A nano-drug delivery system for treating methamphetamine addiction, a preparation method thereof, and a pharmaceutical preparation

Abstract

The application discloses a nano drug delivery system for treating methamphetamine addiction and a preparation method and a pharmaceutical preparation thereof, and belongs to the technical field of medical preparations, wherein the nano drug delivery system takes an exosome derived from a human umbilical vein endothelial cell (HUVEC) subjected to hypoxic pretreatment and transcriptional activation protein (TAT) engineering as a nano carrier of cannabidiol (CBD), and compared with a current drug administration mode mainly through intracerebroventricular injection with high invasiveness or high-dose intraperitoneal injection (40-100 mg / kg) for treating METH addiction, the nano drug delivery system is used for treating METH addiction through nasal administration, and the effective concentration thereof is only 2 mg / kg, which is only 1 / 20 of the minimum effective concentration (40 mg / kg) of the intraperitoneal injection of a CBD solution for treating METH addiction.

Description

Technical Field

[0001] This invention belongs to the field of medical preparation technology, specifically relating to a nano-drug delivery system for treating methamphetamine addiction, its preparation method, and the drug formulation thereof. Background Technology

[0002] Methamphetamine (MET) is a potent central nervous system stimulant that induces addictive behavior by impacting neuroinflammation and reward circuit dysfunction. Its high addictiveness and relapse rate pose a significant challenge to global public health. At the pathophysiological level, in addition to synaptic dysfunction and neurotransmitter dysregulation, extensive literature indicates that neuroinflammation is a key pathological driver of MET addiction. Abnormal microglial activation and its mediated pro-inflammatory responses play a crucial role in MET-induced neurotoxicity. Despite decades of research focusing on small-molecule drug development, existing therapies often suffer from poor adherence and severe side effects; therefore, developing novel interventions is urgently needed.

[0003] Cannabidiol (CBD) is one of the most abundant and active substances among natural cannabinoids, and its potential anti-inflammatory and neuroprotective effects have made it an important candidate molecule for treating METH addiction. However, the application of this compound in the treatment of central nervous system diseases is severely limited by its low water solubility, low bioavailability, and poor permeability across the blood-brain barrier (BBB).

[0004] Currently, various nanomedicine delivery systems targeting CBD (including polymer and lipid-based nanoparticles) have been developed; however, these systems generally suffer from low biomembrane penetration efficiency and potential immunogenicity. In contrast, exosomes, with their excellent biocompatibility, low immunogenicity, and good blood-brain barrier penetration, have become an ideal platform for brain-targeted delivery systems. However, natural exosomes lack target specificity, and most exosomes accumulate in the liver and spleen through systemic circulation. Furthermore, there are currently no studies using functional exosomes with anti-inflammatory effects as nanocarriers to load CBD. Additionally, although there are reports of exosomes being administered intranasally to treat neurological diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD), this method has not yet been used to treat methemoglobin (METH) addiction. Summary of the Invention

[0005] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0006] Therefore, the object of the present invention is to overcome the shortcomings of the prior art and provide a nano-drug delivery system for treating methamphetamine addiction.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: the nano-drug delivery system is obtained by loading cannabidiol onto exosomes extracted from hypoxic pretreated human umbilical vein endothelial cells as nanocarriers and then functionalizing them.

[0008] The functionalization modification involves modifying cannabidiol-loaded exosomes using the transcription activator TAT from human immunodeficiency virus.

[0009] As a preferred embodiment of the nano-drug delivery system for treating methamphetamine addiction according to the present invention, wherein: the cannabidiol loading rate of the nano-drug delivery system is >16%, and the encapsulation rate is >7%.

[0010] As a preferred embodiment of the nanomedicine delivery system for treating methamphetamine addiction according to the present invention, the nanomedicine delivery system inhibits methamphetamine-induced behavioral sensitization and conditioned place preference by inhibiting methamphetamine-induced microglial cell activation and inflammatory factor release in the mPFC and VTA brain regions.

[0011] Beneficial effects of this invention:

[0012] This invention extracts exosomes loaded with cannabidiol (CBD) using an endogenous drug delivery strategy from hypoxic-pretreated human umbilical vein endothelial cells (HUVECs) (HP-Exo-CBD). These exosomes are then functionalized using the transcription activator TAT from human immunodeficiency virus (HIV) to obtain HP-Exo-CBD-TAT. The vector itself (HP-Exo-TAT) possesses the following characteristics:

[0013] 1. At the cellular level, it can inhibit the increase of lipopolysaccharide (LPS)-induced microglial BV-2 inflammatory factors and promote the release of anti-inflammatory factors; 2. It can alleviate methamphetamine-induced behavioral sensitization; reduce the release of inflammatory factor IL-1β in the medial prefrontal cortex (mPFC) and ventral midbrain tegmentum (VTA) induced by methamphetamine; alleviate methamphetamine-induced conditioned position preference; and reduce the activation of microglia in the medial prefrontal cortex (mPFC) and ventral midbrain tegmentum (VTA) induced by methamphetamine.

[0014] The nano-drug delivery system provided by this invention can rapidly accumulate in the brain within 3 hours. The TAT-modified exosomes (HP-Exo-CBD-TAT) showed a 1.4 to 1.6-fold increase in brain accumulation compared to unmodified exosomes (HP-Exo-CBD) at various time points. Furthermore, HP-Exo-CBD-TAT can effectively alleviate methamphetamine addiction, reduce METH-induced behavioral sensitization and conditioned positional preference in mice, and further reduce METH-induced neuroinflammation and microglial activation.

[0015] Another object of the present invention is to provide a method for preparing a nano-drug delivery system for treating methamphetamine addiction.

[0016] To solve the above-mentioned technical problems, the present invention provides the following technical solution: including,

[0017] Human umbilical vein endothelial cells were cultured in DMEM medium until the cells reached 80% confluence, and then incubated for 24 hours in serum-free medium containing cannabidiol under 1% O2 hypoxia. After incubation, cannabidiol-loaded exosomes were obtained through post-processing.

[0018] The post-processing involves centrifuging 300 g of the cell supernatant after incubation for 10 min, centrifuging 1000 g of the obtained supernatant for 20 min to remove cell debris and dead cells, transferring the obtained supernatant and centrifuging 10000 g for 30 min to remove impurities from the sample, and finally centrifuging 100000 g of the obtained supernatant at high speed for 70 min to obtain cannabidiol-loaded exosomes, resuspending the precipitate in PBS and storing it at -80℃.

[0019] Cannabidiol-loaded exosomes were incubated with transcription activator TAT at a mass ratio of 1:10 and then centrifuged to obtain a nano-drug delivery system.

[0020] Another object of the present invention is to provide a pharmaceutical formulation for nasal administration in the treatment of methamphetamine addiction, the pharmaceutical formulation comprising the nano-drug delivery system and further comprising pharmaceutically acceptable excipients.

[0021] It should be noted that nasal administration is a brain-targeted drug delivery method that allows drugs to be absorbed through the trigeminal nerve endings in the nasal mucosa, transported to the brainstem and brain via neuronal cell bodies, and bypassing the blood-brain barrier. It is non-invasive and can reduce systemic exposure and lower the risk of side effects by directly reaching the target site.

[0022] As a preferred embodiment of the pharmaceutical preparation described in this invention, the dosage form of the pharmaceutical preparation includes an injection solution and a lyophilized powder injection.

[0023] Beneficial effects of this invention:

[0024] This invention utilizes a nano-drug delivery system as a drug formulation for nasal administration in the treatment of methamphetamine addiction. Compared to current methods of treating methamphetamine addiction, which mainly involve highly invasive intraventricular injections or high-dose intraperitoneal injections (40-100 mg / kg), nasal administration achieves an effective concentration of only 2 mg / kg, which is only 1 / 20th of the lowest effective concentration (40 mg / kg) for treating methamphetamine addiction with intraperitoneal injection of CBD solution. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The figures show the potential maps of exosomes obtained by mixing HP-Exo-CBD and DSPE-PEG-TAT in different proportions. The data in the figures are expressed as mean ± SEM. The two-tailed unpaired Student's t-test was used for analysis. * in the figures indicates a statistically significant difference compared with HP-Exo-CBD (P < 0.05); ns in the figures indicate no significant difference between the two groups. All experiments were performed independently in triplicate.

[0027] Figure 2 These are transmission electron microscopy (TEM) images of different exosomes from Example 1 of the present invention; wherein, Figure 2 A is a TEM image of exosomes (Exo) of HUVECs under normoxic conditions; Figure 2 B is a TEM image of exosomes (HP-Exo) of HUVECs under hypoxic conditions; Figure 2 C is a TEM image of exosomes loaded with CBD (HP-Exo-CBD) in HUVECs under hypoxic conditions; Figure 2 D is a TEM image of TAT-modified exosomes of HUVECs under hypoxic conditions (HP-Exo-TAT); Figure 2 E is a TEM image of exosomes loaded with CBD (HP-Exo-CBD-TAT) from TAT-modified HUVECs under hypoxic conditions;

[0028] Figure 3 The results of dynamic light scattering (DLS) of different exosomes are shown in Example 1 of the present invention; wherein, Figure 3 A is the DLS diagram of Exo; Figure 3 B is the DLS diagram of HP-Exo; Figure 3C is the DLS diagram of HP-Exo-CBD; Figure 3 D is the DLS diagram of HP-Exo-TAT; Figure 3 E represents the DLS diagram of HP-Exo-CBD-TAT; Figure 3 F represents the Zeta potential of each exosome;

[0029] Figure 4 The nanoflow cytometry results of different exosomes in Example 1 of the present invention are shown; where AE represents the nanoflow cytometry results of Exo, HP-Exo, HP-Exo-CBD, HP-Exo-TAT and HP-Exo-CBD-TAT, respectively.

[0030] Figure 5 The results of Western blot analysis of surface protein markers of exosomes obtained from different exosomes in Example 1 of this invention;

[0031] Figure 6 The results of CBD release from exosomes in Example 2;

[0032] Figure 7 In Example 3, HP-Exo inhibited LPS-stimulated BV2 microglia from polarizing to the M1 type and promoted their transformation to the M2 type, thus inhibiting excessive activation of inflammation; wherein, Figure 7 A is a marker of the M1 state. IL-1b Changes in mRNA levels; Figure 7 B is a marker of the M1 state. iNOS Changes in mRNA levels; Figure 7 C is a marker of the M1 state. IL-6 Changes in mRNA levels; Figure 7 D is a marker of the M2 state. Arg-1 Changes in mRNA levels; Figure 7 E is a marker of the M2 state. IL-4 Changes in mRNA levels;

[0033] Data in the figure are expressed as mean ± SEM. Analysis was performed using a two-tailed unpaired Student's t-test. * indicates a statistically significant difference (P < 0.05) between the treated group and the LPS model group; ** indicates a significant statistical difference (P < 0.01) between the treated group and the LPS model group; *** indicates a highly significant statistical difference (P < 0.001) between the treated group and the LPS model group; and ns indicates no significant difference between the treated group and the LPS model group.

[0034] In the figure, # indicates that the LSP group is significantly different from the blank control group (P<0.05); ## indicates that the LSP group is significantly different from the blank control group (P<0.01); and ### indicates that the LSP group is significantly different from the blank control group (P<0.001). All experiments were performed independently three times.

[0035] Figure 8 This document describes the in vivo distribution of DID-labeled exosomes HP-Exo-TAT and HP-Exo-CBD-TAT after nasal drops of DID-labeled exosomes in PBS at the same concentration, as shown in Example 4. Figure 8 A represents the distribution of exosomes in mice at different time points after administration to each group; Figure 8 B is a statistical graph of the fluorescence intensity of exosomes in the brain at various time points; Figure 8 C shows the images of the isolated organs of mice in each group after 24 h; Figure 8 Data in B are expressed as mean ± SEM and analyzed using a two-tailed unpaired Student's t-test. * indicates statistical difference compared to the HP-Exo-CBD group (P < 0.05), and ** indicates significant difference compared to the HP-Exo-CBD group (P < 0.01). The number of animals used in the experiment was 3.

[0036] Figure 9 In Example 5, HP-Exo-CBD-TAT inhibited methamphetamine-induced behavioral sensitization and the release of inflammatory factors in the mPFC and VTA brain regions; Figure 9 A represents the experimental procedure; Figure 9 B represents the formation of METH-induced behavioral sensitization in mice during the formative period, accompanied by the administration of exosomes. Figure 9 C represents the post-withdrawal ignition period, during which exosomes can effectively block METH-induced ignition behavior. Figure 9 D represents the mouse mPFC brain region. IL-1b Quantitative analysis; Figure 9 E represents the VTA brain region in mice. IL-1b Quantitative analysis;

[0037] The data in the figure are all expressed as mean ± SEM, where Figure 9 Data B was analyzed using a two-tailed paired Student's t-test. Figure 9 C- Figure 9The E data were analyzed using a two-tailed unpaired Student's t-test. In the figure, * indicates a statistically significant difference compared to the METH treatment group (P < 0.05), ** indicates a significant difference compared to the METH treatment group (P < 0.01), and *** indicates a highly significant statistical difference compared to the METH treatment group (P < 0.001).

[0038] In the figure, # indicates a statistically significant difference compared to the vehicle group (P < 0.05), ## indicates a significant difference compared to the vehicle group (P < 0.01), and ### indicates a highly significant statistical difference compared to the vehicle group (P < 0.001).

[0039] In the figure, '&' indicates a statistically significant difference (P < 0.05) compared to the METH + HP-Exo-TAT group, and '&&' indicates a highly significant statistical difference (P < 0.001) compared to the METH + HP-Exo-TAT group; 'ns' indicates no significant difference between the two groups. Figure 9 B. Figure 9 The number of animals used in experiment C was 6. Figure 9 D、 Figure 9 The number of animals used in experiment E was 3.

[0040] Figure 10 In Example 5, HP-Exo-CBD-TAT inhibited methamphetamine-induced microglial activation in mouse CPP, mPFC, and VTA brain regions; Figure 10 A represents the experimental procedure; Figure 10 B represents exosome inhibition of METH-induced increase in CPP scores in mice; Figure 10 C is a schematic diagram of Iba1+ immunofluorescence in the mPFC brain region of mice in each group; Figure 10 D represents the number of Iba1-positive cells per unit area in the mPFC brain region of mice in each group; Figure 10 E is a schematic diagram of Iba1+ immunofluorescence in the VTA brain region of mice in each group; Figure 10 F represents the number of Iba1-positive cells per unit area in the VTA brain region of mice in each group;

[0041] All data in the figure are expressed as mean ± SEM, where Figure 10 Data B was analyzed using a two-tailed unpaired student's t-test. Figure 10 D, Figure 10F was analyzed using one-tailed unpaired Student's t-test. In the figure, * indicates a statistically significant difference compared to the METH treatment group (P<0.05), ** indicates a significant difference compared to the METH treatment group (P<0.01), and *** indicates a highly significant statistical difference compared to the METH treatment group (P<0.001).

[0042] In the figure, # indicates a statistically significant difference compared to the vehicle group (P < 0.05), ## indicates a significant difference compared to the vehicle group (P < 0.01), and ### indicates a highly significant statistical difference compared to the vehicle group (P < 0.001); ns indicates no significant difference between the two groups. Figure 10 Experiment B used 6 animals. Figure 10 D、 Figure 10 The number of animals used in experiment E was 3.

[0043] Figure 11 This is a schematic diagram of the preparation process of the drug delivery system of the present invention and the mechanism of treatment of methamphetamine addiction. The present invention uses HP-Exo and cannabidiol to construct a novel nano-drug delivery system HP-Exo-CBD-TAT, which is used for the treatment of methamphetamine addiction. Detailed Implementation

[0044] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0045] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0046] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0047] Unless otherwise specified, all raw materials used in this invention are commercially available in the field.

[0048] The invention uses iNOS, IL-1b, IL-6, IL-4, Arg-1 and RPL27 The primer sequences for the gene are shown in Table 1.

[0049] Table 1 Primer sequence listing

[0050] ,

[0051] Example 1: This example provides a method for preparing a nanoparticle-based drug delivery system for nasal administration in the treatment of methamphetamine addiction. The nanoparticle-based drug delivery system uses exosomes extracted from hypoxic-pretreated human umbilical vein endothelial cells as nanocarriers loaded with cannabidiol, followed by functionalization modification. Specifically:

[0052] HUVECs cells were cultured in DMEM medium until they reached 80% confluence, then replaced with serum-free basal medium and incubated under the following conditions:

[0053] A. Incubate under normal oxygen conditions for 24 hours;

[0054] B. Incubate for 24 hours under hypoxic (1% O2) conditions with serum-free medium containing 5 μM CBD;

[0055] C. Incubate for 24 hours under hypoxic (1% O2) conditions with serum-free medium without CBD.

[0056] After incubation, the cell supernatant was centrifuged at 300 g for 10 min, then centrifuged at 1000 g for 20 min to remove cell debris and dead cells. The transferred supernatant was centrifuged at 10000 g for 30 min to remove impurities from the sample. The centrifuged supernatant was then ultracentrifuged at 100000 g for 70 min to obtain a high-purity exosome precipitate. The precipitate was resuspended in PBS and stored at -80℃. Exosomes isolated from HUVECs cultured under incubation condition A were labeled as Exo, exosomes under incubation condition B were labeled as HP-Exo-CBD, and exosomes under incubation condition C were labeled as HP-Exo.

[0057] HP-Exo-CBD and DSPE-PEG-TAT were mixed uniformly at mass ratios of 100:1, 10:1, and 1:1. The mixture was then brought to a final volume of 1 mL with PBS and incubated at 37 °C for 30 minutes. The mixture was transferred to the inner tube of a 10 kDa ultrafiltration tube and centrifuged at 4000 g for 30 minutes at 4 °C. The filtrate from the outer tube was discarded. The mixture was then centrifuged inverted mode at 2000 g for 2 minutes at 4 °C to obtain HP-Exo-CBD-TAT. The Zeta potentials of different binding products were measured, and the results are as follows: Figure 1 And as shown in Table 2.

[0058] Table 2. Zeta potential results of different exosome binding products

[0059] ,

[0060] Figure 1The figures show the potential maps of exosomes obtained by mixing HP-Exo-CBD and DSPE-PEG-TAT in different proportions. The data in the figures are expressed as mean ± SEM. The two-tailed unpaired Student's t-test was used for analysis. * in the figures indicates a statistically significant difference compared with HP-Exo-CBD (P < 0.05); ns in the figures indicate no significant difference between the two groups. All experiments were performed independently in triplicate.

[0061] from Figure 1 As can be seen from the results in Table 2, the optimal mixing ratio of HP-Exo-CBD with DSPE-PEG-TAT at a mass ratio of 10:1 is the best. Therefore, the mixing ratio of HP-Exo-CBD to DSPE-PEG-TAT is selected as 10:1.

[0062] HP-Exo and DSPE-PEG-TAT are mixed at a mass ratio of 10:1, using the same method as HP-Exo-CBD, to obtain HP-Exo-TAT.

[0063] Exosomes were identified by observation using transmission electron microscopy (TEM), dynamic light scattering (DLS), nanoflow cytometry, and Western blotting (WB).

[0064] The CBD loading in exosomes was quantified by high-performance liquid chromatography (HPLC) at a wavelength of 254 nm. Specifically, 25 mg of sample (HP-Exo-CBD) was placed in a 1.5 mL centrifuge tube, 600 mL of chloroform was added, the mixture was vortexed, sonicated, and then centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected, filtered through a 0.22 mm filter membrane, and 20 mL was injected. Chromatographic conditions: C18 column (4.6 × 250 mm, 5 mm), mobile phase: methanol-water (95:5), column temperature: 25 °C, detection wavelength: 220 nm.

[0065] The formulas for calculating the load factor and encapsulation rate of CBD are as follows:

[0066] CBD loading rate (%) = (CBD mass in exosomes / total exosomal protein mass) × 100%

[0067] CBD encapsulation rate (%) = (mass of CBD in exosomes / mass of added CBD) × 100%

[0068] Figure 2 Transmission electron microscopy (TEM) images of different exosomes show that the exosomes are typically disc-shaped or spherical.

[0069] Figure 3 Dynamic light scattering (DLS) results for different exosomes Figure 3The particle size distribution peaks of AE), Exo, HP-Exo, HP-Exo-CBD, HP-Exo-TAT and HP-Exo-CBD-TAT are 128.3±0.5 nm, 131.1±1.4 nm, 147.6±2.0 nm, 185.7±4.9 nm and 186.5±3.4 nm, respectively.

[0070] Table 3. Zeta potential results of different exosomes

[0071] ,

[0072] It can be seen that the Zeta potentials of different exosomes were -8.0±0.6 mV, -8.1±0.4 mV, -7.6±0.7 mV, -3.7±0.5 mV and -3.9±0.9 mV, respectively. Figure 3 F and Table 3).

[0073] Figure 4 The average particle size of different exosomes was determined by nanoflow cytometry, and it can be seen that they are 58.9 nm, 62.2 nm, 57.9 nm, 62.4 nm and 58.9 nm, respectively.

[0074] In summary, although the specific particle size varies due to the different detection principles of different technologies, the particle size range of each group of exosomes all conforms to the standard exosome size range of 30-200 nm.

[0075] The expression of exosome-specific markers (TSG101, Alix, CD63, CD81) and the loss of the negative protein GM130 were detected by Western blotting. The results are as follows: Figure 5 As shown, the above experiments confirm the successful extraction of exosomes.

[0076] According to tests and calculations, the CBD loading rate and encapsulation efficiency of the HP-Exo-CBD-TAT nanoparticle drug delivery system prepared in this embodiment are 16.32% and 7.82%, respectively.

[0077] Example 2: This example provides an in vitro release assay of exosomes, specifically:

[0078] CBD-loaded exosomes (HP-Exo-CBD) were placed in a 12 kDa dialysis bag and immersed in a beaker containing 40 mL of PBS (containing 0.5% Tween-80). The mixture was stirred at 37°C. At predetermined time points, 1 mL of the solution was collected to replenish an equal volume of release medium. The CBD release was quantitatively determined by HPLC. The results are shown below. Figure 6 And as shown in Table 4.

[0079] Table 4. Percentage of CBD Release at Each Data Collection Time Point

[0080] ,

[0081] The results show that HP-Exo-CBD releases rapidly in the first 12 hours, then gradually slows down, with a cumulative release rate of 53.31±1.37% over 24 hours. It should be noted that the TAT modification in this invention does not change the encapsulation efficiency, drug loading rate, or exosome drug loading characteristics such as drug release. The peptide modification is to improve transport and cellular uptake. Therefore, HP-Exo-CBD-TAT has the same in vitro release effect as HP-Exo-CBD.

[0082] Example 3: This example verifies the anti-inflammatory effects of different exosomes. Specifically:

[0083] This embodiment verifies the anti-inflammatory effects of different exosomes. Microglial polarization plays an important role in central nervous system (CNS) inflammation. Under LPS stimulation, microglia transition from the M0 resting state to the activated M1 state, releasing pro-inflammatory factors and exacerbating the inflammatory response. Conversely, microglia can also transition from the M1 state to the M2 state, releasing anti-inflammatory factors and inhibiting the inflammatory response.

[0084] Therefore, to evaluate the anti-inflammatory effect of exosomes derived from HUVECs under hypoxia treatment (HP-Exo), Exo and HP-Exo were co-cultured with microglia (BV-2). BV-2 cells were seeded in 6-well plates at 4 × 10⁶ cells per well. 5 Cells were cultured for 24 h, and the supernatant was removed. The blank control group received only basal medium. The LPS model group and the drug-treated group received basal medium containing 20 ng / mL LPS. The drug-treated group received basal medium containing 100 μg / mL Exo or HP-Exo for 6 h. Total RNA was extracted using the TRIzol method and reverse transcribed according to the kit instructions. Gene fragments were amplified using this RNA as templates. Primer sequences are shown in Table 1. RPL27 was used as an internal control. Amplification was performed using the SYBR Green method on a TOptical real-time quantitative PCR instrument (Analytik Jena, Germany). Data were analyzed using the ΔΔCt method and normalized to RPL27. Results are as follows: Figure 7 As shown in Table 5.

[0085] Table 5

[0086] ,

[0087] Figure 7 In Example 3, HP-Exo inhibited LPS-stimulated BV2 microglia from polarizing to the M1 type and promoted their transformation to the M2 type, thus inhibiting excessive activation of inflammation; wherein, Figure 7 A is a marker of the M1 state. IL-1b Changes in mRNA levels; Figure 7 B is a marker of the M1 state. iNOS Changes in mRNA levels; Figure 7 C is a marker of the M1 state. IL-6 Changes in mRNA levels; Figure 7 D is a marker of the M2 state. Arg-1 Changes in mRNA levels; Figure 7 E is a marker of the M2 state. IL-4 Changes in mRNA levels;

[0088] Data in the figure are expressed as mean ± SEM. Analysis was performed using a two-tailed unpaired Student's t-test. * indicates a statistically significant difference (P < 0.05) between the treated group and the LPS model group; ** indicates a significant statistical difference (P < 0.01) between the treated group and the LPS model group; *** indicates a highly significant statistical difference (P < 0.001) between the treated group and the LPS model group; and ns indicates no significant difference between the treated group and the LPS model group.

[0089] In the figure, # indicates a statistically significant difference between the LSP model group and the blank control group (P < 0.05); ## indicates a statistically significant difference between the LSP model group and the blank control group (P < 0.01); and ### indicates a highly statistically significant difference between the LSP model group and the blank control group (P < 0.001). All experiments were performed independently three times.

[0090] The results showed that after 6 h of LPS stimulation, qRT-PCR analysis revealed a significant increase in the mRNA levels of M1 markers IL-1β, iNOS, and IL-6 in BV-2 cells, while the levels of M2 markers Arg-1 and IL-4 mRNA significantly decreased, indicating that BV-2 cells were predominantly in the M1 state. HP-Exo treatment suppressed the mRNA levels of M1 markers, while increasing the levels of M2 markers Arg-1 and IL-4 mRNA, indicating that inflammation was suppressed. However, the M1 / M2 polarization of Exo-treated BV-2 cells remained unchanged, suggesting that exosomes secreted by HUVECs cultured under hypoxic conditions have anti-inflammatory effects, while exosomes derived from HUVECs cultured under normoxic conditions do not.

[0091] Example 4: This example investigates the distribution of exosomes in mice before and after TAT modification. Specifically:

[0092] Nude mice were obtained from commercially available sources. All mice were housed in an environment with controlled temperature (18-21℃) and light (12-hour light-dark cycle, light time 08:00-20:00) and were acclimatized for at least one week before the experiment.

[0093] Nude mice (25-30 g, n=3) were administered 30 mL intranasally of DiD-labeled PBS, HP-Exo-CBD, or HP-Exo-CBD-TAT. At 3, 6, 9, 12, and 24 h post-administration, mice were anesthetized by inhalation of isoflurane. The distribution of exosomes in the mice was monitored using an in vivo imaging system. Mice were sacrificed 24 hours after administration, and organs such as the brain, heart, liver, spleen, lungs, and kidneys were collected for fluorescence and white light imaging. The structures are shown in the image. Figure 8 As shown in Table 6.

[0094] Table 6

[0095] ,

[0096] It can be seen that after intranasal administration of DiD-labeled HP-Exo-CBD or HP-Exo-CBD-TAT to mice, both groups of exosomes reached the brain region within 3 hours. Figure 8 As shown in Table 6.

[0097] Figure 8 This document describes the in vivo distribution of DID-labeled exosomes HP-Exo-TAT and HP-Exo-CBD-TAT after nasal drops of DID-labeled exosomes in PBS at the same concentration, as shown in Example 4. Figure 8 A represents the distribution of exosomes in mice at different time points after administration to each group; Figure 8 B is a statistical graph of the fluorescence intensity of exosomes in the brain at various time points; Figure 8 C shows the images of the isolated organs of mice in each group after 24 h; Figure 8 Data in B are expressed as mean ± SEM and analyzed using a two-tailed unpaired Student's t-test. * indicates statistical difference compared to the HP-Exo-CBD group (P < 0.05), and ** indicates significant difference compared to the HP-Exo-CBD group (P < 0.01). The number of animals used in the experiment was 3.

[0098] The results showed that the HP-Exo-CBD-TAT group had higher fluorescence intensity in brain regions at all time points, and TAT modification increased brain enrichment by 1.4-1.6 times. The fluorescence intensity of exosomes in both groups peaked at 12 hours. In vitro organ imaging showed significantly enhanced brain signal in the TAT-modified group. Figure 8 C). No fluorescence was observed in the PBS group in vivo or in isolated organs.

[0099] Example 5: This example explores the treatment and mechanism of HP-Exo-CBD-TAT on METH addiction in mice. Specifically:

[0100] ①The therapeutic effect of HP-Exo-CBD-TAT on METH-induced behavioral sensitization in mice and the regulation of inflammatory factors in the mPFC and VTA brain regions:

[0101] The behavioral sensitization test in mice was conducted in an open field box (40 cm × 40 cm × 40 cm). Mice were randomly divided into five groups: Vehicle group, METH group, METH + HP-Exo-TAT, METH + HP-Exo-CBD-TAT, and METH + CBD, with 6 mice in each group.

[0102] See the experimental procedure. Figure 9 A, is divided into the following stages:

[0103] Pre-adaptation (Day -2 to Day 0): Mice were placed in an activity box and allowed to move freely for 1 hour each day.

[0104] Formation period (Day 1 to Day 7): Mice that have adapted well were intraperitoneally injected with METH (1 mg / kg) daily for the next 7 days. Mice in the treatment group were given HP-Exo-TAT (2 mg / kg) intranasally, HP-Exo-CBD-TAT (2 mg / kg), or CBD (2 mg / kg) intraperitoneally before the injection of METH. The mice were then placed in an open field to explore freely for 1 h and their movement trajectories were recorded.

[0105] Transition period (Day 8-Day 14): After the transition period, mice in each group were returned to their original cages and fed normally for 7 days without any drug treatment.

[0106] Triggering phase (Day 15): On day 15, mice were intraperitoneally injected with METH (1 mg / kg) to induce behavioral sensitization and their movement trajectories were recorded to assess the effects of continued treatment.

[0107] Following behavioral testing, mice were anesthetized by intraperitoneal injection of the appropriate dose of the anesthetic azedarach based on their body weight and then perfused with PBS. Tissue samples from the medial prefrontal cortex (mPFC) and ventral tegmental area (VTA) were collected, homogenized using a handheld tissue homogenizer with 200 μL of TRIzol™ reagent, and then further homogenized with 800 μL of TRIzol™ reagent. Total RNA was extracted using the standard phenol / chloroform method, and cDNA synthesis and PCR amplification were performed as described above. Detection was performed using a TOptical real-time quantitative PCR instrument. IL-1b (by RPL27 (For internal reference), primer sequences are shown in Table 1, and data were analyzed using the ΔΔCt method.

[0108] Table 7

[0109] ,

[0110] Table 8

[0111] ,

[0112] Experimental results are as follows Figure 9 B and its table 7 are shown. Figure 9 B represents the period of formation accompanied by exosome administration to inhibit the formation of METH-induced behavioral sensitization in mice; all data in the figure are expressed as mean ± SEM. Analysis was performed using a two-tailed paired Student's t-test. In the figure, ** indicates a statistically significant difference compared to the METH-treated group (P < 0.01); *** indicates a highly statistically significant difference compared to the METH-treated group (P < 0.001); ### indicates a highly statistically significant difference compared to the vehicle group (P < 0.001); &&& indicates a highly statistically significant difference compared to the METH + HP-Exo-TAT group (P < 0.001); ns indicates no significant difference between the two groups. The number of animals used in the experiment was 6.

[0113] Compared with the control group (Vehicle), the METH model group mice showed a gradual increase in movement distance. Intranasal administration of 2 mg / kg of HP-Exo-TAT and HP-Exo-CBD-TAT significantly inhibited METH-induced formation-phase sensitization behavior (P<0.01, P<0.001). Figure 9 C and as shown in Table 8, Figure 9 C represents the post-withdrawal ignition period. Exosomes can effectively block METH-induced ignition behavior. Data in the figure are expressed as mean ± SEM. Two-tailed unpaired Student's t-test analysis was used. In the figure, ** indicates a statistically significant difference compared to the METH treatment group (P < 0.01); *** indicates a highly statistically significant difference compared to the METH treatment group (P < 0.001); ### indicates a highly statistically significant difference compared to the vehicle group (P < 0.001); and &&& indicates a highly statistically significant difference compared to the METH + HP-Exo-TAT group (P < 0.001). The number of animals used in the experiment was 6.

[0114] The results showed that after 7 days of abstinence, HP-Exo-TAT and HP-Exo-CBD-TAT effectively blocked METH-induced ignition behavior (P<0.001, P<0.001), while intraperitoneal injection of the same concentration of CBD had no effect. During the formation and ignition phases, the inhibitory effect of the HP-Exo-TAT carrier itself on the distance of travel was lower than that of the HP-Exo-CBD-TAT group (P<0.001).

[0115] After completing behavioral tests on day 15, the mice were euthanized and the mPFC and VTA were dissected. IL-1b Quantitative analysis, results as follows Figure 9 As shown in D, 9E, and Tables 9 and 10.

[0116] Table 9

[0117] ,

[0118] Table 10

[0119] ,

[0120] Figure 9 D represents the mouse mPFC brain region. IL-1b Quantitative analysis; Figure 9 E represents the VTA brain region in mice. IL-1b Quantitative analysis; data in the figures are expressed as mean ± SEM. Two-tailed unpaired Student's t-test analysis was used. In the figures, * indicates a statistically significant difference compared to the METH treatment group (P < 0.05), *** indicates a highly significant statistical difference compared to the METH treatment group (P < 0.001); # indicates a statistically significant difference compared to the vehicle group (P < 0.05), ## indicates a significant difference compared to the vehicle group (P < 0.01); & indicates a statistically significant difference compared to the METH + HP-Exo-TAT group (P < 0.05); ns indicates no significant difference between the two groups. The number of animals used in the experiment was 3.

[0121] It can be seen that chronic METH treatment leads to mPFC ( Figure 9 D and Table 9) and VTA ( Figure 9 E and Table 10) Area IL-1b Increased expression of these cytokines was observed, while HP-Exo-CBD-TAT significantly inhibited cytokine overexpression in these regions. HP-Exo-TAT also showed activity in the mPFC and VTA regions. IL-1b The level decreased by 29-42%.

[0122] ②The effects of HP-Exo-CBD-TAT on the treatment of METH-induced conditioned positional preference in mice and the regulation of microglial activation in the mPFC and VTA brain regions:

[0123] The conditional position preference (CPP) experiment lasted 12 days. Mice were randomly assigned to five groups: Vehicle group, METH group, METH + HP-Exo-TAT group, METH + HP-Exo-CBD-TAT group, and METH + CBD group. The experimental procedure is as follows: Figure 10 A. Conduct an 8-day training program (4 rounds in total), divided into the following stages:

[0124] Pre-acclimatization (Day-2 to Day 0): Allow mice to freely explore the equipment for 15 minutes to adapt to the environment. On Day 0, place the mice in the middle box, remove the partition, and allow them to move freely for 15 minutes. Observe the time the mice spend in each box and analyze the time each mouse spends in each compartment. The compartment with the longer stay time is the mouse's preferred box, and the other is the non-preferred box.

[0125] Formation period (Day 1–Day 8): The compartment exits were blocked with black acrylic plates. After daily administration, mice were placed in their respective compartments for 30 minutes of training. On days 3, 5, 7, and 9, mice were placed in a non-preference chamber after intraperitoneal injection of METH (2 mg / kg). On days 4, 6, 8, and 10, mice were placed in a preference chamber after intraperitoneal injection of saline (5 mL / kg). In the treatment groups, DID-labeled exosomes (2 mg / kg) were administered intranasally or CBD (2 mg / kg) intraperitoneally daily before METH or saline injection.

[0126] Expression testing period (Day 9): 24 hours after the last training, the CPP test was conducted. The movement trajectory of the mice was recorded during 15 minutes of free exploration. The CPP score was calculated by subtracting the time spent in the preferred box from the time spent in the non-preferred box.

[0127] Following the CPP test, mice were anesthetized with azithromycin and perfused with PBS. Brain tissue was harvested, fixed in 4% paraformaldehyde for 24 h, dehydrated with 30% sucrose, embedded in OCT, and then flash-frozen in liquid nitrogen at -80°C. Coronal brain slices (40 μm thick) containing mPFC and VTA were excised using a cryostat (HM525NX, ThermoFisher, USA). After washing three times with PBS, the slices were blocked with 1% BSA / 0.3% Triton X-100 for 30 min, followed by overnight incubation with primary antibody (anti-Iba1, 1:1000, ab5076, Abcam) at 4°C. After washing three times with PBS, the slices were incubated with Alexa Fluor-conjugated secondary antibody (1:1000; Jackson ImmunoResearch, USA) at room temperature for 1 h, and counterstained with DAPI (Servicebio, China) for 10 min. Images were acquired using a SLIDEVIEW VS200 confocal microscope (Olympus, JPN).

[0128] Table 11

[0129] ,

[0130] Table 12

[0131] ,

[0132] Table 13

[0133] ,

[0134] Figure 10 A is the flowchart of this experiment. Figure 10 B represents exosome inhibition of METH-induced increase in CPP scores in mice; Figure 10 C is a schematic diagram of Iba1+ immunofluorescence in the mPFC brain region of mice in each group; Figure 10 D represents the number of Iba1-positive cells per unit area in the mPFC brain region of mice in each group; Figure 10 E is a schematic diagram of Iba1+ immunofluorescence in the VTA brain region of mice in each group; Figure 10 F represents the number of Iba1-positive cells per unit area in the VTA brain region of mice in each group;

[0135] All data in the figure are expressed as mean ± SEM, where Figure 10 Data B was analyzed using a two-tailed unpaired student's t-test. Figure 10 D, Figure 10F was analyzed using one-tailed unpaired Student's t-test. In the figure, * indicates a statistically significant difference compared to the METH treatment group (P<0.05), ** indicates a significant difference compared to the METH treatment group (P<0.01), and *** indicates a highly significant statistical difference compared to the METH treatment group (P<0.001).

[0136] In the figure, # indicates a statistically significant difference compared to the vehicle group (P < 0.05), ## indicates a significant difference compared to the vehicle group (P < 0.01), and ### indicates a highly significant statistical difference compared to the vehicle group (P < 0.001); ns indicates no significant difference between the two groups. Figure 10 Experiment B used 6 animals. Figure 10 D、 Figure 10 The number of animals used in experiment E was 3.

[0137] As the result Figure 10 As shown in Table 11 (B), after an 8-day training period, the CPP scores of mice were significantly higher than those of the control group, suggesting that METH induced the establishment of CPP behavior in mice. The CPP scores of the HP-Exo-CBD-TAT group were significantly lower than those of the METH group, while there was no statistically significant difference between the HP-Exo-TAT group and the CBD-only treatment group. Figure 10 CF, Iba1 immunofluorescence staining analysis was performed on brain sections of the medial prefrontal cortex (mPFC) and ventral tegmental area (VTA). mPFC ( Figure 10 C, D, Table 12) and VTA brain region ( Figure 10 Immunofluorescence analysis (E, F, Table 13) showed that both HP-Exo-TAT and HP-Exo-CBD-TAT treatments significantly inhibited METH-induced microglial activation. In contrast, CBD alone did not show significant inhibitory effects in these brain regions.

[0138] Figure 11This paper presents a schematic diagram illustrating the mechanism of HP-Exo-CBD-TAT in inducing METH addiction in mice. The invention utilizes HP-Exo and cannabidiol to construct a novel nanoparticle drug delivery system, HP-Exo-CBD-TAT. Intranasal administration of a low concentration (2 mg / kg) of HP-Exo-CBD-TAT effectively inhibited METH-induced microglial activation and inflammatory cytokine release in the mPFC and VTA brain regions, and suppressed METH-induced behavioral sensitization and CPP in mice. Notably, the HP-Exo-CBD-TAT drug delivery system of this application was able to suppress METH-induced behavioral sensitization to baseline levels at a concentration of 2 mg / kg, effectively controlling conditioned place preference in mice. It effectively inhibited microglial activation and IL-1β release in the mPFC and VTA. Equivalent concentrations of CBD had no effect, indicating a synergistic effect between the carrier and CBD. In in vivo experiments, the HP-Exo-TAT carrier can alleviate METH-induced behavioral sensitization to a certain extent and significantly reduce METH-induced central nervous system inflammation, demonstrating the functionality of this carrier.

[0139] Comparative Example 1 compares the effects of different nanocarriers in the study of anti-metamine addiction in the prior art. The specific prior art and its anti-addiction effects, characteristics, mechanisms of action and sources are shown in Table 14.

[0140] Table 14

[0141] ,

[0142] As can be seen from Table 14, although there are studies on the resistance of nanomaterials to methamphetamine in the prior art, the study of their mechanism is not in-depth and the assembly structure varies greatly. Therefore, this invention has very significant innovation.

[0143] In summary, this invention provides a nano-drug delivery system for treating methamphetamine addiction. This nano-drug delivery system uses exosomes derived from human umbilical vein endothelial cells (HUVECs) engineered with hypoxia pretreatment and transcription activator protein (TAT) as nanocarriers for cannabidiol (CBD). Compared to current methods of treating methamphetamine addiction, which mainly involve highly invasive intraventricular injections or high-dose intraperitoneal injections (40-100 mg / kg), this invention treats methamphetamine addiction via nasal administration, achieving an effective concentration of only 2 mg / kg, which is only 1 / 20th of the lowest effective concentration (40 mg / kg) for treating methamphetamine addiction with intraperitoneal injection of CBD solution.

[0144] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A nanocarrier drug delivery system for the treatment of methamphetamine addiction, characterized by: The nano-drug delivery system is obtained by loading cannabidiol on exosomes extracted from hypoxia pretreated human umbilical vein endothelial cells and then functional modification; The functional modification is that the exosomes loaded with cannabidiol are modified by transcriptional activator TAT in human immunodeficiency virus, and the mass ratio of the exosomes loaded with cannabidiol to the transcriptional activator TAT is 10:

1. The nano-drug delivery system is used for nasal administration for treating methamphetamine addiction.

2. The nano drug delivery system for the treatment of methamphetamine addiction according to claim 1, wherein: The drug loading rate of cannabidiol in the nano-drug delivery system is greater than 16%, and the encapsulation rate is greater than 7%.

3. The nano drug delivery system for the treatment of methamphetamine addiction according to claim 1, wherein: The nano-drug delivery system can inhibit methamphetamine-induced behavioral sensitization and conditioned place preference by inhibiting the activation of microglial cells and the release of inflammatory factors in the mPFC and VTA brain regions induced by methamphetamine.

4. The method for preparing the nano-drug delivery system for treating methamphetamine addiction as described in any one of claims 1 to 3, characterized in that: The application relates to a nano-drug delivery system for treating methamphetamine addiction. The human umbilical vein endothelial cells are cultured in DMEM medium until the cells reach 80% confluence, and then the cells are incubated in 5 muM cannabidiol-containing serum-free medium under 1% O2 hypoxic conditions for 24 hours, and the exosomes loaded with cannabidiol are obtained after post-treatment. The exosomes loaded with cannabidiol are incubated with the transcriptional activator TAT at a mass ratio of 10:1, and then centrifuged to obtain the nano-drug delivery system.

5. A pharmaceutical preparation for intranasal administration for the treatment of methamphetamine addiction, characterized by: The application also relates to a pharmaceutical preparation containing the nano-drug delivery system.

6. The pharmaceutical preparation according to claim 5, characterized in that: The dosage form of the pharmaceutical preparation includes injection solution and freeze-dried powder injection.

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