Nanometer drug-loading delivery system for treating methamphetamine addiction and preparation method and pharmaceutical preparation of nanometer drug-loading delivery system

Abstract

The invention discloses a nano drug-loading delivery system for treating methamphetamine addiction and a preparation method and a pharmaceutical preparation thereof, and belongs to the technical field of medical configuration products. According to the nano drug-loaded delivery system, exosome from HUVECs (human umbilical vein endothelial cells) engineered and modified by hypoxia pretreatment and transcription activator protein (TAT) is used as a nano carrier of cannabidiol CBD, and compared with a drug delivery mode of high-invasiveness intraventricular injection or high-dose intraperitoneal injection (40-100 mg / kg) in treatment of METH addiction at present, the nano drug-loaded delivery system has the advantages that the drug delivery efficiency is greatly improved; the nano drug-loading delivery system treats METH addiction in a nasal delivery mode, and the effective concentration of the nano drug-loading delivery system is only 2 mg / kg and is only 1 / 20 of the minimum effective concentration (40 mg / kg) of intraperitoneal injection CBD solution for treating METH addiction.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical preparations, and particularly relates to a nano-drug delivery system for treating methamphetamine addiction, a preparation method thereof, and a pharmaceutical preparation. Background Art

[0002] Methamphetamine (METH) is a potent central nervous system stimulant that induces addictive behaviors by triggering neuroinflammation and dysfunction of the reward circuitry. It has a strong addiction potential and a high relapse rate, posing a major challenge in the global public health field. At the pathophysiological mechanism level, in addition to synaptic dysfunction and neurotransmitter dysregulation, a large number of studies have shown that neuroinflammation is a key pathological driver of METH addiction. The abnormal activation of microglia and the pro-inflammatory responses mediated by them play an important role in the neurotoxicity induced by METH. Although decades of research have focused on the development of small molecule drugs, existing therapies usually suffer from problems such as poor drug compliance and strong side effects. Therefore, the development of new intervention methods is extremely urgent.

[0003] Cannabidiol (CBD) is one of the most important active substances in natural cannabinoids and has become an important candidate molecule for treating METH addiction due to its potential anti-inflammatory and neuroprotective effects. 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 through the blood-brain barrier (BBB).

[0004] Currently, a variety of nano-drug delivery systems for CBD (including polymer and lipid-based nanoparticles) have been developed, but these systems generally suffer from problems such as low biofilm penetration efficiency and potential immunogenicity. In contrast, exosomes, with their excellent biocompatibility, low immunogenicity, and good blood-brain barrier penetration ability, have become an ideal platform for brain-targeted delivery systems. However, natural exosomes lack targeting specificity, and most exosomes accumulate in the liver and spleen through the systemic circulation. Moreover, there is no research in the prior art on using functional exosomes with anti-inflammatory effects as nano-carriers to load CBD. At the same time, although there are literature reports on the treatment of neurological diseases such as AD and PD by intranasal administration of exosomes, there is no application of this method for the treatment of METH addiction. Summary of the Invention

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

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

[0007] To solve the above technical problems, the present invention provides the following technical solutions: The nano-drug delivery system is obtained by functionalizing exosomes extracted from hypoxic preconditioned human umbilical vein endothelial cells as nano-carriers loaded with cannabidiol. Among them, the functional modification is to modify the exosomes loaded with cannabidiol using the transcriptional activator TAT in the human immunodeficiency virus.

[0008] As a preferred scheme of the nano-drug delivery system for treating methamphetamine addiction described in the present invention, wherein: the drug loading rate of cannabidiol in the nano-drug delivery system > 16%, and the encapsulation rate > 7%.

[0009] As a preferred scheme of the nano-drug delivery system for treating methamphetamine addiction described in the present invention, wherein: the nano-drug delivery system inhibits methamphetamine-induced behavioral sensitization and conditioned place preference by inhibiting methamphetamine-induced microglial activation and the release of inflammatory factors in the mPFC and VTA brain regions.

[0010] Beneficial effects of the present invention: The present invention extracts exosomes (HP-Exo-CBD) loaded with cannabidiol (CBD) through an endogenous drug loading strategy from hypoxic preconditioned human umbilical vein endothelial cells (HUVECs), and then uses the transcriptional activator TAT in the human immunodeficiency virus (HIV) to functionally modify these exosomes, and finally obtains HP-Exo-CBD-TAT. The carrier itself (HP-Exo-TAT) has the following characteristics: 1. At the cellular level, it can inhibit the increase of inflammatory factors in microglial BV-2 induced by lipopolysaccharide LPS and promote the release of anti-inflammatory factors; 2. Alleviate methamphetamine-induced behavioral sensitization; reduce the release of inflammatory factor IL-1b in the medial prefrontal cortex (mPFC) and ventral tegmental area (VTA) induced by methamphetamine; alleviate methamphetamine-induced conditioned place preference; reduce the activation of microglia in the medial prefrontal cortex (mPFC) and ventral tegmental area (VTA) induced by methamphetamine.

[0011] The nano-drug delivery system provided by the present invention can rapidly accumulate in the brain within 3 h. The exosomes modified with TAT (HP-Exo-CBD-TAT) have a 1.4 - 1.6 times higher enrichment degree in the brain than the unmodified exosomes (HP-Exo-CBD) at each time point. In addition, HP-Exo-CBD-TAT can effectively alleviate methamphetamine addiction, can reduce methamphetamine-induced behavioral sensitization and conditioned place preference in mice, and can further reduce methamphetamine-induced neuroinflammation and microglial activation.

[0012] Another object of the present invention is to provide a preparation method of a nano-drug delivery system for treating methamphetamine addiction.

[0013] To solve the above technical problems, the present invention provides the following technical solutions: including, Human umbilical vein endothelial cells are cultured in DMEM medium until the cells reach 80% confluence, and then incubated in a serum-free medium containing 5 μM cannabidiol under hypoxic conditions of 1% O2 for 24 hours. After incubation, exosomes loaded with cannabidiol are obtained through post-treatment; Among them, the post-treatment is to centrifuge the cell supernatant after incubation at 300 g for 10 min, and the obtained supernatant is centrifuged at 1000 g for 20 min to remove cell debris and dead cells. The supernatant obtained again is transferred and centrifuged at 10000 g for 30 min to remove impurities in the sample. The supernatant obtained after the final centrifugation is ultracentrifuged at 100000 g for 70 min to obtain exosomes loaded with cannabidiol. The precipitate is resuspended with PBS and stored at -80°C; The exosomes loaded with cannabidiol are incubated with the transcriptional activator TAT at a mass ratio of 1:10 and centrifuged to obtain the nano-drug delivery system.

[0014] Another object of the present invention is to provide a pharmaceutical preparation for treating methamphetamine addiction by nasal administration. The pharmaceutical preparation contains the nano-drug delivery system and also includes pharmaceutically acceptable excipients.

[0015] It should be noted that nasal administration is a brain-targeted drug delivery method that enables drugs to be absorbed through the trigeminal nerve endings of the nasal mucosa, transported through the neuronal cell bodies to the brainstem and the brain, bypassing the blood-brain barrier. It has the characteristics of non-invasiveness, and can directly reach the target site, reducing the systemic exposure and the risk of side effects.

[0016] As a preferred embodiment of the pharmaceutical preparation of the present invention, wherein: the dosage form of the pharmaceutical preparation includes injection and freeze-dried powder injection.

[0017] Advantages of the present invention: The present invention uses the nano-drug delivery system as a pharmaceutical preparation for treating methamphetamine addiction by nasal administration. Compared with the current administration methods for treating METH addiction, which mainly rely on highly invasive intracerebroventricular injection or high-dose intraperitoneal injection (40 - 100 mg / kg), the effective concentration for treating METH addiction by nasal administration is only 2 mg / kg, which is only 1 / 20 of the lowest effective concentration (40 mg / kg) of intraperitoneal injection of CBD solution for treating METH addiction. Description of the Drawings

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0019] Figure 1 It is the potential map of exosomes obtained by mixing HP-Exo-CBD and DSPE-PEG-TAT in different proportions. The data in the figure are expressed as mean ± SEM. Analyzed by two-tailed unpaired Student’s t-test, * in the figure indicates P < 0.05 compared with HP-Exo-CBD, with statistical significance; ns in the figure indicates no significant difference between the two groups. All experiments were independently performed 3 times.

[0020] Figure 2 It is the transmission electron microscopy (TEM) image of different exosomes in Example 1 of the present invention; among them, Figure 2 A is the TEM image of exosomes (Exo) of HUVECs under normoxic conditions; Figure 2 B is the TEM image of exosomes (HP-Exo) of HUVECs under hypoxic conditions; Figure 2 C is the TEM image of exosomes of HUVECs loaded with CBD (HP-Exo-CBD) under hypoxic conditions; Figure 2 D is the TEM image of exosomes of HUVECs modified with TAT under hypoxic conditions (HP-Exo-TAT); Figure 2 E is the TEM image of exosomes of HUVECs modified with TAT loaded with CBD under hypoxic conditions (HP-Exo-CBD-TAT); Figure 3 It is the dynamic light scattering (DLS) results of different exosomes in Example 1 of the present invention; among them, Figure 3 A is the DLS image of Exo; Figure 3 B is the DLS image of HP-Exo; Figure 3 C is the DLS image of HP-Exo-CBD; Figure 3 D is the DLS image of HP-Exo-TAT; Figure 3 E is the DLS image of HP-Exo-CBD-TAT; Figure 3 F is the Zeta potential of each exosome; Figure 4 It is the nano-flow cytometry results of different exosomes in Example 1 of the present invention; among them, A-E respectively represent the nano-flow cytometry results of Exo, HP-Exo, HP-Exo-CBD, HP-Exo-TAT, and HP-Exo-CBD-TAT; Figure 5 Western blot analysis results of surface protein markers of exosomes obtained from different exosomes in Example 1 of the present invention; Figure 6 In vitro release results of CBD in exosomes in Example 2; Figure 7 In Example 3, HP-Exo inhibited the polarization of LPS-stimulated BV2 microglia into the M1 type, promoted their transformation into the M2 type, and inhibited the excessive activation of inflammation; among them, Figure 7 A is the marker of the M1 state IL-1b mRNA level change; Figure 7 B is the marker of the M1 state iNOS mRNA level change; Figure 7 C is the marker of the M1 state IL-6 mRNA level change; Figure 7 D is the marker of the M2 state Arg-1 mRNA level change; Figure 7 E is the marker of the M2 state IL-4 mRNA level change; Data in the figure are expressed as mean ± SEM and analyzed by two-tailed unpaired Student’s t-test. * in the figure indicates that compared with the LPS model group, P<0.05, with statistical significance; ** in the figure indicates that compared with the LPS model group, P<0.01, with significant statistical significance; *** in the figure indicates that compared with the LPS model group, P<0.001, with extremely significant statistical significance; ns in the figure indicates that there is no significant difference between the administration group and the LPS model group; # in the figure indicates that compared with the blank control group, P<0.05, with statistical significance for the LSP group; ## in the figure indicates that compared with the blank control group, P<0.01, with significant statistical significance for the LSP group; in the figure indicates that compared with the blank control group, P<0.001, with extremely significant statistical significance for the LSP group; all experiments were independently performed 3 times.

[0021] Figure 8 In Example 4, the in vivo distribution after nasal instillation of DID-labeled exosomes HP-Exo-TAT and HP-Exo-CBD-TAT and PBS with the same concentration of DID; among them, Figure 8 A is the distribution in mice at different time points after administration of exosomes in each group; Figure 8 B is the statistical chart of the fluorescence intensity of exosomes in the brain at each time point in each group; Figure 8 C is the in vitro organ imaging of each group of mice after 24 h; Figure 8Data in B are presented as mean ± SEM and analyzed by two-tailed unpaired Student’s t-test, where * indicates P < 0.05 compared with the HP-Exo-CBD group, showing a statistically significant difference, and ** indicates P < 0.01 compared with the HP-Exo-CBD group, showing a significant difference. The number of animals used in the experiment is 3.

[0022] Figure 9 To implement that HP-Exo-CBD-TAT inhibits methamphetamine-induced behavioral sensitization and the release of inflammatory factors in the mPFC and VTA brain regions in Example 5; Figure 9 A is the experimental procedure; Figure 9 B is that the concomitant administration of exosomes during the formation period inhibits the formation of METH-induced behavioral sensitization in mice; Figure 9 C is the ignition period after withdrawal, and exosomes can effectively block the ignition behavior induced by METH; Figure 9 D is for the IL-1b quantitative analysis of the mouse mPFC brain region; Figure 9 E is for the IL-1b quantitative analysis of the mouse VTA brain region; Data in the figures are presented as mean ± SEM, where Figure 9 Data in B are analyzed by two-tailed paired Student’s t-test, Figure 9 C- Figure 9 Data in E are analyzed by two-tailed unpaired Student’s t-test. In the figures, * indicates P < 0.05 compared with the METH treatment group, showing a statistically significant difference, ** indicates P < 0.01 compared with the METH treatment group, showing a significant difference, and *** indicates P < 0.001 compared with the METH treatment group, showing a highly significant statistical difference; In the figures, # indicates P < 0.05 compared with the vehicle group, showing a statistically significant difference, ## indicates P < 0.01 compared with the vehicle group, showing a significant difference, and indicates P < 0.001 compared with the vehicle group, showing a highly significant statistical difference; In the figures, & indicates P < 0.05 compared with the METH + HP-Exo-TAT group, showing a statistically significant difference, and &&& indicates P < 0.001 compared with the METH + HP-Exo-TAT group, showing a highly significant statistical difference; ns indicates no significant difference between the two groups, Figure 9 B, Figure 9 The number of animals used in the C experiment is 6, Figure 9 D, Figure 9 The number of animals used in the E experiment is 3.

[0023] Figure 10 To demonstrate that HP-Exo-CBD-TAT in Example 5 inhibits methamphetamine-induced conditioned place preference (CPP) in mice and the activation of microglia in the medial prefrontal cortex (mPFC) and ventral tegmental area (VTA) Figure 10 A shows the experimental procedure. Figure 10 B shows that exosomes inhibit the increase in methamphetamine-induced CPP scores in mice. Figure 10 C shows the immunofluorescence schematic diagram of Iba1+ in the mPFC of mice in each group. Figure 10 D shows the statistical analysis of Iba1-positive cells per unit area in the mPFC of mice in each group. Figure 10 E shows the immunofluorescence schematic diagram of Iba1+ in the VTA of mice in each group. Figure 10 F shows the statistical analysis of Iba1-positive cells per unit area in the VTA of mice in each group. Data in the figures are presented as mean ± SEM, where Figure 10 Data in B were analyzed by two-tailed unpaired Student’s t-test, Figure 10 D, Figure 10 F were analyzed by one-tailed unpaired Student’s t-test. In the figures, * indicates P < 0.05 compared with the METH treatment group, showing a statistically significant difference; ** indicates P < 0.01 compared with the METH treatment group, showing a significant difference; *** indicates P < 0.001 compared with the METH treatment group, showing a highly significant statistical difference. In the figures, # indicates P < 0.05 compared with the vehicle group, showing a statistically significant difference; ## indicates P < 0.01 compared with the vehicle group, showing a significant difference; indicates P < 0.001 compared with the vehicle group, showing a highly significant statistical difference; ns indicates no significant difference between the two groups. Figure 10 The number of animals used in experiment B was 6. Figure 10 D, Figure 10 The number of animals used in experiments E was 3.

[0024] Figure 11 This is a schematic diagram of the preparation process of the drug delivery system of the present invention and the mechanism of treating methamphetamine addiction. The present invention constructs a novel nano-drug delivery system HP-Exo-CBD-TAT using HP-Exo and cannabidiol and uses it for the treatment of methamphetamine addiction. Detailed implementation manners

[0025] To make the above objects, features, and advantages of the present invention more obvious and understandable, the following detailed description of the specific implementation manners of the present invention will be given in conjunction with the embodiments of the specification.

[0026] In the following description, numerous specific details are set forth to provide a thorough understanding of the present invention. However, the present invention may be practiced in other ways than those specifically described herein. Those skilled in the art can make similar generalizations without departing from the spirit of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0027] Secondly, as used herein, an "embodiment" or "embodiments" refers to specific features, structures, or characteristics 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 separate or alternative embodiment that is mutually exclusive of other embodiments.

[0028] Unless otherwise specified, the raw materials used in the present invention are commercially available and commonly used in the art.

[0029] The primer sequences of the iNOS, IL-1b, IL-6, IL-4, Arg-1 and RPL27 genes used in the present invention are shown in Table 1.

[0030] Table 1 Primer Sequence Table , Example 1 provided a preparation method of a nano-drug delivery system for treating methamphetamine addiction by nasal administration. The nano-drug delivery system was obtained by functional modification after loading cannabidiol with exosomes extracted from hypoxic preconditioned human umbilical vein endothelial cells. Specifically: HUVECs cells were cultured in DMEM medium until the cell confluence reached 80%, and then the culture medium was replaced with serum-free basal medium and incubated under the following conditions respectively: A. Incubate for 24 hours under normoxic conditions; B. Incubate for 24 hours in serum-free medium containing 5 μM CBD under hypoxic (1% O2) conditions; C. Incubate for 24 hours in serum-free medium without CBD under hypoxic (1% O2) conditions.

[0031] After incubation, the cell supernatant was centrifuged at 300 g for 10 min, and then the supernatant was 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 in the sample. The centrifuged supernatant was ultracentrifuged at 100000 g for 70 min to obtain a high-purity exosome precipitate. The precipitate was resuspended with PBS and stored at -80°C. The exosomes isolated from HUVECs cultured under condition A were labeled as Exo, the exosomes under condition B were labeled as HP-Exo-CBD, and the exosomes under condition C were labeled as HP-Exo; HP-Exo-CBD and DSPE-PEG-TAT were uniformly mixed at a mass ratio of 100:1, 10:1, and 1:1, made up to 1 mL with PBS, incubated at 37 °C for 30 minutes, transferred the mixture to the inner tube of a 10 kDa ultrafiltration tube, centrifuged at 4000 g for 30 minutes at 4 °C, discarded the filtrate in the outer tube of the ultrafiltration tube, and centrifuged in an inverted position at 2000 g for 2 minutes at 4 °C to obtain HP-Exo-CBD-TAT. The Zeta potential of different binding products was measured, and the results are as Figure 1 shown in Table 2.

[0032] Table 2 Zeta potential results of different exosome binding products , Figure 1 is the potential map of exosomes obtained by mixing HP-Exo-CBD and DSPE-PEG-TAT at different ratios. The data in the figure are expressed as mean ± SEM. Analyzed by two-tailed unpaired Student’s t-test, * in the figure indicates P < 0.05 compared with HP-Exo-CBD, with statistical significance; ns in the figure indicates no significant difference between the two groups. All experiments were independently performed 3 times.

[0033] From Figure 1 the results in and Table 2, it can be seen that the best mixing result of HP-Exo-CBD and DSPE-PEG-TAT is at a mass ratio of 10:1. Therefore, the mixing ratio of HP-Exo-CBD and DSPE-PEG-TAT was selected as 10:1.

[0034] HP-Exo and DSPE-PEG-TAT were mixed at a mass ratio of 10:1, and the method was the same as that of HP-Exo-CBD to obtain HP-Exo-TAT.

[0035] Exosomes were identified by transmission electron microscopy (TEM), dynamic light scattering (DLS), nanoflow cytometry, and western blot (WB).

[0036] The CBD loading in exosomes was quantified by high performance liquid chromatography (HPLC) at a wavelength of 254 nm. Specifically, 25 mg of the sample (HP-Exo-CBD) was placed in a 1.5 mL centrifuge tube, 600 mL of chloroform was added, vortexed and sonicated, and then centrifuged at 12,000 rpm for 10 minutes. The supernatant was filtered through a 0.22 mm filter membrane and 20 mL was injected. Chromatographic conditions: C18 chromatographic column (4.6×250 mm, 5 mm), mobile phase was methanol-water (95:5), column temperature was 25 °C, and the detection wavelength was 220 nm.

[0037] The calculation formulas for the loading rate and encapsulation rate of CBD are as follows: CBD loading rate (%) = (mass of CBD in exosomes / total mass of exosome proteins) × 100% CBD encapsulation rate (%) = (mass of CBD in exosomes / mass of CBD input) × 100% Figure 2 Figures show the transmission electron microscopy (TEM) images of different exosomes, indicating that exosomes are typically disc-shaped or spherical.

[0038] Figure 3 Figure shows the dynamic light scattering (DLS) results of different exosomes ( Figure 3 A - E). The peak particle size distributions of 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.

[0039] Table 3 Zeta potential results of different exosomes , It can be seen that the Zeta potentials of different exosomes are -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 ( Figure 3 F and Table 3).

[0040] Figure 4 Figure shows the average particle size of different exosomes detected by nano-flow cytometry. It can be seen that they are 58.9 nm, 62.2 nm, 57.9 nm, 62.4 nm, and 58.9 nm, respectively.

[0041] In summary, although there are differences in the specific particle sizes due to the different detection principles of different technologies, the particle size ranges of each group of exosomes all meet the size range of standard exosomes, which is 30 - 200 nm.

[0042] The expressions of exosome-specific markers (TSG101, Alix, CD63, CD81) and the absence of the negative protein GM130 were detected by immunoblotting. The results are as Figure 5 shown. The above experiments confirmed the successful extraction of exosomes.

[0043] After testing and calculation, the drug loading rate and encapsulation rate of the nano-drug delivery system HP-Exo-CBD-TAT prepared in this example for CBD are 16.32% and 7.82%, respectively.

[0044] Example 2. This example provides an in vitro release test of exosomes. Specifically: The 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), and stirred at a constant temperature of 37°C. 1 mL of the sample was taken at a predetermined time point and an equal volume of the release medium was added. The release amount of CBD was quantitatively determined by HPLC, and the results are as Figure 6 shown in Table 4.

[0045] Table 4 Release percentage of CBD at each collection time point , It can be seen from the results that the release of HP-Exo-CBD is rapid in the first 12 h, and then tends to be flat after 12 h. The cumulative release rate at 24 h is 53.31 ± 1.37%. It should be noted that the TAT modification in the present invention does not change the encapsulation rate and drug loading rate of the drug delivery system, as well as the drug loading characteristics of exosomes such as drug release. The modification of the polypeptide is to improve the transport effect and cell uptake function. Therefore, HP-Exo-CBD-TAT has the same in vitro release effect as HP-Exo-CBD.

[0046] Example 3. This example verifies the anti-inflammatory effects of different exosomes. Specifically: This example verifies the anti-inflammatory effects of different exosomes. Microglial polarization plays an important role in central nervous system (CNS) inflammation. Under LPS stimulation, microglia transform from the M0 resting state to the activated M1 state, releasing pro-inflammatory factors and exacerbating the inflammatory response. On the contrary, microglia can also transform from the M1 state to the M2 state, releasing anti-inflammatory factors and inhibiting the inflammatory response.

[0047] Therefore, in order to evaluate the anti-inflammatory effect of exosomes derived from hypoxic-treated HUVECs (HP-Exo), Exo and HP-Exo were co-cultured with microglia (BV-2). BV-2 cells were seeded in 6-well plates at 4 × 10 5 cells per well. After culturing for 24 h, the supernatant was removed. The blank control group was only added with the basic medium, the LPS model group and the administration group were added with the basic medium containing 20 ng / mL LPS, and the administration group was added with the basic medium containing 100 μg / mL Exo or HP-Exo and treated for 6 hours. Total RNA was extracted using the TRIzol method, reverse transcription was performed according to the kit instructions, and gene fragments were amplified using this as a template. The primer sequences are shown in Table 1. RPL27 was used as an internal reference. Amplification was performed using the SYBR Green method on a TOptical real-time quantitative PCR instrument (Analytik Jena, Germany), and the data was analyzed by the ΔΔCt method and normalized to RPL27. The results are as Figure 7as shown in Table 5.

[0048] Table 5 , Figure 7 In Example 3, HP-Exo inhibited the polarization of LPS-stimulated BV2 microglia into the M1 type, promoted their transformation into the M2 type, and inhibited the excessive activation of inflammation; among them, Figure 7 A is the mRNA level change of the M1 state marker IL-1b ; Figure 7 B is the mRNA level change of the M1 state marker iNOS ; Figure 7 C is the mRNA level change of the M1 state marker IL-6 ; Figure 7 D is the mRNA level change of the M2 state marker Arg-1 ; Figure 7 E is the mRNA level change of the M2 state marker IL-4 ; Data in the figure are expressed as mean ± SEM and analyzed by two-tailed unpaired Student’s t-test. * in the figure indicates that compared with the LPS model group, P < 0.05 for this administration group, with statistical significance; ** in the figure indicates that compared with the LPS model group, P < 0.01 for this administration group, with significant statistical significance; *** in the figure indicates that compared with the LPS model group, P < 0.001 for this administration group, with extremely significant statistical significance; ns in the figure indicates that there is no significant difference between this administration group and the LPS model group; # in the figure indicates that compared with the blank control group, P < 0.05 for the LSP model group, with statistical significance; ## in the figure indicates that compared with the blank control group, P < 0.01 for the LSP model group, with significant statistical significance; in the figure indicates that compared with the blank control group, P < 0.001 for the LSP model group, with extremely significant statistical significance; all experiments were independently performed 3 times.

[0049] The results showed that after 6 h of LPS stimulation in BV-2 cells, it was found by qRT-PCR that the mRNA levels of M1 markers IL-1β, iNOS, and IL-6 in BV-2 cells increased significantly, while the mRNA levels of M2 markers Arg-1 and IL-4 decreased significantly, indicating that BV-2 cells were more in the M1 state. After HP-Exo treatment, the mRNA levels of M1 markers were all inhibited, and the mRNA levels of M2 markers Arg-1 and IL-4 increased, indicating that the inflammation was inhibited. However, there was no change in the M1 / M2 polarization of BV-2 cells treated with Exo, indicating that exosomes secreted by HUVECs cultured under hypoxic conditions had anti-inflammatory effects, while exosomes derived from HUVECs cultured under normoxic conditions had no anti-inflammatory effects.

[0050] Example 4. In this example, the distribution of exosomes in mice before and after TAT modification was explored. Specifically: Nude mice were obtained commercially. All mice were housed in an environment with temperature (18 - 21 °C) and light control (12-hour light-dark cycle, light time 08:00 - 20:00), and were acclimated for at least 1 week before the experiment.

[0051] Nude mice (25 - 30 g, n = 3) were intranasally administered 30 μL of DiD-labeled PBS, HP-Exo-CBD, and HP-Exo-CBD-TAT. After 3, 6, 9, 12, and 24 h of administration, the mice were anesthetized by inhaling isoflurane, and the distribution of exosomes in the nude mice was monitored by an in vivo imaging system. The mice were sacrificed 24 hours after administration, and organs such as the brain, heart, liver, spleen, lung, and kidney were collected for fluorescence and white light imaging, as shown in Figure 8 and Table 6.

[0052] Table 6 , 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 at 3 h, as shown in Figure 8 and Table 6.

[0053] Figure 8 For the in vivo distribution of nasal drops of DID-labeled exosomes HP-Exo-TAT and HP-Exo-CBD-TAT and PBS with the same concentration of DID in Example 4; among them, Figure 8 A shows the distribution of exosomes in mice at different time points after administration of each group; Figure 8 B shows the statistical chart of the fluorescence intensity of exosomes in the brain at each time point of each group; Figure 8 C shows the ex vivo organ imaging of each group of mice after 24 h; Figure 8Data in B are presented as mean ± SEM and analyzed by two-tailed unpaired Student’s t-test, where * indicates P < 0.05 compared with the HP-Exo-CBD group, showing statistical significance, and ** indicates P < 0.01 compared with the HP-Exo-CBD group, showing significant difference. The number of animals used in the experiment was 3.

[0054] As can be seen from the results, the fluorescence intensity in the brain regions of the HP-Exo-CBD-TAT group was higher at each time point. TAT modification increased the brain enrichment by 1.4 - 1.6 times. The fluorescence intensity of exosomes in both groups reached the peak at 12 hours. Ex vivo organ imaging showed that the brain signal of the TAT modification group was significantly enhanced ( Figure 8 C). There was no fluorescence distribution in the PBS group in vivo and in ex vivo organs.

[0055] Example 5. This example explores the treatment and mechanism of HP-Exo-CBD-TAT on METH addiction in mice. Specifically: ① Treatment of HP-Exo-CBD-TAT on METH-induced behavioral sensitization in mice and regulation of inflammatory factors in the mPFC and VTA brain regions: The mouse behavioral sensitization test was carried out in an open field box (40 cm × 40 cm × 40 cm). The 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.

[0056] The experimental procedure is shown in Figure 9 A and is divided into the following stages: Pre-adaptation (Day -2~Day 0): Mice were placed in the activity box and allowed to move freely for 1 h every day.

[0057] Forming period (Day 1~Day 7): The adapted mice were intraperitoneally injected with METH (1 mg / kg) daily for the next 7 days. Mice in the treatment groups were intranasally administered HP-Exo-TAT (2 mg / kg), HP-Exo-CBD-TAT (2 mg / kg), or intraperitoneally injected with CBD (2 mg / kg) before injecting METH. Then the mice were placed in the open field and allowed to explore freely for 1 h, and their movement trajectories were recorded.

[0058] Conversion period (Day 8~Day 14): After the forming period, the mice in each group were returned to the original cage and normally raised for 7 d without any drug treatment.

[0059] Excitation period (Day 15): On the 15th day, mice were intraperitoneally injected with METH (1 mg / kg) to induce behavioral sensitization, and the locomotor trajectories were recorded to evaluate the effect of continuous treatment.

[0060] After the behavioral test, the mice were anesthetized by intraperitoneal injection of the corresponding dose of anesthetic avertin according to the body weight of the mice and perfused with PBS. Tissues of the medial prefrontal cortex (mPFC) and ventral tegmental area (VTA) were taken, 200 μL of TRIzolTM reagent was added, homogenized using a handheld tissue homogenizer, and then 800 μL of TRIzolTM reagent was added and mixed evenly. Total RNA was extracted by the standard phenol / chloroform method, and cDNA synthesis and PCR amplification were as described above. Detection was performed using a TOptical real-time quantitative PCR instrument IL-1b (using RPL27 as an internal reference), primer sequences are shown in Table 1, and the data were analyzed by the ΔΔCt method.

[0061] Table 7 , Table 8 , The experimental results are as shown in Figure 9 Figure B and Table 7. Figure 9 In Figure B, co-administering exosomes during the formation period inhibited the formation of METH-induced behavioral sensitization in mice; the data in the figure are all expressed as mean ± SEM, analyzed by two-tailed paired Student’s t-test. In the figure, ** indicates P < 0.01 compared with the METH treatment group, showing a significant difference, *** indicates P < 0.001 compared with the METH treatment group, showing an extremely significant statistical difference; in the figure indicates P < 0.001 compared with the vehicle group, showing an extremely significant statistical difference; &&& in the figure indicates P < 0.001 compared with the METH + HP-Exo-TAT group, showing an extremely significant statistical difference; ns indicates no significant difference between the two groups. The number of animals used in the experiment was 6.

[0062] Compared with the control group (Vehicle), the locomotor distance of mice in the METH model group gradually increased. Nasal administration of 2 mg / kg of HP-Exo-TAT and HP-Exo-CBD-TAT significantly inhibited the sensitization behavior during the formation period induced by METH (P < 0.01, P < 0.001). As shown in Figure 9 Figure C and Table 8. Figure 9Group C is the post-withdrawal kindling period. Exosomes can effectively block the kindling behavior induced by METH. The data in the figures are expressed as mean ± SEM and analyzed by two-tailed unpaired Student’s t-test. In the figures, ** indicates P < 0.01 compared with the METH treatment group, showing a significant difference; *** indicates P < 0.001 compared with the METH treatment group, showing a highly significant statistical difference; in the figures indicates P < 0.001 compared with the vehicle group, showing a highly significant statistical difference; &&& in the figures indicates P < 0.001 compared with the METH + HP-Exo-TAT group, showing a highly significant statistical difference. The number of animals used in the experiment was 6.

[0063] As can be seen from the results, after 7 days of withdrawal, HP-Exo-TAT and HP-Exo-CBD-TAT can effectively block the kindling behavior induced by METH (P < 0.001, P < 0.001), while intraperitoneal injection of the same concentration of CBD has no effect. During the formation period and the kindling period, the inhibitory effect of the HP-Exo-TAT vector itself on the locomotor distance is lower than that of the HP-Exo-CBD-TAT group (P < 0.001).

[0064] After the behavioral tests were completed on the 15th day, the mice were sacrificed and the mPFC and VTA were dissected for IL-1b quantitative analysis. The results are shown in Figure 9 Figures 9D, 9E, Tables 9 and 10.

[0065] Table 9 , Table 10 , Figure 9 Figure 9D is the IL-1b quantitative analysis of the mPFC brain region of the mice; Figure 9 Figure 9E is the IL-1b quantitative analysis of the VTA brain region of the mice; The data in the figures are expressed as mean ± SEM and analyzed by two-tailed unpaired Student’s t-test. In the figures, * indicates P < 0.05 compared with the METH treatment group, showing a statistical difference; *** indicates P < 0.001 compared with the METH treatment group, showing a highly significant statistical difference; # indicates P < 0.05 compared with the vehicle group, showing a statistical difference; ## indicates P < 0.01 compared with the vehicle group, showing a significant difference; & indicates P < 0.05 compared with the METH + HP-Exo-TAT group, showing a statistical difference; ns indicates no significant difference between the two groups. The number of animals used in the experiment was 3.

[0066] It can be seen that chronic METH treatment leads to increased expression in the mPFC ( Figure 9 D and Table 9) and VTA ( Figure 9 E and Table 10) regions IL-1b while HP-Exo-CBD-TAT can significantly inhibit the overexpression of cytokines in these regions, and HP-Exo-TAT can reduce the IL-1b levels in the mPFC and VTA regions by 29 - 42%.

[0067] ② Treatment of METH-induced conditioned place preference in mice by HP-Exo-CBD-TAT and regulation of microglial activation in the mPFC and VTA brain regions: The conditioned place preference (CPP) experiment lasted for 12 days. Mice were randomly divided into five groups: Vehicle group, METH group, METH + HP-Exo-TAT, METH + HP-Exo-CBD-TAT, METH + CBD. The experimental procedure is shown in Figure 10 A and underwent an 8-day training (a total of 4 rounds of cycles), which was divided into the following stages: Pre-adaptation (Day -2 to Day 0): Let the mice freely explore the device for 15 min to adapt to the environment. On Day 0, the mice were placed in the middle box, the partition was removed, and they were allowed to move freely for 15 min. Observe the time the mice stayed in each box, and analyze the time each mouse stayed in each compartment. The compartment with the longer stay time is the preferred box for the mouse, and the other is the non-preferred box.

[0068] Formation period (Day 1 to Day 8): Use a black acrylic board to block the compartment exits. After daily drug administration, the mice were placed in the corresponding compartments for 30 min of training. On the 3rd, 5th, 7th, and 9th days, METH (2 mg / kg) was injected intraperitoneally and the mice were placed in the non-preferred box. On the 4th, 6th, 8th, and 10th days, normal saline (5 mL / kg) was injected intraperitoneally and the mice were placed in the preferred box. The drug administration groups were given DID-labeled exosomes (2 mg / kg) by nasal administration or CBD (2 mg / kg) by intraperitoneal administration before injecting METH or normal saline every day.

[0069] Expression test period (Day 9): The CPP test was performed 24 hours after the last training. Record the movement trajectories of the mice during 15 min of free exploration, and calculate the CPP score by subtracting the time the mouse spent in the preferred box from the time it spent in the non-preferred box.

[0070] After CPP testing, the mice were anesthetized with avertin and perfused with PBS. The brain tissues were taken, fixed with 4% paraformaldehyde for 24 h, dehydrated with 30% sucrose, embedded in OCT, and snap-frozen in liquid nitrogen and stored at -80 °C. Coronal brain slices (40 μm thick) containing the mPFC and VTA were cut using a cryostat (HM525NX, ThermoFisher, USA). After washing three times with PBS, they were blocked with 1% BSA / 0.3% Triton X-100 for 30 minutes, and then incubated overnight at 4 °C with the primary antibody (anti-Iba1, 1:1000, ab5076, Abcam). After washing three times with PBS, they were incubated with an Alexa Fluor-conjugated secondary antibody (1:1000; Jackson ImmunoResearch, USA) for 1 hour at room temperature, and counterstained with DAPI (Servicebio, China) for 10 minutes. Images were acquired using a SLIDEVIEW VS200 confocal microscope (Olympus, JPN).

[0071] Table 11 , Table 12 , Table 13 , Figure 10 A is the flow chart of this experiment, Figure 10 B shows that exosomes inhibit the increase in CPP scores induced by METH in mice; Figure 10 C is the schematic diagram of Iba1+ immunofluorescence in the mPFC brain region of mice in each group; Figure 10 D is the statistics of Iba1-positive cells per unit area in the mPFC brain region of mice in each group; Figure 10 E is the schematic diagram of Iba1+ immunofluorescence in the VTA brain region of mice in each group; Figure 10 F is the statistics of Iba1-positive cells per unit area in the VTA brain region of mice in each group; Data in the figures are presented as mean ± SEM, where Figure 10 Data in B were analyzed by two-tailed unpaired Student’s t-test, Figure 10 D, Figure 10 F were analyzed by one-tailed unpaired Student’s t-test. * in the figures indicates P < 0.05 compared with the METH treatment group, showing a statistically significant difference, ** indicates P < 0.01 compared with the METH treatment group, showing a significant difference, and *** indicates P < 0.001 compared with the METH treatment group, showing an extremely significant statistical difference; In the figure, # indicates P < 0.05 compared with the vehicle group, showing a statistically significant difference; ## indicates P < 0.01 compared with the vehicle group, showing a significant difference; indicates P < 0.001 compared with the vehicle group, showing an extremely significant statistical difference; ns indicates no significant difference between the two groups. Figure 10 The number of animals used in Experiment B was 6. Figure 10 D, Figure 10 The number of animals used in Experiment E was 3.

[0072] As shown in the results Figure 10 As shown in B (Table 11), after an 8-day training period, the CPP scores of the mice were significantly higher than those of the control group, indicating 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 statistical difference between the HP-Exo-TAT group and the CBD treatment alone. As Figure 10 C-F, 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 regions ( Figure 10 E, F, Table 13) immunofluorescence analysis showed that both the HP-Exo-TAT and HP-Exo-CBD-TAT treatment groups could significantly inhibit METH-induced microglial activation. In contrast, CBD alone did not show a significant inhibitory effect in these brain regions.

[0073] Figure 11 The figure shows the mechanism of HP-Exo-CBD-TAT of the present invention on METH addiction in mice. The present invention constructs a novel nano-drug delivery system HP-Exo-CBD-TAT using HP-Exo and cannabidiol. Nasal administration of low-concentration (2 mg / kg) HP-Exo-CBD-TAT can effectively inhibit METH-induced microglial activation and the release of inflammatory factors in the mPFC and VTA brain regions, and inhibit METH-induced behavioral sensitization and CPP in mice. Notably, the drug delivery system HP-Exo-CBD-TAT of the present application can inhibit METH-induced behavioral sensitization to the baseline level at a concentration of 2 mg / kg, effectively control the conditioned place preference of mice, and can effectively inhibit the activation of microglia and the release of IL-1β in the mPFC and VTA. The same concentration of CBD has no effect, indicating that there is a synergistic effect between the carrier of the present application and CBD. The carrier HP-Exo-TAT can alleviate METH-induced behavioral sensitization to a certain extent in in vivo experiments and can fully reduce the inflammation of the central nervous system induced by METH, demonstrating the functionality of this carrier.

[0074] Comparative Example 1. In this comparative example, the relevant effects of different nanocarriers in the prior art on methamphetamine addiction research were compared. The specific prior art, its anti-addiction effects, characteristics, action mechanisms, and sources are shown in Table 14.

[0075] Table 14 , As can be seen from Table 14, although there are studies on methamphetamine in the prior art using nanomaterials, the mechanism research is not deep enough, and the assembled structures vary greatly. Therefore, the present invention has very significant innovation.

[0076] In summary, the present invention provides a nano-drug delivery system for treating methamphetamine addiction. The nano-drug delivery system uses exosomes derived from human umbilical vein endothelial cells (HUVECs) pretreated with hypoxia and engineered with transcriptional activator protein (TAT) as the nanocarrier of cannabidiol (CBD). Compared with the current administration methods for treating METH addiction, which mainly rely on highly invasive intracerebroventricular injection or high-dose intraperitoneal injection (40 - 100 mg / kg), the present invention treats METH addiction by nasal administration, with an effective concentration of only 2 mg / kg, which is only 1 / 20 of the lowest effective concentration (40 mg / kg) of intraperitoneal injection of CBD solution for treating METH addiction.

[0077] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A nano-drug delivery system for the treatment of methamphetamine addiction, characterized in that: The nano-drug delivery system is obtained by functional modification after loading cannabidiol with exosomes extracted from hypoxic preconditioned human umbilical vein endothelial cells as nano-carriers; Among them, the functional modification is to modify the exosomes loaded with cannabidiol using the transcriptional activator TAT in the human immunodeficiency virus.

2. The nano-drug delivery system for treating methamphetamine addiction according to claim 1, characterized in that: The drug loading rate of cannabidiol in the nano-drug delivery system > 16%, and the encapsulation rate > 7%.

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

4. The preparation method of the nano-drug delivery system for treating methamphetamine addiction according to any one of claims 1 to 3, characterized in that: Including, Human umbilical vein endothelial cells are cultured in DMEM medium until the cells reach 80% confluence, and then incubated in a serum-free medium containing 5 μM cannabidiol under hypoxic conditions of 1% O2 for 24 hours. After incubation, exosomes loaded with cannabidiol are obtained through post-treatment; Among them, the post-treatment is to centrifuge the cell supernatant after incubation at 300 g for 10 min. The obtained supernatant is centrifuged at 1000 g for 20 min to remove cell debris and dead cells. The supernatant obtained again is transferred and centrifuged at 10000 g for 30 min to remove impurities in the sample. The supernatant obtained after final centrifugation is ultracentrifuged at 100000 g for 70 min to obtain exosomes loaded with cannabidiol. The precipitate is resuspended with PBS and stored at -80°C; The exosomes loaded with cannabidiol and the transcriptional activator TAT are incubated and centrifuged at a mass ratio of 10:1 to obtain the nano-drug delivery system.

5. A pharmaceutical preparation for treating methamphetamine addiction by nasal administration, characterized in that: The pharmaceutical preparation contains the nano-drug delivery system described in claim 1, and also includes pharmaceutically acceptable excipients.

6. The pharmaceutical preparation according to claim 5, characterized in that: The dosage forms of the pharmaceutical preparation include injection solutions and freeze-dried powder injections.

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