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

Abstract

Disclosed in the present application are noble metal clusters for low-dose X-ray-induced photodynamic therapy and radiotherapy. An application method of the noble metal clusters is directed to tumors. The low-dose X-ray has a photon energy of kV and MV, with a cumulative dose of 2-2.5 Gy, and the specific type of the noble metal clusters is selected according to the requirements of different fluorescence emission wavelengths. A sensitizer of the noble metal clusters exhibits good blood-brain barrier penetration performance, and has the characteristic of improving the targeting and enrichment of radiotherapy sensitizers in in-situ brain gliomas. Under the energy of the low-dose X-ray (kV and MV photon energy), equivalent inhibition of in-situ brain gliomas is achieved at a total radiation dose far lower than the clinical total radiation dose, thereby reducing toxic and side effects on normal organisms.

Description

Noble metal clusters for low-dose X-ray induced photodynamic therapy and radiotherapy

[0001] Related applications

[0002] This application claims priority to Chinese patent application No. 202510122071.7, filed on January 26, 2025, entitled “Noble metal clusters for low-dose X-ray induced photodynamic therapy and radiotherapy”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of biomedical diagnostic and therapeutic technology, specifically to noble metal clusters for low-dose X-ray induced photodynamic therapy and radiotherapy. Background Technology

[0004] 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.

[0005] 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.

[0006] Public content

[0007] 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.

[0008] According to the first aspect of this disclosure, a method for applying noble metal clusters in low-dose X-ray induced photodynamic therapy and radiotherapy is proposed. The application method targets 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.

[0009] Furthermore, the tumors specifically include gliomas, medulloblastomas, meningiomas, vestibular schwannomas, and lymphomas originating in the central nervous system.

[0010] Furthermore, the specific types of the noble metal clusters include gold nanoclusters, silver nanoclusters, platinum group metal clusters, or a variety of alloy clusters.

[0011] Furthermore, the noble metal cluster is preferably a gold nanocluster cluster (AuNCs).

[0012] 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.

[0013] According to a second aspect of this disclosure, 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 after the brain tissue is made 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 the noble metal clusters and the tumor.

[0014] According to a third aspect of this disclosure, an apparatus for evaluating cells using a clinical radiotherapy device is provided. The apparatus includes an accelerator treatment bed, a cell culture dish, an equivalent solid water solution, and a tissue compensator. The apparatus excites an X-ray induced photodynamic therapy (X-PDT) response against brain tumor cells under a clinical linear accelerator. Specifically, this includes: placing an equivalent solid water solution on the accelerator treatment bed, and then placing the cell culture dish on the solid water solution to increase the radiation dose at the bottom of the cell culture dish; placing the tissue compensator on the cell culture dish; and administering 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 across the cell region, and the radiation effect is evaluated.

[0015] According to a fourth aspect of this disclosure, 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. 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 (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.

[0016] 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.

[0017] According to a fifth aspect of this disclosure, 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 includes a sensitizer application device, a bioluminescence imaging device, a quantitative analysis device, an MRI device, and an HE staining reagent. The system is specifically implemented as follows: 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.

[0018] Compared with the prior art, the advantages of this disclosure are as follows:

[0019] 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

[0020] Figure 1 shows a morphological analysis diagram according to an embodiment of the present disclosure;

[0021] Figure 2 shows a particle size distribution diagram according to a specific embodiment of the present disclosure;

[0022] Figure 3 shows a spectral analysis diagram according to a specific embodiment of the present disclosure;

[0023] Figure 4 shows a spectral analysis of X-ray irradiation excitation according to a specific embodiment of the present disclosure;

[0024] Figure 5 illustrates a cell uptake experiment according to a specific embodiment of the present disclosure;

[0025] Figure 6 shows a cell activity assessment diagram according to a specific embodiment of the present disclosure;

[0026] Figure 7 illustrates the detection of intracellular tumor cells by an SOSG probe according to a specific embodiment of the present disclosure. 1 Image showing the O2 generation results;

[0027] Figure 8 shows the experimental results of DNA damage in U87 MG cells according to a specific embodiment of the present disclosure;

[0028] Figure 9 shows the experimental results of DNA damage in C6 cells according to a specific embodiment of the present disclosure;

[0029] Figure 10 shows the results of a U87 MG cell colony formation experiment according to a specific embodiment of the present disclosure;

[0030] Figure 11 shows the results of a C6 cell colony formation experiment according to a specific embodiment of the present disclosure;

[0031] Figure 12 shows a fluorescence imaging result of a tumor targeting experiment according to a specific embodiment of the present disclosure;

[0032] Figure 13 shows the in vivo microscopic imaging results of a RAR effective crossing of the blood-brain barrier experiment according to a specific embodiment of the present disclosure;

[0033] Figure 14 shows the results of a 3D light-screen microscopy of the whole brain of a mouse after transparency treatment in a tumor targeting experiment according to a specific embodiment of the present disclosure.

[0034] Figure 15 shows the results of a rat whole-brain 3D magnetic resonance imaging and light sheet microscopy-2 experiment on tumor targeting according to a specific embodiment of the present disclosure;

[0035] Figure 16 illustrates bioluminescence imaging of an animal following a 2 Gy low-dose radiotherapy according to a specific embodiment of the present disclosure;

[0036] Figure 17 shows a line graph of animal BLI monitoring results after 2 Gy low-dose radiotherapy according to a specific embodiment of the present disclosure;

[0037] Figure 18 illustrates a schematic diagram of a clinical linear accelerator applied to the evaluation of glioma cells according to a specific embodiment of the present disclosure;

[0038] Figure 19 shows a flowchart of X-PDT evaluation of a clinical linear accelerator applied to rats with orthotopic glioma according to a specific embodiment of the present disclosure;

[0039] Figure 20 shows an MRI imaging monitoring result diagram according to a specific embodiment of the present disclosure;

[0040] Figure 21 shows a statistical line graph of tumor volume according to a specific embodiment of the present disclosure;

[0041] Figure 22 illustrates bioluminescence imaging before and after treatment and monitoring according to a specific embodiment of the present disclosure;

[0042] Figure 23 shows a line graph of signal quantification results according to a specific embodiment of the present disclosure;

[0043] Figure 24 shows a tissue staining result diagram according to a specific embodiment of the present disclosure;

[0044] Figure 25 shows a survival curve of rats according to a specific embodiment of the present disclosure;

[0045] Figure 26 shows the results of immunohistochemical analysis according to a specific embodiment of the present disclosure;

[0046] Figure 27 shows the results of dark toxicity and low-dose radiotherapy effects of different concentrations of RAR on patient-derived glioma cells according to a specific embodiment of the present disclosure;

[0047] Figure 28 shows a graph of singlet oxygen results in patient-derived glioma cells according to a specific embodiment of the present disclosure;

[0048] Figure 29 shows the results of DNA damage detection after different treatments of patient-derived glioma cells according to a specific embodiment of the present disclosure;

[0049] Figure 30 shows the results of a patient-derived glioma cell clonogenic experiment according to a specific embodiment of the present disclosure;

[0050] Figure 31 shows the results of intratumoral enrichment in a mouse bearing a patient-derived glioma after tail vein injection, according to a specific embodiment of the present disclosure.

[0051] Figure 32 shows the treatment results of a patient-derived orthotopic glioma animal model according to a specific embodiment of the present disclosure - Figure 1;

[0052] Figure 33 shows a magnetic resonance imaging (MRI) image of the treatment outcome of a patient-derived orthotopic glioma animal model according to a specific embodiment of the present disclosure, and the corresponding HE result image-2. Detailed Implementation

[0053] 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 relevant disclosure and not intended to limit the scope of the disclosure. Furthermore, it should be noted that, for ease of description, only the parts relevant to the disclosure are shown in the accompanying drawings.

[0054] 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.

[0055] In the embodiments of the noble metal cluster sensitizer disclosed herein, gold nanoclusters (AuNCs) are selected as the noble metal cluster. A photosensitizer, Bengal rose red (RB), which is compatible with AuNCs, 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 problems to be solved and the basic technical effects of this disclosure rely are also applicable to the selection of one of the following noble metal clusters: gold nanoclusters, silver nanoclusters, platinum group metal clusters, or multiple alloy clusters. Similarly, they are applicable to the selection of one of the following photosensitizers: phthalocyanine, chlorophyll, porphyrin, or pigment. Furthermore, they are applicable to the selection of one of the following RGD peptides, iRGD peptides, or photosensitizer-binding polypeptides. 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.

[0056] In the specific embodiments of this disclosure, the detection of the effect of the sensitizer RAR on low-dose X-ray excited photodynamic therapy (X-PDT) was based on the radiation instrument parameters being set to 160kV; the animal bioluminescence imaging and BLI monitoring of the mouse model treatment experiment were performed under low-dose X-ray conditions with the radiation instrument 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 performed under 6MV low-dose X-rays; the evaluation of patient-derived glioma cells and the detection of the treatment effect on the patient-derived orthotopic glioma animal model were both based on 160kV low-dose X-rays.

[0057] Synthesis of precious metal cluster sensitizers

[0058] 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).

[0059] Performance testing of the sensitizer RAR

[0060] Figure 1 shows the high-resolution HTEM images of (A) AuNCs, (B) RB-AuNCs, and (C) RAR. The images show that AuNCs, RB-AuNCs, and RAR exhibit good dispersion, consistent morphology, and uniform 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 shows the particle size distribution of (A) AuNCs, (B) RB-AuNCs, and (C) RAR. The prepared RAR particles are uniform in size and exhibit good monodispersity, with an average particle size of 11 nm. This indicates that the RAR preparation method is stable and feasible, and can be further applied in biological fields.

[0061] As shown in Figure 3, the results of the UV-Vis absorption spectra of RB, RB-AuNCs and RAR and the X-ray excitation optical emission curve 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.

[0062] The X-ray excitation optical emission curves were detected using a QE Pro-FL high-sensitivity fluorescence spectrometer while the sample was excited by a miniature X-ray tube, as shown in Figure 4. The results of the X-ray excited AuNCs (black curve) and RAR (blue line) show that the X-ray excited fluorescence and photosensitizer RB underwent effective fluorescence resonance energy transfer (FRET). After the X-ray excited RAR, the photons generated by the scintillator AuNCs were effectively absorbed by RB, forming a photodynamic process, thereby ensuring the efficient activation of the photosensitizer and the generation of singlet oxygen.

[0063] The targeted uptake efficiency of RAR by tumor cells was analyzed by examining the fluorescence intensity at different time points after incubation with RAR. The fluorescence intensity of tumor cells co-cultured with RAR at different time points was tested, as shown in Figure 5. (AC) Flow cytometry with the PE channel was used to analyze the uptake of RAR by tumor cells (U87 MG, GL261, and C6); Figure 5(D) shows that the average fluorescence intensity of cells increased over time. The three malignant glioma cell lines showed high uptake efficiency of RAR, reflecting the strong tumor targeting of RAR.

[0064] Efficacy of the sensitizer RAR in low-dose X-ray excited photodynamic therapy (X-PDT)

[0065] The effects of X-PDT on three malignant tumor cell lines (U87 MG, GL261, and C6) under different irradiation doses are shown in Figure 6. (AB) U87 MG and GL261 cells were co-cultured with RAR (0-200 μg / mL) and irradiated with X-rays at doses 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 at doses 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.

[0066] After co-incubation of U87 MG cells with RAR and subsequent irradiation with 2 Gy X-rays, compared to the PBS+X-ray group alone, as shown in Figure 7(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. As shown in Figure 7(B), after co-incubation of C6 cells with RAR and subsequent irradiation with 2 Gy X-rays, compared to the X-ray group alone, the X-PDT group showed significantly stronger singlet oxygen (green) fluorescence signal, indicating that the X-PDT effect can generate more singlet oxygen within tumor cells, thus killing tumor cells. 1 O2 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.

[0067] 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 shown in Figure 8. In X-PDT-treated U87 MG tumor cells, green fluorescence was enhanced, and γH2AX (green) expression was significant. Compared with the X-ray-only group, which damaged tumor cell DNA, X-PDT had a better effect on damaging tumor cell DNA. The results on C6 cells are shown in Figure 9. Similarly, the X-PDT group showed enhanced green fluorescence and significant γH2AX (green) expression in tumor cells, indicating that X-PDT had a better effect on damaging tumor cell DNA compared with the X-ray-only group.

[0068] The effects of X-PDT on the long-term survival and proliferation of tumor cells were further evaluated using a clonogenic assay. The results are shown in Figures 10 and 11. The (AB) clonogenic assay measured the cell proliferation capacity of U87 MG cells and C6 cells 10 days after radiotherapy (RT (X-ray) and X-PDT treatment, 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 determined 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 weaker proliferation capacity, indicating that X-PDT more effectively inhibited the tumor cell colony formation ability.

[0069] Brain tumor targeted experiment

[0070] Figure 12(A) shows in vivo fluorescence imaging at different time points after tail vein injection of RAR in a mouse model of orthotopic glioma (U87 MG-luc). The fluorescence signal in the brain region is significantly stronger than in other areas, indicating that RAR can effectively target and accumulate in the brain tumor. Figure 12(B) shows the quantitative analysis of the fluorescence signal intensity of the DiR signal in the mouse brain region (ROI) at different time points. The quantitative results of the average fluorescence signal at different time points show that the tumor aggregation peak is reached 2 hours after tail vein injection of RAR, and then slowly decreases. Figure 12(C) shows the results of ex vivo fluorescence imaging of various organs and the brain tumor 4 hours after tail vein injection of RAR. Compared with normal brain tissue, the fluorescence signal at the tumor location in the brain tissue is significantly stronger, indicating that RAR can effectively accumulate at the brain tumor lesion. The ability of RAR to target and accumulate in the tumor was observed at the in vivo macroscopic level.

[0071] To further visualize the real-time delivery of RAR to brain tumors at the microscopic level, a cranial window was constructed in a model mouse brain for in vivo microscopic imaging of the brain tumor region. As shown in Figure 13, 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 tumor cells. This demonstrates the excellent tumor-targeting and aggregation ability of RAR.

[0072] To further visualize the targeted aggregation process of RAR in brain tumor tissue from a 3D perspective, this disclosure 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.

[0073] Figure 14 shows the results of 3D light-screen microscopy of the whole brain of mice after transparency treatment. (A) The process of brain tissue transparency. (B) Whole brain microscopy of mice with orthotopic glioma shows the targeted distribution of RAR in the tumor. The RAR (red) signal and the tumor GFP (green) have good overlap, indicating that RAR is targeted and aggregated inside the brain tumor tissue and distributed throughout the tumor mass.

[0074] Figure 15 shows the results of whole-brain 3D magnetic resonance imaging and light-sheet microscopy of rats. a. T2-weighted MRI images of rat orthotopic gliomas at different levels and three-dimensional 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 tumors; de. Photographs of brain tissue before and after transparency; 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 brain tissue transparency. Gray: brain tissue, red: RAR, green: tumor (C6-GFP); g. Two-dimensional planar image of three-dimensional panoramic fluorescence imaging; h. Quantification of fluorescence intensity in normal tissue and brain tumors. All statistics are expressed as mean ± SD. *The fluorescence intensity in the tumor is significantly different from that in normal tissue. The independent samples t-test was used to obtain statistical significance. The RAR (red) signal and the tumor GFP (green) signal in the figure have good overlap, indicating that RAR is targeted and aggregated inside the brain tumor tissue and distributed throughout the tumor mass.

[0075] Mouse model treatment experiment

[0076] Figures 16 and 17 show the results of bioluminescence imaging and BLI monitoring in animals after 2 Gy low-dose radiotherapy. As can be seen, in the model mice of the low-dose X-ray photodynamic therapy (X-PDT) group, the ROI luminescence signal value in the brain continuously decreased compared to the control group after day 20 of treatment, while the signal in the control group gradually increased. 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 tumor development.

[0077] To more effectively inhibit gliomas in situ and reduce toxic side effects on normal organisms under low-dose radiotherapy conditions, this disclosure 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 shows a schematic diagram of the application of a clinical linear accelerator in glioma cell evaluation, specifically including:

[0078] (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.

[0079] (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.

[0080] Furthermore, this disclosure 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 shows a flowchart of the X-PDT evaluation process using a clinical linear accelerator on rats with orthotopic gliomas, specifically including:

[0081] (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.

[0082] (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.

[0083] (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.

[0084] (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.

[0085] (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.

[0086] Figure 20 shows the MRI imaging monitoring results of rats with orthotopic gliomas at the beginning (day 11) and end of different treatment groups. T2-weighted MRI imaging (axial plane) was performed 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 rat brain tumor at different time points are shown. Compared with the control group, the X-PDT group showed that the brain tumors in the rat models continuously decreased in size after treatment, even disappearing at the endpoint. Figure 21 shows the quantitative results of brain tumor volume in different groups of rats after treatment. The tumor volume results of the PBS, RAR, X-ray, and X-PDT groups showed that the tumor volume of rats in the PBS, RAR, and X-ray groups gradually increased, while the tumor volume of the X-PDT group continuously decreased.

[0087] Figures 22 and 23 show the bioluminescence imaging and signal quantification results before and after treatment. The images and quantitative results show that after treatment, the signal at the brain tumor location in the three control rat models (PBS, RAR, and X-ray) gradually increased, 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.

[0088] As shown in Figure 24, the original tumors in the X-PDT group were eliminated, and the nuclear density of cells at the lesion site was low, indicating that the tumors were effectively suppressed and eliminated. The survival results of the models in different treatment groups are shown in Figure 25. The low-dose X-ray group alone did not effectively improve the survival of the rat model, while the survival of the rat model in the low-dose X-PDT group was greatly increased.

[0089] Rat orthotopic glioma models in different treatment groups were stained with Ki-67 and PCNA in tumor tissue at the monitoring endpoint. As shown in Figure 26, the quantitative fluorescence intensity of Ki-67 in the immunofluorescence images showed that the original tumor in the X-PDT group was eliminated, and the quantitative fluorescence intensity of Ki-67 and the quantitative value of PCNA were significantly reduced, indicating that the X-PDT group can effectively inhibit the proliferation and invasiveness of tumors.

[0090] The dark toxicity of different concentrations of RAR was detected using a CCK-8 assay kit. Under an X-ray irradiation dose of 2 Gy and with the irradiator parameters set to 160 kV, the results of the dark toxicity of different concentrations of RAR on patient-derived glioma cells and the effect of low-dose radiotherapy are shown in Figure 27. In comparison, RAR has good dark toxicity and exhibits better tumor cell killing ability under X-ray irradiation.

[0091] 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 singlet oxygen production in patient-derived glioma cells are shown in Figure 28. Due to the inability of the SOSG singlet oxygen probe to enter cells, a small amount of green fluorescence signal was observed in the control group. In the X-PDT group, RAR cells, under X-ray irradiation, produced a large amount of singlet oxygen, which bound to the SOSG singlet oxygen probe, resulting in a significant green fluorescence signal.

[0092] DNA damage was detected using a DNA damage detection kit (green fluorescence) after different treatments with PBS, RAR, X-ray, and X-PDT, and quantitative analysis was performed. Figure 29 shows the DNA damage detection results of patient-derived glioma cells after different treatments. DNA double-strand disruption 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.

[0093] The effects of RT and X-PDT on inhibiting the proliferation of patient-derived glioma cells were compared using a cell colony formation assay, and quantitative analysis was performed. As shown in Figure 30, X-PDT significantly inhibited the proliferation of patient-derived glioma cells compared to conventional RT.

[0094] Figure 31 shows the intratumoral enrichment results of RAR (100 μg / mL) after tail vein injection in mice bearing gliomas derived from patients. (A) -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.

[0095] PDX orthotopic glioma animal model treatment efficacy test

[0096] To evaluate the therapeutic effect of PDX or PDX orthotopic glioma animal models, this disclosure 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.

[0097] The treatment results of the patient-derived orthotopic glioma animal model are shown in Figure 32. (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; (E) Survival curves of mice in each group during treatment. The single-treatment and double-treatment groups, which received tail vein injection of RAR combined with X-ray, showed significant tumor suppression effects. Both treatment groups effectively inhibited tumor growth, and the body weight of the mice 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 Balb / c nude-mice PDX glioma animal model, it can effectively inhibit tumor growth and prolong median survival.

[0098] Figure 33 shows the magnetic resonance imaging (MRI) images and corresponding hematologic resection (HE) results of the treatment of a PDX orthotopic glioma animal model derived from patients. (A) The treatment effect of the PDX orthotopic glioma animal model was verified by 9.4T MRI and H&E staining. (B) and (C) The treatment effect of mice after treatment was verified by H&E staining, 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.

[0099] 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.

[0100] 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 applying 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-rays is kV and MV, the cumulative dose is 2-2.5Gy, and the specific type of noble metal cluster is selected according to the different requirements of fluorescence emission wavelength.

2. The application method according to claim 1, characterized in that, The tumors specifically include gliomas, medulloblastomas, meningiomas, vestibular schwannomas, and lymphomas originating in the central nervous system.

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

4. The application method according to claim 3, characterized in that, The noble metal cluster is preferably gold nanoclusters (AuNCs).

5. The application method according to claim 1, characterized in that, The noble metal cluster is used in this application by combining it 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.

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

7. A device for evaluating cells using clinical radiotherapy equipment, characterized in that, The device comprises an accelerator treatment bed, a cell culture dish, an equivalent solid water, and a tissue compensator. The device is used to induce an X-ray-induced photodynamic therapy (X-PDT) response against brain tumor cells under a clinical linear accelerator. Specifically, this includes: 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 administering radiation according to the method described in any one of claims 1-5, 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 across the cell region, and the radiation effect is evaluated.

8. A system for evaluating the treatment process and efficacy of orthotopic brain tumors in rats using clinical radiotherapy equipment, characterized in that, The system includes a CT unit, an MRI unit, an accelerator processing bed, and a clinical linear accelerator. The system is specifically evaluated as follows: rigid registration and fusion simulation of 3D 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 then a CBCT scan of the entire skeleton is performed to ensure strict alignment with the localization CT. Radiation is applied according to any one of claims 1-5 to evaluate the therapeutic effect.

9. The system for the treatment process and treatment effect according to claim 8, characterized in that, The clinical radiotherapy equipment is configured to deliver a cumulative dose of 2-2.5 Gy and to utilize 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.

10. A system for evaluating the efficacy of low-dose radiotherapy in animal models of CDX or PDX orthotopic gliomas, characterized in that, The system includes 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 of any one of claims 1-5, performing bioluminescence imaging and quantitative analysis before and after the treatment, and verifying the treatment effect by MRI and HE staining.

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