloromethane, add trifluoroacetic acid, react at room temperature for 4 hours, concentrate to remove dichloromethane and trifluoroacetic acid, add isopropanol and ethyl acetate and heat to dissolve, cool to crystallize, filter, and dry to obtain NH2-PEG1000-LCFA.

[0064] Step 4: Dissolve NH2-PEG1000-LCFA in dichloromethane, add triethylamine and DBCO-NHS, react at room temperature for 6 hours, concentrate to remove dichloromethane, add tap water and stir to dissolve, wash twice with ethyl acetate. Add 15% w / w sodium chloride to the water tank and stir to dissolve. Extract twice with dichloromethane, combine the organic phases, dry with anhydrous sodium sulfate, concentrate, add isopropanol and heat to dissolve, cool to crystallize, filter, and dry to obtain DBCO-PEG1000-LCFA.

[0065] The reaction between DMG-PEG1000-N3 and DBCO-PEG1000-FITC is as follows:

[0066]

[0067] Where n is 22.

[0068] The structural diagram of the prepared mRNA-LNP-LCFA is shown below. Figure 8 As shown.

[0069] Example 3: Preparation and characterization of calcium alginate microspheres encapsulating mRNA-LNP-LCFA lipid nanoparticles.

[0070] Calcium alginate microspheres encapsulating mL NP-LCFA lipid nanoparticles were prepared using droplet microfluidics. The mL NP-LCFA lipid nanoparticles were mixed with 1% sodium alginate, utilizing the reaction of alginate with CaCl2 at pH 6-8. 2+ The cross-linking characteristics of the reaction were investigated, and microcapsules with high encapsulation efficiency and targeted intestinal release properties were prepared by using an aqueous phase to oil phase flow rate ratio of 1:3. The morphology of the microspheres was observed under an optical microscope, and the average particle size of the microspheres was determined to be 20 μm using dynamic light scattering (DLS). The instrument used for the measurements was a Malvern Zetasizer Nano ZS90.

[0071] Based on the understanding of those skilled in the art, replacing alginate with chitosan, microalgae, or polysaccharides can achieve the same objective. Methods for preparing chitosan, microalgae, or polysaccharide hydrogel microspheres are existing technologies.

[0072] Example 4: In vitro simulation of calcium alginate microsphere release in the gastrointestinal tract.

[0073] Calcium alginate microspheres encapsulating Cy3-containing LNP-LCFA lipid nanoparticles were prepared using microfluidic technology. Lipid-ethanol solutions and 0.1 mg / mL CPG-Cy3-citric acid buffer solutions were prepared using the same methods as in Examples 1-3. The in vivo process of the calcium alginate microspheres passing through the stomach and intestines was simulated in vitro, and the cumulative nucleic acid release was measured. Cy3-labeled microspheres (containing 20 μg of Cy3) were placed in simulated gastric and intestinal fluids, with the temperature maintained at 37 ± 1 °C, the volume at 10 mL, and the rotation speed at a constant 100 rpm. The drug release behavior of the calcium alginate microspheres was investigated sequentially in simulated gastric and intestinal fluids. The residence time in simulated gastric fluid was 4 h, and the residence time in simulated intestinal fluid was 20 h. After the experiment began, 100 μL of release medium was taken every hour using a pipette, and then 100 μL of release medium was immediately added to maintain a constant total volume. The fluorescence intensity was measured using a microplate reader (excitation wavelength 550 nm, emission wavelength 570 nm). The amount of nucleic acid released was determined by comparing the result with a standard curve, and a release curve was plotted. The results are as follows: Figure 3 As shown, the nanoparticles (HM@mLNP-Cy3) encapsulated with calcium alginate microspheres showed almost no signal in simulated gastric fluid (pH 1.2), but continuously released energy over time in simulated intestinal fluid (pH 6.8), accumulating to approximately 78% release over 24 hours. Under acidic conditions, the internal structure of the microspheres contracted, enhancing the protective effect on mLNP. In contrast, the unencapsulated mLNP-Cy3 nanoparticles ruptured rapidly in simulated gastric fluid (pH 1.2), releasing the encapsulated Cy3.

[0074] Example 5: Quantitative fluorescence analysis of major organs at different time points after oral administration of Cy3-labeled microspheres

[0075] To elucidate the biodistribution of the oral vaccine in vivo, Cy3-labeled calcium alginate microspheres were used and orally administered to C57BL / 6 mice. Mice were euthanized at different time points over the next 12 hours, and their major organs (stomach, intestine, heart, liver, spleen, lung, kidney, and mesenteric lymph nodes) were collected. The results of in vitro fluorescence intensity measurements are shown below. Figure 4 As shown, a high level of Cy3 enrichment was observed in the mesenteric lymph nodes at 6 h.

[0076] Example 6: Effect of LCFA modification on the penetration of lipid nanoparticles into the intestinal epithelial barrier

[0077] To elucidate the effect of LCFA modification on the penetration of lipid nanoparticles into the intestinal epithelial barrier, a Transwell assay was designed to simulate the intestinal epithelial barrier in vitro. Caco-2 cells and HT29 cells were co-cultured in the upper chamber at a 9:1 ratio for 21 days to establish a complete intestinal epithelial barrier. Then, DC2.4 cells were seeded in the lower chamber, and different groups containing OVA mRNA antigen were added to the upper chamber. After co-culturing for 12 hours, the activation status of DC2.4 cells in the lower chamber was detected by flow cytometry, specifically by measuring CD80 levels. + CD86 + The percentage of DC. Results are as follows: Figure 5 As shown, the LCFA-modified vaccine group significantly increased the activation rate of DC cells compared to other groups. The PBS group refers to the addition of an equal volume of sterile PBS solution; the mTubulin-LNP group refers to the addition of 20 μg of tubulin mRNA (Tubulin); the mOVA-LNP group refers to the addition of 20 μg of chicken ovalbumin mRNA (OVA); and the mOVA-LNP-LCFA group refers to the addition of 20 μg of chicken ovalbumin mRNA (OVA). The preparation methods for mRNA-LNP and mRNA-LNP-LCFA are the same as in Examples 1 and 2, respectively.

[0078] Example 7: In vivo absorption pathway of calcium alginate microsphere oral vaccine

[0079] To investigate the absorption pathway of the LCFA-modified oral vaccine, cyclohexylimide (CXI), a lymphatic transport inhibitor, was administered via intraperitoneal injection (ip) at a dose of 3 mg / kg to female SD rats weighing 200–225 g. Cyclohexylimide was dissolved in physiological saline (0.6 mg / mL). Blood samples (200 μL) were collected from the jugular vein at 0 h, 2 h, 4 h, 8 h, 10 h, 12 h, and 24 h, centrifuged at 4 °C for 15 min, and the serum fluorescence intensity was quantified. The content of Cy3 in peripheral blood was calculated based on the fluorescence intensity at 0 hours for each rat. Female SD rats were randomly divided into 5 groups: (I) PBS, HM@LNP-Cy3, HM@LNP-Cy3+CXI, HM@LNP-Cy3-LCFA, and HM@LNP-Cy3-LCFA+CXI [equivalent to 1 mg / kg of Cy3 dye], respectively; the PBS group was administered an equal volume of sterile PBS solution by gavage; the HM@LNP-Cy3 group was administered an equal volume of sterile PBS solution by gavage. The stomach was treated with conventional LNP-coated Cy3 calcium alginate microspheres; the HM@LNP-Cy3+CXI group required simultaneous treatment with cycloheximide (CXI) before gavage, with other interventions as above; the HM@LNP-Cy3-LCFA group required gavage with LNP-coated Cy3 calcium alginate microspheres modified with LCFA groups; the HM@LNP-Cy3-LCFA+CXI group required simultaneous treatment with cycloheximide (CXI) before gavage, with other interventions as above. I(I) The specific treatment with cycloheximide (CXI) was: rats were intraperitoneally injected with CXI [3 mg / kg] 1 hour before administration. Results were as follows... Figure 6 As shown, LCFA-modified oral vaccines are mainly transported via the intestinal lymphatic system, while unmodified oral vaccines are mainly transported via the portal vein circulation.

[0080] Example 8: Application of the oral vaccine prepared according to the present invention in melanoma and colorectal cancer tumor models.

[0081] C57BL / 6 mice were injected via tail vein with 1.5*10⁻⁶ B16-OVA cells. 5 A mouse model of melanoma lung metastasis was established by injecting 1*10c ... 6A mouse model of colorectal cancer was established. After tumor implantation, mice were administered medication by gavage starting when the tumor diameter reached 5 mm, every 3 days. Each administration consisted of calcium alginate microspheres containing 20 μg of mRNA antigen, suspended in 100 μL of pre-cooled PBS, for a total of 5 gavages. Female C57BL / 6 mice were randomly divided into 4 groups: PBS group (Control), HM@mTubulin-LNP group, HM@mOVA-LNP group, and HM@mOVA-LNP-LCFA group [each mouse was administered medication containing 20 μg of mRNA antigen]. The Control group received an equal volume of sterile PBS solution by gavage; the HM@mTubulin-LNP group received 20 μg of tubulin mRNA by gavage.

[0082] HM@mOVA-LNP group refers to OVA mRNA calcium alginate microspheres encapsulated with conventional LNPs administered by gavage;

[0083] The HM@mOVA-LNP-LCFA group refers to the administration of OVA mRNA calcium alginate microspheres modified with LCFA groups via gavage. Mice in the control group were sacrificed when their tumor diameter reached 1.2 cm. Tumor diameter was observed at the end of treatment, and the results are as follows: Figure 7 As shown in the figure, the oral vaccines prepared in Examples 1-3 can significantly inhibit tumor growth and have good tumor treatment effects.

[0084] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of compound DMG-PEG1000-N3 in combination with compound DBCO-PEG1000-LCFA in the preparation of oral drug carriers targeting mesenteric lymph nodes, wherein the structural formula of compound DMG-PEG1000-N3 is as follows: The structural formula of compound DBCO-PEG1000-LCFA is as follows: .

2. An oral drug delivery system targeting mesenteric lymph nodes, characterized in that... The product comprises a bioactive agent and lipid nanoparticles (LNPs). The bioactive agent is encapsulated by the lipid nanoparticles (LNPs). The PEG end of the lipid nanoparticles (LNPs) is attached with a long-chain fatty acid group (LCFA). The LCFA is a saturated or unsaturated fatty acid with a carbon content of 12 < C ≤ 24. The structural formula of the PEG end-LCFA of the LNP is as follows: .

3. The oral drug delivery system according to claim 2, characterized in that: The oral drug delivery system is also encased in a hydrogel shell, specifically composed of alginate, chitosan, and microalgae.

4. The oral drug delivery system according to claim 2 or 3, characterized in that: The bioactive agent refers to mRNA, siRNA, ASO, miRNA, or Aptamer.

5. A method for preparing an oral drug delivery system according to any one of claims 2-4, characterized in that, The steps include: preparing lipid-ethanol solution and bioactive agent-citric acid buffer, and preparing lipid nanoparticles encapsulating bioactive agent using microfluidic mixing to obtain bioactive agent-LNP lipid nanoparticles; Long-chain fatty acid groups were modified onto bioactive agent-LNP particles to obtain bioactive agent-LNP-LCFA lipid nanoparticles. Bioactive agent-LNP-LCFA lipid nanoparticles were mixed with sodium alginate solution, and a layer of calcium alginate hydrogel shell was coated on the bioactive agent-LNP-LCFA lipid nanoparticles using droplet microfluidics.

6. The preparation method according to claim 5, characterized in that: The molar percentage of the lipid-ethanol solution is SM102:DSPC:DMG-PEG1000-N3:Cholesterol = 46.3:9.4:1.6:42.7, and the flow rate ratio of the lipid-ethanol solution to the bioactive agent-citric acid buffer is 1:

3.

7. The preparation method according to claim 6, characterized in that: Modifying long-chain fatty acid groups on mRNA-LNP particles refers to reacting bioactive agent-LNP particles with DBCO-PEG1000-LCFA, thereby modifying the LNP surface of the bioactive agent-LNP particles with LCFA-containing groups.

Images