A tetrahedral dna / fluoxetine hydrochloride complex, its preparation method and use for treating depression

Abstract

The application provides a tetrahedral DNA / fluoxetine hydrochloride complex, a preparation method thereof and a use thereof in treating depression, and relates to the technical field of medicines.The tetrahedral DNA / fluoxetine complex of the application is a complex formed by embedding fluoxetine or a salt thereof into base pairs between a double helix structure of a tetrahedral DNA molecule in a non-covalent combination mode.The complex of the application has a fast onset time when treating depression, can exert an antidepressant effect of fluoxetine hydrochloride at a low dose, significantly reduces related side effects generated in the process of taking medicine, and can enhance the medicine-taking compliance of patients with depression.The complex of the application has good safety and effectiveness in treating depression, and has a good application prospect in the clinical treatment of depression.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a tetrahedral DNA / fluoxetine hydrochloride complex, a preparation method thereof, and an application thereof in treating depression. Background Art

[0002] In the relevant diagnosis and treatment guidelines for depression, antidepressant medication is the main treatment method. 5-hydroxytryptamine reuptake inhibitors (SSRIs) are a class of antidepressants widely used in clinical practice. They improve the function of monoamine neurotransmitters by increasing the concentration of 5-hydroxytryptamine in the synaptic cleft, thereby exerting an antidepressant effect. Among them, fluoxetine hydrochloride, as a selective 5-hydroxytryptamine reuptake inhibitor, is currently one of the first-line antidepressants in clinical practice. As a traditional antidepressant, fluoxetine hydrochloride has the common problems of traditional antidepressants: (1) Delayed onset: Due to the effect of the blood-brain barrier (BBB) ​​of the central nervous system, traditional antidepressants take a long time to reach effective drug treatment concentrations in the brain tissue, which significantly increases the risk of self-harm and suicide in patients with depression, and increases the burden on society and families; (2) Significant drug dose-related side effects: When using traditional antidepressants for treatment, when the patient's treatment effect is not obvious, the relevant treatment guidelines will recommend increasing the dosage of the antidepressant. This is accompanied by drug dose-related side effects, which lead to adverse drug reactions in patients with depression. The adverse reactions of fluoxetine hydrochloride are mainly manifested as: systemic Numbness, hepatitis, bradycardia, leukopenia, hair loss, etc.; (3) Low medication compliance: Traditional antidepressants have delayed onset and dose-related side effects, which may cause some patients to stop taking the drugs on their own due to inability to see therapeutic effects or obvious adverse drug reactions, resulting in low patient compliance and affecting the treatment effect; (4) Limited drug targets: Depression is a heterogeneous disease with complex etiology mechanisms and multiple etiological hypotheses. Monoamine neurotransmitter dysfunction, neuroimmunity, neuroinflammation and neuroendocrine factors are intertwined in the onset and progression of the disease. Traditional antidepressants only work on the monoamine neurotransmitter system. Therefore, in clinical treatment, some patients have no therapeutic response to traditional antidepressants. The above problems lead to the inability of fluoxetine hydrochloride to exert its efficacy in the treatment of depression, and the therapeutic effect is limited.

[0003] Nanomaterial drug delivery systems are gaining increasing attention to overcome the shortcomings of traditional antidepressant drugs in clinical practice, better help patients achieve clinical treatment goals, and alleviate socioeconomic and personal burdens. Among these, tetrahedral DNA nanostructures (TDNs) are emerging nanomaterials as promising drug delivery systems. TDNs are formed by four single-stranded DNA strands through interstrand base pairing, resembling a tetrahedron. These nanostructures boast high synthesis efficiency, simple synthesis steps, and excellent biosafety and biocompatibility. TDNs offer advantages such as negligible immunogenicity, inherent biocompatibility, structural stability, and unparalleled programmability—prerequisites for effective drug delivery. Furthermore, studies have demonstrated that TDNs possess certain biological activities, such as anti-inflammatory effects and potential benefits for the treatment of neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. Therefore, TDNs have become an important drug delivery system.

[0004] However, due to the unique structure of TDNs and their limited activity, the interaction between TDNs and small molecule drugs, whether synergistic or antagonistic, is unknown, and the effects of different small molecule drugs may vary significantly. Currently, no DNA tetrahedrons have been used to load fluoxetine hydrochloride, and whether DNA tetrahedrons can successfully load fluoxetine hydrochloride and achieve optimal efficacy requires further study. Summary of the Invention

[0005] The present invention aims to provide a tetrahedral DNA / fluoxetine hydrochloride complex, a preparation method thereof and use thereof in treating depression.

[0006] The present invention provides a tetrahedral DNA / fluoxetine complex, which is a complex formed by embedding fluoxetine or a salt thereof between base pairs in a double helix structure of a tetrahedral DNA molecule in a non-covalent binding manner.

[0007] The structure of fluoxetine is

[0008] Furthermore, the aforementioned tetrahedral DNA / fluoxetine complex is a complex obtained by mixing and incubating fluoxetine or a salt thereof with tetrahedral DNA in a solvent;

[0009] During incubation, the molar mass ratio of fluoxetine or its salt to tetrahedral DNA is 1 mol: 500-1000 g.

[0010] Furthermore, during incubation, the molar mass ratio of fluoxetine or its salt to tetrahedral DNA is 1 mol:500 g;

[0011] and / or, the solvent is water;

[0012] And / or, the incubation temperature is 20-40° C., and the incubation time is 5-10 h.

[0013] Furthermore, the salt of fluoxetine is hydrochloride.

[0014] The structure of fluoxetine hydrochloride is

[0015] Furthermore, the tetrahedral DNA is synthesized by self-assembly of four DNA single strands; the sequences of the four DNA single strands are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively.

[0016] Furthermore, the method for synthesizing tetrahedral DNA comprises the following steps: adding four DNA single strands into TM buffer, maintaining at 95° C. for 10 min, annealing to 4° C. for 20 min, and obtaining tetrahedral DNA;

[0017] Preferably, the four DNA single strands are four DNA single strands in equimolar ratio.

[0018] Furthermore, the preparation method of the TM buffer comprises the following steps: dissolving Tris-HCl and MgCl2·6H2O in ultrapure water, mixing evenly, and adjusting the pH value to 8; the concentration of Tris-HCl in the TM buffer is 10 mM, and the concentration of MgCl2·6H2O is 50 mM.

[0019] The present invention also provides a method for preparing the aforementioned tetrahedral DNA / fluoxetine complex, which comprises the following steps:

[0020] The mixture is prepared by incubating fluoxetine or its salt with tetrahedral DNA in a solvent;

[0021] Preferably, the solvent is water;

[0022] And / or, the incubation temperature is 20-40° C., and the incubation time is 5-10 h.

[0023] The present invention also provides use of the aforementioned tetrahedral DNA / fluoxetine complex in preparing a drug for preventing and / or treating depression.

[0024] The present invention also provides a medicine, which is a pharmaceutical preparation prepared by taking the aforementioned tetrahedral DNA / fluoxetine complex as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The complex prepared by carrying fluoxetine hydrochloride on tetrahedral DNA nanostructures can more efficiently pass through the blood-brain barrier, increase the distribution rate of fluoxetine hydrochloride in brain tissue, accelerate the onset time, and achieve an early antidepressant effect.

[0027] 2. While delivering the drug efficiently, the dosage of fluoxetine hydrochloride can be reduced to alleviate the adverse reactions related to drug dosage;

[0028] 3. Give full play to the biological effects of tetrahedral DNA nanomaterials such as anti-inflammatory, anti-oxidative stress, and immune regulation, combine multiple etiology hypotheses, and add new therapeutic targets for antidepressants such as neuroimmunity and neuroinflammation on the basis of the monoamine neurotransmitter hypothesis.

[0029] In summary, the present invention provides a tetrahedral DNA / fluoxetine hydrochloride complex that exhibits rapid onset of action in the treatment of depression and can exert the antidepressant effects of fluoxetine hydrochloride at low doses, significantly reducing medication-related side effects and enhancing medication compliance in patients with depression. The complex has excellent safety and effectiveness in treating depression, and has promising application prospects in the clinical treatment of depression.

[0030] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0031] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the synthesis of tetrahedral DNA nanostructures (TDNs); wherein, a is a schematic diagram of the synthesis of TDNs; b is a schematic diagram of the synthesis of Cy5 fluorescently labeled TDNs.

[0033] Figure 2 Schematic diagram of the synthesis of tetrahedral DNA / fluoxetine hydrochloride complex; wherein a is the chemical formula of fluoxetine hydrochloride; b is the complex formed by intercalation binding between fluoxetine and DNA; c is a schematic diagram of the synthesis of tetrahedral DNA / fluoxetine hydrochloride complex; d is a schematic diagram of the synthesis of Cy5 fluorescently labeled tetrahedral DNA / fluoxetine hydrochloride complex.

[0034] Figure 3 This is the result of polyacrylamide gel electrophoresis (PAGE).

[0035] Figure 4 Transmission electron microscopy results.

[0036] Figure 5 The UV absorption spectrum and Gel-Red fluorescence spectrum of the tetrahedral DNA / fluoxetine hydrochloride complex are shown in Figure 1. a is the UV absorption spectrum; b is the Gel-Red fluorescence spectrum.

[0037] Figure 6 This is the in vivo imaging result of tetrahedral DNA / fluoxetine hydrochloride complex.

[0038] Figure 7 CUMS stress type and modeling duration.

[0039] Figure 8 The following are the behavioral test results and statistical differences of each treatment group.

[0040] Figure 9 It is a comprehensive indicator of behavioral test results for each treatment group. DETAILED DESCRIPTION

[0041] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercial products.

[0042] In the present invention, unless otherwise specified, a solution refers to an aqueous solution.

[0043] Example 1. Preparation of Tetrahedral DNA / Fluoxetine Hydrochloride Complex

[0044] 1. Synthesis of Tetrahedral DNA Nanostructures (TDNs)

[0045] (1) Preparation of TM buffer

[0046] Add 0.605g Tris-HCl and 5.075g MgCl2·6H2O to 30ml of Milli-pure water and vortex thoroughly to mix. Prepare TM buffer (TM buffer has a Tris-HCl concentration of 10mM and a MgCl2·6H2O concentration of 50mM). Measure the pH of the mixed solution using a pH meter and adjust the pH of the mixed solution to 8.0 based on the measurement result. If the pH of the mixed solution is greater than 8.0, adjust the pH of the solution with HCl. If the pH is less than 8.0, adjust the pH of the solution with NaOH.

[0047] (2) Synthesis of TDNs

[0048] Based on the Watson-Crick principle of complementary base pairing, four 63-base DNA strands mechanically assemble into a stable TDN. Each DNA strand contains three block sequences that complement the other three strands. Through DNA hybridization, the four triangular DNA helices form a rigid tetrahedral structure. Each side of the TDN contains 20 base pairs, and every two edges have an oligonucleotide at their vertices, contributing to the TDN's toughness and flexibility. Detailed DNA sequence information is provided in Table 1.

[0049] Table 1. Detailed sequence information of single-stranded DNA required for TDNs synthesis

[0050]

[0051] like Figure 1 As shown, four presynthesized DNA single strands (S1, S2, S3, and S4) were mixed in TM buffer at equal molar ratios and then placed in a PCR instrument for self-assembly. The PCR temperature was set to rapidly increase to 95°C and hold for 10 minutes, followed by annealing to 4°C and cooling for 20 minutes. This successfully synthesized TDNs. For Cy5-labeled TDNs, the DNA single strand (S1) was replaced with S1-Cy5 and Cy5-labeled TDNs were prepared using the same method. The synthesized TDNs should be stored in a refrigerator at 4°C until ready for use.

[0052] 2. Preparation of Tetrahedral DNA / Fluoxetine Hydrochloride Complex

[0053] The specific preparation method of the tetrahedral DNA / fluoxetine hydrochloride complex comprises the following steps:

[0054] TDNs at a concentration of 1000 μM were incubated with a 500 μg / ml fluoxetine hydrochloride solution at room temperature for 6 hours with shaking to form a tetrahedral DNA nanostructure and fluoxetine hydrochloride-loaded complex (TDNs & Flu) solution.

[0055] The chemical structure of fluoxetine hydrochloride contains two benzene ring structures, such as Figure 2 a; Therefore, it has a certain planar structure in space. When these small organic molecules bind to nucleic acids, they will choose to bind non-covalently in an intercalation manner and specifically embed between two stacked base pairs in the double helix structure of the DNA molecule, such as Figure 2 b. The binding force between fluoxetine hydrochloride and DNA comes from the π-π conjugation and hydrophobic interaction between the delocalized π system of the fused ring and the π system of the base pair.

[0056] The beneficial effects of the present invention are demonstrated below through specific test examples.

[0057] Experimental Example 1: Material Characterization of Tetrahedral DNA / Fluoxetine Hydrochloride Complex

[0058] To verify the successful preparation of the tetrahedral DNA / fluoxetine hydrochloride complex (TDNs & Flu complex), the present invention used 8% native polyacrylamide gel electrophoresis (PAGE) to observe the presence of corresponding bands above and below the DNA marker line of similar molecular weight. Furthermore, to further observe the morphology and size of the TDNs, the present invention performed transmission electron microscopy scanning on the TDNs and the TDNs & Flu complex. Furthermore, the present invention used changes in absorbance from ultraviolet-visible absorption spectroscopy (UV-Vis) and Gel-Red fluorescence spectroscopy to further characterize the successful preparation of the TDNs & Flu complex.

[0059] 1. Polyacrylamide gel electrophoresis (PAGE) characterization of TDNs and TDNs&Flu complexes

[0060] TDNs were synthesized according to the method of Example 1, and TDNs & Flu complex was constructed. Equimolar concentrations of DNA single strands (S1-S4), synthesized TDNs, and TDNs & Flu complex were added to the sample wells and run by gel electrophoresis. The results are shown in Figure 1. Figure 3 shown.

[0061] Figure 3 The outermost lanes on the left and right sides are DNA markers. The band in lane 1 represents single-stranded DNA S1, the band in lane 2 represents single-stranded DNA S2, the band in lane 3 represents single-stranded DNA S3, and the band in lane 4 represents single-stranded DNA S4. Lane 5 represents the synthesized TDNs, and lane 6 represents the loaded TDNs & Flu complex. Each single-stranded DNA strand ranges from 40 to 60 bp, while the TDNs correspond to approximately 200 bp. The PAGE gel electrophoresis results demonstrate the successful construction of the TDNs and TDNs & Flu complex.

[0062] 2. Characterization of the micromorphology and size of TDNs and TDNs&Flu complexes

[0063] According to the scale bar of TEM, the sizes of the TDNs and TDNs & Flu complex prepared in Example 1 were measured using Image J software. Figure 4 Among the TDNs, the triangles show the microscopic morphology of the synthesized TDNs, and the size of the TDNs is about 17-18 nm. Figure 4The circle inside the TDNs&Flu sample shows the microscopic morphology of the synthesized TDNs&Flu complex, which is approximately 20 to 22.5 nm in size. These experimental results demonstrate the changes in morphology and particle size of TDNs before and after loading with fluoxetine hydrochloride, demonstrating the successful construction of the TDNs-Flu complex.

[0064] 3. Characterization results of UV-visible absorption spectra of TDNs and TDNs&Flu complexes

[0065] The binding of organic small molecules to DNA will change the original photochemical characteristics of both. Based on this characteristic, the present invention characterized the loaded TDNs & Flu complex (prepared in Example 1) by UV-visible absorption spectroscopy.

[0066] from Figure 5 As can be seen in a, under ultraviolet light irradiation, fluoxetine hydrochloride has an obvious ultraviolet absorption peak at 227nm ( Figure 5 a curve marked as Flu), pure TDNs in the UV spectrum, the UV absorption peak is at 280nm ( Figure 5 a) The curve marked as TDNs. After TDNs were loaded with fluoxetine to form a TDNs & Flu complex, the properties of its UV spectrum changed, and the maximum absorption peak deviated from the original position of TDNs and Flu ( Figure 5 a) The absorption intensity decreases, and a hypochromic effect appears, indicating the successful preparation of the TDNs & Flu complex.

[0067] 4. Characterization results of Gel-Red fluorescence spectra of TDNs and TDNs&Flu complex

[0068] The nucleic acid dyes Gel-Red and Flu compete with each other when binding to TDNs. Based on this characteristic, the present invention performed Gel-Red staining on TDNs (prepared in Example 1) before and after loading with Flu, and analyzed the differences in Gel-Red binding using fluorescence spectroscopy.

[0069] from Figure 5 As can be seen in b, the nucleic acid dye Gel-Red is only bound to TDNs ( Figure 5 b) The curve marked as TDNs) has a fluorescence absorbance of >35 A.U. After the formation of the TDNs & Flu complex, the fluorescence absorbance of the complex decreases significantly due to Flu occupying the binding site of Gel-Red and DNA, and is approximately between 10 and 15 A.U. ( Figure 5 b) Curve labeled as TDNs & Flu. This indicates the successful preparation of the TDNs & Flu complex.

[0070] 5. In vivo imaging results of brain tissue distribution of TDNs & Flu complex

[0071] The in vivo imaging experimental method for the distribution of TDNs & Flu complex in brain tissue includes the following steps:

[0072] ① Place BALB / c nude mice in an anesthesia induction box, introduce isoflurane at 300-500 ml / min, and observe the activity and status of the mice.

[0073] ② Immerse the tail of the anesthetized nude mouse in 40 degrees Celsius warm water for 15 seconds to promote filling of the tail vein to facilitate tail vein injection.

[0074] ③Use 75% alcohol solution to disinfect the mouse tail and fix the nude mouse on the tail vein injection stand.

[0075] ④ Administer 100 μL of a Cy5 fluorescently labeled TDNs & Flu complex solution containing 200 μg / ml fluoxetine hydrochloride into the tail vein.

[0076] Observe the status of TDNs&Flu complex in mice at different time points.

[0077] from Figure 6 The results of in vivo imaging of the back of BALB / c nude mice show that starting from the 5th minute after the tail vein injection of 0.1 ml of the Cy5-labeled TDNs&Flu complex prepared in Example 1, the fluorescently labeled TDNs&Flu complex was already present in the mouse brain tissue; starting from the 10th minute, the fluorescence intensity in the mouse brain tissue further increased; and 15-20 minutes after the administration, the fluorescence intensity and range in the brain tissue increased significantly; as the administration time prolonged, the fluorescence in the mouse body weakened slightly, and at 40 minutes after the administration, the fluorescence intensity in the brain tissue was still higher than that in other tissues and organs.

[0078] 40 minutes after the administration, the anesthetized mice were dissected, and the heart, spleen, lungs, liver, kidneys and whole brain of the mice were separated and fluorescent imaging was performed again. Figure 6 It can be seen that no Cy5-labeled TDNs&Flu complex was found in the mouse heart, spleen, and lungs; while Cy5-labeled TDNs&Flu complex was found in the liver and kidneys, indicating that it was metabolized in the liver and excreted through the kidneys; the anatomically separated brain tissue still showed a higher fluorescence intensity than other tissues and organs.

[0079] Combined with the above results, the Cy5-labeled TDNs&Flu complex showed higher fluorescence intensity in brain tissue than in other tissues and organs in both in vivo imaging and isolated organs, indicating that it has a good distribution effect in brain tissue.

[0080] Experimental Example 2: Study on the Treatment of Depression with Tetrahedral DNA / Fluoxetine Hydrochloride Complex

[0081] 1. Establishing a mouse depression-like model using chronic unpredictable mild stimulation

[0082] The Chronic Unpredicted Mild Stress (CUMS) depression model is based on and improves the experimental paradigm of the Chronic Mild Stress (CMS) model designed by Katz and colleagues in the early 1980s. The currently used experimental paradigm prolongs the duration of stress stimulation and increases the uncertainty of stress stimulation to avoid the model animals from adapting to the continuous modeling overstimulation (such as Figure 7 ).

[0083] The CUMS modeling paradigm exposes experimental animals to a series of mild stress stimuli in an unpredictable manner over a period of time, more closely mimicking various stressful life events in human daily life. These stimuli involve the experimental animals' feeding behavior, biological rhythms, living environment, social relationships, and other stressors that can cause behavioral changes such as fear and avoidance in experimental animals. In the CUMS modeling paradigm, the duration of stimulation needs to last for several weeks or even months (at least 2 weeks). The advantage of this depression model is that the application of long-term chronic unpredictable stress can cause changes in the behavior, neurochemical, neuroimmunological, neuroendocrine and neuroanatomical aspects of experimental animals, ultimately showing neurological dysfunction similar to that observed in patients with depression.

[0084] Please refer to Table 2 for the operation method of the CUMS stressor used in this experiment, and the specific CUMS arrangement is shown in Table 3.

[0085] Table 2. Types and methods of chronic unpredictable mild stimulation stressors

[0086]

[0087]

[0088] Table 3. CUMS modeling arrangement

[0089]

[0090] 2. Group treatment and medication regimen

[0091] The CUMS modeling group was randomly divided into 4 groups. For the convenience of description, English abbreviations are used below to represent the groups. Control-Buffer is the normal control-TM buffer treatment group; Case-Buffer is the CUMS modeling-TM buffer treatment group; Case-TDNs&Flu is the CUMS modeling-TDNs&Flu complex treatment group; Case-TDNs is the CUMS modeling-TDNs treatment group; Case-Flu is the CUMS modeling-Flu treatment group. The body weight was weighed at 9 o'clock every morning and the drug was administered once a day for 14 consecutive days. The concentration and dosage of each treatment group are shown in Table 4. The TDNs and TDNs&Flu complex used in this example were prepared in Example 1.

[0092] Table 4. Treatment groups and medication

[0093]

[0094] 3. Behavioral testing

[0095] According to relevant research literature (LMD Carvalho, Chen WY, Lasek A W. Epigenetic mechanisms underlying stress-induced depression [J]. International Review of Neurobiology, 2020.) and the DSM-5 diagnostic manual, the present invention finally selected 5 behavioral tests, including depression-related indicators, physiological indicators, and anxiety-related indicators, as shown in Table 5.

[0096] Table 5. Behavioral phenotypes selected in this study and their related endophenotypes

[0097]

[0098] (1) Body Weight Measurement

[0099] Body weight, as a physiological indicator, is closely linked to an individual's emotional and physical state. Depression and disturbed eating habits in mice in the CUMS model group can lead to slow or even no weight gain. Therefore, dynamic changes in body weight can provide valuable information on the effectiveness of the CUMS model and the efficacy of drug interventions.

[0100] The body weights of all mice were recorded starting on the first day of the CUMS modeling phase and continuing until the last day of the scheduled treatment phase. The average body weight of each mouse per week and the mean of the average body weights of all mice in each treatment group were calculated. Statistical comparisons were performed at the fifth week of modeling (before treatment) and at the treatment endpoint.

[0101] (2) Sucrose Preference Test (SPT)

[0102] The sucrose preference test is based on the natural preference of rodents for sweets. It is usually necessary to record the mass of sucrose solution consumed by mice within a fixed time. Before the experiment begins, the mice need to be adapted to the corresponding experimental test conditions (such as single cage housing, starting the test at night, and having two drinking bottles to choose from). The sucrose preference experiment is used to test the anhedonia symptoms of mice. Anhedonia, as one of the core symptoms of depression, is manifested as a loss of interest in most activities and a reduction in the pleasure brought by the activities. In depressive-like mice, it manifests as a loss of the inner drive to obtain sugar water and a reduction in the consumption of sugar water. The specific operation of the sucrose preference test in this experiment is as follows:

[0103] ①Sugar water adaptation stage:

[0104] The test mice were separated into cages and housed individually, and sufficient 50 mL centrifuge tubes were prepared.

[0105] Days 1-2: Because rodents tend to eat and move at night, the acclimation experiment began at 8:00 PM that evening. Two centrifuge tubes were pre-filled with 30 mL of 2% sucrose solution, designated S1 and S2. After 12 hours, the tubes were swapped and acclimation continued. Ensure adequate feed.

[0106] Days 2-3: Before the experiment begins, fill one centrifuge tube (W1) with 30 mL of purified water and the other tube (S1) with 30 mL of 2% sucrose solution. Check the stoppers for leaks. Acclimation begins at 8:00 PM. After 12 hours, swap the two tubes and continue acclimation. Ensure adequate feed throughout this period.

[0107] Day 3-4: Starting at 20:00 at night, remove the two centrifuge tubes and feed, and fast for 24 hours.

[0108] ②Sugar water testing stage:

[0109] Days 4-5: After a 24-hour fast, weigh and record the weights of the 30 ml 2% sucrose solution and purified water (W1 / S1) administered to the animals. Place the animals in the cage and add ample feed. After 12 hours, weigh and record the weights of the W1 / S1 pairs and swap their positions. After 24 hours, weigh and record the weights of the sucrose solution and purified water again.

[0110] The sucrose solution and purified water consumption of each mouse was calculated based on the weighing data at three time points: before the test (0 h), 12 hours after the test (12 h), and 24 hours after the test (24 h). The sucrose preference rate (%) of each mouse was calculated using the following formula:

[0111]

[0112] (3) Open Field Test (OFT)

[0113] The open field test is a classic behavioral test used to assess mice's spontaneous activity, anxiety, and stress in unfamiliar environments. The experimental principle is based on the universal haptaxis of rodents, which indicates that in unfamiliar environments, rodents exhibit both fear of open spaces and avoidance behaviors that encourage exploration of new environments and objects.

[0114] During the experiment, the horizontal movement trajectory of the mouse was recorded by the tracking system and the following indicators were statistically analyzed: total distance moved, movement speed, frequency of entering the central area, time spent in the central area, etc. The specific experimental procedures are as follows:

[0115] ① Before the experiment, mice were placed in the behavioral testing room in advance and allowed to acclimate to the testing environment for 1 hour. The lighting intensity in the behavioral testing room was adjusted to approximately 60 lumens. For the open field apparatus (a cube 40 cm high and 50 cm long), a geometric grid was preloaded on the computer screen to define the area of ​​each box as a central area (a square with a side length of 25 cm) and an edge area.

[0116] ② At the beginning of the experiment, place the mice in the lower left corner of the open field apparatus with their backs facing the operator. Record the activity of each mouse within 10 minutes. During the experiment, the test environment must be kept quiet, and the tester must avoid moving around to reduce confounding factors such as sound.

[0117] ③ After the experiment, record the mice's defecation and urine in the open field, clean the mice's feces and urine, and wipe the open field with 75% alcohol. Wait until the alcohol evaporates before conducting the next mouse experiment.

[0118] ④ Record the total distance moved by the mice within 10 minutes, average speed, frequency of entry into the central area, latency of the first entry, cumulative time in the central area, and defecation and urination.

[0119] (4) Tail Suspension Test (TST)

[0120] The tail suspension test is based on the observation of the rodent's initial escape behavior and subsequent immobility when it is suspended from its tail, unable to escape its environment. This easily recognizable immobility, when the mouse realizes it cannot escape and gives up, is described as "desperate" behavior. The specific procedure is as follows:

[0121] ① Using a loop with medical tape, secure the mouse approximately 1.5 cm from the distal end of the tail, then hang it head downward, approximately 20 cm above the tabletop. Each mouse was suspended for 6 minutes, with a camera recording its movements.

[0122] ② During the last 4 minutes, the immobility time of the mice was recorded by an investigator who was blinded to the treatment;

[0123] ③ After the test is completed, the excrement produced by the mice during the tail suspension process must be cleaned up and the mice must be placed in new cages to avoid contact with the mice not tested.

[0124] (5) Forced Swimming Test (FST)

[0125] The forced swim test assesses rodents' desperation behavior by observing their response to the threat of drowning. The animal is placed in a small container and forced to swim. Initially, the animal will struggle to escape, but when the struggle fails, it will give up and remain afloat. The time it takes the mouse to struggle out of the water and remain afloat can be used to assess depressive behavior and the effectiveness of antidepressant medication. The procedure is as follows:

[0126] ① Place the mice in a transparent glass cylinder (20 cm high, 12 cm in diameter) and fill it with 10 cm of water. The water temperature is set at 25±1 degrees Celsius. During the experiment, the mice are placed in the center of the water in the container for 6 minutes, and the experiment is recorded by a camera.

[0127] ② To avoid the influence of factors such as the excretion of feces and urine by the previous test mouse in the water and changes in water temperature, after the test of the previous mouse is completed, replace the water in the test container with clean water of similar temperature before conducting the next animal experiment.

[0128] ③ During the last 4 minutes, the duration of immobility was recorded by an investigator who was blinded to the treatment. A mouse was judged to be immobile when it remained on the water surface without any movement other than that necessary to keep its nose above water.

[0129] ④ During the test, the test conditions of 4 mice were recorded simultaneously, and partitions were used to block the field of view between each other to avoid mutual influence through vision and hearing.

[0130] ⑤ Place the tested mice in a new cage to avoid contact with untested mice. Use a paper towel to wipe off excess moisture from the mice's hair to prevent hypothermia.

[0131] Body weight measurement started on the first day of modeling and continued until the end of treatment; sucrose preference test was performed on the first day after treatment; open field test was performed on the second day after treatment; tail suspension test was performed on the third day after treatment; and forced swimming test was performed on the fifth day after treatment.

[0132] according to Figure 8 、 9 As shown, the Control-Buffer group showed statistically significant differences compared to the Case-Buffer group in all behavioral tests. The Case-TDNs & Flu group showed statistically significant differences in four behavioral tests (compared to the Case-Buffer group); the Case-TDNs group showed statistically significant differences in two behavioral tests (compared to the Case-Buffer group); and the Case-Flu group showed statistically significant differences in only one behavioral test (compared to the Case-Buffer group).

[0133] The Case-TDNs & Flu group showed significant antidepressant effects (with statistically significant differences) compared to the Case-Buffer group in the sucrose preference test (% sucrose consumption), open field test (defecation test), tail suspension test (immobility time), and forced swim test (immobility time). However, such changes were not apparent in the Case-Flu group. Flu treatment alone showed statistically significant differences compared to the untreated group only in the tail suspension test. In addition, after 2 weeks of treatment, the Case-TDNs group showed statistically significant therapeutic effects compared to the Case-Buffer group only in the sucrose preference test and forced swim test. Regarding the evaluation of the effects of antidepressant drugs, relevant literature suggests that at least significant results must be met in the sucrose preference test, tail suspension test, and forced swim test in order to be considered an effective antidepressant treatment. Based on this, the present invention obtained the following results: the Case-TDNs & Flu group showed an improvement in the depressive-like symptoms of CUMS mice after 2 weeks of antidepressant treatment, while the Case-TDNs group and the Case-Flu group failed to show ideal antidepressant effects.

[0134] These results suggest that, despite significant differences in individual behavioral tests, two weeks of TDNs or Flu alone in CUMS mice failed to produce an effective antidepressant effect. However, the composites exhibited a synergistic antidepressant effect, demonstrating significant therapeutic effects across four behavioral dimensions. Therefore, the material-drug complex exhibits a promising antidepressant advantage.

[0135] The Case-TDNs & Flu group showed significant antidepressant effects at two weeks, achieving an earlier onset of efficacy compared to fluoxetine alone. Furthermore, since the TDNs-loaded Flu concentration was 200 μg / kg, lower than the 1000 μg / kg used in conventional treatment, it still demonstrated significant antidepressant effects, potentially improving dose-related side effects experienced by patients during clinical medication use. This approach has promising applications in enhancing medication compliance among patients with depression.

[0136] In summary, the present invention provides a tetrahedral DNA / fluoxetine hydrochloride complex. This complex exhibits rapid onset of action when prepared for depression and can exert the antidepressant effects of fluoxetine hydrochloride at low doses, significantly reducing medication-related side effects and enhancing medication compliance in patients with depression. The complex exhibits excellent safety and efficacy in treating depression, and has promising application prospects in the clinical treatment of depression.

Claims

1. A tetrahedral DNA / fluoxetine complex for preventing and / or treating depression, characterized in that: It is a complex obtained by mixing and incubating fluoxetine or its salt with tetrahedral DNA in a solvent; During incubation, the molar mass ratio of fluoxetine or its salt to tetrahedral DNA is 1 mol: 500-1000 g; The tetrahedral DNA is synthesized by self-assembly of four DNA single strands; the sequences of the four DNA single strands are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively.

2. The tetrahedral DNA / fluoxetine complex according to claim 1, characterized in that: During incubation, the molar mass ratio of fluoxetine or its salt to tetrahedral DNA is 1 mol:500 g; and / or, the solvent is water; And / or, the incubation temperature is 20-40° C., and the incubation time is 5-10 h.

3. The tetrahedral DNA / fluoxetine complex according to claim 1 or 2, characterized in that: The salt of fluoxetine is hydrochloride.

4. The tetrahedral DNA / fluoxetine complex according to claim 1, characterized in that: The method for synthesizing tetrahedral DNA comprises the following steps: adding four DNA single strands into TM buffer, maintaining at 95° C. for 10 minutes, and annealing to 4° C. for 20 minutes to obtain the tetrahedral DNA.

5. The tetrahedral DNA / fluoxetine complex according to claim 4, characterized in that: The four DNA single strands are four DNA single strands in equal molar ratio.

6. The tetrahedral DNA / fluoxetine complex according to claim 5, characterized in that: The preparation method of the TM buffer comprises the following steps: dissolving Tris-HCl and MgCl2·6H2O in ultrapure water, mixing uniformly, and adjusting the pH value to 8; the concentration of Tris-HCl in the TM buffer is 10 mM, and the concentration of MgCl2·6H2O is 50 mM.

7. The method for preparing the tetrahedral DNA / fluoxetine complex according to any one of claims 1 to 6, characterized in that: It includes the following steps: The product is obtained by mixing fluoxetine or its salt with tetrahedral DNA in a solvent and incubating the mixture.

8. The preparation method according to claim 7, characterized in that: The solvent is water; And / or, the incubation temperature is 20-40° C., and the incubation time is 5-10 h.

9. Use of the tetrahedral DNA / fluoxetine complex according to any one of claims 1 to 6 in the preparation of a medicament for preventing and / or treating depression.

10. A drug for preventing and / or treating depression, characterized in that: The invention relates to a pharmaceutical preparation prepared by taking the tetrahedral DNA / fluoxetine complex described in any one of claims 1 to 6 as an active ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

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