CN111906328B - A kind of 177 Lu-labeled gold nanocluster and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of radioisotope labeling, and provides a method for preparing ¹⁷⁷ Lu-labeled gold nanoclusters, including reducing chloroauric acid with glutathione to obtain gold nanoclusters; using ¹⁷⁷ LuCl ₃ hydrochloric acid to obtain gold nano-clusters; The gold nanoclusters were labeled with the eluent to obtain ^177Lu -labeled gold nanoclusters. The gold nanoclusters of the present invention can stably label ¹⁷⁷ Lu; compared with pure nuclides, the nuclides labeled on the gold nanoclusters are more likely to be enriched and retained in tumor cells, and the ¹⁷⁷ Lu-labeled gold Antitumor drugs prepared by nanoclusters have very good application value.

Description

A kind of 177Lu labeled gold nanocluster and its preparation method and application

technical field

The invention relates to the technical field of radioisotope labeling, in particular to a gold nanocluster labeled with ¹⁷⁷ Lu and a preparation method and application thereof.

Background technique

Cancer is still one of the leading causes of human death worldwide. Radiation therapy, including both external radiation therapy (EBRT) and internal radioisotope therapy, has been widely used in clinical cancer treatment despite tremendous advances in cancer biology, oncology, surgical techniques, etc. in recent years. However, high doses of ionizing radiation are often required during radiotherapy, which leads to severe damage to normal tissue adjacent to the tumor; at the same time, the efficacy of radiotherapy is limited by different mechanisms.

With the development of nanotechnology, people have a great interest in enhancing the effect of radiation therapy based on nanomedicine. Radiation-absorbing nanomaterials can be used as radiosensitizers to deposit radiation energy within tumors and enhance therapeutic efficacy or to modulate the tumor microenvironment through nanomaterials to overcome hypoxia-related radioresistance. At the same time, nanocarriers can also deliver therapeutic radioisotopes to tumor sites to achieve internal radiotherapy or synergistic chemotherapy drugs to achieve combination of radiotherapy and chemotherapy. In addition to this, it has been reported that the interaction of radioisotopes with high atomic numbers can generate secondary electrons, which have a significant effect on enhancing internal radiotherapy.

Therefore, research and development of an anti-tumor drug prepared from nuclide-labeled gold nanoclusters has great social value and broad market prospects.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a ¹⁷⁷ Lu-labeled gold nanocluster and a preparation method and application thereof in order to overcome the deficiencies of the prior art. In the present invention, the ¹⁷⁷ Lu radionuclide is stably labeled on the gold nanocluster by the method of chelation. Compared with the pure nuclide, the nuclide labeled on the gold nanocluster is more likely to be enriched and retained at the tumor cell site. .

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides a preparation method of ¹⁷⁷ Lu-labeled gold nanoclusters, comprising the following steps:

1) reducing chloroauric acid with glutathione to obtain gold nanoclusters;

2) The gold nanoclusters are labeled with ¹⁷⁷ LuCl ₃ hydrochloric acid eluent to obtain ¹⁷⁷ Lu labeled gold nanoclusters.

Preferably, the molar ratio of glutathione and chloroauric acid in step 1) is 3-5:4-6, the glutathione is an aqueous solution of glutathione, and the chloroauric acid is chloroauric acid Acid aqueous solution.

Preferably, the reduction temperature in step 1) is 80-100° C., and the time is 25-40 min.

Preferably, the concentration of the glutathione aqueous solution is 40-60 mg/mL, and the concentration of the chloroauric acid aqueous solution is 0.5-1.5 mol/L.

Preferably, after the reduction in step 1), the reaction solution is filtered, and the pH value of the filtrate is adjusted to 2-5, and then centrifuged and concentrated to obtain gold nanoclusters.

Preferably, the activity of the ¹⁷⁷ LuCl ₃ hydrochloric acid eluent in step 2) is 1 to 3 mCi, the concentration of the gold nanoclusters is 30 to 40 mg/mL, and the ¹⁷⁷ LuCl ₃ hydrochloric acid eluent and gold nano-clusters have a concentration of 30 to 40 mg/mL. The volume ratio of the clusters is 3:50-100.

Preferably, in step 2), the labeled ¹⁷⁷ LuCl ₃ hydrochloric acid eluent is adjusted to a pH value of 5-6 before labeling the gold nanoclusters, and the labeling time is 20-40 min.

The present invention also provides ¹⁷⁷ Lu-labeled gold nano-clusters obtained by the preparation method of the ¹⁷⁷ Lu-labeled gold nano-clusters.

The present invention also provides an application of the ¹⁷⁷ Lu-labeled gold nanocluster in preparing antitumor drugs.

Preferably, the tumor is 4T1.

The beneficial effects of the present invention include:

^The177Lu -labeled gold ^nanoclusters of the present invention are formed by chelating the radionuclide177Lu onto glutathione-modified gold nanoclusters. Gold nanoclusters can stably label ¹⁷⁷ Lu; compared with pure nuclides, nuclides labeled on gold nanoclusters are more likely to be enriched and retained in tumor cells. ¹⁷⁷ Lu-labeled gold nanoclusters The prepared antitumor drug has very good application value.

Description of drawings

FIG. 1 is a schematic diagram of the drug prepared by the ¹⁷⁷ Lu-labeled gold nanoclusters of Example 1 being used for anti-tumor.

FIG. 2 is a transmission electron microscope image of the glutathione-modified gold nanoclusters of Example 1. FIG.

FIG. 3 is the stability test of the ¹⁷⁷ Lu-labeled gold nanoclusters of Example 1. FIG.

FIG. 4 is a liquid scintillation test chart of ^the177Lu -labeled gold nanoclusters of Example 1. FIG.

FIG. 5 is a graph of MTT evaluation of gold nanoclusters ¹⁷⁷ Lu labeled with nuclides with different activities in Application Example 1. FIG.

FIG. 6 is a graph showing the tumor inhibition curve of mice injected with ¹⁷⁷ Lu-labeled gold nanoclusters at one time in the tail vein of Application Example 2.

Figure 7 shows the H&E and Tunnel sections of mouse breast cancer tissue obtained by ¹⁷⁷ Lu-labeled gold nanoclusters using different methods.

Detailed ways

The invention provides a preparation method of ¹⁷⁷ Lu-labeled gold nanoclusters, comprising the following steps:

1) reducing chloroauric acid with glutathione to obtain gold nanoclusters;

2) The gold nanoclusters are labeled with ¹⁷⁷ LuCl ₃ hydrochloric acid eluent to obtain ¹⁷⁷ Lu labeled gold nanoclusters.

The molar ratio of glutathione and chloroauric acid in step 1) of the present invention is preferably 3-5:4-6, more preferably 4:5; the glutathione is preferably an aqueous glutathione solution, The chloroauric acid is preferably an aqueous solution of chloroauric acid.

The reduction temperature in step 1) of the present invention is preferably 80-100°C, more preferably 90°C; the reduction time is preferably 25-40 min, more preferably 30 min.

The concentration of the glutathione aqueous solution of the present invention is preferably 40-60 mg/mL, more preferably 50 mg/mL; the concentration of the chloroauric acid aqueous solution is preferably 0.5-1.5 mol/L, more preferably 1 mol/L; The ratio of the volume of the glutathione aqueous solution to the volume of the chloroauric acid aqueous solution is preferably 461-1154:80-360, more preferably 600-800:120-250, more preferably 738:150.

After the reduction in step 1) of the present invention is completed, the reaction solution is preferably filtered, and the pH of the filtrate is preferably adjusted to 2-5, and then centrifuged and concentrated to obtain gold nanoclusters.

The activity of the ¹⁷⁷ LuCl ₃ hydrochloric acid eluent in step 2) of the present invention is preferably 1 to 3 mCi, more preferably 2 mCi; the concentration of the gold nanocluster concentrate is preferably 30 to 40 mg/mL, more preferably 34.08 mg/mL; the volume ratio of the ¹⁷⁷ LuCl ₃ hydrochloric acid eluent to the gold nanoclusters is preferably 3:50-100, more preferably 3:60-80.

In step 2) of the present invention, the labeling is preferably ¹⁷⁷ LuCl ₃ hydrochloric acid eluent, and the pH value is adjusted to 5-6 before labeling the gold nanoclusters. The labeling time is preferably 20-40 minutes, more preferably 30 minutes .

The present invention also provides ¹⁷⁷ Lu-labeled gold nano-clusters obtained by the preparation method of the ¹⁷⁷ Lu-labeled gold nano-clusters.

The present invention also provides an application of the ¹⁷⁷ Lu-labeled gold nanocluster in preparing antitumor drugs.

The tumor of the present invention is preferably 4T1.

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 protection scope of the present invention.

Example 1

738 μL of 50 mg/mL glutathione (GSH) aqueous solution and 150 μL of 1 mol/L chloroauric acid (HAuCl ₄ ·4H ₂ O) aqueous solution were rapidly added to a solution containing 50 mL of deionized water under stirring conditions. In a single-necked bottle, the single-necked bottle was then sealed, rapidly heated to 90° C., and reacted at 90° C. for 30 minutes. After the reaction was completed, the reaction solution was taken out after returning to room temperature and used a 0.45 μm filter to remove larger particles. Then, the pH value of the filtrate was adjusted to 3 using 1 mol/L NaOH aqueous solution, and the ultrafiltration tube was used for centrifugal concentration (3000Da, 5000r/L). min) The product was dispersed in PBS to obtain glutathione-modified gold nanoclusters (GSHAuNCs).

The pH value of the ¹⁷⁷ LuCl ₃ hydrochloric acid eluent with an activity of 2 mCi and a volume of 0.02 mL was adjusted to 6 using NaOH aqueous solution, and then 1 mL of GSHAuNCs with a concentration of 34.08 mg/mL was added. Centrifugation (3000Da, 4500r/min) washes off the unlabeled ¹⁷⁷ Lu to obtain ¹⁷⁷ Lu-labeled gold nanoclusters ( ¹⁷⁷ Lu@AuNCs).

It can be seen from the TEM image in Figure 2 that the particle size of GSHAuNCs is about 2.5 nm.

From the stability test in Figure 3, it can be seen that the labelling stability of gold nanoclusters to ¹⁷⁷ Lu is as high as 88%.

It can be seen from the liquid scintillation test in Fig. 4 that the ¹⁷⁷ Lu in the ¹⁷⁷ Lu-labeled gold nanoclusters can efficiently generate secondary electrons.

Example 2

650 μL of 55 mg/mL glutathione (GSH) aqueous solution and 120 μL of 1.5 mol/L chloroauric acid (HAuCl ₄ ·4H ₂ O) aqueous solution were rapidly added to 50 mL of deionized water under stirring conditions. In a single-necked bottle, the single-necked bottle was then sealed, and the temperature was rapidly raised to 100° C., and the reaction was carried out at 100° C. for 25 minutes. After the reaction was completed, the reaction solution was taken out after returning to room temperature and used a 0.45 μm filter to remove larger particles. Then, the pH value of the filtrate was adjusted to 3 using 1 mol/L NaOH aqueous solution, and the ultrafiltration tube was used for centrifugal concentration (3000Da, 5000r/L). min) The product was dispersed in PBS to obtain glutathione-modified gold nanoclusters (GSHAuNCs).

The pH value of the ¹⁷⁷ LuCl ₃ hydrochloric acid eluent with an activity of 3 mCi and a volume of 0.01 mL was adjusted to 5 using NaOH aqueous solution, and then 1 mL of GSHAuNCs with a concentration of 30 mg/mL was added, and after the reaction was shaken for 40 minutes, centrifugation was performed using an ultrafiltration tube. (3000Da, 4500r/min) to wash off the unlabeled ¹⁷⁷ Lu to obtain ¹⁷⁷ Lu-labeled gold nanoclusters ( ¹⁷⁷ Lu@AuNCs).

The particle size of GSHAuNCs is about 2.8 nm, and the labeling stability of gold nanoclusters to ¹⁷⁷ Lu is as high as 90%. The ¹⁷⁷ Lu in ¹⁷⁷ Lu-labeled gold nanoclusters can effectively generate secondary electrons.

Example 3

900 μL of 45 mg/mL glutathione (GSH) aqueous solution and 200 μL of 0.8 mol/L chloroauric acid (HAuCl ₄ ·4H ₂ O) aqueous solution were rapidly added to a solution containing 50 mL of deionized water under stirring conditions. In a single-necked bottle, the single-necked bottle was then sealed, and the temperature was rapidly raised to 80° C., and the reaction was carried out at 80° C. for 40 minutes. After the reaction was completed, the reaction solution was taken out after returning to room temperature and used a 0.45 μm filter to remove larger particles. Then, the pH value of the filtrate was adjusted to 4 using 1 mol/L NaOH aqueous solution, and the ultrafiltration tube was used for centrifugal concentration (3000Da, 5000r/L). min) The product was dispersed in water to obtain glutathione-modified gold nanoclusters (GSHAuNCs).

The pH value of the ¹⁷⁷ LuCl ₃ hydrochloric acid eluent with an activity of 1 mCi and a volume of 0.015 mL was adjusted to 5 using NaOH aqueous solution, and then 1 mL of GSHAuNCs with a concentration of 40 mg/mL was added. (3000Da, 4500r/min) to wash off the unlabeled ¹⁷⁷ Lu to obtain ¹⁷⁷ Lu-labeled gold nanoclusters ( ¹⁷⁷ Lu@AuNCs).

The particle size of GSHAuNCs is about 2.3 nm, and the labeling stability of gold nanoclusters to ¹⁷⁷ Lu is as high as 90%. The ¹⁷⁷ Lu in ¹⁷⁷ Lu-labeled gold nanoclusters can efficiently generate secondary electrons.

Application Example 1: In vitro antitumor effect of ^177Lu -labeled gold nanoclusters

Mouse breast cancer cells (4T1 cells) were digested and resuspended in culture medium, and 4T1 cells were seeded into 96-well cell culture plates (5.0×10 ³ cells/well, 0.2 mL) after estimating cell density by hemocytometer. ). After they adhered, 0, 0.313μCi, 0.613μCi, 1.25μCi, 2.5μCi, 5μCi, 10μCi gold nanoclusters ¹⁷⁷ Lu with different activities were added to the well plate and co-cultured with the cells for 24 hours. The original medium was then discarded, washed twice with PBS, and 100 μL of complete medium for culturing 4T1 cells and 25 μL of 253-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide were added Salt solution (MTT, 5 mg/mL) was incubated for 4 hours. After carefully removing the supernatant, 100 μL DMSO was added to each well. After incubation for 10 minutes, the crystals were fully dissolved by shaking. Absorbance was used to evaluate cell viability.

It can be seen from Fig. 5 that ^177Lu @AuNCs have better killing effect, and the killing effect is 88.86% when the activity is only 10μCi, but the killing effect of free ^177Lu is only 8.99-15.92%. This provides the basis for anti-tumor in vivo in mice.

It can be seen from Figure 5 that the survival rates of 4T1 mouse breast cancer cells corresponding to the nuclide-labeled gold nanoclusters ¹⁷⁷ Lu with activities of 0, 0.313 μCi, 0.613 μCi, 1.25 μCi, 2.5 μCi, 5 μCi, and 10 μCi, respectively, are: 99.1379951, 97.04243145, 94.05513859, 86.94359813, 74.90525377, 34.73285279, 11.14661514.

Application Example 2: In vivo antitumor effect of ^177Lu -labeled gold nanoclusters

A subcutaneous tumor model was established with mouse breast cancer cells (4T1), and the mice were treated with ¹⁷⁷ Lu@AuNCs (tail vein: 200 μCi, intratumoral: 75 μCi) by a one-time injection into the tail vein and intratumoral injection of 177 Lu@AuNCs.

It can be seen from Figure 6 that compared with free ¹⁷⁷ Lu, ¹⁷⁷ Lu@AuNCs after one-time tail vein administration has a good anti-tumor effect, and the effect of intratumoral injection is better than that of tail vein injection, and intratumoral injection of ¹⁷⁷ Lu@AuNCs can almost completely inhibit tumor growth.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims (5)

1. A kind of ¹⁷⁷ The preparation method of the Lu-labeled gold nanoclusters is characterized by comprising the following steps of:

1) Reducing chloroauric acid by using glutathione to obtain gold nanoclusters;

2) By using ¹⁷⁷ LuCl ₃ The gold nanoclusters are marked by hydrochloric acid eluent to obtain ¹⁷⁷ Lu-labeled gold nanoclusters;

the molar ratio of the glutathione to the chloroauric acid in the step 1) is 3 to 5 to 4 to 6, the glutathione is a glutathione aqueous solution, and the chloroauric acid is a chloroauric acid aqueous solution;

the concentration of the glutathione aqueous solution is 40 to 60mg/mL, and the concentration of the chloroauric acid aqueous solution is 0.5 to 1.5 mol/L;

the temperature of the reduction in the step 1) is 80-100 ℃, and the time is 25-40 min;

step 2) the ¹⁷⁷ LuCl ₃ The activity of the hydrochloric acid eluent is 1 to 3mCi, the concentration of the gold nanocluster is 30 to 40mg/mL, and the gold nanocluster is prepared from the hydrochloric acid eluent ¹⁷⁷ LuCl ₃ The volume ratio of the hydrochloric acid eluent to the gold nanoclusters is 3 to 50-100;

step 2) the marking ¹⁷⁷ LuCl ₃ The pH value of the hydrochloric acid eluent is adjusted to 5~6, and then the gold nanoclusters are marked for 20-40 min.

2. The production method according to claim 1, wherein the reaction solution is filtered after the reduction in step 1), and the gold nanoclusters are obtained by centrifugal concentration after the pH of the filtrate is adjusted to 2~5.

3. The method of claim 1 or 2 ¹⁷⁷ Prepared by the method for preparing Lu-marked gold nanocluster ¹⁷⁷ Lu-labeled gold nanoclusters.

4. The method of claim 3 ¹⁷⁷ The application of the Lu-marked gold nanoclusters in preparing antitumor drugs.

5. The use of claim 4, wherein the tumor is 4T1.

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