A degradable molecular probe for tumor NIR-Ⅱ fluorescence imaging and radiotherapy sensitization-immunotherapy and its application

Abstract

The present invention discloses a degradable molecular probe for tumor NIR-II fluorescence imaging and radiotherapy sensitization-immunotherapy. The probe is a charged IHDC. The probe is composed of an ICG-Hf ligand formed by the combination of indocyanine green (ICG) and hafnium tetrachloride (HfCl4), which is loaded onto a carrier material (DTSPO) containing tetrasulfide bonds to form IHD nanoparticles with a positive surface potential. The probe is then modified with a slightly acidic degradation layer (CB) on the outer layer through a cross-linking reaction between a three-arm phenylboronic acid motif and a three-arm catechol motif to synthesize an IHDC with a negative surface potential. The degradable molecular probe of the present invention can interact with the slightly acidic environment of the tumor site and overexpressed glutathione (GSH), causing degradation of the carrier material and achieving effective release of the ligand at the tumor site.

Description

Technical Field

[0001] The present invention relates to the technical field of charged molecular probes, and in particular to a degradable molecular probe for tumor NIR-II fluorescence imaging and radiotherapy sensitization-immunotherapy combination therapy; in particular, to a nanoparticle capable of achieving tumor microenvironment-responsive charge reversal, and its preparation method and application. Background Art

[0002] Breast cancer is a highly prevalent malignant tumor among women, and its incidence rate has been increasing year by year, posing a serious threat to women's health.

[0003] Surgery, chemotherapy, and radiotherapy are known as the "three horses" of cancer treatment. Among these, surgery is more traumatic and risky, while chemotherapy has poor specificity and significant systemic toxic side effects. Compared to these, radiotherapy has a wider range of indications, greater safety, and higher accuracy. Furthermore, radiotherapy can induce immunogenic cell death (ICD) of tumor cells, activating the immune response. However, radiotherapy also has its limitations, such as limited treatment range and damage to surrounding normal tissues. Therefore, precise delineation of the radiotherapy target is particularly important for achieving effective tumor treatment and protecting normal tissues.

[0004] Near-infrared fluorescence imaging technology enables real-time in vivo visualization of tumor sites with high sensitivity. Compared to near-infrared range I (NIR-I, 700-900nm) fluorescence, near-infrared range II (NIR-II, 1000-1700nm) offers a unique "tissue transparency window," resulting in lower scattering and absorption in tissues. This further improves imaging spatial resolution and tissue penetration depth, "illuminating" tumor sites and providing a reliable basis for delineating treatment targets.

[0005] In addition, radiotherapy resistance and decreased sensitivity of tumor cells to radiation after multiple radiotherapy are also important factors restricting radiotherapy. Improving the radiotherapy sensitivity of breast cancer is an important means to improve the efficacy of breast cancer radiotherapy. Studies have found that elements with high atomic numbers have enhanced high-energy radiation energy deposition and can produce significant radiotherapy sensitization effects. Most current research focuses on lanthanides. Hafnium (Hf) has a higher atomic number than lanthanides, and its oxide hafnium dioxide (HfO2) nanoparticles have entered clinical trials, showing good biosafety and excellent radiotherapy sensitization properties. Metal ions themselves can also exhibit radiation deposition and radiotherapy sensitization properties that are no less than those of oxides. Therefore, the use of Hf ions as radiotherapy sensitizers is considered safe and reliable. Summary of the Invention

[0006] To overcome the shortcomings and deficiencies of the aforementioned prior art, the primary objective of this invention is to provide a diagnostic and therapeutic molecular probe that can be effectively enriched at the tumor site. This probe utilizes a charge reversal strategy to efficiently accumulate at the tumor site. It is then degraded by tumor GSH, releasing ICG-Hf. This probe, combined with a PD-1 antibody, can be used for fluorescence imaging and radiosensitization-immunotherapy. Ultimately, this nanoparticle could be used to establish a comprehensive diagnostic and therapeutic system integrating "therapeutic target delineation, localized radiotherapy immune triggering, and systemic immune activation."

[0007] The specific technical solutions are as follows:

[0008] A biodegradable molecular probe for tumor NIR-II fluorescence imaging and radiosensitization-immunotherapy combination therapy, the probe being ICG-Hf@DTSPO@CB (IHDC). Indocyanine green (ICG) and hafnium tetrachloride (HfCl4) are combined to form an ICG-Hf complex, which is then loaded onto a carrier to form nanoparticles ICG-Hf@DTSPO (Dendritic tetra-sulfde-bridgedporousorganosilica, DTSPO). The final nanoparticles, ICG-Hf@DTSPO@CB, are then modified with a slightly acidic degradation layer (CB) on the outer layer via a cross-linking reaction between a three-arm phenylboronic acid moiety and a three-arm catechol moiety.

[0009] The carrier used to load the ligand ICG-Hf is dendritic mesoporous silica doped with tetrasulfide bonds. The tetrasulfide bonds in the carrier can react with glutathione GSH overexpressed in the tumor site to produce hydrogen sulfide H2S gas, causing degradation of the carrier material and achieving effective release of the ligand in the tumor site.

[0010] Furthermore, the ICG-Hf@DTSPO@CB probe is negatively charged.

[0011] Furthermore, the preparation method of the ICG-Hf@DTSPO@CB probe comprises the following steps:

[0012] S1. Synthesis of DTSPO, a carrier material containing tetrasulfide bonds;

[0013] S2, Hf loading; Hf ions in hafnium chloride HfCl4 can coordinate with the sulfur atoms of the tetrasulfide bonds in DTSPO and thus be loaded on DTSPO to form Hf@DTSPO;

[0014] S3, ICG loading: ICG is added, and ICG can coordinate with the Hf atoms of Hf@DTSPO in S2 to load ICG, forming spherical solid nanoparticles ICG-Hf@DTSPO;

[0015] S4. CB modification: Through the cross-linking reaction of the three-arm phenylboronic acid unit TAB and the three-arm catechol unit TAC, the slightly acidic degradation layer CB was successfully modified on the outer layer of ICG-Hf@DTSPO to synthesize the degradable molecular probe ICG-Hf@DTSPO@CB with a negative charge on the outer layer.

[0016] Furthermore, step S2 includes:

[0017] S21, weigh 6.4mg HfCl4 and 14.2mg DTSPO, dissolve them in 2mL and 6mL anhydrous ethanol respectively, and fully disperse them by ultrasonication.

[0018] S22, place the anhydrous ethanol containing DTSPO in S21 in a magnetic stirring device, stir at a constant speed, use a pipette to draw the above anhydrous ethanol solution containing HfCl4 and quickly add it into it, then continue to stir the whole system for 8-12 hours

[0019] Furthermore, step S3 includes:

[0020] S31. Weigh 7.8 mg of ICG, dissolve it in 8 mL of anhydrous ethanol, and ultrasonically disperse it for 2 minutes.

[0021] S32, use a pipette to draw the anhydrous ethanol containing ICG in S31 and quickly add it to the reaction system of S22, and continue stirring for 1 hour

[0022] S33, centrifuge the reaction solution in S23 at room temperature at 8000 rpm for 7 minutes, discard the supernatant to obtain a precipitate, and redisperse the precipitate in 10 mL of anhydrous ethanol, sonicate for 10 seconds to disperse, centrifuge at room temperature at 8000 rpm for 7 minutes, and discard the supernatant to obtain ICG-Hf@DTSPO nanoparticles.

[0023] Furthermore, step S4 includes:

[0024] S41, weigh 5.0 mg of three-arm catechol moiety (TAC) and 5.0 mg of three-arm phenylboronic acid moiety (TAB), dissolve them in 2.5 mL of methanol and ethanol respectively, and ultrasonically disperse them.

[0025] S42, add the methanol solution of TAC to the S33 system, ultrasonically disperse for 30 seconds, and stir on a magnetic stirrer for 10 minutes. Add the ethanol solution of TAB dropwise to the stirring system using a pipette, and continue stirring for 30 minutes.

[0026] S43, centrifuge the reaction solution at 8000 rpm for 7 min at room temperature, discard the supernatant, and obtain a precipitate.

[0027] S44. Redisperse the precipitate in 10 mL of anhydrous ethanol, sonicate for 10 seconds to disperse, centrifuge at 8000 rpm for 7 minutes at room temperature, and discard the supernatant to obtain a precipitate. Repeat this operation three times to obtain the final degradable molecular probe IHDC with a negatively charged outer layer.

[0028] Furthermore, the degradable molecular probe is used for defining surgical margins of breast cancer and enhancing radiotherapy sensitization, and degrades the carrier material by interacting with the slightly acidic environment of the tumor site and overexpressed glutathione (GSH).

[0029] Furthermore, the slightly acidic environment responsively degrades CB, and the degradation of CB leads to charge reversal from negative to positive.

[0030] Furthermore, the overexpressed glutathione responds to degrade DTSPO.

[0031] The working principle of this invention is as follows: Indocyanine green (ICG) is used as a fluorescent dye, and hafnium tetrachloride (HfCl4) is used as a radiosensitizer to form an ICG-Hf(IV) complex. This complex is loaded onto a tetrasulfide-doped dendritic mesoporous silica (DTSPO) carrier to form solid spherical nanoparticles ICG-Hf@DTSPO (IHD). The tetrasulfide bonds in the carrier react with glutathione (GSH) overexpressed in the tumor site to produce hydrogen sulfide (H2S) gas, which causes the carrier material to degrade and effectively release the complex at the tumor site. The resulting nanoparticles, ICG-Hf@DTSPO(IHD), were successfully modified with a slightly acidic degradation layer (CB) on the outer layer through a cross-linking reaction between three-armed boronic acid (TAB) and three-armed catechol (TAC) moieties to form ICG-Hf@DTSPO@CB(IHDC). The CB modification imparts a negative charge to the outer layer of the molecular probe, which repel macrophages and endothelial cells, which also carry a negative charge on their surfaces. This reduces the molecular probe's phagocytosis and uptake in the bloodstream, maximizing its delivery to tumor sites. Upon entering the tumor site via the enhanced permeability and retention (EPR) effect, the molecular probe degrades in the slightly acidic environment, releasing the nanoparticles. Due to their positive charge, the nanoparticles attract the negatively charged tumor cells, promoting their efficient uptake.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The present invention utilizes a charge reversal strategy to prepare a degradable diagnostic and therapeutic molecular probe that can be efficiently enriched in tumor sites;

[0034] (2) NIR-II fluorescence imaging can accurately delineate the tumor target area. IHDC combined with radiotherapy can produce a radiosensitization effect, allowing for more effective tumor cell killing with a smaller radiation dose within a more precise range, thereby reducing damage to surrounding normal tissues.

[0035] (3) Compared with radiotherapy alone, IHDC can more effectively promote cell immunogenic death, and combined with checkpoint therapy can produce systemic anti-tumor responses and enhance abscopal effects and immune memory effects;

[0036] (4) The application of IHDC has unique advantages in reversing the poor effect of breast cancer radiotherapy and preventing recurrence, and has important clinical significance for improving the quality of life and prognosis of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is the probe pattern diagram and characterization, where:

[0038] A is the coordination pattern diagram of ICG and Hf(IV);

[0039] B is the IHDC synthesis pattern diagram;

[0040] C is DTSPO and IHD electron microscopy and mapping element analysis;

[0041] D is the electron microscope image of IHDC;

[0042] E is the particle size distribution diagram of DTSPO, IHD, and IHDC;

[0043] F and G are the average particle size and potential of DTSPO, IHD, and IHDC;

[0044] Figure 2 The probe consumes GSH to produce H2S gas at the material and cellular levels, where:

[0045] A is the cytotoxicity of different concentrations of IHD on 4T1 cells;

[0046] B is the electron micrograph of DTSPO and DPO without tetrasulfide bond in GSH solution at different reaction times;

[0047] C is the H2S gas produced by the reaction of DTSPO and GSH solution detected by lead acetate test paper;

[0048] D is the detection of H2S gas produced after co-incubation of cells with different concentrations of IHD;

[0049] Figure 3In vitro and in vivo fluorescence imaging and biodistribution of probes, including:

[0050] A is the white light image and fluorescence imaging of ICG, ICG-Hf, IHD, and IHDC;

[0051] B is the fluorescence imaging over time of IHDC intratumoral injection on the right side and subcutaneous injection on the left side;

[0052] C is the fluorescence imaging of ICG, ICG-Hf, IHD, and IHDC injected into subcutaneous tumor-transplanted mice over time;

[0053] D and E are fluorescence imaging and quantitative analysis of fluorescence intensity of the heart, liver, spleen, lung, kidney, and tumor of mice with subcutaneous tumor transplantation at different times after IHDC was injected into the tail vein;

[0054] Figure 4 Identify minimal residual disease and primary tumor lesions for IHDC in vivo, including:

[0055] A: IHDC was injected into the subcutaneous tumor-transplanted mouse model via tail vein, and the tumor was segmented and imaged in vivo and ex vivo;

[0056] B: IHDCs were injected into a mouse model with multiple microtumors through the tail vein, and then imaged in vivo and in vitro;

[0057] Figure 5 The radiosensitization effect and underlying mechanism of IHD at the cellular level include:

[0058] A, B, and C are cytotoxicity, apoptosis, and live-death staining; D is DNA damage γ-H2AX staining;

[0059] E and F show the staining of total ROS and different types of ROS;

[0060] Figure 6 To enhance radiosensitization of IHD, the drug promotes the maturation of ICD and DC at the cellular level, including:

[0061] A and B are immunofluorescence staining of immunogenic death markers CRT and HMGB1; C is in vitro DC cell maturation detection

[0062] Figure 7 It is a unilateral tumor suppression experiment and immune index detection;

[0063] A and B are the curves of tumor growth and body weight changes during treatment; C is the weight of the dissected tumor after treatment.

[0064] D and E are the detection of DC cell maturation markers CD80 and CD86 expression in tumor-draining lymph nodes;

[0065] F shows H&E staining, Ki67 immunohistochemistry, CRT and HMGB1 immunofluorescence staining, and Tunel staining of tumor tissue after treatment;

[0066] Figure 8 It is a bilateral tumor inhibition experiment and immune index detection, including:

[0067] A, B, C, including: orthotopic and distal tumor growth curves and body weight change curves of bilateral tumor mouse model;

[0068] D and E are middle: after the monitoring, the in situ and distal tumors were dissected and weighed;

[0069] F is the detection of CD4 and CD8 positive cells in the spleen;

[0070] G is Granzyme B immunohistochemical staining in the distal tumor;

[0071] H is the ELISA detection of TNF-α, INF-γ, IL-6, and IL-12 cytokines in distal tumor tissue;

[0072] Figure 9 A lung metastasis model was established by surgically removing the primary lesion and injecting tumor cells into the tail vein after treatment of a subcutaneous tumor transplant mouse model. White light images of the lungs, H&E staining, and detection of immune memory cells in the spleen were performed.

[0073] Figure 10 IHDC was injected into healthy mice via tail vein for in vivo biosafety testing at different time points, including:

[0074] A is H&E staining of major organs;

[0075] B and C are blood routine and biochemical tests. DETAILED DESCRIPTION

[0076] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with specific embodiments and with reference to the accompanying drawings. It should be understood that these descriptions are merely illustrative and are not intended to limit the scope of the present invention. In addition, in the following invention, descriptions of well-known structures and technologies are omitted to avoid unnecessary confusion of the concepts of the present invention.

[0077] Example 1

[0078] A degradable molecular probe for tumor NIR-Ⅱ fluorescence imaging and radiosensitization-immunotherapy combined therapy, wherein the probe is ICG-Hf@DTSPO@CB (IHDC), such as Figure 1 As shown in A, indocyanine green ICG can combine with hafnium tetrachloride HfCl4 to form ICG-Hf coordination complex; the specific synthesis process is as follows Figure 1As shown in B, Hf and ICG were first loaded on dendritic mesoporous silica doped with tetrasulfide bonds (DTSPO) to form nanoparticles ICG-Hf@DTSPO (IHD), and then the outer layer was modified with a slightly acidic degradation layer CB through a cross-linking reaction of a three-arm phenylboronic acid unit (TAB) and a three-arm catechol unit (TAC) to synthesize IHDC. Figure 1 The coordination pattern of ICG and Hf(IV) in A shows that ICG is connected to Hf through the sulfonate group. 4+ To coordinate; Figure 1 B shows that the synthesis of IHDC is to first synthesize the carrier structure DTSPO, then load the ICG-Hf ligand on it, and finally modify the CB on the outer layer to finally synthesize IHDC; DTSPO and IHD were analyzed by electron microscopy and mapping elements. The results are as follows Figure 1 As shown in C, the pure DTSPO carrier presents a branched structure, in which there is an S signal due to the presence of tetrasulfide bonds, and there is no Hf 4+ , Hf has almost no signal; and due to the successful loading of ICG-Hf, the spherical structure of IHD is almost solid and has almost no branches. ICG contains S element, the S signal is enhanced, and the Hf signal is generated from nothing. Then the ICG-Hf@DTSPO@CB(IHDC) was analyzed by electron microscopy. Figure 1 The electron microscopy image of D shows that compared with IHD, due to the modification of CB, the outside of IHDC is wrapped in a wreath-like structure, blocking the pores and preventing the leakage of the ligand.

[0079] For example Figure 1 As shown in E, the particle size distribution of DTSPO, IHD, and IHDC is roughly normal and relatively uniform; F, G) The average particle size distribution of DTSPO, IHD, and IHDC is between 200-250 nm, and the particle size increases due to the loading of the contents and the modification of the surface.

[0080] Example 2

[0081] A method for preparing a degradable molecular probe for tumor NIR-II fluorescence imaging and radiotherapy sensitization-immunotherapy combined therapy, comprising the following steps:

[0082] S1. Synthesis of DTSPO, a carrier material containing tetrasulfide bonds;

[0083] The step S1 comprises:

[0084] S11) Dissolve 122 μl of triethylamine (TEA) in 50 mL of ddH2O and stir at 80°C for 0.5 h.

[0085] S12) Add 760 mg of surfactant CTAB and 300 mg of sodium salicylate NaSal to the above solution and continue stirring at 60°C for 2 h

[0086] S13) A mixture of 4 mL of tetraethoxysilane (TEOS) and 3.5 mL of bis-(γ-triethoxysilylpropyl) tetrasulfide (BTES) was added dropwise to the reaction solution, and the mixture was stirred at 80° C. for 12 h.

[0087] S14) Centrifuge at 10000 rpm for 8 min at room temperature, remove the supernatant and collect the precipitate

[0088] S15) The precipitate was redispersed in 10 mL of anhydrous ethanol, sonicated for 1 minute to fully disperse, and centrifuged at 10,000 rpm for 8 minutes at room temperature. The supernatant was removed and the precipitate was collected. This process was repeated three times.

[0089] S16) The product was dispersed in a 2M methanolic hydrochloric acid solution and refluxed at 75° C. with stirring for 6 h to remove excess CTAB surfactant. This process was repeated three times.

[0090] S17) Centrifuge at 10,000 rpm for 8 minutes at room temperature, remove the supernatant, collect the precipitate, add 10 mL of anhydrous ethanol, sonicate for 1 minute to fully disperse, centrifuge at 10,000 rpm for 8 minutes at room temperature, remove the supernatant, and collect the precipitate to obtain DTSPO.

[0091] S2. Loading Hf to form Hf@DTSPO nanoparticles; Hf is loaded on the carrier prepared in S1 to form Hf@DTSPO nanoparticles, wherein the carrier is a dendritic mesoporous silica DTSPO doped with tetrasulfide bonds.

[0092] The step S2 comprises:

[0093] S21, weigh 6.4mg HfCl4 and 14.2mg DTSPO, dissolve them in 2mL and 6mL anhydrous ethanol respectively, and fully disperse them by ultrasonication.

[0094] S22, place the anhydrous ethanol containing DTSPO in S21 in a magnetic stirring device, stir at a constant speed, use a pipette to draw the above anhydrous ethanol solution containing HfCl4 and quickly add it into it, then continue to stir the whole system for 8-12 hours

[0095] S3, loading ICG to form ICG-Hf@DTSPO nanoparticles; ICG is loaded on the Hf@DTSPO prepared in S2 to form solid spherical nanoparticles ICG-Hf@DTSPO.

[0096] The step S3 comprises:

[0097] S31. Weigh 7.8 mg of ICG, dissolve it in 8 mL of anhydrous ethanol, and ultrasonically disperse it for 2 minutes.

[0098] S32, use a pipette to draw the anhydrous ethanol containing ICG in S31 and quickly add it to the reaction system of S22, and continue stirring for 1 hour

[0099] S33, centrifuge the reaction solution in S23 at room temperature at 8000 rpm for 7 minutes, discard the supernatant to obtain a precipitate, and redisperse the precipitate in 10 mL of anhydrous ethanol, sonicate for 10 seconds to disperse, centrifuge at room temperature at 8000 rpm for 7 minutes, and discard the supernatant to obtain ICG-Hf@DTSPO nanoparticles.

[0100] S4. CB modified molecular probe; through the cross-linking reaction of three-arm phenylboronic acid unit TAB and three-arm catechol unit TAC, the slightly acidic degradation layer CB was successfully modified on the outer layer of ICG-Hf@DTSPO to synthesize a degradable molecular probe ICG-Hf@DTSPO@CB (IHDC) with a negative charge on the outer layer.

[0101] The step S4 comprises:

[0102] S41, weigh 5.0 mg of three-arm catechol moiety (TAC) and 5.0 mg of three-arm phenylboronic acid moiety (TAB), dissolve them in 2.5 mL of methanol and 2.5 mL of ethanol respectively, and ultrasonically disperse them.

[0103] S42, add the methanol solution of TAC to the system of step S33, ultrasonically disperse for 30 seconds, place on a magnetic stirrer and stir for 10 minutes; then add the ethanol solution of TAB dropwise to the stirring system with a pipette, and continue stirring for 30 minutes

[0104] S43, centrifuge the reaction solution at 8000 rpm for 7 min at room temperature, discard the supernatant, and obtain a precipitate.

[0105] S44. Redisperse the precipitate in 10 mL of anhydrous ethanol, sonicate for 10 seconds to disperse, centrifuge at 8000 rpm for 7 minutes at room temperature, and discard the supernatant to obtain a precipitate. Repeat this operation three times to obtain the final degradable molecular probe IHDC with a negatively charged outer layer.

[0106] Example 3: Application of IHDC in Breast Cancer Tumor Imaging (I) Intratumoral Injection-Responsive Fluorescence Imaging

[0107] 4T1 tumor cells were implanted in the right hind limb of Balb / c mice to establish a subcutaneous transplant tumor mouse model. Equal amounts of IHDC molecular probes were injected into the right tumor and subcutaneously on the left side. Continuous imaging was performed over time using an NIR-Ⅱ imager to monitor changes in fluorescence intensity and determine the responsive fluorescence recovery effect of the IHDC molecular probe in the tumor. (II) Identification of microresidual lesions and primary lesion imaging

[0108] 1. Imaging for identification of minimal residual disease

[0109] A unilateral subcutaneous xenograft mouse model with 4T1 tumor cells was established in the right hind limb of Balb / c mice. The IHDC molecular probe was injected via the tail vein, and continuous fluorescence imaging was performed over time to determine optimal enrichment and imaging time points. Another group of subcutaneous xenograft mouse models were also established and injected with the IHDC molecular probe via the tail vein. Fluorescence imaging was performed, and the tumors were sectioned and gradually resected. White light and fluorescence signals were collected with each tumor section removed. The minimum residual lesion size that could be detected by fluorescence imaging was gradually determined. Measurements were made with a ruler and both white light and fluorescence imaging were performed.

[0110] The results are as follows Figure 3 As shown in the figure, A shows ICG, ICG-Hf, IHD, and IHDC. The white light image shows that all four show the original green hue of ICG. Since the outer surface of IHDC is modified with CB, CB appears orange-yellow, and IHDC finally appears yellow-green. Since Hf and ICG coordinate to quench the fluorescence of ICG, under the same parameters, ICG-Hf, IHD, and IHDC can hardly see fluorescence compared to ICG. B shows that IHDC was injected into the right tumor and the left subcutaneous tissue. Since CB degrades in the slightly acidic environment of the tumor site, ICG-Hf ligands are released, and proteins bind to them, so that the ICG fluorescence is restored, thereby lighting up the tumor site. However, since there is no tumor microenvironment in the subcutaneous tissue, the fluorescence activation and recovery are The recovery rate is much lower than that of the tumor site; C shows that ICG, ICG-Hf, IHD, and IHDC were injected into mice with subcutaneous tumors through the tail vein. It can be seen that IHDC can be efficiently enriched in the tumor site for a long time compared with ICG, ICG-Hf, and IHD over time, and the fluorescence of the tumor site is clearly visible until 144 hours; D and E show that IHDC was injected into mice with subcutaneous tumors through the tail vein, and the heart, liver, spleen, lung, kidney, and tumor were dissected at 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, and 144 hours respectively for fluorescence imaging and a fluorescence intensity change bar graph was drawn according to the fluorescence intensity. It can be seen that the probe is mainly metabolized by the liver and spleen, and the tumor site can maintain a high fluorescence intensity for a long time.

[0111] 2. Imaging to Identify Tiny Primary Tumors

[0112] A model of multiple microtumors was established by injecting a small number of 4T1-luc cells at multiple sites on the hind limbs and back of Balb / c mice. Intraperitoneal injection of a bioluminescent substrate was performed for bioluminescent imaging to determine the location and number of microtumors. After tail vein injection of IHDCs, fluorescence imaging was performed. Microtumors were then excised and measured in vitro under fluorescence imaging. The excised microtumors were fixed, dehydrated, paraffin-embedded, sectioned, and stained with H&E to investigate the ability of IHDCs to recognize microscopic residual lesions and primary tumor lesions in vivo.

[0113] The results are as follows Figure 4 As shown in Figure 1, A shows that IHDC was injected into a subcutaneous tumor-transplanted mouse model through the tail vein. After gradual resection of the tumor, it was found that residual tumor lesions as small as 1 mm could be identified; B shows that IHDC was injected into a multiple microtumor mouse model through the tail vein, which can effectively identify microtumors as small as 1 mm.

[0114] Example 4: IHDC for breast cancer radiosensitization and combined immunotherapy

[0115] like Figure 5 The figure shows the radiosensitization effect and internal mechanism of IHD at the cellular level: 5A, 5B, and 5C show that IHD has a significant radiosensitization effect compared with radiotherapy alone through cytotoxicity, apoptosis, and live-dead staining experiments; Figure 5 γ-H2AX staining of D showed that radiation could cause DNA damage and generate a large amount of ROS, among which ·O2 - and OH ( Figure 5 E, F).

[0116] Then by Figure 6 This suggests that the radiosensitizing effect of IHD can promote tumor cell ICD and DC maturation at the cellular level. Panels A and B show increased CRT externalization, an immunogenic death marker, and increased HMGB1 migration, indicating that compared with the radiotherapy alone group, IHD combined with radiotherapy resulted in more significant ICD in tumor cells. Panel C shows that bone marrow cells extracted from healthy mice were induced to differentiate using cytokines to generate immature DCs. After 48 hours of incubation with the corresponding treated tumor cell supernatant, DC maturation was assessed. This indicates that the combined effect of IHD and radiotherapy significantly promoted DC maturation compared with the radiotherapy alone group. Based on this study, IHDCs can be used to sensitize breast cancer to radiotherapy.

[0117] (1) IHDC for breast cancer radiosensitization

[0118] Tumor inhibition experiment: A mouse model with unilateral subcutaneous tumor xenografts was established and randomly divided into four groups: PBS, IHDC, RT, and IHDC+RT. The IHDC molecular probe was injected into the tail vein. For the radiotherapy-related group, the tumor site was irradiated after probe injection. Tumor size and body weight were monitored every two days for a total of 18 days. After the final monitoring period, the mice were sacrificed, and tumor tissues were dissected, photographed, and weighed.

[0119] Tissue specimen and immune index analysis: The ipsilateral inguinal lymph nodes of the tumor in the four groups of mice treated with different methods were dissected to detect DC cell maturity. The dissected tumor tissues were fixed, dehydrated, paraffin-embedded, and cut into 4 μm tissue sections for H&E staining, Ki67 immunohistochemical staining, CRT and HMGB1 immunofluorescence staining, and Tunel staining.

[0120] The results are as follows Figure 7 The results of unilateral tumor suppression experiment and immune index detection are shown as follows:

[0121] A and B show that IHDC combined with radiotherapy can significantly inhibit tumor growth compared with radiotherapy alone, and there is no significant change in the weight of mice throughout the whole process; C shows that after the end of treatment, the mice were killed, the tumors were dissected and weighed, and it was found that the final tumor weight of the IHDC combined with radiotherapy group was significantly lower than that of radiotherapy alone; D and E show that after the tumor-draining lymph node cells were extracted and the expression of DC maturation markers CD80 and CD86 were detected, it was found that the DC maturity of the tumor-draining lymph nodes in the radiotherapy sensitization group was significantly higher; F shows that after the tumor was dissected after the treatment, H&E staining showed that radiotherapy sensitization increased tumor tissue necrosis, decreased Ki67 expression, increased CRT eversion, increased HMGB1 migration, and increased expression of apoptosis-related Tunel staining markers.

[0122] (II) IHDC for breast cancer radiosensitization-immunotherapy combination therapy

[0123] Tumor inhibition experiment: Balb / c mice were implanted with 4T1 tumor cells in the right hind limb and an equal number of tumor cells in the left hind limb every other day to establish a bilateral tumor model. The mice were randomly divided into six groups: PBS, IHDC, α-PD-1, RT, IHDC + RT, and IHDC + RT + α-PD-1. The IHDC molecular probe was injected into the tail vein. In the radiotherapy-related group, the right tumor site was irradiated after probe injection, while the left tumor was not irradiated. Tumor size and body weight were monitored every two days for a total of 18 days. After the final monitoring, the mice were sacrificed, and bilateral tumor tissues were dissected, photographed, and weighed.

[0124] Analysis of tissue specimens and immune indicators: The spleens of the six groups of mice treated with different methods were dissected to detect the proportion of CD4 and CD8 positive cells in the spleen. Dissected distal tumor tissues, part of the tissues were fixed, dehydrated, paraffin-embedded, and cut into 4 μm tissue sections for CD4 and CD8 immunofluorescence and Granzyme B immunohistochemistry staining. Part of the tissues were frozen and used for cytokine detection. The results are shown in the figure. Figure 8 shown.

[0125] Figure 8 The results of tumor inhibition experiments and immune index testing in a bilateral tumor mouse model are shown. Figures A, B, and C show that radiosensitization combined with immunotherapy significantly inhibited both in situ and distal tumors in the bilateral tumor mouse model, with no significant weight changes throughout the process. Figures D and E show that after the monitoring period, mice were sacrificed, tumors were dissected, and in situ and distal tumors were weighed. The results showed that radiosensitization combined with immunotherapy reduced both in situ and distal tumors compared to other groups. Figure F shows that spleen cells were isolated from mice, and detection revealed a significant increase in CD4 and CD8 positive cells. Figure G shows an increase in Granzyme B content in distal tumors. Figure H shows an increase in TNF-α, INF-γ, IL-6, and IL-12 cytokine levels in distal tumor tissue.

[0126] (III) IHDC for anti-recurrence after breast cancer treatment

[0127] A unilateral subcutaneous tumor xenograft mouse model was established and randomly divided into six groups: PBS, IHDC, α-PD-1, RT, IHDC + RT, and IHDC + RT + α-PD-1. The IHDC molecular probe was injected intravenously. In the radiotherapy-related group, the tumor site was irradiated after probe injection. Subsequently, the tumor site was surgically resected and the wound sutured. After wound healing, 4T1 tumor cells were injected into the tail vein and survival was monitored. At the same time points, lung tissues were dissected, fixed, dehydrated, paraffin-embedded, sectioned, and stained with H&E to observe lung metastasis. Splenic tissues were dissected to assess the proportion of immune memory cells.

[0128] Figure 9 Mice with subcutaneous tumors were treated with various therapies, followed by surgical resection of the primary lesion and tail vein injection of tumor cells to establish a lung metastasis model. Macroscopically, mice treated with radiosensitization combined with immunotherapy demonstrated superior resistance to lung metastasis compared to the other groups, as confirmed by H&E whole-lung staining. Splenic dissection and analysis of immune memory cell counts revealed higher levels in mice treated with radiosensitization combined with immunotherapy, suggesting a potential mechanism for resistance to lung metastasis.

[0129] Figure 10Shown are the results of in vivo biosafety testing. IHDC was injected into healthy mice via the tail vein at different time points. A shows the dissection of major organs and H&E staining, revealing no significant pathological changes. B and C show routine blood tests and biochemical parameters, showing no significant changes compared to normal mice at day 0, at different times after probe injection, remaining within normal ranges.

[0130] It should be understood that the above-described specific embodiments of the present invention are merely illustrative of the invention or explanation of the principles of the present invention, and do not constitute limitations of the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included within the scope of protection of the present invention. In addition, the appended claims are intended to cover all variations and modifications that fall within the scope and metes and bounds of the appended claims, or equivalents of such scope and metes and bounds.

Claims

1. A degradable molecular probe for tumor NIR-II fluorescence imaging and radiotherapy sensitization-immunotherapy combined therapy, characterized in that: The probe is ICG-Hf@DTSPO@CB, which combines indocyanine green (ICG) and hafnium tetrachloride (HfCl4) to form an ICG-Hf complex, which is loaded onto a carrier to form nanoparticles ICG-Hf@DTSPO. The outer layer is then modified with a slightly acidic degradation layer (CB) through a cross-linking reaction between a three-arm phenylboronic acid moiety and a three-arm catechol moiety to synthesize the final nanoparticles ICG-Hf@DTSPO@CB. The carrier used to load the ligand ICG-Hf is dendritic mesoporous silica doped with tetrasulfide bonds. The tetrasulfide bonds in the carrier can react with glutathione GSH overexpressed in the tumor site to produce hydrogen sulfide H2S gas, causing degradation of the carrier material and achieving effective release of the ligand in the tumor site.

2. A degradable molecular probe for tumor NIR-II fluorescence imaging and radiosensitization-immunotherapy according to claim 1, characterized in that: The ICG-Hf@DTSPO@CB probe is positively charged.

3. A degradable molecular probe for tumor NIR-II fluorescence imaging and radiosensitization-immunotherapy according to claim 1 or 2, characterized in that: The preparation method of the ICG-Hf@DTSPO@CB probe comprises the following steps: S1. Synthesis of DTSPO, a carrier material containing tetrasulfide bonds; S2, Hf loading; Hf ions in hafnium chloride HfCl4 can coordinate with the sulfur atoms of the tetrasulfide bonds in DTSPO and thus be loaded on DTSPO to form Hf@DTSPO; S3, ICG loading: ICG is added, and ICG can coordinate with the Hf atoms of Hf@DTSPO in S2 to load ICG, forming spherical solid nanoparticles ICG-Hf@DTSPO; S4. CB modification; through the cross-linking reaction of the three-arm phenylboronic acid unit TAB and the three-arm catechol unit TAC, the slightly acidic degradation layer CB was successfully modified on the outer layer of ICG-Hf@DTSPO to synthesize the degradable molecular probe ICG-Hf@DTSPO@CB with a negative charge on the outer layer.

4. The biodegradable molecular probe for tumor NIR-II fluorescence imaging and radiosensitization-immunotherapy according to claim 3, characterized in that: The step S2 comprises: S21. Weigh 6.4 mg HfCl4 and 14.2 mg DTSPO, dissolve them in 2 mL and 6 mL anhydrous ethanol, respectively, and disperse them thoroughly by ultrasonication. S22. Place the anhydrous ethanol containing DTSPO in S21 in a magnetic stirring device and stir at a constant speed. Use a pipette to quickly add the anhydrous ethanol solution containing HfCl4, and then continue stirring the entire system for 8-12 hours.

5. The biodegradable molecular probe for tumor NIR-II fluorescence imaging and radiosensitization-immunotherapy according to claim 3, characterized in that: The step S3 comprises: S31. Weigh 7.8 mg of ICG, dissolve it in 8 mL of anhydrous ethanol, and disperse it by ultrasonication for 2 min. S32, use a pipette to draw the anhydrous ethanol containing ICG in S31 and quickly add it to the reaction system of S22, and continue stirring for 1 h; S33. Centrifuge the reaction solution in S23 at 8000 rpm for 7 min at room temperature, discard the supernatant to obtain a precipitate, and redisperse the precipitate in 10 mL of anhydrous ethanol. Ultrasonicate for 10 s to disperse it, centrifuge at 8000 rpm for 7 min at room temperature, and discard the supernatant to obtain ICG-Hf@DTSPO nanoparticles.

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