Gold nano-cluster taking 1-thiomercaptoglucose as ligand and preparation method and application of gold nano-cluster

Abstract

The invention discloses a gold nano-cluster modified by using 1-thiomercaptoglucose as a ligand as well as a preparation method and application of the gold nano-cluster, and belongs to the technical field of biological medicines. The gold nano-cluster has a strong fluorescence emission characteristic in a near-infrared second region (NIR-II, 900-1300nm), can penetrate through a blood brain barrier (BBB) and is specifically enriched at a brain glioma part, so that high-resolution in-vivo imaging is realized. Meanwhile, the gold nanocluster has the functions of radiotherapy sensitization and efficient anti-tumor effect by inducing pyroptosis of tumor cells, and has the functions of diagnosis and treatment. Experiments show that the nano-drug has good biocompatibility, can significantly inhibit the growth of brain glioma of mice, and provides a novel solution for precise diagnosis and treatment of brain tumor.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a gold nanocluster modified with 1-thiomercaptoglucose as a ligand, its preparation method and application. Background Technology

[0002] Gliomas are among the most aggressive malignant tumors of the central nervous system, and their treatment has long been limited by the strict barrier of the blood-brain barrier (BBB). The BBB, through tight junctions of endothelial cells and active efflux mechanisms, prevents most therapeutic drugs from entering the brain parenchyma, resulting in extremely low accumulation efficiency of traditional chemotherapy drugs (such as temozolomide) at the tumor site. Furthermore, commonly used clinical imaging techniques (such as MRI and CT) have insufficient sensitivity and resolution for early-stage small tumors and cannot provide real-time dynamic monitoring of drug distribution and efficacy.

[0003] Although fluorescence imaging techniques (such as near-infrared I imaging, NIR-I) have been attempted for intraoperative navigation, their tissue penetration depth is typically less than 5 mm, and they are severely interfered with by the autofluorescence of biological tissues, making it difficult to achieve high-resolution visualization of deep brain tumors. In recent years, near-infrared II (NIR-II, 1000-1700 nm) imaging technology, due to its low scattering and low absorption characteristics caused by long wavelengths, has shown significant advantages in centimeter-level penetration depth and micrometer-level resolution, providing a new direction for the precise diagnosis of gliomas. However, existing NIR-II probes (such as carbon nanotubes and quantum dots) generally face problems such as poor biocompatibility, slow metabolism, and difficulty in targeted modification, resulting in slow progress in clinical translation. Meanwhile, the exploration of novel cell death mechanisms in the field of tumor therapy continues to deepen. Among them, pyroptosis, a programmed cell death mode mediated by Gasdermin protein, has become a research hotspot for overcoming the bottleneck of traditional chemotherapy resistance because it can trigger the inflammatory microenvironment and activate anti-tumor immune responses. However, currently reported pyroptosis inducers (such as certain chemotherapy drugs or nanomaterials) lack specific targeting ability for gliomas and cannot penetrate the BBB, limiting their application in brain tumors.

[0004] Gold nanoclusters (AuNCs) have attracted much attention in the field of integrated tumor diagnosis and treatment due to their small size, easy surface functionalization, tunable fluorescence, and high biocompatibility. However, current research has focused on their fluorescence emission in the NIR-I region, and their functional designs are relatively simple (such as being used only for imaging or drug delivery). There are no multifunctional gold nanocluster materials that can simultaneously achieve NIR-II deep imaging, efficient BBB penetration, tumor targeting, radiosensitization, and pyroptosis induction.

[0005] In summary, developing a therapeutic nanomedicine that combines NIR-II imaging, autonomous BBB penetration, tumor targeting, and endogenous pyroptosis induction is an urgent need to address the challenges in the clinical diagnosis and treatment of gliomas. Currently, there is no systematic approach to simultaneously fulfilling these multiple functions through ligand-engineered gold nanoclusters. Summary of the Invention

[0006] In view of the technical problems of poor biocompatibility, slow metabolism, difficulty in targeted modification and single function of NIR-II probes in the prior art, the present invention aims to provide a gold nanocluster modified with 1-thiothioglucose as a ligand, its preparation method and application.

[0007] To achieve the above objectives, the present invention employs the following technical solution:

[0008] This invention provides a method for preparing gold nanoclusters modified with 1-thiomercaptoglucose as a ligand, comprising: stirring and mixing HAuCl4 with 1-thiomercaptoglucose to obtain a precursor complex; adding a reducing agent to the precursor complex for reaction, ultrafiltration, dialysis, and drying to obtain gold nanoclusters modified with 1-thiomercaptoglucose as a ligand.

[0009] The molar ratio of HAuCl4 to 1-thiomercaptoglucose is 1:0.5 to 10.

[0010] Furthermore, the molar ratio of HAuCl4 to 1-thiomercaptoglucose is 1:4.

[0011] The stirring speed is 500-800 rpm.

[0012] The molar ratio of HAuCl4 to the reducing agent is 1:0.1 to 2, and the reducing agent is any one of NaBH4, SC, VC, HH and TA.

[0013] Furthermore, the reducing agent is NaBH4.

[0014] Furthermore, the molar ratio of HAuCl4 to the reducing agent is 1:0.25.

[0015] The ultrafiltration rate is 4000-5000 rpm for 10-15 minutes.

[0016] The dialysis molecular weight cutoff is 100–500D, and the dialysis time is 24–48 hours.

[0017] The present invention provides a gold nanocluster modified with 1-thiothioglucose as a ligand obtained by the above preparation method, wherein the gold nanocluster has a particle size ≤5nm.

[0018] The gold nanoclusters exhibit NIR-II fluorescence emission in the 900-1300 nm range.

[0019] The application of the gold nanoclusters modified with 1-thiomercaprolactone as ligands in the preparation of products for the diagnosis and treatment of glioma, wherein the products for the diagnosis and treatment of glioma are fluorescent imaging reagents obtained by using the NIR-II fluorescence emission of gold nanoclusters modified with 1-thiomercaprolactone as ligands.

[0020] The application of the gold nanoclusters modified with 1-thiomercaptoglucose as ligand in the preparation of integrated diagnostic and therapeutic formulations for glioma.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention provides a method for preparing 1-thiomercaptoglucose-modified gold nanoclusters. Through a combination of molecular-level ligand design and a mild reduction process, a rapid one-step synthesis at room temperature (total reaction time <15 minutes) is achieved, realizing the precise synthesis of gold nanoclusters (AuNCs) without the need for complex equipment or high-temperature, high-pressure conditions, thus facilitating their large-scale production. The thiol group (-SH) of 1-thiomercaptoglucose and Au... 3+ Strong coordination occurs, forming a stable [Au-TG] n+ The formation of the complex restricts the free growth of the gold nucleus, providing a "template" for subsequent reduction reactions. The glucose group (-OH) of the thiomercaptoglucose in 1-thiomercaptoglucose forms steric hindrance through hydrogen bonding, inhibiting the aggregation of Au nuclei. The glucose structure of 1-thiomercaptoglucose remains intact, preserving its binding ability to the GLUT1 transporter, laying the foundation for subsequent targeted modification. The addition of the reducing agent further enhances the Au... 3+ Reduced to Au 0 And under the encapsulation of 1-thiomercaptoglucose ligand, ultrasmall gold nanoclusters are formed.

[0023] The 1-thiomercaptoglucose ligand-modified gold nanoclusters provided by this invention have a particle size ≤5nm. Small-particle-size gold nanoclusters have better biocompatibility and permeability, and can more easily cross biological barriers, such as the blood-brain barrier. This is particularly important for their application in the diagnosis and treatment of glioma, as it can increase their enrichment in tumor tissue.

[0024] Furthermore, gold nanoclusters exhibit strong fluorescence emission in the near-infrared II (NIR-II, 900-1300 nm) region. NIR-II fluorescence imaging offers advantages such as large tissue penetration depth and low background fluorescence interference. These characteristics enable gold nanoclusters to provide clearer and more accurate images in in vivo biological imaging, facilitating earlier and more accurate detection of gliomas. TG-AuNCs have a Zeta potential of around -30 mV and exhibit good colloidal stability, ensuring their application in biological environments.

[0025] Furthermore, this gold nanocluster possesses radiosensitizing properties and exerts a highly effective anti-tumor effect by inducing pyroptosis in tumor cells, combining diagnostic and therapeutic functions. Its ability to actively target and identify the tumor microenvironment provides a foundation for its application in tumor diagnosis and treatment, enabling more precise targeting of tumor sites and improving treatment efficacy and diagnostic accuracy. This nanomedicine exhibits good biocompatibility and can significantly inhibit the growth of gliomas in mice, providing a novel solution for the precision diagnosis and treatment of brain tumors.

[0026] The applications provided by this invention, based on their near-infrared II fluorescence properties and ability to actively target the tumor microenvironment, allow gold nanoclusters to be used in the diagnosis and treatment of gliomas, offering new methods and approaches for glioma diagnosis and treatment. Utilizing their NIR-II fluorescence imaging advantages, high-sensitivity, high-resolution imaging of gliomas can be achieved, aiding in early diagnosis and precise tumor localization; furthermore, by modulating the immune response to enhance the body's anti-tumor immunity, a new strategy for the immunotherapy of gliomas is provided. Attached Figure Description

[0027] Figure 1 Physical images and fluorescence images of TG-AuNCs prepared with different ratios of HAuCl4 aqueous solution and 1-mercaptoglucose aqueous solution;

[0028] Figure 2 Fluorescence patterns of TG-Au NCs prepared with different reducing agents;

[0029] Figure 3 Fluorescence patterns of TG-Au NCs prepared with different reducing agent ratios;

[0030] Figure 4 This is a process flow diagram of the preparation of TG-AuNCs in Example 1 of the present invention;

[0031] Figure 5 The UV-Vis absorption spectrum and fluorescence emission spectrum under 808 nm excitation light of TG-Au NCs prepared in Example 1 of this invention are shown.

[0032] Figure 6Transmission electron microscopy image of TG-Au NCs prepared in Example 1 of this invention;

[0033] Figure 7 This is a particle size distribution diagram of TG-Au NCs prepared in Example 1 of the present invention;

[0034] Figure 8 The surface potential diagram of TG-Au NCs prepared in Example 1 of this invention;

[0035] Figure 9 The fluorescence stability diagram of TG-Au NCs prepared in Example 1 of this invention;

[0036] Figure 10 This is a graph showing the efficiency of TG-AuNCs across the blood-brain barrier at different glucose concentrations in an in vitro simulated blood-brain barrier.

[0037] Figure 11 The images show NIR-II imaging of TG-Au NCs targeting tumors across the blood-brain barrier in mice after ingesting different glucose concentrations in an in vivo simulated blood-brain barrier. In the image, A is the imaging image and B is the change in fluorescence intensity.

[0038] Figure 12 Pyroptosis induction diagram of TG-Au NCs prepared in Example 1;

[0039] Figure 13 This is a comparative diagram showing the effects of the supernatant from the pyroptosis of glioma cells (GL261 cells) induced by TG-Au NCs of the present invention on indicators of mouse bone marrow-derived dendritic cells (DC2.4 cells);

[0040] Figure 14 This is a flow cytometry diagram showing the effect of TG-Au NCs of the present invention on the polarization of DC cells in the cervical lymph nodes of a mouse model of glioma.

[0041] Figure 15 This is a dynamic monitoring diagram showing the effect of TG-Au NCs of the present invention on tumor growth in a mouse model of glioma.

[0042] Figure 16 This is an H&E staining image showing the effects of TG-Au NCs of the present invention on various organs and tissues of a mouse model of glioma. Detailed Implementation

[0043] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0044] The present invention will now be described in further detail with reference to the accompanying drawings:

[0045] In this invention, only the ratio of HAuCl4 aqueous solution to 1-mercaptoglucose aqueous solution was changed, and the subsequent steps in the examples were continued to prepare TG-Au NCs. Imaging was performed using the NIR-II small animal imaging system under the same parameters, and the fluorescence intensity was detected using a steady-state transient fluorescence spectrometer. The results are shown in […]. Figure 1 It can be observed that the optimal molar ratio of HAuCl4 aqueous solution to 1-mercaptoglucose aqueous solution is 1:4.

[0046] In this invention, only the type of reducing agent was changed, and the subsequent steps in the examples were continued to prepare TG-Au NCs. Imaging was performed using the NIR-II small animal imaging system under the same parameters, and the fluorescence intensity was detected using a steady-state transient fluorescence spectrometer. The results are shown in […]. Figure 2 It can be observed that TG-Au NCs obtained by NaBH4 participating in the reaction have the best fluorescence intensity.

[0047] In this invention, only the ratio of HAuCl4 aqueous solution to NaBH4 aqueous solution was changed, and the subsequent steps in the examples were continued to prepare TG-Au NCs. Imaging was performed using the NIR-II small animal imaging system under the same parameters, and the fluorescence intensity was detected using a steady-state transient fluorescence spectrometer. The results are shown in […]. Figure 3 It can be observed that the optimal ratio of HAuCl4 aqueous solution to NaBH4 aqueous solution is 1:0.25. I. Specific Implementation Methods

[0049] Example 1

[0050] See appendix Figure 4 This embodiment provides a gold nanocluster modified with 1-thiomercaptoglucose as a ligand, and the specific preparation process is as follows:

[0051] At room temperature (20-25℃), 1 mL of a 13.21 mM HAuCl4 aqueous solution (4.49 mg, molecular weight 339.79 g / mol) was mixed with 4 mL of a 13.20 mM 1-mercaptoglucose aqueous solution (2.88 mg, molecular weight 218.20 g / mol). The solution changed from golden yellow to dark brown. The mixture was stirred at a constant speed of 500 rpm for 10 minutes to form a precursor complex. Subsequently, 0.5 mL of 6.61 mM Na... The reaction of 0.25 mg of BH4 aqueous solution (molecular weight 37.83 g / mol) was instantaneous, generating TG-AuNCs nanoclusters with NIR-II fluorescence characteristics, with no significant change in solution color. The reaction solution was transferred to an ultrafiltration centrifuge tube (molecular weight cutoff 1 kDa) and centrifuged at 4000 rpm for 15 minutes to remove unreacted small molecule impurities. The ultrafiltration solution was then transferred to a 100-500 D dialysis bag and dialyzed with pure water in the dark for 24 hours to obtain a purified TG-AuNCs solution, which was stored at 4°C in the dark.

[0052] The properties of the gold nanoclusters modified with 1-thiomercaptoglucose as ligand prepared in Example 1 were tested. Specific results are shown in the appendix. Figures 5-9 As shown.

[0053] See appendix Figure 5 The TG-AuNCs solution prepared under the conditions of this embodiment was tested using a UV-Vis spectrophotometer and a steady-state transient fluorescence spectrometer. The absorbance (Abs) showed no obvious absorption peak with increasing wavelength (600-1200 nm), remaining at a relatively low level overall and changing slowly with increasing wavelength. The fluorescence intensity (PL) showed a significant fluorescence emission peak in the near-infrared II region, with the peak value located at approximately 1050 nm, indicating strong fluorescence emission capability in this region. Combining the UV absorption and fluorescence emission data, TG-AuNCs exhibits weak absorption in the UV-Vis region but strong fluorescence emission in the near-infrared II region. This characteristic reduces background interference and improves the sensitivity and accuracy of imaging in biomedical imaging. It also provides an optical basis for its application in deep tissue imaging.

[0054] See appendix Figure 6 The structure of TG-AuNCs prepared under the conditions of this embodiment was observed and analyzed by transmission electron microscopy. (See attached...) Figure 6As can be seen, the nanoclusters exhibit an approximately spherical morphology. This spherical structure endows the nanoclusters with high symmetry and stability, allowing for relatively uniform dispersion in solution and reducing aggregation. The relatively smooth surface morphology, lacking obvious edges or sharp structures, helps reduce non-specific adsorption that may occur when the nanoclusters interact with biomolecules or other substances, improving their specificity and biocompatibility in biological applications. Throughout the observation area, the distribution of the nanoclusters is relatively uniform, with no obvious aggregation or clustering. This indicates that the TG-AuNCs obtained during the preparation process have good dispersibility, which is beneficial for maintaining stable performance in subsequent applications.

[0055] See appendix Figure 7 The particle size distribution of TG-AuNCs prepared under the conditions of this embodiment was analyzed using a DLS analyzer. (See attached...) Figure 7 It can be seen that the particle size of TG-Au NCs is mainly concentrated around 2-3 nm, with most nanoclusters having a particle size of around 3 nm, which accounts for the highest proportion. This indicates that the prepared TG-Au NCs have a relatively concentrated particle size distribution.

[0056] See appendix Figure 8 The surface potential of TG-AuNCs prepared under the conditions of this embodiment was analyzed using a Zeta potential analyzer. (See attached...) Figure 8 It is known that the Zeta potential of TG-Au NCs is around -30mV. The relatively high Zeta potential gives AuNCs good colloidal stability, which ensures their application in biological environments.

[0057] See appendix Figure 9 The fluorescence stability of TG-Au NCs prepared under the conditions of this embodiment was analyzed using an NIR-II small animal imaging system and a steady-state and transient fluorescence spectrometer. (See attached...) Figure 9 It can be seen that the fluorescence of TG-Au NCs can maintain good stability within 96 hours.

[0058] Example 2

[0059] This embodiment provides a gold nanocluster modified with 1-thiomercaptoglucose as a ligand, and the specific preparation process is as follows:

[0060] At room temperature (20-25℃), 1 mL of a 13.21 mM HAuCl4 aqueous solution (4.49 mg, molecular weight 339.79 g / mol) was mixed with 5 mL of a 13.20 mM 1-mercaptoglucose aqueous solution (2.88 mg, molecular weight 218.20 g / mol). The solution changed from golden yellow to dark brown. The mixture was stirred at a constant speed of 500 rpm for 10 minutes to form a precursor complex. Subsequently, 1 mL of 6.61 mM NaB2 was rapidly added. The reaction was completed instantaneously in an aqueous H4 solution (0.25 mg, molecular weight 37.83 g / mol), generating TG-AuNCs nanoclusters with NIR-II fluorescence characteristics, with no significant change in solution color. The reaction solution was transferred to an ultrafiltration centrifuge tube (molecular weight cutoff 1 kDa) and centrifuged at 4000 rpm for 15 minutes to remove unreacted small molecule impurities. The ultrafiltration solution was then transferred to a 100-500D dialysis bag and dialyzed with pure water in the dark for 24 hours to obtain a purified TG-AuNCs solution, which was stored at 4°C in the dark.

[0061] II. Performance Verification

[0062] 1. Validation of blood-brain barrier penetration and tumor targeting function of TG-AuNCs

[0063] (1) In vitro blood-brain barrier model experiment

[0064] A blood-brain barrier model was constructed by seeding mouse brain microvascular endothelial cells (bEnd.3) into Transwell cell culture chambers. Transendothelial impedance (TEER) values ​​were periodically measured. When the TEER value exceeded 150 Ω·cm, a blood-brain barrier model was established. 2 This indicates that the in vitro blood-brain barrier model was successfully constructed. The TEER value is an important indicator for measuring the integrity and function of the blood-brain barrier; a higher TEER value means that the cell layer has a better barrier capacity.

[0065] The commonly used culture medium for mouse brain microvascular endothelial cells (bEnd.3) contains a glucose concentration of 4.5 mg / mL. A high-glucose medium with a glucose concentration of 9.455 mg / mL was obtained by mixing 50% glucose injection with the culture medium at a volume ratio of 1:99. TG-AuNCs prepared in Example 1 were added to the upper chambers of the two culture media containing different glucose concentrations. To investigate the effect of glucose concentration on the penetration of TG-AuNCs across the blood-brain barrier, the culture chambers were incubated at 37°C for 12 hours. The fluorescence intensity in the lower chamber solution was then measured. The results are shown in the appendix. Figure 10 As shown.

[0066] From the appendix Figure 10The data show that the fluorescence intensity of the low-glucose group was significantly higher than that of the high-glucose group, with the low-glucose group having a fluorescence intensity of approximately 3200 and the high-glucose group approximately 2300. This indicates that under low glucose concentration, more TG-Au NCs can penetrate the blood-brain barrier and enter the lower ventricle. In contrast, under high glucose concentration, the penetration amount of TG-Au NCs is relatively small. This difference suggests a competitive uptake relationship between TG-Au NCs and glucose. TG-Au NCs can compete with glucose for transport channels on the blood-brain barrier, and their ability to penetrate the blood-brain barrier is affected by glucose concentration. This provides important in vitro experimental evidence for subsequent research on the blood-brain barrier penetration and tumor targeting mechanism of TG-Au NCs in vivo.

[0067] (2) In vivo glioma model experiment

[0068] A glioma model was established in 4-6 week old female C57 mice, with the inoculation site being the right caudate nucleus of the brain. Mice were anesthetized with isoflurane inhalation, their heads were shaved, and their heads were fixed in a prone position on a stereotactic frame. After disinfecting the skin of the top of the head with 75% ethanol, a vertical incision was made behind the eye fissure at the midline of the skull to expose the anterior fontanelle. The skull was then drilled through with a 1mm diameter animal skull drill, 1 mm anterior to the midpoint of the anterior fontanelle and 2.5 mm to the right of the sagittal suture, using a 5mL GL... A microsyringe for 261-luc cell suspension was vertically inserted into the white matter region of the brain through a drilled hole. The insertion depth was 3 mm from the skull surface. Before injection, the needle was slightly withdrawn by about 0.5 mm. The tumor cell suspension was slowly injected at an injection rate of 0.5 μL / min. After a 2-minute pause before injection, the needle was slowly withdrawn. The scalp was sutured, and the area was disinfected with 75% ethanol. Fourteen days after inoculation, mice were randomly divided into two groups (Non-block group and Block group). The Non-block group received direct injection of TG-AuNCs via tail vein. The Block group received glucose injection 4 hours prior to injection, followed by simultaneous tail vein injection of TG-AuNCs (5 mg / kg) with the Non-block group. NIR-II in vivo imaging was performed on all mice at 0, 0.5, 2, 4, 8, 12, 24, and 48 hours after TG-AuNCs injection. The distribution and accumulation of TG-AuNCs in mice were monitored in real time using the near-infrared II fluorescence characteristics. The results are shown in [Figure number missing]. Figure 11 .

[0069] From the appendix Figure 11The results showed that in the Non-block group, fluorescence intensity gradually increased after injection, reaching a peak of approximately 170 a.u. at around 8–12 hours, and then slowly decreased. This indicates that TG-Au NCs successfully penetrated the blood-brain barrier and accumulated at the glioma site, with the accumulation reaching its maximum at 8–12 hours. In the Block group, the increase in fluorescence intensity was significantly lower than that in the Non-block group, with a peak of approximately 110 a.u., and the fluorescence intensity remained lower than that in the Non-block group throughout the observation period. This suggests that pre-injection of glucose solution reduced the number of TG-Au NCs that penetrated the blood-brain barrier and accumulated at the glioma site due to competition between glucose and TG-Au NCs for transport channels on the blood-brain barrier. This indicates that pre-injection of glucose has a significant inhibitory effect on the accumulation of TG-Au NCs at the glioma site, further confirming that TG-Au NCs achieve penetration and targeted accumulation by competing with glucose for transport channels on the blood-brain barrier.

[0070] 3. Verification of pyroptosis induction and cancer cell killing function of TG-AuNCs

[0071] Mouse glioma cells GL261 were cultured in DMEM high glucose medium (with 10% fetal bovine serum and 1% penicillin-streptomycin) and cultured in a cell culture incubator at 37°C and 5% CO2. Cell growth was observed regularly and the cells were passaged to ensure that the cell number and viability met the experimental requirements.

[0072] GL261 cells were divided into two groups: the Ctrl group (control group) and the TG-AuNCs group. TG-AuNCs were added to the culture system of the TG-AuNCs group cells and incubated. After incubation, the morphology of the two groups of cells was observed under a microscope. (See Appendix) Figure 12 Supernatants from both cell groups were collected, and the LDH activity in the supernatants was detected using a lactate dehydrogenase (LDH) assay kit. The results are shown in the appendix. Figure 13 .

[0073] From the appendix Figure 12 It was observed that, using apoptosis and necrosis staining, cell blistering and swelling were clearly visible in the TG-Au NCs group, preliminarily indicating that TG-Au NCs can induce pyroptosis in GL261 cells; LDH is a stable cytoplasmic enzyme widely present in various organisms. Normally, it cannot pass through the cell membrane, but it can be released extracellularly when cells are damaged or die, and the released LDH is found in the culture medium supernatant. (The last sentence appears to be incomplete and possibly refers to a separate topic.) Figure 13 This further confirms that TG-Au NCs can induce membrane rupture in GL261 cells, releasing their contents, and increasing LDH release by about 5 times, meaning that the pyroptosis level in the TG-Au NCs group was significantly higher than that in the control group.

[0074] 4. Validation of the immune activation function induced by TG-AuNCs

[0075] Dendritic cells (DCs) recognize pathogens in the innate immune system and activate immune cells in the adaptive immune system. DCs are activated or mature when pattern recognition receptors (PRRs) on them recognize pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). CD80 (B7-1) and CD86 (B7-2) proteins, located on the surface of DCs and belonging to the cell surface immunoglobulin superfamily, interact with their T cell ligands CD28 and CTLA-4 to regulate T cell activation and tolerance, cytokine expression, cytotoxic T lymphocyte (CTL) production, antigen-presenting cell maturation, and promote their survival and function. After incubating glioma cells (GL 261 cells) at the experimental concentration of TG-AuNCs and inducing pyroptosis, the supernatant was used to incubate mouse bone marrow-derived dendritic cells (DC2.4 cells) for 24 hours. The DC2.4 cells were then incubated with the supernatant of normally cultured GL 261 cells for 24 hours. The differences in DC2.4 cell polarization indices between the two groups were compared. The results are shown in the appendix. Figure 14 The maturity level of DC2.4 was 2.7 times higher than that of the control group.

[0076] A glioma model was established in six 4-6 week old C57 female mice. The mice were randomly divided into two groups: the Ctrl group and the TG-AuNCs group. Seven days after model establishment, the TG-AuNCs group received a tail vein injection of TG-AuNCs at a dose of 5 mg / kg. Three days after injection, all mice were euthanized, and cervical lymph nodes were harvested, thoroughly ground, fixed, stained, and used to observe dendritic cell polarization. The results are shown in the appendix. Figure 14 The maturation level of DC cells in cervical lymph nodes was 6.5 times higher than that in the control group.

[0077] 5. Validation of the antitumor function of TG-AuNCs

[0078] A glioma model was established in 10 female C57 mice aged 4–6 weeks. The mice were randomly divided into two groups: the Ctrl group and the TG-Au NCs group, with an administration dose of 5 mg / kg. Tumor size was monitored every 7 days after treatment. Results are shown in the appendix. Figure 15 .

[0079] From the appendix Figure 15Data showed that during post-drug administration monitoring, glioma growth in mice in the TG-AuNCs group was significantly inhibited compared to the Ctrl group. At day 7, the tumor volume in the TG-AuNCs group was significantly smaller than that in the Ctrl group (P < 0.05); this difference became increasingly significant on days 14 and 21 (P < 0.01). Tumors in some mice in the TG-AuNCs group showed significant shrinkage at day 21, while tumors in the Ctrl group continued to enlarge. This indicates that TG-AuNCs have significant anti-glioma function at a dose of 5 mg / kg.

[0080] 6. Biosafety evaluation of TG-AuNCs

[0081] Six 4-6 week old female C57 mice were randomly divided into two groups: the Ctrl group and the TG-AuNCs group. The dosage was 5 mg / kg. No mice showed death or abnormal behavior within 7 days of administration. The mice were euthanized after 7 days, and H&E staining was performed on the heart, liver, spleen, lung, and kidney tissues of each group. (See attached image). Figure 16 The results showed that the cellular structure of each organ was intact, with no signs of inflammation or necrosis. This result further confirmed that TG-AuNCs did not have any adverse effects on normal tissues of mice at the given dose.

[0082] Based on the survival status, behavioral performance, and histopathological examination results of mice, it can be concluded that TG-Au NCs have no adverse effects on normal tissues at a dosage of 5 mg / kg, demonstrating good biosafety. This provides important safety evidence for the further application of TG-Au NCs in the biomedical field.

[0083] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for preparing gold nanoclusters modified with 1-thiomercaptoglucose as a ligand, characterized in that, include: HAuCl4 and 1-thiomercaptoglucose were stirred and mixed to obtain a precursor complex; a reducing agent was added to the precursor complex for reaction, followed by ultrafiltration, dialysis, and drying to obtain gold nanoclusters modified with 1-thiomercaptoglucose as a ligand.

2. The method for preparing gold nanoclusters modified with 1-thiomercaptoglucose as a ligand according to claim 1, characterized in that, The molar ratio of HAuCl4 to 1-thiomercaptoglucose is 1:0.5 to 10.

3. The method for preparing gold nanoclusters modified with 1-thiomercaptoglucose as a ligand according to claim 1, characterized in that, The stirring speed is 500-800 rpm.

4. The method for preparing gold nanoclusters modified with 1-thiomercaptoglucose as a ligand according to claim 1, characterized in that, The molar ratio of HAuCl4 to the reducing agent is 1:0.1 to 2, and the reducing agent is any one of NaBH4, SC, VC, HH and TA.

5. The method for preparing gold nanoclusters modified with 1-thiomercaptoglucose as a ligand according to claim 1, characterized in that, The ultrafiltration rate is 4000-5000 rpm for 10-15 minutes.

6. The method for preparing gold nanoclusters modified with 1-thiomercaptoglucose as a ligand according to claim 1, characterized in that, The dialysis molecular weight cutoff is 100–500D, and the dialysis time is 24–48 hours.

7. A gold nanocluster modified with 1-thiomercaptoglucose as a ligand, obtained by the preparation method according to any one of claims 1 to 6, characterized in that, The gold nanoclusters have a particle size ≤ 5 nm.

8. The gold nanocluster modified with 1-thiomercaptoglucose as a ligand according to claim 7, characterized in that, The gold nanoclusters exhibit NIR-II fluorescence emission in the 900-1300 nm range.

9. The application of the gold nanocluster modified with 1-thiomercaptoglucose as a ligand as described in claim 7 or 8 in the preparation of products for the diagnosis and treatment of glioma, characterized in that, The product for diagnosing and treating gliomas is a fluorescent imaging reagent obtained by NIR-II fluorescence emission of gold nanoclusters modified with 1-thiomercaptoglucose as a ligand.

10. The use of the gold nanocluster modified with 1-thiomercaptoglucose as a ligand as described in claim 7 or 8 in the preparation of an anti-glioma drug.

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