Preparation method and application of nanoparticles
By preparing nanoparticle HUCNPs with renal targeting function, the water solubility and targeting of urolithin A in AKI treatment were solved, and stronger ROS clearance, reduced inflammatory response and mitochondrial protection were achieved, which significantly improved the therapeutic effect of AKI.
Patent Information
- Application Number
- CN202510625713.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-15
- Publication Date
- 2025-08-12
AI Technical Summary
In the treatment of acute renal injury (AKI), the existing urolithin A is subject to poor water solubility and lack of targeting limitations, making it difficult to effectively target the lesion site, and lacks antioxidant ability.
The nanoparticle HUCNPs were prepared by dissolving hyaluronic acid, dicyclohexylcarbodiimide and 4-dimethylaminopyridine, and the nanoparticle HUCNPs were prepared by dialysis and centrifugation, which had renal targeting function.
HUCNPs can target the kidneys, significantly clear ROS, reduce cellular inflammatory response, protect mitochondrial function, show stronger anti-apoptotic effects, and have unexpected AKI treatment effects.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of AKI treatment, and in particular to a preparation method of nanoparticles and application thereof. Background Art
[0002] Acute kidney injury (AKI) is a clinical syndrome characterized by a rapid decline in renal function within a short period of time. It poses a serious threat to global public health with high morbidity, mortality and poor prognosis.
[0003] Urolithin A (UA) is a urolithin compound with an α-benzocoumarin skeleton. Its most significant biological effect is improving mitochondrial function. UA can alleviate oxidative stress by restoring mitochondrial function and inducing mitophagy, thereby achieving the goal of treating AKI. However, the clinical application of UA is limited by its physicochemical properties, such as poor water solubility and lack of specific targeting ability to lesions.
[0004] Therefore, the development of therapeutic drugs that can directly target the pathological mechanism of AKI, especially nanomedicines with targeted and efficient antioxidant effects, has important clinical significance and research value. Summary of the Invention
[0005] The purpose of the present invention is to provide a method for preparing nanoparticles with kidney-targeting function for treating AKI.
[0006] To achieve the above object, the present invention provides the following technical solutions:
[0007] The present invention provides a method for preparing nanoparticles, comprising the following steps:
[0008] S1, dissolving hyaluronic acid, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine in a dimethyl sulfoxide solution and stirring to form carboxyl groups of HA to prepare a reaction system;
[0009] S2, adding urolithin A to the reaction system, stirring, adding citric acid and cerium nitrate hexahydrate, pouring in ammonia solution, and stirring overnight;
[0010] S3, transferring the solution obtained by stirring overnight into a dialysis bag and dialyzing to remove water-soluble by-products; centrifuging the dialyzed solution to remove water-insoluble by-products; and then freeze-drying to obtain nanoparticles HUCNPs.
[0011] Furthermore, the dialysis bag in step S3 is a dialysis bag with MWCO: 3.5 kDa.
[0012] Furthermore, in step S2, after adding urolithin A to the reaction system, the stirring temperature is 60° C. and the stirring speed is 300 rpm.
[0013] Furthermore, the concentration of the ammonia solution in step S2 is 3M.
[0014] Furthermore, the centrifugal speed in step S3 is 10000 rpm.
[0015] Furthermore, the dimethyl sulfoxide solution in step S1 is a solution in which the volume ratio of dimethyl sulfoxide to water is 1:1.
[0016] Use of nanoparticles prepared by any of the above preparation methods in preparing drugs for treating AKI.
[0017] Compared with the existing technology, the HUCNPs nanoparticles disclosed in the present invention can target damaged kidneys, and compared with the existing urolithin A, the present invention exhibits stronger ROS scavenging ability, more effective reduction of the inflammatory response of HK-2 cells under stimulation, better anti-apoptosis effect, better mitochondrial protection effect, etc., and has unexpected effects in treating AKI.
[0018] Figures in the specification
[0019] Figure 1 Schematic diagram of the synthesis of HUCNPs.
[0020] Figure 2 Schematic diagram of the hemolysis results of HUCNPs at different concentrations.
[0021] Figure 3 (A) and (B) schematic diagrams of the dynamic distribution process of HUCNPs in mouse kidneys and their quantitative analysis results; (C) comparison of the distribution differences of HUCNPs in major organs in healthy mice and mice with acute kidney injury (AKI).
[0022] Figure 4 Schematic diagram of the ability of HUCNPs to target renal tubules, green: renal tubules; red: R6G-HUCNPs; blue: cell nucleus.
[0023] Figure 5 (A) Schematic diagram of the changes in ROS levels in each group; (B) quantitative comparison of ROS relative fluorescence intensity; (C) and (D) changes in SOD and MDA levels in each group.
[0024] Figure 6 Schematic diagram of PCR determination of inflammatory factor levels (TNF-α, IL-6).
[0025] Figure 7 (A) Schematic diagram of the establishment of the cisplatin-induced AKI mouse model; (B) H&E and PAS staining were used to evaluate the pathological damage of renal tissue in each group of mice; (C), (D) Changes in renal function indicators among different treatment groups: BUN, CRE.
[0026] Figure 8 Schematic diagram of the changes in oxidative stress indicators in different groups: SOD and MDA.
[0027] Figure 9 Schematic diagram of the results of flow cytometry evaluation of anti-apoptotic effects.
[0028] Figure 10 Schematic diagram of TUNEL staining to evaluate anti-apoptotic effects.
[0029] Figure 11 Schematic diagram of the results of evaluating mitochondrial membrane potential using the JC-1 fluorescent probe.
[0030] Figure 12 Schematic diagram of the changes in ATP levels in each group.
[0031] Figure 13 Schematic diagram of the effect of co-incubation with different concentrations of HUCNPs on cell viability.
[0032] Figure 14 Schematic diagram of H&E staining results of major organs of mice on days 1, 5, 15, and 30 after injection of therapeutic doses of HUCNPs.
[0033] Figure 15 Schematic diagram of the changes in serum liver and kidney function indicators: aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine (CRE). DETAILED DESCRIPTION
[0034] The technical solutions of the present invention are further described below in conjunction with specific examples. It should be understood that the following examples are merely exemplary illustrations and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope that the present invention is intended to protect.
[0035] Unless otherwise specified, the HUCNPs used in Examples 2-6 were all HUCNPs prepared in the steps of Example 1. The HCNPs used in Examples 5-6 were all HCNPs prepared in the steps of Example 4.
[0036] Example 1
[0037] Prepare HUCNPs by following the steps below:
[0038] 200 mg of hyaluronic acid (HA), 25 mg of dicyclohexylcarbodiimide (DCC), and 4-dimethylaminopyridine (4-DMAP) were dissolved in 15 mL of a mixture of dimethyl sulfoxide (DMSO) and H₂O (v / v, 1:1) and stirred for 2 h to form the carboxyl groups of HA. Then, 20 mg of urolithin A (UA) was added to the reaction system and stirred at 300 rpm at 60°C for 24 h. After the reaction, 240 mg of citric acid and 260 mg of cerium nitrate hexahydrate were added. The mixture was then quickly poured into 50 mL of 3 M ammonia solution and stirred overnight. The resulting solution was transferred to a dialysis bag (MWCO: 3.5 kDa) and dialyzed for 48 h with frequent exchange of deionized water to remove water-soluble byproducts. The dialyzed solution was centrifuged at 10,000 rpm to remove water-insoluble byproducts and finally lyophilized to obtain HUCNPs.
[0039] Schematic diagram of the synthesis process of HUCNPs Figure 1 As shown in the figure, urolithin A (UA) and hyaluronic acid (HA) are connected together by ester bonds under the action of DCC and 4-DAMP, and finally mixed with cerium nitrate hexahydrate and NH3·H2O to obtain HUCNPs nanoparticles.
[0040] Example 2
[0041] Blood compatibility evaluation
[0042] HUCNPs nanoparticles were used for hemolysis test, and the results were as follows: Figure 2 As shown in the results, no obvious hemolysis was observed even when the concentration of HUCNPs was as high as 160 μg / mL, indicating that HUCNPs have good biocompatibility.
[0043] Example 3
[0044] Kidney targeting evaluation
[0045] 1. Dynamic distribution and quantitative analysis of HUCNPs in mouse kidneys
[0046] Rhodamine 6G-labeled HUCNPs were prepared according to the following steps: 0.1 mg HUCNPs were dispersed in 1 mL PBS, an equal volume of 0.01 mg / mL Rhodamine 6G solution was added, and then the mixture was placed on a shaker and slowly shaken for 6-8 h. After the end, it was placed away from light for future use.
[0047] C57BL / 6 mice (male, 6-8 weeks old, 20-22 g) were injected intraperitoneally with cisplatin (20 mg / kg) 24 hours after which rhodamine 6G-labeled HUCNPs (100 μL, 20 μg / mL) were injected via the tail vein. Healthy mice served as the control group. Images were captured using a small animal imaging system (wavelength range: 570 nm-670 nm; excitation intensity: 10%; exposure time: 10 s) at 20, 40, 60, 80, 100, 120, 140, 160, and 180 minutes after administration. Fluorescence intensity analysis was performed using Aura software.
[0048] The results are as follows Figure 3 A and Figure 3 As shown in B, R6G fluorescence was mainly located in the kidney and was stronger in the Cis group, while negligible R6G fluorescence was observed in the kidney of the healthy group.
[0049] 2. Differences in the distribution of HUCNPs in major organs of mice
[0050] The mice were euthanized immediately after the imaging, and the abdominal and thoracic cavities were opened. The heart, liver, spleen, lungs, and kidneys were collected and arranged neatly in this order on black cardboard. Images were acquired using a small animal imaging system (wavelength range: 570 nm-670 nm; excitation intensity: 10%; exposure time: 5 s).
[0051] The results are as follows Figure 3 As shown in C, in the healthy group, the fluorescence was mainly distributed in the heart, liver, lung, and kidney, while in the Cis group, it was almost distributed in the kidney.
[0052] 3. Distribution of HUCNPs in the kidney
[0053] Kidneys were harvested from the AKI model and fixed with 4% paraformaldehyde at room temperature for 4-6 hours, followed by dehydration with 30% sucrose solution until the tissue sank to the bottom. The tissue was embedded in OCT embedding medium and cut into 5-10 μm thick sections using a freezing microtome. The sections were attached to slides and stored at -20°C until ready for use. The sections were removed, left at room temperature for 10 minutes, and then washed three times with PBS for 5 minutes each to remove the OCT embedding medium. The sections were immersed in preheated sodium citrate buffer (pH 6.0) and heated at 95°C for 10-20 minutes. After cooling to room temperature, they were washed three times with PBS for 5 minutes each. The sections were blocked with PBS containing 5% bovine serum albumin (BSA) and 0.3% TritonX-100 at room temperature for 1 hour to reduce nonspecific binding. The blocking solution was aspirated, and the diluted primary antibody LTL (diluted with 1% BSA in PBS) was added and incubated overnight at 4°C. Wash sections three times with PBS (5 minutes each). Add fluorescently labeled secondary antibody (diluted in 1% BSA in PBS) and incubate at room temperature in the dark for 1-2 hours. Wash sections three times with PBS (5 minutes each). Stain with DAPI for 5 minutes at room temperature to label cell nuclei. Wash sections three times with PBS (5 minutes each). Mount sections with anti-fluorescence quenching mounting medium and cover with a coverslip to avoid air bubbles. Scan sections with a slide scanner and acquire images. (DAPI excitation wavelength: 330-380 nm, emission wavelength: 420 nm; FITC excitation wavelength: 465-495 nm, emission wavelength: 515-555 nm).
[0054] The results are as follows Figure 4 As shown in the figure, there is a significant overlap between HUCNPs and the renal tubular area. This result indicates that HUCNPs have renal tubular targeting properties and can be specifically enriched in the renal tubular area, thereby helping to achieve efficient accumulation in the lesion area and improve therapeutic efficacy.
[0055] Example 4
[0056] Therapeutic evaluation of HUCNPs in AKI
[0057] HCNPs were prepared as follows: 20 mg of urolithin A (UA), 40 mg of anhydrous citric acid, and 260 mg of cerium nitrate hexahydrate were dissolved in 15 mL of a dimethyl sulfoxide (DMSO) / H₂O (v / v, 1:1) mixture. After complete dissolution, the mixture was quickly poured into 30 mL of 3 M ammonia solution and stirred overnight. The resulting solution was transferred to a dialysis bag (MWCO: 3.5 kDa) and dialyzed for 48 hours with frequent exchanges of deionized water to remove water-soluble byproducts. The dialyzed solution was centrifuged at 10,000 rpm to remove water-insoluble byproducts and then lyophilized to obtain HCNPs.
[0058] 1. Detection of Cellular ROS Levels
[0059] (1) Seed plate; HK-2 cells were plated at 2×10 5 Each well was inoculated in a 6-well plate;
[0060] (2) Cell treatment: When cells adhered and the density reached about 70%, the same volume of PBS or 500 μM H2O2 (to induce oxidative stress model) was added to each well and stimulated for 12 h;
[0061] (3) Cell grouping and drug administration: The cells were divided into 5 groups: Control group: HK-2 cells + PBS; H2O2 group: H2O2-HK-2 cells + PBS; H2O2+UA group: H2O2-HK-2 cells + UA; H2O2+HCNPs group: H2O2-HK-2 cells + HCNPs; H2O2+HUCNPs group: H2O2-HK-2 cells + HUCNPs. After drug administration, the cells were cultured in the incubator for 24 h.
[0062] (4) Loading the DCFH-DA fluorescent probe: After discarding the old culture medium and washing three times, 5 μM DCFH-DA fluorescent probe was added to each well and incubated for 30 min (protected from light). The cells were then washed three times with PBS, each time for 5 min. Finally, an inverted fluorescence microscope was used to observe and capture images using an excitation wavelength of 495 nm and an emission wavelength of 530 nm. Semi-quantitative analysis was performed using Image J.
[0063] Oxidative stress is the core pathological mechanism of AKI, and its key intervention strategy is to remove excessive ROS to alleviate kidney damage. Figure 5 As shown. Figure 5 A and Figure 5 As shown in B, compared with the control group, HK-2 cells stimulated by H2O2 produced a large amount of ROS. Compared with other groups, HUCNPs showed a stronger ability to scavenge ROS.
[0064] The effects of nanomaterials on oxidative stress were studied by evaluating the changes in SOD and MDA levels. Figure 5 C and Figure 5 As shown in Figure D, when HK-2 cells were treated with H2O2, the SOD level decreased and the MDA level increased. After the H2O2-treated cells were incubated with UA, HCNPs and HUCNPs for 24 hours, the SOD level increased and the MDA level decreased. The SOD level increased and the MDA level decreased in the HUCNPs group were much more significant than those in the other two groups, confirming the antioxidant capacity of HUCNPs.
[0065] 2. Inflammation level detection
[0066] 1) RNA extraction: Collect the cells treated with (1), (2), and (3) above, add 500 μL Trizol reagent to each well, and after the cell lysate is ice-bathed for 30 minutes, ensure sufficient lysis by repeated pipetting and transfer to a 1.5 mL EP tube. Add 200 μL chloroform, shake vigorously to mix, and let stand at room temperature for 5 minutes. Centrifuge at 4°C and 12,000 rpm for 15 minutes to collect the supernatant, add an equal volume of isopropanol to mix, and precipitate at -20°C overnight. The next day, centrifuge at 4°C and 12,000 rpm for 10 minutes to collect the RNA precipitate, wash three times with 75% ethanol (4°C, 7,500 rpm, 5 minutes / time), dry at room temperature, and redissolve in enzyme-free water. RNA concentration and purity were determined by spectrophotometry;
[0067] 2) Reverse transcription: reverse transcribe RNA into cDNA according to the reverse transcription kit operating procedures;
[0068] 3) qPCR reaction: qPCR detection was performed using the SYBR Green method. The reaction system (10 μL) included 2.4 μL enzyme-free water, 0.3 μL forward and reverse primers, and 5 μL SYBR Green Master Mix. These solutions were added to the qPCR plate in sequence and centrifuged to remove bubbles. The qPCR instrument program was set as follows: initial denaturation at 95°C for 5 minutes; amplification at 95°C for 10 seconds, followed by 60°C for 30 seconds, for 40 cycles; melting curve analysis: signal acquisition every 0.5°C from 60°C to 95°C.
[0069] 4) Data analysis: The relative expression of target genes was calculated using the 2^(-ΔΔCt) method.
[0070] The results are as follows Figure 6 As shown in the results, the levels of TNF-α and IL-6 in the H2O2 group increased significantly, while the inflammatory factors of HK-2 cells in the HUCNPs group decreased significantly and were close to normal levels. HUCNPs can effectively reduce the inflammatory response of HK-2 cells under H2O2 stimulation.
[0071] 3. Glycogen (PAS) staining, hematoxylin-eosin (H&E) staining, and detection of BUN and CRE in serum
[0072] C57BL / 6 mice (6-8 weeks old, 20-22 g) were divided into five groups: Saline group: normal mice were injected with PBS through the tail vein; Cis group: Cis-AKI mice were injected with PBS through the tail vein; UA group: Cis-AKI mice were injected with UA through the tail vein; HCNPs group: Cis-AKI mice were injected with HCNPs through the tail vein; HUCNPs group: Cis-AKI mice were injected with HUCNPs through the tail vein. Cisplatin was injected 24 h after administration and the mice were euthanized 48 h after administration. Blood samples and kidneys were collected for subsequent testing.
[0073] The BUN and CRE in serum were detected; the collected kidneys were subjected to glycogen (PAS) staining test and hematoxylin-eosin (H&E) staining test.
[0074] The results are as follows Figure 7 A and Figure 7 As shown in B, the saline group showed normal renal tissue structure, while the Cis group showed renal damage characterized by tubular swelling, blurred brush edges, and shedding of damaged cells. After treatment with UA and HCNPs, although there was some relief, significant damage remained. Compared with the other groups, the HUCNPs group showed significantly less tubular damage and effectively alleviated tubular swelling. In addition, Figure 7 C and Figure 7 As shown in Figure 5(D), after HUCNPs treatment, the levels of serum creatinine and urea nitrogen were significantly restored, approaching the levels of the saline group, further validating the results of PAS and H&E staining.
[0075] 4. SOD activity and MDA content in renal tissue
[0076] Renal cortex samples were taken from the kidneys of the above groups for MDA and SOD testing. The results are shown in Figure 8 As shown, SOD levels in the Cis group decreased significantly, indicating that SOD consumption increases to cope with the increasing oxidative stress levels during the progression of AKI. In contrast, MDA levels increased significantly, indicating that lipid peroxidation becomes increasingly severe during the AKI process. After treatment with HUCNPs, SOD levels increased significantly and MDA levels decreased significantly, indicating that the oxidative stress and lipid peroxidation associated with AKI were alleviated.
[0077] 5. Apoptosis
[0078] HK-2 cells were cultured at 2×10 5 Cells were seeded at 100 μg / well in a 6-well plate; when the cells adhered and the density reached about 70%, the same volume of PBS or 500 μM H2O2 (to induce oxidative stress model) was added to each well and stimulated for 12 hours; cell grouping and drug administration were divided into 5 groups: Control group: HK-2 cells + PBS; H2O2 group: H2O2-HK-2 cells + PBS; H2O2+UA group: H2O2-HK-2 cells + UA; H2O2+HCNPs group: H2O2-HK-2 cells + HCNPs; H2O2+HUCNPs group: H2O2-HK-2 cells + HUCNPs; after drug administration, the cells were cultured in an incubator for 24 hours and flow cytometry detection was performed.
[0079] The results are as follows Figure 9As shown, in the H2O2 group, the apoptosis rate was approximately 13%, and under the treatment of UA, HCNPs and HUCNPs, the apoptosis rate dropped to below 10%. Compared with the other groups, the HUCNPs group showed better anti-apoptotic effect, which reduced the cell apoptosis rate to below 5%.
[0080] 6. TUNEL staining
[0081] Obtained from the AKI model, the tissue was fixed with 4% paraformaldehyde at room temperature for 4-6 hours, then dehydrated with 30% sucrose solution until the tissue sank to the bottom. The tissue was embedded in OCT embedding medium and cut into 5-10 μm thick sections using a freezing microtome. The sections were attached to slides and stored at -20°C until ready for use. The sections were removed, left at room temperature for 10 minutes, and then washed three times with PBS for 5 minutes each to remove the OCT embedding medium. The sections were immersed in preheated sodium citrate buffer (pH 6.0) at 95°C for 10-20 minutes, cooled naturally to room temperature, and washed three times with PBS for 5 minutes each. TUNEL staining was then performed.
[0082] The results are as follows Figure 10 As shown, extensive nuclear changes occurred in the kidneys of the AKI group, consistent with apoptotic cell death, while fewer positive signals were observed in the control group. UA, HCNPs, and HUCNPs were all able to reduce cell apoptosis, with HUCNPs showing a better anti-apoptotic effect.
[0083] Example 5
[0084] Evaluation of HUCNPs in regulating mitochondrial function
[0085] HK-2 cells were cultured at 2×10 5 Cells were seeded at 100 μg / well in a 6-well plate; when the cells adhered and the density reached about 70%, the same volume of PBS or 500 μM H2O2 (to induce oxidative stress model) was added to each well and stimulated for 12 h; cell grouping and drug administration were divided into 5 groups: Control group: HK-2 cells + PBS; H2O2 group: H2O2-HK-2 cells + PBS; H2O2+UA group: H2O2-HK-2 cells + UA; H2O2+HCNPs group: H2O2-HK-2 cells + HCNPs; H2O2+HUCNPs group: H2O2-HK-2 cells + HUCNPs; after drug administration, the cells were cultured in an incubator for 24 h.
[0086] 1. Use JC-1 to perform staining experiments. The results are as follows Figure 11As shown in the figure, after the cells were incubated with H2O2 for 24 h, the red / green fluorescence intensity ratio decreased significantly, indicating that the oxidative stress induced by H2O2 greatly reduced the MMP of HK-2 cells. The red / green fluorescence intensity ratio was significantly enhanced after treatment with UA, HCNPs and HUCNPs. Compared with UA and HCNPs, the HUCNPs group could cause a greater enhancement in the red / green fluorescence intensity ratio, showing a better mitochondrial protection effect.
[0087] 2. Aspirate the culture medium and add 200 μL of lysis buffer to each well. Lyse the cells mechanically (repeatedly pipetting / shaking) to ensure complete lysis. Centrifuge at 12,000 × g for 5 minutes at 4°C, then collect the supernatant. Determine the RLU value according to the Enhanced ATP Assay Kit protocol and calculate the ATP concentration according to the standard formula.
[0088] The results are as follows Figure 12 As shown in the results, compared with the control group, the ATP level in HK-2 cells was significantly decreased after H2O2 stimulation. After treatment with UA, HCNPs and HUCNPs, the ATP levels in each group increased, among which HUCNPs was the most significant, further proving that HUCNPs has a significant mitochondrial protective effect.
[0089] Example 6
[0090] Safety evaluation
[0091] 1. In vitro cytotoxicity assay
[0092] (1) Cell inoculation: HK-2 cells were seeded at an appropriate density (e.g. 5×10 3 / well) were seeded in 96-well plates and cultured for 24 h to adhere to the wall.
[0093] (2) Drug treatment: Add different concentrations of HUCNPs (0-160 μg / mL) and 200 μL of culture medium to each well, and continue incubation for 24 h.
[0094] (3) Reagent addition: Add 20 μL of CCK-8 reagent to each well and incubate in the dark for 2 h.
[0095] (4) Absorbance detection: The absorbance (OD value) of each well was measured using a microplate reader (450 nm wavelength), and the cell viability (%) was calculated as (OD of experimental group / OD of control group) × 100%.
[0096] Results Figure 13 As shown in the results, HUCNPs had no significant effect on cell viability in the concentration range of 0-160 μg / mL, indicating that their cytotoxicity was low.
[0097] 2. In vivo toxicity experiments
[0098] C57BL / 6 male mice (6-8 weeks old, 20-22 g) were injected with therapeutic doses of HUCNPs via the tail vein on days 1, 5, 15, and 30. The mice were divided into four groups based on the cumulative number of doses: a single-dose group (day 1), a two-dose group (days 1 and 5), a three-dose group (days 1, 5, and 15), and a four-dose group (days 1, 5, 15, and 30). At each time point, the mice were anesthetized and blood was collected from the eyeball. Blood samples were allowed to rest at room temperature for 2 hours and then centrifuged at 4000 rpm for 15 minutes to obtain serum. ALT, AST, CRE, and BUN were measured using an automated biochemical analyzer to assess liver and kidney function. At the end of the experiment, the mice were euthanized, and major organs (heart, liver, spleen, lung, kidney, and brain) were collected. The samples were fixed with 4% paraformaldehyde, embedded in paraffin, and stained with H&E. Histopathological analysis was performed to evaluate the in vivo toxicity of HUCNPs.
[0099] Results Figure 14 As shown in the table, no obvious tissue damage was found in the main organs of mice. Figure 15 As shown in the figure, the values were kept within the normal range, and HUCNPs had no effect on the liver and kidney functions of mice.
[0100] Although the specific embodiments of the present invention are described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and are not intended to limit the scope of the present invention. Any modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included within the scope of protection of the claims of the present invention.
Claims
1. A method for preparing nanoparticles, characterized in that: The following steps are involved: S1, dissolving hyaluronic acid, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine in a dimethyl sulfoxide solution and stirring to form carboxyl groups of HA to prepare a reaction system; S2, adding urolithin A to the reaction system, stirring, adding citric acid and cerium nitrate hexahydrate, pouring in ammonia solution, and stirring overnight; S3, transferring the solution obtained by stirring overnight into a dialysis bag and dialyzing to remove water-soluble byproducts; The dialyzed solution was centrifuged to remove water-insoluble byproducts; Then the nanoparticles HUCNPs were obtained by freeze-drying.
2. The method for preparing nanoparticles according to claim 1, wherein: The dialysis bag in step S3 is a dialysis bag with MWCO: 3.5 kDa.
3. The method for preparing nanoparticles according to claim 1, wherein: In step S2, after adding urolithin A to the reaction system, the stirring temperature is 60° C. and the stirring speed is 300 rpm.
4. The method for preparing nanoparticles according to claim 1, wherein: The concentration of the ammonia solution in step S2 is 3M.
5. The method for preparing nanoparticles according to claim 1, wherein: The centrifugal speed in step S3 is 10000 rpm.
6. The method for preparing nanoparticles according to claim 1, wherein: The dimethyl sulfoxide solution in step S1 is a solution in which the volume ratio of dimethyl sulfoxide to water is 1:
1.
7. Use of the nanoparticles prepared by the preparation method according to any one of claims 1 to 6 in preparing a drug for treating AKI.