CN117838874B - Preparation method and application of gadolinium-platinum radiotherapy sensitizer - Google Patents
Preparation method and application of gadolinium-platinum radiotherapy sensitizer Download PDFInfo
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- QHSZLMGBMCOLEU-UHFFFAOYSA-N gadolinium platinum Chemical compound [Gd].[Pt] QHSZLMGBMCOLEU-UHFFFAOYSA-N 0.000 title claims abstract description 28
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0038—Radiosensitizing, i.e. administration of pharmaceutical agents that enhance the effect of radiotherapy
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/12—Macromolecular compounds
- A61K49/126—Linear polymers, e.g. dextran, inulin, PEG
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Abstract
The invention discloses a preparation method and application of a gadolinium-platinum radiotherapy sensitizer, and belongs to the technical field of radiotherapy sensitizers. The preparation method comprises the following steps: s1, preparing a gadolinium-regulated platinum-based nano material Pt@Gd 2O3 by taking gadolinium acetylacetonate and platinum acetylacetonate as raw materials; s2, dispersing Pt@Gd 2O3 and mercapto-polyethylene glycol-methoxy mPEG-SH synthesized in the step S1 in deionized water, and reacting 4-6 h under the magnetic stirring of ice bath and 500 rpm. According to the preparation method and the application of the gadolinium-platinum radiotherapy sensitizer, the gadolinium-platinum radiotherapy sensitizer with MRI (magnetic resonance imaging) capability is synthesized by utilizing the regulation characteristic of gadolinium ions on the growth of platinum nano particles; the gadolinium-platinum radiotherapy sensitizer has CAT activity, can catalyze H 2O2 to decompose and generate O 2, relieve the hypoxia inhibition of tumor parts and enhance the sensitivity of radiotherapy; the gadolinium-platinum radiotherapy sensitizer has POD activity, can catalyze H 2O2 to decompose and produce OH, enhance the curative effect of radiotherapy and promote tumor cell apoptosis.
Description
Technical Field
The invention relates to the technical field of radiotherapy sensitizers, in particular to a preparation method and application of a gadolinium-platinum radiotherapy sensitizer.
Background
Radiotherapy, also known as radiotherapy, is an important means of clinical cancer treatment, which uses high-energy ionizing radiation to treat cancer by applying radiation to specific tumor sites of the body. Currently, about 70% of cancer patients clinically need to receive radiation therapy, and 40% of tumors can be radically cured by radiation therapy.
The damage of radiotherapy to cancer cells is closely related to the concentration of O 2, and O 2 has the characteristic of stabilizing radiation damage DNA and preventing self-repair. However, there is often an imbalance between the support and consumption of O 2 by rapidly proliferating cancer cells and the tortuosity of tumor vessels, which factors together lead to a lack of O 2 inside the tumor, which may be resistant to radiation therapy, resulting in treatment failure.
Currently, the introduction of O 2 into a tumor or the direct generation of O 2 in a tumor using a nano-delivery system alleviates the lack of O 2 in the tumor microenvironment and treats cancer with lower radiation doses and safer methods, however, the ability to accelerate apoptosis of cancer cells is limited by radiotherapy when the cancer cells are sensitized simply by increasing the concentration of O 2 in the tumor microenvironment.
Disclosure of Invention
The invention aims to provide a preparation method and application of a gadolinium-platinum radiotherapy sensitizer, which are used for solving the problem that the capability of accelerating cancer cell apoptosis is limited when the existing nano-delivery system improves the O 2 concentration in the tumor microenvironment to sensitize cancer cells.
In order to achieve the above purpose, the invention provides a preparation method of a gadolinium-platinum radiotherapy sensitizer, which comprises the following steps:
S1, preparing a gadolinium-regulated platinum-based nano material Pt@Gd 2O3 by taking gadolinium acetylacetonate and platinum acetylacetonate as raw materials;
S2, dispersing Pt@Gd 2O3 and mercapto-polyethylene glycol-methoxy mPEG-SH synthesized in the step S1 in deionized water, and reacting for 4-6 h under the magnetic stirring of ice bath and 500 rpm;
And after the reaction is finished, centrifugally collecting the precipitate, washing the precipitate, centrifugally dispersing the precipitate in deionized water, and obtaining the gadolinium-platinum radiotherapy sensitizer Pt@Gd 2O3 -PEG.
Preferably, step S1 is specifically:
S1-1, dissolving gadolinium acetylacetonate and platinum acetylacetonate in diethylene glycol, and stirring at 80 ℃ to react at 400-500 rpm for 40 min; adding polyethylenimine into the solution, and stirring at 80 ℃ and 400-500 rpm for reaction at 20-min; adding triethanolamine solution into the solution, stirring and reacting at 80 ℃ and 400-500 rpm for 30 min; after stirring was completed, the product was transferred to a teflon lined autoclave and maintained at 200 ℃ for 24 h.
S1-2, centrifugally collecting the product in 14000 rpm and 15 min, washing the product with absolute ethyl alcohol and deionized water for 3 times respectively, and dispersing the final product in the deionized water to obtain the gadolinium-regulated platinum-based nano material Pt@Gd 2O3.
Preferably, in the step S1-1, the mass volume ratio of the polyethylenimine to the gadolinium acetylacetonate to the platinum acetylacetonate to the diethylene glycol is 1:40:40:40.
Preferably, in the step S1-1, the mass-volume ratio of triethanolamine, gadolinium acetylacetonate, platinum acetylacetonate and diethylene glycol is 1:50:50:50.
Preferably, in step S2, the volume ratio of pt@gd 2O3, mercapto-polyethylene glycol-methoxy, deionized water is 1:1:1.
Preferably, in step S2, the centrifugally collecting sediment is centrifugally collecting sediment at 14000 rpm, 10min; the washing is carried out by washing with deionized water for 2-3 times.
Preferably, the prepared gadolinium platinum radiotherapy sensitizer Pt@Gd 2O3 -PEG has the particle size of 20 nm.
Therefore, the preparation method and the application of the gadolinium-platinum radiotherapy sensitizer have the following technical effects:
(1) The gadolinium-platinum radiotherapy sensitizer prepared by the invention has MRI capability and good dispersibility, effectively optimizes the stability and the dispersibility of the platinum nanoparticles by utilizing the regulation characteristic of gadolinium ions on the growth of the platinum nanoparticles, and simultaneously endows the platinum nanoparticles with the MRI capability;
(2) The gadolinium-platinum radiotherapy sensitizer prepared by the invention has Catalase (CAT) and Peroxidase (POD) activities, can catalyze hydrogen peroxide (H 2O2) to generate oxygen (O 2), relieves the deficiency of O 2 in tumors, plays a role in radiotherapy sensitization, can catalyze H 2O2 to generate hydroxyl free radicals (OH), and enhances the effect of radiotherapy.
Drawings
FIG. 1 is a transmission electron microscope image of GP obtained in the first embodiment;
FIG. 2 is a Mapping picture of GP obtained in example I;
Fig. 3 is an XPS picture of GP prepared in example one;
FIG. 4 is an ultraviolet-visible absorbance at 652nm of TMB solution after co-incubation of different concentrations of GP with H 2O2;
FIG. 5 shows the cell uptake of MC 38 cells after incubation with GPP at different concentrations at different times under a fluorescence inverted microscope;
FIG. 6 is a graph of the results of a biocompatibility assay of GPP versus HUVEC cells at various concentrations;
FIG. 7 is a graph showing the results of in vitro hemolysis assays for GPP drugs at various concentrations;
FIG. 8 is a graph showing cytotoxicity results of various treatments on MC 38 cells;
FIG. 9 is a fluorescent image of the presence of DCFH-DA fluorescent probes to detect OH after incubation of GPP and MC 38 cells at different concentrations;
FIG. 10 flow cytometry test the ability of different treatments to induce apoptosis of MC 38 cells; wherein part (A) is a flow cytometry scatter plot of different treatment groups; part (B) is a statistical plot of apoptotic cells in different treatment groups;
FIG. 11 shows the test of cell cloning to examine the inhibition of MC 38 cell proliferation capacity by different treatments; wherein part (A) is a crystal violet dyeing result observation diagram of different treatment groups; part (B) is a value-added rate statistical graph of different treatment groups.
Detailed Description
The technical scheme of the invention is further described below through the attached drawings and the embodiments.
Unless defined otherwise, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.
Example 1
Preparation of gadolinium-regulated platinum nanomaterial
Gadolinium acetylacetonate of 20 mg and platinum acetylacetonate of 20 mg were dissolved in diethylene glycol of 20 mL and reacted at 80℃with 500 rpm magnetic stirring to give 40 min.
0.5 G Polyethylenimine (PEI) was added to the solution and reacted at 80℃with 500: 500 rpm magnetic stirring for 20: 20 min.
To the solution was added 0.4 mL triethanolamine solution and reacted at 80℃under 500 rpm magnetic stirring for 30 min.
After stirring was completed, the product was transferred to a teflon lined autoclave and maintained at 200 ℃ for 24 h.
And finally, centrifugally collecting the precipitate at 14000 rpm for 15 min, washing with absolute ethyl alcohol and deionized water for 3 times respectively, and dispersing the final product in the deionized water to obtain the gadolinium-regulated platinum nanomaterial (Pt@Gd 2O3, abbreviated as GP).
Effect example 1
The Pt@Gd 2O3, mg synthesized in example one, 2mg of mercapto-polyethylene glycol-methoxy (mPEG-SH) were dispersed in 2mL deionized water and reacted under magnetic stirring of 500 rpm in an ice bath to give 5 h.
After stirring, centrifugally collecting precipitate at 14000 rpm for 10min, washing with deionized water for 2 times, and dispersing the product in deionized water to obtain gadolinium-regulated platinum nanometer preparation (Pt@Gd 2O3 -PEG, abbreviated as GPP).
Experimental test
(1) As shown in fig. 1, a proper amount of GP material was taken, ultrasonically treated, and then dropped onto a copper mesh, and dried to prepare a transmission electron microscope sample, and the result showed that the GP particle size prepared in example one was about 20 nm.
(2) As shown in fig. 2, a proper amount of pt@gd 2O3 was taken, ultrasonically treated, and then dropped onto a copper mesh, and dried to prepare a transmission electron microscope sample, which was observed with a transmission electron microscope, and the result shows that GP prepared in example one consisted of Gd, pt, N, O element.
(3) As shown in FIG. 3, the presence of Pt, gd, O and N elements in GP was further confirmed by X-ray photoelectron spectroscopy (XPS) analysis of Pt@Gd 2O3.
Peaks at 1188.9 eV (Gd 3 d) and 141.8 eV (Gd 4 d) may be due to the presence of Gd 2O3.
(4) As shown in FIG. 4, the peroxidase-like activity of GP was further evaluated by observing the UV-visible light absorption at 652nm of TMB solution. As the GP dose increased, the absorption at 652nm gradually increased, indicating that GP can effectively catalyze H 2O2 to OH.
(5) As shown in fig. 5, fluorescence inverted microscopy examined the ability of GPP to be taken up by tumor cells.
Pt@gd 2O3 -cy5.5 was incubated with MC 38 cells at a concentration of 10, 40 μg/ml, and uptake of pt@gd 2O3 was detected using a fluorescence inverted microscope after incubation of 1h and 4 h, respectively.
The results showed that the red fluorescence intensity gradually increased with increasing concentration and time, indicating that the uptake of MC 38 into Pt@Gd 2O3 was concentration and time dependent.
(6) As shown in fig. 6, the toxic effect of GPP on normal cells was detected by MTT assay.
HUVEC cells in the logarithmic growth phase were digested and inoculated into 96-well plates at 1X 10 4/well, incubated in a 37℃incubator containing 5% CO 2 for 24 hours, and GPP was incubated with HUVEC cells at final concentrations of 0, 20, 40, 80, 160, 320. Mu.g/mL for 24h, respectively.
Thereafter, the culture broth was aspirated, 100 μl of MTT medium was added to each well for incubation for 3h, then the medium was discarded, 150 μl of DMSO was added to each well, 10 min was shaken to completely dissolve the purple crystals, absorbance values at 490 nm were read with an microplate reader, and the relationship between cell viability and nanomaterial concentration was calculated from the absorbance values.
The results show that the survival rate of HUVEC cells is greater than 80% at nanomaterial concentrations below 320 μg/mL, indicating good biocompatibility of GPP for normal cells.
(7) Fresh blood was obtained by taking blood from the eyeballs of mice, and centrifuged at 3000 rpm and 15min at normal temperature to obtain erythrocytes. 5mL PBS buffer was added, gently swirled, the supernatant was discarded by centrifugation, and the pellet red blood cells were resuspended in 20 mL PBS. GPP was added to red blood cell suspensions at final concentrations of 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100 μg/mL.
The red blood cell suspension diluted by PBS is used as a negative control, the red blood cell suspension diluted by ultrapure water is used as a positive control, and red blood cells with different concentrations GPP are added as an experimental group. Each group of solutions was incubated in a 37 ℃ incubator for 3 hours, then the solutions were centrifuged at 3000 rpm for 15: 15 min, the samples were placed on the same horizontal line, and their hemolysis was photographed with a cell phone or a camera.
100 Mu L of sample supernatant is sucked into a 96-well plate, absorbance of the sample at 542 nm is detected by using an enzyme-labeled instrument, and the hemolysis rate is calculated.
As shown in fig. 7, it was found that when different concentrations of GPP were used to incubate with erythrocytes, substantially complete sinking of erythrocytes was observed, with the supernatant varying little compared to the negative control. This suggests that GPP does not hemolyze erythrocytes and has good blood compatibility.
(8) As shown in fig. 8, the killing effect of GPP on tumor cells was examined by MTT assay.
MC 38 cells in the logarithmic growth phase were digested and inoculated into 96-well plates at 1X 10 4/well, and cultured in a 37℃incubator containing 5% CO 2 for 24 hours. The experiments were divided into Control, radiotherapy (RT), GPP, GPP+RT groups, with final GPP concentrations of 40 μg/mL, and incubated with MC 38 cells, respectively. RT groups were subjected to 3 Gy X-ray irradiation at 6 h incubation.
24 After h, the culture solution is sucked off, 100 mu L of MTT culture medium is added into each hole for incubation for 3 h, then the culture medium is discarded, 150 mu L of DMSO is added into each hole, the purple crystals are completely dissolved by shaking for 10min, the absorbance value of each concentration at 490 and nm is read by an enzyme-labeling instrument, and the relationship between the cell viability and the nanomaterial and RT is calculated from the absorbance value.
The result shows that the survival rate of MC 38 cells is lower than 50% in GPP group at the concentration of nano material of 40 mug/mL, and the survival rate of GPP+RT group cells is lower, which indicates that GPP has good cytotoxicity to MC 38 cells, and radiotherapy enhances the killing effect to MC 38 cells.
(9) As shown in FIG. 9, the ability of GPP to induce ROS production was tested using DCFH-DA fluorescent probes.
Taking MC 38 cells in logarithmic growth phase, digesting with 3010 4/Well MC 38 cells were seeded in 6-well plates and incubated in a 37℃incubator containing 5% CO 2 for 24 hours. GPP was incubated with MC 38 cells at final concentrations of 0, 10, 20, 40. Mu.g/mL, respectively. After 4h, the culture solution was discarded, 1mL DCFH-DA detection working solution was added, and the incubation was performed at 37℃for 20 min in the absence of light. Subsequently, the distribution of green fluorescence was observed under a fluorescence microscope.
The results show that green fluorescence increases with increasing concentration of GPP, indicating that GPP is capable of inducing the production of ROS.
(10) As shown in FIG. 10, the apoptosis of GPP-treated tumor cells in test (8) was detected using an apoptosis kit, and the dyes used in the apoptosis experiments were Annexin-V FITC and 7-AAD.
Part (a) of fig. 10 is a flow cytometry scatter plot, wherein:
q1 represents Annexin-V FITC-/7-AAD+, indicating cell death;
Q2 represents Annexin-V FITC+/7-AAD+, which represents late apoptosis;
Q3 represents Annexin-VFITC +/7-AAD-, representing early apoptosis;
Q4 represents Annexin-V FITC-/7-AAD-, representing normal cells.
Part (B) of fig. 10 shows an apoptotic cell count.
The result shows that after GPP treatment, the apoptosis rate of tumor cells is obviously increased, which indicates that the GPP prepared by the invention has the capability of promoting tumor cell apoptosis, and the apoptosis rate of GPP+RT group is further increased, which indicates that the radiation therapy can enhance the capability of GPP inducing tumor cell apoptosis.
(11) As shown in fig. 11, inhibition of MC 38 proliferation potency by GPP and RT was examined by a colony formation assay.
Taking MC 38 cells in logarithmic growth phase, digesting with 510 3/Well MC 38 cells were seeded in 6-well plates and incubated in a 37℃incubator containing 5% CO 2 for 24 hours. The experiments were divided into Control, radiotherapy (RT), GPP, GPP+RT, GPP final concentration 40. Mu.g/mL, co-incubated with MC 38 cells, respectively, RT groups were subjected to 3 Gy X-ray irradiation at incubation of 6 h.
Cell cloning was then observed. After proliferation, cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15min, then stained with crystal violet for 30min, washed with PBS, and finally the cloning results were analyzed. The result of the dyeing observation is shown in part (A) of FIG. 11, and the statistical chart of the increment rate is shown in part (B) of FIG. 11.
The results show that the GPP group has few clone balls, which indicates that GPP can effectively inhibit MC 38 proliferation, and the GPP+RT group has almost no clone balls, which indicates that radiotherapy enhances MC 38 inhibition capability of GPP.
Therefore, the gadolinium-platinum radiotherapy sensitizer with MRI capability is synthesized by adopting the preparation method and the application of the gadolinium-platinum radiotherapy sensitizer and utilizing the regulation characteristic of gadolinium ions on the growth of platinum nano particles; the gadolinium-platinum radiotherapy sensitizer has CAT activity, can catalyze H 2O2 to decompose and generate O 2, relieve the hypoxia inhibition of tumor parts and enhance the sensitivity of radiotherapy; the gadolinium-platinum radiotherapy sensitizer has POD activity, can catalyze H 2O2 to decompose and produce OH, enhance the curative effect of radiotherapy and promote tumor cell apoptosis.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention and not for limiting it, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that: the technical scheme of the invention can be modified or replaced by the same, and the modified technical scheme cannot deviate from the spirit and scope of the technical scheme of the invention.
Claims (7)
1. The preparation method of the gadolinium-platinum radiotherapy sensitizer is characterized by comprising the following steps of:
S1, preparing a gadolinium-regulated platinum-based nano material Pt@Gd 2O3 by taking gadolinium acetylacetonate and platinum acetylacetonate as raw materials; the step S1 specifically comprises the following steps:
S1-1, dissolving gadolinium acetylacetonate and platinum acetylacetonate in diethylene glycol, and stirring at 80 ℃ to react at 400-500 rpm for 40 min; adding polyethylenimine into the solution, and stirring at 80 ℃ and 400-500 rpm for reaction at 20-min; adding triethanolamine solution into the solution, stirring and reacting at 80 ℃ and 400-500 rpm for 30 min; after stirring, the product was transferred to a teflon lined autoclave and kept at 24 h at 200 ℃;
s1-2, centrifugally collecting the product from 14000 rpm and 15 min, washing with absolute ethyl alcohol and deionized water for 3 times, and dispersing the final product in deionized water to obtain gadolinium-regulated platinum-based nanomaterial Pt@Gd 2O3;Pt@Gd2O3 with particle size of 20 nm
S2, dispersing Pt@Gd 2O3 and mercapto-polyethylene glycol-methoxy mPEG-SH synthesized in the step S1 in deionized water, and reacting for 4-6 h under the magnetic stirring of ice bath and 500 rpm; and after the reaction is finished, centrifugally collecting the precipitate, washing the precipitate, centrifugally dispersing the precipitate in deionized water, and obtaining the gadolinium-platinum radiotherapy sensitizer Pt@Gd 2O3 -PEG.
2. The method for preparing the gadolinium-platinum radiotherapy sensitizer according to claim 1, wherein the method comprises the following steps: in the step S1-1, the mass-volume ratio of the polyethylenimine to the gadolinium acetylacetonate to the platinum acetylacetonate to the diethylene glycol is 1 g:40 mg:40 mg:40 mL.
3. The method for preparing the gadolinium-platinum radiotherapy sensitizer according to claim 1, wherein the method comprises the following steps: in the step S1-1, the mass-volume ratio of triethanolamine, gadolinium acetylacetonate, platinum acetylacetonate and diethylene glycol is 1 mL:50 mg:50 mg:50 mL.
4. The method for preparing the gadolinium-platinum radiotherapy sensitizer according to claim 1, wherein the method comprises the following steps: in the step S2, the volume ratio of Pt@Gd 2O3, mercapto-polyethylene glycol-methoxy and deionized water is 1:1:1.
5. The method for preparing the gadolinium-platinum radiotherapy sensitizer according to claim 1, wherein the method comprises the following steps: in the step S2, the sediment is centrifugally collected in 14000 rpm and 10 min; the washing is carried out by washing with deionized water for 2-3 times.
6. A gadolinium platinum radiotherapy sensitizer prepared by the method for preparing a gadolinium platinum radiotherapy sensitizer according to any one of claims 1 to 5.
7. Use of a gadolinium platinum radiation sensitizer according to claim 6 for radiation sensitization of cancer cells of non-medical interest.
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