Nano material for treating Alzheimer's disease by targeting cell aging and application of nano material

Abstract

The invention provides a nano-material for treating Alzheimer's disease by targeting cell aging, which is characterized in that the nano-material takes extracellular vesicles of a melanoma cell line as a nano-carrier to load curcumin. The pharmaceutical composition has the advantages of high bioavailability and good safety, and can be used as a medicine for clinically treating AD and related symptoms thereof.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a nanomaterial for treating Alzheimer's disease by targeting cell aging and an application thereof. Background Art

[0002] Alzheimer's disease (AD) poses a growing global health challenge due to an aging population and significant social and economic impact. Although the causes of AD remain controversial, aging is widely recognized as a major risk factor.

[0003] Curcumin is a natural anti-aging and anti-inflammatory compound with promise as a therapeutic agent, but despite its considerable potential in AD treatment, its therapeutic development is significantly limited by poor bioavailability, characterized by low water solubility, rapid degradation and clearance in the circulation, and limited ability to penetrate the blood-brain barrier (BBB). Low solubility leads to poor bioavailability and may cause undesirable side effects, such as: restrictions on administration routes and the need for higher doses to achieve therapeutic plasma concentrations.

[0004] Furthermore, elevated curcumin concentrations were associated with cell death / apoptosis and / or inhibition of cell motility. Figure 3 A and 4B, both of which showed that PBS-CUR inhibited BV2 cells or N2a cells in a concentration-dependent manner ( Figure 4 B) proliferation. Statistically significant antiproliferative activity was observed at concentrations of 30 μM or higher in BV2 cells and 60 μM or higher in N2A cells, respectively. Therefore, curcumin has an obvious problem of poor bioavailability. Summary of the invention

[0005] The present invention aims to overcome the above-mentioned defects and develops a new nanodrug EV-CUR by encapsulating curcumin in extracellular vesicles from melanoma cell lines. The EV-CUR significantly improves the bioavailability of curcumin and shows effective blood-brain barrier penetration. Bioinformatics analysis showed that systemic administration of EV-CUR significantly alleviated senescence and neuroinflammation of neural cells, especially microglia, which may play a key role in the development of AD. Notably, unlike curcumin, EV-CUR rescued neural damage, reduced AD-related pathology, and improved cognitive function without obvious toxicity. This innovative nanodrug provides a promising strategy for the treatment of AD and supports anti-aging treatment.

[0006] The present invention provides a nano material for treating Alzheimer's disease by targeting cell senescence, characterized in that the nano material uses extracellular vesicles of a melanoma cell line as nano carriers to load curcumin.

[0007] Furthermore, the present invention provides a nanomaterial for treating Alzheimer's disease by targeting cell senescence, which is also characterized by:

[0008] After obtaining extracellular vesicles of melanoma cell lines by extrusion method, the nanomaterial NP-CUR was prepared by drug loading with a BCA:CUR ratio of 1:0.5-2 by ultrasound method.

[0009] Furthermore, the present invention provides a nanomaterial for treating Alzheimer's disease by targeting cell senescence, which is also characterized by:

[0010] The preparation process of the extracellular vesicles of the melanoma cell line comprises the following steps:

[0011] S1. When the cell density of melanoma cells is greater than 90%, digest the cells and transfer them to a centrifuge tube. Centrifuge at 1000-2000 rpm for 1-10 min, discard the supernatant, and resuspend the cells in a pre-cooled buffer.

[0012] S2. Centrifuge at 1000-2000 rpm for 1-10 min, discard the supernatant, resuspend the cells in pre-cooled buffer, centrifuge again and resuspend in buffer;

[0013] S3. The cell suspension was sequentially extruded through 5, 1, and 0.4 μm track-etched membranes by a liposome extruder, with each extrusion being repeated 10-15 times;

[0014] S4. The final extruded sample was centrifuged at 10000-15000 g for 5-15 min at 4°C and the precipitate was discarded after centrifugation;

[0015] S5. The supernatant was concentrated by centrifugation at 4°C for 10-15 minutes more than once to obtain purified extracellular vesicles of the melanoma cell line.

[0016] Furthermore, the present invention provides a nanomaterial for treating Alzheimer's disease by targeting cell senescence, which is also characterized by:

[0017] The ultrasonic method was carried out under the conditions of 150-200 kW power, 10-20% amplitude, alternating 15-60 seconds ultrasonic treatment and 15-60 seconds pause, for a total of not less than 3 cycles.

[0018] In addition, the present invention also provides the use of the above nanomaterials in preparing drugs for resisting cell aging or alleviating cell aging.

[0019] In addition, the present invention also provides the use of nanomaterials in preparing medicines for alleviating aging of nerve cells and nerve inflammation.

[0020] In addition, the present invention also provides the use of nanomaterials in preparing drugs for improving cognitive function.

[0021] In addition, the present invention also provides the use of nanomaterials in preparing drugs for treating or preventing Alzheimer's disease.

[0022] The above-mentioned Alzheimer's disease is caused by cellular senescence.

[0023] In addition, the present invention also provides a method for establishing an Alzheimer's disease model based on cell senescence, characterized in that:

[0024] Inducing BV2 cell senescence by H2O2;

[0025] The concentration of H2O2 induction was 300-400 μM;

[0026] Dosage: Repeated administration every 4 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 .Bioinformatics evidence of aging and AD. Among them, (A) shows the intersection of up-regulated genes in the elderly group. (B) Venn diagram shows the intersection of down-regulated genes in the elderly group. (BD) Volcano map shows that there are differentially expressed genes in GSE267529, GSE261698 and GSE251888 datasets. (E) GO enrichment of up-regulated genes. (G) Graphene oxide enrichment of down-regulated genes. (F) Double map of GSM6538356 dataset. (G) Expression of common up-regulated genes in the healthy group. (H) Expression of common up-regulated genes in the AD group.

[0028] Figure 2 , H2O2-induced senescence model, where A is the 24-hour cytotoxicity of H2O2 on BV2 (***p<0.001, ****p<0.0001), B is the 4-day cytotoxicity of H2O2 on BV2 (****p<0.0001), C is the senescence staining of BV2 induced by different concentrations of H2O2 (4 days), D is the senescence staining count (4 days, ****p<0.0001), E is the senescence staining and count of BV2 induced by H2O2 on different days (****p<0.0001), F is the expression and relative quantification of P16INK4A protein in BV2 cells induced by H2O2 (*p<0.05);

[0029] Figure 3Preparation, characterization, stability and drug delivery efficacy of EV-CUR. (A) Cell viability analysis of BV2 cells after treatment with different concentrations of free curcumin and EV-CUR for 24 h. (B) Schematic diagram of the preparation of B16-EV and EV-CUR. (C) In vivo BBB model, DID fluorescence signal transduction of DID-labeled B16-EV, Hepa1-6-EV and MC38-EV from isolated brain by in vivo imaging system. (D) Quantification of fluorescence intensity of (C). (E) Protein amount of EV obtained by extrusion and naturally harvested from the same number of cells. (F) Particle distribution of B16-EV and EV-CUR. (G) TEM images of B16-EV and EV-CUR. Scale bar, 100 nm. (H) Average zeta potential of B16-EV and EV-CUR. (I) Stability of EV-CUR compared with free curcumin. (J) Soluble samples of free curcumin and EV-CUR and their appearance after 3 and 8 h of treatment. n=3, data are mean±sd.

[0030] Figure 4 .. Cell viability of N2a cells after treatment with different concentrations of H2O2 (A) and curcumin (B) for 24 h. Data are mean ± sd. Compared with the untreated group, n ≥ 3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0031] Figure 5 .Particle size distribution of extruded EVs and natural EVs.

[0032] Figure 6 Curcumin was loaded at a mass ratio of 1:2 (BCA:CUR) at 8 h.

[0033] Figure 7 .EV-CUR can alleviate brain aging in AD mice. (A) Images of aging markers (green) and activated microglia (red) in the brains of different groups of 3×Tg mice exposed by immunofluorescence staining, and white arrows indicate aging microglia. (B) P16+ cell counts per mm2. (C) CD11b+ cell counts per mm2. (D, E) WB detection of P16INK4A expression and relative quantification. (F, G, H) qPCR detection of the relative expression of IL-1β, IL-6, and TNF-α mRNA in brain tissues. n≥3, data are mean±sd, **p<0.01, ***p<0.001, ****p<0.0001.

[0034] Figure 8.EV-CUR improves microglial senescence. Mouse brain immunofluorescence (n=3). (A) The red fluorescence in the figure is P16INK4A, the green fluorescence is IBA-1, and the white arrow indicates the co-localization of P16INK4A and IBA-1. Scale bar, 75 μm. (B) P16+ cell count per mm2. (C) IBA+ cell count per mm2. (D) Double-positive P16+IBA+ cell count per mm2. (E) Double-positive p16+CD11b+ cell count per mm2. Data are mean ± s. d., n≥3, *p<0.05, **p<0.01.

[0035] Fig. 9 .EV-CUR can improve AD-related pathological changes. (A) Immunofluorescence images of p-Tau205 (red) in brain CA1 and DG. (B) Immunofluorescence images of Aβ (red) in the cerebral cortex. (C) Quantitative analysis of fluorescence intensity in (A). (D) Quantitative analysis of Aβ plaque area in (B). (E, F) WB detection and relative quantification of p-Tau and APP levels in the brain. (G) Nissl staining images of different regions (CA1, CA3, DG) of the hippocampus in different treatment groups. Optical microscope, 20×. (H) Quantitative analysis of Nissl staining. n=3-6, data are mean ± sd, *p<0.05, **p<0.01, ****p<0.0001.

[0036] Fig.10 .EV-CUR can improve cognitive function in AD mice. (A) Representative traces and heat maps of spontaneous alternation test. (B) Spontaneous alternation rate of mice in different groups. (C) Total arm entries of mice in different groups. (D) Representative traces and heat maps of novel arm exploration test. (E) Number of novel arm entries of mice in different groups. (F) Ratio of novel arm to total arm entries of mice in different groups. (G, H) Nesting test and coring. n = 6, data are mean ± sd, *p < 0.05, **p < 0.01, ***p < 0.001.

[0037] Fig.11 .EV-CUR rescue evaluation of neuronal injury and biosafety. (A) Monitoring the body weight of mice during treatment. (B) Laboratory test results of liver and kidney function of mice. (C) HE staining of important organs of mice in different cell groups. Optical microscope, 20×. n=6, data are mean±sd, *p<0.05, ****p<0.0001.

[0038] Fig.12 .Standard curve of curcumin; wherein A is the spectrum scan of the curcumin solution, and B is the standard curve and linear regression equation of the curcumin solution. DETAILED DESCRIPTION

[0039] The present invention is capable of various modifications and various embodiments, and therefore specific embodiments are illustrated and described in the accompanying drawings. However, this is not intended to limit the present invention to specific implementations, but should be understood to include all modifications, equivalents, and even substitutes that fall within the concept and technical scope of the present invention.

[0040] Experimental Example 1. Aging microglia are associated with AD

[0041] Aging and associated inflammation play a crucial role in the development of AD. To explore the genetic mechanisms behind this relationship, this example used three independent microglial transcriptome databases to analyze gene expression changes between old and young mice. By comparing the gene expression patterns of these datasets, several genes that were consistently upregulated in old animals were identified ( Figure 1 AD). Gene ontology (GO) enrichment analysis showed that many of these upregulated genes were involved in inflammatory and immune response pathways ( Figure 1 E). A key inflammation-related gene module, including genes IL1RN, ITGAX, MMP12, CCL8, CXCL13, and PDCD1, was identified due to its association with aging in three datasets. We further examined the expression changes of this gene module in neural tissues of AD patients compared with healthy donors and demonstrated significant upregulation in AD patients, especially in microglia ( Figure 1 FH). These bioinformatic analyses suggest that aging-associated upregulation of inflammatory pathways may be significantly associated with the development of AD, particularly via senescent microglia.

[0042] Experimental Example 2. Microglia-based aging model

[0043] BV2 cells were cultured in 6-well plates and divided into three groups: untreated group (CON), H2O2 group, and CUR+H2O2 group. The H2O2-induced group was treated with complete medium containing 300 μM H2O2 for 4 consecutive days. On the fourth day after induction, the CUR+H2O2 group was given a therapeutic concentration of curcumin. The same classification was performed on N2a cells, where the CUR+H2O2 group received a treatment regimen involving curcumin for 1 day and then exposed to H2O2 for another day.

[0044] The process of establishing the induction scheme for the above H2O2 induction group is as follows:

[0045] Mouse microglial BV2 cells were inoculated into culture dishes, and the complete culture medium was replaced once a day. The BV2 cell density was observed to be 80-90% under the microscope. The culture medium was discarded, and after washing with PBS buffer 3 times, 2 ml of complete culture medium was added again. Then, the BV2 cells were gently blown off the bottom of the dish and transferred to a 15 ml centrifuge tube and mixed by blowing. After taking 10 μl of the cell suspension and counting it by hemocytometer technology, the cell density was adjusted to 5×10 4 / mL. 100μl of the adjusted cell suspension was inoculated in 96-well plates, with 6 replicate wells in each column, and a blank group (no cells were inoculated) was reserved. To prevent evaporation of the culture medium and edge effects, 100μl of PBS buffer 1z was added to the peripheral wells of the experimental group and the blank group. The 96-well plate after inoculation of cells was placed in a cell incubator (37°C, 5% CO2) overnight to allow the cells to adhere to the wall.

[0046] Take 2 μl of 3% H2O2 solution and add it to 880 μl DMEM complete medium to prepare H2O2 stock solution (2000 μM). Dilute the H2O2 stock solution to 50, 100, 200, 300, 400, 600, 800 μM working solution with complete medium, and store in a 4°C refrigerator away from light for later use.

[0047] Observe under the microscope to confirm that the cells are attached to the wall, add 100ul PBS buffer to each well for washing once, then add 100μL of H2O2 working solution of different concentrations in step 2, and take blank culture medium as the control group. Six replicate wells for each concentration were placed in a 37℃, 5% CO2 incubator for overnight incubation. After confirming that the cell density is appropriate, completely discard the culture medium, wash twice with PBS, add 100μl DMEM basal culture medium containing 10% CCK-8 to each well, and continue incubation in the incubator for 1 hour.

[0048] The 96-well plate was placed in a multifunctional microplate reader, and the program was set to linearly shake the plate for 20 seconds and then measure the OD value at a wavelength of 450 nm. According to the formula: cell viability (%) = (OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) × 100%.

[0049] Similarly, complete culture medium and H2O2 working solution of different concentrations were replaced daily, and cell viability was measured after 4 days of culture.

[0050] To ensure that H2O2 can induce BV2 cell senescence at a safe concentration without significantly inducing cell apoptosis, the 24-hour toxicity of H2O2 at different concentrations on BV2 cells was first determined. Figure 2As shown in A. According to the CCK-8 results, it was concluded that the activity of BV2 cells was higher than 80% after 24 hours of induction at a H2O2 concentration of 400 μM. Therefore, in subsequent experiments, concentrations within 400 μM were selected to induce BV2 cell senescence. However, in subsequent experiments inducing senescence, it was found that 24 hours of H2O2 treatment at a concentration of less than 400 μM did not significantly cause BV2 cell senescence, so the induction time of H2O2 was further extended. Subsequently, a toxicity experiment on BV2 cells with continuous administration of H2O2 for 4 days was conducted to determine a safer H2O2 concentration for a longer period of time. The results are shown in Figure 2 As shown in Figure B, after repeated administration of 400 μM H2O2 for 4 days, the viability of BV2 cells decreased significantly to 68.88±2.83%, while there was no significant difference in cell viability at 300 μM. Therefore, the safe concentration range for repeated administration for 4 days was determined to be within 300 μM.

[0051] After that, take 2×10 5 BV2 cells were inoculated into 6-well plates, and the complete medium was replaced once a day. The BV2 cell density was observed to be 50-60% under the microscope. 200, 300, and 400uM H2O2 solutions were added for 4 days and 300μM H2O2 solution was added for 2 days and 4 days, and an untreated control group was reserved. After the treatment, the complete medium was aspirated, washed once with PBS buffer, and fixed with the fixative in the SA-β-gal kit at room temperature for 15 minutes. The fixative was discarded and washed with PBS buffer on a shaker for 5 minutes × 3 times. After washing, 1ml of aging staining solution working solution was added to each well (formula: 10μl staining solution A + 10μl staining solution B + 930μl staining solution C + 50μl X-Gal solution), placed in a sealed bag and incubated overnight in a 37℃ CO2-free oven. On the second day, photos were taken and counted under a fluorescent inverted microscope in the white field.

[0052] After induction by the same method, protein was extracted and P16 INK4A Protein expression determination.

[0053] The β-galactosidase produced by senescent cells is highly active in weakly acidic conditions (pH 6.0) and can catalyze X-Gal to form a blue product, which can be used to determine cell senescence. Figure 2 A. The results of H2O2 cytotoxicity. BV2 cells were treated with 0, 200, 300, and 400 μM H2O2 for 24 hours and then stained with SA-β-gal. It was found that there was no significant difference in the proportion of SA-β-gal positive cells in each group, which means that the acute oxidative stress stimulation caused by H2O2 cannot cause microglial senescence. Therefore, the induction time was extended to 4 days, and fresh culture medium containing H2O2 was replaced every day. The results of SA-β-gal staining after 4 days of induction are shown in Figure 2. Figure 2As shown in C, in the 300μM and 400μM groups, SA-β-gal staining visually showed that the number of blue-stained BV2 cells increased significantly, and the cell morphology changed significantly, and the volume of some cells increased significantly, while there was no obvious SA-β-gal positive staining in the uninduced group and the 200μM group. Further senescent cell counting results showed that the proportion of senescent cells in the 300μM and 400μM concentration groups was 21.62±2.49% and 23.57±6.57%, respectively. There was no significant statistical difference between the groups (p>0.05), but there was a significant statistical difference between the uninduced and 200μM groups (p<0.0001). Combined with H2O2 repeated treatment for 4 days on BV2 cytotoxicity results ( Figure 2 B), the final selected H2O2 concentration was 300μM. On this basis, BV2 cells were induced with 300μM H2O2 for 2 days and 4 days, and SA-β-gal staining and counting analysis were performed ( Figure 2 D, 2E). The results showed that there was no significant difference in the number of senescent BV2 cells induced by 300 μM H2O2 for 2 days compared with the uninduced group, while the number of SA-β-gal positive cells increased significantly at 4 days (p < 0.0001), indicating that chronic and continuous stimulation is the main cause of microglial senescence.

[0054] To further confirm the successful induction of senescence, P16 INK4A Protein expression changes. INK4A Protein is a widely recognized marker of cell senescence and is significantly increased in senescent cells. WB results showed that P16 INK4A The protein expression was significantly increased compared with the untreated group (Figure F), which was consistent with the results of SA-β-gal staining, confirming the senescence of BV2 cells.

[0055] This confirms that chronic or persistent oxidative stress is the important reason for inducing microglial senescence. In recent years, the aging hypothesis has received more and more attention in the pathogenesis of AD, especially microglial senescence may be closely related to the pathological changes of AD. Therefore, the present invention uses the above aging model to explore the effectiveness of new drugs for the treatment of Alzheimer's disease.

[0056] Experimental Example 3. Construction of EV-CUR

[0057] like Figure 3 As shown in B, B16-EV was prepared using a liposome extruder (Avanti, USA) and then loaded with curcumin using an ultrasonic method. The specific method is as follows:

[0058] 1. B16F10 and b.end3 cell culture

[0059] The formula of complete medium for B16F10 cells: 90% double-antibody high-glucose medium (DEME) + 10% fetal bovine serum (FBS, Gibco), the formula of complete medium for b.end3 cells: 89% double-antibody high-glucose medium (DEME) + 10% fetal bovine serum (FBS, Gibco) + 1% sodium pyruvate solution. Except for trypsin digestion required for passaging, the recovery, medium replacement and freezing steps are the same as those for BV2 cell culture in the first part.

[0060] Specific operation of trypsin digestion: aspirate the old culture medium, add 2ml PBS and shake gently to wash 3 times, then add 1ml trypsin, slowly and repeatedly tilt up and down and left and right to allow the trypsin to fully contact the cells, and then place in the cell culture incubator for digestion (B16F10 digestion for 2 minutes, b.end3 cell digestion for 4 minutes). Take out the cell culture dish and add 3ml complete culture medium to terminate the digestion. Use a pipette to blow gently repeatedly to completely detach the cells from the bottom of the dish (you can see that the bottom of the dish changes from cloudy and fuzzy to transparent and clear). Transfer the cell suspension to a 15ml centrifuge tube, centrifuge at 1000rpm for 5 minutes, discard the upper liquid, add 2ml fresh complete culture medium to resuspend the cells, and pass them at a ratio of 1:2.

[0061] 2. B16-EV preparation

[0062] B16-EVs were prepared by liposome extruder. The specific operation was as follows: When the B16F10 cell density was >90% under a microscope, the cells were digested and transferred to a 15 ml centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended with 2 ml of pre-cooled PBS buffer. Centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, the cells were resuspended with 2 ml of pre-cooled PBS buffer, centrifuged again, and finally resuspended with 1 ml of PBS buffer. The liposome extruder was assembled according to the instructions, and the cell suspension was extruded through 5, 1, and 0.4 μm track-etched membranes in sequence, with 15 back and forth extrusions each time. The final extruded sample was centrifuged at 10000 g for 10 minutes at 4 ° C, and the precipitate was discarded after centrifugation. The supernatant was concentrated at 1000 g for 15 minutes at 4 ° C using a 100 kDa centrifugal filter. Concentrated again twice to obtain purified B16-EVs. The protein concentration of the finally obtained B16-EVs was determined using the BCA method, marked with the date and frozen in a -80 ° C refrigerator for future use.

[0063] 3. Preparation of NP-CUR

[0064] According to BCA quantification, take an appropriate amount of B16-EV, add an appropriate amount of curcumin mother solution to the B16-EV solution at a mass ratio (BCA: CUR = 1: 0.5-2), insert the ultrasonic cell disruptor probe to about 5mm below the liquid surface, and use the ultrasonic method to prepare NP-CUR. The specific conditions are: 20% amplitude, 30 seconds of ultrasound + 30 seconds of pause, a total of 6 cycles. After ultrasound, incubate at 37°C for 30 minutes.

[0065] Example 4. Condition screening experiment

[0066] In this example, we used B16-EV as our nanoparticle delivery system because they have excellent stability, biocompatibility, and immune evasion properties compared to synthetic nanoparticles such as liposomes and polymer-based systems. In addition, based on comparative experiments, EVs derived from the highly metastatic tumor cell line B16 showed enhanced BBB penetration ability.

[0067] Specifically, this example compares the BBB penetration of EVs extracted from three different cell lines: two low metastatic lines, Hepa1-6 and MC38, and a highly metastatic line, B16. Fluorescent compounds were encapsulated in EVs from these cell lines, and the fluorescent signal in the brain was quantified 24 hours after intraperitoneal injection. Figure 3 As shown in C and 3D, B16-derived EVs showed a significantly stronger presence in the brain, approximately three times that of EVs derived from Hepa1-6 and MC38.

[0068] In addition, this example solves the challenges of low productivity and high cost associated with clinical translation by optimizing the EV extraction process. The total amount of EVs produced by the optimized procedure was 163.97 ± 9.88 g / 10 ^ 6 cells, while the total amount of EVs obtained using the natural EV isolation method described by Wang et al. was 8.73±1.65g / 10^6 cells ( Figure 3 E). Importantly, the EV quality was comparable between the two methods, with the majority of EVs generated from both methods showing similar sizes, mainly in the range of 100 nm to 300 nm ( Figure 5 ). Notably, the present method did not produce oversized EVs compared with the natural EV extraction method, indicating that the nanoparticles have better consistency and stability.

[0069] In addition, this example uses ultrasound to load curcumin into B16-EVs, resulting in a new nanomedicine named EV-CUR. Various mass ratios of EV to curcumin were tested to optimize loading capacity and stability. Choose 1:1 The ratio is due to its excellent stability, while 1:2 ( Figure 6 ) and 1:3 ratios showed precipitation during incubation. At this optimal mass ratio, EV-CUR maintained a regular round morphology similar to that of empty EVs, with an average size of 217.47±21.54nm, which is comparable to the size of empty EVs (195.88±4.58nm) ( Figure 3F and 3G). As suggested by Zhang et al., the range of 100–300 nm is optimal for the advancement of nanomedicine and is beneficial for the penetration of the blood-brain barrier. This is due to the enhanced transport facilitated by phagocytosis by vascular endothelial cells. In addition, the average zeta potential of EV-CUR was slightly reduced compared to empty EVs, with an average zeta potential of -26.61 ± 1.84 mV for EV-CUR and -18.2 ± 2.93 mV for empty EVs ( Figure 3 H). The decrease in zeta potential indicates a potentially better BBB penetration ability. Notably, EV-CUR exhibited superior stability compared to PBS-CUR at equimolar concentrations. Curcumin is considered to be insoluble in water, and consistent with earlier reports, we confirmed that curcumin exhibited poor solubility and stability in PBS. Figure 3 As shown in Figure 1, after 3 hours of incubation at 37°C, only 44.05±3.78% of soluble curcumin was measured in the PBS-CUR group. EV encapsulation significantly improved solubility and stability, and the soluble curcumin in the EV-CUR group was maintained at 92.16±2.24%. After 8 hours of incubation, only 14.56±3.15% of soluble curcumin was retained in the PBS-CUR group, while 80.67±3.15% of soluble curcumin was retained in the EV-CUR group. In addition, accumulation of curcumin precipitates was observed in the PBS-CUR group, however, the EV-CUR group was not obvious ( Figure 3 J). Interestingly, at higher concentrations (above 30 μM), encapsulation of curcumin within EVs significantly mitigated curcumin-dependent cytotoxicity. This observation suggests that slower release and / or reduced aggregation of the drug could enhance cellular tolerance to curcumin ( Figure 3 A). In summary, this example generated a novel nanomedicine by encapsulating curcumin into B16-derived EVs, which showed potent brain penetration potential and desirable drug developability characteristics that could significantly improve the bioavailability of curcumin.

[0070] Experimental Example 5. Feasibility Analysis of EV-CUR as AD Drug

[0071] 1. EV-CUR reduces neuronal senescence and neuroinflammation in aged AD mice

[0072] The inflammatory microenvironment of the central nervous system (CNS) plays a crucial role in the development and progression of AD, particularly through the involvement of aging microglia, the main immune cells in the brain. The above bioinformatics analysis showed that microglia exhibited the highest expression of aging-related inflammatory genes in AD patients. To evaluate the anti-aging and anti-inflammatory effects of EV-CUR, the APP / PS1 / TAU transgenic (3×Tg) mouse model was utilized, which closely mimics the pathological changes observed in AD patients. Consistent with other reports, p16 was detected in brain lysates of these aged AD mice. Notably, administration of EV-CUR by intraperitoneal injection significantly reduced p16 levels, while PBS-CUR treatment did not, indicating the anti-aging potential of EV-CUR ( Figure 7 D and 7E).

[0073] To further investigate the relationship between aging and inflammation, immunofluorescence staining for p16 as well as CD11b and IBA-1, both established markers of activated microglia, was performed in the cerebral cortex and hippocampus of AD mice. As expected, the number of p16+ and / or CD11b+ neurons was significantly lower in the EV-CUR-treated group compared with the untreated control and PBS-CUR-treated groups ( Figure 7 AC). Consistently, unlike PBS-CUR, EV-CUR treatment significantly reduced the number of p16+ and / or IBA-1+ neurons in the cortex ( Figure 8 AC). In addition, the population of inflammatory senescent microglia was quantified by counting cells expressing p16 and CD11b / IBA-1. Figure 8 As shown in D and E, EV-CUR treatment resulted in a 30-50% reduction in senescent activated microglia in the cortex of AD mice, and p16 / CD11b double positive cells decreased from approximately 120 cells / mm 2 Reduced to 80 cells / mm 2 p16 / IBA-1 double positive cells from 50 cells / mm 2 Reduced to 30 cells / mm 2 In contrast, PBS-CUR treatment did not produce any significant changes in the senescent activated microglial population. Finally, EV-CUR treatment resulted in a significant decrease in the expression of the proinflammatory cytokines IL-1β, IL-6, and TNF-α in whole brain lysates ( Figure 7 FH). In conclusion, EV-CUR showed significant anti-aging and anti-inflammatory effects in the aged AD mouse model, whereas PBS-CUR did not show any significant therapeutic effects. These findings highlight the key role of EV-CUR in improving therapeutic outcomes.

[0074] 2. EV-CUR reduces AD-related pathology and improves cognitive function

[0075] Aβ accumulation and Tau hyperphosphorylation are hallmark features of AD. Here, we evaluated whether reduced neuronal senescence and neuroinflammation by EV-CUR treatment ameliorated AD-related pathology and cognitive deficits. Fig. 9 As shown in A and 9C, the expression of p-Tau in the CA1 and dentate gyrus (DG) regions of the hippocampus was significantly reduced in the EV-CUR-treated group compared with the untreated control and PBS-CUR-treated groups. Similarly, EV-CUR treatment led to a significant decrease in Aβ levels in the cortical region ( Fig. 9 B and 9D). These findings were further validated by western blot analysis, which revealed that only EV-CUR induced the downregulation of p-Tau and amyloid precursor protein (APP), highlighting the critical role of EV delivery in enhancing therapeutic efficacy ( Fig. 9 E and 9F). To evaluate whether EV-CUR could ultimately rescue neuronal damage, we performed Nissl staining on brain sections. Fig. 9 As shown in Figure H, the number of neurons in the hippocampus of the EV-CUR group was significantly higher than that of the other two groups. In addition, the neurons in the EV-CUR-treated group were clearly arranged, with visible nucleoli, obvious Nissl demarcation, and lack of dark pyknotic cells, suggesting the potential of EV-CUR in rescuing neural damage and restoring normal neuronal function.

[0076] In addition, it was evaluated whether EV-CUR treatment could restore cognitive function in aged AD mice. The Y-maze spontaneous alternation test and novel arm exploration test were used to evaluate short-term memory and exploratory behavior. A valid spontaneous alternation was defined as a continuous exploration of three arms. The data showed that the effective spontaneous alternation of AD mice treated with EV-CUR was significantly increased by about 20% ( Fig.10 A and 10B). There was no statistical difference in the total number of arm entries among the three groups, further confirming that the increase in effective spontaneous alternation in the EV-CUR treatment group was due to the recovery of cognitive function ( Fig.10 C).

[0077] In the novel arm exploration test, mice treated with EV-CUR made approximately twice as many entries into the novel arm compared with the other groups during the observation period, resulting in a significant increase in the proportion of novel arm entries to total arm entries ( Fig.10 DF). Consistent with the results of the Y-maze test, AD mice treated with EV-CUR also showed substantial improvement in nesting behavior, as indicated by significantly higher scores ( Fig.10G, H). Enhanced nesting behavior generally reflects improved hippocampal function and overall health of mice. These behavioral results suggest that EV-CUR-treated mice exhibited better cognitive function than mice in other groups. Overall, the above results suggest that EV-CUR has the potential to serve as an effective therapeutic intervention for AD.

[0078] 3.EV-CUR exhibits excellent biosafety in vivo

[0079] Good biosafety is a key prerequisite for the clinical application of nanomedicines. Therefore, a thorough in vivo evaluation of the potential side effects of EV-CUR was performed. In the efficacy study conducted on elderly AD mice, body weight was also monitored as an indicator of overall health, showing no significant changes between the EV-CUR treated group and the untreated control group ( Fig.11 A). In addition, EV-CUR treatment did not result in any significant changes in serum biochemical parameters, including ALT, AST, CREA, UREA, and eGFR ( Fig.11 B). Interestingly, PBS-CUR treatment resulted in a slight decrease in CREA and UREA levels, although this change was not statistically significant compared to the untreated control group. However, it may indicate some underlying metabolic issues, such as different metabolic pathways of the drug before and after loading. Finally, histopathological examination showed no significant alterations in the liver, kidney, heart, lung, spleen, or brain of animals treated with EV-CUR compared to the control group ( Fig.11 C). In general, the above data indicate that EV-CUR has good biosafety characteristics and no significant adverse reactions to long-term repeated dosing regimens.

[0080] Description of the sources of materials used in this example and some experimental methods:

[0081] Materials: Curcumin (Analytical Reagent, C805204, Macklin), CDKN2A / p16INK4a rabbit polyclonal antibody (AF6471, Beyotime), APP (E4H1U) rabbit mAb (76600S, CST), phospho-T205 rabbit mAb (AP1445, ABclonal), anti-phospho-Tau (S202 / T205) rabbit pAb (GB113883, Servicebio), anti-amyloid β40 rabbit pAb (GB111197, Servicebio), CD11b antibody (AF300081, AiFang biological), β-actin rabbit mAb (AC038, ABclonal), anti-rabbit IgG (H+L) (DyLight 8004X PEG conjugate) (5151S, CST), anti-aging β-galactosidase staining kit (C0602, Beyotime).

[0082] Cell lines: BV2, B16F10, Hepa1-6 and MC38 cells were cultured in Dulbecco's modified Eagle medium (DMEM, 12100, MeilunBio) containing 10% fetal bovine serum (FBS, 04-001-1ACS, Biological Industries). N2a cells were cultured in minimal essential medium (MEM) containing NEAA (MA0217, MeilunBio) with 10% FBS. All complete media contained 1% penicillin-streptomycin solution (100X, 15070063, Gibco). The cell culture incubator (ThermoFisher, USA) was set to 37°C, 5% CO2.

[0083] Animals: SPF-grade C57BL / 6 mice and APP / PS1 / TAU (3×Tg) mice were purchased from Beijing HFK Biotechnology Co., Ltd. and kept in the model animal platform of the Institute of Translational Medicine of Shanghai University for self-breeding and genotyping analysis. The mice were kept in an environment of 21-26°C and 50-60% relative humidity, and the circadian rhythm time was set to 12 hours. C57BL / 6 mice were used for in vivo BBB penetration experiments, and all other animal experiments were performed in 3×Tg mice. All animal experiments were supervised by the Ethics Committee of Shanghai Changzheng Hospital.

[0084] Standard curve of curcumin: Curcumin was mixed with DMSO as a 100 μg / μL stock solution, and then diluted with DMSO to 0.001, 0.002, 0.005, 0.01, 0.02, 0.03 and 0.05 μg / μL working solutions. The maximum absorbance of the spectrum from 300 to 700 nm was scanned in a microplate reader (BioTek, USA), and the standard curve (e.g. Fig.12 A, as shown in 12B).

[0085] Cytotoxicity assay: 100 μL of cell suspension at a density of 5×10^4 cells / mL was carefully inoculated into each well of a 96-well plate, ensuring at least three replicate wells for each experimental condition. A FA control group without cell inoculation was also included to account for background signals. After cell adhesion, 100 ul of complete medium containing hydrogen peroxide (H2O2) or curcumin was added to each well at a stable concentration gradient. After a further 24 hours of incubation, the medium used was replaced with a basal medium containing 10% cell counting kit-8 (MA0218, MeilunBio), facilitating a subsequent 1 hour incubation to allow measurement of cell viability. Similarly, complete medium containing H2O2 was renewed every day, and cell viability was determined after 4 days.

[0086] SA-β-Gal staining: The procedure was performed according to the manufacturer's guidelines. Briefly, cells seeded in 6-well plates were fixed at room temperature for 15 min and then rinsed once with PBS. After washing three times with PBS, the staining fixative was completely removed. Subsequently, the prepared staining solution was added, and the sealed six-well plates were incubated overnight at 37 °C in a CO2-free environment.

[0087] Western Blot: The treated cells and brains were carefully collected, and then total protein was extracted using RIPA (P0013B, Beyotime). The total protein concentration in each sample was determined using an enhanced BCA protein assay kit (P0009, Beyotime). The final concentration of each sample was adjusted consistently for each batch before adding 20% ​​protein loading buffer (LT103, Epizyme Biotechnology). The protein samples were loaded into PAGE gels and analyzed according to the full-length method. TM PAGE gels were prepared according to the instructions of the One-Step Color PAGE Gel Rapid Preparation Kit (PG213, Epilepsy Biotechnology) and then transferred to polyvinylidene fluoride transfer membranes (IPVH00010, Millipore). The membranes were blocked and then incubated with primary antibodies overnight at 4°C. After washing, the membranes were incubated with corresponding secondary antibodies for 1 h at room temperature and then washed for chemical or fluorescent imaging.

[0088] Quantitative real-time PCR: Trizol reagent (R 0016, Beyotime) was used to facilitate the extraction of total RNA from the samples. The RNA concentration was then determined using a nanodrop spectrophotometer (Metash, CHN). Subsequently, complementary DNA (cDNA) synthesis was performed according to the protocol provided by Evo M-MLV RT premix (AG11706, Accurate Biology). PCR amplification was performed in a CFX96 real-time PCR detection system (Bio-Rad, USA) using Genious 2X SYBR Green Fast qPCR mix (RK21204, ABclonal) as the reaction mixture and cycling parameters. Each experiment was repeated at least three times. The relative expression level of the gene is usually expressed by calculating the 2-ΔΔCt value. IL-1β primer: F-GCAACTGTTCCTGAACTCAACT, R-ATCTTTTGGGGTCCGTCAACT, IL-6 primer: F-GACAAAGCCAGAGTCCTTCAGAGAGATACAG, R-TGGATGGTCTTGGTCCTTAGCCAC , TNF-α primers: F-CCACCACGCTCTTCTGTCTACTG, R-GCCATAGACTGATGAGAGG, ACTB primers: F-GTCCCTCACCCTCCCAAAAAG, R-GCTGCCCTCAAAAAG, R-GCTGCCTCAACAACCC.

[0089] In vitro stability determination of EV-CUR: After preparing EV-CUR, an equal amount of curcumin was dissolved in PBS buffer using the same ultrasonic procedure to prepare PBS buffer containing curcumin (PBS-CUR), which was stored at 37°C and protected from light. Within 8 hours after preparation, the samples were observed every hour to see if there was any separation or precipitation of curcumin. 5μl EV-CUR and PBS-CUR were added to a 96-well plate with 95μl DMSO. After keeping the plate away from light for 10 minutes, the absorbance of curcumin was measured using a microplate reader. Finally, according to the curcumin standard curve obtained in this article ( Figure 4 ), and the content of curcumin in the solution was calculated.

[0090] Characterization of EV-CUR: The average size and zeta potential of the nanoparticles were measured using a Zano ZS90 instrument (Malvern, UK). 5-10 μl of B16-EV or EV-CUR was placed in a cuvette and 1 ml of ultrapure water was injected. After mixing, the cuvette was placed in a zeta cell for size measurement. The sample was then transferred from the cuvette to a zeta potential cell for zeta potential measurement. The morphology of the nanoparticles was observed using transmission electron microscopy. 10 μl of the sample was placed on a copper grid, allowed to stand for 1 min, and the floats were removed using filter paper. 10 μl of uranyl acetate was carefully added to the copper grid and allowed to settle for 10 min, after which the removed floats were removed. After the sample was dried at room temperature for a few minutes, electron microscopy was performed (Hitachi, JPN) at an operating voltage of 100 kV, and the images were observed and captured.

[0091] In vivo blood-brain barrier penetration test: C57BL / 6 mice around 4 months old were divided into Hepa1-6-EV group, MC38-EV group, and B16-EV group, with 4 mice in each group. Each group of mice was intraperitoneally injected with 200ugEV per kg body weight. EV staining and labeling were performed according to the instructions of DID (C1995S, Beyotime). After 24 hours of treatment, the mice were killed, and their brains were collected, rinsed with saline, and then visualized using an in vivo imaging system (Bio-Illumination Biotechnology, CHN).

[0092] In vivo administration: 18 aged male 3xTg AD model mice of approximately 15-16 months of age were evenly divided into three groups: CON group (received PBS injection), PBS-CUR group (free CUR given at 7.5 mg / kg) and EV-CUR group (EV-CUR injected as curcumin at 7.5 mg / kg). Intraperitoneal injection once every 3 days for a total of 15 doses. The uniformity of the dosing volume for each round of injection was maintained at 300-500 μL. Before each dosing, the weight of the mice was measured and recorded to monitor their health throughout the study.

[0093] Preparation of tissue sections for staining: Collect half of the brain and heart, lung, liver, kidney and spleen and fix them in 4% paraformaldehyde for more than 24 hours. The fully fixed tissues are sequentially dehydrated with graded alcohol, cleared with xylene, embedded in paraffin and sectioned.

[0094] HE staining: The sections were stained with hematoxylin staining solution (G1004, Service Biology), differentiated with differentiation solution (G1039), blued with blue solution (G1040), dehydrated with graded alcohol, stained with eosin staining solution (G1002), and mounted with neutral balsam after dehydration and clearing. In this process, appropriate washing is required to remove the reagents of the previous step before the next step.

[0095] Nissl staining: Dewaxed brain sections were stained with toluidine blue staining solution (G1032, Servicebio), rinsed with ddH2O, and then differentiated with 1% glacial acetic acid. After washing and drying, the sections were washed and mounted in sequence.

[0096] Immunofluorescence staining: Dewaxed sections after antigen extraction with citrate. After washing three times, the sections were blocked with 3% bovine serum albumin (BSA) at room temperature for 30 minutes. The primary antibody was incubated overnight, and the corresponding fluorescent secondary antibody (GB213303, Servicebio) was added after washing three times, and incubated at room temperature in the dark for 50 minutes. Subsequently, the cell nuclei were stained with DAPI, and after washing, they were mounted with anti-fading mounting medium (G1401, serebio). In the double immunofluorescence labeling procedure, the first primary antibody, the corresponding HRP-conjugated goat anti-rabbit immunoglobulin antibody (H+L) (GB23303, Service Bio) and FITC-tyramide working solution were incubated in sequence, followed by washing and blocking, and the second primary antibody, the corresponding HRP secondary antibody and CY3-tyramide working solution were also incubated.

[0097] Y-maze test: The y-maze consisted of 3 arms separated by 120 degrees, and each arm was 28 cm × 6 cm × 18 cm long. Spontaneous alternation task: Each mouse was placed on the starting arm and allowed to explore freely for 8 min, during which the spontaneous alternation rate was assessed. Before testing the next mouse, the arms of the y-maze were cleaned with 75% alcohol to eliminate any residual odor. The spontaneous alternation rate (%) was calculated as [(number of spontaneous alternations) / (total number of arm entries-2)]×100. Novel arm exploration experiment: The three arms of the maze were divided into: novel arm, starting arm, and other arms. Throughout the experiment, the starting arm and the other arm were always open, allowing animals to enter and exit freely. During the training period, the novel arm was closed and the mice were allowed to freely explore the remaining two arms for 10 min. After one hour, the partition was removed and each mouse was given 5 min of free exploration. The number of entries into each arm, especially the novel arm, was recorded. Before injecting the next mouse, 75% alcohol was used to eliminate the odor.

[0098] Nesting test: Mice were housed individually in cages and acclimated for about 1 hour, and then 6 pieces of soft fiberboard with a diameter of about 6×9 cm were placed on one side of each cage as nesting substrates. After 12 hours, the nesting was evaluated on a scale of 1-5 points: 1 point, no obvious interference; 2 points, irregular dispersion of nesting materials; 3 points: the paper was piled on one side with no obvious nest shape; 4 points: a recognizable but flat nest; 5 points, a perfect or nearly perfect nest.

[0099] Liver and kidney function test: Mice were anesthetized with isoflurane and blood was collected from the eye sockets. After coagulation at room temperature, the blood was centrifuged at 3000 rpm for 5 minutes to collect serum. The serum was analyzed for the main indicators of liver and kidney function (alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, creatinine) using an automatic biochemical analyzer.

[0100] Bioinformatics analysis: Datasets GSE267529, GSE261698, GSE251888, GSE253754, and GSM653856 were all derived from the GEO database. Bulk RNA-seq data were processed and analyzed using the “edgeR” package to identify differentially expressed genes. These genes were subsequently visualized as volcano plots using the “ggplot2” package. To identify commonly up-regulated and down-regulated genes in GSE267529, GSE261698, and GSE251888, we used the “VennDetail” package. Genes that were up-regulated or down-regulated in at least two datasets were subjected to graphene oxide enrichment analysis using the “cluster analyzer” package. For single-cell RNA-seq data analysis, the “Seurat” package was used on the GSM653856 dataset. UMAP plots were generated to visualize single-cell distributions, and cells were color-coded according to the expression of genes that were up-regulated in the bulk RNA-seq data.

[0101] Statistical analysis: The graphics were processed using ImageJ, and the obtained data were imported into GraphPad Prism for statistical analysis and graphical representation. All data were normally distributed (n ≥ 3). Student's t-test was used between two independent samples, and one-way ANOVA was used for two-way comparisons between multiple samples. A p value < 0.05 was considered statistically significant.

[0102] Although the above description is centered on the embodiment, it is only an example and does not limit the present invention. It is clear to those skilled in the art that various modifications and applications not illustrated above can be made within the scope of the essential characteristics of the present embodiment. For example, each component specifically shown in the embodiment can be implemented after being modified. Moreover, various differences related to such modifications and applications should be interpreted as being included in the scope of the present invention as defined in the attached claims.

Claims

1. A nanomaterial for treating Alzheimer's disease by targeting cell senescence, characterized in that: The nano material uses the extracellular vesicles of the melanoma cell line as nano carriers and loads curcumin.

2. A nanomaterial for treating Alzheimer's disease by targeting cell senescence as claimed in claim 1, characterized in that: After obtaining extracellular vesicles of melanoma cell lines by extrusion method, the nanomaterial NP-CUR was prepared by drug loading with a BCA:CUR ratio of 1:0.5-2 by ultrasound method.

3. The nanomaterial for treating Alzheimer's disease by targeting cell senescence as claimed in claim 1, characterized in that: The preparation process of the extracellular vesicles of the melanoma cell line comprises the following steps: S1. When the cell density of melanoma cells is greater than 90%, digest the cells and transfer them to a centrifuge tube. Centrifuge at 1000-2000 rpm for 1-10 min, discard the supernatant, and resuspend the cells in a pre-cooled buffer. S2. Centrifuge at 1000-2000 rpm for 1-10 min, discard the supernatant, resuspend the cells in pre-cooled buffer, centrifuge again and resuspend in buffer; S3. The cell suspension was sequentially extruded through 5, 1, and 0.4 μm track-etched membranes by a liposome extruder, with each extrusion being repeated 10-15 times; S4. The final extruded sample was centrifuged at 10000-15000 g for 5-15 min at 4°C and the precipitate was discarded after centrifugation; S5. The supernatant was concentrated by centrifugation at 4°C for 10-15 minutes more than once to obtain purified extracellular vesicles of the melanoma cell line.

4. The nanomaterial for treating Alzheimer's disease by targeting cell senescence as claimed in claim 1, characterized in that: The ultrasonic method was carried out under the conditions of 150-200 kW power, 10-20% amplitude, alternating 15-60 seconds ultrasonic treatment and 15-60 seconds pause, for a total of not less than 3 cycles.

5. Use of the nanomaterial according to any one of claims 1 to 4 in the preparation of drugs for resisting or alleviating cell aging.

6. Use of the nanomaterial according to any one of claims 1 to 4 in the preparation of a drug for alleviating aging of nerve cells and neuroinflammation.

7. Use of the nanomaterial according to any one of claims 1 to 4 in the preparation of a drug for improving cognitive function.

8. Use of the nanomaterial according to any one of claims 1 to 4 in the preparation of a drug for treating or preventing Alzheimer's disease.

9. The use according to claim 8, characterized in that: The Alzheimer's disease is caused by cellular senescence.

10. A method for establishing an Alzheimer's disease model based on cell senescence, characterized in that: Inducing BV2 cell senescence by H2O2; The concentration of H2O2 induction was 300-400 μM; Dosage: Repeated administration every 4 days.

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