Mucosal adhesive lipid material and its use as a gene carrier

Abstract

The application discloses a mucous membrane adhesion auxiliary material and a lipid material with mucous membrane adhesion prepared by using the auxiliary material, the nanoparticle has mucous membrane adhesion and can be adhered to the surface of a mucous membrane and is suitable for mucous membrane administration. The lipid nanoparticle promotes the entry of the lipid nanoparticle into cells by forming a disulfide bond with the sulfydryl on the surface of mucous membrane cells, and the entry of the lipid nanoparticle into cells is a non-endosome-lysosome pathway, so that the encapsulated nucleic acid macromolecule can be avoided from being degraded by enzymes in cells, thereby directly delivering the macromolecular nucleic acid in the cytoplasm to play a role.

Description

Technical Field

[0001] This invention relates to a mucosal adhesive lipid material and the preparation of its lipid nanoparticle composition, as well as the application of the lipid nanoparticles in gene delivery. This delivery carrier can bind to the dithiopentane ring structure on the surface of the lipid nanoparticles via thiol groups on the cell surface, facilitating the entry of the lipid nanoparticles into the cell and bypassing common endosomes for cellular endocytosis.

[0002] —Lysosomal pathway. Background Technology

[0003] Nucleic acid drug delivery systems are a key factor affecting the efficacy of nucleic acid drugs. Current delivery systems often utilize cell-penetrating peptides to improve cell entry efficiency, but these peptides exhibit some toxicity. Lipoic acid, a non-toxic natural compound, possesses free radical scavenging, antioxidant, and heavy metal removal capabilities. Furthermore, its dithiopentyl ring can react with exposed thiol groups on the surface of mucosal cells to form thiol-disulfide bonds, thus achieving mucosal adhesion. Based on this principle, this patent prepares lipoic acid-modified lipid nanoparticles with mucosal adhesion and enhanced endocytosis for nucleic acid drug delivery. Using eGFP plasmid as a model nucleic acid drug, lipid nanoparticles encapsulated with eGFP were prepared using microfluidic technology. The particle size was approximately 80 nm, and the encapsulation efficiency was over 90%. The adhesion of the lipoic acid-modified lipid nanoparticles to the cell surface and the process of promoting endocytosis were also observed using laser confocal microscopy and fluorescence microscopy.

[0004] Currently, the targets of small molecule chemical drugs and large molecule antibody drugs are mostly receptors, kinases, transport carriers, and ion channels. The screening, purification, and production processes for these targets are cumbersome, and many targets remain untargetable by small molecule chemical drugs and large molecule monoclonal antibodies. These drawbacks limit the application of small molecule chemical drugs and large molecule biological drugs in disease treatment. According to the central dogma, mRNA is the bridge between DNA and protein, and the targets of small molecule chemical drugs and large molecule antibody drugs can be converted into corresponding nucleic acid sequences. This means that therapeutic effects can be achieved by deleting, replacing, or silencing the target nucleic acid sequence using nucleic acid drugs. However, nucleic acid drugs have large molecular weights, carry a large amount of negative charge, are highly sensitive to nucleases, and have poor targeting. Developing low-toxicity and highly efficient delivery systems is crucial to enabling nucleic acid drugs to exert their therapeutic effects. Furthermore, the poor targeting of nucleic acid drugs means they tend to accumulate in the liver and kidneys after administration. Currently marketed nucleic acid drugs are mainly concentrated in the areas of liver disease, kidney disease, and hematological diseases.

[0005] Inhalation delivery allows for direct and rapid drug delivery to the lungs, making the process more convenient. This local delivery method for nucleic acid drugs can enhance their targeting effect, avoid accumulation in the liver and kidneys, and reduce side effects caused by insufficient targeting. The surface of lung mucosal cells contains a certain number of thiol groups, and mucosal adhesion based on thiol-thiol interactions has been extensively studied. However, thiol groups are unstable and easily oxidized; therefore, adhesion molecules containing disulfide bonds are widely used in mucosal adhesion research. Studies have shown that the dihedral angle of the disulfide bond is key to the interaction between the disulfide bond and thiol groups on the cell surface, and compounds containing a dithiopentane structure can promote endocytosis. Lipoic acid, a natural compound containing a dithiopentane structure, has good stability. The dithiopentane ring exhibits ring strain, readily reacting with thiol groups on the mucosal cell surface to open the ring, allowing lipoic acid-modified lipid nanoparticles to adhere to the mucosal cell surface and further promoting endocytosis.

[0006] Therefore, this invention uses cationic lipids, DSPC, LA-PEG-DSPC, and cholesterol as excipients, and eGFP plasmid as a model nucleic acid drug, to prepare lipid nanoparticles loaded with eGFP using microfluidic technology. Lipoic acid was used as a functional molecule to modify the LNP as a mucosal adhesion and endocytosis-promoting agent, resulting in LANP nanoparticles. Studies have shown that LANP exhibits better mucosal adhesion than LNP and can promote endocytosis of LANP by cells. In cytotoxicity studies, the LANP prepared in this invention showed significantly better safety than the commercially available transfection reagent Lipo2000. Furthermore, LANP-loaded eGFP exhibited superior gene expression compared to LNP-loaded eGFP. Lung inhalation experiments in mice also demonstrated that LANP adheres better to the lungs. Surprisingly, the lipoic acid-modified LNP can enter cells via a reaction between the dithiopental ring and sulfhydryl groups on the cell surface, rather than through the traditional macropinocytosis process, i.e., the endosome-lysosome pathway. This non-macropoietic pathway allows nucleic acid drugs to enter the cell without being degraded by enzymes in endosomes or lysosomes, directly releasing the nucleic acid into the cytoplasm. Summary of the Invention

[0007] The purpose of this invention is to provide: (1) a mucosal adhesive material and a method for synthesizing the material; (2) a formulation and preparation method of lipoic acid modified lipid nanoparticles (LANP); (3) a method for preparing mucosal adhesive lipid nanoparticles loaded with nucleic acids; and (4) a pulmonary inhalation drug delivery system for nucleic acid drugs.

[0008] This invention designs and synthesizes a mucosal adhesion molecule, LA-PEG-DSPE, which has the structure of Formula I:

[0009]

[0010] This invention discloses a method for preparing the above-mentioned mucosal adhesion molecule, comprising the following steps: first, reacting lipoic acid with carbonyl diimidazole to obtain a lipoic acid intermediate activated by carbonyl diimidazole. Then, α-

[0011] Amino-ω-1,2-distearate-3-phosphatidylethanolamine polyethylene glycol and dimethylaminopyridine catalyst were added and reacted at 40°C for 24 h to obtain the compound with the structure of formula I. This synthetic method is simple and direct, and the reaction conditions are mild and controllable.

[0012] In the above technical solution, the molar ratio of carbonyl diimidazole to lipoic acid is 1:1 to 1.5, the activation temperature is 30℃, and the activation time is 24 hours. The molar ratio of lipoic acid to α-amino-ω-1,2-distearate-3-phosphatidylethanolamine polyethylene glycol is 5:1 to 1:1, and the reaction temperature is 30 to 60℃. Preferably, α-amino-

[0013] The molar ratio of ω-1,2-distearate-3-phosphatidylethanolamine polyethylene glycol, thioctic acid, and carbonyl diimidazole is 1:3:3.6.

[0014] The specific reaction process in the above technical solution is as follows:

[0015] Thioctic acid and carbonyl diimidazole were added to dichloromethane and stirred to dissolve. After reacting at 30°C for 0.5 hours, α-amino-ω-1,2-distearate-3-phosphatidylethanolamine polyethylene glycol and N,N-dimethyl-4-pyridinium amine were added and reacted at 40°C for 24 hours. The reaction solution was then precipitated with ice-cold diethyl ether to obtain a white turbid precipitate. The white precipitate was centrifuged and dried at 40°C to obtain a white powdery solid.

[0016] Lipid nanoparticles, primarily composed of cationic lipids, auxiliary lipids, and PEGylated lipids, are currently the main delivery vehicle for nucleic acid drugs. One siRNA drug and two mRNA drugs currently utilize lipid nanoparticles as delivery carriers. Based on marketed and clinically investigated nucleic acid drugs, lipid nanoparticles are primarily used for mRNA delivery due to limitations in delivery efficiency and toxicity, while siRNA delivery often employs GalNAc technology. Lipid nanoparticles generally consist of three components: cationic lipids, auxiliary lipids, and PEGylated phospholipids. This patent discloses a novel lipoic acid-modified PEGylated phospholipid that, compared to the aforementioned PEGylated lipid materials, possesses mucosal adhesion capabilities. It can adhere to mucosal surfaces and, through the disulfide bonds of lipoic acid, forms disulfide bonds with thiol groups on the cell membrane surface, thereby mediating the lipid nanoparticles bypassing the endosome-lysosome pathway to enter the cell directly into the cytoplasm. Compared to existing lipid nanoparticle delivery technologies, the mucosa-adhesive lipid nanoparticles of this invention can bypass the endosome-lysosome pathway into cells and directly deliver lipid nanoparticles into the cellular lipids where nucleic acid drugs exert their effects through the adhesion of dithiopental rings. This avoids the degradation of nucleic acids by enzymes in the endosomes and significantly improves the delivery efficiency of nucleic acid drugs.

[0017] This invention also discloses a formulation and preparation method for mucosal adhesive lipid nanoparticles. The lipid nanoparticles prepared by this formulation and process possess mucosal adhesive properties and can bypass the endosome-lysosome pathway to enter cells, efficiently delivering nucleic acids to the cytoplasm. The components of the mucosal adhesive lipid nanoparticle formulation are: DSPC, LA-PEG-DSPE, cholesterol, and ALC0315. Their molar ratio is 10:2–20:35–30:40–45. A preferred mucosal adhesive lipid nanoparticle formulation contains: DSPC, LA-PEG-DSPE, cholesterol, and ALC0315. Their molar ratio is 10:10:35:45. The preparation process of the lipid nanoparticles is as follows: weigh each component in the lipid nanoparticle formulation and dissolve it in anhydrous ethanol; dissolve the nucleic acid in a 10 mM pH 4.0 citrate buffer solution; use microfluidics to set the flow rate of the ethanol phase to 2.5 mL / min and the flow rate of the aqueous phase to 7.5 mL / min to mix the two phase solutions; place the obtained lipid nanoparticles in PBS and dialyze to remove the ethanol.

[0018] The present invention has the following advantages:

[0019] This patent discloses a lipid material with mucosal adhesion properties and the preparation of mucosal adhesive lipid nanoparticles using this material, along with the formulation and preparation process of the lipid nanoparticles. Due to the ring strain of the dithiopentane ring, it can react with thiol groups on the cell surface, thereby allowing the lipid nanoparticles to adhere to the cell surface. Furthermore, the adhesion of lipid nanoparticles to the cell surface by the reaction of thiol groups with the dithiopentane ring structure further promotes their entry into the cytoplasm. Currently, the pathway for lipid nanoparticles to enter the cytoplasm is the endosome-lysosome pathway. This method, based on the interaction between thiol groups and the dithiopentane ring, can promote the direct entry of lipid nanoparticles into the cytoplasm, thereby avoiding the degradation of nucleic acids by lysosomes and improving nucleic acid delivery efficiency. This invention is suitable for achieving mucosal immunity through mucosal drug delivery and has good application prospects in the prevention of large-scale epidemic respiratory diseases, such as delivering nucleic acids directly to the lungs via inhalation for the prevention of respiratory epidemics. Attached Figure Description

[0020] Appendix Figure 1 The proton NMR spectrum of LA-PEG-DSPE;

[0021] Appendix Figure 2 The proton NMR spectrum of LA-PEG-DSPE;

[0022] Appendix Figure 3 The proton NMR spectrum of LA-PEG-DSPE;

[0023] Appendix Figure 4 The proton NMR spectrum of LA-PEG-DSPE;

[0024] Appendix Figure 5 Particle size distribution diagrams of mucosal adhesive lipid nanoparticles in Examples 5 to 8;

[0025] Appendix Figure 6 Transmission electron microscopy image of lipid nanoparticles carrying eGFP plasmid in Example 8;

[0026] Appendix Figure 7 The mucosal adhesive lipid nanoparticle anti-nuclease experiment shows the following bands from left to right: EGFP, EGFP+DNase, EGFP@LANP+DNase+EDTA+OP10, EGFP@LANP+DNase+EDTA+OP10, EGFP@LANP+OP10, and EGFP@LANP.

[0027] Appendix Figure 8 Mucosal adhesive lipid nanoparticle transfection experiment, A549 cell density was 5x10⁻⁶ 4Samples / well, eGFP dosage of 5ug / well, after incubation of the sample and cells for 2h, the cells were cultured for another 48h;

[0028] Appendix Figure 9 Intracellular endocytosis assay of mucosal adhesive lipid nanoparticles: Fluorescence microscopy images of A549 cells incubated with Cy5-labeled lipid nanoparticles for different time periods; red indicates Cy5-labeled LNPs; Cy5 concentration was approximately 10 μg / mL.

[0029] Appendix Figure 10 Cytotoxicity of LANP and Lipo2000 on A549 cells. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and embodiments. These embodiments do not limit the scope of the invention.

[0031] Example 1: Synthesis of LA-PEG-DSPE

[0032] Accurately weighed carbonyl diimidazole (6.5 mg, 0.04 mmol) was placed in an anhydrous two-necked round-bottom flask, and dichloromethane (1.5 mL) was added. The reaction system was stirred evenly in an oil bath at 30 °C. Then, lipoic acid (8.3 mg, 0.04 mmol) was dissolved in dichloromethane (1.5 mL) and added to the above solution. DSPE-PEG-NH2 (109.7 mg, 0.04 mmol) was dissolved in dichloromethane (1.5 mL) and added to the above reaction solution. The reaction was carried out at 30 °C for 24 h. After the reaction was completed, the reaction solution was precipitated twice in ice-cold diethyl ether. The precipitate was collected by centrifugation and dried under vacuum in the dark. (78.1 mg, yield 66.2%)

[0033] NMR characterization of LA-PEG-DSPE (500MHz, DMSO-d6) is attached. Figure 1 .

[0034] Example 2: Synthesis of LA-PEG-DSPE

[0035] Accurately weighed carbonyl diimidazole (9.7 mg, 0.06 mmol) was placed in an anhydrous two-necked round-bottom flask, and dichloromethane (1.5 mL) was added. The reaction system was stirred evenly in an oil bath at 30 °C. Then, lipoic acid (8.3 mg, 0.04 mmol) was dissolved in dichloromethane (1.5 mL) and added to the above solution. DSPE-PEG-NH2 (109.7 mg, 0.04 mmol) was dissolved in dichloromethane (1.5 mL) and added to the above reaction solution. The reaction was carried out at 30 °C for 24 h. After the reaction was completed, the reaction solution was precipitated twice in ice-cold diethyl ether. The precipitate was collected by centrifugation and dried under vacuum in the dark. (86.2 mg, yield 73.1%)

[0036] NMR characterization of LA-PEG-DSPE (500MHz, DMSO-d6) is attached. Figure 2 .

[0037] Example 3: Synthesis of LA-PEG-DSPE

[0038] Accurately weighed carbonyl diimidazole (32.4 mg, 0.20 mmol) was placed in an anhydrous two-necked round-bottom flask, and dichloromethane (1.5 mL) was added. The reaction system was stirred evenly in an oil bath at 30 °C. Then, lipoic acid (41.3 mg, 0.20 mmol) was dissolved in dichloromethane (1.5 mL) and added to the above solution. DSPE-PEG-NH2 (109.7 mg, 0.04 mmol) was dissolved in dichloromethane (1.5 mL) and added to the above reaction solution. The reaction was carried out at 30 °C for 24 h. After the reaction was completed, the reaction solution was precipitated twice in ice-cold diethyl ether, and the precipitate was collected by centrifugation and dried under vacuum in the dark. (117.2 mg, yield 77.6%).

[0039] NMR characterization of LA-PEG-DSPE (500MHz, DMSO-d6) is attached. Figure 3 .

[0040] Example 4: Synthesis of LA-PEG-DSPE

[0041] Accurately weighed carbonyl diimidazole (48.7 mg, 0.30 mmol) was placed in an anhydrous two-necked round-bottom flask, and dichloromethane (1.5 mL) was added. The reaction system was stirred evenly in an oil bath at 30 °C. Then, lipoic acid (41.3 mg, 0.20 mmol) was dissolved in dichloromethane (1.5 mL) and added to the above solution. DSPE-PEG-NH2 (109.7 mg, 0.04 mmol) was dissolved in dichloromethane (1.5 mL) and added to the above reaction solution. The reaction was carried out at 30 °C for 24 h. After the reaction was completed, the reaction solution was precipitated twice in ice-cold diethyl ether. The precipitate was collected by centrifugation and dried under vacuum in the dark. (120.2 mg, yield 79.6%).

[0042] NMR characterization of LA-PEG-DSPE (500MHz, DMSO-d6) is attached. Figure 4 .

[0043] Example 5: Preparation of Mucosal Adhesive Lipid Nanoparticles

[0044] The formulations of mucosal adhesive lipid nanoparticles are shown in the table below:

[0045]

[0046] Weigh each component according to the table above and dissolve it in 10 mL of anhydrous ethanol to obtain a lipid solution. The aqueous phase solution is PBS. INano was used. TM An L-shaped rapid nanomedicine preparation system was used, with the lipid solution flow rate set at 3 mL / min and the PBS solution flow rate at 9 mL / min. The two solutions were mixed under these conditions. The resulting mixture was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed with PBS for 12 h. After dialysis, the particle size of the lipid nanoparticles was determined using a dynamic laser light scattering particle size analyzer.

[0047] Example 6: Preparation of Mucosal Adhesive Lipid Nanoparticles

[0048] The formulations of mucosal adhesive lipid nanoparticles are shown in the table below:

[0049]

[0050] Weigh each component according to the table above and dissolve it in 10 mL of anhydrous ethanol to obtain a lipid solution. The aqueous phase solution is PBS. INano was used. TMAn L-shaped rapid nanomedicine preparation system was used, with the lipid solution flow rate set at 3 mL / min and the PBS solution flow rate at 9 mL / min. The two solutions were mixed under these conditions. The resulting mixture was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed with PBS for 12 h. After dialysis, the particle size of the lipid nanoparticles was determined using a dynamic laser light scattering particle size analyzer.

[0051] Example 7: Preparation of Mucosal Adhesive Lipid Nanoparticles

[0052] The formulations of mucosal adhesive lipid nanoparticles are shown in the table below:

[0053]

[0054] Weigh each component according to the table above and dissolve it in 10 mL of anhydrous ethanol to obtain a lipid solution. The aqueous phase solution is PBS. INano was used. TM An L-shaped rapid nanomedicine preparation system was used, with the lipid solution flow rate set at 3 mL / min and the PBS solution flow rate at 9 mL / min. The two solutions were mixed under these conditions. The resulting mixture was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed with PBS for 12 h. After dialysis, the particle size of the lipid nanoparticles was determined using a dynamic laser light scattering particle size analyzer.

[0055] Example 8: Preparation of mucosal adhesive lipid nanoparticles loaded with eGFP plasmid

[0056] The formulations of mucosal adhesive lipid nanoparticles loaded with eGFP plasmid are shown in the table below:

[0057]

[0058] Weigh each component according to the table above and dissolve it in 10 mL of anhydrous ethanol to obtain a lipid solution. The aqueous phase solution is a 10 mM citrate buffer solution with pH 4.0, wherein the eGFP concentration in the citrate buffer solution is 135 μg / mL. INano was used. TM An L-type rapid nanomedicine preparation system was used, with the lipid solution flow rate set at 3 mL / min and the citrate buffer solution flow rate at 9 mL / min. The two solutions were mixed under these conditions. The resulting mixture was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed with PBS for 12 h. After dialysis, the particle size of the lipid nanoparticles was determined using a dynamic laser light scattering particle size analyzer. The encapsulation efficiency was 96.2% as measured by the Ribogreen kit. Transmission electron microscopy showed that the mucosal adhesive lipid nanoparticles encapsulating the eGFP plasmid were uniform in size, approximately 50 nm. (See attached image). Figure 6Because the transmission electron microscope (TEM) captures the particle size of nanoparticles in their dry state, the particle size is smaller than the hydration kinetic particle size detected by the dynamic laser light scattering particle size analyzer. The particle size distributions of the nanoparticles in Examples 4 to 8 are attached. Figure 5 .

[0059] Example 9: Nuclease Resistance Experiment of Mucosal Adhesive Lipid Nanoparticles Loaded with eGFP Plasmid

[0060] 20 μL of the eGFP-loaded lipid nanoparticles prepared in Example 8 were incubated with 2 μL of nuclease (1 U / μL) at 37°C for 2 h. Then, 5 μL of saturated EDTA solution was added and incubated together at room temperature for 10 min. Next, 10 μL of op10 was added and incubated at room temperature for 2 h. Finally, 1.8 μL + 1 μL of loading buffer was taken from the mixture and subjected to agarose gel electrophoresis at 90 V for 20 min. The electrophoretic bands were observed. The anti-nuclease performance of the eGFP-loaded mucosal adhesive lipid nanoparticles was compared with that of the eGFP-loaded mucosal adhesive lipid nanoparticles as controls. See attached gel electrophoresis image. Figure 7 .

[0061] Example 10: Cell Adhesion Experiment of Mucosal Adhesion Lipid Nanoparticles Loaded with eGFP Plasmid

[0062] Cells were cultured using 24-well plate circular cell spreaders. Small circular spreaders were placed in each well of a 24-well plate, with approximately 10 cells seeded per well. 5 1000 μL of A549 cells (10⁵ cells / mL) were cultured until the cells reached approximately 80-90% confluence. The culture medium was then discarded, and the cells were washed with PBS. 100 μL of sample was added to each well, followed by 400 μL of serum-free culture medium. The sample and cells were incubated together for 0.5, 1, and 2 hours. After incubation, the cells were washed three times with 500 μL of PBS. Then, 200 μL of 4% paraformaldehyde-PBS solution was added to each well to fix the cells for 15-30 minutes. After fixation, the cells were washed three times with PBS. Next, 120 μL of DAPI solution (diluted 1000 times with PBS) was added to each well to stain the cell nuclei for 2-5 minutes. After staining, the cells were washed three times with PBS. Finally, the slides were mounted and stored at 4°C. The cell adhesion behavior of mucosal adhesive lipid nanoparticles carrying the eGFP plasmid was observed using a laser confocal microscope.

[0063] Example 11: Transfection Experiment of Mucosal Adhesive Lipid Nanoparticles Loaded with eGFP Plasmid

[0064] A549 cells were cultured routinely in RPMI 1640 medium containing 10% FBS, and seeded at a cell count of 5 x 10⁶ cells / well in 24-well plates. 4For each well, add 500 μL of culture medium and incubate overnight to allow cells to reach approximately 70%–90% confluence at transfection. Discard the old culture medium from the 24-well plate, wash the cells twice with PBS, and add 500 μL of serum-free, antibiotic-free culture medium to each well. Add 5 μg of eGFP-encapsulated mucosal adhesive lipid nanoparticles to each well, with three replicates per sample. Two hours after adding the samples, discard the culture medium and replace it with medium containing FBS and antibiotics. Continue culturing for 48 hours, then observe eGFP expression using a fluorescence microscope.

[0065] Example 12: Cytotoxicity Experiment of Mucosal Adhesive Lipid Nanoparticles

[0066] The cytotoxicity of LANP and Lipo2000 against A549 cells was assessed using the MTT assay. A549 cells were seeded into 96-well plates (80 μL, 5 x 10⁶ cells / well). 3 Cells were cultured in 100 μL of PBS in each well for 24 h until the cell confluence reached 80-90%. 100 μL of PBS was added to the wells around the perimeter of the 96-well plate, and 20 μL of sample was added to each of the remaining wells, with four replicates per sample. Incubation continued for another 24 h. Then, 10 μL of MTT (5 mg / mL) was added to each well, and incubation continued for 4 h. The culture medium was removed, and 150 μL of DMSO was added to each well to dissolve the generated Gazanium crystals. Cell viability was calculated by measuring the absorbance at 492 nm using a microplate reader.

[0067] Example 13: Preparation of a pulmonary inhalation lipid nanoparticle drug delivery system based on mucosal adhesion.

[0068] Lipid nanoparticles with mucosal adhesion properties loaded with eGFP were prepared according to Example 8. The lipid nanoparticles were dialyzed using a 10 mM pH 7.4 Tris buffer solution, and the osmotic pressure was measured. Based on the osmotic pressure measurement results, sodium chloride was added to make the lipid nanoparticles isotonic.

Claims

1. A type of mucosal adhesive lipid nanoparticle, characterized in that: It is composed of a tertiary amine-based cationic lipid material, cholesterol, distearate phosphatidylcholine, a mucosal adhesive lipid material, and nucleotides; the composition of the mucosal adhesive lipid nanoparticles, by molar parts, is as follows: 2-20 parts of mucosal adhesive lipid material, 40-50 parts of tertiary amine cationic lipid, 30-38 parts of cholesterol, 10 parts of distearate phosphatidylcholine, and water for injection to a final volume of 10 mL. The structural formula of the mucosal adhesive lipid material is shown in Formula 1; it consists of three parts: lipoic acid, polyethylene glycol, and phosphoryl ethanolamine phospholipid, and chemical bonds linking these three parts. 。 2. The mucosal adhesive lipid nanoparticles as described in claim 1, characterized in that, The number average molecular weight of the polyethylene glycol portion is 2000-5000; the phosphoryl ethanolamine phospholipid portion is one of 1,2-dicepanoyl-sn-glycerol-3-phosphate ethanolamine, 1-palmitoyl-2-oleoyl ethanolamine, 1-stearoyl-2-linoleic acid-sn-glycerol-3-phosphate ethanolamine, distearyl phosphatidyl ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), and 1,2-dioleoyl-SN-glycerol-3-phosphate ethanolamine (DOPE).

3. The mucosal adhesive lipid nanoparticles as described in claim 1, characterized in that, The preparation method is as follows: Weigh each lipid component and dissolve it in anhydrous ethanol to obtain a lipid solution; the aqueous solution is a PBS solution; use a syringe pump to set the flow rate of the lipid solution to 3 mL / min and the flow rate of the PBS solution to 9 mL / min, and mix the two solutions in a microfluidic chip under these conditions; After obtaining the mixed solution, place it in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyze with PBS for 12 hours.

4. The mucosal adhesive lipid nanoparticles as described in claim 1, characterized in that, The nucleotides include: antisense oligonucleotides, small interfering nucleic acids, small nucleic acids, messenger deoxyribonucleic acid, or combinations thereof.

5. The mucosal adhesive lipid nanoparticles as described in claim 1, characterized in that, The particle size range is 20-300nm.

6. The mucosal adhesive lipid nanoparticles as described in claim 5, characterized in that, The particle size range is 50-150 nm.

7. A nucleic acid drug with mucosal adhesion activity, characterized in that, The drug is composed of mucosal adhesive lipid nanoparticles as described in claim 1, pH adjusters and osmotic pressure adjusters such as potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, and sodium chloride, as well as water for injection. It is administered to the lungs by nebulization using an ultrasonic liquid nebulizer.

8. A nucleic acid drug with mucosal adhesion activity, characterized in that, The formulation of the mucosal adhesive lipid nanoparticles loaded with nucleic acid as described in claim 1 is shown in the table below. Prescription composition: Preparation method: Weigh each component according to the table above, dissolve them in anhydrous ethanol to obtain a lipid solution; the aqueous phase solution is a PBS solution; use INano. TM The L-type rapid nanomedicine preparation system sets the flow rates of the lipid solution and the PBS solution, and uses microfluidics to mix the two solutions. After obtaining the mixed solution, it is placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa, and lipid nanoparticles can be obtained by dialysis with PBS. How to use: After obtaining lipid nanoparticles using the above method, place the lipid nanoparticle solution in an atomizer, atomize it, and then inhale the aerosol through an inhaler.

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