Molecular chaperone, nanocarrier, preparation method and application thereof

Abstract

The present application relates to a kind of molecular chaperone, nano-carrier and its preparation method and application.The molecular chaperone is the recombinant histidine-sulphur oxygen protein coupled with single-stranded DNA, and the cysteine residue is introduced in the protein and coupled with the single-stranded DNA of amino modification.The nano-carrier is DNA tetrahedron, and the DNA tetrahedron is obtained by self-assembly based on base pairing principle from multiple DNA single strands.DNA nano-carrier is used to deliver the molecular chaperone, can realize HTrX to specific subcellular compartment, i.e.the targeted delivery of ribosome, so that the protein in ribosome exerts biological effect, reduces the negative influence of oxidative stress on protein synthesis to reverse the aging process.The present application also discloses the application of the above-mentioned molecular chaperone and nano-carrier in preparing anti-aging drugs.

Description

Technical Field

[0001] This invention relates to the medical field, specifically to a molecular chaperone, a nanocarrier, its preparation method, and its application. Background Technology

[0002] Aging is accompanied by a decline in cellular protein homeostasis, leading to the misfolding of proteins that play a regulatory role within the cell. This phenomenon is the pathological basis of age-related diseases. Studies have shown that in senescent cells, the decline in ribosome function and the increased aggregation of misfolded proteins occur simultaneously. Ribosomal biogenesis is a highly dynamic and coordinated process, precisely regulated by multiple signaling pathways. These pathways respond to growth factors, energy, and nutrients, collectively regulating protein synthesis and thus affecting cells. The nascent polypeptide chains released from ribosomes typically need to fold into their correct three-dimensional conformation in a complex environment; this process is crucial for their biological activity and cellular function. This folding pathway is susceptible to microenvironmental influences, leading to the formation of misfolded intermediates or easily aggregated states, especially under cellular stress conditions. With age, the antioxidant defense system becomes imbalanced, resulting in excessive accumulation of reactive oxygen species (ROS) exceeding the cell's antioxidant capacity. This oxidative stress damages key intracellular macromolecules, including proteins and nucleic acids, impairs ribosome function, promotes protein synthesis defects, and ultimately leads to cellular senescence.

[0003] Molecular chaperones are a class of proteins that do not participate in determining the final folding structure but are crucial for protein biogenesis. By interacting with nascent chains or early folding intermediates, molecular chaperones prevent nonproductive interactions and aggregation, thereby promoting efficient folding pathways and ensuring the efficient generation of correctly folded and functional proteins. Introducing molecular chaperones with protective properties for protein synthesis through rational design and construction is of practical significance for addressing issues related to protein oxidative damage. Developing artificial molecular chaperones with stable and mild antioxidant properties that can assist in the correct folding of proteins could help improve ribosome function and offers potential for reversing the aging process by mitigating the negative effects of oxidative stress on protein synthesis.

[0004] Thioredoxins (TrxA or Trx1) are molecular chaperones that play a crucial role in maintaining cellular redox balance and cell survival. However, overcoming biological barriers, such as cell membranes and tissue barriers, to precisely target thioredoxins to ribosomes remains a challenge. Traditional nanocarriers, such as liposomes, suffer from low drug loading and poor targeting when delivering protein drugs. Therefore, there is an urgent need to develop a novel drug carrier that can precisely deliver thioredoxins to ribosomes, maintain protein homeostasis under cellular stress, and improve cellular senescence. Summary of the Invention

[0005] In one aspect, this invention provides a molecular chaperone and its preparation method. The molecular chaperone is a recombinant histidine-thioredoxin (HTrx). The HTrx protein is expressed by constructing a plasmid, transforming the plasmid into competent cells, inducing protein expression, and finally purifying the recombinant protein using an affinity chromatography column. The introduction of a cysteine ​​residue (-C) into HTrx aims to achieve coupling with single-stranded DNA (ssDNA), which is amino-modified. The sequence of the single-stranded DNA is shown in SEQ ID NO:30, and the amino-modified single-stranded DNA is 5'-NH2-TTCAGAGGCGCT-3'.

[0006] In another aspect, the present invention provides a nanocarrier and a method for preparing the same. The nanocarrier is a DNA tetrahedron, which is obtained by the self-assembly of multiple single-stranded DNA molecules based on base pairing principles. The sequences of the single-stranded DNA molecules are shown in SEQ ID NO:1-28. The carrier is used to deliver histidine-thioredoxin. Further, the carrier is used to deliver the aforementioned molecular chaperone.

[0007] To precisely deliver thioredoxin to ribosomes, the nanocarrier of this invention is an addressable double-stranded DNA tetrahedron capable of targeting ribosomes. The probe sequence targeting the ribosome is shown in SEQ ID NO:29, and the number of probes can be set according to specific needs, with a minimum of one; two or three probes can also be added as needed. Preferably, there are six probes targeting the ribosome. It is particularly important to emphasize that even without adding ribosome-targeting probes, the non-targeting DNA nanocarrier of this invention can overcome biological barriers, deliver the aforementioned molecular chaperones, and enhance their anti-aging effects.

[0008] This invention provides a method for preparing a DNA nanocarrier, wherein the DNA nanocarrier is obtained by self-assembly of multiple DNA single strands based on base pairing principles, and the sequences of the multiple DNA single strands are shown in SEQ ID NO:1-28; the preparation method includes the following steps: 1) The DNA single strands are grouped into V1, V2, V3, and V4, and then mixed separately in buffer solutions. The buffer solution is a common buffer used to dissolve DNA, such as TE buffer, EB buffer, or enzyme-free water. To ensure DNA stability, the buffer solution is a weakly alkaline buffer with a pH of 7.5-8.5. In one embodiment, a 1×TAE / Mg buffer solution is used. 2+ Buffer solution. Based on the base pairing principle, the above DNA single strands are mixed in equal amounts in the buffer solution; The V1 group includes the following sequences, as shown in SEQ ID NO:2-10; The V2 group includes the following sequences: as shown in SEQ ID NO:11-16; The V3 group includes the following sequences: as shown in SEQ ID NO:17-22; The V4 group includes the following sequences, as shown in SEQ ID NO:23-28; In addition to the above sequence, V1, V2, V3 and V4 all contain the sequence YL, which is shown in SEQ ID NO:1; 2) The four groups of DNA single-stranded mixtures were heated to denature and then slowly cooled; the heating temperature was a conventional temperature for denaturing DNA, for example, 90-100°C, or more specifically 93-98°C; in one embodiment, the heating denaturation temperature was 95°C; the cooling temperature was 30-60°C; in one embodiment, the cooling temperature was 37°C. 3) After mixing the four groups of DNA single-stranded mixtures from step 2), add the aforementioned molecular chaperone, heat the mixture at a low temperature, and then slowly cool it to finally obtain the DNA nanocarrier. The low-temperature heating temperature is 30-50℃, more specifically 35-40℃, and in one embodiment, 37℃; the slow cooling time should not be too fast, and generally requires overnight cooling; in one embodiment, the slow cooling time is 12 hours.

[0009] Furthermore, the DNA nanocarrier targets ribosomes, and the probe sequence for targeting ribosomes is shown in SEQ ID NO:29; the probe is added to group V2, V3, or V4 in step 1) and mixed; the number of probes can be set according to specific needs, with a minimum of 1; preferably, there are 6 probes targeting ribosomes. When there are 6 probes targeting ribosomes, 2 probes are added to each of groups V2, V3, and V4.

[0010] On the other hand, this invention provides the application of molecular chaperones in the preparation of anti-aging drugs. The molecular chaperones of this invention can be used to prepare anti-aging drugs that reverse the aging process by improving ribosome function and mitigating the negative effects of oxidative stress on protein synthesis.

[0011] On the other hand, the DNA nanocarrier provided by this invention is used in the preparation of anti-aging drugs. The DNA nanocarrier exhibits good anti-aging effects.

[0012] This invention offers the following advantages: The DNA nanocarrier utilizes the predictable and programmable Watson-Crick base pairing principle of DNA to construct nanostructures with defined sizes, shapes, and topologies with unprecedented precision and control. The DNA nanocarrier exhibits unique biocompatibility and modifiability, precisely anchoring the active ligand—the molecular chaperone HTrx-S—to specific sites on the DNA nanocarrier, thereby constructing an integrated targeting unit-carrier system. The DNA carrier not only enhances the binding affinity between the targeting portion and the surface receptor but also integrates the cellular regulatory functions of different functional molecules. The DNA nanocarrier achieves precise subcellular localization and targeted therapy, precisely designed to navigate in the complex intracellular environment, enabling targeted delivery of HTrX to specific subcellular compartments—namely, ribosomes—allowing the protein to exert its biological function within the ribosome, mitigating the negative impact of oxidative stress on protein synthesis, and reversing the aging process. Attached Figure Description

[0013] Figure 1 A schematic diagram of the molecular chaperone HTrx-S.

[0014] Figure 2 SDS-PAGE analysis results of molecular chaperones HTrx and HTrx-S.

[0015] Figure 3 A schematic diagram of the DNA nanocarrier preparation process of this invention.

[0016] Figure 4 A schematic diagram of DNA strand V-1.

[0017] Figure 5 A schematic diagram of DNA strand V-2.

[0018] Figure 6 A schematic diagram of DNA strand V-3.

[0019] Figure 7 A schematic diagram of DNA strand V-4.

[0020] Figure 8 Atomic force microscopy characterization of the DNA nanocarrier of this invention, scale bar: 50 nm.

[0021] Figure 9 Agarose gel electrophoresis analysis diagram.

[0022] Figure 10 Agarose gel electrophoresis analysis.

[0023] Figure 11 Dynamic light scattering analysis of particle size.

[0024] Figure 12 The BTD method was used to detect the level of oxidation of intracellular protein thiol groups.

[0025] Figure 13 The results of quantitative detection of intracellular oxidized protein products are shown in the figure.

[0026] Figure 14 Figure showing the results of intracellular protein carbonylation level analysis.

[0027] Figure 15 The retention time was measured using a rotating rod experiment.

[0028] Figure 16 The asymmetry score is measured in the cylinder experiment.

[0029] Figure 17 The grid experiment measures the asymmetry index.

[0030] Figure 18 Analysis of protein carbonylation levels in brain tissue.

[0031] Figure 19 Analysis of GSH levels in brain tissue.

[0032] Figure 20 Immunofluorescence analysis of reactive oxygen species (ROS) levels in brain tissue, scale bar: 50 μm.

[0033] Figure 21 H&E staining analysis of mouse brain tissue. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0035] Example 1: Expression and purification of molecular chaperone recombinant histidine-thioredoxin (HTrx) and synthesis of HTrx protein with single-strand DNA modification (His-Thioredoxin-single strand DNA, HTrx-S). The pET-28a-His-Trx-C plasmid was constructed to express the HTrx protein, which carries an N-terminal His6 tag and a C-terminal -(GS)3-C sequence. A cysteine ​​residue (-C) was introduced to enable coupling with single-stranded DNA (ssDNA). This plasmid was transformed into *E. coli* Rosetta-gami competent cells, and protein expression was induced using isopropyl-β-D-thiogalactoside (IPTG, 0.4 mM). After overnight induction (approximately 12 hours), the cells were collected by centrifugation at 5000 rpm for 15 minutes.

[0036] The bacterial pellet collected by centrifugation was resuspended in binding buffer (20 mM Tris-HCl, 150 mM NaCl, pH=8.0) and lysed by sonication. Cell lysates were centrifuged at 4°C and 16,000 rpm for 25 minutes. Recombinant proteins were purified using a Ni-NTA affinity chromatography column (His-Trap HP 5 mL, GE Healthcare) with elution using a linear gradient of 10–250 mM imidazole in the binding buffer. HTrx protein concentration was determined using the Bradford method, and HTrx purity was analyzed by SDS-PAGE.

[0037] HTrx was coupled to the ssDNA strand (5'-NH2-TTCAGAGGCGCT-3') using a sulfo-SMCC crosslinking agent. The amino-modified ssDNA was diluted to 100 μM with PBS buffer (pH=7.4). This amino-modified oligonucleotide solution was mixed with sulfo-SMCC (25 equivalents relative to the amino-modified ssDNA) and reacted at 25°C for 10 h. Subsequently, ultrafiltration was performed three times using a 10 kDa molecular weight cutoff ultrafiltration tube to remove excess sulfo-SMCC, obtaining SMCC-ssDNA. Simultaneously, HTrx protein was displaced into PBS buffer and purified using a HiTrap™ desalting column on an AKTA purification system (GE Healthcare).

[0038] To conjugate reduced HTrx to DNA strands, reduced HTrx was mixed with purified SMCC-ssDNA at a ratio of 2.5:1 to prepare the HTrx-single strand DNA conjugate, HTrx-S. After incubation at room temperature for 6 hours, HTrx-S was purified using an AKTA purification system equipped with a HiLoad 10 / 60 Superdex 200 gel filter column. Chromatograms were monitored at 254 nm and 280 nm wavelengths. The product, HTrx modified with a single strand of DNA, was determined by the concentration of HTrx-S using the BCA method based on the HTrx protein, and the purity of HTrx-S was analyzed by SDS-PAGE.

[0039] like Figure 2 As shown in the SDS-PAGE results, the prepared HTrx-S has high purity, and the band of HTrx-S is significantly lagging, indicating that high-purity HTrx-S was successfully synthesized using this method.

[0040] Example 2: Preparation of DNA Nanocarriers The DNA tetrahedral nanocarriers were assembled according to the method previously reported by our group, with appropriate modifications. To achieve the ordered assembly of functional groups, a different number of modification sites were introduced on the bottom surface of the DNA tetrahedral structure; to achieve the localized loading of histidine-thioredoxin (HTrx), three hybridization sites were extended from the vertices of the DNA tetrahedron. The DNA sequences used for assembly in this study are detailed in Table 1.

[0041] Table 1 DNA sequences in nanocarriers

[0042] Specifically, the sequence YL is present in all four DNA strands V-1, V-2, V-3, and V-4, as shown in SEQ ID NO:1.

[0043] In addition to sequence YL, sequence V-1 also includes the following sequences: as shown in SEQ ID NO:2-10.

[0044] In addition to sequence YL, sequence V-2 also includes the following sequences: as shown in SEQ ID NO:11-16.

[0045] In addition to sequence YL, sequence V-3 also includes the following sequences: as shown in SEQ ID NO:17-22.

[0046] In addition to sequence YL, sequence V-4 also includes the following sequences: as shown in SEQ ID NO:23-28.

[0047] The ribosome-targeting probe sequence is shown in SEQ ID NO:29.

[0048] The specific preparation method of the nanocarrier is as follows: First, the 28 single-stranded DNA and ribosome-targeting probe sequences were synthesized according to their sequences. Then, the single-stranded DNA was grouped into V1, V2, V3, and V4, and the single-stranded DNA was precisely measured and mixed in equal amounts in 1×TAE / Mg. 2+ In a buffer solution (pH=8.3), ensuring a final DNA strand concentration of 100 nM, four sample tubes (V1, V2, V3, and V4) were prepared. It should be further noted that ribosome-targeting probe sequences were added to sample tubes V2, V3, or V4, with two ribosome-targeting probes added to each tube, for a total of six ribosome-targeting probes. The minimum number of ribosome probes is one; however, two or three can be added depending on specific needs. In this embodiment, six ribosome-targeting probes were used. Without the addition of the targeting probe sequence, a targetless DNA nanocarrier was obtained.

[0049] Subsequently, sample tubes V1, V2, V3, and V4 were heated at 95°C for 5 minutes to completely denature the DNA single strands. They were then slowly cooled to 37°C over 1 hour, during which time all single strands formed double strands according to the designed combination. Next, the HTrx-S prepared in Example 1 was mixed with the aforementioned DNA strands in a ratio of 3:1:1:1:1, and the mixture was slowly cooled from 37°C to 25°C over 12 hours. The final prepared nanoparticle drug delivery system was stored at 4°C for later use.

[0050] The nano-drug delivery system was characterized using atomic force microscopy. Experimental results showed that, Figure 8 As shown, the prepared DNA nanocarrier is monodisperse, has a regular structure, and a size of about 20 nm, which is consistent with the design.

[0051] Example 3: Analysis of the binding effect between DNA nanocarriers and ribosomes The DNA nanocarrier prepared in Example 2 was mixed with 28S rRNA in PBS buffer at a molecular ratio of 1:1 (ribosome targeting probe: 28S rRNA). The mixture was incubated at 37°C for 12 hours. After incubation, the mixture was analyzed by agarose gel electrophoresis.

[0052] The experimental results show that the DNA nanocarriers formed clear, single bands, indicating that they assembled as expected, without aggregation or disintegration.

[0053] Example 4: DNA nanocarrier binding experiment with ribosomes Ribosomes were extracted from HEK293 cells using a method described in published literature. The DNA nanocarrier prepared in Example 2 was mixed with different concentrations of the extracted ribosomes (0, 0.1, 1.0, 5.0, 10, and 50 µg / mL). The mixtures were incubated at 37°C with shaking. The incubated products were then analyzed by 1.5% agarose gel electrophoresis.

[0054] Experimental results show that the DNA nanocarrier synthesized using this invention can effectively bind to ribosomes extracted from cells, demonstrating that this drug delivery system has good ribosome targeting function.

[0055] Example 5: Stability Analysis Experiment of DNA Nanocarriers To investigate the stability of the DNA nanocarriers, the DNA nanocarriers prepared in Example 2 were dispersed in PBS or DMEM medium at a concentration of 100 nM. The mixtures were incubated at 37°C with shaking, and samples were taken at different time points for dynamic light scattering measurements to analyze the particle size.

[0056] Experimental results show that the particle size of the DNA nanocarriers prepared by the present invention remains relatively stable after 24 hours of incubation in either PBS or DMEM medium, demonstrating that the DNA nanocarriers of the present invention have good stability.

[0057] Example 6: Analysis of sulfhydryl oxidation levels on intracellular proteins To investigate the anti-aging effect of the DNA nanocarrier of this invention, the level of thiol oxidation on intracellular proteins was measured. The cellular senescence model was obtained by adding 50 mg / mL of D-galactose to adherent HEK293 cells and stimulating the cells at 37°C for 24 h.

[0058] This embodiment is divided into four groups of experiments: 1) Control group: HEK293 cells cultured normally, without any other treatment.

[0059] 2) PBS treatment group: The above-mentioned senescent cell model group was treated with PBS.

[0060] 3) Targetless DNA Nanocarrier Drug Delivery Group: 100 nM of targetless DNA nanocarrier was administered to the above-mentioned senescent cell model group for 24 h. The preparation method of the targetless DNA nanocarrier was the same as in Example 2, except that no ribosome-targeting probe was added during synthesis.

[0061] 4) Targeted DNA nanocarrier drug delivery group: 100 nM of the DNA nanocarrier prepared in Example 2 was administered to the above-mentioned senescent cell model group for 24 h.

[0062] After drug administration, the HEK293 cell pellets from the above four experiments were thoroughly washed with PBS, resuspended in a gradient buffer, and incubated at room temperature for 5 minutes. This buffer contained: 1 mM Biotinylated BTD, 0.1 mg / mL cycloheximide, 1 mM benzamidine, 1 mM PMSF, and a mixture of complete protease inhibitors. Subsequently, proteins were separated by Western blotting. The BTD-labeled proteins were imaged and analyzed using the BioRad imaging system. The sulfonation level of intracellular proteins was analyzed using biotinylated BTD, a benzothiazide derivative probe that covalently binds to the sulfenic acid form of oxidized cysteine, and was separated and detected by Western blotting.

[0063] Experimental results show that, compared with the control group, D-galactose can induce high levels of intracellular protein thiol oxidation, proving the successful establishment of the senescent cell model in this invention. Both the non-targeting DNA carrier and the ribosome-targeting DNA nanocarrier can reduce thiol oxidation, demonstrating that the DNA nanocarrier prepared using this invention can effectively deliver the molecular chaperone HTrx protein of this invention. Furthermore, the ribosome-targeting DNA nanocarrier in this invention can significantly reduce thiol oxidation. This indicates that the DNA nanocarrier of this invention can not only maintain intracellular protein homeostasis by inhibiting protein oxidation in senescent cells under oxidative stress, but also greatly improve the delivery efficiency and antioxidant effect of HTrx due to its excellent ribosome-targeting function.

[0064] Example 7: Quantitative detection of intracellular oxidized protein products To investigate the anti-aging effect of the DNA nanocarrier of the present invention, the intracellular oxidized protein products were quantitatively analyzed using the AOPP detection kit (catalog number: MF00122).

[0065] The experimental grouping and drug administration methods in this embodiment are the same as in Example 6, and will not be repeated here. After drug administration, the HEK293 cell pellets from the four experimental groups were thoroughly washed with PBS, and then the procedures were performed according to the kit instructions. Subsequently, the absorbance at 420 nm was measured using a spectrophotometer.

[0066] Experimental results showed that, compared with the control group, the levels of oxidized protein products in cells treated with D-galactose were significantly increased, proving the successful establishment of the senescent cell model in this invention. DNA nanocarriers without ribosome-targeted modification, due to their inherent HTrx content, possess certain antioxidant activity and can reduce the level of intracellular oxidized protein products. However, DNA nanocarriers with ribosome-targeted modification showed a more significant effect in inhibiting excessive protein oxidation. This indicates that the DNA nanocarriers of this invention not only maintain intracellular protein homeostasis by inhibiting protein oxidation in senescent cells under oxidative stress, but also possess excellent ribosome-targeting function.

[0067] Example 8: Detection of intracellular protein carbonylation levels To investigate the anti-aging effect of the DNA nanocarrier of the present invention, the degree of protein carbonylation was quantitatively detected by OxiBlot method to reflect the level of intracellular oxidative stress.

[0068] The experimental grouping and drug administration methods in this embodiment are the same as in Example 6, and will not be repeated here. After drug administration and incubation, the cells were washed three times with cold PBS, and then transferred to tubes using a cell scraper. PBS was added, and the cells were lysed by sonication. The supernatant was then separated by centrifugation (10,000 rpm) to obtain the protein extract. The fresh protein extracts from the four experimental groups were mixed with 2,4-dinitrophenylhydrazine (DNPH) at a 1:1 ratio and incubated for 10 minutes to derivatize the protein carbonyl group into 2,4-dinitrophenylhydrazine (DNP) hydrazone. Subsequently, the cell lysate was incubated in neutralization solution for 30 minutes and separated and analyzed by Western blotting. After electrophoresis and membrane transfer, the membrane was incubated with anti-protein carbonylation antibody (provided in the kit) at a 1:1000 dilution at room temperature for 1 hour, followed by incubation in blocking buffer with secondary antibody (1:10,000 dilution) at room temperature for 1 hour. Finally, imaging analysis was performed using the BioRad imaging system.

[0069] Experimental results showed that, compared with the control group, the level of carbonylated proteins in cells treated with D-galactose was significantly increased, proving the successful establishment of the senescent cell model in this invention. The DNA nanocarrier with ribosome-targeting modification significantly reduced the level of intracellular protein carbonylation, bringing it close to the state of untreated cells. This indicates that the DNA nanocarrier maintains intracellular protein homeostasis by inhibiting protein oxidation in senescent cells under oxidative stress.

[0070] Example 9: Study on the effect of DNA nanocarriers on inhibiting aging oxidative stress in mice To investigate the inhibitory effect of the DNA nanocarrier of the present invention on aging oxidative stress in mice, this embodiment selected a naturally aging mouse model of 26-month-old aging mice, and demonstrated the effect through behavioral, aging biomarker and immunofluorescence experiments.

[0071] (a) Behavioral Analysis This study used 104-week-old (26-month-old) male C57BL / 6 mice as an aging model. Aging mice were administered either PBS or the targeted DNA nanocarrier prepared in Example 2 via intracerebrospinal injection. The drug concentration of the targeted DNA nanocarrier was 2 μM, and 5 μL / mouse was injected at week 0 and week 1 using a Hamilton syringe with a 33-G needle. During the treatment period, the mice's behavioral performance, routine blood parameters, body weight, and survival status were observed and recorded for 3 weeks.

[0072] To evaluate the anti-aging effect of targeted DNA nanocarriers, behavioral tests were used to assess efficacy during treatment. Rota-rod test, cylinder test, and grid test were performed at weeks 0, 1, 2, and 3. Specific methods for these experiments are described in published literature and will not be repeated in this embodiment.

[0073] Retention time was measured using a rotarod experiment. The results showed that the DNA nanocarrier prepared in this invention effectively increased the residence time of aging mice in the rotarod experiment and improved the behavioral flexibility of aging mice.

[0074] The asymmetry score was measured by the cylinder experiment. The experimental results showed that the DNA nanocarrier prepared in this invention improved the score of aging mice in the cylinder experiment, improved the symmetry of forelimb use in aging mice, and reduced unilateral motor dysfunction.

[0075] The asymmetry index was measured by a grid experiment. The results showed that the DNA nanocarrier prepared by this invention can reduce the asymmetry index in aging mice and improve their motor coordination and muscle control.

[0076] (ii) Detection of protein carbonylation levels in mouse brain tissue After 3 weeks of treatment, mice in the PBS or targeted DNA nanocarrier drug delivery groups of this embodiment were sacrificed, and brain tissue was extracted. Brain tissue from both groups was homogenized, and the homogenate was mixed with 2,4-dinitrophenylhydrazine (DNPH) at a 1:1 ratio and incubated for 10 minutes to derivatize protein carbonyl groups to 2,4-dinitrophenylhydrazine (DNP) hydrazone. Subsequently, the tissue homogenate was incubated in neutralization solution for 30 minutes and separated and analyzed by Western blotting. After electrophoresis and membrane transfer, the membrane was incubated with an anti-protein carbonylation antibody (provided in the kit) at a 1:1000 dilution at room temperature for 1 hour, followed by incubation in blocking buffer with a secondary antibody (1:10000 dilution) at room temperature for 1 hour. Finally, imaging analysis was performed using the BioRad imaging system.

[0077] Experimental results show that the DNA nanocarrier prepared by the present invention can significantly reduce the level of protein carbonylation in the brain tissue of aging mice, indicating that the DNA nanocarrier maintains intracellular protein homeostasis by inhibiting protein oxidation in aging cells under oxidative stress at the in vivo level.

[0078] (iii) Detection of GSH content in tissues Brain tissue was extracted from mice in the PBS or targeted DNA nanocarrier drug delivery groups in this example. The brain tissue samples were homogenized according to the instructions of the glutathione (GSH) detection kit, buffer and indicator were added, and the GSH content in the brain tissue was analyzed using an enzyme-linked immunosorbent assay (ELISA) reader.

[0079] Experimental results show that the DNA nanocarrier prepared in this invention can increase the level of reducing GSH in brain tissue, which is beneficial to reducing the damage to brain cells caused by oxidative stress.

[0080] (iv) Detecting the level of reactive oxygen species (ROS) in tissues Brain tissue was extracted from mice in the PBS or targeted DNA nanocarrier drug delivery groups used in this example, frozen in liquid nitrogen, and then cryosectioned. The sections were stained according to the instructions of the reactive oxygen species (ROS) detection kit, and then imaged using a laser confocal microscope.

[0081] Experimental results show that treatment with DNA self-assembly molecular chaperones can reduce the level of reactive oxygen species (ROS), a marker of oxidative stress, in brain tissue, which is beneficial in reducing the damage caused by oxidative stress to brain cells.

[0082] (V) H&E Analysis Mouse brain tissue was extracted from the PBS or targeted DNA nanocarrier drug delivery groups in this embodiment, fixed with tissue fixative, sectioned, and then processed according to the H&E staining kit. After staining, imaging was performed.

[0083] Experimental results show that in the hippocampus of aging mice, H&E staining typically reveals loose tissue structure, disordered cell arrangement, reduced number of neurons, irregular cell morphology, and degenerative changes such as nuclear pyknosis and karyolysis in some cells. However, treatment with the DNA nanocarrier prepared in this invention alleviated significant apoptosis and degeneration of hippocampal neurons in aging mice.

Claims

1. A molecular chaperone, characterized in that, The molecular chaperone is a recombinant histidine-thioredoxin, which incorporates cysteine ​​residues to couple with single-stranded DNA; the single-stranded DNA is amino-modified, and its sequence is shown in SEQ ID NO:

30.

2. A nanocarrier, characterized in that, The nanocarrier is a DNA tetrahedron, which is obtained by self-assembly of multiple single-stranded DNA molecules based on base pairing principles. The sequence of the single-stranded DNA molecules is shown in SEQ ID NO:1-28. The nanocarrier is an addressable double-stranded DNA tetrahedron, which can target ribosomes. The probe sequence for targeting ribosomes is shown in SEQ ID NO:

29. The number of probes can be set according to specific needs, with a minimum of one. The carrier is used to deliver the molecular chaperone as described in claim 1.

3. The nanocarrier according to claim 2, characterized in that, The number of probes targeting the ribosome is 6.

4. A method for preparing a DNA nanocarrier, characterized in that, The DNA nanocarrier is obtained by self-assembly of multiple DNA single strands based on the base pairing principle, and the sequences of the multiple DNA single strands are shown in SEQ ID NO:1-28; the preparation method includes the following steps: 1) Synthesize 28 DNA single strands according to the sequence, and group the DNA single strands into V1, V2, V3 and V4, and mix them separately in buffer solution; the V1 group includes the following sequences as shown in SEQ ID NO:2-10; the V2 group includes the following sequences as shown in SEQ ID NO:11-16; the V3 group includes the following sequences as shown in SEQ ID NO:17-22; the V4 group includes the following sequences as shown in SEQ ID NO:23-28; in addition to the above sequences, each of the V1, V2, V3 and V4 contains the sequence YL, as shown in SEQ ID NO:1; 2) The four groups of DNA single-stranded mixtures were heated to denature them, and then slowly cooled. 3) Mix the four groups of DNA single-strand mixtures from step 2) together, add the molecular chaperone as described in claim 1, heat the mixture at low temperature and then slowly cool it to obtain the DNA nanocarrier.

5. The preparation method according to claim 4, characterized in that, The DNA nanocarrier targets ribosomes, and the probe sequence for targeting ribosomes is shown in SEQ ID NO:29; the probe is added to group V2, V3 or V4 in step 1) and mixed; the number of probes can be set according to specific needs, with a minimum of 1.

6. The preparation method according to claim 5, characterized in that, The number of probes targeting the ribosome is 6.

7. The use of the molecular chaperone according to claim 1 in the preparation of anti-aging drugs.

8. The application of the nanocarrier according to claim 2 in the preparation of anti-aging drugs.

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