Precious metal clusters for low dose x-ray induced photodynamic therapy and radiotherapy

Abstract

The invention discloses a precious metal cluster for photodynamic therapy and radiotherapy induced by low-dose X-rays, the application method of the precious metal cluster aims at tumors, the photon energy of the low-dose X-rays is kV and MV, the cumulative dose is 2-2.5 Gy, and the specific type of the precious metal cluster is selected according to the requirements of different fluorescence emission wavelengths. The noble metal cluster sensitizer has good performance of penetrating through a blood-brain barrier, and has the characteristics of improving targeting and enrichment of a radiotherapy sensitizer in in-situ brain glioma; under low-dose X-ray energy (kV and MV photon energy), equivalent inhibition on in-situ glioma is realized under the condition of far lower than clinical total radiation dose, and toxic and side effects on normal organisms are reduced.

Description

Technical Field

[0001] This invention relates to the field of biomedical diagnostics and treatment technology, specifically to noble metal clusters for low-dose X-ray induced photodynamic therapy and radiotherapy. Background Technology

[0002] Gliomas are among the most aggressive malignant tumors, typically progressing rapidly after diagnosis, with poor prognosis and a high recurrence rate, making them one of the most challenging brain tumors to treat. Despite decades of advancements in medical technology, improved healthcare, and the development and application of new therapies and combination therapies, including immunotherapy, gene therapy, and tumor stem cell therapy, the actual efficacy is limited. Furthermore, each treatment method or combination therapy has its own limitations, including high total radiation doses (exceeding 50 Gy), high recurrence rates, and the potential for toxic side effects and complications, and ultimately, they cannot effectively improve the survival rate of glioma patients.

[0003] Therefore, developing a safe and effective new method to inhibit gliomas is urgently needed. Preclinical and clinical data show that in gliomas, the blood-brain barrier (BBB) ​​transforms into a blood-tumor barrier, still limiting drug delivery to the brain parenchyma. Most small molecule drugs and large biopharmaceuticals (including growth factors and monoclonal antibodies) cannot effectively cross this barrier to enter the tumor tissue. This significantly reduces the efficacy of drugs targeting gliomas, leading to the failure of numerous clinical trials. To overcome the inefficiency of targeted therapy for gliomas, it is necessary to improve the drug's ability to cross the blood-brain barrier and its targeting specificity to gliomas. Summary of the Invention

[0004] To address the technical problems of low drug penetration through the blood-brain barrier and low targeting in existing technologies, this application proposes a noble metal cluster for low-dose X-ray induced photodynamic therapy and radiotherapy, which improves the drug's ability to penetrate the blood-brain barrier and its targeting and enrichment capabilities in gliomas.

[0005] According to a first aspect of the present invention, a method for applying noble metal clusters in low-dose X-ray induced photodynamic therapy and radiotherapy is proposed, the method being applied to tumors, wherein the photon energy of the low-dose X-rays is kV and MV, the cumulative dose is 2-2.5 Gy, and the specific type of noble metal cluster is selected according to the requirements of different fluorescence emission wavelengths.

[0006] Furthermore, the tumors specifically include gliomas, medulloblastomas, meningiomas, vestibular schwannomas, and lymphomas originating in the central nervous system.

[0007] Furthermore, the specific types of the noble metal clusters include gold nanoclusters, silver nanoclusters, platinum group metal clusters, or a variety of alloy clusters.

[0008] Furthermore, the noble metal cluster is preferably a gold nanocluster cluster (AuNCs).

[0009] Furthermore, in the application, the noble metal cluster is used after being combined with a photosensitizer and a polypeptide conjugate using an EDC / NHS bioconjugation strategy to form a sensitizer; the photosensitizer is selected from one of phthalocyanine photosensitizers, chlorophyll photosensitizers, porphyrin photosensitizers, or pigment photosensitizers; the polypeptide is selected from one of RGD peptides, iRGD peptides, or photosensitizer-binding polypeptides.

[0010] According to a second aspect of the present invention, a 3D optical imaging method for orthotopic brain tumors in mice and rats is provided, the method comprising: administering a sensitizer made of noble metal clusters to mice and rats, and performing 3D light sheet microscopy of the whole brain of mice and 3D magnetic resonance imaging and light sheet microscopy of the whole brain of rats after the brain tissue has become transparent, so as to visualize the overlap between the noble metal clusters and the tumor.

[0011] According to a third aspect of the invention, there is a device for evaluating cells using a clinical radiotherapy apparatus, the device comprising an accelerator treatment bed, a cell culture dish, an equivalent solid water, and a tissue compensator, wherein the device excites an X-ray induced photodynamic therapy (X-PDT) response against brain tumor cells under a clinical linear accelerator, specifically comprising: placing an equivalent solid water on the accelerator treatment bed, and then placing the cell culture dish on the solid water to increase the radiation dose at the bottom of the cell culture dish; placing the tissue compensator on the cell culture dish; and performing radiation according to the application method such that when a low dose of 2-2.5 Gy of X-rays passes through the cell culture dish, the radiation is uniformly distributed in the cell region and the radiation effect is evaluated.

[0012] According to a fourth aspect of the present invention, a system is provided for evaluating the treatment process and efficacy of orthotopic brain tumors in rats using clinical radiotherapy equipment. The system includes a CT device, an MRI device, an accelerator processing bed, and a clinical linear accelerator. Specifically, the evaluation is performed as follows: rigid registration and fusion simulation of three-dimensional reconstructed CT and MRI images of the patient's brain is performed, with the focal point of the registration area being the patient's skeletal location, to determine the total tumor volume (GTV); a target volume volume (PTV) is formed and a ring structure is added to ensure coverage of the target volume PTV exceeds 95%; the patient is placed on the accelerator processing bed, with the center of the patient aligned with the clinical linear accelerator laser, and a CBCT scan of the entire skeleton is performed to ensure strict alignment with the localization CT; radiation is then administered according to the described method to evaluate the treatment efficacy.

[0013] Furthermore, the clinical radiotherapy equipment is configured to deliver a cumulative dose of 2-2.5 Gy and to apply basic radiation technology and SBRT technology in synergy with low-dose X-ray excited X-PDT to target in situ patient-derived animal brain tumor models and animal tumor-bearing models.

[0014] According to a fifth aspect of the present invention, a system is provided for evaluating the therapeutic effect of low-dose radiotherapy in an animal model of CDX or PDX orthotopic glioma, the system comprising a sensitizer application device, a bioluminescence imaging device, a quantitative analysis device, an MRI device, and an HE staining reagent, and the system is specifically implemented in the following manner: administering a sensitizer made of noble metal clusters to mice, performing radiotherapy according to the application method, performing bioluminescence imaging and quantitative analysis before and after the treatment, and verifying the therapeutic effect by MRI and HE staining.

[0015] Compared with the prior art, the beneficial effects of this invention are as follows:

[0016] Noble metal clusters are selected as scintillator materials for X-ray-excited photoluminescence. The high absorption capacity of noble metal atoms for X-rays allows for the use of ultra-low doses (cumulative 2-2.5 Gy) of X-rays, with radiotherapy combined with photodynamic therapy to treat deep brain tumors, while reducing damage to surrounding normal brain tissue, lowering toxic side effects, and improving efficacy. A photosensitizer compatible with the noble metal cluster scintillator is selected, and by combining with a targeting peptide, it specifically targets the αvβ3 integrin overexpressed in gliomas, thereby improving the targeting of the radiosensitizer in situ gliomas, achieving inhibition of gliomas in situ, and reducing toxic side effects on normal tissues. Attached Figure Description

[0017] Figure 1 A morphological analysis diagram according to an embodiment of the present invention is shown;

[0018] Figure 2 A particle size distribution diagram according to a specific embodiment of the present invention is shown;

[0019] Figure 3 A spectral analysis diagram according to a specific embodiment of the present invention is shown;

[0020] Figure 4 A spectral analysis diagram of X-ray irradiation excitation according to a specific embodiment of the present invention is shown;

[0021] Figure 5 A cellular uptake experiment diagram according to a specific embodiment of the present invention is shown;

[0022] Figure 6 A cell viability assessment diagram according to a specific embodiment of the present invention is shown;

[0023] Figure 7 An SOSG probe for detecting tumor cells according to a specific embodiment of the present invention is shown. 1 Image showing the O2 generation results;

[0024] Figure 8 The figure shows the experimental results of DNA damage in U87 MG cells according to a specific embodiment of the present invention;

[0025] Figure 9 The figure shows the experimental results of DNA damage in C6 cells according to a specific embodiment of the present invention;

[0026] Figure 10 The figure shows the results of a U87 MG cell colony formation experiment according to a specific embodiment of the present invention;

[0027] Figure 11 The figure shows the experimental results of C6 cell colony formation according to a specific embodiment of the present invention;

[0028] Figure 12 A fluorescence imaging result of a tumor targeting experiment according to a specific embodiment of the present invention is shown;

[0029] Figure 13 The following is a live microscopic imaging result of an experiment demonstrating the effective crossing of the blood-brain barrier by RAR according to a specific embodiment of the present invention;

[0030] Figure 14 The image shows a 3D light-screen microscopy result of the whole brain of a mouse after transparent treatment in a tumor targeting experiment according to a specific embodiment of the present invention.

[0031] Figure 15 The results of a rat whole-brain 3D magnetic resonance imaging and light-slice microscopy-2 experiment for tumor targeting according to a specific embodiment of the present invention are shown.

[0032] Figure 16 This illustration shows bioluminescence imaging of an animal following a 2 Gy low-dose radiotherapy, according to a specific embodiment of the invention.

[0033] Figure 17 A line graph showing the results of animal BLI monitoring after a 2 Gy low-dose radiotherapy according to a specific embodiment of the present invention is shown.

[0034] Figure 18 A schematic diagram illustrating the application of a clinical linear accelerator in the evaluation of glioma cells according to a specific embodiment of the present invention is shown.

[0035] Figure 19A flowchart illustrating the X-PDT evaluation of a clinical linear accelerator applied to rats with orthotopic gliomas according to a specific embodiment of the present invention is shown.

[0036] Figure 20 An MRI imaging monitoring result diagram according to a specific embodiment of the present invention is shown;

[0037] Figure 21 A statistical line graph of tumor volume according to a specific embodiment of the present invention is shown;

[0038] Figure 22 Bioluminescence imaging before and after treatment and monitoring according to a specific embodiment of the present invention is shown;

[0039] Figure 23 A line graph showing the signal quantification results according to a specific embodiment of the present invention is shown;

[0040] Figure 24 A tissue staining result diagram is shown according to a specific embodiment of the present invention;

[0041] Figure 25 A survival curve of a rat according to a specific embodiment of the present invention is shown;

[0042] Figure 26 An image showing the results of immunohistochemical analysis according to a specific embodiment of the present invention is displayed;

[0043] Figure 27 The diagram illustrates the effects of different concentrations of RAR on the dark toxicity and low-dose radiotherapy efficacy of patient-derived glioma cells according to a specific embodiment of the present invention.

[0044] Figure 28 This diagram illustrates the results of singlet oxygen in patient-derived glioma cells according to a specific embodiment of the present invention.

[0045] Figure 29 The diagram illustrates the DNA damage detection results of patient-derived glioma cells after different treatments, according to a specific embodiment of the present invention.

[0046] Figure 30 The figure shows the results of a clone formation experiment of patient-derived glioma cells according to a specific embodiment of the present invention;

[0047] Figure 31 The image shows the results of intratumoral enrichment of patient-derived glioma-bearing mice after tail vein injection, according to a specific embodiment of the present invention.

[0048] Figure 32Figure 1 shows the treatment results of a patient-derived orthotopic glioma animal model according to a specific embodiment of the present invention;

[0049] Figure 33 The magnetic resonance imaging (MRI) image and corresponding HE result image-2 of the treatment results of a patient-derived orthotopic glioma animal model according to a specific embodiment of the present invention are shown. Detailed Implementation

[0050] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0051] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0052] In the embodiments of this invention concerning noble metal cluster sensitizers, gold nanoclusters (AuNCs) are selected as the noble metal cluster. A photosensitizer, Bengal rose red (RB), is selected to synthesize an RB-AuNCs conjugate. Then, an RGD peptide is selected as the polypeptide, and the RGD peptide is conjugated onto the surface of the RB-AuNCs to obtain RB-AuNCs-RGD(RAR). It should be understood that the innovative solutions upon which the technical problem to be solved and the basic technical effects of this invention rely are also applicable to the selection of gold nanoclusters, silver nanoclusters, platinum group metal clusters, or multiple alloy clusters as the noble metal cluster; similarly, they are applicable to the selection of phthalocyanine photosensitizers, chlorophyll photosensitizers, porphyrin photosensitizers, or pigment photosensitizers as the photosensitizer; and similarly, they are applicable to the selection of RGD peptides, iRGD peptides, or photosensitizer-binding polypeptides as the polypeptide. Although the following specific embodiments are all described using RB-AuNCs-RGD(RAR) as the selection, these embodiments are also applicable to the other selections listed above.

[0053] In specific embodiments of this invention, the detection of the effect of the sensitizer RAR on low-dose X-ray excited photodynamic therapy (X-PDT) was conducted under the condition that the radiation meter parameters were set to 160kV; the animal bioluminescence imaging and BLI monitoring of the mouse model treatment experiment were conducted under the condition of low-dose X-ray with the radiation meter parameters set to 160kV; the MRI imaging monitoring of the rat model treatment experiment and the effect evaluation on the rat orthotopic glioma model were conducted under 6MV low-dose X-ray; the evaluation of patient-derived glioma cells and the detection of the treatment effect of the patient-derived orthotopic glioma animal model were both based on 160kV low-dose X-ray.

[0054] Synthesis of precious metal cluster sensitizers

[0055] Gold nanoclusters (AuNCs) were synthesized using L-glutathione (GSH) and tetrachloroauric acid trihydrate (HAuCl4-3H2O). GSH aqueous solution (6 mM, 10 mL) was added to HAuCl4 aqueous solution (4 mM, 10 mL), and the mixture was stirred at 70 °C for 6–24 hours. The resulting AuNCs were dialyzed through a 10 kDa dialysis bag using ultrapure water. The AuNCs were then concentrated by centrifugation at 9000 RPM using an Amicon Ultra-4 filter and stored at 4 °C in the dark. An EDC / NHS bioconjugation strategy was used to conjugate the AuNCs solution with Bengal rose ruby ​​(RB) solution. The prepared AuNCs solution was added to the Bengal rose ruby ​​(RB) solution and stirred continuously for 12 hours to obtain the RB-AuNCs conjugate. This conjugate was then dialyzed through a 10 kDa dialysis bag in the dark to obtain RB-AuNCs. Using a similar bioconjugation strategy, the RGD peptide was conjugated onto the surface of RB-AuNCs to obtain RB-AuNCs-RGD(RAR).

[0056] Performance testing of the sensitizer RAR

[0057] like Figure 1 As shown, (A) is a high-resolution HTEM image of AuNCs; (B) is a high-resolution HTEM image of RB-AuNCs; and (C) is a high-resolution HTEM image of RAR. The high-resolution electron microscopy images of the three samples—AuNCs, RB-AuNCs, and RAR—show that AuNCs, RB-AuNCs, and RAR exhibit good dispersion, with consistent morphology and particle size. This demonstrates that the bio-template-synthesized scintillator AuNCs, through covalent coupling with the photosensitizer RB and modification with the targeting peptide cRGD, do not affect the one-step synthesis of AuNCs. Figure 2 As shown, (A) the particle size distribution of AuNCs; (B) the particle size distribution of RB-AuNCs; and (C) the particle size distribution of RAR. The prepared RAR particles are uniform in size and have good monodispersity, with an average average particle size of 11 nm. This demonstrates that the method for preparing RAR is stable and feasible, and can be further applied in biological fields.

[0058] like Figure 3 As shown, the results of the UV-Vis absorption spectra of RB, RB-AuNCs and RAR and the X-ray excitation optical emission curves of AuNCs after X-ray irradiation show that the emission peak of AuNCs in the X-ray excitation optical emission spectrum is at 565 nm, which matches the absorption peak of the UV-Vis absorption spectrum of RB well.

[0059] The X-ray excitation optical emission curve was detected using a QE Pro-FL high-sensitivity fluorescence spectrometer while the sample was excited by a miniature X-ray tube. Figure 4 As shown, the results of X-ray irradiation-excited AuNCs (black curve) and RAR (blue line) show that the X-ray irradiation-excited fluorescence and photosensitizer RB undergo efficient fluorescence resonance energy transfer (FRET). After X-ray irradiation excites RAR, the photons generated by the scintillator AuNCs are effectively absorbed by RB, forming a photodynamic process, thereby ensuring the efficient activation of the photosensitizer and the generation of singlet oxygen.

[0060] The targeted uptake efficiency of RAR by tumor cells was analyzed by examining the fluorescence intensity at different time points after co-culturing tumor cells with RAR. The fluorescence intensity of tumor cells and RAR at different time points was tested, such as... Figure 5 As shown, flow cytometry using the PE channel was used to analyze the uptake of RAR in tumor cells (U87 MG, GL261 and C6). Figure 5 (D) The average fluorescence intensity of the cells increased over time. The three malignant glioma cell lines showed high uptake efficiency of RAR, reflecting the strong tumor targeting of RAR.

[0061] Efficacy of the sensitizer RAR in low-dose X-ray excited photodynamic therapy (X-PDT)

[0062] The effects of X-PDT on three malignant tumor cell lines (U87 MG, GL261, and C6) under different irradiation doses, such as... Figure 6 As shown, (AB)U87MG and GL261 cells were co-cultured with RAR (0-200 μg / mL) and irradiated with X-rays of 0 Gy, 0.5 Gy, 1 Gy and 2 Gy. (C)C6 cells were co-cultured with RAR (0-200 μg / mL) and then irradiated with X-rays of 0, 0.5, 1 and 2 Gy. The inhibitory effect on malignant tumor cells was best at a single irradiation of 2 Gy and a RAR concentration of 200 μg / mL.

[0063] After co-incubation of U87 MG cells with RAR, followed by irradiation with 2 Gy of X-rays, compared to the PBS+X-ray group receiving radiotherapy alone, the following results were observed: Figure 7 As shown in (A), the X-PDT group exhibited significantly stronger singlet oxygen (green) fluorescence signal, demonstrating that the X-PDT effect can generate more singlet oxygen within tumor cells, thereby killing tumor cells. Figure 7 As shown in (B), after co-incubation with C6 cells and RAR cells and subsequent irradiation with 2 Gy X-rays, the X-PDT group showed significantly stronger singlet oxygen (green) fluorescence signal compared to the X-ray-only group. 1O2 is the main active factor in photodynamic therapy (PDT), and it also directly proves that low-dose X-ray-induced photodynamic therapy (X-PDT) can generate more singlet oxygen in tumor cells, thereby killing tumor cells.

[0064] U87 MG and C6 cells were divided into PBS, RAR, X-ray, and X-PDT groups (RAR: 100 μg / mL; X-rays: 2 Gy), as follows: Figure 8 As shown, enhanced green fluorescence and significant expression of γH2AX (green) were observed in X-PDT-treated U87 MG tumor cells. Compared with the X-ray-only radiotherapy group, which damaged tumor cell DNA, this indicates that X-PDT is more effective at damaging tumor cell DNA. Results on C6 cells are shown below. Figure 9 As shown, the same findings revealed enhanced green fluorescence and significant expression of γH2AX (green) in tumor cells of the X-PDT group. Compared with the X-ray group alone, which damaged the DNA of tumor cells, this indicates that the X-PDT effect is more effective in damaging the DNA of tumor cells.

[0065] The effects of X-PDT on the long-term survival and proliferation of tumor cells were further evaluated using a clonogenic assay. Results are as follows: Figure 10 and Figure 11 As shown, the (AB) colony formation assay measured the cell proliferation capacity of U87 MG cells and C6 cells 10 days after radiotherapy (RT (X-ray) and X-PDT, respectively. All statistical data are expressed as mean ± SD. Compared with the X-ray group, X-PDT showed a significant difference (P = 0.0001, ***P < 0.001). Statistical significance was obtained using a two-sample t-test. Compared with the X-PDT group, at the same radiation dose, the RT group cells showed a higher survival rate, while the X-PDT-treated cells had a weaker proliferative capacity, indicating that X-PDT more effectively inhibited the tumor cell colony formation capacity.

[0066] Brain tumor targeted experiment

[0067] In vivo fluorescence imaging at different time points after tail vein injection of RAR in a mouse model of orthotopic glioma (U87 MG-luc) as shown in the figure. Figure 12 As shown in (A), the fluorescence signal in the brain region is significantly stronger than in other areas, indicating that RAR can effectively target and accumulate in brain tumors. Figure 12(B) Quantitative analysis of the fluorescence intensity of DiR signals in mouse brain regions (ROIs) at different time points. The quantitative results of the average fluorescence signal at different time points show that the tumor aggregation peak was reached 2 hours after tail vein injection of RAR, followed by a slow decline. Four hours after tail vein injection of RAR, the results of ex vivo fluorescence imaging of various organs and brain tumors in mice are as follows: Figure 12 As shown in (C), the fluorescence signal at the tumor site in the brain tissue is significantly stronger compared to normal brain tissue, indicating that RAR can effectively aggregate at the location of brain tumor lesions. The ability of RAR to target and aggregate in tumors was observed at the in vivo macroscopic level.

[0068] To further achieve real-time visualization of the RAR targeted delivery process to brain tumors at the microscopic level, a cranial window was constructed in a model mouse brain to perform in vivo microscopic imaging of the brain tumor region. For example... Figure 13 As shown, one hour after tail vein injection of RAR, the RAR was mainly located inside the tumor vessels. Four hours later, RAR was clearly visible leaking out of the vessels and targeting and accumulating in the tumor tissue and even within the tumor cells. This demonstrates the excellent tumor-targeting and aggregation ability of RAR.

[0069] To further visualize the targeted aggregation process of RAR in brain tumor tissue from a 3D perspective, this invention provides a 3D optical imaging method for orthotopic brain tumors in mice and rats, specifically including: administering RAR to mice and rats, and after the brain tissue becomes transparent, performing 3D light sheet microscopy of the whole brain of mice and 3D magnetic resonance imaging and light sheet microscopy of the whole brain of rats, so as to visualize the overlap between RAR and tumor.

[0070] The results of 3D light slide microscopy of the whole brain of mice after transparent treatment are as follows: Figure 14 As shown, (A) the process of brain tissue becoming transparent. (B) Whole-brain microscopy of an orthotopic glioma model mouse shows the targeted distribution of RAR in the tumor. The RAR (red) signal and the tumor's GFP (green) signal have good overlap, indicating that RAR is targeted and aggregated inside the brain tumor tissue and distributed throughout the tumor mass.

[0071] Results of whole-brain 3D magnetic resonance imaging and light sheet microscopy in rats: Figure 15As shown, a. T2-weighted MRI images of rat orthotopic gliomas at different levels and 3D reconstructed images; b. Ex vivo fluorescence imaging of orthotopic glioma brain tissue after tail vein injection of RAR; c. Quantification of fluorescence intensity in normal brain tissue and brain tumor; de. Photographs of brain tissue before and after clearing; f. Targeted delivery of RAR to rat gliomas, also confirmed by fluorescence 3D images (light sheet microscopy). Three-dimensional panoramic fluorescence imaging (de) of the whole brain of glioma rats at different angles after clearing of brain tissue. Gray: brain tissue, red: RAR, green: tumor (C6-GFP); g. Two-dimensional planar image of the three-dimensional panoramic fluorescence imaging; h. Quantification of fluorescence intensity in normal tissue and brain tumor. All statistics are expressed as mean ± SD. *The fluorescence intensity in the tumor is significantly different from that in normal tissue. Statistical significance was obtained using an independent samples t-test. The RAR (red) signal and the GFP (green) signal in the tumor show good overlap, indicating that RAR is targeted and aggregated within the brain tumor tissue and distributed throughout the tumor mass.

[0072] Mouse model treatment experiment

[0073] Results of animal bioluminescence imaging and BLI monitoring after 2Gy low-dose radiotherapy are as follows: Figure 16 and Figure 17 It is evident that, after day 20 of treatment, the ROI luminescence signal values ​​in the model mice of the low-dose X-ray photodynamic therapy (X-PDT) group continuously decreased compared to the control group, while the signal gradually increased in the control group. This indicates that RAR can inhibit the proliferation of deep brain tumors through low-dose kV and MV photons of X-rays, and the X-PDT effect can inhibit the tumor development process.

[0074] To more effectively inhibit in situ gliomas and reduce toxic side effects on normal organisms under low-dose radiotherapy conditions, this invention proposes a device for evaluating cells using clinical radiotherapy equipment. This device excites an X-ray-induced photodynamic therapy (X-PDT) response against brain tumor cells using a clinical linear accelerator. Figure 18 This diagram illustrates the application of a clinical linear accelerator in the evaluation of glioma cells, specifically including:

[0075] (1) Several equivalent solid water blocks were placed on the accelerator treatment bed, and then cell culture dishes were placed on the solid water to increase the radiation dose at the bottom of the cell culture dishes. The solid water was purchased from IBA Dosimetry and its density was equivalent to that of liquid water.

[0076] (2) Place a 1 cm tissue compensator on the cell culture dish so that when a low dose of X-rays (MV level) of 2-2.5 Gy passes through the cell culture dish, the radiation is evenly distributed in the cell area and the radiation effect is evaluated.

[0077] Furthermore, this invention proposes a system for evaluating the treatment process and efficacy of orthotopic brain tumors in rats using clinical radiotherapy equipment. The system includes a CT device, an MRI device, an accelerator processing bed, and a clinical linear accelerator. Figure 19 The flowchart illustrates the X-PDT evaluation process using a clinical linear accelerator in rats with orthotopic gliomas, specifically including:

[0078] (1) After anesthesia, the rat was placed on a fixed device, and the corresponding coordinates were recorded to begin CT localization. The laser crosshair was aligned with the center of the rat's brain, and the Y-axis laser was aligned with the rat's sagittal midline. The X-axis laser was used for localization and to draw the crosshair center. The CT slice thickness and interslice spacing were 0.625 mm, the tube current was 200-250 mA, and the tube voltage was 120 kV. After the scan was completed, the DICOM image was transmitted to the radiation physics workstation.

[0079] (2) Simultaneously, T2-weighted MR images of the rat brain were also transmitted to the radiophysics workstation for registration. The scanning range of the MRI images was the same as that of the CT scan, the scanning thickness of the MRI images was 0.8 mm, the interslice spacing was 0 mm, and the scanning parameter domain confirmed that the scanning frame was not selected in all three directions and the angle was 0.

[0080] (3) Rigid registration and fusion simulation of three-dimensional reconstructed CT and MRI images of rat brain: The focal point of the registration area is the location of the rat's skull. The brain lesions shown on the MRI images are delineated layer by layer on the localization CT. The total tumor volume (GTV) is determined, and the location of the tumor lesion is confirmed based on the distance between the tumor lesion and the foramen magnum to avoid omission.

[0081] (4) After completing the rigid registration and fusion simulation of CT and MRI images, a radiotherapy plan is developed. Due to positional errors during radiotherapy, the target volume (PTV) is extended outward by 0.1 cm in three dimensions from the ground-to-ground (GTV) to form the planned target volume (PTV). The outline of normal brain tissue is delineated on the CT image, and an optimized ring structure is added to the target PTV to limit the dose to external normal tissues to a low level. The radiation conditions are set to a 180° single arc, a radiation energy of 6 MV, and a dose rate of 6 Gy / min. In addition, the settings can be further optimized to ensure that the coverage of the PTV exceeds 95%, the maximum dose does not exceed 110% of the prescribed dose, and that the optimized plan's target coverage and normal organ dose both meet the requirements.

[0082] (5) After determining the radiotherapy plan, the rats were brought to the clinical linear accelerator. The animal fixation equipment was adjusted to ensure precise radiotherapy positioning, aligning the center of the three coplanar "crosses" on the rat's thermoplastic membrane with the clinical linear accelerator laser. A CBCT scan of the entire skull was performed, and the CBCT was strictly aligned with the positioning CT to ensure accurate skull positioning. The radiotherapy dose was set to 2 Gy, and radiotherapy was conducted under single-shot conditions.

[0083] MRI imaging monitoring results as follows Figure 20 As shown, T2-weighted MRI imaging of orthotopic gliomas in rats from different treatment groups was performed at the beginning (day 11) and the end. T2-weighted MRI (axial plane) imaging was conducted at different time points, with the same tumor slice section selected to accurately show changes in tumor size at the same location. The dashed line represents the region of interest used to calculate tumor volume. The changes in the same location of the brain tumor in the model rats at different time points are shown. Compared with the control group, the X-PDT results in the rat model showed that the brain tumors in the rat models continuously shrank after treatment, even disappearing at the endpoint. Simultaneously, the quantitative results of brain tumor volume in different groups of rat models after treatment are shown below. Figure 21 As shown, the tumor volume results of different groups of PBS, RAR, X-ray and X-PDT showed that the tumor volume of rats in the PBS, RAR and X-ray groups gradually increased, while the tumor volume in the X-PDT group continuously decreased.

[0084] Bioluminescence imaging and signal quantification results before, during, and after treatment are as follows: Figure 22 and Figure 23 As shown in the images and quantitative results, the signal at the brain tumor location in the three control rat models (PBS, RAR, and X-ray) gradually increased after treatment, but this did not effectively inhibit tumor development. In contrast, the signal at the brain tumor location in the X-PDT group rat model gradually decreased, effectively inhibiting tumor progression.

[0085] like Figure 24 As shown, the original tumor in the X-PDT group was eliminated, and the nuclear density of cells at the lesion site was low, indicating that the tumor was effectively suppressed and eliminated. The model survival results for different treatment groups are as follows: Figure 25 As shown, the low-dose X-ray group alone did not effectively improve the survival of the rat model, while the low-dose X-PDT group significantly increased the survival of the rat model.

[0086] Rat orthotopic glioma models in different treatment groups underwent Ki-67 and PCNA staining of tumor tissue at the monitoring endpoint. The quantitative fluorescence intensity of Ki-67 in the immunofluorescence images was analyzed, as shown in... Figure 26 As shown, the original tumor in the X-PDT group was eliminated, the fluorescence intensity of Ki-67 was significantly reduced, and the quantitative value of PCNA was also significantly reduced, indicating that the X-PDT group can effectively inhibit the proliferation and invasiveness of tumors.

[0087] The dark toxicity of different concentrations of RAR was detected using a CCK-8 assay kit. At an X-ray irradiation dose of 2 Gy and with the irradiator parameters set to 160 kV, the results of the dark toxicity and low-dose radiotherapy efficacy of different concentrations of RAR on patient-derived glioma cells are as follows: Figure 27As shown, in comparison, RAR exhibits good dark toxicity and demonstrates better tumor cell killing ability under X-ray irradiation.

[0088] After treatment with PBS, RAR, X-ray, and X-PDT, the production of intracellular singlet oxygen was monitored using the SOSG singlet oxygen probe (green fluorescence). The results of intracellular singlet oxygen production in patient-derived glioma cells are as follows: Figure 28 As shown, due to the characteristic that SOSG singlet oxygen probes cannot enter cells, a small amount of green fluorescence signal was present in the control group. In the X-PDT group, RAR can generate a large amount of singlet oxygen under X-ray irradiation, which binds to SOSG singlet oxygen probes and produces obvious green fluorescence signal.

[0089] DNA damage to cells treated with PBS, RAR, X-ray, and X-PDT was detected using a DNA damage detection kit (green fluorescence) and quantitatively analyzed. The results of DNA damage detection after different treatments of patient-derived glioma cells are as follows: Figure 29 As shown, DNA double-strand damage is an important marker in the X-PDT process. Comparison revealed that no obvious green fluorescence signal was observed in the three control groups, while a significant green fluorescence signal appeared in the X-PDT group, proving that DNA damage occurred in the X-PDT group.

[0090] The effects of RT and X-PDT on inhibiting the proliferation of patient-derived glioma cells were compared using cell clonogenic assays, and quantitative analysis was performed. Figure 30 As shown, X-PDT significantly inhibited the proliferation of patient-derived glioma cells compared to conventional RT.

[0091] Results of intratumoral enrichment in patient-derived glioma-bearing mice after tail vein injection: Figure 31 As shown, (A) RAR (100 μg mL) -1 (A) Fluorescence imaging was performed in mice bearing gliomas in situ via tail vein administration. (B) Quantitative analysis of the fluorescence imaging results was performed, and ex vivo fluorescence imaging of the mouse brain was also conducted. RAR reached maximum intratumoral enrichment 1 hour after tail vein injection and could continue to accumulate at the tumor site.

[0092] PDX orthotopic glioma animal model treatment efficacy test

[0093] To evaluate the therapeutic effect of PDX or PDX orthotopic glioma animal models, this invention provides a system for evaluating the therapeutic effect of low-dose radiotherapy in these models. The system includes a sensitizer application device, a bioluminescence imaging device, a quantitative analysis device, an MRI device, and an HE staining reagent. Specifically, the system includes administering RAR radiotherapy to mice, performing bioluminescence imaging and quantitative analysis before and after treatment, and verifying the therapeutic effect using MRI and HE staining.

[0094] Treatment outcomes for animal models of orthotopic gliomas derived from patients, such as Figure 32 As shown, (A) bioluminescence imaging of each group during treatment; (B) and (C) quantitative analysis of bioluminescence imaging; (D) body weight curves of mice in each group during treatment; and (E) survival curves of mice in each group during treatment. The single-treatment and double-treatment groups, receiving tail vein injection of RAR combined with X-ray, exhibited significant tumor suppression effects. Both treatment groups effectively inhibited tumor growth, and the mice's body weight did not change significantly. The single-treatment and double-treatment X-PDT groups significantly prolonged survival, demonstrating that RAR can effectively combine photodynamic therapy and radiotherapy. In the Blab / c nude-mice PDX glioma animal model, it can effectively inhibit tumor growth and prolong median survival.

[0095] Magnetic resonance imaging (MRI) images and corresponding HE results of the treatment outcomes of a PDX-derived animal model of glioma in patients are shown below. Figure 33 As shown, (A) the therapeutic effect of RAR on the PDX orthotopic glioma animal model was verified using 9.4T MRI and H&E staining results; (B) and (C) the therapeutic effect was verified by H&E staining of the treated mice, and the number of cell nuclei in each group was statistically analyzed. The H&E results verified that RAR under the X-PDT treatment method can kill tumor tissue in the PDX glioma animal model and effectively inhibit tumor growth.

[0096] The specific embodiments of this application have been described above, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

[0097] In the description of this application, it should be understood that the terms "upper," "lower," "inner," "outer," etc., indicating orientation or positional relationships based on the orientation or positional relationships shown in the accompanying drawings, are used only for the convenience of describing this application and for simplification, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this application. The word "comprising" does not exclude the presence of elements or steps not listed in the claims. The word "a" or "an" preceding an element does not exclude the presence of a plurality of such elements. The simple fact that certain measures are recited in mutually different dependent claims does not indicate that combinations of these measures cannot be used for improvement. Any reference signs in the claims should not be construed as limiting the scope.

Claims

1. A method for using noble metal clusters in low-dose X-ray-induced photodynamic therapy and radiotherapy, characterized in that: The application method is aimed at tumors, the photon energy of the low-dose X-ray is kV and MV, the cumulative dose is 2-2.5 Gy, and the specific type of the precious metal cluster is selected according to the requirements of different fluorescence emission wavelengths.

2. The application method according to claim 1, characterized in that: The tumors specifically include glioma, medulloblastoma, meningioma, vestibular schwannoma and lymphoma originating from the central nervous system.

3. The application method according to claim 1, characterized in that: The specific types of the noble metal clusters include gold nanoclusters, silver nanoclusters, platinum group metal clusters or one of a variety of alloy clusters.

4. The application method according to claim 3, characterized in that: The noble metal clusters are preferably gold nanoclusters AuNCs.

5. The application method according to claim 1, characterized in that: In the application, the noble metal cluster is used after being combined with a photosensitizer and a polypeptide conjugate to form a sensitizer using an EDC / NHS bioconjugation strategy; the photosensitizer is one selected from phthalocyanine photosensitizers, chlorophyll photosensitizers, porphyrin photosensitizers or pigment photosensitizers; and the polypeptide is one selected from RGD peptide, iRGD peptide or photosensitizer-bound polypeptide.

6. A 3D optical imaging method for orthotopic brain tumors in mice and rats, characterized in that: The method comprises: administering a sensitizer made of precious metal clusters to mice and rats, and performing 3D light sheet microscopy of the whole brain of mice and 3D magnetic resonance imaging and light sheet microscopy of the whole brain of rats after the brain tissue is transparent, so as to visualize the overlap between the precious metal clusters and the tumor.

7. A device for evaluating cells using clinical radiotherapy equipment, characterized in that: The device comprises an accelerator treatment bed, a cell culture dish, equivalent solid water and a tissue compensator, wherein the device excites an X-ray induced photodynamic therapy X-PDT reaction for brain tumor cells under a clinical linear accelerator, specifically comprising: placing equivalent solid water on the accelerator treatment bed, and then placing the cell culture dish on the solid water to increase the radiation dose at the bottom of the cell culture dish; placing the tissue compensator on the cell culture dish, and implementing radiation according to the application method described in any one of claims 1 to 5, so that when a low-dose X-ray of 2-2.5Gy passes through the cell culture dish, the radiation is evenly distributed in the cell area and the radiation effect is evaluated.

8. A system for evaluating the treatment process and treatment effect of rat orthotopic brain tumors using clinical radiotherapy equipment, characterized in that: The system includes a CT device, an MRI device, an accelerator treatment bed and a clinical linear accelerator. The system is evaluated in the following manner: rigid registration and fusion simulation of a 3D reconstructed CT image of the brain and an MRI image of the medical subject are performed, the focus of the registration area is the bone position of the medical subject, and the total tumor volume GTV is determined; a target area PTV is formed and a ring structure is added to ensure that the coverage rate of the target area PTV exceeds 95%; the medical subject is placed on the accelerator treatment bed, the center of the medical subject is aligned with the clinical linear accelerator laser, and the CBCT of the entire skeleton is scanned to strictly align it with the positioning CT; Radiation is performed according to the application method described in any one of claims 1 to 5 to evaluate the therapeutic effect.

9. The system for treatment process and treatment effect according to claim 8, characterized in that: The clinical radiotherapy equipment is configured to target and treat in situ patient-derived animal brain tumor models and animal tumor-bearing models with a cumulative dose of 2-2.5 Gy and the application of basic radiation technology and SBRT technology in synergy with low-dose X-ray-excited X-PDT.

10. A system for evaluating the therapeutic effect of low-dose radiotherapy on CDX or PDX orthotopic brain glioma animal models, characterized in that: The system includes a sensitizer application device, a bioluminescence imaging device, a quantitative analysis device, an MRI device and a HE staining reagent, and the system is specifically implemented in the following manner: a sensitizer made of precious metal clusters is administered to mice, radiotherapy is performed according to the application method described in any one of claims 1 to 5, bioluminescence imaging and quantitative analysis are performed before and after the treatment, respectively, and the treatment effect is verified by MRI and HE staining.

Images