A rutin-cerium nanocomposite, its preparation method, and its application in the prevention and treatment of acute kidney injury.

Abstract

This invention discloses a rutin-cerium nanocomposite, its preparation method, and its application in the prevention and treatment of acute kidney injury, belonging to the field of nanobiomedicine technology. The preparation method includes the following steps: using a polyvinylpyrrolidone aqueous solution as the reaction system, first adding a substance that provides Ce to the reaction system... 4+ A water-soluble cerium salt was ultrasonically treated to obtain a precursor solution containing cerium ions; rutin was then added to this precursor solution, and the mixture was stirred for 6–72 h. During the reaction, rutin converted cerium ions into cerium ions. 4+ Reduced to Ce³ + Through coordination and self-assembly of cerium ions with phenolic hydroxyl groups, and stabilization of the coordination bonds with polyvinylpyrrolidone, an ultrasmall, water-dispersible rutin-cerium nanocomposite was obtained. This rutin-cerium nanocomposite can be used for the treatment of acute kidney injury, exerting a nephroprotective effect by remodeling the redox and inflammatory microenvironment of the kidney.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomedicine technology, specifically relating to a rutin-cerium nanocomposite, its preparation method, and its application in the prevention and treatment of acute kidney injury. Background Technology

[0002] Acute kidney injury (AKI) is a common and critical renal syndrome characterized by a rapid decline in renal excretory function, accompanied by elevated blood urea nitrogen and serum creatinine. AKI can be induced by a variety of factors, including ischemia-reperfusion, sepsis, nephrotoxic drugs, trauma, and rhabdomyolysis. Despite the different precipitating factors, AKI typically involves common pathological processes such as renal tubular epithelial damage, oxidative stress, mitochondrial dysfunction, cell death, and inflammatory responses.

[0003] In rhabdomyolysis-related acute kidney injury, muscle injury leads to the release of large amounts of myoglobin, increased heme and iron load, which in turn induce renal tubular obstruction, lipid peroxidation, oxidative DNA damage, and mitochondrial dysfunction. Excessive reactive oxygen species can further disrupt mitochondrial membrane potential, induce renal tubular cell apoptosis, and promote the release of injury-related signals. These injury signals can recruit macrophages and neutrophils, increase myeloperoxidase activity, and promote the production of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), thus forming a vicious cycle of mutually amplified oxidative stress and inflammatory response.

[0004] Currently, the clinical treatment of acute kidney injury mainly relies on supportive measures such as fluid resuscitation, maintaining hemodynamic stability, discontinuing nephrotoxic drugs, and, when necessary, renal replacement therapy. Effective drugs that can directly block oxidative stress and the inflammatory cascade are still lacking. Therefore, developing novel therapeutic drugs that can exert antioxidant and anti-inflammatory effects locally in the damaged kidney is an important need in the prevention and treatment of acute kidney injury. Summary of the Invention

[0005] Rutin is a natural flavonoid glycoside with antioxidant, anti-inflammatory, and cytoprotective effects. Its phenolic hydroxyl group can participate in free radical scavenging, electron or hydrogen transfer, and metal ion coordination. However, free rutin suffers from poor water solubility, low bioavailability, rapid metabolism in vivo, and insufficient accumulation at lesion sites, limiting its application as a treatment for acute kidney injury. While traditional carrier-based nanodelivery systems can improve rutin delivery, they typically suffer from complex composition, limited drug loading, premature drug leakage, and potential long-term safety concerns.

[0006] Cerium has Ce 3+ / Ce 4+The reversible redox cycle capability can mimic the activity of antioxidant enzymes such as superoxide dismutase and catalase, thereby scavenging various reactive oxygen species. Constructing nanocomposites by coordinating natural polyphenol small molecules with cerium ions holds promise for simultaneously improving the water dispersibility of natural small molecules, preserving their bioactivity, and introducing the redox catalytic ability of cerium ions. However, current technology lacks an ultra-small rutin-cerium nanocomposite formed using rutin as a reducing agent and coordinating ligand, cerium ions as metal nodes, and stabilized by polyvinylpyrrolidone, and there are no reports of its use for antioxidant and anti-inflammatory treatment of acute kidney injury.

[0007] Therefore, establishing a new method for constructing rutin-cerium nanocomposites and further proposing the application of this nanocomposite in the prevention and treatment of acute kidney injury is of great innovative significance and application value.

[0008] Based on this, the present invention provides an ultra-small rutin-cerium ion-polyvinylpyrrolidone nanocomposite constructed by rutin-mediated cerium ion reduction and rutin-cerium ion coordination self-assembly, and the application of the nanocomposite in the preparation of drugs for the prevention and treatment of acute kidney injury.

[0009] Specifically, the present invention provides the following technical solution: This invention provides a method for preparing a rutin-cerium nanocomposite, wherein the rutin-cerium nanocomposite comprises rutin, cerium ions, and polyvinylpyrrolidone (PVP). The rutin forms a coordination bond with the cerium ions via a phenolic hydroxyl group, and the PPVP is used to stabilize the coordination bond. The cerium ions are primarily in the form of Ce. 3+ Form exists; The preparation method of the rutin-cerium nanocomposite includes the following steps: (1) Using polyvinylpyrrolidone aqueous solution as the reaction system; wherein, the polyvinylpyrrolidone aqueous solution is prepared by dissolving polyvinylpyrrolidone (abbreviated as PVP) in water (preferably deionized water), and the polyvinylpyrrolidone is a polyvinylpyrrolidone with an average molecular weight of 2~30 kDa. (2) First, add a substance that can provide Ce to the aqueous solution of polyvinylpyrrolidone. 4+ Water-soluble cerium salts are subjected to ultrasonic treatment to obtain a precursor solution (clear solution) containing cerium ions. Rutin was then added to the cerium-containing precursor solution, and the mixture was stirred at room temperature for 6–72 h. During the reaction, rutin converted Ce into cerium ions. 4+ Reduced to Ce³ + Polyvinylpyrrolidone (PVP) coordinates with cerium ions and self-assembles through phenolic hydroxyl groups, and stabilizes the coordination bonds through hydrogen bonding and / or steric hindrance. The mass ratio of polyvinylpyrrolidone, water-soluble cerium salt, and rutin is (100-1000):(50-1000):(10-200). (3) After the stirring reaction is completed, the resulting reaction solution is centrifuged and the supernatant is collected; the supernatant is ultrafiltered and washed to obtain the rutin-cerium nanocomposite.

[0010] This invention describes a preparation method using rutin, cerium salt, and polyvinylpyrrolidone as raw materials. Through rutin-mediated cerium ion reduction, rutin-cerium ion coordination, and polyvinylpyrrolidone stabilization, an ultra-small, well-water-dispersible rutin-cerium nanocomposite is constructed. In this nanocomposite, rutin can reduce cerium ions... 4+ Restored to Ce 3+ Furthermore, by forming a Ce-O coordination structure with cerium ions through phenolic hydroxyl groups, the resulting nanocomposite exhibits broad-spectrum reactive oxygen species scavenging ability and good biocompatibility. This rutin-cerium nanocomposite can enhance accumulation and retention in the kidneys of patients with acute kidney injury. By scavenging reactive oxygen species, reducing lipid peroxidation, restoring antioxidant enzyme levels, inhibiting macrophage and neutrophil infiltration, and reducing inflammatory factor levels, it can reshape the renal redox and inflammatory microenvironment, improve renal function, and alleviate acute kidney injury.

[0011] PVP with an average molecular weight of 2–30 kDa can act as a stabilizer and dispersant during the coordination self-assembly of rutin-cerium ions, reducing particle aggregation and improving the water dispersibility and colloidal stability of rutin-cerium nanocomposites. PVP with an average molecular weight of 10 kDa is preferred (denoted as PVP). 10k This approach can balance spatial stability and the fluidity of the reaction system, which is beneficial for forming rutin-cerium nanocomposites with smaller particle size, more uniform distribution, and better stability.

[0012] In a preferred embodiment, the cerium salt is selected from one or more of cerium sulfate, cerium nitrate, cerium chloride, cerium acetate, cerium ammonium nitrate, cerium ammonium sulfate and their hydrates, more preferably cerium sulfate.

[0013] In a preferred embodiment, the mass ratio of polyvinylpyrrolidone, cerium salt and rutin is (200-500):(200-500):(20-100), and more preferably 300:300:50.

[0014] In a more preferred embodiment, the cerium salt is cerium sulfate, and the mass ratio of polyvinylpyrrolidone, cerium sulfate, and rutin is 300:300:50. Using a mass ratio of 300:300:50 for polyvinylpyrrolidone, cerium sulfate, and rutin achieves a good balance between cerium ion supply, rutin coordination reduction, and the stabilizing effect of polyvinylpyrrolidone, which is beneficial for forming rutin-cerium nanocomposites with small particle size, good dispersibility, and high stability. This ratio can reduce the residual free cerium ions or inorganic precipitation caused by excessive cerium salt, and also reduce the decrease in dispersibility and particle aggregation caused by excessive rutin, while ensuring the stabilizing effect of polyvinylpyrrolidone on the surface of the nanocomposite, thereby improving the reproducibility of the preparation and the stability of the material. Most preferably, the amounts of each component are as follows: the polyvinylpyrrolidone is PVP... 10k PVP 10k The concentrations are 300 mg, cerium sulfate 300 mg, rutin 50 mg, and deionized water 10 mL.

[0015] In a preferred embodiment, the stirring reaction time is 12 to 48 hours, with the most preferred time being 24 hours.

[0016] In step (3), unreacted rutin and large particle precipitates are removed by centrifugation, and the supernatant is collected; then, free cerium ions and residual polyvinylpyrrolidone are removed by ultrafiltration, and the rutin-cerium nanocomposite is obtained after washing.

[0017] In a preferred embodiment, the centrifugation conditions are 10,000 rpm for 10 min, the ultrafiltration device used for ultrafiltration has a molecular weight cutoff of 100 kDa, and the washing is performed 5 times.

[0018] This invention also provides a rutin-cerium nanocomposite prepared by the above method. The nanocomposite comprises rutin, cerium ions, and polyvinylpyrrolidone, wherein rutin can convert cerium ions into cerium nanocomposites. 4+ Restored to Ce 3+ Therefore, cerium ions are mainly in the form of Ce. 3+ It exists in the form of rutin, and rutin forms a Ce-O coordination bond with cerium ions through a phenolic hydroxyl group; polyvinylpyrrolidone stabilizes the coordination bond through hydrogen bonding and / or steric hindrance.

[0019] The rutin-cerium nanocomposite prepared by the above method in this invention exhibits ultra-small size, good water dispersibility, good colloidal stability, and broad-spectrum reactive oxygen species scavenging ability. Furthermore, the rutin-cerium nanocomposite has a uniformly dispersed nanoparticle morphology, with a transmission electron microscope particle size of 3.88 ± 1.16 nm and a dynamic light scattering hydrated particle size of 4.38 ± 0.47 nm. The rutin-cerium nanocomposite can be stably dispersed in deionized water, phosphate-buffered saline (PBS), physiological saline, and Durbeco modified Eagle medium (DMEM).

[0020] This invention also provides the use of the rutin-cerium nanocomposite in the preparation of drugs for the prevention and treatment of acute kidney injury. The acute kidney injury includes, but is not limited to, rhabdomyolysis-related acute kidney injury, ischemia-reperfusion-related acute kidney injury, drug-induced acute kidney injury, inflammation-related acute kidney injury, and oxidative stress-related acute kidney injury. Preferably, the acute kidney injury is glycerol-induced rhabdomyolysis-related acute kidney injury.

[0021] The rutin-cerium nanocomposite of this invention can scavenge 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cationic free radicals (ABTS• + ), 2,2-diphenyl-1-picrylhydrazine radical (DPPH•), hydroxyl radical (•OH), and superoxide anion radical (O2•) It can kill free radicals or reactive oxygen species, and has superoxide dismutase-like and catalase-like activities. At the cellular level, it can reduce the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) in macrophages under inflammatory stimulation, restore superoxide dismutase (SOD) activity, protect mitochondrial membrane potential, inhibit nuclear translocation of nuclear factor κB p65 subunit (NF-κB p65), reduce CD86 expression, and reduce the secretion of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6).

[0022] The rutin-cerium nanocomposite described in this invention, after intravenous administration, is rapidly cleared by the kidneys in healthy mice; in mice with acute kidney injury, it enhances accumulation and prolongs retention in damaged kidneys. In a glycerol-induced rhabdomyolysis-associated acute kidney injury model, it reduces blood urea nitrogen and serum creatinine, alleviates renal tubular pathological damage, reduces positive signals of kidney injury molecule-1 (KIM-1), 8-hydroxydeoxyguanosine (8-OHdG), and apoptosis (TUNEL), decreases dihydroethidium (DHE)-labeled renal tissue ROS levels, reduces MDA levels, restores SOD and catalase (CAT) levels, reduces CD68-positive macrophage infiltration and Ly6G-positive neutrophil recruitment, decreases myeloperoxidase (MPO), TNF-α, IL-1β, and IL-6 levels, and improves the survival rate of mice with acute kidney injury.

[0023] This invention also provides a drug for the prevention and treatment of acute kidney injury, comprising the rutin-cerium nanocomposite described in this invention and pharmaceutically acceptable excipients, wherein the excipients include one or more of solvents, buffer solutions, stabilizers, isotonic adjusters, and pH adjusters; the drug is an injection, preferably an intravenous injection; the dosage of the rutin-cerium nanocomposite is 5-20 mg / kg, preferably 20 mg / kg, wherein mg / kg is a dosage unit calculated based on the body weight of the test subject, representing the corresponding number of milligrams of rutin-cerium nanocomposite administered per kilogram of body weight; for example, 20 mg / kg represents 20 mg of rutin-cerium nanocomposite administered per kilogram of body weight, and the dosage is based on the dry weight of the rutin-cerium nanocomposite.

[0024] Compared with the prior art, the present invention has the following beneficial effects: First, this invention provides a novel method for constructing rutin-cerium nanocomposites. This method differs from traditional nanodelivery systems that encapsulate rutin on a carrier. Instead, it utilizes rutin's own reducing and coordinating abilities to enable it to participate in cerium ion reduction and rutin-cerium coordination self-assembly, thereby forming a bioactive rutin-cerium nanocomposite.

[0025] Secondly, this invention transforms poorly soluble natural small molecule rutin into a stable, water-dispersible ultra-small nanocomposite, which improves the problems of poor water solubility of free rutin and insufficient accumulation in kidney lesions.

[0026] Third, the rutin-cerium nanocomposite of the present invention integrates the natural antioxidant and anti-inflammatory activities of rutin with the redox catalytic ability of cerium ions, which can achieve broad-spectrum scavenging of reactive oxygen species and regulation of inflammatory responses.

[0027] Fourth, this invention is the first to propose the use of the rutin-cerium nanocomposite for the treatment of acute kidney injury, which can exert a nephroprotective effect by remodeling the redox and inflammatory microenvironment of the kidney.

[0028] Fifth, the rutin-cerium nanocomposite of the present invention has good biocompatibility in vivo, and no obvious blood toxicity, hepatotoxicity, nephrotoxicity or major organ pathological damage has been observed, and it has the potential to be further developed into a drug for the treatment of acute kidney injury. Attached Figure Description

[0029] Figure 1 This is a transmission electron microscope (TEM) image of the rutin-cerium nanocomposite (denoted as CRP) in Example 1 of the present invention.

[0030] Figure 2 This is the Ce 3d high-resolution X-ray photoelectron spectrum of CRP in Embodiment 1 of the present invention.

[0031] Figure 3 In Example 1 of this invention, CRP is used to counteract superoxide anion (O2• The results of the cleaning ability test are shown in the figure.

[0032] Figure 4 The figure shows the protective effect of CRP against H2O2-induced oxidative damage and apoptosis in HEK293 cells in Example 1 of this invention.

[0033] Figure 5 This is a fluorescence imaging and tissue distribution result of Cy7-CRP in mice with acute kidney injury in Example 1 of the present invention.

[0034] Figure 6 This is a graph showing the therapeutic effect of CRP on a glycerol-induced rhabdomyolysis-related acute kidney injury model, as assessed by serum blood urea nitrogen (BUN) concentration in Example 1 of this invention.

[0035] Figure 7 This is a graph showing the therapeutic effect of CRP on a glycerol-induced rhabdomyolysis-related acute kidney injury model, as assessed by serum creatinine (Cr) concentration in Example 1 of this invention. Detailed Implementation

[0036] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are used to explain the present invention and should not be construed as limiting the scope of protection of the present invention. Conventional substitutions, equivalent modifications, or parameter adjustments made by those skilled in the art without departing from the concept of the present invention should all fall within the scope of protection of the present invention.

[0037] Example 1 (1) Preparation of rutin-cerium nanocomposite (CRP) Weighing PVP 10k 300 mg was added to 10 mL of deionized water and dissolved completely to obtain PVP. 10kAqueous solution. 300 mg of cerium sulfate was added to this aqueous solution, and after sonication, a clear precursor solution containing cerium ions was formed. Then, 50 mg of rutin powder was added, and the reaction was stirred at room temperature for 24 h. During the reaction, rutin, as a small molecule of natural polyphenol, can, on the one hand, reduce cerium ions... 4+ Restored to Ce 3+ On the other hand, it induces the formation of a rutin-cerium ion coordination structure by forming a Ce-O coordination bond with cerium ions through its phenolic hydroxyl groups. 10K The coordination structure is stabilized through hydrogen bonding and steric hindrance, thus forming an ultrasmall nanostructure and preventing further aggregation or precipitation. After the reaction, the reaction solution was centrifuged at 10,000 rpm for 10 min to remove unreacted rutin and large precipitate particles. The supernatant was collected and purified using a 100 kDa ultrafiltration centrifugal filter to remove free cerium ions and residual PVP. The solution was then washed five times with deionized water and lyophilized to obtain the PVP-stabilized ultrasmall rutin-cerium nanocomposite, abbreviated as CRP. The obtained CRP was stored at 4 °C for later use.

[0038] This embodiment demonstrates that the method of the present invention can construct rutin-cerium nanocomposites in one step under mild conditions through rutin-mediated reduction coordination and polyvinylpyrrolidone stabilization. This method does not rely on traditional inert nanocarriers for encapsulation; instead, it allows rutin itself to participate in the formation of the nanocomposite framework, representing a novel method for constructing rutin-cerium nanomedicines.

[0039] (2) Characterization and performance testing of rutin-cerium nanocomposites 1) Characterization of CRP morphology, particle size and stability The morphology and particle size of CRP were observed using transmission electron microscopy, and the results are as follows: Figure 1 As shown in the figure. The results show that CRP exhibits a uniformly dispersed nanoparticle morphology with no obvious large particle aggregation, and its TEM particle size is 3.88±1.16 nm. The hydrated particle size of CRP was detected by dynamic light scattering (DLS), and the result was 4.38±0.47 nm. The above results indicate that CRP is an ultrasmall nanocomposite with size characteristics suitable for renal processing and renal-related delivery. Free rutin forms a turbid suspension in water due to its poor water solubility; however, CRP prepared by the method of this invention can form a clear yellow to brownish-yellow aqueous dispersion, indicating that the water dispersibility of rutin is significantly improved after coordination with cerium ions and stabilization by PVP.

[0040] CRP was further dispersed in deionized water, PBS, physiological saline, and DMEM, and its dispersion was observed at 1, 3, 5, and 7 days. The results showed that CRP remained clear or stably dispersed in all the above media, with no obvious visible precipitation. DLS analysis further showed that CRP maintained its ultra-small hydrated particle size after 7 days of storage in the above media, indicating its good colloidal stability.

[0041] Zeta potential assays showed that the potentials of CRP in deionized water, physiological saline, PBS, and DMEM were -29.1±2.8 mV, -11.2±1.2 mV, -14.4±1.0 mV, and -21.1±1.3 mV, respectively. These results indicate that CRP can maintain a stable nano-dispersion state under different ionic strengths and protein environments.

[0042] 2) Characterization of the coordination structure and chemical state of CRP Fourier transform infrared spectroscopy (FT-IR) was used to analyze the coordination process in CRP formation. The results showed that, compared to free rutin, approximately 1200 cm⁻¹ of CRP contained higher concentrations of rutin. 1 The C-OH vibration peak at 500-600 cm⁻¹ showed a significant change accompanied by a decrease in peak intensity, suggesting that the hydroxyl group of rutinol participated in the coordination with cerium ions. Meanwhile, CRP showed a significant change at 500-600 cm⁻¹. 1 The presence of absorption peaks in the region related to Ce-O stretching vibrations indicates that Ce-O coordination bonds have formed between rutin and cerium ions.

[0043] The coordination process was further verified using ultraviolet-visible absorption spectroscopy (UV-vis). Free rutin exhibited characteristic absorption peaks at approximately 255 nm and 352 nm, while CRP showed a redshift absorption, suggesting that cerium ion coordination altered the electronic structure and conjugated system of rutin.

[0044] The crystal structure of CRP was determined by X-ray diffraction (XRD). The results showed that CRP did not exhibit obvious diffraction peaks corresponding to crystalline cerium species, indicating that CRP is mainly an amorphous nanocomposite rather than simple crystalline cerium oxide particles.

[0045] X-ray photoelectron spectroscopy (XPS) was used to analyze the elemental composition and cerium valence state of CRP. The full XPS spectrum showed the presence of Ce, O, N, and C signals in CRP, proving that cerium ions, rutin, and PVP were successfully integrated into the nanocomposite. Figure 2 High-resolution Ce3d spectra show that cerium ions are mainly in the form of Ce. 3+ The presence of this form indicates that Ce was present during the preparation process. 4+ Reduced to Ce by rutin 3+The above results demonstrate that the CRP constructed in this invention is not a simple physical mixture of rutin and cerium salt, but a novel nanocomposite formed through rutin reduction, rutin-cerium coordination, and PVP stabilization.

[0046] 3) CRP's broad-spectrum reactive oxygen species scavenging ability In the progression of acute kidney injury, excessive reactive oxygen species (ROS) are a key factor in inducing renal tubular damage, mitochondrial dysfunction, and amplified inflammation. Therefore, this study evaluated the scavenging capacity of CRP against various free radicals and ROS. Electron paramagnetic resonance spectroscopy was used to detect •OH and O2•. Signal. Results showed that after adding CRP, •OH and O2• The characteristic signal was significantly weakened, indicating that CRP can effectively remove the aforementioned highly reactive oxygen species. Using ABTS• + The decolorization assay was used to detect the free radical scavenging ability of CRP. CRP exhibited concentration-dependent ABTS• within 10 min. + The scavenging effect of CRP was assessed, with scavenging rates of 64.65±1.81% and 95.77±2.77% at 80 μg / mL and 160 μg / mL, respectively. The DPPH• scavenging ability of CRP against stable free radicals was detected using a DPPH• decolorization assay. The results showed that the DPPH• scavenging rates of CRP at 80 μg / mL and 160 μg / mL were 61.90±2.77% and 86.81±2.91%, respectively. The hydroxyl radical scavenging ability of CRP against •OH was detected using a hydroxyl radical scavenging assay. The results showed that the •OH scavenging rates of CRP at 160 μg / mL and 320 μg / mL were 74.92±0.16% and 90.46±0.84%, respectively. The superoxide anion scavenging effect of CRP against O2• was detected using a superoxide anion scavenging assay. The ability to clear, the result is as follows Figure 3 The results showed that CRP at 80 μg / mL and 160 μg / mL resulted in O2• The scavenging rates were 77.60±0.38% and 92.57±0.38%, respectively. These results indicate that the rutin-cerium nanocomposite prepared in this invention possesses broad-spectrum reactive oxygen species scavenging ability and can remove ABTS• + DPPH•, •OH and O2• It can kill various free radicals or reactive oxygen species and has antioxidant enzyme mimicry activities similar to superoxide dismutase and catalase.

[0047] 4) Protective effect of CRP against oxidative damage in HEK293 cells To evaluate the uptake and cytoprotective effects of CRP in renal-derived cells, Cy5.5-labeled CRP (denoted as Cy5.5-CRP) was prepared. The specific method was as follows: PVP... 10k30 mg of cerium sulfate and 30 mg of cerium sulfate were dissolved sequentially in 1 mL of deionized water, and a clear precursor solution was obtained by sonication. 1 mg of Cy5.5 and 5 mg of rutin were added, and the mixture was stirred at room temperature in the dark for 24 h. After the reaction, the mixture was centrifuged at 10,000 rpm for 10 min, and the supernatant was collected. Free cerium ions, unloaded Cy5.5, and excess PVP were removed by a 100 kDa ultrafiltration device. After washing 5 times, the mixture was lyophilized to obtain Cy5.5-CRP.

[0048] Human embryonic kidney 293 cells (HEK293 cells) were incubated with Cy5.5-CRP for 1, 4, 12, and 24 h, respectively, and intracellular fluorescence signals were observed using confocal microscopy. The results showed that intracellular Cy5.5 fluorescence increased with increasing incubation time, with a strong signal at 12 h and detectable fluorescence even after 24 h, indicating that CRP can be effectively taken up by HEK293 cells, providing a basis for its intracellular antioxidant activity. CCK-8 staining and live / dead staining were used to evaluate the cytocompatibility of CRP with HEK293 cells. The results showed that even at a concentration of 200 μg / mL, the cell viability of HEK293 cells remained at 97.79±3.61% after 24 h of CRP treatment, and no significant cell death was observed in the live / dead staining, indicating that CRP has good cytocompatibility.

[0049] An oxidative damage model of HEK293 cells was established using H2O2. After stimulation with 250 μM H2O2 for 24 h, the cell viability of HEK293 cells decreased to 53.94 ± 5.22%. Figure 4 As shown, CRP treatment restored cell viability in a concentration-dependent manner, with cell viability recovering to 86.11±4.64% and 95.11±2.29% at 80 μg / mL and 160 μg / mL, respectively. Compared with free rutin or cerium sulfate, CRP showed superior cell protection, indicating that the rutin-cerium nanocomposite has a synergistic protective effect. Further analysis used DCFH-DA to detect intracellular ROS, MitoSOX Red to detect mitochondrial ROS, and JC-1 to detect mitochondrial membrane potential. The results showed that H2O2 stimulation significantly increased intracellular and mitochondrial ROS and induced a decrease in mitochondrial membrane potential; after CRP treatment, the levels of intracellular and mitochondrial ROS were significantly reduced, and the mitochondrial membrane potential was protected.

[0050] Western blot analysis was used to detect apoptosis-related proteins. Results showed that H2O2 stimulation reduced the expression of the key apoptosis protein B-cell lymphoma / leukemia-2 (Bcl-2) and increased the expression levels of Bcl-2-associated X protein (Bax) and cleaved caspase-3. After CRP treatment, Bcl-2 expression recovered, while the levels of Bax and cleaved caspase-3 decreased. These results indicate that CRP can alleviate oxidative stress-induced renal cell damage by reducing intracellular and mitochondrial ROS, protecting mitochondrial membrane potential, restoring Bcl-2 / Bax balance, and inhibiting caspase-3 activation.

[0051] 5) The inhibitory effect of CRP on the inflammatory activation of RAW264.7 macrophages. The anti-inflammatory effect of CRP was evaluated using the mouse macrophage line RAW264.7. First, the cytocompatibility of CRP with RAW264.7 cells was assessed using CCK-8 assay. After treatment with different concentrations of CRP for 24 h or 48 h, RAW264.7 cells maintained high viability, indicating that CRP had no significant cytotoxic effect on macrophages. An inflammation model of RAW264.7 cells was induced using lipopolysaccharide (LPS). Cells were pretreated with 80 μg / mL or 160 μg / mL CRP for 4 h, followed by stimulation with 1 μg / mL LPS for 72 h. Intracellular ROS were detected using DCFH-DA staining. The results showed that LPS significantly enhanced intracellular ROS fluorescence signal, and CRP treatment reduced ROS levels in a concentration-dependent manner. Mitochondrial membrane potential was detected using JC-1 staining. LPS stimulation led to a decrease in mitochondrial membrane potential in RAW264.7 cells, manifested as decreased red fluorescence and increased green fluorescence. CRP treatment restored the red / green fluorescence ratio, indicating that CRP can protect mitochondrial function in macrophages under inflammatory stimulation. Further analysis of MDA and SOD revealed that LPS stimulation significantly increased MDA levels and decreased SOD activity; CRP treatment reduced MDA levels and restored SOD activity, indicating that CRP can inhibit lipid peroxidation and improve macrophage antioxidant defense.

[0052] Immunofluorescence was used to detect NF-κB p65 nuclear translocation. LPS stimulation induced significant NF-κB p65 nuclear translocation, while CRP treatment significantly reduced intranuclear p65 fluorescence signal, indicating that CRP can inhibit the classical NF-κB inflammatory pathway. Further detection of inflammatory phenotypic markers CD86 and inflammatory cytokines IL-1β and TNF-α was performed. Results showed that LPS significantly upregulated the expression of CD86, IL-1β, and TNF-α, while CRP treatment significantly reduced these signals. ELISA results showed that LPS significantly increased the levels of TNF-α, IL-1β, and IL-6 in the culture supernatant of RAW264.7 cells, while CRP treatment significantly reduced the secretion of these pro-inflammatory cytokines. These results indicate that CRP can inhibit macrophage inflammatory activation by reducing ROS, inhibiting lipid peroxidation, protecting mitochondrial function, blocking NF-κB p65 nuclear translocation, reducing CD86 expression, and decreasing the secretion of pro-inflammatory cytokines.

[0053] 6) CRP accumulation in the kidneys of mice with acute kidney injury To evaluate the in vivo distribution and renal accumulation capacity of CRP, Cy7-labeled CRP was prepared. Cy7-CRP was prepared using the same method as Cy5.5-CRP. Cy7-CRP was injected intravenously into healthy mice and mice with glycerol-induced acute kidney injury at a dose of 5 mg / kg. Whole-body fluorescence imaging was performed at 1, 3, 6, 12, and 24 h post-injection, and the heart, liver, spleen, lung, and kidney were collected for ex vivo fluorescence imaging and quantitative fluorescence analysis. In healthy mice, significant bladder fluorescence was observed at 1 and 3 h post-injection, accompanied by transient renal signal; the fluorescence signal gradually weakened thereafter, and significantly decreased at 24 h, indicating that CRP can be rapidly cleared via the kidney-bladder pathway. In mice with acute kidney injury, although bladder fluorescence indicated partial urinary excretion, the fluorescence signal in the injured kidney area was stronger and lasted longer than in healthy mice; renal fluorescence was still detectable at 24 h. Figure 5 As shown in the figure, in vitro organ imaging revealed significantly enhanced CRP accumulation in the kidneys of patients with acute kidney injury compared to healthy kidneys. The fluorescence intensity in the kidneys of patients with acute kidney injury was approximately 5.24 times that of healthy kidneys at 1 hour post-injection, reaching approximately 6.14 times at 6 hours, and still approximately 3.94 times at 24 hours. These results indicate that CRP is rapidly cleared by the kidneys in healthy individuals, but accumulates more significantly and remains longer in the injured kidneys during acute kidney injury. This characteristic is beneficial for CRP to exert sustained antioxidant and anti-inflammatory effects locally in the injured kidney.

[0054] 7) CRP treatment for glycerol-induced rhabdomyolysis-related acute kidney injury A glycerol-induced rhabdomyolysis-related acute kidney injury model was established in male ICR mice. Before modeling, mice were deprived of water for 15 hours but allowed free access to food. After the water deprivation period, 50% glycerol was injected intramuscularly into both hind limbs at a total dose of 8 mL / kg, with equal injection volumes in both sides. Free access to food and water was resumed after glycerol injection, and the acute kidney injury model was considered established 2 hours later.

[0055] Mice were randomly divided into four groups: a healthy control group, an AKI (acute kidney injury) group, an AKI + low-dose CRP group, an AKI + medium-dose CRP group, and an AKI + high-dose CRP group, with five mice in each group. The healthy control group received 200 μL of PBS intravenously; the AKI group received 200 μL of PBS intravenously; and the low, medium, and high-dose CRP groups received 5, 10, and 20 mg / kg of CRP via tail vein injection, respectively, with an injection volume of 200 μL. Mice had free access to food and water after administration, and blood and kidney tissue were collected for analysis 24 h after administration.

[0056] Weight measurement results showed that AKI mice treated with PBS experienced significant weight loss within 24 hours, while CRP treatment alleviated the weight loss, with the high-dose CRP group showing a more pronounced effect. Serum renal function tests showed that the blood urea nitrogen (BUN) in healthy mice was 25.08±6.61 mg / dL, while it increased to 201.28±27.89 mg / dL in the AKI group; after high-dose CRP treatment, BUN decreased to 44.18±13.97 mg / dL. Figure 6 As shown. Regarding serum creatinine (Cr), the level in healthy mice was 23.95±5.11 μmol / L, while it increased to 261.22±42.82 μmol / L in the AKI group; after high-dose CRP treatment, it decreased to 38.09±9.27 μmol / L, close to healthy levels. Figure 7 As shown in the figure. The results indicate that CRP can significantly improve renal function after glycerol-induced acute kidney injury.

[0057] Hematoxylin and eosin (H&E) staining revealed significant renal tubular epithelial destruction, tubular dilation, cast formation, and structural disorder in the renal tissue of the AKI group. CRP treatment dose-dependently alleviated these pathological changes and reduced the renal injury score. KIM-1 detection showed significantly elevated KIM-1 expression in the renal tissue of the AKI group; CRP treatment dose-dependently reduced KIM-1 levels, with the high-dose CRP group approaching healthy levels. TUNEL staining showed a significant increase in TUNEL-positive cells in the renal tissue of the AKI group; CRP treatment significantly reduced TUNEL-positive signals. 8-OHdG detection showed significantly enhanced oxidative DNA damage in the renal tissue of the AKI group; 8-OHdG levels significantly decreased after CRP treatment.

[0058] Further observation of 14-day survival rate was conducted. The mortality rate of PBS-treated AKI mice was 20% on day 2, 40% on day 3, reached 80% on day 5, and was only 20% on day 14. The mortality rate on day 14 was 40% in the low-dose CRP group, 20% in the medium-dose CRP group, and no deaths occurred in the high-dose CRP group during the 14-day observation period. These results indicate that CRP can significantly improve the survival rate of mice with acute kidney injury.

[0059] The above results indicate that the rutin-cerium nanocomposite described in this invention can improve renal function, reduce renal tubular pathological damage, decrease the level of molecular markers of renal injury, reduce oxidative DNA damage and cell apoptosis, and improve the survival rate of mice with acute kidney injury.

[0060] 8) CRP remodels the redox and inflammatory microenvironment of the kidneys in acute kidney injury. DHE staining was used to detect ROS levels in renal tissue. Results showed significantly enhanced DHE fluorescence in the AKI group, indicating significant oxidative stress in the kidney tissue; CRP treatment dose-dependently reduced DHE fluorescence intensity. MDA detection showed significantly increased lipid peroxidation levels in the AKI group, while CRP treatment reduced MDA levels, with the high-dose CRP group approaching healthy levels. Further analysis of SOD and CAT levels in renal tissue revealed that AKI led to a significant decrease in SOD and CAT levels, indicating impaired endogenous antioxidant defense; CRP treatment dose-dependently restored SOD and CAT levels, with the high-dose CRP group approaching healthy control levels. These results demonstrate that CRP can improve the redox status of the kidneys in acute kidney injury by reducing ROS, decreasing lipid peroxidation, and restoring antioxidant enzyme defense.

[0061] Macrophage infiltration was detected by CD68 immunohistochemistry. The number of CD68-positive cells in the renal tissue of the AKI group was significantly increased, and CRP treatment dose-dependently reduced the number of CD68-positive cells. Neutrophil infiltration was detected by Ly6G immunohistochemistry. The number of Ly6G-positive cells in the renal tissue of the AKI group was significantly increased, and CRP treatment significantly reduced the number of Ly6G-positive cells. Inflammatory factors in renal tissue were detected by ELISA. The levels of MPO, TNF-α, IL-1β, and IL-6 were significantly increased in the AKI group; CRP treatment dose-dependently reduced these inflammatory markers, and high-dose CRP brought MPO and pro-inflammatory factor levels close to those of healthy controls.

[0062] The above results indicate that CRP exerts local antioxidant and anti-inflammatory effects by enhancing accumulation and retention in the injured kidney, reducing ROS production, inhibiting lipid peroxidation, restoring SOD and CAT antioxidant defense, reducing macrophage infiltration and neutrophil recruitment, and lowering MPO, TNF-α, IL-1β and IL-6 levels, thereby blocking the pathological cycle of "ROS-lipid peroxidation-immune cell infiltration-inflammatory factor amplification" in acute kidney injury.

[0063] 9) In vivo biosafety evaluation of CRP To evaluate the in vivo safety of CRP, healthy mice were observed for 14 days after intravenous injection of CRP. Complete blood count, serum biochemistry, and histological examination of major organs were performed on days 7 and 14 post-administration. Complete blood count showed that after CRP treatment, the number of white blood cells, lymphocytes, monocytes, neutrophils, eosinophils, basophils, platelets, erythrocytes, hemoglobin, hematocrit, mean corpuscular hemoglobin (MCH), and MCH concentration in mice were comparable to those in the control group and within the normal reference range, indicating that CRP did not cause significant hematologic toxicity, platelet abnormalities, bone marrow suppression, or acute systemic inflammatory response. Serum biochemistry showed that on days 7 and 14 post-CRP administration, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), BUN, and Cr levels in mice were all within the normal range, indicating that CRP did not cause significant liver or kidney damage. H&E staining showed that the heart, liver, spleen, lung, and kidney tissues of CRP-treated mice remained structurally intact, with no obvious inflammatory infiltration, necrosis, hemorrhage, edema, or structural damage. These results indicate that CRP exhibits good in vivo biocompatibility and systemic safety within the tested dosage and observation period.

[0064] Conclusion: The method for preparing rutin-cerium nanocomposites provided in this invention is mild, simple, uses readily available raw materials, and exhibits good reproducibility. The resulting nanocomposites possess ultra-small size, good water dispersibility, colloidal stability, broad-spectrum reactive oxygen species scavenging ability, anti-inflammatory activity, reduced renal accumulation due to injury, and good biocompatibility. These nanocomposites can serve as active pharmaceutical ingredients for the prevention and treatment of acute kidney injury, and can be further prepared into intravenous injections and other drugs for the prevention and treatment of rhabdomyolysis-related acute kidney injury and other oxidative stress and inflammation-related acute kidney injuries, showing promising prospects for industrialization and clinical translation.

[0065] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A method for preparing a rutin-cerium nanocomposite, characterized in that, The rutin-cerium nanocomposite comprises rutin, cerium ions, and polyvinylpyrrolidone (PVP). Rutin forms a coordination bond with cerium ions via a phenolic hydroxyl group, and PPVP stabilizes this coordination bond. The cerium ions are primarily in the form of Ce. 3+ Form exists; The preparation method of the rutin-cerium nanocomposite includes the following steps: (1) Using polyvinylpyrrolidone aqueous solution as the reaction system; wherein, the polyvinylpyrrolidone aqueous solution is prepared by dissolving polyvinylpyrrolidone in water, and the polyvinylpyrrolidone is a polyvinylpyrrolidone with an average molecular weight of 2~30 kDa; (2) First, add a substance that can provide Ce to the aqueous solution of polyvinylpyrrolidone. 4+ Water-soluble cerium salts were subjected to ultrasonic treatment to obtain a precursor solution containing cerium ions; Rutin was then added to the cerium-containing precursor solution, and the mixture was stirred at room temperature for 6–72 h. During the reaction, rutin converted Ce into cerium ions. 4+ Reduced to Ce³ + Polyvinylpyrrolidone (PVP) coordinates with cerium ions and self-assembles through phenolic hydroxyl groups, and stabilizes the coordinate bonds through hydrogen bonding and / or steric hindrance. The mass ratio of polyvinylpyrrolidone, water-soluble cerium salt, and rutin is (100-1000):(50-1000):(10-200). (3) After the stirring reaction is completed, the resulting reaction solution is centrifuged and the supernatant is collected; the supernatant is ultrafiltered and washed to obtain the rutin-cerium nanocomposite.

2. A rutin-cerium nanocomposite, characterized in that, Prepared by the preparation method according to claim 1; The rutin-cerium nanocomposite has a uniformly dispersed nanoparticle morphology. The rutin-cerium nanocomposite has a transmission electron microscope particle size of 3.88±1.16 nm and a dynamic light scattering hydrated particle size of 4.38±0.47 nm.

3. The use of the rutin-cerium nanocomposite according to claim 2 in the preparation of a drug for the prevention and treatment of acute kidney injury.

4. The application according to claim 3, characterized in that, The acute kidney injury refers to rhabdomyolysis-related acute kidney injury, ischemia-reperfusion-related acute kidney injury, drug-induced acute kidney injury, inflammation-related acute kidney injury, or oxidative stress-related acute kidney injury.

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