A nano-preparation for delivering neurotransmitters and a preparation method and application thereof

Abstract

The application provides a nano preparation for efficiently delivering neurotransmitters and a preparation method and application thereof, and the nano preparation is prepared from therapeutic drugs mon 5-hydroxytryptamine and catalase, a protein crown is formed by surface adsorption of human serum albumin, and a nano anti-depression preparation is constructed by connecting a targeting molecule neurotropic virus derived peptide. The surface modification of RVG29 can realize the crossing of the BBB, and the surface modification of HSA can effectively inhibit non-specific protein adsorption and improve the brain targeting efficiency of the nano particles. The nano anti-depression preparation releases 5- HT through weak acid induced degradation under a depressive microenvironment. The increase of 5- HT can universally improve depressive symptoms, and the problem of low response rate of fluoxetine nano delivery system is solved. The CAT in the nano preparation can significantly reduce neuroinflammation and improve depressive symptoms; meanwhile, the CAT can prevent 5- HT from being oxidized and inactivated by ROS in neuroinflammation, improve the curative effect of 5- HT, and multi-target combined treatment of depression, so that the problem of single target of anti-depression drugs is solved.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials and nanobiomedicine, specifically relating to a nano-formulation for efficient delivery of neurotransmitters, its preparation method, and its application. Background Technology

[0002] Depression is a mental illness characterized by depressed mood, anhedonia, and slowed thinking. According to the World Health Organization, it is projected to become the most common mental illness globally by 2030. With further research, depression is no longer just a mood disorder, but a complex network of conditions accompanied by various pathological changes, such as an acidic brain pH, a deficiency of monoamine neurotransmitters, neuroinflammation, and neuronal reduction and damage. The occurrence of depression involves multiple brain regions related to emotion, behavior, and memory; for example, hippocampal atrophy is a common phenomenon in depressed patients.

[0003] Drug therapy remains the first-line treatment for depression. However, current first-line antidepressants have limitations due to their single target, including significant side effects, low response rates, slow onset of action, and poor efficacy. Studies show that 30%-50% of patients do not respond to their first use of antidepressants, and significant individual differences exist among most patients. In 2019, the US FDA approved esketamine for the treatment of treatment-resistant depression in adults. Esketamine is an isomer of ketamine, an N-methyl-d-aspartate receptor antagonist that can rapidly produce significant antidepressant effects. However, its widespread use is limited due to side effects such as hallucinations and coma. Therefore, developing antidepressants with novel targets and formulations is a key issue in the treatment of depression.

[0004] Currently, research on nano-antidepressants is limited. Most studies utilize nano-formulations co-loaded with fluoxetine to increase intracranial fluoxetine concentration and shorten onset time, often combined with photothermal therapy to enhance antidepressant efficacy. Additionally, some studies achieve antidepressant effects by reducing neuroinflammation. While these studies have successfully constructed nano-antidepressants, achieving the goals of shortening onset time and improving efficacy, several issues remain: 1. Overemphasis on the role of fluoxetine while neglecting its limitations, such as low response rate, high relapse rate, and poor efficacy. Furthermore, the use of photothermal technology to enhance antidepressant efficacy can easily raise intracranial temperature, posing serious safety risks. 2. The complexity of depression pathogenesis is still overlooked, resulting in a single therapeutic target. Therefore, developing nano-antidepressants with high response rates and multiple targets remains a critical unresolved issue.

[0005] 5-Hydroxytryptamine (5-HT), also known as serotonin, is a monoamine neurotransmitter. It is synthesized in central neurons and in the enterochromaffin cells of the digestive tract of animals (including humans) via tryptophan hydroxylase to form 5-hydroxytryptamine, which is then synthesized by 5-hydroxytryptamine decarboxylase. 5-HT is mainly found in the gastrointestinal tract, platelets, and central nervous system, and can induce feelings of happiness and pleasure. 2% of 5-HT is present in the brain, and 8%-9% is found in platelets. The majority is located in the mucosal enterochromaffin cells and nerve plexuses, participating in the regulation of intestinal peristalsis.

[0006] The blood-brain barrier (BBB) ​​is a selective barrier between blood vessels and the brain that prevents certain substances from entering the brain from the bloodstream. The cerebral vascular barrier allows almost no substances to pass through, except for oxygen, carbon dioxide, and blood glucose. Most drugs and proteins, due to their large molecular structures, generally cannot pass through. Enabling drugs to better cross the BBB and enter the brain parenchyma is a primary condition for treating all major brain diseases. The BBB is a physiological barrier system existing between the blood and brain tissue, mainly composed of brain capillary cells connected by tight junctions. It maintains a stable environment in the brain and protects the central nervous system, but it prevents 98% of small molecule drugs and almost all large molecule drugs from entering the brain parenchyma. Existing research has constructed nano-fluoxetine delivery systems to target and cross the blood-brain barrier (BBB), increasing the drug concentration in the brain and thus shortening the onset time to achieve antidepressant effects. However, this research overlooks the problems of low response rate and easy relapse associated with fluoxetine. Furthermore, some nano-antidepressant drugs achieve their antidepressant effects by alleviating neuroinflammation, but still suffer from the problem of single-target therapy. Summary of the Invention

[0007] To address the technical problems of slow onset and poor efficacy of existing drugs, this invention constructs a nano-antidepressant with high response rate and multiple targets. By modifying the surface of the nano-formulation, it can achieve targeted crossing of the blood-brain barrier, thereby increasing the concentration of antidepressant in the brain, shortening the onset time, and enhancing the therapeutic effect.

[0008] To achieve the above objectives, the present invention first provides a nano-formulation for efficient delivery of neurotransmitters, comprising serotonin, catalase, human serum albumin, and neurotropic virus-derived peptides. The serotonin polymerizes in a weakly alkaline environment to form covalent bonds. During the polymerization process, the serotonin is co-loaded with catalase to form core particles. Human serum albumin is adsorbed on the surface of the core particles to form a protein crown. Finally, the neurotropic virus-derived peptides are adsorbed on the surface of the core particles to construct the nano-formulation.

[0009] Preferably, the mass ratio of the mixture of 5-hydroxytryptamine and catalase is 8 to 10:1.

[0010] Based on a general inventive concept, the present invention also provides a method for preparing a nano-formulation for efficient delivery of neurotransmitters, comprising the following steps:

[0011] S1. Dissolve 5-hydroxytryptamine in water, add a weak alkaline buffer solution while stirring continuously, then add catalase to react, and then dialyze to remove unreacted raw materials to obtain core particles;

[0012] S2. The kernel particles obtained in step S1 adsorb human serum albumin in PBS, and the unadsorbed human serum albumin is removed by dialysis to obtain kernel particles adsorbed with human serum albumin.

[0013] S3. The core particles of human serum albumin obtained in step S2 are used to adsorb neurotropic virus-derived peptides in PBS. After dialysis to remove unreacted molecules, the nano-formulation is finally obtained by freeze-drying.

[0014] Preferably, the weakly alkaline buffer solution in step S1 is either Tris-HCl buffer solution with a pH of 9.5 or ammonia solution with a pH of 8.5-10.5.

[0015] Preferably, the reaction time in step S1 is 15–20 h, and the dialysis time is 4–6 h.

[0016] Preferably, the adsorption time in step S2 is 1–3 h, and the dialysis time is 3–6 h.

[0017] Preferably, the adsorption time in step S3 is 1-3 hours, and the dialysis time is 2-4 hours.

[0018] Based on a general inventive concept, the present invention also provides the application of a highly efficient neurotransmitter delivery nanoformulation in the preparation of a therapeutic agent for depression.

[0019] The principle behind this invention for treating depression is as follows:

[0020] The auto-oxidation of 5-HT initiates polymerization, leading to the formation of quinone radicals. Subsequently, the quinone radicals react further with the 5-HT parent molecule, generating dimer intermediates through intramolecular rearrangement. The amine groups present in these dimer intermediates can also react with the quinone intermediates to form imine structures. These imine structures exhibit sensitivity to degradation under acidic conditions. Therefore, the degradation process promotes the release of 5-HT.

[0021] Nanoparticles were prepared using the therapeutic drugs serotonin and catalase (CAT). Human serum albumin (HSA) was adsorbed onto the surface to form a protein crown, which was then linked to the neurotropic virus-derived peptide rabies virus glycoprotein 29 (RVG29) to construct a nano-antidepressant drug. The advantages of this nano-antidepressant drug include: 1) Surface modification of RVG29 enables cross-brain boundary (BBB) ​​targeting, while surface modification of HSA effectively inhibits non-specific protein adsorption, improving the brain-targeting efficiency of the nanoparticles; 2) Microenvironment-responsive degradation of the nanocarrier precisely replenishes monoamine neurotransmitters, increasing the levels of monoamine neurotransmitters in the brain and thus broadly improving depressive symptoms, thereby enhancing the responsiveness of the nano-antidepressant drug; 3) The internally loaded CAT improves neuroinflammation and depressive symptoms, while reducing the content of aerobic free radicals and preventing the oxidative inactivation of neurotransmitters, thus improving the efficacy of neurotransmitters. This will achieve multi-target combined therapy, thereby more effectively combating depression. By constructing a series of nanomedicines and further studying their structure-activity relationships, this invention aims to provide new ideas and theoretical foundations for antidepressant treatment.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The preparation method of the nano-formulation provided by the present invention is simple, and the "one-step method" is used to construct nano-antidepressant drugs with high preparation efficiency.

[0024] 2. The antidepressant nanomedicine provided by this invention has a 5-hydroxytryptamine loading of up to 139.99±11.12 μg / mL, and releases 5-HT only through weak acid-induced degradation in a depressive microenvironment, which greatly improves the efficacy of the drug. The increase of 5-HT can generally improve depressive symptoms, overcoming the problem of low response rate of fluoxetine nanodelivery systems in the prior art.

[0025] 3. The CAT in the nano-formulation provided by this invention can significantly reduce neuroinflammation and improve depressive symptoms; at the same time, it prevents 5-HT from being oxidized and inactivated by ROS in neuroinflammation, thereby improving the efficacy of 5-HT. The good targeting effect of RVG29 is conducive to the combined treatment of depression by multiple targets, which can overcome the problem of single target of antidepressant drugs in the prior art.

[0026] 4. Nanoparticle therapy has largely alleviated the over-activated astrocytes. Among these treatments, nanoparticle antidepressants have shown significant effects in reducing the activation state of glial cells. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 The images show the particle size, surface potential, and morphology of the nano-formulation in Experimental Example 1. Figure 1 A represents the particle size of the nano-formulation. Figure 1 B represents the surface potential of the nano-formulation. Figure 1 C represents the morphology of the nano-formulation;

[0029] Figure 2 This describes the microenvironment-responsive degradation and release characteristics of the nano-formulation in Experimental Example 3. Figure 2 A represents HPLC characterization of the release of single molecules of 5-HT from nano-formulations in different buffer solutions; Figure 2 B indicates the sample characterized by ESI-MS degradation; Figure 2 C represents the release curves of the nano-formulation 5-HT in different buffer solutions;

[0030] Figure 3 The X-ray photoelectron spectra for Experimental Example 3 are shown below. A represents the X-ray photoelectron C, N, and O spectra of polymer 5-HT; B represents the X-ray photoelectron C, N, and O spectra of a single 5-HT molecule; and C represents the X-ray photoelectron C, N, and O spectra of degraded polymer 5-HT.

[0031] Figure 4 Fourier transform infrared spectra of polymer 5-HT and 5-HT monomers degraded in Experiment Example 3;

[0032] Figure 5 Fluorescence imaging in vivo after intravenous injection of Cy7.5-labeled nanomedicine into a depressed mouse in Experiment Example 5;

[0033] Figure 6 The effects of key factors CAT and RVG29 in Experiment 5 on a depressed mouse model were investigated. A represents the procedure of the animal behavioral experiment; B represents the sucrose preference test in the behavioral experiment; C represents the mine test; D represents the forced swimming test; and E represents the changes in mouse body weight during the experimental procedure. Significant differences between groups were analyzed using one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001.

[0034] Figure 7 To demonstrate the in vivo efficacy of the 6-nanometer antidepressant preparation in Experiment Example 6, A is a ROS staining image of a brain slice, and B is astrocytes. Detailed Implementation

[0035] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0036] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0037] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the embodiments are all commercially available.

[0038] Example 1

[0039] Preparation of nanoformulations for efficient delivery of neurotransmitters

[0040] S1. Dissolve 100 mg of 5-HT in water and add it to a weakly alkaline buffer solution of Tris-HCl (pH 9.5) while stirring continuously. Then add 10 mg of CAT and react for 20 h. Subsequently, remove unreacted raw materials by dialysis for 6 h to obtain core particles.

[0041] S2. Subsequently, HSA is adsorbed in the core particles in PBS, and unadsorbed HSA is removed by dialysis for 6 hours to obtain core particles adsorbed with human serum albumin.

[0042] S3. The core particles of human serum albumin obtained in step S2 are adsorbed into RVG29 in PBS for 3 hours, and unreacted molecules are removed by dialysis for 4 hours. Finally, the nano-formulation of the present invention is obtained by freeze drying.

[0043] Experimental Example 1

[0044] Investigating the particle size, surface potential, and morphology of nanoformulations

[0045] The particle size and surface potential of the nanomedicine were measured using a Malvern particle size analyzer, and the morphology of the nanomedicine was characterized by transmission electron microscopy. The results are as follows: Figure 1 As shown, Figure 1 A represents the particle size of the nano-formulation. Figure 1 B represents the surface potential of the nano-formulation. Figure 1 C represents the morphology of the nano-formulation. The particle size of the nano-formulation is between 110-170 nm, the surface potential is -20 to -30 mV, and the morphology of the nano-formulation is spherical.

[0046] Experiment Example 2

[0047] The content of 5-HT and CAT loaded in the nano-formulation was investigated.

[0048] The content of 5-HT in the nano-formulation prepared in Example 1 was measured by ultraviolet spectrophotometer, and the content of CAT was detected by enzyme activity kit.

[0049] The results are shown in Table 1. There is a large difference between 5-HT and CAT in the nano-formulation. The nano-formulation prepared in this invention has a 5-HT loading of up to 139.99±11.12 μg / mL and a CAT content of 1.71±0.042 μg / mL.

[0050] Table 1. Content of each component in the nano-formulation (C nano-antidepressant = 1 mg / mL)

[0051] <![CDATA[C 5-HT [μg / mL]]]> <![CDATA[C CAT [μg / mL]]]> <![CDATA[C CAT [μg / mL] / C 5-HT [μg / mL]]]> Nanoparticle antidepressants 139.99±11.12 1.71±0.042 81.90±4.58

[0052] Experimental Example 3

[0053] Investigating the microenvironment-responsive degradation and release characteristics of nano-formulations

[0054] A series of nanomedicines were incubated at pH 7.4, pH 6.4, reactive oxygen species up (ROSUP) buffer, and pH 6.4 + ROSUP buffer for 0, 1, 7, 12, 24, 48, 72, 120, and 168 h. The released monoamine neurotransmitters (5-HT or NE) were obtained by filtration and centrifugation using a 3 kDa ultrafiltration tube. The retention times of the monoamine neurotransmitters were determined by comparing the chromatographic peaks using HPLC, and the separated filtrate was collected. The composition of the degradation products was further analyzed using ESI-MS. Simultaneously, the release curves of the monoamine neurotransmitters under different conditions were measured using HPLC. The mobile phase for HPLC consisted of solvent A (pH 4.0 buffer, 0.01 M sodium acetate - 0.02 M citric acid) and solvent B (chromatographic grade methanol). For the time period t = 0–10 minutes, 95% solvent A and 5% solvent B were used; for the time period t = 10–15 minutes, 5% solvent A and 95% solvent B were used. The flow rate of the mobile phase was 2 mL / min, and the wavelength of the detector was 254 nm.

[0055] The results are as follows Figure 2 As shown, the weakly acid-responsive release of nano-antidepressants... Figure 2 A indicates that the release of 5-HT single molecules from the nano-antidepressant drug in different buffer solutions was characterized by HPLC. The results showed that the release of 5-HT monomers was detected by HPLC under the conditions of pH 6.4, pH 7.4+ROSUP and pH 6.4+ROSUP.

[0056] Figure 2B indicates that during the retention time of 2-4 minutes, the sample separated by HPLC was collected, and the molecular weight of 5-HT single molecules in the degraded sample was characterized by ESI-MS, further confirming that the polymerized 5-HT can degrade and release 5-HT under weak acid conditions.

[0057] Figure 2 C represents the release curve of the nano-antidepressant 5-HT in different buffer solutions. The nano-formulation released the most 5-HT in pH 6.4 + ROSUP buffer, which was significantly higher than in pH 7.4 + ROSUP buffer and pH 6.4 buffer conditions. This indicates that the nano-drug prepared in this invention releases 5-HT only through weak acid-induced degradation in the depressive microenvironment, thus improving the drug delivery efficiency and the accuracy of treatment.

[0058] Experiment Example 4

[0059] Investigating the polymerization and weak acid-responsive degradation of 5-HT

[0060] 5-HT was dissolved in water and added to a weakly alkaline buffer solution (pH 9.5 Tris-HCl) with constant stirring. The solution was purified by dialysis and lyophilized to obtain polymer 5-HT. Polymer 5-HT was incubated in a buffer solution (pH 6.4, citrate-disodium hydrogen phosphate) for 72 hours, and the degraded polymer 5-HT was obtained by ultrafiltration. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy were used to analyze polymer 5-HT, degraded polymer 5-HT, and 5-HT monomers.

[0061] The results are as follows Figure 3 and Figure 4 As shown, Figure 3 X-ray photoelectron spectroscopy revealed four distinct peaks in the C1s region: CH / C-NH2 (284.8 eV), CO / CN (286.1 eV), C=O / C=N (288.2 eV), and π-packing (292.5 eV). Similarly, the N1s region exhibited two peaks in all three samples, corresponding to R-NH2 (399.8 eV) and R-NH-R (401.9 eV). Clearly, the amino groups were prominent in the 5-HT monomer, but their intensity decreased after polymerization. In the O1s region, three peaks were observed, corresponding to O=C (531.4 eV), OC (532.6 eV), and HOC (533.6 eV). Compared to the 5-HT monomer, the polymer 5-HT showed an increased O=C content and a decreased HOC intensity. These findings collectively suggest that the polymerization of 5-HT is caused by an auto-oxidation reaction, in which primarily the amino and phenolic hydroxyl groups are consumed.

[0062] Figure 4This indicates that the degraded polymer 5-HT exhibits a higher degree of oxidation compared to the 5-HT monomer, but still retains some amino and phenolic hydroxyl groups. Fourier transform infrared spectroscopy further confirmed the presence of amino and phenolic hydroxyl groups in polymer 5-HT, degraded polymer 5-HT, and 5-HT monomers.

[0063] Experimental Example 5

[0064] Investigating the effects of key factors CAT and RVG29 on a mouse model of depression.

[0065] Two control nanomedicines were constructed: control nanomedicine 1 (containing CAT but not RVG29) and control nanomedicine 2 (not containing CAT, replaced by human serum albumin (HSA), containing RVG29).

[0066] This invention employs a chronic unpredictable mild stress (CUMS) model to establish a mouse model of depression. This model requires mice (4-week-old male C57bl / 6j mice) to receive mild, unpredictable stimuli daily, simulating low-frequency stimuli in daily life. These stimuli primarily include the following: ① circadian rhythm reversal and light stimulation (e.g., continuous 24-hour light to 24-hour daytime, continuous 24-hour darkness to 24-hour nighttime, stroboscopic stimulation); ② adjustments to food and water supply (e.g., fasting, water restriction); ③ changes in living environment (e.g., wet bedding, overcrowding, tilting); ④ fear and pain (e.g., swimming in ice water, restraint, tail clamping). To avoid repeated use of stressors, the same stressor should not be used twice within a week, and the stimulation should continue for 5 weeks to achieve an unpredictable effect. The depressive state of the mice is characterized by behavioral tests (including the sucrose preference test, forced swimming test, and mine test).

[0067] First, we examined the brain-targeting ability of nanomedicines, using Cy7.5-labeled nanomedicines to investigate their brain-targeting capacity. A subset of depressed mice were given a single injection, and brain targeting was characterized using in vivo imaging at different time points after injection. To characterize the therapeutic effect and modeling effect, we used the following three behavioral experiments: ① Sucrose preference tests (SPT): Sugar water is considered a reward, and depressed patients often lack a response to rewards. We used 1.5% sucrose water to simulate the mice's response to rewards. In a dark and quiet environment, we placed the mice in cages and allowed them to acclimatize for 2 hours, followed by a 2-hour test. On the first day, we placed two bottles of sugar water of equal mass in the cages. On the second day, we replaced one bottle of sugar water with water and allowed the mice to acclimatize for 2 hours. On the third day, we swapped the positions of the sugar water and water bottles from the second day and tested for 2 hours. The formula for calculating the sugar water preference value is: sugar water consumption (g) / (sugar water consumption (g) + water consumption (g)). A decrease in the sugar water preference rate indicates a decrease in the depressed mice's response to rewards. ② Open Field Tests (OFT): In a quiet, dark environment, mice were placed in a 40cm × 40cm × 60cm (length × width × height) dark box to simulate their exploration behavior in a new environment, reflecting their activity level and curiosity. After the mice had adapted for 3 minutes, we used an infrared camera to record their movement routes over 5 minutes and analyzed the data using the behavioral analysis software Smart V3.0. We divided the open field into nine areas and analyzed the mice's activity level in exploring the new environment by calculating the movement time and path in the peripheral and central areas. The shorter the time and path of the mouse's activity in the central area, the more severe its depression. ③ Forced Swimming Tests (FST): Mice were placed in a 30cm × 11cm (height × diameter) transparent cylindrical container filled with water and forced to swim to simulate the survival instinct of animals in a desperate environment. On the first day, we allowed the mice to adapt to the forced swimming test for 15 minutes. The following day, at the same time and under the same conditions, we used a camera to record the mice swimming for 5 minutes and used the behavioral analysis software Smart V3.0 to analyze the time the mice spent at rest during the experiment. The increase in rest time indicated a decreased will to live in the depressed mice.

[0068] Brain-targeting results such as Figure 5 As shown, both the nano-antidepressant and the control nano-drug 2 have good brain targeting properties, demonstrating the good targeting effect of RVG29.

[0069] Animal experiment results such as Figure 6 As shown, Figure 6 A represents the procedure for animal behavioral experiments. Figure 6The B-saccharide preference experiment showed that, compared with the control group mice, Figure 6 B indicates that depressed mice exhibited a reduced preference for sucrose in the SPT, reflecting the anhedonia symptoms in depressed mice. The results of OFT and FST further confirmed the depressive-like state in the model mice. Figure 6 The C mine experiment showed that depressed mice exhibited a reduced distance of movement in open areas during OFT, reflecting anxiety symptoms in the depressed mice; Figure 6 In the forced swimming test (FST), immobility time was used as an indicator of behavioral despair, and the immobility time was significantly increased in depressed mice. Figure 6 B, C, and D indicate that treatment with fluoxetine and the three nanomedicines increased sucrose preference in mice, prolonged the distance they moved within the region, and reduced their immobility time.

[0070] Figure 6 E represents the change in mouse body weight; depressed mice gained significantly less weight than control mice. Furthermore, the differences at 1, 7, and 10 weeks were calculated, showing that the model mice exhibited reduced sucrose preference, decreased distance traveled within the treatment area, increased immobility time, and slowed the trend of weight gain. However, during treatment with the nano-antidepressant drug, depressed mice experienced a significant increase in body weight.

[0071] These results combined indicate that nano-antidepressants delivered via co-delivery of CAT and 5-HT exhibit the most significant antidepressant effects, even surpassing the therapeutic efficacy of fluoxetine.

[0072] Experimental Example 6

[0073] Investigating the in vivo efficacy of the nano-antidepressant formulation of this invention

[0074] Blood was removed from the brain of mice via microperfusion, and the brain was then analyzed for fluorescent staining of reactive oxygen species (ROS) and glial fibrillary acidic protein (GFAP). GFAP belongs to class III intermediate filaments and can serve as a specific marker for astrocytes. Neuroinflammation leads to astrocyte activation.

[0075] The results are as follows Figure 7 As shown, Figure 7 A shows ROS staining of brain slices. The depressed group exhibited significant ROS production, impairing neurological function in mice. The ROS levels in treated mice were significantly lower than in the depressed group. Among all treatments, the nano-antidepressant reduced ROS levels to the normal range, similar to the normal group. Figure 7B represents astrocytes. The GFAP immunofluorescence in the depression group showed a significant increase, indicating astrocyte activation. Nanomedicine therapy largely alleviated the over-activated astrocytes. Among these treatments, nano-antidepressants showed significant effects, reducing the activated state of glial cells.

Claims

1. A nanoformulation for delivery of a neurotransmitter, characterized in that, The formulation comprises 5-hydroxytryptamine, catalase, human serum albumin, and a neurotropic virus-derived peptide. The 5-hydroxytryptamine polymerizes in a weakly alkaline environment to form covalent bonds. During the polymerization process, the 5-hydroxytryptamine is co-loaded with catalase to form core particles. Human serum albumin is adsorbed onto the surface of the core particles to form a protein crown. Finally, the neurotropic virus-derived peptide is adsorbed onto the surface of the core particles to construct a nano-formulation. The neurotropic virus-derived peptide is RVG29.

2. The nanoformulation according to claim 1, characterized in that, The mass ratio of the 5-hydroxytryptamine and catalase mixture is 8~10:

1.

3. A method for the preparation of a nanoformulation according to any one of claims 1-2, characterized in that, Includes the following steps: S1. Dissolve 5-hydroxytryptamine in water, add a weak alkaline buffer solution while stirring continuously, then add catalase to react, and then dialyze to remove unreacted raw materials to obtain core particles; S2. The kernel particles obtained in step S1 adsorb human serum albumin in PBS, and the unadsorbed human serum albumin is removed by dialysis to obtain kernel particles adsorbed with human serum albumin. S3. The core particles of human serum albumin obtained in step S2 are used to adsorb neurotropic virus-derived peptides in PBS. After dialysis to remove unreacted molecules, the nano-formulation is obtained by freeze-drying.

4. The production method according to claim 3, characterized by, In step S1, the weakly alkaline buffer solution is either Tris-HCl buffer solution with a pH of 9.5 or ammonia solution with a pH of 8.5-10.

5.

5. The preparation method according to claim 3, characterized in that, The reaction time in step S1 is 15-20 hours; the dialysis time is 4-6 hours.

6. The preparation method according to claim 3, characterized in that, In step S2, the adsorption time is 1-3 hours; the dialysis time is 3-6 hours.

7. The preparation method according to claim 3, characterized in that, In step S3, the adsorption time is 1-3 hours; the dialysis time is 2-4 hours.

8. The use of a neurotransmitter delivery nanoformulation as described in any one of claims 1-2 or a neurotransmitter delivery nanoformulation prepared by any one of claims 3-7 in the preparation of a therapeutic agent for depression.

Images