CN114288419B - Preparation of a rifampicin nano drug delivery system and its application in the prevention and treatment of Parkinson's disease - Google Patents

Abstract

The invention belongs to the technical field of nano-drug delivery systems, and in particular relates to the preparation of a rifampin nano-drug delivery system and its application in the prevention and treatment of Parkinson's disease. In the present invention, rifampicin is loaded into the PEG-PCL nano-drug delivery system for the first time , preparing it into polymer micelles; and modifying the surface of the polymer micelles with TTBZ molecules, which can specifically bind VMAT ₂ . Compared with the traditional rifampin drug type, the carrier (PEG-PCL) of the present invention has weak cytotoxicity, and the micelles formed by the PEG-PCL affinity block have the structural characteristics of a hydrophobic inner core-hydrophilic outer core, increasing Improve the water solubility of rifampicin, improve the stability of its circulation in the body; have the ability of targeted drug delivery, improve the local concentration of rifampicin in dopamine neurons, thereby improving the bioavailability of the drug and improving its effect on Pa Protective effect of neurological diseases such as Kimson's disease, and reduce its peripheral toxic side effects.

Description

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

technical field

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

Background technique

Rifampicin (Rif) is a semi-synthetic broad-spectrum antibacterial drug of the rifamycin class, common name: rifampicin, English name: RIFAMPICINTABLETS. As a classic, effective and safe anti-tuberculosis drug, rifampicin has antibacterial activity against a variety of pathogenic microorganisms. Rifampicin is well absorbed after oral administration, and the plasma concentration reaches the peak value 1.5 to 4 hours after taking the drug. This product is used in combination with other anti-tuberculosis drugs for the initial treatment and retreatment of various tuberculosis, including the treatment of tuberculous meningitis. In addition, rifampicin also has potential neuroprotective effects. Studies have shown that in the in vitro model of neurotoxic drug-induced Parkinson's disease (Parkinson's disease, PD), rifampicin pretreatment can reverse the neuronal apoptosis caused by toxic factors such as rotenone and MPP+. Further studies have confirmed that rifampicin can up-regulate the expression of endoplasmic reticulum stress-related factor GRP78, regulate the level of apoptosis-related pathway PI3K/Akt/GSK-3β/CREB, and increase the SUMOylation of α-synuclein. Relieve the toxic damage of dopaminergic neurons; at the same time, reduce the level of neuroinflammation by regulating TLR-4 related pathways and inhibiting the activation of NLRP3 inflammasome; The ability of plasma cells to clear pathological proteins.

Some studies have also found that in living animal models, although intraperitoneal injection of rifampicin has a certain neuroprotective effect on PD mice at the molecular pathological level, it has no stable effect on the motor symptoms and behavioral changes after animal modeling. Improvement, and the administration time is long, and the toxic and side effects on model mice are relatively large. According to the analysis, there are still the following deficiencies in the use of rifampicin in the treatment of neurological diseases: (1) rifampicin has strong fat solubility, but its water solubility is low, and its stability in internal circulation is poor; The local concentration of substantia nigra-striatum is much lower than its working concentration; 3) The peripheral toxicity of rifampicin, such as liver toxicity, limits its use at higher concentrations.

Therefore, it is necessary to increase the water solubility of rifampicin, improve its stability in circulation in vivo, increase the targeting ability of drugs, improve its local concentration in intracranial dopamine neurons, and reduce peripheral toxic and side effects.

Contents of the invention

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

In order to achieve the above object, the technical solution adopted in the present invention is:

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

S1. Prepare PEG-PCL@rifampicin (referred to as PEG-PCL@rif) by loading rifampicin into PEG-PCL carrier by self-assembly;

S2. Put mercaptopropionic acid and TTBZ (C ₂₈ H ₃₉ NO ₆ S, whose structural formula is shown in Figure 3) in EDCI [1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride] and DMAP (4-dimethylaminopyridine) catalyzed reaction to generate HS-TTBZ;

S3. Through the sulfhydryl group of HS-TTBZ and the C=C group at the end of PEG-PCL in step S1 PEG-PCL@rifampicin, a click chemical reaction occurs under the catalysis of APS (ammonium persulfate) and Na ₂ SO ₃ , the rifampicin nano drug delivery system can be prepared.

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

S1. Dissolve rifampicin and APEG-PCL lyophilized powder in DMF to form an organic phase, add the organic phase to water, then dialyze with a dialysis bag for more than 12 hours, collect the solution in the dialysis bag, and obtain PEG-PCL after filtration. PCL@rifampicin solution;

S2. Stir and mix mercaptopropionic acid, EDCI and DMAP at low temperature, then add TTBZ and continue to stir for more than 12h to obtain a TTBZ-SH solution;

S3. Mix the TTBZ-SH solution in step S2 and the PEG-PCL@rifampicin solution in step S1, add APS and Na ₂ SO ₃ and stir at low temperature for more than 36 hours, then dialyze overnight with a dialysis bag, and collect the solution in the dialysis bag The rifampicin nano drug delivery system can be obtained.

The present invention uses PEG-PCL polymer micelles as the carrier of the drug delivery system, and loads hydrophobic rifampicin in the inner core to improve its water solubility and stability of circulation in the body, and can specifically bind to dopamine neurons. TTBZ of VMTA ₂ is used as a ligand for the drug delivery system to improve its targeting ability, thereby increasing the local working concentration of the drug, thereby improving its protective effect on neurological diseases such as Parkinson's disease, and reducing peripheral toxic side effects.

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

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

Preferably, the volume ratio of TTBZ-SH solution to PEG-PCL@rifampicin solution is 1:4-6; the solid-liquid ratio of TTBZ-SH solution to APS and Na ₂ SO ₃ is 1mL:1mg:1mg. Specifically, the volume ratio of TTBZ-SH solution to PEG-PCL@rifampicin solution was 1:5.

Preferably, the filtration in step S1 is performed with a 0.45um microporous membrane.

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

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

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

The present invention also provides the application of the above-mentioned rifampicin nano drug delivery system in the preparation of neuroprotective drugs, and the neuroprotective drugs include drugs for preventing and treating Parkinson's disease.

PD is a neurodegenerative disease due to the degeneration and necrosis of dopaminergic neurons in the substantia nigra of the midbrain, and insufficient secretion of dopaminergic neurotransmitters, which leads to extrapyramidal movement disorders such as bradykinesia and dystonia. The current clinical treatment of PD Mainly dopaminergic replacement therapy and DBS surgery. To date, disease-modifying therapeutics targeting the molecular mechanisms of PD pathogenesis are still lacking. The present invention loads common rifampicin, which has been proven to modify PD disease progression in vitro experiments, into PEG-PCL micelles, and modifies TTBZ molecules on the surface of the micelles to target dopamine neurons, thereby improving Its neuroprotective effect.

Of course, except for Parkinson's disease, other neurological diseases that can make the rifampicin nano drug delivery system of the present invention play a protective role are also within the protection scope of the present invention.

Compared with prior art, the beneficial effect of the present invention is:

In the present invention, for the first time, rifampicin is loaded into the PEG-PCL nano-delivery system, and it is prepared into polymer micelles; and TTBZ molecules are modified on the surface of the polymer micelles, which can specifically bind to monoamine in vesicles of dopamine neurons Transporter 2 (Vesicular Monoamine Transporter 2, VMAT ₂ ). Compared with traditional rifampicin medicament types, the rifampicin nano drug delivery system of the present invention has the following advantages: (1) The carrier (PEG-PCL) has weak cytotoxicity, and the safety of PEG has been approved by the U.S. Food and Drug Administration Bureau (FDA) certification; (2) The micelles formed by the PEG-PCL hydrophilic block have the structural characteristics of a hydrophobic inner core and a hydrophilic outer core, which increases the water solubility of rifampicin and improves the stability of its circulation in the body (3) It has the ability of targeted drug delivery, which can increase the local concentration of rifampicin in dopamine neurons, thereby improving the bioavailability of the drug, improving its protective effect on neurological diseases such as Parkinson's disease, and reducing its peripheral neuropathy. toxic side effects.

Description of drawings

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

Figure 2 is a schematic diagram of TTBZ modified PEG-PCL@rif;

Figure 3 is a chemical synthesis route diagram of TTBZ sulfhydryl modification;

Figure 4 is a synthesis diagram of TTBZ modified PEG-PCL@rif;

Figure 5 is a transmission electron microscope image of TTBZ-PEG-PCL@rif;

Figure 6 is the ¹ H NMR measurement results of TTBZ-PEG-PCL@rif;

Fig. 7 is the stability determination result of TTBZ-PEG-PCL@rif;

Fig. 8 is the safety measurement result of TTBZ-PEG-PCL@rif;

Fig. 9 is the targeting ability test result of carrier TTBZ-PEG-PCL;

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

Detailed ways

Specific embodiments of the present invention will be further described below. It should be noted here that the descriptions of these embodiments are used to help understand the present invention, but are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not constitute a conflict with each other.

The experimental methods in the following examples, if no special instructions, are conventional methods, and the test materials used in the following examples, if no special instructions, all can be purchased through conventional commercial channels.

Example 1 Preparation of rifampicin nano drug delivery system (TTBZ-PEG-PCL@rif polymer micelles)

(1) Preparation of PEG-PCL@rifampicin

As shown in Figure 1, rifampicin is loaded into the PEG-PCL carrier by self-assembly (Self-assembly) to prepare a PEG-PCL@rif (PEG-PCL@rifampicin) solution. The specific preparation method is as follows :

Weighed 1mg of rifampicin (rifampicin, rif) and 10mg of Allyl-PEG-PCL (PEG-PCL) lyophilized powder (purchased from Shunna Nano Co., Ltd.) and dissolved them in 1mL of DMF to form an organic phase, and subjected them to ultrasonication (130W ) was added dropwise into 10 mL of deionized water. Put the above-mentioned mixed solution in a dialysis bag with a molecular weight cut-off of 14,000, dialyze with water for more than 12 hours, collect the solution in the dialysis bag, and use a 0.45um microporous membrane to remove hydrophobic drug aggregates to obtain PEG- PCL@rif solution.

2) Preparation of TTBZ-PEG-PCL@rif

As shown in the preparation schematic diagram in Figure 2, the targeting ligand TTBZ was modified on the surface of PEG-PCL@rif under the catalysis of APS and Na ₂ SO ₃ , so that the nanomicelle had targeting ability. The preparation process is divided into two steps. First, under the catalysis of EDCI [1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride] and DMAP (4-dimethylaminopyridine), mercaptopropyl acid (C ₃ H ₆ O ₂ S) and TTBZ [C ₂₈ H ₃₉ NO ₆ S, 3-(2-hydroxy-3-isobutyl-10-methoxy-2,3,4,6,7,11b-hexahydro- 1H-pyrido[2,1-a]isoquinolin- 9-yloxy)propyl 4-methylbenzenesulfonate)] reacts to generate HS-TTBZ (as shown in Figure 3); then the sulfhydryl group of HS-TTBZ and PEG- The C=C group at the end of PCL undergoes a click chemical reaction under the catalysis of ammonium persulfate (APS) and Na ₂ SO ₃ to generate TTBZ-PEG-PCL@rif (as shown in Figure 4). The specific preparation method is as follows:

Weigh 0.6 mg of mercaptopropionic acid, 2.2 mg of EDCI and 1.4 mg of DMAP respectively, stir and mix at 4°C for 6 h at low temperature, then add 1 mg of TTBZ, and continue stirring for 12 h to obtain a TTBZ-SH solution;

Mix 1 mL of the obtained TTBZ-SH solution with 5 mL of the above-mentioned PEG-PCL@rif solution, add 1 mg APS and 1 mg Na ₂ SO ₃ , stir at 4°C for 36 h, and place the resulting mixed solution in a In the dialysis bag, dialyze with water overnight, collect the solution in the dialysis bag to obtain TTBZ-PEG-PCL@rif polymer micelles, that is, the rifampicin nano drug delivery system.

Example 2 TTBZ-PEG-PCL@rif (referred to as drug-loaded micelles) characterization and physical and chemical properties

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

Pipette 10 μL of drug-loaded micelles, and slowly drop them into a 200-mesh copper grid, dry naturally, and then stain the sample with 0.3% phosphotungstic acid for 1 minute. After the staining is completed, use a transmission electron microscope (TEM) to observe its appearance , particle size.

It can be seen from the transmission electron microscope (TEM) in Figure 5 that the particle size of TTBZ-PEG-PCL@rif nanoparticles is about 100nm, and the polymer micelles are in a circular stable distribution. The stable particle size distribution provided the prerequisite for the micelles to target dopamine neuronal vesicular monoamine transporter 2 (VMAT2) through a passive targeting effect to increase the local concentration of rifampicin.

(2) 1HNMR determination

Dissolve 400 μL drug-loaded micelles in 200 μL deuterated chloroform, place at room temperature for 10 minutes, centrifuge at 10,000 rpm for 10 minutes, then take 500 μL supernatant and put it in a nuclear magnetic magnetic tube with an inner diameter of 5 mm, and use the Inova 600MHz resonance spectrometer to call Noesy-presat-1D pulse The sequence determines the hydrogen spectrum, and the formation of the drug-loaded system is confirmed by assigning each peak in the hydrogen spectrum.

The 1H NMR spectrum in Figure 6 shows that TTBZ-PEG-PCL@rif has the characteristic peaks of TTBZ and PEG-PCL respectively, which proves that TTBZ and PEG-PCL have been successfully connected.

(3) Stability determination of TTBZ-PEG-PCL@rif

Pipette 1 mL of drug-loaded micelles, respectively, into 10 mL of PBS and 10 mL of artificial cerebrospinal fluid (abbreviated as aCSF, containing 124.0 mM NaCl, 26.0 mM NaHCO ₃ , 2.5 mM KCl, 2.0 mM CaCl ₂ , 1.0 mM MgCl ₂ , 1.25 mM NaH ₂ PO ₄ and 10.0mM d-glucose), shake at a speed of 100rpm on a constant temperature shaker at 37°C, and measure the particle size of micelles at different time points (0, 2h, 4h, 6h, 8h, 10h) using Malvern ZetasizerNano-ZS .

As shown in Figure 7, the particle size of TTBZ-PEG-PCL@rif micelles in PBS and artificial cerebrospinal fluid (aCSF) was uniform and stable at different time points by dynamic light scattering technology, which proved that the drug-loading system was stable in It is stable in PBS and artificial cerebrospinal fluid, and its characterization is uniform.

(4) Safety determination of TTBZ-PEG-PCL@rif

SH-SY5Y cells (Wuhan Proser Company) were cultured in complete medium (90% DMEM + 10% FBS + 1% double antibody), and seeded in 96-well plate, the cell density was controlled at 5000/well, placed Cultured in a 37°C, 5% _CO2 incubator. After 12 hours, 100 μL of medium containing TTBZ-PEG-PCL@rif nanomicelles was added to the culture plate to make the final concentrations of nanomicelles 100, 200, 300, 400, and 500 μM, respectively. After co-cultivation for 6, 12, 18, and 24 hours respectively, the cell viability was measured by the CCK-8 (CellCountingKit-8) method to verify the biological safety of the drug-loaded micelles.

The CCK-8 assay results in Figure 8 prove that different concentrations of TTBZ-PEG-PCL@rif have little effect on cell apoptosis at different time lengths after acting on SH-SY5Y cells, which proves that the material is safe and reliable.

(5) Verification of targeting ability of TTBZ-PEG-PCL carrier

TTBZ-PEG-PCL@Nile red (Nile red) nanomicelles were prepared according to the method in Example 1.

Use confocal dishes to inoculate SH-SY5Y cells, control the density of inoculated cells at 10 ⁵ cells/dish, and place them in a 37°C, 5% CO ₂ incubator for culture. When the cells grow in pairs, discard the medium , gently add about 1mL of PBS buffer solution to the dish with a pipette gun, discard the waste liquid after shaking gently several times; then add 2mL of 100μM TTBZ-PEG-PCL@Nile Red Polymer Medium for micelles, discard the medium after incubation for 24 hours, wash with PBS solution 3 times, fix with paraformaldehyde for 5 minutes, wash with PBS solution for 3 times, then use 0.3% Triton-100 for 10 minutes, wash with PBS solution for 3 times Then, block with QuickBlock ^TM Immunostaining Blocking Solution (P0260, Biyuntian) for 1 hour, discard the blocking solution, add anti-VMAT ₂ primary antibody (1:500, ab70808, Abcam) and place overnight at 4°C; discard the next day Remove the primary antibody, wash with PBS solution for 3 times, then add AlexaFlour 488 secondary antibody (1:200, ab150077, Abcam) to incubate for 1 hour, wash with PBS solution for 3 times, add DAPI staining for 5min, and finally pass the confocal microscope (Zeiss LSM 710 ) observe and take pictures of the cells, and analyze the targeting ability and intracellular distribution of the polymer micelles to the cells.

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

First, a PD mouse model was constructed by stereotaxic injection of α-synuclein (α-syn) in the left striatum. The specific operation is as follows: firstly intraperitoneally inject 1% pentobarbital sodium (50mg/kg) to anesthetize the mouse, after local disinfection, cut the skin of the skull top of the mouse longitudinally, and fix it in a mouse stereotaxic instrument (Shenzhen Ruiwode Co., Ltd. )superior. Determine the bregna of the mouse, refer to the anatomical atlas of the mouse, determine the coordinates of the left striatum (front+0.2mm, left+2mm, vertical depth+2.6mm), and then drill holes with a No. 4 needle. Then, 8 μg α-syn was slowly injected into the cranium through the microneedle, keeping the needle insertion speed at 0.27 μL/min. In order to prevent the injected α-syn from leaking out, the needle was withdrawn after waiting for 5 minutes after the injection. After the above steps were completed, the incision was sutured, disinfected with povidone iodine, placed on a thermal blanket, and the vital signs were closely observed until the mice recovered.

In order to reduce the damage caused by repeated administration, a subcutaneous micropump (2002W, that is, 200 μL was continuously pumped for 2 weeks, purchased from Shenzhen Ruiwode Company) was implanted into the lateral ventricle to continuously pump TTBZ-PEG-PCL@rif (using simple rifampicin as the control). The specific operation is:

First, the PD model mice constructed above were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg). After local disinfection, the skin of the top of the skull of the mice was cut longitudinally and fixed on a mouse stereotaxic instrument. Determine the anterior fontanel (bregna) of the mouse, and determine the position of the left lateral ventricle (posterior +0.2mm, left +1mm, vertical depth +3.0mm) by referring to the mouse anatomical atlas, and then drill holes with a No. 4 needle. Then, the micropump containing the liquid medicine was buried subcutaneously in the shoulder of the mouse, connected to the infusion cannula through the catheter, and the cannula was implanted into the lateral ventricle along the drilled hole in the skull. After the above steps were completed, the incision was sutured, and iodine was applied. After volt disinfection, place it on a thermal blanket and closely observe the vital signs until the mice recover.

After the liquid injection was completed, the rat brain samples were collected and paraffin sections were made. The specific operation is: intraperitoneal injection of 1% pentobarbital sodium (50mg/kg) to anesthetize the mouse, fix the anesthetized mouse on the dissection table, open the chest to expose the heart, cut the right atrial appendage, and inject ice saline through the left ventricle Rinse quickly until the mouse liver turns pale. Then, ice-cold 4% paraformaldehyde-PBS (0.01M, pH 7.4) was continuously perfused for 2 hours, and the rat brain was quickly removed by craniotomy and fixed in 4% paraformaldehyde for more than 24 hours. Cut off coronally from the front end of the substantia nigra, take the second half of it for paraffin embedding, 5μm serial sections, and finally observe the staining results.

Tyrosine hydroxylase (TH) can reflect the number of dopamine neurons, which can be found by TH immunohistochemical staining (Figure 10). Compared with the simple rifampicin treatment group, the TTBZ-PEG-PCL@rif treatment group TH in the substantia nigra of the midbrain increased significantly, proving that the neural modification effect of TTBZ-PEG-PCL@rif is more significant.

The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions and modifications to these embodiments still fall within the protection scope of the present invention.

Claims (10)

1. A method for preparing a rifampicin nano drug delivery system, which is characterized by comprising the following steps:

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

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

s3, through the sulfhydryl group of HS-TTBZ and the C=C group at the tail end of the PEG-PCL in the PEG-PCL@rifampicin of the step S1, in APS and Na ₂ SO ₃ Carrying out click chemical reaction under the catalysis of (1) to obtain the rifampicin nano drug delivery system;

the TTBZ and PEG-PCL have the following structures:

2. the method for preparing the rifampicin nano-delivery system according to claim 1, comprising the steps of:

s1, dissolving rifampicin and PEG-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 a solution in the dialysis bag, and filtering to obtain a PEG-PCL@rifampicin solution;

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

s3, mixing the TTBZ-SH solution in the step S2 and the PEG-PCL@rifampicin solution in the step S1, and adding APS and Na ₂ SO ₃ Stirring at low temperature for more than 36h, dialyzing overnight with a dialysis bag, and collecting the solution in the dialysis bag to obtain the rifampicin nano drug delivery system.

3. The method for preparing the rifampicin nano drug delivery system according to claim 2, wherein the mass ratio of the rifampicin and the APEG-PCL freeze-dried powder is 1:8-12; the feed ratio of rifampicin to DMF was 1mg:1-3mL.

4. The method for preparing the rifampicin nano-delivery system according to claim 2, wherein the mass ratio of mercaptopropionic acid, EDCI and DMAP is 3:11:7.

5. the method for preparing the rifampicin nano-delivery system according to claim 2, wherein the volume ratio of TTBZ-SH solution to PEG-pcl@rifampicin solution is 1:4-6; TTBZ-SH solution, APS and Na ₂ SO ₃ The feed liquid ratio of (2) is 1mL:1mg:1mg.

6. The method of claim 2, wherein the filtration in step S1 is performed with a microporous membrane of 0.45 um.

7. The method for preparing the rifampicin nano-delivery system according to claim 2, wherein in step S1, the organic phase is added dropwise to water under the action of ultrasound.

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

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

10. The use of the rifampicin nano-delivery system of claim 9 in the manufacture of a neuroprotective medicament, wherein the neuroprotective medicament is a medicament for preventing and treating parkinson's disease.