Tet1-modified siBACE1-encapsulated cationic liposome, preparation method and application

Abstract

The application discloses a Tet1 modified cationic liposome for loading siBACE1, a preparation method and application thereof, and belongs to the technical field of biological medicines. The application solves the problem of limited treatment effect of siBACE1 in treating Alzheimer's disease in the prior art. The application comprises siBACE1, the siBACE1 is externally coated with a lipid composition, and the lipid composition is modified with a Tet1 peptide; the lipid composition is composed of 1,2-dioleoyl-3-trimethylammonium propane, dioleoylphosphatidylethanolamine, cholesterol and distearoylphosphatidylethanolamine-polyethylene glycol 2000. The Tet1 modified cationic liposome for loading siBACE1 can deliver siBACE1 to an action site, improve the action effect, and effectively treat Alzheimer's disease.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to Tet1 modified cationic liposome for loading siBACE1, a preparation method and application thereof. BACKGROUND

[0002] Alzheimer's disease (AD) is a central nervous system degenerative disease with high incidence in the elderly (>65 years old), mainly manifested as progressive cognitive dysfunction and behavioral impairment, and is the most common type of dementia. The main pathological features of AD are extracellular plaques formed by amyloid-beta protein (Aβ) deposition and intracytoplasmic neurofibrillary tangles formed by tau protein hyperphosphorylation, accompanied by neuroinflammation, neuron and synapse loss, mitochondrial dysfunction, oxidative stress and brain atrophy, etc. Studies have confirmed that Aβ deposition is the core factor of early AD pathological progression and plays a key role in the initiation and exacerbation of the disease. Aβ deposition is caused by the abnormal aggregation of small molecules (such as Aβ 1-40 and Aβ 1-42 , etc.) generated by the cleavage of amyloid precursor protein (APP) by β-site APP cleaving enzyme 1 (BACE1) in the brain, usually in the important brain regions related to cognition and memory such as cerebral cortex and hippocampus. Aβ deposition (of which Aβ 1-42 oligomer is the most neurotoxic form in Aβ deposition) can interact with the neuronal membrane, change the structure and function of the membrane, cause the intracellular calcium ion level to rise, activate microglia and astrocytes, trigger an inflammatory response, and change the synaptic electrophysiological properties, interfering with information transmission between neurons. The above pathological changes interact, leading to axonal transport dysfunction, energy metabolism disorder and irreversible neuronal death, ultimately triggering the characteristic cognitive decline of AD. Therefore, reducing the excessive cleavage of BACE1 to amyloid precursor protein to reduce Aβ generation is an important strategy for early treatment of AD.

[0003] Currently, monoclonal antibody drugs targeting Aβ (such as Aducanumab and Donanemab) have made some progress, but the frequent amyloid-related brain imaging abnormalities, uncertainty of efficacy, and potential side effects of brain atrophy limit their clinical application. In recent years, with the rapid development of gene therapy and brain-targeted drug delivery technology, gene therapy has become an important direction of AD treatment research. Gene therapy is a technology that uses viruses and biological materials as carriers to introduce, remove or modify genetic material to treat or prevent diseases. Its core concept is to use genetic engineering technology to repair or replace defective genes, or to regulate the expression of specific genes to restore the normal function of cells or tissues. Small interfering RNA (siRNA) has high specificity, high efficiency, and rapid and simple development of candidate drugs for pathogenic targets, making siRNA unique in the treatment of central nervous system diseases. However, naked siRNA has the following two problems: (1) small particle size and poor stability, easy to be filtered by the kidney and hydrolyzed by nucleic acid endonuclease in the blood and phagocytic degradation by immune cells; (2) Although siRNA itself has strong targeting when silencing genes, it lacks targeting in the delivery process, which is easy to cause off-target effects. Therefore, siRNA needs a suitable carrier to overcome its own shortcomings. SUMMARY

[0004] With the development of nanotechnology, the types of carriers become diversified, which makes it possible to achieve brain delivery of siRNA drugs. The core components of nanomedicine carriers include a variety of materials such as lipids, polymers and metals, which can encapsulate molecules with different chemical properties. In addition, these nanoparticles can promote the protection and delivery of bioactive molecules, thereby reducing their potential toxicity, and thus improving their solubility, stability, biodistribution and pharmacokinetics. The contents encapsulated in nanoparticles are diverse, ranging from small molecules, polypeptides, proteins to genetic material. For chronic central nervous system diseases, it is essential that nanoparticles can deliver bioactive molecules to hard-to-reach tissues. Liposome nanoparticles have amphiphilic properties, high bioavailability, and are easily biodegradable. Their phospholipid bilayer structure is similar to physiological membranes, making it easier to deliver therapeutic molecules into the brain through the BBB. At the same time, surface modification of liposomes such as transferrin, glucose and specific peptide ligands can improve their brain targeting ability and reduce off-target effects.

[0005] Therefore, the application constructs a Tet1 modified cationic liposome loaded with siBACE1, a preparation method and an application, wherein siRNA (siBACE1) inhibiting BACE1 enzyme cutting activity is loaded. The cationic liposome is characterized by having a lipid bilayer membrane, which is usually formed by complexing a positively charged cationic lipid with a neutral auxiliary lipid under appropriate conditions. DOTAP ((2,3-dioleoyl-propyl)-trimethylamine), DOTMA (trimethyl-2,3-dioleyloxypropylammonium chloride), and DC-Chol (3β-[N-(N',N'-dimethylaminoethyl) carbamoyl] cholesteryl hydrochloride) are the most commonly used cationic lipids, but when forming liposomes alone, the stability, membrane fusion and transfection efficiency are not ideal, and often auxiliary lipids need to be added. The most commonly used auxiliary lipid is DOPE (dioleoylphosphatidylethanolamine), and most of the commercially available cationic liposomes use DOPE as their auxiliary lipid. Based on this, the application selects DOTAP as the cationic lipid and DOPE as the auxiliary lipid as the basic lipid raw material for synthesizing the cationic liposome. Considering the clearance of the phagocytic system, in order to prolong the retention time of the cationic liposome in the blood circulation and improve the stability, distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) is added. In order to endow the liposome with the ability to precisely target neurons to protect exogenous nucleic acid substances, DSPE-PEG2000-Tet1 modified with Tet1 polypeptide is added.

[0006] The technical solutions adopted by the application are as follows:

[0007] The Tet1 modified cationic liposome loaded with siBACE1 comprises siBACE1, the outside of the siBACE1 is coated with a lipid composition, and the lipid composition is modified with a Tet1 peptide.

[0008] The lipid composition is composed of 1,2-dioleoyl-3-trimethylammonium propane, dioleoylphosphatidylethanolamine, cholesterol, and distearoylphosphatidylethanolamine-polyethylene glycol 2000.

[0009] As a preference, the molar proportion of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) in the entire lipid composition is 45% to 50%, based on the total amount of the lipid composition being 100%;

[0010] The molar proportion of dioleoylphosphatidylethanolamine (DOPE) in the entire lipid composition is 8% to 12%;

[0011] The molar proportion of cholesterol in the entire lipid composition is 35% to 40%;

[0012] The molar percentage of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) in the entire lipid composition is 1% to 10%.

[0013] Further, the molar percentage of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) in the entire lipid composition is 50% based on the total amount of the lipid composition being 100%;

[0014] The molar percentage of dioleoylphosphatidylethanolamine (DOPE) in the entire lipid composition is 10%;

[0015] The molar percentage of cholesterol in the entire lipid composition is 38.5%;

[0016] The molar percentage of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) in the entire lipid composition is 1.5%.

[0017] As preferred, the N / P molar ratio of 1,2-dioleoyl-3-trimethylammonium propane to siBACE1 is 6 to 8.

[0018] As preferred, the Tet1 ligand is Tet1-distearoylphosphatidylethanolamine-polyethylene glycol 2000, and the amount of Tet1-distearoylphosphatidylethanolamine-polyethylene glycol 2000 is 0.45% to 0.75% of the amount of the lipid composition based on the molar percentage.

[0019] After the technical scheme is adopted, the limited amount of each component in the lipid composition is based on the comprehensive optimization results of the functional synergy of each component, the physicochemical properties of the nanoparticles, and the delivery efficiency in vivo and in vitro. 45% to 50% of DOTAP as the leading cationic lipid ensures sufficient positive charge density to efficiently encapsulate siBACE1, and its double oleoyl chain is highly compatible with the acyl structure of DOPE to form a uniform lipid bilayer core, avoiding siBACE1 leakage caused by phase separation; 8% to 12% of DOPE promotes the fusion of nanoparticles with cell membrane / endosome membrane through its strong membrane fluidity, and its conical molecular structure can induce the lipid bilayer to transform into a non-lamellar hexagonal phase in the acidic endosome environment, which synergistically enhances the endosome escape efficiency with the charge effect of DOTAP; 35% to 40% of cholesterol balances the excessive fluidity of DOPE by filling the gap between lipid molecules, endows the nanoparticles with higher serum stability (change in particle size <10% after 7 days of storage at 4°C), and reduces the cell membrane damage toxicity of cationic lipids; 1% to 10% of DSPE-PEG2000 significantly reduces macrophage phagocytosis by forming a spatial barrier through the hydrophilic PEG chain, and prolongs the in vivo half-life to 8-12 hours.

[0020] 0.45%~0.75% of Tet1-DSPE-PEG2000 can significantly improve the enrichment efficiency of LNP (lipid nanoparticle) in neurons (5-10 times higher than non-targeted LNP), reduce non-specific tissue distribution, and improve the stability of the lipid composition. Therefore, by limiting the molar ratio of each component, the present application can precisely regulate the balance of rigidity-fluidity, charge-shielding effect, targeting-stealth performance, and achieve the optimization of the whole chain of "high drug loading-stable circulation-targeted delivery-endosome escape".

[0021] In constructing Tet1-modified cationic liposome encapsulating siBACE1, the N / P ratio (nitrogen / phosphate molar ratio) of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) to siBACE1 is optimized to 6-8, which is a ratio set based on its advantages in balancing high encapsulation efficiency, stability and biocompatibility. The cationic amino group (N) of DOTAP combines with the phosphate skeleton (P) of siBACE1 through electrostatic interaction. When the N / P ratio is 6-8, it can provide enough positive charge to completely compress siBACE1 to form a dense core, avoid the degradation of nucleic acids by serum nucleases in circulation (encapsulation rate can reach more than 95%), and at the same time avoid excessive positive charge (Zeta potential controlled at +15~+20 mV) caused by excessive lipid composition. This moderate surface charge can not only reduce non-specific adsorption with anionic proteins in the blood, prolonging in vivo circulation time, but also retain enough positive charge to promote the interaction between Tet1-modified cationic liposome encapsulating siBACE1 and the negatively charged cell membrane, enhancing cell uptake efficiency. In addition, the formulation with N / P=6-8 can form nanoparticles with uniform particle size, and its small size is beneficial to pass through the blood-brain barrier gap (usually required <200 nm), while the tight siBACE1 compression state can ensure efficient release of intact gene drugs in target cells. Experimental data show that when the N / P ratio is less than 4, the siBACE1 encapsulation rate decreases significantly (<80%), and the particles are prone to aggregation due to insufficient charge; when the N / P ratio is higher than 8, the encapsulation rate decreases, and N / P higher than 8 also leads to excessive surface positive charge, which can cause red blood cell membrane rupture or complement activation risk, and excessive lipid occupying the surface of the particles hinders the targeting function of Tet1 peptide. Therefore, the setting of N / P=6-8 precisely regulates the charge density and the interaction between the lipid composition and siBACE1, achieving the optimal solution between delivery efficiency, safety and functional modification.

[0022] As a preferred, the siBACE1 has the following gene sequence:

[0023] Sense strand: GCUUUGUGGAGAUGGUGGATT;

[0024] Antisense strand: UCCACCAUCUCCACAAAGCTT.

[0025] A preparation method of a Tet1 modified cationic liposome encapsulating siBACE1, comprising the following steps:

[0026] S1: preparing a lipid composition;

[0027] S2: encapsulating siBACE1 by using the lipid composition to obtain a siBACE1-lipid complex;

[0028] S3: adding a Tet1 ligand to modify the siBACE1-lipid complex to obtain the Tet1 modified cationic liposome encapsulating siBACE1.

[0029] After adopting the technical scheme, in the construction of the Tet1 modified cationic liposome encapsulating siBACE1, the post-insertion of the Tet1 ligand is more preferred than the “one-step method” of directly incorporating in the self-assembly process of the lipid composition. In the post-insertion method, the Tet1 peptide is coupled to the end of DSPE-PEG2000 to obtain Tet1-DSPE-PEG2000, and Tet1-distearoylphosphatidyl ethanolamine-polyethylene glycol 2000 (Tet1-DSPE-PEG2000) is inserted into the particle surface through hydrophobic interaction after the formation of the lipid composition, which can precisely control the ligand density and spatial orientation, and avoid the embedding failure or activity loss of Tet1 caused by lipid phase separation in the one-step method. The post-insertion method can significantly improve the targeting efficiency of Tet1 while maintaining the high encapsulation rate of siBACE1 and the stability of the lipid composition. The advantage is derived from the decoupling design of the ligand modification step and the core drug-loaded structure, which not only ensures the physicochemical controllability of the self-assembly process of the lipid composition, but also ensures that the active motif of Tet1 is fully exposed to the particle surface. Therefore, the post-insertion method has become the preferred process for functionalizing the lipid composition with targeting peptides, especially in scenarios where both drug loading efficiency and targeting specificity are required.

[0030] As preferred, the specific steps of S1 are as follows:

[0031] S101: mixing 1,2-dioleoyl-3-trimethylammonium propane, dioleoylphosphatidyl ethanolamine, cholesterol, and distearoylphosphatidyl ethanolamine-polyethylene glycol 2000 according to the ratio;

[0032] S102: adding chloroform and forming a film by rotary evaporation;

[0033] S103: removing residual chloroform by nitrogen blowing to obtain the lipid composition.

[0034] As preferred, the specific steps of S2 are as follows:

[0035] S201: citric acid buffer is added to the lipid composition obtained in S1, and the lipid composition solution is obtained by stirring at 60 DEG C;

[0036] S202: siBACE1 solution is added to the lipid composition solution, and incubation is carried out at room temperature;

[0037] S203: the solution is subjected to homogenization treatment to obtain siBACE1-lipid complex.

[0038] As preferred, the specific steps of S3 are as follows:

[0039] S301: the siBACE1-lipid complex obtained in S2 is replaced into 4-hydroxyethyl piperazine ethanesulfonic acid buffer to obtain a replaced siBACE1-lipid complex solution;

[0040] S302: Tet1-distearyl phosphatidylethanolamine-polyethylene glycol 2000 is dissolved in 4-hydroxyethyl piperazine ethanesulfonic acid buffer to obtain a Tet1-distearyl phosphatidylethanolamine-polyethylene glycol 2000 solution;

[0041] S303: the Tet1-distearyl phosphatidylethanolamine-polyethylene glycol 2000 solution obtained in S302 is added to the replaced siBACE1-lipid complex solution for light-avoiding incubation;

[0042] S304: free Tet1-distearyl phosphatidylethanolamine-polyethylene glycol 2000 is removed to obtain Tet1-modified siBACE1-loaded cationic liposomes.

[0043] The Tet1-modified siBACE1-loaded cationic liposomes or the Tet1-modified siBACE1-loaded cationic liposomes prepared by the preparation method of the Tet1-modified siBACE1-loaded cationic liposomes are applied to the preparation of drugs for treating Alzheimer's disease.

[0044] In summary, due to the adoption of the above technical solutions, the present application has the following advantages:

[0045] The application develops a new type of neuron-targeting gene therapy nanoparticle (Tet1-CLNP@siBACE1) based on cationic liposome, which can deliver siBACE1 to Alzheimer's disease (AD) neurons through precise targeting mediated by Tet1 peptide, and specifically silence BACE1 gene expression, thereby inhibiting the pathological generation of beta-amyloid (A beta), while limiting the non-specific central diffusion of liposome through the optimization design of the size and surface charge of the liposome, significantly reducing the off-target risk of systemic gene drugs.

[0046] 1. The Tet1 modified cationic liposome prepared by the application can effectively encapsulate siBACE1, and the positive charge of the cationic liposome can enhance its affinity with the cell membrane, improve the cell uptake efficiency of siBACE1, thereby improving the delivery efficiency of siRNA and achieving better gene silencing effect.

[0047] 2. The Tet1 modified cationic liposome prepared by the application has the ability of specific targeting of peripheral nerves through surface modification of Tet1 peptide. Tet1 peptide can recognize and bind to specific receptors on the surface of nerve cells, enhance the targeting of liposomes to peripheral nerves, and help the directional release of siBACE1 in nerve cells, thereby realizing precise gene therapy.

[0048] 3. The cationic liposome prepared in the application has good stability and sustained release, and the synergistic effect of the components of the cationic liposome can effectively reduce the degradation of the liposome and prolong the action time of siBACE1. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 It is a synthesis route diagram of Tet1 modified siBACE1 loaded cationic liposome (Tetl-CLNP@siBACE1);

[0050] Figure 2 It is a mass spectrum diagram of Tet1-DSPE-PEG2000 and Tet1, wherein (A) is a mass spectrum diagram of Tet1, and (B) is a mass spectrum diagram of Tet1-DSPE-PEG2000;

[0051] Figure 3 It is a particle size detection result diagram of Tetl-CLNP@siBACE1;

[0052] Figure 4 It is a potential detection result diagram of Tetl-CLNP@siBACE1;

[0053] Figure 5Figure 6 is a transmission electron microscopy result diagram of Tetl-CLNP@siBACE1, wherein (A) is a transmission electron microscopy result diagram at a magnification of 50 nm, and (B) is a transmission electron microscopy result diagram at a magnification of 20 nm;

[0054] Figure 6 Figure 7 is an encapsulation efficiency result diagram of Tetl-CLNP@siBACE1;

[0055] Figure 7 Figure 8 is an in vitro nerve targeting ability result diagram of Tetl peptide, wherein (A) is a high-content cell imaging diagram of N2a cells, BV2 cells and 293T cells after being co-incubated with Tetl-CLNP@siBACE1 for 6 h, and (B) is a relative fluorescence intensity detection diagram of Cy5-siNC in cells after N2a cells, BV2 cells and 293T cells were co-incubated with Tetl-CLNP@siBACE1 for 6 h;

[0056] Figure 8 Figure 9 is a result diagram of Tetl-CLNP@siBACE1 effectively reducing the expression of β-secretase BACE1 in neuron cells, wherein (A) is a result diagram of detecting the mRNA expression of β-secretase BACE1 by qPCR, (B) is a β-secretase BACE1 protein gel electrophoresis diagram, and (C) is a result diagram of the expression of β-secretase BACE1 protein in each group;

[0057] Figure 9 Figure 10 is a result diagram of in vitro cytotoxicity detection of CLNP, CLNP@siBACE1 and free Tetl-CLNP@siBACE1 on HT22 cells, N2a cells and 293T cells;

[0058] Figure 10 Figure 11 is a result diagram of Morris water maze experiment before administration, wherein (A) is a statistical diagram of the time to find the hidden platform each day during the training period, (B) is a statistical diagram of the number of times of crossing the platform area during the test period, and (C) is a statistical diagram of the time spent in the platform quadrant during the test period;

[0059] Figure 11 Figure 12 is a result diagram of Morris water maze experiment after administration for 4 weeks, wherein (A) is a statistical diagram of the time to find the hidden platform each day during the training period, (B) is a statistical diagram of the latency to reach the platform during the test period, and (C) is a statistical diagram of the number of times of crossing the platform area during the test period;

[0060] Figure 12 Figure 13 is a result diagram of immunohistochemical staining and analysis of Aβ 1-42 Figure 13 is a result diagram of immunohistochemical staining and analysis of Aβ 1-42 Figure 13 is a result diagram of immunohistochemical staining and analysis of Aβ 1-42Figure showing the results of plaque area quantitative analysis; (C) is the hippocampus of each group of mice Aβ 1-42 Figure showing the results of plaque area quantitative analysis;

[0061] Figure 13 Figure showing the HE staining of the main organs of mice using PBS buffer, Tetl-CLNP@siBACE1 and Tetl-CLNP@siNC, respectively. DETAILED DESCRIPTION

[0062] To make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments of the present application, all other embodiments obtained by a person skilled in the art without creative work fall within the scope of protection of the present application.

[0063] The sources of the main raw materials in the embodiments are as follows:

[0064] Tet1-distearylphosphatidylethanolamine-polyethylene glycol 2000 (Tet1-DSPE-PEG2000): Chupeibio custom-made its preparation method: 100 mg Mal-PEG2000-DSPE was weighed in a flask, dissolved in 5 mL dichloromethane, and the solvent was removed by rotary evaporation (40°C, under reduced pressure), a uniform lipid film was formed on the bottle wall, 10 mL pH 7.4 PBS buffer was added, and the solution was completely dissolved by slight shaking to form a transparent micelle solution. Tet1-Cys (molar mass calculated according to Mal-PEG2000-DSPE: Tet1-Cys = 1:1.2) was weighed, and Tet1-Cys was dissolved in 5 mL PBS buffer (pH 7.4) under light protection and N2 protection, and slightly vortexed to dissolve. The Mal-PEG2000-DSPE micelle solution was slowly added to the Tet1-Cys solution (dropping speed: 1 mL / min). Room temperature magnetic stirring (200 rpm, light protection, N2 atmosphere) reaction for 48 h, to ensure that the maleimide and thiol are fully reacted. Then the reaction solution was transferred to a dialysis bag (MWCO 3500 Da), and dialyzed with PBS buffer (pH 7.4) for 24 h (3-4 times of liquid change), to remove free peptides. The dialyzed solution was freeze-dried to obtain white solid Tet1-DSPE-PEG2000, which was stored at -20°C. The mass spectrum is shown in Figure 2 (A) is the mass spectrum of Tet1-Cys, and (B) is the mass spectrum of Tet1-DSPE-PEG2000), from Figure 2It can be seen that Tet1-DSPE-PEG2000 is successfully synthesized in the present application.

[0065] 1,2 -diacyl- 3 -trimethylammonium propane (DOTAP): Avitrol (Shanghai) Pharmaceutical Technology Co., Ltd., CAS No. 132172-61-3;

[0066] Dioleoylphosphatidylethanolamine (DOPE): Avitrol (Shanghai) Pharmaceutical Technology Co., Ltd., CAS No. 4004-05-1;

[0067] Distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000): Sigma-Aldrich (Shanghai) Trading Co., Ltd., CAS No. 385437-57-0;

[0068] Cholesterol: Avitrol (Shanghai) Pharmaceutical Technology Co., Ltd., CAS No. 57-88-5;

[0069] siBACE1: Beijing Donna Pharmaceutical Technology Co., Ltd.;

[0070] The Tet1-modified cationic liposome loaded with siBACE1 includes siBACE1, which is coated with a lipid composition outside, and the lipid composition is modified with a Tet1 peptide, as shown in Figure 1 The preparation method thereof includes the following steps:

[0071] S1: preparing a lipid composition, and the specific process is as follows:

[0072] S101: mixing 7.5 mg / mL of 1,2 -diacyl- 3 -trimethylammonium propane, 7.5 mg / mL of dioleoylphosphatidylethanolamine, 4 mg / mL of cholesterol, and 7.5 mg / mL of distearoylphosphatidylethanolamine-polyethylene glycol 2000; in this embodiment, the molar proportion of 1,2 -diacyl- 3 -trimethylammonium propane (DOTAP) in the whole lipid composition is 50%, the molar proportion of dioleoylphosphatidylethanolamine (DOPE) in the whole lipid composition is 10%, the molar proportion of cholesterol in the whole lipid composition is 38.5%, and the molar proportion of distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2000) in the whole lipid composition is 1.5%, based on the total amount of the lipid composition being 100%;

[0073] S102: adding 5 ml of chloroform and evaporating into a film by a rotary evaporator (40°C, 0.08 MPa, 6 rpm);

[0074] S103: The residual chloroform was removed by nitrogen blowing for 5 minutes to obtain a lipid composition, and the final mass of the lipid composition obtained in this embodiment was 10 mg;

[0075] S2: The siBACE1 was encapsulated by using the lipid composition to obtain a siBACE1-lipid complex, and the specific process was as follows:

[0076] S201: 5 mL of a citric acid buffer with pH 4.5 was added to the lipid composition obtained in S1, and a magnetic stirring was performed at 60°C for 1 hour to obtain a lipid composition solution;

[0077] S202: About 13.7 mg of siBACE1 was added to the lipid composition solution according to the N / P molar ratio of 1,2-dioleoyl-3-trimethylammonium propane to siBACE1 was 6, and the incubation was performed at room temperature for 30 minutes;

[0078] S203: The ice water bath probe ultrasonication was performed (30% power, working for 3 seconds / interval 2 seconds, 15 minutes), and then the filtration was performed through 1.2 μm→0.45 μm→0.22 μm filter heads in sequence to remove large particles to obtain the siBACE1-lipid complex;

[0079] S3: The siBACE1-lipid complex was modified by adding Tet1 ligand to obtain the Tet1 modified cationic liposome encapsulating siBACE1, and the specific process was as follows:

[0080] S301: The siBACE1-lipid complex was filtered by using a 100 kDa ultrafiltration centrifuge tube, and the siBACE1-lipid complex was replaced into a HEPES buffer with pH 7.4 (containing 5% sucrose, 0.1 mM EDTA), and the centrifugation conditions were as follows: 4°C, 4000xg, 10 minutes each time, and repeated for 3 times;

[0081] S302: The Tet1-distearylphosphatidylethanolamine-polyethylene glycol 2000 was dissolved in a 37°C preheated HEPES buffer with pH 7.4 (2 mg / mL), and the ultrasonication was performed for 5 minutes (avoiding light);

[0082] S303: The solution of Tet1-distearylphosphatidylethanolamine-polyethylene glycol 2000 obtained in S302 was added to the siBACE1-lipid complex solution obtained in S301, and the incubation was performed at 37°C under the condition of magnetic stirring for 2 hours (avoiding light operation);

[0083] S304: remove the un-inserted free Tet1-distearylphosphatidylethanolamine-polyethylene glycol 2000 (centrifugation conditions are the same as S301) using 100 kDa ultrafiltration centrifuge tube, and adjust the final volume to 5 mL (PBS buffer containing 10% sucrose) to obtain Tet1 modified siBACE1 loaded cationic liposomes.

[0084] The performance of the obtained Tet1 modified siBACE1 loaded cationic liposomes (Tetl-CLNP@siBACE1) was detected, specifically as follows:

[0085] 1. Tetl-CLNP@siBACE1 particle size and potential research:

[0086] Method: 10 μL of Tet1 modified siBACE1 loaded cationic liposome solution was taken into a 1.5 mL microcentrifuge tube with a micropipette, and 1000 μL of ultrapure water was added, and mixed with a micropipette. The Tet1 modified siBACE1 loaded cationic liposome solution was transferred into the particle size and potential special colorimetric cup in turn, and the particle size and surface potential of the nanoparticles were detected by Zetasizer ZS90 particle size instrument.

[0087] The results are shown in Figures 3-4 and Figure 3 The red, green and blue lines in Figure 4 respectively represent three parallel experiments, Figure 3 show that the size of the Tetl-CLNP@siBACE1 prepared by the application is about 67.46±0.35 nm, and the polydispersity index (PDI) is 0.12, Figure 4 showing that the average potential of Tetl-CLNP@siBACE1 is 10.5±0.27 mV.

[0088] 2. Tetl-CLNP@siBACE1 morphology research

[0089] In the application, transmission electron microscope (TEM) was used to observe the morphological structure of Tetl-CLNP@siBACE1. An appropriate amount of Tetl-CLNP@siBACE1 solution was taken and carefully added to the front of the copper mesh. After standing for 90 s, the remaining solution was gently absorbed on the edge of the copper mesh with filter paper. Then 1% uranyl acetate solution was added for negative staining, and after 60 s, the remaining staining solution was absorbed with filter paper. The copper mesh was naturally dried, and its morphology and particle size were observed by TEM. The results are shown in Figure 5The results are shown (where (A) is a transmission electron microscopy result at 50 nm magnification, and (B) is a transmission electron microscopy result at 20 nm magnification), Figure 5 It can be seen that the prepared Tetl-CLNP@siBACE1 has a typical multilayer vesicle structure, and the particle size distribution is uniform.

[0090] 3. The encapsulation rate of siBACE1 in Tetl-CLNP@siBACE1

[0091] The Quantbit TM RNA HS Assay Kit (Quantbit TM RNA HS Assay Kit) was used to measure the encapsulation rate of siBACE1 in Tetl-CLNP@siBACE1. The detection principle is as follows: first, the amount of free siBACE1 outside the lipid composition particles in the siBACE1 solution is detected, then the lipid composition structure is destroyed with Triton X-100, so that all siBACE1 is released to the outside solution, and the amount of all siBACE1 in the solution is detected. The difference between the two is the amount of siBACE1 encapsulated inside the lipid composition particles, and the amount of encapsulated siBACE1 divided by the total amount of siBACE1 is the encapsulation rate. The specific detection steps refer to the reagent use instruction of Thermo Fisher Company, and the operation steps are briefly described as follows:

[0092] 1) Prepare the RNA detection working solution: take [(sample number)+(2 standard curve)+(1)] μL of Qubit TM RNA HS reagent, add 200x([(sample number)+(2 standard curve)+(1)] μL of Qubit TM RNA HS bufer, prepare the working solution at a ratio of 1:200. For example, 6 samples to be tested, take (6+2+1)-9 μL of QubitM RNA HS reagent, add 200x(6+2+1) L=1800 μL of Qubit TM RNA HS bufer, prepare the working solution at a ratio of 1:200. For example, 6 samples to be tested, take (6+2+1)-9 μL of QubitM RNA HS reagent, add 200x(6+2+1) L=1800 μL of Qubit

[0093] 2) Make a standard curve and calibrate the standard curve on the Qubit 4.0.

[0094] 3) Detect the free RNA content in the LNP synthesis end product. If the concentration is too low to be detected, take 190 μL of working solution, add 10 μL of sample to be tested, mix well, and then detect. Select 1 μ in Qubit 4.0, and then divide the value by 10 to perform 10-fold conversion.

[0095] 4) Use TE buffer or DEPC water to prepare 4% Triton X-100, mix with LNP final product 1:1 (5 μL + 5 μL ready). Use this final concentration of 2% Triton X-100 to break emulsion for 5 minutes.

[0096] 5) Add 1 μL of 2% Triton X-100 in the regular standard curve, and perform calibration on the Qubit 4.0 with the new standard curve containing Triton X-100.

[0097] 6) Measure the RNA concentration after breaking emulsion of the lipid composition.

[0098] 7) Calculate the encapsulation efficiency, formula: encapsulation efficiency (%) = (drug loading amount / reading value after breaking emulsion) x 100%

[0099] The results are shown in Figure 6 , Figure 6 Table 1, which shows that the Tet1 modified cationic liposome loaded with siBACE1 prepared in the present application, when the N / P ratio of the cationic amino group (N) of DOTAP and the phosphate skeleton (P) of siRNA is 4:1, 6:1, 8:1, and 10:1, the encapsulation efficiency is 81.42 ± 0.77%, 95.29 ± 0.34%, 97.49 ± 0.29%, and 89.17 ± 0.64%, respectively. Therefore, when the N / P ratio is 8:1, the encapsulation efficiency of Tetl-CLNP for siBACE1 is the highest.

[0100] 4, Tet1 peptide activity verification

[0101] Neuron binding experiment: co-incubate fluorescently labeled liposomes with neurons, and observe the targeting efficiency under a confocal microscope.

[0102] (1) Cell culture and plating

[0103] Resuscitate and culture neuron cell models N2a cells, microglial cell line BV2 cells, and human embryonic kidney 293T cells (non-neuronal control), using DMEM medium containing 10% FBS (37°C, 5% CO2). Plate in a confocal dedicated culture plate at a density of 5 x 10 4 cells per well, and incubate for 24 hours to 70% confluence.

[0104] (2) Co-culture with cells

[0105] Cy5 (red fluorescent dye) labeled Tetl-CLNP@siNC (its preparation method is exactly the same as Tetl-CLNP@siBACE1) was diluted to the working concentration (such as 50 nM siRNA) with serum-free medium. siNC is a designed non-targeting siRNA, which has no homology with the known genes of the target organism, and is consistent with siBACE1 but has no effect of knocking down β-secretase BACE1. Add 2 ml of medium containing Tetl-CLNP@siNC to each well, and incubate at 37°C for 6 hours.

[0106] (3) Cell fixation and permeabilization

[0107] Aspirate the medium, wash gently with PBS for 3 times; fix with 4% paraformaldehyde (PFA) for 15 minutes at room temperature, and wash with PBS for 3 times again; permeabilize with 0.1% Triton X-100 (prepared in PBS) for 10 minutes (to enhance antibody penetration), and wash with PBS.

[0108] (4) Fluorescent staining

[0109] Nucleus staining: add DAPI (4', 6-diamidino-2-phenylindole) and incubate in the dark for 15 minutes, and wash with PBS for 3 times; cytoskeleton staining: incubate with phalloidin in the dark for 30 minutes (diluted according to the instructions), and wash with PBS for 3 times.

[0110] (5) High-content cell imaging

[0111] Take pictures of the nucleus, cytoskeleton and LNP endocytosis signal of each channel respectively, and take multi-view Z-stack images to ensure covering the three-dimensional structure of the cells, and compare the endocytosis efficiency of each group of cells to Tetl-CLNP@siNC.

[0112] The results of the images taken are shown in Figure 7 (A), the results of the fluorescence intensity are shown in Figure 7 (B), and as shown in Figure 7 After the three kinds of cells were co-incubated with Tetl-CLNP@siNC for 6 hours, the uptake of Cy5-siNC signal in N2a cells was more significant than that in BV2 cells and 293T, indicating that Tetl-CLNP@siNC has a tendency for neuron cells.

[0113] 5. Silencing effect of Tetl-CLNP@siBACE1 on β-secretase BACE1 in neuron cells

[0114] mRNA level (qPCR)

[0115] (1) Tetl-CLNP@siNC and Tetl-CLNP@siBACE1 were added to 12-well plates containing N2a cells, with a siRNA concentration of 100 nM, and incubated at 37 °C for 6 hours. CALNP™ RNAi transfection reagent was used as a control.

[0116] (2) The siRNA-transfection complex solution was prepared according to the CALNP™ RNAi transfection reagent instructions, and 100 μL of the prepared siRNA-transfection complex solution was added to each cell well. The plate was shaken and mixed gently. After incubation at 37 °C for 24 h, the culture solution was aspirated, 0.5 ml of trypsin was added, and the entire solution was transferred to a 1.5 ml centrifuge tube, which was centrifuged at 1000 g / min for 5 min. The trypsin was aspirated, 0.5 ml of cell culture solution was added, and the solution was centrifuged for 5 min. The cell culture solution was aspirated, and 0.5 ml of PBS buffer was added.

[0117] (3) The RNA of N2a cells was extracted using the RNA extraction kit instructions, and the RNA was reverse transcribed into cDNA. The cDNA was subjected to qPCR detection.

[0118] (4) The relative expression of the BACE1 gene was calculated in the Excel table according to the ∆∆Ct method.

[0119] The results are shown in Figure 8 (A). Figure 8 (A) shows that the level of BACE1 mRNA in N2a cells treated with Tetl-CLNP@siBACE1 is significantly reduced, and the gene silencing effect is comparable to that of siBACE1.

[0120] Protein level (Western Blot)

[0121] Experimental method: Tetl-CLNP@siNC and Tetl-CLNP@siBACE1 were added to six-well plates containing N2a cells, with a siRNA concentration of 160 nM, and incubated at 37 °C for 6 hours. CALNP™ RNAi transfection reagent was used as a control. After incubation, the supernatant was discarded, and RIPA mixture was added. The cells were scraped with a cell scraper, centrifuged to obtain the upper protein solution, and placed in a new microcentrifuge tube. The protein concentration was detected. According to the Western Blot detection process, the processes of sample loading, electrophoresis, membrane transfer, blocking, and antibody incubation were completed. Finally, the full-automatic chemiluminescence image analysis system was used for development.

[0122] The results are shown in Figure 8As shown in (B) and (C), the results indicate that the BACE1 protein level in N2a cells treated with Tetl-CLNP@siNC was significantly reduced compared to the Tetl-CLNP@siBACE1 group. Both qPCR and Western blotting results confirmed that Tetl-CLNP@siBACE1 can effectively silence BACE1 expression in N2a cells.

[0123] 6. Cytotoxicity assay

[0124] Log-phase HT22 cells (hippocampal neurons), N2a cells, and 293T cells were transplanted into 96-well plates at a density of 5000 cells per well. The toxicity of LNP-siRNA primarily originates from LNPs; siRNA toxicity is generally low. After 24 h of cell incubation, the culture medium was removed, and different concentrations of empty CLNP (lipid composite), CLNP@siBACE1, and Tetl-CLNP@siBACE1 were added. LNP concentrations were set at 100.0 μmol / L, 50.0 μmol / L, 12.5 μmol / L, 6.3 μmol / L, 3.1 μmol / L, 1.8 μmol / L, and 0.8 μmol / L, respectively, and incubated for 24 h. The culture medium was discarded, and 10% CCK-8 (CellCounting Kit-8) was added to each well for 4 h of incubation. The absorbance was measured at 450 nm using a microplate reader. Cell viability = [(absorbance value of drug-treated group - absorbance value of pure culture medium) / (absorbance value of blank group - absorbance value of pure culture medium)] × 100%.

[0125] The results are as follows Figure 9 As shown in AC, Figure 9 In the figure, A represents the cell survival rate of HT22 cells in different concentrations of empty CLNP (lipid composition), CLNP@siBACE1, and Tetl-CLNP@siBACE1. Figure 9 As can be seen from A in the figure, the survival rate of HT22 cells does not vary much under different conditions, and basically remains at around 90%. Figure 9 In the figure, B represents the cell survival rate of N2a cells in different concentrations of empty CLNP (lipid composition), CLNP@siBACE1, and Tetl-CLNP@siBACE1. Figure 9 As can be seen from B in the figure, the survival rate of N2a cells does not change much under different conditions, and the survival of N2a cells is similar to that of HT22 cells, basically maintained at around 90%. Figure 9C in FIG. 6 shows the cell survival rate of 293T cells in different concentrations of empty CLNP (lipid composition), CLNP@siBACE1, and Tetl-CLNP@siBACE1, from Figure 9 C in FIG. 6 shows the cell survival rate of 293T cells in different concentrations of empty CLNP (lipid composition), CLNP@siBACE1, and Tetl-CLNP@siBACE1, from Figure 9 C in FIG. 6 shows the cell survival rate of 293T cells in different concentrations of empty CLNP (lipid composition), CLNP@siBACE1, and Tetl-CLNP@siBACE1, from

[0126] 7. Cognitive function of AD mice

[0127] Behavioral test: Morris water maze

[0128] (1) Animal selection and grouping

[0129] AD transgenic mice (APP / PS1) and littermate wild-type (WT) controls were selected, with half male and half female, aged 4-6 months, and divided into AD model group, PBS control group (negative control), Tetl-CLNP@siNC group (targeted delivery control), Tetl-CLNP@siBACE1 group (treatment group), and WT control group (baseline control), with 12 mice in each group.

[0130] (2) Drug treatment

[0131] Tetl-CLNP@siNC and Tetl-CLNP@siBACE1 were injected through the tail vein at a dose of siRNA 0.5-1 mg / kg, twice a week for 4 weeks.

[0132] (3) Morris water maze experiment process

[0133] Morris water maze is a widely used behavioral test method for assessing the spatial learning and memory ability of experimental animals (such as mice), and plays an important role in the study of hippocampus-related cognitive function. The main body of the device is a circular pool, which is evenly divided into four quadrants along the circumference. A hidden escape platform is placed at a fixed position in one of the quadrants (usually the third quadrant). Visual markers of different colors and shapes are set around the pool wall, serving as spatial reference cues to assist animals in orientation recognition and spatial memory formation, thereby guiding them to accurately locate the position of the escape platform.

[0134] One day before the formal experiment, the animals were placed in the pool for adaptive swimming to familiarize them with the experimental environment. The following four days were used for positioning navigation training, with four training sessions per day. The time required for the animals to find the platform was automatically recorded by the system. On the fifth day, the spatial exploration test was conducted, mainly to record the memory performance of the animals in the original platform location after the platform was removed.

[0135] (1) Positioning navigation experiment: This phase lasted for 4 days, with 4 training sessions per day. During each training session, the animals were placed in the water facing the pool wall markers in a predetermined order from four different quadrants (1→2→3→4 in clockwise direction), and the video recording system was started simultaneously to record the movement trajectory. If the animal successfully located and climbed onto the platform within 90 seconds and stayed on the platform for at least 3 seconds, the recording was stopped, and the actual escape latency was recorded. If the animal failed to find the platform within the specified time, the experimenter guided it to the platform and stayed for 15 seconds for learning. After the end of the experiment, the animal was dried and placed back in the cage. Through continuous training for several days, the formation process of spatial learning and memory ability could be systematically evaluated.

[0136] (2) Spatial exploration experiment: On the day after the positioning navigation training, the original escape platform in the pool was removed. The animal was gently placed in the water from the fixed entry point (usually the first quadrant), taking care not to directly face the original platform area to reduce the initial orientation interference on the movement trajectory. Within 90 seconds of the experiment, the animal could freely explore the pool, and its movement path was tracked throughout by the overhead camera system. The number of times the animal crossed the original platform center was counted as an indicator of its spatial memory retention ability.

[0137] Before administration, as shown in A-C of Figure 10 , the escape latency of the AD group was significantly longer than that of the WT group, confirming that the AD model mice had spatial learning and memory impairment. After administration, as shown in A-C of Figure 11 , the treatment group effect: the latency of the AD+Tetl-CLNP@siBACE1 group was significantly shorter than that of the AD+PBS group (p=0.001) and the AD+Tetl-CLNP@siNC group (p=0.006), indicating that the siRNA targeting BACE1 could effectively improve cognitive function. There was no significant difference between the Tetl-CLNP@siNC group and the AD+PBS group (p>0.05), ruling out the influence of non-specific LNP. Tetl-CLNP@siBACE1 significantly improved the spatial learning and memory ability of AD mice (shortened latency, optimized cognitive strategy) by targeting and inhibiting BACE1, and the effect was better than that of the non-treatment control group.

[0138] 8. Aβ 1-42 deposition

[0139] To evaluate the clearance effect of Tetl-CLNP@siBACE1 on the deposition of β-amyloid (Aβ) in the brain of AD mice, and to verify that it reduces Aβ by targeting and silencing BACE1 (β-secretase) 1-42 The therapeutic potential generated.

[0140] (1) Select AD transgenic mice (APP / PS1) and littermate wild type (WT) controls, half male and half female, aged 4-6 months, and divide them into WT control group, AD model group, AD+Tetl-CLNP@siNC group, and AD+Tetl-CLNP@siBACE1 group, 6 in each group.

[0141] (2) Tetl-CLNP@siNC and Tetl-CLNP@siBACE1 are injected through the tail vein at a dose of siRNA 0.5-1 mg / kg, twice a week for 4 weeks.

[0142] (3) Brain tissue sampling and sectioning: After anesthesia of the mice, 4% paraformaldehyde (PFA) is used for heart perfusion, the brain is taken and fixed for 24 hours. Gradient sucrose dehydration, paraffin embedding, coronal section (thickness 5 μm), covering the cortical area and hippocampal area.

[0143] (4) Immunohistochemical staining of sections: sodium citrate buffer (pH 6.0) heat repair for 20 minutes, 5% BSA room temperature blocking for 1 hour to reduce non-specific binding; anti-Aβ antibody (such as 6E10, 1:500) 4°C overnight; HRP-labeled secondary antibody (1:1000) room temperature for 1 hour, hematoxylin counterstaining of cell nuclei.

[0144] (5) Microscope shooting: Aβ 1-42 deposition (brown particles) in the cortical area and hippocampal area (such as CA1, DG). ImageJ analyzes the percentage of Aβ 1-42 positive area, and one-way ANOVA is used for comparison between groups.

[0145] Previous studies have shown that Aβ 1-42 plaque number is closely related to cognitive level, and the present application uses immunohistochemistry to observe Aβ 1-42 pathological plaque area in the hippocampus, and representative staining pictures are shown in Figure 12 A, the Aβ 1-42 plaque area of the cortical area of each group of mice is quantitatively analyzed, as shown in Figure 12 B, and the Aβ 1-42 plaque area of the hippocampus of each group of mice is quantitatively analyzed, as shown in Figure 12 C, from Figure 12 A, B, and C, it can be seen that there is no Aβ 1-42 plaque deposition in the hippocampal area and cortical area of the WT control group of mice, and the Aβ 1-42The plaque area increased, and the AD model group and the AD + Tetl-CLNP@siBACE1 group were compared. The AD + Tetl-CLNP@siNC group of mice hippocampus, cortex Aβ 1-42 The plaque area decreased. It is shown that Tetl-CLNP@siBACE1 can reduce the deposition of Aβ in the hippocampus and cortex of mice 1-42 .

[0146] 8. Toxic side effect detection

[0147] In order to explore whether Tetl-CLNP@siBACE1 administration will cause toxic side effects to mice, we respectively intravenously injected equal amounts of PBS buffer, Tetl-CLNP@siNC, Tetl-CLNP@siBACE1, and continuously administered for 4 weeks. The mouse heart, lung, liver, spleen and kidney tissues were taken out 24 hours after the last administration, and HE staining was performed to observe whether there were pathological changes in the organs.

[0148] The observation results are shown in Figure 13 , and the mice treated in each administration group did not cause obvious organic changes in important organs, indicating that Tetl-CLNP@siBACE1 has good safety in vivo and no obvious toxic side effects.

[0149] The above-described examples only express the specific embodiments of the present application, and the description is more specific and detailed, but it cannot be understood as a limitation on the protection scope of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the technical concept of the present application, a number of modifications and improvements can be made, which are all within the protection scope of the present application.

Claims

1. Tetl-modified cationic liposomes encapsulating siBACEl, characterized in that: The siBACE1 is coated outside by a lipid composition, and the lipid composition is modified by a Tet1 peptide; the Tet1 peptide is modified on the lipid composition in the form of a Tet1 ligand, which is Tet1-distearylphosphatidylethanolamine-polyethylene glycol 2000, and the lipid composition is composed of 1,2-dioleoyl-3-trimethylammonium propane, dioleoylphosphatidylethanolamine, cholesterol, and distearylphosphatidylethanolamine-polyethylene glycol 2000; The molar percentage of 1,2-dioleoyl-3-trimethylammonium propane in the whole lipid composition is 50% based on the total amount of the lipid composition being 100%; The molar percentage of dioleoylphosphatidylethanolamine in the whole lipid composition is 10%; The molar percentage of cholesterol in the whole lipid composition is 38.5%; The molar percentage of distearylphosphatidylethanolamine-polyethylene glycol 2000 in the whole lipid composition is 1.5%; The amount of Tet1-distearylphosphatidylethanolamine-polyethylene glycol 2000 is 0.45%-0.75% of the amount of the lipid composition based on the molar percentage; The N / P molar ratio of 1,2-dioleoyl-3-trimethylammonium propane to siBACE1 is 6-8; The Tet1-modified siBACE1-loaded cationic liposome is used for preparing an intravenous injection drug for treating Alzheimer's disease.

2. A method of preparing Tetl-modified siBACEl-encapsulated cationic liposomes as claimed in claim 1, characterized by: The method comprises the following steps: S1: preparing a lipid composition; S2: loading siBACE1 by using the lipid composition to obtain a siBACE1-lipid complex; S3: modifying the siBACE1-lipid complex by adding a Tet1 ligand to obtain the Tet1-modified siBACE1-loaded cationic liposome according to claim 1.

3. The method of claim 2, wherein the Tetl-modified siBACEl-encapsulating cationic liposome is prepared by: The specific steps of S1 are as follows: S101: mixing 1,2-dioleoyl-3-trimethylammonium propane, dioleoylphosphatidylethanolamine, cholesterol, and distearylphosphatidylethanolamine-polyethylene glycol 2000 according to the mixing ratio; S102: adding chloroform and forming a film by rotary evaporation; S103: removing residual chloroform by nitrogen blowing to obtain the lipid composition.

4. The method of claim 2, wherein the Tetl-modified siBACEl-encapsulating cationic liposome is prepared by the following steps of: The specific steps of S2 are as follows: S201: adding a citric acid buffer to the lipid composition obtained in S1 and stirring at 60°C to obtain a lipid composition solution; S202: adding a siBACE1 solution to the lipid composition solution and incubating at room temperature; S203: homogenizing the solution to obtain a siBACE1-lipid complex.

5. The method of claim 2, wherein the Tetl-modified siBACEl-encapsulating cationic liposome is prepared by: The specific steps of S3 are as follows: S301: replacing the siBACE1-lipid complex obtained in S2 into a 4-hydroxyethylpiperazineethanesulfonic acid buffer to obtain a replaced siBACE1-lipid complex solution; S302: dissolving Tet1-distearylphosphatidylethanolamine-polyethylene glycol 2000 in the 4-hydroxyethylpiperazineethanesulfonic acid buffer to obtain a Tet1-distearylphosphatidylethanolamine-polyethylene glycol 2000 solution; S303: The Tet1-distearylphosphatidylethanolamine-polyethylene glycol 2000 solution obtained in S302 is added to the replaced siBACE1-lipid complex solution for light-protected incubation; S304: The free Tet1-distearylphosphatidylethanolamine-polyethylene glycol 2000 is removed to obtain the Tet1-modified siBACE1-loaded cationic liposome.

6. Use of the Tet1-modified siBACE1-loaded cationic liposome of claim 1 or the Tet1-modified siBACE1-loaded cationic liposome prepared by the method of any one of claims 2-5 in the preparation of a medicament for intravenous injection for the treatment of Alzheimer's disease.

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