Phosphatidylcholine gold nano CT probe as well as preparation method and application thereof

Abstract

The invention discloses a phosphatidylcholine gold nano CT (Computed Tomography) probe as well as a preparation method and application thereof, and belongs to the technical field of medical materials. The preparation method comprises the following steps: step S1, preparing a phosphatidylcholine ligand NH2-MPC; step S2, synthesis of a gold nanocluster GS-Au25 is carried out; and S3, the GS-Au25 is modified with a PC ligand, and the PC-Au25 is prepared. The ultra-small gold nano CT probe PC-Au25 with an Au25 gold nano cluster as a core and phosphatidylcholine PC as a surface ligand is obtained by optimizing and screening the core size and the surface ligand property of ultra-small gold nano particles. Wherein the 2-methacryloyloxyethyl phosphorylcholine is used as a PC ligand for the first time to modify the surfaces of the ultra-small gold nanoparticles, so that the surface modification of the ultra-small gold nanoparticles is realized, and the ultra-small gold nanoparticles can be efficiently and freely filtered through the kidney. The CT probe can be applied to the determination of the glomerular filtration rate through a CT imaging or blood test method.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and particularly relates to a phosphatidylcholine gold nanocomposite CT probe, a preparation method thereof, and an application thereof. Background Art

[0002] The glomerular filtration rate (GFR) is the amount of ultrafiltrate produced by both kidneys per unit time (usually per minute). GFR is one of the best indicators for measuring renal function and has important clinical significance for accurately evaluating renal function and early detecting kidney diseases. Clinically, in acute kidney injury caused by surgical ischemia-reperfusion, GFR drops sharply within a short time, indicating impaired renal filtration function; while in chronic kidney disease caused by diabetic nephropathy (DN), early hyperglycemia causes hemodynamic changes, leading to glomerular hyperfiltration and an increase in GFR. As the disease progresses, glomerular sclerosis and nephron loss eventually result in a decrease in GFR. Therefore, by monitoring changes in GFR, doctors can understand the development of kidney diseases and provide more accurate diagnosis and treatment suggestions for patients.

[0003] Currently, the most commonly used GFR detection methods in clinical practice include the following: estimated glomerular filtration rate (eGFR) calculated based on the patient's serum creatinine level; measured glomerular filtration rate (mGFR) based on inulin clearance, which is the gold standard for measuring GFR; iohexol plasma clearance is the latest GFR measurement method recommended by the European Renal Function Consortium in 2024; renal ECT (Emission Computed Tomography) is a new method for measuring renal function using radionuclides. However, all of the above detection methods have corresponding defects: The method of estimating GFR through serum creatinine is simple, but has poor accuracy and latency. This is mainly because: First, creatinine levels are affected by factors such as muscle mass and eating habits; Second, creatinine is not only filtered through the glomeruli, but also undergoes tubular excretion, which affects the assessment of glomerular filtration function. Although inulin renal clearance is the gold standard for measuring GFR, it requires frequent collection of the patient's blood and urine, resulting in low patient acceptance and poor applicability in the clinical environment; Iohexol and other iodinated contrast agents have certain renal toxicity and sensitization, and are not suitable for some populations; Although renal ECT can measure GFR, due to the need to use radionuclide drugs and professional large-scale nuclear imaging equipment during the detection process, the high cost makes it impossible to be popularized and carried out in medical institutions at all levels.

[0004] Compared with the above-mentioned GFR detection methods, Dynamic Contrast-Enhanced CT (DCE-CT) has the advantages of real-time monitoring, fast scanning speed, and deep imaging depth. It is very suitable for real-time monitoring of renal hemodynamic changes to evaluate glomerular filtration function, and thus has become the most widely used detection technology in clinical practice. However, the iodine-based contrast agents commonly used in clinical CT at present (such as iohexol) have certain limitations, including their small X-ray attenuation coefficient, low imaging efficiency, certain nephrotoxicity, and inapplicability to some iodine-allergic patients, which greatly limits the application of DCE-CT in the accurate determination of GFR.

[0005] Aiming at the defects of the existing GFR determination methods in terms of accuracy and applicability, as well as the lack of CT probes that can accurately measure GFR in clinical practice, it is urgent to develop a new type of CT contrast agent that can achieve accurate determination of GFR. Based on the excellent X-ray attenuation ability of gold nanoparticles, the present invention has developed a brand-new ultra-small gold nanosensor that can freely filter through the kidneys, which can accurately measure GFR based on DCE-CT imaging method and blood test method. In order to achieve accurate determination of GFR, it is required that the constructed probe can be efficiently and freely filtered through the kidneys, mainly meeting the following conditions: (1) the core size of the probe is smaller than the glomerular filtration threshold (6 nm); (2) the probe can freely filter through the glomerulus without any interaction with the renal tubules. The present invention realizes its efficient and free filtration through the glomerulus and has no interaction with the renal tubules by regulating the size and surface ligand properties of the gold nanosensor. Summary of the Invention

[0006] The object of the present invention is to provide a phosphatidylcholine gold nanosensor for CT, its preparation method and application, and a gold nanosensor PC-Au modified with phosphatidylcholine and having 25 gold atoms is prepared 25 which is a brand-new ultra-small gold nanosensor that can freely filter through the kidneys, and can accurately measure GFR based on DCE-CT imaging method and blood test method, so as to solve the problems of low accuracy and applicability of the existing GFR determination methods and the lack of CT probes that can accurately measure GFR in the background technology.

[0007] To achieve the above object, the present invention provides a preparation method of a phosphatidylcholine gold nanosensor for CT, which specifically includes the following steps: Step S1, preparation of phosphatidylcholine ligand NH2-MPC Dissolve 2-methacryloyloxyethyl phosphorylcholine in a methanol solution and purge with nitrogen for 15 min; dissolve cysteine hydrochloride in a methanol solution. After complete dissolution, mix the above two solutions, add triethylamine, and stir the reaction at room temperature. After the reaction is completed, rotary evaporate to remove the excess solvent, then dissolve in a dichloromethane-ether mixed solution, let stand to remove the supernatant, and then rotary evaporate to remove the excess solvent. Finally, dissolve the product in distilled water and lyophilize to obtain NH2-MPC; Step S2, synthesis of gold nanoclusters GS-Au 25 Synthesis Dissolve GSH, HAuCl4·3H2O, and borane-tert-butylamine complex in H2O and react at 37 °C. After the reaction is completed, precipitate the gold nanoparticles with saturated NaCl and absolute ethanol, and then centrifuge and purify them in an ultrafiltration tube to obtain GS-Au 25 ; Step S3, modification of GS-Au with PC ligand 25 Preparation of PC-Au 25 Take GS-Au 25 , 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and NH2-MPC and dissolve them separately in a 1×PBS buffer solution for standby; mix the above four solutions and stir the reaction at room temperature. After the reaction is completed, transfer the reaction system to an ultracentrifugation tube for ultrafiltration purification, and lyophilize to obtain the target product phosphatidylcholine gold nanocluster CT probe PC-Au 25 .

[0008] Preferably, in step S1, the mass-volume ratio of 2-methacryloyloxyethyl phosphorylcholine to the methanol solution is 100 mg:10 mL; The mass-volume ratio of cysteine hydrochloride to the methanol solution is 28.7 mg:1 mL; The volume of triethylamine is 10 μL.

[0009] Preferably, in step S1, stir the reaction at 400 rpm for 4 hours at room temperature.

[0010] Preferably, in step S2, the mass-volume ratio of GSH, HAuCl4·3H2O, borane-tert-butylamine complex to H2O is 38.5 mg:10 mg:35 mg:6 mL.

[0011] Preferably, in step S2, react at 37 °C for 12 h, and the cut-off molecular weight of the ultrafiltration tube is 5 kDa.

[0012] Preferably, in step S3, the mass-volume ratio of GS-Au 25 to the 1×PBS buffer solution is 2 mg:1 mL; The mass-volume ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to 1×PBS buffer solution is 7 mg:1 mL; The mass-volume ratio of N-hydroxysuccinimide to 1×PBS buffer solution is 4 mg:1 mL; The mass-volume ratio of NH2-MPC to 1×PBS buffer solution is 3 mg:1 mL.

[0013] Preferably, in step S3, the pH value of the 1×PBS buffer solution is 7.2.

[0014] Preferably, in step S3, the reaction is stirred at room temperature for 6 h, and the cut-off molecular weight of the ultrafiltration centrifuge tube is 5 kDa.

[0015] The present invention also provides a phosphatidylcholine gold nanocomputed tomography (CT) probe prepared by the above preparation method.

[0016] The present invention also provides the application of the above phosphatidylcholine gold nanocomputed tomography (CT) probe in the determination of glomerular filtration rate by CT imaging or blood test method.

[0017] Therefore, a phosphatidylcholine gold nanocomputed tomography (CT) probe, its preparation method and application provided by the present invention have the following beneficial effects: (1) By systematically optimizing and screening the core size and surface ligand properties of ultrasmall gold nanoparticles, the present invention obtains an ultrasmall gold nanocomputed tomography (CT) probe PC-Au with Au 25 gold nanoclusters as the core and phosphatidylcholine PC as the surface ligand. 2-Methacryloyloxyethyl phosphorylcholine is used as the PC ligand to modify the surface of ultrasmall gold nanoparticles for the first time, successfully realizing the surface modification of ultrasmall gold nanoparticles, enabling them to be efficiently and freely filtered through the kidneys. On the one hand, the accurate and rapid determination of bilateral kidney GFR can be achieved by continuous DCE-CT scanning; on the other hand, the GFR can also be measured by the blood test method using an extremely low dose of the probe. 25

[0018] Compared with traditional iodine contrast agents, gold has a higher X-ray attenuation coefficient. Under the condition of the same imaging element mass (equal molar amount of gold and iodine), PC-Au 25 has a higher imaging enhancement contrast than iohexol; in addition, compared with the potential allergic risk of iodine contrast agents, the gold nanocluster core of this probe has good chemical inertness and stability, the probe surface is a biomimetic phosphatidylcholine ligand with good biocompatibility, and the whole probe can be cleared out of the body through urine within a certain period of time. Therefore, PC-Au 25 has good safety.

[0019] ​(3) Compared with the traditional GFR measurement methods, this probe provides two measurement schemes: by using DCE-CT imaging technology, it is not necessary to repeatedly collect blood and urine, and only 2 minutes of continuous CT scanning is required to accurately measure GFR; by measuring GFR through the blood method, the time window is wider, the probe dose is smaller, and the test cost is lower. This probe provides the possibility for doctors and patients to make choices according to the actual situation.

[0020] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is the preparation route diagram of the phosphatidylcholine ligand in the embodiment of the present invention; Figure 2 It is the synthesis route diagram of the ultrasmall gold nanoclusters GS-Au 25 in the embodiment of the present invention; Figure 3 It is the preparation route diagram of preparing PC-Au by modifying GS-Au with the PC ligand in the embodiment of the present invention; 25 Preparation of PC-Au 25 in the embodiment of the present invention; Figure 4 It is the core particle size diagram of the PC-Au 25 prepared in the embodiment of the present invention; Figure 5 It is the infrared absorption spectrum diagram, nuclear magnetic resonance hydrogen spectrum diagram and visible light absorption spectrum diagram of NH2-MPC, GS-Au 25 , PC-Au 25 prepared in the embodiment of the present invention; among them, (a) is the infrared absorption spectrum of GS-Au 25 and PC-Au 25 ; (b) is the nuclear magnetic resonance spectrum of NH2-MPC, GS-Au 25 , PC-Au 25 ; (c) is the ultraviolet-visible light absorption spectrum diagram of GS-Au 25 , PC-Au 25 ; Figure 6 It is the comparison curve diagram of the renal clearance rate of GS-Au 25 and PC-Au 25 prepared in the embodiment of the present invention; Figure 7 It is the metabolic kinetic parameter diagram of GS-Au 25 and PC-Au 25 prepared in the embodiment of the present invention, among which, (a) is the pharmacokinetic curve diagram; (b) is the comparison bar chart of the clearance rate; Figure 8Effect of the renal tubular transport protein inhibitor on PC-Au prepared in the examples of the present invention 25 Graph showing the effect on renal clearance rate; Figure 9 PC-Au prepared in the examples of the present invention 25 Imaging comparison diagrams of the aorta, kidneys, and bladder before and after injection; Figure 10 PC-Au prepared in the examples of the present invention 25 Analysis diagrams of the enhanced signal results of CT probe scanning of the aorta and kidneys; among them, (a) is the curve graph of the aortic signal enhancement value; (b) is the curve graph of the bilateral renal signal enhancement value; (c) is the renal Patlak-Rutland image; Figure 11 PC-Au prepared in the examples of the present invention 25 Comparison diagram of the results of CT probe determination of bilateral renal GFR and GFR determined by FITC-inulin; among them, (a) is the GFR value of a single kidney; (b) is the sum of the GFR values of bilateral kidneys; Figure 12 PC-Au prepared in the examples of the present invention 25 Relationship diagram between the GFR values measured by the probe in normal mice and DN model mice and the measured values of FITC-inulin; Figure 13 After injecting PC-Au prepared in the examples of the present invention 25 Dual-phase fitting curve of the change in the content of gold element in the blood of normal mice; Figure 14 After injecting PC-Au prepared in the examples of the present invention 25 Dual-phase fitting curve of the change in the content of gold element in the blood of DN model mice. Detailed implementation manners

[0022] The technical solutions of the present invention will be further described below with reference to the accompanying drawings and examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application and belong to the protection scope of the present invention.

[0023] References to "embodiments" in this application mean that the specific features, structures, or characteristics described in connection with the embodiments may be included in at least one embodiment of the present application. The term "embodiment" appearing at various positions in the specification does not necessarily refer to the same embodiment, nor does it particularly limit its independence or relevance to other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, the various technical features mentioned in each embodiment can be combined in any way to form corresponding implementable technical solutions.

[0024] Unless otherwise defined, the meanings of the technical terms used in this application are the same as those commonly understood by those skilled in the technical field to which this application belongs; the use of the relevant terms in this application is only for describing specific embodiments and is not intended to limit this application.

[0025] Unless otherwise specified in the present invention, the reagents, instruments, equipment, and performance testing methods used are all the reagents, instruments, equipment, and methods commonly used by those skilled in the art.

[0026] Embodiment This embodiment provides a method for preparing a phosphatidylcholine gold nanocomposite CT probe, which specifically includes the following steps: Step S1, preparation of phosphatidylcholine ligand: Dissolve 100 mg of 2-methacryloyloxyethyl phosphorylcholine in 10 mL of methanol, and purge with nitrogen for 15 min; dissolve 28.7 mg of cysteine hydrochloride in 1 mL of methanol solution. After complete dissolution, mix the above two solutions, then add 10 μL of triethylamine, and stir at 400 rpm at room temperature for 4 hours. After the reaction, remove the excess solvent by a rotary evaporator, then redissolve the product with a 10 mL mixed solution of dichloromethane and ether, let it stand for 30 min, remove the supernatant, collect the product, and finally remove the excess solvent by rotary evaporation and add 1 mL of distilled water to fully dissolve the product. After lyophilization, the phosphatidylcholine ligand NH2-MPC is obtained. The preparation route is as Figure 1 shown.

[0027] Step S2, synthesis of ultrasmall gold nanoclusters GS-Au 25 : Dissolve 38.5 mg of GSH, 10 mg of HAuCl4·3H2O and 35 mg of borane-tert-butylamine complex in 6 mL of H2O, and react at 37 °C for 12 h. Precipitate the gold nanoparticles with saturated NaCl and absolute ethanol, and purify them by centrifugation with a 5 kDa ultrafiltration tube to obtain GS-Au 25 The synthesis route is as Figure 2 shown.

[0028] Step S3, modification of GS-Au with PC ligand 25 : Take 2 mg of GS-Au25 Fully dissolve it in 1×PBS buffer solution (pH = 7.2) for later use. Dissolve 7 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4 mg of N-hydroxysuccinimide in 1 mL of 1×PBS buffer solution (pH = 7.2) respectively for later use. Take 3 mg of phosphatidylcholine ligand and fully dissolve it in 1 mL of 1×PBS buffer solution (pH = 7.2) for later use. Subsequently, mix the above four solutions and stir the reaction at room temperature for 6 h. After the reaction is completed, transfer the reaction system to a 5 kDa ultrafiltration tube for ultrafiltration purification, and obtain the ultrasmall phosphatidylcholine gold nanocomposite CT probe PC-Au after lyophilization. 25 , and the preparation route is as Figure 3 shown.

[0029] Characterize the basic physicochemical properties of the intermediate products and target products prepared in the examples.

[0030] Use a transmission electron microscope (TEM) to measure the core size of PC-Au 25 . The results are as Figure 4 shown. It can be seen from Figure 4 that the core diameter of PC-Au 25 is 0.98 ± 0.17 nm, which is much smaller than the glomerular filtration threshold (6 nm) and can theoretically pass through the glomerular filtration.

[0031] Use a Fourier transform infrared spectrometer (FITR) and a nuclear magnetic resonance spectrometer for hydrogen ( 1 H-NMR) to measure the infrared absorption spectra and nuclear magnetic resonance spectra of hydrogen of the PC ligand (NH2-MPC), GS-Au 25 or PC-Au 25 ; use a UV-visible spectrophotometer (UV-Vis) to measure the UV-visible absorption spectra of GS-Au 25 and the ultrasmall gold nanoparticles PC-Au 25 . The results are as Figure 5 shown. It can be seen from Figure 5 (a) that compared with GS-Au 25 , C=O, P=O, N(CH3) 25 and P-O stretching vibrations appear in PC-Au 3+ . It can be seen from Figure 5 (b) that PC-Au 25 contains hydrogen atoms of both GS-Au 25 and the PC ligand at the same time. It can be seen from Figure 5 (c) that the absorption spectra of GS-Au 25 and PC-Au 25 are consistent.

[0032] The in vivo metabolic behaviors of the intermediate products and target products prepared in the examples were investigated.

[0033] Eight-week-old male C57BL / 6 mice were used as the research objects, and GS-Au prepared in the examples was injected into the tail vein respectively. 25 and PC-Au 25 . The urine of the mice was collected within 24 h, and the mice were dissected and the kidneys were collected after 24 h; after digesting the samples with aqua regia, the gold content in each sample was determined by ICP-MS to determine the renal clearance rate of the probe and its distribution level in the kidneys. The results are as Figure 6 shown. As Figure 6 can be seen, the renal clearance rate results showed that 93.2 ± 2.0% of the PC-Au 25 probe was excreted from the body through urine within 2 hours, while that of GS-Au 25 was only 54.8 ± 6.1%.

[0034] Another group of mice were injected with PC-Au 25 through the tail vein, and blood samples were collected at different time points. The gold element content in each sample was determined by ICP-MS, and the time-concentration curve was plotted to determine the pharmacokinetic parameters of the ultrasmall biomimetic gold nanoparticles. The results are as Figure 7 shown. As Figure 7 shown in (a), 10 minutes after injecting the ultrasmall gold nanoparticles, the concentration of PC-Au 25 in the blood began to be significantly lower than that of GS-Au 25 . As Figure 7 shown in (b), the 24-hour clearance rate of PC-Au 25 was 12.3 times higher than that of GS-Au 25 .

[0035] The above results indicate that the modification of ultrasmall gold nanoparticles with phosphatidylcholine ligands can significantly improve their in vivo metabolic clearance rate, including the pharmacokinetic clearance rate and renal clearance rate. The high in vivo clearance rate of PC-Au 25 lays a theoretical foundation for the accurate determination of the glomerular filtration rate.

[0036] To further explore whether there is room for optimization of the PC-Au 25 probe, the surface ligands were compared and screened. The protein binding rates of ultrasmall biomimetic gold nanoparticles and ultrasmall gold nanoparticles modified with other ligands were tested by agarose gel electrophoresis (AGE) and inductively coupled plasma mass spectrometry (ICP-MS). The renal clearance rate was determined by the above method for testing the renal clearance rate. The results are shown in Table 1 below.

[0037] Table 1 Protein binding rate and renal clearance rate of ultrasmall gold nanoprobes modified with different surface ligands ;

[0038] As can be seen from Table 1, among the ultrasmall gold nanoparticles modified with four different ligands, the protein binding rate of PC-Au 25 is the lowest, indicating that the PC ligand can minimize the interaction between the probe and the body. At the same time, the 24-hour renal clearance efficiency of PC-Au 25 is the highest.

[0039] Furthermore, the core size of the probe was optimized and screened. In addition to the PC-Au 25 prepared in the examples, by adjusting the ratio of HAuCl4·3H2O to GSH in the synthesis raw materials to 5:4, increasing the reaction temperature to 95 °C, and shortening the reaction time to 40 minutes, ultrasmall gold nanoparticles with a core particle size of 2.5 nm were synthesized. After modification with the PC ligand, PC-AuNPs-2.5 nm was obtained. Its pharmacokinetic parameters and renal clearance rate are shown in Table 2 below.

[0040] Table 2 Pharmacokinetic parameters and renal clearance rate of gold nanoprobes of different sizes ;

[0041] As can be seen from Table 2, compared with PC-AuNPs-2.5 nm, the PC-Au 25 prepared in this example is metabolized faster in plasma and has a higher renal clearance efficiency at the same time.

[0042] An experimental proof was carried out on the mechanism of interaction between the target product prepared in the examples and the renal tubules.

[0043] Probenecid and Cimetidine were used to inhibit the organic anion transporter and organic cation transporter of renal tubular cells, and the renal clearance efficiency of the gold nanoprobe before and after inhibition was compared. The results are as Figure 8 shown. As can be seen from Figure 8 , the renal clearance rate of PC-Au 25 did not change significantly before and after the use of the inhibitor, indicating that there is no interaction process between PC-Au 25 and the renal tubules.

[0044] Based on the above systematic study of the pharmacokinetics and renal clearance pathway of PC-Au 25 , as well as the optimization and screening of the surface ligand type and gold nanoparticle core size, the PC ligand used in the examples was determined to be an ideal choice for modifying ultrasmall gold nanoparticles, which can significantly improve their in vivo metabolic behavior; PC-Au 25 can be efficiently and freely filtered through the kidneys, laying a theoretical foundation for the accurate determination of GFR.

[0045] Application Example 1: Using the PC-Au prepared in the examples 25 for CT imaging to measure GFR (1) Eight-week-old C57 BL / 6J mice were selected and injected with PC-Au 25 Before injection, the mice were pre-scanned. The scanning parameters were 70 kV, 114 μA, the scanning window was selected with FOV = 72 mm, the scanning time was 3.9 s, and the detector reset time was about 11 s. Subsequently, PC-Au was injected via the tail vein 25 , with a dose of 450 mg / kg. Continuous scanning was started simultaneously with the injection of PC-Au 25 . The number of scans was 3 times, and contrast images for a total of 120 s were obtained. As Figure 9 shown, it can be seen from Figure 9 that imaging of the abdominal aorta was achieved 15 s after injection of the probe; at 60 s, CT signals of both kidneys could be observed; at 120 s, signal accumulation in the bladder was observed, indicating that the probe was filtered by the kidneys, formed urine and entered the bladder

[0046] (2) GFR calculation method: Outline and calculate the CT signal enhancement values of the aorta and bilateral kidneys. As Figure 10 shown in Figure 10 (a) and 25 (b), after injection of the PC-Au Figure 10 probe, the CT signal of the aorta reached its peak at 15 s, and the signal values of both kidneys gradually increased over time. The data processing software origin was used to calculate the area under the curve of the change in the CT value of the aorta over time, and calculate the ratio of this area to the CT value of the aorta [∫b(t)dt / b(t)]; calculate the ratio of the CT enhancement signal values of the left and right kidneys to the aorta [c(t) / b(t)]; using ∫b(t)dt / b(t) as the abscissa and c(t) / b(t) as the ordinate, after fitting,

[0047] (c) was obtained. Record the slope of the linear equation, multiply by (1 - HCT), and then multiply by the volume of each kidney to finally obtain the GFR (μL / min) of each kidney (the kidney volume was obtained by 3D reconstruction of the kidney using CT software). The sum of the left and right kidneys is the total renal GFR. As Figure 11 shown in Figure 11 (a) is the GFR value of a single kidney, Figure 11 (b) is the sum of the GFR values of both kidneys, that is, the total renal GFR. It can be seen from

[0048] (3) Establish a DN mouse model: 8-week-old C57 BL / 6J mice were fed a high-fat diet for 8 weeks (60% of energy from fat), and streptozotocin (STZ) (40 mg / kg) was intraperitoneally injected for 5 consecutive days in the 9th week, and then continued to be fed with a high-fat diet.

[0049] Four weeks later, PC-Au 25 and CT imaging were used to measure the GFR of normal mice and DN model mice. The methods, parameters, and data processing methods were the same as above, and the results are as Figure 12 shown. As Figure 12 can be seen, the GFR values measured by CT imaging using the PC-Au 25 probe were consistent with those measured by the gold standard FITC-inulin. The R 2 value of the fitting curve of the relationship between the two was 0.914, and the fitting effect was good, indicating the accuracy of the PC-Au 25 probe for measuring GFR in chronic kidney disease by CT imaging. The GFR value of normal mice measured by the PC-Au 25 probe was 218.9 ± 17.9 μL / min. The GFR of DN model mice increased in the early stage of the disease and decreased in the later stage, indicating that the renal function of DN model mice was damaged, and different GFR values reflected different degrees of renal function loss.

[0050] Application Example 2 Use the PC-Au 25 prepared in the example for blood test to measure the GFR of mice (1) Select 8-week-old C57 BL / 6J mice, and inject PC-Au 25 into the tail vein at a dose of 2.5 mg / kg. Reserve the injected sample for later determination of the injection dose (ID). At 2, 5, 10, 15, 20, 40, and 60 minutes after injection, collect mouse blood into a centrifuge tube by orbital ischemia, and record the blood weight. Add 1 mL of aqua regia to each centrifuge tube to digest the PC-Au 25 probe to obtain a gold ion solution, which was diluted and used for ICP-MS quantitative determination of gold element content.

[0051] (2) Calculate the gold element concentration of each blood sample, draw the relationship image between concentration and time, and use the two-phase exponential decay function ExpDec2 to fit the image to obtain the curve parameters A1, t1, A2, t2; calculate the area under the curve AUC = A1·t1 + A2·t2. Finally, calculate GFR = ID / AUC·(1 - Hct), where Hct is the hematocrit value, which is 0.5. As Figure 13 can be seen, after tail vein injection of PC-Au 25, the concentration of gold in the blood was measured after one hour, and a two-phase exponential decay fitting was performed. As shown in Table 3, the GFR value of the mice was calculated to be 223.27 ± 10.50 μL / min. This result is close to the GFR value (209.55 ± 32.00 μL / min) measured by the FITC-inulin method, indicating that the blood test method for measuring GFR in mice has high accuracy.

[0052] Table 3 Parameters of gold content change in blood in the experiment of measuring GFR in normal mice by blood method ;

[0053] (3) Establish a DN mouse model using the same method as in Application Example 1.

[0054] Use PC-Au 25 Measure the GFR of normal mice and DN model mice by the blood method. The method and data processing method are the same as above. It can be seen that Figure 14 after the DN model mice were injected with PC-Au 25 via the tail vein, the concentration of gold in the blood was measured after one hour, and a two-phase exponential decay fitting was performed. As shown in Table 4, the GFR value of the mice was calculated to be 150.53 ± 14.42 μL / min. This result is significantly lower than the GFR value of normal mice (223.27 ± 10.50 μL / min), indicating that the renal function of DN model mice is impaired.

[0055] Table 4 Parameters of gold content change in blood in the experiment of measuring GFR in DN mice by blood method ;

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A preparation method of a phosphatidylcholine gold nanoparticle CT probe, characterized in that, Specifically, it includes the following steps: Step S1: Preparation of phosphatidylcholine ligand NH2-MPC Dissolve 2-methacryloyloxyethyl phosphorylcholine in a methanol solution and purge with nitrogen for 15 min; dissolve cysteine hydrochloride in a methanol solution. After complete dissolution, mix the above two solutions, add triethylamine, and stir at room temperature. After the reaction is completed, rotary evaporate to remove the excess solvent, then dissolve in a dichloromethane-ether mixed solution, let stand to remove the supernatant, and then rotary evaporate to remove the excess solvent. Finally, dissolve the product in distilled water and freeze-dry to obtain NH2-MPC; Step S2, synthesis of gold nanoclusters GS-Au 25 Synthesis Dissolve GSH, HAuCl4·3H2O and borane-tert-butylamine complex in H2O and react at 37 °C. After the reaction, precipitate gold nanoparticles using saturated NaCl and absolute ethanol, and then centrifuge and purify them in an ultrafiltration tube to obtain GS-Au 25 ; Step S3: Modify GS-Au with PC ligand 25 Prepare PC-Au 25 Take GS-Au 25 、1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and NH2-MPC were respectively dissolved in a 1×PBS buffer solution for standby; after mixing the above four solutions, the reaction was stirred at room temperature. After the reaction was completed, the reaction system was transferred to an ultracentrifugation tube for ultrafiltration purification, and freeze-dried to obtain the target product phosphatidylcholine gold nanocomposite CT probe PC-Au 25 .

2. The preparation method of a phosphatidylcholine gold nanoparticle CT probe according to claim 1, characterized in that: In step S1, the mass-volume ratio of 2-methacryloyloxyethyl phosphorylcholine to the methanol solution is 100 mg: 10 mL; The mass-volume ratio of cysteine hydrochloride to the methanol solution is 28.7 mg: 1 mL; The volume of triethylamine is 10 μL.

3. The preparation method of a phosphatidylcholine gold nanocomposite CT probe according to claim 1, characterized in that: In step S1, stir at 400 rpm for 4 hours at room temperature.

4. The preparation method of a phosphatidylcholine gold nanocomposite CT probe according to claim 1, characterized in that: In step S2, the mass-volume ratio of GSH, HAuCl4·3H2O, borane-tert-butylamine complex and H2O is 38.5 mg: 10 mg: 35 mg: 6 mL.

5. The preparation method of a phosphatidylcholine gold nanocomposite CT probe according to claim 1, characterized in that: In step S2, react at 37 °C for 12 h, and the cut-off molecular weight of the ultrafiltration tube is 5 kDa.

6. The preparation method of a phosphatidylcholine gold nanocomposite CT probe according to claim 1, characterized in that: In step S3, the mass-volume ratio of GS-Au 25 to 1×PBS buffer solution is 2 mg: 1 mL; The mass-volume ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to 1×PBS buffer solution is 7 mg: 1 mL; The mass-volume ratio of N-hydroxysuccinimide to 1×PBS buffer solution is 4 mg: 1 mL; The mass-volume ratio of NH2-MPC to 1×PBS buffer solution is 3 mg: 1 mL.

7. The preparation method of a phosphatidylcholine gold nanocomposite CT probe according to claim 1, wherein: In step S3, the pH value of the 1×PBS buffer solution is 7.

2.

8. The preparation method of a phosphatidylcholine gold nanoparticle CT probe according to claim 1, characterized in that: In step S3, stir at room temperature for 6 h, and the cut-off molecular weight of the ultracentrifugation tube is 5 kDa.

9. A phosphatidylcholine gold nanocomposite CT probe, characterized in that, The phosphatidylcholine gold nanocomposite CT probe is prepared by the preparation method described in any one of claims 1-8.

10. The application of a phosphatidylcholine gold nanocomposite CT probe as described in claim 9, wherein: The phosphatidylcholine gold nanocomposite CT probe is applied to the determination of glomerular filtration rate by CT imaging or blood test method.

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