A nano-drug preparation, a preparation method and application thereof

Abstract

The present application relates to the biomedical technology field, and more particularly to a kind of nano drug preparation and its preparation method and application.The nano drug preparation includes rosmarinic acid and nilotinib, and the mass ratio of rosmarinic acid and nilotinib is 1:0.1-2.The present application provides a kind of composition of rosmarinic acid and nilotinib, which can synergistically activate autophagy, has the effect of neuroprotection and antioxidant stress.The drug loaded with rosmarinic acid and nilotinib provided by the present application can effectively protect dopaminergic neurons in substantia nigra, and then improve the motor symptoms of MPTP-induced PD mice, and provide a prospective scheme for PD targeted therapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a nanomedicine formulation, its preparation method, and its application. Background Technology

[0002] Neurodegenerative diseases are major brain diseases that plague the elderly population in modern society. Parkinson's disease (PD) is the second most common neurodegenerative disease, with the main pathological changes being the loss of dopaminergic neurons in the substantia nigra and a decrease in dopamine neurotransmitter levels.

[0003] Studies have shown that most neurodegenerative diseases are accompanied by the abnormal accumulation of misfolded protein aggregates in nerve cells, thereby exerting toxic effects on neurons. α-synuclein is the most important protein in the pathogenesis of Parkinson's disease (PD). The misfolding and aggregation of α-synuclein to form amyloid fibrils is the main pathological process of PD, and α-synuclein aggregation is closely related to the formation of Lewy bodies and the death of dopaminergic neurons. To date, there are no mature and effective drugs or methods to stop or reverse the progression of PD. Developing novel drugs, especially natural extracts, that can prevent and treat PD and have neuroprotective effects will have significant practical social and economic value for protecting human health.

[0004] Autophagy is a molecular mechanism closely related to the pathogenesis of Parkinson's disease (PD) proposed in recent years. Autophagy inducers are widely used as neuroprotective drugs in the development of anti-PD drugs. Autophagy can clear misfolded proteins and reduce protein aggregation in neurons, effectively preventing the occurrence of neurodegenerative diseases such as Parkinson's disease. Beclin1, as an autophagy regulator, provides a platform for interaction, thus playing a unique and important role in autophagy regulation and being a key point for regulating signal convergence. Studies have shown that drug-activated autophagy can reduce the level of intracellular pathogenic α-synuclein protein aggregates, thereby effectively alleviating the toxicity of these protein aggregates to nerve cells. Meanwhile, studies have also shown that oxidative stress, mitochondrial dysfunction, and autophagy defects play important roles in the formation of PD. In PD patients with PINK1 and Parkin gene mutations, autophagy defects occur in mitochondria, leading to oxidative stress and impaired mitochondrial energy supply, which in turn causes dopaminergic neuronal necrosis and PD. In PD animal models, α-synuclein misfolding leads to mitochondrial edema in substantia nigra dopaminergic neurons, thereby causing apoptosis of DA neurons.

[0005] Currently, various autophagy promoters have been publicly reported, but due to their physicochemical properties, these drugs have low oral bioavailability, high in vivo clearance and metabolic rates, and limited bioavailability, failing to induce high levels of autophagy in neurons and thus not achieving good therapeutic effects. In neurodegenerative diseases, nanomedicine combined with synergistic therapies is gradually replacing single-drug regimens, as it can increase BBB crossing efficiency, improve efficacy, and reduce side effects. Therefore, this invention develops an aggressive therapeutic strategy and a safe and effective drug combination delivery system to overcome these clinical limitations. Summary of the Invention

[0006] To address the problems of existing technologies, this invention provides a nanomedicine formulation, its preparation method, and its application. By synergistically clearing ROS and reducing inflammatory responses through rosmarinic acid and nilotinib, it reduces the toxicity to dopaminergic neurons in the pathological process of neurodegenerative diseases, providing a new approach for targeted therapy of PD.

[0007] In a first aspect, the present invention provides a composition comprising: rosmarinic acid and nilotinib;

[0008] The chemical formula of the rosmarinic acid is shown in Formula I:

[0009]

[0010] And / or, the chemical formula of the nilotinib is as shown in Formula II:

[0011]

[0012] Furthermore, the mass ratio of rosmarinic acid to nilotinib is 1:0.1 to 2.

[0013] Furthermore, the mass ratio of rosmarinic acid to nilotinib is 1:1 to 2.

[0014] In a second aspect, the present invention provides a nanomedicine formulation, wherein the active ingredient of the nanomedicine formulation includes the composition.

[0015] Furthermore, the nanoparticles used in the nanomedicine formulation are one or more of PLGA-PEG2000-Ang2, DSPE-PEG2000-Ang2, or mPEG-PLA.

[0016] Furthermore, the nanomedicine formulation also includes pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients are preferably any one or more of excipients, diluents, carriers, flavoring agents, binders, or fillers.

[0017] For example, water or saline solution can be used as a diluent, polylactic acid-glycolic acid copolymer can be used as a carrier, and gelatin or cellulose derivatives can be used as an adhesive.

[0018] Thirdly, the present invention provides a method for preparing the aforementioned nanomedicine formulation, comprising:

[0019] The composition is loaded onto nanoparticles using a nanoemulsion co-precipitation method.

[0020] Furthermore, the preparation method includes the following steps:

[0021] The composition and nanoparticles were placed in a solvent to obtain a nanomaterial solution, and deionized water was added and mixed for 2-8 hours.

[0022] Nanoparticle suspension emulsions were prepared by precipitation method;

[0023] After purification, it was freeze-dried.

[0024] Furthermore, the solvent is an organic solvent, such as dichloromethane, chloroform, methanol, acetonitrile, or dimethyl sulfoxide; preferably dichloromethane.

[0025] Furthermore, the nanoparticle suspension emulsion has a particle size of 20–200 nm.

[0026] Furthermore, the volume ratio of the nanomaterial solution to the deionized water is 1:10 to 50.

[0027] As a preferred embodiment, the present invention provides a method for preparing the nanomedicine formulation, comprising:

[0028] (1) Dissolve PLGA-PEG2000-Ang2 polymer and Nilotinib in DMSO as the organic phase, and dissolve RA in water as the aqueous phase. While stirring, slowly add the organic phase to the aqueous phase and stir until the organic solvent has completely evaporated.

[0029] The mass ratio of the PLGA-PEG2000-Ang2 polymer to the Nilotinib is 10:1 to 20;

[0030] The mass ratio of Nilotinib to RA is 1:1 to 5;

[0031] (2) Centrifuge at 24000-28000 rpm and 2-6℃ for 15-30 min to wash away the unloaded drug, and then purify the nanoparticles by centrifugation in a 100K ultrafiltration tube.

[0032] The present invention further provides the use of the composition, or the nanomedicine formulation, in the preparation of a medicament for the prevention or treatment of a disease;

[0033] The disease in question is either Alzheimer's disease or Parkinson's disease.

[0034] The present invention further provides the use of the composition, or the nanomedicine formulation, in promoting autophagosome formation.

[0035] The present invention further provides the use of the composition, or the nanomedicine formulation, in the preparation of a drug that promotes autophagy formation.

[0036] The present invention further provides the use of the composition, or the nanomedicine formulation, in the preparation of anti-oxidative stress drugs.

[0037] The present invention further provides the use of the composition, or the nanomedicine formulation, in the preparation of a drug that reduces the expression level of α-synuclein.

[0038] The present invention has the following beneficial effects:

[0039] This invention provides a nanomedicine formulation containing both rosmarinic acid and nilotinib. By synergistically combining these two components, it can effectively alleviate motor impairment and improve gait abnormalities in Parkinson's mice, and protect nerve cells by inducing an increase in autophagy levels in brain tissue cells.

[0040] The nanomedicine formulation provided by this invention can improve the motor function of animals and protect the activity of TH neurons in the substantia nigra when used in animal models. The mechanism may be to synergistically promote the degradation of α-synuclein protein by activating the autophagy activity of neurons in the substantia nigra pars compacta and reducing the level of oxidative stress, thereby reducing neuronal damage and achieving effective relief of PD symptoms.

[0041] The nanomedicine formulation provided by this invention can efficiently cross the blood-brain barrier with the assistance of the Angiopep2 peptide, significantly reducing the amount of drug required and thus reducing toxic side effects.

[0042] The nanomedicine formulation provided by this invention regulates autophagy to intervene in Parkinson's disease, achieving efficient and low-toxicity combined drug delivery for brain diseases, high lesion enrichment, controlled release and visualized delivery, providing a prospective solution for targeted therapy of PD. Attached Figure Description

[0043] Figure 1 The diagram shows the TEM and DLS characterization of the P@RNP nanomedicine provided in Example 1 of this invention; the left diagram is the TEM characterization diagram and the right diagram is the DLS characterization diagram.

[0044] Figure 2 This is an in vitro release diagram of rosmarinic acid and nilotinib in the P@RNP nanomedicine provided in Example 2 of the present invention.

[0045] Figure 3 This is an imaging schematic diagram illustrating the ability of the P@RNP nanomedicine provided in Example 3 of the present invention to scavenge ROS levels in SH-SY5Y cells.

[0046] Figure 4 The diagram shows the open field behavior of Parkinson's disease model mice after P@RNP treatment provided in Example 4 of this invention; where a is the mouse movement trajectory diagram, b is the statistical result of the total distance moved by the mouse in the open field test, and c is the statistical result of the time spent on the rotundus in the rotundus test.

[0047] Figure 5 The image shows the gait behavior of a Parkinson's disease model mouse after P@RNP treatment, as provided in Example 4 of this invention; where a is the movement speed, b is the turning speed, c is the three-paw support, and d is the four-paw support.

[0048] Figure 6 This is a schematic diagram illustrating the expression of autophagy-related proteins in the substantia nigra of the brain of PD mice after P@RNP treatment, as provided in Example 5 of this invention.

[0049] Figure 7 This is a schematic diagram of the immunofluorescence analysis results of TH, IBA-1, NeuN and GFAP in the brain tissue of PD mice after P@RNP treatment provided in Example 6 of the present invention. Detailed Implementation

[0050] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0052] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0053] The reagents and instruments used in the following examples were purchased from the following manufacturers:

[0054]

[0055]

[0056] Example 1

[0057] This embodiment provides a method for preparing and characterizing PLGA-loaded rosmarinic acid-nilotinib nanomedicine P@RNP, specifically including the following steps:

[0058] 1. Design, synthesis and identification of Angiopep2 (Ang2) peptide

[0059] The Ang2 peptide TFFYGGSRGKRNN FKTEEYC was synthesized using the Fmoc solid-phase synthesis method. The α-amino protecting group of the amino acid, Fmoc, was rapidly removed under alkaline conditions using a deprotecting agent (hexahydropyridine / DMF, 20%, v / v). The peptide chain was then elongated by reacting excess amino acids activated with N-methylmorpholine (NMM / DMF, 4%, v / v) under the catalysis of the activating agent HBT U. Finally, the resin was lysed in a lysis buffer (92.5% TFA: 2.5% H2O: 2.5% TIS: 2.5%). The purity and molecular weight of the Ang2 peptide were identified by HPLC and ESI-MS mass spectrometry.

[0060] 2. Preparation and characterization of P@RNP nanomedicines

[0061] Synthesis of the functional amphiphilic molecule PLGA-PEG2000-Ang2. Specific steps: Ang2 and PLGA-PEG2000-MAL were weighed at a molar ratio of 1:2, dissolved separately in aqueous solutions, and mixed. The mixture was gently stirred at room temperature for 48 hours. The reaction product PLGA-PEG2000-Ang2 was purified by dialysis using a dialysis bag with a molecular weight cutoff of 3500 Da, and then freeze-dried to obtain a dry powder sample.

[0062] This invention prepares Nilotinib and RA-supported PLGA multifunctional nanoparticles PLGA@Nilotinib / RA (P@RNP) via nanoemulsion co-precipitation, specifically including the following process:

[0063] (1) 100 mg of PLGA-PEG2000-Ang2 polymer and 10 mg of Nilotinib dissolved in 5 mL of dichloromethane were dissolved in DMSO. 20 mg of RA was dissolved in water and 6 mL of PVA (2% w / v) aqueous solution was used as the aqueous phase. Under magnetic stirring, the organic phase was slowly added dropwise to the aqueous phase and stirred overnight to evaporate the organic solvent.

[0064] (2) On the second day, the nanoparticles were centrifuged at 24,000 rpm and 4°C for 20 min, washed with double distilled water to remove unloaded drugs, and purified by centrifugation in a 100K ultrafiltration tube for later use.

[0065] (3) Using the same method as steps (1) and (2), PLGA@Nilotinib (P@N) and PLGA@RA (P@R) loaded with only one drug were prepared as controls. P@RNP (1 mg / mL) was placed in the sample cell and the particle size was measured by dynamic light scattering (DLS).

[0066] (4) Experimental data were processed using Zetasizer Software. The morphology of P@RNPs was characterized by transmission electron microscopy, specifically including the following procedures:

[0067] 2 μL of P@RNP (1 mg / mL) was dropped onto a copper grid supported by a regular carbon film. After 2 minutes, the excess sample was removed by filter paper. Then, 1 μL of 1% uranium acetate was added for negative staining. After 5 minutes, the excess liquid was removed by filter paper. After drying, the morphology of the nanoparticles was observed by transmission electron microscopy at 120 kV.

[0068] The results are as follows Figure 1 As shown, by Figure 1 Dynamic light scattering (DLS) and transmission electron microscopy revealed that P@RNPs were uniform nanomicelle particles, approximately 75 nm in size, consistent with the DLS results. These nanoparticles, with their highly efficient tissue and cell penetration capabilities, demonstrate that drug nanoparticles can reduce or avoid adverse reactions through sustained drug release.

[0069] This embodiment further experimented with changing the dosage of Nilotinib to 1 mg and RA to 10 mg, and then performing the same experiment with both Nilotinib and RA dosages of 10 mg. The resulting nanoparticles showed similar characteristics to... Figure 1 Similar structure.

[0070] Example 2

[0071] This embodiment studies the in vitro drug release of the P@RNP nanoparticles prepared in Example 1. The specific procedure is as follows:

[0072] Place the P@RNP nanoparticle solution (1 mg / mL, 2 mL) in a dialysis bag and then place it in a beaker containing dialysis buffer (pH 7.4 PBS containing 10% serum, 50 mL).

[0073] The release device was placed in a horizontal oscillator and subjected to continuous gentle oscillation at 37°C. At different time points (0, 1, 2, 6, 8, 12, 24, 36, 48 hours), 0.1 mL of the dialysate was taken into a 96-well plate, and the contents of rosmarinic acid and nilotinib were determined by high performance liquid chromatography (HPLC) (rosmarinic acid detection wavelength 330 nm; nilotinib detection wavelength 240 nm).

[0074] The detection conditions for high performance liquid chromatography are as follows: C18 column, mobile phase is methanol-1% formic acid water (60:40, v / v) (38:62), flow rate is 1.0 mL / min, column temperature is 25℃, and the drug release rate is evaluated by dialysis at pH 7.4.

[0075] The results are as follows Figure 2As shown, P@RNP exhibits a high degree of controlled drug release within 0-48 hours at pH 7.4. Instead of an initial burst of drug release, it releases slowly, avoiding rapid clearance from the body, thus improving drug utilization and reducing drug toxicity.

[0076] Example 3

[0077] This embodiment examines the effect of the P@RNP nanoparticles prepared in Example 1 on intracellular ROS levels. The specific procedure is as follows:

[0078] SH-SY5Y cells were seeded in 10 mm glass-bottomed culture dishes. When the cells reached about 60% confluence, they were pretreated with 10 mM 1-methyl-4-phenylpyridine ions (MPP+) for 12 h, and then treated with P@RNP nanoparticles for 24 h.

[0079] The cells were then washed three times with PBS, and culture medium containing 10 μM DCFH-DA probe was added and incubated in an incubator for 30 min. After washing the cells three times with PBS, the images were taken under a confocal microscope.

[0080] The accumulation of reactive oxygen species (ROS) peroxides is a significant pathological cause of disease (PD), and eliminating excess ROS is crucial for maintaining health. This invention first used 2,7-dichlorofluorescein diacetate (DCFH-DA) as a probe to test the ROS scavenging ability of P@RNP nanoparticles. DCFH-DA reacted with ROS, exhibiting strong fluorescence. DCFH-DA was diluted to a concentration of 10 μmol / L with serum-free culture medium. The cell culture medium in the 6-well plates was discarded, and after washing with PBS, 1 mL of DCFH-DA was added to each well. The plates were incubated for 20 min in the dark. This invention detects the ROS scavenging ability of SH-SY5Y cells by monitoring the fluorescence of DCF.

[0081] The results are as follows Figure 3 As shown, compared with the control group, the ROS level in SH-SY5Y cells treated with MPP+ was significantly increased, and the fluorescence intensity in SH-SY5Y cells treated with P@RNP nanoparticles was the lowest. The in vitro experimental results indicate that P@RNP nanoparticles have the ability to effectively scavenge ROS and reduce its damage to neurons, which can reduce ROS-related symptoms in PD.

[0082] Example 4

[0083] This embodiment evaluates the neurobehavioral response of mice treated with P@RNP to induce MPTP-induced PD using the mice prepared in Example 1. The specific procedure is as follows:

[0084] 1. Healthy mice and PD mice were randomly divided into 5 groups:

[0085] (1) Normal healthy mice (normal group, n=7);

[0086] (2) PD mice were intravenously injected with phosphate saline PBS (PBS group, n=7);

[0087] (3) PD mice were intravenously injected with P@N (P@N group, n=7);

[0088] (4) PD mice were intravenously injected with P@R nanoparticles (P@R group, n=7);

[0089] (5) PD mice were intravenously injected with P@RNP nanoparticles (P@RNP group, n=7) at a drug concentration of 5 mg / kg.

[0090] Two weeks after the end of treatment, behavioral observations were conducted, followed by dissection of the mice, separation of brain tissue, and various pharmacological tests.

[0091] 2. The MPTP Parkinson's mouse model exhibits symptoms such as exploratory movement disorders and significant anxiety, which can be detected by the open field test. The specific procedure for the open field test is as follows:

[0092] MPTP-induced PD model mice were tested once before and once after treatment. The mouse open field reaction chamber was 25–30 cm high, with a base length of 72 cm, blackened inner walls, and the bottom surface divided into 64 small squares. The experiment was conducted in a quiet environment. The animal was placed in the center of the chamber's bottom surface, and video recording and timing were performed simultaneously. Video recording was stopped after a certain observation time, which could be determined based on the experiment, generally 3–5 minutes. The animal was then replaced, and the experiment continued. Depending on the computer software design, different observable parameters could be used, such as the time the animal spent in the central square per unit time, the number of squares crossed by a limb (horizontal score), the number of times the hind limb stood (vertical score), and other parameters such as the number of trimmings, urination / defecation frequency, movement speed, movement distance, rest time, distance traveled along the edges, and distance traveled to the center.

[0093] 3. Rotary bar test: MPTP-induced PD model mice were tested once before and once after treatment. The fatigue level and grip strength of the mice were observed using the rotary bar test. The time each mouse spent walking on the rotating bar (30 revolutions / minute) was recorded at least 3 times after training.

[0094] The results are as follows Figure 4 As shown, this invention recorded the behavior of Parkinson's disease mice through rotarod and open field tests. Compared with wild-type control mice, the model mice exhibited significantly altered movement trajectories, which returned to normal after nanomedicine treatment. Compared with normal healthy mice, mice in the PBS group (PD+PBS) showed increased movement distance and significantly abnormal anxiety-like emotional states.

[0095] Figure 4 The results showed that PD mice treated with P@RNP nanoparticles exhibited increased open field movement distance and significantly improved anxiety-like affective state compared to untreated PBS mice, indicating a significant improvement in their PD symptoms. Compared to normal healthy mice, the PD+PBS group mice spent an average of approximately 130 seconds on the rotarod, significantly shorter than normal mice (approximately 210 seconds). The P@RNP group showed a significant improvement, with the time spent on the rotarod approaching that of normal mice (p<0.01). Animal experiments demonstrate that P@RNP treatment can improve motor symptoms in PD mice.

[0096] 4. Gait disturbance is an important indicator of Parkinson's disease. This invention further records the behavior of Parkinson's disease mice using a gait analyzer. Figure 5 Compared with normal healthy mice, the average movement speed of mice in the PBS group (9.7 cm / s) was significantly shorter than that of normal mice (13.9 cm / s). However, a significant therapeutic effect was observed in the P@RNP group, where mice achieved a movement speed of 20 cm / s. Figure 5 (a and b in the text). Swinging speed was also observed to be worse in the PBS group than in normal mice, while the P@RNP group showed improvement. Furthermore, the PBS group showed significantly more three-pronged and four-pronged support than the normal group, while the P@RNP group showed a significant improvement. Figure 5 (c and d in the text).

[0097] These results lead to the conclusion that PD mice treated with P@RNP nanoparticles were more likely to cross the target quadrant compared to untreated mice, indicating a significant improvement in their PD symptoms. This demonstrates that animal experiments show that P@RNP treatment can improve motor symptoms in PD mice.

[0098] Example 5

[0099] This embodiment performs Western Blot (WB) analysis on the P@RNP nanoparticles prepared in Example 1. The specific procedure is as follows:

[0100] Substantia nigra tissue from PD mice was lysed in RIPA lysis buffer; proteins were collected after centrifugation and measured using a BCA protein assay kit (Pierce); samples were separated by SDS-PAGE, then transferred to a PVDF membrane, blocked with skim milk, and incubated with a primary antibody and a secondary antibody; finally, the immunoreaction bands were detected using a chemiluminescent reagent, with β-actin as the internal control.

[0101] Beclin1 is a key factor regulating autophagy. A crucial marker protein on the autophagosome membrane is microtubule-associated protein 1 light chain 3 (LC3), which is divided into LC3-I and LC3-II. Autophagy activity can be reflected by changes in LC3-II levels. Literature reports that α-synuclein clearance is mediated through the Beclin-1 pathway; therefore, changes in Beclin1 levels can reflect changes in α-synuclein protein levels. Figure 6 As shown, compared with the control group, Beclin-1 was upregulated in the substantia nigra of mice in the P@RNP group, and the LC3II / LC3-I ratio was increased, indicating that P@RNP activated autophagy to downregulate α-synuclein and reduced toxicity to dopaminergic neurons.

[0102] Example 6

[0103] This embodiment evaluates the efficacy of the P@RNP nanoparticles prepared in Example 1 using immunofluorescence assay. The specific procedure is as follows:

[0104] After perfusion fixation, the brains of mice were harvested, dehydrated, embedded by OCT, sectioned by cryostat (20-25 μm thick), mounted, and stored at -80°C.

[0105] The specific procedures for staining frozen sections are as follows:

[0106] (1) After the slices are taken out of -80℃, they are equilibrated to room temperature for 30 minutes;

[0107] (2) Wash with PBS for 5 min × 3;

[0108] (3) Treat the sections with 0.2% Triton X-100 for 10 min (to increase cell membrane permeability), then wash with PBS 3 times for 5 min each time;

[0109] (4) Block serum at room temperature for 30 min;

[0110] (5) Incubate the primary antibody mixture at 4°C overnight;

[0111] (6) Wash with PBS at room temperature for 5 min × 3 on the second day;

[0112] (7) Incubate the secondary antibody mixture at room temperature for 30 min;

[0113] (8) Wash with PBS for 5 min × 3;

[0114] (9) DAPI double staining of nuclei;

[0115] (10) Mount the slide with anti-quenching mounting medium and take a picture with a fluorescence microscope.

[0116] To further evaluate the treatment efficacy and determine whether P@RNP has a protective effect against damage to DA neurons after MPTP treatment, this invention assessed the number of tyrosine hydroxylase (TH)-positive neurons using immunofluorescence. The results are as follows: Figure 7 As shown, Figure 7 The results showed that P@RNP treatment could inhibit the MPTP-induced reduction in the number of TH+ neurons. As can be seen from the figure, the number of TH-positive stained neurons in the substantia nigra of the mouse brain was significantly reduced in the other three groups (PD+PBS, PD+P@N and PD+P@R). This indicates that the P@RNP nanoparticles prepared in Example 1 of this invention can alleviate dopaminergic neuron damage in model mice and have a protective effect.

[0117] Furthermore, this invention analyzed the levels of IBA-1, a marker of microglial activation, and GFAP, a marker of astrocyte activation (commonly used to reflect neuroinflammatory responses). The results showed that the expression of IBA-1 and GFAP in the brains of P@RNP group mice was similar to that in normal mice, indicating that the synergistic effect of nilotinib and rosmarinic acid can reduce neuroinflammation caused by oxidative stress in the brain. Conversely, the levels of IBA-1 and GFAP in the other three groups were significantly upregulated compared to the normal group mice. In addition, this invention also found through NeuN staining that P@RNP treatment can reduce MPTP-induced neurotoxicity.

[0118] In summary, experiments 1-6 demonstrate that the rosmarinic acid-nilotinib nanomedicine combination can exert a neuroprotective effect on SH-SY5Y cells. This nanomedicine formulation can significantly reduce MPP+-induced toxicity, decrease ROS generation, activate autophagy, downregulate α-synuclein levels, and thus reduce toxicity to dopaminergic neurons.

[0119] The P@RNP provided by this invention is non-cytotoxic, has few side effects, and has neuroprotective and antioxidant effects.

[0120] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention. sequence list <110> Fujian Medical University Union Hospital, Fujian Medical University <120> A nanomedicine formulation, its preparation method and application <130> KHP211118216.3 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> PRT <213> Artificial Sequence <400> 1 Thr Phe Phe Tyr Gly Gly Ser Arg Gly Lys Arg Asn Asn Phe Lys Thr 1 5 10 15 Glu Glu Tyr Cys 20

Claims

1. A nanomedicine formulation, characterized in that, The active ingredient of the nanomedicine formulation includes a composition comprising: rosmarinic acid and nilotinib; The chemical formula of the rosmarinic acid is shown in Formula I: ； The chemical formula of nilotinib is shown in Formula II: ； The nanoparticles used in the nanomedicine formulation are PLGA-PEG2000-Ang2, and the mass ratio of PLGA-PEG2000-Ang2, rosmarinic acid and nilotinib is 5:1:0.

5.

2. The nanomedicine formulation according to claim 1, characterized in that: The nanomedicine formulation further includes pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients are one or more of excipients, diluents, carriers, flavoring agents, binders, or fillers.

3. The method for preparing nanomedicine formulations as described in claim 1 or 2, characterized in that: The composition is loaded onto nanoparticles using a nanoemulsion co-precipitation method.

4. The use of the nanomedicine formulation as described in claim 1 or 2 in the preparation of a medicament for the prevention or treatment of a disease, wherein the disease is Alzheimer's disease or Parkinson's disease.

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