A method for radiolabeling nanometer quantum dots

Abstract

This invention provides a method for radioisotope-labeled quantum dot nanoparticles, belonging to the field of isotope labeling technology. The method for radioisotope-labeled quantum dot nanoparticles provided by this invention includes the following steps: (1) mixing a quantum dot solution with a radioisotope solution, adding a catalyst, and allowing the reaction to proceed statically; (2) centrifuging and ultrafiltration the reaction product from step (1), and collecting the ultrafiltration product, which is the radioisotope-labeled quantum dot nanoparticle. This invention uses I isotope to label quantum dots, enabling quantitative analysis, which is of great significance for the future clinical application of quantum dot nanomedicines in BNCT (Brain-Necrophage-Related Therapy).

Description

Technical Field

[0001] This invention relates to the field of isotope labeling technology, and more particularly to a method for radioactive isotope labeling of nanoquantum dots. Background Technology

[0002] Radioisotope labeling, as a standard method prescribed by pharmacopoeias, utilizes the binding of radioisotopes into the structure of drug molecules. It provides precise data for the quantitative assessment of the absorption, distribution, metabolism, and / or excretion (ADME) of chemical entities and enables qualitative studies of drug metabolites with high sensitivity. Radioisotope labeling is widely used in biochemistry and molecular biology, playing a crucial role in revealing the secrets of physicochemical processes in vivo and within cells, and elucidating the material basis of life activities. The development of these technologies has enabled biochemistry to move from a static to a dynamic process, and from the cellular level to the molecular level, clarifying a series of significant questions.

[0003] Currently, positron-emitting nuclides used in PET (positron emission tomography) and CT (computed tomography) disease diagnosis and research are mainly based on... 11 C 13 N、 15 O and 18 F is the predominant element, and these nuclides have relatively short half-lives. 11 C:T1 / 2 = 20min; 13 N:T1 / 2=10min; 15 O: T1 / 2 = 2min: 18 F:T1 / 2=110min), is not suitable for molecular probe labeling with complex synthesis processes and long time consumption, nor is it suitable for long-term disease monitoring.

[0004] Iodine radioactive isotopes include 123 I(T1=13.2 hours), 125 I(T1=59.45 days), 124 I(T1=4.18days) and 131 I (T1 = 8.02 days) is widely used for radiolabeling of molecules such as monoclonal antibodies. Among them, 123 I is suitable for disease imaging diagnosis, as it releases gamma rays with an energy of 159 keV, which interact with nuclides. 99 With a similar mTc (140keV), it can be used for low-energy, high-resolution single-photon emission computed tomography (SPECT). However, 123 I has a relatively short half-life, limiting its application to labeling simple, rapidly synthesized molecular probes and diseases with short diagnostic cycles. 123 The preparation of I nuclides is quite difficult. 125I has a relatively long half-life and mainly releases 27 keV X-rays and 35.5 keV gamma rays. Its energy is too low to be suitable for diagnostic imaging. Currently, it is mostly used for antibody or peptide labeling methods and in vitro experiments. 131 Iodine is the most widely used radioactive isotope of iodine. It mainly releases beta rays with an energy of 606 keV and is often used clinically for radiotherapy of thyroid cancer. It can also release gamma rays for SPECT-CT imaging. 124 I has a half-life of 4.15 days, emits 25.6% β+ rays and has an electron capture EC of 74.4%, and can be used for PET / CT imaging, with applications ranging from simple thyroid and parathyroid imaging to complex neurotransmitter receptor imaging.

[0005] Quantum dots, due to their excellent physicochemical properties, have been widely used in the biomedical field. As nanomaterials, biocompatibility evaluation is a necessary prerequisite for their practical application, and tracking the metabolism and distribution of quantum dots in vivo is a crucial step in this process. While quantum dots themselves exhibit fluorescence, the optical signal penetration within living organisms is relatively poor, especially considering the tissue thickness in patients is much greater than in mouse models, making quantitative analysis of the optical signal difficult.

[0006] Radionuclide-labeled nanomaterials mainly consist of two parts: radionuclides and nanomaterials. Almost all nanomaterials can be radionuclide-labeled. However, radionuclide labeling requires consideration of two aspects: ① the selection of radionuclides; ② how to label the radionuclides onto the nanomaterials so that the labeling does not affect the in vivo distribution and metabolic behavior of the nanomaterials, and the labeling method is safe, rapid, and effective.

[0007] Therefore, using radioactive isotope labeling of nanocollar dots may amplify fluorescence detection signals, enabling tissue depth detection. Summary of the Invention

[0008] The purpose of this invention is to provide a method for labeling nano-quantum dots with radioactive isotopes, with the goal of labeling quantum dots using radioactive isotope I.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides a method for radioactive isotope labeling of nanoquantum dots, comprising the following steps:

[0011] (1) Mix the quantum dot solution with the radioactive isotope solution, add a catalyst and let the reaction stand;

[0012] (2) The reaction product of step (1) is subjected to centrifugation and ultrafiltration, and the ultrafiltration product is collected, which is radioisotope labeled nano-quantum dots.

[0013] Preferably, the quantum dot solution is a boron-doped quantum dot (BCD) solution, wherein the BCDs refer to boron-doped carbon quantum dots; the mass activity ratio of the BCDs to the radioactive isotope is 1 mg: 1 μCi to 10 mCi.

[0014] Preferably, the radioactive isotope is 124 I, 125 I or 131 I.

[0015] Preferably, the catalyst is a chloramine T solution.

[0016] Preferably, the concentration of the chloramine T solution is 1-100 mg / ml, and the volume ratio of the chloramine T solution to the radioactive isotope solution is 10-1000:1.

[0017] Preferably, the settling time is 1 min to 20 h.

[0018] Preferably, the centrifugal ultrafiltration is performed at a speed of 3000-20000 rpm for 10-20 min.

[0019] The present invention also provides a radioisotope-labeled nanoquantum dot synthesized by the method.

[0020] This invention also provides an application of the aforementioned radioisotope-labeled nanoquantum dot-based diagnostic reagent.

[0021] This invention uses I isotope labeling of quantum dots, especially boron-containing quantum dots (BCDs), which enables quantitative analysis. Furthermore, I radioactive isotope-labeled BCDs exhibit stronger fluorescence signals, allowing for in-depth analysis. This is of great significance for the future clinical application of BCD nanomedicines in the treatment of BNCT. Attached Figure Description

[0022] Figure 1 The labeling efficiency test results of the centrifuged ultrafiltration products in Example 1;

[0023] Figure 2 The results show the stability of the purified product in Example 2 after incubation in 100% bovine serum at 37°C for 0, 24, and 72 hours.

[0024] Figure 3 The labeling efficiency test results of the ultrafiltration purified product in Example 3;

[0025] Figure 4 The above are the PET / CT imaging results of the intravenous injection of labeled quantum dots 6 hours after Example 3.

[0026] Figure 5The labeling efficiency of the product purified by the PD-10 column in Comparative Example 1 is shown.

[0027] Figure 6 The labeling efficiency of the product before column separation after 10 min of reaction in Comparative Example 2 is as follows;

[0028] Figure 7 The labeling efficiency of the product before column separation after 20 min of reaction in Comparative Example 2 is as follows;

[0029] Figure 8 The labeling efficiency of the product before column separation after 30 min of reaction in Comparative Example 2 is as follows;

[0030] Figure 9 The labeling efficiency of the product before column separation after 70 min of reaction in Comparative Example 2 is as follows;

[0031] Figure 10 The labeling efficiency of the product before column separation after 2 min of reaction in Comparative Example 3 is as follows;

[0032] Figure 11 The labeling efficiency of the product before column separation after 7 min of reaction in Comparative Example 3 is as follows;

[0033] Figure 12 The labeling efficiency of the product before column separation after 10 min of reaction in Comparative Example 3 is as follows;

[0034] Figure 13 The labeling efficiency of the product before column separation after 20 min of reaction in Comparative Example 3 is as follows;

[0035] Figure 14 The labeling efficiency of the product after column separation in Comparative Example 3 after 20 min of reaction is as follows;

[0036] Figure 15 The labeling efficiency of the product before column separation after 10 min of reaction in Comparative Example 4 is as follows;

[0037] Figure 16 The labeling efficiency of the product before column separation after 20 min of reaction in Comparative Example 4 is as follows;

[0038] Figure 17 The labeling efficiency of the product before column separation after 8 hours of reaction in Comparative Example 4 is shown. Detailed Implementation

[0039] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] Example 1

[0041] Transfer 0.5 ml of BCDs (dissolved in PB (0.5 mol / L pH 7.5 phosphate buffer, 2 mg / mL) to a 1.5 ml centrifuge tube, and add Na. 124 I (1 μL, 150 μCi) was mixed, and then 250 μL of chloramine T (dissolved in PB, 20 mg / mL) was added. The mixture was allowed to stand at room temperature for 14 h, then ultrafiltered by centrifugation (3000 g, 15 min) and washed four times with ultrapure water to obtain the final product. 124 I-labeled BCDs. Sampling and TLC (developing solvent: physiological saline) were performed, see [link to TLC]. Figure 1 ,from Figure 1 The labeling efficiency is 62.5%.

[0042] Example 2

[0043] Determination of the preparation in Example 1 124 The stability of I-labeled BCDs was assessed by incubating the purified product from Example 1 in 100% bovine serum (FBS) at 37°C for 0, 24, and 72 h. The activity-to-volume ratio of the purified product to the bovine serum was 1 μCi: 300 μL. TLC was performed, and the results are as follows: Figure 2 No label detachment was detected, indicating that the labeled product has good stability.

[0044] Example 3

[0045] The product of Example 1 124 I-labeled BCDs were ultrafiltered with physiological saline, and the product in the ultrafiltration tube was collected. A sample was taken for TLC analysis (see [link to TLC analysis]). Figure 3 After ultrafiltration purification, the effective labeling rate was 98.5%. The activity of the purified product was adjusted to 100 μCi, and it was intravenously injected into BalB / C nude mice at a volume of 200 μl per mouse. PET / CT imaging was performed 6 hours later, and the results are as follows. Figure 4 As shown, the labeled BCD nanoparticles accumulated in the liver 6 hours after injection and were not taken up in sites such as the thyroid gland.

[0046] Because free isocyanate (I) is readily taken up by the thyroid gland in animals, any large amount of free I in an animal will inevitably accumulate in the thyroid gland. However, in this experiment, mice were injected with... 124 No free I was found to accumulate in the thyroid gland after I-BCDs, indicating that the method provided by this invention... 124 I-BCDs are also very stable in animals.

[0047] Comparative Example 1

[0048] NBS (N-bromosuccinimide) was used as a catalyst.

[0049] Transfer 0.5 ml of BCDs (dissolved in PB, 2 mg / ml) to a 1.5 ml centrifuge tube, and add Na. 124 I (350 μl, 1.121 mCi) was added, followed by 12 μl of NBS (dissolved in PBS, 1 mg / ml), and the mixture was shaken at room temperature for 1 min. The nanoparticles were purified by elution with physiological saline using a PD-10 column, and a 2 ml eluent volume was collected. The activity level measured by the activity meter was 481 μCi. This solution was then used for TLC. TLC instrument: BioScan AR-2000. The results are as follows: Figure 5 As shown. From Figure 5 As can be seen, the labeling rate using NBS as a catalyst was 13.30%, which is much lower than the labeling rate in Example 1.

[0050] Comparative Example 2

[0051] Extend the reaction time catalyzed by NBS.

[0052] Transfer 0.6 ml of BCDs (dissolved in PB, 2 mg / ml) to a 1.5 ml centrifuge tube, and add Na. 125 I (350 μl, 53 μCi) was added, followed by 14 μl of NBS (dissolved in PBS, 0.24 mg / ml). The reaction was carried out at room temperature with shaking. Samples were taken for TLC at 10, 20, 30, and 70 min after the reaction started. The results are as follows. Figures 6-9 As shown.

[0053] As shown in the figure, the TLC expansion has two peaks, with the first peak closest to the origin corresponding to... 125 I-BCDs, and as the reaction time increases, the proportion of the first peak gradually decreases, with a reaction time of 70 min ( Figure 9 At that time, the proportion of the second peak (unlabeled iodine) was already greater than 90%. This indicates that extending the reaction time with NBS as the catalyst reduces the labeling efficiency.

[0054] Comparative Example 3

[0055] Using chloramine T as a catalyst, the effect of different reaction times on the labeling rate was investigated (the time range was determined based on the optimal labeling time of graphene oxide nanomaterials; the optimal reaction time for labeling graphene oxide nanocomposites with cobalt was 3 min, with a labeling rate of 75%).

[0056] Transfer 0.5 ml of BCDs (dissolved in PB, 2 mg / ml) to a 1.5 ml centrifuge tube, and add Na. 125I (1 μl, 153 μCi) was mixed, and then 250 μl of chloramine T (20 mg / ml) was added. The mixture was shaken at room temperature. After the reaction started, samples were taken for TLC at 2, 7, 10, and 20 min. At 20 min, 250 μl of sodium metabisulfite (40 mg / ml) was added to terminate the reaction, and samples were taken for TLC. The results are as follows. Figures 10-13 As shown.

[0057] from Figures 10-13 It can be seen that the labeling rates at 2, 7, 10, and 20 minutes of the reaction were 28.7%, 27.5%, 25.2%, and 20.7%, respectively. Therefore, it is evident that the labeling rates at 2 and 7 minutes of the reaction... 125 The labeling rate of I-BCDs (peaks near the origin) was higher than that at 10 and 20 min of reaction. The product at 20 min of reaction was purified using a PD-10 column, and the elution volume of 1-2 ml (the darkest color) was collected. The activity level was measured at 14 μCi using an activity meter. This solution was then used for TLC. The results are as follows... Figure 14 As shown. From Figure 4 It can be seen that the labeling rate after purification is 18.5%.

[0058] The above results indicate that within a reaction time range of 2–20 minutes, 125 The labeling rates of I-BCDs were all low. However, in order to further improve the labeling rate and labeling stability, the inventors continued to extend the reaction time beyond 20 min. Unexpectedly, they found that when the reaction was carried out for 14 h, not only was the labeling efficiency improved (see Example 1, 62.5%), but the resulting labeled product also had good stability (see Examples 2 and 3).

[0059] Comparative Example 4

[0060] Iodogen (chlorglycine) was used as a catalyst.

[0061] Weigh a certain amount of Iodogen, dissolve it in CH2Cl2 to prepare a solution, then add it to a 1.5 ml centrifuge tube and dry it to form a film. Set aside for later use.

[0062] Add 0.6 ml of BCDs (dissolved in PB, 2 mg / ml) to the prepared Iodogen tube, then add Na. 124 I (350 μl, 1 mCi), reaction at room temperature, samples were taken for TLC at 10, 20 min and 8 h after the reaction started. Results are as follows. Figures 15-17 As shown, the first peak after sample development before column separation is negligible, indicating labeling failure. This demonstrates that Iodogen, as a catalyst, cannot catalyze the reaction. 124 I marks BCDs.

[0063] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A radioisotope-labeled boron-containing quantum nanodot, characterized in that, The radioisotope-labeled boron-containing quantum nanodots were prepared by the following steps: (1) Mix the quantum dot solution with the radioactive isotope solution, add a catalyst and let the reaction stand; (2) The reaction product of step (1) is subjected to centrifugation and ultrafiltration, and the ultrafiltration product is collected, which is radioisotope labeled nano-quantum dots. The quantum dot solution is a boron-containing quantum dot (BCDs) solution, and the mass activity ratio of the BCDs to the radioactive isotope is 1 mg: 1 μCi ~ 10 mCi; the radioactive isotope is... 124 I, 125 I or 131 I; the catalyst is chloramine T solution; the standing reaction time is 14 h.

2. The radioisotope-labeled boron-containing quantum nanodots as described in claim 1, characterized in that, The concentration of the chloramine T solution is 1–100 mg / ml, and the volume ratio of the chloramine T solution to the radioactive isotope solution is 10–1000:

1.

3. The radioisotope-labeled boron-containing quantum nanodots as described in claim 1 or 2, characterized in that, The centrifugal ultrafiltration process is performed at a speed of 3000–20000 rpm for 10–20 min.

4. The application of the radioisotope-labeled boron-containing nanoquantum dots according to any one of claims 1-3 as a diagnostic reagent.

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