Nano preparation with mitochondrial targeting property, preparation method thereof and application of nano preparation in myasthenia gravis

Abstract

The invention discloses a nano preparation with mitochondrial targeting, a preparation method of the nano preparation and application of the nano preparation in myasthenia gravis. According to the method, a nano-carrier is prepared from PLGA and HPMC through an anti-solvent precipitation method, astragaloside and black phosphorus quantum dots are loaded in a manner of forming nanoparticles, and the surfaces of the nanoparticles are modified with mitochondrial targeting micromolecules NH2-PEG-TPP, so that the nano-preparation for mitochondrial targeting treatment of myasthenia gravis is obtained. The nano preparation has a mitochondrial targeting function, can improve the distribution of drugs in muscle cell mitochondria, avoids the too fast degradation of BPQDs in vivo, improves the bioavailability of AS-IV and BPQDs, exerts the function of resisting oxidative stress to protect mitochondria, and provides a nano preparation for effectively treating mitochondrial damage for myasthenia gravis.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomedicine, and in particular to a nano preparation with mitochondrial targeting, a preparation method thereof, and an application thereof in myasthenia gravis. Background Art

[0002] Myasthenia gravis (MG) is a chronic autoimmune disease that occurs at the neuromuscular junction (NMJ) and is mediated by B cells, requires the participation of T cells and complement, and is mediated by autoantibodies. The clinical manifestations are weakness and fluctuating fatigue of the affected muscles. The symptoms worsen after exercise and are relieved after rest. A few patients have thymus function defects. The initial symptoms are diplopia and ptosis, which can gradually affect the muscles of the whole body. In severe cases, it can be life-threatening and cause great harm to patients. Studies have shown that the onset of MG is not only related to the disorder of the immune mechanism, but also may be pathological damage to the mitochondria of muscle cells. In recent years, the incidence of MG has been gradually increasing, and the treatment of myasthenia gravis is mainly based on symptom relief, but it will be accompanied by adverse reactions of varying degrees. The cost of treatment is high and the prognosis is not ideal, which brings great psychological pressure and economic burden to patients. Therefore, new drugs that improve the symptoms of myasthenia gravis and reduce the side effects of clinical drug treatment to achieve better therapeutic effects are urgently needed to be developed.

[0003] Existing research and clinical practice have shown that many active ingredients of traditional Chinese medicine have wide application potential in the treatment of mitochondrial damage. For example, astragaloside IV, the active ingredient of Astragalus, has the effects of regulating immunity, resisting oxidative stress, improving mitochondrial functional damage, regulating mitochondrial quality control and improving MG symptoms. The use of traditional Chinese medicine and its active ingredients for the prevention and treatment of MG has good development prospects and will also be an important research direction in the field of MG treatment in the future. However, the poor water solubility, low bioavailability and poor specific distribution of astragaloside IV limit its clinical efficacy. Using PLGA nanoparticles as drug carriers can significantly improve the solubility of drugs and improve bioavailability.

[0004] The ideal MG drug delivery system must deliver therapeutic drugs to the mitochondrial site in a targeted manner, increase drug concentration, and give full play to the role of regulating mitochondrial quality control. Functionally modified nanocarriers successfully accumulate on target tissues or organelles and overcome common physiological and structural barriers to effectively deliver drugs to designated locations. The mitochondrial targeting small molecule, (2-carboxyethyl)triphenylphosphonium bromide (TPP), is a delocalized lipophilic cation with positive charge properties. The cell membrane and mitochondrial membrane are negatively charged. TPP targets mitochondria through membrane potential. The high negative charge of the mitochondrial membrane and the high positive charge of TPP combine to form a membrane potential-dependent targeted mitochondrial property, which mediates the nanocarrier to overcome the barrier of the cell membrane and mitochondrial membrane, thereby achieving the effect of targeted therapy. Summary of the invention

[0005] In order to overcome the problems existing in the prior art, one of the objectives of the present invention is to provide an application of black phosphorus quantum dots in the preparation of drugs for treating myasthenia gravis.

[0006] The second object of the present invention is to provide a method for preparing a nano preparation for mitochondrial targeted treatment of myasthenia gravis.

[0007] The third object of the present invention is to provide a nano preparation for mitochondrial targeted treatment of myasthenia gravis.

[0008] A fourth object of the present invention is to provide an application of the above-mentioned nanoformulation in the preparation of a drug for treating myasthenia gravis.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] Application of black phosphorus quantum dots in the preparation of drugs for the treatment of myasthenia gravis.

[0011] Furthermore, the black phosphorus quantum dots are obtained by a preparation method comprising the following steps: dispersing black phosphorus powder in N-methylpyrrolidone, avoiding exposure to water and oxygen, ultrasonically treating, and centrifugally separating a supernatant containing black phosphorus quantum dots.

[0012] Furthermore, the ratio of the black phosphorus powder to N-methylpyrrolidone is 1-2 mg: 1-2 mL.

[0013] Furthermore, the conditions of the ultrasonic treatment are: frequency: 19-25 kHz, power: 1200 W, cycle: 5 s, interval: 5 s, first ultrasonic treatment for 4 hours, and then ice bath ultrasonic treatment for 10 hours.

[0014] Furthermore, the centrifugal conditions are: rotation speed: 7000 rpm, time: 20 min.

[0015] A method for preparing a nano preparation for mitochondrial targeted treatment of myasthenia gravis comprises the following steps:

[0016] S1, preparing an organic phase by using an organic solvent to prepare poly(lactic acid-co-glycolic acid) PLGA, astragaloside AS-IV and black phosphorus quantum dots BPQDs;

[0017] S2, HPMC was treated with ddH 2 After dissolving with O and PBS, add the mitochondrial target head TPP-PEG-NH 2 , formulated into a water phase;

[0018] S3. Under stirring, the organic phase is quickly injected into the aqueous phase to prepare the nanoformulation by anti-solvent precipitation method.

[0019] Furthermore, the molecular weight of the PLGA in step S1 is 8K to 10K, preferably 9.5K.

[0020] Furthermore, the organic solvent described in step S1 is any one or more of methanol, ethanol, acetone, and dimethyl sulfoxide (DMSO); preferably dimethyl sulfoxide.

[0021] Furthermore, the ratio of PLGA, AS-IV, BPQDs and organic solvent in step S1 is 10-20 mg: 15-25 mg: 90-110 μg: 1 mL: preferably 15 mg: 20 mg: 100 μg: 1 mL.

[0022] Further, the TPP-PEG-NH 2 The molecular weight of PEG is 1K to 3K, preferably 2K.

[0023] Further, the ddH 2 The volume ratio of O and PBS is 1-2:1-2; preferably 1:1.

[0024] Furthermore, the amount of HPMC added in step S2 is calculated based on its concentration in the aqueous phase being 0.5±0.2 mg / mL.

[0025] Further, the TPP-PEG-NH 2 The addition amount is calculated based on the concentration ratio of HPMC to 1:5 to 1:20, preferably 1:20.

[0026] Furthermore, the ratio of the organic phase to the aqueous phase in step S3 is 1:30 to 70, preferably 1:50.

[0027] Furthermore, the stirring condition in step S3 is: rotation speed: 1000 rpm.

[0028] A nano preparation with mitochondrial targeting is obtained by the above preparation method.

[0029] Application of the above-mentioned nanoformulation with mitochondrial targeting in the preparation of drugs for treating myasthenia gravis.

[0030] Compared with the prior art, the present invention has the following advantages and effects:

[0031] The method of the present invention uses PLGA (9.5k, 75:25) and HPMC (E15) to prepare a nanocarrier by an anti-solvent precipitation method, so as to load astragaloside IV (AS-IV) and black phosphorus quantum dots (BPQDs) in the form of nanoparticles, and modify the surface of the nanoparticles with mitochondrial targeting small molecule NH 2 -PEG2000-TPP, a nanoformulation for mitochondrial targeted treatment of myasthenia gravis is obtained. The nanoformulation has the function of mitochondrial targeting, can improve the distribution of drugs in muscle cell mitochondria, and at the same time avoid BPQDs from being degraded too quickly in the body, improve the bioavailability of AS-IV and BPQDs, and play the function of anti-oxidative stress and protection of mitochondria, providing a nanoformulation for the effective treatment of mitochondrial damage for myasthenia gravis.

[0032] The PLGA nanoparticles with mitochondrial targeting provided by the present invention can significantly improve the bioavailability, specific distribution and controlled drug release of AS-IV and BPQDs; can significantly improve the water solubility of AS-IV, and have a positive effect on the long circulation of the drug in the body; show good biosafety in L6 myoblasts; simultaneously load the drugs AS-IV and BPQDs, and the synergistic effect significantly improves the antioxidant and immunomodulatory functions of the nanoparticles; have mitochondrial targeting, can increase the accumulation of drugs in mitochondria, have the advantage of precise drug delivery, and have good application prospects.

[0033] The materials used in the present invention are derived from auxiliary materials approved for use by FDA, and have excellent biocompatibility and high safety.

[0034] The invention has simple process and fast preparation speed and is suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The TPP-PEG2000-NH prepared in Example 1 2 Characterization diagram; where A is TPP-PEG2k-NH 2Structural formula, B is TPP-PEG2k-NH 2 H-NMR spectrum, C is the mapping test image of TPP-PLGA;

[0036] Figure 2 Characterization diagram of BPQDs prepared in Example 1; wherein A is a transmission electron microscopy image (HRTEM); B is a particle size distribution diagram; C is an X-ray photoelectron spectroscopy (XPS); D is a Raman spectrum; E is a Fourier transform infrared spectrum (FT-IR); F is a picture of BPQDs and BPQDs@PLGA NPs in air at different time points, and G is an ultraviolet absorption spectrum of BPQDs and BPQDs@PLGA NPs at different time points.

[0037] Figure 3 Characterization diagrams of different AS-IV nanoformulations prepared in Example 1; wherein A is a particle size distribution diagram; B is a transmission electron microscopy diagram;

[0038] Figure 4 The diagram is a diagram showing the uptake characteristics and mechanism of black phosphorus-based astragaloside nanoparticles in Example 2; wherein A is the cellular safety of different AS-IV preparations; B is a concentration-dependent uptake diagram of C6@PLGA nanoparticles; C is a time-dependent uptake diagram of C6@PLGA nanoparticles; and D is a diagram showing the uptake mechanism of C6@PLGA nanoparticles;

[0039] Figure 5 This is the co-localization diagram of C6@PLGA nano-mitochondria in Example 3; wherein A is C6@PLGA; B is TPP-C6@PLGA;

[0040] Figure 6 The hemolysis test diagrams of different AS-IV nanoformulations in Example 4; A is AS-IV@PLGA; B is AS-IV / BPQDs@PLGA; C is TPP-AS-IV / BPQDs@PLGA;

[0041] Figure 7 The in vivo pharmacodynamic evaluation diagram of different AS-IV nanoformulations in Example 5; wherein A is a picture of rats after modeling; B is a Lennon score diagram; C is an acetylcholine antibody content diagram; and D is an RNS decay rate diagram;

[0042] Figure 8 These are behavioral evaluation diagrams of different AS-IV nanoformulations in Example 5; wherein A is a gait analysis diagram; B is a forced swimming result diagram; and C is a fatigue rotarod result diagram;

[0043] Fig. 9The results of HE staining of rat gastrocnemius muscle in Example 5 are shown; wherein A is HE staining of the cross section of rat gastrocnemius muscle; B is HE staining of the longitudinal section of rat gastrocnemius muscle;

[0044] Fig.10 This is the result of glycogen staining of rats in Example 5;

[0045] Fig.11 This is the result diagram of gastrocnemius energy metabolism index of rats in Example 5:

[0046] Fig.12 The transmission electron microscopy results of the ultrastructure of the gastrocnemius muscle of the Control, Model, and TPP-AS-IV / BPQDs@PLGA rats in Example 5; wherein the black arrows point to mitochondria, and the red arrows point to autophagosomes;

[0047] Fig.13 This is the result diagram of rat immune cytokines in Example 5:

[0048] Fig.14 This is a blood routine analysis diagram of rats in Example 5;

[0049] Fig.15 This is the blood biochemical analysis diagram of rats in Example 5;

[0050] Fig.16 This is the result diagram of the coefficients of the main organs of rats in Example 5;

[0051] Fig.17 This is the HE staining result of the main organs of rats in Example 5;

[0052] Fig.18 This is the expression diagram of proteins related to mitochondrial quality control in rat gastrocnemius muscle in Example 5; among them, AJ corresponds to OPA1, MFN1, MFN2, DRP1, FIS1, PINK1, Parkin, P62, LC3Ⅱ, and Beclin1 proteins respectively.

[0053] Fig.19 It is a schematic diagram of the process of the present invention. DETAILED DESCRIPTION

[0054] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0055] In the present invention, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects before and after are in an "or" relationship.

[0056] In the present invention, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c", can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple.

[0057] It should be understood that in various embodiments of the present invention, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.

[0058] The terms used in the embodiments of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The singular forms "a", "said" and "the" used in the embodiments of the present invention and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0059] The weight of the relevant components mentioned in the description of the embodiments of the present invention may not only refer to the specific content of each component, but also indicate the weight ratio between the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the description of the embodiments of the present invention, it is within the scope disclosed in the description of the embodiments of the present invention. Specifically, the mass described in the description of the embodiments of the present invention may be a mass unit known in the chemical industry such as μg, mg, g, kg, etc.

[0060] Example 1: Preparation and characterization of black phosphorus-based astragaloside nanoparticles

[0061] (1) Preparation of black phosphorus quantum dots:

[0062] 20mg of bulk black phosphorus (BP) powder was dispersed in 20mL N-methylpyrrolidone (NMP), and a tube and ultrasonic tip were used to prevent exposure to water and oxygen, and then ultrasonicated for 4 hours with a probe ultrasonicator (frequency: 19-25kHz) at a power of 1200W (cycle of 5s, interval of 5s), and then ultrasonicated in an ice bath for 10 hours. Finally, the dispersion was centrifuged at 7000rpm for 20min and the supernatant containing BPQDs was gently aspirated. BPQDs were characterized by HRTEM, XPS, Raman spectroscopy, and FTIR. The results are shown in Figure 2. The distribution of the prepared BPQDs is uniform, with an average particle size of 3-5nm. The XPS results show that clear XPS peaks corresponding to P2p, C1s, N1s and O1s are observed in the entire spectrum. The P2p spectrum shows two main peaks: one centered at 130eV, contributed by P2p1 / 2 and P2p3 / 2, and the other higher binding energy comes from POx due to the surface oxidation of BPQDs. The Raman spectrum results of BPQDs show that the atomic layer number of the BPQDs we prepared is 3-4. The FT-IR results of BPQDs show that the peak at 1615cm -1 There is a large absorption peak at 1467cm -1 There is a small absorption peak at 1115 and 962 cm, which belongs to the O=P-oh group and the P=O group respectively. The PO3 stretching of BP quantum dots occurs at 1115 and 962 cm -1 , with ~920cm -1 The large absorption at the center is attributed to the POP vibration.

[0063] (2) Preparation of black phosphorus-based astragaloside nanoparticles:

[0064] As shown in Tables 1 and 2, AS-IV@PLGA nanoparticles were prepared by anti-solvent precipitation method: stabilizers polyvinyl pyrrolidone (PVP) K90, K29 / 32, hydroxypropyl methylcellulose HPMCE5, E15, E50, poloxamer P188, P407, sodium dodecyl sulfate (SDS) were added to the mixture with ddH 2 O and PBS (volume ratio of 1:1) were dissolved and prepared into the aqueous phase, with the initial concentration of 0.5 mg / mL. Dimethyl sulfoxide was used as the solvent of the organic phase, and 20 mg of PLGA (9.5k), PLGA (10k), PLGA (8K)-NH 2(The PLGA used in the present invention is polymerized from 75% polylactic acid (PLA) and 25% polyglycolic acid (PGA)), and 15 mg of AS-IV, observe the state of the nanoformulation after 48 hours, screen out the appropriate PLGA, and use the same stabilizer to prepare AS-IV@PLGA NPs with 15 mg and 20 mg of AS-IV respectively. Control the speed of the magnetic stirrer at 1000 rpm, and the organic phase and the aqueous phase are in a ratio of 1:50. 0.2 mL of the organic phase is quickly and evenly injected into 10 mL of the aqueous phase to form nanoparticles. Then, the particle size and polydispersity index (PDI) at different time points are monitored with a particle size analyzer to screen out the optimal stabilizer and its concentration and the amount of AS-IV. After screening out the appropriate amount of AS-IV, the mitochondrial target NH 2 -PEG2000-TPP was used to screen the nanoparticles with 0.025 mg / mL, 0.05 mg / mL, and 0.1 mg / mL aqueous solutions, while other conditions remained unchanged. The amount of AS-IV and NH 2 After adjusting the concentration of -PEG-TPP, 100 μg of BPQDs were added to the organic phase to prepare AS-IV@PLGA nanoparticles, AS-IV / BPQDs@PLGA nanoparticles, and TPP-AS-IV / BPQDs@PLGA nanoparticles. The preparation of each particle is as follows:

[0065] AS-IV@PLGA nanoparticles: 15 mg AS-IV and 20 mg PLGA were weighed and dissolved in 1 mL DMSO as the organic phase. 2 O and PBS (volume ratio of 1:1) were used to prepare 0.5 mg / mL E15 as the aqueous phase, the magnetic stirrer speed was controlled at 1000 rpm, the organic phase and the aqueous phase were in a ratio of 1:50, and 0.2 mL of the organic phase was quickly and evenly injected into 10 mL of the aqueous phase to form AS-IV@PLGA nanoparticles.

[0066] AS-IV / BPQDs@PLGA nanoparticles: 15 mg AS-IV and 20 mg PLGA were weighed and dissolved in 1 mL DMSO containing 100 μg BPQDs as the organic phase. 2 O and PBS (volume ratio of 1:1) were used to prepare 0.5 mg / mL E15 as the aqueous phase, the speed of the magnetic stirrer was controlled at 1000 rpm, the ratio of the organic phase to the aqueous phase was 1:50, and 0.2 mL of the organic phase was quickly and evenly injected into 10 mL of the aqueous phase to form AS-IV / BPQDs@PLGA nanoparticles.

[0067] TPP-AS-IV / BPQDs@PLGA nanoparticles: 15 mg AS-IV and 20 mg PLGA were weighed and dissolved in 1 mL DMSO containing 100 μg BPQDs as the organic phase. 2 O and PBS (volume ratio of 1:1) were used to prepare 0.5 mg / mL E15 and 0.025 mg / mL TPP-PEG2000-NH 2 As the aqueous phase, the magnetic stirrer speed was controlled at 1000 rpm, and the organic phase and aqueous phase were in a ratio of 1:50. 0.2 mL of the organic phase was quickly and evenly injected into 10 mL of the aqueous phase to form AS-IV / BPQDs@PLGA nanoparticles. The particle size of the above AS-IV different nanoformulations was measured and the transmission electron microscopy imaging was performed. The results are shown in Figure 3 As shown in the figure, the average particle size of AS-IV@PLGA nanoparticles is about 110nm, the average particle size of AS-IV / BPQDs@PLGA nanoparticles is about 150nm, and the average particle size of TPP-AS-IV / BPQDs@PLGA nanoparticles is about 210nm. The morphology of nanoparticles in each group is spherical. It is worth noting that the BPQDs loaded and modified TPP-PEG2000-NH 2 Afterwards, the particle size of the nanoparticles increased, but the morphology was spherical.

[0068] (3) TPP-PLGA nanoparticles: 20 mg of PLGA was weighed and dissolved in 1 mL of DMSO as the organic phase. 2 O and PBS (volume ratio of 1:1) were used to prepare 0.5 mg / mL E15 and 0.025 mg / mL TPP-PEG2000-NH 2 As the aqueous phase, the magnetic stirrer speed was controlled at 1000 rpm, and the organic phase and aqueous phase were in a ratio of 1:50. 0.2 mL of the organic phase was quickly and evenly injected into 10 mL of the aqueous phase to form TPP-PLGA nanoparticles. 2 The preparation was successful. The distribution of TPP-PEG2000-NH2 on the surface of PLGA nanoparticles was characterized by mapping test ( Figure 1 ).

[0069] (4) BPQDs@PLGA nanoparticles: 20 mg of PLGA was weighed and dissolved in 1 mL of DMSO containing 100 μg of BPQDs as the organic phase. 0.5 mg / mL of E15 was prepared with ddH2O and PBS (volume ratio of 1:1) as the aqueous phase. The magnetic stirrer speed was controlled at 1000 rpm. The ratio of organic phase to aqueous phase was 1:50. 0.2 mL of organic phase was quickly and evenly injected into 10 mL of aqueous phase to form BPQDs@PLGA nanoparticles. The stability was observed by measuring its UV absorption spectrum with an ultra-micro spectrophotometer.

[0070] Table 1 Effects of different organic phases on the state of AS-IV@PLGA NPs after 48 h

[0071]

[0072] Note: n=3.

[0073] Table 2 Effect of different organic phase concentrations on the state of AS-IV@PLGA NPs after 48 h

[0074]

[0075] Note: n=3.

[0076] Table 3 Effect of different TPP concentrations on the state of AS-IV@PLGA NPs after 48 h

[0077]

[0078] Note: n=3.

[0079] Example 2: Cellular uptake test of black phosphorus-based astragaloside nanoparticles

[0080] First, weigh an appropriate amount of coumarin 6 (C6) and prepare a solution with a concentration of 400 μg / mL using DMSO. Then take 1 mL of C6 to dissolve 20 mg of PLGA, so that the organic phase PLGA:C6 is 50:1. The screened stabilizer is used as the aqueous phase. According to the volume ratio of the organic phase to the aqueous phase of 1:50, the organic phase is quickly injected into the aqueous phase at a speed of 1000 rpm / min to prepare C6@PLGA nanoparticles. Pay attention to avoid light during the preparation process. Then soak the cell slide in 75% ethanol for 15 minutes, then wash it three times with PBS (wash off the residual alcohol) and stick it into a 12-well plate. When the L6 myoblasts grow to the logarithmic phase and the density is 80%, the cells are digested and collected by centrifugation. After counting, it is configured into a cell suspension with a density of 4×10 4Each well was seeded into a 12-well plate with a volume of 1 mL. When the cell density grew to about 80%, the upper culture medium was aspirated, and C6@PLGA nanoparticles with concentrations of 20 ng / mL, 40 ng / mL, and 80 ng / mL were incubated for 1 hour, and 40 ng / mL C6@PLGA nanoparticles were incubated with L6 myoblasts in the dark for 0.5 h, 1 h, and 2 h. When the incubation time was up, the culture medium containing C6@PLGA nanoparticles was discarded, and the residual C6@PLGA on the slide was rinsed three times with PBS. Add 4% paraformaldehyde for fixation for 10 minutes. After fixation, aspirate the paraformaldehyde, then add PBS to rinse three times to wash away the residual paraformaldehyde. Add 450 μL of DAPI staining solution and incubate in the dark for 8-10 minutes. After nuclear staining, discard the DAPI staining solution and rinse three times with PBS to wash away the excess staining solution. Finally, prepare a mounting medium (glycerol: PBS = 1:1), drop it on the slide, and place the cell slide upside down on the slide containing the mounting medium. After completion, place the slide under a confocal laser scanning microscope to observe the uptake of L6 myoblasts by each group of preparations containing C6. Figure 4 The results in Figures B and C show that the fluorescence intensity of C6@PLGA is time-dependent and concentration-dependent.

[0081] After the inhibitors were prepared in advance, MβCD powder was prepared to a concentration of 10 μmol / L using DMEM basal medium, chlorpromazine (CPZ) was prepared to 30 μM, and EIPA was prepared to a concentration of 40 μmol / L. Cell slides were prepared and L6 myoblasts were cultured at 4 × 10 4 1 / well was inoculated in a circular slide of a 12-well plate, and then placed in a cell culture incubator for 36 hours. At this time, when the L6 myoblasts grew to about 80% of the bottom area of ​​the circular slide, the supernatant was discarded, and 1 mL of the inhibitor prepared with fresh culture medium was added to the 12-well plate. After culturing in the incubator for 30 minutes, C6@PLGA was then added to make the final concentration 40 ng / mL. After the well plate was placed in a 37°C cell culture incubator for 1 hour in the dark, all the culture medium was discarded, and the cell slide was repeatedly rinsed with PBS 3 times to remove residual drugs. Then 4% paraformaldehyde was added to fix the cells for 10-12 minutes, and washed with PBS 3 times to wash off the excess paraformaldehyde. Finally, DAPI staining solution was added and incubated in the dark for 8 minutes (to stain the cell nucleus), and then the DAPI staining solution was discarded, and the slide was repeatedly rinsed with PBS to wash off the excess staining solution. A small amount of sealing agent was dropped on the slide, and the circular slide was turned upside down on the slide, and the edge of the slide was sealed with a sealing agent. The fluorescence intensity of the different inhibitor groups was compared with that of the control group. Figure 4 The results in D show that the uptake of C6@PLGA by L6 myoblasts is through caveolin-mediated endocytosis.

[0082] Example 3: Mitochondrial targeting effect of black phosphorus-based astragaloside nanoparticles

[0083] When L6 myoblasts grew to the logarithmic phase and the density reached 80%, the cells were digested and collected by centrifugation. After counting, they were prepared into a cell suspension with a density of 4×10 4 Each well was added to a 12-well plate with a volume of 1 mL and placed in a cell culture incubator for 36 hours. The fluorescent probes for marking organelles were diluted according to a certain ratio, wherein the lysosome fluorescent probe (Lysosome-Tracker, Lyso-Tracker) was diluted at a ratio of 1:10000, the mitochondrial fluorescent probe (Mitochondria-Tracker, Mito-Tracker) was diluted at a ratio of 1:3000, and the endoplasmic reticulum fluorescent probe (Endoplasmic reticulum-Tracker, ER-Tracker) was diluted at a ratio of 1:200. Then 1 mL of the diluted fluorescent probe was added to a 12-well plate, and Lyso-Tracker, Mito-Tracker, and ER-Tracker were incubated with L6 myoblasts for 2 hours, 30 minutes, and 30 minutes, respectively. After the incubation was completed, the Tracker was aspirated, the slide was washed 3 times with PBS, and C6@PLGA and TPP-C6@PLGA at a concentration of 40 ng / mL were continued to be incubated with the cells for 1 hour. Then remove the supernatant, wash the cells three times with pre-cooled PBS to remove excess dye, then add DAPI staining solution and incubate the cells in the dark for 10 minutes, rinse the cell slides three times with PBS to wash away excess cells and fluorescent dyes, and finally use a confocal laser microscope to observe the co-localization of cells and subcellular organelles. Figure 5 The results showed that TPP-C6@PLGA nanoparticles could aggregate more in mitochondria, and TPP as a targeting functional group could enhance the mitochondrial targeting effect of nanoparticles.

[0084] Example 4: In vitro hemolysis experiment of black phosphorus-based astragaloside nanoparticles

[0085] In order to evaluate the potential of AS-IV@PLGA, AS-IV@PLGA, AS-IV / BPWDs@PLGA, and TPP-AS-IV / BPWDs@PLGA nanoparticles for in vivo applications, the blood compatibility of different AS-IV nanoformulations was determined using red blood cells (RBC) of SD rats. Distilled water and PBS were used as positive and negative controls, respectively. The hemolysis rate (HR) was calculated as follows:

[0086]

[0087] Figure 6 The results showed that the hemolysis rates of different AS-IV nanoformulations were all lower than 5%, and they could be used for tail vein injection.

[0088] Example 5: Pharmacodynamic evaluation of black phosphorus-based astragaloside nanoparticles in regulating mitochondrial quality control in vivo

[0089] Lewis female rats were randomly divided into 6 groups: ①Control group, ②Model group, ③AS-IV group, ④AS-IV@PLGA group, ⑤AS-IV / BPQDs@PLGA group, ⑥TPP-AS-IV / BPQDs@PLGA group. Except for the normal group, the remaining rats were modeled using the EAMG modeling method. The number of inoculations required for modeling was three times. The first inoculation day was marked as day 0, and the remaining two were days 30 and 45 respectively. The modeling was performed by multi-point subcutaneous injection, and the injection sites included the abdomen, back and foot pads of both lower limbs. Each site was injected with 50μL of immune emulsion. After the modeling was completed and the model was successfully evaluated, the dose of AS-IV in all AS-IV groups was 0.5mg / kg, and the corresponding drugs were injected through the tail vein. The drug was administered once every 2 days, for a total of 10 times. After the end of the drug administration, the RNS decay rate, Lennon score and behavioral experimental evaluation of the rats in each treatment group were performed. Subsequently, the rat gastrocnemius muscles were collected for HE staining, glycogen staining, and ultrastructural observation. The mitochondrial energy metabolism indicators ATP, ROS, SDH, and CCO of the gastrocnemius muscles were detected, and the levels of serum acetylcholine receptor antibodies and immune cytokines were determined. Finally, Western Blot was used to detect the expression levels of mitochondrial quality control-related proteins OPA1, MFN1, MFN2, DRP1, FIS1, PINK1, Parkin, P62, LC3Ⅱ, and Beclin1 in the gastrocnemius muscles.

[0090] After modeling, rats showed symptoms of myasthenia. After administration of different AS-IV preparations, the levels of serum acetylcholine receptor antibodies, Lennon scores, and RNS decay rates of rats decreased. The improvement effect of TPP-AS-IV / BPQDs@PLGA group rats was the most significant. Figure 7 As shown. The behavioral evaluation of rats in each group found that the gait results, fatigue rotarod results, and forced swimming test results of rats in the TPP-AS-IV / BPQDs@PLGA group had significant therapeutic effects, such as Figure 8 HE staining of the cross-section and longitudinal section of the rat gastrocnemius muscle showed that the inflammatory infiltration in the TPP-AS-IV / BPQDs@PLGA group was significantly improved ( Fig. 9 ), glycogen staining showed that the glycogen content in the TPP-AS-IV / BPQDs@PLGA group increased significantly ( Fig.10 ). By detecting the energy metabolism indicators of rat gastrocnemius mitochondria, ATP, ROS, SDH, and CCO, it was found that the energy metabolism capacity of the TPP-AS-IV / BPQDs@PLGA group was significantly improved. Fig.11 The gastrocnemius muscles of the Control group, Model group, and TPP-AS-IV / BPQDs@PLGA group were observed using a transmission microscope. It was found that the muscle fiber rupture, mitochondrial cristae rupture, and vacuolization in the TPP-AS-IV / BPQDs@PLGA group were significantly improved, and more autophagosomes were observed, indicating that the TPP-AS-IV / BPQDs@PLGA group can promote mitochondrial autophagy. Fig.12 The imbalance of immune cytokines IL-4, IL-10, IL-6, and TNF-α in the serum of the TPP-AS-IV / BPQDs@PLGA group was significantly improved. Fig.13 The biosafety evaluation includes blood routine analysis ( Fig.14 )、blood biochemical analysis( Fig.15 )、Organ coefficient( Fig.16 )、HE staining of major organs ( Fig.17 ) The results showed that TPP-AS-IV / BPQDs@PLGA nanoparticles had good biocompatibility. Finally, the results of Western blotting showed that different AS-IV preparations could promote mitochondrial fusion, fission and autophagy, among which the TPP-AS-IV / BPQDs@PLGA group had a more significant effect. Fig.18 shown.

[0091] Finally, it should be noted that the above embodiments are intended to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Application of black phosphorus quantum dots in the preparation of drugs for the treatment of myasthenia gravis.

2. The use according to claim 1, characterized in that: The black phosphorus quantum dots are obtained by a preparation method comprising the following steps: dispersing black phosphorus powder in N-methylpyrrolidone, avoiding exposure to water and oxygen, ultrasonically treating, and centrifugally separating a supernatant containing black phosphorus quantum dots.

3. The use according to claim 2, characterized in that: The ratio of black phosphorus powder to N-methylpyrrolidone is 1-2 mg: 1-2 mL; The ultrasonic treatment conditions are: frequency: 19-25kHz, power: 1200W, cycle: 5s, interval: 5s, ultrasonic treatment for 4 hours, followed by ultrasonic treatment in an ice bath for 10 hours; The centrifugal conditions are: rotation speed: 7000 rpm, time: 20 min.

4. A method for preparing a nanoformulation for mitochondrial targeted treatment of myasthenia gravis, characterized in that: The steps include: S1, preparing an organic phase by using an organic solvent to prepare poly(lactic acid-co-glycolic acid) PLGA, astragaloside IV and black phosphorus quantum dots BPQDs; S2, dissolving hydroxypropyl methylcellulose HPMC with ddH2O and PBS, adding mitochondrial target head TPP-PEG-NH2 to prepare an aqueous phase; S3. Under stirring, the organic phase is quickly injected into the aqueous phase to prepare the nanoformulation by anti-solvent precipitation method.

5. The method for preparing the nano preparation for mitochondrial targeted treatment of myasthenia gravis according to claim 4, characterized in that: The molecular weight of PLGA described in step S1 is 8K to 10K; The organic solvent in step S1 is any one or more of methanol, ethanol, acetone, and dimethyl sulfoxide; The ratio of PLGA, AS-IV, BPQDs, and organic solvent described in step S1 is 10-20 mg: 15-25 mg: 90-110 μg: 1 mL.

6. The method for preparing the nano preparation for mitochondrial targeted treatment of myasthenia gravis according to claim 5, characterized in that: The molecular weight of PLGA described in step S1 is 9.5K; The organic solvent described in step S1 is dimethyl sulfoxide; The ratio of PLGA, AS-IV, BPQDs, and organic solvent described in step S1 is 15 mg: 20 mg: 100 μg: 1 mL.

7. The method for preparing the nano preparation for mitochondrial targeted treatment of myasthenia gravis according to claim 4, characterized in that: The molecular weight of PEG in the TPP-PEG-NH2 described in step S2 is 1K to 3K; The volume ratio of ddH2O and PBS described in step S2 is 1-2:1-2; The amount of HPMC added in step S2 is calculated based on its concentration in the aqueous phase of 0.5±0.2 mg / mL; The amount of TPP-PEG-NH2 added in step S2 is calculated based on a concentration ratio of 1:5 to 1:20 to HPMC; The ratio of the organic phase to the aqueous phase in step S3 is 1:30-70.

8. The method for preparing the nano preparation for mitochondrial targeted treatment of myasthenia gravis according to claim 7, characterized in that: The molecular weight of PEG in TPP-PEG-NH2 described in step S2 is 2K; The volume ratio of ddH2O and PBS described in step S2 is 1:1; The amount of HPMC added in step S2 is calculated based on its concentration in the aqueous phase being 0.5 mg / mL; The amount of TPP-PEG-NH2 added in step S2 is calculated based on a concentration ratio of 1:20 to HPMC. The ratio of the organic phase to the aqueous phase in step S3 is 1:50; The stirring conditions described in step S3 are: rotation speed: 1000 rpm.

9. A mitochondrial targeting nanoparticle preparation, characterized in that: The method is obtained by the preparation method described in any one of claims 4 to 8.

10. Use of the mitochondrial targeting nanoformulation according to claim 9 in the preparation of a drug for treating myasthenia gravis.

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