Hybrid dendrimer nano material loaded with medicine and small interfering RNA (Ribonucleic Acid) as well as preparation method and application of hybrid dendrimer nano material

Abstract

The invention relates to a drug and small interfering RNA loaded hybrid dendrimer nano-material as well as a preparation method and application thereof. The hybrid dendrimer nano-material comprises a hybrid nano-delivery system, a drug and small interfering RNA, wherein the hybrid nano-delivery system is composed of a phosphorus-containing dendrimer at a hydroxyl terminal and a polyamide-amine dendrimer at a third-generation amino terminal; wherein the hybrid nano-delivery system is loaded with a drug and small interfering RNA. The preparation method has the advantages of being simple, easy to operate, good in biocompatibility and the like, and the provided strategy has good development prospects and application values in treatment of Parkinson's disease or other neurodegenerative diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a hybrid dendrimer nanomaterial loaded with drugs and small interfering RNA, and a preparation method and application thereof. Background Art

[0002] With the accelerating aging of the global population, the incidence of neurodegenerative diseases continues to grow, becoming a major societal health concern. Parkinson's disease (PD) is the second most common neurodegenerative disease, with primary symptoms including tremor, muscle rigidity, bradykines, and impaired balance. Current studies have shown that the pathological changes of PD primarily include abnormal aggregation of α-synuclein (α-syn) and the formation of Lewy bodies, as well as loss of dopaminergic neurons in the substantia nigra pars compacta. Furthermore, α-syn aggregates trigger abnormal activation and oxidative stress in microglia and astrocytes, leading to the self-assembly of NLRP3 inflammasomes in glial cells. This promotes the secretion of large amounts of proinflammatory cytokines by microglia, further triggering neuroinflammation and accelerating disease progression (Signal Transduction Targeted Ther. 2023, 8, 3649-3680). Common clinical treatments include medications such as levodopa and dopamine receptor agonists, surgery, and exercise therapy. However, these treatments are limited in effectiveness, can only improve symptoms but cannot prevent disease progression, and are prone to adverse reactions. Therefore, inhibiting the abnormal expression and aggregation of α-syn in neurons, as well as abnormal activation and oxidative stress in glial cells, and thereby controlling neuroinflammation (e.g., by inhibiting inflammasome self-assembly and the secretion of proinflammatory cytokines) has become an effective strategy for treating PD.

[0003] In recent years, small nucleic acid therapeutics have shown great potential in the treatment of neurodegenerative diseases. As a small interfering RNA for α-syn, siSNCA can effectively inhibit α-syn expression in neurons, reducing its abnormal aggregation and Lewy body formation, thereby blocking the progression of PD. It has been widely used in PD treatment research. Curcumin (Cur) has attracted much attention for its excellent anti-inflammatory and antioxidant properties. Studies have shown that Cur not only reduces reactive oxygen species (ROS) levels in cells and restores mitochondrial function, but also inhibits α-syn aggregation. Furthermore, by inhibiting the self-assembly of NLRP3 inflammasomes in microglia, it suppresses the secretion of proinflammatory cytokines, providing a basis for its application in the treatment of neurodegenerative diseases (Heliyon 2023, 9, e16195). Combining curcumin with siSNCA is expected to produce a 1+1>2 therapeutic effect, controlling α-syn formation and neuroinflammation at the source, achieving better therapeutic effects. Liu et al. combined curcumin with siSNCA to treat PD, inhibiting the expression and aggregation of α-syn and maintaining immune homeostasis, achieving good therapeutic effects (Sci. Adv. 2020, 6, eaba3967). However, due to the presence of the blood-brain barrier (BBB), drugs are difficult to effectively deliver to the brain to exert their efficacy, so it is necessary to construct a nano-drug delivery system that can efficiently penetrate the BBB. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a hybrid dendrimer nanomaterial loaded with drugs and small interfering RNA, as well as its preparation method and application, so as to enable drugs to penetrate the blood-brain barrier and improve the bioavailability of genes and drugs, thereby achieving efficient treatment of Parkinson's disease.

[0005] The present invention provides a hybrid dendrimer nanomaterial loaded with drugs and small interfering RNA, wherein the hybrid dendrimer nanomaterial comprises a hybrid nano-delivery system composed of a hydroxyl-terminated phosphorus-containing dendrimer and a third-generation amino-terminated polyamidoamine dendrimer, drugs and small interfering RNA; wherein the hybrid nano-delivery system is loaded with drugs and small interfering RNA.

[0006] Furthermore, the hydroxyl-terminated phosphorus-containing dendrimer is combined with the third-generation amino-terminated polyamide-amine dendrimer through hydrogen bonds and hydrophobic interactions, and the formed hybrid nanodelivery system is used as a carrier, which is further combined with the drug through hydrophobic interactions and loaded with small interfering RNA through electrostatic forces to form a hybrid dendrimer nanomaterial.

[0007] Preferably, the structural formula of the hydroxyl-terminated phosphorus-containing dendrimer is:

[0008] Recorded as ZY-128.

[0009] Preferably, the third generation amino-terminated polyamidoamine dendrimer has the structural formula:

[0010] Denoted as G3PAMAM-NH2.

[0011] Preferably, the drug includes but is not limited to curcumin (Cur).

[0012] Preferably, the small interfering RNA includes but is not limited to α-synuclein small interfering RNA (siSNCA).

[0013] The present invention provides a method for preparing the hybrid dendrimer nanomaterial loaded with drugs and small interfering RNA, comprising the following steps:

[0014] (1) mixing a hydroxyl-terminated phosphorus-containing dendrimer with a third-generation amino-terminated polyamide-amine dendrimer, stirring at room temperature, and centrifuging to prepare hybrid nanoparticles of the hydroxyl-terminated phosphorus-containing dendrimer and the third-generation amino-terminated polyamide-amine dendrimer, thereby forming a hybrid nanodelivery system;

[0015] (2) mixing the drug with the hybrid nanoparticles, stirring at room temperature, and centrifuging to prepare a composite nanomaterial;

[0016] (3) The composite nanomaterial is mixed with small interfering RNA and incubated at room temperature to prepare a hybrid dendrimer nanomaterial.

[0017] Preferably, the mass ratio of the hydroxyl-terminated phosphorus-containing dendrimer to the third-generation amino-terminated polyamide-amine dendrimer in step (1) is 1:0.5 to 1:4.

[0018] Preferably, the mixing in step (1) is specifically: adding dropwise the methanol solution of the hydroxyl-terminated phosphorus-containing dendrimer to the aqueous solution of the third-generation amino-terminated polyamide-amine dendrimer; and the stirring reaction time at room temperature is 12 to 14 hours.

[0019] Preferably, the centrifugation in step (1) is specifically as follows: firstly perform low-speed centrifugation at 1500-2000 rpm for 10-15 min, then take the supernatant and perform high-speed centrifugation at 12000-15000 rpm for 10-15 min, and collect the precipitate.

[0020] Preferably, the mass ratio of the drug to the hybrid nanoparticles in step (2) is 1:2 to 1:15.

[0021] Preferably, the mixing in step (2) is specifically: adding the methanol solution of the drug dropwise into the aqueous solution of the hybrid nanoparticles.

[0022] Preferably, the stirring time at room temperature in step (2) is 12 to 14 hours; the centrifugation is specifically at 1000 to 1200 rpm for 10 to 15 minutes, and the supernatant is collected.

[0023] Preferably, the mass ratio of the composite nanomaterial to the small interfering RNA in step (3) is 10:1 to 100:1.

[0024] Preferably, the mixing in step (3) is specifically: adding the DEPC (Diethypyrocarbonate) aqueous solution of the small interfering RNA to the aqueous solution of the composite nanomaterial.

[0025] Preferably, the incubation time in step (3) is 30 to 40 minutes.

[0026] The present invention provides an application of the hybrid dendrimer nanomaterial loaded with drugs and small interfering RNA in the preparation of anti-inflammatory / antioxidant / gene therapy drugs.

[0027] The present invention also provides a use of the hybrid dendrimer nanomaterial loaded with drugs and small interfering RNA in the preparation of drugs for treating Parkinson's disease.

[0028] The hybrid nanomaterial proposed in the present invention is a hybrid nano-delivery system formed by combining a hydroxyl-terminated phosphorus-containing dendrimer and an amino-terminated third-generation PAMAM dendrimer through hydrogen bonding and hydrophobic interactions. The hybrid nano-delivery system is then physically loaded with the drug curcumin and further loaded with siSNCA by electrostatic force to construct a hybrid dendrimer nano-drug P-ZYS@Cur for the anti-inflammatory / antioxidant combined treatment of PD. Dendrimers, due to their unique molecular structure and properties, can interact specifically with abnormal proteins, thereby blocking their aggregation. Studies have shown that PAMAM, carbosilanes, and phosphorus-containing dendrimers can effectively inhibit the aggregation of α-syn and also have a certain depolymerization effect on the fibers that have been formed. Rekas et al. found that amino-terminal PAMAM can inhibit the formation of α-syn fibers and has a good decomposition effect on existing α-syn aggregates (Macromol.Biosci.2009,9,230-238). Although hydroxyl-terminated phosphorus-containing dendrimers have good BBB penetration, their neutral surface charge makes them unsuitable for gene delivery. While amino-terminated PAMAM dendrimers have good gene delivery capacity, their positive surface charge can lead to strong cytotoxicity. Therefore, hybridizing these two dendrimers to form a hybrid nanodelivery system combines the advantages of both, potentially allowing for BBB penetration and improving the bioavailability of genes and drugs, ultimately achieving efficient PD treatment.

[0029] Beneficial effects

[0030] (1) The process of the present invention is simple, the required reaction conditions are simple, the product is easy to operate and separate, and it has good development prospects.

[0031] (2) The hybrid dendrimer nanomaterials prepared by the present invention have good biocompatibility, can effectively improve the bioavailability of Cur and siSNCA, and enhance their brain delivery efficiency, providing a new idea for the construction of safe and efficient nanomedicines.

[0032] (3) The hybrid dendrimer nanomaterial prepared by the present invention can efficiently penetrate the BBB. On the one hand, it can inhibit the expression and aggregation of α-syn, protect neurons from damage, and reverse neuronal mitochondrial dysfunction to restore neuronal function. On the other hand, it can alleviate microglial oxidative stress, inhibit its overactivation, and regulate the related pathways of NLRP3 inflammasome assembly in microglial cells to inhibit inflammatory responses, thereby effectively treating PD. The strategy provided by the present invention has good development prospects and application value in the treatment of PD or other neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1Schematic diagram of the preparation process of the hybrid dendrimer nanomaterials of the present invention and their use in the treatment of PD.

[0034] Figure 2 This is the ITC titration curve of the interaction between ZY-128 and G3 PAMAM-NH2 in Example 2.

[0035] Figure 3 The infrared absorption spectra of P-ZY, ZY-128 and G3 PAMAM-NH2 in Example 2 are shown.

[0036] Figure 4 The hydrated particle size (A) and surface potential diagram (B) of P-ZY and P-ZYS@Cur in Example 3.

[0037] Figure 5 TEM images of P-ZY (A) and P-ZYS@Cur (B) in Example 3.

[0038] Figure 6 Graph showing the cumulative release curves of Cur from free Cur and P-ZYS@Cur in phosphate buffered saline (PBS) containing 0.5% Tween 80 (v / v) in Example 4.

[0039] Figure 7 Effects of different concentrations of Cur (A) and P-ZY (B) on the viability of SH-SY5Y cells and BV2 cells in Example 5; Cell viability diagram after PS@Cur and P-ZYS@Cur were co-incubated with SH-SY5Y and BV2 cells for 24 hours (C).

[0040] Figure 8 This is the laser confocal analysis of lysosomal escape of Cy5 fluorescently labeled P-ZYS@Cur after co-incubation with SH-SY5Y cells for 1 h, 4 h, and 6 h in Example 6 (A) and the fluorescence colocalization Pearson correlation coefficient quantitative graph (B).

[0041] Figure 9 The in vitro BBB model constructed with the Transwell chamber in Example 7 (A) and the fluorescence intensity analysis graph of bEnd.3 cells in the upper chamber and SH-SY5Y cells in the lower chamber after 12 h of incubation of siSNCA-Cy5+Cur, PS-Cy5@Cur, and P-ZYS-Cy5@Cur with bEnd.3 cells in the upper chamber (B) and the fluorescence quantitative analysis graph of bEnd.3 cells in the upper chamber (C) and SH-SY5Y cells in the lower chamber (D).

[0042] Figure 10 Flow cytometry histogram (A) and fluorescence intensity quantitative analysis diagram (B) of ROS in SH-SY5Y cells after treatment with different materials in Example 8.

[0043] Figure 11 (A) and (B) are the flow cytometric analysis graphs of ROS in BV2 cells after treatment with different materials in Example 9.

[0044] Figure 12 These are laser confocal fluorescence analysis images of JC-1 polymers and JC-1 monomers in SH-SY5Y cells after treatment with different materials in Example 10.

[0045] Figure 13 Figure 11 shows the WB detection results of protein expression in BV2 cells after treatment with different materials in Example 11 (A) and the quantitative graph of ASC (B), Cleaved GSDMD (C), Cleaved caspase-1 (D) and NLRP3 complex (E) protein expression.

[0046] Figure 14 Quantitative analysis of the expression levels of pro-inflammatory cytokines TNF-α (A), IL-1β (B), IL-6 (C) and anti-inflammatory cytokine IL-10 (D) in the supernatant of BV2 cells after treatment with different materials in Example 11.

[0047] Figure 15 Small animal fluorescence imaging analysis of the accumulation of siSNCA-Cy5+Cur and P-ZYS-Cy5@Cur in the mouse brain at 0 h, 4 h, 8 h, 12 h, and 24 h after tail vein injection of MPTP-induced PD mice in Example 12 (A) and fluorescence imaging analysis of the drugs in the mouse brain, heart, liver, spleen, lung, and kidney 12 h after injection (B); quantitative analysis of fluorescence intensity for in vivo imaging (C) and in vitro imaging (D).

[0048] Figure 16 Figure 13 shows the behavioral profile of the open field test in MPTP-induced PD mice after 18 days of treatment with PBS, siSNCA + Cur, P-ZYR, P-ZYS, and P-ZYS@Cur, respectively. It also shows the quantitative analysis of the immobility time in the open field (B), the average movement speed (C), the movement distance (D), and the activity time (E).

[0049] Figure 17 Figure 14 shows the WB detection results of α-syn and aggregated α-syn protein expression in the brain of MPTP-induced PD mice after 18 days of treatment in each group (A) and the quantitative graphs of α-syn (B) and aggregated α-syn (C) protein expression.

[0050] Figure 18The figures show the immunofluorescence staining results of TH-positive neurons in the striatum region of the brains of MPTP-induced PD mice in Example 15 after 18 days of treatment in each group.

[0051] Figure 19 The figures show the immunofluorescence staining results of TH-positive neurons in the substantia nigra region of the brains of MPTP-induced PD mice in Example 15 after 18 days of treatment in each group.

[0052] Figure 20 Figure 16 shows the Western blot analysis of protein expression in the brain tissue of MPTP-induced PD mice after 18 days of treatment with PBS, siSNCA + Cur, P-ZYR, P-ZYS, and P-ZYS@Cur, respectively. (A) Also shown are grayscale quantitative images of NLRP3 complex (B), cleaved GSDMD (C), cleaved caspase-1 (D), and ASC (E) protein expression.

[0053] Figure 21 Quantitative analysis of the expression levels of pro-inflammatory cytokines TNF-α (A), IL-1β (B), IL-6 (C) and anti-inflammatory cytokine IL-10 (D) in the brains of MPTP-induced PD mice in Example 16 after 18 days of treatment in each group. DETAILED DESCRIPTION

[0054] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0055] Unless otherwise specified, all chemical reagents were used without further purification.

[0056] Hydroxylated phosphorus-containing dendrimer ZY-128 was obtained from the research group of Professor JP Majoral of the French National Center for Scientific Research. The third-generation PAMAM dendrimer was obtained from Shandong Weihai Chenyuan Molecular New Materials Co., Ltd. Cur was obtained from Shanghai MacLean Biochemical Technology Co., Ltd. siSNCA was obtained from Shanghai Jima Pharmaceutical Technology Co., Ltd. 1-Methyl-4-phenylpyridinium iodide (MPP +Iodide) was purchased from MedChemExpress. SH-SY5Y cells (human neuroblastoma cell line), BV2 cells (mouse microglial cell line), and bEnd.3 cells (mouse brain microvascular endothelial cells) were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. DMEM medium, fetal bovine serum, penicillin-streptomycin, and trypsin were purchased from Hangzhou Jinuo Biomedical Technology Co., Ltd. Lysosomal green fluorescent probe, JC-1 detection kit, and ROS detection kit were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. CCK-8 was purchased from Wuhan Yacoin Biotechnology Co., Ltd. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP.HCl) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. C57BL / 6 mice were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. Recombinant anti-alpha-synuclein and recombinant anti-cleaved N-terminal GSDMD were purchased from Abcam. NLRP3 polyclonal antibody and ASC / TM(1) polyclonal antibody were purchased from Wuhan Tri-Eagle Biotechnology Co., Ltd. Caspase 1p20 (Cleaved Asp296) polyclonal antibody was purchased from ThermoFisher Scientific (Waltham, MA). Water with a resistivity higher than 18.2 MΩ·cm used in all experiments was purified using a PURIST UV ultrapure water system (Shanghai Ruifulai Biotechnology Co., Ltd.).

[0057] The present invention uses Zeta potential and dynamic light scattering analysis (DLS), ultraviolet spectrophotometer, transmission electron microscope (TEM), Fourier transform infrared spectrometer (FTIR), and isothermal titration calorimetry (ITC) to characterize the physical and chemical properties of the hybrid dendrimer nanodrugs P-ZY and P-ZYS@Cur prepared in the examples. The CCK-8 method was used to analyze and evaluate the cytotoxicity of P-ZYS@Cur and related control materials; the lysosomal escape ability of the gene-carrying nanomaterials was detected by CLSM; an in vitro BBB model was constructed using a Transwell chamber to investigate the ability of the nanomaterials to penetrate the BBB; the effects of the materials on ROS in neurons and microglia were evaluated by flow cytometry; the effects of the materials on the mitochondrial membrane potential (MMP) level of neurons were evaluated by CLSM; the expression of NLRP3, cleaved caspase-1, cleaved-GSDMD and ASC proteins in microglia were detected by Western blot (WB); and the expression levels of inflammatory-related factors TNF-α, IL-1β, IL-6 and IL-10 were detected by enzyme-linked immunosorbent assay (ELISA) kits.

[0058] A PD mouse model was established by intraperitoneal injection of MPTP, and the restorative effect of the material on movement disorders in PD mice was evaluated through behavioral tests. Western blot was used to detect the inhibitory effect of the nanomaterial on α-syn expression in the substantia nigra region of the mouse brain after treatment. Immunofluorescence staining was used to analyze the effect of the nanomaterial on the number of TH-positive neuronal cells in the substantia nigra region and striatum of the PD mouse brain. Western blot was used to detect the inhibitory effect of the nanomaterial on the expression and assembly of NLRP3 complex, cleaved caspase-1, cleaved-GSDMD and ASC protein in microglia in the substantia nigra region of the PD mouse brain. ELISA kits were used to detect the expression levels of inflammatory-related factors TNF-α, IL-1β, IL-6 and IL-10 in the brain tissue of PD mice after treatment.

[0059] Example 1

[0060] The preparation process of hybrid dendrimer nanomaterials in this embodiment is as follows: Figure 1 As shown, the following steps are included:

[0061] (1) 0.5 mg of hydroxyl-terminated phosphorus dendrimer ZY-128 was dissolved in 0.2 mL of methanol and then added dropwise to a 1 mg / mL G3 PAMAM-NH2 aqueous solution (1 mL). The mixture was stirred at room temperature for 12 h until the methanol was completely evaporated. The mixed solution was first centrifuged at 1500 rpm and 4°C for 10 min, the supernatant was collected, and then centrifuged at 12000 rpm and 4°C for 10 min. The precipitate was collected to obtain P-ZY hybrid nanoparticles. In addition, the P-ZY hybrid nanoparticles were freeze-dried, and the calculated yield was 93.64 ± 1.34%;

[0062] (2) 0.2 mg of Cur was dissolved in 100 μL of methanol and added dropwise to a solution containing 2 mg / mL P-ZY (1 mL). The mixture was stirred at room temperature for 12 h. After the reaction was completed, the mixed solution was transferred to a centrifuge tube and centrifuged at 1000 rpm and 4 °C for 10 min. The supernatant was collected to obtain the PAMAM / ZY-128@Cur nanocomposite (denoted as P-ZY@Cur), and the unloaded Cur was precipitated. At the same time, the Cur content in P-ZY@Cur was measured by UV-vis, and the Cur loading rate was calculated to be 4.96% and the encapsulation rate was 54.55%.

[0063] (3) 84 μg of P-ZY@Cur was mixed with 1 μL of siSNCA suspension (1 μg / μL), where the mass ratio of P-ZY to siSNCA was 80:1. After incubation at room temperature for 30 min, P-ZYS@Cur was obtained.

[0064] In addition, control materials were prepared as follows:

[0065] 80 μg of P-ZY was mixed with 1 μL of siRNA (small interfering RNA) suspension (1 μg / μL) at a mass ratio of 80:1. After incubation at room temperature for 30 min, P-ZYR was obtained.

[0066] 0.4 mg Cur was dissolved in 1 mL methanol, and 10 μL was added dropwise into an aqueous solution containing 0.1 mg / mL G3 PAMAM-NH2 (530 μL), with the mass ratio of G3 PAMAM-NH2 to Cur being 53:4. The reaction was stirred openly at room temperature for 12 h to obtain a PAMAM@Cur nanocomplex; 53 μg PAMAM@Cur was mixed with 1 μL siSNCA suspension (1 μg / μL), with the mass ratio of G3PAMAM-NH2 to siSNCA being 53:1, and incubated at room temperature for 30 min to obtain PS@Cur.

[0067] Example 2

[0068] G3 PAMAM-NH2 and ZY-128 were dissolved in 50% volume methanol / water solution with concentrations of 70 μM and 47 μM, respectively. 50 μL of ZY-128 solution was drawn into the syringe, and 300 μL of G3 PAMAM solution was placed in the sample cell. The titration parameters were set to 2 μL per drop, 120 s interval, 25 drops in total, titration temperature of 25 ° C, and stirring speed of 350 r / min. The blank solvent (50% volume methanol / water solution) was titrated with ZY-128 solution as the control group. The titration curve is shown in Figure 2. Figure 2 As shown, the test data were fitted based on the SequentialTwoSite model, and it was found that G3 PAMAM-NH2 had two binding sites with ZY-128, and the corresponding dissociation constant of one of the binding sites was Kd1 = 3.05×10 -8 , enthalpy change ΔH1=-1369, binding constant Ka1=3.28×10 7 , entropy change ΔS = -4.45×10 3 , where ΔH<0, ΔS<0, indicating that the binding site interacts through hydrogen bonds; the corresponding dissociation constant of the other binding site is Kd2=1.00×10 -8 , enthalpy change ΔH2=1291, binding constant Ka2=1.00×10 8 , entropy change ΔS=4.48×10 3 , where ΔH>0 and ΔS>0, indicating that the binding site is driven by hydrophobic interaction. Therefore, there is a strong binding force between G3 PAMAM-NH2 and ZY-128, and they are combined with each other through hydrogen bonds and hydrophobic interactions to form hybrid nanoparticles P-ZY.

[0069] The infrared spectra of G3 PAMAM-NH2, ZY-128 and the P-ZY samples prepared in Example 1 were characterized by Fourier transform infrared spectroscopy. The results are shown in Figure 2. Figure 3 As shown, 3400 cm in P-ZY -1 The characteristic peaks around the left and right sides decreased in wavenumber and red-shifted, indicating the formation of hydrogen bonds in P-ZY. This indicates that there is a hydrogen bond between the terminal amino group of G3 PAMAM-NH2 and the terminal hydroxyl group of ZY-128, which is consistent with the ITC titration results.

[0070] Example 3

[0071] The particle size and potential of P-ZY and P-ZYS@Cur were measured and their morphology was characterized. The P-ZY and P-ZYS@Cur prepared in Example 1 were dissolved in ultrapure water to prepare solutions with a concentration of 0.1 mg / mL, and the hydrated particle size and surface potential were measured. Figure 4As shown in AB, the hydrated particle size of P-ZY is 211.57±1.24nm and the potential is 29.17±0.42mV; after loading Cur and siSNCA, the hydrated particle size increases to 322.30±21.19nm and the potential changes to 22.7±0.36mV. Figure 5 As shown in A, P-ZY is a uniform spherical shape. Figure 5 B shows that when P-ZY is loaded with Cur and siSNCA, the particle size of the P-ZY complex increases and still presents a uniform spherical shape.

[0072] Example 4

[0073] In order to detect the drug release of P-ZYS@Cur, a PBS solution containing Tween 80 (0.5%, v / v) was used as the release solution. 152μg / mL P-ZYS@Cur solution (1mL) and 83μg / mL Cur solution (1mL) were respectively placed in 1000D dialysis bags, and the dialysis bags were placed in the above-mentioned release solution (30mL). Placed in a 37°C constant temperature shaker, 1mL of release solution was drawn at predetermined time points (0, 1, 2, 4, 6, 8, 10, 12, 24, 36, 72h), and 1mL of fresh isothermal release solution was added. The absorption intensity of the release solution drawn at different time points at 426nm was tested by ultraviolet absorption spectroscopy. According to the standard curve, the Cur release curve of P-ZYS@Cur at different time points was drawn. As shown Figure 6 As shown in the data, the release rate of Cur from P-ZYS@Cur in 0.5% Tween 80 PBS solution was slower than that of free Cur, with a cumulative drug release rate of approximately 70.93% over 72 h. This can effectively delay the burst release of Cur and prolong the drug action time. This can be attributed to the strong binding force or encapsulation stability between Cur and the P-ZYS carrier, which delays the release rate of Cur.

[0074] Example 5

[0075] SH-SY5Y and BV2 cells were used as cell models to test the cytotoxicity of P-ZYS@Cur and related materials prepared in Example 1. SH-SY5Y or BV2 cells in the logarithmic growth phase were collected and 1×10 4Cells were seeded at a density of 100 μg / mL in 96-well plates in complete medium supplemented with 100 U / mL penicillin, 100 U / mL streptomycin, and 10% FBS and incubated for 12 h at 5% CO2 and 37°C. The original medium was discarded, and medium containing different concentrations of Cur (0, 1, 2, 4, 6, 8, and 10 μg / mL), P-ZY (0, 1, 5, 10, 20, 40, 60, and 80 μg / mL), PS@Cur, or P-ZYS@Cur (G3 PAMAM-NH2 at 26.67 μg / mL, P-ZY at 80 μg / mL, Cur at 4 μg / mL, and siSNCA at 1 μg / mL) was added to each well of the plate and co-cultured with the cells at 5% CO2 and 37°C for 24 h. PBS was added to the control group (NC group). After that, the plate was removed, the original culture medium was discarded, and the cells were washed three times with PBS. Fresh culture medium containing 10% (v / v) CCK-8 was added and the cells were incubated in the incubator for another 1 hour. Finally, the absorbance of each well was measured at a wavelength of 450nm using a multifunctional microplate reader. The cells treated with PBS were used as blank controls. Figure 7 As shown in A, within the experimental concentration range, with the increase of Cur and P-ZY concentrations, each group showed no obvious cytotoxicity to SH-SY5Y and BV2 cells. It is worth noting that after PS@Cur was co-cultured with SH-SY5Y and BV2 cells for 24 hours, as shown in Figure 7 As shown in B, the cell viability decreased significantly, while after incubation of P-ZYS@Cur with SH-SY5Y and BV2 cells for 24 h, as shown in Figure 7 As shown in Figure C, there was almost no decrease in cell viability. The results showed that after ZY-128 was hybridized with G3 PAMAM-NH2, the cytotoxicity caused by the amino groups on the surface of G3 PAMAM-NH2 was significantly reduced.

[0076] Example 6

[0077] In order to verify the lysosomal escape ability of P-ZYS@Cur nanocomplex, SH-SY5Y cells in the logarithmic growth phase were collected and cultured at a rate of 1×10 5 The cells were seeded at a density of 10 cells per well in a confocal microplate and cultured at 5% CO2 and 37°C for 12 hours. The culture medium was then replaced with DMEM containing P-ZYS-Cy5@Cur (P-ZY concentration was 80 μg / mL, siSNCA was fluorescently labeled with Cy5) and incubated with the cells for 1, 4, and 6 hours, respectively. Subsequently, after labeling lysosomes with a lysosomal green fluorescent probe, the co-localization of siSNCA-Cy5 with lysosomes was detected by CLSM. The results are shown in Figure 2. Figure 8As shown in AB, when incubated for 1 hour, the colocalization Pearson correlation coefficient of Cy5 fluorescence and lysosomal green fluorescence was approximately 0.13. At 4 hours of incubation, the colocalization coefficient reached a maximum of 0.42, and at 6 hours, the colocalization coefficient decreased to approximately 0.26. This indicates that P-ZYS@Cur successfully carried siSNCA to escape the lysosomes of SH-SY5Y cells and enter the cytoplasm, thereby achieving efficient transfection and inhibiting the expression of α-syn protein.

[0078] Example 7

[0079] In order to verify the ability of P-ZYS@Cur nanocomplexes to penetrate the simulated BBB in vitro, an in vitro BBB model was constructed using Transwell chambers, e.g. Figure 9 As shown in A, bEnd.3 cells in the logarithmic growth phase were collected and seeded in the upper chamber and cultured under 5% CO2 and 37°C for 5-7 days. During this period, the transendothelial electrical resistance (TEER) was monitored. When the TEER was 200 Ω·cm 2 When , it indicates that the in vitro BBB model has been successfully constructed; then, SH-SY5Y cells were inoculated in the lower chamber and cultured under 5% CO2 and 37°C for 12 hours. The original culture medium in the upper chamber was discarded and replaced with DMEM culture medium containing siSNCA-Cy5+Cur, PS-Cy5@Cur or P-ZYS-Cy5@Cur (G3 PAMAM-NH2 concentration is 26.67μg / mL, P-ZY concentration is 80μg / mL, Cur concentration is 4μg / mL, siSNCA-Cy5 concentration is 1μg / mL). After culturing for 12 hours, the culture medium in the upper and lower chambers was discarded and washed three times with PBS. The fluorescence intensity in the cells in the upper and lower chambers was observed using a small animal fluorescence imager. The results are as follows Figure 9 As shown in BD, compared with siSNCA-Cy5+Cur and PS-Cy5@Cur, the cells in the upper chamber of the P-ZYS-Cy5@Cur group ( Figure 9 C) Lower chamber cells ( Figure 9 The fluorescence intensity in D) is the strongest, indicating that the hydroxyl groups at the ends of the phosphorus-containing dendrimers in the hybrid complex can promote the uptake of epithelial cells in the upper chamber and penetrate the BBB composed of endothelial cells, thereby increasing the uptake of nerve cells in the lower chamber.

[0080] Example 8

[0081] SH-SY5Y cells were used as a cell model to evaluate the ability of P-ZYS@Cur to alleviate oxidative stress. In order to verify the effect of P-ZYS@Cur nanocomplex on ROS scavenging in SH-SY5Y cells, SH-SY5Y cells in the logarithmic growth phase were collected and cultured at 2×10 5Cells were seeded at a density of 10 cells per well in a 6-well plate and cultured at 5% CO2 and 37°C for 12 h. The culture medium was replaced with DMEM medium containing PBS, siSNCA + Cur, P-ZYR, P-ZYS, or P-ZYS@Cur (P-ZY concentration was 80 μg / mL, Cur concentration was 4 μg / mL, siSNCA-Cy5 concentration was 1 μg / mL) and incubated for 6 h. The original culture medium was discarded and MPP was added. + The cells were stimulated with DMEM medium (concentration of 1mM) and incubated for 18h. The PBS-treated group served as the positive control group, and the untreated cells served as the negative control group (NC group). After trypsin digestion and collection of cells, the cell pellet was resuspended with 1mL PBS, and the ROS fluorescent probe DCFH-DA was added and incubated with the cells at 37°C for 20min. After washing three times with PBS to remove the probe that was not bound to the cells, the cell pellet was resuspended with 300μL PBS and transferred to a flow tube. The effect of the nanomaterial on MPP was evaluated by flow cytometry. + The effect of ROS clearance in SH-SY5Y cells after stimulation. Figure 10 As shown in Figures AB, siSNCA + Cur exhibited significant ROS scavenging activity compared to the positive control, attributed to the antioxidant activity of Cur. P-ZYR exhibited ROS scavenging ability through the inherent anti-inflammatory activity of the hydroxyphosphorus dendrimer. Furthermore, P-ZYS, due to its combined anti-inflammatory activity of the hydroxyphosphorus dendrimer and its ability to inhibit α-syn expression, exhibited enhanced ROS scavenging activity, effectively alleviating excessive oxidative stress in macrophages. Compared to the other treatment groups, the P-ZYS@Cur group exhibited the most significant intracellular ROS scavenging effect. This is due to the synergistic effects of Cur's potent anti-inflammatory / antioxidant properties, combined with the actions of ZY-128 and siSNCA, resulting in a multi-component antioxidant effect that maximally alleviated oxidative stress.

[0082] Example 9

[0083] BV2 cells were used as a cell model to evaluate the ability of P-ZYS@Cur to alleviate oxidative stress. In order to verify the effect of P-ZYS@Cur nanocomplex on ROS scavenging in BV2 cells, BV2 cells in the logarithmic growth phase were collected and cultured at 2×10 5 Cells were seeded at a density of 10 cells per well in a 6-well plate and cultured at 5% CO2 and 37°C for 12 h. The culture medium was replaced with DMEM containing PBS, siSNCA+Cur, P-ZYR, P-ZYS, or P-ZYS@Cur (P-ZY concentration was 80 μg / mL, Cur concentration was 4 μg / mL, siSNCA-Cy5 concentration was 1 μg / mL) and incubated for 6 h. The original culture medium was discarded and MPP was added. +The cells were stimulated with DMEM medium (concentration of 2mM) and incubated for 18h. The PBS-treated group served as the positive control group, and the untreated cells served as the negative control group (NC group). After trypsin digestion and collection of cells, the cell pellet was resuspended with 1mL PBS, and the ROS fluorescent probe DCFH-DA was added and incubated with the cells at 37°C for 20min. After washing three times with PBS to remove the probe that was not bound to the cells, the cell pellet was resuspended with 300μL PBS and transferred to a flow tube. The effect of the nanomaterial on MPP was evaluated by flow cytometry. + The effect of ROS clearance in SH-SY5Y cells after stimulation. Figure 11 As shown in Figures AB, siSNCA + Cur exhibited significant ROS scavenging activity compared to the positive control. P-ZYR and P-ZYS exhibited ROS scavenging capabilities through the inherent anti-inflammatory activity of the hydroxyphosphine dendrimers. Compared to all other treatment groups, the P-ZYS@Cur group exhibited the most pronounced intracellular ROS scavenging effect. This is attributed to the synergistic effects of Cur's potent anti-inflammatory / antioxidant properties and ZY-128's anti-inflammatory activity, synergizing the antioxidant activity of each component to alleviate oxidative stress.

[0084] Example 10

[0085] In order to verify the effect of P-ZYS@Cur nanocomplex on mitochondrial membrane potential of SH-SY5Y cells, SH-SY5Y cells in the logarithmic growth phase were collected and cultured at 2×10 5 Cells were seeded at a density of 10 cells per well in a confocal dish and cultured at 5% CO2 and 37°C for 12 h. The culture medium was replaced with DMEM containing PBS, siSNCA + Cur, P-ZYR, P-ZYS, or P-ZYS@Cur (P-ZY concentration was 80 μg / mL, Cur concentration was 4 μg / mL, siSNCA-Cy5 concentration was 1 μg / mL) and incubated for 6 h. The original culture medium was discarded and MPP was added. + (Concentration is 1mM) DMEM medium was used to stimulate the cells and incubated for 18 hours. The PBS-treated group served as the positive control group, and the untreated cells served as the negative control group (NC group). 0.5mL of JC-1 staining working solution was added to each dish and incubated at 37°C in a cell culture incubator for 20 minutes. After the incubation, the cells were washed twice with JC-1 staining buffer and then covered with PBS. The changes in MMP in different treatment groups were observed using a confocal laser microscope. When MMP is high, JC-1 aggregates in the matrix of mitochondria to form polymers (J-aggregates) and produce red fluorescence. When MMP is low, JC-1 cannot aggregate in the matrix of mitochondria. At this time, JC-1 is a monomer (J-monomer) and produces green fluorescence. The change in mitochondrial membrane potential can be detected by the change in fluorescence color. The results are as follows. Figure 12 As shown, the JC-1 monomer content in the positive control group cells treated with PBS increased, indicating that MPP + It significantly induced a decrease in MMP in SH-SY5Y cells, leading to an imbalance in mitochondrial homeostasis. The number of green fluorescence-positive cells in the siSNCA + Cur, P-ZYR, and P-ZYS groups decreased compared to the positive control group. Cells treated with P-ZYS@Cur exhibited the least green fluorescence and the most red fluorescence, demonstrating its ability to effectively restore MMP and maintain mitochondrial homeostasis.

[0086] Example 11

[0087] In order to explore the molecular mechanism by which P-ZYS@Cur nanocomplexes regulate macrophages to fight against inflammation, BV2 cells in the logarithmic growth phase were collected and cultured at 2×10 5 Cells were seeded at a density of 10 cells per well in a 6-well plate and cultured at 5% CO2 and 37°C for 12 h. The culture medium was replaced with DMEM containing PBS, siSNCA+Cur, P-ZYR, P-ZYS, or P-ZYS@Cur (P-ZY concentration was 80 μg / mL, Cur concentration was 4 μg / mL, siSNCA-Cy5 concentration was 1 μg / mL) and incubated for 6 h. The original culture medium was discarded and MPP was added. + The cells were stimulated with DMEM medium (at a concentration of 2 mM) and incubated for 18 hours. The PBS-treated group served as the positive control group, and the untreated cells served as the negative control group (NC group). Subsequently, the culture medium was collected, the cells were washed twice with PBS, and after trypsin digestion and collection of the cells, the cells were washed twice with PBS to collect the cells. The protein concentration was determined, and then SDS-PAGE electrophoresis, membrane transfer, immunoreaction, and ECL chemical developer fixation experiments were performed in sequence. The protein content of ASC, Cleaved GSDMD, Cleaved caspase-1, and NLRP3 complex in the cells was studied, with β-actin used as an internal reference. The results are shown in Figure 2. Figure 13 AE shows that in MPP + Under stimulation, the expression of ASC, Cleaved GSDMD, Cleaved caspase-1 and NLRP3 complex pro-inflammatory pathway related proteins were upregulated, proving that BV2 cells were stimulated by MPP. +After treatment with P-ZYS@Cur, the levels of ASC, Cleaved GSDMD, Cleaved caspase-1 and NLRP3 complex pro-inflammatory pathway-related proteins in BV2 cells decreased, indicating that P-ZYS@Cur enhances the anti-inflammatory / antioxidant activity of Cur by improving its bioavailability, and at the same time, combines the anti-inflammatory advantages of the hydroxyl-terminated phosphorus dendrimer carrier itself to alleviate the MPP + Induced oxidative stress and inflammatory responses.

[0088] The contents of pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and anti-inflammatory cytokine IL-10 in the collected culture medium were detected using corresponding ELISA kits. Figure 14 AD shows that compared with the normal NC group, MPP + After induction, the levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in BV2 macrophages increased significantly, while the level of the anti-inflammatory cytokine IL-10 decreased significantly. After treatment with each material group, the levels of pro-inflammatory cytokines decreased to varying degrees, while the levels of anti-inflammatory cytokines increased. The P-ZYS@Cur-treated group showed the lowest levels of TNF-α, IL-1β, and IL-6, and the highest IL-10 level (except for the NC group), indicating that P-ZYS@Cur can alleviate the inflammatory response of macrophages by scavenging ROS and inhibiting the self-assembly of the inflammasome NLRP3.

[0089] Example 12

[0090] All animal experiments were approved by the Science and Technology Ethics Committee of Donghua University and were conducted in strict accordance with the standards of the Animal Protection Association. Female SPF-grade 10-week-old C57BL / 6 mice used in the experiment were purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd. In order to explore the ability of P-ZYS@Cur hybrid nanomaterials to penetrate the BBB in PD mice, MPTP (30 mg / kg) was injected intraperitoneally for 7 consecutive days. The mice showed behaviors such as arched back, erect hair, and frequent tremors, indicating that the PD mouse model was successfully established. PD mice were injected with siSNCA-Cy5+Cur or P-ZYS-Cy5@Cur (siSNCA-Cy5 dose of 0.5 mg / kg, 3 mice in each group) through the tail vein. After 0h, 4h, 8h, 12h and 24h, the fluorescence intensity in the mouse brain was analyzed by the small animal fluorescence imaging system. The results are shown in the figure. Figure 15As shown in A and C, compared with the free drug group, the fluorescence intensity in the brain of mice in the P-ZYS-Cy5@Cur group was stronger at all time points; the fluorescence intensity in the brain of mice in the free drug group reached the highest level 8 hours after injection, while the fluorescence intensity in the brain of mice in the P-ZYS-Cy5@Cur group was the highest at 12 hours, and the fluorescence intensity decreased slightly after 24 hours. In order to explore the tissue distribution of P-ZYS-Cy5@Cur in PD mice, mice were euthanized 12 hours after injection, and in vitro fluorescence imaging of the brain, heart, liver, spleen, lungs and kidneys of mice was performed. The results are shown in Figure 15 As shown in Figures B and D, the fluorescence intensity in the brains of mice in the P-ZYS-Cy5@Cur group was significantly higher than that in the siSNCA-Cy5+Cur group, consistent with the in vivo fluorescence imaging results. At this time, the fluorescence intensity in the kidneys of both groups of mice was significantly higher than that in other organs, indicating that the hybrid dendrimer complex successfully loaded the drug and accumulated it in the brains of PD mice, improving the efficiency of drug penetration through the BBB and prolonging the drug's residence time in the brain, ultimately leading to drug metabolism in the mice's kidneys.

[0091] Example 13

[0092] In order to explore the therapeutic effect of P-ZYS@Cur on PD mice, PD mice were randomly divided into 5 groups (6 mice in each group), and 200 μL PBS (positive control group was recorded as PBS group), siSNCA+Cur, P-ZYR, P-ZYS, P-ZYS@Cur (P-ZYS@Cur dose was 40 mg / kg) were injected through the tail vein every 3 days, for a total of 5 injections. The healthy mice in the control group were injected with PBS intraperitoneally (the negative control group was recorded as Normal group). After the end of the treatment (18 days), each group of mice underwent three days of behavioral training, and behavioral tests were performed on the mice after the training. The results of the open field behavioral test of mice are shown in Figure 2. Figure 16 As shown in A, after MPTP induction, PD mice tended to stay in the corners of the open field and walk along the edge. After treatment with siSNCA + Cur, P-ZYR, and P-ZYS, the tendency of PD mice to explore the center of the open field increased. After treatment with P-ZYS@Cur, the tendency of PD mice to explore the center of the open field returned to a level similar to that of normal mice. In addition, as Figure 16 As shown in BE, the average movement speed of PD mice was significantly reduced, and the activity time and movement distance were significantly reduced. After P-ZYS@Cur treatment, compared with other treatment groups, the immobility time of PD mice was significantly reduced, the activity time and movement distance were significantly increased, and the average movement speed was also significantly improved, returning to a level close to that of normal group mice, indicating that P-ZYS@Cur can significantly alleviate the behavioral disorders of PD mice.

[0093] Example 14

[0094] In order to further verify the molecular mechanism of P-ZYS@Cur in treating PD mice, the mice were euthanized after the behavioral test, and the substantia nigra of the brain tissue of each group of mice was taken for processing. 200 μL of tissue lysis solution (RIPA lysis solution containing 1% PMSF) was added to the substantia nigra of each mouse, and ultrasonic homogenization was performed on ice (power of 80HZ, ultrasound for 5s, interval of 10s, 6 rounds). The tissue homogenate was then centrifuged at 12000rpm and 4°C for 10min, and the supernatant solution was taken for protein concentration determination. Subsequently, SDS-PAGE electrophoresis, membrane transfer, immunoreaction, and ECL chemical developer fixation experiments were performed in sequence to detect the expression of α-syn and aggregated α-syn proteins in the tissue, with β-actin used as an internal reference. The results are shown in Figure 2. Figure 17 As shown in Figures AC, under MPTP induction, the expression of α-syn and aggregated α-syn proteins in the substantia nigra of PD mice was significantly upregulated. After treatment with P-ZYS@Cur, the expression of α-syn and aggregated α-syn proteins in the mouse brain tissue was most significantly downregulated compared with other treatment groups. This indicates that P-ZYS@Cur inhibits the abnormal expression and aggregation of α-syn by improving the bioavailability of Cur and siSNCA, and can effectively alleviate the deterioration of the disease process caused by pathological α-syn aggregation, thereby effectively treating PD.

[0095] Example 15

[0096] To further verify the effect of P-ZYS@Cur treatment on the neuronal function of PD mice, the mice were euthanized after the behavioral test, and the brain tissues of mice in different treatment groups were subjected to immunofluorescence staining. Figure 18 As shown in the results, the number of TH-positive neurons in the striatum of PD mice was significantly reduced. After treatment with different drugs, the number of TH-positive neurons increased. Among them, the number of TH-positive neurons in the striatum of the P-ZYS@Cur group increased the most, almost returning to the level of normal mice. At the same time, the number of TH-positive neurons in the substantia nigra of PD mice was also significantly reduced ( Figure 19 ). Treatment with siSNCA+Cur, P-ZYR, and P-ZYS increased the number of TH-positive neurons in the substantia nigra, with P-ZYS@Cur treatment showing the greatest recovery. This result is attributed to P-ZYS@Cur's ability to synergistically enhance the anti-inflammatory / antioxidant activity of Cur and the anti-inflammatory activity of ZY-128, while simultaneously reducing the abnormal expression and aggregation of α-syn, significantly alleviating neuronal oxidative stress and mitochondrial dysfunction, thereby effectively restoring neuronal function.

[0097] Example 16

[0098] In order to further verify the effect of P-ZYS@Cur treatment on the function of microglia in PD mice, the mice were euthanized after the behavioral test, and the substantia nigra and striatum of the brain tissue of each group of mice were taken and minced, and 200 μL of tissue lysis solution (RIPA lysis solution containing 1% PMSF) was added to each mouse. The homogenate was ultrasonically homogenized on ice (power of 80HZ, ultrasonication for 5s, interval of 10s, 6 rounds), and then the tissue homogenate was centrifuged at 12000rpm and 4℃ for 10min. The supernatant solution was taken to determine the protein concentration. Then, SDS-PAGE electrophoresis, transfer, immunoreaction, and ECL chemical developer fixation experiments were carried out in sequence, and the protein contents of ASC, Cleaved GSDMD, Cleaved caspase-1 and NLRP3 complex in the tissue were studied, with β-actin used as an internal reference. The results are shown in Figure 2. Figure 20 As shown in Figures AE, under MPTP stimulation, the expression of NLRP3 complex, Cleaved GSDMD, Cleaved caspase-1, and ASC, proteins involved in the pro-inflammatory pathway, was significantly upregulated, demonstrating that microglia in the brains of PD mice are under severe oxidative stress and intense inflammatory responses. After treatment with P-ZYS@Cur, the levels of these pro-inflammatory pathway-related proteins in the mouse brains were significantly decreased, indicating that P-ZYS@Cur significantly improves the brain accumulation and bioavailability of Cur and siSNCA through BBB penetration mediated by hydroxylated phosphorus-containing dendrimers, enhancing their anti-inflammatory / antioxidant activities. Simultaneously, combined with the anti-inflammatory benefits of the phosphorus-containing dendrimer carrier itself, it effectively reduces the stimulation of abnormal α-syn expression and aggregation on microglia, thereby alleviating excessive oxidative stress and attenuating inflammatory responses in the brains of PD mice.

[0099] The corresponding ELISA kits were used to detect the levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and anti-inflammatory cytokine IL-10 in the brain tissue homogenates of mice in each group. Figure 21 As shown in Figures AD, compared with normal mice, the levels of pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in the brains of PD mice were significantly increased, while the level of the anti-inflammatory cytokine IL-10 was significantly decreased. After treatment in each group, the levels of pro-inflammatory cytokines decreased to varying degrees, while the levels of anti-inflammatory cytokines increased. Among all treatment groups, the P-ZYS@Cur-treated group showed the lowest levels of TNF-α, IL-1β, and IL-6, and the highest levels of IL-10, indicating that P-ZYS@Cur can effectively treat PD by inhibiting the self-assembly of the inflammasome NLRP3 and alleviating the inflammatory response of microglia in the brain.

Claims

1. A hybrid dendrimer nanomaterial loaded with drugs and small interfering RNA, characterized in that: The hybrid dendrimer nanomaterial comprises a hybrid nano delivery system consisting of a hydroxyl-terminated phosphorus-containing dendrimer and a third-generation amino-terminated polyamidoamine dendrimer, a drug and a small interfering RNA; wherein the hybrid nano delivery system is loaded with the drug and the small interfering RNA.

2. The hybrid dendrimer nanomaterial loaded with drugs and small interfering RNA according to claim 1, characterized in that: The structural formula of the phosphorus-containing dendrimer at the hydroxyl end is: The structural formula of the third generation amino-terminated polyamidoamine dendrimer is: The drug includes curcumin; The small interfering RNA includes α-synuclein small interfering RNA.

3. A method for preparing a hybrid dendrimer nanomaterial loaded with a drug and small interfering RNA according to any one of claims 1 to 2, comprising the following steps: (1) mixing a hydroxyl-terminated phosphorus-containing dendrimer with a third-generation amino-terminated polyamide-amine dendrimer, stirring at room temperature, and centrifuging to prepare hybrid nanoparticles of the hydroxyl-terminated phosphorus-containing dendrimer and the third-generation amino-terminated polyamide-amine dendrimer, thereby forming a hybrid nanodelivery system; (2) mixing the drug with the hybrid nanoparticles, stirring at room temperature, and centrifuging to prepare a composite nanomaterial; (3) The composite nanomaterial is mixed with small interfering RNA and incubated at room temperature to prepare a hybrid dendrimer nanomaterial.

4. The preparation method according to claim 3, characterized in that The mass ratio of the phosphorus-containing dendrimer at the hydroxyl end of step (1) to the third-generation amino-terminated polyamide-amine dendrimer is 1:0.5 to 1:

4.

5. The preparation method according to claim 3, characterized in that The mixing in step (1) is specifically as follows: adding dropwise the methanol solution of the hydroxyl-terminated phosphorus-containing dendrimer to the aqueous solution of the third-generation amino-terminated polyamide-amine dendrimer; the stirring reaction time at room temperature is 12 to 14 hours; and the centrifugation is specifically as follows: centrifuging at 1500 to 2000 rpm for 10 to 15 minutes, then taking the supernatant, centrifuging at 12000 to 15000 rpm for 10 to 15 minutes, and collecting the precipitate.

6. The preparation method according to claim 3, characterized in that The mass ratio of the drug to the hybrid nanoparticles in step (2) is 1:2 to 1:

15.

7. The preparation method according to claim 3, characterized in that The mixing in step (2) is specifically as follows: adding the methanol solution of the drug dropwise into the aqueous solution of the hybrid nanoparticles; the stirring time at room temperature is 12 to 14 hours; the centrifugation is specifically as follows: centrifuging at 1000 to 1200 rpm for 10 to 15 minutes, and collecting the supernatant.

8. The preparation method according to claim 3, characterized in that In step (3), the mass ratio of the composite nanomaterial to the small interfering RNA is 10:1 to 100:

1.

9. The preparation method according to claim 3, characterized in that The mixing in step (3) specifically comprises: adding the DEPC aqueous solution of the small interfering RNA into the aqueous solution of the composite nanomaterial; and the incubation time is 30 to 40 minutes.

10. Use of the hybrid dendrimer nanomaterial loaded with drugs and small interfering RNA according to any one of claims 1 to 2 in the preparation of anti-inflammatory / antioxidant / gene therapy drugs.

Images