CN118021989A

CN118021989A - Preparation of ROS-responsive bionic nano vesicle and application of ROS-responsive bionic nano vesicle in treatment of Alzheimer's disease

Info

Publication number: CN118021989A
Application number: CN202410100412.6A
Authority: CN
Inventors: 王子华; 蒋素君; 曾华杰; 胡志远
Original assignee: Fujian Medical University
Current assignee: Fujian Medical University
Priority date: 2024-01-24
Filing date: 2024-01-24
Publication date: 2024-05-14

Abstract

The invention relates to the technical field of biological medicines, in particular to preparation of a bionic nano vesicle responded by ROS and application of the bionic nano vesicle in Alzheimer disease treatment. The ROS-responsive bionic nano vesicle is prepared by mixing stem cell exosomes with drug-loaded liposomes, loading TREM2 plasmid and BACE1siRNA through an extrusion method. The bionic nano vesicle is simple to prepare, has high gene transfection efficiency and good biocompatibility, has the capacity of penetrating the blood brain barrier, reduces the quantity of Abeta plaque by reversing microglial cell phenotype and reduces nervous system inflammation, and provides a new idea for treating neurodegenerative diseases.

Description

Preparation of ROS-responsive bionic nano vesicle and application of ROS-responsive bionic nano vesicle in treatment of Alzheimer's disease

Technical Field

The invention relates to the technical field of biological medicines, in particular to preparation of a bionic nano vesicle responded by ROS and application of the bionic nano vesicle in Alzheimer disease treatment.

Background

With the increase in global aging, degenerative diseases closely related to aging are becoming a serious challenge in jeopardizing social health. Among them, alzheimer's Disease (AD) is a typical neurodegenerative disease, and is mainly characterized by progressive cognitive impairment and hypomnesis. The international Alzheimer's disease society issues a report on Alzheimer's disease worldwide of 2021, which shows that every 3 seconds, a dementia patient is produced worldwide. Currently about 5500 tens of thousands of dementia patients worldwide, it is expected that this number may increase to over 1.52 billion in the middle of this century, of which about 70% are AD patients. The current annual loss of the disease is about $1 trillion, and this figure is expected to double by 2030. The aging problem is aggravated continuously, so that the disease situation of AD becomes extremely severe.

Because of the complexity of the pathogenesis of AD, many hypotheses have been proposed, including the aβ cascade hypothesis, the Tau hypothesis, the cholinergic hypothesis, the neuroinflammatory hypothesis, and the like. The pathological features of AD are mainly represented by the deposition of amyloid plaques (aβ) and the aggregation of phosphorylated Tau protein. Extracellular plaques deposited in the brain parenchyma consist mainly of amyloid β (aβ) polypeptides derived from Amyloid Precursor Protein (APP), whereas NFTs consist of hyperphosphorylated tau proteins aggregated within neurons. In addition to the selective deposition of amyloid, neuroinflammation associated with microglial proliferation is a common feature of many neurodegenerative diseases. There is no clinically effective treatment for AD to date. None of the drugs is able to stop or delay the progression of AD disease. Therefore, finding new AD drug targets and developing targeted drugs have extremely important economic and social significance.

Currently, clinical therapies using acetylcholinesterase inhibitors or N-methyl-D-aspartate receptor antagonists are palliative treatment options that only moderately improve cognition and behavior in patients with alzheimer's disease, but do not slow down the progression of the disease. Some studies have utilized aβ antibodies for the treatment of AD, but clinical applications are limited due to low potency and difficulty in crossing the blood brain barrier. Therefore, development of therapeutic methods for different pathological mechanisms of AD is imperative.

In AD, microglia participate in and regulate neuroinflammatory reactions, monitor the surrounding environment in real time, migrate and aggregate to focal sites, and clear aβ deposits and damaged neurons in the central nervous system, thereby improving AD symptoms and slowing down the process. There is growing evidence that the maintenance of microglial cell function mediated by trigger receptor 2 (TREM 2) expressed in myeloid cells plays a very important protective role in AD, and TREM2 is a new popular target for AD drug development. TREM2 is a membrane protein that is selectively expressed in bone marrow cells (including microglia). Genetic variation and mutation of TREM2 is associated with early and late AD. TREM2 signals through its ligand DAP12 (TYROBP) to regulate key functions of microglia such as metabolism, proliferation, survival, cytokine release and accumulation around amyloid plaques. It was found that increasing TREM2 gene expression levels reprogrammed microglial cell reactivity and improving pathological phenotypes in alzheimer's disease model. Meanwhile, researchers develop TREM2 agonist antibodies to show good treatment effects in AD mice, can activate microglial cells to phagocytose plaques, and improve cognitive functions of the mice. Thus, activating the TREM2 pathway may be an effective option for treating AD.

Amyloid Precursor Protein (APP) lyase 1 (BACE 1) has been identified as a central promoter of amyloid β (aβ) production in the brain, a key pathological hallmark of AD. However, recent studies provide evidence that BACE1 also plays a critical role in metabolic regulation, and by knocking down the BACE1 enzyme at the gene level by siRNA, the production of oligomeric aβ is reduced, further damage to neurons is reduced, and symptoms of AD can be ameliorated.

Gene therapy is an important emerging strategy for treating neurodegenerative diseases, and is particularly suitable for fully validated genetic targets that cannot be treated by traditional therapies. Existing gene delivery methods rely primarily on transgenic technology or intra-brain injection of viral vectors. These methods suffer from unavoidable limitations of clinical transformations, such as genotoxicity, immune or inflammatory responses, and patient non-compliance.

The rapid development of nano technology and nano medicine utilizes the unique physical and chemical advantages of nano materials to encapsulate drugs or bioactive agents to enhance the ability of the drugs to penetrate the blood brain barrier, so that the nano materials have wide application prospects in the diagnosis and treatment of neurodegenerative diseases. The combination of gene therapy with nanotechnology has created a first line of hopes for overcoming these obstacles. Liposomes, which are an effective nanocarrier, exhibit high biocompatibility, non-toxic characteristics in terms of drug delivery, and also possess good surface conjugation capabilities, have the ability to controllably release their payload in response to internal and/or external stimuli, can encapsulate drugs and facilitate targeted delivery across the Blood Brain Barrier (BBB). The exosome is used as a nano-scale lipid bilayer closed structure carrying proteins, lipids, RNA, metabolites, growth factors and cytokines, is a safe and multifunctional natural membranous vesicle, further enhances the drug carrying performance of the liposome, constructs liposome nano-particles with high loading rate and stable properties, and realizes the accurate brain delivery of small-molecule drugs. ROS responsiveness realizes long-distance targeted delivery of the drug, avoids accumulation and loss in nonspecific tissues, and ensures high enrichment of the drug in lesion sites.

Disclosure of Invention

In order to overcome the problems that the conventional anti-AD drugs are low in potency and difficult to cross the blood brain barrier at present and overcome the defects that a single drug possibly cannot achieve enough therapeutic effect and a gene delivery method, the invention provides a ROS-responsive bionic nano vesicle as a combined drug delivery system, realizes the combination of gene therapy and nanotechnology, and provides the application of the bionic nano vesicle in the treatment of neurodegenerative diseases, and the specific invention is as follows.

Firstly, the invention provides a preparation method of a ROS-responsive bionic nano vesicle, which comprises the following steps:

(1) Mixing and reacting the Ang2 polypeptide with DSPE-Se-Se-PEG-Mal to obtain DSPE-Se-Se-PEG-Ang2;

(2) Dissolving DSPE-Se-Se-PEG-Ang2, DOTAP (2, 3-dioleoyl-propyl-trimethyl ammonium chloride), DPPC (dipalmitoyl phosphatidylcholine) and cholesterol in an organic solvent, and removing the organic solvent by reduced pressure evaporation to obtain a liposome membrane;

(3) Dissolving the liposome membrane, TREM2 plasmid and BACE1 siRNA in an aqueous phase, and forming liposome nano particles at the phospholipid phase transition temperature;

(4) And mixing the liposome nano particles with exosomes, and extruding to obtain the ROS-responsive bionic nano vesicles.

According to the invention, the Ang2 polypeptide (Angiopep-2) is connected to the surface of the liposome nano-particles, and the liposome nano-particles are fused with exosomes, so that the prepared bionic nano-vesicle can greatly improve the ability of penetrating through a Blood Brain Barrier (BBB), thereby synergistically improving the administration efficiency of gene therapy. In addition, the invention selects TREM2 plasmid and BACE1 siRNA as a loaded drug combination, prevents the formation of oligomeric Abeta and simultaneously eliminates the generated toxic Abeta plaque, reduces the neuroinflammation, simultaneously reduces the formation of Abeta plaque from the source and eliminates the formed toxic Abeta plaque, finally protects hippocampal neurons from apoptosis, and cooperatively plays a role in treating or improving neurodegenerative diseases. In addition, the "-Se-Se-" bond of DSPE-Se-Se-PEG-Mal in the bionic nano vesicle can be broken under the H ₂O₂ (ROS) environment, so that the bionic nano vesicle is promoted to be broken and disintegrated, an encapsulated substance is released to a target position, and the ROS response characteristic enables the medicine to be accurately released in a lesion area, so that the curative effect of the medicine is further improved.

In some embodiments, the amino acid sequence of the Ang2 polypeptide is: TFFYGGSRGKRNNFKTEEYC (SEQ ID No. 1). Angiopep-2 (Ang 2) is a polypeptide capable of entering the brain through low density lipoprotein receptor-related protein (LRP) receptor-mediated endocytosis expressed on the blood brain barrier.

Preferably, the exosomes are mesenchymal stem cell exosomes.

In some embodiments, the method of preparing the exosomes comprises:

exosomes in the supernatant of mesenchymal stem cells were extracted by differential centrifugation and exosome-related markers (Calnexin, CD9, CD63, CD81, hsp70, TSG 101) were detected by Western Blot, the composition of the extracted material was identified and the morphology of the exosomes was characterized by TEM.

In some embodiments, the liposome membrane is dissolved in an aqueous phase with TREM2 plasmid and BACE1siRNA, and the blocked liposomes are prepared at a phospholipid phase transition temperature, and then the blocked liposomes are passed through a 0.22 μm polycarbonate membrane to form liposome nanoparticles.

In some embodiments, step (3) specifically comprises: the liposome membrane was dissolved in aqueous phase with TREM2 plasmid and BACE1 siRNA, the lipid film was hydrated off at phospholipid phase transition temperature, self-assembled to form closed liposome, and liposome nanoparticles were formed by intermittent ultrasound (preferably, ultrasound 3s apart for 3s for 5 min) and passing the liposome through a 0.22 μm polycarbonate membrane (preferably 3 times).

Preferably, the liposome nanoparticle has a particle size of 200nm or less.

Preferably, the mass ratio of Ang2 polypeptide to DSPE-Se-PEG-Mal is 1: (1.5-2.5).

The invention further discovers that the mass ratio of the Ang2 polypeptide to the DSPE-Se-Se-PEG-Mal can influence the drug release effect of the bionic nano-vesicle in the ROS environment and the capability of the bionic nano-vesicle to penetrate the blood brain barrier, and the effects of the two aspects can be optimized at the same time.

Preferably, the DSPE-Se-Se-PEG-Ang2 is obtained by taking sulfhydrylation Ang2 polypeptide as a raw material through Michael addition reaction.

Preferably, the mole ratio of DSPE-Se-Se-PEG-Ang2, DOTAP, DPPC and cholesterol is (2-4): (18-22): (12-18): (28-32).

Preferably, the organic solvent is chloroform or absolute methanol.

And/or the phospholipid phase transition temperature is 44-46 ℃.

Preferably, the mass ratio of the liposome nano-particles to the exosomes is (8-10): 1.

Under the proportion, the particle size of the bionic nano-vesicles can be controlled within a proper range (130-140 nm), so that the uniformity of the bionic nano-vesicles is good, and the blood brain barrier penetrating capacity and the drug carrying capacity can be well balanced; if the particle size is too large, the ability to penetrate the blood brain barrier will decrease although the drug carrying ability is improved; if the particle size is too small, the ability to penetrate the blood brain barrier is good, but too little drug is loaded, which affects the synergistic effect of the drugs.

Preferably, the volume ratio of TREM2 plasmid to BACE1 siRNA is (1.8-2.2): 1.

Preferably, the concentration of BACE1 siRNA is 1 to 1.5nM; the concentration of the TREM2 plasmid is 0.5-1 mug/mL.

The invention also discovers that when the volume ratio of the two medicaments is controlled within the range, the loading rate and the encapsulation rate of the medicaments can be both the highest.

Further, the present invention provides a ROS-responsive biomimetic nanovesicle produced by the method of any of the above embodiments.

The bionic nano vesicle provided by the invention can obviously improve multiple functions of microglia, and is specifically characterized in that: promote inflammatory response, inhibit apoptosis, promote cell migration, and increase endocytosis and degradation of oligomeric aβ42 by microglia.

The bionic nano vesicle provided by the invention has the advantages of positive biocompatibility, capability of protecting the medicine from being damaged, prolonging the effective medicine maintaining time, reducing the toxic and side effects of the medicine and the like, can be further functionalized to realize safe, effective and tissue-targeted delivery, and provides a new way for treating neurodegenerative diseases.

Further, the present invention provides a pharmaceutical composition comprising: the ROS-responsive bionic nano-vesicles and pharmaceutically acceptable auxiliary materials or auxiliary components.

Preferably, the pharmaceutically acceptable auxiliary or auxiliary ingredient is at least one of an excipient, diluent, carrier, flavoring agent, binder or filler.

Preferably, the vector is a different functional vector heterozygous for exosomes or nanomaterials secreted by cells of biological origin.

Further, the invention provides any one of the preparation methods, or the application of the ROS-responsive bionic nano-vesicle in preparing medicines; the medicament is used for treating or improving neurodegenerative diseases.

Preferably, the neurodegenerative disease comprises at least one of alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis, or huntington's disease.

Compared with the prior art, the invention has the beneficial effects that:

The bionic liposome exosome heterozygous nano vesicle is used as a medicine carrying tool, and has the functions of protecting small molecular substances such as plasmids and the like from enzyme degradation in the transportation process, phagocytic uptake of immune cells and the like under the encapsulation and transfer functions of the nano vesicle, so that long-distance transportation in blood is realized. In addition, the bionic liposome and exosome heterozygous nano vesicle simulate the structural characteristics of a phospholipid bilayer in cells, and the effective uptake of encapsulated substances by the cells is promoted by virtue of the principle of similar compatibility. The ROS response characteristic ensures that the medicament can be accurately released in a lesion area, the curative effect of the medicament is improved, and the nano vesicle has the effects of no toxicity and low stimulation, and avoids the further injury effect on neurons.

In addition, the bionic nano vesicle regulates the transition of microglial cells from M1 to M2 polarization through up-regulating TREM2, promotes the phagocytosis of Abeta plaque by microglial cells, and down-regulates pro-inflammatory cytokines. Meanwhile, siRNA down regulates BACE1 expression, reduces Abeta plaque formation from the source and reduces neuronal damage. The invention provides a safe and efficient non-viral gene transfer system, increases the possibility of clinical application of TREM2 targeted gene therapy, and provides a new method for AD treatment.

Drawings

FIG. 1 is a Western Blot and TEM characterization of exosomes extracted from mesenchymal stem cells.

Fig. 2 is a TEM and DLS characterization of TL, TSL and TESL nanoparticles.

FIG. 3 is a graph showing the results of functional verification of genes carried by TL, TSL and TSEL after uptake by cells.

Fig. 4 is a graph of test results of ROS responsiveness, drug encapsulation efficiency.

FIG. 5 is a graph of the in vitro BBB penetration performance characterization of TL and TSEL.

FIG. 6 is a graph showing the results of the identification of the penetration properties of TSL and TSEL in the brain BBB of AD mice.

FIG. 7 is a graph of immunofluorescent staining identification of TSEL-regulated microglial cell phenotypes.

FIG. 8 is a graph of flow cytometry identification of TSEL-regulated microglial cell phenotypes.

FIG. 9 is a graph showing the results of behavioral assessment of cognitive dysfunction in APP/PS1 mice treated with TSEL.

FIG. 10 is a graph showing the evaluation results of immunofluorescence analysis of Abeta plaque in the brain of an APP/PS1 mouse treated with TSEL.

FIG. 11 is a graph showing the results of drug loading and encapsulation efficiency of the various ratios of TREM2 plasmid and siBACE1 loaded with nanovesicles.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

DSPE-Se-Se-PEG2000-Mal, cholesterol (cholesterol, abbreviated as Chol), DPPC, DOTAP are all available from Siamiliaxi Biotechnology Co. Ang2 peptide was purchased from Anhui Guo Ping pharmaceutical industry. APC anti-mouse CD206 (MMR) antibody (141708), FITC anti-mouse CD16/32 antibody (101305) were purchased from BIOSS. Goat anti-rabbit IgG H & L/AF Cy5 and goat anti-mouse IgG H & L/AF 488 were purchased from BIOSS. Glial BV2 cells, neuroblastoma SH-SY5Y cells and mouse brain endothelial b End.3 cells were purchased from the national academy of sciences cell bank. Cells were cultured in medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 1% (v/v) penicillin/streptomycin, and in humidified incubator at 37℃under 5% CO ₂. APP/PS1 mice were purchased from Beijing HFK Biotech Co.

The sense and antisense strands of the BACE1 siRNA are:

GCUUUGUGGAGAUGGUGGATT (SEQ ID No.2 sequence end +TT); UCCACCAUCUCCACAAAGCTT (SEQ ID No. 3. End of sequence +TT). The TREM2 plasmid is obtained by inserting a TREM2 gene sequence into a pcDNA3.1-EGFP vector;

The TREM2 gene sequence is shown as SEQ ID No. 4.

Example 1 preparation and characterization of mesenchymal Stem cell extraction exosomes

Exosomes were extracted from human mesenchymal stem cell culture supernatant by differential centrifugation. First, 2000 Xg, 4℃for 30min, and the supernatant was collected. Next, 10000 Xg was centrifuged again at 4℃for 45min, and the supernatant was collected. The supernatant was collected by filtration through a 0.45 μm filter. The exosome precipitate was collected at 100000 Xg, 4℃for 70min. The obtained exosome precipitate was suspended in PBS, washed 2 times and stored at-80 ℃. The exosome-associated markers (Calnexin, CD9, CD63, CD81, hsp70, TSG 101) were detected by Western Blot, the composition of the extract was identified, and the exosome morphology was characterized by TEM, see figure 1.

Example 2 preparation and characterization of biomimetic nanovesicles (TSEL)

The liposome is prepared by adopting a film dispersion method. Thiol-modified Ang2 polypeptide and DSPE-Se-Se-PEG2000-Mal are used as raw materials, DSPE-Se-Se-PEG-Ang2 is obtained through Michael addition reaction (reference for synthetic method) A ROS-responsive Targeted Nanoscavenger to promote Mitophagy for the treatment of Alzheimer'sdisease.Small 2023,19,2302284.).

DSPE-Se-Se-PEG-Ang2:Chol: DPPC: DOTAP was weighed in a molar ratio of 3:30:15:20, the total mass was 20mg, and thoroughly shaken in 3mL of CHCl ₃ until completely dissolved. After the mixture was vaporized in a rotary evaporator at 45℃for more than 45min, it was observed that a thin, uniformly distributed lipid film was formed on the inner wall of the round-bottomed flask. Then, nitrogen was injected for 2min to completely dry the liposome membrane.

TREM2 pDNA (TREM 2 pDNA is a free TREM2 plasmid solution without liposome coating) and BACE1 siRNA were dissolved in 3mL PBS solution, TREM2 pDNA concentration was 2.6. Mu.g/3 mL, BACE1 siRNA concentration was 1.25nM, and dissolved liposome membrane was added. At phospholipid transformation temperature, the lipid membrane is hydrated and falls off, self-assembled to form a closed spherical structure, and the closed liposome is formed by oscillating for 30min at 45 ℃. On this basis, liposome nanoparticles were sonicated for 3s at 3s intervals for 5min, repeatedly extruded 3 times with a 0.22 μm polycarbonate film, forming liposome nanoparticles, labeled TSL. By the same method of preparation, liposome nanoparticles coated only with TREM2 pDNA, labeled TL, were obtained.

In order to obtain hybrid nanovesicles of exosomes and liposomes, the obtained liposome nanoparticles and exosomes are mixed according to the mass ratio of 9:1, and the hybrid nanovesicles are extruded for multiple times to obtain the bionic nanovesicles (TSEL).

Adding 2 mu L of the nanoparticle solution into a 300-mesh copper mesh, standing for 10min, carrying out negative dyeing on copper wires by using a 1% uranium acetate solution, and drying at room temperature. The morphology of the particles was analyzed by Transmission Electron Microscopy (TEM). The nanoparticle solution was diluted 1:100. 1mL of the dilution was taken and injected into a Markov sample cell. The size and zeta potential of the nanoparticles were determined using Dynamic Light Scattering (DLS), see fig. 2.

To further verify the function of the gene carried by TSEL after uptake by cells, BV2 and SH-SY5Y cells were seeded in 8-well plates at a density of 1X 10 ⁴ and after 24h incubation experiments were performed. Incubate with TL, TSL and TSEL for 24h, respectively. Protein was extracted for WB experiments. The expression of BACE1 in SH-SY5Y cells was found to decrease from 1.06 to 0.48 (FIG. 3). At the same time, WB results in microglial BV2 indicate that TSL, TSEL, can up-regulate TREM2 protein expression. These results show that the hybrid nanovesicles have higher gene transfer efficiency, can improve expression of TREM2 and reduce expression of BACE1, thereby providing a nontoxic and convenient method for cell gene therapy.

Example 3 ROS responsiveness, encapsulation efficiency, loading identification of biomimetic nanovesicles (TSEL)

TSEL containing 2.6 μg TREM2 pDNA was transferred to dialysis bags with mwco=3500 Da. The dialysis bag was completely immersed in 600mL of PBS buffer containing 30% h ₂O₂. Constant temperature of 37 ℃ and continuously oscillating for 0-24h at 80 r/min. 1mL of the system was added after each 1mL of the solution was taken at regular intervals. The absorbance value (OD) of the sample was then measured at 488nm excitation light and 525nm emission light. Percent released (%) = fluorescence intensity of free small molecules in liquid after release/fluorescence intensity of total small molecules in liquid.

The TREM2 plasmid and BACE1 siRNA modified GFP, cy5 fluorophores, respectively, and TSEL drug contents showed different release efficiencies in 1mM H ₂O₂ and PBS environments. Wherein, when H ₂O₂ is treated for 2 hours, the TREM2 release amount in TSEL is 30.22%, and the TREM2 release amount reaches 83.14% when H ₂O₂ is treated. At 8h in PBS as well, the TREM2 cumulative release in TSEL was only 29.75% (fig. 4B). The same BACE1 siRNA has similar results (fig. 4C). And detecting the encapsulation performance of TSEL on TREM2 plasmid and BACE1 siRNA, wherein the Encapsulation Efficiency (EE)% and Drug Loading (DLC)% of TSEL are 81.65% and 1.09%, respectively (FIG. 4A).

Example 4 in vitro and in vivo identification of the penetration of biomimetic nanovesicles (TSELs) through the BBB Performance

The blood brain barrier is a major challenge in treating most brain diseases. The experiment establishes an in vitro blood brain barrier model by using mouse brain capillary endothelial cells (bEnd.3). An external blood brain barrier model was constructed using Transwell plates. Mouse cerebrovascular endothelial b end.3 cells were seeded into the upper lumen of the Transwell plate. After 2 days of culture, a dense monolayer was formed and SH-SY5Y cells were seeded into the lower chamber. Monolayer b end.3 cells can reasonably be considered an in vitro blood brain barrier. The blood brain barrier model is considered successful when the transendothelial resistance of the cell reaches at least the 200 Ω command. TSEL (2. Mu.g/. Mu.L) was dispersed in fresh medium, added to the top chamber, and the lower layer of the top chamber was added to Transwell normal medium. After incubation for 0.5, 1,2, 4 hours, fluorescence intensity of TSEL was measured with a confocal laser microscope. The recorded images were analyzed with ImageJ software. The mass of TSEL transported to SH-SH5Y cells by the blood brain barrier model was estimated. The results showed that the numbers of b end.3 cells and inferior luminal human neuroblastoma cells (SH-SY 5Y) after TSEL culture were higher compared to TL, i.e., blood brain barrier endocytosis of TSEL was nearly 2-fold better than TL, see FIG. 5.

In vivo experiments, 10 month old APP/PS1 mice (APP/PS 1 mice are double transgenic mice, expressing chimeric mouse/human amyloid precursor protein (Mo/HuAPP 695 swe) and mutant human presenilin 1 (PS 1-dE 9)) were injected with Cy 5-labeled nanoparticles and monitored using a live fluorescence imaging system to assess brain targeting ability of TSEL in vivo, with fluorescent signals in the brain of APP/PS1 mice treated with free siRNA as controls. In vivo imaging showed that TSEL is significantly more fluorescent than TSL in the brain. In contrast, mice treated with free siRNA showed minimal Cy5 fluorescence in the brain, consistent with in vitro observations, see fig. 6. It is demonstrated that TSEL can achieve drug delivery through the BBB with high efficiency. The bionic nano vesicle prepared by the invention can penetrate through the blood brain barrier, has certain active targeting property, enhances the targeting effect of the nano vesicle through synergistic effect after being combined with the targeting polypeptide, and can target AD.

Example 5TSEL treatment of AD model mice reduced aβ burden and neuroinflammation

1. Microglial BV2 cell immunofluorescence staining analysis of phenotype transition

BV2 cells were cultured in 8-well plates at an seeding density of 1X 10 ⁴. After cell adhesion, BV2 cells were pretreated with 1. Mu. M A β42 oligomer for 12h and then incubated with PBS, TL, TSL and TSEL (0.8. Mu.g/. Mu.L), respectively, for 24h. The 4% paraformaldehyde solution was fixed for 15 minutes. Blocking with 5% BSA for 30 min. 0.1% Triton X-100PBS was infiltrated for 15 min. Cells were then incubated overnight at 4℃simultaneously with APC anti-mouse CD206 and FITC anti-mouse CD16/32 antibody (1:500, biolgend). After washing with PBS, the images were obtained by staining with Hoechst 33342 staining solution (1:20 00, solarbio) at 37℃for 10min, followed by observation under CLSM. Brain sections were incubated overnight with recombinant anti-iba 1 antibody (mouse mab) (1:1000, servicebio), anti-Abeta mouse mab (1:1000, servicebio), anti-NeuN rabbit mab (1:1000, servicebio) and anti-GFAP mouse mab (1:1000, servicebio) at 4℃and stained with DAPI solution (1 μg/mL, solarbio) to observe CLSM. After pretreatment of microglial cells BV 24h with Abeta 42 oligomer, immunofluorescence imaging and quantitative analysis show that after stimulation of the oligomer Abeta 42, CD16/32 positive green fluorescence of BV2 cells is stronger, CD206 positive red fluorescence is negligible, and CD16/32 positive green fluorescence after TSEL treatment is slightly weakened. Only intense CD206 positive red fluorescence was observed after TSEL treatment, suggesting that TSEL treatment caused polarization of microglia toward M2, inhibiting the production of neuroinflammation. After 12h of drug induction, microglial phenotype was changed from M1 to M2, iNOS expression was decreased and CD206 expression was increased, see fig. 7. It was demonstrated that TSEL was able to significantly increase endocytosis of oligomeric aβ42 by microglia.

2. Microglial BV2 cell immunofluorescence staining analysis of phenotype transition

BV2 cells were seeded in 6-well plates at a density of 1X 10 ⁶. BV2 cells were pretreated with 1. Mu. M A. Beta.42 oligomer for 12h and then incubated with PBS, TL, TSL and TSEL (0.8. Mu.g/. Mu.L) respectively for 24h. After treatment, the cells are fixed, permeabilized and blocked. Staining with APC anti-mouse CD206 antibody (1:500, biolgend) and FITC anti-mouse CD16/32 antibody (1:500. Staining was performed, cells were then washed three times with PBS, collected, suspended in PBS for flow cytometry detection, stained cells were detected by flow cytometry, analyzed by FlowJo software. Flow cytometry results showed that the proportion of iNOS+BV2 cells increased from 84.1% to 97.4% in the control group after stimulation with oligomeric Aβ42, whereas the proportion of iNOS+BV2 cells decreased to 70.1% after TSEL treatment. The proportion of CD206+BBBV2 cells in the control group was 17.1% with the addition of oligomeric Aβ42, CD206 ⁺ BV2 cells increased to 17.5% without significant change after stimulation with CD206 ⁺ BV2 cells to 24.7% data, indicating that TSEL can reduce the percentage of inflammatory microglia cells, see FIG. 8.

3. Water maze experiments identify the ability of TSEL to improve cognitive/PS 1 mice for cognitive impairment

10 Month old APP/PS1 mice were randomly divided into 5 groups (n=6), and Wild Type (WT) C57BL/6 mice (10 months old) were normal control groups. Each group of mice was intravenously injected with physiological saline, TL, TSL, TSEL (dose of 2. Mu.g/mL nanoparticle 0.2 mL). The WT group was given an equivalent amount of physiological saline only. Every other day for 28 days. After the last dose, training and testing was performed using the Morris water maze video analysis system. This process involved five days of training, guiding the mice to a platform below the water surface. On day six, the platform was evacuated for testing. The monitoring system observes the water entering moment of the mice, records the movement track and escape latency of the mice, and further evaluates the memory capacity of the mice. The results show that APP/PS1 mice have longer escape latency than WT groups after 5 days, with impaired spatial cognition. The escape latency of TSEL treated APP/PS1 mice was significantly shorter than that of APP/PS1 mice group, indicating that TSEL can significantly improve spatial cognitive dysfunction in AD mice, see figure 9.

After the end of the behavioural experiment, mice were euthanized, the major viscera and brain tissues were excised and fixed, the hippocampus of the different treated mice were frozen to 3 μm and immunofluorescent stained with primary antibody at 4 ℃. Immunofluorescence analysis the effect of TSEL on reduction of aβ deposition in the brains of AD mice was evaluated. The results show a significant reduction in aβ plaques in the cortex and hippocampus of TSEL treated mice compared to PBS treated mice. The number of aβ plaques in the cortex and hippocampus of TSEL treated mice was significantly reduced compared to the other groups (fig. 10). These results indicate that TSEL nanovesicles, by delivering pTREM and siBACE1 to the brain, can not only reverse microglial phenotype by TREM2, increase their phagocytosis of toxic aβ plaques, but also down-regulate aβ production by silencing BACE1, thus alleviating the burden of aβ in AD brain.

In conclusion, the bionic nano vesicle transfer system has good biocompatibility and brain targeting. TSEL regulates the transition of microglial cell polarization from M1 to M2 by up-regulating TREM2, promotes microglial phagocytosis of aβ plaques, down-regulates pro-inflammatory cytokines. Meanwhile, siRNA down regulates BACE1 expression, reduces Abeta plaque formation from the source and reduces neuronal damage. In a word, the invention provides a safe and efficient non-viral gene transfer system, increases the possibility of clinical application of TREM2 targeted gene therapy, and provides a new method for AD treatment.

Example 6

Referring to the preparation method of the above example, the present example examined the Encapsulation Efficiency (EE)% and Drug Loading (DLC)% of the biomimetic nanovesicles prepared by the TREM2 plasmid and BACE1 siRNA in different ratios and adjusting the addition amount of exosomes.

Encapsulation efficiency (EE%) = (1-Cf/Ct) ×100%. Wherein Cf is the free drug amount, and Ct is the total drug amount in the suspension in the nano vesicle. After lyophilization, the nanovesicle sample W ₁ mg was dissolved in a 10ml volume bottle and destroyed by the addition of dimethyl sulfoxide. Drug content W ₂ mg was examined. Drug loading (DLC%) =w ₂/W₁ ×100%.

As can be seen from FIG. 11, the encapsulation efficiency and drug loading vary with the dosage of nucleic acid loaded. The dosage of TREM2 plasmid and BACE1 siRNA is adjusted to be 20 mu L and 10 mu L respectively, and the drug loading rate and the encapsulation rate can be the highest.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a biomimetic nano vesicle responsive to ROS, comprising:

(2) Dissolving DSPE-Se-Se-PEG-Ang2, DOTAP, DPPC and cholesterol in an organic solvent, and removing the organic solvent by reduced pressure evaporation to obtain a liposome membrane;

2. The method of claim 1, wherein the exosomes are mesenchymal stem cell exosomes.

3. The preparation method according to claim 1, wherein the mass ratio of Ang2 polypeptide to DSPE-Se-PEG-Mal is 1: (1.5-2.5).

4. The preparation method according to claim 1, wherein the molar ratio of DSPE-Se-PEG-Ang 2, DOTAP, DPPC, cholesterol is (2-4): (18-22): (12-18): (28-32).

5. The method according to claim 1, wherein the organic solvent is chloroform or absolute methanol;

and/or the phospholipid phase transition temperature is 44-46 ℃.

6. The preparation method according to claim 1, wherein the mass ratio of the liposome nanoparticle to the exosome is (8-10) 1;

And/or the volume ratio of TREM2 plasmid to BACE1 siRNA is (1.8-2.2): 1.

7. A biomimetic nano-vesicle responsive to ROS, characterized in that it is produced by the preparation method of any one of claims 1 to 6.

8. A pharmaceutical composition comprising: the ROS-responsive biomimetic nanovesicle of claim 7, wherein the auxiliary or auxiliary component is pharmaceutically acceptable.

9. Use of the preparation method of any one of claims 1 to 6, or the ROS-responsive biomimetic nanovesicle of claim 7, in the preparation of a medicament; the medicament is used for treating or improving neurodegenerative diseases.

10. The use according to claim 9, wherein the neurodegenerative disease comprises at least one of alzheimer's disease, parkinson's disease, amyotrophic lateral sclerosis or huntington's disease.