Hollow virus-like rare earth nano-material, preparation method thereof and application of hollow virus-like rare earth nano-material in radiotherapy sensitization

Abstract

The invention discloses a hollow virus-like rare earth nano material, a preparation method thereof and application of the hollow virus-like rare earth nano material in radiotherapy sensitization. The rare earth nano material is marked as RVLu and comprises Lu-containing hollow rare earth nano particles (namely VLu) and annular polypeptide c (RGDfK) located on the surfaces of the Lu-containing hollow rare earth nano particles. The material is good in biological safety and excellent in in-vivo radiotherapy sensitization effect, and can remarkably improve the sensitivity of tumor cells to radioactive rays, so that the killing ability of radiotherapy to tumors is improved, and the material has important clinical significance for improving prognosis of patients.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a hollow virus-like rare earth nanomaterial, a preparation method thereof, and an application thereof in radiotherapy sensitization. Background Art

[0002] Radiotherapy is one of the main treatments for patients with locally advanced tumors. Some tumor cells are insensitive to radiotherapy due to hypoxia and heterogeneity inside the tumor, becoming the "seeds" of local recurrence and leading to treatment failure. Therefore, how to "improve the sensitivity of tumor radiotherapy" is a key scientific issue to break through the bottleneck of tumor radiotherapy.

[0003] The current mechanism of action of radiosensitizers mainly includes improving the radiosensitivity of tumor hypoxic cells, inhibiting tumor cell DNA damage repair or inducing cell cycle synchronization. Studies have found that the above-mentioned sensitizers have the disadvantages of large tissue toxicity or poor targeting, which limits their clinical application. The new high atomic number Lu element has more X-ray photon capture cross-section and Compton scattering effect, which is stronger than the existing radiosensitizer effect. Summary of the invention

[0004] The present invention provides a hollow virus-like rare earth nanomaterial and a preparation method thereof and application in radiotherapy sensitization. The material has good safety and excellent radiotherapy sensitization effect. After being modified and coupled with c(RGDfK) by 3-aminopropyltriethoxysilane, it has excellent tumor targeting ability. It can be used as a targeting probe that specifically aggregates in tumor tissue. The use of the probe can significantly improve the sensitivity of tumor cells to radiation, thereby increasing the killing ability of radiotherapy on tumors, and solving the problem of tumor recurrence caused by some tumor cells that are insensitive to radiotherapy. In addition, the probe can be quickly degraded into small particles in an acidic environment and excreted from the body through the kidneys, and has high biological safety.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] A rare earth nanomaterial (denoted as RVLu) comprises hollow rare earth nanoparticles containing Lu (namely VLu) and cyclic polypeptide c (RGDfK) located on the surface of the hollow rare earth nanoparticles containing Lu.

[0007] According to an embodiment of the present invention, the cyclic polypeptide c (RGDfK) is coupled to the surface of the Lu-containing hollow rare earth nanoparticles by modifying a silane coupling agent (such as 3-aminopropyltriethoxysilane) on the Lu-containing rare earth nanoparticles.

[0008] According to an embodiment of the present invention, the particle size of the rare earth nanomaterial is 70 to 200 nm, such as 80 to 150 nm, and exemplarily 100 nm.

[0009] According to an embodiment of the present invention, the content of Lu element in the rare earth nanomaterial is 40-70%, such as 50-65%, and exemplarily 55.12%.

[0010] According to an embodiment of the present invention, the content of the cyclic polypeptide c (RGDfK) in the rare earth nanomaterial is 10-30%, such as 15-25%, and exemplarily 19.34%.

[0011] The present invention also provides a method for preparing the rare earth nanomaterial, which comprises the following steps:

[0012] (1) reacting silicon nanoparticles (denoted as VSi), lutetium salt and urotropine in a water solvent to obtain silicon nanoparticles with lutetium compound coated on the surface (denoted as VSi&Lu);

[0013] (2) placing the VSi & Lu in an alkaline solution to obtain hollow rare earth nanoparticles containing Lu (denoted as VLu);

[0014] (3) The VLu and silane coupling agent react in an alcohol solvent to obtain VLu&NH2 nanoparticles;

[0015] (4) The VLu&NH2 nanoparticles, cyclic polypeptide c (RGDfK), EDC and NHS are reacted in an aqueous solvent to obtain the rare earth nanomaterial (denoted as RVLu).

[0016] According to an embodiment of the present invention, the preparation of the VSi comprises: reacting hexadecyltrimethylammonium bromide (CTAB), a base and tetraethyl orthosilicate (TEOS) to obtain the VSi;

[0017] For example, hexadecyltrimethylammonium bromide (CTAB) is dissolved in water, heated at 50-70°C for 20-40 minutes; then a base (such as sodium hydroxide) is added to react for 20-40 minutes; then a cyclohexane mixture of tetraethyl orthosilicate is dripped into the system to form an oil-water layer, and heating and stirring are continued for 50-90 hours; after post-treatment, the VSi is obtained;

[0018] Preferably, the molar ratio of CTAB to TEOS is (20-40):1, for example 31:1;

[0019] In an exemplary scheme, the preparation of VSi includes: dissolving 750 mg of hexadecyltrimethylammonium bromide (CTAB) in ultrapure water, placing it in a round-bottom flask, ultrasonically dissolving it, and heating it in a 60°C oil bath for 30 minutes, stirring it continuously at 300 r / min; after 30 minutes, adding 120 μL of NaOH solution (0.08 M) to continue the reaction, and after 30 minutes, slowly dripping a mixed solution containing 16 mL of tetraethyl orthosilicate (TEOS) and 20 mL of cyclohexane into the bottle along the wall of the bottle to form an oil-water layer, and continuing to heat and stir for 72 hours; after absorbing the upper oil phase solution, centrifuging at 10,000 r / min to obtain the precipitate, and washing it three times with anhydrous ethanol and ultrapure water respectively to obtain VSi for standby use.

[0020] According to an embodiment of the present invention, the mass ratio of VSi to lutetium salt is 1:(1.5-4), for example 1:(2-3).

[0021] According to an embodiment of the present invention, the lutetium salt is a water-soluble lutetium salt, such as lutetium nitrate hexahydrate (Lu(NO3)3·6H2O).

[0022] According to an embodiment of the present invention, the reaction temperature of step (1) is 80-100° C., and the reaction time is 6-12 hours.

[0023] According to an embodiment of the present invention, step (1) comprises: firstly dispersing VSi in water by ultrasonic wave, adding lutetium salt at 80-100°C, stirring for 20-40 minutes, then adding urotropine, reacting for 6-12 hours, and obtaining the VSi&Lu by post-treatment.

[0024] In an exemplary scheme, step (1) includes: taking 100 mg of VSi and dissolving it in ultrapure water, placing it in a 90°C oil bath with stirring and heating after ultrasonic dispersion, then adding 230 mg of lutetium nitrate hexahydrate (Lu(NO3)3·6H2O), stirring for 30 minutes, adding urotropine and continuing the reaction for 8 hours, then placing the solution in a centrifuge for precipitation (4500r / min), and then washing it twice with ultrapure water to obtain VSi&Lu.

[0025] According to an embodiment of the present invention, the alkaline solution in step (2) may be a Na2CO3 solution.

[0026] According to an embodiment of the present invention, step (2) comprises: placing the VSi&Lu in an alkaline solution, stirring at 70-90° C. for 12-20 hours, and washing with water to obtain VLu.

[0027] In an exemplary scheme, step (2) includes: placing the VSi&Lu in a 0.1M Na2CO3 solution, and heating with stirring in an 80°C oil bath for 15 hours, etching away silicon, and washing with ultrapure water to obtain VLu.

[0028] According to an embodiment of the present invention, the silane coupling agent may be 3-aminopropyltriethoxysilane, and / or the alcohol solvent may be (anhydrous) ethanol.

[0029] According to an embodiment of the present invention, step (3) comprises: ultrasonically dispersing the VLu in an alcohol solvent, adding a silane coupling agent in the alcohol solvent at 70 to 80° C., stirring the reaction for 8 to 15 hours, and washing with water to obtain VLu-NH2 nanoparticles.

[0030] In an exemplary scheme, step (3) includes: dissolving VLu in 30 mL of anhydrous ethanol, dispersing and heating in a 75°C oil bath with stirring, then adding 200 μL of 3-aminopropyltriethoxysilane and continuing to stir for 12 hours, and washing with ultrapure water to obtain VLu-NH2 nanoparticles.

[0031] According to an embodiment of the present invention, the mass ratio of the VLu-NH2 nanoparticles to the cyclic polypeptide c(RGDfK) is (5-15):1, for example, 10:1.

[0032] According to an embodiment of the present invention, step (4) comprises: reacting the VLu-NH2 nanoparticles, cyclic polypeptide c (RGDfK), EDC and NHS in an aqueous solvent at 2 to 6°C for 10 to 15 hours to obtain the RVLu.

[0033] In an exemplary scheme, step (4) includes: dissolving 50 mg VLu-NH2 and 5 mg cyclic polypeptide c (RGDfK) in ultrapure water, adding 10 mg EDC and NHS, stirring at 4°C overnight, and washing 3 times with ultrapure water to obtain RVLu.

[0034] The present invention also provides the use of the rare earth nanomaterial as a tumor targeting probe and / or a radiotherapy sensitizer. The rare earth nanomaterial can be used for radiotherapy sensitization of tumors.

[0035] Beneficial effects of the present invention:

[0036] The sensitizer nitroimidazole compounds reported so far have dose-limiting toxicity, which makes it impossible to achieve sufficient drug concentrations required for sensitization during the entire course of fractionated treatment. Although drugs that inhibit HIF and its pathways can inhibit the growth of tumor cells, due to their lack of tumor targeting, some tumor cells can still adapt to the hypoxic environment and proliferate rapidly, ultimately leading to radiotherapy failure. Although platinum and other drugs have good radiosensitization effects, their corresponding toxic side effects are relatively large.

[0037] The inventors have discovered that high atomic number elements, such as lutetium, can produce a dose enhancement effect after being exposed to ionizing radiation and promote apoptosis of tumor cells. Because these elements have more X-ray photon capture cross-sections and Compton scattering effects, they can be used to prepare preparations that are more efficient than other radiosensitizers.

[0038] The hollow virus-like nanoparticles (VLu) synthesized with virus silicon as a hard template in the present invention have good safety. The targeting peptide c (RGDfK) for breast cancer can be modified by 3-aminopropyltriethoxysilane to target and bind to integrin α overexpressed on tumor tissue. v β3, actively transporting RVLu to the tumor site and exerting its excellent radiosensitization effect, has important clinical significance for improving the quality of life and prognosis of patients. In addition, RVLu can be completely degraded into ~5nm nanoparticles and excreted from the body under physiological conditions, which further demonstrates that the material has good biosafety and provides the possibility for its further clinical transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a schematic diagram of the synthesis of the probe RVLu of the present invention;

[0040] Figure 2 TEM images of Experiment 1VSi and RVLu in the embodiment of the present invention;

[0041] Figure 3 Zeta potential of VLu, VLu-NH2 and RVLu in Experiment 1 of the present invention;

[0042] Figure 4 The UV absorption diagrams of VLu, VLu-NH2, RVLu and c(RGDfK) in Experiment 1 of the present invention are shown;

[0043] Figure 5 This is an electron microscopic image of RLu degradation in Experiment 1 in an embodiment of the present invention;

[0044] Figure 6 This is the cell targeting detection of Experiment 2RVLu in the embodiment of the present invention;

[0045] Figure 7 This is the detection of the radiosensitization effect of 3RVLu at the cell level in the experiment of the present invention;

[0046] Figure 8 This is a graph showing the results of flow cytometry detection of the effect of RVLu combined with radiotherapy on cell apoptosis in Experiment 3 of the present invention;

[0047] Fig. 9 This is the change of intracellular ROS production after RVLu combined with radiotherapy detected by confocal microscopy in Experiment 3 of the present invention;

[0048] Fig.10 This is the in vivo tumor targeting detection of RVLu in the CT26 subcutaneous transplanted tumor mouse model in the experiment of the present invention;

[0049] Fig.11 This is the tumor volume detection of the radiosensitization effect of 5RVLu at the animal level in the experiment of the embodiment of the present invention;

[0050] Fig.12 The change in tumor weight of the radiosensitization effect of 5RVLu in the animal level of the experiment in the embodiment of the present invention;

[0051] Fig.13 The weight change of mice in the experiment of 5RVLu's radiosensitization effect at the animal level in the embodiment of the present invention;

[0052] Fig.14 This is a hematological analysis test for the animal-level safety of the experiment 5RVLu in the embodiment of the present invention;

[0053] Fig.15 These are the H&E staining results of mouse organ tissues used to test the animal-level safety of 5RVLu in the examples of the present invention. DETAILED DESCRIPTION

[0054] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0055] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0056] In the present invention, cRGD(fK) and c(RGDfK) represent the same cyclic polypeptide.

[0057] Example 1

[0058] The present invention discloses a hollow virus-like rare earth nanoprobe (ie, RVLu), the synthesis route of which is as follows: Figure 1 The probe is RVLu, which includes hollow virus-like rare earth nanoparticles Lu2O3 synthesized using virus silicon as a hard template, and the outside is modified with 3-aminopropyltriethoxysilane to couple the cyclic peptide c (RGDfK).

[0059] The preparation method of the hollow virus-like rare earth nanoprobe comprises the following steps:

[0060] S1. First, 750 mg of hexadecyltrimethylammonium bromide (CTAB) was dissolved in ultrapure water and placed in a round-bottom flask. After ultrasonic dissolution, it was placed in a 60°C oil bath and heated for 30 minutes, and stirred continuously at 300 r / min. After 30 minutes, 120 μL of NaOH solution (0.08 M) was added to continue the reaction. After 30 minutes, a mixture of 16 mL of tetraethyl orthosilicate (TEOS) and 20 mL of cyclohexane was slowly dripped into the bottle along the bottle wall to form an oil-water layer, and continued to heat and stir for 72 hours. After removing the upper oil phase solution, centrifuge at 10,000 r / min to obtain the precipitate, and wash it three times with anhydrous ethanol and ultrapure water to obtain virus silicon nanoparticles (VSMN) for standby use.

[0061] S2. Subsequently, 100 mg of VSi was dissolved in ultrapure water, ultrasonically dispersed, and placed in a 90°C oil bath with stirring and heating. Subsequently, 230 mg of lutetium nitrate hexahydrate (Lu(NO3)3·6H2O) was added. After stirring for 30 min, hexamethylenetetramine was added and the reaction was continued for 8 h. Subsequently, the solution was placed in a centrifuge for precipitation (4500 r / min), and washed twice with ultrapure water in the same manner as above to obtain virus-like nanoparticles (VSi&Lu) coated with lutetium compounds on the surface.

[0062] S3. Subsequently, the above VSi&Lu were placed in a 0.1M Na2CO3 solution and heated with stirring in an 80°C oil bath for 15 h, the silicon was etched away, and hollow virus-like nanoparticles (VLu) were obtained after washing with ultrapure water.

[0063] S4. VLu was dissolved in 30 mL of anhydrous ethanol, dispersed and placed in a 75°C oil bath with heating and stirring, then 200 μL of 3-aminopropyltriethoxysilane was added and stirring was continued for 12 h. VLu-NH2 nanoparticles were obtained after washing with ultrapure water.

[0064] S5. Dissolve 50 mg VLu&NH2 and 5 mg c(RGDfK) peptide in ultrapure water, add 10 mg EDC and NHS, stir overnight at 4°C, wash three times with ultrapure water to obtain RVLu nanoparticles (i.e., RVLu), and store at room temperature after precipitation.

[0065] This hollow virus-like rare earth nanoprobe can be used to enhance the radiosensitization of tumors.

[0066] Through the following characterization and cell experiments on the prepared RVLu probe, it was found that the probe has a relatively uniform particle size, good safety and specific rectal cancer cell targeting ability; RVLu has a better tumor enrichment effect than VLu, and can be inhibited by excessive c(RGDfK); in the NIR-II region, the probe can accurately distinguish breast tumors from normal tissues, and at the same time, it is applied to breast cancer fluorescence surgical navigation, which can accurately and timely judge the state of the resection margin, reduce the positive rate of the resection margin and thus solve the problem of local recurrence. In addition, the probe can be completely degraded into nanoparticles of ~5nm under physiological conditions and excreted from the body through the kidneys, which further demonstrates that the probe has good biosafety. This targeted probe that can specifically aggregate in tumor tissue can significantly increase the sensitivity of tumor cells to radiation, and has important clinical significance for improving the quality of life and prognosis of patients.

[0067] Experiment 1: Characterization of RVLu

[0068] 1. RVLu morphology and size detection: Take a small amount of VSi, VLu and RVLu and dissolve them in ultrapure water. Take 10μl of the solution and drop it on the ultrathin carbon film, and dry it in an oven at 40℃. Use TEM detection with a voltage of 100kV to observe the morphology, size and dispersion of the sample, and select a clear and pollution-free field of view to take pictures and save.

[0069] 2. Zeta potential determination: To evaluate the coupling of c(RGDfK) peptide, VLu, VLu-NH2 and RVLu were ultrasonically dispersed in deionized water, and the Zeta potential changes of the three were observed using a multi-angle particle size high-sensitivity Zeta potential analyzer;

[0070] 3. Determination of UV absorption peak: VLu, VLu-NH2, RVLu and c(RGDfK) were dispersed in ultrapure water respectively, and the UV absorption peaks of the above four solutions were detected by UV-visible near-infrared spectrophotometer. The similarities and differences of the absorption peaks were compared to further evaluate the coupling of c(RGDfK) peptide.

[0071] 4. Degradation of RVLu in aqueous solutions with different pH values: Take an appropriate amount of RVLu and dissolve it in aqueous solutions with pH values ​​of 5.0, 6.5, and 7.4, and then take 10 μl of the solution and drop it on the ultra-thin carbon film at different time points, and dry it in an oven at 40°C. Use TEM detection with a voltage of 100 kV to observe the morphological changes of the samples, and select appropriate fields of view to take pictures and save them.

[0072] Experiment 1 test results: Figure 2 As shown in the electron microscopy images of VSi (left), VLu (middle) and RVLu (right), the probes have a relatively uniform particle size and good dispersion under the electron microscope (scale = 100 nm). Figure 3As shown in the figure, the Zeta potential results show that the Zeta potential of VLu is -8.843mV, the Zeta potential of VLu-NH2 is 19.14mV, and the Zeta potential of RVLu is 34.65mV. After coupling c(RGDfK), the potential of the nanoparticles changes toward the positive direction, indicating that c(RGDfK) may be successfully connected. Figure 4 As shown in the figure, compared with VLu and VLu-NH2, RVLu has an ultraviolet absorption peak near 258nm, and c(RGDfK) also has an absorption peak here, which also confirms the possible successful connection of c(RGDfK). Figure 5 As shown in the figure, in an aqueous solution of pH 5.0, RVLu can be degraded into small particles after 6 hours; in an aqueous solution of pH 6.5, RVLu can be degraded into small particles after 12 hours; in an aqueous solution of pH 7.4, RVLu can also be slowly degraded, and it can be observed that it begins to degrade into small particles after 48 hours.

[0073] Experiment 2: Cellular targeting detection of RVLu

[0074] 1. Take 0.01mmol VLu and RVLu respectively, ultrasonically disperse them in 15mL deionized water, add 0.8mg ICG, and stir at room temperature for 12h. The obtained products are centrifuged and washed with deionized water until the supernatant is colorless to obtain VLu@ICG and RVLu@ICG.

[0075] 2. CT26 cells were plated and cultured for 24 h, and the above two probes were added respectively, with a final concentration of 40 μg / mL. ICG was used as a control. After incubation for different time periods (0.25, 0.5, 1, 2, 4 h), the cells in each group were collected and the average fluorescence intensity of each group of cells was detected by flow cytometry.

[0076] 3. CT26 cells were cultured on slides for 24 h, and the above two probes were added respectively, with a final concentration of 40 μg / mL, and ICG was used as a control. After incubation for 4 h, the slides were collected, fixed, and sealed after DAPI staining, and photographed under an upright fluorescence microscope.

[0077] 4. CT26 cells were plated and divided into a probe group and a c(RGDfK) blocking group. The probe RVLu@ICG was added at a final concentration of 40 μg / mL. The c(RGDfK) blocking group was added with an equal amount of probe and free c(RGDfK) at a final concentration of 35 μg / mL. After incubation for 4 hours, the cells in each group were collected and flow cytometry was used to detect the average fluorescence intensity of the cells in each group.

[0078] Experiment 2 test results: Figure 6(A) Flow cytometry analysis results of CT26 cells incubated with ICG, VLu@ICG and RVLu@ICG at different time points and Figure 6 (B) Fluorescence microscopy results of CT26 cells after incubation with ICG, VLu@ICG and RVLu@ICG for 4 h (scale bar = 100 μm). CT26 cells have a higher uptake of RVLu@ICG, which can be blocked by excessive c(RGDfK) ( Figure 6 (C) demonstrates that the synthesized RVLu probe has specific breast cancer cell targeting ability.

[0079] Experiment 3: Detection of RVLu's ability to sensitize and kill tumor cells through radiotherapy

[0080] 1. Take 0.01mmol RVLu and disperse it into 15mL deionized water by ultrasonic.

[0081] 2. Clonogenic experiment to evaluate the radiosensitization performance of RVLu: CT26 cells were plated and cultured for 24 hours, and then divided into 4 groups after the cells adhered to the wall: PBS (1 mL of culture medium alone), RVLu (1 mL of culture medium containing 40 μg / mL of RVLu), RT alone, and RVLu+RT. The cells were treated for 4 hours, washed with culture medium three times, and then irradiated with a 4Gy radiotherapy dose, and then continued to be placed in a cell culture incubator at 37°C and 5% CO2 for 10-14 days. The culture was stopped, the cell colonies were gently washed with PBS, fixed with 4% paraformaldehyde, and then stained with 0.5% crystal violet for 20 minutes. After washing, they were dried at room temperature, and colonies consisting of more than 50 cells were counted under a microscope, and the survival score was calculated.

[0082] 3. Flow cytometry was used to detect the effect of RVLu combined with radiotherapy on cell apoptosis: CT26 cells were plated and cultured for 24 hours. After the cells adhered to the wall, they were divided into 4 groups: PBS (1 mL of culture medium alone), RVLu (1 mL of culture medium containing RVLu 40 μg / mL), RT alone, and RT+RT. The cells were treated for 4 hours, washed with culture medium three times, and then irradiated with a 4Gy radiotherapy dose. They were then placed in a cell culture incubator at 37°C and containing 5% CO2 for 24 hours. After digestion, centrifugation, and washing with saline, the cells were collected and dispersed in Binding Buffer, and Annexin V-FITC was added at room temperature. The cells were gently mixed and stained for 15 minutes in the dark. Then, propidium iodide staining solution was added, gently mixed, and stained for 5 minutes. The fluorescence intensity of Annexin V-FITC produced by cell apoptosis was detected by flow cytometry.

[0083] 4. Confocal microscopy was used to detect the changes in intracellular ROS production after RVLu combined with radiotherapy: CT26 cells were plated and cultured for 24 hours. After the cells adhered to the wall, they were divided into 4 groups: PBS (1 mL of culture medium alone), RVLu (1 mL of culture medium containing 40 μg / mL RVLu), RT alone, and RVLu + RT. The cells were treated for 4 hours, washed with culture medium three times, and then irradiated with a 4 Gy radiotherapy dose. 1 mL of the fluorescent probe DCFH-DA diluted 1:1000 was added and incubated for 30 minutes. The cells were washed three times with RPMI 1640-free cell culture medium. After incubating the above cells for another 3 hours, the changing trends of the average fluorescence intensity of each group were analyzed after preparation and photography.

[0084] The test results of Experiment 3: Figure 7 The colonies of PBS (1 mL of culture medium alone), RVLu (1 mL of culture medium containing 40 μg / mL RVLu), RT alone, and RVLu+RT were counted, and the survival scores were calculated. The results showed that the colony count and survival score of the RVLu+RT group were much lower than those of the RT alone group, proving that the synthesized RVLu probe has excellent radiosensitization and killing ability for tumor cells. Figure 8 The cell apoptosis rates of PBS (1 mL of culture medium alone), RVLu (1 mL of culture medium containing 40 μg / mL RVLu), radiotherapy alone, and RVLu+radiotherapy groups showed that the cell apoptosis rate of the RVLu+radiotherapy group was as high as 50%, much higher than the 15% of the radiotherapy alone group, proving that the synthesized RVLu probe has excellent radiosensitization and killing ability for tumor cells. Fig. 9 The changes in intracellular ROS production in the PBS (1 mL of culture medium alone), RVLu (1 mL of culture medium containing 40 μg / mL RVLu), radiotherapy alone, and RVLu+radiotherapy groups showed that the RVLu+radiotherapy group produced more reactive oxygen species, much higher than the radiotherapy alone group, proving that the synthesized RVLu probe has excellent radiotherapy sensitization and killing capabilities for tumor cells.

[0085] Experiment 4: In vivo tumor targeting detection of RVLu in CT26 subcutaneous transplanted tumor mouse model

[0086] Six mice with CT26 subcutaneous transplanted tumors were randomly divided into ICG, VLu@ICG and RVLu@ICG groups. The corresponding probes were injected into the tail vein of each group of mice, and the NIR-II images of mice were collected using the NIR-II small animal imaging device at different time points (1, 6, 12, 24, 36, 48, 72, and 96 h) after injection. The differences in signal-to-background ratios of each group at different time points were analyzed using PSLViewer software.

[0087] Experiment 4 test results: Fig.10NIR-II fluorescence images of the tumor site at different time points (1, 6, 12, 24, 36, 48, 72, 96 h) after injection of ICG (upper), VLu@ICG (middle), and RVLu@ICG (lower) Fig.10 A) and quantitative analysis of TBR at corresponding time points ( Fig.10 As shown in Figure B), after the probe was injected into the tail vein of CT26 tumor-bearing mice, the TBR of the RVLu@ICG group was higher than that of the VLu@ICG group at each time point, and the TBR of the RVLu@ICG group reached the highest point at 48h; Fig.10 As shown in C, the TBR at the optimal uptake time point of the RVLu@ICG group was significantly higher than that of the VLu@ICG group, proving that RVLu@ICG has a better tumor enrichment effect than VLu@ICG.

[0088] Experiment 5: Exploring the effectiveness of RVLu for radiosensitization in a subcutaneous tumor transplant mouse model

[0089] Twenty mice with subcutaneous tumor transplantation model (tumor volume 100 mm 3 ) were randomly divided into four groups: PBS, RVLu, radiotherapy alone, and RVLu + radiotherapy. 3+ Concentration quantification, reference dose is [Lu 3+ ]=40×10 -6 M / kg), and the radiotherapy group received 8Gy irradiation 48h after probe injection. Radiotherapy was performed using a bracket containing a lead collimator while protecting the head, neck, and chest of the mice to prevent radiation interference with normal tissues. The long diameter L and short diameter W of the tumor were measured with a vernier caliper every two days after radiotherapy, and the volume was calculated according to the formula "volume = L×W 2 / 2” to calculate the tumor volume, and measure and record the weight changes every two days. On the 18th day, whole blood was collected from mice to test blood biochemical indicators such as creatinine, urea nitrogen, alanine aminotransferase, and blood cytological indicators such as white blood cells, red blood cells, and platelets. Each group of mice was killed by cervical dislocation immediately after blood collection, the tumor was removed and white light photos were taken, and the final weight of the mouse tumor was weighed and recorded. Finally, the heart, liver, kidney, brain, lung, and spleen were obtained for H&E staining. The pathological status of the organ tissues of each group of mice was observed.

[0090] Experiment 4 test results: Fig.11 As shown in the figure, the tumor size in the PBS group or the RVLu-only treatment group increased rapidly. Therefore, it is difficult to completely eradicate the tumor by treating RVLu alone. Compared with radiotherapy alone, the RVLu+radiotherapy group had a significant tumor growth inhibition effect. The final tumor size ( Fig.12 A, I, II, III, and IV are PBS, RVLu, RT alone, and RVLu+RT groups) and weight ( Fig.12 Figure B) also strongly supports the radiosensitizing effect of RVLu. Fig.13 As shown, the weight changes of mice, Fig.14 Hematological analysis and Fig.15 HE staining results showed that no obvious in vivo toxicity or organ damage caused by RVLu was observed within 18 days after radiotherapy treatment.

[0091] The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A rare earth nanomaterial RVLu, characterized in that: The rare earth nanomaterial RVLu comprises Lu-containing hollow rare earth nanoparticles VLu and cyclic polypeptide c (RGDfK) located on the surface of the Lu-containing hollow rare earth nanoparticles.

2. The rare earth nanomaterial RVLu according to claim 1, characterized in that: The cyclic polypeptide c (RGDfK) is coupled to the surface of the Lu-containing hollow rare earth nanoparticle VLu through a silane coupling agent modified on the Lu-containing rare earth nanoparticle.

3. The rare earth nanomaterial RVLu according to claim 1 or 2, characterized in that: The particle size of the rare earth nanomaterial is 70 to 200 nm; And / or, the content of Lu element in the rare earth nanomaterial RVLu is 40-70%; And / or, the content of the cyclic polypeptide c(RGDfK) in the rare earth nanomaterial RVLu is 10-30%.

4. The method for preparing the rare earth nanomaterial RVLu according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: (1) reacting silicon nanoparticles VSi, lutetium salt and urotropine in a water solvent to obtain silicon nanoparticles VSi&Lu with lutetium compound coated on the surface; (2) placing the silicon nanoparticles VSi&Lu coated with lutetium compounds on the surface in an alkaline solution to obtain hollow rare earth nanoparticles VLu containing Lu; (3) the Lu-containing hollow rare earth nanoparticles VLu react with a silane coupling agent in an alcohol solvent to obtain VLu-NH2 nanoparticles; (4) The VLu-NH2 nanoparticles, cyclic polypeptide c (RGDfK), EDC and NHS are reacted in an aqueous solvent to obtain the rare earth nanomaterial RVLu.

5. The preparation method according to claim 4, characterized in that: The preparation of the VSi comprises: reacting hexadecyltrimethylammonium bromide (CTAB), a base and tetraethyl orthosilicate (TEOS) to obtain the VSi.

6. The preparation method according to claim 4, characterized in that: The mass ratio of VSi to lutetium salt is 1:(1.5-4); and / or, the lutetium salt is a water-soluble lutetium salt; And / or, the reaction temperature of step (1) is 80-100° C., and the reaction time is 6-12 h.

7. The preparation method according to claim 4, characterized in that: The alkaline solution in step (2) is a Na2CO3 solution; In step (3), the silane coupling agent is 3-aminopropyltriethoxysilane, and / or the alcohol solvent is ethanol; The mass ratio of the VLu-NH2 nanoparticles and the cyclic polypeptide c(RGDfK) in step (4) is (5-15):

1.

8. The preparation method according to claim 4, characterized in that: Step (2) comprises: placing VSi&Lu in an alkaline solution, stirring at 70-90° C. for 12-20 hours, and washing with water to obtain VLu; And / or, step (3) comprises: ultrasonically dispersing the VLu in an alcohol solvent, adding a silane coupling agent in the alcohol solvent at 70-80° C., stirring for 8-15 hours, and washing with water to obtain VLu-NH2 nanoparticles; And / or, step (4) comprises: reacting the VLu-NH2 nanoparticles, cyclic polypeptide c (RGDfK), EDC and NHS in an aqueous solvent at 2 to 6° C. for 10 to 15 hours to obtain the RVLu.

9. Use of the rare earth nanomaterial RVLu according to any one of claims 1 to 3 in the preparation of tumor targeting probes and / or radiotherapy sensitizers.

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