A brain-targeting nano-preparation for treating depression and a preparation method and application thereof

Abstract

This invention relates to the field of traditional Chinese medicine research and development technology, specifically providing a brain-targeting nano-formulation for treating depression, its preparation method, and its application. This formulation uses graphene oxide, polyethylene glycol, verbascoside, and ferulic acid as raw materials, and achieves synergistic brain-targeted drug delivery of multiple components by constructing a nano-co-delivery carrier. First, graphene oxide is functionalized using polyethylene glycol. Then, ferulic acid is grafted onto the surface of the modified carrier, and finally, verbascoside is loaded to form a composite nano-formulation. This design fully leverages the brain-targeting function of ferulic acid and the antidepressant efficacy of verbascoside, synergistically enhancing therapeutic effects, reducing drug resistance and adverse reactions, and achieving targeted co-delivery of multiple components within the brain.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine research and development technology, and specifically provides a brain-targeting nano-preparation for the treatment of depression, its preparation method and application. Background Technology

[0002] Depression is a common mental disorder affecting approximately 350 million people worldwide. Its persistent low mood, anhedonia, sleep disturbances, psychomotor retardation, and cognitive impairment severely damage patients' physical and mental health and social functioning, imposing a heavy social burden. This illness not only significantly increases the economic burden on families and medical treatment but is also a leading cause of loss of earning capacity, making it a major public health problem urgently needing global attention.

[0003] In modern medicine, first-line drug treatment for depression mainly includes selective serotonin reuptake inhibitors (SSRIs, such as fluoxetine, paroxetine, and sertraline), serotonin and norepinephrine reuptake inhibitors (SNRIs, such as venlafaxine and duloxetine), norepinephrine and specific serotonergic antidepressants (NaSSAs, such as mirtazapine), and other classes of drugs (such as bupropion). Although these drugs are widely used in clinical practice and have certain efficacy, their limitations are also very prominent. Common adverse reactions include central nervous system symptoms (such as insomnia, dizziness, drowsiness, and tremor), gastrointestinal discomfort (nausea, vomiting, diarrhea, or constipation), autonomic dysfunction (excessive sweating and palpitations), sexual dysfunction, and weight changes. In addition, some patients have a slow response to treatment, incomplete symptom relief, or even discontinue treatment due to drug resistance or intolerance of side effects, which seriously affects medication adherence and long-term prognosis.

[0004] In recent years, various monomeric components of traditional Chinese medicine have shown significant potential in the study of antidepressant mechanisms. For example, ginsenoside Rg1 alleviates depressive-like behaviors through neuroprotective effects; resveratrol improves depressive states by anti-inflammatory effects, regulating neurotransmitters, and inhibiting excessive activation of the HPA axis; and paeoniflorin can reverse reserpine-induced depressive behavior, promote the formation of dendritic spines in the hippocampus, and inhibit neuroinflammatory responses. These components not only have the characteristics of multi-target and multi-pathway action but also show unique therapeutic advantages. However, they generally face pharmaceutical bottlenecks such as low water solubility, poor bioavailability, and difficulty in crossing the blood-brain barrier, which seriously restricts their clinical translation and practical application.

[0005] At the same time, existing chemical antidepressants also have significant limitations, such as long treatment duration, high relapse rate, potential dependence risk, and significant side effects. Against this backdrop, developing novel drug delivery formulations to improve efficacy and reduce adverse reactions has become an increasingly urgent direction in antidepressant treatment research. Summary of the Invention

[0006] The purpose of this invention is to provide a brain-targeting nano-formulation for the treatment of depression, its preparation method and application, thereby developing a novel antidepressant drug.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] First, this invention provides a method for preparing a multi-component brain-targeting nanoparticle formulation of traditional Chinese medicine for treating depression, the preparation method comprising the following steps:

[0009] (1) Preparation of blank nanocarrier PEG-GO:

[0010] Polyethylene glycol (PEG) was added to the graphene oxide (GO) dispersion, mixed thoroughly, and then ultrasonically treated.

[0011] EDC was then added to carry out the reaction, followed by dialysis purification and freeze-drying to obtain PEG-GO lyophilized powder.

[0012] (2) Preparation of a single-component nano-drug delivery system PEG-GO-FA loaded with ferulic acid (FA):

[0013] PEG-GO lyophilized powder was dispersed in water, and FA was added at a mass ratio of FA to PEG-GO of 1:10. The mixture was ultrasonically treated, stirred and reacted, purified by dialysis, and then freeze-dried to obtain PEG-GO-FA lyophilized powder.

[0014] (3) Preparation of a multi-component nano-drug delivery system ACT-PEG-GO-FA loaded with verbascoside (ACT) and ferulic acid (FA):

[0015] Take PEG-GO-FA lyophilized powder, disperse it in water, add ACT at a mass ratio of 1:10 of ACT to PEG-GO-FA, sonicate, stir to react, dialyze to purify, and freeze dry to obtain ACT-PEG-GO-FA solid nano-formulation.

[0016] Preferably, in step (1), the concentration of the graphene oxide dispersion is 1 mg / mL, the amount of polyethylene glycol added is 10 times the mass of the graphene oxide dispersion, and the ultrasonic treatment time is 5 min.

[0017] The EDC was initially added to a final concentration of 5 mM, followed by sonication for 30 min, and then the final concentration of EDC was increased to 20 mM. The reaction conditions were room temperature reaction for 12 h.

[0018] The molecular weight cutoff for dialysis purification was 10 kDa, and the dialysis purification time was 48 h.

[0019] Preferably, in step (2), the ultrasonic treatment time is 30 min, the stirring reaction time is 24 h, the molecular weight cutoff for dialysis purification is 3.5 kDa (consistent with the description in step 1), and the dialysis purification time is 24 h.

[0020] Preferably, in step (3), the ultrasonic treatment time is 30 min, the stirring reaction time is 24 h, the molecular weight of the dialysis purified is 3.5 kDa, and the dialysis purification time is 24 h.

[0021] Secondly, this invention provides a multi-component brain-targeting nano-formulation of traditional Chinese medicine for the treatment of depression, wherein the raw materials for preparing the multi-component brain-targeting nano-formulation of traditional Chinese medicine include:

[0022] Nanocarriers PEG-GO, ferulic acid and verbascoside;

[0023] The nanocarrier PEG-GO has a mass ratio of ferulic acid and verbascoside of 10:1:1.

[0024] Preferably, the therapeutic effect on depression is to improve anhedonia, behavioral hopelessness, and anxiety-like behavior.

[0025] Preferably, the multi-component brain-targeting nano-formulation of traditional Chinese medicine is prepared by the above-described preparation method.

[0026] Furthermore, this invention provides the use of the aforementioned multi-component brain-targeting nano-formulation of traditional Chinese medicine in the preparation of drugs for treating depression.

[0027] Finally, the present invention provides a pharmaceutical composition for treating depression, characterized in that the pharmaceutical composition comprises the above-mentioned multi-component brain-targeting nano-formulation of traditional Chinese medicine, as well as pharmaceutically acceptable excipients or carriers.

[0028] Preferably, in the pharmaceutical composition, the mass content of the multi-component brain-targeting nano-preparation of traditional Chinese medicine is greater than or equal to 8.06%;

[0029] The pharmaceutical composition is administered via intravenous injection.

[0030] The beneficial effects of this invention are as follows:

[0031] This formulation uses graphene oxide (GO) as the core carrier, modified with polyethylene glycol (PEG) to enhance stability and biocompatibility, and ferulic acid (FA) as a brain-targeting ligand to actively mediate the drug's crossing of the blood-brain barrier. Co-loading ACT and FA onto a PEG-modified GO nanocarrier significantly improves drug stability and solubility. The nanocarrier formulation also retains the highly efficient brain-targeting properties of FA and the good antidepressant properties of ACT, ultimately achieving drug delivery across the blood-brain barrier and improving bioavailability. In vitro and in vivo pharmacodynamic studies demonstrate that this formulation exhibits enhanced overall antidepressant efficacy, with the multi-component brain-targeting nanocarrier group showing superior antidepressant effects compared to single-component and traditional formulations. Attached Figure Description

[0032] Figure 1 A schematic diagram illustrating the preparation of a multi-component brain-targeting nano-formulation of traditional Chinese medicine;

[0033] Figure 2 Physical characterization, encapsulation efficiency and drug loading of multi-component brain-targeting nano-formulations of traditional Chinese medicine at different proportions;

[0034] Figure 3 The optical properties, microscopic characterization, and dispersibility under different media of multi-component brain-targeting nano-formulations of traditional Chinese medicine;

[0035] Figure 4 To assess the biocompatibility of multi-component brain-targeting nano-preparations of traditional Chinese medicine;

[0036] Figure 5 This refers to the targeting properties of multi-component brain-targeting nano-formulations of traditional Chinese medicine.

[0037] Figure 6 Evaluation of the antidepressant properties of multi-component brain-targeting nano-formulations of traditional Chinese medicine. Detailed Implementation

[0038] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the embodiments described below are only some representative embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the disclosure of the present invention without creative effort are within the protection scope of the claims of the present invention.

[0039] Example 1

[0040] Preparation of blank nanocarriers (PEG-GO)

[0041] Its preparation method is as follows:

[0042] Take 5 mL of graphene oxide (GO) dispersion with a concentration of 1 mg / mL, add 50 mg of polyethylene glycol (PEG), mix well and then sonicate for 5 min;

[0043] Then EDC was added to a final concentration of 5 mM, and sonication was continued for 30 min. EDC was then added to a final concentration of 20 mM, and the mixture was stirred at room temperature for 12 h.

[0044] The reaction solution was placed in a dialysis bag (molecular weight cutoff 10 kDa) and purified by dialysis for 48 hours to obtain a blank nanocarrier PEG-GO dispersion. After freeze-drying at -80℃, PEG-GO lyophilized powder was obtained.

[0045] Example 2

[0046] Preparation of a single-component nano-drug delivery system loaded with ACT (ACT-PEG-GO)

[0047] Its preparation method is as follows:

[0048] Take 50 mg of the above PEG-GO lyophilized powder, ultrasonically disperse it in 5 mL of ultrapure water, add 5 mg of verbascoside (ACT), ultrasonically treat for 30 min, stir at room temperature for 24 h, place the reaction solution in a dialysis bag (Mw=3.5 kDa) and dialyze for 24 h to purify it, and the single-component nano-drug-loaded formulation ACT-PEG-GO dispersion is obtained. After freeze-drying, ACT-PEG-GO solid nano-formulation is obtained.

[0049] Preparation mode diagram as shown Figure 1 As shown in (A).

[0050] Example 3

[0051] Preparation of a single-component nano-drug delivery system loaded with fatty acids (PEG-GO-FA)

[0052] Its preparation method is as follows:

[0053] Take 50 mg of PEG-GO lyophilized powder, sonicate it to redisperse it in 5 mL of ultrapure water, add 5 mg of FA at a ratio of 1:10 of drug FA to nanocarrier PEG-GO, sonicate for 30 min, stir at room temperature for 24 h, place the reaction solution in a dialysis bag (Mw=3.5kDa) and dialyze for 24 h to purify it, and obtain a single-component nano-drug-loaded formulation PEG-GO-FA dispersion. After freeze-drying, obtain PEG-GO-FA solid nano-formulation.

[0054] Preparation mode diagram as shown Figure 1 As shown in (B).

[0055] Example 4

[0056] Preparation of multi-component nano-drug delivery formulation ACT-PEG-GO-FA loaded with ACT and FA

[0057] Its preparation method is as follows:

[0058] Take 50 mg of PEG-GO-FA lyophilized powder, sonicate it into 5 mL of ultrapure water, add 5 mg of ACT at a ratio of 1:10 for drug ACT to nanocarrier PEG-GO-FA, sonicate for 30 min, stir at room temperature for 24 h, and purify the reaction solution by dialyzing in a dialysis bag (Mw=3.5 kDa) for 24 h to obtain ACT-PEG-GO-FA co-delivered nano-formulation dispersion. After freeze-drying, obtain ACT-PEG-GO-FA solid nano-formulation.

[0059] Example 5

[0060] The nanocarriers and nanoformulations obtained in Examples 1-4 were physically characterized, and the encapsulation efficiency and drug loading of the final nanoformulation products were determined.

[0061] The specific method is as follows:

[0062] PEG-GO, ACT-PEG-GO, PEG-GO-FA, and ACT-PEG-GO-FA powders were mixed with potassium bromide at a mass ratio of 1:100, ground evenly in an agate mortar, pressed into thin slices, and subjected to infrared spectroscopy analysis in the scanning range of 500–4000 cm⁻¹. -1 The resolution is 2cm. -1 .

[0063] PEG-GO, ACT-PEG-GO, PEG-GO-FA and ACT-PEG-GO-FA were determined by high performance liquid chromatography (HPLC).

[0064] Chromatographic conditions: The mobile phase was a methanol-water gradient elution system, with a methanol volume fraction of 45% from 0 to 25 min, a flow rate of 0.9 mL / min, a column temperature of 35 °C, and an injection volume of 10 μL.

[0065] The detection wavelengths were 334 nm (ACT) and 310 nm (FA).

[0066] The reference solution was diluted to prepare a series of standard solutions with concentrations of 1, 5, 10, 20, 30, and 40 μg / mL. The solutions were then injected under the conditions described above, and the peak areas were recorded.

[0067] A standard curve was established by performing a linear regression of peak area (Y) against concentration (C).

[0068] Each group of samples was measured under the same conditions. The obtained peak areas were substituted into the standard curve to calculate the contents of ACT and FA, and the drug loading and encapsulation efficiency were further calculated.

[0069] The results obtained are as follows Figure 2 As shown.

[0070] from Figure 2 The results show that, in optimizing the effect of different ratios of ACT and PEG-GO-FA on nano-formulations, different ratios do not cause significant differences in particle size, potential, and PDI.

[0071] However, as the feed ratio increases, there are significant differences in encapsulation efficiency and drug loading: the encapsulation efficiency gradually increases, while the drug loading gradually decreases. Therefore, this experiment ultimately selected a feed ratio of 1:10 as the optimal feed ratio for co-delivering nanoformulations for subsequent experiments.

[0072] Example 6

[0073] The nanocarriers and nanoformulations obtained in Examples 1-4 were subjected to optical properties, microscopic characterization, and dispersibility determination in different media.

[0074] The specific method is as follows:

[0075] After diluting the sample by an appropriate factor, the particle size, polymer dispersity index (PDI), and zeta potential were determined using a laser particle size analyzer.

[0076] The sample was ultrasonically dispersed in ultrapure water for 15 min, dropped onto a 400-mesh copper mesh and dried at room temperature for 10 min before being observed under a transmission electron microscope. The test voltage was 200 kV.

[0077] The sample was ultrasonically dispersed in water, phosphate buffered saline (PBS), or cell culture medium, and the dispersion was observed after standing for 30 days.

[0078] The results obtained are as follows Figure 3 As shown.

[0079] like Figure 3 A and Figure 3 As shown in B, the particle size determination results of the nano-formulation meet the characteristic requirements of nanomedicines; the Zeta potential test further characterizes its surface electrical properties. Figure 3 C shows the changes in particle size and PDI of the nanoformulation over 7 days, indicating good stability during the test. Figure 3 The infrared spectral analysis shown in D reveals that the nanocarrier and nanoformulation exhibit absorption peaks corresponding to functional groups at specific wavenumbers, confirming that they have been successfully constructed. Figure 3 E is a transmission electron microscope (TEM) image, which further verifies the structural integrity of the nanocarrier and nanoformulation from the perspective of microscopic morphology. Figure 3 F shows the dispersion of each sample in different media (water, PBS, and DMEM cell culture medium) after standing at 4°C for 30 days. ACT-PEG-GO, PEG-GO-FA, and ACT-PEG-GO-FA all exhibited good dispersion stability. The above experimental results demonstrate that the ACT-PEG-GO, PEG-GO-FA, and ACT-PEG-GO-FA nanoformulations prepared in this invention were successfully constructed and possess excellent stability.

[0080] Example 7

[0081] Biocompatibility determination of nanocarriers and nanoformulations in Examples 1-4

[0082] The specific method is as follows:

[0083] To comprehensively evaluate the biocompatibility of this nano-formulation, the present invention conducted systematic experiments in three aspects: in vitro cytotoxicity, blood compatibility, and in vivo histopathological effects.

[0084] First, this invention uses a toxicity experiment of nanocarriers and nanoformulations on astrocytes to detect the effect of materials on cell proliferation and activity (10-200 μg / mL). Astrocyte cells were prepared into 1×10⁻⁶ cells. 5 Cell suspension at a concentration of / mL was pre-added to each well of a 96-well plate with 100 μL of complete culture medium, followed by 100 μL of cell suspension. After 24 h of overnight culture, cells were divided into a blank control group, a GO group, a PEG-GO group, and a PEG-GO-ACT group. The blank control group was replaced with fresh DMEM complete culture medium, while the other three groups were supplemented with nano-formulation at concentrations of 10, 25, 50, 100, and 200 μg / mL, respectively, with six replicates per group. After 24 h of intervention, all groups were replaced with 100 μL of DMEM complete culture medium per well, followed by 10 μL of CCK8 solution, and incubated at 37°C for 2 h. Once the culture medium turned orange-yellow, the absorbance (A) at OD 450 nm was measured using a microplate reader set to 37°C. Cell viability was used to determine the cytotoxicity of each group and to evaluate the in vitro biocompatibility of the nanocarrier and nano-formulation.

[0085] Secondly, this invention analyzes the destructive effect of the materials on the erythrocyte membrane and blood compatibility (10-200 μg / mL) through hemolysis experiments. Mouse blood cells were used to detect the hemolysis of GO, PEG-GO, and PEG-GO-ACT. One ml of fresh blood from a healthy mouse was mixed with PBS, centrifuged at 3000 rpm for 5 min, and the supernatant was removed. The precipitate was washed three times with PBS. 0.2 mL of the erythrocyte suspension was diluted to 10 mL with PBS to obtain the erythrocyte suspension. Two mL of the erythrocyte suspension was taken and mixed with dispersions of different concentrations of GO, PEG-GO, and PEG-GO-ACT (10, 25, 50, 100, and 200 μg / mL) at a ratio of 1:5. The mixture was incubated at 37°C for 4 h and centrifuged at 3000 rpm for 5 min. Erythrocytes suspended in ultrapure water and PBS were used as positive controls (+) and negative controls (-), respectively. The supernatant was collected and its absorbance (A) was measured at 540 nm. The in vitro biocompatibility of the nanocarrier and nanoformulation was evaluated by the hemolysis rate.

[0086] Finally, this invention observed the changes in the tissue morphology and structure of major organs by hematoxylin-eosin (HE) staining. PEG-GO and PEG-GO-ACT dispersions were administered to mice via tail vein injection at a dose of 60 mg / kg for 14 consecutive days. After the 14th day of administration, tissue samples were collected, and histopathological analysis was performed on the major metabolic organs (liver and kidney) of the mice. HE staining was used to observe the toxic effects of PEG-GO and PEG-GO-ACT on mouse organs to evaluate their biosafety in vivo (200 μg / mL).

[0087] The results obtained are as follows Figure 4 As shown.

[0088] from Figure 4 The hemolytic assay results showed that ACT-PEG-GO, PEG-GO-FA and ACT-PEG-GO-FA had no effect on blood cells and good cell compatibility.

[0089] As shown in the astrocyte survival experiment, ACT-PEG-GO, PEG-GO-FA and ACT-PEG-GO-FA at concentrations of 10-200 μg / ml had no significant effect on cell survival and did not exhibit cytotoxicity.

[0090] Histopathological analysis showed that ACT-PEG-GO, PEG-GO-FA, and ACT-PEG-GO-FA did not cause any pathological changes in any of the animal's organs.

[0091] Example 8

[0092] In vitro and in vivo targeting assays were performed on the nanocarriers and nanoformulations obtained in Examples 1-4.

[0093] The specific method is as follows:

[0094] This invention evaluates the targeting performance of nanocarriers and their formulations through in vitro cellular uptake experiments and in vivo small animal imaging technology.

[0095] At the cellular level, to evaluate the cellular uptake behavior of nanomaterials, each sample was uniformly mixed with fluorescein isothiocyanate (FITC) at a mass ratio of 1:5, sonicated for 30 min, and then stirred continuously at room temperature for 24 h to prepare FITC-labeled GO, PEG-GO, PEG-GO-FA, ACT-PEG-GO, and ACT-PEG-GO-FA aqueous dispersions. These dispersions were diluted to a FITC concentration of 10 μg / mL using serum-free culture medium and co-incubated with astrocytes for 3 h (37℃). Subsequently, the endocytosis of nanomaterials within the cells and their uptake efficiency were observed and analyzed using a fluorescence microscopy system.

[0096] At the animal level, to investigate the distribution and targeting of nanoparticles in vivo, samples were mixed with the near-infrared fluorescent dye DIR at a mass ratio of 1:5, sonicated for 30 min, and stirred at room temperature for 24 h to obtain DIR-labeled PEG-GO, ACT-PEG-GO, PEG-GO-FA, and ACT-PEG-GO-FA aqueous dispersions. These dispersions were freeze-dried, redissolved in physiological saline, and the DIR concentration was adjusted to 3 mg / mL. Each group of formulations was injected into mice via tail vein injection. At 3, 6, 12, and 24 h post-injection, the distribution of the nanoparticles throughout the body was monitored in real time using a small animal in vivo imaging system (excitation wavelength 748 nm, emission wavelength 780 nm) to further evaluate their accumulation characteristics and brain-targeting delivery effects in the model animals.

[0097] according to Figure 5 The results of cellular uptake and small animal in vivo imaging experiments show that, in astrocyte uptake, the ACT-PEG-GO-FA group exhibited the highest endocytic efficiency, significantly outperforming other control groups. At the animal level, the ACT-PEG-GO-FA group also showed the strongest fluorescence signal accumulation in mouse brain tissue, which remained stable within the observation time window, indicating significant brain-targeting performance. In conclusion, ACT-PEG-GO-FA demonstrates excellent targeted delivery capabilities at both the cellular and animal levels, particularly exhibiting significant brain-targeting effects, providing a valid basis for its application as a brain-targeting nanomedicine delivery system.

[0098] Example 9

[0099] Evaluation of the antidepressant effect of ACT-PEG-GO-FA

[0100] The specific method is as follows:

[0101] The process of constructing the mouse depression model used in this study is as follows:

[0102] First, the location of the target brain region was determined based on the mouse brain atlas, and three-dimensional coordinate localization was performed using a stereotaxic instrument with the anterior fontanelle as the origin. The target brain region was selected as the prelimbic cortex (PrL) within the medial prefrontal cortex (mPFC).

[0103] Subsequently, the inhibitory chemogenetic viral vectors rAAV-EF1α-DIO-hM4D(Gi)-mCherry and rAAV-fSST-Cre were diluted to a titer of 2E12 and mixed at a 2:1 ratio. The mixture was then microinjected into the bilateral anterior limbic cortex of mice, with a total injection volume of 150 nmol per side at a rate of 40-80 nmol / min. Injection was stopped for 10 minutes after completion. By injecting the virus into the target brain region, the Cre-loxP system specifically targets SST-positive interneurons and expresses the hM4D(Gi) receptor to achieve chemogenetic inhibition, thereby constructing a mouse model of depression with precise neuronal function regulation capabilities.

[0104] The detailed process is as follows:

[0105] This study used 42 four-week-old SPF-grade male C57BL / 6N mice. After one week of acclimatization, the 42 mice were stereotactically injected with a chemically inherited virus to establish an animal model of depression.

[0106] Twenty-one days after viral expression, mice were evenly divided into seven groups (n=6 per group) based on their body weight, sucrose preference test results, and baseline values ​​of the open field test: control group, depression model group, blank nanocarrier group (PEG-GO), single-component brain-targeting nano-preparation group (PEG-GO-FA), multi-component brain-targeting nano-preparation group (ACT-PEG-GO-FA), multi-component physical mixture group (ACT+FA), and lily and rehmannia decoction group (LBRD).

[0107] The control group received stereotactic injection of an empty viral vector into the brain without any other treatment. Except for the blank control group, all other groups received stereotactic injection of the modeling virus into the brain, followed by intravenous injection of the corresponding therapeutic drug via tail vein and intraperitoneal injection of 3 mg / kg of clozapine N-oxide (CNO). The model group received stereotactic injection of the virus into the brain, followed by an intravenous injection of an equal volume of saline solution and an intraperitoneal injection of the same dose of CNO as the other groups. The PEG-GO group served as the blank nanocarrier control group; its virus injection, intraperitoneal injection of CNO, and tail vein injection volumes were consistent with the other treatment groups, with the tail vein injection containing PEG-GO dispersion. The PEG-GO-FA group received stereotactic injection of the corresponding virus into the brain and intraperitoneal injection of CNO, followed by a tail vein injection of PEG-GO-FA, with a total FA dose of 40 mg / kg. The ACT-PEG-GO-FA group received stereotactic injection of the corresponding virus into the brain and intraperitoneal injection of CNO, followed by a tail vein injection of ACT-PEG-GO-FA, with a total ACT dose of 60 mg / kg. mg / kg; In the ACT+FA group, after stereotactic injection of the corresponding virus into the brain and intraperitoneal injection of CNO, a mixed solution of ACT and FA was injected into the tail vein, with a total dose of ACT of 60 mg / kg and a total dose of FA of 40 mg / kg; The LBRD group, as a positive control treatment group for traditional Chinese medicine, after stereotactic injection of the corresponding virus into the brain and intraperitoneal injection of CNO, was administered via gavage, with a total dose of 6.25 mg / kg.

[0108] The treatment lasted 14 days. During the last 4 days of treatment, behavioral tests were conducted in groups, using the following methods:

[0109] Sugar water preference experiment:

[0110] Each cage of mice was given one bottle of 1% sucrose solution and one bottle of mineral water for 24 hours to acclimatize. During the 12-hour period, the two bottles were switched. On the second day after the acclimatization period, the mice were deprived of water but not food for 4 hours. The two bottles of 1% sucrose solution and mineral water were weighed and placed together. After 3 hours, their positions were switched. The mice were allowed free access to water for 6 hours. The two bottles were weighed and the sugar water intake was recorded. Sugar water preference = sugar water intake / (ordinary water intake + sugar water intake) × 100%.

[0111] Forced swimming experiment:

[0112] The mice were gently placed in a plastic bucket filled with warm water at 23-24°C. After the mice adapted for 30 seconds, their struggling was recorded for 4 minutes. After the test, the mice were dried and kept warm. The time spent struggling, floating, and remaining still for 4 minutes was statistically analyzed.

[0113] Open field experiment:

[0114] After the mice were allowed to move freely in the open field box for 30 seconds, the time the mice spent in the center of the open field box within 4 minutes was recorded. After each mouse was tested, their feces were removed and the open field box was wiped with 75% ethanol to avoid residual odors interfering with subsequent experiments. The movement time of the mice in the central area within 4 minutes was statistically analyzed.

[0115] Cross-shaped elevated maze experiment:

[0116] The mice were placed headfirst into the center of the elevated cross maze with their heads facing the closed arms. After the mice were allowed to move freely for 30 seconds, the time and number of times they entered the open arms within 4 minutes were recorded. After each mouse was tested, the experimental area was wiped with 75% ethanol to avoid leaving any odor that might interfere with subsequent experiments. The time and number of times the mice entered the open arms within 4 minutes were then statistically analyzed.

[0117] After the behavioral tests were completed, statistical evaluation was performed using GraphPad Prism software. All measurements are expressed as mean ± standard deviation. For comparisons of two groups of measurement data that conform to a normal distribution and homogeneous variance, t-tests were used. Analysis of variance was used for multiple groups of measurement data. The LSD method was used for pairwise comparisons of multiple groups of measurement data. P < 0.05 was considered statistically significant, and P < 0.01 was considered statistically significant.

[0118] At the same time, the treatment improvement rate (the percentage increase or decrease compared to the model group) was calculated using the model group as a baseline.

[0119] The results obtained are as follows Figure 6 As shown in Table 1.

[0120] Table 1. Differences in treatment improvement rates among different treatment groups (%)

[0121]

[0122] from Figure 6 As clearly shown in Table 1, the ACT-PEG-GO-FA group demonstrated the best antidepressant efficacy across multiple behavioral evaluation indicators. Specifically, in the sucrose preference test, this group significantly increased mice's preference for sweet stimuli, reflecting effective relief of anhedonia symptoms; in the forced swimming test, their immobility time was significantly shortened, indicating a reduction in behavioral despair-like behavior; in the open field test, the mice's total movement distance and central area dwell time were significantly increased, suggesting enhanced spontaneous activity and reduced anxiety-like behavior; and in the elevated cross maze test, the increased number of open arm entries and dwell time further confirmed its anti-anxiety effect. In summary, the treatment effect of the ACT-PEG-GO-FA group was significantly superior to that of the other control groups.

[0123] Further analysis shows that the effect of the ACT-PEG-GO-FA group is significantly better than the effect of using PEG-GO and ACT+FA alone, proving that the ACT-PEG-GO-FA prepared by combining PEG-GO and ACT+FA in this invention can enable the combination of PEG-GO and ACT+FA to exert a synergistic effect in treating depression.

[0124] More notably, compared to using the PEG-GO carrier or the ACT+FA drug combination alone, the ACT-PEG-GO-FA group not only demonstrated superior efficacy but also showed a significantly greater degree of improvement than the simple sum of the effects of either drug alone. This result strongly suggests that the ACT-PEG-GO-FA nanoformulation constructed in this invention is not a mechanical mixture of components, but rather achieves functional synergy between the PEG-GO nanocarrier, the active ingredient ACT, and the targeting ligand FA through rational structural design. This significantly enhances the bioavailability and neuropharmacological effects of ACT, thereby exhibiting excellent comprehensive antidepressant and anti-anxiety efficacy at the behavioral level. This finding fully validates the scientific rigor and superiority of the nanoformulation design of this invention, providing new strategies and experimental evidence for the precision treatment of depression.

Claims

1. A method for preparing a multi-component brain-targeting nanoparticle formulation of traditional Chinese medicine for treating depression, characterized in that, The preparation method includes the following steps: (1) Preparation of blank nanocarrier PEG-GO: Polyethylene glycol (PEG) was added to the graphene oxide (GO) dispersion, mixed thoroughly, and then ultrasonically treated. EDC was then added to carry out the reaction, followed by dialysis purification and freeze-drying to obtain PEG-GO lyophilized powder. (2) Preparation of a single-component nano-drug delivery system PEG-GO-FA loaded with ferulic acid (FA): PEG-GO lyophilized powder was dispersed in water, and FA was added at a mass ratio of FA to PEG-GO of 1:

10. The mixture was ultrasonically treated, stirred and reacted, purified by dialysis, and then freeze-dried to obtain PEG-GO-FA lyophilized powder. (3) Preparation of a multi-component nano-drug delivery system ACT-PEG-GO-FA loaded with verbascoside (ACT) and ferulic acid (FA): Take PEG-GO-FA lyophilized powder, disperse it in water, add ACT at a mass ratio of 1:10 of ACT to PEG-GO-FA, sonicate, stir to react, dialyze to purify, and freeze dry to obtain ACT-PEG-GO-FA solid nano-formulation.

2. The preparation method according to claim 1, characterized in that, In step (1), the concentration of the graphene oxide dispersion is 1 mg / mL, the amount of polyethylene glycol added is 10 times the mass of the graphene oxide dispersion, and the ultrasonic treatment time is 5 min. The reaction by adding EDC involves first adding EDC to a final concentration of 5 mM, continuing sonication for 30 min, and then adding more EDC to a final concentration of 20 mM. The reaction conditions are room temperature reaction for 12 h. The molecular weight cutoff for dialysis purification was 10 kDa, and the dialysis purification time was 48 h.

3. The preparation method according to claim 2, characterized in that, In step (2), the ultrasonic treatment time is 30 min, the stirring reaction time is 24 h, the molecular weight cutoff for dialysis purification is 3.5 kDa, and the dialysis purification time is 24 h.

4. The preparation method according to claim 3, characterized in that, In step (3), the ultrasonic treatment time is 30 min, the stirring reaction time is 24 h, the molecular weight cutoff for dialysis purification is 3.5 kDa, and the dialysis purification time is 24 h.

5. A multi-component brain-targeting nano-formulation of traditional Chinese medicine for the treatment of depression, characterized in that, The raw materials for preparing the multi-component brain-targeting nano-preparation of traditional Chinese medicine include: Nanocarriers PEG-GO, ferulic acid, and verbascoside; The mass ratio of the nanocarrier PEG-GO, ferulic acid, and verbascoside is 10:1:

1. The multi-component brain-targeting nano-preparation of traditional Chinese medicine is prepared by the preparation method described in any one of claims 1-4.

6. The use of a multi-component brain-targeting nano-formulation of traditional Chinese medicine as described in claim 5 in the preparation of a drug for treating depression.

7. A pharmaceutical composition for treating depression, characterized in that, The pharmaceutical composition comprises the multi-component brain-targeting nano-formulation of traditional Chinese medicine as described in claim 5, and pharmaceutically acceptable excipients; In the pharmaceutical composition, the mass content of the multi-component brain-targeting nano-preparation of traditional Chinese medicine is greater than or equal to 8.06%; The pharmaceutical composition is administered via intravenous injection.

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