Gallium ion-quercetin coordination nanoparticle as well as preparation method and application thereof

Abstract

The invention discloses gallium ion-quercetin coordination nanoparticles (GQNPs) as well as a preparation method and application thereof, specifically, quercetin, gallium nitrate hydrate and polyvinylpyrrolidone are respectively dissolved in an absolute methanol solution to prepare a quercetin solution, a gallium nitrate hydrate solution and a polyvinylpyrrolidone solution; secondly, fully mixing the quercetin solution and the polyvinylpyrrolidone solution, dropwise adding the gallium nitrate hydrate solution, and rapidly changing the color of the mixed solution from faint yellow to orange yellow in the dropwise adding process; and finally, dialyzing the orange-yellow mixed solution to remove excessive gallium ions and polyvinylpyrrolidone, thereby finally obtaining the GQNPs. The prepared GQNPs and deferiprone show excellent treatment effects on cell and living body levels, are expected to be applied to treatment of Parkinson's disease through multi-dimensional anti-ferroptosis, do not show obvious toxic and side effects in the embodiment, and are expected to be applied to further preclinical research.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanoparticle preparation and application, and specifically relates to gallium ion-quercetin coordinated nanoparticles (GQNPs) and a preparation method and application thereof. Background Art

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide. Although its pathogenesis remains incompletely understood, multiple pathological processes, such as pathological aggregation of α-synuclein, mitochondrial dysfunction, oxidative stress, and dysregulated iron homeostasis, have been implicated in disease progression. Elevated iron levels, oxidative stress, and accumulation of lipid peroxides are three hallmarks of ferroptosis, all of which are closely associated with PD pathology. The susceptibility of dopaminergic neurons to ferroptosis is related to their structural characteristics. Their large axons and long synapses require a high energy supply, and ATP is primarily produced by mitochondria, whose physiological activity is strongly dependent on iron. However, iron overload not only induces mitochondrial dysfunction by increasing mitochondrial membrane potential and reactive oxygen species but also may induce lipid peroxidation of cell membranes through the Fenton reaction. Furthermore, the high lipid content and oxygen demand of tissues further contribute to the sensitivity of dopaminergic neurons to oxidative damage. Thus, ferroptosis is widely involved in PD-related pathological processes and is a key driver of PD pathogenesis and progression.

[0003] Currently, targeted therapies for ferroptosis in PD are mainly based on reducing cellular iron content and oxidative stress levels, resulting in the development of iron regulation strategies (iron chelators, the use of drugs to regulate iron metabolism proteins) and antioxidant strategies (scavenging reactive oxygen species, reducing lipid peroxides). However, the treatment angles of existing methods are relatively single, and the combination of multiple strategies is rarely achieved, making it difficult to cope with the complex pathological environment of PD. To overcome this difficulty, it is necessary to develop new ferroptosis inhibitors to achieve the combination of multiple ferroptosis inhibition strategies within a single platform. Gallium ions have similar ionic radius, ionization potential, and electron affinity to iron ions and are generally considered to be redox-inert iron substitutes. By interfering with iron metabolism, they are used in antibacterial and anti-tumor research, and therefore are expected to be applied to the inhibition of ferroptosis in PD. In order to reduce the side effects of pure gallium ions themselves and enrich the dimensions of ferroptosis inhibition, the use of the polyphenol drug quercetin to coordinate with gallium ions is expected to give the nanoplatform excellent antioxidant effects. Summary of the Invention

[0004] In response to the problems existing in the prior art, the present invention provides a gallium ion-quercetin coordinated nanoparticle (GQNPs) and its preparation method and application. The gallium ion-quercetin coordinated nanoparticles are rich in functions, with good iron-lowering effects, reactive oxygen and lipid peroxide scavenging effects and mitochondrial protection functions. At the same time, they can also have good therapeutic effects in animal models. They are expected to serve as a new type of ferroptosis inhibitor to break through the limitations of the relatively single existing ferroptosis inhibition strategy, which is conducive to further improving the treatment effect of Parkinson's disease.

[0005] In order to solve the above technical problems and achieve the above technical effects, the present invention is implemented through the following technical solutions:

[0006] A method for preparing gallium ion-quercetin coordinated nanoparticles (GQNPs) comprises the following steps:

[0007] Step 1) weighing quercetin, hydrated gallium nitrate, and polyvinyl pyrrolidone according to a certain mass ratio;

[0008] Step 2) dissolving weighed quercetin, hydrated gallium nitrate, and polyvinyl pyrrolidone in corresponding volumes of anhydrous methanol solution to prepare quercetin solution, hydrated gallium nitrate solution, and polyvinyl pyrrolidone solution, respectively;

[0009] Step 3) mixing the prepared quercetin solution and polyvinyl pyrrolidone solution and stirring at room temperature for a period of time to obtain a quercetin-polyvinyl pyrrolidone mixed solution;

[0010] Step 4) adding the prepared hydrated gallium nitrate solution dropwise to the uniformly mixed quercetin-polyvinyl pyrrolidone mixed solution, wherein the color of the mixed solution rapidly changes from light yellow to orange-yellow during the addition process, and then stirring the orange-yellow mixed solution at room temperature for a period of time;

[0011] Step 5) dialyzing the orange-yellow mixed solution in a dialysis bag for a period of time to remove excess gallium ions and polyvinylpyrrolidone, and the resulting solution is gallium ion-quercetin coordinated nanoparticles (GQNPs);

[0012] The gallium ion-quercetin coordination nanoparticles have red-shifted ultraviolet absorption ability compared with quercetin;

[0013] The gallium ion-quercetin coordination nanoparticles have the ability of spontaneous fluorescence.

[0014] Furthermore, in step 1, the mass ratio of quercetin, hydrated gallium nitrate and polyvinyl pyrrolidone is 8:40:100.

[0015] Furthermore, in step 2, the volume ratio of the anhydrous methanol solution used to dissolve quercetin, hydrated gallium nitrate and polyvinyl pyrrolidone is 1.5:4:3.

[0016] Furthermore, the hydrated particle size of the gallium ion-quercetin coordinated nanoparticles is 51.1 nm.

[0017] A gallium ion-quercetin coordinated nanoparticle is prepared by adopting the above preparation method.

[0018] The invention discloses an application of gallium ion-quercetin coordinated nanoparticles prepared by the above preparation method as a ferroptosis inhibitor in the treatment of Parkinson's disease.

[0019] The invention discloses an application of gallium ion-quercetin coordinated nanoparticles prepared by the above preparation method in the preparation of a ferroptosis targeted drug for Parkinson's disease.

[0020] Furthermore, the complete in vitro experimental dose of the ferroptosis inhibitor or the Parkinson's disease ferroptosis targeted drug is 20 μg / mL.

[0021] Furthermore, the ferroptosis inhibitor or the Parkinson's disease ferroptosis-targeted drug can improve cellular oxidative stress and significantly reduce the total level of reactive oxygen species in cells.

[0022] Furthermore, the ferroptosis inhibitor or the Parkinson's disease ferroptosis-targeted drug can enhance the antioxidant capacity of cells and reduce lipid peroxides in cells.

[0023] Furthermore, the ferroptosis inhibitor or the Parkinson's disease ferroptosis-targeted drug can significantly reduce the iron ion level of cells.

[0024] Furthermore, the ferroptosis inhibitor or the Parkinson's disease ferroptosis-targeted drug can reduce excess iron in cells and lower the iron content in cells by regulating changes in iron metabolism-related proteins (ferrotransferrin receptor and iron storage protein).

[0025] Furthermore, the ferroptosis inhibitor or the Parkinson's disease ferroptosis-targeted drug can improve mitochondrial function by regulating the activity of mitochondrial respiratory chain complexes.

[0026] After preparing the gallium ion-quercetin coordinated nanoparticles, the present invention conducted in vitro and in vivo experiments on Parkinson's animal models to evaluate the ferroptosis inhibitory effect on the Parkinson's model.

[0027] Furthermore, the Parkinson's animal model is a Parkinson's mouse model.

[0028] The beneficial effects of the present invention are:

[0029] The present invention provides a method for preparing gallium-based nanoparticles (GQNPs) and their application. The GQNPs provided by the present invention have the ability to inhibit Parkinson's disease ferroptosis. Because the nanoparticles combine the advantages of gallium ions and quercetin, they exhibit excellent multi-dimensional ferroptosis inhibition effects in Parkinson's disease models, scavenging reactive oxygen species (ROS) and lipid peroxides, and enhancing the antioxidant system. Furthermore, in terms of reducing cellular iron content, they utilize the competitive binding of gallium ions with iron ions and further reduce intracellular iron content by regulating iron metabolism-related proteins.

[0030] The GQNPs and deferiprone (DFP) prepared by the present invention exhibited excellent therapeutic effects at both the cellular and in vivo levels, and are expected to be used in the treatment of Parkinson's disease through multidimensional anti-ferroptosis. In addition, they did not show obvious toxic side effects in the examples and are expected to be used for further preclinical studies.

[0031] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the invention and to implement it according to the contents of the description, the following preferred embodiments of the present invention are described in detail with reference to the accompanying drawings. The specific implementation methods of the present invention are given in detail by the following embodiments and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0033] Figure 1 Characterization diagram of GQNPs in Example 1 of the present invention: a is a GQNPs synthesis flow chart, b is an electron microscopy image of GQNPs, c is an electron microscopy particle size statistics diagram of GQNPs, and d is a hydrated particle size statistics diagram of GQNPs.

[0034] Figure 2 These are the X-ray photoelectron spectroscopy results of GQNPs in Example 1 of the present invention: a is the energy spectrum result diagram of GQNPs, and b is the peak fitting result diagram of the Ga 3d orbital of GQNPs.

[0035] Figure 3 The cytotoxicity test results of GQNPs in Example 2 of the present invention are as follows: a is the drug 1-methyl-4-phenylpyridinium iodide (MPP) used for in vitro PD modeling. + ) is a graph showing the cytotoxic effects of different doses; b is a graph showing the cytotoxic effects of different doses of GQNPs.

[0036] Figure 4These are the reactive oxygen species monitoring results in Example 3 of the present invention: wherein a is a fluorescence image taken under a laser confocal scanning microscope (using the green fluorescence channel to detect total intracellular reactive oxygen species, and the blue fluorescence channel to detect cell nuclei), b is a fluorescence image taken under a confocal microscope (using the red fluorescence channel to detect mitochondrial reactive oxygen species levels, and the blue fluorescence channel to monitor cell nuclei), c is a graph showing the total intracellular reactive oxygen species level, and d is a graph showing the intracellular superoxide anion level.

[0037] Figure 5 This is a graph showing the changes in malondialdehyde (MDA) levels in different treatment groups in Example 4 of the present invention.

[0038] Figure 6 These are the Western Blot test results of the antioxidant system in Example 5 of the present invention: a is a graph showing the expression changes of glutathione peroxidase 4 (GPX4) in different treatment groups, and b is a graph showing the expression changes of nuclear factor-related factor 2 (NRF2) in different treatment groups.

[0039] Figure 7 These are the results of changes in total iron content levels in cells in different treatment groups in Example 6 of the present invention: a is the result of the effect of pure gallium ions on total iron content in cells, and b is the result of detecting the effect of GQNPs on total iron content in cells.

[0040] Figure 8 These are the results of changes in intracellular ferrous iron content levels in different treatment groups in Example 7 of the present invention: a is a confocal staining fluorescence image of ferrous ions; b is a semi-quantitative analysis result based on the fluorescence signal of the confocal image.

[0041] Figure 9 These are the results of changes in intracellular iron metabolism-related proteins in different treatment groups in Example 8 of the present invention: a is a Western Blot result diagram of TFR1, FTH1, DMT1 and FPN, and b is a grayscale statistical diagram of the protein expression levels of TFR1, FTH1, DMT1 and FPN.

[0042] Figure 10 This is a graph showing the results of detecting the activity of complex III using a mitochondrial respiratory chain complex III activity detection kit in Example 9 of the present invention.

[0043] Figure 11 Graph showing the therapeutic effects of different treatment groups in Example 10 of the present invention on Nissl bodies and tyrosine hydroxylase (TH) in the brains of Parkinson's mice.

[0044] Figure 12These are the quantitative analysis results of tyrosine hydroxylase (TH), dopamine enzyme (DA) and malondialdehyde enzyme (MDA) in different treatment groups in Example 11 of the present invention, wherein a is the quantitative analysis result graph of TH, b is the quantitative analysis result graph of DA, and c is the quantitative analysis result graph of MDA.

[0045] Figure 13 This is a graph showing the results of blood routine and blood biochemistry data analysis of mice in different treatment groups in Example 12 of the present invention. DETAILED DESCRIPTION

[0046] The following will be described in detail with reference to the accompanying drawings to better understand the purpose, features and advantages of the invention. It should be understood that the embodiments shown in the accompanying drawings are not intended to limit the scope of the invention, but are only intended to illustrate the essential spirit of the technical solution of the invention.

[0047] In the following description, for the purpose of illustrating the various disclosed embodiments, certain specific details are set forth in order to provide a thorough understanding of the various disclosed embodiments. However, those skilled in the relevant art will recognize that the embodiments may be practiced without one or more of these specific details. In other cases, well-known devices, structures, and techniques associated with this application may not be shown or described in detail to avoid unnecessarily obscuring the description of the embodiments.

[0048] Unless the context requires otherwise, throughout the specification and claims, the word "comprise" and variations such as "include" and "have" should be construed in an open, inclusive sense, that is, should be interpreted to mean "including, but not limited to."

[0049] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any manner in one or more embodiments.

[0050] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should be noted that the term "or" is generally employed in its sense including "and / or" unless the context clearly dictates otherwise.

[0051] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other. Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

[0052] The present invention develops gallium-based nanoparticles formed by coordination of gallium ions and quercetin, namely gallium ion-quercetin coordinated nanoparticles (GQNPs). The GQNPs include quercetin, gallium ions and polyvinyl pyrrolidone.

[0053] The GQNPs have red-shifted ultraviolet absorption compared to quercetin.

[0054] The GQNPs have autofluorescence capability.

[0055] The hydrated particle size of the GQNPs is 51.1 nm.

[0056] The GQNPs are prepared by mixing quercetin, polyvinyl pyrrolidone, and gallium nitrate in methanol. Figure 1 As shown in a, the main preparation steps are as follows:

[0057] Step 1) weighing quercetin, hydrated gallium nitrate, and polyvinyl pyrrolidone in a mass ratio of 8:40:100;

[0058] Step 2) Dissolving weighed quercetin, hydrated gallium nitrate, and polyvinyl pyrrolidone in three parts of anhydrous methanol solution with a volume ratio of 1.5:4:3 to prepare a quercetin solution, a hydrated gallium nitrate solution, and a polyvinyl pyrrolidone solution, respectively;

[0059] Step 3) mixing the prepared quercetin solution and polyvinyl pyrrolidone solution and stirring at room temperature for 1 hour to obtain a quercetin-polyvinyl pyrrolidone mixed solution;

[0060] Step 4) using a pipette, the prepared hydrated gallium nitrate solution was added dropwise to the uniformly mixed quercetin-polyvinyl pyrrolidone mixed solution. During the addition, the color of the mixed solution quickly changed from light yellow to orange-yellow. The orange-yellow mixed solution was then stirred at room temperature for 6 hours.

[0061] Step 5) The orange-yellow mixed solution was dialyzed in a dialysis bag for 18 hours to remove excess gallium ions and polyvinyl pyrrolidone. The final solution obtained was gallium ion-quercetin coordinated nanoparticles (GQNPs).

[0062] The authenticity and feasibility of the present invention are described and verified in detail below using a specific gallium ion-quercetin coordinated nanoparticle (GQNPs) preparation case and related test experiments.

[0063] The materials and reagents used in this case and its related test experiments are as follows:

[0064] Hydrated gallium nitrate (Ga(NO)3·xH2O) was purchased from Anhui Zesheng Co., Ltd.;

[0065] Polyvinylpyrrolidone (40 kDa) was purchased from Beijing Solebow Technology Co., Ltd.;

[0066] Quercetin was purchased from Jiuding Chemical Technology Co., Ltd.;

[0067] Anhydrous methane was purchased from Sinopharm Chemical Reagent Suzhou Co., Ltd.;

[0068] 1-Methyl-4-phenylpyridinium iodide (MPP + ) was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.;

[0069] Deferiprone (DFP) was purchased from Shanghai Taoshu Company;

[0070] SH-SY5Y human neuroblastoma cells were purchased from Wuhan Punosai Biotechnology Co., Ltd.;

[0071] Mice were purchased from Weitonglihua Experimental Animal Co., Ltd.

[0072] Example 1 Preparation of GQNPs:

[0073] See also Figure 1 As shown in Figure a, a method for preparing gallium ion-quercetin coordinated nanoparticles (GQNPs) comprises the following steps:

[0074] (1) Weigh 8 mg of quercetin, 40 mg of hydrated gallium nitrate, and 100 mg of polyvinylpyrrolidone and dissolve them in 1.5 mL, 4 mL, and 3 mL of anhydrous methanol, respectively. First, mix the quercetin solution with the polyvinylpyrrolidone solution and stir at room temperature for 1 h. Then, add the gallium nitrate solution dropwise using a pipette. During this process, the color of the solution changes from light yellow to orange-yellow. The mixed solution is then stirred at room temperature for 6 h.

[0075] (2) The solution was dialyzed using a dialysis bag for 18 h to remove excess gallium ions and polyvinylpyrrolidone, and the final solution obtained was GQNPs.

[0076] The electron microscopy and hydration particle size characterization results of GQNPs are as follows Figure 1 As shown, from Figure 1 As can be seen from the electron microscope image b, the morphology of the obtained GQNPs is circular and has good monodispersity; Figure 1 The particle size statistics of electron microscopy in c show that the electron microscopy size of GQNPs is 40.5±10.1nm. Figure 1 d The statistical results of the hydrated particle size show that the hydrated particle size of GQNPs is 51.1 nm.

[0077] The results of X-ray photoelectron spectroscopy of GQNPs are shown in Figure 2 As shown, from Figure 2 From the energy spectrum results of a, we can see that there is Ga 3d orbital. Figure 2 The peak fitting results of b show that the analysis of Ga 3d orbitals of GQNPs yields two binding energy peaks, 19.35 eV and 20.2 eV, which belong to Ga δ+ and Ga 3+ , Ga δ+ The formation of further proves the occurrence of coordination.

[0078] Example 2 Cytotoxicity Detection:

[0079] 5×10 per well 3 SH-SY5Y cells were plated into sterile 96-well plates at a density of 100 cells / dish and cultured in a cell culture incubator for 24 h to ensure complete cell attachment. The old complete culture medium was aspirated and discarded, and 0, 4, 6, 8, 10, 15, 20 μg / mL GQNPs solution or 0, 1, 2, 4, 6, 8, 10 mM MPP were added. + The solution was incubated in the cell culture incubator for 24 h.

[0080] After incubation, remove the added sample and wash with PBS to remove any residue. Add 20 μL of CCK8 reagent and 180 μL of culture medium to each well to be tested, and return the 96-well plate to the cell culture incubator for 1 hour. Finally, set the microplate reader to a wavelength of 450 nm and measure the absorbance of the sample. Calculate the cell viability according to the cell viability formula provided in the kit:

[0081] Cell viability (%) = [A(treated group) - A(blank group)] / [A(control group) - A(blank group)] × 100%;

[0082] Among them, A (treated group) represents the absorbance of cells after incubation with drugs or nanoparticles, A (blank group) represents the average absorbance without drugs and cells, and A (control group) represents the average absorbance of cells without any treatment.

[0083] The results of cytotoxicity assays were as follows Figure 3 As shown, from Figure 3 In vitro PD modeling of a drug MPP + The results of the cytotoxicity effect of the dose showed that the cell viability was 60%, and MPP was used + The appropriate dose for establishing an in vitro PD model is 4 mM; Figure 3 From the results of b on the cytotoxicity of GQNPs, it can be seen that 20 μg / ml (based on the gallium ion concentration) of GQNPs has no obvious toxicity to SH-SY5Y cells and can be used in subsequent experiments.

[0084] Example 3 Active Oxygen Species Monitoring:

[0085] For the determination of total intracellular reactive oxygen species: 5×10 4 SH-SY5Y cells were seeded into the confocal dish at a density of 10 cells / dish. After the cells were well attached, 20 μg / mL GQNPs solution and 10 μM DFP were added to the confocal dish for pre-incubation for 4 h. Then the culture medium was removed and 4 mM MPP was added. + Incubate for 24 hours with fresh culture medium. Wash away the old culture medium with PBS, then add 200 μL of 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe to the confocal dish and incubate in the dark for 30 minutes in a cell culture incubator. Then wash three times with PBS to remove the probe, add 10 μg / mL nuclear dye, and incubate in the dark for 15 minutes in an incubator. Finally, wash away the nuclear dye with PBS. Observe and capture fluorescence images under a laser confocal scanning microscope. Figure 4 As shown in a, the green fluorescence channel was used to detect total intracellular reactive oxygen species, and the blue fluorescence channel was used to detect cell nuclei. The mean fluorescence intensity of the acquired images was quantified using Image J software.

[0086] For the determination of superoxide anion levels: cells were treated in the same manner as above before incubation with the probe. After washing away the old culture medium with PBS, 200 μL of Mito SOX Red mitochondrial superoxide indicator was added to the confocal dish and incubated in a dark place at 37°C for 10 minutes. Unabsorbed probes were washed away with PBS, and nuclear dye was added and incubated with the cells for 15 minutes. The nuclear probe was washed away again with sterile PBS. Fluorescence images were taken under a confocal microscope, see [see ]. Figure 4 As shown in (b), mitochondrial ROS levels were detected using the red fluorescence channel, and cell nuclei were observed using the blue fluorescence channel. Confocal images were semi-quantitatively analyzed using ImageJ software to determine the mean fluorescence intensity of each cell group.

[0087] The experimental results can be found in Figure 4 As shown in c-4d, MPP + It can significantly increase the total level of reactive oxygen species in cells. The use of 20μg / mL GQNPs can significantly inhibit the production of reactive oxygen species in PD cells, but 10μM DFP does not seem to have a significant inhibitory effect on the total intracellular ROS. The generation of reactive oxygen species is inseparable from mitochondrial metabolism. Since mitochondria are the main site of ROS production, and excessive ROS will further lead to mitochondrial homeostasis imbalance, metabolic disorders, and the production of superoxide anions. The experimental results show that, consistent with the trend of changes in the total intracellular ROS level, MPP +In treated cells, mitochondrial superoxide anion levels increased significantly, and GQNPs were able to significantly suppress superoxide anion levels, while DFP had no significant scavenging effect on superoxide anions. These results demonstrate that GQNPs have superior antioxidant capacity compared to clinical iron chelators, which facilitates their multidimensional ferroptosis inhibition function.

[0088] Example 4 Determination of intracellular MDA levels:

[0089] (1) Preparation of cell samples: SH-SY5Y cells were cultured at 5×10 6 The cells were inoculated into cell culture flasks at a density of 1000 cells / dish for at least 24 h, and 20 μg / mL GQNPs solution and 10 μM DFP solution were added for pre-incubation for 4 h, then removed and replaced with 4 mM MPP solution. + Continue incubating with fresh culture medium for 24 hours. Disrupt the cells with cell lysis buffer on ice for half an hour. Centrifuge the sample at 12,000 g for 10 minutes at 4°C, and collect the supernatant for subsequent experiments.

[0090] (2) Preparation of the assay working solution: Prepare the required malondialdehyde (MDA) assay working solution from a certain volume of TBA stock solution, its diluent, and an antioxidant. The volume ratios are as specified in the kit instructions. Place the mixture at 70°C to facilitate complete dissolution of the reagents. The MDA assay working solution should be prepared and used immediately.

[0091] (3) Dilute the standard solution: Use ultrapure water to dilute the standard solution to 1, 2, 5, 10, 20, and 50 μM in sequence.

[0092] (4) Preparation and determination after sample addition: First, poke several small holes in the cap of the 1.5mL centrifuge tube with a needle. Then, add 0.1mL of PBS solution as a blank control tube, 0.1mL of standard solution of different concentrations for standard curve determination, and 0.1mL of supernatant of different groups of cell samples to different centrifuge tubes. Then, add 0.2mL of MDA detection working solution to each centrifuge tube. After thorough mixing, heat the solution at 100℃ until the color of the solution changes. After heating, cool the sample in a water bath and then centrifuge it at 1000g for 10 minutes at room temperature. Transfer 200μL of supernatant to a 96-well plate. Finally, use an enzyme-linked microplate reader to measure the absorbance value of the sample to be tested at a wavelength of 532nm. The standard curve is constructed based on the relationship between the concentration of the standard and the corresponding absorbance for subsequent analysis to obtain the molar concentration of MDA contained in the sample.

[0093] (5) BCA protein quantification: Dilute the supernatant obtained from the cell sample preparation by 5 times, take 20 μL and drop it on a 96-well plate, take another 20 μL of ready-to-use protein standards of different concentrations and drop it on the same 96-well plate, and then add 180 μL of BCA working solution (prepared in a ratio of 50:1 between solution A and solution B). After incubation at 37°C for 40 minutes, use a microplate reader to read the absorbance of the sample and the standard. A fitting curve is constructed based on the relationship between the absorbance of the standard and the protein concentration to calculate the protein concentration in the sample. The final MDA concentration should be normalized based on the protein concentration of the sample and the unit is μmol / g protein.

[0094] The experimental results can be found in Figure 5 As shown in the figure, the quantitative results showed that compared with the control group, MPP + The results showed that the MDA level in SH-SY5Y cells increased significantly when treated with 10 μM DFP, but the difference was not statistically significant. The MDA level in cells was significantly inhibited by 20 μg / mL GQNPs, which demonstrated that GQNPs under these conditions could significantly reduce the content of lipid peroxides and have a significant inhibitory effect on ferroptosis.

[0095] Example 5 Detection of GPX4 and NRF2 expression:

[0096] The expression of GPX4 and NRF2 was detected by Western Blot. Cell culture and treatment were the same as above. The remaining experimental steps are as follows:

[0097] (1) Protein lysis and extraction: Each group was treated with cell lysis buffer containing protease inhibitors, and the cells were then placed on ice for 30 minutes. After lysis, the cells and debris were collected and centrifuged at 15,000 rpm for 10 minutes in a four-degree centrifuge. The supernatant was used as the sample for subsequent experiments.

[0098] (2) Protein quantification and sample preparation: The protein concentration in the sample was determined by the BCA assay. The remaining protein sample, except for the portion used for protein quantification, was used to prepare the Western Blot loading sample. A volume of 5× Loading buffer corresponding to the sample volume was added, vortexed, and then heated at 97°C for 10 min for denaturation. After denaturation, the sample can be stored in a -80°C freezer.

[0099] (3) Electrophoresis: After determining the molecular weight of the target protein, select a precast gel with the optimal separation gel concentration based on its size. Pour the electrophoresis solution into the electrophoresis tank. Then, add the protein marker and loading buffer to the two lanes. Then, based on the measured sample protein concentration, determine the appropriate loading volume and add the protein sample to the middle lane. The electrophoresis conditions are to run at a constant voltage of 80V for 25 minutes, then switch to 120V until the end.

[0100] (4) Transfer: Take a polyvinylidene fluoride membrane of appropriate size and activate it in anhydrous ethanol for 5 minutes. At the same time, soak a filter paper-free sponge pad in the transfer solution and set aside. Trim the excess gel after the electrophoresis step and sandwich the filter paper-free sponge pad, gel, and fully activated membrane together. Finally, place it in the transfer electrode tank and pour in the transfer solution. The transfer conditions are a constant current of 400mA for 40 minutes.

[0101] (5) Blocking step: After the transfer is completed, wash with 1×TBST on a shaker, and then incubate the membrane with protein-free rapid blocking solution on a shaker for 30 minutes to complete the blocking treatment.

[0102] (6) Incubation with primary antibody: After blocking, wash the membrane with 1×TBST and then dilute the primary antibody in universal antibody diluent or 1×TBST. Soak the membrane in the primary antibody and incubate overnight at 4°C on a shaker.

[0103] (7) Incubation with secondary antibody: Wash the membrane with 1×TBST, then dilute the secondary antibody appropriately with universal antibody diluent or 1×TBST, and incubate the membrane with the diluted secondary antibody on a shaker at room temperature for 1 h.

[0104] (8) Development: The membrane was washed with 1× TBST, and then excess water was absorbed with absorbent paper. The developing solution was allowed to fully contact the membrane under light-proof conditions. Finally, the membrane was exposed to light using a chemiluminescence imaging instrument. The grayscale values ​​of the obtained bands were quantitatively analyzed using ImageJ software.

[0105] The experimental results can be found in Figure 6 As shown, from Figure 6 The results of GPX4 expression changes in a showed that, compared with the control group, MPP +Treatment resulted in a significant decrease in GPX4 expression in SH-SY5Y cells, and although DFP had a certain upregulation effect on GPX4, there was no statistical difference; PD cells pretreated with GQNPs were able to significantly upregulate the expression of GPX4, which was consistent with the superior antioxidant capacity of GQNPs shown in the confocal microscopy results. NRF2 is a master transcription factor that regulates a series of genes involved in exogenous detoxification and antioxidant defense. This transcription factor is considered an important therapeutic target for neurodegenerative diseases. Activated NRF2 binds to antioxidant response elements, thereby upregulating other antioxidant genes and proteins. Figure 6 As shown in the results of NRF2 expression changes in (b), the Western Blot results were consistent with those of GPX4. DFP did not significantly restore the level of NRF2 in cells, but GQNPs had the ability to enhance the expression of NRF2 in cells. This result suggests that GQNPs can upregulate the expression of GPX4 by activating NRF2 in PD cells.

[0106] Example 6 Detection of total iron content in cells:

[0107] The total iron content in the cells was detected by the cell iron content kit. The specific experimental steps are as follows:

[0108] (1) Sample preparation: Add the treated cells to the cell extract in the kit and then lyse them in an ice bath for half an hour. The lysed cells are further disrupted by ultrasound.

[0109] (2) Test: Add 20 μL of sample to a 96-well plate (add an equal volume of distilled water to the blank control group and an equal volume of 0.5 μmol / mL iron standard solution to the standard wells), then add 180 μL of the test reagent, mix thoroughly, and let it stand at room temperature for 10 min. Then, use an enzyme-linked microplate reader to detect the absorbance value of the test well at a wavelength of 510 nm.

[0110] The experimental results can be found in Figure 7 As shown in the figure, since gallium ions mainly play a role by competing with iron ions to bind to transferrin, to verify this view, PD cells were pretreated with free gallium ions and the changes in total iron levels in the cells were detected. Figure 7 The results of the changes in total cellular iron content in a show that the experimental results show that pretreatment with 20 μg / mL free gallium ions can significantly reduce the iron accumulation in PD cells, proving that gallium ions can interfere with iron uptake in the PD model; then further verification was conducted to determine whether GQNPs have the same ability to reduce cellular iron content as free gallium ions, and comparison was made with DFP. Figure 7The results of detecting the effect of GQNPs on the total iron content in cells shown in b show that both 10 μM DFP and 20 μg / mL GQNPs can reduce the total iron content of PD cells.

[0111] Example 7 Detection of changes in intracellular ferrous iron content levels:

[0112] 5×10 4 SH-SY5Y cells were seeded onto confocal microplates at a density of 100 cells / dish and cultured in a cell culture incubator for at least 24 hours to ensure good cell adhesion and growth. 10 μM DFP or 20 μg / mL GQNPs were added to different confocal microplates and co-cultured with cells for 4 hours, then the drugs were washed off with PBS and 4 mM MPP was added. + The solution was added and cultured for 24 hours. After the drug incubation was completed, 300 μL of 5 μM ferrous ion probe FeRhoNox-1 was added to the confocal dish and incubated in a cell culture incubator for 60 minutes. The dye was washed off with PBS, and then 300 μL of live cell nuclear dye was added. The sample was placed in a cell culture incubator and incubated in the dark for 15 minutes. Next, PBS was used to wash to remove excess dye, and PBS was added to the confocal dish to prevent the cells in the confocal dish from being dehydrated and affecting their normal state. Immunofluorescence images of the cells were taken using a laser confocal microscope. The orange channel detects ferrous ions, and the blue channel is the nuclear dye channel. Finally, the fluorescence signal intensity was semi-quantitatively analyzed using ImageJ.

[0113] The experimental results can be found in Figure 8 As shown, from Figure 8 Confocal images of a and Figure 8 b The semi-quantitative results showed that MPP + The ferrous ion level in the established in vitro PD model was significantly increased, while the DFP and GQNPs treatment groups showed a significant decrease in ferrous content.

[0114] Example 8 Detection of changes in cellular iron metabolism-related protein levels:

[0115] Western Blot was used to detect changes in the levels of proteins related to cellular iron metabolism. The experimental steps are as follows:

[0116] SH-SY5Y cells were seeded in a T25 cell culture flask. When the cell density in the cell culture flask was about 80%, 10 μM DFP or 20 μg / mL GQNPs were added. After incubation for 4 h, the medium was replaced with 4 mM MPP. +After incubation for 24 hours, the cells were lysed and proteins were extracted. Western Blot experiments were then performed according to the steps of Example 4 to detect the expression changes of iron metabolism-related proteins TFR1, FTH1, DMT1 and FPN.

[0117] Western Blot results can be found in Figure 9 a, quantitative results see Figure 9 b. MPP + In PD cells treated with GQNPs, TFR1 expression was significantly upregulated, promoting excessive iron uptake, and GQNPs pretreatment could downregulate TFR1 expression, which proves that GQNPs can reduce iron influx. + The expression of GQNPs was significantly reduced in the treated cells, indicating that the iron storage capacity of PD cells was impaired. After pretreatment, GQNPs and DFP restored the expression of FTH1, indicating enhanced iron storage. Considering that quercetin is a key component of GQNPs, it has been reported that quercetin stabilizes ferritin and regulates iron metabolism, and its role in FTH1 upregulation helps to inhibit iron-dependent apoptosis, so the upregulation of FTH1 is the contribution of quercetin in this gallium-based nanoparticle. DMT1 is a divalent metal transporter that plays an important role in regulating intracellular iron levels. In our study, MPP + Unexpectedly, it led to the downregulation of DMT1, while GQNPs significantly upregulated the expression of DMT1, which indicates that DMT1 still has a therapeutic effect in PD cell pathology. We quantified the changes in FPN expression for the iron efflux function of cells. The expression analysis of FPN showed that there was no significant change between the groups, which indicated that MPP + The iron accumulation phenomenon in treated PD cells was not associated with alterations in the iron export mechanism.

[0118] Example 9 Detection of Complex III Activity:

[0119] The activity of complex III was detected using the mitochondrial respiratory chain complex III activity detection kit. The specific experimental steps are as follows:

[0120] (1) Sample preparation: After the treated cells are centrifuged, 1 mL of the extract solution is added to the pellet, and the cell pellet is quickly ground 20 times with a pestle in an ice bath. Subsequently, the pellet is centrifuged at 600 g for 10 min in a 4°C centrifuge, and the supernatant is further centrifuged at 18,000 g for 15 min at 4°C. The supernatant cytoplasmic extract is removed, and 200 μL of the extract solution is added to the pellet, which is then broken in an ice bath using an ultrathinner.

[0121] (2) Preheat the microplate reader to 37°C. After the temperature stabilizes, add 160 μL of working solution to the test wells in the 96-well plate. Add 20 μL of Reagent 3 to the test wells and no additional reagent to the control wells. Incubate in the microplate reader at 37°C for 2 min. Then, add 20 μL of sample to the test wells and 20 μL of ultrapure water to the control wells. After mixing, measure the absorbance A1 at 550 nm and the absorbance A2 after reacting at 37°C for 10 min.

[0122] (3) The remaining sample was used to measure the protein concentration in the mitochondria using a BCA kit. Finally, the activity of mitochondrial respiratory chain complex III was calculated according to the formula provided in the instructions.

[0123] The experimental results can be found in Figure 10 As shown in the figure, since studies have shown that DMT1 downregulation can disrupt mitochondrial iron transport, leading to dysregulation of the respiratory chain complex, and then promoting mitochondrial ROS accumulation and membrane potential abnormalities. We speculate that the changes in DMT1 levels mentioned above may be related to this. The experimental results show that compared with the control group, MPP + The activity of complex III was significantly elevated in the control group, while pretreatment with GQNPs effectively reduced this trend. Downregulation of complex III impairs mitochondrial respiratory function and accumulates ROS, thus aligning with the previously reported results on mitochondrial superoxide anion levels. These results demonstrate that GQNPs possess mitochondrial protective properties in an in vitro Parkinson's disease model.

[0124] Example 10 Detection of the therapeutic effect of Nissl bodies and TH in the brain of Parkinson's mice:

[0125] After acclimation for 7 days, C57BL / 6 mice were intraperitoneally injected with MPTP at a dose of 35 mg / kg for 8 consecutive days to establish a PD model. A control group received intraperitoneal injections of normal saline. The mice were randomly divided into four groups: Ctrl (healthy mice), PD (untreated PD mice), DFP (DFP-treated PD mice), and NPs (GQNPs-treated PD mice). DFP (1 mM) and GQNPs (1.5 mg / mL) were intranasally injected once every other day after grouping, with an injection volume of 20 μL, for a total of three doses. Mice were euthanized, and brain tissue was removed after transcardial perfusion with PBS and fixed in 4% paraformaldehyde for 48 hours. Nissl body staining and immunofluorescence staining for TH were then performed.

[0126] Nissl bodies are involved in neuronal protein synthesis and are related to the dopamine secretion function of neurons. TH is the rate-limiting factor in the biosynthesis of catecholamines (such as dopamine, epinephrine, and norepinephrine). Therefore, changes in the number of Nissl bodies and TH expression levels can serve as important indicators of PD pathological changes. Figure 11 As shown, the number of Nissl bodies in the hippocampus of PD mice was significantly reduced, while the number of Nissl bodies in the treatment group increased significantly and was densely arranged. Immunofluorescence sections were used to observe TH expression in the substantia nigra of mice from different groups. The results clearly show that TH density decreased significantly in the PD group, while TH fluorescence intensity in the DFP- and GQNP-treated groups recovered to a certain extent compared to the PD group.

[0127] Example 11 Quantitative analysis of TH, DA and MDA:

[0128] Three mice were randomly euthanized in each group. After perfusion, the brain tissue was removed and the midbrain was further isolated on ice. The mouse midbrain tissue was weighed and placed in a 2 mL centrifuge tube. Pre-cooled PBS was added at a weight-to-volume ratio of 1:9. Two 3 mm zirconium oxide grinding balls were added to the centrifuge tube and fully crushed using a high-speed tissue grinder with parameters of 60 Hz, running for 10 seconds, pausing for 5 seconds, and 12 cycles. To fully lyse tissue cells, an ultrasonic disruptor can be used for further crushing. Finally, the tissue homogenate was centrifuged at 12,000 rpm for 10 minutes using a four-degree centrifuge, and the supernatant was used as the sample for subsequent testing. Subsequent operations were carried out according to the instructions of the TH, DA, and MDA enzyme-linked immunosorbent assay kits.

[0129] TH, DA and MDA were quantitatively analyzed by ELISA. The experimental results are shown in Figure 12 As shown, from Figure 12 As can be seen from a, GQNPs can significantly restore the expression of TH in PD mice, further proving its therapeutic effect on PD mice. Although DFP treatment has a certain effect on TH, it is not enough to cause statistical differences. Figure 12 b As can be seen from the figure, the GQNPs treatment group can significantly increase the DA content in the midbrain, which is reduced in PD mice. These results strongly prove that GQNPs enhance the expression of TH and promote the production of DA by restoring the function of dopaminergic neurons. In order to further verify that the therapeutic effect of GQNPs on PD mice is related to the ferroptosis we speculated above, the MDA level in the midbrain of mice was detected. Figure 12 As shown in Figure 3, GQNPs significantly reduced the increased MDA content in PD mice, while DFP, due to its single iron chelation effect, had no significant effect on MDA clearance. These results demonstrate that GQNPs are significantly effective in treating PD animal models, which is closely related to the nanoparticles' ability to integrate multiple ferroptosis inhibition strategies.

[0130] Example 12 Routine blood test and blood biochemistry test:

[0131] Blood collection from the retroorbital venous plexus: Three mice were randomly selected from each group. After anesthesia with isoflurane, their whiskers were trimmed unilaterally. The neck was then compressed to exophthalmos, and blood was collected from the retroorbital venous plexus. Blood for biochemical analysis was collected in a coagulant tube in an ice bath. The blood was then centrifuged at 3000 g for 5 minutes, and the upper serum layer was collected as the test sample. Samples for routine blood tests were collected in 1.5 mL anticoagulant tubes on ice. Results were analyzed using an automated animal blood and body fluid analyzer.

[0132] The experimental results can be found in Figure 13 As shown in the results, the analysis of blood routine parameters (WBC, RBC, HGB, PLT) and biochemical indicators (ALT, ALB, CREA, TP) showed that there were no significant differences between the experimental groups and the normal control group. These results indicate that GQNPs have good biosafety and biocompatibility and are suitable for further therapeutic applications.

[0133] The present invention takes the inhibition of ferroptosis in Parkinson's disease as its starting point and constructs gallium ion-quercetin coordination nanoparticles (GQNPs). The above-mentioned related test experiments can prove that in terms of reducing iron overload, the gallium ions in the GQNPs prepared by the present invention can competitively bind with transferrin, reducing the cell's uptake of trivalent iron; at the same time, the GQNPs prepared by the present invention can regulate the expression of iron metabolism-related proteins to restore iron homeostasis. In addition, quercetin also gives gallium-based nanoparticles excellent antioxidant capacity and mitochondrial protection function. In animal experiments, GQNPs successfully restored the motor and cognitive functions of PD mice, which shows that the multidimensional ferroptosis inhibition strategy based on GQNPs provides a promising alternative for the treatment of Parkinson's disease.

[0134] Therefore, the gallium ion-quercetin coordination nanoparticles (GQNPs) prepared by the present invention are expected to be used as a new type of multidimensional ferroptosis inhibitor, or to be used to prepare ferroptosis-targeted drugs for Parkinson's disease.

[0135] The in vitro safe dose of this multidimensional ferroptosis inhibitor or Parkinson's disease ferroptosis-targeted drug is 20 μg / mL.

[0136] This multidimensional ferroptosis inhibitor or Parkinson's disease ferroptosis targeted drug can not only improve cellular oxidative stress and significantly reduce the total level of reactive oxygen species in cells, but also enhance the antioxidant capacity of cells and reduce lipid peroxides in cells. It can also reduce excess iron in cells by regulating changes in iron metabolism-related proteins (transferrin receptors and iron storage proteins), significantly reduce the iron content in cells, and improve mitochondrial function by regulating the activity of mitochondrial respiratory chain complexes.

[0137] The gallium ion-quercetin coordination nanoparticles (GQNPs) and deferiprone (DFP) prepared by the present invention exhibited excellent therapeutic effects at both the cellular and in vivo levels, and are expected to be used in the treatment of Parkinson's disease through multidimensional anti-ferroptosis. In addition, they did not show obvious toxic side effects in the examples and are expected to be used for further preclinical research.

[0138] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing gallium ion-quercetin coordinated nanoparticles, characterized in that: The following steps are involved: Step 1) weighing quercetin, hydrated gallium nitrate, and polyvinyl pyrrolidone according to a certain mass ratio; Step 2) dissolving weighed quercetin, hydrated gallium nitrate, and polyvinyl pyrrolidone in corresponding volumes of anhydrous methanol solution to prepare quercetin solution, hydrated gallium nitrate solution, and polyvinyl pyrrolidone solution, respectively; Step 3) mixing the prepared quercetin solution and polyvinyl pyrrolidone solution and stirring at room temperature for a period of time to obtain a quercetin-polyvinyl pyrrolidone mixed solution; Step 4) adding the prepared hydrated gallium nitrate solution dropwise to the uniformly mixed quercetin-polyvinyl pyrrolidone mixed solution, wherein the color of the mixed solution rapidly changes from light yellow to orange-yellow during the addition process, and then stirring the orange-yellow mixed solution at room temperature for a period of time; Step 5) dialyzing the orange-yellow mixed solution in a dialysis bag for a period of time to remove excess gallium ions and polyvinylpyrrolidone, and the final solution obtained is the gallium ion-quercetin coordinated nanoparticles; The gallium ion-quercetin coordination nanoparticles have red-shifted ultraviolet absorption ability compared with quercetin; The gallium ion-quercetin coordination nanoparticles have the ability of spontaneous fluorescence.

2. The preparation method according to claim 1, characterized in that In step 1, the mass ratio of quercetin, hydrated gallium nitrate and polyvinyl pyrrolidone is 8:40:

100.

3. The preparation method according to claim 1, characterized in that In step 2, the volume ratio of the anhydrous methanol solution used to dissolve quercetin, hydrated gallium nitrate and polyvinyl pyrrolidone is 1.5:4:

3.

4. The preparation method according to claim 1, characterized in that The hydrated particle size of the gallium ion-quercetin coordinated nanoparticles is 51.1 nm.

5. A gallium ion-quercetin coordinated nanoparticle, characterized in that: The method is as described in any one of claims 1 to 4.

6. Use of the gallium ion-quercetin coordinated nanoparticles prepared by the preparation method according to any one of claims 1 to 4 and / or the gallium ion-quercetin coordinated nanoparticles according to claim 5 as a ferroptosis inhibitor in the treatment of Parkinson's disease.

7. The use according to claim 6, characterized in that The ferroptosis inhibitor can improve cellular oxidative stress and reduce the total level of reactive oxygen species in cells.

8. The use according to claim 6, characterized in that The ferroptosis inhibitor can enhance the antioxidant capacity of cells and reduce lipid peroxides in cells.

9. The use according to claim 6, characterized in that The ferroptosis inhibitor can reduce the iron content in cells by regulating changes in iron metabolism-related proteins.

10. The use according to claim 6, characterized in that The ferroptosis inhibitor can improve mitochondrial function by regulating the activity of mitochondrial respiratory chain complexes.

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