Engineered exosomes targeting alzheimer's disease lesions and clearing beta amyloid plaques, methods of making and uses thereof

Abstract

The application provides an engineered exosome targeting Alzheimer's disease lesions and removing beta amyloid plaques, a preparation method and application thereof, the engineered exosome being a core-shell structure with a photothermal nanoparticle as a core of the engineered exosome, a hybrid exosome after fusion of brain microvascular endothelial cell exosomes and macrophage exosomes as a shell, an A beta recognition probe modified on the surface of the core-shell structure, and a small molecule drug loaded in the core-shell structure. The preparation method comprises the following steps: mixing the photothermal nanoparticle with a small molecule drug solution, incubating overnight, adding brain microvascular endothelial cell exosomes and macrophage exosomes, ultrasonicating and incubating to obtain the hybrid exosome loaded with the photothermal nanoparticle and the small molecule drug; adding the A beta recognition probe for incubation and centrifugation. The engineered exosome has good biocompatibility, high blood-brain barrier permeability, lesion site targeting and multi-target intervention capacity, and can be used for treating Alzheimer's disease.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to an engineered exosome for targeting Alzheimer's disease lesions and removing beta amyloid plaques as well as a preparation method and application thereof. BACKGROUND

[0002] Alzheimer's disease (AD) is a neurodegenerative disease. According to statistics, about 55 million people worldwide are affected by AD. However, there is still no effective method to cure AD, which may be due to the complexity of the pathogenesis of AD, and the treatment drugs targeting a single pathogenic factor are not enough to improve the multiple pathological changes related to AD. Therefore, it is of great practical significance to develop multifunctional AD treatment drugs.

[0003] Due to the special structure and function of the blood-brain barrier (BBB), most therapeutic drugs cannot effectively penetrate the BBB, which negatively affects the bioavailability of the drug and the therapeutic effect. Existing works mostly use synthetic nanoparticles (such as gold-based nanomaterials, carbon materials, and polymers) as drug delivery carriers to treat AD. However, these nanoparticles generally have problems such as complex synthesis, poor biocompatibility, easy induction of immune response, and poor BBB permeability, which limit their further application in vivo. Therefore, it is imperative to develop drug delivery carriers with good biocompatibility and BBB permeability.

[0004] Exosomes, as an endogenous extracellular vesicle, have the ability to act as a drug delivery carrier due to their natural vesicular structure. Compared with other synthetic nanoparticle drug carriers, exosomes have lower immunogenicity and toxicity, and have the ability to naturally cross the BBB to deep brain tissues. Moreover, some exosomes can perform directional tropism migration with the help of their self-generated membrane surface receptors or extracellular matrix binding proteins to be recognized and taken up by specific receptor cells, and are a promising brain drug delivery carrier. Studies have found that exosomes mainly accumulate non-specifically in the spleen and liver after systemic administration. Although some exosomes have therapeutic effects in intervening disease development, their functions are relatively single. It can be seen that it is far from enough to rely only on the intrinsic properties of exosomes to develop exosome-based treatments. Therefore, it is urgent to reasonably engineer and modify exosomes for AD intervention needs to obtain engineered exosomes with specific functions to achieve effective intervention of AD. SUMMARY

[0005] The technical problem to be solved by the present application is to overcome the poor biocompatibility and low BBB permeability of anti-AD drugs or delivery carriers in the prior art, and the poor targeting of exosomes in AD treatment, single function and other deficiencies, and to provide an engineered exosome targeting Alzheimer's disease lesions and removing beta amyloid plaques, and a preparation method and application thereof.

[0006] The technical solution adopted by the present application to solve the technical problem is:

[0007] The photothermal nanoparticles are used as the core of the engineered exosome to endow the engineered exosome with good photothermal properties, enabling controllable release of loaded drugs and enabling photothermal depolymerization of A beta (A beta) aggregates; the natural homing effect of brain microvascular endothelial cell exosomes and the natural inflammation tropism of macrophage exosomes are utilized, and the two kinds of exosomes are fused by membrane fusion technology to obtain hybrid exosomes, the hybrid exosomes are used as the shell and A beta recognition probes are modified on the surface of the hybrid exosomes by membrane targeting modification technology, which avoids the influence of modification density and steric hindrance when two or more than two ligands are modified on the exosomes, and improves the BBB penetration ability and lesion site (inflammation area and A beta aggregate enrichment area in the brain) enrichment level of the engineered exosomes. In addition, small molecule drugs are simultaneously loaded in the cavity of the exosome and the natural phospholipid bilayer of the exosome membrane, which improves the drug loading capacity of the engineered exosome, and delivers therapeutic drugs to the brain injury site in an efficient and low-toxicity manner. The engineered exosome restores the function of microglial cells by promoting the clearance of A beta, scavenging reactive oxygen species and reducing the expression of nitric oxide synthase (iNOS), restores the activity of neurons by photothermal depolymerization of A beta aggregates, provides a new strategy for AD treatment, and lays a systematic and scientific foundation for the application of engineered exosomes in AD treatment.

[0008] The present application provides an engineered exosome targeting Alzheimer's disease lesions and removing beta amyloid plaques, the engineered exosome comprising photothermal nanoparticles, hybrid exosomes, A beta recognition probes and small molecule drugs; the engineered exosome takes the photothermal nanoparticles as the core of the engineered exosome, takes the hybrid exosomes as the shell to form a core-shell structure, the A beta recognition probes are modified on the surface of the core-shell structure, and the small molecule drugs are loaded in the cavity of the engineered exosome and / or the natural phospholipid bilayer of the membrane; the hybrid exosomes are obtained by membrane fusion of brain microvascular endothelial cell exosomes and macrophage exosomes.

[0009] The above-mentioned engineered exosome further comprises one of polydopamine nanoparticles, pyrrolopyrrole dione polymer, gold nanorods, black phosphorus nanomaterials and copper sulfide nanoparticles.

[0010] The engineered exosome, further, the small molecule drug is one or more of resveratrol, curcumin, and quercetin.

[0011] The engineered exosome, further, the brain microvascular endothelial cells are bEnd.3 cells, and the macrophages are RAW264.7 cells.

[0012] The engineered exosome, further, the Aβ recognition probe is an Aβ aptamer, an antibody, or a polypeptide.

[0013] The gene sequence of the Aβ aptamer is shown in SEQ ID NO. 1.

[0014] The antibody is 1F12.

[0015] The polypeptide is one of KLVFF, KLVFFADE, LPFFD, and LVFFA.

[0016] The engineered exosome, further, the Aβ recognition probe is labeled with cholesterol.

[0017] The engineered exosome, further, the mass ratio of the photothermal nanoparticles to the small molecule drug is 1-4:1.

[0018] The engineered exosome, further, the weight ratio of the brain microvascular endothelial cell exosomes to the macrophage exosomes is 1:1.

[0019] The engineered exosome, further, the mass ratio of the hybrid exosomes to the photothermal nanoparticles is 1:1.

[0020] Based on one general technical concept, the present application also provides a preparation method of the engineered exosome.

[0021] S1, mixing photothermal nanoparticles and a small molecule drug solution, incubating overnight to obtain a mixed solution;

[0022] S2, adding brain microvascular endothelial cell exosomes and macrophage exosomes to the mixed solution, performing ultrasonic treatment and incubation to obtain hybrid exosomes loaded with photothermal nanoparticles and small molecule drugs;

[0023] S3, adding an Aβ recognition probe for incubation, and centrifuging to obtain engineered exosomes.

[0024] The preparation method, further, the ultrasonic treatment in S2 is performed under ice bath conditions at 100W for 2s, with an interval of 2s, and the incubation is performed at 37°C for 1h.

[0025] The preparation method further includes that the incubation time in S3 is 30 minutes, and the incubation temperature is 37 DEG C.

[0026] Based on the overall technical concept, the application further provides application of the engineered exosome in preparation of a drug for targeting Alzheimer's disease lesions and / or removing beta amyloid plaques.

[0027] Based on the overall technical concept, the application further provides application of the engineered exosome in preparation of a drug for promoting microglial cells to remove A beta aggregates.

[0028] Based on the overall technical concept, the application further provides application of the engineered exosome in preparation of a drug for removing active oxygen in microglial cells and reducing iNOS expression in microglial cells.

[0029] Based on the overall technical concept, the application further provides application of the engineered exosome in preparation of a drug for photothermally disaggregating A beta aggregates and restoring neuron activity.

[0030] The engineered exosome has high BBB permeability, can target A beta aggregates and inflammatory sites in AD brain lesions, reduces non-specific loss, and has enhanced multiple targeting ability.

[0031] The engineered exosome can promote microglial cells to remove A beta, restore microglial cell function by removing active oxygen and reducing iNOS expression, and restore neuron activity by photothermally disaggregating A beta aggregates.

[0032] Compared with the prior art, the application has the following advantages:

[0033] The application provides an engineered exosome, which is safe and non-toxic, has high biocompatibility, and can solve the problems of low drug efficacy, large adverse reactions and low drug delivery efficiency in AD treatment. The preparation process of the engineered exosome obtained by engineering the exosome is simple and mature, has the effect of protecting the loaded drug from being phagocytosed by immune cells, can penetrate the BBB, targets A beta aggregates and brain inflammatory sites, and delivers therapeutic drugs to the brain in a high-efficiency and low-toxicity manner, so as to realize multi-target combined treatment of AD. The method provides a new strategy for AD treatment. BRIEF DESCRIPTION OF DRAWINGS

[0034] In order to make the purpose, technical scheme and advantages of the embodiments of the application clearer, the technical scheme of the embodiments of the application will be described clearly and completely below with reference to the drawings of the embodiments of the application.

[0035] Figure 1Synthesis path of the engineered exosome of embodiment 1 of the present application and the principle of its targeting Alzheimer's disease lesions and removing beta amyloid plaques

[0036] Figure 2 TEM characterization diagram of the engineered exosome of embodiment 1 of the present application.

[0037] Figure 3 DLS characterization diagram of the engineered exosome of embodiment 1 of the present application.

[0038] Figure 4 CLSM characterization diagram of the engineered exosome of embodiment 1 of the present application.

[0039] Figure 5 Ultraviolet absorption spectrum characterization diagram of the engineered exosome of embodiment 1 of the present application.

[0040] Figure 6 SDS-PAGE characterization diagram of the engineered exosome of embodiment 1 of the present application.

[0041] Figure 7 Photothermal property identification result diagram of the engineered exosome of embodiment 1 of the present application.

[0042] Figure 8 Release rate identification result diagram of resveratrol in the engineered exosome of embodiment 1 of the present application.

[0043] Figure 9 Identification result diagram of the engineered exosome of embodiment 1 of the present application escaping phagocytosis by macrophages.

[0044] Figure 10 Identification result diagram of the engineered exosome of embodiment 1 of the present application penetrating BBB.

[0045] Figure 11 Identification result diagram of the engineered exosome of embodiment 1 of the present application targeting inflammatory sites.

[0046] Figure 12 Identification result diagram of the engineered exosome of embodiment 1 of the present application targeting Aβ.

[0047] Figure 13 Identification result diagram of the engineered exosome of embodiment 2 of the present application promoting the ability of microglial cells to remove Aβ aggregates.

[0048] Figure 14 Identification result diagram of the engineered exosome of embodiment 3 of the present application removing reactive oxygen species in microglial cells.

[0049] Figure 15 Identification result diagram of the engineered exosome of embodiment 3 of the present application reducing iNOS expression in microglial cells by immunofluorescence staining.

[0050] Figure 16 Figure for AFM imaging and ThT fluorescence identification of in vitro photothermal depolymerization of Aβ aggregates by engineered exosomes of Example 4 of the present application.

[0051] Figure 17 Figure for identification of the ability of Example 4 of the present application to decompose Aβ aggregates at the cellular level.

[0052] Figure 18 Figure for identification of the ability of Example 4 of the present application to restore the viability of neurons.

[0053] Figure 19 Figure for identification of the ability of Example 4 of the present application to remove Aβ-induced reactive oxygen species in SH-SY5Y cells. DETAILED DESCRIPTION

[0054] The present application will be further described in conjunction with specific preferred embodiments, but the scope of protection of the present application is not limited thereby.

[0055] The materials, reagents and instruments used in the following examples can be obtained from commercial channels. The experimental methods in the following examples are conventional methods in the art, unless otherwise specified. The dopamine hydrochloride is purchased from Biotang Technology Co., Ltd.; the resveratrol is purchased from Aladdin Reagent (Shanghai) Co., Ltd.; the cholesterol-labeled Aβ aptamer is synthesized by Genechem Technology Co., Ltd.; the amyloid Aβ is purchased from Gill Biochemical (Shanghai) Co., Ltd.; the iNOS antibody and CoraLite 647-labeled goat anti-rabbit secondary antibody are purchased from Proteintech; the reactive oxygen species detection kit is purchased from Biyun Tian Biological Technology Co., Ltd.; the FITC is purchased from Beijing Solabio Technology Co., Ltd.; the Thioflavin T (ThT) is purchased from Sigma Company; the human neuroblastoma cell line SH-SY5Y is purchased from China Typical Culture Collection Center; the bEnd.3 cell line and RAW264.7 cell line are purchased from the Cell Bank of Chinese Academy of Sciences.

[0056] EMBODIMENT

[0057] An engineered exosome targeting Alzheimer's disease lesions and removing β-amyloid plaques, a core-shell structure with a photothermal nanoparticle as the core of the engineered exosome and a hybrid exosome after fusion of brain microvascular endothelial cell exosomes and macrophage exosomes as the shell, an Aβ recognition probe modified on the surface of the core-shell structure, and a small molecule drug loaded in the cavity of the exosome and the natural phospholipid bilayer of the exosome membrane.

[0058] Figure 1The application discloses an engineering exosome and a preparation method thereof, and relates to the field of Alzheimer's disease (AD) treatment.

[0059] Therefore, the engineering exosome can effectively intervene AD through various ways, including: ① promoting microglial cells to remove Aβ and reducing the Aβ load of the brain; ② down-regulating iNOS expression and removing active oxygen to normalize the function of microglial cells; and ③ photothermal depolymerization of toxic Aβ aggregates to enhance the viability of neurons.

[0060] Further, the photothermal nanoparticles are one or more of polydopamine nanoparticles, pyrrolopyrrole diketone polymers, gold nanorods, black phosphorus nanomaterials and copper sulfide nanoparticles. The photothermal nanoparticles can depolymerize Aβ aggregates and controllably release small molecule drugs, and therefore, nanoparticles with photothermal properties can produce the same or similar technical effects.

[0061] Further, the small molecule drugs are one or more of resveratrol, curcumin and quercetin. The small molecule drugs can remove active oxygen and relieve neural inflammation.

[0062] Further, the brain microvascular endothelial cells are bEnd.3 cells, and the macrophages are RAW264.7 cells.

[0063] Further, the Aβ recognition probe is an Aβ aptamer, an antibody or a polypeptide.

[0064] The gene sequence of the Aβ aptamer is shown in SEQ ID NO. 1.

[0065] The antibody is 1F12.

[0066] The polypeptide is one of KLVFF, KLVFFADE, LPFFD and LVFFA.

[0067] Further, the Aβ recognition probe is labeled with cholesterol. The role of cholesterol labeling is that cholesterol as a lipid-soluble cross-linking agent can anchor the insertion into the phospholipid bilayer of the exosome, so that the recognition probe is stably retained on the exosome membrane.

[0068] Example 1

[0069] An engineered exosome targeting Alzheimer's disease lesions and removing β-amyloid plaques is a core-shell structure with polydopamine nanoparticles (PDA) as the core of the engineered exosome and hybrid exosomes as the shell. The hybrid exosomes are obtained by fusing brain microvascular endothelial cell (bEnd.3 cell) exosomes and macrophage (RAW264.7 cell) exosomes. The cholesterol-labeled Aβ recognition probe is modified on the surface of the core-shell structure, and the small molecule drug resveratrol (Res) is loaded in the exosome cavity and the natural phospholipid bilayer of the exosome membrane. Res can remove reactive oxygen species and relieve neuroinflammation.

[0070] The sequence of the cholesterol-labeled Aβ recognition probe (chol-Apt40) is shown in SEQ ID NO. 1, specifically: 5'-TTTTTTTTGCTGCCTGTGGTGTTGGGGCGGGTGCG-3'.

[0071] The preparation method comprises the following steps:

[0072] (1) Extraction of brain microvascular endothelial cell exosomes and macrophage exosomes.

[0073] 1.1, Preparation of exosome-free DMEM complete medium: ultracentrifugation method (100000xg, 4℃, 4h) is used to remove exosomes in fetal bovine serum (FBS), the precipitate is removed, and exosome-free serum is obtained. The exosome-free serum is used to prepare DMEM complete medium, which contains 10% exosome-free FBS by volume and 1% penicillin-streptomycin by volume.

[0074] 1.2, Obtain culture medium rich in brain microvascular endothelial cell exosomes and macrophage exosomes: use DMEM containing 10% fetal bovine serum as culture medium, culture brain microvascular endothelial cells and macrophages at 37℃, 5% CO2, when the cells adhere to the culture dish for 60%, replace the exosome-free DMEM complete medium and continue to culture at 37℃ for 48h, then collect the culture medium of the two kinds of cells into centrifuge tubes, respectively, to obtain culture medium rich in brain microvascular endothelial cell exosomes (bEnd.3Exo) and macrophage exosomes (RAW264.7Exo).

[0075] 1.3, Extraction of exosomes: centrifugation at 2000xg for 10 min at 4°C to remove dead cells, centrifugation at 10000xg for 60 min to remove cell debris, filtration through 0.22 pm filter to remove microvesicles with a diameter greater than 200 nm, centrifugation at 100000xg for 70 min, discard the supernatant, resuspend the pellet with PBS, and obtain bEnd.3Exo and RAW 264.7Exo, store at -80°C for later use.

[0076] (2) Synthesis of PDA: 90 mL of ultrapure water, 40 mL of anhydrous ethanol, and 3 mL of ammonia water were sequentially added to the reaction bottle, and stirred at 30°C for 30 min. 10 mL of 50 mg / mL dopamine hydrochloride aqueous solution was added to the stirred solution, and continued to stir for 24 h. The precipitate was collected by centrifugation at 12000 rpm, and purified with deionized water to obtain PDA, which was freeze-dried and weighed for later use.

[0077] (3) Preparation of engineered exosomes:

[0078] 3.1, 200 pg / mL PDA and 80 pg / mL Res solution were mixed and incubated overnight, bEnd.3Exo and RAW 264.7Exo (bEnd.3Exo and RAW 264.7Exo weight ratio 1:1, 200 pg / mL) were added, and ultrasonic treatment (100W, ultrasonic 2s interval 2s) was performed for 10 min under ice bath condition to make the two exosome membranes fuse and load PDA and Res therein. After incubation at 37°C for 1 h, the precipitate was collected after centrifugation and washing.

[0079] 3.2, The precipitate was resuspended in PBS solution (maintaining PDA concentration at 200 pg / mL), and cholesterol-labeled chol-Apt40 (600 nM) was added. After incubation at 37°C for 30 min with shaking at 300 rpm, the precipitate was collected after centrifugation and washing.

[0080] 3.3, The precipitate was resuspended in PBS solution to obtain engineered exosomes (RPDA@Rb-A) targeting Alzheimer's disease lesions and removing beta-amyloid plaques.

[0081] Experiment one: characterization of RPDA@Rb-A.

[0082] 1.1, TEM characterization.

[0083] Transmission electron microscopy (TEM) characterized the morphology of RPDA@Rb-A, and the results are shown in Figure 2The images show that the two types of exosomes are cup-shaped, which proves the successful extraction of exosomes; PDA is spherical and well dispersed; the characterization image of RPDA@Rb-A shows obvious membrane coating on PDA, which has a typical core-shell structure, indicating the successful synthesis of engineered exosomes.

[0084] 1.2 DLS characterization.

[0085] The dimensions of PDA, PDA@Rb, and RPDA@Rb-A were determined using dynamic light scattering (DLS). See the results below. Figure 3 The figures show that the particle sizes of PDA, RPDA@Rb (engineered exosomes without chol-Apt40 modification), and RPDA@Rb-A are 86.61±5.16 nm, 108.67±6.32 nm, and 117.37±4.58 nm, respectively. PDA@Rb is approximately 22 nm larger than PDA, indicating successful encapsulation of PDA by the exosomes. RPDA@Rb-A is approximately 8 nm larger than RPDA@Rb, indicating successful modification of the PDA@Rb surface with chol-Apt40, thus demonstrating the successful synthesis of engineered exosomes.

[0086] 1.3 CLSM characterization.

[0087] RAW 264.7Exo and bEnd.3Exo were labeled with Dil and DiD dyes, respectively, and the fusion of the two exosome membranes was verified using laser confocal microscopy (CLSM). Results are shown below. Figure 4 As can be seen from the figure, RAW 264.7Exo and bEnd.3Exo exhibit green and red fluorescence, respectively. When RAW 264.7Exo and bEnd.3Exo are fused at a protein weight ratio of 1:1, RPDA@Rb-A shows obvious fluorescent colocalization, indicating the successful fusion of the two exosome membranes.

[0088] 1.4. Ultraviolet absorption spectroscopy characterization.

[0089] The engineered exosomes were characterized using ultraviolet absorption spectroscopy. The results are shown in [reference needed]. Figure 5 As can be seen from the figure, the UV absorption spectrum of RPDA@Rb-A has characteristic absorption peaks of both PDA and Res, which further proves the successful synthesis of engineered exosomes.

[0090] 1.5. SDS-PAGE characterization.

[0091] The full protein profile of exosomes in RPDA@Rb-A was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and compared with that of RAW 264.7Exo, bEnd.3Exo, and hybrid exosomes (Rb). See [link to results].Figure 6 The figure shows that RPDA@Rb-A matches the protein bands of two types of exosomes and hybrid exosomes, indicating that PDA was successfully encapsulated by exosomes and may have the same biological functions as exosomes, proving the successful synthesis of engineered exosomes.

[0092] Experiment 2: Investigation and identification of the photothermal properties of engineered exosomes.

[0093] RPDA@Rb-A was diluted to different concentrations and analyzed using an 808nm near-infrared laser (1.0W / cm²). 2 The RPDA@Rb-A was exposed to light for 10 minutes, and the temperature change over time was recorded. The photothermal stability of RPDA@Rb-A was evaluated by an on / off cyclic irradiation experiment.

[0094] Figure 7 The results show the photothermal properties of engineered exosomes; Figure A represents the temperature changes of different concentrations of RPDA@Rb-A under near-infrared (NIR) irradiation, and Figure B represents the photothermal stability results. As can be seen from the figures, compared to aqueous solutions, different concentrations of RPDA@Rb-A at 1.0 W / cm²... 2 Under NIR irradiation, a significant temperature increase was observed in all cases, indicating that the engineered exosomes possess excellent photothermal conversion capabilities. During four irradiation cycles, the four temperature peaks of the RPDA@Rb-A solution were 44.9℃, 44.6℃, 44.2℃, and 44.1℃, showing only minor differences, indicating that the engineered exosomes exhibit good photothermal stability.

[0095] Experiment 3: Identification of resveratrol release rate, encapsulation rate and loading in engineered exosomes.

[0096] RPDA@Rb-A was placed in a dialysis bag (MWCO = 3500 Da), immersed in 50 mL of PBS (containing 1% w / v Tween 80), and stirred at 37°C (100 rpm). At specified time intervals, 1 mL was taken and added to the dialysis system each time. In the NIR light irradiation group, a near-infrared laser (808 nm, 1.0 W / cm²) was used. 2 Irradiate the sample for 20 minutes.

[0097] Figure 8The concentration of Res was determined by UV-Vis absorption spectroscopy. The figure shows that the cumulative release of Res from RPDA@Rb-A was 42% after 28 hours. After near-infrared laser irradiation, the release rate of Res increased to 64%, indicating that this engineered exosome can achieve controlled drug release under near-infrared light irradiation, reducing the toxic side effects of the drug on normal tissues and cells and increasing the drug concentration at the lesion site. The encapsulation performance of Res, with encapsulation efficiency and drug loading of 46.7% and 27.1%, respectively.

[0098] Experiment 4: Investigating the ability of engineered exosomes to evade macrophage phagocytosis.

[0099] RAW 264.7 cells were seeded in 24-well plates at a density of 4 × 10⁶ cells / well. 5 Cells / wells were incubated for 24 hours, followed by incubation with FITC-labeled RPDA (PDA loaded with Res), RPDA@RA (RPDA coated with RAW 264.7Exo and modified with chole-Apt40), RPDA@bA (RPDA coated with bEnd.3Exo and modified with chole-Apt40), and RPDA@Rb-A for 4 hours. Cells were observed under CLSM. Simultaneously, the fluorescence intensity of the cells was quantified by flow cytometry.

[0100] Figure 9 The figure shows the results of evaluating the ability of engineered exosomes to evade macrophage phagocytosis. Figure A represents the CLSM results, while B and C represent the fluorescence intensity results obtained by flow cytometry. As can be seen from the figure, compared to the significant green fluorescence signal in the cytoplasm after PDA treatment, almost no fluorescence signal was observed in cells treated with RPDA@Rb-A, indicating that these engineered exosomes have the potential to evade immune clearance. Flow cytometry analysis showed that the fluorescence intensity of cells treated with RPDA@Rb-A was significantly reduced compared to those treated with RPDA, further confirming this result.

[0101] Experiment 5: Investigating the ability of engineered exosomes to penetrate the BBB.

[0102] bEnd.3 cells were seeded into the lower chamber of a Transwell plate and cultured until a dense cell monolayer was formed, establishing an in vitro BBB model. The upper chamber medium was then discarded, and DMEM medium containing FITC-labeled RPDA, RPDA@RA, RPDA@bA, and RPDA@Rb-A were added, respectively. The effect of NIR light irradiation on the penetration performance of RPDA@Rb-ABBB (RPDA@Rb-A+NIR) was investigated. At different time points, the culture medium in the lower chamber was collected, and fluorescence intensity was measured using a fluorometer.

[0103] Figure 10The figure shows the results of the engineered exosomes' BBB penetration performance evaluation. As can be seen from the figure, RPDA@Rb-A has a higher BBB penetration efficiency than RPDA, and near-infrared laser irradiation can further enhance the engineered exosomes' ability to penetrate the BBB, with a penetration rate approximately twice that of RPDA. This indicates that the engineered exosomes have a high BBB penetration rate.

[0104] Experiment 6: Investigating the performance of engineered exosomes in targeting inflammatory sites.

[0105] To evaluate the in vitro inflammatory tropism of engineered exosomes, BV2 cells were treated with Aβ aggregates for 12 h to simulate a cellular inflammatory microenvironment, and then incubated with FITC-labeled RPDA, RPDA@RA, RPDA@bA, and RPDA@Rb-A for 1 h, respectively. Cells were observed using CLSM. Simultaneously, the fluorescence intensity of the cells was quantified using flow cytometry.

[0106] Figure 11 The figure shows the performance evaluation results of engineered exosomes targeting inflammatory sites. Figure A represents the CLSM results, while B and C represent the fluorescence intensity results of cells measured by flow cytometry. The figure shows that RPDA@Rb-A only exhibited enhanced uptake in inflammatory cells, significantly higher than RPDA uptake. This indicates that the engineered exosomes inherit the natural inflammatory targeting ability of macrophage exosomes and have the potential to precisely target inflammatory sites in brain lesions. Flow cytometry analysis results are consistent with the CLSM results, further confirming this finding.

[0107] Experiment 7: Investigating the performance of engineered exosomes in targeting Aβ.

[0108] Aβ monomers (AβM), Aβ oligomers (AβO), and Aβ filaments (AβF) were co-incubated with RPDA@Rb or RPDA@Rb-A at room temperature for 1 h. The mixture was then centrifuged (12000 rpm, 10 min) to remove engineered exosomes and adsorbed Aβ. The concentration of Aβ in the supernatant was determined using a BCA kit.

[0109] Figure 12 The figure shows the results of the performance evaluation of engineered exosomes targeting Aβ. As can be seen from the figure, RPDA@Rb-A modified with chol-Apt40 exhibits better targeting ability for Aβ aggregates (AβO and AβF).

[0110] Example 2

[0111] The application of an engineered exosome of Example 1 in the preparation of a drug that promotes the clearance of Aβ aggregates by microglia.

[0112] Its application method is as follows: 1×10 5BV-2 cells were seeded in confocal dishes. After cell adhesion, they were incubated for 8 hours in medium containing AβO with or without PDA, RPDA, RPDA@Rb, RPDA@Rb-A, and RPDA@Rb-A+NIR (RPDA@Rb-A was subjected to near-infrared illumination). Then, they were incubated for another 4 hours with FITC-AβO instead of AβO. The cells were stained with LysoTracker Red and Hoechst 33342, and the effect of engineered exosomes on the phagocytosis of Aβ aggregates by microglia was observed using CLSM.

[0113] Figure 13 The figure shows the results of identifying the ability of engineered exosomes to promote the clearance of Aβ aggregates by microglia. As can be seen from the figure, stronger orange fluorescence was observed in microglia incubated with AβO and RPDA@Rb-A, indicating greater co-localization of Aβ and lysosomes. This demonstrates that engineered exosomes can promote Aβ clearance through the microglia-lysosomal pathway.

[0114] Example 3

[0115] The application of an engineered exosome of Example 1 in the preparation of a drug that scavenges reactive oxygen species in microglia and reduces iNOS expression in microglia.

[0116] Experiment 8: Engineered exosomes scavenge reactive oxygen species in microglia.

[0117] Its application method is as follows: 1×10 5 BV-2 cells were seeded overnight in confocal dishes. After cell adhesion, they were incubated for 24 h in medium containing AβO with or without PDA, RPDA, RPDA@Rb, RPDA@Rb-A, or RPDA@Rb-A+NIR. Cells were washed with PBS buffer and incubated with the DCFH-DA probe for 30 min. Intracellular ROS levels were observed using CLSM. Cells were then seeded in 12-well plates and treated as described above. After incubation, cells were washed three times with PBS buffer, digested with trypsin, collected, centrifuged, washed, and dispersed in PBS for flow cytometry analysis.

[0118] Figure 14The image shows the results of engineered exosomes scavenging reactive oxygen species (ROS) in microglia. Figure A represents CLSM imaging results, while figures B and C represent flow cytometry analysis results. The image shows that cells treated with AβO exhibited strong fluorescence, indicating the generation of large amounts of ROS in these cells. The addition of PDA and RPDA did not significantly change the fluorescence intensity, but the addition of RPDA@Rb and RPDA@Rb-A (with or without NIR illumination) resulted in a significant decrease in fluorescence intensity, demonstrating the ability of engineered exosomes to scavenge intracellular ROS. The flow cytometry analysis results were consistent with the CLSM results, further confirming this finding.

[0119] Experiment 9: Engineered exosomes reduce iNOS expression in microglia.

[0120] Its application method is as follows: 1×10 5 BV-2 cells were seeded in confocal dishes. After cell adhesion, BV-2 cells were pretreated with AβO for 36 h to induce a pro-inflammatory state in microglia. Then, they were incubated with PBS, PDA, RPDA, RPDA@Rb, RPDA@Rb-A, and RPDA@Rb-A+NIR for 24 h, respectively. After incubation, they were fixed with methanol at -20°C for 5 min, infiltrated with 0.1% Triton X-100 for 15 min, blocked with 5% BSA for 30 min, incubated overnight at 4°C with iNOS antibody, washed three times with PBST and PBS, incubated with CoraLite 647-labeled goat anti-rabbit secondary antibody for 2 h, washed with PBS, stained with DAPI at 37°C for 10 min, and imaged using CLSM.

[0121] Figure 15 Immunofluorescence staining results of engineered exosomes reducing iNOS expression in microglia. The figure shows that the presence of AβO leads to increased iNOS expression, indicating a pro-inflammatory state in microglia. After treatment with RPDA@Rb-A, a significant decrease in iNOS expression was observed in the cells, demonstrating that engineered exosomes can alleviate the inflammatory response in microglia, inhibit microglia dysfunction, and accelerate Aβ clearance.

[0122] Example 4

[0123] Application of an engineered exosome of Example 1 in the preparation of a drug that photothermally depolymerizes Aβ aggregates and restores neuronal vitality.

[0124] Experiment 10: Investigating the in vitro photothermal depolymerization of Aβ aggregates from engineered exosomes.

[0125] Alzheimer's disease (AD) is mainly caused by Aβ misfolding. To confirm the photothermal depolymerization effect of engineered exosomes on Aβ aggregates, the morphological changes of AβF after near-infrared irradiation were studied using atomic force microscopy (AFM). AβF was incubated with PBS, PDA, RPDA, RPDA@Rb, and RPDA@Rb-A for 24 h with and without NIR (Aβ concentration 50 μM, nanoparticle concentration 200 μg / mL, 808 nm laser power 1.0 W / cm²). 2 (Illumination time 10 min). Then, the sample was dropped onto fresh mica, dried, and the morphological changes of AβF were observed using AFM. Simultaneously, the above samples were mixed with ThT solution. The fluorescence intensity of ThT was detected using a fluorescence spectrometer to evaluate its ability to depolymerize AβF.

[0126] Figure 16 The figures show the AFM imaging and ThT fluorescence identification results of the in vitro photothermal depolymerization of Aβ aggregates by engineered exosomes. Figure A represents the AFM imaging, while figures B and C represent the ThT fluorescence identification results. As can be seen from the figures, the AβF structure was almost completely destroyed after the addition of RPDA@Rb-A and NIR treatment, demonstrating the effectiveness of the 808nm near-infrared photothermal effect in decomposing Aβ aggregates. Furthermore, when AβF with different treatments was mixed with ThT solution, the results of the RPDA@Rb-A addition and NIR treatment group and the PBS group were essentially the same. This indicates that engineered exosomes combined with near-infrared irradiation exhibit good function in decomposing AβF.

[0127] Experiment 11: Evaluating the ability of engineered exosomes to break down Aβ aggregates at the cellular level.

[0128] SH-SY5Y cells were co-incubated with AβF-containing medium for 24 h in the presence or absence of PDA, RPDA, RPDA@Rb, RPDA@Rb-A, and RPDA@Rb-A+NIR. The ThT fluorescence intensity was observed by CLSM.

[0129] Figure 17 The figure shows the results of identifying the ability of engineered exosomes to break down Aβ aggregates at the cellular level. As can be seen from the figure, SH-SY5Y cells treated with AβF alone exhibit strong ThT green fluorescence. When co-incubated with RPDA@Rb-A, ThT fluorescence significantly decreased, and in the 808nm combined photothermal treatment group, green fluorescence almost completely disappeared, indicating that the β-sheet structure was almost completely destroyed, thus verifying the ability of engineered exosomes to break down Aβ aggregates at the cellular level.

[0130] Experiment 12: Evaluating the ability of engineered exosomes to restore neuronal vitality.

[0131] Using Aβ-induced apoptosis in SH-SY5Y cells as an AD experimental model, the therapeutic effect of engineered exosomes on AD was investigated at the cellular level. SH-SY5Y cells were seeded in 96-well plates. After cell adhesion, cells were treated with AβF for 6 h, followed by further incubation with PBS, PDA, RPDA, RPDA@Rb, and RPDA@Rb-A for 24 h with or without NIR. After treatment with MTT for 4 h, the culture medium was removed, and 150 μL of DMSO was added to each well. Samples were measured at 490 nm using a microplate reader. The Aβ-induced cytotoxicity model was analyzed using a live / dead staining method. SH-SY5Y cells were incubated at 2 × 10⁶ cells per well. 5 The cells were seeded at a density of 1 / 25 mm in 35 mm culture dishes and incubated at 37 °C for 24 h. Then, cells with or without AβF and different materials were added and incubated for another 24 h. Cells were stained with Calcein-AM / PI and then photographed with CLSM.

[0132] Figure 18 The figure shows the results of the assessment of the ability of engineered exosomes to restore neuronal viability. Figure A represents the results of the Calcein-AM / PI live / dead cell double staining experiment, Figure B represents the statistical analysis results of Figure A, and Figure C represents the cell survival rate with and without NIR. The figure shows that the survival rate of SH-SY5Y cells decreased to 43% after treatment with AβF, while the survival rates increased to 65%, 71%, 83%, 85%, and 95% respectively after treatment with PDA, RPDA, RPDA@Rb, RPDA@Rb-A, and RPDA@Rb-A+NIR. This indicates that engineered exosomes can effectively inhibit Aβ-induced cytotoxicity and restore neuronal viability. The Calcein-AM / PI live / dead cell double staining experiment also corroborates these results.

[0133] Experiment Thirteen: Engineered exosomes scavenging Aβ-induced reactive oxygen species in SH-SY5Y cells.

[0134] 1×10 5 SH-SY5Y cells were seeded in confocal dishes and incubated overnight. After cell adhesion, the cells were incubated for 24 h in medium containing AβF with or without PDA, RPDA, RPDA@Rb, RPDA@Rb-A, or RPDA@Rb-A+NIR. After washing the cells with PBS buffer, the DCFH-DA probe was added and incubated for 30 min. The intracellular ROS content was observed using CLSM.

[0135] Figure 19The figure shows the results of engineered exosomes scavenging Aβ-induced intracellular reactive oxygen species (ROS) in SH-SY5Y cells. As can be seen from the figure, the fluorescence intensity of the RPDA@Rb-A treatment group was basically the same as that of the control group, but significantly lower than that of the AβF group. This indicates that engineered exosomes can eliminate abnormal ROS, maintain normal intracellular ROS levels, and protect cells from Aβ-induced neural damage.

[0136] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. An engineered exosome targeting Alzheimer's disease lesions and clearing β-amyloid plaques, characterized in that, The engineered exosome comprises a photothermal nanoparticle, a hybrid exosome, an A beta recognition probe and a small molecule drug; The engineered exosome takes the photothermal nanoparticle as the core of the engineered exosome, takes the hybrid exosome as the shell to form a core-shell structure, the A beta recognition probe is modified on the surface of the core-shell structure, and the small molecule drug is loaded in the cavity and / or the natural phospholipid bilayer of the membrane of the engineered exosome; the hybrid exosome is obtained by fusion of brain microvascular endothelial cell exosome and macrophage exosome; The A beta recognition probe is an A beta aptamer, an antibody or a polypeptide; the gene sequence of the A beta aptamer is shown in SEQ ID NO. 1; The antibody is 1F12; The polypeptide is one of KLVFF, KLVFFADE, LPFFD and LVFFA.

2. The engineered exosome of claim 1, wherein, The photothermal nanoparticle is one of polydopamine nanoparticle, pyrrolopyrrole dione polymer, gold nanorod, black phosphorus nanomaterial and copper sulfide nanoparticle; And / or, the small molecule drug is one or more of resveratrol, curcumin and quercetin; And / or, the brain microvascular endothelial cell is bEnd.3 cell; and the macrophage is RAW264.7 cell.

3. The engineered exosome of claim 1, wherein, The A beta recognition probe is labeled with cholesterol.

4. The engineered exosome of any one of claims 1 to 3, wherein, The mass ratio of the photothermal nanoparticle to the small molecule drug is 1-4:1, the mass ratio of the brain microvascular endothelial cell exosome to the macrophage exosome is 1:1, and the mass ratio of the hybrid exosome to the photothermal nanoparticle is 1:

1.

5. A method of producing the engineered exosome of any one of claims 1 to 4, characterized in that, The preparation method comprises the following steps: S1, mixing the photothermal nanoparticle with the small molecule drug solution, incubating overnight to obtain a mixed solution; S2, adding the brain microvascular endothelial cell exosome and the macrophage exosome to the mixed solution, performing ultrasonic treatment and incubation to obtain the hybrid exosome loaded with the photothermal nanoparticle and the small molecule drug; S3, adding the A beta recognition probe for incubation, and centrifuging to obtain the engineered exosome.

6. The production method according to claim 5, characterized by The ultrasonic treatment in S2 is performed under ice bath condition at 100 W for 2 s with 2 s interval, and the incubation is performed at 37 DEG C for 1 h; And / or, the incubation time in S3 is 30 min, and the incubation temperature is 37 DEG C.

7. Use of the engineered exosome in any one of claims 1-4 in the preparation of a drug for targeting Alzheimer's disease lesions.

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