Method for preparing targeted nuclear drug by reducing nuclide lutetium 177 through biological agent
By using the radiation-resistant Mycobacterium tumefaciens vector to load lutetium-177 to prepare targeted radiopharmaceuticals, the problem of insufficient lutetium-177 supply was solved, achieving efficient and safe targeted therapy, reducing treatment costs and significantly reducing tumor volume.
Patent Information
- Application Number
- CN202511384451.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-26
- Publication Date
- 2025-11-21
AI Technical Summary
Lutetium-177 isotope drugs have shown significant efficacy in treating gastrointestinal and pancreatic neuroendocrine tumors and metastatic prostate tumors, but insufficient supply leads to high treatment costs. Existing technologies make it difficult to prepare highly efficient, safe, and targeted nuclear drugs.
Using *Radiata spp.* as a carrier, lutetium-177 was loaded onto the *Radiata spp.* through a process of cultivation, washing, and incubation. By utilizing the characteristics of its cell membrane and the reducing properties of its red pigment, lutetium-177 attracted to the heterogeneous charge of tumor cells, achieving targeted aggregation.
The prepared targeted nuclear drug has high biosafety and strong targeting, which can effectively reduce treatment costs, improve the accessibility of lutetium-177, significantly reduce the volume of solid tumors, and reduce the white blood cell and lymphocyte counts in mouse blood.
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Figure CN120983666A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of radio-pharmaceuticals, and particularly relates to a method for preparing a targeted radio-pharmaceutical by reducing the radionuclide Lutetium-177. BACKGROUND
[0002] Deinococcus radiodurans was first discovered by American scientist Anderson in 1956 in canned meat sterilized by high-dose ionizing radiation. It is a red non-pathogenic bacterium, and the optimal growth temperature thereof is 30 DEG C. In the stable growth period, the growth of Deinococcus radiodurans is relatively stable, and each cell contains 4 similar genomes, so that a reliable template can be provided when DNA repair occurs, and the cells in the logarithmic growth phase grow vigorously, and each cell contains 8-10 genomes. Studies have shown that the acute irradiation half lethal dose of human is only 3-5 Gy, while Deinococcus radiodurans can still survive under the condition of acute irradiation of 15 kGy, and the radiation resistance is 3000 times that of human. When Deinococcus radiodurans is subjected to a dose of 60 Gy / h, it can still grow normally, showing that Deinococcus radiodurans has super strong resistance to ionizing radiation. In addition to ionizing radiation resistance, Deinococcus radiodurans can still survive under ultraviolet irradiation conditions, showing super strong ultraviolet radiation resistance. At the same time, Deinococcus radiodurans also shows strong resistance to chemical mutagens such as mitomycin C, and can survive in a desiccator for six years and maintain a 10% survival rate. Due to its super strong ionizing radiation resistance, ultraviolet resistance, chemical mutagen resistance and desiccation resistance, Deinococcus radiodurans is used as a model organism for resistance mechanism research. 2 Deinococcus radiodurans was first discovered by American scientist Anderson in 1956 in canned meat sterilized by high-dose ionizing radiation. It is a red non-pathogenic bacterium, and the optimal growth temperature thereof is 30 DEG C. In the stable growth period, the growth of Deinococcus radiodurans is relatively stable, and each cell contains 4 similar genomes, so that a reliable template can be provided when DNA repair occurs, and the cells in the logarithmic growth phase grow vigorously, and each cell contains 8-10 genomes. Studies have shown that the acute irradiation half lethal dose of human is only 3-5 Gy, while Deinococcus radiodurans can still survive under the condition of acute irradiation of 15 kGy, and the radiation resistance is 3000 times that of human. When Deinococcus radiodurans is subjected to a dose of 60 Gy / h, it can still grow normally, showing that Deinococcus radiodurans has super strong resistance to ionizing radiation. In addition to ionizing radiation resistance, Deinococcus radiodurans can still survive under ultraviolet irradiation conditions, showing super strong ultraviolet radiation resistance. At the same time, Deinococcus radiodurans also shows strong resistance to chemical mutagens such as mitomycin C, and can survive in a desiccator for six years and maintain a 10% survival rate. Due to its super strong ionizing radiation resistance, ultraviolet resistance, chemical mutagen resistance and desiccation resistance, Deinococcus radiodurans is used as a model organism for resistance mechanism research.
[0003] Lutetium-177 has high LET rays, short range, high stability and good drug forming property, and has remarkable effect on treating gastrointestinal pancreatic neuroendocrine tumors, metastatic prostate tumors and the like compared with other isotope drugs. The cost of medical isotope diagnosis and treatment is relatively high. Limited by the shortage of Lutetium-177 supply, the drug is in short supply.
[0004] The application explores a method for preparing precise targeted radio-pharmaceuticals by using Deinococcus radiodurans carrying radionuclides, so as to improve the availability of Lutetium-177 and reduce the treatment cost. SUMMARY
[0005] The application aims to provide a method for preparing a targeted radio-pharmaceutical by reducing the radionuclide Lutetium-177, and the Deinococcus radiodurans obtained in the method has strong adsorption effect and combination capacity on Lutetium-177, and can effectively reduce the preparation cost of the targeted radio-pharmaceutical; the prepared targeted radio-pharmaceutical has high biological safety and enhanced targeting.
[0006] The technical scheme for achieving the object of the application is as follows:
[0007] The application discloses a method for preparing a targeted nuclear medicine by reducing a radionuclide of Lutetium-177, and the method comprises the following steps:
[0008] Step 1, activating and culturing of a strain of Deinococcus radiodurans to obtain a Deinococcus radiodurans cell body;
[0009] Step 2, incubating the Deinococcus radiodurans cell body in a radionuclide of Lutetium Chloride 177 to obtain a Deinococcus radiodurans targeted nuclear medicine loaded with Lutetium-177.
[0010] Further, the step 1 comprises the following steps:
[0011] Step 1.1, activating and culturing of a strain of Deinococcus radiodurans;
[0012] Step 1.2, collecting a Deinococcus radiodurans cell body;
[0013] Step 1.3, washing the Deinococcus radiodurans cell body to remove impurities.
[0014] Further, the step 1.1 is specifically as follows: a strain of Deinococcus radiodurans is activated, a single colony is picked and inoculated in a TGY culture medium, constant-temperature shock culture is carried out, the absorbance value of Deinococcus radiodurans at 600 nm wavelength is determined regularly, and the culture is ended when the absorbance value of Deinococcus radiodurans is OD600=0.8-1.2.
[0015] Further, in the step 1.1, the formula of the TGY culture medium is as follows: in terms of mass ratio, peptone: yeast: glucose = 5:3:1; and the culture condition parameters are as follows: the temperature is 28-34 DEG C, and the rotation speed of a shaking table is 200 rpm.
[0016] Further, in the step 1.1, the cycle of determining the absorbance value of Deinococcus radiodurans at 600 nm wavelength is 20-40 min.
[0017] Further, the step 1.2 is specifically as follows: the Deinococcus radiodurans cell body is collected by using a centrifugal method; and the centrifugal condition parameters are as follows: the rotation speed is 6000-8000 rpm, and the time is 8-12 min.
[0018] Further, the step 1.3 is specifically as follows: the Deinococcus radiodurans cell body is washed by using a phosphate buffer to remove impurities; the concentration of the phosphate buffer is 6X, and the pH value is 6.0-8.0.
[0019] Further, the components of the phosphate buffer are as follows: NaCl 120-150 mM; KCl 2.2-3.2 mM; Na2HPO4 7.5-8.5 mM; and KH2PO4 1.5-1.9 mM.
[0020] Further, in the step 2, the mass ratio of Deinococcus radiodurans bacteria and the nuclide Lutetium Chloride is 1000:1.
[0021] Further, in the step 2, the condition parameters of the incubation are as follows: the incubation temperature is 18-28 DEG C, the incubation time is 0.5-3 hours, and the shaking speed during the incubation is 80-120 rpm.
[0022] The beneficial technical effects of the present application are as follows:
[0023] 1. The targeted nuclear medicine prepared by the present application has high biological safety: Deinococcus radiodurans is a non-pathogenic bacterium, and its gene fragment has been found in the intestinal metagenome of a long-lived population, which provides safety basis for the Deinococcus radiodurans targeted with Lutetium-177.
[0024] 2. The targeted nuclear medicine prepared by the present application has enhanced targeting: Deinococcus radiodurans cell membrane surface has metal ion channels, and Deinococcus radiodurans has red pigments with reducing property. After Lutetium-177 ions contact with Deinococcus radiodurans, the Lutetium-177 ions can be adsorbed on the sugar coating layer, and the Lutetium-177 metal ions react with the pigments with reducing property to be reduced into metal monomers. Deinococcus radiodurans cell membrane contains a large amount of cationic phospholipids, and the head of the phospholipid molecules has positive charge. This structure makes the whole cell membrane have strong positive charge, so that the Lutetium-177 nuclear medicine prepared by Deinococcus radiodurans has positive charge. Deinococcus radiodurans has the ability to resist 15kGy acute irradiation, which enables Deinococcus radiodurans to carry Lutetium-177 metal ions. The solid tumor is negatively charged after lactic acidification, and the Lutetium-177 nuclear medicine can be highly concentrated in the tumor area by using the property of attracting each other by heterocharge. Cancer cells lose normal regulation, continuously divide, and gather to form tumors, and after continuous stacking, an anaerobic environment is formed, and cancer cells cannot obtain sufficient oxygen and secrete a large amount of lactic acid in a relatively closed environment of the solid tumor. The injection into the solid tumor realizes tumor-specific recognition by using the property of attracting each other by heterocharge.
[0025] 3. The Deinococcus radiodurans in the targeted nuclear medicine prepared by the present application has strong adsorption effect and combination ability to Lutetium-177: the OD 600 of the cultured Deinococcus radiodurans is 0.8-1.2, at this time, the Deinococcus radiodurans is in the logarithmic growth phase and has the highest activity. At this time, the Deinococcus radiodurans has the strongest adsorption effect to Lutetium-177. The cultured Deinococcus radiodurans is washed by phosphate buffer (pH=6.0-8.0) to ensure that the culture medium is completely removed, thereby effectively improving the adsorption and combination ability of Deinococcus radiodurans to Lutetium-177. BRIEF DESCRIPTION OF DRAWINGS
[0026] Figure 1 It is an external coating structure diagram of Deinococcus radiodurans bacteria in the embodiments of the present application. DETAILED DESCRIPTION
[0027] The application will be further described in detail below in combination with the accompanying drawings and examples.
[0028] The application provides a method for preparing a targeted nuclear medicine by reducing radionuclide Lutetium-177 by a biological agent, which specifically comprises the following steps:
[0029] Step 1, activation and culture of Deinococcus radiodurans strain to obtain Deinococcus radiodurans cell
[0030] Step 1.1, activation and culture of Deinococcus radiodurans strain
[0031] The Deinococcus radiodurans strain is activated, a single colony is picked and inoculated in TGY culture medium, and constant temperature shaking culture is carried out under the condition that the temperature is 28-34℃ and the shaking speed is 200 rpm. The absorbance value of Deinococcus radiodurans at 600 nm wavelength is determined regularly by a spectrophotometer, that is, the absorbance OD 600 When the absorbance of Deinococcus radiodurans is OD 600 =0.8-1.2, the culture is ended.
[0032] The formula of TGY culture medium is that the mass ratio of proteose peptone, yeast and glucose is 5:3:1. In a specific embodiment, the formula of TGY culture medium is 5 g of proteose peptone, 3 g of yeast and 1 g of glucose.
[0033] When the absorbance value of Deinococcus radiodurans at 600 nm wavelength is determined by a spectrophotometer, the absorbance value is determined every 20-40 min because it takes several hours for Deinococcus radiodurans to breed a generation.
[0034] Step 1.2, collection of Deinococcus radiodurans cell
[0035] The Deinococcus radiodurans cell is collected by centrifugation at a speed of 6000-8000 rpm for 8-12 min.
[0036] Step 1.3, washing of Deinococcus radiodurans cell to remove impurities
[0037] The Deinococcus radiodurans cell is washed with phosphate buffer solution at a concentration of 6X and a pH value of 6.0-8.0 at 20-30℃ until the supernatant after centrifugation of the phosphate buffer solution is colorless from the light yellow of the culture medium, so as to remove the impurities in the culture medium.
[0038] In a specific embodiment, the phosphate buffer solution is washed for 3 times to remove the interference of the culture medium. The specific operation method is that after the first centrifugal collection, the cell is suspended by blowing with a buffer solution by using a pipette gun, and the supernatant is discarded after centrifugation, which is repeated for 3 times.
[0039] The components of the phosphate buffer are: NaCl 120-150 mM; KCl 2.2-3.2 mM; Na2HPO4 7.5-8.5 mM; KH2PO4 1.5-1.9 mM.
[0040] Step 2, incubate the Deinococcus radiodurans bacteria in the lutetium chloride solution (LuCl3) to obtain the Deinococcus radiodurans targeted nuclear medicine loaded with lutetium-177. 177 Step 2, incubate the Deinococcus radiodurans bacteria in the lutetium chloride solution (LuCl3) to obtain the Deinococcus radiodurans targeted nuclear medicine loaded with lutetium-177.
[0041] The mass ratio of Deinococcus radiodurans bacteria to the nuclide lutetium chloride is 1000:1. According to the mass ratio, the Deinococcus radiodurans bacteria is added to the nuclide lutetium chloride solution for incubation. The incubation temperature is 18-28°C, and the incubation time is 0.5-3 hours. The Deinococcus radiodurans targeted nuclear medicine loaded with lutetium-177 is obtained. During the incubation process, the shaking bed is required, and the shaking speed is 80-120 rpm to allow the lutetium-177 ions to fully contact and adsorb the Deinococcus radiodurans. If the adsorption is insufficient, the treatment effect will be affected.
[0042] In a specific embodiment, considering that one patient needs 100 mCi of lutetium-177 for one treatment, and the Deinococcus radiodurans has no toxic side effects, the lutetium-177 metal ions are fully adsorbed. The adsorption effect is detected by measuring the amount of lutetium-177 in the supernatant after centrifugation. When the OD of the cultured Deinococcus radiodurans is 1.0, about 1×10 600 9 Deinococcus radiodurans are contained in 1 ml of culture solution. 100 mCi of lutetium-177 is required for the treatment of one patient, which is about 3.09×10 15 atoms of lutetium-177, i.e., about 9.085×10 -7 g of lutetium-177. According to the adsorption capacity of Deinococcus radiodurans, which is about 100 mg / g, 1 / 100 (1 mg / g) of the treatment capacity is taken, and 9.085×10 -7 g of lutetium-177 requires 9.08×10 -4 g of Deinococcus radiodurans (i.e., 0.908 mg of bacteria).
[0043] During the incubation process, the lutetium chloride metal ions are reduced to metal elements after being adsorbed to the surface of the Deinococcus radiodurans and adhering to the outside of the cell membrane or entering the cell membrane through the metal ion channel. Figure 1 is a schematic diagram of the cell membrane structure of Deinococcus radiodurans, as shown in Figure 1 The cell membrane of Deinococcus radiodurans includes six multi-layer structures, from the outside to the inside, which are the sugar coating layer, the S layer, the outer membrane layer, the periplasmic space layer, the peptidoglycan layer, and the inner membrane layer.
[0044] Lutetium-177 metal ions are incubated with Deinococcus radiodurans, the surface of Deinococcus radiodurans has a metal ion channel (between two outer sugar layers), and the reduced lutetium-177 metal element can enter the Deinococcus radiodurans cells to form a mixture of Deinococcus radiodurans loaded with lutetium-177 metal elements, and the lutetium-177 metal elements are in the cytoplasm, and since Deinococcus radiodurans has strong radiation resistance and DNA damage repair ability, it will not be radiated to death. The tumor cells with weak radiation resistance will be irradiated by beta rays, causing DNA single-strand damage, and the DNA replication, RNA transcription, protein translation and post-translational modification of cancer cells will be blocked, eventually leading to the death of cancer cells.
[0045] The prepared Deinococcus radiodurans loaded with lutetium-177 is suspended in 0.9% physiological saline for nuclear medicine, and injected into a mouse solid tumor model to perform a mouse model experiment.
[0046] After two weeks of observation, the tumor size and blood index changes of the mice are observed.
[0047] Mouse SPECT-CT shows that the drug has high aggregation degree in the body, the retention time is about 8 days, and the solid tumor is significantly smaller. The white blood cell and lymphocyte counts in the blood of the mice are significantly lower than those of the control group after 10 days of injection.
[0048] The size change of the solid tumor is observed by imaging, and if the tumor continues to shrink, it indicates that the Deinococcus radiodurans loaded with lutetium-177 nuclear medicine achieves a therapeutic effect.
[0049] The mechanism of action of the Deinococcus radiodurans loaded with lutetium-177 prepared in the application is as follows:
[0050] Solid tumors have high energy demand due to rapid proliferation, and anaerobic respiration can provide energy faster to meet the demand of rapid proliferation; tumor tissues are usually insufficiently supplied with blood vessels, resulting in low internal oxygen levels, in which case cells turn to anaerobic respiration to produce lactic acid; even in the case of sufficient oxygen, tumor cells tend to perform anaerobic metabolism, preferentially decomposing glucose to produce lactic acid. The production of a large amount of lactic acid causes the solid tumor to become lactic acidized, making it negatively charged.
[0051] Deinococcus radiodurans can synthesize a red pigment in vivo, and the pigment has a reducing function. The reducing substances on the cell membrane and secreted outside the cell make Deinococcus radiodurans have reducing properties. The reducing properties can reduce metal ions in contact with them to metal elements, and at the same time, the reduced metal elements are chelated on the cell membrane surface or in the cells of Deinococcus radiodurans. In addition, it has 8-10 sets of genome copies, and its DNA damage repair ability is very strong, showing strong radiation resistance. Therefore, Deinococcus radiodurans can be used to prepare a nuclear medicine loaded with positively charged lutetium-177 elements.
[0052] The prepared Deinococcus radiodurans nuclear medicine is injected into a solid tumor, under the action of mutual attraction of different hetero-charges, the Deinococcus radiodurans nuclear medicine is combined to the surface of the solid tumor, the cancer cells are caused to occur DNA double-strand / single-strand break by the beta rays emitted by the Lutetium-177 nuclide, the cancer cells cannot complete DNA replication, and the cancer cells are immediately apoptotic.
[0053] The application is described in detail above in combination with the drawings and examples, but the application is not limited to the above examples, and various changes can be made within the knowledge of those skilled in the art without departing from the purpose of the application. The contents not described in detail in the application can adopt the prior art.
Claims
1. A method for preparing targeted radiopharmaceuticals by reducing the radionuclide lutetium-177 with a biological agent, characterized in that, The method includes: Step 1: Activate and culture the *Radiata-resistant* strain to obtain *Radiata-resistant* cells; Step 2: Add the radiation-resistant *Gastrodinium tumefaciens* cells to the radionuclide lutetium chloride solution. 177 Incubation in LuCl3 solution yielded a radiation-resistant, targeted radiopharmaceutical loaded with lutetium-177 from *Morphococcus radiodurans*.
2. The method for preparing targeted radiopharmaceuticals by reducing the radionuclide lutetium-177 with a biological agent according to claim 1, characterized in that, Step 1 includes: Step 1.1: Activation and culture of *Radiata resistant* strain; Step 1.2: Collect *Radiata-resistant* cells; Step 1.3: Wash the radiation-resistant bacteria to remove impurities.
3. The method for preparing targeted radiopharmaceuticals by reducing the radionuclide lutetium-177 with a biological agent according to claim 2, characterized in that, Step 1.1 specifically involves: activating the *Radiataegilops* strain, picking a single colony and inoculating it into TGY medium, performing isothermal shaking culture, periodically measuring the absorbance of *Radiataegilops* at a wavelength of 600 nm, and culturing until the absorbance of *Radiataegilops* reaches OD0.
05. 600 When the concentration reaches 0.8-1.2, the culture is terminated.
4. The method for preparing targeted radiopharmaceuticals by reducing the radionuclide lutetium-177 with a biological agent according to claim 3, characterized in that, In step 1.1, the TGY culture medium formula is as follows: peptone: Yeast: glucose = 5:3:1; culture conditions: temperature 28-34℃, shaker speed 200 rpm.
5. The method for preparing targeted radiopharmaceuticals by reducing the radionuclide lutetium-177 with a biological agent according to claim 3, characterized in that, In step 1.1, the absorbance of *Bacillus radiodurans* at a wavelength of 600 nm is measured every 20-40 minutes.
6. The method for preparing targeted radiopharmaceuticals by reducing the radionuclide lutetium-177 with a biological agent according to claim 2, characterized in that, Step 1.2 specifically involves collecting *Radiata toxicaria* cells using centrifugation. The centrifugation parameters are: rotation speed of 6000-8000 rpm and time of 8-12 min.
7. The method for preparing targeted radiopharmaceuticals by reducing the radionuclide lutetium-177 with a biological agent according to claim 2, characterized in that, Step 1.3 specifically involves washing the *Bacillus radiata* cells with a phosphate buffer solution to remove impurities; the phosphate buffer solution concentration is 6X, and the pH value is 6.0-8.
0.
8. The method for preparing targeted radiopharmaceuticals by reducing the radionuclide lutetium-177 with a biological agent according to claim 7, characterized in that, The phosphate buffer solution has the following components: NaCl 120–150 mM; KCl 2.2–3.2 mM; Na₂HPO₄ 7.5–8.5 mM; KH₂PO₄ 1.5–1.9 mM.
9. The method for preparing targeted radiopharmaceuticals by reducing the radionuclide lutetium-177 with a biological agent according to claim 1, characterized in that, In step 2, the mass ratio of radiation-resistant *Gastrodinium radiata* cells to lutetium chloride is 1000:
1.
10. The method for preparing targeted radiopharmaceuticals by reducing the radionuclide lutetium-177 with a biological agent according to claim 1, characterized in that, In step 2, the incubation conditions are as follows: incubation temperature is 18-28℃, incubation time is 0.5-3 hours, and the shaking speed during incubation is 80-120 rpm.