Nanoparticle with anti-oxidation and anti-ferroptosis functions as well as preparation and application thereof

Abstract

The invention provides a nanoparticle with anti-oxidation and anti-ferroptosis functions as well as preparation and application of the nanoparticle. The nanoparticles are cerium element-containing carbon dots (Ceuim-GSHCDs, Ce-CDs) synthesized on the basis of microwaves, and a small molecule compound, namely, tripterine (Cel), is loaded on the Ce-CDs through non-covalent binding, so that the efficient antioxidant and ferroptosis regulator Ce-CDs (at) Cel is prepared. The size of the nanoparticles is about 3 nm, and the nanoparticles are in a uniform sphere shape, good in dispersity and clear in crystal lattice. The nano material prepared by the invention can effectively inhibit ferroptosis and oxidative stress caused by high oxalic acid, reduce calculus formation and improve the treatment effect of calcium oxalate calculus.

Description

Technical field:

[0001] The present invention relates to the field of biomedicine, and in particular to a nanoparticle with anti-oxidation and anti-ferroptosis functions and a preparation method and application thereof. Background technology:

[0002] Kidney stones are one of the most common diseases of the urinary system due to their high prevalence and recurrence rates. Patients with kidney stones face the risk of pain, loss of renal function, and even sepsis. Currently, there is still no drug that can effectively prevent the recurrence of kidney stones. Recent studies have shown that ferroptosis, a new type of cell death caused by free iron overload and massive accumulation of intracellular lipid reactive oxygen species (ROS), is closely related to the occurrence of kidney stones. Several significant and well-defined characteristics of kidney stones, such as abnormal accumulation of lipid reactive oxygen species (ROS) in renal tubular epithelial cells, decreased glutathione (GSH) levels, and inactivation of glutathione peroxidase 4 (GPX4), are consistent with the characteristics of ferroptosis. Ferroptosis is not only the result of the development and progression of kidney stones, but also its main pathogenic factor. Targeted regulation of the ferroptosis state of the renal microenvironment may be an effective strategy for the prevention and treatment of stones in the future. Summary of the invention:

[0003] 1. Technical issues to be resolved

[0004] The present invention aims at the problem that there is no drug that can effectively prevent the recurrence of kidney stones, and proposes nanoparticles with antioxidant and anti-ferroptosis functions and their preparation and application. The nanoparticles can effectively inhibit ferroptosis and oxidative stress caused by high oxalate, reduce stone formation, and improve the treatment effect of kidney stones.

[0005] (II) Technical solution

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A nanoparticle with antioxidant and anti-ferroptosis functions, wherein the nanoparticle is an antioxidant and ferroptosis regulator Ce-CDs@Cel, wherein the Ce-CDs@Cel is Ce-CDs loaded with a small molecule compound celastrol Cel through non-covalent binding, wherein the Ce-CDs is a carbon dot containing cerium element synthesized based on microwave, and its English name is Ceruim-GSHCDs, abbreviated as Ce-CDs.

[0008] The preparation method of Ce-CDs@Cel comprises the following steps:

[0009] S1, citric acid, glutathione GSH and a certain amount of cerium nitrate were dissolved in ultrapure water, heated in a microwave oven for 4 min, and then 10 mL of ultrapure water was added to disperse the mixture, centrifuged at 10000 rpm for 10 min, large particles of precipitate were removed, and Ce-CDs were obtained after freeze-drying;

[0010] S2, dissolving Cel in anhydrous ethanol to obtain Cel in anhydrous ethanol, dispersing Ce-CDs in ultrapure water and adding the Cel in anhydrous ethanol solution, thoroughly mixing and rotary evaporating to remove anhydrous ethanol, removing the precipitate by low-speed centrifugation to obtain Ce-CDs@Cel, and storing at 4°C.

[0011] Preferably, the mass ratio of citric acid, glutathione GSH, cerium nitrate and tripterygium wilfordii Cel is 20-30: 5-10: 0.1-5: 0.1-1.

[0012] Preferably, the mass ratio of citric acid, glutathione GSH, cerium nitrate and celastrol Cel is 27:8:0.7:1.

[0013] Preferably, the nanoparticles Ce-CDs@Cel are used in the preparation of drugs for treating kidney stones.

[0014] Preferably, the kidney stones are calcium oxalate stones.

[0015] The present invention also provides a nanocarrier, which is a carbon dot Ce-CDs containing cerium element synthesized based on microwaves, and the components of the Ce-CDs are citric acid, glutathione GSH and cerium oxide.

[0016] (III) Beneficial effects

[0017] The nanoparticles with antioxidant and anti-ferroptosis functions proposed in the present application are based on cerium-containing ceruim-GSHCDs synthesized by microwave, and are loaded with the small molecule compound celastrol Cel by covalent binding. They can not only effectively reduce the toxicity of celastrol Cel, but also effectively improve its effect in the treatment of kidney stones. Moreover, the nanoparticles have a size of about 3 nm, a uniform spherical shape, good dispersion, a clear lattice, and a simple preparation method. They can effectively inhibit ferroptosis and oxidative stress caused by high oxalic acid, reduce stone formation, and improve the therapeutic effect of calcium oxalate stones, and are suitable for large-scale promotion and application. Description of the drawings:

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below.

[0019] Figure 1 This is the state diagram of HK2 cells after administration of Ce-CDs;

[0020] Figure 2 The results of CCK-8 experiments of Ce-CDs on HK-2 cells;

[0021] Figure 3 Characterization electron microscope image of Ce-CDs@Cel;

[0022] Figure 4 This is the spectrum analysis diagram of Ce-CDs@Cel;

[0023] Figure 5 It is a comparison chart of total SOD activity of Ce-CDs solution with different concentrations;

[0024] Figure 6 Comparison of total antioxidant capacity of Ce-CDs solutions with different concentrations (ABTS method);

[0025] Figure 7 This is a test diagram of cytotoxicity detected by CCK-8 method;

[0026] Figure 8 This is a comparison chart of the absorbance calculation results of the hemolysis rate measured by the microplate reader at a wavelength of 545nm;

[0027] Fig. 9 This is a blood biochemical test chart of mouse renal function;

[0028] Fig.10 HE staining micrograph of mouse renal function;

[0029] Fig.11 The cellular uptake of Ce-CDs and Ce-CDs@Cel was observed using a confocal microscope;

[0030] Fig.12 This is a picture showing the formation of calcium oxalate crystals observed by pizzalato staining;

[0031] Fig.13 This is a comparison of the therapeutic effects of PAS staining on mouse kidney inflammation;

[0032] Fig.14 This is the immunohistochemical evaluation of KIM-1, SOD, and OPN molecular detection in mouse kidney;

[0033] Fig.15 This is a picture of TUNEL experiment in mouse kidney;

[0034] Fig.16 DHE staining shows the detection of reactive oxygen species in mouse kidneys;

[0035] Fig.17 The morphology of mitochondrial shrinkage and mitochondrial cristae in mouse renal tubular epithelial cells induced by glyoxylate was observed by TEM electron microscopy.

[0036] Fig.18 Western blot detection of mouse kidney ferroptosis pathway;

[0037] Fig.19 for Fig.16 Statistical comparison chart of China Railway Death Pathway Detection Results; Specific implementation method:

[0038] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0039] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0040] Example

[0041] 1. Preparation method:

[0042] Citric acid, GSH and a certain amount of cerium nitrate were dissolved in ultrapure water, heated in a microwave oven for 4 minutes, and then 10 mL of ultrapure water was added to disperse the mixture, centrifuged at 10000 rpm for 10 minutes, large particles of precipitates were removed, and Ce-CDs were obtained after freeze-drying. Tripterine was dissolved in anhydrous ethanol to obtain a tripterine ethanol solution, Ce-CDs were dispersed in ultrapure water and the tripterine ethanol solution was added, and after thorough mixing, the anhydrous ethanol was removed by rotary evaporation, and the precipitate was removed by low-speed centrifugation to obtain Ce-CDs@Cel, which was stored at 4°C.

[0043] After Ce-CDs were prepared with citric acid, glutathione GSH, cerium nitrate and tripterygium wilfordii in a ratio of 27:8:3.5:1, the cytotoxicity was evaluated on cells. Microscopic observation showed that a large number of cells died after adding 1 mg / mL Ce-CDs, and no cell death was observed after adding 1 mg / mL CDs. Subsequently, it was mixed with undoped Ce CDs at different ratios for administration. No cell death was observed when CDs:Ce-CDs was 2:1, as shown in Fig. Figure 1 The Ce-CDs were prepared by adjusting the dosage of cerium nitrate with this ratio as a reference value, and the ratio of citric acid, glutathione GSH, cerium nitrate and tripterygium wilfordii Cel was adjusted to 27:8:1.16 / 0.7 / 0.35:1. CCK-8 assay was performed on HK-2 cells to evaluate cytotoxicity. Figure 2 The ratio of citric acid, glutathione GSH, cerium nitrate and celastrol Cel was selected as 27:8:0.7:1 as the final feed ratio.

[0044] 2. Characterization of Ce-CDs@Cel:

[0045] It can be clearly observed in the field emission transmission electron microscope that the size of Ce-CDs is about 3nm, with a uniform spherical shape, good dispersion, and clear lattice (0.199nm). The size and lattice remain basically unchanged after adding tripterygium wilfordii. Figure 3 . Dynamic light scattering (DLS). The optical properties were studied by UV-visible spectrophotometer and fluorescence spectrometer. An absorption peak at 330nm was detected in the aqueous solution of Ce-CDs and Ce-CDs@Cel, representing the n→π* electronic transition of the surface carbonyl group. The FL spectrum showed that the optimal excitation and emission wavelengths of Ce-CDs were 350.8nm and 420.6nm, respectively. After loading Cel, they shifted to 359.3nm and 425.6nm. This change in excitation and emission wavelengths indicated that Cel was adsorbed on the carbon dots, causing changes in the orbital energy of the system. In addition, both Ce-CDs and Ce-CDs@Cel exhibited energy-dependent excitation. When the wavelength of the excitation light (310nm-400nm) was increased or decreased on the basis of 350nm, the emission intensity of Ce-CDs and Ce-CDs@Cel decreased accordingly, as shown in Figure 2. Figure 4 .

[0046] 3. Application scenarios and functional verification:

[0047] 1. SOD-like activity: Superoxide anion detection kit (Biyuntian Biotechnology Co., Ltd.) was used to measure 2- Ce-CDs solutions of different concentrations (0-200 μg / mL) were added to the working solution, reacted at 37°C for 30 minutes, and the absorbance at 450 nm was measured using an enzyme marker. Figure 5 and Figure 6 .

[0048] Results: In order to verify the ability of Ce-CDs to scavenge ROS, the superoxide anion detection kit was used to detect the O 2- Compared with CDs without Ce incorporation, Ce-CDs and Ce-CDs@Cel showed a significant inhibitory effect on O 2- The ability of free radicals was enhanced, indicating that Ce components mediated SOD-like activity. 2- The scavenging activity gradually increased.

[0049] 2. Cytotoxicity: HK-2 cells were seeded at a density of 1×104 cells per well in a 96-well plate. After incubation for 24 h, the culture medium was replaced with a culture medium containing different concentrations (0-1 mg / mL) of drugs (Cel, Ce-CDs, Ce-CDs@Cel). After the plate was incubated for 24 h, 10 μL of CCK8 solution was added. After incubation for 2 h, the absorbance at a wavelength of 450 nm was recorded using a microplate reader (Varioskan LUX, Thermo Fisher). Figure 7 .

[0050] Results: CCK-8 assay results showed that the survival rate of HK-2 cells remained above 90% after incubation with 0-1 mg / mL Ce-CDs / Ce-CDs@Cel for 24 h.

[0051] 3. Hemolysis rate: Whole blood was collected from healthy C57 mice using EDTA anticoagulant tubes, 10 times the volume of normal saline was added, red blood cells (RBC) were separated by centrifugation (2000 rpm, 10 min), different concentrations (0.1-10 mg / mL) of Ce-CDs / Ce-CDs@Cel were mixed with 400 μL of 5% RBC suspension, Ce-CDs / Ce-CDs@Cel was replaced with H2O as a positive control, and normal saline was used as a negative control. After incubation at 37°C for 2 h, centrifugation (2000 rpm, 5 min) was performed, the supernatant was collected, and the absorbance at a wavelength of 545 nm was measured using an ELISA reader. The hemolysis rate was calculated by the following formula:

[0052] Hemolysis rate (%) = (absorbance of test sample - absorbance of negative control) / (absorbance of positive control - absorbance of negative control) × 100%, see Figure 8 .

[0053] Results: In addition, the biosafety was further investigated by evaluating the effect of Ce-CDs / Ce-CDs@Cel on cell hemolysis. When the concentration of Ce-CDs / Ce-CDs@Cel was 0-10 mg / mL, the hemolysis rate was less than 5% and no hemolysis occurred, indicating that Ce-CDs / Ce-CDs@Cel had high biocompatibility.

[0054] 4. Biocompatibility: After administration, approximately 0.3 mL of blood was collected from each mouse for blood biochemical examination. Serum alanine aminotransferase (ALT), alanine aminotransferase (AST), and creatinine (CRE) levels were measured using a blood biochemical test kit to evaluate liver and kidney function. The mice were euthanized, and the main organs (heart, liver, spleen, lungs, and kidneys) were collected, fixed with 4% paraformaldehyde, and embedded in paraffin. Subsequently, hematoxylin-eosin (H&E) staining was used, and the tissue morphology was observed under a microscope. Fig. 9 and Fig.10 .

[0055] Results: Routine blood tests and liver and kidney function indices were within normal ranges, and no obvious pathological changes were observed in the H&E-stained sections of major organs, indicating that Ce-CDs@Cel has high biocompatibility.

[0056] 5. Cell uptake: HK-2 cells were seeded into 12-well plates at a density of 5×104 cells per well and cultured overnight. Ce-CDs@Cel (1 mg / mL) was added and incubated for 2-24 hours. Cells were fixed with paraformaldehyde and then stained with cell membrane (DiD). Confocal microscopy was used for observation. Fig.11 .

[0057] Results: The cellular uptake of Ce-CDs and Ce-CDs@Cel was observed by confocal microscopy. CDs emitted blue light when excited. In the initial stage of co-incubation of drugs and cells (2 h), the intracellular drug fluorescence was weak. Over time, the fluorescence in the cytoplasm increased, indicating that the cellular uptake of drugs increased. There was no significant difference in fluorescence between 10 h and 24 h of co-incubation, indicating that the cell uptake was basically saturated at 10 h.

[0058] 6. Effect and mechanism of nanomedicine in preventing and treating stones: Calcium oxalate stone mice were modeled by intraperitoneal injection of glyoxylate (Gly), and treated by tail vein injection of Ce-CDs@Cel. At the same time, the other two groups of mice were treated by tail vein injection of Cel and oral administration of potassium sodium hydrogen citrate as reference. The formation of calcium oxalate crystals was observed under polarizing microscope and pizzalato staining, see Fig.12 The results showed that calcium oxalate crystals were significantly produced in the Gly modeling group, while calcium oxalate crystals were significantly reduced after treatment with Ce-CDs@Cel. The results of PAS staining showed that the renal inflammation of mice in the Ce-CDs@Cel treatment group was significantly reduced compared with the modeling group and other treatment groups. Fig.13 To further evaluate the tubular injury, we evaluated the expression of KIM-1 in the kidney by immunohistochemistry. Similarly, the expression of KIM-1 in the kidney of the Ce-CDs@Cel treated mice was significantly reduced compared with that in the GOX model group. Fig.14 The results of TUNEL experiments showed that the Ce-CDs@Cel treatment group could inhibit renal cell death. Fig.15 The above data show that Ce-CDs@Cel can effectively reduce the formation of calcium oxalate stones, reduce the inflammation of the renal microenvironment, alleviate the damage to renal tubular cells and protect the normal morphology of renal tubules.

[0059] See Fig.17TEM observation showed that the mitochondria of the renal tubular epithelial cells of mice induced by glyoxylate shrank and the morphology of mitochondrial cristae was destroyed, which was consistent with the changes in the ultrastructure of mitochondria in ferroptotic cells. Western blot experiments detected the expression of Nrf2, Slc7a11, and GPX4 in different groups. The results showed that the expression of Nrf2, slc7a11, and GPX4 in the kidneys of mice in the calcium oxalate stone group was significantly increased, while it was significantly reduced in the kidneys of mice in the Ce-CDs@Cel treatment group. Fig.18 and Fig.19 Similarly, immunohistochemical detection of SOD molecules revealed that the expression of SOD in the Ce-CDs@Cel treatment group was significantly increased, and DHE staining showed that the reactive oxygen species in the Ce-CDs@Cel treatment group was significantly reduced. Fig.16 . This indicates that Ce-CDs@Cel alleviates oxidative stress and intervenes in the state of renal tubular ferroptosis by regulating the expression of slc7a11 and GPX4 molecules, thereby reducing the formation of stones.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the present invention and are not intended to limit the protection scope of the present invention. In addition, after reading the technical content of the present invention, those skilled in the art may make various changes, modifications or variations to the present invention, and all of these equivalent forms also fall within the protection scope defined by the present application.

Claims

1. A nanoparticle with antioxidant and anti-ferroptosis functions, characterized in that: The nanoparticles are antioxidants and ferroptosis regulators Ce-CDs@Cel, the Ce-CDs@Cel are Ce-CDs loaded with a small molecule compound celastrol Cel through non-covalent binding, the Ce-CDs are carbon dots containing cerium elements synthesized based on microwaves, and the English name is Ceruim-GSHCDs, abbreviated as Ce-CDs.

2. The nanoparticle according to claim 1, characterized in that Preparation method of Ce-CDs@Cel The following steps are involved: S1, citric acid, glutathione GSH and a certain amount of cerium nitrate were dissolved in ultrapure water, heated in a microwave oven for 4 min, and then 10 mL of ultrapure water was added to disperse the mixture, centrifuged at 10000 rpm for 10 min, large particles of precipitate were removed, and Ce-CDs were obtained after freeze-drying; S2, dissolving Cel in anhydrous ethanol to obtain Cel in anhydrous ethanol, dispersing Ce-CDs in ultrapure water and adding the Cel in anhydrous ethanol solution, thoroughly mixing and rotary evaporating to remove anhydrous ethanol, removing the precipitate by low-speed centrifugation to obtain Ce-CDs@Cel, and storing at 4°C.

3. The method for preparing Ce-CDs@Cel according to claim 2, characterized in that: The mass ratio of the citric acid, glutathione GSH, cerium nitrate and tripterygium wilfordii Cel is 20-30:5-10:0.1-5:0.1-1.

4. The method for preparing Ce-CDs@Cel according to claim 3, characterized in that: The preferred mass ratio of the citric acid, glutathione GSH, cerium nitrate and celastrol Cel is 27:8:0.7:

1.

5. Use of the nanoparticle Ce-CDs@Cel according to any one of claims 1 to 4 in the preparation of a drug for treating kidney stones.

6. The use according to claim 5, characterized in that: The kidney stones are calcium oxalate stones.

7. A nanocarrier, characterized in that: The nano-carrier is a carbon dot Ce-CDs containing cerium element synthesized based on microwave, and the components of the Ce-CDs are citric acid, glutathione GSH and cerium oxide.

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