Liposome preparation capable of improving anti-depression effect of fluoxetine and preparation method thereof

Abstract

The invention provides a lipidosome preparation capable of performing brain-targeted drug delivery in a depression model, a preparation method of the lipidosome preparation and application of the lipidosome preparation in improving the anti-depression effect of fluoxetine, and belongs to the technical field of pharmaceutical preparations. The composition disclosed by the invention consists of DOPG, DSPE-PEG2000-PGP, DSPE-PEG2000, cholesterol, fluoxetine and celecoxib, and can be used for improving the anti-depression effect of fluoxetine. The double-drug-loading liposome with high entrapment efficiency and uniform particle size is prepared through a film hydration-film extrusion method, has good stability and biocompatibility, has an obvious targeting drug delivery effect in a chronic social contusion stress model, is suitable for a combined treatment strategy of depression, and has a good clinical application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical preparations, in particular, to a liposome preparation with brain-targeting drug delivery characteristics to improve the antidepressant effect of fluoxetine in a depression model and a preparation method thereof. BACKGROUND

[0002] Depression is one of the most common mental illnesses, and its clinical manifestations include low mood, reduced consciousness, impaired cognitive function, and body pain and discomfort, and even suicidal tendencies, which severely reduces the quality of life and endangers physical and mental health. According to the World Health Organization's 2023 report on depression, depression is a major cause of global mental and physical disability and has a significant impact on global disease burden. Depression patients are distributed all over the world, and an estimated 3.8% of the global population is affected, including 5% of adults and 5.7% of adults over 60 years old, with a total of about 280 million people suffering from depression.

[0003] The onset of depression is usually gradual, but it can also be sudden. The duration of the attack, the number of attacks in a lifetime, and the pattern of the attack are all variable. Severe depression is a recurring lifelong illness, and more than 50% of people affected by severe depression recover within 6 months, and about 3 / 4 recover within a year, but some (as high as 27%) patients do not recover and continue to develop into chronic depression, which is related to the patient's treatment environment.

[0004] Selective serotonin reuptake inhibitors (SSRI) are currently more commonly used in clinical antidepressants. SSRI selectively inhibits the reuptake of 5-hydroxytryptamine at the presynaptic membrane, and has less effect on norepinephrine and dopamine. Commonly used SSRI drugs in clinical treatment include fluoxetine, paroxetine, trazodone, fluvoxamine, citalopram, etc. Due to high tolerance and broad-spectrum efficacy, SSRI drugs are used as first-line drugs for treating depression in patients of different ages. However, their clinical effects are still only effective for some patients, and they need to be used for a period of time before they take effect. The main reason for this may be that the drug concentration in the relevant brain area is not enough, resulting in slow onset and poor efficacy.

[0005] Clinical studies have shown that combining classic antidepressants with nonsteroidal anti-inflammatory drugs (NSAIDs) can enhance antidepressant effects. For example, in a trial of 24 patients who did not respond to SSRIs initially, a combination of SSRIs (160 mg / day) for 4 weeks significantly improved response rates and remission. Four trials (N=160) investigated celecoxib in combination with antidepressants; three trials used 400 mg / day for 6 weeks, and one trial used 200 mg / day for 8 weeks. All studies supported a significant improvement in antidepressant efficacy when combined with celecoxib.

[0006] Exploring the simultaneous encapsulation of fluoxetine and the anti-inflammatory drug celecoxib for the treatment of depression could provide a more favorable strategy. Liposome delivery systems have become one of the most studied and widely used drug delivery systems. For example, triserline liposomes can enhance antidepressant effects. Therefore, liposomal formulations may be an important means of administration to improve the antidepressant effects of SSRIs.

[0007] The chronic social defeat stress (CSDS) model is a primary method for inducing depressive moods and behaviors in experimental animals. It employs both physiological and psychological stress, and assesses drug effectiveness using commonly used depression assessment indicators. This model effectively simulates the inevitable depressive behaviors that humans experience in daily social life due to excessive stress and intense frustration caused by bullying or humiliation. This model is now widely used in researching the pathological mechanisms of depression and in the development of antidepressants. Therefore, this paper proposes a liposomal formulation that can enhance the antidepressant effect of fluoxetine and its preparation method. Summary of the Invention

[0008] The technical problem solved by this invention is to overcome the clinical problem that some patients do not respond well or respond slowly during recurrent episodes of depression. A liposomal formulation has been invented that can improve drug efficacy by increasing the concentration of the formulation in relevant brain regions during recurrent episodes of depression, thereby enhancing the antidepressant effect.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: a liposomal formulation with brain-targeted drug delivery characteristics that can enhance the antidepressant effect of fluoxetine, wherein the liposomal formulation comprises DOPG, DSPE-PEG2000-PGP, DSPE-PEG2000, cholesterol, fluoxetine, and celecoxib.

[0010] As a preferred embodiment of the present invention, the molar ratio of DOPG, cholesterol, DSPE-PEG2000 and DSPE-PEG2000-PGP is (62~68:27~33:3~7) (mol / mol), wherein (mol / mol) is preferred.

[0011] As a preferred embodiment of the present invention, the liposome formulation has a particle size between 120nm and 130nm and a potential between -15mV and 9.45mV.

[0012] As a preferred embodiment of the present invention, the ratio of fluoxetine to DOPG is 6:20-7:20.

[0013] As a preferred technical solution of the present invention, the ratio of the amount of celecoxib to the amount of DOPG is 0.38:20-0.5:20.

[0014] As a preferred technical solution of the present invention, the liposome formulation includes dual-drug-loaded conventional liposomes (fluoxetine / celecoxib liposome, FXT / CLX-Lip) and dual-drug-loaded targeted liposomes (fluoxetine / celecoxib PGP-liposome, FXT / CLX-PGP-Lip).

[0015] A method for preparing a liposomal formulation that can enhance the antidepressant effect of fluoxetine includes the following steps:

[0016] Step 1: Weigh 20mg of DOPG, and weigh cholesterol and DSPE-PEG2000 / DSPE-PEG2000-PGP according to the ratio of DOPG:cholesterol:DSPE-PEG2000 / DSPE-PEG2000-PGP = (mol / mol);

[0017] Step 2: Accurately weigh 10.00 mg of fluoxetine and 0.60 mg of celecoxib, and dissolve them together with phospholipids and cholesterol in an appropriate amount of chloroform.

[0018] Step 3: Evaporate the solvent by rotary evaporation in a 37°C water bath, add 4 ml of PBS solution, and hydrate at 37°C.

[0019] Step 4: Pass the liposome solution through 400nm and 200nm polyester carbonate membranes in sequence to obtain dual-drug-loaded ordinary liposomes or dual-drug-loaded targeted liposomes.

[0020] As a preferred technical solution of the present invention, the method further includes the step of preparing DSPE-PEG2000-PGP: DSPE-PEG2000-MAL and PGP (mol / mol = 1:1.5) are weighed and dissolved in PBS solution. After stirring and reacting in a sealed, light-protected environment at room temperature for 24 hours, the mixture is dialyzed for 48 hours at room temperature in the light-protected environment using a 2000D dialysis bag. The dialysis medium is ultrapure water. After dialysis, the liquid in the dialysis bag is removed and placed in a -80℃ freezer for freezing. The mixture is then freeze-dried in a freeze dryer to obtain the product DSPE-PEG2000-PGP, which is stored at -20℃.

[0021] The application of a liposomal formulation with brain-targeted drug delivery characteristics in the preparation of a drug to enhance the therapeutic effect of fluoxetine on a model animal of chronic social frustration-induced depression.

[0022] A method for characterizing the physicochemical properties of a liposomal formulation with brain-targeted drug delivery characteristics, comprising the following steps:

[0023] Step 1: Take the dual-drug-loaded liposomes, dilute them 5 times with pure water, and use a Zetasizer Nano-ZS laser particle size analyzer to measure the particle size and zeta potential of the dual-drug-loaded liposomes.

[0024] Step 2: Dilute the dual-drug-loaded liposomes and drop them onto a copper grid. After air drying, add 2% phosphotungstic acid for negative staining for 1 minute. After absorbing excess stain with filter paper, observe the morphology of the liposomes using a transmission electron microscope.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: the liposomes are surface modified by using DSPE-PEG2000-PGP, which enables the liposomes to target and cross the blood-brain barrier, significantly improves the distribution concentration of the drug in the central nervous system, and further enhances its antidepressant activity.

[0026] This invention enhances the antidepressant therapeutic effect by co-encapsulating fluoxetine and celecoxib in liposomes and utilizing their synergistic pharmacological effects. Celecoxib, as a COX-2 inhibitor, has anti-inflammatory and neuroprotective effects, which help improve depression-related neuroinflammatory responses, thereby improving the therapeutic efficacy of fluoxetine.

[0027] This invention, by precisely controlling the ratio of DOPG, cholesterol, and DSPE-PEG2000 / DSPE-PEG2000-PGP (mol / mol), not only improves the physical stability of liposomes but also significantly enhances the encapsulation efficiency of fluoxetine and celecoxib, ensuring the controllability and effectiveness of drug release in vivo. The resulting liposomes have a particle size controlled between 120 and 130 nm and a zeta potential of -15 mV to -9 mV, meeting the requirements for intravenous injection formulations, which is beneficial for prolonging blood circulation time and reducing rapid clearance by the reticuloendothelial system.

[0028] The preparation method provided by this invention is simple to operate and operates under mild conditions. It includes a thin-film hydration method combined with membrane extrusion technology, which can efficiently obtain liposomes with uniform particle size and good morphology, making it suitable for large-scale production and clinical translation. The materials used are all commonly used pharmaceutical excipients, such as DOPG, cholesterol, and DSPE-PEG2000, which have no toxic side effects, good biocompatibility, and are suitable for long-term use as carriers for antidepressants.

[0029] This invention is the first to co-encapsulate fluoxetine and celecoxib in targeted liposomes, providing a novel formulation platform for the combination treatment of depression and serving as a reference example for the development of other multi-target antipsychotic drugs. Experimental results show that the liposomal formulation of this invention exhibits significantly better antidepressant behavioral improvement effects than free fluoxetine in an animal model of chronic social frustration stress (CSDS), demonstrating a clear preclinical research basis. Attached Figure Description

[0030] Figure 1 This is a physicochemical characterization diagram of the dual-drug-loaded liposomes of the present invention;

[0031] Figure 2 This is a chromatogram of the HPLC specificity results of celecoxib according to the present invention;

[0032] Figure 3 This is a chromatogram showing the HPLC specificity of fluoxetine in this invention.

[0033] Figure 4 This is a diagram showing the distribution of the liposomes of the present invention in a mouse model of secondary depression after multiple administrations.

[0034] Figure 5 This is a schematic diagram of the sampling time for the secondary depression model of the present invention;

[0035] Figure 6 This is a distribution diagram of the liposomes of the present invention in the hippocampal region of mice in primary and secondary depression models;

[0036] Figure 7 The distribution of liposomes in the mouse amygdala at each stage of the primary and secondary depression models of this invention;

[0037] Figure 8 This is a graph showing the behavioral evaluation results of mice in the depression model after multiple doses of the present invention.

[0038] Figure 9 This is a diagram showing the changes in Nissl bodies in the hippocampus after multiple drug administrations according to the present invention.

[0039] Figure 10 This is a diagram showing the HE staining results of various tissues after multiple drug administrations in the secondary depression model of this invention.

[0040] Figure 11 This is a diagram showing the drug loading and encapsulation efficiency of the dual-drug-loaded liposomes of the present invention. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0042] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely illustrates some embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. It should be noted that, in the absence of conflict, the embodiments and features and technical solutions in the embodiments of the present invention can be combined with each other. It should be noted that similar reference numerals and letters in the following figures indicate similar items; therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0043] The reagents involved in the embodiments of this invention are as follows: dioleoylphosphatidylglycerol (DOPG), distearate-rac-glycerol-polyethylene glycol 2000 (DSPE-PEG2000), phosphatidylethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG2000-MAL): Aivito (Shanghai) Pharmaceutical Technology Co., Ltd., China;

[0044] Ac-Pro–Gly–Pro(PGP) peptide: Hubei Qiangyao Biotechnology Co., Ltd., China;

[0045] Cholesterol: Shanghai Maclean Biotechnology Co., Ltd., China;

[0046] Fluoxetine, Celecoxib: Shanghai Aladdin Biochemical Technology Co., Ltd., China;

[0047] Chloroform, Tetrahydrofuran: Sinopharm Reagent, China;

[0048] Methanol (MeOH), Acetonitrile (ACN): Fisher Scientific International Inc., USA;

[0049] Sephadex TM G-50 Medium: GE Healthcare, USA;

[0050] [Example 1] Preparation and property evaluation of dual drug-loaded liposomes

[0051] Preparation of dual-drug-loaded liposomes: DSPE-PEG2000-MAL and PGP (mol / mol = 1:1.5) were weighed and dissolved in PBS solution. The mixture was stirred and reacted in a sealed, light-protected environment at room temperature for 24 h. Then, the mixture was dialyzed for 48 h at room temperature in the light-protected environment using a 2000D dialysis bag. The dialysis medium was ultrapure water. After dialysis, the liquid in the dialysis bag was removed and frozen in a -80℃ freezer. The product DSPE-PEG2000-PGP (PGP) was then freeze-dried in a freeze dryer and stored at -20℃.

[0052] Weigh 20 mg of DOPG. Weigh cholesterol and DSPE-PEG2000 / DSPE-PEG2000-PGP in a DOPG:cholesterol:DSPE-PEG2000 / DSPE-PEG2000PGP ratio (mol / mol). Accurately weigh 10.00 mg of fluoxetine (FXT) and 0.60 mg of celecoxib (CLX). Dissolve them together with the phospholipids and cholesterol in an appropriate amount of chloroform. Evaporate the solvent by rotary evaporation in a 37°C water bath. Add 4 ml of PBS solution and hydrate at 37°C. Pass the liposome solution sequentially through 400 nm and 200 nm polyester carbonate membranes to obtain dual-drug-loaded conventional liposomes (fluoxetine / celecoxib liposome, FXT / CLX-Lip) and dual-drug-loaded targeted liposomes (fluoxetine / celecoxib PGP-liposome, FXT / CLX-PGP-Lip).

[0053] Physicochemical characterization of liposomes: Two types of dual-drug-loaded liposomes (FXT / CLX-Lip, FXT / CLX-PGP-Lip) were diluted 5 times with pure water. The particle size and zeta potential of the dual-drug-loaded liposomes were measured using a Zetasizer Nano-ZS laser particle size analyzer. The diluted dual-drug-loaded liposomes (FXT / CLX-Lip, FXT / CLX-PGP-Lip) were dropped onto a copper grid and allowed to air dry. Then, 2% phosphotungstic acid was added for negative staining for 1 min. After absorbing excess stain with filter paper, the morphology of the liposomes was observed using a transmission electron microscope (TEM). The results showed that the particle size of ordinary liposomes was approximately 124.3 nm, and the zeta potential was approximately -9.45 mV; the particle size of the dual-drug-loaded targeted liposomes was approximately 129.0 nm, and the zeta potential was approximately -14.80 mV. This indicates that target modification had no significant effect on the particle size and zeta potential of the liposomes. TEM observation showed that both types of liposomes were approximately spherical. Figure 1 ).

[0054] Celecoxib HPLC determination conditions: using A 100RP-8 column was used with a mobile phase of methanol:water = 85:15, an ultraviolet detector at a detection wavelength of 254 nm, a flow rate of 1 ml / min, a column temperature of 25 ℃, and an injection volume of 10 μl.

[0055] Accurately weigh 10.00 mg of celecoxib reference standard, place it in a 10 ml volumetric flask, add an appropriate amount of methanol to dissolve it, continue to add methanol to make up to volume, shake well, and obtain a 1.00 mg / ml celecoxib reference standard stock solution.

[0056] Accurately transfer 2 ml of the mother liquor to a 10 ml volumetric flask, and dilute to the mark with methanol to obtain a 500 μg / ml celecoxib standard solution. Repeat the same procedure to obtain a 31.25 μg / ml celecoxib standard solution. Inject 10 μl of the standard solution directly into the liquid chromatograph and record the chromatogram. Figure 2 .

[0057] Add 250 μl of 5% Triton X-100 to blank liposomes to break the emulsion, then dilute with methanol to obtain the excipient negative control. Inject 10 μl of the negative control solution into the liquid chromatograph and record the chromatogram. Figure 2 .

[0058] Take blank liposomes, add 250 μl of 5% Triton X-100 to demulsify, then add celecoxib stock solution with a concentration of 1.00 mg / mL to obtain the sample solution. Inject 10 μl of standard solution into the liquid chromatograph and record the chromatogram. Figure 2 .

[0059] Linearity study of celecoxib: A 1.00 mg / ml celecoxib stock solution was diluted with methanol to prepare a series of standard solutions of 500, 250, 125, 62.5, 31.25, 31.25, 15.625 and 7.8125 μg / ml. 10 μl of each solution was injected under the above chromatographic conditions, and the peak area was recorded. Linear regression was performed with concentration on the x-axis and peak area on the y-axis, yielding the regression equation y = 25.484x + 271.8 (r = 0.998). The results showed good linearity in the range of 175-1000 μg / ml.

[0060] Fluoxetine content determination method by HPLC: A 100RP-8 column was used with a mobile phase of tetrahydrofuran:methanol:triethylamine buffer (pH=5.8)=35:10:55. An ultraviolet detector was used with a detection wavelength of 227nm, a flow rate of 1ml / min, a column temperature of 25℃, and an injection volume of 10μl.

[0061] Accurately weigh 10.00 mg of fluoxetine standard, place it in a 10 ml volumetric flask, add an appropriate amount of methanol to dissolve it, add methanol to make up to volume, shake well, and obtain a 1.00 mg / ml fluoxetine standard stock solution.

[0062] Accurately transfer 400 μl of the 1.00 mg / ml fluoxetine standard stock solution and dilute with 600 μl of methanol to obtain a 400.00 μg / ml fluoxetine standard solution.

[0063] Take blank liposomes, add methanol to demulsify and obtain a negative control, record the chromatogram, such as... Figure 3 .

[0064] Take blank liposomes, add fluoxetine standard stock solution, and dilute with methanol to obtain a 100 μg / ml fluoxetine sample solution. Record the chromatogram, as shown below. Figure 3 .

[0065] like Figure 3 As shown in the figure, the fluoxetine standard solution eluted at 8 min, while the blank excipient did not show a peak at that time. The fluoxetine sample solution eluted at 8 min, indicating that the excipient did not interfere with the elution of fluoxetine. This condition can be used for subsequent detection.

[0066] Linearity of fluoxetine:

[0067] A 1.00 mg / ml fluoxetine standard stock solution was diluted with methanol to prepare a series of standard solutions of 200, 100, 20, 10, and 1 μg / ml. 10 μl of each solution was injected under chromatographic conditions, and the peak area was recorded. Linear regression was performed with the injection concentration as the x-axis and the response peak area as the y-axis, yielding the regression equation y = 18.812x + 13.825 (r = 0.999), indicating a good linear relationship within the range of 1-200 μg / ml.

[0068] Establishment of a method for determining the encapsulation efficiency of dual-drug-loaded liposomes:

[0069] Dual-drug-loaded liposomes were loaded onto a Sephadex™ MG-50 Medium chromatography column (60 mm inner diameter, 145 mm packing material). 3 ml of liposome solution was loaded, and eluted with PBS to remove free drug. The eluent flow rate was controlled at 1 ml / min, 1 ml / tube, and 10 tubes were collected. The eluent was then diluted 10-fold with methanol. The contents of fluoxetine and celecoxib were determined by HPLC, and the encapsulation efficiency and drug loading were calculated. The results are shown below. Figure 11 .

[0070] Encapsulation efficiency = (Material mass of drug in liposomes / Total amount of drug in liposomes) × 100%

[0071] Drug loading capacity = (Mass of drug in liposomes / (Mass of drug in liposomes + Mass of lipids)) × 100%

[0072] [Effective Example 1] Brain-targeted distribution of liposomes in a secondary depression animal model:

[0073] Establishment of a single-dose CSDS depression model and multiple-dose regimen

[0074] After CD1 mice were housed individually for 7 days to develop territorial awareness, C57BL / 6 mice were placed in the CD1 cages for 3 minutes. If a CD1 mouse initiated multiple attacks, exhibiting aggressive behavior every minute, with an attack latency of less than 1 minute, it was considered aggressive and could be used as an aggressive mouse in the CSDS model. The C57BL / 6 mice used to screen for aggressive mice were not subsequently used to establish the CSDS model.

[0075] C57BL / 6 mice were placed in CD1 cages for 10 consecutive days. After being challenged for 5 minutes, they were isolated for 24 hours using a perforated transparent acrylic plate. New CD1 challenge mice were introduced daily to prevent C57BL / 6 adaptation. If a CD1 mouse showed decreased aggression or exhibited social behaviors such as grooming, the challenge mouse was culled, and a new challenge mouse was used for the experiment. The control group did not have contact with the challenge mice and was housed in the same environment as the experimental group. After 10 days of model establishment, all mice were housed individually. On day 11, the control and model groups underwent a social interaction test (SIT) to verify the successful establishment of the CSDS model. Starting on day 12, mice were administered dual-drug liposomes or PBS for 10 consecutive days. Control group mice were housed in the same environment as the model group mice, without stress stimulation or medication.

[0076] Establishment of a secondary CSDS depression model (rCSDS depression model) and multiple dosing regimen

[0077] Mice that successfully established a CSDS model for the first time were administered the drug for 10 consecutive days, followed by another 10 days of stress stimulation to induce a secondary CSDS-induced depression model. Starting on day 12, mice were administered dual-drug-loaded liposomes or PBS for 6 consecutive days. Control group mice were housed in the same environment as the model group mice, without stress stimulation or drug administration. A schematic diagram of the administration regimen is shown below. Figure 4 A.

[0078] Distribution of liposomes in a secondary CSDS depression model after multiple administrations: On day 8 after successful secondary CSDS modeling, C57BL / 6 mice were administered Lip@DiD, PGP-Lip@DiD, or PBS solution via tail vein, with 4 mice per group. Thirty minutes after administration, mice were anesthetized with isoflurane using an air anesthesia machine and placed in an InVivo Imaging Systems (IVIS) to observe the distribution of liposomes in the model mice. After imaging, each mouse was perfused sequentially with PBS solution and 4% paraformaldehyde solution via the heart. The brain, heart, liver, spleen, lung, and kidney of the mice were removed and placed on black cardstock. The distribution of liposomes in different isolated tissues was observed using the InVivo Imaging Systems. Results of liposome distribution in vivo after multiple administrations following successful secondary CSDS depression modeling. Figure 4 Figure 5 As shown in B. Thirty minutes after administration, significant fluorescent signals were observed in the brains of mice in both the Lip@DiD group and the PGP-Lip@DiD group. Figure 5 Figure 4 B), based on the statistical results ( Figure 5 Figure 4 C) It can be seen that the overall fluorescence intensity of both groups was significantly higher than that of the PBS group, and the PGP-targeted group was significantly higher than that of the ordinary liposome group. This indicates that both the ordinary liposome group and the targeted liposome group could reach the brain after multiple administrations in the secondary depression animal model, with the targeted formulation showing better efficacy. Obvious fluorescence signals were observed in the liver and spleen of both the Lip@DiD group and the PGP-Lip@DiD group. Figure 5 Figure 4 D) indicates that liposomes are distributed in the liver and spleen.

[0079] Distribution of liposomes in different brain regions after multiple administrations to different CSDS depression model animals: C57BL / 6 mice, day 8 after successful modeling (first / Day 8); day 8 after successful modeling (second / Day 8). Figure 6 As shown, mice were injected via tail vein with Lip@DiD, PGP-Lip@DiD, or PBS solution (n=3, dosage calculated based on lipids: 50 mg / kg). Thirty minutes after administration, mice were anesthetized with 5% chloral hydrate, and the heart was perfused sequentially with PBS solution and 4% paraformaldehyde solution. Brain tissue was then harvested. The experimental protocol is detailed below. Figure 5 .

[0080] The obtained brain tissue was placed in 4% paraformaldehyde and fixed at 4°C for at least 24 hours. After removal, it was dehydrated sequentially by a gradient of 20% sucrose solution and 30% sucrose solution, then embedded in OCT embedding medium, coronally sectioned using a cryostat, and mounted on an adhesive glass slide to obtain a frozen section of the target brain region with a thickness of 10 μm.

[0081] Cryosections of brain tissue were washed three times with PBS (1X) to remove embedding medium, then stained with DAPI and incubated at room temperature in the dark for 5 min. The sections were then washed three times with PBS and mounted with anti-fluorescence quenching mounting medium. The distribution of liposomes throughout the brain, as well as in the hippocampus and amygdala, was observed using an Olympus VS200 digital scanning system.

[0082] The distribution of different liposomes in the hippocampus and the results of their semi-quantitative statistics are as follows: Figure 6 Figure 7 As shown in AD. In the first depression model, after 6 consecutive days of administration starting from day 2 post-modeling (First / Day 8), and in the second depression model, after 6 consecutive days of administration starting from day 2 post-modeling (Second / Day 8), enrichment of both Lip@DiD and PGP-Lip@DiD liposomes was observed in the hippocampus. Semi-quantitative statistical results showed ( Figure 7 Figure 6 In the E and F models, after multiple administrations in a single depression model, PGP-Lip@DiD showed a higher total fluorescence intensity in the hippocampus than Lip@DiD, but in the secondary depression model, after multiple administrations, there was no difference in the total fluorescence intensity between the two.

[0083] The distribution of different liposomes in the amygdala region and the results of their semi-quantitative statistics are as follows: Figure 7 As shown. In both the first depression model and the second depression model, two types of liposomes were observed in the amygdala region after 6 consecutive days of drug administration starting from day 2 after model establishment (First / Day 8). Semi-quantitative statistical results showed ( Figure 7 In the first depression model, after multiple administrations, the total fluorescence intensity of PGP-Lip@DiD in the amygdala region was significantly higher than that of Lip@DiD. In the second depression model, there was no significant difference in fluorescence between PGP-Lip@DiD and Lip@DiD.

[0084] [Effective Example 2] Results of multiple drug administration in a single depression model:

[0085] Efficacy results of multiple dosing in a single CSDS depression model: In the tail suspension experiment ( Figure 8 Compared with the free drug group, the immobility time of mice in the FXT / CLX-PGP-Lip group was significantly reduced, indicating that PGP-modified liposomes (PGP-Lip) can significantly improve the antidepressant effect of FXT / CLX, and targeted delivery can provide a powerful means for the treatment of depression.

[0086] [Effective Example 3] Effects of repeated drug administration on neurons in a secondary depression model:

[0087] Effects of repeated drug administration on hippocampal neurons in a secondary depression model:

[0088] Nissl staining results of the hippocampus in brain slices at various stages are as follows: Figure 10 After a single modeling treatment for 6 consecutive days, the number of Nissl bodies in the PBS group was significantly reduced compared to the normal group. Figure 9 Figure AB) indicates that CSDS stress damages neurons. The number of Nissl bodies in the hippocampus was significantly increased in the FXT / CLX-Lip group and the FXT / CLX-PGP-Lip group compared with the PBS group (Figure B). There was no significant difference between the FXT / CLX-Lip group and the FXT / CLX-PGP-Lip group, indicating that FXT / CLX-Lip and FXT / CLX-PGP-Lip have a restorative effect on hippocampal neurons.

[0089] After six consecutive days of administration following the second modeling, there was no significant difference in the number of Nissl bodies in the hippocampus among the groups (Figure C). It is speculated that the treatment with FXT / CLX-Lip and FXT / CLX-PGP-Lip prior to the second modeling may have a preventive effect on neuronal damage caused by CSDS stress. Comparing the number of Nissl bodies in the hippocampus during the two phases, the PBS group showed a significant increase in Nissl bodies after six consecutive days of administration following both the first and second modeling. This may be due to the spontaneous repair of neurons during the interval between the two modeling phases, and because the mice were desensitized to external stimuli after the first modeling, the number of Nissl bodies did not decrease significantly after the second modeling.

[0090] [Effective Example 4] Safety evaluation results of multiple doses in a secondary depression model:

[0091] The results of hematoxylin-eosin staining (HE) in a secondary depression model with multiple drug administration are as follows: Figure 10 As shown, the cardiac muscle fibers in each group were smooth, the edges of liver, spleen, and lung cells were clear and intact, and the kidney cells were smooth and rounded. No obvious lesions were observed in any tissue, indicating that the liposomes showed good safety with repeated administration.

[0092] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described herein. Although the present invention has been described in detail with reference to the above embodiments, the present invention is not limited to the specific embodiments described above. Therefore, any modifications or equivalent substitutions to the present invention, as well as all technical solutions and improvements that do not depart from the spirit and scope of the invention, are covered within the scope of the claims of the present invention.

Claims

1. A liposomal formulation with brain-targeted drug delivery characteristics, which enhances the antidepressant effect of fluoxetine, characterized in that, The liposome formulation contains DOPG, DSPE-PEG2000-PGP, DSPE-PEG2000, cholesterol, fluoxetine, and celecoxib.

2. The liposome formulation with brain-targeted drug delivery characteristics according to claim 1, characterized in that, The molar ratio of DOPG, cholesterol, DSPE-PEG2000 and DSPE-PEG2000-PGP is (62~68:27~33:3~7), of which the preferred ratio is (mol / mol).

3. The liposome formulation with brain-targeted drug delivery characteristics according to claim 1, characterized in that, The liposome formulation has a particle size between 120 nm and 130 nm and a potential between -15 mV and 9 mV.

4. The liposome formulation with brain-targeted drug delivery characteristics according to claim 1, characterized in that, The ratio of fluoxetine to DOPG is 6:20-7:

20.

5. The liposome formulation with brain-targeted drug delivery characteristics according to claim 1, characterized in that, The ratio of celecoxib to DOPG was 0.38:20 to 0.5:

20.

6. The liposome formulation with brain-targeted drug delivery characteristics according to claim 1, characterized in that, The liposome formulations include dual-drug-loaded conventional liposomes (fluoxetine / celecoxib liposome, FXT / CLX-Lip) and dual-drug-loaded targeted liposomes (fluoxetine / celecoxib PGP-liposome, FXT / CLX-PGP-Lip).

7. A method for preparing a liposome formulation with brain-targeted drug delivery characteristics, characterized in that, Includes the following steps: Step 1: Weigh 20mg of DOPG, and weigh cholesterol and DSPE-PEG2000 / DSPE-PEG2000-PGP according to the ratio of DOPG:cholesterol:DSPE-PEG2000 / DSPE-PEG2000-PGP = (62~68:27~33:3~7) (mol / mol); Step 2: Accurately weigh 10.00 mg of fluoxetine and 0.60 mg of celecoxib, and dissolve them together with phospholipids and cholesterol in an appropriate amount of chloroform. Step 3: Evaporate the solvent by rotary evaporation in a 37°C water bath, add 4 ml of PBS solution, and hydrate at 37°C. Step 4: Pass the liposome solution through 400nm and 200nm polyester carbonate membranes in sequence to obtain dual-drug-loaded ordinary liposomes or dual-drug-loaded targeted liposomes.

8. A method for preparing a liposome formulation with brain-targeted drug delivery characteristics according to claim 7, characterized in that, The procedure also includes the preparation of DSPE-PEG2000-PGP: DSPE-PEG2000-MAL and PGP (mol / mol = 1:1~2) were weighed and dissolved in PBS solution. The mixture was stirred and reacted in a sealed, light-protected environment at room temperature for 24 hours. Then, dialysis was performed using a 2000D dialysis bag at room temperature in the light-protected environment for 48 hours. The dialysis medium was ultrapure water. After dialysis, the liquid in the dialysis bag was removed and placed in a -80℃ freezer for freezing. The product DSPE-PEG2000-PGP was then freeze-dried in a freeze dryer and stored at -20℃.

9. The use of a liposomal formulation with brain-targeted drug delivery characteristics according to any one of claims 1 to 6 in the preparation of a drug for improving the treatment of fluoxetine in animal models of chronic social frustration stress depression.

10. A method for characterizing the physicochemical properties of a liposomal formulation with brain-targeted drug delivery characteristics according to any one of claims 1 to 6, characterized in that, Includes the following steps: Step 1: Take the dual-drug-loaded liposomes, dilute them 5 times with pure water, and use a Zetasizer Nano-ZS laser particle size analyzer to measure the particle size and zeta potential of the dual-drug-loaded liposomes. Step 2: Dilute the dual-drug-loaded liposomes and drop them onto a copper grid. After air drying, add 2% phosphotungstic acid for negative staining for 1 minute. After absorbing excess stain with filter paper, observe the morphology of the liposomes using a transmission electron microscope.

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