Diagnosis and treatment integrated rifampicin nanoparticles for Parkinson's disease as well as preparation method and intranasal administration application of rifampicin nanoparticles

Abstract

The invention provides diagnosis and treatment integrated rifampicin nano-particles for Parkinson's disease as well as a preparation method and intranasal administration application of the rifampicin nano-particles. The nano-particle comprises a TTBZ targeting molecule modified polycaprolactone-polyethylene glycol copolymer carrier, a ferroferric oxide nano-particle and rifampicin, wherein the rifampicin and the ferroferric oxide are encapsulated in the polycaprolactone-polyethylene glycol copolymer carrier. The nano-particles provided by the invention can realize more effective targeted enrichment in a Parkinson's disease related brain region. Meanwhile, the nano-particles are suitable for a nasal administration route, can bypass a blood brain barrier through a nasal-brain pathway and realize delivery of drugs to a central nervous system, so that trauma and infection risks caused by an invasive administration mode are avoided, patient compliance is improved, and the nano-particles are more suitable for long-term treatment of chronic neurodegenerative diseases such as Parkinson's disease and the like.

Description

Technical Field

[0001] This invention relates to the field of drug-loaded nanoparticles, and particularly to a rifampicin nanoparticle for the diagnosis and treatment of Parkinson's disease, its preparation method, and its intranasal administration application. Background Technology

[0002] Parkinson's disease (PD) is a common neurodegenerative disease of the central nervous system, characterized by progressive damage and loss of dopaminergic neurons in the substantia nigra. Current drug treatments for PD still face many challenges, the most prominent being the difficulty in effectively crossing the blood-brain barrier, low brain targeting efficiency, and the lack of technologies for real-time monitoring of drug distribution and efficacy. Therefore, improving drug accumulation efficiency in the brain lesion area and achieving visualized monitoring of the treatment process has become an important research direction in the field of PD treatment. Rifampin, a small molecule drug with neuroprotective potential, has been shown in in vitro studies to inhibit α-synuclein aggregation and exert neuroprotective effects. However, in practical applications, rifampin still faces challenges such as poor water solubility, insufficient in vivo stability, difficulty in achieving effective therapeutic concentrations in the brain, and potential hepatotoxicity. These factors severely limit its further application in the treatment of Parkinson's disease. Therefore, developing a drug delivery system that can improve the efficiency of rifampin delivery to the brain is of great significance.

[0003] In recent years, nanomedicine delivery technology has provided a feasible approach to solving the aforementioned problems. By constructing nanoscale drug carriers, the solubility and stability of drugs can be improved, and their in vivo distribution can be enhanced to some extent. Currently reported rifampicin nanomedicine delivery systems mainly include polymer micelles, liposomes, and polylactic-co-glycolic acid copolymers as nanocarriers. However, existing technologies still generally suffer from the following shortcomings.

[0004] First, the construction of some targeted nanomedicines relies on complex multi-step organic synthesis reactions. Such techniques typically require multiple chemical coupling steps to link the target molecule to a polymer support, resulting in long reaction routes, low overall yields, and stringent reaction conditions. This leads to complex experimental procedures, poor reproducibility, and difficulty in large-scale preparation, thus limiting their practical application value.

[0005] Secondly, most existing nanomedicine delivery systems only have a single drug delivery function and lack imaging or tracking capabilities. During treatment, doctors find it difficult to observe the distribution, accumulation, and clearance of drugs in the body, especially in brain lesions, in real time, thus making it impossible to adjust the treatment plan in a timely manner and achieve precise and personalized treatment.

[0006] In addition, to increase the local concentration of drugs in brain tissue, some studies have employed invasive administration methods such as lateral ventricle injection or intrathecal injection. While these methods can increase the level of drugs in the brain to some extent, they are highly invasive, carry a high risk of infection, and have poor patient compliance, making them unsuitable for chronic diseases such as Parkinson's disease that require long-term treatment.

[0007] Therefore, developing a nanomedicine delivery system that is easy to prepare, can achieve efficient and active brain-targeted delivery, and has both diagnostic and therapeutic functions is of great significance for improving the treatment effect of Parkinson's disease and promoting the clinical translation of related technologies. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention proposes a rifampicin nanoparticle for the diagnosis and treatment of Parkinson's disease, its preparation method, and its intranasal administration application.

[0009] This invention provides a therapeutic rifampicin nanoparticle, comprising: TTBZ-targeted molecularly modified polycaprolactone-polyethylene glycol copolymer carrier, iron oxide nanoparticles, and rifampin; Rifampicin and ferric oxide are encapsulated within the polycaprolactone-polyethylene glycol copolymer carrier.

[0010] In this invention, polycaprolactone (PCL) serves as the hydrophobic segment, which encapsulates hydrophobic rifampicin and iron oxide nanoparticles through hydrophobic interactions, forming a stable nanoparticle core and ensuring the structural integrity of the drug delivery system. Polyethylene glycol (PEG) serves as the hydrophilic segment, forming a hydrophilic shell for the nanoparticles. This effectively reduces phagocytosis by the reticuloendothelial system in vivo, prolongs the in vivo circulation time of the nanoparticles, and simultaneously improves the water solubility and biocompatibility of the nanoparticles, solving the problems of poor water solubility and insufficient in vivo stability of rifampicin. TTBZ can specifically bind to vesicle monoamine transporter 2 (VMAT2) on the synaptic vesicle membrane of dopaminergic neurons. VMAT2 is highly expressed in brain regions related to Parkinson's disease lesions. By modifying the PEG terminus with TTBZ, the nanoparticles acquire active targeting capabilities, enabling them to recognize and bind to dopaminergic neurons in the diseased brain regions, achieving precise enrichment of drugs to brain lesions and improving brain targeting efficiency.

[0011] In some embodiments, the average particle size of the nanoparticles is 50 nm to 200 nm. Nanoparticles within this size range can balance in vivo circulation stability and brain delivery efficiency. A particle size of less than 200 nm can avoid being intercepted by pulmonary capillaries and can pass through the mucosal epithelial gaps of the nasobrain pathway to achieve brain transport after nasal administration. A particle size greater than 50 nm can reduce rapid in vivo leakage of nanoparticles and ensure the structural stability of the drug delivery system before delivery to the lesion.

[0012] The present invention also provides a method for preparing the rifampicin nanoparticles, comprising the following steps: S1. Dissolve TTBZ in a mixed solvent of methanol and tetrahydrofuran, add magnesium powder and ammonium chloride to react and obtain TTBZ-diol; Magnesium powder and ammonium chloride form a mild reducing system that can specifically remove the p-toluenesulfonate protecting group at the 9-position of the TTBZ molecule, yielding TTBZ-diol containing a secondary alcohol at the 2-position and a phenolic hydroxyl group at the 9-position. This provides an active site for subsequent carboxylation modification, and the mild reaction conditions prevent the destruction of the TTBZ molecular structure.

[0013] S2. Add succinic anhydride and 4-dimethylaminopyridine (DMAP) to the TTBZ-diol described in S1, dissolve in dichloromethane and react. After extraction with dichloromethane in an acidic buffer, TTBZ-COOH is obtained and dissolved in anhydrous N,N-dimethylformamide. 4-Dimethylaminopyridine, as a catalyst, can promote the selective esterification reaction between succinic anhydride and the secondary alcohol at the 2-position of TTBZ-diol, introducing a free carboxyl group (-COOH) to convert TTBZ into an active form (TTBZ-COOH) that can undergo a coupling reaction with PCL-PEG-COOH. Acidic buffer extraction can remove reaction byproducts and ensure the purity of TTBZ-COOH.

[0014] S3: Dissolve polycaprolactone-polyethylene glycol-carboxyl copolymer PCL5000-PEG2000-COOH in anhydrous N,N-dimethylformamide, add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 4-dimethylaminopyridine to obtain solution 1, add TTBZ-COOH described in S2 dropwise to solution 1, react with the reaction solution, precipitate with ether, filter and dry to obtain PCL-PEG-TTBZ; EDC, acting as a condensing agent, can activate the carboxyl groups at the ends of PCL-PEG-COOH, causing them to undergo a condensation reaction with the carboxyl groups of TTBZ-COOH to form mixed anhydride bonds, thus achieving covalent coupling between TTBZ and the PCL-PEG copolymer. DMAP, acting as a catalyst, can improve the efficiency and selectivity of the coupling reaction and reduce the occurrence of side reactions. Ether precipitation can precipitate water-soluble PCL-PEG-TTBZ from the reaction system, achieving the purification of the targeted polymer.

[0015] S4: PCL-PEG-TTBZ, rifampicin, and iron oxide nanoparticles were dissolved in chloroform and an emulsion was formed under ice-water bath and ultrasonic conditions. The rifampicin nanoparticles were obtained by centrifugation and washing.

[0016] The emulsification-solvent evaporation method was employed, using chloroform as the organic solvent to dissolve PCL-PEG-TTBZ, rifampin, and iron oxide nanoparticles. Ice-water bath and ultrasonic conditions enabled the organic phase to form uniformly sized droplets in the aqueous phase, and the ice-water bath reduced the evaporation rate of the organic solvent, preventing droplet aggregation. Subsequent magnetic stirring caused the chloroform to evaporate slowly, and the droplets gradually solidified to form nanoparticles. Centrifugation and washing removed unencapsulated drugs and impurities, resulting in high-purity therapeutic nanoparticles.

[0017] In some embodiments, in step S1, the mass ratio of TTBZ, magnesium powder, and ammonium chloride is 1:(0.4~1.2):(0.2~0.8); preferably 1:0.6:0.3. This ensures that the deprotection reaction of TTBZ proceeds fully, the excess magnesium powder as a reducing agent prevents incomplete reaction, and the ammonium chloride can adjust the acidity and alkalinity of the reaction system, optimizing the reduction reaction conditions. Under the preferred ratio, the yield and purity of the deprotection reaction are optimal.

[0018] In some embodiments, in step S2, the amount of succinic anhydride used is 1.5 to 3.0 equivalents of the molar amount of TTBZ; preferably 2.0 equivalents. This ensures that the carboxylation reaction of TTBZ-diol proceeds fully, avoids unreacted TTBZ-diol residue, and the preferred equivalent amount can reduce the waste of excess succinic anhydride while ensuring complete reaction, thus reducing the difficulty of subsequent purification.

[0019] In some embodiments, in step S3, the amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide used is PCL. 5000 -PEG 2000 The molar amount of the -COOH terminal carboxyl group is 1.5 to 3.0 equivalents; The amount of 4-dimethylaminopyridine used is 0.05~0.2 equivalents. Excess EDC can fully activate the terminal carboxyl group of PCL-PEG-COOH and improve the coupling efficiency with TTBZ-COOH; DMAP, as a catalyst, can exert a high catalytic effect at low equivalents, while excess can easily lead to side reactions. This ratio range can achieve high yield and high selectivity of the coupling reaction.

[0020] In some embodiments, in step S4, the mass percentage of PCL-PEG-TTBZ is 85-95 wt%; the mass percentage of rifampicin is 3-9 wt%; and the mass percentage of iron oxide nanoparticles is 2-6 wt%. This mass percentage ensures the structural stability of the nanoparticles and the synergistic effect of their diagnostic and therapeutic functions. PCL-PEG-TTBZ, as the carrier, has the highest proportion, providing a stable core-shell structure for the nanoparticles. The proportion of rifampicin ensures that the nanoparticles have an effective therapeutic concentration, while avoiding insufficient carrier encapsulation capacity and nanoparticle aggregation due to drug overdose. The proportion of iron oxide nanoparticles ensures the signal intensity of MRI imaging, meeting the requirements for real-time tracking, and does not affect the carrier's encapsulation and targeted delivery of rifampicin.

[0021] In some embodiments, in step S3, the TTBZ-COOH is added dropwise to solution 1 at a rate of 0.1~0.5 mL / min; slow dropwise addition allows TTBZ-COOH to fully contact the activated PCL-PEG-COOH, avoiding polymer agglomeration or side reactions caused by excessively high local concentrations, ensuring the uniformity of the coupling reaction. Within this rate range, efficient coupling can be achieved within a reasonable reaction time, improving the quality of the targeted polymer.

[0022] The present invention also provides the use of the rifampicin nanoparticles in the preparation of medicaments for the diagnosis and / or treatment of Parkinson's disease.

[0023] The nanoparticles of this invention can achieve precise enrichment of rifampin in the brain regions affected by Parkinson's disease through active targeting, thereby increasing the effective concentration of the drug in the brain and exerting therapeutic effects such as inhibiting α-synuclein aggregation and neuroprotection. At the same time, the iron oxide nanoparticles can achieve real-time MRI imaging tracking, dynamically monitoring the distribution and enrichment of the drug in the brain, providing a basis for clinical diagnosis and treatment plan adjustment, and realizing the integration of diagnosis and treatment.

[0024] In some embodiments, the drug is administered via the nasal cavity. The nasal mucosa is rich in capillaries and the nasobrain pathway. Nasal administration allows nanoparticles to bypass the blood-brain barrier and be directly transported to the central nervous system via the nasobrain pathway, significantly improving the efficiency of drug delivery to the brain. At the same time, nasal administration is a non-invasive method of drug delivery, which is simple to operate, minimally invasive, has a low risk of infection, and has high patient compliance. It is more suitable for chronic neurodegenerative diseases such as Parkinson's disease that require long-term treatment, and solves the clinical application defects of traditional invasive drug delivery methods.

[0025] In summary, compared with the prior art, the present invention achieves the following technical effects: (1) The innovative “three-step” synthesis route of this invention involves simple structural modification of TTBZ molecules followed by coupling with PCL-PEG copolymer to construct a TTBZ-targeted polymer carrier. Compared with the preparation route of existing technologies that relies on multi-step organic coupling and complex click chemical reactions, the synthesis route of this invention has fewer steps, milder reaction conditions, and simpler and more stable operation. This route is highly efficient and reproducible, paving the way for industrial-scale production.

[0026] (2) The nanoparticles constructed in this invention can not only deliver drugs, but also perform imaging tracing, thereby achieving the integration of "treatment-imaging-monitoring", which helps to dynamically monitor the accumulation and clearance process of drugs at the lesion site and provides a basis for precision treatment.

[0027] (3) The nanoparticles of the present invention can achieve more effective targeted enrichment in brain regions related to Parkinson's disease. At the same time, the nanoparticles of the present invention are suitable for nasal administration, which can bypass the blood-brain barrier through the nasobrain pathway to deliver drugs to the central nervous system, thereby avoiding the trauma and infection risks of invasive administration, improving patient compliance, and being more suitable for the long-term treatment of chronic neurodegenerative diseases such as Parkinson's disease.

[0028] (4) After nasal administration of the nanoparticles of the present invention, the latency period in mice during the rotarod test was significantly prolonged, and the performance in the pole climbing test and the wire mesh suspension test was significantly improved, indicating that their motor coordination, balance and muscle strength were effectively protected. It can not only improve motor function, but also effectively alleviate cognitive impairment and emotional abnormalities related to Parkinson's disease. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 The images shown are transmission electron microscope images of Embodiment 1 of the present invention; A shows the overall distribution of nanoparticles at a scale of 500 nm, B shows the morphological details of nanoparticles at a scale of 200 nm, and C shows the microstructure of a single nanoparticle at a scale of 100 nm.

[0031] Figure 2Examples of the present invention, Example 2, show brain imaging of PEG-PCL@rif@TTBZ nanoparticles based on MRI; A is a conventional T2-weighted MRI image of the brain of control mice; B is a T2-weighted gradient echo MRI image of the brain of control mice; C is a conventional T2-weighted MRI image of the brain of mice 2 hours after intranasal instillation of the nanomedicine; D is a T2-weighted gradient echo MRI image of the brain of mice 2 hours after intranasal instillation of the nanomedicine; E is a semi-quantitative analysis of the average pixel value of the right striatum in conventional T2-weighted images in both groups; F is a semi-quantitative analysis of the average pixel value of the left striatum in conventional T2-weighted images in both groups; G is a semi-quantitative analysis of the average pixel value of the left striatum in conventional T2-weighted images in both groups. Semi-quantitative analysis of the average pixel value of the right striatum in weighted gradient echo sequence images; H represents the T2 value in the two groups. Semi-quantitative analysis of the average pixel value of the right striatum in a weighted gradient echo sequence image.

[0032] Figure 3 Example 2 of this invention shows the cellular uptake of nanoparticles by SH-SY5Y cells using Prussian blue staining; A is Prussian blue staining of the untreated control group under 20x magnification; B is Prussian blue staining of the untreated control group under 40x magnification; C is Prussian blue staining of the nanoparticle-treated group under 20x magnification; D is Prussian blue staining of the nanoparticle-treated group under 40x magnification.

[0033] Figure 4 The images show the expression changes of HMC3 cells after treatment with nanoparticles in Example 2 of this invention; B is the low-power immunofluorescence image of VMAT2 in the blank control group cells; C is the low-power immunofluorescence image of VMAT2 in the nanoparticle-treated group cells; and D is the high-power immunofluorescence image of VMAT2 in the nanoparticle-treated group cells.

[0034] Figure 5 The figures show the behavioral evaluation results of Parkinson's disease model mice after treatment with nanoparticles in Example 3 of this invention; A is the quantitative statistics of latency in the rotarod test; B is the quantitative statistics of indicators in the pole climbing test; C is the quantitative statistics of indicators in the wire mesh suspension test; D / E is the quantitative statistics of stride length of the mouse's two hind limbs; F~I is the quantitative statistics of time the mouse's four limbs touched the ground; J is the statistics of time spent in the central region; K is the quantitative statistics of spontaneous alternation rate; L is the quantitative statistics of time spent at rest in forced swimming; M is the quantitative statistics of time spent at rest in the tail suspension test.

[0035] Figure 6 The images show immunofluorescence staining of TH in the substantia nigra of mice in each group of the present invention in Example 3; A is the sham-operated group, B is the PD model group, C is the intranasal nanotherapy group, and D is the ordinary rifampicin administration group.

[0036] Figure 7These are immunofluorescence staining images of pS129-α-syn in the brains of mice in each group in Example 3 of this invention.

[0037] Figure 8 This is a diagram showing the expression and quantitative analysis of key proteins in the panapoptotic pathway in mice, as described in Example 3 of this invention.

[0038] Figure 9 The images show Prussian blue staining of various brain layers in mice after nanoparticle uptake in Example 3 of this invention; A is a Prussian blue staining image of the olfactory bulb layer; B is a Prussian blue staining image of the striatum layer; C is a Prussian blue staining image of the classic hippocampus layer; D is a Prussian blue staining image of the hypothalamus layer; E is a Prussian blue staining image of the largest surface of the substantia nigra pars compacta layer; and F is a Prussian blue staining image of the cerebellar cortex layer.

[0039] Figure 10 Figure 3 shows the safety evaluation results of mice after treatment with nanoparticles in Example 3 of this invention; A is a bar chart of quantitative statistical analysis of serum ALT, AST and TBIL-Z levels in each group of mice; B~E are H&E stained light micrographs. Detailed Implementation

[0040] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0041] This invention innovatively designs a simple, efficient, and repeatable preparation process, which mainly consists of the following two stages: Phase 1: Three-Step Chemical Synthesis of Targeted Polymers This stage aims to firmly and specifically attach TTBZ to the ends of the commercially available PCL-PEG-COOH polymer, and the key lies in the ingenious "activation-linkage" design of the TTBZ molecule: Deprotection: The TTBZ raw material (C 28 H 39 NO6S) was deprotected under mild conditions (Mg / NH4Cl, 40°C) by removing the p-toluenesulfonate protecting group at the 9-position to obtain TTBZ-diol containing a secondary alcohol at the 2-position and a phenolic hydroxyl group at the 9-position.

[0042] Carboxylation: By selectively reacting succinic anhydride with the secondary alcohol at the 2-position of TTBZ-diol, a free carboxyl group is introduced as a "linking handle," yielding TTBZ-COOH. This step is crucial, as it converts TTBZ into an active form capable of highly efficient coupling with the polymer terminus.

[0043] Coupling: TTBZ-COOH and PCL-PEG-COOH were reacted in a classic EDC / DMAP coupling system. By optimizing the material ratio, feeding sequence, and quenching method, side reactions were effectively avoided, and the final product PCL-PEG-TTBZ-targeted polymer linked by mixed anhydride bonds was prepared in high yield.

[0044] Phase Two: One-Step Co-encapsulation Method for Therapeutic Nanoparticles The PCL-PEG-TTBZ polymer prepared above was dissolved together with rifampicin and iron oxide nanoparticles in an organic solvent (such as chloroform). Using an emulsification-solvent evaporation method, nanoparticles were formed in one step in the aqueous phase. During this process, the hydrophilic PEG-TTBZ segments face the aqueous phase to form the outer shell and targeting surface, while the hydrophobic PCL segments fold inward, simultaneously encapsulating rifampicin and Fe3O4 within the core, ultimately yielding water-dispersed, uniformly sized therapeutic nanoparticles.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents used are commercially available.

[0046] Composition formulation: Carrier material: PCL 5000 -PEG 2000 -COOH.

[0047] Target molecule: TTBZ, chemically named 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, with the molecular formula C 28 H 39 NO6S has a molecular weight of 517.66 g / mol.

[0048] Treatment medication: Rifampin.

[0049] Example 1: Preparation and characterization of the optimized targeting polymer PCL-PEG-TTBZ a) Preparation of TTBZ-diol: Weigh 100 mg of TTBZ and dissolve it in a mixed solvent of 0.5 mL methanol and 0.5 mL tetrahydrofuran. Add 60 mg of magnesium powder and 30 mg of ammonium chloride, and sonicate at 40°C for 30 minutes. After the reaction is complete, dilute with 0.5 mL of water and vortex for 10 seconds. Filter the solution through 0.45 μm and 0.22 μm filter membranes, respectively. Dry the filtrate under nitrogen at ≤35°C to obtain a light yellow oily substance, TTBZ-diol, which can be used directly in the next step.

[0050] b) Preparation of TTBZ-COOH: Add 40 mg (2.0 equivalent) of succinic anhydride, 5 mg of DMAP, and 0.5 mL of anhydrous dichloromethane to the product obtained in the previous step, and stir at room temperature for 6 hours. After drying the solvent, to remove residual DMAP•HCl from the crude product and to avoid hydrolysis of succinic acid monoester under strong acid conditions, vortex 0.5 mL of citrate buffer (pH 3.5) with 0.5 mL of DCM for 10 seconds, then immediately separate the contents, and dry the DCM phase to obtain crude TTBZ-COOH.

[0051] c) Preparation of PCL-PEG-TTBZ: PCL 5000 -PEG 2000 300 mg of TTBZ-COOH was dissolved in 2 mL of anhydrous DMF, and activated with EDC (2.0 equivalents) and DMAP (0.1 equivalents). The crude TTBZ-COOH was first removed by rotary evaporation under reduced pressure at 37 °C to completely remove residual DCM, and then redissolved in 1 mL of anhydrous DMF.

[0052] The DMF solution of TTBZ-COOH was slowly added dropwise to the activated polymer solution using a syringe (26G needle) at a rate of approximately 0.2 mL / min. After stirring at room temperature for 12 hours, 0.1 mL of water was added to quench the reaction for 10 minutes. The reaction solution was poured into 30 mL of ice-cold diethyl ether to precipitate the product, filtered, and dried under vacuum to obtain a white flocculent solid product, PCL-PEG-TTBZ.

[0053] Example 2: Preparation, characterization, and efficacy evaluation of therapeutic nanoparticles 1. Preparation of nanoparticles: 50 mg PCL-PEG-TTBZ, 2.8 mg rifampin, and 2 mg iron oxide nanoparticles were dissolved together in 3 mL of chloroform.

[0054] Under ice-water bath and ultrasonic conditions (180W power, 40kHz frequency), ensure that the liquid level in the ice-water bath completely submerges the water level in the ultrasonic tank by at least 2 cm to guarantee effective cooling during the emulsification process. Then, slowly add the above organic phase dropwise (approximately 5 minutes) to 8 mL of deionized water to form a white emulsion. Transfer the emulsion to an open container and magnetically stir at 600 rpm for 3 hours to evaporate and remove chloroform.

[0055] The solution was first centrifuged at 4000 rpm for 5 minutes to remove large particles; the supernatant was then centrifuged at 12000 rpm to collect the precipitate. The precipitate was washed three times with deionized water and finally dispersed in 10 mL of deionized water to obtain the final product, TTBZ-PEG-PCL@Rif / Fe3O4 nanoparticle aqueous dispersion.

[0056] 2. Characterization and targeting validation results: (1) Morphology and particle size: The nanoparticles exhibited a unimodal distribution in 0.1 mg / mL aqueous phase, with an average hydrodynamic diameter of 135.5 ± 9.4 nm (PDI = 0.21 ± 0.02), and the particle size distribution was concentrated in the range of 120–150 nm. Transmission electron microscopy (TEM) showed that the nanoparticles were regular spherical, uniformly distributed, and had an average diameter of 95 ± 12 nm in the dry state. Figure 1 ).

[0057] (2) MRI imaging function: Under 1.0 T MRI, this product showed significant T2-weighted signal attenuation. Two hours after mice received the nano-drug via nasal ingestion (Nano group), the bilateral striatum showed significant attenuation on both conventional T2-weighted and T2-weighted images. The signal value of the weighted gradient echo sequence decreased by approximately 20% compared to the control group (con group), confirming it as a highly effective MRI contrast agent. Figure 2 This study demonstrates that TTBZ-PEG-PCL@Rif / Fe3O4 nanomedicine can significantly reduce MRI signal in the striatum region of the brain, thus creating a clear contrast in images. This indicates that TTBZ-PEG-PCL@Rif / Fe3O4 nanomedicine is a highly effective MRI contrast agent.

[0058] (3) In vitro targeting assessment: a. Verification of cellular uptake of nanoparticles: To verify whether nanoparticles could be effectively uptaken by target cells, a Prussian blue staining experiment was performed. For example... Figure 3 As shown, the cells in the control group without nanoparticles had normal morphology. Figure 3 In cells A and B, no blue granules were observed deposited in the cytoplasm, ruling out interference from endogenous iron. However, after treatment with 100 μg / mL TTBZ-PEG-PCL@Rif / Fe3O4 nanoparticles for 24 hours, a large number of clear blue granules appeared in the cytoplasm of the vast majority of cells in the experimental group. Figure 3(C and D in the original text) Some particles aggregated around the cell nucleus. High-power microscopy confirmed that these particles were located within the cytoplasm, rather than on the cell membrane surface, demonstrating that the nanoparticles were effectively internalized by the cells. This result visually demonstrates that the target cells have a good uptake capacity for the nanoparticles of this invention.

[0059] b. Validation of co-localization between nanoparticles and VMAT2 (vesicle monoamine transporter 2): VMAT2 is mainly expressed on the synaptic vesicle membrane of dopaminergic neurons, responsible for the active transport of monoamine neurotransmitters from the cytoplasm into the vesicles for storage, so that they can be subsequently released into the synaptic cleft to participate in nerve signal transmission. To further confirm that the uptake targeting originates from the specific binding of TTBZ on the nanoparticle surface to VMAT2 on the cell surface, VMAT2 immunofluorescence staining was performed. Figure 4 The results showed that Figure 4 The control cells (A and C) showed only weak, diffuse basal VMAT2 green fluorescence. In contrast, Figure 4 Cells treated with nanoparticles B and D showed significantly enhanced VMAT2 fluorescence signal intensity, exhibiting numerous bright, "gritty" particles densely distributed in the perinuclear cytoplasm, consistent with the distribution characteristics of the Golgi apparatus and vesicle structures. Under high magnification, these fluorescent particles, ranging in diameter from 0.2 to 0.5 μm, highly overlapped with the intracellular uptake regions of nanoparticles. Quantitative analysis revealed that the number of VMAT2-positive particles in the treated cells (average 12-15 per cell) significantly exceeded that in the control group (<3). This result strongly demonstrates that TTBZ-PEG-PCL@Rif / Fe3O4 nanoparticles can promote active uptake and intracellular accumulation of VMAT2 through the specific interaction between TTBZ and VMAT2.

[0060] Example 3: Study on the neuroprotective efficacy and mechanism of nanoparticles administered via nasal administration in a Parkinson's disease model. To verify the actual efficacy and mechanism of action of the therapeutic nanoparticles (TTBZ-PEG-PCL@Rif / Fe3O4) described in this invention, a systematic in vivo pharmacodynamic evaluation was conducted.

[0061] 1. Experimental Methods: (1) Model establishment: C57BL / 6J mice were used to construct a Parkinson's disease (PD) model by stereotactic injection of a pre-formed α-synuclein oligomer suspension (α-syn suspension, 5 μg) into the left striatum.

[0062] (2) Grouping and administration: The mice after modeling were randomly divided into five groups: ① sham-operated control group (Con); ② PD model group (α-syn); ③ intranasal nanoparticle treatment group (α-syn+Nano), that is, the nanoparticles of the present invention were administered via the nose; ④ intraperitoneal rifampicin control group (α-syn+rif), that is, free rifampicin solution was injected intraperitoneally.

[0063] (3) Behavioral assessment: Motor function tests (rotating rod test, pole climbing test, wire mesh suspension test) were conducted at 1, 3, 6 and 9 months after modeling; non-motor function tests (Y maze to assess spatial memory, open field test to assess anxiety-like behavior, tail suspension test and forced swimming test to assess depression-like behavior) and gait analysis were conducted at 9 months.

[0064] (4) In vivo distribution: The distribution of nanoparticles in the brain was traced by magnetic resonance imaging (MRI) and Prussian blue staining of brain tissue.

[0065] (5) Histological and molecular biological analysis: After the behavioral test, the brain was perfused and harvested. The following key indicators in the midbrain substantia nigra-striatal pathway and hippocampus were detected by immunohistochemistry and Western blotting: tyrosine hydroxylase (TH, a marker of dopamine neurons), phosphorylated α-synuclein (p-α-syn, a pathological protein), and key proteins such as apoptosis and necroptosis (including RIPK1, RIPK3, p-MLKL, Caspase-8, CleavedCaspase-3, etc.).

[0066] Safety assessment: Serum was collected for blood biochemistry analysis, and liver samples were taken for H&E staining to assess systemic toxicity.

[0067] 2. Results: (1) Significant improvement in behavioral function Motor function: Compared with the PD model group, mice in the intranasal nanoparticle treatment group showed a significantly prolonged latency in the rotarod test. Figure 5 In the pole climbing experiment (A), Figure 5 (B) and wire mesh suspension test ( Figure 5 The performance in the C group was significantly better (p<0.01), indicating that their motor coordination, balance, and muscle strength were effectively protected and improved. The improvement effect of intraperitoneal rifampin was not significant. The stride length of both hind limbs of the PD model group mice was significantly shortened (p<0.01). Figure 5 In DE), the time of limb contact with the ground was significantly prolonged ( Figure 5 The FI (fibrillation index) indicated difficulty in initiating movement, limb stiffness, and increased time required to support the body. These indicators significantly improved after nanomedicine treatment, while little change was observed in the conventional rifampicin treatment group.

[0068] Non-motor function: In the Y-maze test ( Figure 5 In the K group, the spontaneous alternation rate in mice in the nanotherapy group was significantly higher than that in the model group (p<0.05), suggesting that cognitive function was protected. In the open field (… Figure 5 J in the middle), tail ( Figure 5 M in the experiment and forced swimming experiment ( Figure 5 In the L group, the anxiety and depression-like behaviors of mice in the nanotherapy group were also significantly alleviated.

[0069] (2) Neuropathology and molecular pathology were effectively reversed. ① Protection of dopaminergic neurons: Immunofluorescence results showed that, compared with the sham-operated group ( Figure 6 Compared to A in the model group, the PD model group lost more than 50% of TH-positive neurons; while compared to the PD model group ( Figure 6 In the B group, mice in the intranasal nanotherapy group ( Figure 6 In the C) substantia nigra pars compacta, the number of TH-positive neurons decreased by approximately 40%, while in the conventional rifampicin group ( Figure 6 The degree of loss of TH-positive neurons in the D group was comparable to that in the PD model group, proving that the nanoparticles of this invention can effectively protect dopaminergic neurons from α-synucleotoxic damage. Figure 6 ).

[0070] ② Pathological protein aggregation was inhibited: Immunofluorescence analysis showed that the level of abnormal p-α-syn aggregation in the brain tissue of the nanotherapy group decreased by about 50%, indicating that it can effectively inhibit the progression of PD core pathology. Figure 7 ).

[0071] ③ Synergistic inhibition of apoptosis signaling pathways: Molecular mechanism studies revealed the core mechanism of action of the nanoparticles in this invention. In the PD model group, key proteins related to necrosis and apoptosis (p-MLKL, Cleaved Caspase-8, Cleaved Caspase-3) were significantly upregulated. Figure 8 (A) In this invention, after treatment with the nanoparticles, the key activation signals of all the above-mentioned panapoptotic pathways were synergistically and significantly downregulated (A). Figure 8 The figures BD in the graphs represent the quantitative analysis statistics of the relative average density of Cleaved Caspase-3, Cleaved Caspase-8, and p-MLKL staining signals in the four mouse groups, respectively. This indicates that its neuroprotective effect is achieved by simultaneously inhibiting multiple programmed cell death pathways, thereby broadly protecting neurons.

[0072] (2) Target distribution and safety confirmation ① Targeted distribution in the brain: MRI imaging and Prussian blue staining of brain tissue confirmed that nanoparticles administered via the nose or lateral ventricle can be effectively distributed and enriched in target brain regions such as the cerebral cortex, striatum, substantia nigra, and hippocampus, which highly overlap with the expression regions of VMAT2. Figure 9 In the image, AF represents Prussian blue staining images of the olfactory bulb, striatum, classical hippocampus, hypothalamus, largest surface of the substantia nigra pars compacta, and cerebellar cortex of mice 2 hours after intranasal instillation of PEG-PCL@rif@TTBZ nanoparticles.

[0073] ② Good biocompatibility: The serum liver enzyme (ALT / AST) levels in the nanotherapy group mice were not significantly different from those in the control group. Figure 10 AC), and no pathological changes were observed in liver tissue sections ( Figure 10 (D), proving that the nanoparticles of the present invention do not produce significant hepatotoxicity at effective doses and have good biosafety.

[0074] In summary, the therapeutic nanoparticles TTBZ-PEG-PCL@Rif / Fe3O4 prepared in this invention can be effectively enriched and taken up by neurons in the brain, regardless of whether they are administered non-invasively via nasal administration or intraventricular administration, thereby achieving highly efficient targeted delivery to lesion areas in the brain. Animal experiments show that these nanoparticles can significantly improve motor and non-motor function deficits in Parkinson's disease model mice, with therapeutic effects far exceeding those of free rifampin. Simultaneously, these nanoparticles can exert multi-pathway neuroprotective effects by synergistically inhibiting a "pan-apoptotic" network composed of multiple programmed cell death pathways, including necroptosis, apoptosis, and pyroptosis, thereby reducing the loss of dopaminergic neurons and inhibiting the pathological accumulation and spread of α-synuclein. Furthermore, safety evaluation results show that these nanoparticles did not exhibit significant hepatotoxicity at effective doses, demonstrating good biocompatibility. Therefore, the nanoparticles constructed in this invention have promising applications in the modification and treatment of Parkinson's disease and provide reliable in vivo experimental evidence for neuroprotective strategies based on nanotechnology.

[0075] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rifampicin nanoparticle for integrated diagnosis and treatment, characterized in that, include: TTBZ-targeted molecularly modified polycaprolactone-polyethylene glycol copolymer carrier, iron oxide nanoparticles, and rifampin; Rifampicin and ferric oxide are encapsulated within the polycaprolactone-polyethylene glycol copolymer carrier.

2. The rifampicin nanoparticles according to claim 1, characterized in that, The average particle size of the nanoparticles is 50 nm to 200 nm.

3. The method for preparing rifampicin nanoparticles according to claim 1 or 2, characterized in that, Includes the following steps: S1. Dissolve TTBZ in a mixed solvent of methanol and tetrahydrofuran, add magnesium powder and ammonium chloride to react and obtain TTBZ-diol; S2. Add succinic anhydride and 4-dimethylaminopyridine to the TTBZ-diol described in S1, dissolve in dichloromethane and react. After extraction with dichloromethane in an acidic buffer solution, TTBZ-COOH is obtained and dissolved in anhydrous N,N-dimethylformamide. S3: Polycaprolactone-polyethylene glycol-carboxyl copolymer PCL 5000 -PEG 2000 -COOH was dissolved in anhydrous N,N-dimethylformamide, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 4-dimethylaminopyridine were added to obtain solution 1. The TTBZ-COOH of S2 was added dropwise to solution 1 to react. After ether precipitation, filtration and drying, PCL-PEG-TTBZ was obtained. S4: PCL-PEG-TTBZ, rifampicin, and iron oxide nanoparticles were dissolved in chloroform and an emulsion was formed under ice-water bath and ultrasonic conditions. The rifampicin nanoparticles were obtained by centrifugation and washing.

4. The preparation method according to claim 3, characterized in that, In step S1, the mass ratio of TTBZ, magnesium powder and ammonium chloride is 1:(0.4~1.2):(0.2~0.8).

5. The preparation method according to claim 3, characterized in that, In step S2, the amount of succinic anhydride used is 1.5 to 3.0 equivalents of the molar amount of TTBZ.

6. The preparation method according to claim 3, characterized in that, In step S3, the amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide used is PCL. 5000 -PEG 2000 The molar amount of the -COOH terminal carboxyl group is 1.5 to 3.0 equivalents; The amount of 4-dimethylaminopyridine used is 0.05~0.2 equivalents.

7. The preparation method according to claim 3, characterized in that, In step S4, the mass percentage of PCL-PEG-TTBZ is 85~95 wt%; the mass percentage of rifampicin is 3~9 wt%; and the mass percentage of iron oxide nanoparticles is 2~6 wt%.

8. The preparation method according to claim 3, characterized in that, In step S3, the TTBZ-COOH is added dropwise to solution 1 at a rate of 0.1~0.5 mL / min.

9. The use of the rifampicin nanoparticles according to claim 1 or 2 in the preparation of medicaments for the diagnosis and / or treatment of Parkinson's disease.

10. The application according to claim 9, characterized in that, The drug is administered via the nasal cavity.

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