CN114288419A

CN114288419A - Preparation of rifampicin nano drug delivery system and application of rifampicin nano drug delivery system in prevention and treatment of Parkinson's disease

Info

Publication number: CN114288419A
Application number: CN202111656866.4A
Authority: CN
Inventors: 陶恩祥; 李耀; 林淡钰; 卜璐璐; 梁嫣然; 陈颖; 井秀娜; 彭素丹
Original assignee: Eighth Affiliated Hospital of Sun Yat Sen University
Current assignee: Eighth Affiliated Hospital of Sun Yat Sen University
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2022-04-08
Anticipated expiration: 2041-12-30
Also published as: CN114288419B

Abstract

The invention belongs to the technical field of nano drug delivery systems, and particularly relates to preparation of a rifampicin nano drug delivery system and application of the rifampicin nano drug delivery system in prevention and treatment of Parkinson's disease; the surface of the polymer micelle is modified with TTBZ molecules which can be specificIncorporating VMAT₂. Compared with the traditional rifampicin medicament type, the carrier (PEG-PCL) of the invention has weak cytotoxicity, and the micelle formed by PEG-PCL hydrophilic blocks has the structural characteristics of a hydrophobic inner core and a hydrophilic outer core, so that the water solubility of rifampicin is increased, and the stability of the rifampicin in vivo circulation is improved; has targeting administration ability, and can improve local aggregation concentration of rifampicin in dopamine neuron, thereby improving bioavailability of the medicine, improving its protective effect on nervous diseases such as Parkinson disease, and reducing its peripheral toxic and side effects.

Description

Preparation of rifampicin nano drug delivery system and application of rifampicin nano drug delivery system in prevention and treatment of Parkinson's disease

Technical Field

The invention belongs to the technical field of nano drug delivery systems, and particularly relates to preparation of a rifampicin nano drug delivery system and application of the rifampicin nano drug delivery system in prevention and treatment of Parkinson's disease.

Background

Rifampin (Rifampicin, Rif) is a semi-synthetic broad-spectrum antibacterial of rifamycins, with the common name: rifampin, english name: rifavicintiebelets. As a classic, effective and safe antitubercular drug, rifampicin has antibacterial activity against a variety of pathogenic microorganisms. The rifampicin is well absorbed by oral administration, and the blood concentration reaches the peak value 1.5-4 hours after the rifampicin is taken. The product can be used in combination with other anti-tuberculosis drugs for the primary treatment and secondary treatment of tuberculosis, including the treatment of tubercular meningitis. In addition, rifampicin also has a potential neuroprotective effect. Research shows that in a Parkinson's Disease (PD) in-vitro model induced by neurotoxic drugs, the rifampicin pretreatment can reverse the neuronal apoptosis phenomenon caused by toxic factors such as rotenone, MPP + and the like. Further research proves that rifampicin can relieve the toxic damage of dopaminergic neurons by means of up-regulating the expression of endoplasmic reticulum stress-related factor GRP78, regulating the level of apoptosis-related pathway PI3K/Akt/GSK-3 beta/CREB, improving SUMO of alpha-synuclein and the like; simultaneously, the nerve inflammation level is reduced by regulating a TLR-4 related channel and inhibiting the activation of NLRP3 inflammatory corpuscle; and enhance the clearance of glial cells from pathological proteins by affecting the level of autophagy-lysosomal pathways in microglia.

Research also finds that, in a living animal model, the intraperitoneal injection of rifampicin has a certain neuroprotective effect on a PD mouse on a molecular pathological layer, but does not stably improve the movement symptoms and the behavioral changes generated after animal modeling, has long administration time and has larger toxic and side effects on the model mouse. The analysis shows that the following defects exist in the treatment of the neurological diseases by utilizing the rifampicin: (1) rifampicin has strong lipid solubility, but low water solubility and poor stability in vivo circulation; (2) the local concentration of rifampicin in the intracranial substantia nigra-striatum is much lower than its working concentration at which it works; 3) the peripheral toxicity of rifampicin, such as hepatotoxicity, limits its use at higher concentrations.

Therefore, there is a need to increase the water solubility of rifampicin, improve its stability in vivo circulation, increase the targeting ability of the drug, increase its local concentration of accumulation in intracranial dopamine neurons, and reduce peripheral toxic side effects.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a preparation method of a rifampicin nano drug delivery system, and the prepared rifampicin nano drug delivery system effectively increases the water solubility of rifampicin, improves the stability of rifampicin in-vivo circulation, increases the targeting ability of the rifampicin, and improves the local aggregation concentration of rifampicin in intracranial dopamine neurons, thereby improving the protective effect of rifampicin on neurological diseases such as Parkinson's disease and the like, and reducing the peripheral toxic and side effects.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides a preparation method of a rifampicin nano drug delivery system, which comprises the following steps:

s1, loading rifampicin in a PEG-PCL carrier in a self-assembly mode to prepare PEG-PCL @ rifampicin (simply called PEG-PCL @ rif);

s2, mixing mercaptopropionic acid and TTBZ (C)₂₈H₃₉NO₆S, the structural formula of which is shown in figure 3) reacts under the catalysis of EDCI [ 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride ] and DMAP (4-dimethylaminopyridine) to generate HS-TTBZ;

s3 reacting thiol of HS-TTBZ and C ═ C group at tail end of PEG-PCL in PEG-PCL @ rifampicin of step S1 in APS (ammonium persulfate) and Na₂SO₃The rifampicin nano drug delivery system can be prepared by click chemical reaction under catalysis.

As a preferred embodiment of the present invention, the preparation method of the rifampicin nano-drug delivery system comprises the following steps:

s1, dissolving rifampicin and APEG-PCL freeze-dried powder in DMF to form an organic phase, adding the organic phase into water, dialyzing for more than 12 hours by using a dialysis bag, collecting the solution in the dialysis bag, and filtering to obtain a PEG-PCL @ rifampicin solution;

s2, stirring and uniformly mixing mercaptopropionic acid, EDCI and DMAP at low temperature, adding TTBZ, and continuously stirring for more than 12 hours to obtain TTBZ-SH solution;

s3, mixing the TTBZ-SH solution of the step S2 and the PEG-PCL @ rifampicin solution of the step S1, and adding APS and Na₂SO₃Stirring at low temperature for more than 36h, dialyzing with a dialysis bag overnight, and collecting the solution in the dialysis bag to obtain the rifampicin nano drug delivery system.

The invention takes PEG-PCL polymer micelle as a carrier of a drug delivery system, loads hydrophobic rifampicin into an inner core to improve the water solubility and the stability of in vivo circulation of the rifampicin, and can be specifically combined with VMTA in dopamine neuron₂TTBZ is used as a ligand of a drug delivery system to improve the targeting capability of the TTBZ and further increase the local working concentration of the drug, thereby improving the protective effect of the TTBZ on neurological diseases such as Parkinson's disease and reducing the peripheral toxic and side effects.

Preferably, the mass ratio of the rifampicin to the APEG-PCL freeze-dried powder is 1: 8-12; the feed-liquid ratio of rifampicin to DMF was 1 mg: 1-3 mL. Specifically, the mass ratio of rifampicin to APEG-PCL freeze-dried powder is 1: 10; the feed-liquid ratio of rifampicin to DMF was 1 mg: 1 mL.

Preferably, the mass ratio of mercaptopropionic acid, EDCI, and DMAP is 3: 11: 7.

preferably, the volume ratio of the TTBZ-SH solution to the PEG-PCL @ rifampicin solution is 1: 4-6; TTBZ-SH solution, APS and Na₂SO₃The feed-liquid ratio of (1 mL): 1 mg: 1 mg. Specifically, the volume ratio of the TTBZ-SH solution to the PEG-PCL @ rifampicin solution is 1: 5.

preferably, the filtration of step S1 is a filtration with a 0.45um microfiltration membrane.

Preferably, in step S1, the step of adding the organic phase to the water is to drop the organic phase to the water gradually under the action of ultrasound.

Preferably, the molecular weight cut-off of the dialysis bag in steps S1 and S3 is 14,000.

The invention also provides the rifampicin nano drug delivery system prepared by the preparation method of the rifampicin nano drug delivery system.

The invention also provides application of the rifampicin nano drug delivery system in preparation of neuroprotective drugs, wherein the neuroprotective drugs comprise drugs for preventing and treating Parkinson's disease.

PD is a neurodegenerative disease of extrapyramidal movement disorders such as bradykinesia and dystonia caused by degeneration and necrosis of mesencephalic nigral dopaminergic neurons and insufficient secretion of dopaminergic neurotransmitters, and currently, clinical treatment of PD is mainly dopaminergic replacement therapy and DBS surgical treatment. To date, there is still a lack of disease modifying therapies directed at the molecular mechanisms of the pathogenesis of PD. The invention loads the common rifampicin which is proved to have the function of modifying the PD disease progress in vitro experiments into the PEG-PCL micelle, and modifies the TTBZ molecule on the surface of the micelle to lead the rifampicin to target dopamine neurons, thereby improving the neuroprotective effect of the rifampicin.

Of course, other neurological diseases, besides Parkinson's disease, that would enable the rifampicin nano-delivery system of the present invention to exert its protective effect are also within the scope of the present invention.

Compared with the prior art, the invention has the beneficial effects that:

the rifampicin is loaded in a PEG-PCL nano drug delivery system for the first time and is prepared into a polymer micelle; and the surface of the polymer micelle is modified with TTBZ molecules which can be specifically combined with dopamine neuron inner vesicle Monoamine Transporter 2(VMAT2)₂). Compared with the traditional rifampicin medicament type, the rifampicin nano drug delivery system has the following advantages: (1) the carrier (PEG-PCL) has weak cytotoxicity, wherein the safety of the PEG is certified by the United states Food and Drug Administration (FDA); (2) the micelle formed by the PEG-PCL hydrophilic block has the structural characteristics of a hydrophobic inner core and a hydrophilic outer core, increases the water solubility of rifampicin, and improves the stability of the rifampicin in-vivo circulation; (3) has the targeting administration capability, improves the local concentration of rifampicin in dopamine neurons, thereby improving the bioavailability of the medicament, improving the protective effect of the medicament on neurological diseases such as Parkinson's disease and the like, and reducing the toxic and side effects of the periphery of the medicament.

Drawings

FIG. 1 is a flow chart of the preparation of PEG-PCL @ rif;

FIG. 2 is a schematic representation of TTBZ-modified PEG-PCL @ rif;

FIG. 3 is a diagram of the chemical synthesis scheme for the sulfhydryl modification of TTBZ;

FIG. 4 is a diagram showing the synthesis of TTBZ-modified PEG-PCL @ rif;

FIG. 5 is a transmission electron micrograph of TTBZ-PEG-PCL @ rif;

FIG. 6 shows TTBZ-PEG-PCL @ rif¹H NMR measurement results;

FIG. 7 shows the results of the stability assay of TTBZ-PEG-PCL @ rif;

FIG. 8 shows the safety assay results of TTBZ-PEG-PCL @ rif;

FIG. 9 shows the results of the targeting ability test of the vector TTBZ-PEG-PCL;

FIG. 10 shows the modified effect of TTBZ-PEG-PCL @ rif.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.

Example 1 preparation of Rifampicin NanoDeliver System (TTBZ-PEG-PCL @ rif Polymer micelle)

(1) Preparation of PEG-PCL @ rifampicin

As shown in FIG. 1, rifampicin is loaded in PEG-PCL carrier by Self-assembly (Self-assembly) to prepare PEG-PCL @ rifaxin solution, the specific preparation method is as follows:

1mg of rifampicin (rifampicin, rif) and 10mg of Allyl-PEG-PCL (PEG-PCL) lyophilized powder (available from Shunhner Nano Co.) were each weighed and dissolved in 1mL of DMF to form an organic phase, and the resulting organic phase was added dropwise to 10mL of deionized water under sonication (130W). And (3) putting the obtained mixed solution into a dialysis bag with the molecular weight cutoff of 14,000, dialyzing with water for more than 12h, collecting the solution in the dialysis bag, and removing hydrophobic drug aggregates by using a 0.45um microporous filter membrane to obtain the PEG-PCL @ rif solution.

2) Preparation of TTBZ-PEG-PCL @ rif

As shown in the preparation scheme of FIG. 2, the targeting ligand TTBZ is at APS and Na₂SO₃The surface of the PEG-PCL @ rif is modified under catalysis, so that the nano micelle has targeting capability. The preparation process includes two steps, and mercapto propionic acid (C) is catalyzed by EDCI [ 1- (3-dimethylaminopropyl) -3-ethyl carbodiimide hydrochloride ] and DMAP (4-dimethylaminopyridine)₃H₆O₂S) and TTBZ [ C ]₂₈H₃₉NO₆S， 3-(2-hydroxy-3-isobutyl-10-methoxy-2,3,4,6,7,11b-hexahydro-1H-pyrido[2,1-a]isoquinolin-9-yloxy) propyl 4-methyllbenzenesufonate) to produce HS-TTBZ (shown in FIG. 3); then the sulfydryl of HS-TTBZ and the C ═ C group at the tail end of PEG-PCL in PEG-PCL @ rif are added in Ammonium Persulfate (APS) and Na₂SO₃Under catalysis ofClick chemistry occurred to generate TTBZ-PEG-PCL @ rif (shown in FIG. 4). The preparation method comprises the following steps:

respectively weighing 0.6mg of mercaptopropionic acid, 2.2mg of EDCI and 1.4mg of DMAP, uniformly stirring at a low temperature of 4 ℃ for 6 hours, adding 1mg of TTBZ, and continuously stirring for 12 hours to obtain a TTBZ-SH solution;

1mL of the resulting TTBZ-SH solution was mixed with 5mL of the above-mentioned PEG-PCL @ rif solution, and 1mg of APS and 1mg of Na were added₂SO₃Stirring at 4 ℃ for 36h, placing the obtained mixed solution into a dialysis bag with the molecular weight cut-off of 14,000, dialyzing with water overnight, and collecting the solution in the dialysis bag to obtain the TTBZ-PEG-PCL @ rif polymeric micelle, namely the rifampicin nano drug delivery system.

Example 2 characterization and physicochemical Properties of TTBZ-PEG-PCL @ rif (drug loaded micelle for short)

(1) Determination of TTBZ-PEG-PCL @ rif morphology characterization

And transferring 10 mu L of the drug-loaded micelle, slowly dripping the drug-loaded micelle into a 200-mesh copper mesh, naturally drying, dyeing the sample for 1 minute by using 0.3% phosphotungstic acid, and observing the appearance and the particle size of the sample by using a Transmission Electron Microscope (TEM) after dyeing.

As can be seen from a Transmission Electron Microscope (TEM) in FIG. 5, the particle size of the TTBZ-PEG-PCL @ rif nanoparticles is about 100nm, and the polymer micelles are in circular stable distribution. The stable particle size distribution provides a prerequisite for micelles to target the dopamine neuron vesicular monoamine transporter 2(VMAT2) by a passive targeting effect to increase the local concentration of rifampicin.

(2)1HNMR assay

Dissolving 400 mu L of drug-loaded micelle in 200 mu L of deuterated chloroform, placing the mixture for 10min at room temperature, centrifuging the mixture for 10min at 10000rpm, then placing 500 mu L of supernatant in a nuclear magnetic tube with the inner diameter of 5mm, calling a Noesy-presat-1D pulse sequence by using an Inova 600MHz resonance spectrometer to determine a hydrogen spectrum, and attributing each peak in the hydrogen spectrum to confirm the formation of a drug-loaded system.

FIG. 6 shows the 1HNMR atlas of TTBZ-PEG-PCL @ rif has characteristic peaks of TTBZ and PEG-PCL, respectively, and the successful connection of TTBZ and PEG-PCL is proved.

(3) Stability assay for TTBZ-PEG-PCL @ rif

Transferring 1mL of the drug-loaded micelle, and placing the micelle in 10mL of PBS and 10mL of artificial cerebrospinal fluid (aCSF for short, containing 124.0 mM NaCl and 26.0mM NaHCO)₃、2.5mM KCl、2.0mM CaCl₂、1.0mM MgCl₂、1.25mMNaH₂PO₄And 10.0mM d-glucose) were shaken at 100rpm on a constant temperature shaker at 37 ℃ and the size of the micelles was measured at different time points (0, 2h, 4h, 6h, 8h, 10h) using Malvern zetasizer nano-ZS.

As shown in FIG. 7, the particle sizes of TTBZ-PEG-PCL @ rif micelles in PBS and artificial cerebrospinal fluid (aCSF) are uniformly and stably shown by a dynamic light scattering technology at different time points, and the drug delivery system is proved to be stable in properties and uniform in characterization of the PBS and the artificial cerebrospinal fluid.

(4) Safety assay for TTBZ-PEG-PCL @ rif

SH-SY5Y cells (Wuhan Proxel Co.) were cultured in complete medium (90% DMEM + 10% FBS + 1% diabody) and seeded in 96-well plates with a cell density of 5000 cells/well and with a CO content of 5% at 37 ℃ in a 96-well plate₂Culturing in an incubator. After 12h, 100. mu.L of the culture medium containing TTBZ-PEG-PCL @ rif nano-micelles was added to the culture plate so that the final concentrations of the nano-micelles were 100, 200, 300, 400 and 500. mu.M, respectively. After co-culturing for 6, 12, 18 and 24 hours, the Cell activity was measured by using the CCK-8(Cell counting kit-8) method to verify the biological safety of the drug-loaded micelles.

The CCK-8 method determination result of FIG. 8 proves that TTBZ-PEG-PCL @ rif with different concentrations has small influence on apoptosis under different time lengths after acting on SH-SY5Y cells, and the material is proved to be safe and reliable.

(5) Targeting capability verification of TTBZ-PEG-PCL vector

TTBZ-PEG-PCL @ Nile Red (Nile red) nanomicelles were prepared according to the method of example 1.

SH-SY5Y cells are inoculated on a confocal dish, and the density of the inoculated cells is controlled to be 10⁵Placing the rice in a dish at 37 deg.C and 5% CO₂Culturing in incubator, discarding culture medium when cells grow in pairs, and adding into dish with pipette1mL of PBS buffer solution is slightly shaken for several times, and then waste liquid is discarded; adding 2mL of a culture medium containing 100 mu M of TTBZ-PEG-PCL @ Nile red polymer micelle into the dish, incubating for 24 hours, discarding the culture medium, washing with PBS (phosphate buffer solution) for 3 times, fixing polyformaldehyde for 5min, washing with PBS for 3 times, breaking the membrane with 0.3% Triton-100 for 10 minutes, washing with PBS for 3 times, and then washing with QuickBlock^TMSealing with immunostaining blocking solution (P0260, Byunyian) for 1 hr, discarding blocking solution, and adding anti-VMAT₂Primary antibody (1: 500, ab70808, Abcam) was left overnight at 4 ℃; the primary antibody is discarded in the next day, washed 3 times by PBS, then AlexaFlours 488 secondary antibody (1: 200, ab150077, Abcam) is added for incubation for 1 hour, after washing 3 times by PBS, DAPI staining is added for 5min, and finally the cells are observed and photographed by a confocal microscope (Zeiss LSM 710) to analyze the targeting ability of the polymer micelle to the cells and the distribution condition of the cells in the cells.

(6) Neuroprotective effect of TTBZ-PEG-PCL @ rif

First, a PD mouse model was constructed by stereotactic injection of the left striatum of alpha-synuclein (alpha-syn). The specific operation is as follows: first, a 1% pentobarbital sodium (50mg/kg) is injected into the abdominal cavity to anaesthetize the mouse, after local disinfection, the cranial vertex skin of the mouse is longitudinally incised and fixed on a mouse stereotaxic apparatus (Shenzhen Riwold Co.). Mouse bregma was determined, and left striatal coordinates (anterior +0.2mm, left +2mm, vertical depth +2.6mm) were determined by reference to the mouse anatomical map, followed by drilling with a 4-gauge needle. Then 8 mu g of alpha-syn is slowly injected into the cranium by the micro-needle, the needle inserting speed is kept at 0.27 mu L/min, and in order to prevent the injected alpha-syn from seeping, the needle is withdrawn after 5min of stopping after the injection. After the above steps are completed, the incision is sutured, sterilized with iodophor, placed on a heat preservation blanket and closely observed for vital signs until the mice are resuscitated.

To reduce the damage from repeated dosing, TTBZ-PEG-PCL @ rif (control with rifampin alone) was continuously pumped through the lateral ventricle by subcutaneous micropump (2002W, i.e., 200 μ L continuously pumped for 2 weeks, purchased from shenzhen ruiwade). The specific operation is as follows:

firstly, 1% pentobarbital sodium (50mg/kg) is injected into the abdominal cavity to anaesthetize the constructed PD model mouse, after local disinfection, the cranial vertex skin of the mouse is longitudinally cut and fixed on a mouse stereotaxic apparatus. Mouse bregma was determined, and left lateral ventricle position (posterior +0.2mm, left +1mm, vertical depth +3.0mm) was determined by reference to mouse anatomical atlas, followed by drilling with a 4-gauge needle. Then the micro pump with the liquid medicine is embedded into the shoulder subcutaneous part of the mouse and connected to the infusion cannula through the catheter, the cannula is implanted into the lateral ventricle along the drill hole of the skull, after the steps are completed, the incision is sutured, the mouse is placed on the heat preservation blanket after being disinfected by iodophor, and the vital signs are closely observed until the mouse is recovered.

After the injection of the liquid medicine is completed, a rat brain specimen is collected and a paraffin section is made. The specific operation is as follows: the mice were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (50mg/kg), fixed on a dissecting table, the heart was exposed by opening the chest, the right auricle was cut open, and rapidly washed with ice physiological saline through the left ventricle until the liver of the mice became white. Then, ice 4% paraformaldehyde-PBS (0.01M, pH 7.4) was continuously perfused for 2 hours, the rat brain was rapidly craniotomized and removed, and fixed in 4% paraformaldehyde for over 24 hours. The distal end of the substantia nigra was cut into a coronal shape, the latter half was embedded in paraffin, and 5 μm serial sections were obtained, and the staining results were observed.

Tyrosine Hydroxylase (TH) can reflect the number of dopamine neurons, and TH immunohistochemical staining can show that the TTBZ-PEG-PCL @ rif treatment group has significantly increased mesencephalic substantia nigra TH compared with a simple rifampicin treatment group, thereby proving that the nerve modification effect of TTBZ-PEG-PCL @ rif is more significant.

The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and still fall within the scope of the invention.

Claims

1. A preparation method of a rifampicin nano drug delivery system is characterized by comprising the following steps:

s1, loading rifampicin in a PEG-PCL carrier in a self-assembly mode to prepare PEG-PCL @ rifampicin;

s2, reacting mercaptopropionic acid with TTBZ under the catalysis of EDCI and DMAP to generate HS-TTBZ;

s3 thiol group through HS-TTBZ and C ═ C group at APS and Na with PEG-PCL tail end in PEG-PCL @ rifampicin of step S1₂SO₃The rifampicin nano drug delivery system can be prepared by click chemical reaction under catalysis.

2. The method of preparing a rifampicin nano-delivery system of claim 1, comprising the following steps:

3. The preparation method of a rifampicin nano drug delivery system according to claim 2, wherein the mass ratio of rifampicin to APEG-PCL lyophilized powder is 1: 8-12; the feed-liquid ratio of rifampicin to DMF was 1 mg: 1-3 mL.

4. The method of claim 2, wherein the mass ratio of mercaptopropionic acid, EDCI, and DMAP is 3: 11: 7.

5. the method of claim 2, wherein the volume ratio of TTBZ-SH solution to PEG-PCL @ rifampicin solution is 1: 4-6; TTBZ-SHSolution with APS, Na₂SO₃The feed-liquid ratio of (1 mL): 1 mg: 1 mg.

6. The method of claim 2, wherein the step S1 is performed by using a 0.45um microporous membrane.

7. The method of claim 2, wherein the step of adding the organic phase to the water in step S1 is performed by gradually adding the organic phase to the water under the action of ultrasound.

8. The method of claim 2, wherein the dialysis bag of steps S1 and S3 has a molecular weight cut-off of 14,000.

9. The rifampicin nano drug delivery system prepared by the method for preparing the rifampicin nano drug delivery system according to any one of claims 1 to 8.

10. Use of the rifampicin nano-drug delivery system of claim 9 for the preparation of neuroprotective drugs, wherein said neuroprotective drugs comprise drugs for the prevention and treatment of parkinson's disease.