Diagnosis and treatment integrated bimodal nano-particles for ECT imaging and radiosensitization treatment as well as preparation method and application of diagnosis and treatment integrated bimodal nano-particles

Abstract

The invention discloses diagnosis and treatment integrated bimodal nanoparticles for ECT imaging and radiosensitization treatment as well as a preparation method and application of the diagnosis and treatment integrated bimodal nanoparticles, and belongs to the technical field of tumor diagnosis and treatment medicines. The nanoparticle is obtained by marking a detectable marker with a small-size hafnium-gallic acid nanoparticle; the small-size hafnium-gallic acid nanoparticles are formed by assembling polyvinylpyrrolidone, hafnium tetrachloride and gallic acid through coordination. The bimodal nanoparticles provided by the invention can be used for ECT imaging and radiosensitization treatment at the same time, have good biological safety and excellent radiosensitization performance, have good metabolic characteristics in vivo, and overcome the defect that conventional nanoparticles can be retained in vivo.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor diagnosis and treatment drugs, and specifically relates to a dual-modal nanoparticle for diagnosis and treatment that is simultaneously used for ECT imaging and radiosensitization therapy, and a preparation method and use thereof. Background Art

[0002] Tumor is one of the major diseases that endanger human health. Traditional therapies for tumors (such as surgery and chemotherapy) fail to completely eradicate tumors, leading to tumor metastasis, which is the main cause of death in cancer patients. Radiotherapy (RT) is the most widely used first-line treatment for solid tumors in clinical practice. Radiotherapy uses high-energy X-rays to directly damage DNA and ionize water to produce reactive oxygen species (ROS) to induce tumor cell apoptosis. Studies have shown that radiotherapy can induce innate immunity by activating the cGAS-STING pathway. At the same time, neoantigens produced by radiotherapy-induced gene mutations can activate CD8+T cell-mediated adaptive immunity and eliminate unirradiated distal metastases. However, radiotherapy-mediated distal tumor regression is not common in clinical practice. This is mainly because the therapeutic effect of radiotherapy is often limited by the inherent physiological barriers of tumors. Due to the poor ability of low-Z atoms to absorb energy (tumor soft tissue is mainly composed of low-Z organic matter), tumors often show innate resistance to ionizing radiation, resulting in a low apoptosis rate of tumor cells after radiotherapy and a small amount of tumor-associated antigen release, thereby weakening the immune response induced by radiotherapy. In addition, radiotherapy resistance and decreased sensitivity of tumor cells to radiation after multiple radiotherapy are also important factors restricting radiotherapy. Therefore, improving radiotherapy sensitivity is an important means to improve the efficacy of tumor radiotherapy.

[0003] In tumor radiotherapy, the application of nanotechnology is developing rapidly. On the one hand, nanoparticles can act as radiosensitizers for tumor cells to increase the sensitivity of tumor cells to radioactivity. On the other hand, they can change the microenvironment of tumor tissue, especially the hypoxic environment, thereby increasing the sensitivity of tumor tissue to ionizing radiation. At present, a large number of nanomedicines have been used in clinical trials. Researchers have prepared more effective radiosensitizers or radioprotectors through clever design. For example, ionizing radiation changes the tumor microenvironment, thereby allowing nanoparticles to diffuse in tumor tissue; ionizing radiation kills tumor vascular endothelial cells, further expanding the tumor tissue's increased penetration and retention effect (EPR effect) of nanoparticles; ionizing radiation destroys chemical bonds in nanoparticles that are sensitive to redox reactions, which is beneficial to the release of chemotherapeutic drugs loaded in nanostructures, etc.

[0004] At present, the most researched at home and abroad are heavy metal nanoparticles such as gold nanoparticles, silver nanoparticles and iron oxide nanoparticles; nanoparticles that produce oxygen free radicals (ROS) such as ferroferric oxide and polymer nanomaterials; nanoparticles with photothermal conversion capabilities such as copper sulfide and iron sulfide. Studies have found that elements with high atomic numbers have the effect of enhancing the energy deposition of high-energy rays and can produce obvious radiotherapy sensitization effects. Most of the current research focuses on lanthanide elements. Hafnium (Hf) has a higher atomic number than lanthanide elements, and its oxide hafnium dioxide (HfO2) nanoparticles have entered clinical trials, showing good biosafety and excellent radiotherapy sensitization performance. Metal ions themselves can also show radiation deposition and radiotherapy sensitization performance that is no less than that of oxides. Therefore, the use of Hf ions as radiotherapy sensitizers is considered safe and reliable.

[0005] Although nanoformulations can absorb high-energy ionizing radiation (X-rays) as radiosensitizers to improve the efficacy of radiotherapy. However, after administration, a large amount of nanomaterials will be retained in the body, causing people to worry about their potential toxicity, which has hindered the clinical transformation of many nanomaterials. Based on this, the present invention designs an ultra-small-sized Hf-GA nanoparticle that has no long-term toxicity and can be rapidly metabolized. Compared with other M-GA nanodots, Hf-GA nanoparticles have a higher absorption peak in the infrared region and a stronger photothermal conversion ability. Due to the presence of high-Z elements such as Hf to absorb rays, it can be used as an effective sensitizer for radiotherapy, significantly improving the killing effect of ionizing radiation on tumor cells. In addition, the present invention labels radionuclides on Hf-GA nanoparticles for ECT imaging, further expanding its scope of application, making it a dual-modal nanoparticle for diagnosis and treatment that is simultaneously used for ECT imaging and radiosensitization therapy. Summary of the invention

[0006] One object of the present invention is to provide a dual-modal nanoparticle for diagnosis and treatment, which can be used for ECT imaging and radiosensitization therapy at the same time; a second object of the present invention is to provide a method for preparing the dual-modal nanoparticle for diagnosis and treatment; and a further object of the present invention is to provide a use of the dual-modal nanoparticle for diagnosis and treatment of tumors.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] In a first aspect, the present invention provides a dual-modal nanoparticle for diagnosis and treatment, characterized in that it is obtained by labeling a detectable marker with a small-sized hafnium-gallic acid nanoparticle. The small-sized hafnium-gallic acid nanoparticle is formed by coordination assembly of polyvinyl pyrrolidone (PVP), hafnium tetrachloride (HfCl4) and gallic acid (GA).

[0009] The mass ratio of polyvinyl pyrrolidone, hafnium tetrachloride and gallic acid is (40-45):(3.2-3.6):(1-2).

[0010] In a specific embodiment of the present invention, the mass ratio of polyvinyl pyrrolidone, hafnium tetrachloride and gallic acid is 40:3.2:2.

[0011] The detectable marker described in the present invention is a detectable substance with imaging function, including but not limited to at least one of radioactive nuclides, paramagnetic metal ions, fluorescent or chemiluminescent molecules, and biotin.

[0012] The radionuclide ions are selected from 99m Tc, 125 I. 131 I. 117 Sn, 111 In, 97 Such as 67 Ga, 89 Zr, 177 Lu, 47 Sc, 105 Rh, 188 Re, 60 Cu, 62 Cu, 64 Cu, 67 Cu, 90 Y. 159 Gd, 149 Pr or 166 Ho.

[0013] The paramagnetic metal ion is selected from Gd 3+ , Mn 2+ , Yb 3+ or Fe 3+ .

[0014] In a specific embodiment of the present invention, the detectable marker is a radionuclide, specifically, the radionuclide is selected from 99m Tc, 125 I or 131 I.

[0015] Most preferably, the radionuclide is selected from 99m Tc.

[0016] The formation principle of the dual-modal nanoparticles provided by the present invention is as follows: first, the amide bond of polyvinyl pyrrolidone and Hf ions form a chelate through coordination; the phenolic hydroxyl group in the gallic acid structure coordinates with the Hf ions on the chelate to form small-sized hafnium-gallic acid nanoparticles; finally, the radioactive nuclide and gallic acid further coordinate to form dual-modal nanoparticles. The dual-modal nanoparticles of the present invention can be used for ECT imaging and radiosensitization therapy at the same time, and the ECT imaging includes SPECT imaging and PET imaging.

[0017] In a second aspect, the present invention provides a method for preparing the diagnosis and treatment dual-modal nanoparticles according to the first aspect of the present invention, characterized in that the method comprises:

[0018] (1) Preparation of small-sized hafnium-gallic acid nanoparticles;

[0019] (2) Labeling radionuclides.

[0020] In a specific embodiment of the present invention, the radionuclide is selected from 99m Tc, 125 I or 131 I. The labeling method comprises adding a radioactive nuclide to the small-sized hafnium-gallic acid nanoparticle system prepared in step (1).

[0021] Specifically, the small-sized hafnium-gallic acid nanoparticles are prepared by the following method:

[0022] S1: dissolve polyvinyl pyrrolidone in the solution to form a colorless solution;

[0023] S2: slowly adding gallic acid and triethylamine to the polyvinyl pyrrolidone solution to form a first solution, and incubating for 5-10 minutes;

[0024] S3: slowly adding the prepared hafnium tetrachloride solution to the first solution to prepare a second solution, and incubating for 1-1.5 hours;

[0025] S4: The second solution is dialyzed, ultrafiltered and washed to obtain small-sized hafnium-gallic acid nanoparticles.

[0026] Preferably, the solution in step S1 is selected from water, methanol, ethanol or a combination of two or more thereof. In a specific embodiment of the present invention, the solution is water.

[0027] In a specific embodiment of the present invention, the weight average molecular weight of the polyvinyl pyrrolidone is 8000, and can be selected from PVP K16-18.

[0028] Preferably, the amount of triethylamine added in step S2 is to adjust the pH value of the system to 8-10.

[0029] Preferably, the mass ratio of polyvinyl pyrrolidone, hafnium tetrachloride and gallic acid is (40-45):(3.2-3.6):(1-2).

[0030] More preferably, the mass ratio of polyvinyl pyrrolidone, hafnium tetrachloride and gallic acid is 40:3.2:2.

[0031] Preferably, step S3 specifically comprises dialyzing with an 8-14 kDa dialysis bag for 24 hours, centrifuging with a 10k ultrafiltration tube, and washing with deionized water for 2-3 times.

[0032] In a third aspect, the present invention provides a use of the dual-modal nanoparticles for diagnosis and treatment according to the first aspect of the present invention in at least one of the following:

[0033] (1) Use in the preparation of drugs for tumor radiosensitization;

[0034] (2) Use in the preparation of a drug for ECT imaging;

[0035] (3) Use in the preparation of drugs for simultaneous ECT imaging and radiosensitization therapy.

[0036] The dual-modal nanoparticles for diagnosis and treatment provided by the present invention have the following technical advantages:

[0037] The bimodal nanoparticles are prepared by labeling radioactive elements with small-sized hafnium-gallic acid nanoparticles. Elements with high atomic numbers have the effect of enhancing the energy deposition of high-energy rays and can produce obvious radiotherapy sensitization effects. The hafnium-gallic acid nanoparticles prepared by the present invention using hafnium (Hf) exhibit good biosafety and excellent radiotherapy sensitization performance. In addition, the particle size of the hafnium-gallic acid nanoparticles is 7-10nm, and the small particle size enables the nanoparticles to have good metabolic characteristics in the body, overcoming the defect that conventional nanoparticles will be retained in the body. The hafnium-gallic acid nanoparticles prepared by the present invention have a higher absorption peak in the infrared region and a stronger photothermal conversion ability, further improving their radiotherapy sensitization effect. Based on the hafnium-gallic acid nanoparticles, the present invention technicians use the coordination principle to label radioactive nuclides, so that the nanoparticles further have imaging functions, forming a nanoparticle that can simultaneously perform ECT imaging and radiosensitization therapy. After further screening, the present invention technicians unexpectedly found that the hafnium-gallic acid nanoparticles have a strong affinity for radioactive nuclides. 99m Tc has the highest labeling rate and the best adaptability. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 TEM image of hafnium-gallic acid nanoparticles.

[0039] Figure 2Particle size characterization of hafnium-gallic acid nanoparticles.

[0040] Figure 3 Fourier transform infrared spectrum of hafnium-gallic acid nanoparticles.

[0041] Figure 4 Figure 2. Cytotoxicity test results of hafnium-gallic acid nanoparticles.

[0042] Figure 5 A diagram showing the radiosensitization effect of hafnium-gallic acid nanoparticles. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Example 1 Preparation of Hafnium-Gallic Acid Nanoparticles

[0045] Polyvinyl pyrrolidone (pvp, 8kDa, 40mg) was dissolved in 40ml of deionized water, and 0.2mL of gallic acid (GA, 10mg / mL) and 0.01mL of triethylamine were slowly added dropwise to the polyvinyl pyrrolidone aqueous solution, and its color quickly changed to light brown, and incubated for 5min; 0.1mL of freshly prepared hafnium tetrachloride (HfCl4, 32mg / mL) was slowly added to the above solution and incubated for 1h; dialyzed with an 8-14kDa dialysis bag for 24h, centrifuged with a 10k ultrafiltration tube, and washed twice with deionized water to obtain hafnium-gallic acid nanoparticles (Hf-GA).

[0046] The hafnium-gallic acid nanoparticles prepared by the present invention were characterized, and the results were as follows:

[0047] (1) Transmission electron microscopy (TEM) detection

[0048] The Hf-GA prepared by the present invention was dissolved in deionized water to prepare a 0.1 mg / ml solution, 10 μl of which was dropped on a copper mesh covered with a carbon film, and this operation was repeated twice after the liquid evaporated. The morphology of the nanoparticles was observed under a transmission electron microscope. The results are as follows: Figure 1 shown.

[0049] (2) Particle size detection

[0050] The nanoparticle size of the hafnium-gallic acid nanoparticles prepared by the present invention was detected. The results are as follows Figure 2 As shown. Combined Figure 1 and Figure 2It can be seen from the characterization results that the particle size of the hafnium-gallic acid nanoparticles prepared by the present invention is 7-9 nm.

[0051] (3) Fourier transform red spectrum of Hf-GA

[0052] The infrared spectrum of the hafnium-gallic acid nanoparticles (Hf-GA) prepared by the present invention is as follows: Figure 3 As shown in the figure, compared with the infrared spectrum of gallic acid (GA), the hafnium-gallic acid nanoparticles have a cm-1 The characteristic peak appears at , indicating that hafnium is successfully coordinated.

[0053] (4) Biocompatibility testing of hafnium-gallic acid nanoparticles

[0054] Different concentrations of Hf-GA solution were prepared in culture medium and incubated with Hela (breast cancer), U251 (glioma), and CT26 (colon cancer). The cytotoxicity at different concentrations was tested using MTT kit. Figure 4 As shown in the figure, since the cell activity was still good at a concentration of 200 μg / ml, it proved that Hf-GA had good biocompatibility, so this concentration was selected for all cell experiments.

[0055] (5) Detection of the sensitization effect of hafnium-gallic acid nanoparticles

[0056] The experiment was conducted using CT26 cells, which were divided into 4 groups. Group 1 was named control group, group 2 was Hf-GA group, group 3 was X-ray group, and group 4 was Hf-GA+X-ray group. Hafnium-gallic acid nanoparticles at a concentration of 200 μg / ml were added to the cells in group 2 and group 4, and the same volume of PBS was added to group 1 and group 3; group 1 and group 2 were placed under normal conditions, and group 3 and group 4 were subjected to radiotherapy. After the experiment, the four groups of cells were stained with DAPI, λ-H2AX, and Merge, respectively.

[0057] The steps of fluorescence staining are as follows:

[0058] a) Take out cells of appropriate density, wash once with 1X PBS buffer, and then fix with 4% paraformaldehyde at room temperature for 15 minutes (1 ml / dish);

[0059] b) Discard the paraformaldehyde solution in the dish, add 1X PBS buffer to the dish and wash for 3 times, 5 minutes each time, and then add PBS solution containing 0.5% TritonX-100 (1 ml / well, shake at room temperature for 10 minutes);

[0060] c) Add 1 ml of immunofluorescence blocking solution to each dish and block the permeabilized cells for 1 hour at room temperature;

[0061] d) Use an aspirator to remove the blocking solution, then dilute the primary antibody for immunofluorescence at a ratio of 1:400, take out the slide and incubate it in a refrigerator at 4°C overnight;

[0062] e) Recover the primary antibody, wash each dish with 1 ml of PBS containing 0.1% Tween 20, wash three times for 5 minutes each time;

[0063] f) Dilute the fluorescent secondary antibody at a ratio of 1:1500, incubate the slides in the dark at room temperature for 2 h, and then wash with PBS containing 0.1% Tween 20;

[0064] g) Place half a drop of DAPI-containing mounting medium on the slide, place the coverslip upside down on the slide with the mounting medium, seal the slide with nail polish, and observe with a confocal microscope at 60×.

[0065] The results are as follows Figure 5 As shown, compared with the control group, the addition of Hf-GA alone does not cause obvious cell death, indicating that the hafnium-gallic acid nanoparticles prepared by the present invention have good biocompatibility and high safety. Compared with the radiotherapy group alone, the hafnium-gallic acid nanoparticles + radiotherapy have the strongest killing effect on cells, indicating that the hafnium-gallic acid nanoparticles prepared by the present invention have obvious radiotherapy sensitization effect.

[0066] Example 2 Radionuclide Labeled Hafnium-Gallic Acid Nanoparticles

[0067] (1) 99m Tc labeling

[0068] Weigh 10 mg of sodium borohydride and dissolve it in 2 ml of PBS solution to obtain a 5 mg / ml sodium borohydride PBS solution. First, activate 100 μl of the solution with 5 mCi 99m Tc 5-10min, then mix evenly with 2ml Hf-GA solution (freeze-dried Hf-GA powder dissolved in 2ml ddH2O, greater than 1g / ml, the material concentration is high enough), wait for reaction for 1 hour, filter and wash 3 times using a 10k ultrafiltration centrifuge tube, measure the dosage and obtain the labeling rate of the material is 67%.

[0069] (2) 131 I Mark

[0070] Weigh 10 mg of sodium borohydride and dissolve it in 2 ml of PBS solution to obtain a 5 mg / ml sodium borohydride PBS solution. First, activate 100 μl of the solution with 5 mCi 131I 5-10min, then mixed evenly with 2ml Hf-GA solution (freeze-dried Hf-GA powder dissolved in 2ml ddH2O), allowed to react for 1 hour, filtered and washed 3 times using a 10k ultrafiltration centrifuge tube, and the dosage was measured to obtain a labeling rate of 27%.

[0071] (3) 125 I Mark

[0072] Weigh 10 mg of sodium borohydride and dissolve it in 2 ml of PBS solution to obtain a 5 mg / ml sodium borohydride PBS solution. First, activate 100 μl of the solution with 5 mCi 125 I 5-10min, then mixed evenly with 2ml Hf-GA solution (freeze-dried Hf-GA powder dissolved in 2ml ddH2O), allowed to react for 1 hour, filtered and washed 3 times using a 10k ultrafiltration centrifuge tube, and the labeling rate of the material was measured to be 34%.

[0073] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A dual-modal nanoparticle for diagnosis and treatment, characterized in that: The method is obtained by labeling a detectable marker with small-sized hafnium-gallic acid nanoparticles; the small-sized hafnium-gallic acid nanoparticles are formed by coordination assembly of polyvinyl pyrrolidone, hafnium tetrachloride and gallic acid.

2. The diagnosis and treatment integrated dual-modal nanoparticle according to claim 1, characterized in that: The detectable marker is a detectable substance with imaging function, including at least one of radioactive nuclides, paramagnetic metal ions, fluorescent or chemiluminescent molecules, and biotin.

3. The diagnosis and treatment integrated dual-modal nanoparticle according to claim 1, characterized in that: The radionuclide ions are selected from 99m Tc, 125 I. 131 I. 117 Sn, 111 In, 97 Such as 67 Ga, 89 Zr, 177 Lu, 47 Sc, 105 Rh, 188 Re, 60 Cu, 62 Cu, 64 Cu, 67 Cu, 90 Y. 159 Gd, 149 Pr or 166 Ho.

4. The diagnosis and treatment integrated dual-modal nanoparticle according to claim 1, characterized in that: The paramagnetic metal ion is selected from Gd 3+ , Mn 2+ , Yb 3+ or Fe 3+ .

5. The diagnosis and treatment integrated dual-modal nanoparticle according to claim 1, characterized in that: The detectable marker is a radionuclide selected from 99m Tc, 125 I or 131 I.

6. The diagnosis and treatment integrated dual-modal nanoparticle according to claim 1, characterized in that: The mass ratio of polyvinyl pyrrolidone, hafnium tetrachloride and gallic acid is (40-45): (3.2-3.6): (1-2).

7. The method for preparing the diagnosis and treatment dual-modal nanoparticles according to any one of claims 1 to 6, characterized in that: The method comprises: (1) Preparation of small-sized hafnium-gallic acid nanoparticles; (2) Labeling radionuclides.

8. The preparation method according to claim 7, characterized in that: The small-sized hafnium-gallic acid nanoparticles are prepared by the following method: S1: dissolve polyvinyl pyrrolidone in the solution to form a colorless solution; S2: slowly adding gallic acid and triethylamine to the polyvinyl pyrrolidone solution to form a first solution, and incubating for 5-10 minutes; S3: slowly adding the prepared hafnium tetrachloride solution to the first solution to prepare a second solution, and incubating for 1-1.5 hours; S4: The second solution is dialyzed, ultrafiltered and washed to obtain small-sized hafnium-gallic acid nanoparticles.

9. The preparation method according to claim 8, characterized in that: The solution described in step S1 is selected from one or a combination of water, methanol, and ethanol; step S3 specifically comprises dialyzing with an 8-14kDa dialysis bag for 24 hours, centrifuging with a 10k ultrafiltration tube, and washing with deionized water for 2-3 times.

10. Use of the diagnosis and treatment integrated dual-modal nanoparticles according to any one of claims 1 to 6 in at least one of the following: (1) Use in the preparation of drugs for tumor radiosensitization; (2) Use in the preparation of a drug for ECT imaging; (3) Use in the preparation of drugs for simultaneous ECT imaging and radiosensitization therapy.

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