Cerium-doped polypyrrole nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses cerium-doped polypyrrole nanoparticles as well as a preparation method and application thereof, and relates to the technical field of medicines. The preparation method of the cerium-doped polypyrrole nanoparticles comprises the following steps: (1) preparing a mixed solution of polyvinyl alcohol and pyrrole, with the concentration of the polyvinyl alcohol being 0.05-5 g / mL and the concentration of the pyrrole being 5-20 [mu] L / mL; and (2) adding a Ce (SO4) 2 solution into the mixed solution of polyvinyl alcohol and pyrrole obtained in the step (1), and reacting under a stirring condition to obtain the cerium-doped polypyrrole nanoparticles. The CePPy nanoparticles synthesized by the invention have efficient and broad-spectrum free radical scavenging activity, including OH, O2 <-> and H2O2. In the nano-particles, unique positive surface charges of (+) CePPy are preferentially combined with negatively charged cell membranes, and efficient enrichment in damaged kidneys is realized. And active targeting and enrichment in mitochondria, which is a main place for generating ROS, are realized, so that the treatment efficiency is greatly improved, and the off-target effect is reduced.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a cerium-doped polypyrrole nanoparticle, its preparation method, and its application. Background Technology

[0002] Acute kidney injury (AKI) is a severe clinical syndrome characterized by a rapid decline in kidney function and is closely associated with high morbidity and mortality. Currently, clinical treatment for AKI primarily relies on renal replacement therapy (such as dialysis) and nutritional support. However, these methods are all alternative and supportive treatments, aimed at maintaining the patient's vital signs rather than repairing or preventing kidney damage at its pathological root. To date, there are no specific drugs that can effectively treat AKI, highlighting the urgent need to develop novel drug treatment strategies.

[0003] The pathogenesis of acute kidney injury (AKI) is complex, with oxidative stress widely recognized as a key component. Proximal tubular epithelial cells (PTECs) in the kidney are particularly sensitive to oxidative damage due to their abundant mitochondria and high oxygen consumption. In AKI models such as ischemia / reperfusion (I / R) injury, intracellular redox homeostasis is severely disrupted, triggering oxidative stress and leading to excessive production of reactive oxygen species (ROS) in the kidney tissue. These ROS include superoxide anion radicals (O2). - • Hydrogen peroxide (H2O2), highly reactive hydroxyl radicals (•OH), etc. These factors work together to drive the progression of kidney damage. Mitochondria, as the main source of intracellular ROS, contribute nearly 90% of total intracellular ROS. Excessive ROS triggers a vicious cycle of pathological events, including mitochondrial dysfunction, cellular energy depletion, and renal tubular cell death. Therefore, antioxidant therapy targeting oxidative stress has become a promising strategy for intervening in acute kidney injury (AKI).

[0004] Recent studies have shown that in AKI treatment, positively charged nanoparticles (such as cationic dextran nanoparticles) can achieve more efficient renal accumulation and cellular uptake than neutral or anionic particles through electrostatic attraction with negatively charged cell membranes, demonstrating superior therapeutic potential.

[0005] However, existing technologies have the following drawbacks:

[0006] 1. Although the concept of antioxidant therapy is clear, traditional antioxidants have many limitations in clinical application, such as low bioavailability, poor ability to scavenge ROS, poor renal targeting, insufficient stability, and difficulty in achieving effective concentrations at the lesion site.

[0007] 2. The synthesis of simple cerium-based nanomaterials is usually complex and may involve organic solvents, posing potential biosafety risks. More importantly, these materials generally lack the ability to actively target damaged kidneys, especially renal tubular cell mitochondria, resulting in limited therapeutic efficacy and potential systemic side effects.

[0008] 3. Simple cationic nanoparticles: These model particles (such as cationic dextran) usually do not possess strong, multifunctional enzyme-mimicking antioxidant activity and have no therapeutic effect. Summary of the Invention

[0009] To address the technical problems existing in the prior art, embodiments of the present invention provide cerium-doped polypyrrole nanoparticles, their preparation method, and applications. The technical solution is as follows:

[0010] A method for preparing cerium-doped polypyrrole nanoparticles, the method comprising:

[0011] (1) Prepare a mixed solution of polyvinyl alcohol and pyrrole, wherein the concentration of polyvinyl alcohol is 0.05-5 g / mL and the concentration of pyrrole is 5-20 μL / mL;

[0012] (2) Add Ce(SO4)2 solution to the mixed solution of polyvinyl alcohol and pyrrole obtained in step (1) and react under stirring conditions to obtain the cerium-doped polypyrrole nanoparticles.

[0013] Optionally, in step (2), the concentration of the Ce(SO4)2 solution is 10-50 mg / mL;

[0014] And / or, in step (2), the mass ratio of Ce(SO4)2 solution to the mixed solution of polyvinyl alcohol and pyrrole is 1:1 to 4:1.

[0015] Optionally, in step (2), the reaction time is 12-24 hours;

[0016] And / or, in step (2), the reaction is carried out at room temperature;

[0017] And / or, in step (2), the stirring speed is 500-1500 rpm.

[0018] Optionally, the preparation method further includes:

[0019] (A) Dialyze the mixed solution of cerium-doped polypyrrole nanoparticles obtained in step (2); concentrate the solution using an ultrafiltration centrifuge tube and collect positively charged cerium-doped polypyrrole nanoparticles.

[0020] Optionally, the preparation method further includes:

[0021] (B) The solution of positively charged cerium-doped polypyrrole nanoparticles obtained in step (A) is added to DSPE-PEG2000 solution and concentrated using an ultrafiltration centrifuge tube to obtain negatively charged cerium-doped polypyrrole nanoparticles.

[0022] The cerium-doped polypyrrole nanoparticles obtained by the aforementioned preparation method.

[0023] Optionally, the cerium-doped polypyrrole nanoparticles include: positively charged cerium-doped polypyrrole nanoparticles and negatively charged cerium-doped polypyrrole nanoparticles.

[0024] The application of the cerium-doped polypyrrole nanoparticles in the preparation of drugs for treating acute kidney injury and / or the progression of acute kidney injury to chronic kidney disease.

[0025] A pharmaceutical composition for treating acute kidney injury and / or progression of acute kidney injury to chronic kidney disease, the pharmaceutical composition comprising: the cerium-doped polypyrrole nanoparticles and a carrier.

[0026] Optionally, the drug is cerium-doped polypyrrole nanoparticles with a positive charge.

[0027] The beneficial effects of the technical solutions provided by the embodiments of the present invention include at least the following:

[0028] This invention overcomes the shortcomings of the prior art and provides cerium-doped polypyrrole nanoparticles, their preparation method, and their application in the treatment of acute kidney injury.

[0029] The CePPy nanoparticles synthesized in this invention possess highly efficient and broad-spectrum free radical scavenging activity, including •OH and O2. - • and H2O2. Preferably, the present invention further synthesizes positively charged (+)CePPy by modifying the surface charge to increase the active targeting ability of renal tubular cell mitochondria. In the nanoparticles of the present invention, the unique positive surface charge of (+)CePPy enables it to preferentially bind to negatively charged cell membranes through electrostatic interactions, and utilizes the enhanced permeability and retention effect of damaged kidneys to achieve efficient accumulation in damaged kidneys. Furthermore, due to the strong attraction between the positive charge and the high negative mitochondrial membrane potential (~-180 mV), it actively targets and accumulates in mitochondria—the main site of ROS production—achieving targeted treatment, greatly improving treatment efficiency and reducing off-target effects.

[0030] Furthermore, the cerium-doped polypyrrole nanoparticles of this invention are synthesized in a one-step aqueous phase using Ce(SO4)2 as both an oxidant and a dopant source, avoiding organic solvents. This process is simple, environmentally friendly, and better meets the requirements for clinical translation. The cerium doping level is extremely low (only 0.1%), significantly reducing the potential toxicity of heavy metals and the risk of particle aggregation. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 These are schematic diagrams and transmission electron microscope images of CePPy nanoparticle synthesis provided in Example 1 of this invention; wherein, Figure 1 A in the diagram is a schematic diagram of the preparation method of CePPy nanoparticles; Figure 1 Images B through C are transmission electron microscopy (TEM) images of (+) CePPy nanoparticles and (-) CePPy nanoparticles.

[0033] Figure 2 These are characterization diagrams of CePPy nanoparticles with different charges; among them Figure 2 Figure A shows the dynamic light scattering results of (+)CePPy nanoparticles and (-)CePPy nanoparticles; Figure 2 Figure B shows the zeta potential measurement results for (+)CePPy nanoparticles and (-)CePPy nanoparticles;

[0034] Figure 3 This is the energy-dispersive X-ray spectrum of (+)CePPy nanoparticles;

[0035] Figure 4 This is a graph showing the in vitro antioxidant capacity of CePPy nanoparticles with different charges; among which... Figure 4 A in the middle is CePPy, which scavenges superoxide anions (O2). - )picture; Figure 4 Figure B is a diagram showing the removal of hydrogen peroxide (H2O2) by CePPy; Figure 4 C in the diagram represents (+) CePPy scavenging hydroxyl radicals (•OH); Figure 4 D in the diagram represents (-)CePPy scavenging hydroxyl radicals (•OH); Figure 4 E in the middle is the CePPy cleaned-up free radical graph;

[0036] Figure 5 This is a diagram showing the effect of the positive surface charge of CePPy nanoparticles on proximal tubular uptake, as provided in Example 2 of this invention; wherein, Figure 5 In the image, A and C are in vivo imaging images of (+) CePPy nanoparticles and (-) CePPy nanoparticles in various organs; Figure 5 Figures B and D are the results of quantitative fluorescence analysis of (+) CePPy nanoparticles and (-) CePPy nanoparticles in ex vivo organs; Figure 5 E and F are high-content imaging images of (+) CePPy nanoparticles and (-) CePPy nanoparticles in renal tubular epithelial cells at different time points;

[0037] Figure 6 The diagram shows the results of CePPy nanoparticles' positive surface charge determining mitochondrial targeting; among which... Figure 6 Figures A to C show the co-localization results of (+) CePPy nanoparticles and (-) CePPy nanoparticles with organelle markers in PTECs;

[0038] Figure 7 This is a diagram showing the results of CePPy nanoparticles provided in Example 3 of the present invention alleviating IRI-induced AKI in mice, wherein... Figure 7 Figure A shows the experimental procedure of administering Sham and ischemia-reperfusion induced AKI mice as controls and (+) CePPy nanoparticles or (-) CePPy nanoparticles; Figure 7 Figure B shows the creatinine levels in AKI mice after injection of (+) CePPy nanoparticles or (-) CePPy nanoparticles. Figure 7 C is a PAS staining image of the renal tubules of AKI mice after injection of (+) CePPy nanoparticles or (-) CePPy nanoparticles; Figure 7 Figure D shows the renal tubular injury scores of AKI mice after injection of (+) CePPy nanoparticles or (-) CePPy nanoparticles. Figure 7 E is an immunofluorescence staining image of KIM1, an indicator of renal tubular damage, in AKI mice after injection of (+) CePPy nanoparticles or (-) CePPy nanoparticles; Figure 7 The figure in Figure F is a quantitative analysis of the expression area of ​​KIM1 in AKI mice after injection of (+) CePPy nanoparticles or (-) CePPy nanoparticles. Figure 7 Figure G is a Western blot result of apoptosis protein expression in AKI mice after injection of (+) CePPy nanoparticles or (-) CePPy nanoparticles; Figure 7 Figure H in the middle is the result of detecting the expression level of KIM1 in AKI mice after injection of (+) CePPy nanoparticles or (-) CePPy nanoparticles; Figure 7 Figure I shows the results of BAX expression level detection in AKI mice after injection of (+) CePPy nanoparticles or (-) CePPy nanoparticles; Figure 7 Figure J shows the results of detecting the expression level of cleaved caspase-3 in AKI mice after injection of (+) CePPy nanoparticles or (-) CePPy nanoparticles; where sham represents the sham-operated group. Detailed Implementation

[0039] The technical solution of the present invention will now be described with reference to the accompanying drawings.

[0040] This invention provides cerium-doped polypyrrole nanoparticles, a method for preparing the cerium-doped polypyrrole nanoparticles, and the application of cerium-doped polypyrrole nanoparticles in the preparation of drugs for treating acute kidney injury and its progression to chronic kidney disease (CKD).

[0041] This invention utilizes polyvinyl alcohol as a stabilizer and cerium sulfate (Ce(SO4)2) as an oxidant to induce the oxidative polymerization of pyrrole monomers to form cerium-doped polypyrrole nanoparticles. This invention also provides cerium-doped polypyrrole nanoparticles with positive surface charge (+)CePPy nanoparticles and cerium-doped polypyrrole nanoparticles with negative surface charge (-CePPy nanoparticles).

[0042] The CePPy nanozyme synthesized in this invention exhibits highly efficient and broad-spectrum free radical scavenging activity, including •OH and O2. - • and H2O2. Further by modifying the surface charge, positively charged (+)CePPy was synthesized to enhance the active targeting ability of renal tubular cell mitochondria.

[0043] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0044] Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods, performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions.

[0045] Unless otherwise specified, all materials and reagents described in the following examples are commercially available.

[0046] In the following embodiments, the detection experimental methods, such as transmission electron microscopy and dynamic light scattering analysis, are conventional experimental methods well known to those skilled in the art. Where no specific conditions are specified in the experimental methods, they are usually operated according to conventional conditions.

[0047] In this invention, DSPE-PEG2000 is an amphiphilic block copolymer composed of distearate phosphatidylethanolamine (DSPE) and polyethylene glycol (PEG) with a molecular weight of 2000, which is available commercially.

[0048] Example 1

[0049] 1. Preparation of (+)CePPy nanoparticles

[0050] Weigh 1g of polyvinyl alcohol (PVA, purchased from Aladdin, 0588 low viscosity type) and dissolve it in 10mL of deionized water. Stir at 92°C for 1 hour to prepare a 10% PVA solution. Measure 100μL of pyrrole monomer and add it to the above PVA solution. Stir continuously at room temperature for 1 hour.

[0051] 200 mg Ce(SO4)2 was dissolved in 10 mL of deionized water to prepare a solution. The Ce(SO4)2 solution was then slowly added dropwise to the pyrrole-PVA mixture while stirring. After the addition was complete, the reaction was continued with stirring at room temperature for 20 hours.

[0052] The resulting suspension was transferred to a dialysis bag (MWCO: 10-14 kDa) and dialyzed with deionized water for 24 hours to remove unreacted impurities. After dialysis, the product was concentrated using an ultrafiltration centrifuge tube, and the collected product was the (+)CePPy nanoparticle aqueous dispersion.

[0053] 2. The preparation method of (-)CePPy nanoparticles is as follows:

[0054] (+)CePPy was dispersed in DMSO to prepare a 20 mg / mL solution. 250 μL of the (+)CePPy solution was added to 200 μL of a DMSO solution of DSPE-PEG2000 (100 mg / mL), and mixed thoroughly. The mixture was then slowly added dropwise to 5 mL of vigorously stirred deionized water, and stirring was continued for 5 minutes. The product was concentrated using an ultrafiltration centrifuge tube, and the collected product was the (-)CePPy nanoparticle aqueous dispersion.

[0055] 3. Transmission electron microscopy was used to analyze the obtained (+)CePPy nanoparticles and (-)CePPy nanoparticles. Dynamic light scattering analysis, energy-dispersive X-ray spectroscopy, and inductively coupled plasma mass spectrometry (ICP-MS) were performed, and the zeta potential was measured. All the above experimental methods were conventional and could be performed according to the techniques or conditions described in the literature in this field.

[0056] The following free radical scavenging activities were detected in (+)CePPy nanoparticles and (-)CePPy nanoparticles: •OH, O2 - • H2O2 was detected using a commercially available detection kit, such as those from Beyotime Biotechnology Co., Ltd. See the kit instructions for detailed detection procedures.

[0057] Experimental results:

[0058] Experimental results are as follows Figure 1-4 As shown. Among them,

[0059] Figure 1A in the diagram is a schematic diagram of the preparation method of CePPy nanoparticles.

[0060] Figure 1 Images B through C are transmission electron microscopy (TEM) images of (+)CePPy nanoparticles and (-)CePPy nanoparticles. As can be seen from the images, both (+)CePPy and (-)CePPy nanoparticles exhibit a monodisperse, near-spherical structure.

[0061] Figure 2 Figure A shows the dynamic light scattering results of (+)CePPy nanoparticles and (-)CePPy nanoparticles. As can be seen from the figure, the hydration dynamic diameter of the (+)CePPy nanoparticles is 86.87±2.45 nm, and that of the (-)CePPy nanoparticles is 96.77±1.04 nm. These results indicate that both types of nanoparticles have a roughly uniform size distribution, and their sizes are in the nanometer range.

[0062] Figure 2 Figure B shows the Zeta potential measurements of (+)CePPy nanoparticles and (-)CePPy nanoparticles. The figure shows that the Zeta potential of (+)CePPy nanoparticles is 7.29 ± 0.35 mV. Furthermore, after PEGylation, the Zeta potential of (-)CePPy nanoparticles changed from positive to negative, recorded as -14.06 ± 1.39 mV. These results indicate that there is a difference in positive and negative charges between (+)CePPy and (-)CePPy nanoparticles.

[0063] Figure 3 Energy-dispersive X-ray spectroscopy confirmed the presence of C, N, O, and Ce elements in (+)CePPy nanoparticles. The results demonstrate the definite presence of these characteristic elements, consistent with the design and synthesis principles of (+)CePPy.

[0064] Figure 4 In the middle, A represents the superoxide anion O2 of (+)CePPy nanoparticles and (-)CePPy nanoparticles. - Results of O2 scavenging ability test. As shown in the figure, both types of nanoparticles exhibit excellent O2 scavenging ability in vitro. - The scavenging activity is enhanced in a dose-dependent manner.

[0065] Figure 4B shows the results of hydrogen peroxide (H2O2) scavenging activity detection for (+) CePPy nanoparticles and (-) CePPy nanoparticles. As can be seen from the figure, both types of nanoparticles exhibit excellent H2O2 scavenging activity in vitro, and the scavenging effect increases in a dose-dependent manner.

[0066] In Figure 4, C represents the hydroxyl radicals of (+) CePPy nanoparticles. The results of the scavenging ability test are shown in the figure. As can be seen from the figure, (+)CePPy nanoparticles exhibit excellent scavenging ability in vitro. The scavenging activity is enhanced in a dose-dependent manner.

[0067] In Figure 4, D represents the hydroxyl radicals of (-) CePPy nanoparticles. The results of the scavenging ability test are shown in the figure. As can be seen from the figure, (-)CePPy nanoparticles exhibit excellent scavenging ability in vitro. The scavenging activity is enhanced in a dose-dependent manner.

[0068] Figure 4 shows the free radical scavenging activity of (+) CePPy nanoparticles and (-) CePPy nanoparticles (E). As can be seen from the figure, both types of nanoparticles exhibit excellent free radical scavenging activity in vitro, and this scavenging effect increases in a dose-dependent manner.

[0069] The results of detection using relevant assay kits show that, regardless of charge, CePPy nanoparticles exhibit significant, concentration-dependent O2 activity in vitro. - •, H2O2 and The scavenging activity indicates that CePPy nanoparticles have good antioxidant activity against various ROS in vitro.

[0070] Example 2 Evaluation of (+) CePPy uptake by proximal renal tubules and mitochondrial targeting

[0071] 1. Establishing a mouse model of AKI induced by renal ischemia-reperfusion:

[0072] After approval by the Experimental Animal Ethics Committee of the General Hospital of the Chinese People's Liberation Army, 8-week-old male C57BL / 6J mice (purchased from Spefair Company) were anesthetized by intraperitoneal injection of sodium pentobarbital. The skin and muscle tissue were incised with ophthalmic scissors along the spine and lower ribs on the back, revealing the red kidneys. Both renal arteries were clamped with arterial clamps, causing the kidneys to change color from red to white and then to dark purple. Ischemia was maintained for 23 minutes while the mice's body temperature was kept at 37°C. The arterial clamps were then released, and renal blood perfusion was observed to recover. The muscle and skin tissues were then sutured. After the animals recovered, they were returned to the animal facility for continued rearing. Blood perfusion was maintained for 24 hours, thus establishing the AKI model. Mice whose kidneys were exposed through abdominal incision but whose renal arteries were not clamped were designated as the Sham group (sham surgery group).

[0073] 2. Preparation of (+)CePP-Cy5 nanoparticles and (-)CePP-Cy5 nanoparticles: The (+)CePPy nanoparticles and (-)CePPy nanoparticles prepared in Example 1 were loaded with fluorescent molecules Cy5 through electrostatic adsorption to prepare (+)CePP-Cy5 nanoparticles and (-)CePP-Cy5 nanoparticles, respectively.

[0074] 3. (+) CePP-Cy5 nanoparticles and (-) CePP-Cy5 nanoparticles were injected via tail vein, respectively. The main organs (heart, liver, spleen, lung, kidney, intestine) of mice were placed in a small animal imaging instrument for observation and photography, and quantitative fluorescence analysis of the isolated organs was performed.

[0075] Experimental results:

[0076] Experimental results are as follows Figure 5 As shown in diagrams A through D. Wherein,

[0077] Figure 5 In Figures A and C, we see in vivo imaging of (+) and (-) CePPy nanoparticles in various organs. The results show that one hour after injection, Cy5 fluorescence mainly accumulated in the liver, with significant secondary localization in the kidneys. This indicates that the liver and kidneys are the main organs where CePPy nanoparticles accumulate.

[0078] Figure 5 Figures B and D show the results of in vitro organ fluorescence quantitative analysis of (+)CePPy nanoparticles and (-)CePPy nanoparticles. The results show that the renal uptake efficiency of (+)CePPy is 3.05 times higher than that of (-)CePPy (p=0.007), confirming that positive charge enhances renal uptake capacity.

[0079] 4. To verify this charge-dependent uptake in vitro, a time-dependent kinetic study was conducted in renal tubular epithelial cells incubated with (+) / (-) CePPy-Cy5, and the results were monitored using high-content imaging. The specific experimental steps were as follows: Renal tubular epithelial cells were cultured, and images were taken immediately after adding 20 μg / ml of (+) CePPy-Cy5 and (-) CePPy-Cy5, respectively. High-content imaging was set to take images every 5 minutes for a total of 120 minutes.

[0080] Experimental results:

[0081] Experimental results are as follows Figure 5 As shown in Figures E to F. Wherein, Figure 5E and F are high-content imaging images of (+) CePPy nanoparticles and (-) CePPy nanoparticles in renal tubular epithelial cells at different time points. As can be seen from the figures, (+) CePPy exhibits rapid internalization, reaching its peak mean fluorescence intensity (MFI) at an early time point (within 40 minutes) and then stabilizing. In contrast, the uptake of (-) CePPy is significantly delayed and attenuated, reaching a significantly lower peak MFI at a later time point (120 minutes). These results confirm that the positive surface charge greatly enhances the rate and extent of uptake by proximal tubular cells.

[0082] 5. To elucidate the subcellular localization of internalized (+) CePPy, we used confocal laser scanning microscopy to assess its co-localization with organelle markers in primary renal tubular epithelial cells (PTECs). The specific experimental steps were as follows: Primary PTECs were cultured, and after the cells reached a suitable density, mitochondrial-specific labeling dye MitoTracker and lysosome-specific labeling dye LysoTracker were added to the cells, respectively. The cells were incubated for an appropriate time to allow the mitochondria and lysosomes of the PTECs to be specifically labeled and exhibit green fluorescence under a confocal microscope. Subsequently, (+) CePPy-Cy5 (Cy5-labeled (+) CePPy, exhibiting red fluorescence) was added to both groups of cells at a final concentration of 20 μg / ml. After further incubation, images were observed and acquired using a confocal laser scanning microscope. The degree of overlap between the red fluorescence of (+) CePPy-Cy5 and the green fluorescence of mitochondria / lysosomes was analyzed to determine the subcellular localization of (+) CePPy within PTECs.

[0083] Experimental results:

[0084] Experimental results are as follows Figure 6 As shown in diagrams A through C. Wherein, Figure 6 Figures A to C show the co-localization results of (+) CePPy nanoparticles and (-) CePPy nanoparticles with organelle markers in PTECs.

[0085] As shown in the figure, after 1 hour of incubation, the (+)CePPy-Cy5 fluorescence exhibited significant co-localization with MitoTracker, but very little overlap with LysoTracker. These results indicate that (+)CePPy nanoparticles preferentially accumulate in mitochondria.

[0086] Example 3: Evaluation of the effect of (+) CePPy nanoparticles on AKI improvement

[0087] Sham and ischemia-reperfusion induced AKI mice were administered 5 mg / kg of (+) or (-) CePPy nanoparticles intravenously as controls (physiological saline) and mice, respectively.

[0088] 1. Collect the above mouse serum and use the kit purchased from Nanjing Jiancheng Company to detect serum creatinine levels. Refer to the kit instructions for specific experimental procedures.

[0089] Experimental results:

[0090] Experimental results are as follows Figure 7 As shown in sections A to B. Among them, Figure 7 Figure A shows the experimental procedure of administering Sham and ischemia-reperfusion induced AKI mice as controls and (+) CePPy nanoparticles or (-) CePPy nanoparticles.

[0091] Figure 7 Figure B shows the creatinine levels in AKI mice after injection of (+) CePPy nanoparticles or (-) CePPy nanoparticles. The figure shows that (+) CePPy significantly reduced the increase in creatinine levels in AKI mice.

[0092] 2. Use PAS staining to assess renal tubular damage and score the renal tubule damage. The specific steps are as follows: (1) Dewaxing the sections to water: Place the sections in xylene I (10 minutes) → xylene II (15 minutes) → 100% ethanol (5 minutes) → 95% ethanol (5 minutes) → distilled water (5 minutes) to completely remove paraffin.

[0093] (2) Periodic acid oxidation: Immerse the slices in periodic acid solution and incubate at room temperature for 30 minutes, then rinse with distilled water 3 times for 2 minutes each time.

[0094] (3) Schiff reagent color development: Add Schiff reagent to cover the slide, incubate at room temperature in the dark for 15-20 minutes until the tissue turns purple-red, rinse with distilled water 3 times, 2 minutes each time.

[0095] (4) Counterstaining cell nuclei: Immerse in hematoxylin staining solution for 5 minutes to make the cell nuclei turn blue, which makes it easier to distinguish tissue morphology. Then rinse with distilled water.

[0096] (5) Differentiation and blueing: Differentiate with 0.5% hydrochloric acid ethanol for 10-15 seconds, then immediately rinse with running water for 10 minutes to blue.

[0097] (6) Dehydration and clearing: The slide is dehydrated and cleared in sequence by passing through 80% ethanol (3 minutes) → 90% ethanol (3 minutes) → 100% ethanol (3 minutes) → xylene (5 minutes), and finally neutral resin is added and covered with a coverslip.

[0098] Experimental results:

[0099] Experimental results are as follows Figure 7 As shown in C to D. Wherein, Figure 3 Image C shows PAS staining of renal tubules in AKI mice after injection of (+)CePPy nanoparticles or (-)CePPy nanoparticles. The image shows that, compared to the simple model group and the (-)CePPy group, the (+)CePPy group exhibited significantly reduced renal tubular epithelial cell necrosis, shedding, and casts, as well as less luminal dilation. Figure 3 Figure D shows the renal tubular injury scores of AKI mice after injection of (+) CePPy nanoparticles or (-) CePPy nanoparticles. As can be seen from the figure, compared with the model group and the (-) CePPy group, the renal tubular injury score of the (+) CePPy group was significantly reduced. These results indicate that (+) CePPy nanoparticles can significantly alleviate AKI.

[0100] 2. The renal tubular injury marker KIM1 and apoptosis-related markers were detected by immunofluorescence staining and Western blot. The specific steps are as follows:

[0101] (1) Western blot detection steps: Take the kidney tissue of each group of mice, add RIPA lysis buffer (containing protease inhibitor), grind and lyse on ice, collect the supernatant by centrifugation, and determine the protein concentration by BCA method.

[0102] Mix equal volumes of protein sample with loading buffer, denature in a boiling water bath for 5 minutes, and separate proteins by SDS-PAGE gel electrophoresis. Transfer the gel proteins to a PVDF membrane and block with 5% skim milk at room temperature for 1 hour. Incubate the membrane overnight at 4°C with KIM1, BAX, and caspase-3 cleavage primary antibodies (diluted proportionally). After rinsing with TBST, add HRP-labeled secondary antibody and incubate at room temperature for 1 hour. Develop using an ECL chemiluminescence kit, take photos using a gel imaging system, and quantify the band grayscale values ​​using ImageJ software.

[0103] (2) Immunofluorescence staining detection steps: Dewax tissue sections to water (same as PAS staining pretreatment steps). Citrate buffer (pH 6.0) for 10 minutes at high temperature, then rinse 3 times with PBS after cooling.

[0104] Block with 5% bovine serum albumin (BSA) at room temperature for 30 minutes to inhibit non-specific binding. Add KIM1, BAX, and caspase-3 cleavage primary antibody (diluted according to instructions), incubate overnight at 4°C, and wash three times with PBS. Add fluorescently labeled secondary antibody, incubate at room temperature in the dark for 1 hour, and wash three times with PBS. Stain the nucleus with DAPI, incubate at room temperature in the dark for 5 minutes, wash with PBS, mount with anti-fluorescence quencher, and observe the image under a confocal microscope.

[0105] Experimental results:

[0106] Experimental results are as follows Figure 7 As shown in E to J. Wherein,

[0107] Figure 7 Image E shows the immunofluorescence staining of KIM1, an indicator of renal tubular cell damage, in AKI mice after injection of (+) CePPy nanoparticles or (-) CePPy nanoparticles. As can be seen from the image, compared with the model group and the (-) CePPy group, the area of ​​KIM1 expression in renal tubular epithelial cells of mice in the (+) CePPy group was significantly reduced.

[0108] Figure 7 Figure F shows the quantitative analysis of KIM1 expression area in AKI mice after injection of (+) CePPy nanoparticles or (-) CePPy nanoparticles. The figure shows that (+) CePPy nanoparticles significantly reduced KIM1 expression levels.

[0109] Figure 7 Figure G shows the Western blot results of protein expression in AKI mice after injection of (+) CePPy nanoparticles or (-) CePPy nanoparticles. The figure shows that (+) CePPy nanoparticles significantly reduced the expression levels of KIM1 and apoptosis-related proteins BAX and cleaved caspase-3.

[0110] Figure 7 Figure H shows the quantitative analysis results of KIM1 expression levels in AKI mice after injection of (+) CePPy nanoparticles or (-) CePPy nanoparticles. The figure shows that (+) CePPy nanoparticles significantly reduced KIM1 expression levels.

[0111] Figure 7 Figure I shows the quantitative analysis results of BAX expression levels in AKI mice after injection of (+) CePPy nanoparticles or (-) CePPy nanoparticles. The figure shows that (+) CePPy nanoparticles significantly reduced BAX expression levels.

[0112] Figure 7 Figure J shows the quantitative analysis results of cleaved caspase-3 expression levels in AKI mice after injection of (+) CePPy nanoparticles or (-) CePPy nanoparticles. The figure shows that (+) CePPy nanoparticles significantly reduced the expression level of cleaved caspase-3.

[0113] The results show that (+)CePPy nanoparticles significantly reduced KIM1 expression and decreased the levels of apoptosis-related proteins such as cleaved caspase-3 and BAX. Therefore, (+)CePPy nanoparticles have a significant ameliorative effect on AKI.

[0114] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing cerium-doped polypyrrole nanoparticles, characterized in that, The preparation method includes: (1) Prepare a mixed solution of polyvinyl alcohol and pyrrole, wherein the concentration of polyvinyl alcohol is 0.05-5 g / mL and the concentration of pyrrole is 5-20 μL / mL; (2) Add Ce(SO4)2 solution to the mixed solution of polyvinyl alcohol and pyrrole obtained in step (1) and react under stirring conditions to obtain the cerium-doped polypyrrole nanoparticles.

2. The preparation method according to claim 1, characterized in that, In step (2), the concentration of the Ce(SO4)2 solution is 10-50 mg / mL; And / or, in step (2), the mass ratio of Ce(SO4)2 solution to the mixed solution of polyvinyl alcohol and pyrrole is 1:1 to 4:

1.

3. The preparation method according to claim 1, characterized in that, In step (2), the reaction time is 12-24 hours; And / or, in step (2), the reaction is carried out at room temperature; And / or, in step (2), the stirring speed is 500-1500 rpm.

4. The preparation method according to claim 1, characterized in that, The preparation method further includes: (A) Dialyze the mixed solution of cerium-doped polypyrrole nanoparticles obtained in step (2); concentrate the solution using an ultrafiltration centrifuge tube and collect positively charged cerium-doped polypyrrole nanoparticles.

5. The preparation method according to claim 4, characterized in that, The preparation method further includes: (B) The solution of positively charged cerium-doped polypyrrole nanoparticles obtained in step (A) is added to DSPE-PEG2000 solution and concentrated using an ultrafiltration centrifuge tube to obtain negatively charged cerium-doped polypyrrole nanoparticles.

6. Cerium-doped polypyrrole nanoparticles obtained by the preparation method according to any one of claims 1-5.

7. The cerium-doped polypyrrole nanoparticles according to claim 6, characterized in that, The cerium-doped polypyrrole nanoparticles include: positively charged cerium-doped polypyrrole nanoparticles and negatively charged cerium-doped polypyrrole nanoparticles.

8. The use of cerium-doped polypyrrole nanoparticles according to any one of claims 6-7 in the preparation of medicaments for treating acute kidney injury and / or the progression of acute kidney injury to chronic kidney disease.

9. A pharmaceutical composition for treating acute kidney injury and / or the progression of acute kidney injury to chronic kidney disease, characterized in that, The pharmaceutical composition comprises: cerium-doped polypyrrole nanoparticles and a carrier according to any one of claims 6-7.

10. The pharmaceutical composition according to claim 9, characterized in that, The drug is cerium-doped polypyrrole nanoparticles with a positive charge.

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