CN114272264A - Application of prussian calcium nanoparticles in preparation of medicines for treating and/or preventing acute kidney injury diseases - Google Patents
Application of prussian calcium nanoparticles in preparation of medicines for treating and/or preventing acute kidney injury diseases Download PDFInfo
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- CN114272264A CN114272264A CN202111357513.4A CN202111357513A CN114272264A CN 114272264 A CN114272264 A CN 114272264A CN 202111357513 A CN202111357513 A CN 202111357513A CN 114272264 A CN114272264 A CN 114272264A
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Abstract
The invention discloses an application of prussian calcium nano-particles in preparation of a medicine for treating and/or preventing acute kidney injury diseases. The prussian calcium nano-particle has the functions of nano-enzyme activity, oxidation resistance and iron death inhibition, can effectively treat and/or prevent acute kidney injury, is used as a novel nano-material, and can be applied to the rescue of oxidative injury such as acute kidney injury and the like and various free radical injury diseases.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to application of prussian calcium nanoparticles in preparation of medicines for treating and/or preventing acute kidney injury diseases.
Background
Prussian Blue (PB) is also called ferric ferrocyanide (chemical formula Fe)4[Fe(CN)6]3) Is a long-history blue dye, Fe3+And Fe2+The coexistence gives the PB particular physical, chemical, optical and magnetic advantages. Due to the special molecular structure and chemical properties of PB, researchers have attracted great interest in the past decade, and have been widely used in various fields such as electrochemistry, gas storage, magnetism, biomedicine, catalysis, batteries, and sensors, and the application thereof in biomedicine field is particularly interesting.
With the continuous development of nano science and nano technology, the nano material has wide application due to the unique advantages of simple and controllable preparation, easy surface functionalization and functional assembly, good stability, higher drug loading rate, targeting property and the like, and the PB nano particles (PBNPs) also have the unique properties. In addition, PBNPs have a Fe-like structure3O4The nanometer particle has nanometer enzyme characteristic and can be used in the fields of disease treatment, etc.
Acute Kidney Injury (AKI) is a common clinical symptom, which refers to rapid renal injury and hypofunction caused by various reasons, the incidence rate of AKI is high, and is closely related to the occurrence of chronic kidney disease and the adverse prognosis of severe patients, and the severe stage is Acute Renal Failure (ARF) which needs to receive Renal Replacement Therapy (RRT). Intervention in time at the initial stage of AKI can reduce kidney injury to the maximum extent and promote renal function recovery. Therefore, the development of effective therapeutic and prophylactic agents for AKI-related diseases is of great importance.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides the application of the novel Prussian calcium nanoparticles in preparing the medicines for treating and/or preventing acute kidney injury diseases, and provides a novel method for treating AKI diseases.
The invention also provides application of the prussian calcium nano-particles in preparation of a medicament for inhibiting iron death.
According to one aspect of the invention, the application of the prussian calcium nanoparticles in preparing the medicine for treating and/or preventing acute kidney injury diseases is provided.
According to a specific embodiment of the invention, at least the following advantages are achieved: the Prussian calcium nano-particles have nano-enzyme activity, in-vivo and in-vitro oxidation resistance and anti-iron death capability, are used as a novel nano-material, and can be applied to the rescue of oxidative damage such as acute kidney injury and the like and various free radical injury diseases.
In some embodiments of the present invention, the structure of the prussian calcium nanoparticle has at least one of the following characteristics (i) to (iii): (i) the average particle size is 3-20 nm; (ii) contains divalent calcium (II) and trivalent iron (III); (iii) contains cyanide ions.
In some embodiments of the invention, the prussian calcium nanoparticles are prepared by: and adding a potassium cyanide solution into a calcium chloride and polyvinylpyrrolidone solution, uniformly stirring and dialyzing to obtain the prussian calcium nano-particles.
In some preferred embodiments of the present invention, the prussian calcium nanoparticles are prepared by the following method: 10-20 mL of 5.0-10.0 mM potassium cyanide solution (pH value about 1) is added dropwise to 10-20 mL of 5.0-10.0 mM CaCl containing 250-500 mg of polyvinylpyrrolidone (PVP, average molecular weight: 30,000-40,000)2At room temperature, a transparent light yellow solution is generated; after stirring at a constant speed for 20 minutes, adding the solution into a dialysis bag (the molecular weight cut-off value (MWCO) is approximately equal to 12,000), stirring for 2 hours, and replacing distilled water for dialysis after 30 minutes each time to prepare the Prussian calcium nanoparticles.
In some embodiments of the invention, the prussian calcium nanoparticles have antioxidant activity. Specifically, the prussian calcium nanoparticles have in-vivo and in-vitro antioxidant activity.
In some embodiments of the invention, the prussian calcium nanoparticles have nanoenzyme activity comprising at least one of superoxide dismutase activity, catalase activity, peroxidase activity, and glutathione peroxidase activity.
In some embodiments of the invention, the prussian calcium nanoparticles are capable of inhibiting iron death. The iron death inhibition effect of the prussian calcium nanoparticles is proved in some embodiments of the invention, and Acute Kidney Injury (AKI) is one of serious renal dysfunction syndromes, the pathological mechanism of which is derived from the overproduction of endogenous reactive oxygen/nitrogen species (RO/NSs), so that iron death is induced and the renal structural function is damaged, and therefore, the inhibition of iron death can effectively treat and/or prevent Acute kidney injury.
In some embodiments of the present invention, the pharmaceutical formulation is a capsule, a tablet, a pill, a granule, an oral liquid, or an injection.
In some embodiments of the invention, the medicament further comprises a pharmaceutically acceptable carrier.
In some preferred embodiments of the present invention, the pharmaceutically acceptable carrier refers to a pharmaceutical carrier conventional in the pharmaceutical field, such as: diluents, excipients such as water, etc., fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; humectants such as glycerol; disintegrating agents such as agar, calcium carbonate and sodium bicarbonate; absorption enhancers such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and bentonite clay; lubricants such as talc, calcium stearate and magnesium stearate, and polyethylene glycol, and the like. Other adjuvants such as sweetener, flavoring agent, etc. can also be added into the composition.
In the present invention, the term "treating" includes alleviating, inhibiting or ameliorating the symptoms or conditions of a disease; inhibiting the generation of complications: ameliorating or preventing underlying metabolic syndrome; inhibiting the development of a disease or condition, such as controlling the development of a disease or condition; alleviating the disease or symptoms; regression of the disease or symptoms; alleviating a complication caused by the disease or symptom, or preventing or treating a symptom caused by the disease or symptom. As used herein, administration can result in an improvement in a disease, symptom, or condition, particularly an improvement in severity, delay in onset, slow progression, or decrease in duration of a condition.
According to a further aspect of the invention, the application of the prussian calcium nanoparticles in preparing the medicine for inhibiting iron death is provided.
In some embodiments of the invention, the agent that inhibits iron death is an agent that treats and/or prevents acute kidney injury disease.
Drawings
The invention is further described with reference to the following figures and examples, in which:
FIG. 1 is a drawing showing the preparation and characterization of Prussian calcium nanoparticles in example 1 of the present invention, in which (a) is a drawing showing the preparation method of Prussian calcium nanoparticles, (b) is a drawing showing a Transmission Electron Microscope (TEM) of Prussian calcium nanoparticles, (c) is a size distribution diagram of Prussian calcium nanoparticles, (d) is an XPS spectrum diagram of Prussian calcium nanoparticles, and (e) is a drawing showing Ca content in Prussian calcium nanoparticles2+(f) absorption spectrum of Prussian calcium nanoparticles dispersed in water, and the middle panel shows different solutions of Prussian calcium nanoparticles (H)2O, 1640, FBS and DMEM), (f) is an FTIR spectrogram of Prussian calcium nanoparticles, (h) is a Zeta potential diagram of Prussian calcium nanoparticles in different solutions, and (i) is a Ca-in-Prussian calcium nanoparticle effect photograph2+And Fe in the ambient2+Ion exchange between ions;
FIG. 2 is a graph showing the results of experiments on the ability of pullulanase nanoparticles to mimic the activity of enzymes in example 2 of the present invention, wherein (a) is a graph showing the results of SOD-like activity of the pullulan calcium nanoparticles, (b) is a graph showing the results of CAT-like activity of the pullulan calcium nanoparticles, (c) is a graph showing the results of POD-like activity of the pullulan calcium nanoparticles, and (d) is a graph showing the results of H-like activity at different concentrations in the pullulan calcium nanoparticles2O2Absorption of TMB in the presence, (e) GPx-type activity results for Prussian calcium nanoparticles, (f) O2 -ESR result graph of elimination, (g) ESR result graph of-OH elimination, (h) RNS elimination capacity graph of Prussian calcium nanoparticles to (i) ONOO-RNS cleaning capacity graph of (a);
FIG. 3 is a graph showing the results of the antioxidant activity and cytoprotective effect of Prussian calcium nanoparticles in vitro according to example 3 of the present invention, wherein (a) L929 cells were protected from LPS-induced oxidative stress for Prussian calcium nanoparticles, and (b) L929 cells were protected from H for Prussian calcium nanoparticles2O2Induced oxidative stress, (c) prussian calcium nanoparticles protected L929 cells from Fenton reagent-induced oxidative stress, (d) prussian calcium nanoparticles protected L929 cells from ultraviolet radiation-induced oxidative stress, (e) CLSM plots and corresponding quantitative analysis plots for L929 cells after staining with Calcein-AM/PI, DCFH-DA and DAF-FM DA (n ═ 3, data represent mean ± SD, ns represents no statistical difference,.; p; "p<0.001 and<0.0001)；
FIG. 4 is the exchange capacity of Prussian calcium nanoparticles for Fe ions in example 4 of the present invention;
fig. 5 is a graph showing rescue of prussian calcium nanoparticles against iron death-inducing agent Fin 56-induced cell death and changes in expression of related proteins in example 4 of the present invention, wherein (a) is a graph showing the activity and fluorescence absorption of prussian calcium nanoparticle iron death-inducing agent Fin 56-induced cells, and (b) is a graph showing the expression of related proteins of prussian calcium nanoparticle iron death-inducing agent Fin 56-induced cells;
fig. 6 is a graph of the kidney protection of IR-induced AKI mice by prussian calcium nanoparticles in example 5 of the present invention, wherein (a) is the protocol for prussian calcium nanoenzyme treatment of AKI mice, (b) is the fluorescence image of cy 5.5-labeled prussian calcium nanoparticles in the kidneys of ischemia-reperfusion (IR) mice, (c) is the body weight change in the mice over 5 days, (d) is the H & E staining of the kidneys in different groups, (E) is the TUNEL detection and Ki67 immunohistochemical staining of different treated AKI mice;
fig. 7 is a graph of the results of iron poisoning and inflammation measurements of prussian calcium nanoparticles of example 5 of the present invention, wherein (a) is the immunohistochemical staining of GPx4, ACSL4 and PTGS2 in mice with different treatments, (b) is the bio-TEM image of mitochondria in the kidney, (c) is the change in the lipid peroxidation levels in mice with different treatments, and (d) is the expression of cytokines IL-1 β, IFN- γ, TNF- α and IL-10 in mice with different treatments (n is 6, data represent mean ± SD, p < 0.05).
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.
In the present example, an ultraviolet-visible (UV-vis) absorption spectrum was measured by an ultraviolet-visible spectrophotometer. Electron Spin Resonance (ESR) spectra were measured by a brook EMX plus model spectrometer at the X-band frequency (9.8 GHz). Transmission Electron Microscope (TEM) imaging was performed by Tecnai G220(Shimadzu, Japan) at 200 keV. The samples were dropped onto a 230 mesh copper TEM grid covered with a carbon film. Fourier transform Infrared Spectroscopy (FTIR) was performed by an FTIR-8300 series spectrometer (Shimadzu, Japan). XPS was performed from ESCALB 250Xi Mg (Thermo Scientific, Japan) X-ray resources. Fluorescence imaging was performed by CarlZeiss LSM710(ZEISS, germany) or Leica DM 6B (Leica, germany). Fluorescence intensity was measured by SpectraMax iD5(Molecular Devices, USA). Westernblot imaging was performed by AI600(GE, USA). Cell cycle analysis was performed by FACS Canto II (molecular devices, usa).
Example 1: preparation, fluorescent labeling and characterization of prussian calcium nanoparticles
1.1 preparation of Prussian calcium nanoparticles
This example prepared a prussian calcium nanoparticle, which was synthesized by a simple aqueous phase displacement method, as shown in fig. 1 (a), by adding potassium cyanide solution to calcium chloride and polyvinylpyrrolidone solution, stirring uniformly (Uniform stirring) and dialyzing (Dialysis) to obtain prussian calcium nanoparticle.
The specific process is as follows: dripping 10.0mM potassium cyanide solution (pH value is approximately equal to 1) with the volume of 10-20 mL into 10-20 mL of 5.0-10.0 mM CaCl2 containing 250-500 mg polyvinylpyrrolidone (PVP, average molecular weight: 30,000-40,000), and generating transparent light yellow solution at room temperature; after stirring at a constant speed for 20 minutes, adding the solution into a dialysis bag (the molecular weight cut-off value (MWCO) is approximately equal to 12,000), stirring for 2 hours, and replacing distilled water for dialysis after 30 minutes each time to prepare the Prussian calcium nanoparticles.
1.2 preparation of PEI-modified Prussian calcium nanoparticles
Using the prepared prussian calcium nanoparticles, and modifying by Polyethyleneimine (PEI), to obtain PEI modified prussian calcium nanoparticles: and adding a PEI solution (5-10 mL, 4mg/mL and pH 5) into a Prussian calcium nanoparticle solution (10-20 mL, 1mg/mL) under stirring at room temperature to modify the synthesized Prussian calcium nanoparticles. Stirring for 4 hours, centrifuging and drying for 12 hours to obtain PEI modified Prussian calcium nanoparticles
1.3 Cy5.5 fluorescent labeling of Prussian calcium nanoparticles
Dripping Cy5.5 aqueous solution (5-10 mL; 100mg/mL) into the previously obtained PEI-modified Prussian calcium nanoparticle dispersion liquid (5-10 mL), and then stirring at a constant speed for 24 hours; all of the above syntheses are carried out in the dark, since Cy5.5 degrades rapidly under illumination. The Cy5.5 molecules are absorbed by the PEI modified Prussian calcium nano-particles through electrostatic interaction to obtain Cy5.5 fluorescence labeled Prussian calcium nano-particles.
1.4 Prussian calcium nanoparticle Structure and Property characterization
This exampleThe prepared prussian calcium nanoparticles are uniform in size, and the properties of the prussian calcium nanoparticles are characterized, and the results are shown in figure 1: wherein (b) is a Transmission Electron Microscope (TEM) image of prussian calcium nanoparticles, (c) is a size distribution map of prussian calcium nanoparticles, Transmission Electron Microscope (TEM) images and Dynamic Light Scattering (DLS) measurements show that prussian calcium nanoparticles have an average hydrodynamic diameter of 5.3nm, close to spherical, which enables them to pass through glomerular filtration membranes, ensuring absorption and excretion by the kidney; (d) XPS spectrum of Prussian calcium nano-particles, (e) Ca in Prussian calcium nano-particles2+The XPS spectrum and X-ray photoelectron spectroscopy (XPS) analysis show that the successfully synthesized Prussian calcium nano-particles contain calcium (II) and iron (III); (f) the absorption spectrum of the prussian calcium nano-particles dispersed in water is shown in the middle panel, and the prussian calcium nano-particles are in different solutions (H)2Tyndall effect in O, 1640, FBS and DMEM), a pale yellow solution of prussian calcium nanoparticle aqueous suspension showed strong absorbance absorption in the Ultraviolet (UV) region, in various physiological solutions, including H2O, Roswell Park clinical Institute-1640(RPMI-1640), Fetal Bovine Serum (FBS) and Dulbecco's Modified Eagle Medium (DMEM), has good stability; (g) as an FTIR spectrum of Prussian calcium nanoparticles, Fourier transform Infrared Spectroscopy (FTIR) of Prussian calcium nanoparticles showed 2094cm-1And 1635cm-1Corresponds to the C ≡ N and C ═ O tensile vibratory stretching of the PVP unit; (h) is Zeta potential diagram of Prussian calcium nanoparticles in different solutions, in H2O, RPMI-1640, FBS, DMEM and Phosphate Buffered Saline (PBS), the surface charge of the Prussian calcium nanoparticles was measured as-7.27, -3.77, -3.05, -6.69 and-2.40 mV, respectively; (i) is Ca in prussian calcium nano-particles2+And Fe in the ambient2+Ion exchange between ions, the calcium ions of prussian calcium nanoparticles can be replaced by iron through ion exchange reactions, revealing the potential to inhibit iron death.
Example 2: antioxidant capacity of prussian calcium nanoparticles
In this example, the nano-enzyme activity (antioxidant enzyme activity) and the radical scavenging ability of the prussian calcium nano-particles prepared in example 1 were verified, and the specific process was as follows:
2.1 Prussian calcium pseudo-enzyme Activity test
This example verifies the performance of superoxide dismutase (SOD), Catalase (CAT), Peroxidase (POD) and glutathione peroxidase (GPx) of prussian calcium nanoparticles, as follows:
1) and (3) detecting the SOD activity: SOD-like capacity was measured by SOD detection kit and spectrophotometric measurement of the mixture at 450nm using UV-visible spectroscopy.
2) POD activity assay: POD sample capacity was measured by UV-visible absorption spectroscopy to obtain the absorbance of TMB solution. H is to be2O2The solution (200. mu.L, 40mM) was added to a cuvette with nanoparticles (20. mu.L) and TMB (0.8mM) and the absorbance was monitored at 652 nm.
3) And (3) CAT activity detection: CAT sample Capacity O was obtained by using dissolved oxygen meter2The concentration of the solution is detected. H2O2The solution (200. mu.L, 40mM) was mixed with nanoparticles (20. mu.L) in a glass vial and O was monitored over 30 minutes2The concentration of the solution.
4) And (3) GPx activity detection: GPx-like capacity was measured by using a GPx detection kit and the reaction was monitored spectrophotometrically at 340nm using uv-vis spectroscopy.
The results are shown in FIGS. 2(a) - (e), wherein (a) is the SOD activity results of prussian calcium nanoparticles, (b) is the CAT activity results of prussian calcium nanoparticles, (c) is the POD activity results of prussian calcium nanoparticles, and (d) is the H activity results of prussian calcium nanoparticles and different concentrations of H2O2Absorption of TMB in the presence, (e) GPx-type activity results of prussian calcium nanoparticles. From the results, prussian calcium nanoparticles have the properties of SOD, CAT, POD and GPx.
2.2 RONS scavenging ability test
Measurement of O by ESR Spectroscopy with BMPO as trapping agent2 -(scavenging ability of oxygen free radical). Mixing KO with water2(35.56. mu.g) was mixed with 18-crown-6 in DMSO solution (200. mu.L, 0.35mM) and BMPO (1mg) was further added under sonication. ESR signals were then collected in the absence and presence of prussian calcium nanoparticles to estimate O2 -The ability to purge. the-OH scavenging ability was investigated by ESR spectroscopy. At H2O2BMPO (1mg) was added to (1mM) or Phosphate Buffered Saline (PBS) buffer (200. mu.L, 10mM) for radical capture. The fenton reagent is then dissolved in this solution to produce-OH. The removal capacity was evaluated by the intensity of ESR amplitude with/without prussian calcium nanoparticles. To generate ONOO-Adding NaNO to the solution2Solution (10mL, 50mM) with H2O2(10mL, 25mM) and the mixture was stirred for 3 minutes. Hydrochloric acid (5mL, 1M) and NaOH (5mL, 1.5M) were then added over 1 second with stirring until the mixture turned pale yellow. The whole reaction system was kept in an ice-water bath. The scavenging ability was studied by the absorbance at 302 nm of a UV-visible spectrometer. For DPPH clearance, prussian calcium nanoparticles were mixed with DPPH in ethanol (200 μ L, 25mM) while measuring with uv-vis spectrometer.
The results are shown in FIGS. 2 (f) to (i) where (f) is O2 -ESR results of Elimination, (g) ESR results of-OH Elimination (concentration of 10. mu.g/mL using Prussian calcium nanoparticles), (h) RNS Elimination Capacity Curve of Prussian calcium nanoparticles, (i) ONOO Elimination of Prussian calcium nanoparticles-RNS cleaning capacity graph of (a). The experimental results show that the prussian calcium nanoparticles can effectively eliminate various active oxygen and nitrogen free Radicals (RONS), and are beneficial to protecting the normal functions of cells and tissues and organs.
Example 3: in-vitro oxidation resistance and cell protection experiment of prussian calcium nanoparticles
In this example, the prussian calcium nanoparticles prepared in example 1 were studied for their in vitro antioxidant properties and cytoprotective properties, and the specific process was as follows:
3.1 in vitro antioxidant assay
Two cells (RAW264.7 and L929 cells) in 48-well cell culture plexusCulturing for 24 hours in the medium, wherein the cell density is 60-80%; the cell culture medium was then replaced with fresh medium containing different concentrations of prussian calcium nanoparticles for 12 hours, followed by LPS, H, respectively2O2And treating the cells under different conditions. For LPS treatment, 10. mu.g/mL LPS was co-cultured with the cells for 24 hours; and the hydrogen peroxide reagent is H2O2Treatment (400. mu.M) was continued for 4 hours. After the treatment, the activity of the cells is detected by a CCK-8 activity determination kit. The medium was replaced with FBS-free medium containing CCK-8 reagent (10: 1), and the cells were co-cultured with the pretreated cells in an incubator for 1 hour. The viability of the cells was then determined using SpectraMax iD5 (n-4 for each group).
3.2 live-dead staining
Before the preparation of the dyes, with ddH2O dilute 10X assay buffer to 1X assay buffer. Fluorescent dyes were then prepared by adding 5 μ L of LCalcein-AM solution and 15 μ L of propidium iodide solution to 5mL of 1X detection buffer. After different kinds of pretreatment, the cells were washed 3 times with PBS and the medium was removed. The fluorochrome was co-incubated with the cells for 15 minutes and then washed 3 times with PBS. Finally, fluorescence imaging was performed using CarlZeiss LSM 710.
The live cell/dead cell staining kit is used for staining live cells and dead cells by adopting a Calcein-AM/PI double cell staining method. Calcein-AM is a cell staining reagent that can fluorescently label living cells, and it can easily penetrate living cell membranes because of its enhanced hydrophobicity. When it enters the cytoplasm, esterase will hydrolyze it into Calcein, which is left in the cell and emits strong green fluorescence. The experimental results are shown in FIG. 3, in which the Prussian calcium nanoenzyme protects L929 cells from (a) LPS and (b) H2O2(c) Fenton reagent and (d) oxidation pressure caused by ultraviolet irradiation; (e) CLSM-map and corresponding quantitative analysis of L929 cells after staining with Calcein-AM/PI, DCFH-DA and DAF-FMDA (n-3, data represent mean. + -. SD, ns represents no statistical difference,. SP<0.001 and<0.0001). The experimental result shows that the prussian calcium nano-particles have in-vitro oxidation resistance and cell protection effect.
Example 4: iron death inhibition capacity of prussian calcium nanoparticles
This example demonstrates the iron death suppressing ability of the prussian calcium nanoparticles prepared in example 1.
Iron death (Ferroptosis) is an iron-dependent, novel programmed cell death modality distinguished from apoptosis, necrosis, and autophagy. The main mechanism of iron death is that under the action of ferrous iron or ester oxygenase, unsaturated fatty acid which is highly expressed on cell membranes is catalyzed, lipid peroxidation is carried out, and cell death is induced; in addition, it is also shown that the regulation core enzyme GPX4 of the antioxidant system (glutathione system) is reduced. Acute Kidney Injury (AKI) is one of the severe syndromes of renal dysfunction, the pathological mechanism of which results from the overproduction of endogenous reactive oxygen/nitrogen species (RO/NSs), which in turn induces iron death and impairs renal structural function.
The iron death inducer Fin56(CAS No.1083162-61-1) can reduce GPx4 expression in cells. Fin56 can bind to and further activate squalene synthetase (SQS), thereby increasing the sensitivity of the cell to iron death.
The specific experimental steps are as follows:cells were cultured in an incubator for 12 hours to reach a cell density of 50% before induction of iron death. Fin56 was then added to fresh medium along with different concentrations of Prussian calcium nanoparticles to give 10. mu.M Fin 56. Fin56 medium was added to replace the original cell culture medium at 37 ℃ and 5% CO2The cells were cultured in the incubator for another 48 hours. After the co-culture was completed, the viability of the cells was examined by the CCK-8 method.
The experimental results are shown in fig. 4 and 5, and fig. 4 shows Ca in prussian calcium nanoparticles2+And Fe in the ambient2+Ion exchange between ions, the calcium ions of prussian calcium nanoparticles can be replaced by iron through ion exchange reactions, revealing the potential to inhibit iron death. FIG. 5 shows rescue of Prussian calcium nanoparticles from iron death-inducing agent Fin 56-induced cell death and related protein expression, where (a) is Prussian calcium nanoparticle iron death-inducingA cell activity map and a fluorescence absorption map induced by the agent Fin56, and (b) a related protein expression map of cells induced by the Prussian calcium nanoparticle iron death inducer Fin 56. Experimental results show that after the prussian calcium nanoparticles are added, the cell activity can be obviously improved, the effect of an iron death inducer Fin56 is inhibited, and the cell protection effect is improved.
Example 5: acute kidney injury treatment effect of prussian calcium nanoparticles
In this example, the effect of the prussian calcium nanoparticles prepared in example 1 on the treatment of acute kidney injury is studied, and the specific process is as follows:
5.1 construction of ischemia reperfusion injury
Balb/c mice (male, 6 weeks, 20-25 g) were purchased from Shanghai Slek laboratory animals Co., Ltd and bred at Shanghai university laboratory animal center. The animal is raised in the center of the laboratory animal of Shanghai Tongji university. The mice freely obtain standard feed and water in a dark-light period of 12 hours under the environmental conditions of room temperature (20-24 ℃) and relative humidity (45-55%). All animal experiments were performed according to the guidelines for laboratory animal Care and use of the national institutes of health, USA, and were approved by the ethical Committee of the tenth national Hospital affiliated with the college of medicine of the same university. Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital at a concentration of 50 mg/kg. Renal ischemia is induced by entrapment of the renal stem. During ischemia, mice were placed on a 37 ℃ heating table to maintain body temperature. After 30 minutes, the clamps were released and mice were injected intravenously with prussian calcium nanoparticles.
5.2 analysis of the results
Further experimental studies were performed on the above ischemia reperfusion injury model mice. Experimental results as shown in fig. 6 and 7, fig. 6 is a graph of renal protection of prussian calcium nanoparticles on IR-induced AKI mice, wherein (a) is an experimental protocol for prussian calcium nanoenzyme treatment of AKI mice, (b) is a graph of fluorescence imaging of mouse kidneys, and 2 hours after intravenous injection, the cy5.5 modified prussian calcium nanoparticles have a fluorescent signal in the kidneys of Ischemia Reperfusion (IR) mice, and the control group (not injected) has no fluorescent signal; (c) the weight change of the mice within 5 days is shown in a figure, the weight change recorded within 5 days (n is 6, data represent the average value +/-SD), and the weight of the mice injected with the prussian calcium nanoparticles is obviously increased compared with that of a control group, which indicates that the disease condition is obviously improved; (d) for H & E staining of kidneys in different groups, kidney tissue indicated glomeruli with black pentagons and brush borders of tubules with red triangles, (E) TUNEL detection (brown nucleolus) and Ki67 immunohistochemical staining (brown) of differently treated AKI mice. Compared with the control group, the kidney condition of the mice injected with the prussian calcium nanoparticles is improved, and the prussian calcium nanoparticles have a better treatment effect on AKI mice.
Fig. 7 is a graph of iron poisoning and inflammation measurements for prussian calcium nanoparticles, wherein (a) immunohistochemical staining of GPx4, ACSL4, and PTGS2 (all brown) for different treated AKI mice, (b) bio-TEM images of mitochondria in the kidney, broken mitochondria are indicated in red boxes, (c) altered lipid peroxidation levels for different treated AKI mice, and (d) expression of cytokines IL-1 β, IFN- γ, TNF- α, and IL-10 (n ═ 6, data representing mean ± SD,. p <0.05) for different treated AKI mice. The experimental result shows that the prussian calcium nano-particles can relieve iron poisoning and inflammation conditions and have a treatment effect on acute kidney injury.
The invention further researches the oxidation resistance of the Prussian calcium nano-particles, explores the application of the Prussian calcium as nano-enzyme, and explores the free radical removing performance and the iron death inhibiting capacity of the Prussian calcium nano-particles; in vitro and in vivo experiments prove that the oxidation resistance of the prussian calcium nanoparticles is well explored, and the treatment effect evaluation is comprehensively carried out on Acute Kidney Injury (AKI). Through in vivo and in vitro experiments, the invention verifies the oxidation resistance of the prussian calcium nanoparticles and further explores the effect of the prussian calcium nanoparticles in treating AKI. Meanwhile, the prussian calcium nano-particles have the capabilities of nano-enzyme and inhibiting iron death, and the prussian calcium serving as a novel nano-material can be applied to the rescue of oxidative damage such as acute kidney injury and the like and various free radical injury diseases.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
Claims (10)
1. Application of Prussian calcium nanoparticles in preparation of medicine for treating and/or preventing acute kidney injury diseases is provided.
2. The use according to claim 1, wherein the structure of the prussian calcium nanoparticles has at least one of the following characteristics (i) to (iii):
(i) the average particle size is 3-20 nm;
(ii) contains divalent calcium and trivalent iron;
(iii) contains cyanide ions.
3. The use according to claim 1, wherein the prussian calcium nanoparticles are prepared by the following method:
and adding a potassium cyanide solution into a calcium chloride and polyvinylpyrrolidone solution, uniformly stirring and dialyzing to obtain the prussian calcium nano-particles.
4. The use according to claim 1, wherein the prussian calcium nanoparticles have antioxidant activity.
5. The use of claim 1, wherein the prussian calcium nanoparticles have nanoenzyme activity comprising at least one of superoxide dismutase activity, catalase activity, peroxidase activity, and glutathione peroxidase activity.
6. The use according to claim 1, wherein the prussian calcium nanoparticles are capable of inhibiting iron death.
7. The use of claim 1, wherein the medicament is in the form of capsules, tablets, pills, granules, oral liquid or injections.
8. The use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable carrier.
9. An application of Prussian calcium nanoparticles in preparing medicines for inhibiting iron death is provided.
10. The use according to claim 9, wherein the iron death suppressing drug is a drug for the treatment and/or prevention of acute kidney injury diseases.
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| US20080145448A1 (en) * | 2005-01-10 | 2008-06-19 | Secretary, Department Of Atomic Energy Government Of India | Calcium Potassium Ferrocyanide, a Prophylactic Mixture Comprising this Compound and the Use Thereof for Decorporation of Radiocesium in Subjects Affected by Nuclear Radiation |
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| US20080145448A1 (en) * | 2005-01-10 | 2008-06-19 | Secretary, Department Of Atomic Energy Government Of India | Calcium Potassium Ferrocyanide, a Prophylactic Mixture Comprising this Compound and the Use Thereof for Decorporation of Radiocesium in Subjects Affected by Nuclear Radiation |
| US20200016086A1 (en) * | 2017-02-17 | 2020-01-16 | Kent State University | Nanoparticulate Materials and Methods for Targeting Iron Acquisition and Metabolism for Treating Bacterial Infections |
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