A kind of ergothioneine gold nanoparticles and preparation method and application thereof

Abstract

The present invention relates to a kind of ergothioneine gold nanoparticles and its preparation method and application, belonging to the field of biomedical technology. The present invention provides the application of ergothioneine gold nanoparticles in the preparation of contrast agents and / or antioxidants, the ergothioneine gold nanoparticles include nanogold particles, ergothioneine is connected to the nanogold particles by gold-sulfur bonds, and the antioxidant includes a drug for preventing and / or treating acute kidney injury. The present invention designs and synthesizes ergothioneine gold nanoparticles with antioxidant activity by combining an antioxidant natural product ergothioneine with a higher imaging contrast of the gold element, which is an ultra-small gold nanoparticle, realizes the combination of CT contrast imaging and antioxidant effect, is suitable for kidney contrast imaging, is particularly suitable for early diagnosis and treatment of acute kidney injury, realizes integrated diagnosis and treatment, avoids the contrast agent toxicity of conventional nanomaterials, reduces serum creatinine and urea nitrogen levels, and reduces the degree of renal tubular damage.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to ergothioneine gold nanoparticles and a preparation method and application thereof. Background Art

[0002] Acute Kidney Injury (AKI) refers to a clinical syndrome caused by a rapid decline in renal function due to a variety of causes. It can occur in people without previous kidney disease or on the basis of existing chronic kidney disease. AKI seriously endangers human health. According to surveys, about 5% of hospitalized patients may develop AKI, and the incidence of AKI in the intensive care unit (ICU) is as high as 30%. Although the nephrology community pays more and more attention to AKI, there is still no specific treatment method. Its mortality rate is high and it is an acute and critical disease among kidney diseases. "Early diagnosis and early treatment" of AKI is extremely important for the prognosis of patients. However, due to the serious hysteresis of serum creatinine, a commonly used renal function test indicator in clinical practice, the creatinine level will often increase significantly only when the patient's glomerular filtration rate decreases by more than 50%. At this time, the kidney function has been irreversibly damaged. Therefore, it is urgent to realize the early diagnosis of AKI and timely treatment when the kidney function is less damaged, effectively protect the kidney function, reduce the mortality rate of AKI patients, and inhibit the patient's transformation to chronic kidney disease (CKD). During the progression of AKI, cell damage caused by intracellular oxidative stress is one of the main causes of AKI progression. Therefore, the development of antioxidant drugs targeting reactive oxygen species (ROS) can counteract the progression of AKI and achieve effective protection of renal function.

[0003] CT technology is currently a widely used detection technology in clinical practice. Compared with other imaging technologies (MRI, PET), CT has low cost, fast detection speed, and high clinical popularity; however, due to the limitation of tissue X-ray attenuation ability, CT's imaging and diagnostic capabilities in bones and calcification are far higher than those in organs. Therefore, in order to achieve high-contrast imaging of organs, the use of CT contrast agents for enhanced scanning has become a widely used method in clinical practice; among them, iodine contrast agents are currently the CT contrast agents used in clinical practice.

[0004] At present, with the continuous development of nanomaterials, many nanomaterials have been synthesized and applied in the diagnosis and treatment of AKI; for example, nanoprobes with fluorescent or luminescent properties have been developed through changes in the AKI microenvironment to achieve non-invasive detection of renal function; in addition, substances with antioxidant activity are loaded into nanomaterials to target the oxidative stress pathological state of the kidneys in the case of AKI to achieve the treatment of AKI.

[0005] Currently, the new diagnostic methods developed for the early diagnosis of AKI are mainly fluorescence imaging or urine analysis technology. Fluorescence imaging is limited by wavelength and is difficult to apply in practice in humans. Although urine analysis can achieve early diagnosis of AKI, it lacks intuitive imaging data.

[0006] Although the use of iodinated contrast agents in CT can achieve enhanced imaging of the kidneys, they are limited by the physical and chemical properties of iodinated contrast agents themselves and there is a risk of contrast-induced kidney injury. Contrast-induced kidney injury is a type of drug-induced kidney damage, specifically acute renal failure caused by the use of contrast agents. The use of iodinated contrast agents should be avoided as much as possible for the elderly, diabetics, and those with pre-existing chronic kidney disease, especially those with chronic renal insufficiency. Therefore, CT diagnosis of kidney injury cannot be achieved using iodinated contrast agents. Summary of the invention

[0007] The purpose of the present invention is to overcome the shortcomings of the prior art and provide ergothioneine gold nanoparticles with contrast agent imaging and antioxidant activity and a preparation method and application thereof.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] The invention provides use of the ergothioneine gold nanoparticles in preparing a contrast agent and / or an antioxidant. The ergothioneine gold nanoparticles comprise nanogold particles, on which ergothioneine is connected via a gold-sulfur bond. The antioxidant comprises a drug for preventing and / or treating acute kidney injury.

[0010] The ergothioneine gold nanoparticles of the present invention are ultra-small gold nanoparticles with contrast imaging and antioxidant effects, are suitable for kidney contrast imaging, and are particularly suitable for early diagnosis and treatment of acute kidney injury, thus realizing integrated diagnosis and treatment.

[0011] Furthermore, the contrast agent is a kidney contrast agent.

[0012] In a specific embodiment of the present invention, the drug reduces serum creatinine and urea nitrogen levels and alleviates the degree of renal tubular damage.

[0013] Furthermore, the average particle size of the ergothioneine gold nanoparticles is 1.516-3.210 nm.

[0014] Furthermore, the hydrated particle size of the ergothioneine gold nanoparticles is 1.726-3.462 nm.

[0015] Furthermore, the ergothioneine gold nanoparticles are spherical or quasi-spherical nanoparticles.

[0016] Furthermore, the preparation method of the thioneine gold nanoparticles is: reacting an thioneine alkaline solution with a tetrachloroauric acid solution to obtain thioneine gold nanoparticles.

[0017] Furthermore, the molar ratio of tetrachloroauric acid to ergothioneine is tetrachloroauric acid: ergothioneine = 1: (0.7-1.5), preferably 1: 0.7. The molar ratio of tetrachloroauric acid to ergothioneine is 1: 0.7-1.5, and ultra-small gold nanoparticles can be synthesized, but the molar ratio of tetrachloroauric acid to ergothioneine is 1: 0.5 and 1: 2, and ultra-small gold nanoparticles cannot be synthesized.

[0018] Further, the concentration of the ergothioneine is 0.2-1.0 mg / mL, preferably 0.55 mg / mL.

[0019] Furthermore, the concentration of tetrachloroauric acid is 0.5-1.5 M, preferably 1 M.

[0020] Furthermore, the reaction temperature is 50-100°C, preferably 70°C.

[0021] Furthermore, the stirring speed is 200-1000 rpm, preferably 420 rpm.

[0022] Furthermore, the reaction time is 5 to 20 h, preferably 15 h. When the reaction time is 15 h, the fluorescence intensity of the nanoparticles of ergothioneine gold nanoparticles prepared is the largest.

[0023] Further, the pH value of the thioneine alkaline solution is 8 to 12, preferably 12. The reagent for adjusting the pH of the thioneine alkaline solution can be at least one of NaOH, KOH, ammonia and Na2CO3. In a specific embodiment of the present invention, the reagent used is NaOH.

[0024] Furthermore, the ergothioneine gold nanoparticles are purified after the reaction.

[0025] In a specific embodiment of the present invention, the purification method is ultrafiltration centrifuge tube purification.

[0026] Further, purification is performed using an ultrafiltration centrifuge tube with a cut-off of 1000 to 5000 Daltons, preferably 5000 Daltons.

[0027] Furthermore, the purification is performed 1 to 3 times, preferably 3 times.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] Compared with iodine, gold has a higher atomic number and a larger relative atomic mass. Therefore, at the same molar concentration, gold has a higher X-ray attenuation coefficient. The present invention uses the higher imaging contrast of gold and combines an antioxidant natural product, ergothioneine, to design and synthesize ergothioneine gold nanoparticles with antioxidant activity. The ultra-small gold nanoparticles combine CT contrast imaging with antioxidant effects, are suitable for kidney contrast imaging, and are particularly suitable for early diagnosis and treatment of acute kidney injury. The invention realizes integrated diagnosis and treatment, avoids the contrast agent toxicity of conventional nanomaterials, reduces serum creatinine and urea nitrogen levels, and alleviates the degree of renal tubular damage.

[0030] Ergothioneine is a natural product widely present in food with good safety assurance. Ultra-small gold nanoparticles have excellent kidney targeting ability, which can increase the enrichment of materials in the kidneys and reduce non-specific uptake by other organs, thereby improving efficacy while reducing side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The synthesis principle and appearance of ergothioneine gold nanoparticles (EGT-AuNPs). A is the synthesis principle; B is the appearance.

[0032] Figure 2 Ultraviolet-visible spectrophotometer (UV-Vis) spectra of ergothioneine gold nanoparticles (EGT-AuNPs) prepared with different ratios of tetrachloroauric acid and ergothioneine.

[0033] Figure 3 Fluorescence spectra of ergothioneine gold nanoparticles (EGT-AuNPs) prepared at different reaction times.

[0034] Figure 4 is the core diameter of ergothioneine gold nanoparticles (EGT-AuNPs).

[0035] Figure 5 is the hydrodynamic diameter of ergothioneine gold nanoparticles (EGT-AuNPs).

[0036] Figure 6 Excitation / emission spectra and UV-visible absorption spectra of ergothioneine gold nanoparticles (EGT-AuNPs).

[0037] Figure 7 In vitro antioxidant activity of ergothioneine gold nanoparticles (EGT-AuNPs).

[0038] Figure 8 Figure 2 shows the in vitro CT signal intensity of ergothioneine gold nanoparticles (EGT-AuNPs) and iopromide.

[0039] Fig. 9 This is the imaging effect of ergothioneine gold nanoparticles (EGT-AuNPs) in mice (0-30 min). A is the bladder area; B is the kidney area.

[0040] Fig.10 This is the imaging effect of iopromide and ergothioneine gold nanoparticles (EGT-AuNPs) in mice (0-10 min). A is iopromide; B is ergothioneine gold nanoparticles.

[0041] Fig.11 The CT images of kidneys in normal (Normol) and acute kidney injury (AKI) mouse models. A is a blank image without injection of ergothioneine gold nanoparticles (EGT-AuNPs); B is an image 10 minutes after injection of ergothioneine gold nanoparticles (EGT-AuNPs); C is an image 30 minutes after injection of ergothioneine gold nanoparticles (EGT-AuNPs).

[0042] Fig.12 Profile signal distribution of kidneys in normal (Normol) and acute kidney injury (AKI) mouse models after 10 min injection of ergothioneine gold nanoparticles (EGT-AuNPs).

[0043] Fig.13 Serum creatinine and urea nitrogen levels in acute kidney injury (AKI) mouse model after treatment with ergothioneine gold nanoparticles (EGT-AuNPs). A is serum creatinine; B is urea nitrogen.

[0044] Fig.14 The kidney tissue pathological sections and renal tubular injury scores of the acute kidney injury (AKI) mouse model after treatment with ergothioneine gold nanoparticles (EGT-AuNPs). A is the pathological section; B is the renal tubular injury score. DETAILED DESCRIPTION

[0045] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific examples. Other materials, reagents, etc. used in the examples, unless otherwise specified, can be obtained from commercial sources.

[0046] Example 1 Preparation of ergothioneine gold nanoparticles with antioxidant activity

[0047] like Figure 1 As shown in A, it is the synthesis principle of ergothioneine gold nanoparticles, such as Figure 1 As shown in B, the appearance of ergothioneine gold nanoparticle powder and solution is ultra-small spherical nanoparticles.

[0048] 1. Dissolve 27.5 mg of ergothioneine (EGT) in 50 mL of 0.1 mM NaOH aqueous solution, quickly add 150 μL of 1 M tetrachloroauric acid aqueous solution at 70°C and 420 rpm stirring speed, maintain 70°C and 420 rpm stirring speed for 15 h to obtain a reaction solution. The molar ratio of tetrachloroauric acid to ergothioneine is 1:0.7.

[0049] 2. The reaction solution in step 1 was cooled to room temperature, and purified three times using an ultrafiltration centrifuge tube with a cutoff of 5000 Daltons to obtain a retentate; the retentate was freeze-dried to obtain ergothioneine gold nanoparticles (EGT-AuNPs).

[0050] Example 2 Effect of different ratios of tetrachloroauric acid and ergothioneine and reaction time on EGT-AuNPs

[0051] 1. Experimental Methods

[0052] 1. The molar ratios of tetrachloroauric acid and ergothioneine in the reaction system were set to 1:0.5, 1:0.7, 1:1.0, 1:1.2, 1:1.5 and 1:2.0, respectively. The volumes of 1 M tetrachloroauric acid solution added in the above different ratios were 100, 150, 200, 250, 300 and 400 μL, respectively. The other parameters and methods for preparing EGT-AuNPs were the same as those in Example 1. EGT-AuNPs were prepared, and the characteristic absorption of EGT-AuNPs was characterized by ultraviolet-visible spectrophotometer (UV-Vis). Whether there are large-sized nanoparticles is determined by whether an absorption peak appears at 500-600 nm. Large-sized nanoparticles will absorb at 500-600 nm. Particles with a diameter of ≤3 nm are ultra-small-sized particles.

[0053] 2. The reaction time was set to 5, 10, 15 and 20 h respectively. The other parameters and methods for preparing EGT-AuNPs were the same as those in Example 1. EGT-AuNPs were prepared and the fluorescence properties of EGT-AuNPs were characterized by fluorescence spectrometer (FL).

[0054] 2. Experimental Results

[0055] 1. If Figure 2 As shown, ultra-small gold nanoparticles can be synthesized when the molar ratio of tetrachloroauric acid to ergothioneine is in the range of 1:0.7~1.5, but ultra-small gold nanoparticles cannot be synthesized when the molar ratio of tetrachloroauric acid to ergothioneine is 1:0.5 and 1:2.

[0056] 2. If Figure 3As shown, when the reaction time is 15 h, the fluorescence intensity of the prepared EGT-AuNPs nanoparticles is the highest, so the optimal reaction time is 15 h.

[0057] Example 3 Physicochemical properties of EGT-AuNPs

[0058] 1. Experimental Methods

[0059] 1. The diameter and morphology of the EGT-AuNPs prepared in Example 1 were characterized by transmission electron microscopy (TEM), and the average diameter was calculated as the average particle size (i.e., core diameter) of the EGT-AuNPs. The hydration kinetic diameter of the EGT-AuNPs prepared in Example 1 was characterized by dynamic light scattering (DLS), and the average value was calculated as the hydrated particle size of the EGT-AuNPs. The characteristic absorption of the EGT-AuNPs prepared in Example 1 was characterized by an ultraviolet-visible spectrophotometer (UV-Vis). The fluorescence properties of the EGT-AuNPs prepared in Example 1 were characterized by a fluorescence spectrometer (FL).

[0060] 2. The antioxidant activity of the EGT-AuNPs prepared in Example 1 was detected by free radical scavenging experiment using 2,2'-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS), as follows:

[0061] (1) ABTS was dissolved in a sodium acetate solution with a pH value of 4.5 to prepare an ABTS solution with a concentration of 7.4 mmol / L. The ABTS solution was mixed with a 2.6 mmol / L potassium persulfate solution in a volume ratio of 1:1. The mixture was allowed to stand at room temperature in the dark for 12 h to obtain a reaction solution.

[0062] (2) Detection method: EGT-AuNPs phosphate buffer solution (PBS) of different concentrations (0.1, 0.5, 1, 2, 5 and 10 mg / mL) were mixed with the reaction solution of step (1) at a volume ratio of 1:9. After reacting in the dark for 30 min, the absorbance was detected at 734 nm and adjusted to zero with distilled water. The total antioxidant activity at different concentrations was calculated.

[0063] 2. Experimental Results

[0064] 1. (1) If Figure 4 As shown, the core diameter of the EGT-AuNPs prepared in Example 1 is 2.363±0.847 nm and is spherical, which is in line with the size range of ultrasmall gold nanoparticles.

[0065] (2) If Figure 5 As shown, the hydrated particle size of the EGT-AuNPs prepared in Example 1 is 2.594±0.868 nm, which is consistent with the size range of ultrasmall gold nanoparticles.

[0066] (3) If Figure 6 As shown, the EGT-AuNPs prepared in Example 1 have fluorescence properties, the best excitation wavelength is 460 nm, and the maximum emission wavelength is 710 nm. In addition, within the range of 400-500 nm, the nanoparticles do not have obvious UV-Vis absorption, proving that there is no surface plasmon resonance and they are ultra-small size nanoparticles.

[0067] 2. If Figure 7 As shown, the EGT-AuNPs prepared in Example 1 have good antioxidant activity.

[0068] Example 4 Imaging capability of EGT-AuNPs

[0069] 1. Experimental Methods

[0070] The CT imaging capability of the EGT-AuNPs prepared in Example 1 was characterized in vitro and in vivo using a small animal in vivo tomography imaging system (Micro-CT, Quantum GX2, manufacturer: PerkinElmer).

[0071] 1. Prepare a 100 mg / mL EGT-AuNPs (prepared in Example 1, gold concentration is 20 mM) solution with PBS. Take 100 μL of EGT-AuNPs solution and add it to a 96-well plate. Use iodine contrast agent solution (iopromide, iodine concentration is 20 mM) prepared with PBS and PBS as controls to compare the difference in signal values ​​under the same amount of gold and iodine, and clarify the CT enhanced imaging capability of EGT-AuNPs.

[0072] 2. (1) Inject 200 μL each of EGT-AuNPs solution, iodine contrast agent solution and PBS into the tail vein of mice.

[0073] (2) Micro-CT was used for CT scanning with scanning parameters of 90 kV and scanning time of 2 min. A blank scan was performed before contrast agent injection to obtain unenhanced images of the kidney and bladder regions. After contrast agent injection, 15 consecutive scans were performed for a total of 30 min to obtain images of the kidney and bladder regions.

[0074] (3) In order to compare the imaging effects of the two contrast agents, EGT-AuNPs and Iopromide, the signal intensity of the renal pelvis and renal parenchyma was circled using the CT image analysis software Analyzer, and the imaging enhancement effects of the two contrast agents in the entire kidney area and in the renal pelvis and renal parenchyma were compared.

[0075] 2. Experimental Results

[0076] 1. If Figure 8As shown, EGT-AuNPs have a signal intensity 1.58 times higher than that of lopromide.

[0077] 2. If Fig. 9 A and Fig. 9 As shown in Figure 2B, EGT-AuNPs effectively enhanced the signal intensity in the kidney and bladder regions and maintained the imaging contrast of the kidney and bladder within 30 min after injection.

[0078] 3. If Fig.10 A and Fig.10 As shown in Figure B, compared with Iopromide, EGT-AuNPs has better kidney enhancement imaging effect and clearer kidney structure.

[0079] Example 5 Diagnostic and therapeutic effects of EGT-AuNPs on AKI

[0080] 1. Experimental Methods

[0081] 1. Inject mice with 50% glycerol by volume into the lower limbs of the muscle at a dose of 8 mL / kg to induce rhabdomyolysis-induced AKI mouse model. Perform a renal CT scan 2 h after glycerol injection to obtain a blank scan image. Inject the EGT-AuNPs prepared in Example 1 into the mouse model via the tail vein at a dose of 200 mg / kg. Perform continuous scanning with Micro-CT at 2 min / time for a total of 15 times. Compare the differences between renal imaging under normal conditions and under AKI conditions, and use software to analyze the signal intensity in the renal region to perform non-invasive diagnosis of AKI.

[0082] 2. 24 h after EGT-AuNPs injection, the blood renal function biochemical indicators (creatinine, urea nitrogen) and renal pathological sections of the mouse model were detected to determine the degree of AKI and the treatment effect.

[0083] 24 hours after EGT-AuNPs injection, the mouse model was anesthetized with isoflurane gas, and blood was collected from the heart by puncture. The blood was naturally coagulated and then the serum was separated. The levels of serum creatinine and urea nitrogen were detected by automatic biochemical analyzer. The abdomen was opened longitudinally along the middle of the abdomen, and the kidneys were removed. The tissues were fixed with 4% paraformaldehyde and embedded in paraffin. The paraffin sections of the kidneys were obtained and stained with hematoxylin and eosin (H&E) to analyze the degree of tubular injury. Ten high-power microscopic fields were randomly selected from each kidney section and scored according to the following criteria: 1 point for obvious tubular dilation and flattened cells; 1 point for brush border damage and 2 points for shedding; 2 points for casts, and 1 point for shedding and necrotic cells (not casts or cell fragments) in the lumen of the tubules; the highest score for each field was 4 points, and the lowest score was 0 points.

[0084] 2. Experimental Results

[0085] 1. If Fig.11 As shown in Figure A, in the early stage of AKI (2 h after intramuscular injection of glycerol), the renal cortical signal of AKI mice was more obvious, while the renal pelvis and medulla signals were lower than those of normal mice. Fig.11 C) and 10min ( Fig.11 Compared with (B), mice in AKI condition continued to maintain obvious signals, while the overall signal intensity in the kidneys of normal mice decreased.

[0086] 2. If Fig.12 As shown, 10 min after EGT-AuNPs injection, there was a significant difference in the signal distribution in the renal cross-section (from the cortex to the renal pelvis) between normal and AKI conditions.

[0087] 3. After 24 h of EGT-AuNPs treatment, the serum creatinine of mice treated with EGT-AuNPs was significantly higher than that of mice without treatment. Fig.13 A) and urea nitrogen level ( Fig.13 B) decreased significantly and returned to normal levels.

[0088] 4. If Fig.14 A and Fig.14 As shown in B, H&E staining results showed that the degree of renal tubular damage in mice treated with EGT-AuNPs was significantly reduced.

[0089] 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 the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. The application of ergothioneine gold nanoparticles in the preparation of contrast agents and / or antioxidants, is characterized in that, The ergothioneine gold nanoparticles include nanogold particles, and ergothioneine is connected to the nanogold particles via a gold-sulfur bond, and the antioxidant includes a drug for preventing and / or treating acute kidney injury; The contrast agent is a kidney contrast agent; The preparation method of the thioneine gold nanoparticles comprises: reacting an thioneine alkaline solution with a tetrachloroauric acid solution to obtain the thioneine gold nanoparticles; The molar ratio of tetrachloroauric acid to ergothioneine is tetrachloroauric acid:ergothioneine=1:(0.7-1.5).

2. The use according to claim 1, characterized in that: The average particle size of the ergothioneine gold nanoparticles is 1.516-3.210 nm.

3. The use according to claim 1, characterized in that: The hydrated particle size of the ergothioneine gold nanoparticles is 1.726-3.462 nm.

4. The use according to claim 1, characterized in that: The ergothioneine gold nanoparticles are spherical or quasi-spherical nanoparticles.

5. The use according to claim 1, characterized in that: The reaction time is 5 to 20 h.

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