Application of gold cluster fluorescent probe in detection of hematopoietic stem cells of radiation-damaged animals

Abstract

The invention discloses application of a gold cluster fluorescent probe in detection of hematopoietic stem cells of radiation-damaged animals, and belongs to the technical field of nano-drugs. In order to solve the technical problem that in the prior art, accurate detection of radiation-damaged animal hematopoietic stem cells is difficult, the invention provides application of a gold cluster fluorescent probe in detection of radiation-damaged animal hematopoietic stem cells, a gold cluster fluorescent molecule is composed of an Au atom, other metal atoms M and an outer ligand L, coupling the gold cluster fluorescent molecule with an antibody through a chemical cross-linking method to prepare a gold cluster fluorescent probe coupled with the antibody; the gold cluster fluorescent probe shows good biocompatibility, relatively strong photobleaching resistance, good light stability, high sensitivity and high specificity, has the targeting characteristic of hematopoietic stem cells, can be applied to detection and monitoring of hematopoietic stem cells in vivo, and can be applied to detection and monitoring of hematopoietic stem cells in vivo. And detecting the residual volume of the hematopoietic stem cells after radiation injury and evaluating the injury condition.

Description

Technical Field

[0001] The invention belongs to the technical field of nano-drugs, and relates to the application of a gold cluster fluorescent probe in the detection of hematopoietic stem cells in radiation-damaged animals. Background Art

[0002] Studies have shown that exposure to ionizing radiation exceeding a certain dose threshold can cause damage to the animal body and even affect life. Among them, a one-time whole-body exposure to 100-800rad ionizing radiation can cause bone marrow radiation sickness in the human body, leading to damage to hematopoietic tissue, thereby causing a series of lesions such as infection and bleeding. Due to hematopoietic dysfunction, skin and mucosal barrier damage, decreased peripheral blood neutrophils, weakened phagocyte function and blocked antibody production, the body's ability to resist infection is significantly reduced. The treatment principle for the above symptoms is to treat the symptoms comprehensively according to the characteristics of each stage of the course of the disease. In the early stage, anti-radiation drugs that can improve hematopoietic damage should be used as early as possible. Blood transfusion, especially hematopoietic stem cell transplantation, is an important treatment measure for bone marrow radiation sickness. Therefore, the detection of the residual amount of hematopoietic stem cells after exposure to radiation is one of the means to judge the degree of radiation damage, and the proliferation detection of the residual amount of hematopoietic stem cells after hematopoietic stem cell transplantation is one of the means to judge whether the operation is successful, which is crucial for the treatment of acute radiation sickness. However, it is still a very challenging research task to accurately grasp the distribution and content of hematopoietic stem cells in the body.

[0003] Compared with traditional infrared zone I (NIR-I, 750-900nm), the penetration depth of NIR-II has reached the centimeter level, which greatly reduces the absorption and scattering of biological tissues and improves the tissue penetration depth. NIR-II fluorescence imaging technology has greatly promoted the early detection of diseases, precise navigation during surgery, and real-time monitoring of treatment progress. Using NIR-II fluorescent probes, doctors can more accurately identify and locate abnormal tissues deep in the body, such as tiny tumors, so as to intervene in the early stages of the disease, greatly improving the possibility of cure. During surgery, it can help surgeons clearly distinguish healthy tissues from diseased tissues, achieve precise resection, avoid unnecessary damage to normal tissues, and reduce surgical risks. In addition, doctors can also use NIR-II imaging technology to monitor the effect of disease treatment in real time, adjust treatment plans in time, and ensure that patients receive the most appropriate treatment. NIR-II fluorescence imaging technology can show more detailed information about physiological and pathological functions, which not only deepens doctors' understanding of disease development, but also helps to design personalized treatment plans. However, traditional infrared zone II fluorescent probes have problems such as low brightness and poor biosafety; for example, organic small molecule fluorescent probes have low fluorescence efficiency and short half-life, and semiconductor polymers, carbon nanotubes, quantum dots, and rare earth-doped nanoparticles have poor water solubility and biocompatibility.

[0004] Therefore, those skilled in the art are eager to develop a method for accurately detecting hematopoietic stem cells in vivo and monitoring treatment of animals after radiation damage. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides the application of gold cluster fluorescent probe in the detection of hematopoietic stem cells in radiation-damaged animals.

[0006] One of the purposes of the present invention is to provide a gold cluster fluorescent probe for use in detecting radiation-damaged animal hematopoietic stem cells.

[0007] In a preferred embodiment of the present invention, the hematopoietic stem cell detection refers to detecting the residual amount of hematopoietic stem cells in radiation-damaged animals.

[0008] In a preferred embodiment of the present invention, the method for preparing the gold cluster fluorescent probe comprises the following steps:

[0009] S1: Prepare HAuCl with a concentration range of 10-60mM 4 solution, and then prepare the outer layer ligand L solution with a concentration range of 20-100mM. HAuCl 4The solution and the outer ligand L solution are added to deionized water in a mass ratio of 1:(1-3) and mixed evenly to obtain a mixed solution; under stirring, a 0.1-3M alkaline solution is added to adjust the pH, and then a reducing gas is introduced for 2 minutes. The mixed solution is stirred at room temperature for 2-24 hours, and then the precipitate is collected by a rotary evaporator, washed with an organic solvent for 3-5 times, and vacuum dried to obtain a gold cluster fluorescent molecular powder;

[0010] S2: The gold cluster fluorescent molecule powder obtained in S1 is placed in deionized water to obtain a gold cluster fluorescent molecule solution, and the above solution is mixed with a coupling activator in a mass ratio of 1:(2-5), stirred at room temperature for 5-60 min, and ultrafiltered for 10-40 min twice, and then 10-80 μL of antibody is added, stirred at 4°C for 1-5 h, and ultrafiltered 3 times to obtain a gold cluster fluorescent probe coupled to the antibody.

[0011] In a preferred embodiment of the present invention, the general formula of the gold cluster fluorescent molecule in S1 is Au x M y L z , x represents the number of Au atoms contained in the fluorescent molecule, M represents other metal atoms contained in the fluorescent molecule, y represents the number of other metal atoms, L represents the outer layer ligands contained in the fluorescent molecule, and z represents the number of outer layer ligands; the x:y:z atom number ratio is (25-n):n:18, and n represents a positive integer between 0 and 24.

[0012] In a preferred embodiment of the present invention, M is one or more of calcium, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, gallium, germanium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, indium, tin, antimony, tellurium, barium, lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, thallium, bismuth or polonium.

[0013] In a preferred embodiment of the present invention, L is one or more of glutathione, cysteine, captopril, 3-mercaptopropionic acid, p-mercaptobenzoic acid, 6-mercaptohexanoic acid, thioglycolic acid or lipoic acid.

[0014] In a preferred embodiment of the present invention, the pH adjustment range in S1 is 9-13.

[0015] In a preferred embodiment of the present invention, the reducing gas in S1 is one or more of hydrogen, carbon monoxide, hydrogen sulfide or sulfur monoxide; the organic solvent in S1 is one or more of ethanol, methanol, acetone, ethyl acetate, dichloromethane, chloroform, n-hexane, benzene, toluene, dimethyl sulfoxide, tetrahydrofuran, isopropanol, acetonitrile, dimethylformamide or ether.

[0016] In a preferred embodiment of the present invention, the antibody in S2 is one or more of anti-CD3e, anti-CD43, anti-CD49 (integrin α6), anti-CD90 (Thy-1), anti-c-Kit / CD117, anti-Ly-6A / E (Sca-1), anti-CD105, anti-CD36, anti-CD44, anti-CD73, anti-Nestin, anti-STRO-1, anti-CD11b, anti-CD14, anti-CD19, anti-CD34, anti-CD79a or anti-HLA-DR antibodies.

[0017] In a preferred embodiment of the present invention, the coupling activator in S2 is one or more of Ynamides, EDC, NHS, HOBt, HBTU, HATU, PyBOP, DCC or DIC.

[0018] Beneficial effects of the present invention: The present invention provides an application of a gold cluster fluorescent probe in the detection of radiation-damaged animal hematopoietic stem cells, wherein the general formula of the gold cluster fluorescent molecule is Au x M y L z The gold cluster fluorescent molecule comprises other metal atoms M and outer ligands L. The gold cluster fluorescent molecule is coupled with an antibody by a chemical cross-linking method to prepare a gold cluster fluorescent probe coupled with an antibody.

[0019] The gold cluster fluorescent probe coupled to the antibody provided by the present invention still shows two extremely significant optical characteristic absorption peaks at 450nm and 670nm, and the coupling yield is as high as 94.1%; the gold cluster fluorescent probe has excellent near-infrared second-zone luminescence performance, can specifically bind to hematopoietic stem cells and emit infrared second-zone fluorescence under the excitation of a specific light source, and the fluorescence can penetrate biological tissues and be received by an infrared camera to image the hematopoietic stem cells. The gold cluster fluorescent probe coupled to the antibody provided by the present invention shows good biocompatibility, strong resistance to photobleaching, good photostability, high sensitivity and high specificity, and has the targeting characteristics of hematopoietic stem cells.

[0020] The antibody-coupled gold cluster fluorescent probe provided by the present invention can detect the residual amount of hematopoietic stem cells in the body through fluorescence intensity, thereby achieving accurate detection and monitoring of hematopoietic stem cells, and realizing the detection of the residual amount of hematopoietic stem cells and injury assessment after radiation damage, which is of great significance for promoting research in the field of imaging of hematopoietic stem cell-related diseases and clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a comparison analysis diagram of the gold cluster fluorescent molecules and the gold cluster fluorescent probe coupled to the near-infrared second region antibody; a is the absorption spectrum diagram, and b is the statistical diagram of the coupling reaction yield and purity;

[0022] Figure 2 The fluorescence spectrum and infrared second region fluorescence image thereof in Example 3, a is the near infrared second region fluorescence spectrum, b is the infrared second region fluorescence image;

[0023] Figure 3 : is a photostability detection diagram of the gold cluster fluorescent probe coupled to the antibody in Example 4; a is an infrared second zone fluorescence image, b is a fluorescence quantitative statistical diagram; ce are respectively three repeated infrared second zone fluorescence quantitative normalized result diagrams;

[0024] Figure 4 The infrared second-zone fluorescence images of the gold cluster fluorescent probe coupled to the antibody in Example 5 on hematopoietic stem cells in animals with different degrees of radiation damage;

[0025] Figure 5 These are statistical graphs of the in vivo fluorescence quantification of hematopoietic stem cells in animals with different degrees of radiation damage by the gold cluster fluorescent probe conjugated with antibodies in Example 5, a is a quantitative graph of the fluorescence changes of the gold cluster fluorescent probe conjugated with antibodies in the left leg of mice over time, and b is a quantitative graph of the fluorescence changes of the gold cluster fluorescent probe conjugated with antibodies in the right leg of mice over time. DETAILED DESCRIPTION

[0026] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0027] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific implementation methods and the accompanying drawings of the specification. The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by those skilled in the art through commercial channels.

[0028] The sources of raw materials and instrument models used in the following examples are described:

[0029] Chloroauric acid trihydrate (HAuCl 4 ·3H 2 O, 1 g, 99%) were purchased from Sigma;

[0030] Reduced glutathione, cysteine, captopril, 3-mercaptopropionic acid and other ligands were purchased from Shanghai Aladdin Reagent Co., Ltd.

[0031] Organic solvents such as ethanol, methanol, and acetone were purchased from Tianjin Yuanli Chemical Co., Ltd.;

[0032] The UV spectrophotometer was a UV-2600 UV-visible spectrophotometer purchased from Shimadzu Corporation;

[0033] The fluorescence spectrometer is a Fluorolog-3 fluorescence spectrometer manufactured by HORIBA JobinYvon of the United States;

[0034] The near-infrared zone II optical imaging light path is a self-built imaging system, in which the near-infrared zone II camera model is NIRvana:640ST, manufactured by Princeton, USA, and the laser model used is FLC300-0808-8W-F200, manufactured by Frankfurt Laser Company, Germany.

[0035] Example 1: Preparation of gold cluster fluorescent molecules

[0036] In this example, 3.75 mL of 40 mM HAuCl 4 The solution and 4.5 mL of 50 mM glutathione GSH solution were added to 141.75 mL of ultrapure water. Under stirring, 0.1-3 M NaOH was added to adjust the pH to 11, and then CO was introduced for 2 min. The mixture was stirred at room temperature for 24 h (500 rpm), and the precipitate was collected by rotary evaporator, washed with methanol 3 times (3500 rpm, 10 min), and vacuum dried to finally obtain 40 mg of gold cluster fluorescent molecules with GSH ligands.

[0037] In this example, an ultraviolet spectrophotometer was used to detect the optical characteristic absorption peaks of the gold cluster fluorescent molecules. The results are as follows: Figure 1 As shown in a, the gold cluster fluorescent molecules prepared in this example exhibit two extremely obvious optical characteristic absorption peaks at 450nm and 670nm.

[0038] Example 2: Preparation of gold cluster fluorescent probe conjugated to antibody

[0039] In this example, 12 mg of EDC coupling activator and 12 mg of NHS coupling activator were added to 2 mL of the gold cluster fluorescent molecule solution prepared in Example 1 with a concentration of 2 mg / mL, stirred at room temperature for 20 min (500 rpm), ultrafiltered (10K, 3000 rpm, 15 min), and repeated twice; 40 μL of Thy1 / CD90 antibody was added to the above solution, stirred at 500 rpm for 2 h at 4°C, and ultrafiltered twice (30K, 3000 rpm, 10 min) to obtain a gold cluster fluorescent probe coupled to the antibody.

[0040] In this example, the gold cluster fluorescent probe coupled to the antibody in the near-infrared region II was subjected to near-infrared region II optical imaging detection. The results are as follows: Figure 1 As shown in a, the gold cluster fluorescent probe coupled to the antibody still exhibits two very significant optical characteristic absorption peaks at 450nm and 670nm, which are consistent with the optical characteristic absorption peaks of the gold cluster fluorescent molecules. This example quantitatively analyzes the fluorescent molecules before and after the characteristic peak reaction of the gold cluster fluorescent probe coupled to the antibody in the near-infrared region II. The results are shown in Figure 1 As shown in b, the coupling yield of the gold cluster fluorescent probe coupled to the antibody in the near-infrared region II is as high as 94.1%.

[0041] Example 3: Fluorescence spectroscopy detection of gold cluster fluorescent probes coupled to antibodies

[0042] The gold cluster fluorescent molecules obtained in Example 1 and the gold cluster fluorescent probe coupled to the antibody obtained in Example 2 were respectively dissolved in 2.5 mL of deionized water, and the absorption values ​​of the aqueous solutions of the two were determined to be 0.5 at 808 nm using a UV-visible spectrophotometer. The emission spectra of the two were measured under excitation at 808 nm using a Fluorolog-3 fluorescence spectrometer.

[0043] The results are as follows Figure 2 As shown, under 808nm excitation, the gold cluster fluorescent molecules obtained in Example 1 and the gold cluster fluorescent probe coupled to antibodies obtained in Example 2 exhibit characteristic emission peaks at 1050nm, indicating that both have excellent near-infrared second-region luminescence properties.

[0044] Example 4: Photostability detection of gold cluster fluorescent probes coupled to antibodies in the near-infrared region II

[0045] In this example, the photostability of the gold cluster fluorescent probe coupled to the antibody obtained in Example 2 was tested. The above 0.59 mg / mL gold cluster fluorescent probe was dissolved in water, PBS and plasma, respectively. The absorption value was set to 0.1 at a wavelength of 808 nm using a UV-visible spectrophotometer. Our self-built second-zone infrared camera was used to excite at a wavelength of 808 nm, set the power to 25 mW / cm2, and set the exposure time to 100 ms. The fluorescence intensity changes of the gold cluster fluorescent probe coupled to the antibody after incubation for 0 h, 2 h, 4 h, 6 h, 8 h, 12 h and 24 h at an ambient temperature of 37 ° C were observed.

[0046] The results are as follows Figure 3As shown, at 37°C ambient temperature at 0h, the gold cluster fluorescent probe of the conjugated antibody has a strong fluorescence intensity, and the fluorescence values ​​of the gold cluster fluorescent probe of the conjugated antibody in water, PBS and plasma can reach about 20,000. After incubation for 0h, 2h, 4h, 6h, 8h, 12h and 24h, the gold cluster fluorescent probe of the conjugated antibody still has a high fluorescence intensity value measured at each time point, indicating that the gold cluster fluorescent probe of the conjugated antibody maintains good photostability within 24h.

[0047] Example 5: Application of Antibody-Conjugated Gold Cluster Fluorescent Probe in Detection of Radiation-Injured Animal Hematopoietic Stem Cells

[0048] In this example, C57 male mice purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. were used as experimental subjects. 12 C57 male mice were divided into 4 groups and subjected to different doses of radiation treatment, namely 0 Gy-R, 4 Gy-R, 6 Gy-R and 8 Gy-R. The gold cluster fluorescent probe coupled with the antibody obtained in Example 2 was dissolved in deionized water to obtain a 10 mg / mL gold cluster fluorescent probe solution. The abdominal cavity was pierced at a 45° angle on the left side of the lower abdomen of the C57 male mice after the above-mentioned different doses of radiation treatment, and 300 μL of 3.5% chloral hydrate was injected to anesthetize the C57 male mice. The C57 male mice were depilated under anesthesia. 200 μL of the gold cluster fluorescent probe solution was injected into the depilated C57 male mice through the tail vein, and the C57 male mice were fixed on the infrared zone II camera stand, and the power used was 25 mW / cm 2 With 808nm laser excitation, under the action of 1000nm long-pass filter, the infrared two-zone camera detector can receive the two-zone fluorescence emitted by the fluorescent molecular probe, and the fluorescence signal of the femur of C57 male mice was recorded by near-infrared camera at different times after injection.

[0049] The results are as follows Figure 4 As shown in the figure, different doses of radiation treatment caused certain damage to the hematopoietic stem cell function of C57 male mice, and the degree of damage to hematopoietic stem cells gradually increased with the increase of irradiation dose. The intensity of the fluorescence signal reflects the function of the residual hematopoietic stem cells. From the fluorescence results, it can be seen that the number of residual hematopoietic stem cells in C57 male mice treated with 4Gy radiation is more than that in C57 male mice treated with 6Gy radiation, and the number of residual hematopoietic stem cells in C57 male mice treated with 8Gy radiation is the least.

[0050] This embodiment also carries out fluorescence quantitative analysis, and the result is as follows Figure 5As shown in the figure, the fluorescence intensity at the femur of C57 male mice treated with 4Gy radiation was the highest, followed by the fluorescence intensity at the femur of C57 male mice treated with 6Gy radiation, and the fluorescence signal of this group was the weakest because the hematopoietic stem cells of C57 male mice treated with 8Gy radiation were most damaged. In summary, the results of fluorescence quantitative analysis intuitively show that with the continuous increase of irradiation dose, the fluorescence signal intensity at the femur of C57 male mice gradually weakens, indicating that the gold cluster probe coupled with the antibody can be associated with the hematopoietic stem cells at the femur of C57 male mice, and the number of hematopoietic stem cells in mice can be effectively detected by the intensity of the fluorescence signal.

[0051] The contents not described in detail in the specification of the present invention are well-known technologies for those skilled in the art. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be based on the definition of the claims.

Claims

1. Application of a gold cluster fluorescent probe in the detection of hematopoietic stem cells in radiation-damaged animals.

2. The reference according to claim 1, characterized in that: The hematopoietic stem cell detection refers to the detection of the residual amount of hematopoietic stem cells in the radiation damaged animal.

3. The reference according to claim 1, characterized in that: The preparation method of the gold cluster fluorescent probe comprises the following steps: S1: Prepare a HAuCl4 solution with a concentration range of 10-60mM, and then prepare an outer layer ligand L solution with a concentration range of 20-100mM, add the HAuCl4 solution and the outer layer ligand L solution into deionized water at a volume ratio of 1:(1-3) at room temperature and mix them evenly to obtain a mixed solution; under stirring, add a 0.1-3M alkaline solution to adjust the pH, and then introduce a reducing gas for 2 minutes, stir the mixed solution at room temperature for 2-24 hours, collect the precipitate by a rotary evaporator, wash it with an organic solvent for 3-5 times, and vacuum dry it to obtain a gold cluster fluorescent molecular powder; S2: The gold cluster fluorescent molecule powder obtained in S1 is placed in deionized water to obtain a gold cluster fluorescent molecule solution, and the above solution is mixed with a coupling activator in a mass ratio of 1:(2-5), stirred at room temperature for 5-60 min, and ultrafiltered for 10-40 min twice, and then 10-80 μL of antibody is added, stirred at 4°C for 1-5 h, and ultrafiltered 3 times to obtain a gold cluster fluorescent probe coupled to the antibody.

4. The use according to claim 3, characterized in that: The general formula of the gold cluster fluorescent molecule described in S1 is Au x M y L z , x represents the number of Au atoms contained in the fluorescent molecule, M represents other metal atoms contained in the fluorescent molecule, y represents the number of other metal atoms, L represents the outer layer ligands contained in the fluorescent molecule, and z represents the number of outer layer ligands; the x:y:z atom number ratio is (25-n):n:18, and n represents a positive integer between 0 and 24.

5. The use according to claim 4, characterized in that: The M is one or more of calcium, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, gallium, germanium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, indium, tin, antimony, tellurium, barium, lanthanum, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, thallium, bismuth or polonium.

6. The use according to claim 4, characterized in that: The L is one or more of glutathione, cysteine, captopril, 3-mercaptopropionic acid, p-mercaptobenzoic acid, 6-mercaptohexanoic acid, thioglycolic acid or lipoic acid.

7. The use according to claim 3, characterized in that: The pH adjustment range described in S1 is 9-13.

8. The use according to claim 3, characterized in that: The reducing gas in S1 is one or more of hydrogen, carbon monoxide, hydrogen sulfide or sulfur monoxide, and the organic solvent in S1 is one or more of ethanol, methanol, acetone, ethyl acetate, dichloromethane, chloroform, n-hexane, benzene, toluene, dimethyl sulfoxide, tetrahydrofuran, isopropanol, acetonitrile, dimethylformamide or ether.

9. The use according to claim 3, characterized in that: The antibody described in S2 is one or more of anti-CD3e, anti-CD43, anti-CD49 (integrinα6), anti-CD90 (Thy-1), anti-c-Kit / CD117, anti-Ly-6A / E (Sca-1), anti-CD105, anti-CD36, anti-CD44, anti-CD73, anti-Nestin, anti-STRO-1, anti-CD11b, anti-CD14, anti-CD19, anti-CD34, anti-CD79a or anti-HLA-DR antibodies.

10. The use according to claim 3, characterized in that: The coupling activator in S2 is one or more of Ynamides, EDC, NHS, HOBt, HBTU, HATU, PyBOP, DCC or DIC.

Images