A phosphatidylcholine gold nano-CT probe and its preparation method and application

Abstract

The present invention discloses a phosphatidylcholine gold nano-CT probe and its preparation method and application, which belongs to the field of medical material technology. The preparation method comprises: step S1, preparation of phosphatidylcholine ligand NH2-MPC; step S2, preparation of gold nano-clusters GS-Au 25 Synthesis; Step S3, PC ligand modification of GS-Au 25 Preparation of PC‑Au 25 By optimizing the core size and surface ligand properties of ultrasmall gold nanoparticles, Au 25 Ultrasmall gold nanoCT probe PC‑Au with gold nanoclusters as core and phosphatidylcholine PC as surface ligand 25 2-Methacryloyloxyethylphosphocholine was first used as a PC ligand to modify the surface of ultrasmall gold nanoparticles, achieving surface modification of the ultrasmall gold nanoparticles, enabling efficient and free filtration by the kidneys. This CT probe can be used to measure glomerular filtration rate through CT imaging or blood tests.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and in particular to a phosphatidylcholine gold nano-CT probe and a preparation method and 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 of renal function and is clinically important for accurately assessing renal function and detecting kidney disease early. Clinically, in acute kidney injury caused by surgical ischemia-reperfusion, GFR decreases rapidly over a short period of time, indicating impaired renal filtration function. In chronic kidney disease caused by diabetic nephropathy (DN), early hyperglycemia causes hemodynamic changes, leading to glomerular hyperfiltration and elevated GFR. As the disease progresses, glomerulosclerosis and nephron loss eventually lead to a decrease in GFR. Therefore, by monitoring GFR changes, physicians can understand the progression of kidney disease and provide more accurate diagnosis and treatment recommendations for patients.

[0003] Currently, the most commonly used GFR testing methods in clinical practice include the following: estimated glomerular filtration rate (eGFR), calculated based on a patient's serum creatinine level; measured glomerular filtration rate (mGFR), based on inulin clearance, is the gold standard for GFR determination; iohexol plasma clearance is the most recently recommended GFR determination method by the European Renal Consortium in 2024; and renal ECT (Emission Computed Tomography) is a new examination method that uses radionuclides to measure renal function. However, all of these testing methods have corresponding drawbacks: While estimating GFR based on serum creatinine is simple, it has poor accuracy and lags behind. This is primarily due to the following: 1. Creatinine levels are affected by factors such as muscle mass and dietary habits; 2. 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 blood and urine collection from patients, has low patient acceptance, and is less applicable in clinical settings; iodine contrast agents such as iohexol have certain renal toxicity and sensitization effects and are not suitable for some people; although renal ECT can measure GFR, the high cost of using radioactive nuclide drugs and professional large-scale nuclide imaging equipment during the test prevents it from being widely used in medical institutions at all levels.

[0004] Compared with the aforementioned GFR measurement methods, dynamic contrast-enhanced CT (DCE-CT) offers the advantages of real-time monitoring, fast scanning speed, and deep imaging depth, making it ideal for real-time monitoring of renal hemodynamic changes and assessment of glomerular filtration function. Therefore, it has become the most widely used clinical testing technique. However, the iodine contrast agents commonly used in clinical CT (such as iohexol) have certain limitations, including their low X-ray attenuation coefficient, low imaging efficiency, certain nephrotoxicity, and incompatibility with some individuals with iodine allergies. These limitations significantly limit the application of DCE-CT for accurate GFR measurement.

[0005] In view of the defects of existing GFR measurement methods in terms of accuracy and applicability, as well as the lack of CT probes that can accurately measure GFR in clinical practice, there is an urgent need to develop a new CT contrast agent that can achieve accurate GFR measurement. Based on the excellent X-ray attenuation ability of gold nanoparticles, the present invention has developed a new ultra-small gold nanoprobe that can be freely filtered by the kidney, which can accurately measure GFR based on DCE-CT imaging and blood test methods. In order to achieve accurate measurement of GFR, the constructed probe is required to be able to be efficiently and freely filtered by the kidney, mainly meeting the following conditions: (1) the probe core size is smaller than the glomerular filtration threshold (6nm); (2) the probe is freely filtered by the glomerulus without any interaction with the renal tubules. The present invention achieves the effect of efficient and free filtration by the glomerulus without any interaction with the renal tubules by regulating the size and surface ligand properties of the gold nanoprobe. Summary of the Invention

[0006] The purpose of the present invention is to provide a phosphatidylcholine gold nano-CT probe and its preparation method and application, and to prepare a phosphatidylcholine-modified gold nano-probe PC-Au with 25 gold atoms. 25 It is a new ultra-small gold nanoprobe that can be freely filtered by the kidneys. It can accurately measure GFR based on DCE-CT imaging and blood test methods to solve the problems of low accuracy and applicability of existing GFR measurement methods proposed in the background technology, as well as the lack of CT probes that can accurately measure GFR in clinical practice.

[0007] To achieve the above object, the present invention provides a method for preparing a phosphatidylcholine gold nano-CT probe, which specifically comprises the following steps:

[0008] Step S1: Preparation of phosphatidylcholine ligand NH2-MPC

[0009] 2-Methacryloxyethyl phosphorylcholine was dissolved in a methanol solution and nitrogen was passed through for 15 minutes. Cysteine ​​hydrochloride was dissolved in a methanol solution. After fully dissolved, the two solutions were mixed, triethylamine was added, and the mixture was stirred at room temperature to react. After the reaction was completed, excess solvent was removed by rotary evaporation, and then a dichloromethane-ether mixed solution was added to dissolve the mixture. The supernatant was removed after standing, and excess solvent was removed by rotary evaporation again. Finally, distilled water was added to dissolve the product, and lyophilized to obtain NH2-MPC.

[0010] Step S2, gold nanoclusters GS-Au 25 Synthesis

[0011] GSH, HAuCl4·3H2O and borane-tert-butylamine complex were dissolved in H2O and reacted at 37°C. After the reaction, saturated NaCl and anhydrous ethanol were used to precipitate gold nanoparticles, which were then placed in an ultrafiltration tube and centrifuged to obtain GS-Au. 25 ;

[0012] Step S3: PC ligand modification of GS-Au 25 Preparation of PC-Au 25

[0013] Take GS-Au 25 , 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and NH2-MPC were dissolved in 1×PBS buffer solution respectively for later use; the above four solutions were mixed and stirred at room temperature for reaction. After the reaction was completed, the reaction system was transferred to an ultracentrifuge tube for ultrafiltration purification and freeze-dried to obtain the target product phosphatidylcholine gold nano-CT probe PC-Au 25 .

[0014] Preferably, in step S1, the mass volume ratio of 2-methacryloyloxyethyl phosphorylcholine and methanol solution is 100 mg:10 mL;

[0015] The mass volume ratio of cysteine ​​hydrochloride and methanol solution is 28.7 mg:1 mL;

[0016] The volume of triethylamine was 10 uL.

[0017] Preferably, in step S1, the reaction is stirred at 400 rpm at room temperature for 4 hours.

[0018] Preferably, 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.

[0019] Preferably, in step S2, the reaction is carried out at 37° C. for 12 h, and the molecular weight cut-off of the ultrafiltration tube is 5 kDa.

[0020] Preferably, in step S3, GS-Au 25 The mass volume ratio of the 1× PBS buffer solution is 2 mg:1 mL;

[0021] The mass volume ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1×PBS buffer solution is 7 mg:1 mL;

[0022] The mass volume ratio of N-hydroxysuccinimide and 1× PBS buffer solution is 4 mg:1 mL;

[0023] The mass volume ratio of NH2-MPC and 1×PBS buffer solution is 3 mg:1 mL.

[0024] Preferably, in step S3, the pH value of the 1×PBS buffer solution is 7.2.

[0025] Preferably, in step S3, the reaction is stirred at room temperature for 6 hours, and the molecular weight cut-off of the ultracentrifuge tube is 5 kDa.

[0026] The present invention also provides a phosphatidylcholine gold nano-CT probe prepared by the above preparation method.

[0027] The present invention also provides the use of the above-mentioned phosphatidylcholine gold nano-CT probe in measuring glomerular filtration rate through CT imaging or blood test.

[0028] Therefore, the phosphatidylcholine gold nano-CT probe provided by the present invention and its preparation method and application have the following beneficial effects:

[0029] (1) The present invention systematically optimizes and screens the core size and surface ligand properties of ultrasmall gold nanoparticles to obtain Au 25 Ultrasmall gold nanoCT probe PC-Au with gold nanoclusters as core and phosphatidylcholine PC as surface ligand 25 In this system, 2-methacryloyloxyethyl phosphorylcholine was used for the first time as a PC ligand modified onto the surface of ultrasmall gold nanoparticles, successfully achieving surface modification of the ultrasmall gold nanoparticles, enabling them to be efficiently and freely filtered by the kidneys. On the one hand, continuous DCE-CT scanning can achieve accurate and rapid measurement of bilateral renal GFR; on the other hand, GFR can also be measured through blood tests using extremely low-dose probes.

[0030] (2) Compared with traditional iodine contrast agents, gold has a higher X-ray attenuation coefficient. Under the same imaging element mass (gold and iodine are equimolar), PC-Au 25Compared with iohexol, it has higher imaging contrast enhancement; in addition, compared with the potential allergenic risk of iodine contrast agents, the gold nanocluster core of the probe has good chemical inertness and stability, the probe surface is a biomimetic phosphatidylcholine ligand, has good biocompatibility, and the probe as a whole can be cleared out of the body through urine within a period of time. Therefore, PC-Au 25 Has very good security.

[0031] (3) Compared with traditional GFR measurement methods, this probe provides two measurement options: using DCE-CT imaging technology, there is no need to repeatedly collect blood and urine, and only 2 minutes of continuous CT scanning is required to accurately measure GFR; measuring GFR through blood method has a wider time window, smaller probe dosage, and lower testing cost. This probe provides doctors and patients with the possibility of choosing according to their actual situation.

[0032] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a preparation route for the phosphatidylcholine ligands in the embodiments of the present invention;

[0034] Figure 2 The ultra-small gold nanoclusters GS-Au in the embodiment of the present invention 25 Synthesis route map;

[0035] Figure 3 PC ligand modified GS-Au in the embodiment of the present invention 25 Preparation of PC-Au 25 Preparation roadmap;

[0036] Figure 4 PC-Au prepared in the embodiment of the present invention 25 Core particle size diagram;

[0037] Figure 5 NH2-MPC and GS-Au prepared in the embodiment of the present invention 25 、PC-Au 25 Infrared absorption spectrum, nuclear magnetic resonance hydrogen spectrum and visible light absorption spectrum; among them, (a) is GS-Au 25 and PC-Au 25 Infrared absorption spectra of NH2-MPC and GS-Au; (b) 25 、PC-Au 25 NMR spectrum of GS-Au; (c) 25 、PC-Au 25 UV-visible absorption spectrum of ;

[0038] Figure 6 GS-Au prepared in the embodiment of the present invention 25 and PC-Au 25 Comparison curve of renal clearance of

[0039] Figure 7 GS-Au prepared in the embodiment of the present invention 25 and PC-Au 25 Metabolic kinetic parameter diagram, where (a) is the pharmacokinetic curve diagram; (b) is the clearance comparison bar graph;

[0040] Figure 8 The PC-Au prepared in the embodiment of the present invention is treated with a renal tubular transporter inhibitor 25 Effect diagram of renal clearance;

[0041] Figure 9 PC-Au prepared in the embodiment of the present invention 25 Comparison of images of the aorta, kidney, and bladder before and after injection;

[0042] Figure 10 PC-Au prepared in the embodiment of the present invention 25 Analysis of the enhanced signal results of the aorta and kidneys scanned by the CT probe; (a) is the aortic signal enhancement value curve; (b) is the bilateral kidney signal enhancement value curve; (c) is the kidney Patlak-Rutland image;

[0043] Figure 11 PC-Au prepared in the embodiment of the present invention 25 Comparison of bilateral renal GFR measured by CT probe and FITC-inulin; (a) is the GFR value of a single kidney; (b) is the sum of the GFR values ​​of both kidneys;

[0044] Figure 12 PC-Au prepared in the embodiment of the present invention 25 The relationship between the GFR values ​​of normal mice and DN model mice measured by probe and the values ​​measured by FITC-inulin;

[0045] Figure 13 For injecting PC-Au prepared in the embodiment of the present invention 25 The two-phase fitting curve of the change of gold content in the blood of normal mice;

[0046] Figure 14 For injecting PC-Au prepared in the embodiment of the present invention 25 Two-phase fitting curve of the changes in gold content in the blood of post-DN model mice. DETAILED DESCRIPTION

[0047] The technical solution of the present invention is further described below by means of the accompanying drawings and examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Any other changes, modifications, substitutions, combinations, and simplifications made without violating the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the protection scope of the present invention. In addition, it should be understood that after reading the contents 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 claims attached to this application and belong to the scope of protection of the present invention.

[0048] References to "embodiments" herein mean that the specific features, structures, or characteristics described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the word "embodiment" in various places 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 this application, as long as there are no technical contradictions or conflicts, the various technical features mentioned in the embodiments can be combined in any manner to form a corresponding implementable technical solution.

[0049] Unless otherwise defined, the technical terms used herein have the same meanings as those generally understood by those skilled in the art to which this application belongs; the use of relevant terms herein is only for describing specific embodiments and is not intended to limit this application.

[0050] Unless otherwise specified in the present invention, the reagents, instruments, equipment and performance testing methods used are those commonly used by those skilled in the art.

[0051] Example

[0052] This embodiment provides a method for preparing a phosphatidylcholine gold nano-CT probe, which specifically includes the following steps:

[0053] Step S1, preparation of phosphatidylcholine ligand: 100 mg of 2-methacryloyloxyethyl phosphorylcholine was dissolved in 10 mL of methanol and nitrogen was passed through for 15 min; 28.7 mg of cysteine ​​hydrochloride was dissolved in 1 mL of methanol solution. After fully dissolved, the two solutions were mixed, and then 10 uL of triethylamine was added. The reaction was stirred at 400 rpm at room temperature for 4 hours. After the reaction was completed, the excess solvent was removed by rotary evaporation, and then the product was redissolved in a mixed solution of 10 mL of dichloromethane and ether. After standing for 30 min, the supernatant was removed and the product was collected. Finally, the excess solvent was removed by rotary evaporation, and 1 mL of distilled water was added to fully dissolve the product. After freeze-drying, the phosphatidylcholine ligand NH2-MPC was obtained. The preparation route is as follows: Figure 1 shown.

[0054] Step S2, ultra-small gold nanoclusters GS-Au 25 Synthesis: 38.5 mg of GSH, 10 mg of HAuCl4·3 H2O and 35 mg of borane-tert-butylamine complex were dissolved in 6 mL of H2O and reacted at 37 °C for 12 h. Gold nanoparticles were precipitated with saturated NaCl and anhydrous ethanol and purified by centrifugation through a 5 kDa ultrafiltration tube to obtain GS-Au. 25 , the synthetic route is as follows Figure 2 shown.

[0055] Step S3: PC ligand modification of GS-Au 25 :Take 2 mg GS-Au 25 Dissolve thoroughly in 1×PBS buffer solution (pH=7.2) and set aside. 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) separately and set aside. Take 3 mg of phosphatidylcholine ligand and dissolve it thoroughly in 1 mL of 1×PBS buffer solution (pH=7.2) and set aside. Then mix the above four solutions and stir them at room temperature for 6 hours. After the reaction is completed, transfer the reaction system to a 5 kDa ultracentrifuge tube for ultrafiltration purification and freeze-dry to obtain the ultrasmall phosphatidylcholine gold nanoCT probe PC-Au. 25 , the preparation route is as follows Figure 3 shown.

[0056] The basic physical and chemical properties of the intermediate products and target products obtained in the examples were characterized.

[0057] PC-Au was determined using transmission electron microscopy (TEM) 25 The core size of Figure 4 As shown by Figure 4 It can be seen that PC-Au 25 The core diameter of the nanoparticles is 0.98±0.17 nm, which is much smaller than the glomerular filtration threshold (6 nm) and can theoretically pass through glomerular filtration.

[0058] Fourier transform infrared spectrometer (FITR) and nuclear magnetic resonance 1H spectrometer ( 1 H-NMR) determination of PC ligand (NH2-MPC), GS-Au 25 or PC-Au 25 The infrared absorption spectrum and nuclear magnetic resonance hydrogen spectrum of GS-Au were measured by ultraviolet-visible spectrophotometer (UV-Vis). 25 and ultrasmall gold nanoparticles PC-Au 25 The UV-visible absorption spectrum of Figure 5 As shown. Figure 5(a) It can be seen that compared with GS-Au 25 , PC-Au 25 C=O, P=O, N(CH3) appeared 3+ And PO stretching vibration. Figure 5 (b) It can be seen that PC-Au 25 Contains GS-Au 25 and the hydrogen atoms of the PC ligands. Figure 5 (c) It can be seen that GS-Au 25 and PC-Au 25 The absorption spectra remain consistent.

[0059] The in vivo metabolic behavior of the intermediate products and target products prepared in the examples was investigated.

[0060] Eight-week-old C57BL / 6 male mice were used as research subjects and the GS-Au prepared in Example 1 was injected into the tail vein. 25 and PC-Au 25 , mouse urine was collected within 24 hours, and the mice were dissected and kidneys were collected after 24 hours; after the samples were digested with aqua regia, the gold content in each sample was determined by ICP-MS to determine the renal clearance rate of the probe and the distribution level in the kidney. The results are as follows Figure 6 As shown. Figure 6 It can be seen that the renal clearance results showed that 93.2±2.0% of PC-Au was eliminated within 2 hours. 25 The probe is excreted through urine, while GS-Au 25 Only 54.8±6.1%.

[0061] Another mouse was injected with PC-Au via tail vein. 25 Afterwards, blood samples were collected at different time points, and the gold content in each sample was determined by ICP-MS. Time-concentration curves were drawn to determine the pharmacokinetic parameters of ultrasmall biomimetic gold nanoparticles. The results are as follows: Figure 7 As shown. Figure 7 (a) It can be seen that 10 minutes after the injection of ultrasmall gold nanoparticles, the PC-Au 25 The concentration began to be significantly lower than that of GS-Au 25 .Depend on Figure 7 (b) It can be seen that PC-Au 25 The 24-hour clearance rate of GS-Au was 25 Increased by 12.3 times.

[0062] The above results indicate that phosphatidylcholine ligand-modified ultrasmall gold nanoparticles can significantly improve their metabolic clearance rate in vivo, including pharmacokinetic clearance and renal clearance. 25 The efficient clearance rate in the body lays a theoretical foundation for the accurate determination of glomerular filtration rate.

[0063] To further explore PC-Au 25 To determine whether there was room for probe optimization, a comparative screening of surface ligands was conducted. The protein binding rates of ultrasmall biomimetic gold nanoparticles and other ligand-modified ultrasmall gold nanoparticles were tested using agarose gel electrophoresis (AGE) and inductively coupled plasma mass spectrometry (ICP-MS). Renal clearance was determined using the aforementioned method. The results are shown in Table 1.

[0064] Table 1 Protein binding rate and renal clearance rate of ultrasmall gold nanoprobes modified with different surface ligands

[0065]

[0066] As shown in Table 1, among the four ultrasmall gold nanoparticles modified with different ligands, PC-Au 25 The protein binding rate of PC-Au is the lowest, indicating that PC ligand can minimize the interaction between the probe and the body. 25 The renal clearance efficiency is highest within 24 hours.

[0067] Furthermore, the core size of the probe was optimized and screened. 25 In addition, by adjusting the ratio of HAuCl₄·3H₂O to GSH in the synthetic 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 diameter of 2.5 nm were synthesized. PC-AuNPs-2.5 nm were then modified with PC ligands. The pharmacokinetic parameters and renal clearance of PC-AuNPs are shown in Table 2.

[0068] Table 2 Metabolic kinetic parameters and renal clearance of gold nanoprobes of different sizes

[0069]

[0070] As shown in Table 2, compared with PC-AuNPs-2.5 nm, the PC-Au prepared in this example 25 It is metabolized more rapidly in plasma and has a higher renal clearance efficiency.

[0071] The target product obtained in the embodiment was experimentally demonstrated to have a tubular interaction mechanism.

[0072] Probenecid and cimetidine were used to inhibit the organic anion transporters and organic cation transporters of renal tubular cells, and the renal clearance efficiency of gold nanoprobes before and after inhibition was compared. Figure 8 As shown. Figure 8It can be seen that PC-Au 25 The renal clearance of PC-Au did not change significantly. 25 There is no interaction with the renal tubules.

[0073] Based on the above, PC-Au 25 Through systematic studies of metabolic kinetics and renal clearance pathways, as well as optimization screening of surface ligand types and gold nanocore sizes, 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 It can be filtered efficiently and freely through the kidneys, laying a theoretical foundation for the accurate measurement of GFR.

[0074] Application Example 1: PC-Au prepared in Example 25 Used for CT imaging to measure GFR

[0075] (1) 8-week-old C57 BL / 6J mice were selected and injected with PC-Au 25 The mice were pre-scanned with the scanning parameters of 70 kV, 114 μA, FOV=72 mm, scanning time of 3.9 s, and detector reset time of about 11 s. 25 , the dose was 450 mg / kg, after injection of PC-Au 25 Continuous scanning was started at the same time, the number of scans was 3, and a total of 120 s of contrast images were obtained. Figure 9 As shown by Figure 9 It can be seen that imaging of the abdominal aorta was achieved 15 seconds after the probe was injected; at the 60th second, CT signals of the kidneys on both sides could be observed; at the 120th second, signal accumulation in the bladder was observed, indicating that the probe was filtered through the kidneys, formed urine and entered the bladder.

[0076] (2) GFR calculation method: circle and calculate the CT signal enhancement value of the aorta and bilateral kidneys. Figure 10 (a) and Figure 10 (b) It can be seen that the injection of PC-Au 25 After the probe was inserted, the aortic CT signal reached its peak at 15 seconds, and the bilateral kidney signal values ​​gradually increased over time. The data processing software origin was used to calculate the area under the curve of the aortic CT value over time, and the ratio of this area divided by the aortic CT value [∫b(t)dt / b(t)] was calculated; the ratio of the left and right kidney CT enhancement signal values ​​to the aortic CT value [c(t) / b(t)] was calculated; with ∫b(t)dt / b(t) as the horizontal coordinate and c(t) / b(t) as the vertical coordinate, the fitting was obtained. Figure 10 (c).

[0077] Record the slope of the linear equation, multiply it by (1-HCT), and then multiply it by the volume of each kidney to finally obtain the GFR (μL / min) of each kidney (kidney volume is obtained by 3D reconstruction of the kidney using CT software). The sum of the left and right kidneys is the total renal GFR. 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. Figure 11 It can be seen that there is good consistency between the calculated GFR and the GFR determined by the gold standard FITC-inulin method.

[0078] (3) Construction of DN mouse model: 8-week-old C57 BL / 6J mice were fed a high-fat diet (60% fat energy supply) for 8 weeks. At the 9th week, streptozotocin (STZ) (40 mg / kg) was injected intraperitoneally for 5 consecutive days and the mice continued to be fed a high-fat diet.

[0079] After 4 weeks, PC-Au 25 The GFR of normal mice and DN model mice was measured by CT imaging. The methods, parameters and data processing were the same as above. The results are shown in the figure. Figure 12 As shown. Figure 12 It can be seen that using PC-Au 25 The GFR value measured by the probe through CT imaging is consistent with the value measured by the gold standard FITC-inulin, and the R value of the relationship fitting curve between the two is 2 The value is 0.914, and the fitting effect is good, which shows that PC-Au 25 The accuracy of the probe in determining GFR in chronic kidney disease using CT imaging. 25 The probe measured the GFR value of normal mice to be 218.9±17.9 μL / min, while 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 impaired, and different GFR values ​​reflected different levels of renal function loss.

[0080] Application Example 2: PC-Au prepared in Example 25 Used for blood test to measure GFR in mice

[0081] (1) 8-week-old C57 BL / 6J mice were selected and PC-Au was injected into the tail vein. 25 The dose was 2.5 mg / kg, and a sample was reserved for later determination of the injection dose (ID). Blood was collected from the mice by orbital ischemia at 2, 5, 10, 15, 20, 40, and 60 minutes after injection, and the blood weight was recorded. 1 mL of aqua regia was added to each centrifuge tube to digest the PC-Au. 25The probe was used to obtain a gold ion solution, which was diluted and used for ICP-MS quantification of gold content.

[0082] (2) Calculate the gold concentration of each blood sample, plot the relationship 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, and t2; calculate the area under the curve AUC = A1·t1+A2·t2. Finally, calculate GFR = ID / AUC·(1-Hct), where Hct is the hematocrit, which is 0.5. Figure 13 It can be seen that PC-Au was injected into the tail vein of mice 25 One hour later, the gold concentration in the blood was measured and fitted with a two-phase exponential decay. The calculated GFR value for the mouse was 223.27±10.50 μL / min, as shown in Table 3. This result is close to the GFR value measured by the FITC-inulin method (209.55±32.00 μL / min), demonstrating the high accuracy of the blood test method for measuring mouse GFR.

[0083] Table 3 Parameters of changes in blood gold content in the experiment of measuring GFR of normal mice by blood method

[0084]

[0085] (3) Construct a DN mouse model using the same method as in Application Example 1.

[0086] Using PC-Au 25 The GFR of normal mice and DN model mice was determined by blood method, and the method and data processing were the same as above. Figure 14 It can be seen that PC-Au was injected into the tail vein of DN model mice. 25 One hour later, the gold concentration in the blood was measured and a two-phase exponential decay fitting was performed. As shown in Table 4, the GFR value of the mouse was calculated to be 150.53±14.42 μL / min. This result was significantly lower than the GFR value of normal mice (223.27±10.50 μL / min), indicating that the renal function of the DN model mice was impaired.

[0087] Table 4 Parameters of changes in blood gold content in the experiment of measuring GFR in DN mice by blood method

[0088]

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements 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 a phosphatidylcholine gold nano-CT probe, characterized in that: The specific steps include: Step S1: Preparation of phosphatidylcholine ligand NH2-MPC 2-Methacryloxyethyl phosphorylcholine was dissolved in a methanol solution and nitrogen was passed through for 15 minutes; cysteine ​​hydrochloride was dissolved in a methanol solution, and after being fully dissolved, the two solutions were mixed, triethylamine was added, and the mixture was stirred at room temperature for reaction. After the reaction was completed, excess solvent was removed by rotary evaporation, and then a dichloromethane-ether mixed solution was added to dissolve the mixture. The supernatant was removed after standing, and excess solvent was removed by rotary evaporation again. Finally, distilled water was added to dissolve the product, and lyophilized to obtain NH2-MPC; Step S2, gold nanoclusters GS-Au 25 Synthesis GSH, HAuCl4·3H2O and borane-tert-butylamine complex were dissolved in H2O and reacted at 37°C. After the reaction, saturated NaCl and anhydrous ethanol were used to precipitate gold nanoparticles, which were then placed in an ultrafiltration tube and centrifuged to obtain GS-Au. 25 ; Step S3, NH2-MPC ligand modification of GS-Au 25 Preparation of PC-Au 25 Take GS-Au 25 , 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and NH2-MPC were dissolved in 1×PBS buffer solution respectively for later use; the above four solutions were mixed and stirred at room temperature for reaction. After the reaction was completed, the reaction system was transferred to an ultracentrifuge tube for ultrafiltration purification and freeze-dried to obtain the target product phosphatidylcholine gold nano-CT probe PC-Au 25 ; Among them, GS-Au 25 The mass volume ratio of the 1× PBS buffer solution is 2 mg:1 mL; The mass volume ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1×PBS buffer solution is 7 mg:1 mL; The mass volume ratio of N-hydroxysuccinimide and 1× PBS buffer solution is 4 mg:1 mL; The mass volume ratio of NH2-MPC and 1×PBS buffer solution is 3 mg:1 mL.

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

3. The method for preparing a phosphatidylcholine gold nano-CT probe according to claim 1, characterized in that: In step S1, the reaction was stirred at 400 rpm at room temperature for 4 hours.

4. The method for preparing a phosphatidylcholine gold nano-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 method for preparing a phosphatidylcholine gold nano-CT probe according to claim 1, characterized in that: In step S2, the reaction was carried out at 37° C. for 12 h, and the molecular weight cut-off of the ultrafiltration tube was 5 kDa.

6. The method for preparing a phosphatidylcholine gold nano-CT probe according to claim 1, characterized in that: In step S3, the pH value of the 1×PBS buffer solution is 7.

2.

7. The method for preparing a phosphatidylcholine gold nano-CT probe according to claim 1, characterized in that: In step S3, the reaction was stirred at room temperature for 6 h, and the molecular weight cut-off of the ultracentrifuge tube was 5 kDa.

8. A phosphatidylcholine gold nano-CT probe, characterized in that: The phosphatidylcholine gold nano-CT probe is prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the phosphatidylcholine gold nano-CT probe according to claim 8 in the preparation of a probe for detecting glomerular filtration rate.

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