A mitochondrial miRNA-204 nano probe and a preparation method and application thereof

Abstract

The application relates to the technical field of nanobiomaterial synthesis, in particular to a mitochondrion-targeting mi RNA-204 nanoprober as well as a preparation method and application thereof. The mitochondrion-targeting mi RNA-204 nanoprober comprises CeO2-NH2 nanomaterial; the surface of the CeO2-NH2 nanomaterial is modified with a mitochondrion-targeting molecule and a DNA-RNA hybrid chain; the DNA-RNA hybrid chain comprises a DNA chain and an RNA chain, and the DNA chain is complementary to part or all of the bases in the sequence of the mi RNA-204. The nanoprober has good biocompatibility, can highly detect mitochondrion mi RNA-204 and influence the expression of mitochondrion mi RNA-204, and can be applied to the preparation of AD diagnosis and treatment drugs.

Description

Technical Field

[0001] This invention relates to the field of nanobiomaterial synthesis technology, and in particular to a nanoprobe targeting mitochondrial miRNA-204, its preparation method, and its application. Background Technology

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease, primarily characterized by memory loss and cognitive impairment. With the increasing aging of the global population and the rising proportion of elderly people, the incidence of Alzheimer's disease is also showing a corresponding upward trend, placing a heavy psychological and economic burden on families and society. Because the pathogenesis of AD is very complex and its specific mechanisms remain uncertain, the development of drugs for AD still faces significant challenges. Therefore, early diagnosis, intervention, and treatment of AD are extremely important.

[0003] Studies have found that autophagy plays a crucial role in the pathogenesis of Alzheimer's disease (AD), with miRNA-204 controlling mitochondrial energy output and triggering autophagy. However, in the context of AD, overexpression of mitochondrial miRNA-204 leads to abnormal activation of autophagy, preventing cells from effectively clearing harmful proteins, triggering apoptosis, and exacerbating neurodegeneration. Furthermore, cellular dysfunction also affects energy metabolism, leading to excessive production of reactive oxygen species (ROS), further aggravating mitochondrial damage and dysfunction. Therefore, the detection and regulation of mitochondrial miRNA-204 are particularly important for the diagnosis and treatment of AD.

[0004] Cerium dioxide (CeO2) possesses excellent thermal and chemical stability and can interact with various components. It is commonly used as a carrier material or additive to enhance the stability and activity of nanomaterials. The unique Ce4+ / Ce3+ redox cycle in CeO2 nanomaterials endows them with the activity of natural redox enzymes. Therefore, CeO2 nanomaterials can eliminate reactive oxygen species and prevent cell damage during autophagy.

[0005] Current research on Alzheimer's disease (AD) aims to develop drugs that can treat AD by activating mitophagy while also regulating ROS levels. However, identifying molecules that can selectively remove defective mitochondria and reduce damage to normal mitochondrial respiration remains an important area of ​​research. Summary of the Invention

[0006] To address the problems mentioned above in the background art, the first objective of this application is to provide a nanoprobe that targets mitochondrial miRNA-204. This nanoprobe has good biocompatibility, can accurately detect mitochondrial miRNA-204 and affect its expression, and can also eliminate reactive oxygen species. It can be used to prepare drugs for the diagnosis and treatment of AD.

[0007] The solution adopted by this application to solve its technical problem is: including CeO2-NH2 nanomaterials;

[0008] The CeO2-NH2 nanomaterials are surface-modified with mitochondrial targeting molecules and DNA-RNA hybrid chains with fluorescent groups on their surface;

[0009] The DNA-RNA hybrid chain comprises a DNA chain and an RNA chain, wherein the DNA chain is complementary to the nucleotide sequence in the miRNA-204, and the nucleic acid sequence of the DNA chain is TTT TTA GAA AAA CGC CAG ACC CGA ACG ATT TTT-COOH.

[0010] The second objective of this application is to provide a method for preparing a nanoprobe targeting mitochondrial miRNA-204, comprising the following steps:

[0011] The CeO2-NH2 nanomaterials were washed, and then the washed CeO2-NH2 nanomaterials were mixed evenly with DNA-RNA hybrid chains and mitochondrial targeting molecules and incubated for 10-14 h to obtain mitochondrial miRNA-204 nanoprobes. The molar ratio of DNA-RNA hybrid chains to mitochondrial targeting molecules was 1:1.

[0012] Preferably, the CeO2-NH2 nanomaterial is prepared by a method comprising the following steps:

[0013] S1, Cerium acetate and oleylamine are dissolved in xylene to obtain mixed solution one, wherein the mass ratio of cerium acetate (g), oleylamine (g), and xylene (ml) is (4.0-4.5):(30-35):(145-155);

[0014] S2, Mixed solution one is stirred vigorously at room temperature for 12 hours, then heated to 90°C under vacuum, and then deionized water is rapidly injected into the heated mixed solution one. The volume ratio of deionized water to mixed solution one is (1-13):(1-17) to obtain the reaction solution.

[0015] S3, incubate the obtained reaction solution for 2-4 hours at a temperature of 90-95°C until the reaction solution becomes transparent, and then cool the reaction solution to room temperature;

[0016] S4. Take 15-17 ml of the reaction solution treated in step S3, then precipitate the nanoparticles with 100 ml of acetone and add it to the reaction solution. Then centrifuge to obtain crude CeO2 nanoparticles.

[0017] S5, add chloroform to the crude CeO2 nanoparticles until the concentration of the crude CeO2 nanoparticles is 10 mg / mL, and then centrifuge to obtain intermediate CeO2 nanoparticles.

[0018] S6, the amination reagent and intermediate CeO2 nanoparticles are placed in chloroform and mixed evenly to obtain mixed solution two. Then, the mixed solution two is subjected to rotary evaporation under vacuum conditions at an evaporation temperature of 80-85℃ and an evaporation time of 1-1.5h to obtain an intermediate. The ratio of the mass (mg) of the intermediate CeO2 nanoparticles to the volume (ml) of the amination reagent and the volume (ml) of chloroform is (7-9):(2-5):(20-30).

[0019] S7. Deionized water is poured into a container containing the intermediate and then sonicated to obtain a suspension. The suspension is then filtered through a filter with a pore size of 0.2 μm until it becomes transparent.

[0020] S8. The transparent suspension was filtered using a centrifugal filter with a molecular weight cutoff of 50 kDa, and then dialyzed for 24 hours using a dialysis box with a molecular weight cutoff of 10 kDa to obtain pure CeO2 nanoparticles.

[0021] S9. Pure CeO2 nanoparticles were modified with a silane coupling agent for 10–14 h. The modified product was then activated with 20 μL of SPDP (3-(2-pyridyl dithio)propionate succinimide ester) within 30 min to obtain CeO2-NH2 nanomaterials.

[0022] Preferably, the DNA-RNA hybrid strand is prepared by a method comprising the following steps:

[0023] S1. Prepare DNA and RNA chain solutions. Place the DNA and RNA chain solutions in a test tube under light-protected conditions and mix thoroughly. Then place the test tube in a thermostat. Raise the temperature of the thermostat to 65°C and maintain it for 2 minutes at a rate of 2°C / min. Then raise the temperature to 95°C and maintain it for 10 minutes at a rate of 20°C / min to obtain the DNA-RNA hybrid chain.

[0024] Preferably, the mitochondrial targeting molecule is prepared by a method comprising the following steps:

[0025] Mitochondrial-targeting molecules were synthesized using a nucleic acid synthesizer.

[0026] The third objective of this application is to provide an application of a mitochondrial miRNA-204 nanoprobe in the preparation of drugs for the diagnosis and treatment of Alzheimer's disease.

[0027] Preferably, the dosage form of the drug includes tablets, powders, suspensions, granules, capsules, injections, sprays, solutions, enemas, emulsions, films, suppositories, patches, nasal drops, or pills.

[0028] The fourth objective of this application is to provide an application of a mitochondrial miRNA-204-targeting nanoprobe in the preparation of miRNA-204 detection drugs.

[0029] In summary, the beneficial effects of the present invention are as follows:

[0030] The nanoprobe targeting mitochondrial miRNA-204 is loaded with a DNA-RNA hybrid strand for detecting mitochondrial miRNA-204. It can highly recognize overexpressed miRNA-204 in mitochondria and silence miRNA-204 gene expression.

[0031] By silencing the expression of mitochondrial miRNA-204, the autophagy pathway was mediated by targeting mitochondrial miRNA-204 nanoprobes, restoring the mitochondrial membrane potential to normal levels.

[0032] The mitochondrial miRNA-204 nanoprobe has good biocompatibility, can cross the blood-brain barrier to target mitochondria, and can utilize CeO2's unique reactive oxygen species scavenging mechanism to treat AD.

[0033] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0034] Appendix Figure 1 This is a TEM image of the CeO2-DRP / MA nanomaterial from Example 1;

[0035] Appendix Figure 2 The crystal structure diagram of CeO2-DRP / MA nanomaterials in Example 1 is shown.

[0036] Appendix Figure 3 Elemental composition analysis of CeO2-DRP / MA nanomaterials in Example 1; 15 nm;

[0037] Appendix Figure 4Fluorescence intensity at different time points during co-incubation of CeO2-DRP / MA and miRNA-204;

[0038] Appendix Figure 5 A graph showing the specificity of CeO2-DRP / MA in detecting mitochondrial miRNA-204;

[0039] Appendix Figure 6 A graph showing the sensitivity of CeO2-DRP / MA for detecting mitochondrial miRNA-204;

[0040] Appendix Figure 7 This study aimed to detect the ability of CeO2-DRP / MA to detect mitochondrial miRNA-204 in nerve cells. Scale bar: 50 μm.

[0041] Appendix Figure 8 A study on the ability of CeO2-DRP / MA to restore mitochondrial membrane potential in damaged nerve cells, scale bar 50 μm;

[0042] Appendix Figure 9 The biological transmission electron microscopy image of CeO2-DRP / MA inhibiting autophagosome production, scale bar 1 μm;

[0043] Appendix Figure 10 Research on the ability of CeO2-DRP / MA to cross the blood-brain barrier;

[0044] Appendix Figure 11 The swimming trajectory diagrams of mice in the water maze experiment for each group;

[0045] Appendix Figure 12 A graph showing the number of intersecting grids traversed by mice in the open field experiment for each group;

[0046] Appendix Figure 13 A graph showing the number of defecations in each group of mice during the open field experiment;

[0047] Appendix Figure 14 Nissl-stained sections of the hippocampus of mice in different experimental groups, scale bar 50 μm;

[0048] Appendix Figure 15 Stained sections of hippocampal Aβ protein from different experimental groups of mice. Scale bar: 50 μm. Detailed Implementation

[0049] To make the content of this invention easier to understand, the invention will be further described below with reference to specific embodiments and accompanying drawings.

[0050] This application provides a mitochondrial miRNA-204 targeting nanoprobe (CeO2-DRP / MA), comprising CeO2-NH2 nanomaterials; the surface of the CeO2-NH2 nanomaterials is modified with mitochondrial targeting molecules and DNA-RNA hybrid chains modified with fluorescent groups on the surface; the DNA-RNA hybrid chains include DNA chains and RNA chains, the surface of the DNA chain is modified with fluorescein, the surface of the RNA chain is modified with a fluorescence quencher, and the DNA chain is complementary to all the bases of the sequence in miRNA-204.

[0051] In this application, the fluorescent group modified at the end of the DNA strand is FAM, and the fluorescence quencher group modified at the end of the RNA strand is BHQ. FAM emits green fluorescence upon photoexcitation. When the DNA and RNA strands hybridize, the distance between FAM and BHQ decreases, and the fluorescence of FAM is quenched by BHQ. However, when miRNA-204 is present in the mitochondria, FAM and BHQ separate, and the fluorescence signal is recovered to a certain extent, thus achieving in-situ imaging of miRNA-204 in the mitochondria. This application describes CeO2-DRP / MA obtained by modifying the surface of CeO2-NH2 nanomaterials with mitochondrial targeting molecules and DNA-RNA hybrid chains. The mitochondrial targeting molecules can recognize and bind to mitochondria, and the DNA strand in the DNA-RNA hybrid chain is complementary to the bases of the key functional sequence in miRNA-204. Therefore, CeO2-DRP / MA can specifically detect miRNA-204 and silence the expression of the miRNA-204 gene through the DNA-RNA hybrid chain, mediating the occurrence of autophagy pathway, restoring the mitochondrial membrane potential to a normal level, and simultaneously synergizing with CeO2's unique reactive oxygen species scavenging mechanism to reduce cell damage and dysfunction, thereby improving the therapeutic effect of AD.

[0052] In this application, there is no particular limitation on the particle size of CeO2-DRP / MA, but 40 nm is preferred.

[0053] This application also provides a method for preparing a nanoprobe targeting mitochondrial miRNA-204, comprising the following steps:

[0054] The CeO2-NH2 nanomaterials were washed, and then the washed CeO2-NH2 nanomaterials were mixed evenly with DNA-RNA hybrid chains and mitochondrial targeting molecules and incubated for 10-14 h to obtain mitochondrial miRNA-204 nanoprobes. The molar ratio of DNA-RNA hybrid chains to mitochondrial targeting molecules was 1:1.

[0055] In this application, there are no particular restrictions on the order in which CeO2-NH2 nanomaterials, DNA-RNA hybrid chains, and mitochondrial targeting molecules are added. The molar ratio of DNA-RNA hybrid chains to mitochondrial targeting molecules is preferably 1:1. There are no particular restrictions on the number of times CeO2-NH2 nanomaterials are washed, but preferably not less than 3 times.

[0056] In this application, the CeO2-NH2 nanomaterials are prepared by a method comprising the following steps:

[0057] S1, Cerium acetate and oleylamine are dissolved in xylene to obtain mixed solution one, wherein the mass ratio of cerium acetate (g), oleylamine (g) to xylene (ml) is (4.0~4.5):(30~35):(145~155);

[0058] S2, Mixed solution one is vigorously stirred at room temperature for 10-14 hours, then heated to 90°C under vacuum, and then deionized water is rapidly injected into the heated mixed solution one, with the volume ratio of deionized water to mixed solution one being 10-14 hours, to obtain the reaction solution; when heating under vacuum, there is no particular limitation on the heating rate, but the preferred heating rate is 2°C / min;

[0059] S3, incubate the obtained reaction solution for 2-4 hours at a temperature of 90-95℃ until the reaction solution becomes transparent, and then cool the reaction solution to room temperature;

[0060] S4. Take 15-17 ml of the reaction solution treated in step S3, then precipitate the nanoparticles with 100 ml of acetone and add it to the reaction solution. Then centrifuge to obtain crude CeO2 nanoparticles.

[0061] S5, add chloroform to the crude CeO2 nanoparticles until the concentration of the crude CeO2 nanoparticles is 10 mg / mL, and then centrifuge to obtain intermediate CeO2 nanoparticles.

[0062] S6, the amination reagent and intermediate CeO2 nanoparticles are placed in chloroform and mixed evenly to obtain mixed solution two. Then, the mixed solution two is subjected to rotary evaporation under vacuum conditions at an evaporation temperature of 80-85℃ and an evaporation time of 1-1.5h to obtain an intermediate. The ratio of the mass (mg) of the intermediate CeO2 nanoparticles to the volume (ml) of the amination reagent and the volume (ml) of chloroform is (7-9):(2-5):(20-30).

[0063] S7. Deionized water is poured into a container containing the intermediate and then sonicated to obtain a suspension. The suspension is then filtered through a filter with a pore size of 0.2 μm until it becomes transparent.

[0064] S8. The transparent suspension was filtered using a centrifugal filter with a molecular weight cutoff of 50 kDa, and then dialyzed for 24 hours using a dialysis box with a molecular weight cutoff of 10 kDa to obtain pure CeO2 nanoparticles.

[0065] S9. Pure CeO2 nanoparticles with a concentration of 5-7 mM were modified with a silane coupling agent for 10-14 h. The modified product was activated with 20 μL of SPDP (3-(2-pyridyl dithio)propionate succinimide ester) within 30 min to obtain CeO2-NH2 nanomaterials.

[0066] In this application, the DNA-RNA hybrid strand is prepared by a method comprising the following steps:

[0067] S1. Prepare DNA and RNA chain solutions. Place the DNA and RNA chain solutions in a test tube under light-protected conditions and mix thoroughly. Then place the test tube in a thermostat. Raise the temperature of the thermostat to 65°C and maintain it for 2 minutes at a rate of 2°C / min. Then raise the temperature to 95°C and maintain it for 10 minutes at a rate of 20°C / min to obtain the DNA-RNA hybrid chain.

[0068] The mitochondrial targeting molecule was prepared by a method including the following steps:

[0069] Mitochondrial-targeting molecules were synthesized using a nucleic acid synthesizer.

[0070] This application also provides the application of a mitochondrial miRNA-204-targeting nanoprobe or a mitochondrial miRNA-204-targeting nanoprobe prepared by a preparation method in the preparation of Alzheimer's disease diagnostic and therapeutic drugs or in the detection of miRNA-204. The dosage form of the drug includes tablets, powders, suspensions, granules, capsules, injections, sprays, solutions, enemas, emulsions, films, suppositories, patches, nasal drops, or pills.

[0071] To further illustrate the present invention, the following embodiments provide a detailed description. The experimental materials used in the following embodiments of the present invention can be purchased commercially or prepared according to conventional preparation methods well known to those skilled in the art.

[0072] Example 1

[0073] 1) Preparation of CeO2-NH2 nanomaterials:

[0074] S1, Cerium acetate and oleylamine are dissolved in xylene to obtain mixed solution one, wherein the mass of cerium acetate is 0.43 g, the mass of oleylamine is 3.25 g, and the volume of xylene is 15 ml;

[0075] S2, Mixed solution one is stirred vigorously at room temperature for 12 hours, then heated to 90°C under vacuum at a rate of 2°C / min, and then 1 ml of deionized water is rapidly injected into the heated mixed solution one to obtain the reaction solution;

[0076] S3, the obtained reaction solution is incubated for 3 hours at 90°C until the reaction solution becomes transparent, and then the reaction solution is cooled to room temperature;

[0077] S4. Precipitate the nanoparticles with 100 ml of acetone, then add them to the reaction solution obtained in step S3, and then centrifuge to obtain crude CeO2 nanoparticles.

[0078] S5, add chloroform to the crude CeO2 nanoparticles until the concentration of the crude CeO2 nanoparticles is 10 mg / mL to obtain intermediate CeO2 nanoparticles.

[0079] S6. The amination reagent and intermediate CeO2 nanoparticles were placed in chloroform and mixed evenly to obtain mixed solution two. Then, the mixed solution two was subjected to rotary evaporation under vacuum conditions at a temperature of 80°C for 1 hour to obtain an intermediate. The mass of the intermediate CeO2 nanoparticles was 8 mg, the volume of the amination reagent was 3 ml, and the volume of chloroform was 25 ml.

[0080] S7. Deionized water is poured into a container containing the intermediate and then sonicated to obtain a suspension. The suspension is then filtered through a filter with a pore size of 0.2 μm until it becomes transparent.

[0081] S8. The transparent suspension was filtered using a centrifugal filter with a molecular weight cutoff of 50 kDa, and then dialyzed for 24 hours using a dialysis box with a molecular weight cutoff of 10 kDa to obtain pure CeO2 nanoparticles.

[0082] S9. Pure CeO2 nanoparticles with a concentration of 5-7 mM were modified with a silane coupling agent for 12 h. The modified product was activated with 20 μL of SPDP (3-(2-pyridyl dithio)propionate succinimide ester) within 30 min to obtain CeO2-NH2 nanomaterials.

[0083] 2) Preparation of DNA-RNA hybrid strands:

[0084] S1. Prepare DNA and RNA chain solutions. Place the DNA and RNA chain solutions in a test tube under light-protected conditions and mix thoroughly. Then place the test tube in a thermostat. Raise the temperature of the thermostat to 65°C and maintain it for 2 minutes at a rate of 2°C / min. Then raise the temperature to 95°C and maintain it for 10 minutes at a rate of 20°C / min to obtain the DNA-RNA hybrid chain.

[0085] 3) Preparation of mitochondrial-targeting molecules:

[0086] Mitochondrial-targeting molecules were synthesized using a nucleic acid synthesizer.

[0087] 4) Preparation of mitochondrial miRNA-204 nanoprobes

[0088] The CeO2-NH2 nanomaterials were washed, and then the washed CeO2-NH2 nanomaterials were mixed evenly with DNA-RNA hybrid chains and mitochondrial targeting molecules and incubated for 12 h to obtain mitochondrial miRNA-204 nanoprobes. The molar ratio of DNA-RNA hybrid chains to mitochondrial targeting molecules was 1:1.

[0089] In this embodiment, the nucleic acid sequence required for the synthesis of CeO2-DRP / MA is as follows:

[0090] miRNA-204: CUU UUU GCG GUC UGG GCU UGC;

[0091] DNA strand: FAM-TTT TTA GAA AAA CGC CAG ACC CGA ACG ATT TTT-COOH;

[0092] RNA strand: BHQ-UUG CGG UCU GGG C;

[0093] Mitochondrial targeting molecules: CAC, CAC, GAU, CAC, GGU, UUC, CCU, CGC, AGG, UAA, GGU, GUA, GA-COOH

[0094] The method described in this application yields a mitochondrial miRNA-204-targeting nanoprobe, such as... Figure 1 As shown, the morphology is a nanocluster material with a morphology of about 40 nm.

[0095] like Figure 2 As shown, compared with unmodified CeO2 nanoparticles, no significant changes were observed in the crystal structure of CeO2-DRP / MA nanoclusters modified with mitochondrial targeting molecules and DNA / RNA hybrid chains.

[0096] like Figure 3As shown, the presence of the three characteristic elements Ce, N, and P confirms the successful synthesis of CeO2-DRP / MA nanomaterials and the effective connection between mitochondrial targeting molecules and DNA-RNA hybrid chains.

[0097] Performance testing

[0098] 1) Study on the ability of in vitro targeted detection of mitochondrial miRNA-204.

[0099] First, CeO2-DRP / MA was added to a phosphate-buffered saline (PBS) solution containing mitochondrial miRNA-204, and the fluorescence intensity of the FAM fluorescent group was detected using a UV spectrophotometer at 0, 5, 10, 15, 30, 45 and 60 minutes.

[0100] The result is as follows Figure 4 As shown, the fluorescence intensity of the FAM fluorescent group in the nanomaterial gradually increases with increasing reaction time. This confirms the effectiveness of CeO2-DRP / MA nanomaterials in detecting mitochondrial miRNA-204.

[0101] Figure 5 The relative fluorescence intensity change F / F0 showed a good linear relationship with the logarithm of miRNA-204 concentration, covering a concentration range from 0.1 pmol / L to 1 nmol / L. The limit of detection (LOD), calculated by three times the standard deviation of the blank control, was 64.9 fmol / L, indicating that CeO2-DRP / MA nanomaterials have high sensitivity for detecting mitochondrial miRNA-204 at extremely low concentrations.

[0102] CeO2-DRP / MA was reacted with different mitochondrial miRNAs to detect the detection specificity of the nanomaterials. The results are as follows: Figure 6 As shown, CeO2-DRP / MA nanomaterials exhibited significant fluorescence recovery only in the presence of mitochondrial miRNA-204. In contrast, the FAM fluorescence intensity of the nanomaterials for non-target miRNAs-155 and miRNA-181 was comparable to that of the blank control group, further confirming the high specificity of this nanomaterial for detecting mitochondrial miRNA-204.

[0103] SY5Y cells were further cultured and incubated with β-amyloid (Aβ)-treated SY5Y cells in a 1 mmol / L CeO2-DRP / MA solution for 4 h. The detection ability of the nanomaterials for mitochondrial miRNA-204 was then assessed using confocal laser scanning microscopy. The results are as follows: Figure 7As shown, strong green fluorescence (FAM) was observed in Aβ-treated SY5Y cells, indicating a higher expression level of mitochondrial miRNA-204 in AD cells. Normal cells exhibited weak green fluorescence (FAM), indicating a significant reduction in mitochondrial miRNA-204 expression compared to AD cells. This confirms that CeO2-DRP / MA possesses excellent detection performance in cells.

[0104] 2) Examine its ability to restore mitochondrial membrane potential in vitro.

[0105] The ability of nanomaterials to restore mitochondrial membrane potential was observed using a mitochondrial membrane potential assay kit. The results are as follows: Figure 8 As shown, SY5Y+Aβ cells treated with CeO2-DRP / MA nanomaterials exhibited very few green fluorescent positive cells, while untreated cells showed a strong green fluorescent positive population. This indicates that CeO2-DRP / MA nanomaterials significantly increased the membrane potential of mitochondria in cells, thereby helping to restore damaged mitochondria to a normal state.

[0106] 3) Research on the in vitro inhibition of autophagy.

[0107] Biological transmission electron microscopy was used to observe whether nanomaterials could inhibit the production of autophagosomes. The results are as follows: Figure 9 As shown, a distinct double-membrane autophagosome structure was observed in SY5Y+Aβ cells (marked by red arrows). However, no autophagosome formation was observed in SY5Y+Aβ cells treated with CeO2-DRP / MA, indicating that CeO2-DRP / MA nanomaterials effectively inhibited autophagosome production.

[0108] Study on the in vivo therapeutic effects of CeO2-DRP / MA nanomaterials for AD

[0109] The use of laboratory animals in relevant experiments was approved by the Animal Ethics Committee of Shenzhen Longgang Central Hospital (approval number LCH-IACUC-2024-008).

[0110] 1) The ability to perform fluorescence imaging of mitochondrial miRNA-204 in vivo.

[0111] Brains were collected from 3×Tg-AD mice after equal volumes of 1 mM CeO2-DRP / MA were injected into them at 12 and 24 hours, respectively. Fluorescence images of the mouse brains were captured using an IVIS spectroscopy system. The results are as follows: Figure 10 As shown, the nanomaterials effectively crossed the blood-brain barrier to reach the mouse brain, enabling effective detection in mice.

[0112] 2) Research on improving the behavioral abilities of mice.

[0113] Four-month-old 3×Tg-AD mice and wild-type (WT) mice (C57BL / 6) were obtained from Beijing Victoria Laboratory Animal Technology Co., Ltd. (China). Animal experiments were conducted in three groups:

[0114] (1) Wild-type group: 10 C57BL / 6 mice;

[0115] (2) AD control group: 10 3×Tg AD mice were injected with PBS via tail vein every two days;

[0116] (3) CeO2-DRP / MA treatment group: 10 3×Tg AD mice were injected with CeO2-DRP-MA at a concentration of 1mM via the tail vein every two days.

[0117] Morris Water Maze: All mice underwent daily training for the first five days. During the experiment, they were tested three times daily using various random paths, each starting from a different quadrant of the maze. All tests were completed within one minute. On day seven, each mouse was tested to determine its response to platform removal. Data was collected using a camera and analyzed using software. The results are as follows: Figure 11 As shown, the 3×Tg-AD model mice mainly performed mechanical swimming along the edge of the water maze within 1 minute of the experiment and failed to effectively locate the specific position of the previous platform; the mice treated with CeO2-DRP / MA showed obvious purposeful swimming behavior and showed similarity to wild-type mice in their ability to remember the position of the previous platform.

[0118] Field Experiment: Mice were first placed in a 30×30×20cm chamber and given time to acclimatize. The chamber was free of other interfering factors. Data was collected using a camera during 5 minutes of exploration of the open area. It is important to note that the ground should be cleaned with 70% ethanol each time. The results are as follows... Figure 12 and Figure 13 As shown, during the 5-minute observation period, compared with 3×Tg-AD mice, WT mice and CeO2-DRP / MA-treated mice significantly increased the number of times they crossed the intersecting grid (p<0.01); CeO2-DRP / MA-treated mice showed significantly improved anxiety behavior, such as reduced defecation frequency.

[0119] 3) Investigate the repair ability of CeO2-DRP / MA nanomaterials on damaged neurons in 3×Tg-AD mice.

[0120] The morphology and density of mouse neurons were observed using Nissl staining. The results are as follows: Figure 14As shown, compared with wild-type mice, neurons in the hippocampus of 3×Tg-AD model mice exhibited pathological features such as nuclear shrinkage and hypocellularity. In contrast, neurons in the hippocampus of mice treated with CeO2-DRP / MA maintained intact morphology, and their cell density was similar to that of wild-type mice, with no significant difference. This indicates that CeO2-DRP / MA nanomaterials effectively alleviated the degeneration and necrosis of neurons in 3×Tg-AD mice, demonstrating their potential therapeutic effects in promoting neuronal cell repair and maintaining the structural integrity of the nervous system.

[0121] 4) To further evaluate the potential impact of CeO2-DRP / MA on Aβ protein deposition in the brains of 3×Tg-AD mice.

[0122] The distribution and content of Aβ protein in the hippocampus of mouse brain were observed using immunohistochemical experiments. The results are as follows: Figure 15 As shown, a large amount of Aβ protein expression was observed in the hippocampus of 3×Tg-AD model mice, which was in stark contrast to the control wild-type mice. Tissue sections of the hippocampus region of mice treated with CeO2-DRP / MA showed a significant reduction in the amount of Aβ protein deposited, indicating that CeO2-DRP / MA effectively reduced the deposition of Aβ protein in the mouse brain and promoted its clearance, thereby inhibiting the formation of Aβ protein and confirming its significant therapeutic effect in vivo.

[0123] The embodiments described above are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and modifications made by those skilled in the art based on the invention shall fall within the scope of protection of the present invention.

Claims

1. A nanoprobe targeting mitochondrial miRNA-204, characterized in that, Including CeO2-NH2 nanomaterials; The CeO2-NH2 nanomaterial is surface-modified with mitochondrial targeting molecules and DNA-RNA hybrid chains. The nucleic acid sequence of the mitochondrial targeting molecules is CAC CAC GAU CAC GGU UUC CCU CGC AGG UAA GGU GUA GA-COOH. The DNA-RNA hybrid chain comprises a DNA chain and an RNA chain, wherein the DNA chain is complementary to the nucleotide sequence in the miRNA-204, and the nucleic acid sequence of the DNA chain is TTT TTA GAA AAA CGC CAG ACC CGA ACG ATT TTT-COOH.

2. The nanoprobe targeting mitochondrial miRNA-204 according to claim 1, characterized in that, The nanoprobe has a particle size of 40 nm.

3. A method for preparing the mitochondrial miRNA-204 nanoprobe of claim 1 or 2, characterized in that, Includes the following steps: The CeO2-NH2 nanomaterials were washed, and then the washed CeO2-NH2 nanomaterials were mixed evenly with DNA-RNA hybrid chains and mitochondrial targeting molecules and incubated for 10-14 h to obtain mitochondrial miRNA-204 nanoprobes. The molar ratio of DNA-RNA hybrid chains to mitochondrial targeting molecules was 1:

1.

4. The preparation method according to claim 3, characterized in that, The CeO2-NH2 nanomaterial is prepared by a method comprising the following steps: S1, Cerium acetate and oleylamine are dissolved in xylene to obtain mixed solution one, wherein the mass ratio of cerium acetate (g), oleylamine (g), and xylene (ml) is (4.0-4.5):(30-35):(145-155); S2, Stir the mixed solution one at room temperature for 10-14 hours, then heat it to 90°C under vacuum, and then inject deionized water into the heated mixed solution one. The volume ratio of deionized water to mixed solution one is (1-13):(1-17) to obtain the reaction solution. S3, incubate the reaction solution for 2-4 hours at a temperature of 90-95°C until the reaction solution becomes transparent, and then cool the reaction solution to room temperature; S4. Take 15-17 ml of the reaction solution treated in step S3, then precipitate the nanoparticles with 100 ml of acetone and add it to the reaction solution. Then centrifuge to obtain crude CeO2 nanoparticles. S5, add chloroform to the crude CeO2 nanoparticles and stir until the concentration of the crude CeO2 nanoparticles is 10 mg / mL to obtain intermediate CeO2 nanoparticles. S6, the amination reagent and intermediate CeO2 nanoparticles are placed in chloroform and mixed evenly to obtain mixed solution two. Then, the mixed solution two is subjected to rotary evaporation under vacuum conditions at an evaporation temperature of 80-85℃ and an evaporation time of 1-1.5h to obtain an intermediate. The ratio of the mass (mg) of the intermediate CeO2 nanoparticles to the volume (ml) of the amination reagent and the volume (ml) of chloroform is (7-9):(2-5):(20-30). S7. Deionized water is poured into a container containing the intermediate and then sonicated to obtain a suspension. The suspension is then filtered through a filter with a pore size of 0.2 μm until it becomes transparent. S8. The transparent suspension was filtered using a centrifugal filter with a molecular weight cutoff of 50 kDa, and then dialyzed for 24 hours using a dialysis box with a molecular weight cutoff of 10 kDa to obtain pure CeO2 nanoparticles. S9. Pure CeO2 nanoparticles were modified with a silane coupling agent for 10-14 h. The modified product was activated with 20 μL of SPDP (3-(2-pyridyl dithio)propionate succinimide ester) within 30 min to obtain CeO2-NH2 nanomaterials.

5. The preparation method according to claim 3, characterized in that, The DNA-RNA hybrid strand is prepared by a method comprising the following steps: S1. Prepare DNA and RNA chain solutions. Place the DNA and RNA chain solutions in a test tube under light-protected conditions and mix thoroughly. Then place the test tube in a thermostat. Raise the temperature of the thermostat to 65°C and maintain it for 2 minutes at a rate of 2°C / min. Then raise the temperature to 95°C and maintain it for 10 minutes at a rate of 20°C / min to obtain the DNA-RNA hybrid chain.

6. The preparation method according to claim 3, characterized in that, The mitochondrial targeting molecule is prepared by a method comprising the following steps: Mitochondrial-targeting molecules were synthesized using a nucleic acid synthesizer.

7. The application of a nanoprobe in the preparation of drugs for the diagnosis and treatment of Alzheimer's disease, characterized in that, The mitochondrial miRNA-204 nanoprobe as described in claim 1 or 2 is used.

8. The application of the nanoprobe according to claim 7 in the preparation of drugs for the diagnosis and treatment of Alzheimer's disease, characterized in that: The dosage forms of the drug include tablets, powders, suspensions, granules, capsules, injections, sprays, solutions, enemas, emulsions, films, suppositories, patches, nasal drops, or pills.

9. The application of a nanoprobe in the preparation of miRNA-204 detection drugs, characterized in that, The mitochondrial miRNA-204 nanoprobe as described in claim 1 or 2 is used.

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