A KIM-1 actively targeted gold nanoparticle, its preparation method and application

Abstract

This invention discloses a KIM-1 actively targeted gold nanoparticle, its preparation method, and its applications, belonging to the field of medical materials technology. The gold nanoparticles are modified with serine as a targeting ligand, exhibiting high affinity and specificity for the kidney-damaging molecule KIM-1. Preparation involves first synthesizing GS-AuNPs, then dissolving them in PBS, adjusting the pH, adding EDC and NHS, vortexing, adding serine, adjusting the pH again, vortexing, and finally ultrafiltration and centrifugation to obtain the product. These particles can be used as a CT contrast agent to detect intrarenal KIM-1. They can also be combined with phosphatidylcholine-modified silver nanoparticles to quantify intrarenal KIM-1 via urine analysis, achieving early non-invasive diagnosis of acute kidney injury, with low toxicity and good biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, and in particular to a KIM-1 actively targeted gold nanoparticle, its preparation method, and its application. Background Technology

[0002] Acute kidney injury (AKI) is a common clinical syndrome, and early diagnosis is crucial for improving patient prognosis. Currently, clinical detection of AKI mainly relies on biomarkers of kidney injury. However, some common renal function indicators have certain limitations. For example, neutrophil gelatinase-associated lipocalin (NGAL) lacks specificity, is expressed in multiple organs besides the kidneys (lungs, small intestine, etc.), and shows non-specific elevations in systemic inflammation (such as sepsis), easily leading to false positives. Additionally, NAG (N-acetyl-β-D-glucosidase), while reflecting renal tubular damage, is easily affected by urine pH and the inhibitory activity of metal ions, exhibiting poor stability. Even the most relied-upon renal function indicator, serum creatinine (sCr), suffers from low sensitivity and poor specificity, typically only significantly increasing 48-72 hours after kidney injury, and is influenced by various factors such as age, sex, and muscle mass, failing to provide early warning.

[0003] Kidney injury molecule-1 (KIM-1) exhibits high tubule specificity. While it is almost entirely absent in healthy kidneys, it is significantly upregulated within 12 hours of proximal tubular epithelial cell damage (e.g., ischemia, nephrotoxic injury) and remains highly expressed throughout the entire injury-to-repair process. Compared to NGAL and other markers, KIM-1 possesses tubule specificity, ultra-early warning capabilities, high stability, and strong prognostic correlation, making it the optimal biomarker for kidney injury currently available. It can effectively avoid false-positive interference from systemic inflammation and other factors.

[0004] High levels of KIM-1 expressed in damaged kidneys are shed and cleared into the urine over time. Therefore, while urine KIM-1 levels are currently used clinically to reflect kidney function, issues such as time lag remain. Thus, accurate and non-invasive detection of intrarenal KIM-1 expression levels is crucial for early detection of kidney disease. Currently, clinical detection of KIM-1 relies on enzyme-linked immunosorbent assay (ELISA) to detect urine samples. However, this method has several limitations: firstly, it is time-consuming (>6 hours); secondly, antibodies are expensive and easily affected by biological matrices; thirdly, it only provides in vitro, static concentration information, unable to locate the intrarenal distribution of KIM-1 or assess its intrarenal content and damage progression in real time; and fourthly, cross-reactions caused by non-specific binding of antibodies to similar biomolecule fragments often affect the test results.

[0005] Computed tomography (CT) is one of the most commonly used imaging techniques in clinical practice. Renal CT contrast agents hold promise for non-invasive assessment of intrarenal KIM-1 levels. Currently used clinical CT contrast agents are mainly iodine-containing small-molecule compounds (such as iohexol and diatrizoate). These contrast agents enhance imaging by increasing tissue density contrast, but they have three major drawbacks: First, they lack specificity and cannot reflect intrarenal molecular levels, such as KIM-1. Second, they have nephrotoxicity issues; over 90% of iodine-containing contrast agents are excreted through the kidneys, and high concentrations can induce renal vasoconstriction and damage to renal tubular epithelial cells, leading to contrast-induced nephropathy (CIN). This risk of acute kidney injury is significantly increased, especially in patients with renal insufficiency. Third, iodine-containing compounds have a short circulation time in the body and are rapidly cleared (usually within 24 hours), resulting in a short imaging window and making continuous dynamic monitoring difficult.

[0006] Therefore, developing a method that can actively target KIM-1 to achieve early, non-invasive, in vivo diagnosis of AKI is of great significance and value. Summary of the Invention

[0007] The purpose of this invention is to provide KIM-1 actively targeted gold nanoparticles, their preparation method, and applications, thereby solving the problems in the background art.

[0008] To achieve the above objectives, this invention provides a method for preparing KIM-1 actively targeted gold nanoparticles, specifically including the following steps:

[0009] Step S1: Synthesis of GS-AuNPs

[0010] (1) Dissolve glutathione (GSH) in deionized water and then add tetrachloroauric acid. Heat to 95°C and stir to obtain a reaction solution.

[0011] (2) Cool to room temperature, add saturated sodium chloride to the above reaction solution, stir thoroughly, add anhydrous ethanol, shake well, centrifuge, and collect the precipitate to obtain GS-AuNPs;

[0012] Step S2: Synthesis of S-Au-Agent gold nanoparticles with KIM-1 active targeting

[0013] (1) Dissolve GS-AuNPs in PBS buffer solution, add hydrochloric acid solution to adjust pH to 4.5-5.5, then add activator EDC and NHS, and vortex using a vortex apparatus;

[0014] (2) After vortexing, add serine to the reaction system, add sodium hydroxide aqueous solution to adjust the pH value to 7.8, and then vortex using a vortex apparatus;

[0015] (3) Collect the reaction liquid after vortexing and purify it by ultrafiltration and centrifugation. Collect the upper liquid to obtain the target product S-Au-Agent.

[0016] Preferably, in step S1 (1), the mass-to-volume ratio of glutathione, tetrachloroauric acid and deionized water in the mixed system is 36.87 mg: 59.07 mg: 50 mL.

[0017] Preferably, in step S1 (2), the volume ratio of saturated sodium chloride to anhydrous ethanol is 5 mL:55 mL.

[0018] Preferably, in step S2 (1), the mass-to-volume ratio of GS-AuNPs to PBS is 10 mg: 5 mL, wherein the pH of the PBS buffer solution is 7.4; the mass ratio of EDC to NHS is 50 mg: 60 mg; and the molar concentration of the hydrochloric acid solution is 1 M.

[0019] Preferably, in step S2 (2), the mass of serine is 100 mg; the molar concentration of the sodium hydroxide aqueous solution is 10 M.

[0020] The present invention also provides a KIM-1 actively targeted gold nanoparticle prepared by the above preparation method.

[0021] The present invention also provides the above-mentioned gold nanoparticles as a CT contrast agent for detecting intrarenal KIM-1 by CT imaging.

[0022] This invention also provides a method for quantitative analysis of renal KIM-1 content using urine analysis, combining the above-mentioned gold nanoparticles and phosphatidylcholine-modified silver nanoparticles.

[0023] Preferably, the preparation of phosphatidylcholine-modified silver nanoparticles includes the following steps:

[0024] Step S1: Synthesis of GS-AgNPs

[0025] Add a mixed solution of AgNO3 and GSH to deionized water, stir until turbid, then add NaOH solution to adjust the pH to 4.9-5.1, stir constantly until clear, then add NaBH4, stir the reaction at room temperature, collect the reaction solution and filter to obtain GS-AgNPs;

[0026] Step S2: Synthesis of phosphatidylcholine ligand NH2-MPC

[0027] 2-Methacryloxyethylphosphocholine was dissolved in methanol and deoxygenated by passing nitrogen gas. Then it was mixed with a methanol solution of cysteine ​​hydrochloride and triethylamine was added to catalyze the reaction. After the reaction was completed, excess solvent was removed by rotary evaporation. The product was then dissolved and purified with a mixed solution of dichloromethane and diethyl ether and finally lyophilized to obtain NH2-MPC.

[0028] Step S3: Synthesis of phosphatidylcholine-modified silver nanoparticles PC-AgNPs

[0029] GS-AgNPs were dispersed in 1×PBS buffer solution and reacted with activators EDC and NHS dissolved in 1×PBS buffer solution to activate the carboxyl groups. Then, NH2-MPC dissolved in 1×PBS buffer solution was added and mixed. The pH was adjusted to 7.8 with sodium hydroxide aqueous solution and the reaction was stirred at room temperature to couple the phosphatidylcholine ligands to the nanoparticles. After the reaction was completed, the nanoparticles were purified by ultrafiltration and centrifugation and then lyophilized to obtain PC-AgNPs.

[0030] Preferably, in step S1, the mass ratio of AgNO3, GSH and NaBH4 is 34 mg: 61.4 mg: 38 mg;

[0031] In step S2, the mass-to-volume ratio of 2-methacryloyloxyethyl phosphocholine to methanol is 100 mg: 10 mL; the mass-to-volume ratio of cysteine ​​hydrochloride to methanol is 28.7 mg: 1 mL.

[0032] In step S3, the mass-to-volume ratio of GS-AgNPs to 1×PBS buffer solution is 2 mg:1 mL, wherein the pH of the 1×PBS buffer solution is 4.5;

[0033] The mass-to-volume ratios of EDC, NHS, and 1×PBS buffer solution were 7 mg:1 mL and 4 mg:1 mL, respectively, with the pH of the 1×PBS buffer solution being 4.5.

[0034] The mass-to-volume ratio of NH2-MPC to 1×PBS buffer solution was 3 mg: 1 mL, wherein the pH of the 1×PBS buffer solution was 7.8.

[0035] Therefore, the KIM-1 actively targeted gold nanoparticle, its preparation method, and its application provided by this invention have the following beneficial effects:

[0036] (1) High imaging performance and targeting of S-Au-Agent

[0037] Gold has an atomic number of 79, while iodine has an atomic number of 53. The X-ray mass absorption coefficient of gold (5.16 cm² / g) is approximately 2.7 times that of iodine (1.94 cm² / g). At the same dose, S-Au-Agent provides clearer CT image contrast, particularly suitable for imaging obese mice or deep kidney tissues. Furthermore, the ultra-small size of S-Au-Agent (core diameter 2.19±0.86 nm, hydration kinetic diameter 3.97±1.31 nm) has a longer elimination half-life in vivo compared to iodine contrast agents, extending the imaging window for renal CT imaging to over 30 minutes, facilitating continuous dynamic monitoring. Additionally, the use of serine as a ligand endows S-Au-Agent with active targeting capabilities for KIM-1, enabling its specific accumulation in areas of kidney injury (especially the proximal tubules of the cortex), enhancing the microscopic contrast of the injured site, thus facilitating early diagnosis of kidney injury and observing the distribution of KIM-1 in the kidney using non-invasive imaging methods.

[0038] (2) Non-invasiveness and accuracy of urine element ratio analysis

[0039] This invention designs and synthesizes another inorganic silver nanoparticle, PC-AgNPs, with an internal control function. Its inorganic interaction properties ensure that its metabolism in the kidney is unaffected by KIM-1 levels, effectively offsetting the interference of individual differences in the body (such as fluctuations in glomerular filtration rate and metabolic rate differences) on the detection results. Through the active targeting ability of S-Au-Agent to renal KIM-1 and the stable internal control function of PC-AgNPs, the content and ratio of silver and gold in urine can be calculated using elemental mass spectrometry. The ratio of silver to gold in urine increases with the increase of renal KIM-1 content, thereby quantitatively assessing the renal KIM-1 content through urine analysis. This achieves non-invasive quantitative assessment of renal KIM-1 levels, detecting acute kidney injury at least 42 hours earlier than traditional urine KIM-1 detection (which shows significant differences only after 48 hours of renal ischemia-reperfusion), avoiding the time lag and heterogeneity of urine detection. This method is simple to operate, requiring no complex tissue sampling; detection can be completed using only urine samples.

[0040] (3) Low toxicity and clinical applicability

[0041] Both S-Au-Agent and PC-AgNPs are ultra-small nanoparticles with surfaces modified by serine and phosphatidylcholine, respectively. They have good biocompatibility and can be eliminated through normal renal metabolism, avoiding accumulation and toxicity in the body. Compared with iodine contrast agents, S-Au-Agent can achieve good imaging results without high-concentration injections, significantly reducing the risk of contrast agent nephropathy (CIN), and is especially suitable for high-risk groups with renal insufficiency.

[0042] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0043] Figure 1 This is a synthetic route diagram of ultrasmall gold nanoparticles using serine as a ligand in an embodiment of the present invention;

[0044] Figure 2 This is a diagram showing the core diameter of the S-Au-Agent obtained in an embodiment of the present invention.

[0045] Figure 3 The hydrodynamic diameter diagram of the S-Au-Agent obtained in an embodiment of the present invention;

[0046] Figure 4 The excitation / emission spectrum and ultraviolet-visible absorption spectrum of S-Au-Agent obtained in an embodiment of the present invention are shown below.

[0047] Figure 5 This is an agarose gel electrophoresis image of the S-Au-Agent prepared according to an embodiment of the present invention;

[0048] Figure 6 The images show in vitro CT signal intensity maps of S-Au-Agent and iodine contrast agents prepared according to embodiments of the present invention. In these images, A represents CT images of S-Au-Agent and iopromide solutions at different concentrations; B represents the linear relationship between the concentrations of the two contrast agents and CT enhancement, with the slope indicating the efficiency of contrast agent enhancement as the concentration increases.

[0049] Figure 7 The image shows the kidney imaging effect of S-Au-Agent prepared in the embodiment of the present invention in mice in the sham-operated group;

[0050] Figure 8 This is an image showing the renal imaging effect of S-Au-Agent prepared in an embodiment of the present invention in mice undergoing ischemia-reperfusion.

[0051] Figure 9 This is a flowchart illustrating the quantitative assessment of intrarenal KIM-1 and detection of acute kidney injury using urine analysis of S-Au-Agent and PC-AgNPs prepared in this embodiment of the invention.

[0052] Figure 10 Figure showing the KIM-1 content in the kidneys and urine of mice after different times of ischemia-reperfusion;

[0053] Figure 11 Immunohistochemical images of the kidneys of mice in the sham-operated group and 6 h after ischemia-reperfusion injury are shown. In the image, A is the immunohistochemical image of mice in the sham-operated group and the group with renal ischemia-reperfusion injury 6 h later; B is the quantitative analysis of the KIM-1 positive area in the image A.

[0054] Figure 12 The table shows the concentrations of gold and silver in the kidneys and their ratio, where A represents the concentration of silver in the kidneys; B represents the concentration of gold in the kidneys; and C represents the ratio of the concentrations of silver and gold in the kidneys.

[0055] Figure 13 The table shows the content of gold and silver elements in urine and their ratio, where A represents the content of silver in urine; B represents the content of gold in urine; and C represents the ratio of the content of silver and gold in urine.

[0056] Figure 14 The graph shows the linear correlation between the ratio of silver to gold in the kidney and urine and the content of KIM-1 in the kidney. In the graph, A is the linear correlation between the content of KIM-1 in the kidney and the ratio of silver to gold in the kidney; B is the linear correlation between the content of KIM-1 in the kidney and the ratio of silver to gold in urine. Detailed Implementation

[0057] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention. Furthermore, it should be understood that after reading the contents of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims and are all within the protection scope of the present invention.

[0058] In this document, the term "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The term "embodiment" appearing in various places throughout the specification does not necessarily refer to the same embodiment, nor does it specifically limit its independence or connection with other embodiments. In principle, in this application, as long as there are no technical contradictions or conflicts, the technical features mentioned in each embodiment can be combined in any way to form corresponding implementable technical solutions.

[0059] Unless otherwise defined, the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the use of related terms herein is merely for the purpose of describing particular embodiments and is not intended to limit this application.

[0060] Unless otherwise specified, the reagents, instruments, equipment, and performance testing methods used in this invention are all commonly used by those skilled in the art.

[0061] Example

[0062] This embodiment provides a method for preparing KIM-1 actively targeted gold nanoparticles, specifically including the following steps:

[0063] (1) Dissolve 36.87 mg of glutathione in 50 mL of water in a three-necked flask, and then add 59.07 mg of tetrachloroauric acid to the reaction system. Heat the mixture at 95°C and stir at 420 rpm for 40 min.

[0064] (2) After the reaction has cooled to room temperature, add 5 mL of saturated sodium chloride to the reaction solution, stir thoroughly, add 55 mL of anhydrous ethanol, shake well, place in a 50 mL centrifuge tube, centrifuge at 4200 rpm for 10 min and collect the precipitate to obtain GS-AuNPs.

[0065] (3) Dissolve 10 mg of GS-AuNPs in 5 mL of PBS (pH=7.4) in a vial, and adjust the pH to 4.5-5.5 with 1M hydrochloric acid. Then add 50 mg of EDC and 60 mg of NHS to the system and vortex at 600 rpm for 2 hours.

[0066] (4) Add 100 mg of serine to the vortexed reaction system, add 40 μL of sodium hydroxide aqueous solution (10 M) to adjust the pH to 7.8, and then vortex at 600 rpm for 3 h.

[0067] (5) Collect the reaction solution and filter it using a 3000 Da ultracentrifuge tube to remove unreacted EDC, NHS and serine. After filtering 5 times, collect the upper liquid to obtain S-Au-Agent.

[0068] Steps (3)-(5) involve the synthesis of ultrasmall gold nanoparticles with serine as a ligand, and the schematic diagram of the synthesis route is shown below. Figure 1 As shown.

[0069] The basic physicochemical properties of the target product S-Au-Agent obtained in the examples were characterized.

[0070] The core size and morphology of the nanoparticles were characterized using transmission electron microscopy (TEM), and the results are as follows: Figure 2 As shown. By Figure 2 It can be seen that the core diameter of S-Au-Agent is 2.19±0.86 nm, which is spherical and conforms to the size range of ultra-small gold nanoparticles.

[0071] The hydration dynamics diameter of nanoparticles was characterized using dynamic light scattering (DLS), and the results are as follows: Figure 3 As shown. By Figure 3 It can be seen that the hydrodynamic diameter (hydration particle size) of S-Au-Agent is 3.97±1.31 nm, which is consistent with the size range of ultrasmall gold nanoparticles.

[0072] The characteristic absorption of the nanoparticles was characterized using a UV-Vis spectrophotometer, and the fluorescence properties of the nanoparticles were characterized using a fluorescence spectrometer (FL). The results are as follows: Figure 4 As shown. By Figure 4 It is known that S-Au-Agent exhibits fluorescence properties, with an optimal excitation wavelength of 452 nm and a maximum emission wavelength of 812 nm. Furthermore, the nanoparticles do not exhibit significant UV-Vis absorption in the 400-500 nm range, indicating the absence of surface plasmon resonance and classifying them as ultra-small nanoparticles.

[0073] Comparative analysis of S-Au-Agent and GS-AuNPs was performed by agarose gel electrophoresis, and the results are as follows: Figure 5 As shown. By Figure 5 As can be seen from the agarose gel electrophoresis experiment, the synthesis of S-Au-Agent was successfully demonstrated. Its slower migration speed in electrophoresis compared to GS-AuNPs proved that serine was successfully attached to the surface of gold nanoparticles.

[0074] Application Example 1: The serine-modified ultrasmall gold nanoparticles S-Au-Agent prepared in the examples were used as a CT contrast agent for CT imaging to detect intrarenal KIM-1.

[0075] 1. Performance evaluation of S-Au-Agent in in vitro CT imaging

[0076] The CT imaging capability of S-Au-Agent was characterized in vitro using small animal Micro-CT. Specifically, S-Au-Agent was dissolved in PBS to prepare a 100 mg / mL solution. 100 μL of this solution was added to individual 96-well plates. Using iodine and PBS as controls (equal molar amounts of gold), the signal values ​​of the iodine contrast agent (iopromide) and gold nanoparticles were compared under the same mass of imaging elements (gold and iodine) to clarify the CT-enhanced imaging capability of the gold nanoparticles. CT scans were performed using a small animal Micro-CT scanner with a scan parameter of 70 kV and a scan time of 2 min. A pre-scan was performed before nanoparticle injection to obtain images of the unenhanced kidney region. After nanoparticle injection, 15 consecutive scans were performed over a total of 30 minutes to obtain images of the kidney region enhanced by the gold nanoparticles. The results are as follows: Figure 6 As shown. By Figure 6It can be seen that, under the condition of equal mass of imaging elements (equal molar amounts of gold and iodine), S-Au-Agent has a signal intensity that is 3.22 times higher than that of iodine contrast agent.

[0077] 2. Evaluation of S-Au-Agent's in vivo CT imaging performance

[0078] A mouse model of ischemia-reperfusion (IRI) kidney injury induced by elevated KIM-1 levels was established. Incisions were made at the bilateral renal sites on the dorsal side of the mice, and the renal arteries were clamped for 30 min. The clamps were then released to restore blood flow and reperfusion was induced. Renal CT imaging was performed 6 h later. A sham-operated group was established for comparison; in this group, only the corresponding skin area on the dorsal side of the mice was sutured. S-Au-Agent was administered intravenously at a dose of 1.8 mg / mouse to both the sham-operated group and the IRI-injured mice. Continuous scanning was performed using a small animal Micro-CT scanner at 2 min intervals, for a total of 15 scans. Differences in renal imaging between normal and diseased conditions were compared, and the signal intensity in the renal region was analyzed using software. The results are as follows: Figure 7 and Figure 8 As shown. By Figure 7 It was found that when S-Au-Agent (Au elemental mass) was injected into sham-operated mice, S-Au-Agent was rapidly transported within the mice and clearly illuminated the renal pelvis. This is because KIM-1 is almost not expressed in the kidneys of sham-operated mice, therefore S-Au-Agent is rapidly cleared from the kidneys and accumulates significantly in the renal pelvis. Figure 8 It was observed that, compared to the sham-operated group, the IRI group mice showed significant cortical accumulation of S-Au-Agent in the renal cortex in the early post-injection period (within the first 10 minutes). Subsequently, at 20 and 30 minutes post-injection, S-Au-Agent gradually migrated from the cortex to the entire kidney, and was finally cleared after 4 hours. This early enrichment in the cortical region was highly consistent with the spatial distribution of high KIM-1 expression in the proximal tubules of the kidney (located in the cortex), demonstrating the excellent KIM-1 targeting ability of S-Au-Agent. Based on this, it can be concluded that the upregulation of KIM-1 can be detected using non-invasive imaging techniques with S-Au-Agent, and early diagnosis of kidney injury can be achieved, enabling in vivo, non-invasive, and dynamic assessment of KIM-1 expression levels.

[0079] 3. Comparison and evaluation of S-Au-Agent non-invasive CT imaging modality and renal KIM-1 content.

[0080] (1) After CT imaging, mice were euthanized, and their kidneys were collected. The KIM-1 content in the kidneys was measured using enzyme-linked immunosorbent assay (ELISA), and the spatial distribution of KIM-1 within the kidneys was quantified and stained using immunohistochemistry. The correlation between the spatial distribution of S-Au-Agent in the kidneys during CT imaging and the distribution of KIM-1 in immunohistochemistry was compared. This enables early diagnosis and microstructural localization of kidney diseases.

[0081] (2) At the same time, mouse serum was collected and kidney function indicators such as serum creatinine and blood urea nitrogen were measured to compare with CT imaging to determine whether kidney damage could be detected earlier through non-invasive imaging.

[0082] (3) After the kidneys were removed, they were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned for hematoxylin and eosin (H&E) staining to analyze the degree of renal tubular damage. Ten high-power fields were randomly selected from each kidney section and scored according to the following criteria: obvious tubular dilation and cell flattening were scored as 1 point; brush border damage was scored as 1 point and detachment as 2 points; casts were scored as 2 points, and detached or necrotic cells (not forming casts or cell fragments) in the tubular lumen were scored as 1 point. The highest score for each field of view was 4 points, and the lowest score was 0 points. CT detection was also used to compare with pathological analysis. CT imaging technology was used to reflect the pathological changes caused by kidney damage.

[0083] Application Example 2: The S-Au-Agent prepared in the previous example, combined with phosphatidylcholine-modified silver nanoparticles, was used to quantitatively analyze the intrarenal KIM-1 content and detect early renal damage using urine analysis.

[0084] The phosphatidylcholine-modified silver nanoparticles possess metabolic internal control function, and their preparation method includes the following steps:

[0085] (1) Synthesis of GS-AgNPs: 100 mL of deionized water was added to a three-necked flask, and 34 mg of AgNO3 and 61.4 mg of GSH solution were added at 900 rpm. The solution became turbid. Then, 60 μL of 5M NaOH solution was added to adjust the pH of the solution to 4.9-5.1, and the solution was stirred constantly until it became clear. At this time, 38 mg of NaBH4 was quickly added to the three-necked flask, and the reaction was carried out at 900 rpm for 12 h with vigorous stirring at room temperature. The reaction solution was collected and purified by dialyzing with a 1500 Da dialysis bag overnight to synthesize GS-AgNPs.

[0086] (2) Synthesis of phosphatidylcholine ligand NH2-MPC: 100 mg of 2-methacryloyloxyethyl phosphocholine was dissolved in 10 mL of methanol and purged with nitrogen for 15 min; 28.7 mg of cysteine ​​hydrochloride was dissolved in 1 mL of methanol solution. After complete dissolution, the two solutions were mixed, and 10 μL of triethylamine was added. The mixture was stirred at 400 rpm for 4 hours at room temperature. After the reaction was completed, excess solvent was removed by rotary evaporation. The product was then redissolved in a mixture of 10 mL of dichloromethane and diethyl ether. After standing for 30 min, the supernatant was removed, and the product was collected. Finally, excess solvent was removed by rotary evaporation, and 1 mL of distilled water was added to fully dissolve the product. After lyophilization, phosphatidylcholine ligand NH2-MPC was obtained.

[0087] (3) Synthesis of phosphatidylcholine-modified silver nanoparticles PC-AgNPs: 2 mg of GS-AgNPs were fully dissolved in 1×PBS buffer (pH=4.5) for later use. 7 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4 mg of N-hydroxysuccinimide were each dissolved in 1 mL of 1×PBS buffer (pH=4.5) for later use. The above systems were mixed and stirred at room temperature for 2 h. Then, 3 mg of NH2-MPC was fully dissolved in 1 mL of 1×PBS buffer (pH=7.8) for later use. Subsequently, the above four solutions were mixed, and 40 μL of sodium hydroxide aqueous solution (10 M) was added to adjust the pH to 7.8. The reaction was then stirred at room temperature for 6 h. After the reaction was completed, the reaction system was transferred to a 3 kDa ultracentrifuge tube for ultrafiltration purification, and PC-AgNPs were obtained after lyophilization.

[0088] The flowchart for urine testing is as follows: Figure 9 As shown in the figure. Mouse models and sham-operated groups were constructed at different time points (0, 30 min, 2, 6, 12, 24, 48 h) after IRI, using the method described above. Each group of mice was simultaneously injected via tail vein with S-Au-Agent and PC-AgNPs (20 μg each). Thirty minutes after injection, urine was collected from the mice, and then the mice were sacrificed, and the kidneys were removed. A portion of the kidney tissue was used to quantify KIM-1 content by ELISA and immunohistochemistry. The results are shown in the figure. Figure 10 and Figure 11 As shown in the figure. Another portion of kidney tissue and all urine samples were nitrated with nitric acid, and the content of gold (Au) and silver (Ag) was determined by mass spectrometry. The results are shown in the figure. Figure 12As shown. The principle of using the elemental ratio method to reflect renal KIM-1 levels is as follows: when KIM-1 is highly expressed in the kidneys, the accumulation of S-Au-Agent in the kidneys increases, while the amount cleared into the urine decreases. However, silver nanoparticles without any specific targeting are not affected by the upregulation of KIM-1. Therefore, the level of KIM-1 in the kidneys can be reflected by analyzing the Au / Ag ratio in the urine. Furthermore, the ratio method has the advantage of removing interference from the organism, resulting in higher accuracy.

[0089] Depend on Figure 10 It was found that the detection of KIM-1 in urine had a lag effect, and a significant difference from that in normal mouse urine was only observed 48 hours after reperfusion. However, the KIM-1 content in mouse kidneys increased significantly 30 minutes after reperfusion, but remained within the normal range. Therefore, the early stage of significant KIM-1 elevation, i.e., 6 hours after reperfusion, was selected as the experimental subject for CT imaging detection.

[0090] Depend on Figure 11 Immunohistochemical staining was performed on the kidneys of mice in the sham-operated group and mice that had undergone ischemia-reperfusion for 6 hours. The results showed that kidney injury led to high expression of KIM-1 in the proximal tubules. Figure 11 The A in the sample resulted in a 2.72-fold increase in the area of ​​KIM-1 positivity in the cortex compared to the sham-operated group. Figure 11 (B in the middle).

[0091] Depend on Figure 12 It can be seen that the accumulation of the two types of nanoparticles in the kidneys 30 minutes after injection shows that the accumulation of silver does not change significantly with the extension of renal reperfusion time, and only slightly increases in the experimental group after 48 hours of reperfusion. This is because the damage has caused organic lesions in the kidneys and a decrease in glomerular filtration rate. Figure 12 (A in the text), but in reality, the amount of gold accumulated had already increased after 6 hours of reperfusion ( Figure 12 The B value in this figure is consistent with previous KIM-1 level measurements. This is because elevated KIM-1 levels increase the proximal tubules' ability to take up S-Au-Agent, and similarly, the ratio of the two in the kidney reflects the same trend. Figure 12 (B in the middle).

[0092] Further analysis of the clearance efficiency of gold and silver nanoparticles in urine and their ratio yielded the following results: Figure 13 As shown, because the accumulation in the kidneys increases, the clearance efficiency relative to urine decreases, and silver nanoparticles hardly reflect this trend, demonstrating the stability of the internal control. Figure 13(A) Gold nanoparticles exhibit this phenomenon after 12 hours of reperfusion, showing a decrease in clearance efficiency as renal KIM-1 levels increase. Figure 13 (B in the original text). By calculating the ratio of each reperfusion time period using the ratio method, it can be observed that this method can detect the occurrence of acute kidney injury at least 6 hours earlier, avoiding the heterogeneity of urine tests ( ). Figure 13 (C in the middle).

[0093] The correlation curves between the ratio of silver to gold in the kidneys and urine and the kidney KIM-1 content of mice in all experimental groups are shown in the figure below. Figure 14 As shown, a linear fit was performed with the kidney KIM-1 content as the horizontal axis and the ratio of silver to gold in urine and kidney as the vertical axis. It can be clearly observed that the ratio of silver to gold in the kidney is negatively correlated with the kidney KIM-1 content, which is due to the increased relative accumulation of gold in the kidney. The opposite is true for urine, that is, the ratio of silver to gold in urine is negatively correlated with the kidney KIM-1 content. Both have a good correlation. This linear relationship can be used to quantitatively assess the KIM-1 content in the kidney through urine analysis and to achieve early detection of kidney damage.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for preparing KIM-1 actively targeted gold nanoparticles, characterized in that, Specifically comprising the following steps: Step S1, synthesis of GS-AuNPs (1) After dissolving glutathione in deionized water, add tetrachloroauric acid, heat to 95°C and stir to obtain a reaction solution; (2) Cool to room temperature, then add saturated sodium chloride to the above reaction solution, stir thoroughly, then add anhydrous ethanol, shake thoroughly, centrifuge, and collect the precipitate to obtain GS-AuNPs; Step S2, synthesis of gold nanoparticles S-Au-Agent with KIM-1 active targeting (1) Dissolve GS-AuNPs in PBS buffer solution, add hydrochloric acid solution to adjust the pH to 4.5-5.5, then add activators EDC and NHS, and use a vortex instrument to vortex; (2) Add serine to the vortexed reaction system, adjust the pH to 7.8 with sodium hydroxide solution, and then vortex using a vortex instrument; (3) Collect the vortexed reaction solution and purify by ultrafiltration centrifugation, and collect the upper liquid to obtain the target product S-Au-Agent; The gold nanoparticles prepared in step (3) are used as CT contrast agents to detect KIM-1 in the kidney by CT imaging; Or the gold nanoparticles prepared in step (3) and phosphatidylcholine modified silver nanoparticles are used together to quantitatively analyze the content of KIM-1 in the kidney by urine analysis method.

2. The preparation method of KIM-1 active targeting gold nanoparticles according to claim 1, characterized in that: In step S1(1), the mass-volume ratio of glutathione, tetrachloroauric acid and deionized water in the mixed system is 36.87 mg:59.07 mg:50 mL.

3. The preparation method of KIM-1 active targeting gold nanoparticles according to claim 1, characterized in that: In step S1(2), the volume ratio of saturated sodium chloride and anhydrous ethanol is 5 mL:55 mL.

4. The preparation method of KIM-1 active targeting gold nanoparticles according to claim 1, characterized in that: In step S2(1), the mass-volume ratio of GS-AuNPs and PBS is 10 mg:5 mL, wherein the pH of the PBS buffer solution is 7.4; the mass ratio of EDC and NHS is 50 mg:60 mg; and the molar concentration of the hydrochloric acid solution is 1M.

5. The preparation method of KIM-1 active targeting gold nanoparticles according to claim 1, characterized in that: In step S2(2), the mass of serine is 100 mg; and the molar concentration of the sodium hydroxide solution is 10M.

6. The preparation method of KIM-1 active targeting gold nanoparticles according to claim 1, characterized in that, The preparation of phosphatidylcholine modified silver nanoparticles comprises the following steps: Step S1, synthesis of GS-AgNPs Add a mixed solution of AgNO3 and GSH in deionized water, stir until turbid, then add NaOH solution to adjust the pH to 4.9-5.1, constantly stir until clear, then add NaBH4, stir at room temperature, collect the reaction solution and filter to obtain GS-AgNPs; Step S2, synthesis of phosphatidylcholine ligand NH2-MPC Dissolve 2-methacryloyloxyethylphosphocholine in methanol and remove oxygen under nitrogen, then mix with a methanol solution of cysteine hydrochloride, add triethylamine as a catalyst, remove the excess solvent by rotary evaporation after the reaction is complete, then dissolve in a mixed solution of dichloromethane and diethyl ether and purify the product, and finally freeze-dry to obtain NH2-MPC; Step S3, synthesis of phosphatidylcholine modified silver nanoparticles PC-AgNPs The GS-AgNPs were dispersed in 1×PBS buffer solution, mixed with the activated carboxyl groups of EDC and NHS dissolved in another two 1×PBS buffer solutions respectively, then mixed with NH2-MPC dissolved in another 1×PBS buffer solution, added with sodium hydroxide aqueous solution to adjust the pH to 7.8, and stirred at room temperature to couple the phosphatidylcholine ligand with the nanoparticles. After the reaction was completed, the PC-AgNPs were purified by ultrafiltration centrifugation and freeze-dried.

7. The preparation method of KIM-1 active targeting gold nanoparticles according to claim 6, characterized in that, In step S1, the mass ratio of AgNO3, GSH and NaBH4 was 34 mg:61.4 mg:38 mg; In step S2, the mass-volume ratio of 2-methacryloyloxyethyl phosphorylcholine and methanol was 100 mg:10 mL; the mass-volume ratio of cysteine hydrochloride and methanol was 28.7 mg:1 mL; In step S3, the mass-volume ratio of GS-AgNPs and 1×PBS buffer solution was 2 mg:1 mL, wherein the pH of the 1×PBS buffer solution was 4.5; The mass-volume ratio of EDC, NHS and another two 1×PBS buffer solutions was 7 mg:1 mL and 4 mg:1 mL respectively, wherein the pH of the 1×PBS buffer solution was 4.5; and the mass to volume ratio of the other 1 x PBS buffer solution is 3 mg: 1 mL, wherein the pH of the 1 x PBS buffer solution is 7.

8.

8. A KIM-1 actively targeted gold nanoparticle, characterized in that, The gold nanoparticles are prepared by the method of any one of claims 1-7.

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