Cannabinoid receptor 1 type mediated brain targeting lipid nano-capsule drug delivery system and preparation and application thereof

Abstract

The application discloses a cannabinoid 1 type receptor-mediated brain-targeting lipid nanocapsule drug loading system and a preparation and application thereof, and belongs to the technical field of targeted drugs. The brain-targeting lipid nanocapsule drug loading system comprises a drug-loaded lipid nanocapsule, the surface of the lipid nanocapsule is modified with a ligand compound of a CB1 receptor, and the ligand compound is one of quebrachamine, methoxylated myristicin, chelerythrine, ostruthol or cannabigerol. The application utilizes the lipid nanocapsule carrier to load a compound X with neuroprotective activity, prolongs the circulation time of the compound X in the body, adsorbs a ligand compound Y on the surface of the lipid nanocapsule, can specifically recognize the CB1 receptor highly expressed on the brain nerve epidermal cells, can transport across the blood-brain barrier, and improves the drug concentration in the brain tissue and cells. The preparation method provided by the application is simple in operation, easy to popularize, can be used for preparing drugs crossing the blood-brain barrier, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of targeted drug technology, specifically to a brain-targeting lipid nanocapsule drug delivery system mediated by cannabinoid type 1 receptor, its preparation method, and its application. Background Technology

[0002] Currently, approximately 1.5 billion people worldwide suffer from varying degrees of central nervous system diseases, which has become a major threat to human life and health. Epidemiological surveys show that the prevalence of neurodegenerative diseases among people aged 65 and above in my country is 5.56% ("China Mental Disorders Burden and Health Service Utilization" project study). Currently, there are no effective drugs or methods for treating neurodegenerative diseases.

[0003] The blood-brain barrier (BBB), a unique biological barrier between the bloodstream and the central nervous system, protects the brain from bloodborne pathogens such as viruses, bacteria, and parasites, strictly limiting the entry of fluids and ions to ensure the optimal state of the central nervous system. However, it also restricts the intrabrain transport of most drugs. Statistics show that approximately 98% of small molecule compounds and almost 100% of large molecule drugs, including protein peptides and gene therapy drugs, are difficult to penetrate the brain. This severely hinders the clinical drug treatment of central nervous system diseases such as brain tumors, Parkinson's disease, and Alzheimer's disease, which pose serious threats to human health. Therefore, the blood-brain barrier has become a bottleneck in the drug treatment of central nervous system diseases.

[0004] To enhance drug permeability across the blood-brain barrier, various strategies have been explored to increase intrabrain delivery. Nanomaterials, especially surface-functionalized nanomaterials, have become an effective tool for enhancing drug transport from the blood to the brain. Researchers at Tufts University have developed a neurotransmitter-derived lipidoid (NT-lipidoids) that utilizes neurotransmitters to help lipid nanoparticles (LNPs) carrying small molecule drugs, macromolecules, and even gene-editing proteins cross the blood-brain barrier into the mouse brain (Neurotransmitter-derived lipidoids (NT-lipidoids) for enhanced brain delivery through intravenous injection, Science Advances, 2020 Jul 24; 6(30)).

[0005] Lipid nanocapsules (LNCs) are a type of nano-formulation that has attracted attention in recent years. Their structure consists of a lipid core and a surfactant shell. As a drug carrier, they are characterized by being solvent-free, low-energy, relatively stable, and easily absorbed. Furthermore, lipid nanocapsules exhibit high encapsulation efficiency and good drug loading capacity.

[0006] Cannabinoid receptors are a class of specialized proteins located on or within cell membranes. They bind to specific extracellular signaling molecules, activating a series of intracellular biochemical reactions that enable cells to respond to external stimuli. Currently, the recognized cannabinoid receptors are cannabinoid type 1 (CB1) and cannabinoid type 2 (CB2) receptors. CB1 receptors are primarily located in the brain, spinal cord, and peripheral nervous system, and are also known as central cannabinoid receptors. In the brain, CB1 receptors are mainly distributed in the basal ganglia (substantia nigra, globus pallidus, lateral striatum), the CA pyramidal cell layer of the hippocampus, the cerebellum, and the cerebral cortex. CB2 receptors are mainly distributed in the periphery, such as in the limbic region of the spleen, immune cells, tonsils, and thymus, and are also known as peripheral cannabinoid receptors. Since CB1 receptors are primarily located in the brain, spinal cord, and peripheral nervous system, and ligands for CB1 receptors exist in natural products, it is theoretically possible to modify nanoparticle drug delivery systems using compounds with affinity for CB1 receptors, potentially achieving brain-targeted drug delivery. However, there are currently no reports of lipid nanocapsule drug delivery systems being able to be given the ability to cross the blood-brain barrier through surface modification.

[0007] Developing a CB1 receptor-mediated brain-targeting lipid nanocapsule drug delivery system that simultaneously achieves cross-blood-brain barrier transport and maintains the efficacy of the encapsulated neuroprotective drugs is a problem that needs to be solved by those skilled in the art. Summary of the Invention

[0008] The purpose of this invention is to provide a brain-targeting lipid nanocapsule drug delivery system based on cannabinoid type 1 (CB1) receptor to solve the problem that drugs or nutrients cannot enter brain tissue to exert neuroprotective effects due to the blood-brain barrier's resistance to exogenous substances.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A brain-targeting lipid nanocapsule drug delivery system mediated by cannabinoid type 1 receptor includes lipid nanocapsules encapsulating drugs, wherein the surface of the lipid nanocapsules is modified with a ligand compound of cannabinoid type 1 receptor, wherein the ligand compound is one of styracil, methoxycapsulin, chelidonine, falciferol, or cannabinol.

[0011] This invention utilizes lipid nanocapsules as drug carriers to encapsulate drugs or nutrients with neuroprotective effects. By modifying the surface of the lipid nanocapsules with ligand compounds targeting cannabinoid type 1 receptors, the ligand compounds specifically recognize CB1 receptors. Through CB1 receptor-mediated endocytosis, the lipid nanocapsules carrying drugs or nutrients effectively enter the brain, increasing the concentration of drugs or nutrients in brain tissue and cells. Studies have shown that modifying these nanocapsules with styraxamine, methoxycapsulin, chelidonine, falcinol, or cannabinoids can achieve the dual functions of drug transport across the blood-brain barrier and targeted therapy, solving the technical problems of insufficient targeting and excessively high dosages in existing brain disease drugs.

[0012] Furthermore, the drug is selected from compounds with neuroprotective activity. Preferably, the drug encapsulated in lipid nanocapsules is one of doxorubicin, paclitaxel, verbascoside, echinacoside, quercetin, or anthocyanin.

[0013] LNCs are composed of oil, water, surfactants, and co-surfactants, and are prepared by phase inversion. Commonly used oil phases include caprylic / capric triglycerides and ethyl palmitate; the aqueous phase is mostly sodium chloride aqueous solution; emulsifiers commonly used include lecithin, Span 80, and Tween 80; and commonly used surfactants include dodecanoic acid and polyethylene glycol 12-hydroxystearate.

[0014] Preferably, the LNCs consist of: medium-chain triglycerides, polyethylene glycol 12-hydroxystearate, and Span 80. The lipid nanocapsules are prepared using the phase transition temperature method. Span 80 has a low phase transition temperature, and the phase transition heating and cooling process can be controlled within 70℃-45℃ to achieve the preparation of lipid nanocapsules, effectively reducing high-temperature degradation of the drug during encapsulation.

[0015] This invention also provides a method for preparing the aforementioned cannabinoid type 1 receptor-mediated brain-targeting lipid nanocapsule drug delivery system, comprising the following steps:

[0016] (1) LNCs loaded with drug X were prepared by phase inversion method;

[0017] (2) The LNCs containing drug X are mixed with an ethanol solution of ligand compound Y and stirred until the ethanol is completely evaporated to obtain the brain-targeting lipid nanocapsule drug delivery system mediated by cannabinoid type 1 receptor. The ligand compound Y is one of cinnamylamine, methoxycapsulin, chelidonine, falciferol or cannabinol.

[0018] In step (1), drug X, Span 80, distilled water, sodium chloride, medium-chain triglycerides and polyethylene glycol 12-hydroxystearate are mixed evenly. Under stirring conditions, the mixture undergoes three alternating heating and cooling processes. When the phase transition temperature is reached during the last cooling process, distilled water at 0°C is added, and stirring continues at room temperature to obtain lipid nanocapsules loaded with drug X.

[0019] Preferably, by mass percentage, the concentration of polyethylene glycol 12-hydroxystearate is 20-30%, the concentration of medium-chain triglycerides is 15-20%, the concentration of Span 80 is 1.5-2%, the concentration of sodium chloride is 1.5-2%, the concentration of drug X is 1-2%, and water is the balance.

[0020] More preferably, by mass percentage, the concentration of polyethylene glycol 12-hydroxystearate is 30%, the concentration of medium-chain triglycerides is 15%, the concentration of Span 80 is 1.5%, the concentration of sodium chloride is 1.5%, and the concentration of drug X is 2%.

[0021] The medium-chain triglyceride is caprylic / capric triglyceride.

[0022] Preferably, the heating and cooling process is as follows: from room temperature to 70°C to 45°C to 70°C to 45°C to 70°C to 55°C; the heating or cooling rate is 4°C / min, and the next heating or cooling process begins immediately after reaching the temperature point.

[0023] Preferably, the stirring rate is 500–600 rpm, the weight of 0°C distilled water is 5–10 times the weight of the mixture, and 0°C distilled water is added when the temperature drops to 55°C for the last time. Stirring continues at room temperature for 5–10 min. The resulting LNCs-X solution is then passed through a gel permeation chromatography column to remove sodium chloride and unencapsulated X.

[0024] In step (2), lipid nanocapsules loaded with drug X are mixed with an ethanol solution of ligand compound Y and magnetically stirred at 250 rpm for more than 24 hours at room temperature to allow the ethanol to evaporate completely. During the stirring process, ligand compound Y adsorbs onto the surface of the lipid nanocapsules, giving the lipid nanocapsules the ability to cross the blood-brain barrier.

[0025] Preferably, the lipid nanocapsules encapsulating drug X are mixed with an ethanol solution of ligand compound Y at a volume ratio of 3:1, wherein the concentration of the ethanol solution of Y is 1-5 mg / mL.

[0026] The lipid nanocapsules loaded with drug X prepared in this invention are modified with ligand compound Y to form Y-LNCs-X. By utilizing Y via CB1 receptor-mediated endocytosis, the intrabrain transport of X is increased, enhancing the blood-brain barrier permeability of X, thus providing a brain-targeting nanoparticle drug formulation. Therefore, this invention provides the application of the aforementioned brain-targeting lipid nanocapsule drug delivery system in the preparation of drugs that cross the blood-brain barrier.

[0027] X is a drug or nutrient with neuroprotective activity. Due to the obstruction of the blood-brain barrier, the concentration of X in the brain is limited. After the modification of the preparation method of this invention, X can cross the blood-brain barrier, increase its accumulation concentration in brain tissue and cells, enhance its neuroprotective effect, and delay aging.

[0028] The present invention also provides the application of the aforementioned cannabinoid type 1 receptor-mediated brain-targeting lipid nanocapsule drug delivery system in the preparation of drugs for treating Alzheimer's disease or anti-aging.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] 1) The Y-LNCs-X prepared in this invention utilizes lipid nanocapsules to encapsulate compound X, which has neuroprotective activity, thereby prolonging its circulation time in vivo. The ligand compound Y adsorbed on the surface of the lipid nanocapsules can specifically recognize the CB1 receptor highly expressed on brain neuroepidermal cells and can be transported across the blood-brain barrier, increasing the drug concentration in brain tissue and cells.

[0031] 2) The Y-LNCs-X preparation method provided by this invention is simple to operate and easy to promote. It can be used to prepare drugs that cross the blood-brain barrier, such as drugs for treating Alzheimer's disease or anti-aging. It has broad clinical application prospects and significant socio-economic value. Attached Figure Description

[0032] Figure 1 The structure of compound Y, the ligand compound of the CB1 receptor selected in Example 1, is shown below. Detailed Implementation

[0033] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0035] Example 1: Screening of ligand compound Y with CB1 receptor that crosses the blood-brain barrier

[0036] I. Screening for ligand compounds Y with CB1 receptors that cross the blood-brain barrier

[0037] 1. Screening for compounds Y with CB1 affinity

[0038] The names and affinities of the specific compounds (compiled based on various literature reports) are shown in Table 1, and the structural formulas of the compounds are shown in [reference needed]. Figure 1 .

[0039] Table 1. Affinity of selected cannabinoid receptor compounds

[0040]

[0041]

[0042] 2. Preparation of Y-modified lipid nanocapsules loaded with the fluorescent substance Dir

[0043] (1) First, 20 mg of fluorescent dye Dir, 300 mg of polyethylene glycol 12-hydroxystearate, 150 mg of caprylic / capric triglyceride, 15 mg of Span 80, 15 mg of sodium chloride, and 500 mg of distilled water were mixed to make the total weight of the system 1 g. The mixture was magnetically stirred at 500 rpm while simultaneously undergoing a temperature increase and decrease process at room temperature—70℃—45℃—70℃—45℃—70℃—55℃ (heating or cooling at a rate of 4℃ per minute, and starting the next round of heating or cooling immediately after reaching the temperature point). When the phase transition temperature (55℃) was reached during the last cooling, 5 g of distilled water at 0℃ was added, and stirring was continued at room temperature for 5 min to obtain a lipid nanocapsule (LNCs-Dir) solution loaded with Dir. The prepared LNCs-Dir solution was then passed through a gel permeation chromatography column to remove sodium chloride and unencapsulated Dir.

[0044] (2) The prepared LNCs-Dir was mixed with a Y ethanol solution with a concentration of 4 mg / mL at a ratio of 3:1 (v / v), and the mixture was magnetically stirred at 250 rpm at room temperature until the ethanol was completely evaporated to obtain Y-LNCs-Dir.

[0045] 3. BALB / c mice were used in the experiment, with 3 mice in each group. They were divided into a drug treatment group (Y-LNCs-Dir), a control group (LNCs-Dir), and a blank group (intravenous injection of physiological saline (Sal) and fluorescent dye (Dir)). After injecting 150 μL of different drug solutions via the tail vein, the mice were sacrificed 12 hours later, dissected, and their intact brains were harvested. The differences in fluorescence intensity in the brains of the mice from each group were semi-quantitatively analyzed using an in vivo imaging system. Excitation light was 740 nm, and emission light was 780 nm. The ability of compound Y to cross the blood-brain barrier was determined based on the differences in fluorescence intensity in the mouse brains.

[0046] 4. Results

[0047] Table 2. Fluorescence intensity in mouse brains 12 hours after tail vein injection of Y-LNCs-Dir

[0048]

[0049]

[0050] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05).

[0051] Table 2 shows that the levels of the fluorescent substance Dir entering the brain varied after different Y modifications, with no significant difference between the Sal and Dir groups. This indicates that Dir has a very low ability to cross the blood-brain barrier. Compared with LNC-Dir, modifications with styracin (Voa), methoxycapsaicin (Yan), celandine (Che), falciferol (Fal), or cannabinol (Cbg) significantly increased the amount of Dir entering the brain, with styracin (Voa) showing the greatest increase. Although cannabidiol (Cbd), oleuropein (Oli), curcumin (Cur), magnolol (Mag), and sanguisorbin (San) also have affinity for CB1, the levels of Dir entering the brain after modification with these substances were not significantly different from those of LNC-Dir.

[0052] 5. Conclusion

[0053] (1) The fact that the compound has an affinity for the CB1 receptor does not mean that it can cross the blood-brain barrier.

[0054] (2) Voa, Yan, Chelidonine, Fal, or Cbg have the ability to cross the blood-brain barrier. Among them, Voa has the strongest ability to cross the blood-brain barrier.

[0055] II. Verification of whether compound Y crosses the blood-brain barrier via the CB1 receptor.

[0056] 1. Verification Method

[0057] BALB / c mice were used in the experiment, with 3 mice in each group. Experimental groups (Y-LNCs-Dir and LNCs-Dir) and a control group were established. Thirty minutes before the tail vein injection of 150 μL of different drug solutions, the experimental groups received a tail vein injection of 150 μL of rimonaban solution. Because rimonaban (Ki = 1.8 nM) has a very strong affinity for the CB1 receptor, its pre-injection allows it to bind to the CB1 receptor. Compared to rimonaban, Y-LNCs have a lower affinity for the CB1 receptor and cannot compete with it for binding. Therefore, this method can be used to verify whether Y-LNCs can cross the blood-brain barrier via the CB1 receptor.

[0058] 2. Results

[0059] Table 3. Fluorescence intensity in the brains of Y-LNCs-Dir mice after rimonaban injection 30 min in advance.

[0060]

[0061] Note: Different lowercase letters in the same line indicate significant differences (P < 0.05).

[0062] As shown in Table 3, injecting rimonaban 30 minutes in advance did not significantly affect the fluorescence intensity of LNC-Dir, San-LNC-Dir, Mag-LNC-Dir, Cur-LNC-Dir, and Oli-LNC-Dir in the brain. However, injecting rimonaban 30 minutes in advance significantly reduced the fluorescence intensity of Voa-LNC-Dir, Yan-LNC-Dir, Che-LNC-Dir, Fal-LNC-Dir, and Cbg-LNC-Dir in the brain.

[0063] 3. Conclusion

[0064] Voa-LNC, Yan-LNC, Che-LNC, Fal-LNC, Cbg-LNC, and Cbd-LNC cross the blood-brain barrier via the CB1 receptor.

[0065] Example 2: Study on the formulation process of Voa-modified ECH lipid nanocapsules (Voa-LNCs-ECH)

[0066] The formulation and process of Voa-modified echinacoside (ECH) lipid nanocapsules were investigated using a single-factor study. The preparation method was the same as in Example 1, except that the fluorescent dye Dir was replaced with echinacoside (ECH). With other preparation conditions kept constant, only the amount of distilled water was adjusted to ensure the total mass of the system was 1 g. The formulation and process of Voa-LNCs-ECH were evaluated using particle size and encapsulation efficiency as indicators.

[0067] (1) Investigation on the dosage of echinacoside

[0068] LNCs-ECH were prepared by adding 10, 15, 20, 25, and 30 mg of ECH, respectively, while keeping the proportions of other reagents constant. The results are shown in Table 4. The encapsulation efficiency of LNCs-ECH was highest when the amount of ECH was 20 mg.

[0069] Table 4. Effect of ECH dosage on LNCs-ECH particle size and encapsulation efficiency

[0070]

[0071] Note: Different lowercase letters in the same line indicate significant differences (P < 0.05).

[0072] (2) Investigation into the dosage of Span 80

[0073] Span 80 was added at concentrations of 10, 15, 20, 25, and 30 mg, respectively. The proportions of other reagents remained constant, and LNCs-ECH were prepared. The results are shown in Table 5. The highest encapsulation efficiency of LNCs-ECH was achieved when 15 mg of Span 80 was used.

[0074] Table 5. Effect of Span 80 dosage on LNCs-ECH particle size and encapsulation efficiency

[0075]

[0076] Note: Different lowercase letters in the same line indicate significant differences (P < 0.05).

[0077] (3) Investigation on the dosage of polyethylene glycol 12-hydroxystearate

[0078] LNCs-ECH were prepared by adding polyethylene glycol 12-hydroxystearate at concentrations of 100, 200, 300, 400, and 500 mg, respectively, while keeping the proportions of other reagents constant. The results are shown in Table 6. The highest encapsulation efficiency of LNCs-ECH was achieved when the amount of polyethylene glycol 12-hydroxystearate was 300 mg.

[0079] Table 6. Effect of polyethylene glycol 12-hydroxystearate dosage on particle size and encapsulation efficiency of LNCs-ECH

[0080]

[0081] Note: Different lowercase letters in the same line indicate significant differences (P < 0.05).

[0082] (4) Investigation on the dosage of caprylic acid / capric acid triglycerides

[0083] The mass of caprylic / capric triglyceride was 100, 125, 150, 175, and 200 mg, respectively. The proportions of other reagents remained unchanged to prepare LNCs-ECH. The results are shown in Table 7. The highest encapsulation efficiency of LNCs-ECH was achieved when the amount of caprylic / capric triglyceride was 150 mg.

[0084] Table 7. Effect of caprylic / capric triglyceride dosage on particle size and encapsulation efficiency of LNCs-ECH

[0085]

[0086] Note: Different lowercase letters in the same line indicate significant differences (P < 0.05).

[0087] (5) Investigation of Voa solution concentration

[0088] The prepared LNCs-ECH was mixed with Voa solutions of 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, and 5 mg / mL at a ratio of 3:1 (v / v), and stirred at 250 rpm for 24 h at room temperature to prepare Voa-LNCs-ECH. The results are shown in Table 8. Voa was adsorbed on the LNCs surface; the larger the particle size, the more Voa was adsorbed. When the particle size no longer changed, it indicated that adsorption had reached saturation. When the Voa solution concentration was 4 mg / mL and 5 mg / mL, the particle size of the prepared Voa-LNCs-ECH was not significantly different. From a cost perspective, a Voa solution concentration of 4 mg / mL was chosen.

[0089] Table 8. Effect of Voa solution concentration on particle size

[0090]

[0091]

[0092] Note: Different lowercase letters in the same line indicate significant differences (P < 0.05).

[0093] Example 3 Preparation and characterization of lipid nanocapsules modified with Voa compound X (echinacoside (ECH), quercetin (QUE), doxorubicin (DOX), paclitaxel (PTX), or verbascoside (ACT)).

[0094] 1. Preparation method

[0095] First, 20 mg of compound X, 300 mg of polyethylene glycol 12-hydroxystearate, 150 mg of caprylic / capric triglyceride, 15 mg of Span 80, 15 mg of sodium chloride, and 500 mg of distilled water were mixed to a total weight of 1 g. The mixture was magnetically stirred at 500 rpm while simultaneously undergoing a temperature fluctuation process: room temperature – 70°C – 45°C – 70°C – 45°C – 70°C – 55°C. At the final cooling point, when the phase transition temperature (55°C) was reached, 5 g of distilled water at 0°C was added, and stirring was continued at room temperature for 5 min to obtain a lipid nanocapsule (LNCs-X) solution encapsulated with X. The prepared LNCs-X solution was then passed through a gel permeation chromatography column to remove sodium chloride and unencapsulated compound X.

[0096] The prepared LNCs-X was mixed with a Voa solution with a concentration of 4 mg / mL at a ratio of 3:1 (v / v), and the mixture was magnetically stirred at 250 rpm at room temperature until the ethanol was completely evaporated to obtain Voa-LNCs-X.

[0097] 2. Results

[0098] The particle size, polydispersity index (PDI), zeta potential, and encapsulation efficiency of Voa-LNCs-ECH, Voa-LNCs-QUE, Voa-LNCs-DOX, Voa-LNCs-PTX, and Voa-LNCs-ACT are shown in Table 9.

[0099] Table 9. Particle size, zeta potential, and encapsulation efficiency of Y-modified lipid nanocapsules

[0100] LNCs Particle size (nm) PDI Zeta(mV) Encapsulation efficiency (%) Voa-LNCs-ECH 108.6±2.17 0.14±0.01 -11.2±0.25 91.1±1.17 Voa-LNCs-QUE 102.2±2.03 0.12±0.02 -9.6±0.12 95.4±1.32 Voa-LNCs-DOX 101.4±2.02 0.17±0.04 -8.2±0.23 93.6±1.24 Voa-LNCs-PTX 103.1±2.01 0.13±0.05 -9.3±0.57 93.4±1.24 Voa-LNCs-ACT 106.2±1.09 0.11±0.02 -7.6±0.97 93.6±1.23

[0101] As can be seen from the table above, echinacoside (ECH), quercetin (QUE), doxorubicin (DOX), paclitaxel (PTX), or verbascoside (ACT) have similar polydispersity index (PDI) and zeta potential.

[0102] 3. Conclusion

[0103] This drug delivery system can encapsulate different neuroprotective active compounds, exhibiting good stability and high encapsulation efficiency.

[0104] Example 4: Effect of Voa-modified echinacoside lipid nanocapsules (Voa-LNCs-ECH) on ECH content in mouse brains

[0105] 1. Test Methods

[0106] Determination of Echinacoside (ECH) Concentration in Mouse Brain Tissue: Thirty-six Kunming mice were randomly divided into three groups: ① free ECH; ② LNCs-ECH; ③ Voa-LNCs-ECH. Mice were fasted for 12 hours before the experiment but allowed free access to water. Each group of mice was administered 20 mg / kg of ECH via a single tail vein injection. Mice were sacrificed at 0.5, 2, 4, 6, 12, and 24 hours post-administration, and brain tissue was collected. The brain tissue was washed with PBS to remove blood adhering to the organs and weighed. Brain tissue homogenates were prepared, and the concentration of ECH in the brain tissue was determined.

[0107] ECH was detected using a Waters Alliance 2695 high-performance liquid chromatograph with a Hypersil ODS (C18) column (4.6 mm × 250 mm, 5 μm). The mobile phase was acetonitrile-0.4% phosphoric acid aqueous solution gradient elution (mobile phase A was 0.4% phosphoric acid aqueous solution, B was acetonitrile, gradient conditions were 0–5 min 83% A-17% B; 5–20 min 80% A-20% B; 20–25 min 80% A-20% B; 25–30 min 83% A-17% B), flow rate was 1.0 mL / min, detection wavelength was 333 nm, column temperature was 30 °C, and injection volume was 20 μL.

[0108] 2. Test Results

[0109] Brain tissue distribution: The content of ECH in brain tissue at different time points after tail vein injection of free ECH, ECH-LNCs, or Voa-LNCs-ECH in mice was determined. The results are shown in Table 10. 0.5 h after administration, the content of ECH in the free ECH group was significantly higher than that in the ECH-LNCs group. However, at 4 h, the ECH content in the brain tissue of the free ECH group decreased rapidly, and by 24 h, ECH was undetectable, possibly due to drug metabolism. Furthermore, at 4, 6, 12, and 24 h, the ECH content in the brain of the ECH-LNCs group was higher than that of the free ECH group. At 2 h, the ECH content in the brain tissue of the Voa-LNCs-ECH group was significantly higher than that of the LNCs-ECH group, and this trend continued until 24 h. This further demonstrates that Voa modification increased the targeting of LNCs-ECH to brain tissue and effectively improved drug accumulation in the brain.

[0110] Table 10. Quantitative analysis of ECH content in brain tissue at different time points

[0111]

[0112]

[0113] Note: Different lowercase letters in the same line indicate significant differences (P < 0.05).

[0114] 3. Conclusion

[0115] The ECH content in the brains of mice in the Voa-LNCs-ECH group was significantly increased, further demonstrating that Voa modification of the LNCs-ECH surface significantly enhanced the intracranial concentration of ECH and improved its brain-targeting ability. Example 5: Therapeutic effect of Voa-modified echinacoside lipid nanocapsules (Voa-LNCs-ECH) on APP / PS1 double transgenic AD mice.

[0116] 1. Test Methods

[0117] AD model mouse grouping and administration: APP / PS1 double transgenic AD mice were maintained at approximately 9 months of age. Treatment was administered at week 30. Twenty mice were divided into four groups (n=5 per group) and treated with intravenous injections of saline (Sal), echinacoside (ECH), LNCs-ECH, and Voa-LNCs-ECH (20 mg / kg), respectively, twice weekly (Tuesday and Friday). After 6 weeks of continuous administration, the learning and cognitive abilities of the AD mice were assessed using the Morris water maze test.

[0118] Morris Water Maze Test: The Morris water maze test was used to assess the learning and memory abilities of mice. The Morris water maze consisted of a circular pool (120 cm in diameter, 60 cm high) with a black inner wall divided into four equal quadrants, filled with water (25°C) to a depth of 30 cm. An escape platform (10 cm in diameter) was placed in one quadrant and submerged approximately 1 cm below the water surface. The escape latency of the mice was observed in each of the four quadrants for 5 consecutive days. If a mouse could not reach the platform within 60 seconds, it was guided to the platform and allowed to remain there for 10 seconds. After 5 days of training, the platform was removed, and a spatial exploration experiment was conducted. Mice from each group were placed in the water from the same entry point, and the number of times they crossed the original platform and the time spent in the quadrant containing the original platform were observed.

[0119] 2. Test Results

[0120] The results of the water maze experiment are shown in Tables 11-13. After 5 days of orientation and navigation training, compared with the Sal group, the escape latency of the Voa-LNCs-ECH, LNCs-ECH, and ECH groups was significantly shortened. Furthermore, the Voa-LNCs-ECH group showed a more significant effect compared to the LNCs-ECH and ECH groups. After the orientation and navigation experiment, the platform was removed, and a spatial exploration experiment was conducted to assess the memory ability of AD mice. The results showed that after Voa-LNCs-ECH, LNCs-ECH, and ECH treatment, the percentage of time spent crossing the platform relative to the original platform quadrant was significantly longer than in the Sal group, with the Voa-LNCs-ECH group showing the strongest effect. The experimental results indicate that Voa modification can significantly enhance the learning and memory abilities of AD mice improved by ECH.

[0121] Table 11. Escape latency of mice in each group

[0122] time Sal ECH LNCs-ECH Voa-LNCs-ECH Day 1 <![CDATA[69±3.14 a ]]> <![CDATA[57±2.12 b ]]> <![CDATA[59±3.16 c ]]> <![CDATA[53±3.34 d ]]> Day 2 <![CDATA[63±2.16 a ]]> <![CDATA[55±1.14 b ]]> <![CDATA[52±3.18 c ]]> <![CDATA[48±2.24 d ]]> Day 3 <![CDATA[59±1.15 a ]]> <![CDATA[45±2.16 b ]]> <![CDATA[43±2.16 c ]]> <![CDATA[36±2.24 d ]]> Day 4 <![CDATA[46±1.16 a ]]> <![CDATA[36±2.18 b ]]> <![CDATA[34±1.15 c ]]> <![CDATA[23±1.26 d ]]> Day 5 <![CDATA[42±2.15 a ]]> <![CDATA[31±1.22 c ]]> <![CDATA[23±1.16 c ]]> <![CDATA[16±1.12 d ]]>

[0123] Note: Different lowercase letters in the same line indicate significant differences (P < 0.05).

[0124] Table 12. Comparison of the number of times mice crossed the platform in each group.

[0125] Group Sal ECH LNCs-ECH Voa-LNCs-ECH frequency <![CDATA[2±0.04 a ]]> <![CDATA[3±0.04 b ]]> <![CDATA[5±0.05 c ]]> <![CDATA[8±0.12 d ]]>

[0126] Note: Different lowercase letters in the same line indicate significant differences (P < 0.05).

[0127] Table 13. Percentage of time mice spent in the quadrant where the original platform was located in each group

[0128] Group Sal ECH LNCs-ECH Voa-LNCs-ECH percentage <![CDATA[11.4±0.12 a ]]> <![CDATA[17.9±0.16 b ]]> <![CDATA[23.2±0.15 c ]]> <![CDATA[35.7±0.18 d ]]>

[0129] Note: Different lowercase letters in the same line indicate significant differences (P < 0.05).

[0130] 4. Conclusion

[0131] Compared with free ECH, LNCs-ECH can improve the learning and memory abilities of APP / PS1 double transgenic AD mice; after Voa modification, the effect of LNCs-ECH on improving learning and memory abilities is further enhanced.

[0132] Example 6: Potential application of Voa-modified echinacoside lipid nanocapsules in anti-aging effects

[0133] In this experiment, Voa-LNCs-ECH was added to maize culture medium to feed fruit flies in order to observe the anti-aging effect of Voa-LNCs-ECH.

[0134] 1. Experimental Methods

[0135] Drosophila melanogaster were housed in culture tubes containing 0.2% (by mass) Voa-LNCs-ECH, LNCs-ECH, or ECH medium, with a normal culture medium serving as a blank control group. Four groups were formed. Each dosage group contained approximately 200 flies, half male and half female, with four replicates per group. Observations were conducted twice daily, morning and evening, recording the number of dead and surviving flies until all flies had died. Lifespan was compared between groups, recording the median time to death, maximum lifespan, and average lifespan.

[0136] The time it takes for half of the fruit flies to die in each treatment group is considered the half survival time. The average lifespan of the last 10 fruit flies to die in each group is the maximum lifespan. The arithmetic mean of the lifespans of all fruit flies is the average lifespan. The lifespan extension rate is calculated as (average lifespan of the experimental group - average lifespan of the control group) / average lifespan of the control group × 100%.

[0137] The culture medium formula is as follows: 76g water, 10g corn flour, 1.5g agar, 0.7g yeast powder, 13.5g sugar, and 0.5mL propionic acid. The preparation method is as follows: Take half the required amount of water, add agar, boil and stir until dissolved, then add sugar; take the other half of the water, mix the corn flour into a paste, add it to the agar water, stir and boil until a paste forms, add 0.5ml propionic acid and stir, dispense into culture flasks, and plug with cotton plugs; autoclave for 20 minutes, cool, and then add yeast.

[0138] 2. Results

[0139] (1) Compared with the group without added drugs in the culture medium, Voa-LNCs-ECH, LNCs-ECH, or free ECH all prolonged the half-life, average lifespan, and maximum lifespan of Drosophila to varying degrees. The free ECH group, LNCs-ECH group, and Voa-LNCs-ECH group prolonged the average lifespan of male and female Drosophila by 20.6%, 27.2%, and 41.9%, respectively (Table 14).

[0140] Table 14. Effects on fruit fly lifespan

[0141]

[0142] Note: Different lowercase letters in the same column indicate significant differences (P < 0.05).

[0143] 3. Conclusion

[0144] Fruit fly survival experiments have shown that Fal-ACT-LNCs have anti-aging effects.

Claims

1. A brain-targeting lipid nanocapsule drug delivery system mediated by cannabinoid type 1 receptor, characterized in that, The invention includes lipid nanocapsules encapsulating drug X, wherein the surface of the lipid nanocapsules is modified with a ligand compound Y of a cannabinoid type 1 receptor, wherein the ligand compound Y is argentinium chloride; The preparation method of the drug delivery system includes the following steps: (1) Mix drug X, Span 80, distilled water, sodium chloride, medium-chain triglycerides and polyethylene glycol 12-hydroxystearate evenly. Under stirring conditions, after three alternating heating and cooling processes, when the phase transition temperature point is reached in the last cooling, add distilled water at 0°C and continue stirring at room temperature to obtain lipid nanocapsules loaded with drug X. By mass percentage, the concentration of polyethylene glycol 12-hydroxystearate is 20-30%, the concentration of medium-chain triglycerides is 15-20%, the concentration of Span 80 is 1.5-2%, the concentration of sodium chloride is 1.5-2%, and the concentration of drug X is 1-2%. The heating and cooling process is as follows: room temperature—70℃—45℃—70℃—45℃—70℃—55℃; the heating or cooling rate is 4℃ / min; the stirring rate is 500~600 rpm. (2) The lipid nanocapsules containing drug X are mixed with an ethanol solution of ligand compound Y at a volume ratio of 3:1, wherein the concentration of the ethanol solution of Y is 1~5 mg / mL. The mixture is stirred until the ethanol is completely evaporated at a stirring rate of 250 rpm to obtain the brain-targeting lipid nanocapsule drug delivery system mediated by cannabinoid type 1 receptor.

2. The cannabinoid type 1 receptor-mediated brain-targeting lipid nanocapsule drug delivery system as described in claim 1, characterized in that, The lipid nanocapsules encapsulate one of the following drugs: doxorubicin, paclitaxel, verbascoside, echinacoside, quercetin, or anthocyanin.

3. The cannabinoid type 1 receptor-mediated brain-targeting lipid nanocapsule drug delivery system as described in claim 1, characterized in that, By mass percentage, the concentration of polyethylene glycol 12-hydroxystearate is 30%, the concentration of medium-chain triglycerides is 15%, the concentration of Span 80 is 1.5%, the concentration of sodium chloride is 1.5%, and the concentration of drug X is 2%.

4. The cannabinoid type 1 receptor-mediated brain-targeting lipid nanocapsule drug delivery system as described in claim 1, characterized in that, The medium-chain triglyceride is caprylic / capric triglyceride.

5. The use of the cannabinoid type 1 receptor-mediated brain-targeting lipid nanocapsule drug delivery system as described in any one of claims 1-4 in the preparation of drugs that cross the blood-brain barrier.

6. The use of the cannabinoid type 1 receptor-mediated brain-targeting lipid nanocapsule drug delivery system as described in any one of claims 1-4 in the preparation of drugs for treating Alzheimer's disease or for anti-aging.

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