Internal and external source dual nano sensitizer, preparation method thereof and application of internal and external source dual nano sensitizer in preparation of brain glioma drugs

Abstract

The invention particularly relates to an endogenous and endogenous dual nano sensitizer, a preparation method of the endogenous and endogenous dual nano sensitizer and application of the endogenous and endogenous dual nano sensitizer in preparation of brain glioma drugs. The endogenous and exogenous dual nano sensitizer comprises a glycosylated dendrimer Gal-G5 modified compound norcantharidin (DMC) and nano particles internally wrapped with gold (Au). The preparation method and the application comprise the following steps: preparation of Gal-G5, preparation of Gal-G5-DMC, and preparation of the internal and external source dual nano sensitizer Gal-G5 (Au)-DMC. The preparation method is simple, the required raw materials are easy to obtain, the synthesis process is simple and convenient, separation and purification are easy, and the synthesized endogenous and endogenous dual nano sensitizer has the capability of crossing a blood brain barrier to reach brain glioma, can be used for dual sensitization of brain glioma radiotherapy, can also be combined with an immune checkpoint inhibitor (anti-PD-L1 antibody) for combined application of radiation-immunotherapy, and has broad application prospects. And potential application prospects are realized in the aspects of radiotherapy and immunotherapy.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-glioma drugs, and in particular to an endogenous and exogenous dual nanosensitizer and a preparation method thereof, a pharmaceutical composition containing the sensitizer, and use of the sensitizer and the pharmaceutical composition in preparing anti-glioma drugs. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Glioma (GBM), the most aggressive primary malignant tumor of the central nervous system, faces dual challenges in its clinical treatment: on the one hand, the physical barrier of the blood-brain barrier (BBB) significantly limits the efficiency of drug delivery; on the other hand, the diffuse infiltrating growth characteristics of tumor cells make it difficult to completely remove the lesions through surgery. The current standard treatment for GBM is a comprehensive treatment model that combines surgery with radiotherapy and chemotherapy, but the median survival of patients is still only 14.6 months, and the five-year survival rate is less than 10%. Among them, radiotherapy, as a core treatment method, is limited by the inherent radioresistance of glioma tissue and the tolerance threshold of normal brain tissue to radiation dose, making it difficult to completely eliminate the tumor. Studies have shown that glioma cells often further weaken the effect of radiotherapy by activating DNA damage repair capacity, leading to treatment failure.

[0004] To address the aberrant activation of DNA damage repair pathways in these radioresistance mechanisms, researchers are focusing on developing novel radiosensitizers to reshape the radiation response threshold of tumor tissues and block the self-repair capacity of tumor cells, thereby establishing a novel dual-track therapeutic strategy of "enhancing killing and inhibiting repair." For example, gold (Au) nanoparticles, due to their high atomic number, enhance physical energy deposition, leading to the generation of large amounts of ROS and thus exacerbating DNA double-strand breaks. Meanwhile, the small molecule drug norcantharidin (DMC), a PP2A inhibitor, inhibits DNA damage repair responses by regulating the ATM / ATR signaling axis, while also interfering with cell cycle checkpoints and promoting activation of apoptosis pathways. Combined use of these two agents could create a cascade effect of "enhancing physical damage and inhibiting biochemical repair," significantly enhancing radiosensitivity. Furthermore, norcantharidin can inhibit regulatory T cells and promote the immune response of effector T cells, potentially enhancing the efficacy of radiotherapy-immunotherapy for tumors. However, single gold nanoparticles or DMC generally have problems such as low delivery efficiency and poor targeting when crossing the blood-brain barrier, resulting in insufficient effective concentration in the tumor, which seriously restricts the clinical translation potential of the combination of the two.

[0005] Addressing the critical bottleneck of inefficient blood-brain barrier delivery, the diversified development of nanocarrier systems offers new avenues for overcoming this barrier in the delivery of radiosensitizers. Among these, fifth-generation (G5) polyamidoamine (PAMAM) dendrimers, commercially available cationic nanocarriers, demonstrate significant advantages in the diagnosis and treatment of gliomas due to their unique protein-mimicking branching structure, abundant internal cavities, monodisperse nanoscale properties, and 128 functionalizable amino groups on their surfaces. Previous work has leveraged the surface-modifiable properties of G5 PAMAM dendrimers to develop dendrimer-based copper complexes for magnetic resonance imaging and chemodynamic therapy of in situ gliomas (Song C. et al. Nano Today. 2021, 12, 101325). The inventors believe that radioresistance and immune surveillance evasion mechanisms in glioma treatment severely limit the effectiveness of radiotherapy. Providing a multifunctional sensitizer to improve these inhibitory mechanisms is crucial for improving glioma treatment outcomes. Summary of the Invention

[0006] In response to the above research background, the present invention is designed to provide a fifth-generation (G5) polyamidoamine (PAMAM) dendrimer as a carrier, which, through grafting functional side groups and internally encapsulating metal nanoparticles, combines with immune checkpoint inhibitors to significantly enhance the radiotherapy-immunotherapy effect of in situ brain gliomas, thereby achieving spatiotemporal synergy between local tumor radiodynamic killing and systemic immune activation.

[0007] Based on the above technical effects achieved by the present invention, the following technical solutions are provided: In a first aspect, the present invention provides an endogenous and exogenous dual nanosensitizer, which is a positively charged nanomaterial, with an amino-terminated polyamide-amine dendrimer (G5) as a carrier, grafted with galactose (Gal) and demethylcantharidin (DMC), and loaded with gold nanoparticles.

[0008] The present invention provides a sensitizer designed to reduce radiotherapy resistance mechanisms, enhance glioma targeting, and synergistically enhance radiotherapy-immunotherapy activity. In the first aspect of the sensitizer, G5 serves as a nanocarrier, possessing abundant drug-loading cavities and grafting sites. Gal can enter the brain and target gliomas through active transport via the glucose transporter 1 receptor in the blood-brain barrier. Gal-grafted G5 (Gal-G5) has been shown to exhibit enhanced blood-brain barrier permeability in both in vitro and in vivo studies.

[0009] Norcantharidin (DMC) is an active ingredient in anti-glioma drugs that inhibits DNA damage repair and arrests the cell cycle. Due to its poor water solubility and short half-life, the present invention grafts it onto the surface of G5, effectively enhancing DMC's bioavailability. The sensitizer designed in this invention effectively improves the ability of DMC and gold nanoparticles to penetrate the blood-brain barrier, increasing their intratumoral concentrations. Furthermore, the present invention's research has found that the grafting of Gal and DMC effectively enhances the sensitizer's resistance to protein adsorption, allowing it to prolong its blood circulation time in the body and further increase its intratumoral concentration.

[0010] The hydrated particle size of the above sensitizer is 260~320 nm, and the Zeta potential is 25~32 mV.

[0011] In some preferred embodiments, the G5 is connected to Gal via "COC", and 12 to 18 molecules of Gal and 26 to 30 molecules of DMC are grafted onto the surface of each molecule of G5.

[0012] In a second aspect, a method for preparing the endogenous and exogenous dual nanosensitizer according to the first aspect is provided, comprising the following steps: (1) Add the G5 solution to the activated Gal solution and stir the reaction at room temperature for 2.5 to 3.5 days. The component with a molecular weight cutoff of 5000 Da and above is Gal-G5; (2) Add pyridine to the Gal-G5 solution under stirring in a water bath, then add DMC solution dropwise, and stir for 6-8 h. Retain components with a molecular weight of more than 3500 Da to obtain Gal-G5-DMC. (3) Adding chloroauric acid aqueous solution to the aqueous solution of Gal-G5-DMC under ice bath conditions, stirring for 15-30 min under ice bath, adding a strong reducing agent and continuing the reaction in ice bath for 3-4 h, and retaining components above 5000 Da in the obtained product, which is the endogenous and exogenous dual nanosensitizer (Gal-G5(Au)-DMC).

[0013] The above preparation method does not require high temperature and high pressure, does not require special reaction vessels, and does not have complicated purification or post-processing steps. The preparation process is simple, the raw materials are easily available and the cost is economical, and it has high operational safety for production technicians.

[0014] Furthermore, the present invention also provides the following preferred technical solutions: In step (1): 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) is added to the Gal solution and stirred at room temperature for 25-35 minutes. N-Hydroxysuccinimide (NHS) is added and stirred for 2-4 hours for activation. In this embodiment, the molar ratio of Gal to EDC and NHS is 1:10-20:10-20.

[0015] The molar ratio of G5 to Gal is 1:30-40.

[0016] In step (2): The temperature of the water bath is 28-32°C.

[0017] The dosage ratio of Gal-G5 to pyridine is 10 mg: 0.8-1.2 mL.

[0018] The molar ratio of the G5-Gal and DMC is 1:100-200.

[0019] In step (3): The molar ratio of the Gal-G5-DMC to chloroauric acid is 1:50-75.

[0020] Examples of the strong reducing agent include sodium borohydride, hydrazine hydrate, ascorbic acid, etc.; sodium borohydride is preferably used, as the generated gold nanoparticles are smaller in size and more stable in properties. The molar ratio of chloroauric acid to sodium borohydride is 1:3~4.

[0021] In the product Gal-G5(Au)-DMC, the molar ratio of Gal, G5, DMC and Au is 12-17:1:25-30:25-75.

[0022] In addition, in the above steps (1), (2), and (3), the components with corresponding molecular weights are retained, preferably using a cellulose dialysis membrane, and dialyzed in ultrapure water for 2 to 3 days, with the water being changed 2 to 4 times a day, with 1.5 to 2.5 L of ultrapure water each time.

[0023] In steps (1), (2) and (3), the “solution” is prepared using an organic reagent as a solvent unless otherwise specified. The organic reagent should have corresponding solubility, preferably a non-toxic and non-volatile reagent, such as dimethylformamide (DMF) and dimethyl sulfoxide (DMSO).

[0024] The third aspect of the present invention provides a pharmaceutical composition comprising an active dose of the endogenous and exogenous dual nanosensitizer according to the first aspect.

[0025] In the third aspect, the "active dose" refers to the dosage of the pharmaceutical composition that can prevent, improve, treat a disease or improve prognosis after being administered to a subject. Since the present invention has confirmed the in vivo and in vitro therapeutic activity of the above-mentioned endogenous and exogenous dual nanosensitizers for brain glioma, clarifying the specific "active dose" value is a technical content that can be obtained by conventional research methods for those skilled in the art.

[0026] In some embodiments, the pharmaceutical composition, in addition to the endogenous and exogenous dual nanosensitizer, further comprises an anti-glioma active ingredient, an auxiliary ingredient, or a pharmaceutically acceptable carrier.

[0027] The anti-glioma active ingredients include, but are not limited to, natural plant extracts (such as baicalein, curcumin, and triptolide), small molecule compounds (temozolomide, carmustine, and cisplatin), targeted therapeutic drugs (bevacizumab, everolimus, gefitinib, and erlotinib), and immune checkpoint inhibitors. In one embodiment validated by the present invention, the pharmaceutical composition includes the endogenous and exogenous dual nanosensitizer described in the first aspect and also includes a PD-L1 antibody.

[0028] The auxiliary ingredients include antiemetic drugs, granulocyte colony stimulating factor, thrombopoietin, liver protection ingredients, immune enhancers, intestinal flora regulators, etc.

[0029] The pharmaceutically acceptable carrier includes, but is not limited to, absorption enhancers, disintegrants, osmotic pressure regulators, solubilizers, solubilizers, emulsifiers, adhesives, diluents, wetting agents, pH regulators, antioxidants, colorants or flavoring agents, etc.

[0030] In a fourth aspect, the present invention provides the use of the endogenous and exogenous dual nanosensitizer described in the first aspect and the pharmaceutical composition described in the third aspect in the preparation of brain glioma drugs.

[0031] The purposes of the above applications include but are not limited to any of the following: 1. Provide a nanomedicine that can dually enhance the sensitivity of brain glioma radiotherapy; 2. Improve the efficacy of combined radiotherapy and immunotherapy; 3. Activate the subject's immune system and promote the immune response of effector T cells.

[0032] Preferably, the glioma drug is an injection, further preferably, an intravenous injection. The present invention also provides a method for using the glioma drug with better effect, wherein the subject is irradiated with X-rays 20 to 40 minutes after intravenous injection of the above-mentioned endogenous and exogenous dual nanosensitizer or pharmaceutical composition.

[0033] Compared with the prior art, the present invention has the following beneficial effects: The present invention synthesizes the endogenous and exogenous dual nanosensitizer Gal-G5 (Au) -DMC, utilizes galactose for glucose transporter 1-mediated BBB crossing, and achieves GBM targeting. On the one hand, the gold nanoparticles loaded by the present invention are used to directly enhance DNA damage in tumor cells, and DMC is used to regulate the relevant pathways inside tumor cells to inhibit DNA damage repair, thereby achieving a dual sensitization effect of external enhanced killing and internal inhibition of DNA repair. On the other hand, the present invention enhances the immunogenic cell death of tumors through radiotherapy sensitization, further activating the immune system of mice, and DMC inhibits regulatory T cells and promotes the immune response of effector T cells. When used in combination with the immune checkpoint inhibitor anti-PD-L1 antibody, it further induces systemic immunity and enhances the overall immunotherapy effect.

[0034] Through the rational design and application of dendrimer nanomaterials, this invention overcomes some of the defects of dendrimers in crossing the blood-brain barrier and in radiosensitization-immunotherapy applications. It will also have potential guiding significance for the two major problems of clinical radiosensitization and limited immunotherapy effects, and has potential application prospects in achieving tumor radiotherapy and immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0036] Figure 1 The synthesis and treatment flow chart of the endogenous and exogenous dual nanosensitizers of the present invention; Figure 1 Figure A is a flow chart of the synthesis process of the endogenous and exogenous dual nanosensitizer; Figure 1 Middle B is a flow chart showing the radio-immunotherapy effect of the endogenous and exogenous dual nanosensitizers; Figure 2 The morphology characterization results of Gal-G5 prepared in the present invention; Figure 2 A in the middle 1 H NMR spectrum, Figure 2 Middle B is the Fourier transform near infrared spectrum; Figure 3 The phagocytic status of GL261 cells after being treated with G5-Cy5 and Gal-G5-Gy5 prepared by the present invention for 4 hours at different concentrations; Figure 3 A in the middle is the flow cytometry result. Figure 3 Middle B is the quantitative analysis graph; Figure 4 The results of in vivo imaging of the distribution of G5-Cy5 and Gal-G5-Gy5 prepared by the present invention in healthy mice; Figure 4 A in the middle is a live fluorescence imaging image, Figure 4Middle B is the in vitro fluorescence imaging of brain tissue, Figure 4 Middle C is a quantitative analysis of the fluorescence intensity of brain tissue in vitro; Figure 5 The G5-DMC and Gal-G5-DMC prepared by the present invention are 1 H NMR spectrum; Figure 5 A in the middle is G5-DMC 1 H NMR spectrum, Figure 5 B in the middle is Gal-G5-DMC 1 H NMR spectrum; Figure 6 The UV-visible absorption spectra of Gal-G5-DMC and Gal-G5(Au)-DMC prepared in the present invention; Figure 7 The morphology and particle size characterization results of Gal-G5(Au)-DMC prepared by the present invention are shown in FIG. Figure 7 A in the middle is a high-resolution transmission electron microscopy (TEM) image of Gal-G5(Au)-DMC. Figure 7 B is the particle size distribution histogram; Figure 8 The particle size characterization results of Gal-G5, Gal-G5-DMC and Gal-G5(Au)-DMC prepared in the present invention; Figure 8 A is the hydrodynamic diameter diagram, Figure 8 B is the surface potential diagram; Figure 9 This is a graph showing the results of anti-protein adsorption experiments of G5, Gal-G5, Gal-G5-DMC and Gal-G5(Au)-DMC prepared in the present invention under different mass conditions; Figure 10 This is a graph showing the cell viability of GL261 cells after treatment with Gal-G5, Gal-G5-DMC and Gal-G5(Au)-DMC prepared in the present invention at different concentrations for 24 hours; Figure 11 The cytotoxicity results of Gal-G5(Au), Gal-G5-DMC and Gal-G5(Au)-DMC prepared in the present invention are shown; Figure 11 A in the middle shows the cell activity results of GL261 cells treated with different concentrations for 24 hours and then cultured for another 24 hours after X-ray irradiation. Figure 11 Middle B is the cell viability result of GL261 cells treated for 24 hours and then cultured for 48 hours after X-ray irradiation; Figure 12γ-H2AX immunofluorescence staining of GL261 cells treated with Gal-G5(Au), Gal-G5-DMC and Gal-G5(Au)-DMC prepared in the present invention for 24 hours at certain concentrations and then cultured for 48 hours after X-ray irradiation; Figure 13 Figure 2 shows the results of a clone formation experiment in which GL261 cells were treated with Gal-G5(Au), Gal-G5-DMC, and Gal-G5(Au)-DMC prepared in the present invention for 24 hours at certain concentrations and then cultured for 14 days after X-ray irradiation; Figure 14 This is a CRT immunofluorescence staining image of GL261 cells treated with Gal-G5(Au)-DMC prepared in the present invention and its control group under certain concentration conditions for 24 hours and then cultured for 48 hours after X-ray irradiation; Figure 15 The results of dendritic cell maturation after Gal-G5(Au)-DMC prepared by the present invention and treating GL261 cells; Figure 15 A in the middle is the flow cytometry result. Figure 15 Middle B is the quantitative analysis graph; Figure 16 This is a graph showing the blood compatibility test results of Gal-G5(Au)-DMC prepared in the present invention; Figure 17 The results of activity imaging of the distribution of Cy5-Gal-G5(Au)-DMC in mice with orthotopic brain glioma; Figure 17 A in the middle is the in vivo fluorescence imaging. Figure 17 Middle B is a quantitative analysis of brain fluorescence intensity. Figure 17 Middle C is the distribution map of major organs and tissues; Figure 18 This is a graph showing the results of a study on the in vivo anti-glioma activity of Gal-G5(Au)-DMC prepared in the present invention; Figure 18 A in the middle is the animal experimental treatment flow chart, Figure 18 Middle B is the imaging image of living mice (bioluminescence mode), Figure 18 Middle C is the graph of mouse weight changes. Figure 18 Middle D is the survival rate statistics chart; Figure 19 The results of brain tissue extraction, sectioning, and fluorescent staining in mice with orthotopic gliomas are shown; Figure 19 A in the middle is the hematoxylin-eosin (H&E) staining picture. Figure 19 Middle B shows Ki-67 immunofluorescence staining, TUNEL (TdT-mediated dUTP Nick-End Labeling) staining, γ-H2AX immunofluorescence staining, and CRT immunofluorescence staining of the tumor site; Figure 20 Figure 2 shows the anti-tumor process and effect of Gal-G5(Au)-DMC in combination with anti-PD-L1 antibody in mice bearing orthotopic brain glioma. Figure 20 A in the middle is the animal experimental treatment flow chart, Figure 20 Middle B is the graph of the weight change of mice. Figure 20 Middle C is the bioluminescence imaging of mouse tumor. Figure 20 Middle D is the survival rate statistics chart; Figure 21 The results are as follows: Characterization of immune cells in cervical lymph nodes and spleen tissues of orthotopic GBM mice after co-administration of Gal-G5(Au)-DMC and anti-PD-L1 antibody; Figure 21 A in the middle is a flow cytometry analysis of mature dendritic cells in cervical lymph nodes. Figure 21 Middle B is the quantitative analysis result of mature dendritic cells. Figure 21 Middle C is the flow cytometry analysis of Treg cells in spleen tissue. Figure 21 D in the middle is the quantitative analysis result of Treg cells. Figure 21 E in the middle is CD8 + T and CD4 + Flow cytometry analysis of T cells. Figure 21 F in the middle is CD8 + Cell quantitative analysis results of the ratio of T cells to Treg cells; Figure 22 This is a diagram showing the effect of H&E staining of heart, liver, spleen, lung, and kidney tissue sections extracted from healthy mice after intravenous injection of the Gal-G5(Au)-DMC prepared by the present invention. DETAILED DESCRIPTION

[0037] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0038] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0039] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0040] In the following examples and comparative examples, unless otherwise noted, all chemical reagents are commercially available and used directly without further purification. The fifth-generation amino-terminated polyamidoamine dendrimer G5.NH2 was purchased from Weihai Chenyuan Molecular New Materials Co., Ltd. (Weihai, China). All other raw materials or processing techniques, unless otherwise specified, are commercially available and are based on conventional techniques in the art.

[0041] Example 1 In this embodiment, a dual-source nanosensitizer is provided, and its preparation method is as follows: (1) Weigh 2.21 mg of Gal, 19.95 mg of EDC·HCl, and 12.01 mg of NHS and dissolve them in 2 mL of DMSO. Then, add the EDC·HCl solution dropwise to the Gal solution. Stir at room temperature for 30 min, then add the NHS solution dropwise to the mixed solution. Continue stirring at room temperature for 3 h to obtain the activated Gal solution. Weigh 10 mg of G5 and dissolve it in 5 mL of DMSO. Add the activated Gal solution dropwise to the G5 solution. Continue stirring at room temperature for 3 days. Transfer the obtained product to a dialysis bag with a molecular weight cutoff of 5000 Da and dialyze it in ultrapure water for three days (2 L × 3 times / day). Then, freeze-dry it to obtain Gal-G5 powder, which is stored at -20 °C for future use.

[0042] (2) Weigh 10 mg of the Gal-G5 obtained in step (1) above and 5.3 mg of DMC and dissolve them in 5 mL of DMSO. Add 1 mL of pyridine to the Gal-G5 solution under stirring in a water bath at 30 °C, then add the DMC solution dropwise. Stir and react for 6-8 h. Transfer the obtained product to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze it in ultrapure water for three days (2 L × 3 times / day). Then, freeze-dry it to obtain Gal-G5-DMC powder, which is stored at -20 °C for future use.

[0043] (3) Weigh 10 mg of the Gal-G5-DMC obtained in step (2) above and dissolve it in 2 mL of ultrapure water. Add 191.44 μL of chloroauric acid aqueous solution (30 mg / mL) dropwise in an ice bath and stir for 15-30 min. Then, quickly pour 1.58 mL of sodium borohydride aqueous solution (1 mg / mL) into the mixed solution and continue stirring in an ice bath for 3-4 h. Transfer the obtained product to a dialysis bag with a molecular weight cutoff of 5000 Da and dialyze it in ultrapure water for three days (2 L × 3 times / day). Then, freeze-dry it to obtain Gal-G5(Au)-DMC powder, which is stored at -20 °C for future use.

[0044] Comparative Example 1 In this embodiment, a G5-DMC is provided, and the preparation method is as follows: 10 mg of G5 and 5.8 mg of DMC were weighed and dissolved in 5 mL of DMSO. 1 mL of pyridine was added to the G5 solution, followed by the DMC solution, with stirring in a 30°C water bath. The reaction was stirred for 6–8 h. The resulting product was transferred to a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed in ultrapure water for three days (2 L × 3 times / day). The product was then freeze-dried to obtain G5-DMC powder, which was stored at -20°C until use.

[0045] Comparative Example 2 In this embodiment, a Gal-G5(Au) is provided, and the preparation method is as follows: 10 mg of Gal-G5 obtained in step (1) of Example 1 was weighed and dissolved in 2 mL of ultrapure water. 214.53 μL of chloroauric acid aqueous solution (30 mg / mL) was added dropwise in an ice bath, and the mixture was stirred on ice for 15-30 min. Subsequently, 1.77 mL of sodium borohydride aqueous solution (1 mg / mL) was quickly poured into the mixed solution, and the reaction was continued by stirring on ice for 3-4 h. The obtained product was transferred to a dialysis bag with a molecular weight cutoff of 5000 Da and dialyzed in ultrapure water for three days (2 L × 3 times / day). The product was then freeze-dried to obtain Gal-G5(Au) powder, which was stored at -20°C for future use.

[0046] Performance Characterization (1) Gal-G5 1. Morphology characterization The Gal-G5 prepared in step (1) of Example 1 was characterized by nuclear magnetic resonance and Fourier transform infrared spectroscopy (FI-IR). 1 The H NMR characterization results are as follows Figure 2 As shown in Figure A: 2.2~3.4 ppm is the characteristic proton peak of G5.NH2, and 3.5~4.5 ppm is the characteristic proton peak of Gal. Based on the ratio of their integrated areas, it is calculated that each G5 is connected to 15 Gal molecules. The FI-IR characterization results are shown in Figure 1. Figure 2 Shown in B: Gal-G5 at 1080 cm -1 There is a COC absorption peak at , while the G5 dendrimer alone does not, which proves the successful synthesis of Gal-G5.

[0047] 2. In vitro activity characterization (1) Characterization of targeting performance in vitro Mouse glioma (GL261) cells were used as model cells to characterize the targeting performance of Gal-G5 in vitro, and the targeting effect of Gal-G5 dendrimers was evaluated by detecting the amount of Gal-G5 phagocytosis by cells. First, GL261 cells were seeded in a 12-well plate at a density of 100,000 cells / well. After overnight culture, the culture medium was replaced with a sugar-free culture medium containing G5 and Gal-G5 labeled with the fluorescent dye Cy5 (denoted as G5-Gy5 and Gal-G5-Gy5, respectively) (material concentrations were 1 μM and 2 μM, respectively), and co-cultured with GL261 cells at 37°C for 4 h. The cells were then washed twice with PBS buffer solution, digested with trypsin, collected by centrifugation (1200 rpm, 3 min), and resuspended in an appropriate amount of PBS. Finally, the fluorescence intensity of the cells was detected by flow cytometry. The results are shown in Figure 2. Figure 3 As shown, compared with the normal cell group (control group), the fluorescence intensity of GL261 cells treated with two concentrations of G5-Gy5 almost did not increase, while the fluorescence intensity of GL261 cells treated with Gal-G5 increased significantly, indicating that Gal-G5 has the ability to target GL261 cells.

[0048] (2) Characterization of the ability to cross the blood-brain barrier (BBB) in vivo Male C57BL / 6 healthy mice aged 6-8 weeks were selected (purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (Beijing, China). All animal experiments were carried out in strict accordance with the standards of the Animal Protection Association, the same below). First, the healthy mice were randomly divided into two groups and fasted for 24 hours. Then, 30 minutes after intraperitoneal injection of 100 μL of 20% glucose solution, 100 μL of the same concentration of G5-Cy5 and Gal-G5-Gy5 were injected into the tail vein, and in vivo fluorescence imaging of the mice was performed at 0, 15, 30, 45, 60, 90, 120 and 240 minutes after injection (IVIS SPECTRUM CT, PerkinElmer, USA). The results are shown in Figure 3. Figure 4 As shown in center A, 15 minutes after injection, fluorescence signals were observed in the brains of both groups of mice, and the fluorescence signals gradually weakened over time. However, within a certain observation time range (0-240 minutes), the fluorescence intensity of the Gal-G5-Gy5 brain was always higher than that of the G5-Cy5 group.

[0049] In order to better observe the accumulation of G5-Cy5 and Gal-G5-Gy5 in the mouse brain, 100 μL of the same concentration of G5-Cy5 or Gal-G5-Gy5 was injected into the tail vein of healthy mice. The mice were then killed at 0, 15, 30, 45, 60, 90, 120, and 240 min, and their brain tissues were extracted and subjected to in vitro fluorescence imaging. Figure 4As shown in Figures BC, 15 minutes after injection, the brain fluorescence intensity in the Gal-G5-Cy5 group reached its peak and was significantly higher than that in the G5-Cy5 group. However, 30 minutes after injection, the brain fluorescence intensity in the G5-Cy5 group reached its peak and was lower than that in the Gal-G5-Cy5 group. These data demonstrate that galactose modification significantly enhances the efficiency of G5 dendrimers in crossing the BBB.

[0050] (2) Gal-G5-DMC, Gal-G5(Au)-DMC, and G5-DMC 1. Morphology characterization The Gal-G5-DMC and G5-DMC prepared in step (2) of Example 1 and Comparative Example 1 were characterized by nuclear magnetic resonance. 1 The H NMR characterization results are as follows Figure 5 As shown in Figure 2, 2.2-3.4 ppm is the characteristic proton peak of G5.NH2, and 1.7-1.8 ppm is the characteristic proton peak of DMC. Based on the ratio of their integrated areas, it is calculated that each G5 is connected to 40 DMC molecules ( Figure 5 Middle A), each Gal-G5 is linked to 28 DMC molecules ( Figure 5 Middle B).

[0051] 2. Characterization of optical properties Gal-G5-DMC and Gal-G5(Au)-DMC were prepared into a 0.1 mg / mL uniform aqueous solution and scanned in the full wavelength range of 200-900 nm using UV-vis. Figure 6 As shown, the Gal-G5(Au)-DMC solution exhibits a significant characteristic absorption peak in the 500-550 nm wavelength range, with a maximum absorption peak at around 520 nm. This characteristic peak corresponds to the surface plasmon resonance absorption peak of gold nanoparticles. In contrast, no characteristic absorption peak was observed for Gal-G5-DMC in this wavelength range, demonstrating that the synthesized Gal-G5(Au)-DMC was successfully loaded with gold nanoparticles.

[0052] 3. Transmission electron microscopy characterization The Gal-G5(Au)-DMC prepared in step (3) of Example 1 was characterized using a transmission electron microscope (Talos F200i, Thermo Fisher Scientific, USA). First, the Gal-G5(Au)-DMC was prepared into a 0.5 mg / mL solution, and then 5 μL was dropped onto an ultra-thin carbon-supported copper grid and observed after drying. The results are shown in Figure 2. Figure 7 As shown in the figure, the gold nanoparticles in Gal-G5(Au)-DMC were distributed in a regular spherical shape. The core particle size was 1.9 ± 0.03 nm according to statistical analysis by ImageJ software.

[0053] 4. Characterization of hydrated particle size and surface potential G5-Gal, Gal-G5-DMC and Gal-G5(Au)-DMC were prepared into a 1 mg / mL uniform aqueous solution, and then the hydrodynamic diameter and Zeta potential of the materials were measured using a Malvern laser particle size analyzer (Zetasizer Pro, Malvern, UK). Figure 8 As shown in the data, the hydrated particle sizes of Gal-G5, Gal-G5-DMC and Gal-G5(Au)-DMC were 259.9 ± 15.9 nm, 336.6 ± 9.8 nm and 302.1 ± 6.7 nm, respectively, which are in line with the typical size range of cellular uptake; the Zeta potentials of G5-Gal, Gal-G5-DMC and Gal-G5(Au)-DMC were all positive, namely 34.6 ± 1.9 mV, 28.4 ± 1.1 mV and 28.9 ± 1.1 mV, respectively, indicating that the materials are positively charged and can promote cellular uptake and internalization.

[0054] 5. Characterization of anti-protein adsorption performance Bovine serum albumin aqueous solution (2 mg / mL) was mixed with G5, G5-Gal, Gal-G5-DMC, and Gal-G5(Au)-DMC aqueous solutions at different concentrations (0.5, 1, 2, and 4 mg / mL) at a volume ratio of 1:1. The mixture was incubated at 37°C for 4 h and then centrifuged (8000 rpm, 5 min) to collect the supernatant. The protein content in the supernatant was determined using a BCA quantitative kit according to standard specifications. The results are shown in Figure 2. Figure 9 As shown in the figure, compared with the G5, G5-Gal and Gal-G5-DMC groups, the supernatant protein concentration of Gal-G5(Au)-DMC was the highest at all tested concentrations, indicating that Gal-G5(Au)-DMC had the best anti-protein adsorption performance.

[0055] 6. In vitro activity evaluation GL261 cells were used as model cells to evaluate the in vitro activities of G5-Gal, Gal-G5-DMC and Gal-G5(Au)-DMC.

[0056] (1) CCK8 method First, GL261 cells were cultured at 8×10 3 The cells were seeded at a density of 100 cells / well in a 96-well plate and cultured in DMEM high-glucose medium (named DMEM) containing 10% fetal bovine serum and 1% double-antibody (100 U / mL penicillin and 100 U / mL streptomycin). +++) and cultured overnight at 37°C, 5% CO2. The culture medium was then replaced with medium containing different concentrations (0-4 μM) of G5-Gal, Gal-G5-DMC, and Gal-G5(Au)-DMC, with G5 concentrations of 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, and 4 μM, respectively. The culture was then continued for 24 hours. The culture medium was discarded, and cell viability was detected using a CCK-8 kit according to the manufacturer's instructions. The results are shown in Figure 2. Figure 10 As shown in Figure A, G5-Gal had no obvious toxicity to GL261 cells at all tested concentrations, and the cell survival rate was greater than 90%, indicating good cell compatibility; while Gal-G5-DMC and Gal-G5(Au)-DMC showed concentration-dependent cytotoxicity, indicating that these two materials had a significant inhibitory effect on GL261 cells due to the presence of DMC.

[0057] (2) In vitro radiosensitization activity of Gal-G5(Au)-DMC To further evaluate the in vitro therapeutic effect of Gal-G5(Au)-DMC dual radiosensitization on GL261 cells, based on the same experimental system, cells were treated with different concentrations of Gal-G5(Au)-DMC (0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, and 4 μM) for 24 hours and then irradiated with 6 Gy (in Gy) of X-rays (RS2000PRO, Rad Source Technologies, USA). Cell viability was then measured after 24 and 48 hours of culture. The PBS group, the radiotherapy group alone, and the different concentrations of Gal-G5(Au) + 6 Gy and Gal-G5-DMC + 6 Gy served as controls. Results are shown in the table. Figure 11 As shown, the Gal-G5(Au)-DMC + 6 Gy group exhibited more significant cytotoxicity compared to the other groups, and this was concentration-dependent. Notably, the cytotoxicity in the 48-h culture group was more pronounced than in the 24-h group, particularly at a concentration of 1 μM. This fully demonstrates the synergistic therapeutic effect of Gal-G5(Au)-DMC as a dual radiosensitizer. These results not only confirm the direct cytotoxicity of Gal-G5(Au)-DMC on GL261 cells but also demonstrate its significant radiosensitization effect.

[0058] (3) γ-H2AX immunofluorescence staining GL261 cells were used as model cells. γ-H2AX immunofluorescence staining was used to evaluate the effects of Gal-G5(Au), Gal-G5-DMC and Gal-G5(Au)-DMC combined with 6 Gy X-ray irradiation on DNA damage in GL261 cells. GL261 cells were cultured at a density of 1×105 The cells were seeded at a density of 100 / well in a confocal culture dish using DMEM. +++ The culture medium was cultured overnight at 37 °C and 5% CO2. The culture medium was then replaced with a culture medium containing 1 μM Gal-G5(Au), Gal-G5-DMC or Gal-G5(Au)-DMC (500 μL / well). After 24 h of culture, 6 Gy X-ray irradiation was given and culture was continued for 48 h. Among them, the PBS group and the 6 Gy irradiation group alone served as controls. After the culture was completed, the culture medium was washed away, and the DNA damage sites were labeled with anti-γ-H2AX antibody, and the cell nucleus was labeled with the nuclear dye DAPI. After thorough washing, 300 μL PBS was added to each well to maintain cell morphology. The expression of γ-H2AX protein in each group was observed by laser confocal microscopy, and fluorescence quantitative analysis was performed. For results, see Figure 12 As shown in the data, compared with the other groups, a small amount of γ-H2AX focus formation was observed in the X-ray irradiation group alone, confirming that radiotherapy can induce DNA double-strand breaks; and the Gal-G5(Au)-DMC+6 Gy group showed the strongest red fluorescence signal, indicating that Gal-G5(Au)-DMC has the most significant radiosensitization effect.

[0059] (4) Clone formation experiment GL261 cells were used as model cells. The clone formation assay was used to evaluate the effects of Gal-G5(Au), Gal-G5-DMC and Gal-G5(Au)-DMC combined with 6 Gy X-ray irradiation on the proliferation of GL261 cells. GL261 cells were cultured at a density of 1×10 5 The cells were seeded at a density of 1000 cells / well in a 12-well plate using DMEM. +++ The culture medium was incubated at 37°C and 5% CO2 for 24 hours. The culture medium was then replaced with a medium containing 1 μM Gal-G5(Au), Gal-G5-DMC, or 1 μM Gal-G5(Au)-DMC (500 μL / well). After 24 hours of culture, the cells were irradiated with 6 Gy X-rays and cultured for 14 days. The PBS group and the 6 Gy irradiation group served as controls. After culture, cell colony formation was observed by fixation with 4% paraformaldehyde and staining with 1% crystal violet, and quantitative analysis was performed using Image J software. Results are shown in the table. Figure 13 As shown in the figure, compared with the control group, the colony formation rates of the other groups decreased in order, with the lowest in the Gal-G5(Au)-DMC+6 Gy group, which significantly enhanced the inhibitory effect of X-rays on tumor cell proliferation. This result further validated the synergistic therapeutic effect of Gal-G5(Au)-DMC as a dual radiotherapy sensitizer from the perspective of cell proliferation.

[0060] (5) Immunofluorescence staining GL261 cells were used as model cells, and immunofluorescence staining was performed using anti-calreticulin (CRT) antibody. Laser confocal microscopy was used to evaluate the immunogenic cell death effect of GL261 cells after Gal-G5(Au)-DMC combined with X-ray irradiation. GL261 cells were cultured at a density of 1×10 5 The cells were seeded at a density of 100 / well in a confocal culture dish using DMEM. +++ The culture medium was cultured overnight at 37 °C and 5% CO2. The culture medium was then replaced with a culture medium containing 1 μM Gal-G5(Au)-DMC (500 μL / well), and after 24 h of culture, 6 Gy X-ray irradiation was given, and the culture was continued for 48 h after irradiation. Among them, the PBS group was used as the control, and the experimental groups were divided into a single 6 Gy irradiation group, a single Gal-G5(Au)-DMC group, and a Gal-G5(Au)-DMC+6 Gy irradiation group. After the culture was completed, the culture medium was washed away, and the CRT protein and cell nucleus were stained with anti-CRT antibody and cell nuclear dye DAPI, respectively. Then, the CRT expression levels of cells in different treatment groups were observed using a laser confocal microscope. Results are shown in Figure 14 As shown in the figure, compared with the control group, obvious green fluorescence signals could be observed after X-ray irradiation; moreover, the Gal-G5(Au)-DMC+6 Gy irradiation group showed the strongest green fluorescence, which indicated that the GL261 cells treated with Gal-G5(Au)-DMC combined with X-ray irradiation underwent obvious immunogenic cell death.

[0061] (6) Flow cytometry GL261 cells and mouse dendritic cells (DCs) were used as model cells. Fluorescent (PE)-labeled CD80 antibody was used to quantitatively analyze the effect of Gal-G5(Au)-DMC combined with X-ray irradiation on the maturation of GL261 cells by flow cytometry. 5 Cells were seeded at a density of 100 cells / well in the upper chamber of a Transwell-12 well plate (0.4 μm), and 0.5 mL of DMEM was added to each well. ++ At the same time, dendritic cells were cultured at a rate of 1×10 5 Cells were seeded at a density of 100 cells / well in the lower chamber of a transwell-12 well plate, and the cells seeded in the upper and lower chambers were separated and cultured in a 5% CO2, 37°C incubator. After GL261 cells were cultured overnight, the culture medium in the upper chamber was replaced with 0.5 mL of DMEM containing Gal-G5(Au)-DMC. +++The cells were cultured in the culture medium and supplemented with 6 Gy X-ray irradiation after 24 hours. The upper and lower chambers were then combined and cultured for 48 hours. The PBS group was used as the control, and the experimental groups were divided into a single 6 Gy irradiation group, a single Gal-G5(Au)-DMC group, and a Gal-G5(Au)-DMC+6 Gy irradiation group. After the culture was completed, the DCs were removed, digested and centrifuged, and PBS was added to resuspend the cells. According to standard specifications, the cells were labeled with PE-CD80 antibody, and the fluorescence intensity was detected by flow cytometry. The results are shown in Figure 2. Figure 15 As shown in the results, compared with the PBS group, X-ray irradiation increased the expression of CD80 on the surface of DCs, reflecting the degree of DC maturation. Moreover, the DCs in the Gal-G5(Au)-DMC+6 Gy irradiation group showed the highest degree of DC maturation, indicating that the immunogenic cell death of cancer cells induced by Gal-G5(Au)-DMC+6 Gy irradiation can promote the maturation of DCs.

[0062] (7) Blood compatibility characterization The blood compatibility of the Gal-G5(Au)-DMC prepared in step (3) was characterized. First, PBS was used as a solvent to prepare Gal-G5(Au)-DMC solutions with a concentration gradient of 100, 200, 300, 400, 500, 750, 1000, 1500 and 2000 nM. PBS and 1% Triton X-100 were used as negative and positive controls, respectively. Then, blood was collected from the eyeballs of healthy mice, and 1.5 mL of blood was collected in an anticoagulant tube. After washing with PBS three times, the blood was resuspended in 5 mL of PBS to prepare a red blood cell suspension. Subsequently, 100 μL of the red blood cell suspension was thoroughly mixed with 900 μL of each solution of the above concentration, incubated at 37 °C for 2 h, and centrifuged at 2000 rpm for 5 min to obtain a hemolyzed sample. Finally, the supernatant was taken and its absorbance at a wavelength of 540 nm was measured using a UV-visible spectrophotometer. The results are shown in Figure 2. Figure 16 As shown in the data, within a certain concentration range, the hemolysis rate of Gal-G5(Au)-DMC was lower than 8%, which indicated that Gal-G5(Au)-DMC had good blood compatibility within the tested concentration range and could meet the safety requirements of in vivo experiments.

[0063] 7. In vivo activity evaluation (1) In vivo imaging Using luciferase-labeled GL261 (Luc-GL261) cells as model cells, the distribution of Gal-G5(Au)-DMC in an orthotopic glioma model mouse model was evaluated using small animal in vivo imaging. First, an orthotopic glioma mouse model was established by slowly injecting 5 μL of PBS solution (containing 5×10 5 Luc-GL261 cells were added to the mouse model. Seven days later, the model was successfully constructed using an in vivo small animal imaging system (bioluminescence mode). Then, three tumor-bearing mice were randomly selected and fasted for 24 hours. 100 μL of 20% glucose solution was injected intraperitoneally. 30 minutes later, 100 μL of Cy5-labeled Gal-G5(Au)-DMC (dose: 10 mg / kg) was injected into the tail vein. In vivo small animal imaging (fluorescence mode) was performed at 0, 0.5, 0.75, 1, 2, 4, 8, 12, and 24 hours after injection. In vitro fluorescence imaging of the mouse brain tissue and heart, liver, spleen, lung, and kidney was performed at 0.5 and 24 hours. Results are shown in the table. Figure 17 As shown, within a specific timeframe, brain fluorescence intensity reached its peak at 0.5 hours after dosing, demonstrating the excellent blood-brain barrier penetration of Gal-G5(Au)-DMC. Fluorescence signals were still observed in the brain at 24 hours after dosing, indicating that Cy5-Gal-G5(Au)-DMC persists in the brain, facilitating sustained therapeutic effects during radiotherapy. Furthermore, in vitro fluorescence imaging of mice revealed high brain fluorescence at 0.5 hours after dosing, and continued fluorescence in the brain (notably in the tumor site) at 24 hours, demonstrating Gal-G5(Au)-DMC's rapid and efficient brain accumulation and sustained tumor retention. Furthermore, Gal-G5(Au)-DMC was distributed throughout major organs at 0.5 hours after dosing, and fluorescence significantly decreased across all organs at 24 hours, indicating ongoing metabolism. However, strong fluorescence signals remained in the kidneys, suggesting that the kidneys may be a primary metabolic organ.

[0064] (2) Anti-tumor effect of sensitized radiotherapy Using Luc-GL261 cells as model cells, a mouse orthotopic glioma model was established according to the established protocol ( Figure 18Middle A) was injected to evaluate the anti-tumor effect of the designed Gal-G5(Au)-DMC sensitized radiotherapy. Orthotopic glioma model mice were randomly divided into four groups (n=5 in each group for tumor monitoring, n=10 for survival analysis), namely control group, radiotherapy alone (6 Gy) group, Gal-G5(Au)-DMC group and Gal-G5(Au)-DMC+6 Gy group. All groups were fasted for 24 hours before administration, and 100 μL of 20% glucose solution was injected intraperitoneally 30 minutes before administration, followed by 100 μL of PBS or Gal-G5(Au)-DMC (dose: 10 mg / kg) injected into the tail vein. 30 minutes later, the mouse brain was irradiated with a dose of 6 Gy of X-rays. Subsequently, the tumor size was monitored on the 3rd, 8th, 13th and 18th days using a small animal in vivo imaging (bioluminescence mode) system. The mouse weight was weighed every 2 days (recorded 10 times in total), and the survival status was continuously recorded. Results are shown in Figure 18 As shown in Figure 2, compared with the other groups, the tumor growth of the Gal-G5(Au)-DMC+6 Gy group was the slowest, showing the best tumor inhibition effect, followed by the Gal-G5(Au)-DMC group ( Figure 18 Middle B). Figure 18 Figure C shows that the weight of mice in the control group and radiotherapy group decreased to varying degrees. No significant weight loss was observed in the Gal-G5(Au)-DMC treatment group, while the weight of mice in the Gal-G5(Au)-DMC combined with radiotherapy group remained stable or even increased slightly. In addition, compared with the control group, the survival time of mice in the other treatment groups was prolonged ( Figure 18 (D in the figure). For example, on day 18 of treatment, all mice in the control group had died, while the survival rates of the 6 Gy group, the Gal-G5(Au)-DMC group, and the Gal-G5(Au)-DMC + 6 Gy group were 60%, 90%, and 100%, respectively. Furthermore, on day 45 of treatment, the survival rates of the control group, the 6 Gy group, and the Gal-G5(Au)-DMC group were all 0, while the survival rate of the Gal-G5(Au)-DMC + 6 Gy group remained at 50%. This demonstrates that the Gal-G5(Au)-DMC prepared by the present invention has excellent in vivo antitumor activity and a radiosensitizing effect, enhancing the in vivo therapeutic effect.

[0065] After 15 days of treatment, the mice were euthanized and their brain tissues were dissected and sliced. H&E staining, immunofluorescence staining, and TUNEL assays were performed to evaluate cell necrosis, apoptotic proliferation, DNA damage (γ-H2A expression), and immunogenic cell death (CRT expression) in the tumor site. Figure 19 As shown, the Gal-G5(Au)-DMC+6 Gy group showed the smallest tumor distribution ( Figure 19A), which resulted in the greatest degree of tumor cell apoptosis, proliferation inhibition, DNA damage, and CRT expression ( Figure 19 (B) These phenomena also fully demonstrate that the Gal-G5(Au)-DMC prepared by the present invention can enhance radiotherapy sensitivity and achieve the purpose of enhancing the therapeutic effect.

[0066] (3) Radiotherapy-immunotherapy combination therapy Using Luc-GL261 cells as model cells, a mouse orthotopic glioma model was established according to the established protocol ( Figure 20 Middle A) Injection was performed to evaluate the antitumor and immune effects of combined radioimmunotherapy with the designed Gal-G5(Au)-DMC for glioma. This study evaluated the antitumor effect of radiosensitization therapy using Gal-G5(Au)-DMC in combination with radiotherapy and the immune checkpoint inhibitor anti-PD-L1 antibody (aPD-L1). To enhance the antitumor effect of radiotherapy, an orthotopic glioma model was established in 6-8 week-old male C57BL / 6 mice using stereotactic injection to systematically evaluate the antitumor effect of radioimmunotherapy. GBM mice were randomly divided into four groups (n=3 per group for tumor monitoring and n=10 per group for survival analysis): PBS control, 6 Gy + aPD-L1, Gal-G5(Au)-DMC + aPD-L1, and Gal-G5(Au)-DMC + 6 Gy + aPD-L1. All groups were fasted for 24 hours before administration, and 100 μL of 20% glucose solution was injected intraperitoneally 30 minutes before administration (recorded as day 0). Then 100 μL of PBS or Gal-G5(Au)-DMC (dose: 10 mg / kg) was injected into the tail vein. 30 minutes later, the mouse brain was irradiated with a dose of 6 Gy of X-rays. Anti-PD-L1 antibody was injected intraperitoneally on days 1, 3, 5, and 7. Tumor size was monitored on days 3, 8, 13, and 18 using a small animal in vivo imaging (bioluminescence mode) system. The mouse weight was weighed every 2 days (a total of 10 times), and survival status was continuously recorded. Results are shown in the table. Figure 20 As shown in Figures BD, compared with other groups, Gal-G5(Au)-DMC+6 Gy+aPD-L1 showed the best tumor inhibitory effect and improved the survival rate of GBM mice within a certain range.

[0067] On the 10th day of treatment, the mice were euthanized and the cervical lymph node tissues were extracted by dissection to prepare single cell suspensions. + and CD86 + Antibodies were used to detect the maturity of dendritic cells in cervical lymph node tissues of each group by flow cytometry; at the same time, the spleen of mice was extracted and single cell suspension was prepared, and fluorescently labeled CD4 + 、CD25 + and CD8 +Antibodies were used to detect the content of various T cells in the spleen tissue of each group by flow cytometry to evaluate the degree of T cell activation and analyze the systemic immune response of mice induced by treatment. Figure 21 As shown in the figure, the proportion of mature dendritic cells in the cervical lymph nodes of mice in the Gal-G5(Au)-DMC+6 Gy+aPD-L1 group was significantly higher than that in the other groups, and the CD8 + The proportion of T cells was the highest and the proportion of Treg cells was the lowest, indicating that the G5(Au)-DMC+6 Gy+aPD-L1 group could induce a significant systemic immune response effect.

[0068] (4) Biosafety evaluation The biosafety of the Gal-G5(Au)-DMC prepared in step (3) was evaluated. First, 6-8 week old male C57BL / 6 healthy mice were randomly divided into two groups (PBS group and Gal-G5(Au)-DMC group) and fasted for 24 h. Then, 100 μL of 20% glucose solution was injected intraperitoneally 30 min later, and then 100 μL of PBS and Gal-G5(Au)-DMC (dose: 10 mg / kg) were injected into the tail vein. At 24 h, the mice were euthanized and the main organs were extracted by dissection for sectioning and H&E staining. The results are shown in Figure 2. Figure 22 As shown, each group of preparations did not produce any obvious cardiac toxicity, liver and kidney damage, lung toxicity and spleen infiltration, which indicates that the Gal-G5(Au)-DMC prepared by the present invention has good biocompatibility.

[0069] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An endogenous and exogenous dual nanosensitizer, characterized in that: The sensitizer is a positively charged nano material, which uses amino-terminated polyamide-amine dendrimer as a carrier, is grafted with galactose and norcantharidin, and is loaded with gold nanoparticles.

2. The endogenous and exogenous dual nanosensitizer according to claim 1, characterized in that: The hydrated particle size of the sensitizer is 260-320 nm, and the Zeta potential is 25-32 mV.

3. The endogenous and exogenous dual nanosensitizer according to claim 1, characterized in that: The amino-terminated polyamide-amine dendrimer is connected to galactose via COC, and 12 to 18 molecules of galactose and 26 to 30 molecules of norcantharidin are grafted onto the surface of each molecule of the amino-terminated polyamide-amine dendrimer.

4. A method for preparing the endogenous and exogenous dual nanosensitizer, characterized in that: The steps include: (1) Add activated Gal to the G5 solution and stir the reaction at room temperature for 2.5 to 3.5 days. The component with a molecular weight cutoff of 5000 Da and above is Gal-G5; (2) Add pyridine to the Gal-G5 solution under stirring in a water bath, add DMC solution dropwise, and react with stirring for 6-8 hours, and retain components with a molecular weight of more than 3500 Da to obtain Gal-G5-DMC; (3) Adding aqueous chloroauric acid solution to the aqueous solution of Gal-G5-DMC under ice bath conditions, stirring for 15-30 min under ice bath, adding a strong reducing agent and continuing the reaction under ice bath for 3-4 h, the resulting product retains components above 5000 Da, namely Gal-G5(Au)-DMC.

5. The preparation method according to claim 4, wherein In step (1), EDC·HCl is added to the Gal solution and stirred at room temperature for 25-35 min, and NHS is added and stirred for 2-4 h for activation; the molar ratio of the Gal to EDC and NHS is 1:10-20:10-20; the molar ratio of the G5 to the Gal feed is 1:30-40.

6. The preparation method according to claim 4, wherein In step (2), the temperature of the water bath is 28-32° C.; the dosage ratio of the Gal-G5 to pyridine is 10 mg:0.8-1.2 mL; and the molar ratio of the G5-Gal to DMC is 1:100-200.

7. The preparation method according to claim 4, wherein In step (3), the molar ratio of the Gal-G5-DMC and chloroauric acid is 1:50-75; the strong reducing agent is selected from sodium borohydride, hydrazine hydrate or ascorbic acid; preferably, it is sodium borohydride, and the molar ratio of the chloroauric acid to sodium borohydride is 1:3-4; in the product Gal-G5(Au)-DMC, the molar ratio of Gal, G5, DMC and Au is 12-17:1:25-30:25-75.

8. A pharmaceutical composition, characterized in that The composition comprises an active dose of the endogenous and exogenous dual nanosensitizer according to any one of claims 1 to 3; and further comprises a PD-L1 antibody.

9. Use of the endogenous and exogenous dual nanosensitizer according to any one of claims 1 to 3 and the pharmaceutical composition according to claim 8 in the preparation of drugs for treating brain gliomas.

10. The use according to claim 9, characterized in that The brain glioma drug is an injection, and further, is an intravenous injection.

Images