Rare earth ion doped gold nanocluster contrast agent for X-ray imaging and preparation method thereof

Abstract

The invention provides a rare earth ion doped gold nanocluster contrast agent for X-ray imaging and a preparation method of the rare earth ion doped gold nanocluster contrast agent. The molecular formula of the rare earth ion doped gold nanocluster contrast agent is Au24Er1SR18, and SR represents a ligand glutathione. The rare earth ion doped gold nanocluster contrast agent has the characteristics of low toxicity, ultra-small size, good biocompatibility and the like, and meanwhile, the doping of rare earth ions further improves the stability of the gold nanocluster without changing the original structure of the gold cluster. The rare earth ion doped gold nanocluster can be used for a stomach X-ray imaging contrast agent, realizes X-ray imaging of soft tissues such as gastrointestinal tracts and the like, and has a good application prospect in the fields of detection of gastrointestinal diseases and the like.

Description

Technical Field

[0001] The present invention relates to the field of X-ray imaging, and in particular to a rare earth ion-doped gold nanocluster contrast agent for X-ray imaging and a preparation method thereof. Background Art

[0002] X-ray imaging, as a non-invasive imaging examination, has been widely used in the field of medical diagnosis. Since X-rays have strong penetrating power and different tissues in the body absorb X-rays, X-ray imaging has become an effective means to visualize the internal tissues of the body. According to the principle of X-ray absorption, the absorption of X-rays by atoms with larger atomic numbers is stronger, so bones are easily detected by X-ray imaging. However, some soft tissues are difficult to detect due to their weak absorption of X-rays, especially for the detection of gastrointestinal diseases. X-ray contrast agents are a type of material that has a strong absorption capacity for X-rays, can enhance the contrast of different tissues or structures in X-ray imaging, and has the characteristics of low toxicity and good biocompatibility. These contrast agents usually contain elements with higher atomic numbers such as iodine, barium and gold. In biomedical research and medical diagnosis, by enhancing the contrast of X-ray imaging, organs, blood vessels and soft tissue structures in the body can be observed more clearly. At present, X-ray imaging contrast agents such as barium meal and iodine-based compounds have been developed. Although they can improve the resolution and sensitivity of gastrointestinal X-ray imaging, they still have serious side effects. For example, barium meal is an insoluble substance and may be retained in the gastrointestinal tract after examination, making it difficult to metabolize. In addition, barium meal is not suitable for patients with gastrointestinal perforation, and iodine-based compounds may cause allergic reactions and have nephrotoxicity. In recent years, with the development of nanomaterials, it has gradually become a substitute for traditional X-ray contrast agents. Therefore, it is of great significance to develop a small-sized, low-toxic, and biocompatible nanomaterial as an X-ray imaging contrast agent.

[0003] Gold nanoclusters (AuNCs) are nanomaterials composed of several to dozens of gold atoms. Due to their ultra-small size (2-3 nm), low toxicity, excellent biocompatibility and unique physical and chemical properties, they have attracted extensive research interest in the biomedical field. In addition, the high atomic number (Z=79) and K edge value (80.7keV) of gold make gold nanoclusters have good X-ray imaging capabilities. Existing gold-based X-ray imaging contrast agents are mainly gold nanoparticles, whose particle size exceeds 10 nm, exceeding the clearance threshold of the kidney (about 5.5 nm), and are easily accumulated in the body to produce toxicity.

[0004] Based on this, the present invention uses glutathione as a ligand to synthesize gold nanoclusters with ultra-small size (about 2 nm), and doping erbium ions further improves its stability. The synthesized rare earth ion-doped gold nanoclusters have good stability, biocompatibility and X-ray imaging ability. The mice were fed with 0.2 ml of gold nanoclusters (50 mg / mL) by oral gavage. The mice after gavage were irradiated with X-rays, and the gastrointestinal contours of the mice in different time periods could be clearly recorded by the X-ray flat panel detector, realizing real-time monitoring of gastrointestinal motility. In summary, the rare earth ion-doped gold nanoclusters developed by the present invention are used as gastric X-ray imaging contrast agents, which can realize X-ray imaging of soft tissues such as the gastrointestinal tract. Compared with the existing commercial contrast agent iohexol, the synthesized gold nanoclusters of the present invention have significantly enhanced absorption capacity for X-rays, improved its sensitivity and resolution, and provide ideas for evaluating gastrointestinal motility disorders and realizing early diagnosis of gastrointestinal diseases. Summary of the invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the present invention provides a rare earth ion doped gold nanocluster contrast agent for X-ray imaging and a preparation method thereof. The present invention provides a gold nanocluster protected by glutathione and doped with rare earth ions, which has strong X-ray imaging ability, good stability and good biocompatibility.

[0006] The present invention is achieved through the following technical solutions.

[0007] A rare earth ion doped gold nanocluster contrast agent for X-ray imaging: the molecular formula of the rare earth ion doped gold nanocluster contrast agent is Au 24 1aS 18 , where SR represents the ligand glutathione; the particle size of rare earth ion-doped gold nanoclusters is 2 nm.

[0008] Furthermore, the rare earth ion doped gold nanocluster contrast agent is a gold nanocluster Au 25 SR 18 Doped with rare earth ions Er.

[0009] Furthermore, the gold nanoclusters Au 25 SR 18 It is a core-shell structure, composed of Au 13 The core is composed of six (-SR-Au-SR-Au-SR-) motifs; among them, thirteen Au atoms are connected by Au-Au bonds to form an Au with an icosahedral structure. 13The core, the thiol group in the glutathione ligand SR forms an Au-S bond with the Au atom, where three S atoms and two Au atoms are arranged to form a (-SR-Au-SR-Au-SR-) motif structure, and the six motif structures as the shell are bonded to the Au with an icosahedral structure through S-Au and Au-Au bonds. 13 Core combination to form Au 25 SR 18 Core-shell structure.

[0010] Furthermore, the rare earth ion Er is doped at the position on the motif structure.

[0011] A method for preparing a rare earth ion-doped gold nanocluster contrast agent material for X-ray imaging comprises the following steps: (1) Under stirring at room temperature, a chloroauric acid solution and an erbium nitrate solution are reacted with a ligand glutathione in a solvent; (2) adding sodium hydroxide solution to adjust the pH of the system to alkaline; (3) Slowly add sodium borohydride solution and continue to stir the reaction; (4) After the reaction is completed, purification is performed by ultrafiltration centrifugation; (5) The purified sample is placed in a freeze dryer for freeze drying to obtain a solid sample, which is the rare earth ion-doped gold nanocluster contrast agent.

[0012] Preferably, the solvent in step (1) is ultrapure water.

[0013] Preferably, the molar concentration ratio of the chloroauric acid solution and the erbium nitrate solution in step (1) is 1:1, and the molar concentration ratio of the ligand glutathione solution to the chloroauric acid solution is 1:4; the molar concentration ratio of the sodium hydroxide solution to the chloroauric acid solution in step (2) is 50:1; and the molar concentration ratio of the reducing agent sodium borohydride solution to the chloroauric acid solution in step (3) is 5.68:1.

[0014] Preferably, the amounts of the chloroauric acid solution and the erbium nitrate solution in step (1) are 0.24 mL and 0.01 mL respectively, and the amount of the ligand glutathione solution is 2 mL; the amount of the sodium hydroxide solution in step (2) is 0.3 mL; and the amount of the sodium borohydride solution in step (3) is 0.1 mL.

[0015] Preferably, in step (1), chloroauric acid and rare earth ions are first added to ultrapure water at room temperature and then the ligand glutathione is added, and the stirring reaction time is 5 minutes; in step (2), sodium hydroxide is added to adjust the pH to a strong alkaline and the stirring reaction time is continued for 2 minutes; in step (3), the reducing agent sodium borohydride is added and the stirring reaction time is continued for 12 hours. The reaction temperature in all the above (1)-(4) is room temperature 25°C, the stirring speed is 500rpm, and all reactions are carried out in a light-proof environment.

[0016] Preferably, in step (4), the operation of purifying by ultrafiltration centrifugation at room temperature comprises: first using a 50 kDa ultrafiltration centrifuge tube to remove gold nanoparticles with larger particle sizes during the reaction after the stirring reaction is completed, the ultrafiltration centrifugation time is 15 min, the rotation speed is 6000 rpm, and the process is repeated three times; using a 3 kDa ultrafiltration centrifuge tube to remove unreacted substrate and concentrate the solution after the large particles are removed, the ultrafiltration centrifugation time is 15 min, the rotation speed is 5500 rpm, the solvent added for washing is ultrapure water, and ultrafiltration concentration is performed after the washing is completed.

[0017] Application: The application of the rare earth ion doped gold nanocluster contrast agent material for gastric X-ray imaging comprises the following steps: First, the prepared rare earth ion-doped gold nanoclusters were re-dispersed with ultrapure water and diluted to an appropriate concentration, and then the mice were treated by oral gavage. After gavage, the gastrointestinal images of the mice were clearly recorded under X-ray irradiation using an X-ray flat-panel detector.

[0018] Preferably, the dilution concentration of the rare earth ion-doped gold nanocluster material is 50 mg / mL, the power of the X-ray is 10 W (50 kV, 200 μA), the irradiation time is 10 s, and the image is taken every 15 minutes.

[0019] The present invention adopts an aqueous phase method for synthesis, takes glutathione as a template, dissolves chloroauric acid in a strong alkaline solution, and uses a reducing agent, sodium borohydride, to reduce trivalent gold ions to zero valence, and dopes rare earth ions at the same time, synthesizes rare earth ion-doped gold nanoclusters under suitable temperature, stirring speed and purification conditions, and the synthesis process is simple and easy for industrial production. The gold nanoclusters synthesized by the method have low toxicity, excellent stability and good biocompatibility, and due to the heavy atom effect of gold, the rare earth ion-doped gold nanoclusters prepared by the method can be used as gastric X-ray imaging contrast agents, and have good application prospects in the fields of gastrointestinal disease detection.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects: (1) The rare earth ion-doped gold nanoclusters synthesized in the present invention are synthesized by a room temperature aqueous phase method, which has a simple synthesis process, low cost and is easy for industrial production.

[0021] (2) The rare earth ion-doped gold nanoclusters synthesized in the present invention have good biocompatibility, low toxicity and good stability.

[0022] (3) The rare earth ion-doped gold nanoclusters synthesized in the present invention have strong X-ray imaging capabilities due to the heavy atom effect and can be used for gastric imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Transmission electron microscopy image (a) and particle size distribution (b) of rare earth ion-doped gold nanoclusters. Figure 2 Emission spectra (a) and absorption spectra (b) of rare earth ion-doped gold nanoclusters; Figure 3 X-ray photoelectron spectrum of rare earth ion-doped gold nanoclusters; Figure 4 Fourier transform infrared spectroscopy of rare earth ion doped gold nanoclusters; Figure 5 Stability test of rare earth ion doped gold nanoclusters; Figure 6 Cell Counting Kit-8 (CCK-8), used to analyze the viability of 4T1 (mouse breast cancer cells) cells incubated with rare earth ion-doped gold nanoclusters; Figure 7 Comparison of X-ray absorption capacity between rare earth ion-doped gold nanoclusters and the commercial contrast agent iohexol; Figure 8 Schematic diagram of stomach X-ray imaging; Fig. 9 Rare earth ion-doped gold nanoclusters used as gastric X-ray contrast agents for imaging.

[0024] Specific implementation examples A rare earth ion-doped gold nanocluster material for gastric X-ray imaging: the gold nanocluster is a core-shell structure, composed of Au 13 The core is composed of six (-SR-Au-SR-Au-SR-) "staple" motifs (SR represents the ligand glutathione). Specifically, thirteen Au atoms are connected by Au-Au bonds to form an Au with an icosahedral structure. 13In the core, the thiol group in the glutathione ligand forms an Au-S bond with the Au atom, where three S atoms and two Au atoms are arranged to form a (-SR-Au-SR-Au-SR-) "staple" motif. The six "staple" motifs act as a shell and are bonded to the Au with an icosahedral structure through S-Au and Au-Au bonds. 13 Core combination to form Au 25 SR 18 The rare earth ion doping position is on the "staple" motif, and its precise structure is Au 24 1aS 18 ; The size of a single-particle gold nanocluster is about 2 nm; the gold nanomaterial has a strong X-ray attenuation capability.

[0025] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0026] Example 1 The preparation method of the rare earth ion-doped gold nanocluster contrast agent material for gastric X-ray imaging is as follows: 0.24 mL of chloroauric acid aqueous solution (20 mM) and 0.01 mL of erbium nitrate aqueous solution (20 mM) were added to a glass bottle containing 2.35 mL of ultrapure water at room temperature and in the dark, and then 2 mL of glutathione aqueous solution (5 mM) was added at a speed of 500 rpm, and stirring continued for 5 min. Then 0.3 mL of sodium hydroxide solution (1M) was added and stirring continued for 2 min, and finally 0.1 mL of sodium borohydride solution (4.3 mg / mL) was slowly added dropwise and stirring continued for 12 h.

[0027] After the reaction, the ultrafiltration centrifugation method was used for purification at room temperature. The specific operation included: first, the solution after the reaction was removed by using a 50 kDa ultrafiltration centrifuge tube to remove the gold nanoparticles with larger particle size during the reaction. The ultrafiltration centrifugation time was 15 minutes and the speed was 6000 rpm, which was repeated three times; the solution from which the large particles had been removed was then removed by using a 3 kDa ultrafiltration centrifuge tube to remove the unreacted substrate and concentrate. The ultrafiltration centrifugation time was 15 minutes and the speed was 5500 rpm. The solvent added for washing was ultrapure water. After washing, ultrafiltration and concentration were performed. The purified and concentrated sample was placed in a freeze dryer for freeze drying to obtain a solid sample, which was the rare earth ion-doped gold nanocluster contrast agent Au. 24 1aS 18 .

[0028] Depend on Figure 1 (a) The transmission electron microscopy image of rare earth ion-doped gold nanoclusters shows that their particle size is about 2 nm, with good dispersion and uniform size distribution. The particle size distribution diagram ( Figure 1 The particle size of the synthesized gold nanoclusters shown in (b) is 2 nm, which is consistent with the results of transmission electron microscopy. Figure 2 As shown in (a), the characteristic emission peak of the synthesized gold nanoclusters is around 1100 nm, and the emission intensity is significantly enhanced after doping with rare earth ions, as shown in Figure 2 As shown in (b), the absorption peaks of the synthesized gold nanoclusters are 450nm and 670nm, which is attributed to their unique interband transitions, belonging to Au 25 In addition, the position of the characteristic absorption peak did not change after rare earth ion doping, indicating that the doping of rare earth ions did not change the structure of the gold cluster. Figure 3 As shown in Figure 2, through the analysis of its X-ray photoelectron spectroscopy, it was found that the gold atoms in the synthesized gold nanoclusters mainly exist in a 0-valent state, and there are also some oxidized 1-valent gold atoms. Figure 4 As shown in the Fourier transform infrared spectrum of the gold cluster, the -1 and 1394 cm -1 There is a characteristic vibration peak of -COO- in the ligand glutathione at 2556 cm -1 There is no characteristic vibration peak of -SH at 1900 cm -1 -2300cm -1 This is a stray peak caused by instrument reasons). Figure 5 As shown in the figure, the stability of the gold clusters was tested, and the luminescence intensity remained almost unchanged after seven days. The luminescence intensities of the seven days were 47216, 46935, 46373, 47746, 46098, 45035, and 45335 (the excitation light source was 808 nm laser power of 2W, and the slit width of the fluorescence spectrometer was 5nm), indicating that the synthesized gold clusters had good stability. Figure 6 As shown, the cell experiment was analyzed by a standard cell counting kit (CCK-8). The results showed that after 4T1 cells were incubated with different concentrations of gold clusters (0, 1, 10, 20, 40, 50, 60 mg / mL) for 24 h, the cell viability remained above 95%, indicating that the synthesized gold clusters have good biocompatibility. Figure 7 As shown in the figure, at the same concentration, the gold cluster has a better ability to block X-rays than iohexol, indicating that the synthesized gold cluster has a stronger ability to absorb X-rays and can improve the sensitivity and resolution of imaging. The above characterization fully demonstrates the successful synthesis and excellent performance of rare earth ion-doped gold nanoclusters.

[0029] Example 2 Rare earth ion-doped gold nanoclusters are used to construct gastric X-ray imaging technology. The synthesized and purified gold nanoclusters are dissolved in ultrapure water (concentration of 50 mg / mL) to obtain a brown solution. Figure 8 As shown in the figure, the prepared rare earth ion doped gold nanoclusters were orally administered to normal mice. The specific operation method was to feed the mice 0.2 ml of gold nanoclusters (50 mg / mL) by oral gavage. After gavage, the mice were exposed to X-rays (power was 10 W), and the X-ray flat panel detector could clearly record the gastrointestinal contour of the mice, realizing real-time monitoring of the gastrointestinal tract. Fig. 9 As shown, (a) is an image of mice not fed with gold nanoclusters, from which it can be seen that only the bones of the mice can be seen but soft tissues such as the gastrointestinal tract cannot be seen; (bf) are images of mice after intragastric administration, from which it can be seen that the image information of the mouse stomach can be seen 5 minutes after intragastric administration, and the entire outline of the gastrointestinal tract of the mouse can be gradually clearly seen as time goes by, realizing real-time monitoring of the gastrointestinal tract of the mouse. From the above data, it can be seen that the rare earth ion-doped gold nanoclusters of the present invention have stable X-ray imaging capabilities, good biocompatibility, low toxicity, etc., and can be used to detect gastrointestinal diseases.

[0030] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A rare earth ion-doped gold nanocluster contrast agent for X-ray imaging, characterized in that: The molecular formula of the rare earth ion doped gold nanocluster contrast agent is Au 24 1aS 18 , where SR represents the ligand glutathione; the particle size of rare earth ion-doped gold nanoclusters is 2 nm.

2. The rare earth ion-doped gold nanocluster contrast agent for X-ray imaging according to claim 1, characterized in that: The rare earth ion doped gold nanocluster contrast agent is a gold nanocluster Au 25 SR 18 Doped with rare earth ions Er.

3. The rare earth ion-doped gold nanocluster contrast agent for X-ray imaging according to claim 2, characterized in that: The gold nanoclusters Au 25 SR 18 It is a core-shell structure, composed of Au 13 The core is composed of six (-SR-Au-SR-Au-SR-) motifs; among them, thirteen Au atoms are connected by Au-Au bonds to form an Au with an icosahedral structure. 13 The core, the thiol group in the glutathione ligand SR forms an Au-S bond with the Au atom, where three S atoms and two Au atoms are arranged to form a (-SR-Au-SR-Au-SR-) motif structure, and the six motif structures as the shell are bonded to the Au with an icosahedral structure through S-Au and Au-Au bonds. 13 Core combination to form Au 25 SR 18 Core-shell structure.

4. The rare earth ion-doped gold nanocluster contrast agent for X-ray imaging according to claim 3, characterized in that: The location of rare earth ion Er doping is on the motif structure.

5. The method for preparing the rare earth ion-doped gold nanocluster contrast agent for X-ray imaging according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Under stirring at room temperature, chloroauric acid solution and erbium nitrate solution are reacted with ligand glutathione in water; (2) adding sodium hydroxide solution to adjust the pH of the system to alkaline; (3) adding sodium borohydride solution dropwise and continuing to stir the reaction; (4) After the reaction is completed, purification is performed by ultrafiltration centrifugation; (5) The purified sample is placed in a freeze dryer for freeze drying to obtain a solid sample, which is the rare earth ion-doped gold nanocluster contrast agent.

6. The preparation method according to claim 5, characterized in that: In step (1), the molar concentration ratio of the chloroauric acid solution to the erbium nitrate solution is 1:1, and the molar concentration ratio of the ligand glutathione solution to the chloroauric acid solution is 1:4; in step (2), the molar concentration ratio of the sodium hydroxide solution to the chloroauric acid solution is 50:1; and in step (3), the molar concentration ratio of the sodium borohydride solution to the chloroauric acid solution is 5.68:

1.

7. The preparation method according to claim 5, characterized in that: In step (1), the amounts of chloroauric acid solution and erbium nitrate solution are 0.24 mL and 0.01 mL respectively, and the amount of glutathione solution is 2 mL; in step (2), the amount of sodium hydroxide solution is 0.3 mL; and in step (3), the amount of sodium borohydride solution is 0.1 mL.

8. The preparation method according to claim 5, characterized in that: In step (1), the reaction time is 5 min; in step (2), sodium hydroxide solution is added to adjust the pH to strong alkalinity and the reaction is continued with stirring for 2 min; in step (3), the reaction is continued with stirring for 12 h.

9. The preparation method according to claim 5, characterized in that: All reactions in steps (1) to (4) were carried out at room temperature (25° C.), with a stirring speed of 500 rpm, and all reactions were carried out in a light-proof environment.

10. The preparation method according to claim 5, characterized in that: The purification operation in step (4) specifically includes: first, using a 50 kDa ultrafiltration centrifuge tube to remove the gold nanoparticles with larger particle size during the reaction, the ultrafiltration centrifuge time is 15 min, the rotation speed is 6000 rpm, and this is repeated three times; using a 3 kDa ultrafiltration centrifuge tube to remove the unreacted substrate and concentrate the solution after the large particles are removed, the ultrafiltration centrifuge time is 15 min, the rotation speed is 5500 rpm, the solvent added for washing is ultrapure water, and ultrafiltration concentration is performed after the washing is completed.

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