CN117679527A - Tetrahedron DNA/fluoxetine hydrochloride compound, preparation method thereof and application thereof in treating depression - Google Patents
Tetrahedron DNA/fluoxetine hydrochloride compound, preparation method thereof and application thereof in treating depression Download PDFInfo
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Abstract
The invention provides a tetrahedron DNA/fluoxetine hydrochloride compound, a preparation method thereof and application thereof in treating depression, and relates to the technical field of medicines. The tetrahedral DNA/fluoxetine complex is a complex formed by embedding fluoxetine or salt thereof between base pairs in a duplex structure of a tetrahedral DNA molecule through a non-covalent bonding mode. The compound has quick response time in treating depression, can exert the antidepressant effect of fluoxetine hydrochloride at low dosage, obviously reduces the related side effects generated in the process of medication, and can enhance the medication compliance of patients with depression. The compound has good safety and effectiveness in treating depression, and has good application prospect in clinic treatment of depression.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a tetrahedron DNA/fluoxetine hydrochloride compound, a preparation method thereof and application thereof in treating depression.
Background
In the relevant diagnosis and treatment guidelines for depression, antidepressant drug treatment is the main treatment means. 5-hydroxytryptamine reuptake inhibitors (SSRIs) are a widely used class of antidepressant drugs in clinic, which improve monoamine neurotransmitter insufficiency by increasing the concentration of 5-hydroxytryptamine in the synaptic cleft, thereby exerting an antidepressant effect. Wherein, fluoxetine hydrochloride is used as a selective 5-hydroxytryptamine reuptake inhibitor, which is one of the current clinical first-line antidepressant drugs. As a conventional antidepressant drug, fluoxetine hydrochloride has the common problems of the conventional antidepressant drug: (1) onset time delay: due to the Blood Brain Barrier (BBB) effect of the central nervous system, the traditional antidepressant can take a long time to reach the effective therapeutic concentration of the drug in brain tissues, so that the risk of self-injury and suicide of depression patients is obviously increased, and social and family burdens are increased; (2) significant drug dose related side effects: when the traditional antidepressant drug is used for treatment, when the treatment effect of a patient is not obvious, the related treatment guidelines recommend increasing the use dose of the antidepressant drug, and the side effect related to the drug dose is associated with the use dose, so that the patient suffering from depression has adverse reaction of taking medicine, and the adverse reaction of fluoxetine hydrochloride mainly appears as follows: general numbness, hepatitis, sinus bradycardia, leukopenia, alopecia, etc.; (3) low medication compliance: the traditional antidepressant drug has delayed onset and dose-related side effects, so that partial patients can stop drug automatically due to incapability of seeing the treatment effect or obvious adverse reaction of the drug, the treatment compliance of the patients is low, and the treatment effect is influenced; (4) limited drug action targets: depression is a kind of heterogeneous disease with complex etiology mechanism, which relates to the hypothesis of various etiologies, and factors such as monoamine neurotransmitter insufficiency, neuroimmunity, neuroinflammation, neuroendocrine and the like are interwoven in the disease onset and progress, and the traditional antidepressant drugs only act on the monoamine neurotransmitter system, so that some patients do not respond to the traditional antidepressant drugs in clinical treatment. The problems cause that fluoxetine hydrochloride cannot exert efficacy in time when being used for treating depression, and the treatment effect is limited.
In order to break through the short board of the clinical application of the traditional antidepressant, the purpose of clinical treatment of the patient suffering from depression is better helped, and the social economy and personal burden are lightened. Nanomaterial drug delivery systems are receiving increasing attention. Among them, tetrahedral DNA nanostructures (Tetrahedral DNA nanostructures, TDNs) are a very potential drug delivery system in emerging nanomaterials. TDNs are formed from 4 single-stranded DNA strands by interchain base complementary pairing, morphologically resembling tetrahedra. The nanostructure has high synthesis efficiency, simple synthesis steps and good biological safety and biocompatibility. Advantages of TDNs include negligible immunogenicity, natural biocompatibility, structural stability and unparalleled programmability, which are a prerequisite for effective drug carriers. Meanwhile, the research shows that the TDNs have certain biological activity, such as anti-inflammatory effect, potential effect of improving neurodegenerative diseases such as Parkinson disease, alzheimer disease and the like. TDNs are therefore an important drug carrier.
However, since the TDNs have special structures and certain activities, it is unknown how the TDNs interact with the small molecule drugs to perform synergistic or antagonistic actions, and the effects of different small molecule drugs may be greatly different. At present, the DNA tetrahedron is not used for carrying fluoxetine hydrochloride, and whether the DNA tetrahedron can successfully carry fluoxetine hydrochloride or not ensures that the DNA tetrahedron plays a better effect needs to be further researched.
Disclosure of Invention
The invention aims to provide a tetrahedron DNA/fluoxetine hydrochloride compound, a preparation method thereof and application thereof in treating depression.
The invention provides a tetrahedral DNA/fluoxetine compound, which is a compound formed by embedding fluoxetine or salt thereof between base pairs in a duplex structure of a tetrahedral DNA molecule in a non-covalent bonding mode.
The fluoxetine has the structure that
Further, the aforementioned tetrahedral DNA/fluoxetine complex is a complex obtained by mixing fluoxetine or a salt thereof with tetrahedral DNA in a solvent and incubating;
during incubation, the molar mass ratio of the fluoxetine or salt thereof to the tetrahedral DNA is 1mol: 500-1000 g.
Further, upon incubation, the molar mass ratio of fluoxetine or salt thereof to tetrahedral DNA is 1mol:500g;
and/or the solvent is water;
and/or the incubation temperature is 20-40 ℃ and the incubation time is 5-10 h.
Further, the salt of fluoxetine is the hydrochloride.
The fluoxetine hydrochloride has the structure that
Further, the tetrahedral DNA is synthesized by self-assembly of four DNA single strands; the sequences of the four DNA single strands are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.
Further, the method for synthesizing tetrahedral DNA comprises the steps of: adding four DNA single strands into TM buffer, maintaining at 95deg.C for 10min, and annealing to 4deg.C for 20 min;
preferably, the four DNA single strands are four DNA single strands in equimolar ratio.
Further, the preparation method of the TM buffer solution comprises the following steps: tris-HCl and MgCl 2 ·6H 2 O is dissolved in ultrapure water, evenly mixed and the pH value is regulated to 8, thus obtaining the product; the concentration of Tris-HCl in the TM buffer is 10mM, mgCl 2 ·6H 2 The concentration of O is50mM。
The invention also provides a preparation method of the tetrahedron DNA/fluoxetine compound, which comprises the following steps:
mixing fluoxetine or salt thereof and tetrahedron DNA in a solvent for incubation to obtain the product;
preferably, the solvent is water;
and/or the incubation temperature is 20-40 ℃ and the incubation time is 5-10 h.
The invention also provides application of the tetrahedron DNA/fluoxetine compound in preparing a medicament for preventing and/or treating depression.
The invention also provides a medicine which is a medicine preparation prepared by taking the tetrahedron DNA/fluoxetine compound as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.
Compared with the prior art, the invention has the beneficial effects that:
1. the compound prepared by carrying fluoxetine hydrochloride on the tetrahedron DNA nano structure can pass through the blood brain barrier more efficiently, so that the distribution speed of fluoxetine hydrochloride in brain tissues is improved, the acting time is shortened, and the early anti-depression effect is achieved.
2. The drug administration dosage of fluoxetine hydrochloride can be reduced while the drug is efficiently delivered, so as to reduce adverse reactions related to the drug dosage;
3. the novel therapeutic target of antidepressants such as neuroimmunity, neuroinflammation and the like is added on the basis of monoamine neurotransmitter hypothesis by combining multiple etiology hypotheses through exerting the biological effects such as anti-inflammatory, anti-oxidative stress and immunity regulation of the tetrahedron DNA nanomaterial.
In summary, the invention provides a tetrahedron DNA/fluoxetine hydrochloride compound which has quick response time in treating depression, can exert the anti-depression effect of fluoxetine hydrochloride at low dosage, obviously reduces the related side effects generated in the process of medication, and can enhance the medication compliance of depression patients. The compound has good safety and effectiveness in treating depression, and has good application prospect in clinic treatment of depression.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 is a schematic diagram of Tetrahedral DNA Nanostructures (TDNs) synthesis; wherein a is a synthetic schematic diagram of TDNs; b is a schematic diagram of the synthesis of Cy5 fluorescent-labeled TDNs.
FIG. 2 is a schematic diagram of tetrahedral DNA/fluoxetine hydrochloride complex synthesis; wherein a is the chemical formula of fluoxetine hydrochloride; b is that fluoxetine and DNA form a complex in an insertion combination mode; c is a synthetic schematic diagram of tetrahedral DNA/fluoxetine hydrochloride complex; d is a schematic diagram of the synthesis of Cy5 fluorescence-labeled tetrahedron DNA/fluoxetine hydrochloride complex.
FIG. 3 is a diagram showing the result of polyacrylamide gel electrophoresis (PAGE).
Fig. 4 is a graph of transmission electron microscope results.
FIG. 5 shows the results of ultraviolet absorption spectrum and Gel-Red fluorescence spectrum of tetrahedral DNA/fluoxetine hydrochloride complex; wherein a is ultraviolet absorption spectrum; b is Gel-Red fluorescence spectrum.
FIG. 6 shows the results of in vivo imaging of tetrahedral DNA/fluoxetine hydrochloride complex.
FIG. 7 is a graph showing CUMS stress category and modeling duration.
Fig. 8 shows the results of the behavioral tests and the statistical differences for each treatment group.
Fig. 9 is a comprehensive index of the behavioral test results of each treatment group.
Detailed Description
The materials and equipment used in the embodiments of the present invention are all known products and are obtained by purchasing commercially available products.
In the present invention, the solution means an aqueous solution unless otherwise specified.
EXAMPLE 1 preparation of tetrahedral DNA/Fluoxetine hydrochloride Complex
1. Synthesis of tetrahedral DNA nucleic acid nanostructures (TDNs)
(1) Preparation of TM buffer
0.605g Tris-HCl and 5.075g MgCl 2 ·6H 2 O was added to 30ml of Milli-ultra-pure water, and the mixture was shaken well by shaking with a vortex mixer to prepare a TM buffer (Tris-HCl concentration in the TM buffer was 10mM, mgCl) 2 ·6H 2 O concentration was 50 mM). The pH of the mixed solution was measured by using a pH detector, and the preparation of the TM buffer was completed by adjusting the pH of the mixed solution to 8.0 according to the measurement result. If the pH value of the mixed solution is more than 8.0, HCl is used for adjusting the pH value of the solution, and if the pH value is less than 8.0, naOH is used for adjusting the pH value of the solution.
(2) Synthesis of TDNs
According to Watson-Crick base pairing theory, 4 DNA single strands 63 bases in length are mechanically assembled into a stable TDNs. Each DNA single strand contains 3 block sequences, which are complementary to the other 3 single strands. By DNA hybridization, 4 triangular DNA helices make up a rigid tetrahedral solid structure, each side of the TDNs contains 20 base pairs, with one oligonucleotide at the apex of each 2 sides, helping to enhance the toughness and flexibility of the TDNs. For detailed sequence information on single strands of DNA, refer to Table 1.
TABLE 1 detailed sequence information of Single-stranded DNA required for TDNs Synthesis
As shown in FIG. 1, four DNA single strands S1, S2, S3, S4 synthesized in advance were uniformly mixed in a TM buffer at the same molar ratio, and then put into a PCR apparatus for self-assembly preparation. The PCR temperature program is set to be quickly raised to 95 ℃ and kept for 10 minutes, and then the PCR temperature program is annealed to 4 ℃ and cooled for 20 minutes, so that the TDNs can be successfully synthesized. For TDNs requiring labeling of Cy5, it is necessary to replace the DNA single strand S1 with S1-Cy5, and then prepare Cy 5-labeled TDNs in the same manner. The synthesized TDNs were placed in a 4 degree celsius refrigerator for use.
2. Preparation of tetrahedral DNA/fluoxetine hydrochloride complex
The specific preparation method of the tetrahedral DNA/fluoxetine hydrochloride complex comprises the following steps:
TDNs at a concentration of 1000. Mu.M were incubated with a solution of fluoxetine hydrochloride at 500. Mu.g/ml for 6 hours with shaking at room temperature to form a solution of complexes of tetrahedral DNA nanostructures with fluoxetine hydrochloride (TDNs & Flu).
The chemical structure of fluoxetine hydrochloride contains 2 benzene ring structures, as shown in figure 2a; so that it has a certain planar structure in space. Such small organic molecules, when bound to nucleic acids, will be selected to bind non-covalently in an intercalating manner and to intercalate specifically between two stacked base pairs in the double helix structure of the DNA molecule, as shown in fig. 2b. The forces of fluoxetine hydrochloride binding to DNA result from pi-pi conjugation and hydrophobic interactions formed between the delocalized pi system of fused rings and the pi system of base pairs.
The beneficial effects of the present invention are demonstrated by specific test examples below.
Test example 1 characterization of tetrahedral DNA/Fluoxetine hydrochloride Complex material
To verify successful preparation of tetrahedral DNA/fluoxetine hydrochloride complex (TDNs & Flu complex), the present invention selects 8% non-denaturing polyacrylamide gel electrophoresis (PAGE) for running to see if the corresponding bands appear above and below dnagarker horizontal lines of similar molecular weight. Meanwhile, in order to further observe the morphology and the size of the TDNs, the invention performs transmission electron microscope scanning on the TDNs and the TDNs & Flu compound. In addition, the invention further characterizes the successful preparation of TDNs & Flu complexes by using ultraviolet-visible absorption spectrum (UV-Vis) and Gel-Red fluorescence spectrum absorbance changes.
1. Polyacrylamide gel electrophoresis (PAGE) characterization of TDNs and TDNs & Flu complexes
TDNs were synthesized and TDNs & Flu complexes were constructed according to the procedure of example 1. Each equimolar concentration of DNA single strand (S1 to S4), the synthesized TDNs and TDNs & Flu complex were added to the spot wells, and gel electrophoresis was performed to run the gel, and the results are shown in FIG. 3.
In FIG. 3, the outer-left lane and the outer-right lane are DNAMmarker, the band labeled lane 1 represents DNA single strand S1, the band labeled lane 2 represents DNA single strand S2, the band labeled lane 3 represents DNA single strand S3, the band labeled lane 4 represents DNA single strand S4, lane 5 represents synthesized TDNs, and lane 6 represents carried TDNs & Flu complex. Each single-stranded DNA has a corresponding range of 40-60 bp and TDNs has a corresponding range of about 200 bp. The successful construction of TDNs and TDNs & Flu can be demonstrated by a PAGE gel electrophoresis result diagram.
2. Characterization of the microscopic morphology and size of the TDNs and TDNs & Flu complexes
The sizes of the TDNs and TDNs & Flu complexes prepared in example 1 were measured using Image J software according to the TEM scale. In the TDNs of fig. 4, the triangle is in a microscopic form for synthesizing TDNs, and the TDNs has a size of about 17 to 18nm; in the TDNs & Flu of FIG. 4, the microscopic morphology of the synthesized TDNs & Flu complex is shown in a circle, and the TDNs & Flu size is about 20-22.5 nm. The experimental result shows the morphology and particle size change of the TDNs before and after carrying fluoxetine hydrochloride, and also shows the successful construction of the TDNs-Flu.
3. Characterization result of ultraviolet-visible absorption spectrum of TDNs and TDNs & Flu compound
The organic small molecule and DNA are combined to change the original photochemical characteristics of the organic small molecule and the DNA, and according to the characteristics, the ultraviolet-visible absorption spectrum characterization is carried out on the TDNs & Flu complex (prepared in example 1) after being carried.
As can be seen from fig. 5a, under the irradiation of ultraviolet light, a distinct ultraviolet absorption peak (curve marked Flu in fig. 5 a) is visible at 227nm, a mere TDNs is at 280nm (curve marked TDNs in fig. 5 a), after the TDNs is carried with fluoxetine to form a TDNs & Flu complex, the property of the ultraviolet spectrum is changed, the maximum absorption peak deviates from the positions of the original TDNs and Flu (curve marked TDNs & Flu in fig. 5 a), the absorption intensity is reduced, and a color reduction effect appears. Indicating successful preparation of the TDNs & Flu complex.
4. Characterization result of TDNs and TDNs & Flu complex Gel-Red fluorescence spectrum
According to the feature that the nucleic acid dyes Gel-Red and Flu have a competitive relationship when combined with each other, the invention performs Gel-Red staining on TDNs (prepared in example 1) before and after Flu is carried out, and adopts fluorescence spectrum to analyze the difference of Gel-Red combination.
As can be seen from FIG. 5b, the absorbance of fluorescence of the nucleic acid dye Gel-Red when bound by TDNs alone (FIG. 5b, curve labeled TDNs) is >35A.U., whereas after formation of the TDNs & Flu complex, the absorbance of fluorescence of the complex drops significantly, approximately between 10 and 15A.U., curve labeled TDNs & Flu, due to Flu occupying the binding site of Gel-Red to DNA. Indicating successful preparation of the TDNs & Flu complex.
5. TDNs & Flu complex brain tissue distribution in vivo imaging results
The TDNs & Flu complex brain tissue distribution living body imaging experimental method comprises the following steps:
(1) BALB/c nude mice are placed in an anesthesia induction box, isoflurane 300-500ml/min is introduced, and the activities and states of the mice are observed.
(2) The tail of the anesthetized nude mice is immersed in warm water at 40 ℃ for 15 seconds, and tail vein filling is promoted, so that tail vein injection is facilitated.
(3) Mice were sterilized at the tail using 75% alcohol solution and the nude mice were mounted on a tail vein injection rack.
(4) 100. Mu.L of Cy5 fluorescent-labeled TDNs & Flu complex solution with 200. Mu.g/ml fluoxetine hydrochloride was administered to the tail vein.
The TDNs & Flu complexes were observed in mice at different time points.
From the back in vivo imaging results of BALB/c nude mice of FIG. 6, it can be seen that the fluorescence-labeled TDNs & Flu complex is already present in brain tissue of the mice starting at 5 minutes after 0.1ml of the Cy 5-labeled TDNs & Flu complex prepared in example 1 was administered by tail vein injection; the fluorescence intensity in the brain tissue of the mice increased further by the beginning of 10 minutes; and after 15-20 minutes of administration, the fluorescence intensity and the range in brain tissues are obviously increased; with prolonged administration time, the in vivo fluorescence of mice is slightly reduced, and the mice can still display fluorescence intensity higher than other tissues and organs in brain tissues at 40 minutes of administration.
Anesthetized mice were dissected 40 minutes after dosing, hearts, spleens, lungs, livers, kidneys and whole brains of the mice were isolated and re-fluorescence imaged, as seen in fig. 6, with Cy 5-labeled TDNs & Flu complexes not seen in the hearts, spleens, lungs of the mice; whereas the Cy 5-labeled TDNs & Flu complex is visible in the liver and kidney, indicating its metabolism in the liver and excretion through the kidney; the dissected brain tissue still shows higher fluorescence intensity than other tissue organs.
In combination with the above results, the Cy 5-labeled TDNs & Flu complex shows higher fluorescence intensity in brain tissues than other tissue organs in living body imaging and isolated organs, thus showing better distribution effect in brain tissues.
Test example 2 study of tetrahedral DNA/Fluoxetine hydrochloride Complex for treatment of depression
1. Construction of mouse depression-like model using chronic unpredictable mild stimulation
The model of chronic unpredictable mild stimulation (Chronic Unpredicted Mild Stress, CUMS) depression is based on and improves on the model-related experimental paradigm of chronic mild stimulation (Chronic Mild Stress, CMS) designed by Katz and colleagues early in the 20 th century 80 years. The experimental paradigm currently applied extends the time and uncertainty of the stress stimulus, avoiding adaptation of the model animal in the sustained modeling overstimulation (see fig. 7).
The CUMS modeling paradigm exposes experimental animals to a range of mild stress stimuli in an unpredictable manner over a period of time, more closely mimicking the various stress life events in the daily life of humans. These stimuli relate to the ingestion behavior, biorhythm, living environment, social relationship, and other stressors that can cause behavioral changes such as fear and avoidance in the experimental animal. In the CUMS modeling paradigm, the duration of stimulus application takes weeks or even months (at least 2 weeks). The advantage of this model of depression is that the application of chronic, sustained and unpredictable stress causes changes in the behavior, neurobiochemistry, neuroimmunity, neuroendocrine and neuroanatomy of the experimental animal, ultimately exhibiting neurological dysfunction similar to that observed in depressed patients.
The method of operation of the CUMS stressor used in this experiment is shown in Table 2, with the specific CUMS arrangement shown in Table 3.
TABLE 2 type of stress source for chronically unpredictable mild stimulation and method of operation
TABLE 3 CUMS modeling arrangement
2. Grouping treatment and dosing regimen
The CUMS building modules are randomly divided into 4 groups, and for convenience of description, english shorthand representation grouping is used below, and Control-Buffer is a normal contrast-TM Buffer treatment group; case-Buffer is CUMS modeling-TM Buffer treatment group, case-TDNs & Flu is CUMS modeling-TDNs & Flu complex treatment group; case-TDNs are cure modeling-TDNs treatment group; case-Flu is the CUMS model-Flu treatment group. The weight of each treatment group was weighed 9 a day and administered 1 time a day for 14 days continuously, and the concentration and dose of each treatment group are shown in table 4. The TDNs and TDNs & Flu complexes used in this example were prepared for example 1.
TABLE 4 grouping of treatments and administration
3. Behavioural testing
According to the relevant research literature (LMD Carvalho, chen W Y, lasek A W.epigenetic mechanisms underlying stress-induced depression [ J ]. International Review of Neurobiology,2020 ]) and DSM-5 diagnostic manual, the invention finally selects 5 behavioural tests including depression emotion related index, physiological index, anxiety emotion related index, as shown in Table 5.
TABLE 5 selected behaviours and associated internal phenotypes for this study
(1) Body weight measurement (Body Weight Measurement)
Body weight is a physiological index that is closely related to the emotional and physical state of an individual. The CUMS model mice may have a slow or even no increase in body weight due to depressed mood and eating disorders. Therefore, the dynamic change of the body weight can provide the referential information such as CUMS model construction effect, medicine intervention effect and the like.
The experiment starts recording the body weights of all mice on the first day of the CUMS modeling phase until it completes the last day of the scheduled treatment phase. The average weekly weight of each mouse was calculated as the average of the average weight of all mice in each treatment group. The fifth week of modeling (pre-treatment) and treatment endpoint were selected for statistical comparison.
(2) Sucrose preference test (Sucrose Preference Test, SPT)
Sucrose preference tests are based on the natural nature of rodents' preference for confections. It is often desirable to record the mass of sucrose solution taken by mice over a fixed period of time. Before the experiment starts, the mice need to be adapted to the corresponding experimental test conditions (e.g. single cage rearing, beginning the test at night, 2 alternative drinking bottles, etc.). Sucrose preference experiments were used to test mice for symptoms of loss of hedonia. Loss of pleasure (Anhedonia) is one of the central symptoms of depression, manifested by loss of interest in most activities while a reduction in pleasure brought about by the activity. Loss of mental drive to gain sugar water and reduced consumption of sugar water were shown in depression-like mice. The sucrose preference test of this experiment is specifically operated as follows:
(1) and (3) a syrup adaptation stage:
test mice were caged, single-caged, and prepared for a sufficient quantity of 50mL centrifuge tubes.
Day 1-2: because rodents have the habit of feeding and activity at night, the adaptation experiment is started at 20:00 of the night on the same day, 30mL of 2% sucrose solution is respectively marked as (S1 and S2) in advance filled into the 2 branch off-tube, and the positions of the 2 branch off-tube are exchanged after 12 hours, so that the adaptation is continued. Ensuring the sufficiency of feed.
Day 2-3: before the experiment, one of the separation tubes (W1) is filled with 30mL of purified water, the other tube (S1) is filled with 30mL of 2% sucrose solution, and whether the bottle stopper is dripped or not is checked. Adaptation was started at 20:00 a.m. and after 12 hours the positions of the 2 support tubes were exchanged and continued. The period ensures that the feed is sufficient.
Day 3-4: at night 20:00, 2 branch off tubes and feed were removed, and no food was consumed for 24 hours.
(2) And (3) sugar water testing:
day 4-5: after 24 hours of fasting, 30ml of 2% sucrose solution and purified water, which were previously poured, were weighed and recorded (W1/S1), respectively, and then put into a rearing cage together, and sufficient feed was added. After 12 hours the W1/S1 was weighed, recorded and the positions exchanged, respectively. After 24 hours the weight of sucrose solution and purified water was again weighed and recorded.
The sucrose preference rate (%) of each mouse was calculated from the weight data of 3 time points before the test (0 h), after the test (12 h), after the test (24 h) to calculate the consumption of sucrose solution and purified water of each mouse,
(3) Open Field Test (OFT)
The open field test is a classical behavioral test that evaluates mice' spontaneous activity in unfamiliar environments and their anxiety and stress states. The experimental principle is based on the ubiquitous touch of rodents, namely, in strange environment, rodents have fear of spacious fields and exploring new environment and the evasive behavior of new things.
During the experiment, the horizontal movement track of the mice was recorded by the tracking system and the following indexes were statistically analyzed: total distance moved, speed moved, frequency entered into the central zone, residence time in the central zone, etc. The specific experimental operation is as follows:
(1) before the experiment, the mice are placed in a behavioural test room in advance, the test environment is adapted for 1 hour, the illumination intensity of the behavioural test room is adjusted to be about 60 lumens, and for open field devices (cubes with the height of 40 cm and the side length of 50 cm), the area of each frame is defined as a central area (square with the side length of 25 cm) and an edge area by a geometric grid which is needed to be preloaded on a computer screen.
(2) At the beginning of the experiment, the mice are uniformly placed into the lower left corner of the open field device back to the operator, the activity condition of each mouse within 10 minutes is recorded, the test environment is required to be kept quiet in the experiment, the testers avoid walking, and the noise and other confounding factors are reduced.
(3) After the experiment is finished, the condition of the mice in the open field for defecation and urine is recorded, the excrement and urine of the mice are removed, 75% alcohol is used for wiping the open field, and the next mouse experiment is carried out after the alcohol volatilizes.
(4) The total distance traveled by the mice over 10 minutes, average speed, frequency of entry into the central zone, latency of first entry, cumulative time in the central zone, and bowel movement were recorded.
(4) Tail suspension test (Tail Suspension Test TST)
The tail-suspension test is based on the inability of rodents to escape from the environment when they are suspended from the tail, and the hopeless behavior of depressed mice is assessed by observing the escape behavior they initially exhibit, and thereafter exhibit a state of immobility. This readily identifiable immobilized state is described as a "desperate" behavior when the mouse is aware of the inability to escape and give up. The specific operation is as follows:
(1) after the tail of the far end of the mouse is fixed by using a ring buckle stuck with medical adhesive tape, the head is hung downwards, and the hanging height is about 20cm away from the tabletop. Each mouse is hung for 6 minutes, and the movement state of the mouse is recorded by a camera;
(2) during the last 4 minutes, the immobility time of the mice was recorded by the investigator blinded to the treatment;
(3) after the test is completed, the excreta generated by the mice in the tail suspension process are cleaned, and the mice are placed in a new cage position to avoid contacting the mice to be tested.
(5) Forced swimming test (Forced Swimming Test FST)
Forced swimming tests evaluate the hopeless behavior of rodents by observing their response to a drowning threat, placing them in a narrow container to force them to swim, initially the animals struggle to try to escape, and after struggling is ineffective they will give up escaping, remaining in a floating state in the water. The treatment effect of the antidepressant is judged by calculating the time taken by the mice to struggle out water and float at rest. The specific operation is as follows:
(1) the mice were placed in a clear glass cylinder (20 cm high, 12 cm diameter) filled with 10 cm of water, the water temperature was set at 25.+ -. 1 ℃ and the mice were placed in the center of the water in the container for 6 minutes in the test and recorded by the camera.
(2) In order to avoid the influence of the factors such as excrement, urine and water temperature change of the previous test mouse in water, when the previous test of the mouse is finished, the next animal experiment is carried out after the water with the temperature close to that of the clean water in the test container is replaced.
(3) The time of immobility was recorded by a treatment-unaware researcher during the last 4 minutes. A mouse is judged to be stationary when it is resting on the water surface without any action other than that necessary to keep the nose exposed to the water surface.
(4) In the test process, the test conditions of 4 mice are recorded simultaneously, and the visual fields are blocked by using the partition boards, so that mutual influence of the two mice through audio-visual interaction is avoided.
(5) The mice after the test were placed in a new cage to avoid contact with the untested mice, and the excess moisture in the hair of the mice was wiped dry using paper towels to avoid hypothermia of the mice.
Weight measurement starts on day 1 of modeling and continues until treatment ends; sucrose preference test was performed on day 1 after the end of treatment; open field testing was performed on day 2 after treatment was completed; tail suspension testing was performed on day 3 after treatment was completed; forced swim testing was performed on day 5 after the end of treatment.
According to FIGS. 8 and 9, the Control-Buffer group showed statistical differences in all behavioural tests compared to the Case-Buffer group. The Case-TDNs & Flu group showed statistical differences in 4 behavioural tests (compared to the Case-Buffer group); the Case-TDNs group showed statistical differences in 2 behaviors (compared to Case-Buffer group); the Case-Flu group showed only differences in 1 behavioural test (compared to the Case-Buffer group).
The Case-TDNs & Flu group showed significant antidepressant effect (statistically different results) in both the sugar water preference test (percent sugar water consumption), the open field test (bowel movement test), the tail suspension test (immobility time) and the forced swimming test (immobility time) compared to the Case-Buffer group. However, such changes were not apparent in the Case-Flu group, and the single Flu treatment showed statistically significant differences only in the tail-suspension test in the untreated group. In addition, the Case-TDNs group showed a statistically different therapeutic effect in the sugar water preference test, forced swimming test, only, compared to the Case-Buffer group, after 2 weeks of administration of the treatment. For evaluation of antidepressant drug effects, relevant literature suggests that at least 3 significant results of sucrose preference test, tail suspension test and forced swimming test are required to be satisfied to consider antidepressant treatment effective; the following results were obtained according to the present invention: the Case-TDNs & Flu group showed an improvement effect on the depressive-like symptoms of the CUMS mice after 2 weeks of antidepressant treatment, whereas the Case-TDNs group and the Case-Flu group failed to show an ideal antidepressant effect.
The above results illustrate: treatment with TDNs or Flu alone for 2 weeks with CUMS mice, while there were significant differences in individual behavioural tests, the overall outcome failed to exert an effective antidepressant effect; the compound prepared into the composition has synergistic antidepressant effect, and has obvious therapeutic effect in 4-dimensional behavioural tests. The material-drug complex thus exhibits satisfactory antidepressant benefits.
The Case-TDNs & Flu groups all show obvious antidepressant effect in 2 weeks, and compared with the treatment of fluoxetine alone, the early onset of action is achieved. In addition, since the concentration of TDNs carried Flu is 200 mug/kg and is lower than that of the conventional treatment of 1000 mug/kg, the TDNs still have remarkable antidepressant effect, and are expected to improve the dose-related side effect generated by patients in the clinical medication process. The medicine taking compliance of depression patients is enhanced, and the application prospect is good.
In conclusion, the invention provides the tetrahedron DNA/fluoxetine hydrochloride compound, which has quick response time when being used for preparing depression, can exert the antidepressant effect of fluoxetine hydrochloride at low dosage, obviously reduces the related side effects generated in the process of medication, and can enhance the medication compliance of depression patients. The compound has good safety and effectiveness in treating depression, and has good application prospect in clinic treatment of depression.
Claims (10)
1. A tetrahedral DNA/fluoxetine complex, characterized in that: it is a complex formed by embedding fluoxetine or salt thereof between base pairs in a double-helix structure of a tetrahedral DNA molecule through a non-covalent binding mode.
2. The tetrahedral DNA/fluoxetine complex according to claim 1, wherein: the method is a compound obtained by mixing fluoxetine or salt thereof and tetrahedron DNA in a solvent for incubation;
during incubation, the molar mass ratio of the fluoxetine or salt thereof to the tetrahedral DNA is 1mol: 500-1000 g.
3. The tetrahedral DNA/fluoxetine complex according to claim 2, wherein: during incubation, the molar mass ratio of the fluoxetine or salt thereof to the tetrahedral DNA is 1mol:500g;
and/or the solvent is water;
and/or the incubation temperature is 20-40 ℃ and the incubation time is 5-10 h.
4. A tetrahedral DNA/fluoxetine complex according to any of claims 1 to 3, wherein: the fluoxetine salt is hydrochloride.
5. A tetrahedral DNA/fluoxetine complex according to any of claims 1 to 3, wherein: the tetrahedral DNA is synthesized by self-assembly of four DNA single strands; the sequences of the four DNA single strands are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.
6. The tetrahedral DNA/fluoxetine complex according to claim 5, wherein: the synthesis method of the tetrahedral DNA comprises the following steps: adding four DNA single strands into TM buffer, maintaining at 95deg.C for 10min, and annealing to 4deg.C for 20 min;
preferably, the four DNA single strands are four DNA single strands in equimolar ratio.
7. The tetrahedral DNA/fluoxetine complex according to claim 6, wherein: the preparation method of the TM buffer solution comprises the following steps: tris-HCl and MgCl 2 ·6H 2 O is dissolved in ultrapure water, evenly mixed and the pH value is regulated to 8, thus obtaining the product; the concentration of Tris-HCl in the TM buffer is 10mM, mgCl 2 ·6H 2 The concentration of O was 50mM.
8. The method for producing a tetrahedral DNA/fluoxetine complex according to any of claims 1 to 7, wherein: it comprises the following steps:
mixing fluoxetine or salt thereof and tetrahedron DNA in a solvent for incubation to obtain the product;
preferably, the solvent is water;
and/or the incubation temperature is 20-40 ℃ and the incubation time is 5-10 h.
9. Use of a tetrahedral DNA/fluoxetine complex according to any of claims 1 to 7 for the preparation of a medicament for the prevention and/or treatment of depression.
10. A medicament, characterized in that: the tetrahedron DNA/fluoxetine compound of any one of claims 1 to 7 is used as an active ingredient, and pharmaceutically acceptable auxiliary materials or auxiliary ingredients are added to prepare the pharmaceutical preparation.
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