Double-encapsulated lipid nanoparticles entering brain through nose as well as preparation method and application of double-encapsulated lipid nanoparticles

Abstract

The invention relates to double-encapsulated lipid nanoparticles entering brain through nose and a preparation method and application thereof, the double-encapsulated lipid nanoparticles comprise lipid nanoparticles, drugs in the lipid nanoparticles and rana odorata lectin coupled to the surfaces of the lipid nanoparticles, and the drugs comprise hydrophilic drugs and hydrophobic drugs. According to the invention, the rana odorata lectin and the lipid nanoparticles are coupled, and the rana odorata lectin can be specifically combined with L-fucose residues expressed by olfactory epithelial cells, so that the adhesion of the lipid nanoparticles to olfactory epithelium during nasal administration is enhanced, and the brain entering efficiency of the lipid nanoparticles is finally improved. After entering the brain, the lipid nanoparticles respond to diseased regions with high active oxygen content so as to release drugs. In addition, double-drug encapsulation can target different pathogenesis of central nervous system diseases, and the limitation that a traditional treatment drug is single in action target is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to a double-encapsulated lipid nanoparticle for delivery to the brain through the nose, and a preparation method and application thereof. Background Art

[0002] With the continued acceleration of socioeconomic development and the aging of the population, central nervous system (CNS) diseases are increasingly impacting people's quality of life. Common CNS diseases include Alzheimer's disease (AD), Parkinson's disease (PD), depression, brain tumors, cerebral hemorrhage, and stroke. Currently, CNS diseases affect over one-third of the world's population and are a leading cause of illness and disability. Therefore, actively developing new therapeutic approaches for CNS diseases is crucial. One of the current challenges in treating CNS diseases is that most drugs are unable to penetrate the blood-brain barrier (BBB), preventing them from reaching the brain and exerting their effects. Currently, two strategies have been proposed to increase drug concentrations in the brain. One is to encapsulate drugs in nanocarriers and perform targeted modifications to enhance their BBB penetration. The other is to employ therapies that directly bypass the BBB, primarily through nasal administration. Once administered into the nasal cavity, drugs cross the nasal epithelium and are transported via the olfactory nerve through the olfactory region to the olfactory bulb, or via the respiratory region along the trigeminal nerve to the pons. Through these two pathways, drugs can bypass the blood-brain barrier and enter the brain directly. Nasal drug delivery to the brain has proven to be a promising method for drug delivery. Despite significant progress in this field, nasal drug delivery still presents some disadvantages due to the physicochemical and biological properties of conventional therapeutic drugs. For example, large molecular weight and highly hydrophobic drugs cannot penetrate the nasal mucosa; various enzymes in the nasal mucosa can degrade poorly stable drugs; and the periodic beating of cilia in the nasal mucosa can accelerate drug clearance. The application of nano-drug delivery technology effectively addresses these problems. Nanotechnology can enhance drug transport to the brain without disrupting the structure of the brain or the physiological functions of the nasal epithelium. Currently, a variety of nano-carriers, such as dendrimers, liposomes, exosomes, and nano-gels, have been used for intranasal drug delivery to treat various CNS diseases.

[0003] To date, lipid nanoparticle (LNP) technology, as a key component of lipid carrier drug delivery systems, has become a major technological breakthrough in oligonucleotide-based therapeutics. The main components of LNPs include oligonucleotides (mRNA or siRNA), ionizable lipids, neutral auxiliary lipids, sterol lipids, and PEGylated lipids. LNPs have inherent advantages such as easy surface modification, good biocompatibility, and high-throughput production. In LNPs, hydrophilic oligonucleotide drugs are encapsulated in the hydrophilic core formed by ionizable lipids, while hydrophobic drugs are encapsulated in the hydrophobic hydrocarbon chain region of the lipid bilayer. However, there is no precedent for using LNPs to simultaneously encapsulate oligonucleotides and hydrophobic drugs. Given the complex pathogenesis and diverse pathological manifestations of CNS diseases, the encapsulation of a single type of drug by nanomaterials has limited effect on disease improvement. Using LNPs to simultaneously encapsulate oligonucleotide drugs and hydrophobic drugs can achieve multi-target treatment of CNS diseases.

[0004] Although nasal administration of nanomedicines can effectively enhance drug concentration in the brain, the efficiency of current nano-drug delivery systems for intranasal administration still needs to be improved. The glycosylation pattern on the surface of olfactory epithelial cells has a significant impact on the interaction between drug delivery systems and the olfactory epithelium. Although lectins have shown great biological potential for drug targeting and delivery, natural lectins may raise immunogenicity and toxicity issues due to their large molecular weight.

[0005] Therefore, providing a drug for treating the central nervous system through the nose and brain with high efficiency and no potential safety hazards has become an urgent problem to be solved in this field. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a double-encapsulated lipid nanoparticle for intranasal administration to the brain and its preparation method and application.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a double-encapsulated lipid nanoparticle for intranasal administration to the brain, wherein the double-encapsulated lipid nanoparticle comprises lipid nanoparticles, a drug in the lipid nanoparticles, and a frog lectin coupled to the surface of the lipid nanoparticles, wherein the drug comprises a hydrophilic drug and a hydrophobic drug.

[0009] The present invention provides a dual-encapsulated lipid nanoparticle for nasal administration that responds to reactive oxygen species. These dual-encapsulated lipid nanoparticles improve drug delivery to the brain and release rate to lesions, while simultaneously enabling multi-targeted treatment of CNS diseases. The hydrophilic oligonucleotide drug is encapsulated in a hydrophilic core formed by ionizable lipids, while the hydrophobic drug is encapsulated in the hydrophobic hydrocarbon chain region of the lipid bilayer.

[0010] Oxidative stress is a core factor in the pathogenesis of many central nervous system diseases. Studies have shown that the brain is prone to oxidative stress due to its high lipid content and high oxygen consumption, resulting in excessive production of reactive oxidative species (ROS) that attack neurons, leading to oxidative damage and cell apoptosis. The chemical structure of thioketal (TK) contains a thioether bond (-S-), which is easily oxidized and broken in a high concentration of ROS environment. In ROS-responsive lipid nanoparticles, TK is used as a linker to connect the hydrophilic part (such as PEG) and the hydrophobic part (such as DSPE). Under normal physiological conditions, TK remains stable, allowing lipid nanoparticles to remain intact in the blood circulation. When lipid nanoparticles reach diseased tissues with high ROS levels, TK undergoes oxidative cleavage, resulting in the destruction of the nanoparticle structure, thereby triggering the release of drugs.

[0011] Lectins are proteins or glycoproteins that can bind to sugar molecules. Based on the specificity of protein-sugar interactions, different lectins can serve as carrier molecules to specifically target drugs to different cells expressing different sugar molecules. Therefore, by utilizing lectins to specifically bind to sugar molecules on the surface of olfactory epithelial cells, drug delivery systems can be delivered to the nasal cavity for prolonged retention and facilitate drug transport and absorption from the nasal cavity to the brain. This strategy helps improve drug bioavailability and efficacy in the brain. Odorranalectin (OL) is a novel lectin isolated from the skin secretions of the five-fingered odor frog (Odorranalectin). To date, OL has been shown to be the smallest molecular weight (1700 Da) peptide with lectin-like activity, specifically recognizing L-fucose, which is widely distributed in the olfactory epithelium of the nasal mucosa. OL is composed of 17 amino acid residues, with cysteine ​​residues at positions 6 and 16 linked by a disulfide bond to form a cyclic peptide structure. Compared to natural lectins, which are large in molecular weight, complex in structure, difficult to extract, and expensive, OL, with its small molecular weight and single disulfide bond, is amenable to large-scale chemical synthesis via peptide biosynthesis. This invention, for the first time, couples OL to the surface of lipid nanoparticles. OL has up to five L-fucose binding sites (located at residues K5, C6, F7, C16, and T17), enabling it to specifically bind to L-fucose expressed by olfactory epithelial cells, prolonging the retention of lipid nanoparticles in the nasal cavity and promoting their transport and absorption from the nasal cavity to the brain.

[0012] Preferably, the raw materials for preparing the lipid nanoparticles include ionizable lipids, DOPE, cholesterol, DSPE-TK-PEG and DSPE-TK-PEG-MAL.

[0013] Preferably, the molar ratio of the ionizable lipid, DOPE, cholesterol, DSPE-TK-PEG and DSPE-TK-PEG-MAL is (30-60):(10-20):(20-50):(0.5-2):(0.5-2).

[0014] Among them, the specific point values ​​in (30-60) can be selected from 30, 32, 35, 38, 40, 42, 45, 48, 50, 52, 55, 58, 60, etc., the specific point values ​​in (10-20) can be selected from 11, 12, 14, 16, 18, 20, etc., the specific point values ​​in (20-50) can be selected from 20, 22, 25, 28, 30, 32, 35, 38, 40, 42, 45, 48, 50, etc., the specific point values ​​in (0.5-2) can be selected from 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, etc., and other specific point values ​​within the above numerical range can be selected, so they will not be listed here one by one.

[0015] Preferably, the molecular weight of PEG is 2000-5000, such as 2000, 3400, 5000, etc., more preferably 2000.

[0016] Preferably, the molar ratio of the frog lectin to the DSPE-TK-PEG-MAL in the raw material for preparing lipid nanoparticles is (1.2-3):1; wherein the specific point values ​​in (1.2-3) can be selected from 1.2, 1.5, 1.8, 2, 2.2, 2.5, 2.8, 3, etc., and other specific point values ​​within the above numerical range can be selected, which will not be repeated here.

[0017] Preferably, the mass ratio of the hydrophobic drug to the raw material for preparing lipid nanoparticles is 1:(15-20), and the specific point values ​​in (15-20) can be selected from 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be repeated here.

[0018] Preferably, the molar ratio of phosphorus in the hydrophilic drug to nitrogen in the ionizable lipids in the lipid nanoparticles is 1:(3-8), more preferably 1:(4-6).

[0019] The specific point values ​​in (3-8) can be selected as 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, etc. The specific point values ​​in (4-6) can be selected as 4, 4.2, 4.5, 4.8, 5, 5.2, 5.5, 5.8, 6, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be listed here one by one.

[0020] Preferably, the frog lectin is linked to the lipid nanoparticles via a thioether bond.

[0021] By adding a thiol-containing (-SH) Cys residue to the N-terminus of OL, OL can undergo a Michael addition reaction with the maleimide group in DSPE-TK-PEG-MAL. In this reaction, the thiol group attacks the conjugated double bond of the maleimide group, forming a stable thioether bond, thereby forming DSPE-TK-PEG-OL.

[0022] Preferably, the hydrophilic drug comprises mRNA or siRNA.

[0023] Preferably, the mRNA includes NGF mRNA, MBP mRNA or BDNF mRNA.

[0024] Preferably, the siRNA includes BACE1 siRNA, ApoE4 siRNA or MAPT siRNA.

[0025] Preferably, the sense strand sequence of the BACE1 siRNA is shown as SEQ ID No. 1, and the antisense strand sequence is shown as SEQ ID No. 2.

[0026] SEQ ID No. 1: 5'-GAACCUAUGCGAUGCGAAU-3'.

[0027] SEQ ID No. 2: 5'-AUUCGCAUCGCAUAGGUUC-3'.

[0028] Preferably, the hydrophobic drug comprises any one of quercetin, curcumin or rutin, or a combination of at least two thereof.

[0029] In a second aspect, the present invention provides a method for preparing the double-encapsulated lipid nanoparticles for nasal delivery to the brain according to the first aspect, the preparation method comprising:

[0030] (1) mixing a hydrophobic drug, a raw material for preparing lipid nanoparticles, and a first solvent to obtain an organic phase;

[0031] mixing a hydrophilic drug with a second solvent to obtain an aqueous phase;

[0032] (2) mixing the aqueous phase and the organic phase by microfluidics, collecting the crude product, mixing the crude product with a second solvent, and removing the first solvent by ultrafiltration to obtain lipid nanoparticles encapsulating the drug;

[0033] (3) Mixing the frog lectin with a buffer solution to obtain a lectin solution, mixing the drug-encapsulated lipid nanoparticles with the lectin solution, and ultrafiltration to obtain a product.

[0034] Preferably, the concentration of the hydrophobic drug in the organic phase is 0.5-1 mg / mL, for example, 0.5 mg / mL, 0.55 mg / mL, 0.6 mg / mL, 0.65 mg / mL, 0.7 mg / mL, 0.75 mg / mL, 0.8 mg / mL, 0.85 mg / mL, 0.9 mg / mL, 0.95 mg / mL, 1 mg / mL, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be repeated here.

[0035] Preferably, the concentration of the raw material for preparing lipid nanoparticles in the organic phase is 1-10 mg / mL, for example, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, etc. Other specific values ​​within the above numerical range can be selected and will not be repeated here.

[0036] Preferably, the first solvent comprises anhydrous ethanol.

[0037] Preferably, the second solvent comprises an aqueous citric acid solution.

[0038] Preferably, the molar concentration of citric acid in the citric acid aqueous solution is 50-100 mM, and the pH is 4-5. The molar concentration can be selected from 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, etc., and the pH can be selected from 4, 4.2, 4.4, 4.6, 4.8, 5, etc. Other specific point values ​​within the above numerical range can be selected, and they will not be repeated here.

[0039] Preferably, the concentration of the hydrophilic drug in the aqueous phase is 40-120 μg / mL, for example, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 110 μg / mL, 120 μg / mL, etc. Other specific point values ​​within the above numerical range can be selected and will not be repeated here.

[0040] Preferably, the flow rate of the aqueous phase in the microfluidics is 6-12 mL / min, the flow rate of the organic phase is 2-4 mL / min, and the flow rate ratio of the organic phase to the aqueous phase is 1:(2-5).

[0041] The flow rate of the aqueous phase can be selected from 6mL / min, 6.5mL / min, 7mL / min, 7.5mL / min, 8mL / min, 8.5mL / min, 9mL / min, 9.5mL / min, 10mL / min, 10.5mL / min, 11mL / min, 11.5mL / min, 12mL / min, etc., and the flow rate of the organic phase can be selected from 2mL / min, 2.5mL / min, 3mL / min, 3.5mL / min, 4mL / min, etc. The specific point values ​​in (2-5) can be selected from 2, 2.5, 3, 3.5, 4, 4.5, 5, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be repeated here.

[0042] Preferably, after removing the first solvent in step (2), the process further comprises mixing the product with a buffer solution and ultrafiltration.

[0043] Preferably, the process of mixing the product with a buffer and ultrafiltration is repeated 3-4 times.

[0044] Preferably, the mass percentage of ethanol in the drug-encapsulated lipid nanoparticles is less than 0.5%, for example 0.01%, 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, etc. Other specific point values ​​within the above numerical range can be selected and will not be repeated here.

[0045] Preferably, the molecular weight of the ultrafiltration tube used in the ultrafiltration is independently 10-100 kDa, such as 10 kDa, 30 kDa, 50 kDa, 100 kDa, etc.

[0046] Preferably, the mixing temperature in step (3) is 15-25° C. and the mixing time is 2-6 h.

[0047] The temperature can be selected as 15℃, 18℃, 20℃, 22℃, 25℃, etc., and the time can be selected as 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be listed here.

[0048] Preferably, the buffer comprises PBS buffer.

[0049] In a third aspect, the present invention provides a use of the double-encapsulated lipid nanoparticles for nasal delivery to the brain according to the first aspect in the preparation of drugs for treating central nervous system diseases.

[0050] Preferably, the central nervous system disease includes Alzheimer's disease.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] The present invention couples stinking frog lectin with lipid nanoparticles. The stinking frog lectin specifically binds to L-fucose residues expressed by olfactory epithelial cells, thereby enhancing the adhesion of lipid nanoparticles to the olfactory epithelium during nasal administration, ultimately improving the efficiency of lipid nanoparticles in brain delivery. Once inside the brain, the lipid nanoparticles respond to lesions with high levels of reactive oxygen species, releasing the drug. Furthermore, dual-drug encapsulation can target different pathogenesis mechanisms of central nervous system diseases, overcoming the limitations of traditional therapeutic drugs that often only target a single target. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 This is the particle size distribution result of OL-LNP@QU / siBACE1 in Example 1 of the present invention.

[0054] Figure 2 This is the morphology of OL-LNP@QU / siBACE1 in Example 1 of the present invention under a transmission electron microscope.

[0055] Figure 3 It is the intracellular fluorescence intensity imaging of Caco-2 cells after uptake of L-fucose+LNP@QU / siBACE1, LNP@QU / siBACE1, L-fucose+OL-LNP@QU / siBACE1, and OL-LNP@QU / siBACE1.

[0056] Figure 4 Fluorescence intensity imaging in the mouse brain after intranasal administration of PBS solution, OL-LNP@QU / siBACE1 solution, and LNP@QU / siBACE1 solution into the nasal cavity of wild-type C57BL / 6 mice.

[0057] Figure 5 is the release rate of quercetin from OL-LNP@QU / siBACE1 under different H2O2 concentrations.

[0058] Figure 6 OL-LNP@QU / siBACE1 improves cognitive impairment in AD mice, where Figure a shows the percentage of time mice enter the central area in the open field test, and Figure b shows the percentage of times mice enter the new arm in the maze test. DETAILED DESCRIPTION

[0059] The technical solution of the present invention is further described below by way of specific embodiments. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0060] The sources of the functional ingredients contained in the products involved in the following examples and comparative examples are as follows (only the functional ingredients are reflected, and the necessary auxiliary ingredients contained in other commercially available raw materials are not repeated):

[0061] The hydrophilic oligonucleotide drug described in the embodiment of the present invention is BACE1-siRNA, purchased from Jima Gene; the hydrophobic drug is quercetin, purchased from Sigma-Aldrich; the lectin is stinking frog lectin (OL), purchased from Meiluo Technology; and the CNS disease is Alzheimer's disease.

[0062] The mice used in the embodiments of the present invention are wild-type C57BL / 6 mice, 8 months old, male, weighing about 30-40 g, purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd., with a production license number of SCXK (Su) 2020-0009. APP / PS1 transgenic mice, 8 months old, male, weighing about 30-40 g, were purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. with a production license number of SCXK (Su) 2020-0009. The animal experiments of the present invention have been reviewed and approved by the Experimental Animal Welfare Committee of the National Tobacco Quality Supervision and Inspection Center, and the ethical review approval number is CTQTC-SYXK-20240021.

[0063] SM-102 represents an ionizable lipid, with the structural formula shown in Formula 1; DOPE represents a neutral auxiliary lipid, with the structural formula shown in Formula 2; cholesterol represents a sterol lipid, with the structural formula shown in Formula 3; DSPE-TK-PEG2000 represents a pegylated lipid derivative, with the structural formula shown in Formula 4; the structural formula of ALC-0315 is shown in Formula 5; and the structural formula of Dlin-MC3-DMA is shown in Formula 6.

[0064]

[0065]

[0066]

[0067] Example 1

[0068] This embodiment provides a double-encapsulated lipid nanoparticle for intranasal delivery to the brain, wherein the lipid nanoparticle is prepared by a method comprising the following steps:

[0069] 1. Compound lipid-ethanol solution:

[0070] SM-102, DOPE, DSPE-TK-PEG2000, and DSPE-TK-PEG2000-MAL were dissolved in anhydrous ethanol to prepare solutions with a concentration of 5 mg / mL;

[0071] Quercetin was dissolved in anhydrous ethanol to prepare a 1 mg / mL ethanol solution, and cholesterol was then dissolved in the ethanol solution to prepare a 5 mg / mL cholesterol ethanol solution.

[0072] 427.5 μL of SM-102, 89.6 μL of DOPE, 172.2 μL of cholesterol, 50.6 μL of DSPE-TK-PEG2000, and 52.2 μL of DSPE-TK-PEG2000-MAL were pipetted and mixed to adjust the molar ratio of SM-102, DOPE, cholesterol, DSPE-TK-PEG2000, and DSPE-TK-PEG2000-MAL to 50:10:37:1.5:1.5 to obtain an organic phase.

[0073] 2. Preparation of BACE1-siRNA citric acid solution

[0074] BACE1-siRNA powder was prepared into a 58 μg / mL aqueous solution with 100 mM, pH 4.0 citric acid solution as the aqueous phase. The sense strand sequence of BACE1 siRNA is shown in SEQ ID No. 1, and the antisense strand sequence is shown in SEQ ID No. 2.

[0075] SEQ ID No. 1: 5'-GAACCUAUGCGAUGCGAAU-3'.

[0076] SEQ ID No. 2: 5'-AUUCGCAUCGCAUAGGUUC-3'.

[0077] 3. Microfluidic mixing

[0078] (1) Clean the microfluidic dual-channel syringe pump tubing with 100 mM, pH 4.0 citric acid solution and anhydrous ethanol, so that the microfluidic chip tubing is filled with citric acid solution and anhydrous ethanol;

[0079] (2) Use a 1 mL syringe to draw 500 μL of the organic phase and a 2 mL syringe to draw 1500 μL of the aqueous phase. Set the dual-channel flow rate on the microfluidic dual-channel syringe pump interface to a flow rate of 9 mL / min for the aqueous phase and 3 mL / min for the organic phase, for a total flow rate of 12 mL / min. After setting, save the program, adjust the positions of the components, and then run the program to collect the crude LNP@QU / siBACE1 solution.

[0080] (3) The crude product solution was immediately diluted with 6 mL of 100 mM citric acid solution, pH 4.0, and then transferred to an ultrafiltration tube with a molecular weight of 50 kDa and centrifuged at 3000 g at 4°C to 1 mL.

[0081] (4) Add 1× PBS to 4 mL and centrifuge again at 3000 g at 4°C for 1 mL. Repeat this step 4 times.

[0082] (5) Collect the sample after the last centrifugation to obtain the LNP@QU / siBACE1 final product solution.

[0083] 4. Synthesis of OL-LNP@QU / siBACE1

[0084] 223 μg of OL was dissolved in 500 μL of PBS. The LNP@QU / siBACE1 final product solution and OL solution were mixed and reacted at room temperature for 4 hours. The mixture was then transferred to a 50 kDa ultrafiltration tube and centrifuged at 3000 g at 4°C to a final volume of 1 mL to obtain the OL-LNP@QU / siBACE1 final product solution. The OL-LNP@QU / siBACE1 final product solution was stored at 4°C. The amino acid sequence of OL is shown in SEQ ID No. 3.

[0085] SEQ ID No. 3: CYASPKCFRYPNGVLACT.

[0086] The particle size of OL-LNP@QU / siBACE1 was tested using a nanoparticle size analyzer. The results are as follows: Figure 1 As shown in the figure, the particle size distribution of OL-LNP@QU / siBACE1 is relatively uniform, with the median particle size of 99.37nm. The morphology of OL-LNP@QU / siBACE1 was characterized using transmission electron microscopy, and the results are shown in Figure 2 As shown in the figure, OL-LNP@QU / siBACE1 is spherical or nearly spherical, with regular morphology and a smooth surface without obvious protrusions or depressions.

[0087] Example 2

[0088] This example provides a double-encapsulated lipid nanoparticle for transnasal administration to the brain. The only difference between this example and Example 1 is that SM-102 is replaced with ALC-0315 in equal molar mass, and other operations remain unchanged.

[0089] Example 3

[0090] This example provides a double-encapsulated lipid nanoparticle for intranasal administration to the brain. The only difference between this example and Example 1 is that SM-102 is replaced with Dlin-MC3-DMA in equal molar mass, and other operations remain unchanged.

[0091] Test Example 1

[0092] Particle size, PDI, Zeta potential, and encapsulation efficiency tests

[0093] Test method:

[0094] Particle size, PDI, and Zeta potential test methods:

[0095] The particle size, PDI, and Zeta potential were measured using a nanoparticle size and potential analyzer. The prepared OL-LNP@QU / siBACE1 stock solution was diluted (20 μL of the stock solution was added to 1 mL of ultrapure water) and added to the particle size sample cell. The particle size and PDI of OL-LNP@QU / siBACE1 were measured by dynamic light scattering (DLS). The test conditions were: 1 minute equilibrium time, 10 second measurement interval, and a temperature of 25°C. The sample was then added to the Zeta potential sample cell to measure the Zeta potential of OL-LNP@QU / siBACE1.

[0096] Quercetin encapsulation efficiency test method:

[0097] A standard curve was established by high performance liquid chromatography (HPLC) based on quercetin concentration-peak area.

[0098] 50 μL of OL-LNP@QU / siBACE1 was added to 50 μL of 2% Triton X-100 for demulsification. Ultrasonication was performed for 5 minutes to complete demulsification. The quercetin content was calculated using a standard curve and recorded as the total quercetin content. 200 μL of OL-LNP@QU / siBACE1 was centrifuged at 5000 rpm for 5 minutes. 50 μL of the supernatant was added to 50 μL of 2% Triton X-100 for demulsification. Ultrasonication was performed for 5 minutes to complete demulsification. The quercetin content was calculated using a standard curve and recorded as the quercetin encapsulation amount.

[0099] Quercetin encapsulation efficiency (%) = quercetin encapsulation amount / quercetin total amount × 100%

[0100] BACE1 siRNA encapsulation efficiency test method:

[0101] Quant-iT TM RiboGreen TM The RNA reagent has ultra-sensitive detection characteristics for 1-200ng nucleic acid. Take 1μL of OL-LNP@QU / siBACE1 and add it to 99μL of 2% TritonX-100 to break the emulsion, which is recorded as total siRNA. Take 1μL of OL-LNP@QU / siBACE1 and add it to 99μL of LTE buffer (1×), which is recorded as free siRNA. Use Quant-iT TM RiboGreen TM RNA kit was used to determine the siRNA encapsulation efficiency.

[0102] siRNA encapsulation efficiency (%) = (total siRNA - free siRNA) / total siRNA × 100%

[0103] Table 1

[0104]

[0105] Test Example 2

[0106] OL modification enhances the uptake of LNP@QU / siBACE1 by Caco-2 cells

[0107] In order to verify that OL modification can increase the specific uptake of LNP by nasal mucosal epithelial cells and ultimately increase the drug concentration of LNP in the brain, the present invention uses Caco-2 cells that specifically express L-fucose to simulate the nasal epithelial barrier for in vitro cell uptake verification.

[0108] In this test example, siBACE1 labeled with the fluorescent dye FAM was used to prepare LNP@QU / siBACE1 and OL-LNP@QU / siBACE1. Other preparation conditions were the same as in Example 1, that is, FAM-labeled lipid nanoparticles were obtained to facilitate subsequent fluorescence intensity analysis.

[0109] The specific operation steps are as follows: Caco-2 cells were seeded in 24-well plates at a cell density of 150,000 cells / well, and the cells were cultured until they reached 70-80% confluence. L-fucose (10 μmol / L) + LNP@QU / siBACE1 (QU = 15 μmol / L, siBACE1 = 115.9 nmol / L), LNP@QU / siBACE1 (QU = 15 μmol / L, siBACE1 = 115.9 nmol / L), L-fucose (10 μmol / L) + OL-LNP@QU / siBACE1 (QU = 15 μmol / L, siBACE1 = 115.9 nmol / L) and OL-LNP@QU / siBACE1 (QU = 15 μmol / L, siBACE1 = 115.9 nmol / L) were diluted in serum-free medium and incubated with cells for 4 h. After incubation, cells were stained with Hoechst33342 solution for 10 minutes, washed twice with PBS solution, and then observed using a high-content cell imaging system. The results are shown in Figure 2. Figure 3 The results showed that OL modification enhanced the uptake of LNP@QU / siBACE1 by Caco-2 cells.

[0110] Test Example 3

[0111] OL modification enhances the concentration of LNP@QU / siBACE1 in the mouse brain after intranasal administration

[0112] To verify that OL modification can enhance the brain drug concentration after intranasal administration, wild-type C57BL / 6 mice were used to monitor the brain drug concentration after intranasal administration.

[0113] In this test example, siBACE1 labeled with the fluorescent dye Cy5.5 was used to prepare LNP@QU / siBACE1 and OL-LNP@QU / siBACE1. Other preparation conditions were the same as in Example 1, and Cy5.5-labeled lipid nanoparticles were obtained to facilitate subsequent fluorescence intensity analysis.

[0114] The specific operation steps are as follows: 20 μL PBS solution, LNP@QU / siBACE1 (QU = 2.83 mmol / L, siBACE1 = 24.61 μmol / L) solution and OL-LNP@QU / siBACE1 (QU = 2.83 mmol / L, siBACE1 = 24.61 μmol / L) solution were administered intranasally into the nasal cavity of wild-type C57BL / 6 mice (n = 3). The fluorescence intensity of the drug in the brain was observed using a small animal in vivo imaging system 1 hour after administration. The results are shown in Figure 2. Figure 4 The results showed that compared with LNP@QU / siBACE1 administration, the fluorescence intensity in the brain was stronger when OL-LNP@QU / siBACE1 was administered, indicating that OL modification enhanced the drug concentration in the mouse brain after intranasal administration.

[0115] Test Example 4

[0116] ROS-responsiveness of OL-LNP@QU / siBACE1

[0117] In order to prove that OL-LNP@QU / siBACE1 can effectively release quercetin at the lesion site with high ROS content, the ROS responsiveness of LNP was characterized. The product prepared in Example 1 was incubated with H2O2 at different molar ratios (1:2, 1:5, 1:10) for 4 h, and the release rate of quercetin at different H2O2 concentrations was detected by high performance liquid chromatography (HPLC). The results are shown in Figure 2. Figure 5 The results showed that the release rate of quercetin increased with the increase of H2O2 concentration, which were 25.15±2.15%, 38.1±1.6% and 63.55±1.05% respectively.

[0118] Test Example 5

[0119] Intranasal administration of OL-LNP@QU / siBACE1 can improve cognitive impairment in APP / PS1 mice

[0120] Due to the complex pathogenesis and pathological manifestations of AD, single-drug and single-target therapies have limited efficacy in improving the disease. This invention utilizes LNPs to simultaneously encapsulate BACE1-siRNA and quercetin, enabling multi-targeted treatment of CNS diseases. BACE1-siRNA inhibits BACE1 enzyme activity in the brain, reducing Aβ production and plaque formation; quercetin regulates redox homeostasis in the AD brain through its antioxidant effects.

[0121] To verify that the improvement effect of dual-drug encapsulation is greater than that of single-drug encapsulation, negative control siRNA (i.e., siNC) was used instead of siBACE1 to prepare OL-LNP@QU / siNC encapsulating only quercetin; in addition, OL-LNP@siBACE1 encapsulating only siBACE1 was also prepared. The preparation method is referenced to Example 1.

[0122] The nucleotide sequence of the positive strand of siNC is shown in SEQ ID No.4.

[0123] SEQ ID No. 4: 5′-UUCUCCGAACGUGUCACGU-3′.

[0124] The nucleotide sequence of the antisense strand of siNC is shown in SEQ ID No.5.

[0125] SEQ ID No. 5: 5′-ACGUGACACGUUCGGAGAA-3′.

[0126] The test method is as follows:

[0127] APP / PS1 transgenic AD mice were randomly divided into four groups (n=10) and administered intranasally with PBS, OL-LNP@QU / siNC (QU=2.83mmol / L, siNC=24.61μmol / L), OL-LNP@siBACE1 (siBACE1=24.61μmol / L), and OL-LNP@QU / siBACE1 (QU=2.83mmol / L, siBACE1=24.61μmol / L), respectively. Ten wild-type C57BL / 6 mice were administered the same way with PBS. Drugs were administered intranasally every two days in a 40μL volume. After 15 doses, behavioral tests were performed, including a Y-maze test and an open field test. The behavioral results of the experiments were used to assess the efficacy of OL-LNP@QU / siBACE1 in improving cognitive impairment in AD mice.

[0128] The results are as follows Figure 6As shown in the results, the improvement effect of OL-LNP@QU / siBACE1 administration on cognitive impairment in AD mice was greater than that of OL-LNP@QU / siNC and OL-LNP@siBACE1 administration.

[0129] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the present invention's dual-encapsulated lipid nanoparticles for transnasal delivery to the brain, their preparation methods, and applications. However, the present invention is not limited to the above-mentioned embodiments, and it does not mean that the present invention must rely on the above-mentioned embodiments for implementation. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for various raw materials in the present invention's products, addition of auxiliary ingredients, and selection of specific methods, etc., fall within the scope of protection and disclosure of the present invention.

[0130] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0131] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A double-encapsulated lipid nanoparticle for intranasal administration, characterized in that: The double-encapsulated lipid nanoparticles include lipid nanoparticles, drugs in the lipid nanoparticles and frog lectin coupled to the surface of the lipid nanoparticles, wherein the drugs include hydrophilic drugs and hydrophobic drugs.

2. The double-encapsulated lipid nanoparticles for nasal administration to the brain according to claim 1, characterized in that The raw materials for preparing the lipid nanoparticles include ionizable lipids, DOPE, cholesterol, DSPE-TK-PEG and DSPE-TK-PEG-MAL; Preferably, the molar ratio of the ionizable lipid, DOPE, cholesterol, DSPE-TK-PEG and DSPE-TK-PEG-MAL is (30-60):(10-20):(20-50):(0.5-2):(0.5-2); Preferably, the molecular weight of PEG is 2000-5000; Preferably, the molar ratio of the stinking frog lectin to the DSPE-TK-PEG-MAL in the raw material for preparing lipid nanoparticles is (1.2-3):1; Preferably, the molar ratio of phosphorus in the hydrophilic drug to nitrogen in the ionizable lipids in the lipid nanoparticles is 1:(3-8), more preferably 1:(4-6).

3. The double-encapsulated lipid nanoparticles for nasal administration to the brain according to claim 1 or 2, characterized in that: The mass ratio of the hydrophobic drug to the raw materials for preparing the lipid nanoparticles is 1:(15-20); Preferably, the frog lectin is linked to the lipid nanoparticles via a thioether bond.

4. The double-encapsulated lipid nanoparticles for nasal administration to the brain according to any one of claims 1 to 3, characterized in that: The hydrophilic drug includes mRNA or siRNA; Preferably, the mRNA includes NGF mRNA, MBP mRNA or BDNF mRNA; Preferably, the siRNA comprises BACE1 siRNA, ApoE4 siRNA or MAPT siRNA; Preferably, the hydrophobic drug comprises any one of quercetin, curcumin or rutin, or a combination of at least two thereof.

5. The method for preparing double-encapsulated lipid nanoparticles for nasal administration to the brain according to any one of claims 1 to 4, characterized in that: The preparation method comprises: (1) mixing a hydrophobic drug, a raw material for preparing lipid nanoparticles, and a first solvent to obtain an organic phase; mixing a hydrophilic drug with a second solvent to obtain an aqueous phase; (2) mixing the aqueous phase and the organic phase by microfluidics, collecting the crude product, mixing the crude product with a second solvent, and removing the first solvent by ultrafiltration to obtain lipid nanoparticles encapsulating the drug; (3) Mixing the frog lectin with a buffer solution to obtain a lectin solution, mixing the drug-encapsulated lipid nanoparticles with the lectin solution, and ultrafiltration to obtain a product.

6. The method for preparing double-encapsulated lipid nanoparticles for nasal administration to the brain according to claim 5, characterized in that: The concentration of the hydrophobic drug in the organic phase is 0.5-1 mg / mL; Preferably, the concentration of the raw material for preparing lipid nanoparticles in the organic phase is 1-10 mg / mL; Preferably, the first solvent comprises anhydrous ethanol; Preferably, the second solvent comprises an aqueous solution of citric acid; Preferably, the molar concentration of citric acid in the citric acid aqueous solution is 50-100 mM and the pH is 4-5.

7. The method for preparing double-encapsulated lipid nanoparticles for nasal administration to the brain according to claim 5 or 6, characterized in that: The concentration of the hydrophilic drug in the aqueous phase is 40-120 μg / mL; Preferably, the flow rate of the aqueous phase in the microfluidic system is 6-12 mL / min, the flow rate of the organic phase is 2-4 mL / min, and the flow rate ratio of the organic phase to the aqueous phase is 1:(2-5); Preferably, after removing the first solvent in step (2), the process further comprises mixing the product with a buffer solution and ultrafiltration; Preferably, the process of mixing the product with a buffer and ultrafiltration is repeated 3-4 times.

8. The method for preparing double-encapsulated lipid nanoparticles for nasal delivery to the brain according to any one of claims 5 to 7, characterized in that: The mass percentage of ethanol in the drug-encapsulated lipid nanoparticles is less than 0.5%; Preferably, the molecular weight of the ultrafiltration tube used in the ultrafiltration is independently 10-100 kDa; Preferably, the mixing temperature in step (3) is 15-25° C. and the mixing time is 2-6 h; Preferably, the buffer comprises PBS buffer.

9. Use of the double-encapsulated lipid nanoparticles for nasal administration to the brain according to any one of claims 1 to 4 in the preparation of drugs for treating central nervous system diseases.

10. The use according to claim 9, characterized in that The central nervous system diseases include Alzheimer's disease.

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