Mesenteric lymph node targeting oral drug delivery system and preparation method thereof

Abstract

The invention discloses an oral drug delivery system for targeting mesenteric lymph nodes, a novel LNP oral delivery carrier for simulating release of chyloparticle drugs is prepared, and a way and a method for targeting intestinal chylolymphatic vessels are explored by performing targeted group modification on a carrier material. Compared with the prior art, the intestinal epithelial barrier penetrating capacity of the nano-drug can be remarkably improved, the targeting property of the nano-drug is enhanced, and the oral drug has remarkable advantages in the aspects of improving vaccine stability, enhancing immune response and reducing side effects through wrapping of the calcium alginate hydrogel.

Description

Technical Field

[0001] The invention relates to an oral drug delivery system targeting mesenteric lymph nodes and a preparation method and application thereof, belonging to the technical field of biopharmaceuticals. Background Art

[0002] Fatty acid transporter protein 4 (FATP4) on the surface of intestinal mucosal cells can transport long-chain fatty acids (LCFA) in the intestinal lumen into the cells, and discharge LCFA from the basolateral side of the intestinal mucosal epithelium to the lymphatic system through the endoplasmic reticulum, thereby achieving specific absorption of LCFA. They enter the blood circulation through the lymphatic system. The absorption rate of LCFA in our daily diet exceeds 95%, which proves the high efficiency of this transport pathway. Among intestinal transport proteins, the expression level of FATP4 is also the highest. Therefore, the FATP4 transporter is an ideal and reasonable target for exploring the absorption of active substances in the intestinal epithelium.

[0003] Currently, most tumor vaccines in clinical trials are administered by intramuscular or subcutaneous injection. Compared with injection, oral administration is generally considered to have better safety, better patient compliance and lower medical costs. The intestine is the largest immune organ, containing about 70% of the body's immune cells. Oral tumor vaccines can trigger a strong anti-tumor immune response and are a good alternative. However, oral tumor vaccines are limited by the presence of multiple physical and biological barriers in the gastrointestinal tract, including the harsh acidic environment in gastric juice, multiple digestive enzymes, thick mucosal layers, and challenges of tightly arranged intestinal epithelial cells and intestinal epithelial barriers. Therefore, how to make oral tumor 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 the present invention is to provide a preparation strategy for an oral drug delivery system targeting mesenteric lymph nodes.

[0005] The technical solution adopted by the present invention is:

[0006] A compound DMG-PEG1000-N3, whose structural formula is:

[0007]

[0008] A compound DBCO-PEG1000-LCFA, its structural formula is:

[0009]

[0010] The aforementioned compound DMG-PEG1000-N3 is used in combination with the compound DBCO-PEG1000-LCFA in the preparation of an oral drug carrier 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 LNP, wherein the bioactive agent is encapsulated by the lipid nanoparticles LNP, and the PEG end of the lipid nanoparticles LNP is connected to a long-chain fatty acid group LCFA, wherein the long-chain fatty acid group LCFA is a saturated fatty acid or an unsaturated fatty acid with 16≤C≤24.

[0012] Preferably, the structural formula of the PEG-LCFA of the LNP is

[0013]

[0014] Preferably, the oral drug delivery system is further wrapped with a layer of hydrogel shell, specifically sodium alginate, chitosan, microalgae, and polysaccharide.

[0015] Preferably, the bioactive agent is 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-citrate buffer, preparing lipid nanoparticles encapsulating the bioactive agent by a microfluidic mixing method, and obtaining bioactive agent-LNP lipid nanoparticles;

[0017] Modifying the bioactive agent-LNP particles with long-chain fatty acid groups to obtain bioactive agent-LNP-LCFA lipid nanoparticles;

[0018] The bioactive agent-LNP-LCFA lipid nanoparticles were mixed with sodium alginate solution and the alginate was reacted with CaCl2 at pH 6-8 by droplet microfluidics. 2+ The cross-linking properties of the reaction encapsulate a layer of calcium alginate hydrogel shell around 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-citrate 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 invention also discloses the application of the oral drug delivery system in the preparation of drugs targeting intestinal cells or tissues.

[0022] Preferably, the cells are antigen-presenting DC cells, and the tissue is intestinal lymphoid tissue.

[0023] Beneficial effects:

[0024] Improve the bioavailability of drugs: The carrier prepared by the present invention can simulate the natural behavior of chylomicrons by modifying LNP with LCFA groups, effectively transport drugs from the intestine to the mesenteric lymphatic system, thereby bypassing the first-pass metabolism of the liver and improving the bioavailability of drugs. Enhance the targeting of drugs: Modifying the carrier material with targeting groups is conducive to the drug breaking through the intestinal barrier, allowing the drug to reach the lymphocytes more accurately, reducing the distribution of drugs in non-target areas, thereby reducing side effects and improving the therapeutic effect. Promote the accumulation of drugs in the lymphatic system. Simulating the drug release mechanism of chylomicrons helps the accumulation of drugs in the mesenteric lymphatic system. LNP nanoparticles modified with LCFA are more likely to pass through the intestinal epithelial barrier, thereby improving the drug administration effect.

[0025] Using a droplet microfluidic chip to wrap a layer of calcium alginate hydrogel shell around the bioactive agent-LNP-LCFA can improve the stability and bioavailability of the vaccine: the calcium alginate hydrogel shell can protect the vaccine components from degradation by gastric acid, thereby improving the stability and bioavailability of the vaccine in the gastrointestinal tract. Enhanced immune response: By enhancing the intestinal permeability of antigens and the uptake of macrophages, vaccines wrapped in calcium alginate hydrogel shells can enhance the system's immune response and induce a stronger immune response. Reduced side effects: Due to the slow release of the vaccine in the intestine, local irritation or side effects caused by the rapid and large-scale release of antigens may be reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Shown is an exploration of the modification efficiency of long-chain fatty acid groups (LCFA) on the surface of lipid nanoparticles LNP.

[0027] Figure 2 Shown are the morphology of calcium alginate microspheres prepared by droplet microfluidics (A) and the particle size distribution of calcium alginate microspheres (B).

[0028] Figure 3 Shown is the drug release test result of calcium alginate microspheres.

[0029] Figure 4 Shown is the biodistribution diagram of oral calcium alginate microspheres in vivo.

[0030] Figure 5Figure 2 shows the penetration of LCFA-modified lipid nanoparticles into the intestinal epithelial barrier.

[0031] Figure 6 Shown is a diagram of the intestinal lymphatic system transport test for oral vaccines.

[0032] Figure 7 Shown is the anti-tumor effect of oral vaccine.

[0033] Figure 8 Shown is a schematic diagram of the structure of mRNA-LNP-LCFA. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the present application more clear, the present invention is further described in detail below. However, it should be understood that the description herein is only used to explain the present invention and is not intended to limit the scope of the present invention.

[0035] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention, and the terms used in the specification are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The reagents and instruments used herein are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art, which will not be repeated herein.

[0036] In order to further understand the present invention, the present invention is further described in detail below in conjunction with the best embodiment.

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

[0038] The four components of conventional LNP delivery vectors are ionizable lipid SM-102 (CAS: 2089251-47-6), auxiliary lipid DS PC (CAS: 816-94-4), cholesterol (CAS: 57-88-5) and pegylated lipid DMG-PEG2000 (CAS: 160743-62-4). The DMG-PEG2000 component is replaced with "DMG-PEG 1000-N3", and the other three components remain unchanged. First, prepare lipid-ethanol solution (SM102: DSPC: DMG-PEG1000-N3: Cholesterol = 46.3: 9.4: 1.6: 42.7, molar percentage) and pH 4.0 citric acid buffer with a concentration of 0.125 mg / mL OVA mRNA (Yeasen Biotechnology (Shanghai) Co., Ltd.; 17103ES80), where the nitrogen-phosphorus ratio is 6:1. Prepare lipid nanoparticles encapsulating mRNA by microfluidic mixing. Assemble a suitable syringe sleeve, use a syringe to draw the prepared lipid phase and nucleic acid phase (0.125 mg / mL OVA mRNA in citric acid buffer), exhaust the air bubbles and connect to the chip with a flow rate ratio of 1:3. Plan to synthesize 1 mL of final product and 0.2 mL of pre-waste liquid (i.e., 1.2 mL of total product). Assemble the syringe, and modify or confirm the syringe brand and parameters in the software. Set the synthesis parameters. Flow rate ratio 1:3; total flow rate 12mL / min, front waste liquid 0.2mL, rear waste liquid 0mL.

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

[0040]

[0041] Where n is 22.

[0042] Step 1:

[0043] The raw material was added to TOL 120 degrees to remove water, cooled to room temperature, added 10V DCM to dissolve, added 3.5eq TEA, cooled to 0 degrees and added 3eq MsCl, heated to room temperature and stirred overnight. After the reaction was completed, the temperature was lowered to 0℃, and 10 times the volume of MsCl was added to EtOH to quench the reaction, concentrated to dryness at 35℃, dissolved in 10V water, stripped twice with 5V EA, 15% NaCl was added to the aqueous phase to stir and dissolve, extracted three times with 5V DCM, dried the DCM phase, filtered, concentrated to dryness at 35℃, added 30V IPA to heat at 50℃, crystallized in a high and low temperature circulation bath at -10℃, filtered, and vacuum dried to obtain the product.

[0044] Step 2:

[0045] The raw material was dissolved in 5V MeOH, and Pd / C in an amount equal to the raw material was added, and hydrogen was introduced, and the mixture was stirred and ventilated for 3 times, and stirred at room temperature overnight. After the reaction was completed, the mixture was filtered through celite, and it was taken care not to filter it dry to prevent fire, and the product was concentrated at 45°C to obtain the product.

[0046] Step 3:

[0047] The raw material was dissolved in 10V THF, and 3eq of TBAF and 3eq of TMS-N3 were added in sequence, and the temperature was raised to 65 degrees and stirred overnight. After the reaction was completed, the mixture was concentrated to dryness at 40 degrees, dissolved in 10V water, stripped 3 times with 5V EA, 15% NaCl was added to the aqueous phase and stirred to dissolve, extracted 2 times with 5V DCM, and the DCM phase was dried, 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 10eq of NaOH and 30eq of epichlorohydrin were added in sequence, and a small amount of BHT was used for protection, and the temperature was raised to 70°C and refluxed overnight. After the reaction was completed, the temperature was lowered to room temperature, filtered, and 10% NaH2PO4 was added to dilute 10V water to adjust the pH to about 7, THF was concentrated at 40°C, 5V EA was back-extracted 4 times, 15% NaCl was added to the aqueous phase and stirred to dissolve, and 5V DCM was extracted twice, 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 10V 2mol / L KOH aqueous solution and stirred at room temperature overnight. After the reaction was completed, 15% NaCl was added to the reaction solution and stirred to dissolve, extracted with 5V DCM for 3 times, dried with DCM, filtered, and concentrated at 35°C to obtain the product.

[0052] Step 6:

[0053] The raw materials were dissolved in 10V DCM, 2.8eq tetradecanoic acid and 0.4eq DMAP were added in sequence, the temperature was ventilated and cooled to 0°C, 2.8eq DCC (dissolved in 1V DCM) was added dropwise, after the addition was completed, the temperature was raised to room temperature and stirred overnight. After the reaction was completed, the reaction solution was passed through celite and concentrated at 35°C to obtain a crude product.

[0054] Example 2 CPG-Cy3-LNP lipid nanoparticles were modified with long-chain fatty acid groups (LCFA).

[0055] First, according to the method of Example 1, CPG-Cy3-citric acid buffer was used to replace the mRNA-citric acid buffer to prepare a CPG-Cy3-LNP lipid nanoparticle solution (concentration: 100 ng / uL), specifically, a lipid-ethanol solution (SM102: DSPC: DMG-PEG1000-N3: Cholesterol = 46.3: 9.4: 1.6: 42.7, molar percentage) and a CPG-Cy3 nucleic acid phase with a concentration of 0.125 mg / mL prepared by a pH 4.0 citric acid buffer. Then, at room temperature, the DBCO-PEG1000-LCFA solution was slowly added dropwise to the CPG-Cy3-LNP lipid nanoparticle solution with an azide group on the surface, and slightly shaken to modify the LCFA group to the surface of the lipid nanoparticle LNP through a click chemistry reaction. Flow cytometry analysis was used to explore the changes in the modification ratio of LNPs obtained by adding components at different concentrations. The results are shown in FIG. Figure 1 As shown in the figure, DMG-PEG1000-N3 and DBCO-PEG1000-FITC were added at a molar ratio of 1:0.001. As the concentration of the added components increased, the modification ratio of the components to LNPs also increased. When the molar ratio of DMG-PEG1000-N3 and DBCO-PEG1000-FITC was 1:1.5, the modification ratio reached 98.7%.

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

[0057] The synthetic route of 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 to stir and 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 over anhydrous sodium sulfate, concentrate, add isopropanol, heat to dissolve, cool and crystallize, filter, and dry to obtain TBoc-PEG1000-OH.

[0061] Step 2: Dissolve TBoc-PEG1000-OH in dichloromethane, add DCC and DMAP, add hexadecanedioic acid under ice bath conditions and react for 16 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 over 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 after concentration, 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 to stir and dissolve, and 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 over anhydrous sodium sulfate, concentrate, add isopropanol, heat to dissolve, cool and crystallize, filter, and dry to obtain DBCO-PEG1000-LCFA.

[0065] The reaction of DMG-PEG1000-N3 and DBCO-PEG1000-FITC was as follows:

[0066]

[0067] Where n is 22.

[0068] The schematic diagram of the structure of the prepared mRNA-LNP-LCFA is shown in Figure 8 shown.

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

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

[0071] Based on the knowledge of those skilled in the art, replacing alginic acid with chitosan, microalgae or polysaccharide can also achieve the purpose of the present invention. The preparation method of chitosan, microalgae or polysaccharide hydrogel microspheres is a prior art.

[0072] Example 4 simulates the release of calcium alginate microspheres in the gastrointestinal tract in vitro.

[0073] Calcium alginate microspheres of LNP-LCFA lipid nanoparticles encapsulating Cy3 were prepared by microfluidic technology. Lipid-ethanol solution and 0.1mg / mL CPG-Cy3-citrate buffer were prepared, and the preparation method was the same as that of Examples 1-3. The in vivo process of calcium alginate microspheres passing through the stomach and intestines was simulated in vitro, and the cumulative release of nucleic acids was measured. The Cy3-labeled microspheres (containing Cy320ug) were placed in simulated artificial gastric juice and artificial intestinal juice, and the temperature was always maintained at 37±1°C, the volume was 10mL, and the rotation speed was constant at 100rpm. The drug release behavior of nuclear calcium alginate microspheres was investigated in artificial gastric juice and artificial intestinal juice, respectively. Among them, the residence time of artificial gastric juice is 4h, and the residence time of artificial intestinal juice is 20h. After the experiment started, 100 μL of release medium was taken every hour using a pipette, and 100 μL of release medium was immediately added to keep the total volume of release medium unchanged. The fluorescence intensity was measured by an ELISA instrument (excitation wavelength 550 nm, emission wavelength 570 nm), and the release amount of nucleic acid was determined by comparing with the standard curve, and the release curve was drawn. Figure 3 As shown in the figure, the nanoparticles (HM@mLNP-Cy3) encapsulated by calcium alginate microspheres have almost no signal in artificial gastric juice (pH 1.2), but are continuously released over time in artificial intestinal juice (pH 6.8), with a cumulative release of about 78% at 24 hours. Under acidic conditions, the internal structure of the microspheres is in a contracted state, which enhances the protective effect on mLNP. The nanoparticles mLNP-Cy3 that are not encapsulated by calcium alginate microspheres quickly rupture and release the encapsulated Cy3 in artificial gastric juice (pH 1.2).

[0074] Example 5 Fluorescence quantification of major organs at different time points after oral administration of Cy3-labeled microspheres

[0075] To elucidate the biodistribution of oral vaccines in vivo, Cy3-labeled calcium alginate microspheres were used and orally administered to C57BL / 6 mice. Over the next 12 hours, the mice were euthanized at different time points, and their major organs, including stomach, intestine, heart, liver, spleen, lung, kidney, and mesenteric lymph nodes, were collected. The results of the in vitro fluorescence intensity measurement are shown in Figure 2. Figure 4 As shown, higher levels of Cy3 enrichment were 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] In order to clarify the effect of LCFA modification on the penetration of lipid nanoparticles into the intestinal epithelial barrier, a Transwell experiment 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 ratio of 9:1 for 21 days to form a complete intestinal epithelial barrier. Then, DC2.4 cells were inoculated in the lower chamber, and different groups containing OVA mRNA antigens were added to the upper chamber. After 12 hours of co-culture, flow cytometry was used to detect the activation of DC2.4 in the lower chamber, that is, to detect CD80 + 、CD86 + The result is as follows: Figure 5 As shown, the LCFA-modified vaccine group significantly increased the activation ratio of DC cells compared with 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 20ug tubulin mRNA (Tubulin); the mOVA-LNP group refers to the addition of 20ug chicken ovalbumin mRNA (OVA); and the mOVA-LNP-LCFA group refers to the addition of 20ug chicken ovalbumin mRNA (OVA). The preparation methods of mRNA-LNP and mRNA-LNP-LCFA are the same as those of 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 oral vaccines modified with LCFA, cycloheximide (CXI), a lymphatic transport inhibitor, was injected intraperitoneally (ip) into 200-225 g female SD rats at 3 mg / kg. Cycloheximide was dissolved in saline (0.6 mg / mL). Blood (200 μL) was 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 fluorescence intensity of the serum was quantified. The Cy3 content in peripheral blood was calculated according to the fluorescence intensity at 0 h of each rat. Female SD rats were randomly divided into 5 groups: (I) PBS, HM@LNP-Cy3, HM@LNP-Cy3+CXI, HM@LNP-Cy3-LCFA, HM@LNP-Cy3-LCFA+CXI [equivalent to 1 mg / kg Cy3 dye]; the PBS group was gavaged with an equal volume of sterile PBS solution; the HM@LNP-Cy3 group was gavaged with The stomach was treated with Cy3 calcium alginate microspheres coated with conventional LNP; the HM@LNP-Cy3+CXI group refers to the need to be treated with cycloheximide (CXI) before oral administration, and other intervention measures are the same as above; the HM@LNP-Cy3-LCFA group refers to the Cy3 calcium alginate microspheres coated with LNP modified with LCFA groups by oral administration; the HM@LNP-Cy3-LCFA+CXI[group refers to the need to be treated with cycloheximide (CXI) before oral administration, and other intervention measures are the same as above. I(I) The specific treatment measures of cycloheximide (CXI) are as follows: rats are intraperitoneally injected with CXI [3 mg / kg] 1 hour before administration. The results are shown in Figure 6 As shown, LCFA-modified oral vaccines are mainly transported through the intestinal lymphatic system, while unmodified oral vaccines are mainly transported through the hepatic portal vein circulation.

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

[0081] 1.5*10 B16-OVA cells were injected into the tail vein of C57BL / 6 mice. 5 A mouse melanoma lung metastasis model was established. MC38 cells (1×10) were injected into the axilla of C57BL / 6 mice. 6A mouse colorectal cancer model was established. After tumor implantation, mice were gavaged when the tumor diameter reached 5 mm, once every 3 days. Each dose was calcium alginate microspheres containing 20 μg mRNA antigen, suspended in 100 μL pre-cooled PBS, and gavage was performed 5 times in total. 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 gavaged with 20 μg mRNA antigen]; the Control group refers to gavage with an equal volume of sterile PBS solution; the HM@mTubulin-LNP group refers to gavage with 20ug tubulin mRNA (Tubulin);

[0082] The HM@mOVA-LNP group refers to the OVAmRNA calcium alginate microspheres encapsulated with conventional LNPs administered orally;

[0083] The HM@mOVA-LNP-LCFA group refers to the oral administration of OVAmRNA calcium alginate microspheres coated with LNP modified with LCFA groups. The mice in the control group (Control) were killed when the tumor diameter reached 1.2 cm. The tumor diameter of the mice was observed at the end of the treatment. The results are as follows: Figure 7 It can be seen that the oral vaccines prepared in Examples 1-3 can significantly inhibit the growth of tumors and have good tumor treatment effects.

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

Claims

1. A compound DMG-PEG1000-N3, characterized in that Its structural formula is:

2. A compound DBCO-PEG1000-LCFA characterized by its structural formula:

3. Use of the compound DMG-PEG1000-N3 described in claim 1 in combination with the compound DBCO-PEG1000-LCFA described in claim 2 in the preparation of an oral drug carrier targeting mesenteric lymph nodes.

4. An oral drug delivery system targeting mesenteric lymph nodes, characterized in that The invention comprises a bioactive agent and a lipid nanoparticle LNP, wherein the bioactive agent is wrapped by the lipid nanoparticle LNP, and the PEG end of the lipid nanoparticle LNP is connected with a long-chain fatty acid group LCFA, and the long-chain fatty acid group LCFA is a saturated fatty acid or an unsaturated fatty acid with 12<C≤24.

5. The oral drug delivery system according to claim 4, characterized in that: The structural formula of the PEG-LCFA of the LNP is 6. The oral drug delivery system according to claim 5, characterized in that: The oral drug delivery system is also wrapped with a layer of hydrogel shell, specifically refers to alginate, chitosan, and microalgae.

7. The oral drug delivery system according to any one of claims 4 to 6, characterized in that: The bioactive agent refers to mRNA, siRNA, ASO, miRNA or Aptamer.

8. The method for preparing the oral drug delivery system according to any one of claims 4 to 7, characterized in that: The steps include: preparing a lipid-ethanol solution and a bioactive agent-citrate buffer, preparing lipid nanoparticles encapsulating the bioactive agent by a microfluidic mixing method, and obtaining bioactive agent-LNP lipid nanoparticles; Modifying the bioactive agent-LNP particles with long-chain fatty acid groups to obtain bioactive agent-LNP-LCFA lipid nanoparticles; The bioactive agent-LNP-LCFA lipid nanoparticles were mixed with a sodium alginate solution, and a layer of calcium alginate hydrogel shell was wrapped around the bioactive agent-LNP-LCFA lipid nanoparticles using droplet microfluidics technology.

9. The preparation method according to claim 8, 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-citrate buffer is 1:

3.

10. The preparation method according to claim 9, characterized in that: The modification of mRNA-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.

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