Preparation method and application of bismuth ion chelated ultra-small gold nanoparticles cleared through kidney

Abstract

The invention discloses a preparation method and application of bismuth ion chelated ultra-small gold nanoparticles cleared through kidneys, and belongs to the technical field of CT contrast agents. The preparation method comprises the following steps: S1, preparing sulfydryl chelating ligand protected ultra-small gold nanoparticles Au (at) DTDTPA through a chemical reduction method; and S2, the bismuth element is connected to the surface of Au and DTDTPA prepared in the step S1 through the metal chelation effect, and the bismuth ion chelated ultra-small gold nanoparticles Au and DTDTPA-Bi are prepared. According to the preparation method and application of the bismuth ion chelated ultra-small gold nanoparticles cleared through the kidney, the prepared nanoparticles not only meet the requirements of glomerular filter membrane metabolism in the aspect of the size effect, but also are successfully chelated through positive trivalent bismuth ions in the aspect of the charge effect; the nano-particles with negative charges are converted into nano-particles with positive charges, so that the in-vivo retention time is relatively prolonged, the kidney CT imaging time window is prolonged, and the imaging effect is improved.

Description

Technical Field

[0001] The invention relates to the field of CT contrast agents, and in particular to a preparation method and application of bismuth ion chelated ultrasmall gold nanoparticles cleared by kidneys. Background Art

[0002] At present, various imaging technologies such as ultrasound imaging (US), computed tomography (CT), and magnetic resonance imaging (MRI) are widely used in clinical practice. Among them, CT occupies an important position in emergency diagnosis due to its advantages such as clear imaging, fast speed, wide applicability, and few contraindications, and has become a common means for doctors to detect human body structure.

[0003] CT contrast agents are key auxiliary substances in CT imaging technology. Their main function is to enhance the contrast of CT scan images and help doctors observe the internal structures and lesions of the body more clearly. In medical diagnosis, different tissues and organs of the human body have different absorption degrees of X-rays, but this difference is sometimes not enough for doctors to accurately judge the condition of the lesion. The emergence of CT contrast agents solves this problem. It can change the X-ray absorption characteristics of the target tissue or organ, increase the contrast between the lesion and normal tissue, and enable doctors to more accurately observe the location, shape, size and relationship of the lesion with surrounding tissues, effectively improving the early detection and diagnosis accuracy of the disease. In order to improve CT contrast agents, some researchers have proposed a new type of metal nano CT contrast agent based on high atomic number metal elements and special metal elements, which has the advantages of targeted imaging, renal imaging, and vascular imaging.

[0004] At present, iodine-containing CT contrast agents widely used in clinical practice, such as iopromide and iohexol, have enhanced imaging contrast to a certain extent, but have many disadvantages, including:

[0005] (1) Potential renal toxicity: The potential renal toxicity of iodinated contrast agents has always been a major hidden danger in clinical applications. During their metabolism in the body, they may have adverse effects on the normal physiological functions of the kidneys, especially for patients who already have certain problems with their renal function. The risk of renal damage after using iodinated contrast agents is significantly increased.

[0006] (2) Limited contrast improvement: The contrast of iodine contrast agents is limited. When diagnosing some tiny lesions or lesions with little contrast difference from surrounding tissues, iodine contrast agents may not provide sufficiently clear images, making it difficult for doctors to accurately judge the nature and extent of the lesions.

[0007] (3) The imaging time is short; the iodine contrast agent is cleared from the blood vessels quickly, which makes the imaging time shorter and increases the difficulty and uncertainty of diagnosis.

[0008] In addition to the above problems, iodine contrast agents may also cause thyroid problems and affect the normal function of the thyroid gland. For patients with abnormal thyroid function, the use of iodine contrast agents may aggravate the condition. In addition, iodine contrast agents also have the risk of allergic reactions. The severity of allergic reactions varies, ranging from mild rash and itching to severe anaphylactic shock, which poses a threat to the patient's life safety. Summary of the invention

[0009] The purpose of the present invention is to provide a preparation method and application of bismuth ion chelated ultrasmall gold nanoparticles that are cleared by the kidneys, so as to solve the problems of kidney toxicity, limited contrast enhancement, short imaging time, etc. of current iodine contrast agents.

[0010] To achieve the above object, in a first aspect, the present invention provides a method for preparing bismuth ion chelated ultrasmall gold nanoparticles that are cleared by the kidney, comprising the following steps:

[0011] S1. Preparation of ultrasmall gold nanoparticles Au@DTDTPA protected by thiol chelating ligands by chemical reduction method;

[0012] S2. Bismuth element is attached to the surface of Au@DTDTPA prepared in step S1 through metal chelation to prepare bismuth ion-chelated ultrasmall gold nanoparticles Au@DTDTPA-Bi.

[0013] Preferably, in step S1, the preparation of the thiol chelating ligand comprises:

[0014] S21, dissolving diethylenetriaminepentaacetic acid (DTPA) in DMF, stirring at 70° C. to obtain a DTPA solution;

[0015] S22, dissolving cysteamine hydrochloride in DMF, ultrasonically mixing, dropping triethylamine, mixing, and rapidly adding to the DTPA solution of step S21, stirring at 70° C. for 13 hours;

[0016] S23. After the stirring in step S22 is completed, the mixture is placed in an ice bath for 20 minutes, the precipitate is filtered out, the filtrate is concentrated by rotary evaporation to one time, and then dripped into a chloroform solution with a volume exceeding 10 times, the precipitated white precipitate is collected, and the white precipitate is dried to obtain the product thiol chelating ligand DTDTPA.

[0017] Preferably, in step S21, the mass volume ratio of diethylenetriaminepentaacetic acid:DMF is 50 mg:1 mL.

[0018] Preferably, in step S22, the mass volume ratio of cysteamine hydrochloride:DMF:triethylamine is 101.5 mg:3.875 mL:108.6 μL.

[0019] Preferably, in step S1, the preparation of Au@DTDTPA comprises:

[0020] S31, dissolve tetrachloroauric acid in methanol solution to prepare 1.67 mg / mL Au 3+ Methanol solution;

[0021] S32, dissolving the thiol chelate ligand in methanol solution, adding acetic acid, mixing well, and then adding the Au 3+ The mixture was stirred in methanol solution at room temperature for 20 min to obtain reaction solution Ⅰ;

[0022] S33, adding 1.37 mg / mL sodium borohydride aqueous solution to reaction solution I, and stirring at room temperature for 1 hour;

[0023] S34. After the stirring in step S33 is completed, the precipitate is collected by centrifugation, and the precipitate is washed with 0.01M hydrochloric acid, water, and ether in sequence. The precipitate is added into 0.01M sodium hydroxide and dissolved to obtain an Au@DTDTPA solution.

[0024] Preferably, in step S32, thiol chelating ligand: methanol solution: acetic acid: Au 3+ The mass volume ratio of the methanol solution is 48.2 mg:4 mL:200 μL:12 mL.

[0025] Preferably, in step S33, the volume ratio of sodium borohydride aqueous solution: reaction solution I is 1.4 mL:16.2 mL.

[0026] Preferably, in step S2, the preparation of Au@DTDTPA-Bi comprises:

[0027] S41, adding an equal volume of water to the Au@DTDTPA solution, stirring at 95°C for 5 min, to obtain reaction solution II;

[0028] S42, dissolve bismuth nitrate pentahydrate with 5% nitric acid by ultrasonication, add 800 μL of water to obtain Bi 3+ Solution;

[0029] S43, according to Bi 3+ Solution: reaction solution II volume ratio 0.8mL: 8mL Bi 3+ The solution was added to reaction solution II and stirred at 95 °C for 20 min;

[0030] After the stirring reaction in step S44 and step S43 is completed, the solution is washed and purified with a 3000Da ultrafiltration tube to obtain an Au@DTDTPA-Bi aqueous solution, which is then freeze-dried to obtain an Au@DTDTPA-Bi powder.

[0031] Preferably, in step S42, the mass volume ratio of bismuth nitrate pentahydrate: 5% nitric acid solution: water is 15.4 mg: 200 μL: 800 μL.

[0032] In a second aspect, the present invention provides an application of bismuth ion chelated ultrasmall gold nanoparticles prepared by the above-mentioned preparation method in CT imaging for non-therapeutic or diagnostic purposes.

[0033] Therefore, the preparation method and application of bismuth ion chelated ultrasmall gold nanoparticles cleared by kidneys of the present invention have the following beneficial effects:

[0034] (1) Ultra-small gold nanoparticles protected by thiol metal chelates (Au@DTDTPA) were prepared by chemical reduction. Then, high atomic number bismuth elements were attached to the surface of ultra-small gold nanoparticles through metal chelation to prepare ultra-small gold nano-CT contrast agents chelated with bismuth ions (Au@DTDTPA-Bi). The nanoparticles not only meet the requirements of glomerular filtration membrane metabolism (particle size <6mn) in terms of size effect, but also successfully chelate trivalent bismuth ions in terms of charge effect, transforming the original negatively charged nanoparticles into positively charged nanoparticles, which relatively prolongs the in vivo retention time, prolongs the renal CT imaging time window, and improves the imaging effect.

[0035] (2) After Au@DTDTPA nanoparticles chelate bismuth ions, although Au@DTDTPA-Bi is positively charged as a whole, it can still effectively resist serum protein adsorption, escape liver recognition and capture, and maintain good renal metabolic clearance ability;

[0036] (3) Through tail vein injection, it was found that the metal chelate Au@DTDTPA-Bi could be largely metabolized through urine 3 hours after injection, and the renal accumulation was low 24 hours after tail vein injection;

[0037] (4) Both gold and bismuth are high atomic number metals with strong absorption of X-rays, which can provide good CT imaging contrast; however, there are certain defects when used alone. The combined effect of the two makes up for the poor stability of bismuth and the expensive cost of gold, achieving complementary disadvantages and maximizing advantages; through in vitro X-ray absorption capacity test, it was found that the CT signal value of gold nanoparticles after chelating bismuth ions was significantly improved compared with iodine and single gold;

[0038] (5) Through in vivo CT imaging experiments, it was found that bismuth ion chelated ultrasmall gold nanoparticles have the advantages of slowly arriving at the kidneys and having a longer peak retention time in kidney imaging. The slowly arriving contrast agent is suitable for delayed scanning, which is convenient for observing the renal parenchyma and collecting system. It does not require precise control of the scanning timing and does not need to worry about missing the optimal imaging time. In addition, the imaging effect of the nanoparticles in the renal parenchyma area is better than that of iopromide used clinically.

[0039] (6) The bismuth ion chelated ultrasmall gold nanoparticles of the present invention have high stability, high renal clearance efficiency and high X-ray absorption capacity, providing a new strategy for solving and improving the defects of clinical CT contrast agents such as fast clearance time and potential nephrotoxicity.

[0040] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0042] Figure 1 Schematic diagram of the structure of Au@DTDTPA-Bi nanoparticles;

[0043] Figure 2 The UV absorption spectra of DTDTPA, DTDTPA-Bi, Au-DTDTPA, and Au-DTDTPA-Bi;

[0044] Figure 3 is the Zeta potential of Au@DTDTPA and Au@DTDTPA-Bi;

[0045] Figure 4 Agarose gel electrophoresis comparison of Au@DTDTPA and Au@DTDTPA-Bi;

[0046] Figure 5 TEM images of Au@DTDTPA and Au@DTDTPA-Bi, where (a) is Au@DTDTPA; (b) is Au@DTDTPA-Bi;

[0047] Figure 6 Agarose gel electrophoresis diagrams of the interaction between Au@DTDTPA and Au@DTDTPA-Bi and serum proteins, where (a) is Au@DTDTPA; (b) is Au@DTDTPA-Bi;

[0048] Figure 7 The distribution of Au@DTDTPA-Bi in the main tissues in the body;

[0049] Figure 8 The distribution of Au@DTDTPA-Bi in urine;

[0050] Fig. 9Comparison of the X-ray absorption capacity of iopromide, Au@DTDTPA and Au@DTDTPA-Bi, where (a) is the X-ray scanning diagram; (b) is the linear curve of CT signal value and ion concentration;

[0051] Fig.10 The kidney and bladder imaging effects under CT after Au@DTDTPA-Bi was injected into the tail vein of normal ICR mice, where (a) is the kidney; (b) is the bladder;

[0052] Fig.11 Au@DTDTPA-Bi and iopromide were injected into the tail vein of normal ICR mice to achieve kidney imaging time contrast, where (a) is iopromide; (b) is Au@DTDTPA-Bi;

[0053] Fig.12 Comparison of the peak retention times of Au@DTDTPA-Bi and iopromide, where (a) is iopromide; (b) is Au@DTDTPA-Bi;

[0054] Fig.13 The imaging area of ​​the renal parenchyma of normal ICR mice injected with Au@DTDTPA-Bi and iopromide through the tail vein, where (a) is iopromide; (b) is Au@DTDTPA-Bi;

[0055] Fig.14 Data comparison of Au@DTDTPA-Bi and iopromide in the renal parenchyma area, where (a) is the single-peak fitting curve obtained according to the renal parenchyma CT signal values ​​at different time points; (b) is the area under the single-peak fitting curve obtained according to the renal parenchyma CT signal values ​​at different time points. DETAILED DESCRIPTION

[0056] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0057] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0058] The instruments, equipment, reagents and materials used in the examples were all obtained through commercial channels.

[0059] Embodiment 1

[0060] The synthesis of ultrasmall gold nanoparticles protected by diethylenetriaminepentaacetic acid (DTPA) chelating agent and the bismuth ion chelation process are as follows:

[0061] S1. Preparation of thiol chelating ligand DTDTPA:

[0062] 125 mg of DTPA was dissolved in 2.5 mL of DMF and stirred at 70° C. for 5 min to obtain a DTPA solution.

[0063] 101.5 mg of cysteamine hydrochloride was dissolved and mixed by ultrasonication in 3.875 mL of DMF, 108.6 μL of triethylamine was added dropwise to the solution, and after mixing, it was quickly added to the DTPA solution, and stirring was continued at 70° C. for 13 hours.

[0064] After the reaction is completed, the mixture is placed in an ice bath for 20 minutes to remove the precipitate. The remaining product is concentrated by rotary evaporation to one-fold and then dripped into a chloroform solution with a volume exceeding 10 times. The precipitated white precipitate is collected and dried to obtain the product DTDTPA for use.

[0065] S2. Preparation of Au@DTDTPA NPs:

[0066] Dissolve 20 mg of tetrachloroauric acid in 12 mL of methanol to obtain a solution containing trivalent gold ions (Au 3+ ) in methanol;

[0067] Dissolve 48.2 mg DTDTPA in 4 mL methanol and add 200 μL acetic acid. Mix well and add to Au 3+ The mixture was added to a methanol solution of 1.4 mL of water and stirred at room temperature for 20 minutes. The solution quickly changed from golden transparent to orange turbidity and then to clear transparent to obtain reaction solution I. 19.2 mg of sodium borohydride was dissolved in 1.4 mL of water and added to reaction solution I. Stirring was continued for 1 hour. The solution quickly changed to brown-black.

[0068] After the reaction was completed, the precipitate was collected by centrifugation and washed with 0.01 M hydrochloric acid, water, and ether in sequence, and finally 4 mL of 0.01 M sodium hydroxide was added to dissolve the precipitate to obtain the Au@DTDTPA solution.

[0069] S3. Preparation of Au@DTDTPA-Bi NPs:

[0070] An equal volume of water was added to the Au@DTDTPA solution synthesized in step S2, and the mixture was stirred at 95° C. for 5 min to obtain reaction solution II.

[0071] 15.4 mg of bismuth nitrate pentahydrate was dissolved by ultrasonication in 200 μL of 5% nitric acid, and then 800 μL of water was added to obtain Bi 3+ After mixing, take 800 μL of the solution and add it to reaction solution II, and continue stirring at 95 °C for 20 min.

[0072] After the reaction is completed, the Au@DTDTPA-Bi aqueous solution is obtained by washing and purification with a 3000Da ultrafiltration tube, and then freeze-drying is performed to obtain a powder sample (bismuth-chelated ultrasmall gold nanoparticles).

[0073] Schematic diagram of the structure of Au@DTDTPA-Bi nanoparticles Figure 1 shown.

[0074] Embodiment 2

[0075] The preparation process of DTDTPA-Bi is as follows:

[0076] S1. Preparation of thiol chelating ligand DTDTPA:

[0077] 125mg DTPA was dissolved in 2.5mL DMF and stirred at 70℃ to obtain DTPA solution. 101.5mg cysteamine hydrochloride was dissolved in 3.875mL DMF by ultrasonic mixing, 108.6μL triethylamine was added dropwise to the solution, mixed and quickly added to the DTPA solution, and continued to stir at 70℃ for 13 hours. After the reaction was completed, ice bath was added for 20min to remove the precipitate, the remaining product was concentrated by rotary evaporation to one time, and then dropped into a chloroform solution with a volume of more than 10 times, the precipitated white precipitate was collected, and dried to obtain the product DTDTPA for use.

[0078] S2. Preparation of DTDTPA-Bi:

[0079] Weigh 20 mg of DTDTPA synthesized in step S1 and dissolve it in 4 mL of water. Ultrasonic dissolve 15.4 mg of bismuth nitrate pentahydrate in 200 μL of 5% nitric acid, and then add 800 μL of water to obtain Bi 3+ The solution was mixed and 800 μL was added to the DTDTPA solution. The mixture was stirred at room temperature for 20 min. After the reaction was completed, DTDTPA-Bi was obtained.

[0080] Test Example 1

[0081] The UV absorption spectra of DTDTPA, DTDTPA-Bi, Au@DTDTPA and Au-DTDTPA-Bi are shown in Figure 2. Figure 2 As shown in the figure, the gold nanoparticles protected by DTDTPA chelator (Au@DTDTPA) and the gold nanoparticles chelated by bismuth ions (Au@DTDTPA-Bi) did not have a surface plasmon resonance absorption peak at a wavelength of 500-550nm, indicating that both are ultra-small gold nanoparticles. In addition, the ultraviolet absorption of Au@DTDTPA-Bi showed the characteristic absorption peak of DTDTPA-Bi, indicating that the bismuth ions were successfully chelated on Au@DTDTPA.

[0082] Zeta potentials of Au@DTDTPA and Au@DTDTPA-Bi are shown in Figure 3 As shown in the figure, the potential of AuNPs after bismuth ion chelation changes from negative to positive. This is because the positive trivalent bismuth ions are attached to the surface of AuNPs through chelation, making the particles positively charged as a whole.

[0083] Further characterization by agarose gel electrophoresis showed that the negatively charged Au@DTDTPA moved toward the positive electrode, and the negatively charged Au@DTDTPA-Bi moved toward the negative electrode, as shown in Figure 4 Combined with the above results, it is shown that the above method can successfully prepare ultra-small gold nanoparticles chelated with bismuth ions.

[0084] Through transmission electron microscopy (TEM) observation, Figure 5 As shown, it can be clearly observed that the synthesized Au@DTDTPA and Au@DTDTPA-Bi are both monodisperse nanoparticles with ultrasmall core sizes. The core size of Au@DTDTPA-Bi is less than 3 nm, and the size and monodispersity of the gold nanoparticles are not changed after chelating bismuth ions.

[0085] Test Example 2

[0086] Agarose gel electrophoresis was used to investigate the interaction between Au@DTDTPA-Bi and serum proteins. Two experimental groups were prepared: Au@DTDTPA and Au@DTDTPA-Bi nanoparticles co-incubated with mouse serum; and three control groups: Au@DTDTPA and Au@DTDTPA-Bi pure nanoparticles groups and pure mouse serum groups. All groups were incubated at 37°C for 30 minutes before the experiment.

[0087] like Figure 6 As shown in (a), the performance of the nanoparticles after co-incubation with serum in agarose gel electrophoresis is not significantly different from that of the nanoparticles without serum added, indicating that Au@DTDTPA-Bi has anti-protein adsorption ability and is stable in serum.

[0088] like Figure 6 As shown in (b), the fluorescence of the gold nanoparticles is enhanced after chelating the bismuth ions, which is due to the bismuth ions chelating on the surface of the gold nanoparticles, which enhances the ion resonance frequency, further proving the successful preparation of the ultra-small gold nanoparticles chelated with bismuth ions in Example 1.

[0089] Test Example 3

[0090] From test examples 1 to 2, it can be seen that Au@DTDTPA-Bi has anti-serum protein binding ability and its core size is less than 3nm, which allows the nanoparticles to be effectively cleared from the blood to the kidneys and then excreted from the body through urine.

[0091] like Figure 7As shown, normal ICR mice were selected and 200 μL Au 3+ After 24 hours of tail vein injection of Au@DTDTPA-Bi at a concentration of 10 mg / mL (solvent: 1 M HEPES buffer), the gold and bismuth contents in the kidneys were 16.01% ID / g and 6.99% ID / g, respectively, and there was very low accumulation in tissues such as the spleen and liver.

[0092] like Figure 8 As shown, 3 hours after tail vein injection of Au@DTDTPA-Bi, the urinary clearance rates of gold and bismuth were as high as 40.83%ID and 55.04%ID, respectively; 24 hours after injection, more than 47%ID of Au@DTDTPA-Bi could be excreted through urine.

[0093] The above results indicate that Au@DTDTPA-Bi can be metabolized by kidneys.

[0094] Test Example 4

[0095] CT in vitro experiments show that bismuth ion chelated ultra-small gold nanoparticles Au@DTDTPA-Bi have stronger absorption ability for X-rays

[0096] Different concentration gradients of iopromide (I), Au@DTDTPA and Au@DTDTPA-Bi (0, 0.25, 0.5, 1, 2, 4 mg / mL) were set, and their absorption capacity of X-rays was tested by X-ray scanning. The results are shown in Figure 7 shown.

[0097] like Fig. 9 As shown in (a), the brightness comparison between iopromide (I), Au@DTDTPA and Au@DTDTPA-Bi shows that (the higher the brightness, the higher the HU value of the CT signal and the stronger the X-ray absorption ability). Under the same scanning mode and the same concentration, Au@DTDTPA-Bi has the strongest X-ray absorption ability, followed by Au@DTDTPA and finally iopromide.

[0098] The linear curve of CT signal value and ion concentration is made by ΔHU value, such as Fig. 9 As shown in (b), it is numerically more intuitive to illustrate that Au@DTDTPA-Bi theoretically has better CT imaging effect.

[0099] The above results show that the absorption capacity of gold nanoparticles to X-rays is significantly enhanced after bismuth ion chelation.

[0100] Test Example 5

[0101] Au@DTDTPA-Bi was injected into the tail vein of normal ICR mice, and real-time CT scanning was performed. The CT scanning mode was selected at 70kV, 88mA, and the single scanning time was 2min. The imaging effects of kidney and bladder were observed before scanning injection, and at 5min, 15min, 30min, 45min, 1 hour, 1.5 hours and 2 hours after injection.

[0102] like Fig.10 As shown in (a), after injection via the tail vein, Au@DTDTPA-Bi can be seen passing through the kidneys with the naked eye at 15 minutes, and the kidney imaging effect is still visible at 45 minutes.

[0103] like Fig.10 As shown in (b), the mouse bladder began to be imaged clearly at 15 minutes. The above phenomenon is consistent with the experimental results in test 3, further verifying that Au@DTDTPA-Bi is metabolized by the kidneys after injection into the body and eliminated from the body in the form of urine. This shows that bismuth-chelated ultrasmall gold nanoparticles can be used as CT contrast agents to achieve kidney imaging.

[0104] Test Example 6

[0105] Normal ICR mice were injected with iopromide (20 μM) through the tail vein, and the CT scanning mode was selected at 70 kV, 88 mA, and a single scan time of 2 min. The renal imaging effect was observed before scanning injection, half an hour after injection, 45 minutes, and 1 hour. Normal ICR mice were injected with the same dose (20 μM) of Au@DTDTPA-Bi through the tail vein, and the CT scanning mode and scanning time point were the same as above, and the renal imaging effect was observed.

[0106] like Fig.11 As shown in the figure, iopromide quickly reaches the kidneys, and kidney signals can be seen 2 minutes after injection, which is the peak. Then the signal decays immediately and disappears 45 minutes after injection. Au@DTDTPA-Bi starts to light up the kidneys 12 minutes after injection, reaches the peak at 14 minutes, and continues until 20 minutes when it begins to decay. Kidney signals can still be seen 45 minutes after injection, and disappear 1 hour later.

[0107] like Fig.12 As shown in the figure, a single peak fitting curve is obtained according to the renal pelvic CT signal value at different time points. The data show that the peak retention time of iopromide is very short, and the peak retention time of Au@DTDTPA-Bi is relatively long. This shows that the iodine contrast agent is metabolized and cleared very quickly by the kidney, while Au@DTDTPA-Bi is metabolized more slowly by the kidney.

[0108] The above results show that Au@DTDTPA-Bi has the advantages of slower arrival at the kidney and longer peak retention time in renal imaging.

[0109] Test Example 7

[0110] Normal ICR mice were injected with iopromide (20 μM) through the tail vein, and the CT scanning mode was selected at 70 kV, 88 mA, and a single scan time of 2 min. The renal imaging effect was observed before scanning injection, half an hour after injection, 45 minutes, and 1 hour. Normal ICR mice were injected with the same dose (20 μM) of Au@DTDTPA-Bi through the tail vein, and the CT scanning mode and scanning time point were the same as above, and the renal imaging effect was observed.

[0111] like Fig.13 As shown, the renal parenchyma areas at different time points were divided and their CT signal values ​​were compared.

[0112] like Fig.14 As shown, the single-peak fitting curve and the area under the curve were obtained according to the renal parenchyma CT signal values ​​at different time points. The CT signal value of Au@DTDTPA-Bi in the renal parenchyma area was significantly higher than that of iopromide.

[0113] The above results show that, at the same dose, Au@DTDTPA-Bi has more advantages than iopromide in renal parenchyma imaging.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. 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 solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing bismuth ion chelated ultrasmall gold nanoparticles that are cleared by the kidneys, characterized in that: The steps include: S1. Preparation of ultrasmall gold nanoparticles Au@DTDTPA protected by thiol chelating ligands by chemical reduction method; S2. Bismuth element is attached to the surface of Au@DTDTPA prepared in step S1 through metal chelation to prepare bismuth ion-chelated ultrasmall gold nanoparticles Au@DTDTPA-Bi.

2. The method for preparing bismuth ion chelated ultrasmall gold nanoparticles cleared by kidneys according to claim 1, characterized in that: In step S1, the preparation of the thiol chelating ligand comprises: S21, dissolving diethylenetriaminepentaacetic acid (DTPA) in DMF, stirring at 70° C. to obtain a DTPA solution; S22, dissolving cysteamine hydrochloride in DMF, ultrasonically mixing, dropping triethylamine, mixing, and rapidly adding to the DTPA solution of step S21, stirring at 70° C. for 13 hours; S23. After the stirring in step S22 is completed, the mixture is placed in an ice bath for 20 minutes, the precipitate is filtered out, the filtrate is concentrated by rotary evaporation to one time, and then dripped into a chloroform solution with a volume exceeding 10 times, the precipitated white precipitate is collected, and the white precipitate is dried to obtain the product thiol chelating ligand DTDTPA.

3. The method for preparing the bismuth ion chelated ultrasmall gold nanoparticles cleared by the kidney according to claim 2, characterized in that: In step S21, the mass volume ratio of diethylenetriaminepentaacetic acid:DMF is 50 mg:1 mL.

4. The method for preparing bismuth ion chelated ultrasmall gold nanoparticles cleared by the kidney according to claim 2, characterized in that: In step S22, the mass volume ratio of cysteamine hydrochloride:DMF:triethylamine is 101.5 mg:3.875 mL:108.6 μL.

5. The method for preparing bismuth ion chelated ultrasmall gold nanoparticles cleared by kidney according to claim 1, characterized in that: In step S1, the preparation of Au@DTDTPA includes: S31, dissolve tetrachloroauric acid in methanol solution to prepare 1.67 mg / mL Au 3+ Methanol solution; S32, dissolving the thiol chelate ligand in methanol solution, adding acetic acid, mixing well, and then adding the Au 3+ The mixture was stirred in methanol solution at room temperature for 20 min to obtain reaction solution Ⅰ; S33, adding 1.37 mg / mL sodium borohydride aqueous solution to reaction solution I, and stirring at room temperature for 1 hour; S34. After the stirring in step S33 is completed, the precipitate is collected by centrifugation, and the precipitate is washed with 0.01M hydrochloric acid, water, and ether in sequence. The precipitate is added into 0.01M sodium hydroxide and dissolved to obtain an Au@DTDTPA solution.

6. The method for preparing bismuth ion chelated ultrasmall gold nanoparticles cleared by kidney according to claim 5, characterized in that: In step S32, thiol chelate ligand: methanol solution: acetic acid: Au 3+ The mass volume ratio of the methanol solution is 48.2 mg:4 mL:200 μL:12 mL.

7. The method for preparing bismuth ion chelated ultrasmall gold nanoparticles cleared by kidney according to claim 5, characterized in that: In step S33, the volume ratio of sodium borohydride aqueous solution: reaction solution I is 1.4 mL:16.2 mL.

8. The method for preparing bismuth ion chelated ultrasmall gold nanoparticles cleared by kidneys according to claim 1, characterized in that: In step S2, the preparation of Au@DTDTPA-Bi includes: S41, adding an equal volume of water to the Au@DTDTPA solution, stirring at 95°C for 5 min, to obtain reaction solution II; S42, dissolve bismuth nitrate pentahydrate with 5% nitric acid by ultrasonication, add 800 μL of water to obtain Bi 3+ Solution; S43, according to Bi 3+ Solution: reaction solution II volume ratio 0.8mL: 8mL Bi 3+ The solution was added to reaction solution II and stirred at 95 °C for 20 min; After the stirring reaction in step S44 and step S43 is completed, the solution is washed and purified with a 3000Da ultrafiltration tube to obtain an Au@DTDTPA-Bi aqueous solution, which is then freeze-dried to obtain an Au@DTDTPA-Bi powder.

9. The method for preparing bismuth ion chelated ultrasmall gold nanoparticles cleared by kidneys according to claim 8, characterized in that: In step S42, the mass volume ratio of bismuth nitrate pentahydrate: 5% nitric acid solution: water is 15.4 mg: 200 μL: 800 μL.

10. Use of bismuth ion chelated ultrasmall gold nanoparticles prepared by the preparation method according to any one of claims 1 to 9 in CT imaging for non-therapeutic or diagnostic purposes.

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