A lanthanide rare earth nanoprobes, preparation method and application thereof

Abstract

The application discloses a lanthanide rare earth nano probe and a preparation method and application thereof, the probe is RENPs, the RENPs take NaNdF4:Yb@NaLuF4 as an imaging element, and the plerixafor is a targeting element.The application provides a lanthanide rare earth nano probe and a preparation method and application thereof, takes NaNdF4:Yb@NaLuF4 as an imaging element, and the small-molecule drug plerixafor is a targeting element, both are combined radiotherapy sensitization elements, a rare earth nano probe RENPs with good biocompatibility and stable NIR-Ⅱ luminescence is constructed, the probe can visualize breast cancer before radiotherapy, so that precise radiotherapy target delineation can be carried out, the sensitivity of tumor cells to radiation is improved during radiotherapy, so that the sensitivity of radiotherapy is improved, the anti-tumor immunity of the body is activated, and the treatment of the anti-PD-L1 immune checkpoint inhibitor in combination can effectively improve the immunotherapy responsiveness, and the prognosis of metastatic and recurrent triple-negative breast cancer patients who lose the opportunity of surgery is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a lanthanide rare earth nanometer probe and a preparation method and application thereof. BACKGROUND

[0002] Breast cancer has surpassed lung cancer to become the world's first cancer, among which triple-negative breast cancer (TNBC) has a relatively poor prognosis due to the lack of effective treatment targets, and therefore has a high degree of malignancy, a high recurrence rate, a strong invasion and metastasis, and a short survival period of patients. In the face of the high incidence of breast cancer and the grim situation of poor prognosis of TNBC, the social demand for improving the diagnosis and treatment level of breast cancer, especially TNBC, is increasingly urgent. With the development of immunotherapy, immune checkpoint therapy (ICT) brings a glimmer of hope for the treatment of TNBC, but its effective rate is only 20%-30%, and for insensitive patients, ICT not only cannot show the expected effect but also causes waste of time and money and delays the treatment opportunity. Studies have found that radiotherapy-induced immune enhancement can activate the tumor immune microenvironment (TME) and optimize the treatment effect of ICT, thereby preventing tumor progression. Radiotherapy is an effective way for the treatment of recurrent and metastatic breast cancer, especially for breast cancer patients who have lost the opportunity for surgery, and can significantly reduce the risk of local recurrence of TNBC and to a certain extent, reduce the rate of distant metastasis, and improve the disease-free survival rate and overall survival rate of patients. Although radiotherapy is effective in local control, there is still a phenomenon of radiotherapy resistance leading to local recurrence. Therefore, it is urgent to find a method for accurately and intuitively delineating the tumor target area and to strive to improve the sensitivity of the radiotherapy area and activate anti-tumor immunity, so as to optimize the effect of radiotherapy and immunotherapy combination. SUMMARY

[0003] The present application is a kind of lanthanide rare earth nanometer probe and its preparation method and application for solving the above problems.

[0004] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0005] A lanthanide rare earth nanometer probe, the probe is RENPs, the RENPs take NaNdF4:Yb@NaLuF4 as an imaging element, and plerixafor as a targeting element.

[0006] A preparation method of a lanthanide rare earth nanometer probe, the method comprises the following steps:

[0007] S1, oleic acid, octadecene, Nd(CH3COO)3 and Yb(CH3COO)3 are mixed and stirred, and core particles NaNdF4:Yb are prepared by high-temperature co-precipitation method;

[0008] S2, the core particles NaNdF4:Yb are subjected to epitaxial growth to prepare core-shell structure NaNdF4:Yb@NaLuF4;

[0009] S3, adding DSPE-PEG to NaNdF4:Yb@NaLuF4 2000 -COOH, and obtaining carboxyl-functionalized RENPs-PEG through rotary evaporation, drying, centrifugation, and washing;

[0010] S4, obtaining a probe RENPs by targeting modification of RENPs-PEG through use of plerixafor as a targeting element.

[0011] Preferably, step S1 specifically comprises:

[0012] S11, adding 6 mL of oleic acid, 15 mL of octadecene, 0.85 mmol of Nd(CH3COO)3, and 0.15 mmol of Yb(CH3COO)3 to a three-necked flask, stirring, and then vacuumizing for 10 min;

[0013] S12, heating the solution after vacuumizing in S11 to 120°C and reacting for 20 min until all particles are dissolved to obtain a clear solution;

[0014] S13, releasing the vacuum after cooling the clear solution obtained in S12 to 50°C to obtain solution a;

[0015] S14, dissolving 0.5 M NaOH and 0.4 M NH4F in methanol solutions respectively and mixing them uniformly to obtain solution b;

[0016] S15, adding solution b to solution a, maintaining 50°C, continuously stirring for 30 min, then heating to 100°C and maintaining for 40 min, vacuumizing for 10 min and aerating for 2-3 times, heating to 300°C and reacting for 1.5 h, and then rapidly cooling to room temperature after the reaction is completed to obtain solution c;

[0017] S16, precipitating solution c by adding an ethanol solution, centrifuging at a rotation speed of 8000 rpm for 10 min, and washing, repeating 3 times to obtain core particles NaNdF4:Yb, and then redissolving the core particles NaNdF4:Yb in 4 mL of cyclohexane.

[0018] Preferably, step S2 specifically comprises:

[0019] S21, adding 6 mL of oleic acid, 15 mL of octadecene, and 0.5 mmol of Lu(CH3COO) 3， stirring, and then vacuumizing for 10 min;

[0020] S22, the solution after vacuumizing in S21 is heated to 120℃ and reacted for 20 min until all the particles are dissolved to obtain a clear solution;

[0021] S23, the clear solution obtained in S22 is cooled to 50℃ and vacuum is released to obtain solution d;

[0022] S24, 0.5 M NaOH and 0.4 M NH4F are respectively dissolved in methanol solution and mixed uniformly to obtain solution e;

[0023] S25, the core particles NaNdF4:Yb obtained in S1 and solution e are added to solution d, and after maintaining 50℃, continuous stirring is performed for 30 min, then heating to 100℃ and maintaining for 40 min, vacuum extraction for 10 min and air exchange for 2-3 times, then heating to 300℃ and reacting for 1.5 h, after the reaction is completed, rapid cooling to room temperature to obtain solution f;

[0024] S26, ethanol solution is added to solution f to precipitate, centrifuged at 8000 rpm for 10 min, washed, repeated for 3 times to obtain oil phase core-shell structure NaNdF4:Yb@NaLuF4, and then the core-shell structure NaNdF4:Yb@NaLuF4 is re-dissolved in 4 mL cyclohexane.

[0025] Preferably, step S3 specifically comprises:

[0026] S31, 0.1 mmol NaNdF4:Yb@NaLuF4 synthesized in S2 is taken, centrifuged after adding ethanol, and re-dispersed in 15 mL chloroform solution, and 20 mg DSPE-PEG 2000 -COOH is added to obtain solution g;

[0027] S32, solution g is placed in a fume hood and stirred at a speed of 800 rpm until the chloroform is completely evaporated, and then placed in an oven at 60℃ for 12 h to obtain solution h;

[0028] S33, 15 mL deionized water is added to solution h and ultrasonicated for 2 h, then centrifuged at a speed of 15000 rpm for 10 min, and washed with deionized water for 3 times to remove unbound DSPE-PEG 2000 -COOH, and then the carboxyl functionalized aqueous RENPs-PEG is dispersed in deionized water.

[0029] Preferably, step S4 specifically comprises:

[0030] S41, 20 mg EDC and 60 mg NHS are respectively dissolved in MES buffer to obtain EDC solution and NHS solution;

[0031] S42, centrifuging the carboxyl-functionalized RENPs-PEG dispersed in deionized water obtained in step S33 to obtain a RENPs-PEG solid, and then dispersing the RENPs-PEG solid in 1 mL of MES buffer to obtain solution i;

[0032] S43, EDC solution was added to solution i, and the mixture was stirred for 5 min and ultrasonicated for 25 min, and then NHS solution was added, and the mixture was stirred for 15 min and ultrasonicated for 2 min to obtain solution j.

[0033] S44. Centrifuge solution j at 15,000 rpm for 10 min and resuspend in 1 mL of MES buffer. Add 200 µg of plerixafor and stir at 4°C for 12 h. Ultrasonicate for 5 min. After the reaction is complete, centrifuge at 15,000 rpm for 10 min and resuspend in 1 mL of deionized water. Wash and centrifuge again and resuspend in 1 mL of deionized water to obtain probe RENPs loaded with targeting elements.

[0034] Application of a lanthanide rare earth nanoprobe in the preparation of probe reagents for precise radiotherapy target delineation.

[0035] The invention discloses an application of a lanthanide rare earth nanoprobe in the preparation of a probe reagent for improving the sensitivity of tumor cells to radiation.

[0036] The invention relates to the application of a lanthanide rare earth nanoprobe in the preparation of a probe reagent for anti-tumor immune activation.

[0037] After adopting the above technical solution, the present invention has the following advantages compared with the background technology:

[0038] The present invention provides a lanthanide rare earth nanoprobe and its preparation method and application. NaNdF4:Yb@NaLuF4 is used as an imaging element and the small molecule drug plerixafor is used as a targeting element. The two are combined as a radiotherapy sensitization element to construct a rare earth nanoprobe RENPs with good biocompatibility and stable NIR-Ⅱ luminescence. The probe can visualize breast cancer before radiotherapy, thereby accurately delineating the radiotherapy target area; during radiotherapy, it can increase the sensitivity of tumor cells to radiation, thereby increasing the sensitivity of radiotherapy; activate the body's anti-tumor immunity, and combined with the treatment of anti-PD-L1 immune checkpoint inhibitors, it can effectively improve the responsiveness of immunotherapy and improve the prognosis of patients with metastatic and recurrent triple-negative breast cancer who have lost the opportunity for surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram, electron microscopy, DLS, emission spectrum, photostability and penetration depth of the RENPs probe of the present invention;

[0040] Figure 2 Cell targeting of the inventive probe RENPs;

[0041] Figure 3 In vitro radiotherapy sensitization and immune activation of the inventive probe RENPs;

[0042] Figure 4 Animal targeting and radiotherapy sensitization of the inventive probe RENPs;

[0043] Figure 5 In vivo immune activation of the inventive probe RENPs;

[0044] Figure 6 HE staining of important organs in the in vivo immune activation experiment of the inventive probe RENPs to verify the safety of RENPs;

[0045] Figure 7 Blood routine, blood biochemistry and mouse weight monitoring to verify the safety of RENPs. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application. EMBODIMENT

[0047] As shown in Figures 1 to 7 , the present application discloses a lanthanide rare earth nano probe, the probe is RENPs, the RENPs take NaNdF4:Yb@NaLuF4 as the imaging element, and AMD3100 as the targeting element.

[0048] As shown in Figure 1 , the modified RENPs are purple clear transparent aqueous liquid, have uniform element distribution and spherical structure, stable near-infrared fluorescence (at 979 nm, 1065 nm and 1330 nm) and do not occur aggregation quenching, and the RENPs also have a certain penetration depth (8 mm), which lays a solid foundation for subsequent breast cancer tumor imaging using the NIR-II nano probe.

[0049] A preparation method of a lanthanide rare earth nano probe, the method comprises the following steps:

[0050] S1, OA (oleic acid), ODE (octadecene), Nd(CH3COO)3 (acetic acid neodymium) and Yb(CH3COO)3 (acetic acid ytterbium) are mixed and stirred, and core particles NaNdF4:Yb are prepared by high temperature co-precipitation method;

[0051] S2, epitaxial growth is performed on the core particle NaNdF4:Yb to prepare a core-shell structure NaNdF4:Yb@NaLuF4;

[0052] S3, DSPE-PEG is added to the NaNdF4:Yb@NaLuF4 2000 -COOH (carboxyl phospholipid polyethylene glycol), and through rotary evaporation, drying, centrifugation, and washing, the carboxyl functionalized RENPs-PEG is obtained;

[0053] S4, the RENPs-PEG is targetedly modified by AMD3100 (plerixafor) as a targeting element to obtain a probe RENPs.

[0054] Step S1 specifically includes:

[0055] S11, 6 mL of OA, 15 mL of ODE, 0.85 mmol of Nd(CH3COO)3, and 0.15 mmol of Yb(CH3COO)3 are added to a three-necked flask, and after stirring, vacuum is extracted for 10 min;

[0056] S12, the solution after vacuum extraction of S11 is warmed to 120°C and reacted for 20 min until the particles are completely dissolved to obtain a clear solution;

[0057] S13, the clear solution obtained in S12 is cooled to 50°C, and the vacuum is released to obtain solution a;

[0058] S14, 0.5 M NaOH and 0.4 M NH4F are respectively dissolved in methanol solution and mixed uniformly to obtain solution b;

[0059] S15, solution b is added to solution a, and after maintaining 50°C, continuous stirring is performed for 30 min, then it is warmed to 100°C and maintained for 40 min, vacuum is extracted for 10 min and aerated for 2-3 times, then it is warmed to 300°C and reacted for 1.5 h, and after the reaction is completed, it is quickly cooled to room temperature to obtain solution c;

[0060] S16, ethanol solution is added to solution c to precipitate, and after centrifugation at a speed of 8000 rpm for 10 min, it is washed, and after repeating 3 times, the core particle NaNdF4:Yb is obtained, and then the core particle NaNdF4:Yb is redissolved in 4 mL of cyclohexane.

[0061] Step S2 specifically includes:

[0062] S21, 6 mL of OA, 15 mL of ODE, and 0.5 mmol of Lu(CH3COO) 3， are added to a three-necked flask, and after stirring, vacuum is extracted for 10 min;

[0063] S22, the solution after vacuumizing in S21 is heated to 120℃ and reacted for 20 min until all the particles are dissolved to obtain a clear solution;

[0064] S23, the clear solution obtained in S22 is cooled to 50℃ and vacuum is released to obtain solution d;

[0065] S24, 0.5 M NaOH (sodium hydroxide) and 0.4 M NH4F (ammonium fluoride) are respectively dissolved in methanol solution and then mixed uniformly to obtain solution e;

[0066] S25, the core particles NaNdF4:Yb obtained in S1 and solution e are added to solution d, and after maintaining 50℃, continuous stirring is performed for 30 min, then heating to 100℃ and maintaining for 40 min, vacuum extraction for 10 min and air exchange for 2-3 times, then heating to 300℃ and reacting for 1.5 h, after the reaction is completed, rapid cooling to room temperature to obtain solution f;

[0067] S26, ethanol solution is added to solution f to precipitate, centrifugation is performed at 8000 rpm for 10 min, and after washing, the operation is repeated for 3 times to obtain core-shell structure NaNdF4:Yb@NaLuF4, and then the oil phase core-shell structure NaNdF4:Yb@NaLuF4 is redissolved in 4 mL cyclohexane.

[0068] Step S3 specifically comprises:

[0069] S31, 0.1 mmol of NaNdF4:Yb@NaLuF4 synthesized in S2 is taken, centrifuged after adding ethanol, and then redispersed in 15 mL chloroform solution, and 20 mg of DSPE-PEG 2000 -COOH is added to obtain solution g;

[0070] S32, solution g is placed in a fume hood and stirred at a speed of 800 rpm until the chloroform is completely evaporated, and then placed in an oven at 60℃ for 12 h to obtain solution h;

[0071] S33, 15 mL of deionized water is added to solution h, and ultrasonic treatment is performed for 2 h, then centrifugation is performed at a speed of 15000 rpm for 10 min, and then washed with deionized water for 3 times to remove unbound DSPE-PEG 2000 -COOH, and then the carboxyl functionalized aqueous RENPs-PEG is dispersed in deionized water.

[0072] Step S4 specifically comprises:

[0073] S41, 20 mg EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide) and 60 mg NHS (N-hydroxysuccinimide) were dissolved in MES buffer solution respectively to obtain EDC solution and NHS solution;

[0074] S42, the carboxyl-functionalized RENPs-PEG dispersed in deionized water prepared in step S33 was centrifuged to obtain RENPs-PEG solid, and then the RENPs-PEG solid was dispersed in 1 mL MES buffer solution to obtain solution i;

[0075] S43, EDC solution was added to solution i, and stirred for 5 min and ultrasonic for 25 min, then NHS solution was added, and stirred for 15 min and ultrasonic for 2 min to obtain solution j,

[0076] S44, solution j was centrifuged at a speed of 15000 rpm for 10 min, and then resuspended in 1 mL MES buffer solution, 200 μg AMD3100 was added, and stirred at 4°C for 12 h, then ultrasonic for 5 min, after the reaction was completed, centrifuged at a speed of 15000 rpm for 10 min and resuspended in 1 mL deionized water, then washed and centrifuged again and resuspended in 1 mL deionized water, finally the probe RENPs loaded with targeting elements was obtained.

[0077] With the continuous exploration of anti-tumor mechanisms and TNBC immune characteristics, immunotherapy with immune checkpoint inhibitors (ICIs) as the core has brought new opportunities for the treatment of TNBC with lower toxicity and higher pathological remission rate, and a series of clinical and preclinical trials have promoted the application of ICT in breast cancer. Among them, the treatment effect of ICIs is closely related to TME. As the most immunogenic breast cancer, TNBC has more tumor-infiltrating lymphocytes (TILs), and about 40-65% of patients have relatively high expression levels of programmed death ligand-1 (PD-L1), so this type of breast cancer is most sensitive to ICT. However, due to the relatively insufficient TIL infiltration, weak tumor-induced inflammatory response, and low expression of target PD-L1, the response rate of ICT is very low. Studies have shown that the special tumor killing mechanism of radiotherapy can take advantage of the tumor in situ antigen exposure effect, use radiotherapy as an immunomodulator, and at the same time combine with immunotherapy to reduce the failure rate of treatment, and ultimately improve the recurrence-free survival rate of breast cancer patients. And a large number of clinical studies have also proved that the combination of radiotherapy and immunotherapy can achieve a synergistic effect, activate anti-tumor immunity, and thus prevent tumor progression, which has evolved into a new tumor treatment model.

[0078] Radiation therapy is one of the common palliative treatment methods for recurrent and metastatic advanced TNBC. It is worth noting that the recurrence rate of TNBC patients after radiotherapy is not significantly lower than that of other breast cancer subtypes. More than 33% of TNBC patients relapse and metastasize after radiotherapy, mainly because radiotherapy can only kill cancer cells sensitive to radiation, while tumor cells insensitive to radiation will continue to survive. Increasing the dose of radiotherapy is the main means to improve its efficacy. However, when the radiation dose exceeds a certain threshold, high-dose radiotherapy can cause serious damage to normal tissues, causing corresponding toxic side effects. Therefore, the radiation dose must be controlled within a certain range to minimize the irreversible damage of X-rays to normal tissues, and it is essential to leave only tumor tissue to absorb most of the ionizing radiation. Lanthanide elements with high atomic number, such as lutetium (Lu) and hafnium (Hf), have more X-ray photon capture cross-sections and Compton scattering effects, which can produce a dose-enhancing effect after receiving ionizing radiation, thereby promoting tumor cell apoptosis.

[0079] It is essential to accurately identify tumors, improve tumor cell sensitivity to ionizing radiation, and avoid damage to normal tissues before radiotherapy. In order to accurately delineate the target area before radiotherapy, a nano-platform with good stability, biocompatibility, imaging resolution, and penetration depth can be constructed. Molecular imaging technology can non-invasively image the body's activities at the subcellular level, and optical imaging has become an ideal means of molecular imaging due to its non-radiation, high sensitivity, and simple operation. Compared with the visible light region and the near-infrared region (NIR-I, 700-900 nm), near-infrared region II (NIR-II, 1000-1400 nm) fluorescence imaging has the characteristics of "tissue transparent window", with the advantages of less photon scattering and absorption, and low endogenous tissue autofluorescence background, thereby achieving higher contrast and signal-to-background ratio (SBR). The light emitted by rare earth nanomaterials is very stable and has strong anti-quenching ability. The introduction of tumor-targeting elements to construct nanoparticles can accurately and intuitively identify tumors at the molecular level, providing a new idea for radiotherapy target delineation. Data from the human TNBC sample library show that CXCR4 expression in TNBC is significantly higher than that in normal population, Luminal-type breast cancer, and HER-2 positive breast cancer patients. Further genomic analysis shows that CXCR4 is also an independent risk factor for poor prognosis in patients. Therefore, CXCR4 can be used as an effective target for precise positioning of tumors at the molecular level.

[0080] Based on the above, the probe RENPs proposed in the application can visualize breast cancer before radiotherapy, so as to accurately delineate the radiotherapy target area; improve the sensitivity of tumor cells to radiation during radiotherapy, so as to improve the sensitivity of radiotherapy; activate the body's anti-tumor immunity, and combined with the treatment of anti-PD-L1 immune checkpoint inhibitors, the immunotherapy responsiveness can be effectively improved, and the prognosis of metastatic and recurrent triple-negative breast cancer patients who have lost the opportunity for surgery can be improved.

[0081] The application of a lanthanide rare earth nano probe in the preparation of a probe reagent for accurately delineating a radiotherapy target area.

[0082] Evaluation of the targeting imaging ability of RENPs at the cell and animal levels:

[0083] 1) Verify the difference in the uptake ability of the probe containing AMD3100 for cell lines with different CXCR4 expression levels at the cell level.

[0084] 2) Further verify the targeting ability of the probe by the AMD3100 targeted modification / non-modification group and the 4T1 cell with AMD3100 blocking CXCR4 in advance.

[0085] 3) Construct a 4T1-luc cell breast cancer subcutaneous tumor mouse, and inject the probe RENPs (containing AMD3100) and RENPs-control (not containing AMD3100) into the tail veins of the targeted group and the non-targeted group, respectively. After anesthesia, imaging is performed in the small animal NIR-II fluorescence imaging system, and the difference in fluorescence signal of the tumor area of the two groups is observed after 808 nm laser irradiation, and the imaging SBR of the tumor area is calculated.

[0086] The experimental evaluation results are shown in Figure 2 The probe RENPs have the ability to target the breast tumor surface receptor CXCR4, RENPs can effectively accumulate in the cytoplasm, and the comparison of RENPs and RENPs-control proves that AMD3100 can indeed increase the enrichment of the probe in breast cancer cells.

[0087] The application of a lanthanide rare earth nano probe in the preparation of a probe reagent for improving the sensitivity of tumor cells to radiation.

[0088] Evaluation of the radiotherapy sensitization ability of RENPs at the cell and animal levels:

[0089] 1) At the cell level, evaluate the radiotherapy sensitization characteristics of RENPs through CCK8 experiments, clonogenic assays and apoptosis experiments, and further verify the radiotherapy sensitization mechanism of the probe through ROS generation experiments.

[0090] 2) At the small animal level, 4T1 tumor-bearing mice were divided into PBS group, RENPs group, PBS + RT group and RENPs + RT group. RENPs were injected via tail vein and mice were placed in a small animal live irradiator, and three times of radiotherapy were performed at a dose of 6 Gy every other day. The body weight and tumor volume of mice were continuously monitored for 20 days, and bioluminescence imaging was performed before the mice were sacrificed, and the fluorescence signal at the tumor site was quantified.

[0091] Experimental evaluation results are shown in Figure 3 RENPs have good radiotherapy sensitization performance. The clonogenic assay shows that the proliferation ability of tumor cells is significantly inhibited under the combined action of sensitizers and radiation. In addition, RENPs produce a large amount of ROS after radiotherapy, causing DNA damage and leading to cell death. The results of the apoptosis experiment show that RENPs combined with radiotherapy can significantly increase the proportion of early and late apoptotic cells, and exhibit the radiotherapy sensitization effect mediated by RENPs.

[0092] The application of a lanthanide rare earth nano probe in the preparation of a probe reagent for anti-tumor immune activation. RENPs are evaluated for their ability to activate the immune system at the cellular and animal levels:

[0093] 1) At the cellular level, the exposure of tumor antigens was detected by DC maturation experiments.

[0094] 2) At the small animal level, two different cell densities of subcutaneous transplanted tumors were inoculated on both sides of female BALB / c mice, referred to as the proximal (right) tumor and the distal (left) tumor, and 4T1 double tumor-bearing mice were divided into five groups, including Group A (PBS), Group B (PBS + ICT), Group C (RENP + ICT), Group D (PBS + RT + ICT), and Group E (RENP + RT + ICT). RENPs were administered via tail vein every other day (1, 3, and 5 days), and radiotherapy was performed at a dose of 6 Gy 3 hours after administration (1, 3, and 5 days), and the anti-PD-L1 drug Atezolizumab was administered via intraperitoneal injection every other day (2, 4, and 6 days). After the end of the combined radiotherapy and immunotherapy, the mice were sacrificed and further analyzed for immune system activation.

[0095] Experimental evaluation results are shown in Figure 4 The subcutaneous transplanted tumor mice of breast cancer have good tumor enrichment effect in near-infrared two-zone imaging, and the tumor is significantly reduced under the combined action of RENPs and radiation.

[0096] As shown in Figure 5As shown, the RENPs were involved in the radioimmunotherapy, the mouse anti-tumor immunity was obviously activated, the proportion of cytotoxic T cells was obviously increased, and the related anti-tumor immune cytokines, including TNF-α and IL-12p70 secreted by mature dendritic cells, and IL-6 and IFN-γ secreted by activated CTL cells were also obviously increased.

[0097] As shown in the above table, the RENPs were injected into the normal BALB / c mice through the tail vein, and the mice were killed at different time periods, and the important organs were taken out for HE staining and observation, and the results showed that the organs of the mice injected with the RENPs at different time periods were not obviously damaged. Figure 6 As shown in the above table, the RENPs were injected into the normal BALB / c mice through the tail vein, and the mice were killed at different time periods, and the important organs were taken out for HE staining and observation, and the results showed that the organs of the mice injected with the RENPs at different time periods were not obviously damaged.

[0098] Figure 7 As shown in the above table, the RENPs were injected into the normal BALB / c mice through the tail vein, and the mice were killed at different time periods, and the important organs were taken out for HE staining and observation, and the results showed that the organs of the mice injected with the RENPs at different time periods were not obviously damaged.

[0099] The above is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited to this, any person skilled in the art can easily think of the changes or replacements within the technical range disclosed by the present application, which should be covered in the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.​

Claims

1. A lanthanide rare earth nanoprobes characterized in that: The probe is RENPs, the RENPs take NaNdF4:Yb@NaLuF4 as an imaging element, and the targeting element is plerixafor, the targeting element plerixafor is connected with the imaging element NaNdF4:Yb@NaLuF4 through a carboxyl coupling mode, the modified RENPs are purple clear transparent aqueous liquids, have uniform element distribution and spherical structure, stable near-infrared fluorescence and do not occur aggregation quenching, and the RENPs also have a penetration depth.

2. The method for preparing the lanthanide rare earth nanoprobe according to claim 1, wherein: The method comprises the following steps: S1, mixing and stirring oleic acid, octadecene, Nd(CH3COO)3 and Yb(CH3COO)3, and preparing core particles NaNdF4:Yb by high-temperature co-precipitation; Step S1 specifically comprises: S11, adding 6 mL of oleic acid, 15 mL of octadecene, 0.85 mmol of Nd(CH3COO)3 and 0.15 mmol of Yb(CH3COO)3 into a three-necked flask, stirring, and vacuumizing for 10 min; S12, heating the solution after vacuumizing in S11 to 120 DEG C and reacting for 20 min until the particles are completely dissolved to obtain a clear solution; S13, releasing the vacuum after cooling the clear solution obtained in S12 to 50 DEG C to obtain solution a; S14, dissolving 0.5 M NaOH and 0.4 M NH4F in methanol solution respectively and mixing uniformly to obtain solution b; S15, adding solution b into solution a, maintaining 50 DEG C, continuously stirring for 30 min, then heating to 100 DEG C and maintaining for 40 min, vacuumizing for 10 min and aerating for 2-3 times, then heating to 300 DEG C and reacting for 1.5 h, rapidly cooling to room temperature after the reaction is completed, and obtaining solution c; S16, adding an ethanol solution into solution c to precipitate, centrifuging at a speed of 8000 rpm for 10 min, washing, repeating for 3 times, obtaining core particles NaNdF4:Yb, and then redissolving the core particles NaNdF4:Yb in 4 mL of cyclohexane; S2, performing epitaxial growth on the core particles NaNdF4:Yb to prepare core-shell structure NaNdF4:Yb@NaLuF4; S3, adding DSPE-PEG to NaNdF4:Yb@NaLuF4 2000 -COOH, and by rotary evaporation, drying, centrifugation, washing, obtaining carboxyl functionalized RENPs-PEG; S4, performing targeted modification on RENPs-PEG by taking plerixafor as a targeting element to obtain the probe RENPs.

3. The method for preparing a lanthanide rare earth nanoprobe according to claim 2, wherein: Step S2 specifically comprises: S21, to a three-necked flask was added 6 mL of oleic acid, 15 mL of octadecene and 0.5 mmol of Lu(CH3COO) 3， After stirring, vacuum was applied for 10 min; S22, heating the solution after vacuumizing in S21 to 120 DEG C and reacting for 20 min until the particles are completely dissolved to obtain a clear solution; S23, releasing the vacuum after cooling the clear solution obtained in S22 to 50 DEG C to obtain solution d; S24, dissolving 0.5 M NaOH and 0.4 M NH4F in methanol solution respectively and mixing uniformly to obtain solution e; S25, the core particles NaNdF4:Yb and solution e obtained in S1 are added to solution d, after maintaining 50℃, continuous stirring is performed for 30 min, then the temperature is increased to 100℃ and maintained for 40 min, vacuum extraction is performed for 10 min and air exchange is performed 2-3 times, then the temperature is increased to 300℃ and reaction is performed for 1.5 h, after the reaction is completed, rapid cooling to room temperature is performed, and solution f is obtained; S26, ethanol solution is added to solution f to precipitate, centrifugation is performed at a rotation speed of 8000 rpm for 10 min, and washing is repeated 3 times, and core-shell structure NaNdF4:Yb@NaLuF4 is obtained, and then the oil phase core-shell structure NaNdF4:Yb@NaLuF4 is redissolved in 4 mL cyclohexane.

4. The method for preparing a lanthanide rare earth nanoprobe according to claim 2, wherein: Step S3 specifically comprises: S31, take 0.1 mmol of the synthesized NaNdF4:Yb@NaLuF4 of S2, after centrifugation with ethanol, re-disperse in 15 mL chloroform solution, and add 20 mg of DSPE-PEG 2000 -COOH, to obtain solution g; S32, solution g is placed in a fume hood and stirred at a rotation speed of 800 rpm until chloroform is completely evaporated, and then placed in an oven at 60℃ for 12 h, and solution h is obtained; S33. To solution h, 15 mL of deionized water was added and sonicated for 2 h, then centrifuged at 15000 rpm for 10 min, and washed with deionized water 3 times to remove unbound DSPE-PEG 2000 -COOH, followed by functionalizing the carboxyl group of the aqueous RENPs-PEG dispersion in deionized water.

5. The method for preparing a lanthanide rare earth nanoprobe according to claim 2, wherein: Step S4 specifically comprises: S41, 20 mg EDC and 60 mg NHS are respectively dissolved in MES buffer to obtain EDC solution and NHS solution; S42, the carboxyl-functionalized RENPs-PEG dispersed in deionized water prepared in step S33 is centrifuged to obtain RENPs-PEG solid, and the RENPs-PEG solid is dispersed in 1 mL MES buffer to obtain solution i; S43, EDC solution is added to solution i, and stirring is performed for 5 min and ultrasonic treatment is performed for 25 min, then NHS solution is added, and stirring is performed for 15 min and ultrasonic treatment is performed for 2 min, and solution j is obtained, S44, solution j is centrifuged at a rotation speed of 15000 rpm for 10 min, and resuspended in 1 mL MES buffer, then 200 µg of plerixafor is added, and placed in a 4℃ environment and stirred for 12 h, then ultrasonic treatment is performed for 5 min, after the reaction is completed, centrifugation is performed at a rotation speed of 15000 rpm for 10 min and resuspended in 1 mL deionized water, then washing, centrifugation and resuspension are performed again in 1 mL deionized water, and finally the probe RENPs loaded with a targeting element is obtained.

6. Application of the lanthanide rare earth nanoprobes in the preparation of a probe reagent for precise radiotherapy target delineation according to claim 1.

7. Application of the lanthanide rare earth nanoprobes in the preparation of a probe reagent for improving the sensitivity of tumor cells to radiation according to claim 1.

8. Application of the lanthanide rare earth nanoprobes in the preparation of a probe reagent for anti-tumor immune activation according to claim 1.

Images