Targeted mitochondrial miRNA-204 nano probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of synthesis of nano biological materials, in particular to a mitochondria targeting mi RNA-204 nano probe as well as a preparation method and application thereof, and the mitochondria targeting mi RNA-204 nano probe comprises a CeO2-NH2 nano material; the surface of the CeO2-NH2 nano material is modified with a mitochondrial 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 with a part of or all bases of the sequence in the mi RNA-204. The nano probe has good biocompatibility, can highly detect mitochondria mi RNA-204 and influence the expression of the mitochondria mi RNA-204, and can be applied to preparation of AD diagnosis and treatment drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of nano-biomaterial synthesis, and in particular to a mitochondrial miRNA-204 targeted nano-probe and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is a common neurodegenerative disease, which is mainly manifested by memory loss and cognitive dysfunction. With the aging of the global population and the increasing proportion of the elderly population, the incidence of Alzheimer's disease has also shown a corresponding upward trend, bringing heavy mental pressure and economic burden to families and society. Because the pathogenic mechanism of AD is very complex, the specific pathogenesis of AD is still uncertain, and the development of its drugs still faces major challenges. Therefore, early diagnosis, intervention and treatment of AD are extremely important.

[0003] Studies have found that cellular autophagy plays a key role in the pathogenesis of AD, among which miRNA-204 can control mitochondrial energy output and trigger cellular autophagy. However, in the context of AD, overexpression of mitochondrial miRNA-204 will lead to abnormal activation of autophagy function, and cells will be unable to effectively remove harmful proteins, triggering cell apoptosis mechanisms and exacerbating neurodegeneration. In addition, cellular dysfunction can also affect energy metabolism, leading to excessive production of reactive oxygen species (ROS), further exacerbating mitochondrial damage and dysfunction. Therefore, the detection and regulation of mitochondrial miRNA-204 is particularly important for the diagnosis and treatment of AD.

[0004] Cerium dioxide (CeO2) has excellent thermal and chemical stability and can interact with a variety of components. It is usually used as a carrier material or additive to improve the stability and activity of nanomaterials. The unique Ce4+ / Ce3+ redox cycle in CeO2 nanomaterials gives it natural oxidoreductase activity. Therefore, CeO2 nanomaterials can eliminate reactive oxygen species and prevent cell damage during autophagy.

[0005] Current research on AD focuses on developing drugs that can treat AD by activating mitochondrial autophagy while regulating ROS levels. However, how to selectively remove defective mitochondria and reduce damage to normal mitochondrial respiration remains an important direction of current research. Summary of the invention

[0006] In order to solve the problems in the above-mentioned background technology, the first purpose of this application is to provide a nanoprobe targeting mitochondrial mi RNA-204. The nanoprobe has good biocompatibility, can accurately detect mitochondrial mi RNA-204 and affect the expression of mitochondrial miRNA-204, and can also eliminate reactive oxygen, and can be used to prepare AD diagnostic and therapeutic drugs.

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

[0008] The surface of the CeO2-NH2 nanomaterial is modified with mitochondrial targeting molecules and a DNA-RNA hybrid chain modified with a fluorescent group;

[0009] The DNA-RNA hybrid chain includes a DNA chain and an RNA chain, and the DNA chain is complementary to a part or all of the bases in the sequence of the mi RNA-204.

[0010] The second purpose of this application is to provide a method for preparing a mitochondrial mi RNA-204 targeted nanoprobe, comprising the following steps:

[0011] The CeO2-NH2 nanomaterials were washed, and then the washed CeO2-NH2 nanomaterials were evenly mixed with the DNA-RNA hybrid chain and the mitochondrial targeting molecule and incubated for 10-14 hours to obtain the mitochondrial mi RNA-204 targeted nanoprobe, and the molar ratio of the DNA-RNA hybrid chain to the mitochondrial targeting molecule was 1:1.

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

[0013] S1, dissolving cerium acetate and oleylamine in xylene to obtain a mixed solution 1, wherein the ratio of the mass (g) of cerium acetate, the mass (g) of oleylamine and the volume (ml) of xylene is (4.0-4.5):(30-35):(145-155);

[0014] S2, vigorously stirring the mixed solution 1 at room temperature for 12 hours, then heating to 90° C. under vacuum conditions, and then quickly injecting deionized water into the heated mixed solution 1, wherein the volume ratio of deionized water to the mixed solution 1 is (1-13):(1-17), to obtain a reaction solution;

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

[0016] S4, taking 15-17 ml of the reaction solution treated in step S3, precipitating nanoparticles with 100 ml of acetone and adding the precipitated nanoparticles to the reaction solution, followed by centrifugation to obtain crude CeO2 nanoparticles;

[0017] S5, adding chloroform to the crude CeO2 nanoparticles until the concentration of the crude CeO2 nanoparticles is 10 mg / mL, and then centrifuging to obtain intermediate CeO2 nanoparticles;

[0018] S6, placing the amination reagent and the intermediate CeO2 nanoparticles in chloroform and mixing them evenly to obtain a mixed solution II, and then subjecting the mixed solution II to rotary evaporation under vacuum conditions at an evaporation temperature of 80 to 85°C and an evaporation time of 1 to 1.5 hours to obtain an intermediate, wherein 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 to 9): (2 to 5): (20 to 30);

[0019] S7, pouring deionized water into the container containing the intermediate, then subjecting it to ultrasonic treatment to obtain a suspension, and then filtering the suspension through a filter with a pore size of 0.2 μm until the suspension is transparent;

[0020] S8, filtering the transparent suspension using a centrifugal filter with a molecular weight cutoff of 50 kDa, and then dialyzing it 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 to 14 h, and the modified product was activated with 20 μL of SPDP (3-(2-pyridyldithio) propionic acid succinimidyl ester) within 30 min to obtain CeO2-NH2 nanomaterials.

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

[0023] S1, prepare DNA chain solution and RNA chain solution, put the DNA chain solution and RNA chain solution into a test tube under light-proof conditions and mix them evenly, then put the test tube into a constant temperature instrument, then raise the temperature of the constant temperature instrument to 65°C and maintain it for 2 minutes, the heating rate is 2°C / min, then raise the temperature to 95°C and maintain it for 10 minutes, the heating rate is 20°C / min, to obtain a DNA-RNA hybrid chain.

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

[0025] Synthesize mitochondrial targeting molecules using a nucleic acid synthesizer.

[0026] The third purpose of this application is to provide a nanoprobe targeting mitochondrial miRNA-204 for use 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 the present application is to provide a nanoprobe targeting mitochondrial miRNA-204 for use in miRNA-204 detection.

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

[0030] The mitochondrial miRNA-204-targeted nanoprobe is loaded with a DNA-RNA hybrid chain for detecting mitochondrial miRNA-204, which can highly recognize overexpressed miRNA-204 in mitochondria and silence miRNA-204 gene expression;

[0031] By silencing the expression of mitochondrial miRNA-204, the mitochondrial miRNA-204 nanoprobe was targeted to mediate the occurrence of the autophagy pathway and restore the mitochondrial membrane potential to normal levels;

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

[0033] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the following specifically cites a preferred embodiment and describes it in detail with the accompanying drawings as follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Attached Figure 1 TEM image of CeO2-DRP / MA nanomaterial of Example 1;

[0035] Attached Figure 2 The crystal structure diagram of CeO2-DRP / MA nanomaterial of Example 1;

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

[0037] Attached Figure 4The fluorescence intensity graphs at different time points when CeO2-DRP / MA and miRNA-204 were co-incubated;

[0038] Attached Figure 5 Specificity graph for CeO2-DRP / MA in detecting mitochondrial miRNA-204;

[0039] Attached Figure 6 The sensitivity diagram of CeO2-DRP / MA for detecting mitochondrial miRNA-204;

[0040] Attached Figure 7 This is a study on the ability of CeO2-DRP / MA to detect mitochondrial miRNA-204 in neural cells. The scale bar is 50 μm.

[0041] Attached Figure 8 This is a study on the ability of CeO2-DRP / MA to restore the mitochondrial membrane potential of damaged nerve cells. The scale bar is 50 μm.

[0042] Attached Fig. 9 Biological transmission electron microscopy images of CeO2-DRP / MA inhibiting the production of autophagosomes, the scale bar is 1 μm;

[0043] Attached Fig.10 To study the ability of CeO2-DRP / MA to cross the blood-brain barrier;

[0044] Attached Fig.11 The swimming trajectories of the mice in each group in the water maze experiment;

[0045] Attached Fig.12 This is a graph showing the number of cross-grid ranges crossed by mice in each group in the open field test;

[0046] Attached Fig.13 This is a graph showing the number of defecation in the open field test for each group of mice;

[0047] Attached Fig.14 These are Ni ssl-stained sections of the hippocampus of mice in different experimental groups, with a scale bar of 50 μm;

[0048] Attached Fig.15 These are the staining images of Aβ protein sections in the hippocampus of mice in different experimental groups. The scale bar is 50 μm. DETAILED DESCRIPTION

[0049] In order to make the content of the present invention more clearly understood, the present invention is further described below based on specific embodiments in conjunction with the accompanying drawings.

[0050] The present application provides a mitochondrial miRNA-204 targeted nanoprobe (CeO2-DRP / MA), comprising a CeO2-NH2 nanomaterial; the surface of the CeO2-NH2 nanomaterial is modified with a mitochondrial targeting molecule and a DNA-RNA hybrid chain modified with a fluorescent group on the surface; the DNA-RNA hybrid chain comprises a DNA chain and an RNA chain, the surface of the DNA chain is modified with a fluorescent element, the surface of the RNA chain is modified with a fluorescent quencher, and the DNA chain is complementary to the bases of all sequences in miRNA-204.

[0051] In the present application, the fluorescent group modified at the end of the DNA chain is FAM, and the fluorescent quenching group modified at the end of the RNA chain is BHQ. FAM emits green fluorescence when excited by light. When the DNA chain and the RNA chain are hybridized, the distance between FAM and BHQ becomes closer, and the fluorescence of FAM is quenched by BHQ. However, when mi RNA-204 exists in mitochondria, FAM and BHQ separate, and the fluorescence signal is restored. The fluorescence signal is restored to a certain extent, thereby realizing in situ imaging of mi RNA-204 in mitochondria. The present application obtains CeO2-DRP / MA by modifying the surface of CeO2-NH2 nanomaterials with mitochondrial targeting molecules and DNA-RNA hybrid chains, wherein the mitochondrial targeting molecules can recognize and bind to mitochondria, and the DNA chains in the DNA-RNA hybrid chains are complementary to the bases of the key functional sequence parts in mi RNA-204. Therefore, CeO2-DRP / MA can specifically detect mi RNA-204 and silence the expression of mi RNA-204 gene through the DNA-RNA hybrid chains, mediate the occurrence of autophagy pathway, restore the mitochondrial membrane potential to normal levels, and at the same time cooperate with the unique reactive oxygen scavenging mechanism of CeO2 to reduce cell damage and dysfunction, thereby improving the therapeutic effect of AD.

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

[0053] The present application also provides a method for preparing a mitochondrial mi RNA-204 targeted nanoprobe, comprising the following steps:

[0054] The CeO2-NH2 nanomaterials were washed, and then the washed CeO2-NH2 nanomaterials were evenly mixed with the DNA-RNA hybrid chain and the mitochondrial targeting molecule and incubated for 10-14 hours to obtain the mitochondrial mi RNA-204 targeted nanoprobe, and the molar ratio of the DNA-RNA hybrid chain to the mitochondrial targeting molecule was 1:1.

[0055] In the present application, there is no particular restriction on the order of adding CeO2-NH2 nanomaterials, DNA-RNA hybrid chains, and mitochondrial targeting molecule liquids. The molar ratio of DNA-RNA hybrid chains to mitochondrial targeting molecules is preferably 1:1. There is no particular restriction on the number of times the CeO2-NH2 nanomaterials are washed, preferably not less than 3 times.

[0056] In the present application, the CeO2-NH2 nanomaterial is prepared by a preparation method comprising the following steps:

[0057] S1, dissolving cerium acetate and oleylamine in xylene to obtain a mixed solution 1, wherein the ratio of the mass (g) of cerium acetate, the mass (g) of oleylamine and the volume (ml) of xylene is (4.0-4.5):(30-35):(145-155);

[0058] S2, vigorously stirring the mixed solution 1 at room temperature for 10 to 14 hours, then heating to 90°C under vacuum conditions, and then quickly injecting deionized water into the heated mixed solution 1, the volume ratio of deionized water to the mixed solution 1 being 10 to 14 hours, to obtain a reaction solution; when heating under vacuum conditions, there is no particular restriction on the heating rate, and the preferred heating rate is 2°C / min;

[0059] S3, incubating the obtained reaction solution for 2 to 4 hours at a temperature of 90-95° C. until the reaction solution becomes transparent, and then cooling the reaction solution to room temperature;

[0060] S4, taking 15-17 ml of the reaction solution treated in step S3, precipitating nanoparticles with 100 ml of acetone and adding the precipitated nanoparticles to the reaction solution, followed by centrifugation to obtain crude CeO2 nanoparticles;

[0061] S5, adding chloroform to the crude CeO2 nanoparticles until the concentration of the crude CeO2 nanoparticles is 10 mg / mL, and then centrifuging to obtain intermediate CeO2 nanoparticles;

[0062] S6, placing the amination reagent and the intermediate CeO2 nanoparticles in chloroform and mixing them evenly to obtain a mixed solution II, and then subjecting the mixed solution II to rotary evaporation under vacuum conditions at an evaporation temperature of 80 to 85°C and an evaporation time of 1 to 1.5 hours to obtain an intermediate, wherein 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 to 9): (2 to 5): (20 to 30);

[0063] S7, pouring deionized water into the container containing the intermediate, then subjecting it to ultrasonic treatment to obtain a suspension, and then filtering the suspension through a filter with a pore size of 0.2 μm until the suspension is transparent;

[0064] S8, filtering the transparent suspension using a centrifugal filter with a molecular weight cutoff of 50 kDa, and then dialyzing it 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 5-7mM were modified with a silane coupling agent for 10-14h. The modified product was activated with 20μL of SPDP (3-(2-pyridyldithio) propionic acid succinimidyl ester) within 30min to obtain CeO2-NH2 nanomaterials.

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

[0067] S1, prepare DNA chain solution and RNA chain solution, put the DNA chain solution and RNA chain solution into a test tube under light-proof conditions and mix them evenly, then put the test tube into a constant temperature instrument, then raise the temperature of the constant temperature instrument to 65°C and maintain it for 2 minutes, the heating rate is 2°C / min, then raise the temperature to 95°C and maintain it for 10 minutes, the heating rate is 20°C / min, to obtain a DNA-RNA hybrid chain.

[0068] The mitochondrial targeting molecule is prepared by a preparation method comprising the following steps:

[0069] Synthesize mitochondrial targeting molecules using a nucleic acid synthesizer.

[0070] The present application also provides a targeted mitochondrial mi RNA-204 nanoprobe or a targeted mitochondrial miRNA-204 nanoprobe prepared by a preparation method for use in the preparation of Alzheimer's disease diagnostic and therapeutic drugs or miRNA-204 detection, and 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] In order to further illustrate the present invention, the following examples are provided for detailed description. The experimental raw materials used in the following examples of the present invention can be purchased from the market 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, dissolving cerium acetate and oleylamine in xylene to obtain a mixed solution 1, 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, vigorously stirring the mixed solution 1 at room temperature for 12 h, then heating to 90° C. under vacuum conditions at a heating rate of 2° C. / min, and then quickly injecting 1 ml of deionized water into the heated mixed solution 1 to obtain a reaction solution;

[0076] S3, incubating the obtained reaction solution for 3 hours at a temperature of 90° C. until the reaction solution becomes transparent, and then cooling the reaction solution to room temperature;

[0077] S4, using 100 ml of acetone to precipitate nanoparticles, and then adding the acetone to the reaction solution obtained in step S3, followed by centrifugation to obtain crude CeO2 nanoparticles;

[0078] S5, adding 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, placing the amination reagent and the intermediate CeO2 nanoparticles in chloroform and mixing them evenly to obtain a mixed solution 2, and then subjecting the mixed solution 2 to rotary evaporation under vacuum conditions at a temperature of 80° C. for 1 h to obtain an intermediate, wherein the mass of the intermediate CeO2 nanoparticles is 8 mg, the volume of the amination reagent is 3 ml, and the volume of the chloroform is 25 ml;

[0080] S7, pouring deionized water into the container containing the intermediate, then subjecting it to ultrasonic treatment to obtain a suspension, and then filtering the suspension through a filter with a pore size of 0.2 μm until the suspension is transparent;

[0081] S8, filtering the transparent suspension using a centrifugal filter with a molecular weight cutoff of 50 kDa, and then dialyzing it 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 5-7mM were modified with a silane coupling agent for 12h. The modified product was activated with 20μL of SPDP (3-(2-pyridyldithio) propionic acid succinimidyl ester) within 30min to obtain CeO2-NH2 nanomaterials.

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

[0084] S1, prepare DNA chain solution and RNA chain solution, put the DNA chain solution and RNA chain solution into a test tube under light-proof conditions and mix them evenly, then put the test tube into a constant temperature instrument, then raise the temperature of the constant temperature instrument to 65°C and maintain it for 2 minutes, the heating rate is 2°C / min, then raise the temperature to 95°C and maintain it for 10 minutes, the heating rate is 20°C / min, to obtain a DNA-RNA hybrid chain.

[0085] 3) Preparation of mitochondrial targeting molecules:

[0086] Synthesize mitochondrial targeting molecules using a nucleic acid synthesizer.

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

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

[0089] In this embodiment, the nucleic acid sequences required for synthesizing CeO2-DRP / MA are

[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 molecule: CAC CAC GAU CAC GGU UUC CCU CGC AGG UAA GGU GUA GA-COOH

[0094] The mitochondrial mi RNA-204 targeted nanoprobe prepared by the preparation method of the present application is as follows: Figure 1 As shown, the morphology is a nanocluster material of about 40 nm.

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

[0096] like Figure 3As shown, the presence of three characteristic elements, Ce, N and P, confirmed 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 mi RNA-204.

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

[0100] The results are as follows Figure 4 As shown in the figure, the fluorescence intensity of the FAM fluorescent group in the nanomaterial gradually increased with the extension of the reaction time, which confirmed the effectiveness of CeO2-DRP / MA nanomaterials in detecting mitochondrial miRNA-204.

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

[0102] CeO2-DRP / MA was reacted with different mitochondrial miRNAs to examine the detection specificity of the nanomaterials. Figure 6 As shown in the figure, CeO2-DRP / MA nanomaterials showed obvious fluorescence recovery only in the presence of mitochondrial miRNA-204. In contrast, for non-target miRNA-155 and miRNA-181, the FAM fluorescence intensity of the nanomaterials was the same as that of the blank control group, further confirming the highly specific detection ability of the nanomaterials for mitochondrial miRNA-204.

[0103] SY5Y cells were further cultured, and after incubation of SY5Y cells treated with β-amyloid protein (Aβ) with CeO2-DRP / MA at a concentration of 1 mmol / L for 4 h, confocal laser scanning microscopy was used to detect the ability of nanomaterials to detect mitochondrial miRNA-204. The results are shown in Figure 7As shown in the figure, obvious strong green fluorescence of FAM was observed in SY5Y cells treated with Aβ, indicating that the expression level of mitochondrial miRNA-204 in AD cells was high. Weak green fluorescence of FAM was observed in normal cells, indicating that the expression level of mitochondrial miRNA-204 in normal cells was significantly reduced compared with that in AD cells. This confirms that CeO2-DRP / MA has excellent detection performance in cells.

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

[0105] The ability of the nanomaterials to restore mitochondrial membrane potential was observed using a mitochondrial membrane potential test kit. Figure 8 As shown, there are almost fewer green fluorescence positive cells in SY5Y+Aβ cells treated with CeO2-DRP / MA nanomaterials, and cells not treated with CeO2-DRP / MA present a strong green fluorescence positive population. This indicates that CeO2-DRP / MA nanomaterials significantly increase the membrane potential of mitochondria in cells, thereby helping to restore damaged mitochondria to normal state.

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

[0107] Biological transmission electron microscopy was used to observe whether nanomaterials could inhibit the production of autophagosomes. Fig. 9 As shown in the figure, in the SY5Y+Aβ group cells, obvious double-layer membrane autophagosome structures were observed (indicated by red arrows). However, in the SY5Y+Aβ cells treated with CeO2-DRP / MA, no autophagosome formation was observed, indicating that CeO2-DRP / MA nanomaterials effectively inhibited the generation of autophagosomes.

[0108] Study on the diagnostic and therapeutic effect of CeO2-DRP / MA nanomaterials on AD in vivo

[0109] The use of experimental 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 of in vivo mitochondrial miRNA-204 fluorescence imaging.

[0111] After an equal volume of 1 mM CeO2-DRP / MA was injected into 3×Tg-AD mice at 12 hours and 24 hours, their brains were collected. Fluorescence images of the mouse brains were captured using the IVIS Spectrum system. The results are shown in Figure 2. Fig.10 As shown, the nanomaterials effectively crossed the blood-brain barrier and reached the mouse brain, achieving effective detection in the mouse.

[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 Experimental Animal Technology Co., Ltd. (China). Animal experiments were divided into three groups:

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

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

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

[0117] Morris water maze: All mice received daily training for the first five days. During the experiment, three tests were performed each day using various random paths, and the starting point of each test was a different quadrant of the maze. All tests were completed within 1 minute. On the seventh day, each mouse was tested to determine their response to the removal of the platform. Data was collected using a camera and analyzed using software. The results are shown in Fig.11 As shown, the 3×Tg-AD model mice mainly performed mechanical swimming along the edge of the water maze during the 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 similarities with wild-type mice in the ability to remember the previous platform position.

[0118] Field test: First, the mice were placed in a 30×30×20 cm chamber and given time to adapt to the environment. The chamber had no other interference factors. A camera was used to collect data on the mice exploring the open area for 5 minutes. It is worth noting that 70% ethanol should be used to clean the floor each time. The results are shown in Figure 2. Fig.12 and Fig.13 As shown, during the 5-min observation period, compared with 3×Tg-AD mice, the number of times WT mice and mice in the CeO2-DRP / MA-treated group crossed the cross grid increased significantly (p < 0.01); the anxiety behavior of mice in the CeO2-DRP / MA-treated group was significantly improved, such as a decrease in the number of defecation.

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

[0120] The morphology and density of mouse neuronal cells were observed using Nissl staining. Fig.14As shown in the figure, compared with wild-type mice, neurons in the hippocampus of 3×Tg-AD model mice showed pathological characteristics of nuclear shrinkage and low cellularity. In the hippocampus of mice in the CeO2-DRP / MA treatment group, the morphology of neuronal cells remained intact, and the cell number density was similar to that of wild-type mice, with no significant difference. This shows that CeO2-DRP / MA nanomaterials effectively alleviate the degeneration and necrosis of neurons in 3×Tg-AD mice, proving its potential therapeutic effect in promoting neuronal cell repair and maintaining the structural integrity of the nervous system.

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

[0122] Immunohistochemistry was used to observe the distribution and content of Aβ protein in the hippocampus of mice. Fig.15 As shown in the figure, a large amount of Aβ protein expression was observed in the hippocampus of 3×Tg-AD model mice, which was in sharp contrast to the wild-type mice in the control group. Tissue sections of the hippocampus region of mice treated with CeO2-DRP / MA showed that the amount of Aβ protein deposition was significantly reduced, 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, confirming its significant therapeutic effect in vivo.

[0123] The embodiments described above are only preferred implementation modes of the present invention and cannot be used to limit the protection scope of the present invention. Any non-substantial changes and modifications made by those skilled in the art on the basis of the invention shall fall within the protection scope of the present invention.

Claims

1. A nanoprobe targeting mitochondrial miRNA-204, characterized in that: Including CeO2-NH2 nanomaterials; The surface of the CeO2-NH2 nanomaterial is modified with a mitochondrial targeting molecule and a DNA-RNA hybrid chain, and the nucleic acid sequence of the mitochondrial targeting molecule is CAC CAC GAU CAC GGU UUC CCU CGC AGG UAA GGU GUA GA-COOH; The DNA-RNA hybrid chain includes a DNA chain and an RNA chain, and the DNA chain is complementary to a part or all of the bases in the sequence of the miRNA-204.

2. A mitochondrial miRNA-204 targeting nanoprobe according to claim 1, characterized in that: The particle size of the nanoprobe is 40 nm.

3. A method for preparing the mitochondrial miRNA-204 targeting nanoprobe according to claim 1 or 2, characterized in that: The following steps are involved: The CeO2-NH2 nanomaterials were washed, and then the washed CeO2-NH2 nanomaterials were evenly mixed with the DNA-RNA hybrid chain and the mitochondrial targeting molecule and incubated for 10-14 hours to obtain the mitochondrial targeted miRNA-204 nanoprobe, and the molar ratio of the DNA-RNA hybrid chain to the mitochondrial targeting molecule was 1:

1.

4. The preparation method according to claim 3, characterized in that: The CeO2-NH2 nanomaterial is prepared by a preparation method comprising the following steps: S1, dissolving cerium acetate and oleylamine in xylene to obtain a mixed solution 1, wherein the ratio of the mass (g) of cerium acetate, the mass (g) of oleylamine and the volume (ml) of xylene is (4.0-4.5):(30-35):(145-155); S2, stirring the mixed solution 1 at room temperature for 10 to 14 hours, then heating to 90° C. under vacuum conditions, and then injecting deionized water into the heated mixed solution 1, wherein the volume ratio of deionized water to the mixed solution 1 is (1 to 13): (1 to 17), to obtain a reaction solution; S3, incubating the reaction solution for 2 to 4 hours at a temperature of 90 to 95° C. until the reaction solution becomes transparent, and then cooling the reaction solution to room temperature; S4, taking 15-17 ml of the reaction solution treated in step S3, precipitating nanoparticles with 100 ml of acetone and adding the precipitated nanoparticles to the reaction solution, followed by centrifugation to obtain crude CeO2 nanoparticles; S5, adding chloroform to the crude CeO2 nanoparticles and stirring evenly until the concentration of the crude CeO2 nanoparticles is 10 mg / mL, thereby obtaining intermediate CeO2 nanoparticles; S6, placing the amination reagent and the intermediate CeO2 nanoparticles in chloroform and mixing them evenly to obtain a mixed solution II, and then subjecting the mixed solution II to rotary evaporation under vacuum conditions at an evaporation temperature of 80 to 85°C and an evaporation time of 1 to 1.5 hours to obtain an intermediate, wherein 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 to 9): (2 to 5): (20 to 30); S7, pouring deionized water into the container containing the intermediate, then subjecting it to ultrasonic treatment to obtain a suspension, and then filtering the suspension through a filter with a pore size of 0.2 μm until the suspension is transparent; S8, filtering the transparent suspension using a centrifugal filter with a molecular weight cutoff of 50 kDa, and then dialyzing it 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, and the modified product was activated with 20 μL of SPDP (3-(2-pyridyldithio) propionic acid succinimidyl ester) within 30 min to obtain CeO2-NH2 nanomaterials.

5. The preparation method according to claim 3, characterized in that: The DNA-RNA hybrid chain is prepared by a preparation method comprising the following steps: S1, prepare DNA chain solution and RNA chain solution, put the DNA chain solution and RNA chain solution into a test tube under light-proof conditions and mix them evenly, then put the test tube into a constant temperature instrument, then raise the temperature of the constant temperature instrument to 65°C and maintain it for 2 minutes, the heating rate is 2°C / min, then raise the temperature to 95°C and maintain it for 10 minutes, the heating rate is 20°C / min, to obtain a DNA-RNA hybrid chain.

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

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

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

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

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