Application of bitter melon-derived nanovesicles in the preparation of brain-targeting drugs

Abstract

This invention discloses the application of bitter melon-derived nanovesicles in the preparation of brain-targeted drugs, belonging to the field of biomedical technology. Specifically, this invention discloses that bitter melon-derived nanovesicles, used as carriers to load active substances, can be orally administered to deliver the active substances to the brain in a targeted manner, avoiding the inconvenience and risks of injection administration and providing a more convenient and more compliant drug delivery method for the treatment of brain diseases. Furthermore, bitter melon-derived nanovesicles have the advantages of good biocompatibility, no immunogenicity, safe metabolism in vivo, and long-term use.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of bitter melon-derived nanovesicles in the preparation of brain-targeting drugs. Background Technology

[0002] Brain diseases are a serious threat to human health, encompassing various types such as sleep disorders and neurodegenerative diseases. Their incidence is increasing year by year with population aging and changes in lifestyle. Among them, insomnia is the most common sleep disorder, affecting approximately 10%-30% of adults worldwide, with chronic insomnia patients accounting for more than 10%. Long-term insomnia can induce a series of complications such as anxiety, depression, and cognitive decline.

[0003] The blood-brain barrier (BBB) ​​is a crucial physiological barrier protecting the stability of the brain's microenvironment. Composed of brain capillary endothelial cells, astrocyte foot processes, and the basement membrane, it exhibits high selective permeability, effectively preventing harmful substances from the bloodstream from entering the brain. However, the BBB has also become a major technical bottleneck in the treatment of brain diseases. Over 98% of small-molecule drugs and almost all large-molecule drugs struggle to penetrate the BBB, resulting in excessively low drug concentrations in the brain and failing to achieve effective therapeutic effects. To increase drug concentrations in the brain, clinicians often need to increase the dosage, which not only increases systemic adverse reactions but may also lead to drug resistance.

[0004] In recent years, naturally derived nanovesicles (especially plant-derived nanovesicles) have attracted widespread attention in the field of targeted delivery due to their unique advantages. Plant-derived nanovesicles are characterized by good biocompatibility, low immunogenicity, wide availability, and low cost. However, after being orally administered into the intestines, nanovesicles readily accumulate in abdominal organs such as the liver, spleen, and pancreas via the intestinal lymphatic network or portal vein system, making it difficult for them to cross the blood-brain barrier and reach the brain. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes the application of bitter melon-derived nanovesicles in the preparation of brain-targeting drugs.

[0006] The application of bitter melon-derived nanovesicles according to a first aspect of the present invention in the preparation of a brain-targeting drug, wherein the brain-targeting drug is an oral brain-targeting drug.

[0007] The application of the present invention, according to embodiments thereof, has at least the following beneficial effects: Bitter melon-derived nanovesicles are derived from bitter melon, a food and medicine source. They exhibit good biocompatibility, are non-immunogenic, and are safe for in vivo metabolism, allowing for long-term use. After oral administration, these nanovesicles can penetrate the blood-brain barrier and specifically accumulate in brain tissue. This allows for targeted delivery of active ingredients that cannot otherwise cross the blood-brain barrier to the brain, significantly increasing the concentration and duration of action of these active ingredients. This avoids the inconvenience and risks of injection-based administration, providing a more convenient and patient-compliant method for treating brain diseases. It also offers a novel technological solution for treating brain-related diseases such as sleep disorders, expanding the application areas of bitter melon-derived nanovesicles and possessing significant scientific value, clinical translational value, and broad commercial prospects.

[0008] According to some embodiments of the present invention, the bitter melon-derived nanovesicles have a circular or cup-shaped appearance and a disc-shaped concave structure.

[0009] According to some embodiments of the present invention, the particle size distribution range of the bitter melon-derived nanovesicles is 50 nm-500 nm. For example: it can be 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360nm, 370 nm, 380 nm, 390 nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470nm, 480 nm, 490 nm or 500 nm nm.

[0010] According to some embodiments of the present invention, the zeta potential of the bitter melon-derived nanovesicles is -8.0 mV to 8.0 mV. For example, it can be -8.07 mV, -7.5 mV, -7 mV, -6.5 mV, -6 mV, -5.5 mV, -5 mV, -4.5 mV, -4 mV, -3.5 mV, -3 mV, -2.5 mV, -2 mV, -1.5 mV, -1 mV, -0.5 mV, 0.5 mV, 1 mV, 1.5 mV, 2 mV, 2.5 mV, 3 mV, 3.5 mV, 4 mV, 4.5 mV, 5 mV, 5.5 mV, 6 mV, 6.5 mV, 7 mV, 7.5 mV, or 8 mV.

[0011] According to some embodiments of the present invention, the zeta potential of the bitter melon-derived nanovesicles is -8.0 mV to -2.0 mV.

[0012] According to some embodiments of the present invention, the method for preparing the bitter melon-derived nanovesicles includes: separating the bitter melon-derived nanovesicles from the bitter melon homogenate by at least one of differential centrifugation, ultracentrifugation, density gradient centrifugation, ultrafiltration, and size exclusion chromatography.

[0013] According to some embodiments of the present invention, the preparation method of the bitter melon-derived nanovesicles specifically includes the following steps: Centrifuge the supernatant of the bitter melon homogenate at 500×g-1500×g for 5 min-15 min, and collect the first supernatant; centrifuge at 2000×g-4000×g for 20 min-40 min, and collect the second supernatant; centrifuge at 8000×g-12000×g for 40 min-80 min, and collect the third supernatant; centrifuge at 100000×g-200000×g for 90 min-150 min to obtain the first precipitate; The first precipitate was centrifuged in a gradient sucrose solution of 6w / v%-10w / v%, 20w / v%-40w / v%, 40w / v%-50w / v%, and 55w / v%-65w / v% for 100,000×g-200,000×g for 90 min-150 min, and the resulting second precipitate was the bitter melon-derived nanovesicles. The white band at the interface of the 40w / v%-50w / v% and 55w / v%-65w / v% sucrose solutions was the second precipitate.

[0014] According to some embodiments of the present invention, the brain-targeting drug comprises at least one of an active substance and a pharmaceutically acceptable excipient.

[0015] According to some embodiments of the present invention, a method for loading the active substance onto the bitter melon-derived nanovesicles includes at least one of electroporation, microinjection, and ultrasonic treatment.

[0016] According to some embodiments of the present invention, the method for loading the active substance onto the bitter melon-derived nanovesicles is ultrasonic treatment.

[0017] According to some embodiments of the present invention, the ultrasound treatment is pulsed ultrasound treatment. The ultrasound treatment includes the following steps: preparing a mixture of the bitter melon-derived nanovesicles and active substances, performing pulsed ultrasound treatment to remove free active substances, thereby obtaining the brain-targeting drug.

[0018] According to some embodiments of the present invention, the mass ratio of the active substance to the number of bitter melon-derived nanovesicles is (0.5-2) mg: 10 mg. 10For example: 0.5 mg: 10 10 0.6 mg: 10 10 0.7 mg: 10 10 0.8 mg: 10 10 0.9 mg: 10 10 1 mg: 10 10 1.1 mg: 10 10 1.2 mg: 10 10 1.3mg: 10 10 1.4 mg: 10 10 1.5 mg: 10 10 1.6 mg: 10 10 1.7 mg: 10 10 1.8 mg: 10 10 1.9 mg: 10 10 One or 2 mg: 10 10 indivual.

[0019] According to some embodiments of the present invention, the ultrasonic power of the ultrasonic treatment is 80 kW-100 kW. For example, it can be 80 kW, 81 kW, 82 kW, 83 kW, 84 kW, 85 kW, 86 kW, 87 kW, 88 kW, 89 kW, 90 kW, 91 kW, 92 kW, 93 kW, 94 kW, 95 kW, 96 kW, 97 kW, 98 kW, 99 kW, or 100 kW.

[0020] According to some embodiments of the present invention, the temperature of the ultrasonic treatment is 2℃-6℃. For example, it can be 2℃, 2.5℃, 3℃, 3.5℃, 4℃, 4.5℃, 5℃, 5.5℃ or 6℃.

[0021] According to some embodiments of the present invention, the ultrasonic treatment time is 30 min to 60 min. For example, it can be 30 min, 32 min, 34 min, 36 min, 38 min, 40 min, 42 min, 44 min, 46 min, 48 min, 50 min, 52 min, 54 min, 56 min, 58 min or 60 min.

[0022] According to some embodiments of the present invention, the pulse pattern of the ultrasonic treatment is 1 s-10 s on, 2 s-15 s off. Preferably, the pulse pattern is 2 s-5 s on, 3 s-8 s off; more preferably, the pulse pattern is 3 s on, 5 s off.

[0023] According to some embodiments of the present invention, the method for removing free active substances includes ultrafiltration. The ultrafiltration method involves using an ultrafiltration membrane with a molecular weight cutoff of 50 kDa to 300 kDa, preferably 100 kDa. The centrifugation conditions for the ultrafiltration method are 8000×g to 12000×g for 25 min to 35 min. For example, the molecular weight cutoff of the ultrafiltration membrane can be 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, or 300 kDa.

[0024] According to some embodiments of the present invention, the brain-targeting drug further includes pharmaceutically acceptable excipients.

[0025] According to some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of fillers, antioxidants, pH adjusters, osmotic pressure adjusters, solubilizers, cosolvents, antioxidants, antibacterial agents, lyophilization protectants, suspending agents, and flavoring agents.

[0026] According to some embodiments of the present invention, the dosage form of the brain-targeting drug includes at least one of tablets, granules, liquid formulations and capsules, powders, and nanosuspensions.

[0027] According to some embodiments of the present invention, the brain-targeting drug is used to treat sleep disorders.

[0028] According to some embodiments of the present invention, the sleep disorder is selected from at least one of insomnia, excessive dreaming, sleep apnea syndrome, sleep disorder caused by circadian rhythm disorder, sleep disorder caused by anxiety, and sleep disorder caused by stress.

[0029] According to some embodiments of the present invention, the active substance includes at least one of neuromodulatory components and neuroprotective components.

[0030] According to some embodiments of the present invention, the neuromodulatory components include, but are not limited to, at least one of γ-aminobutyric acid (GABA), magnesium glycine (MG), melatonin, and serotonin; According to some embodiments of the present invention, the neuroprotective ingredients include, but are not limited to, at least one of vitamin C, vitamin E, glutathione, and curcumin.

[0031] According to some embodiments of the present invention, the active substances include γ-aminobutyric acid (GABA) and magnesium glycine. GABA and / or magnesium glycine can significantly reduce the frequency of spontaneous activity, shorten the sleep latency, and prolong the duration of sleep. Furthermore, the sleep aid product composed of GABA and magnesium glycine is significantly more effective than that containing a single active ingredient.

[0032] According to some embodiments of the present invention, the mass ratio of γ-aminobutyric acid to magnesium glycine is 1:(0.5-2). For example, it can be 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9 or 1:2.

[0033] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the separation and purification process of bitter melon-derived nanovesicles prepared in Example 1; Figure 2 The results are obtained by dynamic light scattering detection of the size distribution of bitter gourd-derived nanovesicles prepared in Example 1; Figure 3 This is a transmission electron microscope (TEM) image of the bitter melon-derived nanovesicles prepared in Example 1; Figure 4 These are the results of the oral brain targeting study of the bitter melon-derived nanovesicles prepared in Example 1; Figure 5 This is a graph showing the results of the sleep-aiding effect of oral administration of free γ-aminobutyric acid and GABA-loaded bitter melon-derived nanovesicles in Test Example 2. Figure 6 This is a graph showing the results of the study on the sleep-aiding effects of different oral medications in Test Example 3. Detailed Implementation

[0035] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0036] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0037] In the description of this invention, the use of terms such as first, second, third, etc., is only for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of technical features indicated, or implicitly indicating the order of the technical features indicated.

[0038] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0039] Unless otherwise specified, the steps for testing encapsulation efficiency are as follows: After sonication, the mixture was separated into retentate and filtrate using a 100 kDa ultrafiltration centrifuge tube. The concentration of free GABA / glycine magnesium in the filtrate was determined by HPLC, and the mass of free drug was calculated. Encapsulation efficiency = (total drug mass - free drug mass) / total drug mass × 100%.

[0040] Preparation Example 1 This example demonstrates the preparation of bitter melon-derived nanovesicles (BDNVs) using the following method, as shown in the flowchart below. Figure 1 As shown. The specific steps are as follows: (1) Raw material processing: Take 1 kg of fresh bitter melon, wash it clean, cut it into small pieces of about 3 cm, keep the peel, add 2L of sterile PBS buffer (0.02 mol / L, pH 7.3; the same below), put it into a high-speed homogenizer, homogenize it at 10000 rpm for 5 min to obtain bitter melon homogenate; filter it through a 300 mesh filter membrane and collect about 1.8 L of clear filtrate.

[0041] (2) Preliminary purification: Transfer the filtrate to a centrifuge tube, centrifuge at 1000×g for 10 min and take the supernatant; centrifuge at 3000×g for 30 min and take the supernatant; centrifuge at 10000×g for 60 min and take the supernatant; centrifuge at 150000×g for 120 min and collect the bottom precipitate.

[0042] (3) Purification: The precipitate was resuspended in 50 mL of sterile PBS buffer. The resuspended solution was slowly added to a centrifuge tube pre-filled with a gradient of 8 w / v%, 30 w / v%, 45 w / v%, and 60 w / v sucrose solutions. The tube was centrifuged at 150,000 × g for 120 min at 4 °C. The white band at the interface between the 45 w / v% and 60 w / v% sucrose solutions was collected and washed three times with sterile PBS buffer pre-cooled at 4 °C, centrifuged at 10,000 × g for 30 min each time. Finally, the precipitate was resuspended in about 10 mL of sterile PBS buffer to obtain the bitter gourd-derived nanovesicle suspension.

[0043] (4) Quality control testing: like Figure 2-3 As shown. Dynamic light scattering analysis revealed that the bitter melon-derived nanovesicles ranged in size from 80.75 nm to 336.1 nm, with an average particle size of 111.9 nm and a PDI of 0.19; the Zeta potential was -3.6 mV. Nanoparticle tracking analysis showed a particle concentration of 1.2 × 10⁻⁶. 11 Nanovesicles per mL. Transmission electron microscopy revealed that the nanovesicles were typically round or cup-shaped with a concave disc-shaped structure and uniform morphology.

[0044] Example 1 This example demonstrates the preparation of a drug loaded with magnesium glycine using the following method, with the specific steps outlined below: (1) Take 5 mL of bitter melon-derived nanovesicle suspension (particle concentration 1.2 × 10⁻⁶). 11 Add glycine magnesium powder (number of particles / mL) to make the mass ratio of glycine magnesium to nanovesicles 1 mg: 1.2 × 10⁻⁶. 10 One sample was magnetically stirred for 30 minutes to obtain a mixture.

[0045] (2) Ultrasonic loading and purification: The mixture was placed in an ice bath and subjected to pulsed sonication at a power of 90 kW and a temperature of 4 °C for 45 min (3 s working time followed by 5 s intervals). The sonicated mixture was then added to a 100 kDa ultrafiltration centrifuge tube and centrifuged at 10,000 × g for 30 min to remove free magnesium glycine. The retentate was collected. The retentate was resuspended in 5 mL of sterile PBS buffer to obtain BDNVs. MG Store at 4℃ away from light.

[0046] The PDI was measured to be 0.30, the Zeta potential to be -3.2 mV, and the glycine magnesium encapsulation efficiency to be 65.8%.

[0047] Example 2 This example demonstrates the preparation of a drug loaded with γ-aminobutyric acid using the following method, with the specific steps outlined below: (1) Take 5 mL of bitter melon-derived nanovesicle suspension (particle concentration 1.2 × 10⁻⁶). 11 Add γ-aminobutyric acid powder (gABA) to make the mass ratio of γ-aminobutyric acid to nanovesicles 1 mg: 1.2 × 10⁻⁶ / mL, so that the mass ratio of γ-aminobutyric acid to nanovesicles is 1 mg: 1.2 × 10⁻⁶ / mL. 10 One sample was magnetically stirred for 30 minutes to obtain a mixture.

[0048] (2) Ultrasonic loading and purification: The mixture was placed in an ice bath and subjected to pulsed sonication at a power of 90 kW and a temperature of 4 °C for 45 min (3 s working time followed by 5 s intervals). The sonicated mixture was then added to a 100 kDa ultrafiltration centrifuge tube and centrifuged at 10,000 × g for 30 min to remove free γ-aminobutyric acid (GABA). The retentate was collected. The retentate was resuspended in 5 mL of sterile PBS buffer to obtain BDNVs. GABA Store at 4℃ away from light.

[0049] The PDI was measured to be 0.32, the Zeta potential to be -3.6 mV, and the γ-aminobutyric acid encapsulation efficiency to be 68.5%.

[0050] Example 3 This example demonstrates the preparation of a drug loaded with γ-aminobutyric acid and magnesium glycine using the following method, with the specific steps outlined below: (1) Take 5 mL of bitter melon-derived nanovesicle suspension (particle concentration 1.2 × 10⁻⁶). 11 Add γ-aminobutyric acid (GABA) and magnesium glycine powder (mass ratio 1:1) to make the ratio of the total mass of GABA and magnesium glycine to the number of nanovesicles 1 mg: 1.2 × 10⁻⁶. 10 One sample was magnetically stirred for 30 minutes to obtain a mixture.

[0051] (2) Ultrasonic loading and purification: The mixture was placed in an ice bath and subjected to pulsed sonication at a power of 90 kW and a temperature of 4 ℃ for 45 min (3 s working time followed by 5 s intervals). The sonicated mixture was then added to a 100 kDa ultrafiltration centrifuge tube and centrifuged at 10,000 × g for 30 min to remove free γ-aminobutyric acid and magnesium glycine. The retentate was collected. The retentate was resuspended in 5 mL of sterile PBS buffer to obtain BDNVs. MG+GABA Store at 4℃ away from light.

[0052] The test results showed a PDI of 0.33, a Zeta potential of -3.4 mV, and a total active ingredient encapsulation rate of 67.2%.

[0053] Test Example 1 The DID dye was mixed with BDNVs (final concentration of DID dye was 1 μM, and final concentration of BDNVs was 1 × 10⁻⁶). 11(Number of nanovesicles per mL), incubated at 4℃ in the dark for 30 min, and then the free dye was removed by ultrafiltration centrifuge tubes to obtain DID fluorescent dye-labeled bitter melon-derived nanovesicles. The organ distribution characteristics of unlabeled and DID-labeled bitter melon-derived nanovesicles in mice were compared to clarify the brain-targeting delivery capability of BDNVs.

[0054] Grouping and administration: Six 8-week-old male BALB / c mice (weighing 18-22g) were acclimatized for one week and then randomly divided into two groups of three mice each: the BDNVs group and the BDNVs group. DID Both groups received oral administration, with a dosage volume of 0.2 mL per animal (BDNVs). DID The BDNVs group received a DID-labeled bitter melon-derived nanovesicle solution, while the BDNVs group received an equal volume of unlabeled bitter melon-derived nanovesicle solution. Both labeled and unlabeled bitter melon-derived nanovesicles were resuspended in sterile PBS buffer, with a concentration of 1×10⁻⁶ for both groups. 11 (Number of organs per mL). Twelve hours after administration, mice were sacrificed, and nine organs—lung, heart, spleen, kidney, brain, testis, lymph nodes, thymus, and liver—were completely isolated and placed in a small animal in vivo fluorescence imaging system for fluorescence signal scanning. The fluorescence intensity distribution of each organ was quantitatively analyzed.

[0055] Fluorescence imaging results of organ distribution in vivo Figure 4 As shown.

[0056] No specific fluorescence signals were detected in any organ in the BDNVs group (background control without DID labeling). DID In the brain tissue of the group, DID fluorescence signals were observed to increase cumulatively over time, exhibiting obvious targeted enrichment characteristics; a certain fluorescence distribution was observed in the liver, consistent with the normal physiological characteristics of nanocarrier metabolism via the reticuloendothelial system. This indicates that bitter melon-derived nanovesicles possess natural and excellent brain-targeting capabilities. After oral administration, they can penetrate the blood-brain barrier and specifically accumulate in brain tissue, serving as brain-targeting carriers to efficiently deliver active ingredients to brain tissue via oral administration, while reducing distribution in non-target organs.

[0057] Test Example 2 Eight-week-old male healthy BALB / c mice (weighing 18-22g) were acclimatized for one week and then randomly divided into three groups of three mice each. Mice in the GABA group were orally administered a sterile PBS solution containing dissolved γ-aminobutyric acid (GABA) at a dose of 50 mg / kg based on the active ingredient (GABA); BDNVs GABAMice in the control group were orally administered a sterile PBS solution containing the drug prepared in Example 2, with a dosage of 50 mg / kg based on the active ingredient (GABA). Mice in the PBS control group were orally administered an equal volume of sterile PBS buffer.

[0058] A small animal spontaneous activity monitoring system was used to record the number of spontaneous activities of mice at five time points: 1 h, 2 h, 4 h, 6 h, and 12 h after drug administration (the number of horizontal movements across the infrared sensor line was used as the indicator).

[0059] The results are as follows Figure 5 As shown. One hour after drug administration, the activity frequency of mice in all three groups was at a low level; among them, there was no significant difference in activity frequency (ns) between the GABA group and the PBS control group, BDNVs GABA There was a significant difference in activity frequency between the group and the PBS control group (*) P <0.05). From 2 h to 12 h after drug administration, the activity frequency of mice in the PBS and GABA groups significantly increased and remained high over time; while BDNVs... GABA The activity frequency of mice in the group was significantly lower than that in the PBS group and the GABA group (****) P <0.0001), and lasted from 2 h to 12 h, showing a significant long-term central inhibitory effect.

[0060] This indicates that oral administration of bitter melon nanovesicles can significantly prolong and enhance the central inhibitory effect of γ-aminobutyric acid (GABA), thus exhibiting a long-lasting sleep-inducing effect. Free GABA has limited delivery efficiency to the brain after oral administration, while BDNVs... GABA The group can continuously reduce the activity frequency of mice.

[0061] Test Example 3 Twenty-one 8-week-old male healthy BALB / c mice (weighing 18-22g) were acclimatized for one week and then randomly divided into 7 groups of 3 mice each. The specific drug administration details for each group are as follows: (1) Blank control group: oral administration of sterile PBS by gavage; (2) MG group: Oral administration of sterile PBS solution containing magnesium glycinate dissolved in glycine, with a dose of 50 mg / kg based on the active ingredient (MG) dosage.

[0062] (3) GABA group: oral gavage with sterile PBS solution containing γ-aminobutyric acid, the dose was 50 mg / kg based on the active ingredient (GABA).

[0063] (4) MG+GABA group: oral gavage with sterile PBS solution containing magnesium glycinate and γ-aminobutyric acid. The dosage of magnesium glycinate and γ-aminobutyric acid was 50 mg / kg based on the dosage of active ingredients (MG and GABA).

[0064] (5) BDNVs MG Group: Oral administration of a sterile PBS solution containing a resuspended drug prepared in Example 1, administered at a dose of 50 mg / kg based on the active ingredient (MG) dosage; (6) BDNVs GABA Group: Oral administration of sterile PBS solution containing the drug prepared in Example 2, resuspended by gavage, at a dose of 50 mg / kg based on the active ingredient (GABA).

[0065] (7) BDNVs MG+GABA Group: Oral administration of sterile PBS solution containing the drug prepared in Example 3, with the dosage of magnesium glycinate and γ-aminobutyric acid both being 50 mg / kg based on the dosage of the active ingredients (MG and GABA).

[0066] A small animal spontaneous activity monitoring system was used to record the number of spontaneous activities of mice in each group 12 hours after drug administration (indicated by the number of horizontal movements across the infrared sensor line).

[0067] The results are as follows Figure 6 As shown. Bitter melon-derived nanovesicles, as a natural delivery carrier, have no significant sleep-inducing pharmacological activity. In the blank control group and the free component group (MG, GABA, MG+GABA), the activity frequency of mice was high (150-180 times), with no significant sleep-inducing effect. The single-drug-loaded group (BDNVs) MG BDNVs GABA The activity frequency of mice was significantly reduced (approximately 80-90 times), significantly lower than that of the corresponding free component group (****). P <0.0001). This demonstrates that bitter melon-derived nanovesicles can significantly enhance the intrabrain delivery efficiency of single components. (BDNVs) MG+GAB The mice exhibited the lowest activity frequency (approximately 55 times), significantly lower than the single drug delivery system group (****). P <0.0001), showing a synergistic sleep-aiding effect.

[0068] In summary, bitter melon-derived nanovesicles, as oral brain-targeting carriers, can significantly enhance the bioavailability and central nervous system effects of sleep-aiding active ingredients such as γ-aminobutyric acid (GABA) and magnesium glycine; the compound active ingredient drug delivery system (BDNVs) Compared to single-component drug delivery systems, the +GABA combination exhibits a more significant sleep-aiding effect. This demonstrates that the drug delivery system of this invention achieves superior sleep intervention through synergistic component interaction.

[0069] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. The application of bitter melon-derived nanovesicles in the preparation of brain-targeting drugs, characterized in that, The brain-targeting drug is an oral brain-targeting drug.

2. The application according to claim 1, characterized in that, The bitter melon-derived nanovesicles have a particle size distribution range of 50 nm to 500 nm; and / or, the zeta potential of the bitter melon-derived nanovesicles is -8.0 mV to 8.0 mV.

3. The application according to claim 1, characterized in that, The method for preparing the bitter melon-derived nanovesicles includes: separating the bitter melon-derived nanovesicles from the bitter melon homogenate by at least one of differential centrifugation, ultracentrifugation, density gradient centrifugation, ultrafiltration, and size exclusion chromatography.

4. The application according to claim 1, characterized in that, The brain-targeting drug includes at least one of the following: an active substance and a pharmaceutically acceptable excipient.

5. The application according to claim 4, characterized in that, The method for loading the active substance onto the bitter melon-derived nanovesicles includes at least one of electroporation, microinjection, and ultrasonic treatment.

6. The application according to claim 4, characterized in that, The pharmaceutically acceptable excipients include at least one of the following: fillers, antioxidants, pH adjusters, osmotic pressure adjusters, solubilizers, cosolvents, antioxidants, antibacterial agents, lyophilization protectants, suspending agents, and flavoring agents.

7. The application according to claim 1, characterized in that, The dosage forms of the brain-targeting drugs include at least one of tablets, granules, liquid preparations, capsules, powders, and nanosuspensions.

8. The application according to claim 1, characterized in that, The brain-targeting drug is used to treat sleep disorders.

9. The application according to claim 4, characterized in that, The active substance includes at least one of neuromodulatory components and neuroprotective components; preferably, the neuromodulatory components include, but are not limited to, at least one of γ-aminobutyric acid, magnesium glycine, melatonin, and serotonin; preferably, the neuroprotective components include, but are not limited to, at least one of vitamin C, vitamin E, glutathione, and curcumin.

10. The application according to claim 4, characterized in that, The active substances include γ-aminobutyric acid and magnesium glycine; preferably, the mass ratio of γ-aminobutyric acid to magnesium glycine is 1:(0.5-2).

Images