Nasal Brain-Targeted Nanodrug Delivery System for Central Nervous System and Its Preparation Method and Application

Abstract

The present invention relates to the field of biopharmaceutical technology, and specifically discloses a nasal-to-brain targeted central nervous system nano-drug delivery system, its preparation method and application. The nasal-to-brain targeted central nervous system nano-drug delivery system includes a biotherapeutic drug and a lectin-conjugated liposome nanoparticle wrapped on the surface of the biotherapeutic drug; the biotherapeutic drug is a biomacromolecule encoded by mRNA; the lectin is AAL, and the raw materials of the liposome nanoparticle include SM102, DSPC, cholesterol, DMG-pSar25 and DSPE-PEG-MAL. By conjugating AAL with LNP of specific components, the present invention specifically recognizes and binds to the receptor fucose of target cells, which helps the nanomaterials to be endocytosed through the olfactory mucosa and delivered to the brain through the nose-brain pathway, targeting the delivery of biotherapeutic drugs, breaking through the blood-brain barrier, and significantly improving the targeting efficiency and nasal delivery efficiency.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a transnasal brain-targeted central nervous system nano-drug delivery system, a preparation method thereof, and an application thereof. Background Art

[0002] There are huge challenges in the drug research and development for central nervous system degenerative diseases. An important reason is the existence of the blood-brain barrier (BBB). The blood-brain barrier is a cell complex with extremely low permeability between the blood and the nerve tissue of the central nervous system, featuring tight junctions. Coupled with the presence of transporters and metabolic enzymes, it causes approximately 98% of chemical drugs and almost 100% of macromolecular drugs (including protein polypeptides and gene drugs) to be difficult to enter the brain to reach the therapeutic concentration. The drug delivery strategies for the central nervous system can be achieved through several administration routes: systemic administration (such as intravenous injection), invasive local administration (such as intrathecal injection, intracerebral parenchymal administration). Systemic administration is an invasive method. Intravenous injection provides a minimally invasive opportunity for drugs to enter the brain, but this requires crossing the blood-brain barrier. Local or non-systemic administration routes are usually also invasive. Currently, one strategy for non-invasive methods is to administer drugs through the intranasal route.

[0003] Intranasal administration can deliver therapeutic drugs to the brain by bypassing the blood-brain barrier through the olfactory region of the nose. It has the characteristics of easy use, rapid absorption and onset, high bioavailability, non-invasive and convenient administration, less damage to the body, avoidance of liver metabolism, and high patient compliance. The olfactory region of the nose is located at the top of the nasal cavity and consists of olfactory mucosa, lamina propria, and olfactory bulb. As the only part of the central nervous system directly exposed to the external environment, the olfactory region of the nose is considered the most direct and rapid route for drugs to bypass the blood-brain barrier and enter the cerebrospinal fluid. Therefore, the nasal drug delivery system can directly deliver drugs to the brain tissue via the nasal-brain pathway. Based on the special anatomical connection between the nasal cavity and the brain, the drug transport pathway from the nose to the brain can be divided into three major parts: the olfactory nerve pathway, the trigeminal nerve pathway, and the blood circulation pathway. When the drug reaches the olfactory region, it can be transported into the brain through the intraneuronal pathway: the drug interacts with the nerve endings of olfactory receptor neurons, passes through the cribriform plate along the axonal nerve bundle of olfactory neurons to reach the olfactory bulb, and then diffuses into the interstitial fluid, cerebrospinal fluid, and brain tissue. Compared with the slower olfactory neuron intraneuronal transport pathway, in the extraneuronal transport pathway, the drug can be rapidly transported to different regions of the brain through the tight junctions between supporting cells or the channels between olfactory neurons and supporting cells. Once the drug diffuses to the trigeminal nerve branches in the olfactory and respiratory regions, it can be transported along the intraneuronal pathway of the axon and be transported through the nose via the trigeminal nerve pathway. The blood circulation pathway is an indirect way for drugs to enter the brain through the nose. After intranasal administration, the drug can be absorbed into the systemic blood circulation through the rich capillaries under the nasal mucosa, and then cross the blood-brain barrier with the help of blood circulation to enter the central system.

[0004] Currently, in the research of nanodrug delivery systems, the main methods to bypass the blood-brain barrier are to use surfactants or nanoparticle encapsulation to enhance delivery. Nanostructured lipids (LNP), nanoemulsions, and chitosan coatings can be used to change the surface properties of nanoparticles to improve delivery to the brain; biodegradable polymer materials such as polylactic acid, polyglycolic acid, PLGA, and polyanhydride sebacate can encapsulate and increase the stability of drugs; through the functionalization of cell-penetrating peptides and proteins, brain-targeted uptake and delivery can be further achieved.

[0005] However, no precedent has been found for using nanoliposome particle carriers for intranasal administration. The nasal drug delivery system is limited by the surface area of the nasal cavity and the characteristics of the nasal mucosa, which weakens the effective absorption of drugs. Mucociliary clearance is a major challenge when delivering biologics. Biopharmaceuticals are much larger than traditional small molecule drugs and have a very low membrane permeability, so it is difficult to achieve effective absorption or to extend the therapeutic effect. How to open up the nasal mucosa epithelial pathway, effectively improve the nasal delivery efficiency of the nasal drug delivery system, target the best treatment area, prevent penetration into the lungs, avoid irritating the nasal mucosa, ensure sufficient retention time, improve the ability of liposome nanoparticles to adsorb to the nasal mucosa, and improve their internalization efficiency still needs further research. Summary of the Invention

[0006] In view of the above problems, the present invention provides a nasal-to-brain targeted central nervous system nano drug delivery system, its preparation method and application. The present invention creatively uses liposome nanoparticle (LNP) technology and bioconjugation technology to open up the nasal mucosa epithelial pathway, so as to efficiently and precisely deliver biologic macromolecule drugs into the central nervous system, realize nasal-to-brain delivery of the loaded drugs, and significantly improve the adhesion time and adsorption ability of the drug nanomaterials on the nasal mucosa and the delivery ability in the central nervous system.

[0007] To solve the above technical problems, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a nasal-to-brain targeted central nervous system nano drug delivery system, including a biotherapeutic drug and lectin-conjugated liposome nanoparticles, and the lectin-conjugated liposome nanoparticles are wrapped on the surface of the biotherapeutic drug;

[0009] The biotherapeutic drug is a biologic macromolecule encoded by mRNA; the lectin in the lectin-conjugated liposome nanoparticles is Aleuria aurantia lectin, and the raw materials of the liposome nanoparticles include SM102, DSPC, cholesterol, DMG-pSar25, and phospholipid polyethylene glycol maleimide.

[0010] It should be noted that SM102 represents an ionizable amino lipid, and its structural formula is shown in Formula 1; DSPC represents a liposome compound, and its structural formula is shown in Formula 2; the structural formula of cholesterol is shown in Formula 3; DMG-pSar25 represents a polymer polyethylene glycol (PEG) derivative, and its structural formula is shown in Formula 4.

[0011]

[0012] Formula 1

[0013]

[0014] Formula 2

[0015]

[0016] Formula 3

[0017]

[0018] Formula 4

[0019] Compared with the prior art, the transnasal brain-targeting central nervous system nano-drug delivery system provided by the present invention couples Aleuria aurantia lectin (AAL) with liposome nanoparticles (LNP) of specific components, so as to specifically recognize and bind to the receptor of target cells, fucose, target the delivery of biotherapeutic drugs, break through the blood-brain barrier, and significantly improve the targeting efficiency and transnasal delivery efficiency.

[0020] The present invention creatively uses liposome nanoparticle technology and bioconjugation technology to open up a transnasal brain entry channel, so as to efficiently and precisely deliver biologic macromolecule drugs into the central nervous system. The delivery of LNP-encapsulated mRNA is used to deliver nucleic acids (such as mRNA) to cells. LNP is a small spherical particle composed of a lipid bilayer and a hydrophobic core, containing ionizable lipids, phospholipids, cholesterol, and lipid-anchored PEG and other components as excipients, which contribute to the structural integrity, stability, and intracellular mRNA delivery of LNP. mRNA is encapsulated in the hydrophobic core, which can prevent its degradation in the blood. In the present invention, AAL is first coupled with LNP, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (DSPE-PEG-MAL) is used to modify LNP to facilitate the coupling of LNP with AAL. The lectin AAL of citrus peel fungi is selected as the ligand. AAL contains up to 5 fucose-binding sites, so it has a wider specific binding with the specific receptor fucose of nasal mucosal cells, is designed to target specific cells, significantly improves the adhesion time and adsorption capacity of the nano-material loaded with drugs on the nasal mucosa, helps the nano-material to be endocytosed through the olfactory mucosa, and is delivered to the brain through the nasal-brain pathway without passing through the blood-brain barrier, thereby improving the delivery efficiency of the nano-drug delivery system.

[0021] It should be noted that the present invention does not limit the specific type of biotherapeutic drugs, and any biotherapeutic agent that can target the cerebral cortex is applicable to the present invention, such as BDNF, Klotho, etc.

[0022] Preferably, the molar ratio of the SM102, the DSPC, the cholesterol, the DMG-pSar25, and the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] is (45-50):(10-15):(38-40):(1-2):(0.3-0.7).

[0023] In the present invention, the dosage of DSPE-PEG-MAL is only about 0.3% - 0.7% of the molar amount of LNP, which can ensure that AAL is subsequently conjugated to LNP, and then can specifically bind to fucose, improve the adhesion time and adsorption capacity of the transnasal brain-targeting central nervous system nano-drug delivery system on the nasal mucosa, and ensure that the biotherapeutic drug has a high delivery efficiency.

[0024] Preferably, the molar ratio of nitrogen in the ionizable cationic lipid of the liposome nanoparticles to phosphorus in the mRNA of the biotherapeutic drug is 4:1 to 8:1, more preferably 5:1 to 7:1.

[0025] In the present invention, the drug loading amount of the biotherapeutic drug is controlled by controlling the above nitrogen-phosphorus ratio, so that the encapsulation rate of the mRNA of the biotherapeutic drug reaches the highest, thereby improving the targeting efficiency. If the nitrogen-phosphorus ratio is too low, the encapsulation rate of the mRNA of the biotherapeutic drug will decrease; if the nitrogen-phosphorus ratio is too high, it will cause LNP vacuolization and the drug loading amount of the biotherapeutic drug will decrease.

[0026] Preferably, the mass ratio of the sum of the mass of the liposome nanoparticles and the biotherapeutic drug to the mass of the lectin is 0.8:1 to 1.2:1.

[0027] In the present invention, by controlling the dosage of the lectin, the specific binding ability with fucose on nasal mucosal cells can be further improved, and the adhesion time and adsorption ability of the nasal-to-brain targeted central nervous system nano-drug delivery system on the nasal mucosa can be further improved, thereby further improving the delivery efficiency of the nano-drug delivery system.

[0028] Preferably, the particle size of the nasal-to-brain targeted central nervous system nano-drug delivery system is 70 nm to 200 nm.

[0029] In a second aspect, the present invention provides a preparation method of a nasal-to-brain targeted central nervous system nano-drug delivery system, comprising the following steps:

[0030] S1, assembly of liposome nanoparticles and biotherapeutic drug:

[0031] S1-1, Weigh each component according to the raw material ratio, dissolve SM102, DSPC, cholesterol, DMG-pSar25 and phospholipid polyethylene glycol maleimide in a first solvent to obtain a first mixed system;

[0032] Dissolve the biopolymer encoded by mRNA in a second solvent to obtain a second mixed system;

[0033] S1-2, Add the first mixed system and the second mixed system to a microfluidic mixer, remove the first solvent, and then dissolve it in a third solvent to obtain an mRNA@LNP solution;

[0034] S2, prepare thiolated Aleuria aurantia lectin:

[0035] Mix the Aleuria aurantia lectin solution with the S-acetylmercaptoacetic acid N-hydroxysuccinimide ester solution for an amide reaction; mix the obtained reaction solution with the hydroxylamine hydrochloride solution for a deacetamide reaction to obtain a thiolated Aleuria aurantia lectin solution;

[0036] S3. Conjugation of mercapto - Aleuria aurantia lectin with mRNA@LNP:

[0037] Mix the mRNA@LNP solution and the mercapto - Aleuria aurantia lectin solution to carry out a conjugation reaction, obtaining a nasal - to - brain - targeted central nervous system nano - drug delivery system.

[0038] It should be noted that there is no sequential order between S1 and S2. The present invention does not have special requirements for the preparation of the biological macromolecule encoded by mRNA, and conventional methods in the art can be used.

[0039] The preparation method of the nasal - to - brain - targeted central nervous system nano - drug delivery system provided by the present invention uses microfluidic technology to assemble the biological macromolecule encoded by mRNA with liposomes to obtain an mRNA@LNP solution in which LNP encapsulates a biotherapeutic drug; uses DSPE - PEG - MAL to modify LNP, introducing a maleimide group into the original 4 lipids of LNP. At the same time, use S - acetylmercaptoacetic acid N - hydroxysuccinimide ester (SATA) to modify AAL to introduce a mercapto group (forming AAL - SH), and utilize the thiol - maleimide reaction to form a thioether bond to couple LNP with AAL, adding AAL to mRNA@LNP to obtain a nasal - to - brain - targeted central nervous system nano - drug delivery system conjugated with AAL.

[0040] Preferably, in S1 - 1, the first solvent is ethanol.

[0041] The present invention does not limit the dosage of ethanol, as long as it can completely dissolve the above - mentioned lipids.

[0042] Exemplarily, in S1 - 1, first dissolve SM102, DSPC, cholesterol, DMG - pSar25, and DSPE - PEG - MAL in the first solvent respectively, and then mix the 5 mixed solutions to obtain a first mixed system.

[0043] Preferably, in S1 - 1, the second solvent is a sodium acetate solution with a pH of 3.5 - 4.5 and a concentration of 45 mM - 55 mM.

[0044] Preferably, in S1 - 1, the volume ratio of the biological macromolecule encoded by mRNA to the second solvent is 0.9:1 - 1.1:1.

[0045] Preferably, in S1 - 2, the volume ratio of the first mixed system to the second mixed system is 1:2.5 - 1:3.5.

[0046] Exemplarily, in S1 - 2, the microfluidic mixer is NanoAssemblr ®Ignite+™ (PrecisionNanosystems) mixer.

[0047] The present invention does not limit the method for removing the first solvent in S1-2, and conventional means in the art can be adopted.

[0048] Preferably, in S1-2, the third solvent is a mixed solution of 12 mM to 18 mM Tris-HCl and 12 mM to 18 mM sodium acetate.

[0049] Exemplarily, S1-2 includes the following steps:

[0050] Add the first mixing system and the second mixing system to a microfluidic mixer, set the program of the microfluidic mixer (FRR = 3, TFR = 12 mL / min), insert an NxGEN microfluidic chip, tighten the syringe, perform LNP microfluidic preparation to obtain mRNA@LNP;

[0051] Dilute the obtained mRNA@LNP with a third solvent having a volume 25 times that of mRNA@LNP, then perform ultrafiltration for buffer exchange, concentrate by buffer exchange to the target volume, and then perform 0.2 μm sterile filtration to obtain an mRNA@LNP solution.

[0052] Preferably, in S1-2, the concentration of the mRNA@LNP solution is 450 ng / μL to 500 ng / μL.

[0053] The present invention uses microfluidic technology to assemble a biomacromolecule encoded by mRNA with liposomes. Through specific first and second solvents, a specific amount of liposomes and mRNA are assembled and finally dissolved in a specific third solvent to obtain an mRNA@LNP solution. By controlling various conditions in the microfluidic technology, the present invention improves the encapsulation efficiency of mRNA of a biotherapeutic drug, and further improves the delivery efficiency of the nanodelivery system.

[0054] Preferably, in S2, the solvent of the Aleuria aurantia lectin solution is a fourth solvent, and the fourth solvent is a PBS buffer solution with a pH of 7.2 to 7.6.

[0055] Exemplarily, in S2, the concentration of Na2HPO4 in the fourth solvent is 8 mM to 12 mM, the concentration of NaH2PO4 is 1.5 mM to 2 mM, the concentration of potassium chloride is 2.5 mM to 3 mM, and the concentration of sodium chloride is 0.12 M to 0.15 M.

[0056] Preferably, in S2, the concentration of the Aleuria aurantia lectin solution is 1.5 mg / mL to 2 mg / mL.

[0057] Preferably, in S2, the solvent of the S-acetylmercaptoacetic acid N-hydroxysuccinimide ester solution is the fifth solvent, and the fifth solvent is dimethyl sulfoxide.

[0058] Preferably, in S2, the concentration of the S-acetylmercaptoacetic acid N-hydroxysuccinimide ester solution is 15 mM to 20 mM.

[0059] Preferably, in S2, the molar ratio of the Aleuria aurantia lectin in the Aleuria aurantia lectin solution to the S-acetylmercaptoacetic acid N-hydroxysuccinimide ester in the S-acetylmercaptoacetic acid N-hydroxysuccinimide ester solution is 1:8 to 1:10.

[0060] By controlling the above molar ratio, the reaction rate of the amidation reaction can be further increased, making the reaction proceed more thoroughly.

[0061] Preferably, in S2, the temperature of the amidation reaction is 10°C to 35°C, and the time is 25 min to 35 min.

[0062] Preferably, in S2, the pH of the hydroxylamine hydrochloride solution is 7 to 7.5.

[0063] Preferably, in S2, the solvent of the hydroxylamine hydrochloride solution is the sixth solvent, and the sixth solvent is a PBS buffer solution containing 23 mM to 27 mM ethylenediaminetetraacetic acid.

[0064] Preferably, in S2, the volume molar ratio of the hydroxylamine hydrochloride in the reaction solution to the hydroxylamine hydrochloride in the hydroxylamine hydrochloride solution is 200 μL:0.45 mol to 200 μL:0.55 mol.

[0065] Preferably, in S2, the volume ratio of the reaction solution to the hydroxylamine hydrochloride solution is 9:1 to 11:1.

[0066] By the above dosage limitation, the reaction rate of the deacetamidation reaction can be further increased, making the reaction proceed more thoroughly.

[0067] Preferably, in S2, the temperature of the deacetamidation reaction is 10°C to 35°C, and the time is 25 min to 35 min.

[0068] Preferably, S2 includes the following preparation steps:

[0069] S2-1, dissolve AAL in the fourth solvent to obtain an AAL solution;

[0070] Dissolve SATA in the fifth solvent to obtain a SATA solution;

[0071] Dissolve hydroxylamine hydrochloride in the sixth solvent, and adjust the pH to 7 to 7.5 with NaOH to obtain a hydroxylamine hydrochloride solution;

[0072] S2-2. Mix the AAL solution with the SATA solution and carry out an amide reaction to obtain an AAL-SATA solution.

[0073] S2-3. Mix the AAL-SATA solution with the hydroxylamine hydrochloride solution and carry out a deacetamide reaction to obtain a mercapto-aurantia arcta lectin solution.

[0074] Preferably, in S2-2, after the amide reaction is completed, ultrafiltration is also required. Take the filtrate, change the solution 3 to 4 times, centrifuge, change the solution and make up the volume to obtain an AAL-SATA solution.

[0075] Exemplarily, in S2-2, a 10K ultrafiltration tube is used for ultrafiltration.

[0076] Further preferably, in S2-2, the solvent used for changing the solution is a PBS buffer solution with a pH of 7.2 to 7.5.

[0077] Exemplarily, in S2-2, the centrifugation temperature is 5°C to 10°C, the centrifugation time is 5 min to 10 min, and the centrifugation speed is 900 rpm to 1000 rpm.

[0078] Further preferably, in S2-2, the concentration of the AAL-SATA solution is 35 μM to 45 μM.

[0079] After changing the solution with a specific solvent in the present invention, it is beneficial to the subsequent deacetamide reaction.

[0080] Preferably, in S2-3, after the deacetamide reaction is completed, ultrafiltration is also required. Take the filtrate, change the solution 3 to 4 times, centrifuge, change the solution and make up the volume to obtain an AAL-SH solution.

[0081] Exemplarily, in S2-3, a 10K ultrafiltration tube is used for ultrafiltration.

[0082] Further preferably, in S2-3, the solvent used for changing the solution is a PBS buffer solution containing 8 mM to 12 mM ethylenediaminetetraacetic acid.

[0083] Exemplarily, in S2-3, the centrifugation temperature is 5°C to 10°C, the centrifugation time is 5 min to 10 min, and the centrifugation speed is 900 rpm to 1000 rpm.

[0084] Further preferably, in S2-3, the concentration of the AAL-SH solution is 25 μM to 30 μM.

[0085] After changing the solution with a specific solvent in the present invention, it is beneficial to the subsequent coupling reaction.

[0086] The present invention selects specific fourth, fifth, and sixth solvents and their contents, which can improve the smooth progress of the amide reaction and the deacetamide reaction, and further increase the reaction rate. In the amide reaction, the succinimide group on SATA is removed, and AAL is labeled with SATA in the form of an amide bond; in the deacetamide reaction, hydroxylamine hydrochloride is used to remove the acetamide, exposing the thiol group for subsequent thiol-maleimide reaction to form a thioether bond.

[0087] Preferably, in S3, the mass ratio of mRNA@LNP in the mRNA@LNP solution to Aleuria aurantia lectin in the Aleuria aurantia lectin solution is 0.8:1 to 1.2:1.

[0088] Preferably, in S3, the temperature of the coupling reaction is 10°C to 35°C, and the time is 1.8 h to 2.3 h.

[0089] Preferably, after the coupling reaction in S3, it is also necessary to stay overnight at 2°C to 5°C, followed by purification to obtain the intranasal-to-brain targeted central nervous system nano drug delivery system.

[0090] The present invention allows the coupling reaction to proceed more thoroughly by staying overnight at 2°C to 5°C; the present invention has no special requirements for the purification step, and conventional methods in the art can be used.

[0091] In the present invention, the olefin structure in the maleimide group has high reactivity, which enables the reaction between the thiol group and maleimide to proceed without a catalyst in a highly polar solvent environment such as water and dimethyl sulfoxide (DMSO) (chemical reactions preferably occur at neutral pH), and the reaction rate between maleimide and thiol is relatively fast.

[0092] In a third aspect, the present invention provides an application of the above intranasal-to-brain targeted central nervous system nano drug delivery system or an intranasal-to-brain targeted central nervous system nano drug delivery system prepared by the preparation method of the above intranasal-to-brain targeted central nervous system nano drug delivery system in a drug for treating or preventing central nervous system degenerative diseases.

[0093] Preferably, the administration method of the drug is intranasal administration.

[0094] Preferably, the central nervous system degenerative diseases include Alzheimer's disease, Parkinson's disease, multiple sclerosis, Huntington's disease, or amyotrophic lateral sclerosis.

[0095] The nasal-to-brain targeted central nerve nano-drug delivery system provided by the present invention can be widely applied to the field of drugs for treating or preventing central nervous system degenerative diseases. Any biotherapeutic agent that can target the cerebral cortex is applicable to the present invention, providing a new idea for the preparation of novel nasal-to-brain targeted drugs and having broad application prospects in improving the delivery efficiency of the nano-drug delivery system and other aspects. Description of the Drawings

[0096] Figure 1 It is the particle size light intensity distribution diagram of the nasal-to-brain targeted central nerve nano-drug delivery system in Example 1 of the present invention;

[0097] Figure 2 It is the Western blot of the protein expressed by the in vitro transcribed mRNA of mature BDNF modified by m1Pis in Caco2 cells in Application Example 1 of the present invention; among them, a represents the PBS blank control, b represents BDNF mRNA, c represents LNP, d represents EGFP mRNA@LNP, e represents the BDNF protein positive control, and f~k respectively represent the translational expression of BDNF mRNA@LNP in Caco2 cells;

[0098] Figure 3 It is the expression of Fluc mRNA@AAL / LNP and Fluc mRNA@LNP labeled with Dio in Caco2 cells in Application Example 2 of the present invention;

[0099] Figure 4 It is the bioluminescence image of wild-type ICR mice in Application Example 3 of the present invention; from left to right, the drugs are PBS buffer, Fluc mRNA@LNP, Fluc mRNA@LNP, Fluc mRNA@AAL / LNP, and Fluc mRNA@AAL / LNP;

[0100] Figure 5 It is the schematic diagram of the preparation process of the Ai9 / Cre mRNA mouse model in Application Example 4 of the present invention;

[0101] Figure 6 It is the fluorescence image of Cre mRNA@LNP in region E (hippocampus of the brain, hypothalamic region) in Application Example 4 of the present invention;

[0102] Figure 7 It is the fluorescence image of Cre mRNA@AAL / LNP in region E (hippocampus of the brain, hypothalamic region) in Application Example 4 of the present invention. Detailed Embodiments

[0103] To make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0104] For those not specified with specific conditions in the present invention, they are carried out according to conventional conditions or conditions recommended by the manufacturer; for reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0105] The in vitro transcription, capping and methylation of the mRNA of the biotherapeutic drug include the following steps:

[0106] Sa, Insert the coding sequence of the biotherapeutic drug into the pmRVac vector using the Gibson assembly method to generate a DNA template for in vitro transcription; the pmRVac vector contains a T7 promoter sequence, a 5’UTR, a 3’UTR, an 110nt segmented polyA and a SapI restriction enzyme site for transcription termination.

[0107] Sb, Cap1 mRNA is generated by the T7 in vitro transcription method, followed by enzymatic capping and methylation.

[0108] To produce uncapped in vitro RNA transcripts, mix the linear DNA template with T7 RNA polymerase, nucleotide triphosphates (NTPs) and a magnesium-containing buffer to establish an in vitro transcription reaction; the reaction mixture is Cap1 mRNA generated using the vaccinia capping system.

[0109] After denaturing the uncapped transcript by heating, add vaccinia capping enzyme, 2'-O methyltransferase, GTP, S-adenosylmethionine (SAM) and capping buffer to establish a capping reaction. Incubate the reaction mixture at 37°C for 1 h; then remove the template DNA by DNase I treatment and incubate at 37°C for 2 h.

[0110] For the synthesis of m1Psi-modified transcripts, use m1Psi instead of UTP for in vitro transcription.

[0111] Sc, Further purify the capped transcript using magnetic beads, and the isolated mRNA is eluted with acidic buffer and stored at -80°C. Measure the mRNA concentration and purity on a UV-visible spectrophotometer; measure the integrity of the mRNA by denaturing agarose gel analysis; identify the mRNA molecules using mRNA sequencing.

[0112] In the embodiments of the present invention, the mice used were healthy male Ai9 mice, 4 - 6 weeks old, with a body weight of about 15 g - 20 g, purchased from Guangzhou Bojin Biotechnology Co., Ltd., and the production unit license number is SYXK (Guangdong) 2023 - 0343. Wild - type ICR mice, 4 - 6 weeks old, with a body weight of about 20 - 30 g, were purchased from Guangzhou Bojin Biotechnology Co., Ltd., and the production unit license number is SYXK (Guangdong) 2023 - 0343. The animal experiments of the present invention have been reviewed and approved by the Animal Welfare Committee of Yunzhou Biotechnology, and the ethical review approval number is BG - AMS - 20240801LN01 - Y.

[0113] To better illustrate the present invention, further examples are given below through embodiments.

[0114] Example 1

[0115] This example provides a nasal - to - brain targeted central nervous system nano - drug delivery system, and its preparation method includes the following steps:

[0116] S1, Assembly of liposome nanoparticles and biotherapeutic drugs:

[0117] S1 - 1, First, dissolve SM102, DSPC, cholesterol, DMG - pSar25, and DSPE - PEG - MAL in absolute ethanol respectively, and then mix the 5 mixed solutions evenly to obtain a first mixed system. The molar ratio of SM102, DSPC, cholesterol, DMG - pSar25, and DSPE - PEG - MAL is 50:10:38:1.5:0.5.

[0118] Dissolve the mRNA - encoded biopolymer (BDNF mRNA) in a sodium acetate solution with pH = 4 and a concentration of 50 mM, and mix evenly to obtain a second mixed system. The volume ratio of BDNF mRNA to the sodium acetate solution is 1:1.

[0119] S1 - 2, Add the first mixed system and the second mixed system to a microfluidic mixer. The molar ratio of nitrogen in the ionizable cationic lipid of the first mixed system to phosphorus in the mRNA of the second mixed system is 6:1; remove ethanol, and then dissolve it in a solution containing 15 mM Tris - HCl and 15 mM sodium acetate, and mix evenly to obtain a BDNFmRNA@LNP solution with a concentration of 481 ng / μL.

[0120] S2, Preparation of thiolated AAL:

[0121] S2 - 1, Dissolve 288 μg of AAL in a PBS buffer solution with pH = 7.4, and mix evenly to obtain an AAL protein solution with a concentration of 1.8 mg / mL.

[0122] Dissolve SATA in DMSO and mix well to obtain a SATA solution with a concentration of 18 mM.

[0123] Dissolve 0.5 mol of hydroxylamine hydrochloride in PBS buffer containing 25 mM EDTA, and adjust the pH to 7.3 with NaOH to obtain 20 μL of hydroxylamine hydrochloride solution.

[0124] S2-2. Mix the AAL protein solution and the SATA solution. The molar ratio of SATA in the SATA solution to AAL in the AAL protein solution is 9:1. Carry out the amide reaction at room temperature for 30 min. After the reaction, perform ultrafiltration. Take the filtrate and exchange the solution 3 - 4 times with PBS buffer at pH = 7.3, centrifuge, and make up the volume to 200 μL with the above PBS buffer to obtain the AAL-SATA solution.

[0125] S2-3. Mix the hydroxylamine hydrochloride solution and the AAL-SATA solution evenly, and carry out the deacetylation reaction at room temperature for 30 min. After the reaction, perform ultrafiltration. Take the filtrate and exchange the solution 3 - 4 times with PBS buffer containing 10 mM EDTA, centrifuge, and make up the volume to 300 μL with the above PBS buffer to obtain the AAL-SH solution.

[0126] S3. Coupling of AAL-SH with BDNF mRNA@LNP:

[0127] Mix the BDNF mRNA@LNP solution and the AAL-SH solution. The mass ratio of BDNF mRNA@LNP in the BDNF mRNA@LNP solution to AAL used in the AAL-SH solution is 1:1. Carry out the coupling reaction at room temperature for 2 h, then overnight at 4 °C, and purify to obtain a nasal-to-brain targeted central nervous system nano-drug delivery system, denoted as BDNF mRNA@AAL / LNP.

[0128] Perform particle size analysis and testing on BDNF mRNA@AAL / LNP, and observe the morphological characteristics. The results are as Figure 1 shown. It can be seen from Figure 1 that the median particle size of BDNF mRNA@AAL / LNP in this example is 87.47 nm.

[0129] Example 2

[0130] This example provides a nasal-to-brain targeted central nervous system nano-drug delivery system, and its preparation method includes the following steps:

[0131] S1. Assembly of liposome nanoparticles and biotherapeutic drugs:

[0132] S1-1. First, dissolve SM102, DSPC, cholesterol, DMG-pSar25, and DSPE-PEG-MAL in absolute ethanol respectively, and then mix the five mixed solutions evenly to obtain a first mixed system. The molar ratio of SM102, DSPC, cholesterol, DMG-pSar25, and DSPE-PEG-MAL is 48:12:38.3:1:0.7.

[0133] Dissolve the mRNA-encoded biopolymer (Klotho mRNA) in a sodium acetate solution with pH = 3.5 and a concentration of 55 mM, and mix evenly to obtain a second mixed system. The volume ratio of Klotho mRNA to the sodium acetate solution is 0.95:1.

[0134] S1-2. Add the first mixed system and the second mixed system to a microfluidic mixer. The molar ratio of nitrogen in the ionizable cationic lipid of the first mixed system to phosphorus in the mRNA of the second mixed system is 5.2:1; remove ethanol, and then dissolve it in a solution containing 12 mM Tris-HCl and 12 mM sodium acetate, and mix evenly to obtain a Klotho mRNA@LNP solution with a concentration of 500 ng / μL.

[0135] S2. Prepare thiolated AAL:

[0136] S2-1. Dissolve 290 μg of AAL in a PBS buffer with pH = 7.2, and mix evenly to obtain an AAL protein solution with a concentration of 2 mg / mL.

[0137] Dissolve SATA in DMSO, and mix evenly to obtain a SATA solution with a concentration of 20 mM.

[0138] Dissolve 0.55 mol of hydroxylamine hydrochloride in a PBS buffer containing 25 mM EDTA, and adjust the pH to 7.3 with NaOH to obtain 21 μL of hydroxylamine hydrochloride solution.

[0139] S2-2. Mix the AAL protein solution and the SATA solution. The molar ratio of SATA in the SATA solution to AAL in the AAL protein solution is 8:1, and carry out an amide reaction at room temperature for 25 min; after the reaction is completed, perform ultrafiltration, take the filtrate, and exchange the solution 3 - 4 times with a PBS buffer with pH = 7.2, centrifuge, and volume-fix it to 200 μL with the above PBS buffer to obtain an AAL-SATA solution.

[0140] S2-3. Mix the hydroxylamine hydrochloride solution and the AAL-SATA solution evenly, and carry out a deacetamide reaction at room temperature for 25 min; after the reaction is completed, perform ultrafiltration, take the filtrate, and exchange the solution 3 - 4 times with a PBS buffer containing 8 mM EDTA, centrifuge, and volume-fix it to 300 μL with the above PBS buffer to obtain an AAL-SH solution.

[0141] S3, Coupling of AAL-SH and Klotho mRNA@LNP:

[0142] Mix the Klotho mRNA@LNP solution and the AAL-SH solution. The mass ratio of Klotho mRNA@LNP in the Klotho mRNA@LNP solution to AAL used in the AAL-SH solution is 0.8:1. Conduct the coupling reaction at room temperature for 1.8 h, then incubate overnight at 2 °C, and purify to obtain a nano-drug delivery system targeting the central nervous system via nasal-to-brain route, denoted as Klotho mRNA@AAL / LNP.

[0143] Example 3

[0144] This example provides a nano-drug delivery system targeting the central nervous system via nasal-to-brain route, and its preparation method includes the following steps:

[0145] S1, Assembly of liposome nanoparticles and biotherapeutic drugs:

[0146] S1-1, First, dissolve SM102, DSPC, cholesterol, DMG-pSar25, and DSPE-PEG-MAL in absolute ethanol respectively, and then mix the 5 mixed solutions evenly to obtain a first mixed system. The molar ratio of SM102, DSPC, cholesterol, DMG-pSar25, and DSPE-PEG-MAL is 45:14:39:1.7:0.3.

[0147] Dissolve the mRNA-encoded biopolymer (BDNF mRNA) in a sodium acetate solution with pH = 4.5 and a concentration of 45 mM, and mix evenly to obtain a second mixed system. The volume ratio of BDNF mRNA to the sodium acetate solution is 1.1:1.

[0148] S1-2, Add the first mixed system and the second mixed system to a microfluidic mixer. The molar ratio of nitrogen in the ionizable cationic lipid in the first mixed system to phosphorus in the mRNA in the second mixed system is 6.8:1; remove ethanol, and then dissolve it in a solution containing 18 mM Tris-HCl and 18 mM sodium acetate, and mix evenly to obtain a BDNF mRNA@LNP solution with a concentration of 450 ng / μL.

[0149] S2, Preparation of thiolated AAL:

[0150] S2-1, Dissolve 288 μg of AAL in a PBS buffer solution with pH = 7.6, and mix evenly to obtain an AAL protein solution with a concentration of 1.5 mg / mL.

[0151] Dissolve SATA in DMSO and mix evenly to obtain a SATA solution with a concentration of 15 mM.

[0152] Dissolve 0.45 mol of hydroxylamine hydrochloride in PBS buffer containing 25 mM EDTA, and adjust the pH to 7.3 with NaOH to obtain 19 μL of hydroxylamine hydrochloride solution.

[0153] S2-2. Mix the AAL protein solution and the SATA solution. The molar ratio of SATA in the SATA solution to AAL in the AAL protein solution is 10:1. Carry out the amide reaction at room temperature for 35 min. After the reaction, perform ultrafiltration. Take the filtrate and exchange the solution 3 - 4 times with PBS buffer at pH = 7.5, centrifuge, and make up the volume to 200 μL with the above PBS buffer to obtain the AAL-SATA solution.

[0154] S2-3. Mix the hydroxylamine hydrochloride solution and the AAL-SATA solution evenly, and carry out the deacetamide reaction at room temperature for 35 min. After the reaction, perform ultrafiltration. Take the filtrate and exchange the solution 3 - 4 times with PBS buffer containing 12 mM EDTA, centrifuge, and make up the volume to 300 μL with the above PBS buffer to obtain the AAL-SH solution.

[0155] S3. Coupling of AAL-SH with BDNF mRNA@LNP:

[0156] Mix the BDNF mRNA@LNP solution and the AAL-SH solution. The mass ratio of BDNF mRNA@LNP in the BDNF mRNA@LNP solution to AAL used in the AAL-SH solution is 1.2:1. Carry out the coupling reaction at room temperature for 2.3 h, then overnight at 5 °C, and purify to obtain a nano-drug delivery system for targeting the central nervous system through nasal administration to the brain, denoted as BDNF mRNA@AAL / LNP.

[0157] Comparative Example 1

[0158] This comparative example provides a drug-loaded liposome nanoparticle (BDNF mRNA@LNP), whose preparation method is similar to that of Example 1, except that: DMG-pSar25 is replaced with an equal molar amount of DMG-PEG2000. The remaining steps are the same as those in S1 of Example 1 and will not be elaborated here.

[0159] The structural formula of DMG-PEG2000 is shown in Formula 5.

[0160]

[0161] Formula 5

[0162] Comparative Example 2

[0163] This comparative example provides a drug-loaded liposomal nanoparticle (BDNF mRNA@LNP), and its preparation method is similar to that of Example 1, except that: DMG-pSar25 is replaced with an equimolar amount of ALC-0159 PEG. The remaining steps are the same as S1 of Example 1 and will not be elaborated here.

[0164] The structural formula of ALC-0159 PEG is shown in Formula 6.

[0165]

[0166] Formula 6

[0167] Comparative Example 3

[0168] This comparative example provides a drug-loaded liposomal nanoparticle (BDNF mRNA@LNP), and its preparation method is similar to that of Example 1, except that: SM102 is replaced with an equimolar amount of ALC-0315. The remaining steps are the same as S1 of Example 1 and will not be elaborated here.

[0169] The structural formula of ALC-0315 is shown in Formula 7.

[0170]

[0171] Formula 7

[0172] The present invention uses a dynamic light scattering particle size analyzer (Zetasizer Ultra) to perform physicochemical analysis on the BDNF mRNA@LNP provided in Examples 1 to 3 and Comparative Examples 1 to 3, and the results are shown in Table 1. PDI represents the polymer dispersity index.

[0173] Table 1 Physicochemical data of BDNF mRNA@LNP in Example 1 and Comparative Examples 1 to 3

[0174]

[0175] The main function of PEG lipids is to stabilize and prolong the administration time of liposomal nanoparticles by reducing protein-binding interactions. However, the inventors found through a large number of experiments that repeated infusion of liposomal nanoparticles would generate anti-PEG antibodies, resulting in the interaction between liposomal nanoparticles and anti-PEG antibodies, accelerating blood clearance, and thus reducing the delivery efficiency of liposomal nanoparticles; at the same time, anti-PEG antibodies can also cause severe allergic reactions. As can be seen from Table 1, DMG-PSar25 can completely replace DMG-PEG2000, and the use effect is better than that of liposomal nanoparticles composed of DMG-PEG2000; the comprehensive effect of liposomal nanoparticles formulated with SM102, DSPC, cholesterol, and DMG-pSar25 is the best.

[0176] Application Example 1: Expression of BDNF mRNA on Caco2 mouse nasal mucosal cells

[0177] To verify that mRNA@LNP can be translated into protein, BDNF mRNA@LNP was transfected into Caco2 cells in this invention. One day before transfection, Caco2 cells were seeded in 12-well plates at a seeding density of 2.2×10 5 cells / well. On the day of transfection, BDNF mRNA@LNP was added to the wells, and water was added to set up negative control wells. After 24 h of transfection, the cells were lysed in RIPA lysis buffer. The cell lysates were separated on 4% - 20% SDS-polyacrylamide gels and analyzed with rabbit recombinant anti-BDNF monoclonal antibody. The results are as Figure 2 shown. The results showed that Figure 2 a 13.5 kd band was shown, indicating the expression of BDNF, and there was a good correlation between the transfection amount of BDNF mRNA@LNP and the BDNF protein expression level. Lane e was the BDNF protein positive control, and lanes f - k were the translational expressions of BDNF mRNA@LNP in Caco2 cells, which were carried out in a dose-dependent manner, being 0.5 μg, 1 μg, 2 μg, 0.5 μg, 1 μg, and 2 μg of BDNF mRNA@LNP in sequence; while BDNF protein was not detected in the cell lysates of BDNF mRNA (lane b), LNP (lane c), and EGFP mRNA@LNP (lane d).

[0178] Application Example 2: In vitro cellular uptake of a nano-drug delivery system targeting the central nervous system via nasal-to-brain route by Caco2 cells

[0179] Caco2 cells were used to simulate the in vitro transport of olfactory epithelial cells to evaluate the uptake of a nano-drug delivery system targeting the central nervous system via nasal-to-brain route. Dio is a cell membrane green fluorescent probe used to label LNP to track the uptake of LNP by Caco2 cells. First, Dio was dissolved in DMSO to make a stock solution of 1 mM - 5 mM; then it was dissolved and diluted with PBS buffer to make a working solution of 1 μM - 5 μM; the concentration of Dio was 1%. After Caco2 cells were treated with DiO-labeled LNP for 2 h, fluorescence imaging was performed, and the internalization of DiO-labeled LNP by Caco2 cells was qualitatively observed through fluorescence microscopy imaging. The results are as Figure 3 shown. The results showed that the fluorescence intensity of Fluc mRNA@AAL / LNP was significantly higher than that of Fluc mRNA@LNP.

[0180] The physicochemical analysis of Dio-labeled Fluc mRNA@AAL / LNP and Fluc mRNA@LNP was performed using a dynamic light scattering particle size analyzer (Zetasizer Ultra), and the results are shown in Table 2.

[0181] It should be noted that the preparation method of Fluc mRNA@AAL / LNP (and Fluc mRNA@LNP) is similar to that of Example 1, except that in S1-1, the biomacromolecule encoded by mRNA was replaced with luciferase (FlucmRNA) to facilitate the subsequent observation of fluorescence intensity and the analysis of the expression level of mRNA@LNP on cells. The remaining conditions are the same as those in Example 1 and will not be elaborated here.

[0182] Table 2 Physicochemical data of Dio-labeled Fluc mRNA@AAL / LNP and Fluc mRNA@LNP

[0183]

[0184] Application Example 3 Monitoring the expression of Fluc mRNA@AAL / LNP in mice by intranasal administration

[0185] Fluc mRNA@AAL / LNP was centrifuged to remove unbound Fluc mRNA@LNP, and then 10 μg of the drug was intranasally administered into the nasal cavity of wild-type ICR mice using a nanodrug delivery system targeting the central nervous system through the nose to the brain (administering PBS buffer, Fluc mRNA@LNP, and Fluc mRNA@AAL / LNP respectively).

[0186] Before the experiment, the mice were anesthetized with isoflurane. After the respiration changed from shallow to deep, the next step was started. Before dropping the liquid, hold the mouse's head up with the thumb fingertip on the mouse's mandible. Keep the thumb away from the mouse's throat to avoid the swallowing reflex. After the mouse maintained a stable respiratory rhythm (and at the end of exhalation and the beginning of inhalation), drop the liquid on one nostril drop by drop. After dropping the liquid, moist rales in the mouse's lungs could be heard. Massage the mouse to promote the redistribution of the liquid, keep it lying on its back for 10 min, and finally put it back into the cage. After 6 h, D-luciferin was used as a substrate to monitor and visualize the BLI signal, and the results are as Figure 4 shown. The results showed that compared with Fluc mRNA@LNP, the BLI signal of Fluc mRNA@AAL / LNP remained in the nostrils for a longer time (more than 24 h).

[0187] Application Example 4 Cross-sectional slices of mouse brain tissue

[0188] Ai9 mice can stably express a fixed stop codon upstream of the tdTomato cassette, and tdTomato is produced only after the stop cassette is removed by Cre recombinase (cyclization recombinase). To evaluate the delivery efficiency of a nasal-to-brain targeted central nervous system nanomedicine delivery system, see Figure 5 , an Ai9 / Cre mRNA mouse model was prepared. If the nasally instilled Cre mRNA@AAL / LNP can be delivered into the brain and express Cre in the brain, it can excise LoxP, thereby expressing red fluorescence.

[0189] It should be noted that the preparation method of Cre mRNA@AAL / LNP (and Cre mRNA@LNP) is similar to that of Example 2, except that in S1-1, the biomacromolecule encoded by mRNA was replaced with Cre mRNA to facilitate the subsequent observation of red fluorescence. The remaining conditions are the same as those in Example 2 and will not be elaborated here.

[0190] After anesthetizing the Ai9 mice with drugs, nasal instillation or spraying was administered. The instillation volume of Cre mRNA@AAL / LNP was 10 μg (or 0.5 mg / kg); 3 days (48 h) after administration, cross-sectional cryosections of the mouse brain tissue were taken. The position of the brain tissue was Region E, which characterizes the hippocampus and hypothalamus regions of the brain. The expression of tdTomato-labeled fluorescence was observed under a microscope, and the results are as Figures 6 - 7 shown. Fluorescence imaging showed that compared with the control groups of PBS buffer and Cre mRNA@LNP, the tdTomato fluorescence signals in all Cre mRNA@AAL / LNP groups were more robust and selective. The results confirmed that Cre recombinase mRNA was specifically expressed in the brain cells of mice after nasal administration to the brain, and strong expression of Tomato reporter fluorescence appeared in the brain, verifying that the nasal-to-brain pathway was opened.

[0191] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, or improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A nano drug delivery system targeting the central nervous system through the nose and brain, characterized in that: It includes a biotherapeutic drug and a lectin-coupled liposome nanoparticle, wherein the lectin-coupled liposome nanoparticle is coated on the surface of the biotherapeutic drug; The biotherapeutic drug is a biomacromolecule encoded by mRNA; the lectin in the lectin-coupled liposome nanoparticles is a Dictyostelium aurantium lectin, and the raw materials of the liposome nanoparticles include SM102, DSPC, cholesterol, DMG-pSar25 and phospholipid polyethylene glycol maleimide; The phospholipid polyethylene glycol maleimide is DSPE-PEG-MAL, and the amount of DSPE-PEG-MAL is 0.3% to 0.7% of the molar amount of the liposome nanoparticles; The molar ratio of the nitrogen element in the ionizable cationic lipid of the liposome nanoparticle to the phosphorus element in the mRNA of the biotherapeutic drug is 4:1 to 8:1; The mass ratio of the sum of the masses of the liposome nanoparticles and the biotherapeutic drug to the mass of the lectin is 0.8:1 to 1.2:

1.

2. The nano drug delivery system targeting the central nervous system through the nose and into the brain as claimed in claim 1, characterized in that: The molar ratio of the SM102, the DSPC, the cholesterol, the DMG-pSar25 and the phospholipid polyethylene glycol maleimide is (45-50):(10-15):(38-40):(1-2):(0.3-0.7).

3. The nano drug delivery system targeting the central nervous system through the nose and into the brain as claimed in claim 1, characterized in that: The particle size of the nano drug delivery system for targeting the central nervous system through the nose and into the brain is 70nm to 200nm.

4. The method for preparing the nano drug delivery system targeting the central nervous system through the nose into the brain according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1, Assembly of liposomal nanoparticles with biotherapeutic drugs: S1-1, weighing each component according to the raw material ratio, dissolving SM102, DSPC, cholesterol, DMG-pSar25 and phospholipid polyethylene glycol maleimide in a first solvent to obtain a first mixed system; dissolving the biomacromolecule encoded by the mRNA in a second solvent to obtain a second mixed system; S1-2, adding the first mixed system and the second mixed system into a microfluidic mixer, removing the first solvent, and then dissolving in a third solvent to obtain an mRNA@LNP solution; S2, preparation of thiolated Dictyosporium aurantium agglutinin: The Dictyostelium aurantium agglutinin solution is mixed with the S-acetylthioacetic acid N-hydroxysuccinimide ester solution to carry out an amide reaction; the obtained reaction solution is mixed with the hydroxylamine hydrochloride solution to carry out a deacetamide reaction to obtain a thiolated Dictyostelium aurantium agglutinin solution; S3, coupling of thiolated Dictyosporium aurantium lectin with mRNA@LNP: The mRNA@LNP solution and the thiolated Dictyosporium aurantium lectin solution are mixed to carry out a coupling reaction, thereby obtaining a nano drug delivery system that targets the central nervous system through the nose and into the brain.

5. The method for preparing the nano drug delivery system targeting the central nervous system through the nose and brain as claimed in claim 4, characterized in that: In S1-1, the first solvent is ethanol; In S1-1, the second solvent is a sodium acetate solution with a pH of 3.5 to 4.5 and a concentration of 45 mM to 55 mM; In S1-2, the volume ratio of the first mixed system to the second mixed system is 1:2.5 to 1:3.5; In S1-2, the third solvent is a mixed solution of 12mM~18mM Tris-HCl and 12mM~18mM sodium acetate.

6. The method for preparing the nano drug delivery system targeting the central nervous system through the nose and into the brain as claimed in claim 4, characterized in that: In S2, the solvent of the Dictyostelium aurantium agglutinin solution is a PBS buffer solution with a pH of 7.2 to 7.5; In S2, the solvent of the S-acetylthioglycolic acid N-hydroxysuccinimide ester solution is dimethyl sulfoxide; In S2, the molar ratio of the Dictyostelium aurantium lectin in the Dictyostelium aurantium lectin solution to the S-acetylthioglycolic acid N-hydroxysuccinimide ester in the S-acetylthioglycolic acid N-hydroxysuccinimide ester solution is 1:8-1:

10.

7. The method for preparing the nano drug delivery system targeting the central nervous system through the nose and into the brain as claimed in claim 4, characterized in that: In S2, the solvent of the hydroxylamine hydrochloride solution is a PBS buffer containing 23mM~27mM ethylenediaminetetraacetic acid, and the pH of the hydroxylamine hydrochloride solution is 7~7.5; In S2, the volume molar ratio of hydroxylamine hydrochloride in the reaction solution and the hydroxylamine hydrochloride solution is 200 μL:0.45 mol to 200 μL:0.55 mol; In S2, the volume ratio of the reaction solution to the hydroxylamine hydrochloride solution is 9:1 to 11:1; In S3, the mass ratio of mRNA@LNP in the mRNA@LNP solution to Dictyostelium aurantium lectin in the Dictyostelium aurantium lectin solution is 0.8:1-1.2:

1.

8. The method for preparing the nano drug delivery system targeting the central nervous system through the nose and into the brain as claimed in claim 4, characterized in that: In S2, the temperature of the amide reaction is 10°C to 35°C, and the time is 25min to 35min; In S2, the temperature of the deacetamidation reaction is 10°C to 35°C, and the time is 25min to 35min; In S3, the coupling reaction is carried out at a temperature of 10°C to 35°C and for a time of 1.8h to 2.3h.

9. Use of the nano drug delivery system for nasal administration to the brain targeting the central nervous system as claimed in any one of claims 1 to 3 or the nano drug delivery system for nasal administration to the brain targeting the central nervous system prepared by the preparation method of the nano drug delivery system for nasal administration to the brain targeting the central nervous system as claimed in any one of claims 4 to 8 in the preparation of drugs for treating or preventing degenerative diseases of the central nervous system.

10. Use of the nano drug delivery system targeting the central nervous system through the nose to the brain as claimed in claim 9 in the preparation of a drug for treating or preventing degenerative diseases of the central nervous system, characterized in that: The drug is administered intranasally.

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