A DNA tetrahedron framework-based nanocomplex, a preparation method thereof and application thereof in improving depression

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a DNA tetrahedral framework-based nanocomposite, its preparation method, and its application in improving depression. This invention successfully synthesized the nanocomposite material tFNAs-miR146a by loading miR-146a onto a tetrahedral framework nucleic acid with sticky ends. This nanocomposite material can efficiently cross the blood-brain barrier and has been applied to chronic restraint stress-induced depression-like mice, exhibiting biosafety. It shows a synergistic effect in alleviating depression-like behavior and depression-induced neuroinflammation, demonstrating promising potential in the preparation of drugs for treating depression.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a DNA tetrahedral framework-based nanocomposite, its preparation method, and its application in improving depression. Background Technology

[0002] Depression is a common mental disorder characterized by persistent low mood, loss of interest, cognitive impairment, and decreased social functioning. With increasing social pressure and an aging population, the incidence of depression is rising annually, becoming a significant public health problem globally that seriously impacts human health and quality of life. Although selective serotonin reuptake inhibitors (SSRIs) are commonly used clinically for treatment, these drugs typically have a long onset of action, and some patients do not respond well to existing treatments. Therefore, developing new antidepressant treatment strategies is of great importance.

[0003] MicroRNAs (miRNAs) are a class of non-coding RNA molecules approximately 20–24 nucleotides in length that participate in the regulation of various physiological and pathological processes by modulating the stability or translation of target gene mRNAs. Among them, miR-146a is considered an important inflammatory regulatory molecule, playing a negative feedback regulatory role in various inflammation-related diseases. Studies have shown that miR-146a can inhibit the activation of the NF-κB signaling pathway by targeting and inhibiting the expression of interleukin-1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6). Furthermore, miR-146a has been reported to regulate multiple target genes related to inflammatory responses and immune regulation, including STAT1, CXCR4, and Notch1, thereby participating in the regulation of inflammatory factors (such as IL-1β and TNF-α) expression and the maintenance of immune homeostasis. Therefore, miR-146a plays an important role in the regulation of neuroinflammation and may participate in the development of neurological diseases such as depression by regulating inflammatory responses, microglial activation, and related signaling pathways. Utilizing miR-146a to regulate neuroinflammatory responses may provide a new strategy for the intervention of depression. However, miR-146a is easily degraded by nucleases in the body fluid environment, has a short half-life, and is difficult to stably exist in the in vivo circulation system. Moreover, the blood-brain barrier (BBB) ​​exists in the central nervous system, which has highly selective permeability to most nucleic acid molecules, severely limiting the entry of miRNAs into brain tissue. Currently, some studies use invasive drug delivery methods such as intracerebral injection to achieve delivery, but these methods have problems such as complex operation, easy induction of inflammatory responses, and infection risks, making them difficult to meet the needs of clinical application. Therefore, how to achieve safe and effective brain delivery of miR-146a remains an urgent technical challenge to be solved.

[0004] Tetrahedral framework nucleic acids (tFNAs) are three-dimensional nanostructures formed by the self-assembly of four single-stranded DNA molecules with specific sequences through complementary base pairing. Previous studies have shown that tFNAs possess certain cross-biological barrier capabilities and exhibit superior properties compared to traditional nucleic acid molecules in cell membrane transport and in vivo distribution, suggesting their potential application in nucleic acid delivery. Furthermore, tFNAs are increasingly attracting attention in neurological research, with studies reporting certain biological effects in nerve injury repair, neuroinflammation regulation, and improvement of neurological function. However, their application in crossing the blood-brain barrier, achieving efficient delivery of nucleic acid molecules to brain tissue, and exerting stable biological functions still requires further research and optimization.

[0005] Therefore, constructing a composite material based on tFNAs for more efficient improvement or treatment of depression is a problem facing this field. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a DNA tetrahedral framework-based nanocomposite, its preparation method, and its application in improving depression.

[0007] This invention provides a tetrahedral framework nucleic acid composite material, which is composed of tetrahedral framework nucleic acid with sticky ends A and microRNA with sticky ends B; the sticky ends A and B are single-stranded DNA with complementary base pairing, and the number of nucleotides is 5-8 respectively. The molar ratio of the tetrahedral framework nucleic acid to microRNA is 1:4; The sequence of the microRNA is shown in SEQ ID NO. 5.

[0008] Preferably, the number of nucleotides at the sticky end A and the sticky end B are 6 each.

[0009] Preferably, in the tetrahedral framework structure of the tetrahedral framework nucleic acid, each vertex contains 3-6 non-base-complementary deoxyribonucleotides, and each edge contains 18-22 base pairs.

[0010] Preferably, in the tetrahedral framework structure of the tetrahedral framework nucleic acid, each vertex has 3 non-complementary deoxyribonucleotides, and each edge has 20 base pairs.

[0011] Preferably, the nucleotide sequences of the sticky ends A and B are selected from AAAAAA or TTTTTT.

[0012] Preferably, the nucleotide sequence of the sticky end A is TTTTTT, and the nucleotide sequence of the sticky end B is AAAAAA.

[0013] Preferably, the nucleotide sequence of the microRNA with a sticky B-terminus is shown in SEQ ID NO. 6.

[0014] Preferably, the tetrahedral framework nucleic acid with sticky ends A is composed of four single-stranded nucleic acids S1, S2, S3 and S4 with sticky ends A through base complementary pairing; the molar ratio of S1 to S2, S3 and S4 is 1:1:1:1.

[0015] Preferably, the nucleotide sequences of single-stranded nucleic acids S1, S2, S3 and S4 with sticky A ends are shown in SEQ ID NO. 1-4.

[0016] Preferably, it is prepared by the following steps: mixing and incubating a tetrahedral framework nucleic acid with sticky ends A with a microRNA with sticky ends B.

[0017] Preferably, the conditions for the mixed incubation include: incubation in TM-buffer at 25-37°C for 30-60 min.

[0018] The present invention provides a method for preparing the composite material as described in any of the preceding claims, comprising: mixing and incubating a tetrahedral framework nucleic acid with sticky ends A and a microRNA with sticky ends B.

[0019] The present invention provides the use of the composite material as described in any of the preceding claims in the preparation of antidepressant and / or anti-neuroinflammatory medicaments.

[0020] The present invention provides an antidepressant and / or anti-neuroinflammatory drug, characterized in that it is obtained by using the composite material described in any of the preceding claims as the active ingredient, plus pharmaceutically acceptable excipients or auxiliary ingredients.

[0021] In this invention, tFNAs can not only serve as nucleic acid delivery vectors, but also play a role in the intervention of central nervous system diseases (such as neuroinflammation induced by depression).

[0022] This invention screened the methods, synthesis steps, and nucleic acid sequences in the nanocomposite loaded with tetrahedral framework nucleic acids (tFNAs-miR146a), providing a DNA tetrahedral framework-based nanocomposite, its preparation method, and its application in improving depression. This invention successfully synthesized the nanocomposite tFNAs-miR146a by loading miR-146a onto tetrahedral framework nucleic acids with sticky ends. It can efficiently cross the blood-brain barrier and has been applied to chronic restraint stress-induced depression-like mice, exhibiting biosafety. It shows a synergistic effect in alleviating depression-like behavior and depression-induced neuroinflammation, demonstrating promising potential in the preparation of drugs for treating depression.

[0023] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0024] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0025] Figure 1 Figure showing the results of high-efficiency capillary electrophoresis determination of the synthesis of tFNAs-miR146a nanocomposite; Figure 2 Transmission electron microscopy image of tFNAs-miR146a nanocomposite; Figure 3 Figure 1 shows the particle size and zeta potential of the tFNAs-miR146a nanocomposite; Figure 2 shows the zeta potential and Figure 3 shows the particle size. Figure 4 Figure 1 shows the uptake results of the in vitro blood-brain barrier model and the tFNAs-miR146a nanocomposite; Figure 2 shows the schematic diagram of the in vitro blood-brain barrier model, Figure 3 shows the fluorescence image obtained by laser confocal microscopy, and Figure 4 shows the statistical graph of the uptake results. Figure 5 The results of multidimensional behavioral tests evaluating the antidepressant efficacy of tFNAs-miR146a are shown in the figure. Among them, Figure (A) is the result of the sucrose preference test, Figure (B) is the result of the forced swimming test, Figure (C) is the result of the tail suspension test, Figure (D) is the result of the open field test, and Figure (E) is the heat map of the movement trajectory of mice in the open field test. "CRS-tFNAs+miR146a" is the same as "CRS-tFNAs-miR146a". Figure 6Figure 1 shows the biosafety assessment results of the nucleic acid nanocomposite tFNAs-miR146a; Figure 2 shows the effect on ALT, Figure 3 shows the effect on AST, Figure 4 shows the effect on CR, Figure 5 shows the effect on UREA, Figure 6 shows the effect on UA, Figure 7 shows the effect on RBC, Figure 8 shows the effect on WBC, Figure 9 shows the effect on PLT, and Figure 1 shows the HE staining results of the heart, liver, spleen, lungs and kidneys. Figure 7 The nucleic acid nanocomplex tFNAs-miR146a was used to reduce the level of inflammatory factors. Among them, Figure (A) shows the effect of the drug on the inflammatory factor IL-1β, Figure (B) shows the effect of the drug on the inflammatory factor TNF-α, and Figure (C) shows the effect of the drug on the inflammatory factor IL-6. Figure 8 The nucleic acid nanocomposite tFNAs-miR146a inhibits the activation of microglia in the hippocampus. Figure (A) shows the immunofluorescence of Iba-1, a marker of microglia activation in brain tissue, and Figure (B) shows the statistical results of the quantitative comparison of the fluorescence intensity of Iba-1, a marker of microglia activation. Detailed Implementation

[0026] Unless otherwise specified, all reagents and materials used in the following examples and experimental cases are commercially available.

[0027] Example 1: tFNAs-miR146a nanocomposite based on tetrahedral framework nucleic acids I. Synthesis of tFNAs with sticky ends tFNAs are nucleic acid nanomaterials with a tetrahedral structure formed by four single-stranded DNA strands (ssDNA) folding together in pairs according to the principle of complementary base pairing.

[0028] Four single-stranded ssDNAs (S1, S2, S3, and S4) with sticky ends were dissolved separately in TM-buffer (10 mM Tris-HCl, 50 mM MgCl2, pH 8.0) to prepare a system with a concentration of 1 μmol / L for each ssDNA. The four DNA single strands (1 μL each) were thoroughly mixed with 96 μL of TM-buffer, then rapidly heated to 95°C and held for 10 min. The mixture was then rapidly cooled to 4°C and held for at least 20 min to complete the folding and assembly of tFNAs, synthesizing tFNAs with sticky ends at four vertices.

[0029] The nucleotide sequences of S1, S2, S3, and S4 with sticky ends are shown in Table 1 below: Table 1 II. Synthesis of tFNAs-miR146a Adding 4 μL of miR-146a (with sticky ends, sequence shown in SEQ ID NO. 6) at a concentration of 1 μmol / L to 100 μL of synthesized tFNAs (with sticky ends) and incubating at 37 °C for 30 min can promote complementary pairing and binding of the sticky ends to form tFNAs-miR146a.

[0030] The technical solution of the present invention will be further illustrated by the following experiments. The samples tFNAs-miR146a tested in the following experimental examples were all prepared using the method of Example 1.

[0031] The tFNAs appearing alone in the following experimental examples are tFNAs without sticky ends, which were synthesized according to the method in Example 1. Among them, S1, S2, S3, and S4 do not have sticky ends, and the sequences are shown below: Table 2 In the following experimental example, the S1 chain of the fluorescent dye Cy5-labeled nucleic acid nanocomplex Cy5-tFNAs-miR146a was replaced with Cy5-labeled S1 (Cy5 labeling at the 5' end) during the synthesis process to evaluate cross-artificial blood-brain barrier and hippocampal neuronal uptake analysis.

[0032] Example 1: Characterization of the nucleic acid nanocomposite tFNAs-miR146a I. Experimental Methods 1. High-performance capillary electrophoresis experiment Following the high-performance capillary electrophoresis (HPLC) instructions, DNA single strands (S1, S2, S3, S4), tFNAs, and the nanocomposite tFNAs-miR146a were loaded and processed to obtain simulated gel images based on instrument analysis.

[0033] 2. Morphology determined by transmission electron microscopy (TEM). Dilute the tFNAs-miR146a solution 3-5 times, take a small amount and drop it onto a copper grid, stain with dye for 2 minutes, and then dry it. Use an instrument to observe the morphology and size of the nanocomposite.

[0034] 3. Dynamic light scattering (DLS) determination of particle size and zeta potential of tFNAs and tFNAs-miR146a. The tFNAs and tFNAs-miR146a solutions were diluted 10-20 times. 1 mL of sample was added to both a potentiometric dish and a particle size analyzer. The resulting data were then analyzed.

[0035] 4. Determination of cellular uptake efficiency This experiment constructed an in vitro artificial blood-brain barrier model to evaluate the cross-barrier efficiency of tFNAs delivering miR-146a: Mouse brain microvascular endothelial cells bEND.3 were seeded in the upper layer of a 24-well Transwell chamber (pore size 0.4 μm), and mouse hippocampal neurons HT-22 cells were seeded in the lower layer. After the upper layer formed a dense monolayer, Cy5-labeled miR-146a (Cy5-miR146a) and Cy5-labeled tFNAs loaded with miR-146a (Cy5-tFNAs-miR146a) were added to the upper chamber, respectively. The concentration of Cy5-tFNAs-miR146a was 1000 nM (based on tFNAs), and Cy5-miR146a was set at 4 times the miRNA dosage. The cells were incubated for 4 days. After h, HT-22 cells from the lower chamber were collected, washed with PBS, fixed with 4% paraformaldehyde, stained with DAPI for nuclear staining, labeled with FITC for cytoskeleton, and the distribution of Cy5 fluorescence signal was observed by laser confocal microscopy. The intracellular Cy5 fluorescence intensity was quantitatively analyzed using image analysis software (n = 10 per group) to evaluate the differences in the efficiency of different nanomaterials crossing the blood-brain barrier and being taken up by neurons.

[0036] II. Experimental Results 1. High-performance capillary electrophoresis experiment The results are as follows Figure 1 As shown in the figure, the position and banding of tFNAs in the lanes were observed by high-performance capillary electrophoresis. It can be seen that the simple miR-146a has the smallest molecular weight and is located at the forefront of the electrophoresis, followed by four ssDNAs. The tetrahedral framework nucleic acid tFNAs are located at approximately 180 bp in lane 3 from top to bottom in the left image (lane 2 on the right in the right image), without obvious impurities. The nanocomposite successfully loaded with miR-146a is located further back, indicating a significant increase in molecular weight, proving the successful synthesis of the tFNAs-miR146a nanocomposite.

[0037] 2. Morphological characteristics of the nanocomposite tFNAs-miR146a TEM images of tFNAs-miR146a are as follows Figure 2 As shown, the nanocomposite tFNAs-miR146a exhibits a tetrahedral nanostructure as observed using transmission electron microscopy.

[0038] 3. Particle size and Zeta potential results The particle size distributions of tFNAs and tFNAs-miR146a are shown below. Figure 3 As shown in (A), the Zeta potential diagrams of tFNAs and tFNAs-miR146a are as follows: Figure 3 As shown in (B), it can be seen that after tFNAs are loaded with miR-146a, they form a nanocomposite tFNAs-miR146a. The Zeta potential changes from -9.41±1.13mV to -16.89±2.16mV, and the particle size changes from 11.01±0.98nm to 24.62±2.09nm.

[0039] 4. In vitro Transwell model simulates the cross-blood-brain barrier uptake of tFNAs-miR146a. The results are as follows Figure 4 As shown in the image, under the equivalent dose of miR-146a, the Cy5-tFNAs-miR146a group exhibited a stronger red fluorescence signal in HT-22 cells. Confocal images showed that it was more widely distributed in the cytoplasm and its intensity was significantly enhanced. Figure 4 (B) shows that the mean fluorescence intensity (MFI) of Cy5 in the Cy5-tFNAs-miR146a group was significantly higher than that in the Cy5-miR146a group (P<0.01), suggesting that tFNAs, as a delivery carrier, can significantly improve the cellular uptake efficiency of miR-146a after crossing the artificial blood-brain barrier, thereby enhancing its delivery ability in neurons. Figure 4 As shown in (C).

[0040] The above results demonstrate that the present invention successfully synthesized the nucleic acid nanocomplex tFNAs-miR146a, which has a tetrahedral nanostructure and can efficiently cross the blood-brain barrier.

[0041] Experiment Example 2: Evaluation of Antidepressant Efficacy and Organ Safety Assessment I. Experimental Methods 1. Chronic restraint stress-induced depression-like mouse model Male C57BL / 6 mice (6-8 weeks old) were purchased from Chengdu Jicui Pharmaceutical Experimental Animal Co., Ltd. All mice were acclimatized for one week in an SPF-grade animal facility (temperature 22 ± 2℃, 12-hour light-dark cycle, free access to food and water) before being used in the experiments. A depressive-like model was established using chronic restraint stress (CRS): mice were placed in a well-ventilated restraint device and restrained for 2 hours each in the morning and afternoon for 4 consecutive weeks. Healthy control mice were not restrained and were fed normally. After modeling, subsequent intervention was initiated.

[0042] 2. Nasal delivery of tFNAs-miR146a Mice were randomly divided into 5 groups: a healthy control group (CON-VEH group, healthy mice + Vehicle™ buffer), a chronic restraint stress group (CRS-VEH group, CRS mice + Vehicle™ buffer), a CRS-miR146a group (CRS mice + miR146a), a CRS-tFNAs group (CRS mice + tFNAs), and a CRS-tFNAs-miR146a nanocomposite group (CRS mice + tFNAs-miR146a). Intervention was performed via nasal administration: after mild fixation, the medication was slowly dripped into the nasal cavity for natural inhalation. The administration regimen was as follows: 50 μL twice daily for 2 weeks. The concentration of drug tFNAs-miR146a in the nanocomposite group was 1000 nM (based on tFNAs). To ensure equivalence in the number of miRNA molecules, the concentration of drug miR146a in the CRS+ miR146a group was set to 4 times that of tFNAs; the concentration of drug tFNAs in the CRS+ tFNAs group was consistent with that in the nanocomposite group, at 1000 nM. The control group received an equal volume of TM buffer (VEH). During the drug administration intervention, mice in the CRS-VEH group, CRS-miR146a group, CRS-tFNAs group, and CRS-tFNAs-miR146a nanocomposite group continued to undergo CRS treatment according to the modeling method.

[0043] 3. Evaluation of the antidepressant efficacy of nucleic acid nanocomplexes using multidimensional behavioral tests in mice. After drug administration, mice in each group (n=10 / group) underwent multidimensional behavioral assessments to evaluate the antidepressant effect. The sucrose preference test was used to assess anhedonia, recording the percentage of sucrose intake relative to total fluid intake; the forced swimming and tail suspension tests were used to assess hopeless behavior, recording the cumulative immobility time (s) within a specified time; the open field test was used to assess voluntary activity and anxiety levels, recording the total distance (cm) moved by the mice in the open field. All behavioral experiments were conducted in a quiet environment, and the experimenters were blinded to minimize bias.

[0044] 4. Organ safety assessment After a two-week administration period, mice were euthanized, and blood and major organs were collected for safety assessment. Blood samples were used for complete blood count and blood biochemistry analysis. Complete blood count parameters included red blood cell count (RBC), white blood cell count (WBC), and platelet count (PLT). Blood biochemistry parameters included alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CR), urea (UREA), and uric acid (UA). Simultaneously, heart, liver, spleen, lung, and kidney tissues were collected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). Morphological changes were observed under an optical microscope to assess the in vivo biocomplex safety.

[0045] 5. Data Statistics All experiments were independently repeated at least three times to ensure the stability and reliability of the results. Statistical analysis of the experimental data was performed using GraphPad Prism. Differences between groups were assessed using either independent samples t-tests or one-way ANOVA, depending on the experimental design. Statistical significance was expressed as a p-value, where... This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001. This means P < 0.0001.

[0046] II. Experimental Results 1. The nanocomposite tFNAs-miR146a significantly improved CRS-induced depressive-like behavior. After 6 weeks of cumulative CRS (with modeling continuing during the 2-week treatment period), compared with the healthy control CON-VEH group, the CRS-VEH group showed significant depressive-like behaviors, decreased sucrose preference (p<0.0001), significantly reduced survival behavior, significantly increased cumulative immobility time in forced swimming and tail suspension tests (p<0.01; p<0.01), significantly reduced activity in open field tests, and decreased total distance traveled (p<0.0001); indicating that the depressive-like model induced by chronic restraint stress was successfully established. Meanwhile, the nucleic acid nanocomplex tFNAs-miR146a, after 2 weeks of intranasal treatment, improved anhedonia symptoms in CRS-treated mice, increased sucrose intake (p<0.0001), enhanced survival behavior in a state of despair, reduced cumulative immobility time in forced swimming and tail suspension tests (p<0.01; p<0.05), and increased activity in open field tests (p<0.001). Compared with tFNAs alone and miR-146a alone, the nanocomposite tFNAs-miR146a showed significant antidepressant efficacy, and its antidepressant effect was significantly better than the sum of the effects of tFNAs alone and miR-146a alone, showing a synergistic antidepressant effect.

[0047] 2. The nanocomposite tFNAs-miR146a exhibits good tissue and organ safety. Organ safety assessment results such as Figure 6 As shown in the figure, there were no significant differences in blood routine indicators (RBC, WBC, PLT, n = 5 mice / group) and blood biochemical indicators (ALT, AST, CR, UREA, UA, n = 5 mice / group) among the groups of mice (P>0.05), suggesting that different treatments did not have a significant effect on the body's blood system and liver and kidney function. Further HE staining of heart, liver, spleen, lung, and kidney tissues showed that the tissue structure of each group was intact, with no obvious cell damage, inflammatory cell infiltration, or pathological changes such as nuclear fragmentation or nuclear pyknosis. These results indicate that the tFNAs-miR146a nanocomposite has good in vivo biocompatibility under the experimental conditions.

[0048] The above results indicate that the nanocomposite tFNAs-miR146a is biosafe and exhibits significant antidepressant efficacy. Its antidepressant effect is superior to the combined effects of tFNAs alone and miR-146a alone, demonstrating a synergistic antidepressant effect.

[0049] Experiment Example 3: Detection of Inflammatory Factors and Microglial Cell Activation Status in Brain Tissue I. Experimental Methods 1. Enzyme-linked immunosorbent assay (ELISA) for detecting changes in inflammatory factors in brain tissue. Animal modeling and drug administration were carried out according to the method in Experiment Example 2.

[0050] After drug administration, mice were sacrificed and brain tissue was collected (n = 8 mice / group). The hippocampus was rapidly isolated and washed with pre-cooled PBS to remove blood residue. Then, lysis buffer was added for homogenization. The homogenate was centrifuged at 4°C (12,000 rpm, 15 min), and the supernatant was collected for subsequent detection. Commercially available ELISA kits were used to detect the expression levels of the inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). The experimental groups were the same as in Experiment 2: healthy control group (CON-VEH group), chronic restraint stress group (CRS-VEH group), CRS-miR146a group, CRS-tFNAs group, and CRS-tFNAs-miR146a group. Following the instructions of the kit, the absorbance values ​​of each well were measured on an ELISA reader, and the concentrations of each inflammatory factor were calculated based on the standard curve. The final results were expressed as pg / mg protein and were used to evaluate the regulatory effects of different treatments on the inflammatory response in brain tissue.

[0051] 2. Immunofluorescence detection of microglia (Iba-1) in brain tissue Animal modeling and drug administration were carried out according to the method in Experiment Example 2.

[0052] After the experiment, mice were fixed by cardiac perfusion with saline and 4% paraformaldehyde, and their brains were harvested and fixed again in 4% paraformaldehyde, followed by dehydration. Brain tissue was cut into 30 μm thick coronal sections (mid-hippocampal region) for immunofluorescence staining. After washing with PBS, the tissue sections were permeabilized with 0.3% Triton X-100 and non-specific binding sites were blocked with 5% bovine serum albumin. They were then incubated with primary antibodies: anti-Iba-1 antibody (microglia marker) and anti-NeuN antibody (neuronal marker) overnight at 4°C. The next day, the corresponding fluorescently labeled secondary antibodies were added and incubated for 1 h in the dark. Nuclear staining was then performed using DAPI. After staining, the sections were mounted and observed and images were acquired using a laser confocal microscope. The activation level of microglia was assessed by analyzing the intensity and morphological changes of the Iba-1 positive signal, while neuronal distribution was observed using NeuN labeling. Quantitative analysis of fluorescence intensity was performed using image analysis software to compare the differences in neuroinflammation levels among the groups. The experimental groups are the same as in Experiment Example 2.

[0053] II. Experimental Results 1. The nanocomposite tFNAs-miR146a inhibits the increase of CRS-induced inflammation-related factors. The results are as follows Figure 7 As shown, miR-146a can target the mRNA of Traf6 and Irak1, leading to the binding and degradation of the mRNA by the silencing complex, thereby inhibiting the synthesis of downstream inflammatory factors IL-1β and TNF-α. Two weeks after treatment, brain tissue supernatant analysis revealed significant differences in all three inflammatory factors between the CON-VEH and CRS-VEH groups (all p < 0.0001). The nanocomposite CRS-tFNAs-miR146a showed significant anti-inflammatory effects against IL-1β, TNF-α, and IL-6, with statistically significant differences compared to CRS-VEH (p < 0.0001; p < 0.01; p < 0.01). In contrast, tFNAs and miR-146a alone did not show effective inhibition of inflammatory factors, showing no statistically significant differences compared to CRS-VEH, but statistically significant differences compared to CON-VEH and CRS-tFNAs-miR146a. The results showed that the nanocomposite CRS-tFNAs-miR146a was significantly better than the sum of the effects of tFNAs alone and miR-146a alone in reducing inflammatory factors, demonstrating a synergistic effect.

[0054] 2. The nanocomposite tFNAs-miR146a inhibits CRS-induced microglial cell activation. The results are as follows Figure 8 As shown, chronic restraint stress (CRS-VEH) can increase neuroinflammation and cause excessive activation of microglia. Compared with the healthy control group CON-VEH, the mean fluorescence intensity of Iba-1 in the CRS-VEH group was significantly increased (p<0.0001). After 2 weeks of intranasal administration of the nucleic acid nanocomplex tFNAs-miR146a, the mean fluorescence intensity of Iba-1 was effectively reduced, showing a significant difference compared with CRS-VEH (p<0.0001), but no statistical difference compared with the healthy control group CON-VEH. Although administration of tFNAs or miR146a alone showed a certain degree of reduction in the mean fluorescence intensity of Iba-1, it still showed a significant statistical difference compared with the healthy control group CON-VEH (p<0.001; p<0.05). This also indicates that tFNAs alone also play a role in inhibiting CRS-induced excessive activation of microglia, but the effect is poor. The results showed that the nanocomposite CRS-tFNAs-miR146a was significantly more effective than the combined effects of tFNAs alone and miR-146a alone in inhibiting microglia overactivation, demonstrating a synergistic effect.

[0055] The above results indicate that the nanocomposite CRS-tFNAs-miR146a has a synergistic effect in inhibiting CRS-induced inflammation.

[0056] As can be seen from the above embodiments and experimental examples, this invention provides a DNA tetrahedral framework-based nanocomposite, its preparation method, and its application in improving depression. This invention successfully synthesized the nanocomposite material tFNAs-miR146a by loading miR-146a onto a tetrahedral framework nucleic acid with sticky ends. This nanocomposite material can efficiently cross the blood-brain barrier and has been applied to chronic restraint stress-induced depression-like mice, exhibiting biosafety. It shows a synergistic effect in alleviating depressive-like behavior and CRS-induced inflammation, demonstrating excellent potential in the preparation of drugs for treating depression.

Claims

1. A tetrahedral framework nucleic acid composite material, characterized in that: The composite material consists of a tetrahedral framework nucleic acid with sticky ends A and microRNA with sticky ends B; the sticky ends A and B are single-stranded DNA with complementary base pairing, and the number of nucleotides is 5-8 respectively. The molar ratio of the tetrahedral framework nucleic acid to microRNA is 1:4; The sequence of the microRNA is shown in SEQ ID NO.

5.

2. The composite material according to claim 1, characterized in that: In the tetrahedral framework structure of the aforementioned tetrahedral framework nucleic acid, each vertex contains 3-6 non-complementary deoxyribonucleotides, and each edge contains 18-22 base pairs.

3. The composite material according to claim 1, characterized in that: The nucleotide sequences of the sticky ends A and B are selected from AAAAAA or TTTTTT.

4. The composite material according to claim 1, characterized in that: The tetrahedral framework nucleic acid with sticky A ends is composed of four single-stranded nucleic acids S1, S2, S3 and S4 with sticky A ends, which are formed by complementary base pairing; the molar ratio of S1 to S2, S3 and S4 is 1:1:1:

1.

5. The composite material according to claim 4, characterized in that: The nucleotide sequences of single-stranded nucleic acids S1, S2, S3 and S4 with sticky A ends are shown in SEQ ID NO. 1-4.

6. The composite material according to any one of claims 1-5, characterized in that, It is prepared by mixing and incubating a tetrahedral framework nucleic acid with sticky ends A with a microRNA with sticky ends B.

7. The composite material according to claim 6, characterized in that: The conditions for the mixed incubation include: incubation in TM-buffer at 25-37°C for 30-60 min.

8. A method for preparing the composite material according to any one of claims 1-7, characterized in that, It includes: Tetrahedral framework nucleic acids with sticky A ends are mixed and incubated with microRNAs with sticky B ends.

9. The use of the composite material according to any one of claims 1-7 in the preparation of medicaments for antidepressant and / or anti-neuroinflammatory purposes.

10. A drug for treating depression and / or neuroinflammation, characterized in that: It is obtained by using the composite material described in any one of claims 1-7 as the active ingredient, plus pharmaceutically acceptable excipients or auxiliary ingredients.

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