A nano-regulator for treating cerebral hemorrhage and its preparation method and application

Abstract

The application belongs to the technical field of biological medicine, and discloses a nano regulator for treating cerebral hemorrhage, a preparation method and application thereof. First, a DNA nanoflower containing a hybridization site is prepared based on RCA technology, and is hybridized with a T structure containing a target transferrin receptor, so that the obtained carrier has the ability to cross the blood-brain barrier and gather at the damage site. Then, the nano regulator TNF@Gin is obtained by loading ginkgo flavones through non-covalent interaction. The obtained nano regulator can promote the polarization of microglial cells to the anti-inflammatory M2 phenotype, thereby reducing the level of pro-inflammatory factors and relieving excessive inflammatory response. In addition, the nano regulator can effectively clear ROS and Nrf2 / GPX4 signal pathway, and inhibit the ferroptosis of hippocampal neuron cells. The nano regulator shows high and safe treatment results, and provides a new strategy for treating cerebral hemorrhage.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nano-regulator for treating cerebral hemorrhage, its preparation method, and its application. Background Technology

[0002] Intracerebral hemorrhage (ICH), also known as spontaneous intracerebral hemorrhage, is an acute cerebrovascular disease caused by the rupture of blood vessels within the brain parenchyma without external trauma, leading to bleeding that compresses surrounding brain tissue and causes neurological damage. It is a severe subtype of stroke, characterized by rapid onset, rapid progression, high mortality, and high disability rates. The pathological damage from ICH is mainly divided into primary and secondary damage, with secondary damage being the primary cause of disease progression. Its pathological mechanism is complex, primarily driven by oxidative stress and excessive inflammatory responses, resulting in extremely high mortality and disability rates. Given the limited efficacy of single-target therapies, there is an urgent need to develop novel treatment strategies that can simultaneously regulate multiple aspects of this pathophysiological network.

[0003] Following a brain hemorrhage, the hematoma dissolves, releasing a large amount of hemoglobin. This hemoglobin is degraded by heme oxygenase-1, producing free iron ions. These iron ions enter nerve cells and glial cells via transferrin receptors and divalent metal ion transporter 1 (DMT1), causing local iron overload. Excessive Fe 2+ The Fenton reaction induces a massive generation of reactive oxygen species (ROS), triggering lipid peroxidation of cell membranes. Simultaneously, glutathione (GSH) depletion and decreased glutathione peroxidase 4 (GPX4) activity lead to an imbalance in the antioxidant system, ultimately triggering an iron-dependent lipid peroxidation cascade that results in irreversible cell death. Meanwhile, hematoma and its degradation products, as potent risk-associated molecular patterns, initiate a robust and sustained inflammatory response. At its core is the rapid activation and infiltration of immune cells, particularly microglia and peripherally derived macrophages, which polarize into different functional states. The classically activated M1 phenotype releases pro-inflammatory cytokines, exacerbating neuroinflammation and secondary damage; the alternately activated M2 phenotype secretes anti-inflammatory cytokines, promoting tissue repair, debris clearance, and angiogenesis. The balance among these macrophage phenotypes is a key determinant of ICH prognosis.

[0004] The blood-brain barrier (BBB) ​​is a crucial physiological barrier for maintaining homeostasis within the central nervous system (CNS) and protecting brain tissue from external damage. It is composed of brain microvascular endothelial cells, tight junctions, the subendothelial basement membrane, pericytes, and astrocytes, forming a complete three-dimensional barrier structure. While the BBB effectively prevents the invasion of pathogens such as bacteria, protecting brain tissue from infection, this characteristic also makes it difficult for antibodies, antibiotics, and most therapeutic drugs in the blood to penetrate the barrier and enter the brain parenchyma, posing a significant challenge to the treatment of brain diseases.

[0005] DNA nanoflowers (DNFs) are flower-like nanostructures formed by the self-assembly of long-chain DNA. They have high surface area and high porosity, and their unique properties make them promising for a wide range of applications in the biomedical field, such as drug delivery, cell imaging, and biosensing.

[0006] Ginkgetin is a flavonoid compound extracted from the dried leaves of Ginkgo biloba, a plant in the Ginkgoaceae family. It has antioxidant, blood circulation improvement and vascular protection, neuroprotection and cognitive function improvement, anti-inflammatory and anti-tumor effects. Summary of the Invention

[0007] To address the deficiencies in existing technologies, this invention proposes a nano-regulator for treating cerebral hemorrhage, its preparation method, and its application. The nano-regulator is constructed by using DNA nanoflowers prepared with a T-structure containing a target transferrin receptor as a carrier, and loading Ginkgo biloba flavonoids through non-covalent interactions.

[0008] The technical solution adopted in this invention: A nanomodulator for treating cerebral hemorrhage, characterized in that it comprises a DNA nanoflower carrier containing a transferrin receptor and ginkgo biflavonoids loaded on the DNA nanoflower carrier.

[0009] The present invention also provides a method for preparing the aforementioned nano-regulator, characterized by the following specific steps: (1) A linear DNA template containing hybridization sites was prepared by solid-phase synthesis; the phosphorylated linear DNA template and an equal amount of primers were mixed in 1×T4 DNA ligase buffer and annealed, and then T4 DNA ligase was added and incubated at 14~18℃ for 10~14h to obtain a circular DNA template; the ligated circular DNA template was incubated with dNTPs and phi29 DNA polymerase in the corresponding reaction buffer at 25~35℃ for 1~3h to perform RCA reaction to obtain DNA nanoflowers; the nucleotide sequence of the phosphorylated linear DNA template is shown in SEQ ID NO.1; the nucleotide sequence of the primers is shown in SEQ ID NO.2; (2) DNA strands were prepared and purified by solid-phase synthesis, the DNA strands comprising L, S1-1, S1-2, S1-3, S1, S2, S3, 1-4, 1-5 and 1-6; the targeting aptamer sequence for the transferrin receptor was integrated into S1, S2 and S3 respectively; then all DNA strands were synthesized at the same molar ratio in 1×TAE / Mg 2+ The mixture is prepared in a buffer solution and stored at 90-100°C for 3-5 minutes, then cooled to 25-35°C and held for 22-26 hours to self-assemble into a Y-shaped T-structure intermediate. The nucleotide sequence of L is shown in SEQ ID NO. 4; the nucleotide sequence of S1-1 is shown in SEQ ID NO. 5; the nucleotide sequence of S1-2 is shown in SEQ ID NO. 6; the nucleotide sequence of S1-3 is shown in SEQ ID NO. 7; the nucleotide sequence of S1 is shown in SEQ ID NO. 8; the nucleotide sequence of S2 is shown in SEQ ID NO. 9; the nucleotide sequence of S3 is shown in SEQ ID NO. 10; the nucleotide sequences of 1-4 are shown in SEQ ID NO. 11; the nucleotide sequences of 1-5 are shown in SEQ ID NO. 12; and the nucleotide sequences of 1-6 are shown in SEQ ID NO. 13. (3) The 10 nM T structure intermediate was mixed with DNA nanoflowers and incubated to obtain the nanoflower carrier TNF; after the hybridization was completed, the ginkgo biflavonoid component was directly added and incubated for 6 h, the supernatant was discarded by centrifugation, and the nano-regulator TNF@Gin was obtained.

[0010] In one alternative embodiment, in step (2), the target transferrin receptor aptamer sequence is shown in SEQ ID NO.3.

[0011] In one optional embodiment, in step (3), the concentration of nanoflower is 500 μg / mL and the concentration of ginkgo biflavonoids is 1.5 mg / mL.

[0012] The use of the nanomodifier of claim 1 in the preparation of drugs for the intervention or treatment of cerebral hemorrhage.

[0013] Compared with the prior art, the present invention has the following beneficial effects: 1. The nanomodulator obtained in this invention can stimulate microglia to polarize to the anti-inflammatory M2 phenotype and inhibit neuronal ferroptosis through the Nrf2 / GPX4 signaling pathway. By targeting the inflammatory response and ferroptosis, it provides a novel and promising strategy for the synergistic treatment of cerebral hemorrhage.

[0014] 2. The DNA nanoflower carrier loaded with ginkgo biflavonoids constructed in this invention integrates a target transferrin receptor, which can penetrate the blood-brain barrier to deliver the drug to the site of injury, and achieves a highly efficient therapeutic effect through a synergistic intervention mechanism. Attached Figure Description

[0015] Figure 1 This is a route diagram for the preparation of TNF@Gin; Figure 2 A schematic diagram of the construction of a T structure containing a transferrin receptor targeting aptamer; Figure 3 This is a graph showing the drug loading efficiency. Figure 4 TEM image of TNF@Gin; Figure 5 AFM plot of atomic forces for TNF@Gin; Figure 6 Figures showing particle size characterization, surface charge characterization, and stability analysis of TNF@Gin; Figure 7 This is a drug release curve; Figure 8 This is a graph showing cell viability analysis. Figure 9 This is a graph showing the intracellular ROS level analysis. Figure 10 Plot showing the expression levels of GPX4 and Nrf2 proteins; Figure 11 This is a graph showing the levels of inflammatory factors TNF-α, IL-1β, IL-10, and TGF-β. Figure 12 Horizontal diagrams of M1 and M2 cells; Figure 13 This is a graph showing the targeted fluorescence analysis in mice. Figure 14 Statistical analysis of tomographic examination of mouse paws after 28 days of treatment; Figure 15 Statistical analysis of the rotarod test in mice treated for 28 days; Figure 16H&E staining images of brain sections from ICH mice after different treatments; Figure 17 Immunofluorescence images of ROS in brain slices after different treatments. Detailed Implementation

[0016] The technical solutions of the embodiments of this application will be further described clearly and completely below with reference to the accompanying drawings. It should be noted that the described embodiments are only some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0017] To make the inventive objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be further described in detail below with reference to the accompanying drawings: In order to better understand the above-mentioned objectives, features, and advantages of this invention, the advantages of this invention will be further illustrated below by comparing the embodiments with the accompanying drawings and specific implementation methods.

[0018] The method for preparing the nano-regulator TNF@Gin provided by this invention first involves obtaining DNA nanoflowers via an RCA reaction, then self-assembling a Y-type T-structure intermediate containing transferrin targeting; hybridizing the DNA nanoflowers with the T-structure intermediate to obtain the nanoflower carrier TNF, and then loading ginkgo biloba flavonoids through non-covalent interaction to obtain TNF@Gin. The preparation route is as follows: Figure 1 As shown.

[0019] I. Preparation method of nano-regulator TNF@Gin

[0020] Example 1: Preparation of Nanoflowers using RCA Technology A linear DNA template containing a hybridization site (GTGCTACTCCAGTTC) with a phosphate group at its 5' end was prepared by solid-phase synthesis. 2 μM of the 5' phosphorylated DNA template and an equal volume of primers were mixed and annealed in 1×T4 DNA ligase buffer (50 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, and 1 mM adenosine triphosphate (ATP), pH=7.5), followed by T4 ligase. DNA ligase (10 U / μL) was incubated at 16 °C for 12 h to obtain a circular DNA template. The ligated circular DNA template (0.3 μM) was then incubated with dNTPs (1 mM) and phi29 DNA polymerase (1 U / μL) in the appropriate reaction buffers (50 mM Tris-HCl, 10 mM MgCl2, 66 mM KCl, 0.04% (v / v) Tween 20, 4 mM DTT, pH=7.5) at 30 °C for 18 h to perform an RCA reaction to obtain DNA nanoflowers. The sequences of the template and primers are shown in Table 1.

[0021] Table 1. Nucleotide sequences of templates and primers

[0022] Example 2: Design of the T-structure DNA strands L, S1-1, S1-2, S1-3, S1, S2, S3, 1-4, 1-5, and 1-6 were prepared and purified using solid-phase synthesis. Detailed DNA sequences are shown in Table 1. The transferrin receptor targeting aptamer sequence was integrated into DNA strands S1, S2, and S3. The purified DNA strands (L, S1-1, S1-2, S1-3, S1, S2, S3, 1-4, 1-5, and 1-6) were then synthesized in an equimolar ratio using a 1×TAE / Mg2+ solution. 2+ The mixture was prepared in a buffer solution (40 mM Tris, 20 mM acetic acid, 2 mM EDTA, and 12.5 mM magnesium acetate, pH=8.3) to a final concentration of 1 μM. The assembly solution was incubated at 95°C for 3–5 min, and then slowly cooled from 95°C to 25°C over 24 h. This allowed the extended DNA strands S1, S2, and S3 to self-assemble into a Y-shaped T-structure intermediate according to the pre-defined base pairing. Each strand formed a double helix at one end with the other two strands in the central region, and each end had a single-stranded overhang. The structure was as follows: Figure 2 As shown.

[0023] Table 2. Aptamers and DNA sequences of T-structure intermediates

[0024] Next, the 10 nM T-structure intermediate was mixed with DNA nanoflowers and incubated at 25 °C for 9 h to obtain the nano-carrier TNF.

[0025] Example 3: Optimizing Load Efficiency Different amounts of ginkgo biloba extract were loaded into TNF@Gin at concentrations of 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, and 1.5 mg / mL. After incubation, the cells were washed with PBS, centrifuged at 8500 rpm for 5 min, and stored at 4℃. The concentration of the highest loading was determined by UV absorption spectroscopy. Different incubation times (1 h, 3 h, and 6 h) were then determined based on the obtained concentrations. The optimal drug loading efficiency (DLE) was then determined by UV absorption spectroscopy based on the optimal concentration and optimal incubation time, using the following formula:

[0026] Figure 3 The figures show the drug loading efficiency analysis, where graph a represents the loading amount of ginkgo biloba extract at different concentrations, and graph b represents the loading rate of ginkgo biloba extract at different incubation times. As shown in graph a, the loading amount of ginkgo biloba extract is highest when the concentration reaches 1.5 mg / mL; graph b shows that with an input concentration of 1.5 mg / mL, the loading rate gradually increases with time. Finally, an input concentration of 1.5 mg / mL and an incubation time of 6 h were selected, resulting in the highest drug loading efficiency of 47.83%.

[0027] Example 4: Preparation of TNF@Gin TNF@Gin was obtained by directly adding 1.5 mg / mL of Ginkgo biloba extract to a 500 μg / mL DNA nanoflower carrier and culturing for 6 h.

[0028] II. Characterization and efficacy determination of TNF@Gin 1. Transmission electron microscopy characterization of TNF@Gin The TNF@Gin obtained in Example 3 (concentration of 1 µg / mL in ddH2O) was dropped onto a copper mesh and deposited for 0.5 h. Excess sample solution was then removed. The copper mesh was dried overnight and then coated with gold before observation. Images were acquired and processed using a Hitachi SU8220 scanning electron microscope.

[0029] Figure 4 Here is a transmission electron microscope (TEM) image of TNF@Gin, as shown. Figure 4 It can be seen that the synthesized TNF@Gin exhibits a typical three-dimensional flower-like microsphere structure. Each microsphere is self-assembled from a large number of ultrathin two-dimensional nanosheets. The nanosheets are radially stacked and grow outward from the center to form an open porous network structure with stable structure and uniform size.

[0030] 2. Atomic force microscopy characterization of TNF@Gin The DNA nanoflowers obtained in Example 1 and TNF@Gin (1 µg / mL) obtained in Example 3 were dropped onto a mica sheet and deposited for 3 min. The mica sheet surface was then rinsed with 1 mL ddH2O and allowed to air dry naturally. The mica sheets were then imaged in ScanAsyst-Air mode using a Bruker MultiMode 8 AFM.

[0031] Figure 5 This is an atomic force microscopy (AFM) image of TNF@Gin. Figure 5 The sample exhibits multiple uniformly distributed bright white raised particles. The particles are spherical with clear edges and no obvious aggregation, indicating that the synthesized TNF@Gin has good dispersibility on the substrate.

[0032] 3. Stability analysis of TNF@Gin The DNA nanoflowers obtained in Example 1 and TNF@Gin (10 µg / mL) obtained in Example 3 were incubated in DMEM medium containing 10% FBS at 37°C for 0 to 24 h. Subsequently, the particle size and distribution of the samples were measured by DLS at 25°C, and their aggregation and degradation were verified by 0.8% agarose gel electrophoresis.

[0033] Figure 6 Figures show the particle size, surface charge, and stability analysis of TNF@Gin. Figure a shows the scattered light intensity distribution, figure b shows the zeta potential, and figure c shows the particle size stability. Figure a shows that TNF@Gin has no significant difference in particle size compared to DNA nanoflowers, and the particle size distribution of TNF@Gin is uniform and well-dispersed. Figure b shows that the zeta potential of DNA nanoflowers is approximately -20 mV (negatively charged), while the zeta potential of TNF@Gin is approximately +20 mV (positively charged). This indicates that TNF@Gin successfully altered the surface load of the nanoflowers, and the surface is positively charged, which is beneficial for targeting negatively charged tumor tissue through electrostatic interaction, while also possessing a certain degree of physical stability. Figure c shows that regardless of whether it was continuously shaken for 48 hours or kept still for 48 hours, the particle size of TNF@Gin remained at approximately 150 nm, without significant fluctuations or aggregation, demonstrating excellent physical stability.

[0034] 4. Drug release test The TNF@Gin obtained in Example 3 was resuspended in 1 mL of PBS and sealed in a pre-soaked dialysis bag (MWCO: 5 kDa). The dialysis bag was then completely immersed in 5 mL of PBS with different pH values ​​(6.0, 7.4, and 8.3), and incubated with gentle stirring at 37°C. The concentration of drug released from the collected samples was quantitatively determined using UV-Vis spectrophotometry (λ=352 nm).

[0035] Figure 7 The figure shows the drug release curve, which indicates that TNF@Gin exhibits time-dependent sustained-release behavior, suggesting that the modulator can be continuously released to maintain an effective drug concentration, thereby ensuring sustained pharmacological activity.

[0036] 5. Cell viability assay An in vitro ICH model was constructed by stimulating hippocampal neurons (BV2 cells) with 20 μM oxyhemoglobin (OxyHb) and 5% CO2 at 37°C. ICH cells were then distributed in wells at a density of 7.5 × 102. 4 Cells were seeded at a density of 1000 cells per well in a 96-well plate and divided into a control group, an induction group, and a TNF@Gin group. After incubation with the corresponding drugs for 48 hours, the cells were detected using a CCK-8 assay kit, and the OD value was measured at a wavelength of 450 nm.

[0037] Figure 8 This is a cell viability analysis graph, by Figure 8 It can be seen that the cell viability of the induced group was significantly reduced compared with that of the control group, indicating that the model was successfully constructed. OxyHb-induced damage reduces the cell viability of BV2 cells and causes cell damage. In contrast, the cell viability of the TNF@Gin group was significantly increased compared with that of the induced group. This shows that TNF@Gin can effectively reverse the decline in cell viability caused by damage and restore cell viability to near normal levels, proving that it has a cell protective effect.

[0038] 6. ROS level measurement ICH cells were planted at a density of 1 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells in confocal culture plates and divided into control group, induction group, nanoflower group, ginkgo biloba flavonoid group, and TNF@Gin group. The corresponding drugs were added after cell adhesion. After 12 h of incubation, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe was added for further incubation. Cells were washed three times with cold PBS and imaged under a fluorescence microscope (Ex / Em = 488 / 525 nm).

[0039] Figure 9 This is a graph showing the intracellular ROS level analysis, by... Figure 9 The results showed that ROS levels were significantly higher in the induced group compared to the control group, indicating successful model construction. OxyHb induction increases ROS levels in BV2 cells, causing oxidative stress damage. After TNF@Gin intervention, ROS levels significantly decreased to near the level of the normal control group. This indicates that TNF@Gin can efficiently scavenge excess ROS, significantly reduce oxidative stress levels, and has excellent antioxidant activity.

[0040] 7. Effects on ferroptosis ICH cells were planted at a density of 1 × 10⁶ cells per well. 5Cells were seeded at a density of [number] cells in confocal culture plates. After cell adhesion, the cells were divided into control group, induction group, nanoflower group, ginkgo biloba flavonoid group, and TNF@Gin group, with the corresponding drugs added after cell adhesion. After 24 hours of incubation, the supernatant was collected, and the protein expression levels of Nrf2 (a major regulator of antioxidant responses) and GPX4 (a central enzyme inhibiting lipid peroxidation) were quantitatively detected according to the specific experimental steps in the enzyme-linked immunosorbent assay (ELISA) kit instructions.

[0041] Figure 10 This is a plot showing the expression levels of GPX4 and Nrf2 proteins. Figure 10 The results showed that, compared with the nanoflower group and the ginkgo biflavonoid group, the protein levels of the key factor Nrf2 and its downstream effector GPX4 were significantly increased after TNF@Gin intervention, indicating that TNF@Gin can effectively reduce ferroptosis in HT22 cells by activating the Nrf2 / GPX4 signaling pathway.

[0042] 8. Measurement of inflammatory factor levels ICH cells were planted at a density of 1 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells in confocal culture plates. After cell adhesion, the cells were divided into control group, induction group, nanoflower group, ginkgo biloba flavonoid group, and TNF@Gin group, with the corresponding drugs added after cell adhesion. After 24 hours of incubation, the supernatant was collected, and the levels of inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-10 (IL-10), and transforming growth factor-β (TGF-β) were quantitatively detected according to the specific experimental procedures in the ELISA kit instructions.

[0043] Figure 11 The figures show the levels of inflammatory factors. Figures a and b show the levels of pro-inflammatory factors TNF-α and IL-1β in the supernatant of ICH cells; figures c and d show the levels of anti-inflammatory factors IL-10 and TGF-β in the supernatant of ICH cells. Figures a and b show that, compared with the control group, OxyHb stimulation induced a strong inflammatory response, leading to a significant increase in the secretion of TNF-α and IL-1β. Although treatment with nanoflowers or free ginkgo biloba extract resulted in a moderate reduction of these pro-inflammatory factors, the TNF@Gin group showed the most effective anti-inflammatory effect, with a statistically significant reduction. Figures a and d show that, compared with nanoflowers or free ginkgo biloba extract, TNF@Gin significantly increased the secretion of anti-inflammatory factors IL-10 and TGF-β. This indicates that TNF@Gin has excellent anti-inflammatory effects.

[0044] 9. Effects on M1 / M2 phenotypic balance ICH cells were taken and added to DMEM / F12 complete medium containing 10% FBS and 1% penicillin antibiotics. The cells were then cultured in a 37°C, 5% CO2 incubator at a rate of 1×10⁻⁶. 5 Cells were seeded per well in 24-well plates and cultured for 24 hours until adherence. Cells were divided into a control group (culture medium + PBS) and a TNF@Gin group (culture medium + TNF@Gin obtained in Example 1), with the corresponding drugs added to each. Immunofluorescence was used to detect M1 macrophages (Iba1...). + CD16 / 32 + ) and M2 macrophages (Iba1 + CD206 + ).

[0045] Figure 12 This is a horizontal map of M1 and M2 cells, created by... Figure 12 It was found that TNF@Gin effectively reversed the trend of ICH, significantly reducing M1 cell levels and significantly increasing M2 cell levels. The results indicate that TNF@Gin can significantly inhibit the activation and infiltration of M1 pro-inflammatory macrophages after cerebral hemorrhage, while promoting the polarization of M2 anti-inflammatory / repair macrophages, thus achieving a shift from a pro-inflammatory phenotype to an anti-inflammatory and repair phenotype.

[0046] 10. In vivo distribution and targeted efficacy evaluation experiments An ICH mouse model was induced in the striatum of 6-week-old C57BL / 6 mice by stereotactic injection of collagenase VII (0.08U, dissolved in 1.0μL saline).

[0047] Seven days later, the mice were randomly divided into two groups (targeted group and non-targeted group). 50 μL of TNF@Gin obtained in Example 3 and TNF@Gin without targeting the transferrin receptor were injected via the tail vein, respectively. The dosage was calculated as 2 mg / kg of ginkgo biloba extract. After resting for 10-20 minutes, the mice were anesthetized, and small animal fluorescence imaging was performed. The obtained imaging signals were statistically analyzed.

[0048] Figure 13 The images show targeted fluorescence analysis in mice. Image a is a fluorescence imaging image of the mouse, and image b is a fluorescence signal intensity image. Figure 13 As can be seen, there was almost no fluorescent signal in the brain of mice without targeting, and the signal intensity was extremely low, indicating that TNF@Gin without targeting the transferrin receptor could not be effectively enriched in the brain. In contrast, mice in the targeted group showed strong fluorescent signals in their brains, with the signal concentrated in the head region and the signal intensity significantly increased to about 35, indicating that TNF@Gin after integrating the transferrin receptor can specifically target brain tissue.

[0049] 11. Detection of the effect of in vivo treatment An ICH mouse model was induced in the striatum of 6-week-old C57BL / 6 mice by stereotactic injection of collagenase VII (0.08 U, dissolved in 1.0 μL of physiological saline).

[0050] Seven days later, mice were randomly divided into five groups (control group, model group, nanoflower group, ginkgo biloba flavonoid group, and TNF@Gin group), and the corresponding formulations (50 μL) were injected via tail vein on days 0, 2, and 4. The dosage was calculated based on ginkgo biloba flavonoids at 2 mg / kg. Throughout the treatment period (28 days), the mice's weight and behavioral deficits were monitored. The error rate was calculated using the following formula, where the total steps were the total number of foreleg movements, and the error steps were the number of times the foreleg slipped through the grid gaps and could not support the body.

[0051]

[0052] Figure 14 This is a statistical analysis chart of foot tomography in mice treated for 28 days, by... Figure 14 It was found that, compared with the control group, the model group showed a sharp increase in foot error rate and severe motor function impairment in mice 3 days after treatment, proving the successful establishment of the ICH model. Compared with the model group, the treatment effect was cumulative in the initial stage of treatment (days 7-21). As treatment progressed, the continuous drug effect produced strong results on day 28. Compared with nanoflower and free ginkgo biloba flavonoids, the TNF@Gin intervention significantly reduced the foot error rate in mice, indicating a significant improvement in motor function.

[0053] Figure 15 Statistical analysis of the rotarod test in mice treated for 28 days, as shown in the figure. Figure 15 As shown, compared with the control group, the model group showed significantly impaired motor coordination and balance in mice 3 days after treatment, and a sharp shortening of the fall latency, proving the successful construction of the ICH model. TNF@Gin intervention significantly prolonged the fall latency in ICH mice, improved motor coordination and balance deficits, and promoted long-term motor function recovery, with effects superior to nanoflowers and free ginkgo biflavonoids.

[0054] The study confirmed that TNF@Gin has a significant protective effect against neurological damage after cerebral hemorrhage, which is highly consistent with its mechanisms of regulating M1 / M2 polarization, reducing inflammatory response, resisting oxidative stress and inhibiting ferroptosis.

[0055] 12. Staining of tissue sections Mice were euthanized after treatment, and brain tissue was collected, fixed in 4% paraformaldehyde at room temperature, embedded in paraffin, and cut into 5 μm thick sections. The sections were then stained with hematoxylin and eosin (H&E) for morphological examination. Separately, the collected brain tissue was also cut into 5 μm thick sections and mounted on glass slides for immunofluorescence assays to detect ROS in the brain tissue. After antigen retrieval, the paraffin sections were blocked, incubated with primary antibody at 4°C overnight, washed, and incubated with secondary antibody at room temperature for 1 h. The cell nuclei were then counterstained with DAPI for 5 min, washed with PBS, mounted, and images were acquired under a fluorescence microscope.

[0056] Figure 16 H&E staining images of brain sections from ICH mice after different treatments, by Figure 16 It can be seen that the model group suffered severe neuronal damage, with a large number of red blood cells exuding, significant edema, tissue disorder, and significant inflammatory cell infiltration, indicating that the model was successfully established. The damage level in the nanoflower group was similar to that in the model group. In comparison, TNF@Gin treatment provided a large amount of neuroprotection, preserved normal cell structure, reduced the degree of tissue damage, and had a better protective effect than free ginkgo biloba flavonoids.

[0057] Figure 17 Immunofluorescence images of ROS in brain slices, by Figure 17 It was found that ROS was significantly upregulated in the brain tissue of the model group and the nanoflower group, and the red fluorescence signal was dense and widely distributed, indicating that excessive ROS accumulation after cerebral hemorrhage caused oxidative stress damage. In contrast, ROS in the brain tissue of the ginkgo biflavonoid group decreased and the intensity of the red fluorescence signal was weakened, indicating a certain intervention effect. After TNF@Gin intervention, the accumulation of ROS in the brain tissue decreased sharply, and the red fluorescence signal was sparse and weak, further confirming its effective anti-oxidative stress effect in vivo.

[0058] The results show that the nanomodulator TNF@Gin provided by this invention exhibits excellent physicochemical properties, including uniform morphology, high loading capacity, excellent stability, and good biocompatibility. It can specifically target and accumulate in the brain, providing effective neuroprotection. In terms of its mechanism of action in treating ICH, TNF@Gin effectively scavenges reactive oxygen species, polarizes microglia from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype, and inhibits neuronal ferroptosis by activating the Nrf2 / GPX4 signaling pathway. Therefore, TNF@Gin is a safe and effective nanotherapeutic strategy for cerebral hemorrhage.

[0059] Although preferred embodiments of this application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this application.

[0060] Obviously, those skilled in the art can make various modifications and variations to this application without departing from the spirit and scope of this application. Therefore, if such modifications and variations fall within the scope of the claims of this application and their equivalents, this application also intends to include such modifications and variations.

Claims

1. A nanomodulator TNF@Gin for treating cerebral hemorrhage, characterized in that, This includes a DNA nanoflower carrier containing a transferrin receptor and ginkgo biflavonoids loaded on the DNA nanoflower carrier.

2. The preparation method of the nano-regulator TNF@Gin according to claim 1, characterized in that, The specific steps are as follows: (1) A linear DNA template containing hybridization sites was prepared by solid-phase synthesis; the phosphorylated linear DNA template and an equal amount of primers were mixed in 1×T4 DNA ligase buffer and annealed, and then T4 DNA ligase was added and incubated at 14~18℃ for 10~14h to obtain a circular DNA template; the ligated circular DNA template was incubated with dNTPs and phi29 DNA polymerase in the corresponding reaction buffer at 25~35℃ for 1~3h to perform RCA reaction to obtain DNA nanoflowers; the nucleotide sequence of the phosphorylated linear DNA template is shown in SEQ ID NO.1; the nucleotide sequence of the primers is shown in SEQ ID NO.2; (2) DNA strands were prepared and purified by solid-phase synthesis, the DNA strands comprising L, S1-1, S1-2, S1-3, S1, S2, S3, 1-4, 1-5 and 1-6; the targeting aptamer sequence for the transferrin receptor was integrated into S1, S2 and S3 respectively; then all DNA strands were synthesized at the same molar ratio in 1×TAE / Mg 2+ The mixture is prepared in a buffer solution and stored at 90-100°C for 3-5 minutes, then cooled to 25-35°C and held for 22-26 hours to self-assemble into a Y-shaped T-structure intermediate. The nucleotide sequence of L is shown in SEQ ID NO. 4; the nucleotide sequence of S1-1 is shown in SEQ ID NO. 5; the nucleotide sequence of S1-2 is shown in SEQ ID NO. 6; the nucleotide sequence of S1-3 is shown in SEQ ID NO. 7; the nucleotide sequence of S1 is shown in SEQ ID NO. 8; the nucleotide sequence of S2 is shown in SEQ ID NO. 9; the nucleotide sequence of S3 is shown in SEQ ID NO. 10; the nucleotide sequences of 1-4 are shown in SEQ ID NO. 11; the nucleotide sequences of 1-5 are shown in SEQ ID NO. 12; and the nucleotide sequences of 1-6 are shown in SEQ ID NO.

13. (3) The DNA nanoflower carrier TNF was obtained by mixing and incubating a 10 nM T-structure intermediate with DNA nanoflowers; After hybridization, the ginkgo biflavonoid component was added directly and incubated for 6 hours. The supernatant was then discarded by centrifugation to obtain the nano-regulator TNF@Gin.

3. The preparation method according to claim 2, characterized in that, In step (2), the target transferrin receptor aptamer sequence is shown in SEQ ID NO.

3.

4. The preparation method according to claim 2, characterized in that, In step (3), the concentration of DNA nanoflower carrier is 500 μg / mL and the concentration of ginkgo biflavonoids is 1.5 mg / mL.

5. The use of the nanomodifier of claim 1 in the preparation of drugs for the intervention or treatment of cerebral hemorrhage.

Images