Application of cerium oxide nanoclusters in improving chronic kidney disease

Abstract

The present invention discloses an application of cerium oxide nanoclusters in improving chronic kidney disease. The cerium oxide nanoclusters have good pharmacokinetic parameters, which include prolonged blood circulation time, stable blood drug concentration and low organ accumulation.

Description

Technical Field

[0001] The present invention relates to the technical field of nano-biomaterials, and in particular to an application of cerium oxide nanoclusters with ultra-high active oxygen scavenging ability and good pharmacokinetic parameters in improving chronic kidney disease. Background Art

[0002] Chronic kidney disease refers to a glomerular filtration rate that is persistently lower than 60 mL / min / 1.73 m 2 Chronic kidney disease (CKD) is a progressive renal parenchymal injury characterized by long-term and multiple symptoms, including retention of toxic metabolites, imbalanced water and electrolyte balance, acid-base imbalance, and internal system damage, resulting in abnormal kidney structure or function, and an albumin / creatinine ratio persistently elevated above 30 mg / g for more than three months. It is one of the most prevalent chronic diseases, with a high incidence rate. End-stage CKD requires kidney transplantation or dialysis, but kidney shortages, prolonged treatment, and high costs place a heavy emotional and financial burden on patients, their families, and society.

[0003] Oxidative stress and inflammation are mediators and driving factors in the development of chronic kidney disease. They promote and amplify each other, ultimately leading to renal fibrosis, apoptosis, and the progression of the disease. For this reason, several reactive oxygen species scavengers, particularly small-molecule natural antioxidants, have been used to treat chronic kidney disease. However, these antioxidants have weak reactive oxygen species scavenging capabilities, require high concentrations, and require frequent administration. Accumulation of these antioxidants in other organs can lead to toxic damage, making them unsuitable for the treatment of chronic diseases. Therefore, new treatments are urgently needed to ameliorate the progression of chronic kidney disease.

[0004] Cerium oxide nanoparticles can 3+ and Ce 4+ Cerium oxide nanoparticles can undergo rapid and regenerative redox cycles between cells and tissues, mimicking enzyme activity such as SOD, and effectively scavenging reactive oxygen species. Therefore, they have been widely used in the biomedical field, especially in ROS-related diseases such as acute lung injury, osteoporosis, ischemia, and heart disease. Currently, there are no reports on the use of cerium oxide nanoparticles to improve chronic kidney disease. Summary of the Invention

[0005] The first purpose of the present invention is to provide a method for preparing cerium oxide nanoclusters with ultra-high reactive oxygen species scavenging ability and good pharmacokinetic parameters, which can be used to improve the progression of chronic kidney disease, that is, to alleviate the development of chronic kidney disease.

[0006] The present invention provides a method for preparing cerium oxide nanoclusters having ultra-high active oxygen scavenging ability and good pharmacokinetic parameters, comprising:

[0007] Dissolving cerium nitrate, oleic acid, and tri-n-octylphosphine oxide in ethanol at 70-80° C., then adding octadecene, and reacting at 170-190° C. for 25-35 minutes, cooling, and then washing to obtain a cerium oxide nanocluster body;

[0008] The activated ligand, sodium alendronate and sodium carbonate aqueous solution are mixed and reacted overnight, and then reacted with the cerium oxide nanoclusters at 75-82° C. for 7-9 hours, followed by separation and precipitation to obtain the cerium oxide nanoclusters.

[0009] It is further defined that the molar ratio of the ligand to the cerium oxide nanocluster body is 4 to 5:1.

[0010] It is further defined that the ligand is succinic acid, PEG600 (polyethylene glycol 600) or PEG2000 (polyethylene glycol 2000).

[0011] It is further defined that the activated ligand is prepared by the following steps: the ligand, N-hydroxysulfosuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are reacted in dichloromethane at room temperature.

[0012] Beneficial effects of the present invention:

[0013] Because chronic kidney disease is a chronic disease accompanied by systemic inflammation caused by local inflammation, hypertension, diabetes, and other factors, it requires long-term, multiple doses. Therefore, drugs used to treat chronic kidney disease should not only have ultra-high reactive oxygen species scavenging capabilities, but also possess favorable pharmacokinetic parameters, such as long blood circulation time, stable blood concentrations, and low organ accumulation, to ensure long-term therapeutic activity and avoid possible cumulative toxicity.

[0014] The ligand-modified cerium oxide nanoclusters prepared using the above method have a particle size of approximately 1.2 nm. Compared to mesoporous cerium oxide nanoclusters (approximately 45 nm), the former have a smaller particle size and exhibit a higher ROS scavenging ability in vitro. In vivo, they have a longer blood circulation time, more stable blood concentrations, and lower organ accumulation, with the performance being particularly pronounced in the spleen and liver. In mice with chronic kidney disease, cerium oxide nanoclusters scavenge ROS through the Nrf2 / Keap1 pathway and reduce inflammation through the NF-κB / p65 / p-p65 pathway, thereby reducing renal fibrosis and renal cell apoptosis, effectively improving the progression of chronic kidney disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the preparation process and characterization diagram of the cerium oxide nanoclusters prepared in Example 1;

[0016] Figure 2 Evaluation of the pharmacokinetic parameters of the cerium oxide nanoclusters prepared in Example 1;

[0017] Figure 3 The therapeutic effect of the cerium oxide nanoclusters prepared in Example 1 on chronic kidney disease;

[0018] Figure 4 The cerium oxide nanoclusters prepared in Example 1 inhibited oxidative stress in chronic kidney disease mice;

[0019] Figure 5 The cerium oxide nanoclusters prepared in Example 1 inhibited the inflammatory response in chronic kidney disease mice;

[0020] Figure 6 The cerium oxide nanoclusters prepared in Example 1 inhibited fibrosis in chronic kidney disease mice;

[0021] Figure 7 The cerium oxide nanoclusters prepared in Example 1 inhibited renal cell apoptosis in chronic kidney disease mice. DETAILED DESCRIPTION

[0022] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0023] Example 1

[0024] Preparation and Characterization of Cerium Oxide Nanoclusters

[0025] (1) Preparation of cerium oxide nanoclusters, such as Figure 1 As shown in Figure (a):

[0026] ①The cerium oxide nanoclusters were synthesized by thermal decomposition method.

[0027] 434 mg of cerium nitrate hexahydrate, 564 mg of oleic acid, and 774 mg of tri-n-octylphosphine oxide were dissolved in 2.0 mL of ethanol at 80°C. 5.0 mL of octadecene was added to the mixture, which was then heated to 180°C in an open container for 30 minutes. After cooling to room temperature, the mixture was washed several times with acetone and toluene to remove byproducts. The synthesized cerium oxide nanoclusters were dispersed in 10 mL of tetrahydrofuran and stored at 4°C.

[0028] ② Preparation of mesoporous cerium oxide nanoparticles as a control.

[0029] 217 mg of cerium nitrate hexahydrate and 290 mg of tri-n-octylphosphine oxide were dissolved in 2.0 mL of ethanol at 80°C. 5.0 mL of octadecene was added to the mixture, and the mixture was heated to 180°C in an open container for 30 minutes. The remaining steps were identical to those for the synthesis of the cerium oxide nanoclusters.

[0030] (2) Ligand modification of cerium oxide nanoclusters:

[0031] ① A certain amount of ligand (succinic acid: 109 mg, PEG600: 554 mg, PEG2000: 1847 mg), 105 mg of N-hydroxysulfosuccinimide, and 175 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide were dissolved in 10 mL of dichloromethane and stirred at room temperature for 8 hours. After removing the solvent by rotary evaporation, a mixture containing 100 mg of sodium alendronate and 250 mg of sodium carbonate dissolved in water was added to the activated ligand and allowed to react overnight at room temperature. 1 mL of the synthesized cerium oxide nanoclusters bulk was diluted in 5 mL of tetrahydrofuran and mixed with 5 mL of the ligand solution. The mixture was stirred at 80°C for 8 hours. After cooling to room temperature, the aqueous layer containing the cerium oxide nanoclusters was separated and precipitated by adding acetone. The surface-modified cerium oxide nanoclusters were dialyzed against ultrapure water for 24 hours using a 30 kD dialysis tubing. The obtained cerium oxide nanoclusters were named NC-SA, NC-PEG600, and NC-PEG2000, respectively.

[0032] ② Ligand modification of mesoporous cerium oxide nanoparticles: The same method was used to modify the surface of mesoporous cerium oxide nanoparticles, which were named NP-SA, NP-PEG600 and NP-PEG2000 respectively.

[0033] The NC-SA, NC-PEG600, NC-PEG2000, NP-SA, NP-PEG600 and NP-PEG2000 prepared in this example were characterized as follows:

[0034] ① Morphology and size of cerium oxide nanoclusters. Cerium oxide nanoclusters dispersed in toluene were dropped onto an ultrathin copper mesh and their morphology was observed using a FEI Talos F200X transmission electron microscope at 200 kV. The size distribution of the nanomaterials was measured using nanomeasurer software.

[0035] ② Thermogravimetric analysis: TA SDT650 instrument was used for detection and analysis at a heating rate of 20°C / min.

[0036] ③ X-ray photoelectron spectroscopy (XPS) analysis was performed on a SCIENTIFIC ESCALAB 250 instrument. The binding energy (875 eV to 925 eV) was recorded and calibrated with the Ce3d peak at 883.20 eV. XPS Peak Fit 4.0 software was used for analysis. The software set the Ce 3+ The binding energy peaks of Ce concentration are 880.4eV, 885.5eV, 898.8eV and 903.7eV respectively. 4+The binding energy peaks of the concentrations are 882.7eV, 888.96eV, 898.2eV, 901.3eV, 907eV and 916.7eV, respectively.

[0037] ④ Evaluation of in vitro ROS scavenging ability. 800 μL EDTA-Na2 (0.1 M), 300 μL NBT (2 mM), 200 μL riboflavin (0.6 mM), and 11.4 mL sodium phosphate buffer (10 mM, pH 7.8) were mixed to prepare a working solution. The surface-modified cerium oxide nanoclusters were diluted to the specified concentration in sodium phosphate buffer (10 mM, pH 7.8) to prepare a test solution. After mixing the test solution (50 μL) and the working solution (100 μL) in a 96-well plate and shaking for 60 seconds, the sample was placed under white light for 90 seconds. Excess ROS reacted with NBT to generate a blue color, and the absorbance was measured at 560 nm. The ROS inhibition rate was determined by calculating the decrease in absorbance at 560 nm.

[0038] The results are as follows Figure 1 As shown, Figure 1 (b) Electron microscopy and particle size statistics of cerium oxide nanoclusters; (c) Thermogravimetric images of cerium oxide nanoclusters modified with three different ligands; (d) In vitro reactive oxygen species scavenging test, compared with mesoporous cerium oxide nanoparticles; (eg) XPS test of cerium oxide nanoclusters modified with three different ligands.

[0039] Depend on Figure 1 It can be seen that, through the preparation method disclosed in Example 1, cerium oxide nanoclusters with a particle size of about 1.2 nm were successfully prepared, and had ultra-high active oxygen scavenging ability in vitro.

[0040] Example 2

[0041] Study on the pharmacokinetic parameters of NC-PEG600 and NP-PEG600 prepared in Example 1.

[0042] Mice were randomly divided into NC-PEG600 and NP-PEG600 groups, with 15 mice in each group. Each group received the corresponding cerium oxide nanoclusters or mesoporous cerium oxide nanoparticles at a dose of 10 mg / kg body weight. Orbital blood was collected at 0.25, 0.5, 1, 2, 6, 12, 24, and 48 hours after administration, and organ samples were collected at 24, 48, and 72 hours. Blood samples (10 μL) were digested in concentrated nitric acid (190 μL) at 60°C for 8 hours. Organ samples were digested with concentrated nitric acid (20 mL) in a CEM MARS6 microwave digestion system using the instrument's standard tissue digestion procedure. Sample concentrations were determined using a PE AVIO200 ICP-AES analyzer.

[0043] Figure 2Figure (a) is a schematic diagram of collecting blood samples and organ samples; Figure (b) shows the blood drug concentration within 48 hours; Figure (c) shows the drug accumulation in organ samples.

[0044] Depend on Figure 2 As shown in Figures (b) and (c), in vivo, compared with NP-PEG600 (mesoporous cerium oxide), NC-PEG600 (cerium oxide nanoclusters) has good pharmacokinetic parameters, longer blood circulation time, more stable blood drug concentration, and lower organ accumulation.

[0045] Example 3

[0046] Establishment of chronic kidney disease mouse model and drug administration

[0047] The animals were housed in a pathogen-free environment with free access to water and food. After acclimation, the mice were randomly divided into five groups: control group, model group, NC-SA group, NC-PEG600 group, and NC-PEG2000 group. The control group was fed a normal diet, while the remaining groups were fed a diet containing 0.2% (w / w) adenine to induce the construction of chronic kidney disease mice. After 4 weeks, all groups were fed a normal diet, and the three groups of mice in the treatment group were intraperitoneally injected with the corresponding cerium oxide nanoclusters (1 mg / kg) once a day for four consecutive weeks. The control and model groups were intraperitoneally injected with an equal volume of normal saline.

[0048] Changes in mouse body weight during chronic kidney disease modeling and cerium oxide nanocluster treatment Figure 3 As shown in Figure (a).

[0049] Example 4

[0050] Cerium oxide nanoclusters improve the progression of chronic kidney disease

[0051] (1) Renal function test:

[0052] Mice were anesthetized with 3% isoflurane and blood was collected from the eye socket. The blood samples were centrifuged at 4000 rpm for 10 min, and the upper serum was collected for blood urea nitrogen (BUN) and blood creatinine (Cr) testing. Figure 3 (f) and (g) show the serum creatinine and urea nitrogen tests, respectively.

[0053] (2) Tissue staining:

[0054] Kidneys were collected and fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5-μm sections. The sections were stained with hematoxylin and eosin (HE), Masson's red, and Sirius red, respectively. Images were collected and scanned on a SOPTOP HS6 instrument, and the area of ​​renal fibrosis was determined using Image J software. For CD31 and F4 / 80 immunohistochemistry, sections were immersed in blocking solution for 20 minutes, rinsed three times with PBS, and incubated with monoclonal antibodies against CD31 and F4 / 80, respectively, overnight at 4°C. Sections were then rinsed three times with PBS and incubated with secondary antibodies for 30 minutes at room temperature. Finally, sections were rinsed three times with PBS, developed with DAB solution, and coverslipped. For TUNEL staining, paraffin sections were dewaxed twice in xylene for 5-10 minutes. Dewaxed sections were then treated with anhydrous ethanol for 5 minutes, 90% ethanol for 2 minutes, 70% ethanol for 2 minutes, and distilled water for 2 minutes. Dewaxed renal cortical sections were incubated with 20 μg / mL proteinase K at room temperature for 20 min and then incubated with the TdT reaction mixture at 37°C in the dark for 60 min. After rinsing three times with PBS, sections were counterstained with 4′,6-diaminodiphenylmethane dihydrochloride (DAPI) and images were acquired using an Olympus Slide VS200 whole-slide scanner.

[0055] The collected kidney images are as follows Figure 3 Figure (b) shows a macroscopic image of the kidney (top) and its homogenate (bottom); (c) shows CD31 staining of the kidney (top) and the section staining of the positive area (bottom); (d) shows the HE-stained kidney (top) and the magnified cortex (middle) and medulla (bottom); (e) shows the statistics of CD31-positive areas in (c); (h) KIM-1 mRNA expression level.

[0056] (3) Western blot test

[0057] Kidneys were homogenized in pre-chilled RIPA lysis buffer, and the lysate was centrifuged at 12,000 g for 10 minutes at 4°C to isolate proteins. Protein concentration was determined using a BCA protein assay kit. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 10% gel) and transferred to a PVDF membrane. After blocking with 5% skim milk for 1 hour, the membrane was incubated with the primary antibody overnight at 4°C and the secondary antibody for 2 hours. The membrane was then washed three times with TBST. Finally, bands were visualized using a Bio-Rad ChemiDoc MP imaging system.

[0058] (4) qRT-PCR detection

[0059] Kidneys were homogenized in pre-chilled Trizol, and total RNA was extracted according to the manufacturer's instructions. RNA concentration was determined on a BioDrop μLite instrument, and 1 μg of RNA was reverse transcribed into cDNA using a reverse transcription kit. The reaction mixture containing cDNA was subjected to PCR amplification using specific primers for KIM-1, HO-1, NOX2, IL-1β, IL-6, TNF-α, NF-κB, α-SMA, COL-1, TGF-β1, and Caspase-3.

[0060] (5) Malondialdehyde and total superoxide dismutase detection

[0061] Kidneys were homogenized in buffer, and protein concentration was determined using a BCA protein assay kit to facilitate calculation of malondialdehyde and total superoxide dismutase content per unit weight of tissue. MDA and total superoxide dismutase assay procedures were performed according to the manufacturer's instructions, and malondialdehyde and total superoxide dismutase levels per unit weight were calculated using a standard curve.

[0062] Figure 4 The cerium oxide nanoclusters prepared in Example 1 inhibit oxidative stress in chronic kidney disease mice: (a) (b) HO-1 immunohistochemical staining and statistical graphs; (c) MDA concentration in kidney homogenate supernatant; (d) T-SOD concentration in kidney homogenate supernatant; (e) (f) HO-1 and NOX2 mRNA expression levels; (g) Nrf2 and Keap1 protein expression levels;

[0063] Figure 5 Example 1: Cerium oxide nanoclusters inhibit inflammatory response in chronic kidney disease mice: (a), (b) F4 / 80 immunohistochemical staining and statistical graph of positive areas; (cf) mRNA expression levels of inflammatory factors IL-1β, IL-6, TNF-α, and NF-κB; (g) protein expression levels of p65 and p-p65;

[0064] Figure 6 Example 1: Cerium oxide nanoclusters inhibit fibrosis in chronic kidney disease mice: (ad) Masson staining and Sirius red staining, and statistical graphs of fibrosis areas; (eg) mRNA expression levels of fibrosis-related factors α-SMA, COL-1, and TGF-β1;

[0065] Figure 7 Example 1: Cerium oxide nanoclusters inhibit renal cell apoptosis in chronic kidney disease mice: (a), (b) TUNEL fluorescence staining and statistical graph of its positive area; (c) Caspase-3 mRNA expression level; (d) Bax and Bcl2 protein expression levels.

[0066] From the above, it can be seen that in mice with chronic kidney disease, cerium oxide nanoclusters scavenged reactive oxygen species through the Nrf2 / Keap1 pathway and alleviated the inflammatory response through the NF-κB / p65 / p-p65 pathway, thereby reducing renal fibrosis and apoptosis of renal cells, effectively improving the development of chronic kidney disease.

[0067] In summary, the cerium oxide nanoclusters with a particle size of about 1.2 nm prepared by the preparation method disclosed in the present invention have ultra-high reactive oxygen species scavenging ability and good pharmacokinetic parameters (1. Longer blood circulation time; 2. More stable blood concentration; 3. Lower organ accumulation), can effectively improve the development process of chronic kidney disease (1. Clear reactive oxygen species; 2. Reduce inflammatory response; 3. Improve the process of renal fibrosis; 4. Improve cell apoptosis in the kidney), and are worthy of promotion and use.

[0068] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing cerium oxide nanoclusters with ultra-high active oxygen scavenging ability and good pharmacokinetic parameters, characterized in that: The preparation method comprises: Cerium nitrate, oleic acid, and tri-n-octylphosphine oxide are dissolved in ethanol at 70-80°C, followed by addition of octadecene, and reaction at 170-190°C for 25-35 minutes. The mixture is cooled and then washed to obtain a cerium oxide nanocluster body. An activated ligand, sodium alendronate, and an aqueous sodium carbonate solution are mixed and reacted overnight, and then reacted with the cerium oxide nanocluster body at 75-82°C for 7-9 hours. The mixture is then separated and precipitated to obtain the cerium oxide nanocluster. The ligand is succinic acid, PEG600 or PEG2000; The particle size of the cerium oxide nanoclusters is 1.0-1.5 nm.

2. The preparation method according to claim 1, characterized in that The molar ratio of the ligand to the cerium oxide nanocluster body is 4-5:

1.

3. The preparation method according to claim 1, characterized in that The activated ligand is prepared by the following steps: the ligand, N-hydroxysulfosuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide are reacted in dichloromethane at room temperature.

4. A cerium oxide nanocluster prepared by the preparation method according to any one of claims 1 to 3, characterized in that: The particle size of the cerium oxide nanoclusters is 1.0-1.5 nm.

5. Use of the cerium oxide nanoclusters according to claim 4 in preparing a drug for improving the progression of chronic kidney disease, characterized in that: The cerium oxide nanoclusters have good pharmacokinetic parameters, which include prolonged blood circulation time, stable blood drug concentration and low organ accumulation.

6. Use of the cerium oxide nanoclusters according to claim 5 in preparing a drug for improving the progression of chronic kidney disease, characterized in that: The chronic kidney disease is induced by adenine.

7. Use of the cerium oxide nanoclusters according to claim 5 in preparing a drug for improving the progression of chronic kidney disease, characterized in that: The chronic kidney disease is a systemic inflammatory disease caused by local inflammation, hypertension or diabetes.

8. Use of the cerium oxide nanoclusters according to claim 5 in preparing a drug for improving the progression of chronic kidney disease, characterized in that: The drug further includes a pharmaceutically acceptable carrier.

9. Use of the cerium oxide nanoclusters according to claim 5 in preparing a drug for improving the progression of chronic kidney disease, characterized in that: The improvement of the progression of chronic kidney disease is mainly achieved by scavenging reactive oxygen species through the Nrf2 / Keap1 pathway and alleviating inflammatory responses through the NF-κB / p65 / p-p65 pathway.

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