A triple-target 10 B4C nanosheets, their preparation methods, and applications

Abstract

This invention relates to the fields of biomedicine and nanomaterials technology, specifically to a triple-targeting method. 10 B4C nanosheets, their preparation methods, and applications. The technical solution of this invention is a triple-targeting... 10 B4C nanosheets, including 10 B4C nanosheets and modifications 10 Targeting peptides on the surface of B4C nanosheets; the targeting peptides include NGR peptide, NLS peptide, and PD-L1 targeting peptide. Preparation method: [The text abruptly ends here, so the translation stops as well.] 10 B4C powder was ball-milled to obtain 10 B4C nanosheets; using glycidol for... 10 B4C nanosheets were surface functionalized to obtain 10 B4C-PG nanosheets; will 10 B4C-PG nanosheets, NGR peptide, NLS peptide, and PD-L1 targeting peptide undergo a coupling reaction in the presence of a coupling agent to obtain a triple targeting peptide. 10 B4C nanosheets. The main application of this invention is to provide a new option for BNCT boron delivery agents.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and nanomaterials technology, specifically to a triple-targeting method. 10 B4C nanosheets, their preparation methods, and applications. Background Technology

[0002] Boron neutron capture therapy (BNCT) is a promising binary targeted radiotherapy strategy. Its mechanism of action is based on stable isotopes. 10 B undergoes nuclear fission after capturing a thermal neutron, producing alpha particles with high linear energy transfer (LET). 7 Li recoil nuclei. These particles have an extremely short range in biological tissues (about 5–9 μm), comparable to the diameter of a single cell, thus enabling them to precisely kill cells undergoing neutron capture reactions while causing minimal damage to surrounding normal tissues.

[0003] However, the efficacy of BNCT is highly dependent on the efficient and specific enrichment of boron drugs at the tumor site. An ideal boron delivery agent for BNCT must meet the following conditions: (1) in tumor tissue... 10 The concentration of B should not be less than 20 μg / g; (2) the ratio of tumor to normal tissue (T / N) and tumor to blood (T / B) 10 The B concentration ratio is greater than 3, thus ensuring the selectivity and safety of the treatment. Early clinical studies of BNCT used boric acid and its simple derivatives, but their clinical application was severely limited due to the inherent defects of these small molecule drugs, such as poor targeting, rapid clearance in vivo, and high toxicity to normal tissues.

[0004] BPA is mainly enriched in certain types of tumors via the amino acid transporter LAT1, but its targeting ability remains limited and its intratumoral distribution is uneven. BSH, on the other hand, is mainly distributed within blood vessels and has difficulty effectively penetrating cell membranes to enter tumor cells, resulting in a typically low T / B ratio, which limits its application in the treatment of most solid tumors. Therefore, developing novel and efficient boron delivery systems is crucial to achieve… 10 The high concentration and specific accumulation of B in tumor cells remains a core challenge for the further development of BNCT.

[0005] On the other hand, tumor immunotherapy, especially immune checkpoint blockade therapy targeting programmed death protein-1 (PD-1) and its ligand (PD-L1), has made significant progress in recent years. This therapy activates the body's own anti-tumor immune response by relieving the immunosuppression of T cells by the tumor microenvironment. However, its clinical application still faces problems such as limited response rates and the easy development of drug resistance.

[0006] Given the limitations of monotherapy, combination therapy has become an important direction for the development of cancer treatment. However, most existing BNCT boron delivery agents are mainly based on single-target or single-therapeutic functions, and there is a lack of combination therapy boron delivery systems that can integrate multiple targets and multimodal therapies. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a triple targeting method. 10 B4C nanosheets address the issues of poor targeting, high toxicity to normal tissues, single targeting, and inability to achieve multimodal synergistic therapy associated with existing BNCT boron delivery agents.

[0008] The technical solution of the present invention is as follows:

[0009] This invention provides a triple targeting 10 B4C nanosheets, including 10 B4C nanosheets and modifications in 10 Targeted peptides on the surface of B4C nanosheets;

[0010] The targeted peptides include NGR peptide, NLS peptide, and PD-L1 targeted peptide.

[0011] Through the synergistic effect of NGR peptide (targeting tumor angiogenesis) and NLS peptide (targeting the cell nucleus), it was achieved 10 B4C nanosheets enable precise delivery to tumor cell nuclei, overcoming the poor targeting issues of existing BNCT boron delivery agents. This design holds promise for significantly improving… 10 The enrichment concentration of B near the tumor cell nucleus satisfies the requirements of BNCT. 10 High requirements for B concentration and T / N and T / B ratios. Combining BNCT, immune checkpoint blockade therapy (achieved through PD-L1 targeted peptides) with photothermal therapy (based on...) 10 The B4C nanosheet integrates three functions: photothermal properties, catalytic activity, and immunogenicity. Its synergistic mechanism lies in: the high-LET particles generated by BNCT can precisely kill tumor cells; the PD-L1 targeting peptide can block immune checkpoint pathways and reverse immunosuppression; simultaneously, the photothermal effect generated by the nanosheet under near-infrared laser irradiation can directly ablate tumors. This synergistic effect promises to achieve an organic combination of physical killing and immune activation. This triple targeting... 10 B4C nanosheets with 10Using B4C nanosheets as a carrier, the surface is co-modified with NGR peptide, PD-L1 targeting peptide, and NLS peptide, achieving hierarchical targeting of tumor tissue, PD-L1, and tumor cell nuclei. Based on this design, the nanosheets can not only precisely locate boron neutron capture therapy (BNCT) to the cell nucleus, but also have the functions of immune checkpoint blockade and photothermal therapy, realizing multimodal synergistic therapy and solving the problem that existing BNCT boron delivery agents cannot achieve multimodal synergistic therapy.

[0012] In summary, this triple targeting 10 B4C nanosheets integrate tumor tissue targeting, immune checkpoint targeting, and nuclear targeting into a single nanoplatform, possessing triple targeting capabilities. This enables highly efficient enrichment of boron drugs in tumor cell nuclei, simultaneously exerting synergistic therapeutic effects of immunomodulation and photothermal killing. It also overcomes the limitation of existing BNCT boron delivery agents by focusing solely on a single target, breaking through the functional limitations of current BNCT boron delivery agents.

[0013] Furthermore, the triple targeting 10 The average hydrated particle size of B4C nanosheets is 194 nm.

[0014] Furthermore, the aforementioned 10 The surface of B4C nanosheets was functionalized with glycidol to obtain glycidol-functionalized nanosheets. 10 B4C nanosheets (denoted as B4C nanosheets) 10 B4C-PG nanosheets);

[0015] The glycidol functionalized 10 Surface modification of the target peptide with B4C nanosheets yields triple-targeted peptides. 10 B4C nanosheets.

[0016] Triple Targeting 10 The surface of B4C nanosheets is functionalized with glycidol to form a hydrophilic layer, giving them good dispersibility and stability in aqueous solutions.

[0017] This invention also provides the above-mentioned triple targeting 10 The preparation method of B4C nanosheets includes the following steps:

[0018] S1, to 10 B4C powder was ball-milled to obtain 10 B4C nanosheets;

[0019] S2, Using glycidol on the above 10 B4C nanosheets were surface functionalized to obtain glycidol-functionalized products. 10 B4C nanosheets, i.e. 10 B4C-PG nanosheets;

[0020] S3, the above 10 B4C-PG nanosheets, NGR peptide, NLS peptide, and PD-L1 targeting peptide undergo a coupling reaction in the presence of a coupling agent to obtain the triple targeting peptide. 10 B4C nanosheets.

[0021] This preparation method mainly includes three steps: ball milling, surface functionalization, and coupling reaction. It eliminates the need for complicated procedures and boasts the advantage of a simple preparation process. The ball-milled... 10 B4C powder was prepared into 10 B4C nanosheets are beneficial 10 Surface functionalization and coupling with other substances on B4C nanosheets. NGR peptides, NLS peptides, and PD-L1 targeting peptides were integrated into the nanosheets via coupling reactions. 10 On the surface of B4C-PG nanosheets, a triple-targeted structure was finally prepared. 10 B4C nanosheets have a triple targeting function, enabling precise identification and enrichment of tumor cells. While improving the therapeutic effect, they also reduce damage to normal tissues, solving the problem of high toxicity to normal tissues of existing BNCT boron delivery agents.

[0022] Furthermore, S1 includes: taking 10 B4C powder was added to ultrapure water, ball-milled, centrifuged once, the supernatant was collected, filtered to obtain the filtrate, and then dried once to obtain... 10 B4C nanosheets;

[0023] Furthermore, S2 includes: directing... 10 B4C nanosheets were mixed with glycidol, dispersed by ultrasonication in a water bath, and then subjected to a functionalization reaction. After cooling to room temperature, ultrapure water was added, and the mixture was ultrasonically dispersed again. A second centrifugation was performed, and the supernatant was collected, which contained the glycidol-functionalized nanosheets. 10 The crude product of B4C nanosheets was dispersed, centrifuged a third time, the precipitate was collected, washed, and dried to obtain the final product. 10 B4C-PG nanosheets.

[0024] Furthermore, S3 includes: taking 10 B4C-PG nanosheets, NGR peptide, NLS peptide, PD-L1 targeting peptide, coupling agent and catalyst are dissolved in the reaction solvent to carry out a coupling reaction.

[0025] After the coupling reaction was completed, a fourth centrifugation was performed, the precipitate was collected, resuspended, and repeated 5 times. The product was collected and then dried to obtain the triple-targeted product. 10 B4C nanosheets.

[0026] The preparation method of this invention has outstanding advantages such as simple preparation process, mild conditions, and reliable purification. Under the presence of a coupling agent and in combination with a reaction solvent, a coupling reaction is carried out to efficiently integrate NGR peptide (targeting tumor angiogenesis), NLS peptide (targeting the cell nucleus), and PD-L1 targeting peptide (immune checkpoint blockade therapy) into a single product. 10 On the surface of B4C-PG nanosheets, surface-modified with targeting peptides was successfully constructed. 10 B4C nanosheets (triple targeting) 10 B4C nanosheets avoid the complex process of multi-step modification, significantly improving preparation efficiency. Furthermore, the entire reaction is carried out under mild solution conditions, effectively protecting the nanosheet structure and the bioactivity of each peptide. Combined with a repeated centrifugation-resuspending washing strategy, unreacted peptides, coupling agents, catalysts, and byproducts are thoroughly removed, ensuring the high purity of the final product.

[0027] Furthermore, in S1, the ball milling is performed using a planetary ball mill with a rotation speed of 400~600 r / min and a time of 20~28 h; the first centrifugation is performed with a rotation speed of 3000 r / min and a time of 10 min; the filtration uses a 200~250 nm microporous filter membrane; and the first drying is freeze drying.

[0028] Furthermore, in S2, the functionalization reaction is carried out in an oil bath at 120-160 °C for 20-48 h; the second centrifugation is performed at a speed of 3000 r / min for 30 min; the dispersion solvent is ultrapure water; the third centrifugation is performed at a speed of 12000-16000 r / min for 2 h; and the second drying is freeze drying.

[0029] Furthermore, in step S3, the coupling reaction is carried out under inert conditions, the temperature of the coupling reaction is 35 ℃~40 ℃, and the time is 20 h~28 h; the fourth centrifugation speed is 8000 rpm, the time is 15 min, and the molecular cutoff of the ultrafiltration centrifuge tube used is 100 kDa; the resuspension solvent is ultrapure water; and the third drying is freeze drying.

[0030] Preferably, in step S1, the ball milling uses agate beads as the grinding medium, employs a planetary ball mill, a rotation speed of 500 r / min, and a time of 24 h; the filtration uses a 220 nm microporous filter membrane.

[0031] Preferably, in step S2, the functionalization reaction is carried out in an oil bath at 140 °C for 24 h; the third centrifugation is performed at a speed of 14000 r / min for 2 h.

[0032] Preferably, in step S3, the inert condition is nitrogen gas, and the coupling reaction is specifically a constant temperature oscillation reaction at 37 °C for 24 hours.

[0033] Furthermore, in S1, the 10 The ratio of B4C powder to ultrapure water is 3 g: 10 mL;

[0034] Furthermore, in S2, the 10 The ratio of B4C nanosheets to glycidol is 0.1 g: 20 mL.

[0035] Furthermore, in S3, the 10 The ratio of B4C-PG nanosheets, NGR peptide, NLS peptide, PD-L1 targeting peptide, coupling agent, catalyst and reaction solvent is 100 mg: 10 mg: 10 mg: 10 mg: 12 mg ~ 13 mg: 8.5 mg ~ 12 mg: 8 mL.

[0036] Furthermore, the coupling agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride or N,N'-dicyclohexylcarbodiimide; the catalyst is 4-dimethylaminopyridine or 1-hydroxybenzotriazole; and the reaction solvent is anhydrous N,N-dimethylformamide.

[0037] This invention also provides the above-mentioned triple targeting 10 B4C nanosheets or triple-targeted materials prepared by the above methods 10 Application of B4C nanosheets in the preparation of boron drugs.

[0038] Furthermore, the boron drug is used for boron neutron capture therapy, immune checkpoint blockade, or photothermal therapy.

[0039] Furthermore, the preparation method of the boron drug includes the following steps: [details of the steps are missing here, likely related to a specific drug or process]. 10 B4C nanosheets are mixed with pharmaceutically acceptable carriers to prepare formulations suitable for tumor delivery.

[0040] Furthermore, the boron drug is used to treat tumors, and the treatment includes at least one of boron neutron capture therapy (BNCT), immunotherapy, and photothermal therapy (PTT).

[0041] Furthermore, the boron drug is used to prepare a drug for the combined treatment of tumors, wherein the combined treatment includes at least two of boron neutron capture therapy, immunotherapy, and photothermal therapy.

[0042] Furthermore, when performing the photothermal therapy, it is combined with 808 nm near-infrared laser irradiation.

[0043] Furthermore, the tumor is liver cancer, lymphoma, or head and neck tumor.

[0044] Furthermore, the drug is administered via intravenous injection or intratumoral injection.

[0045] When boron drugs are used to treat tumors, they can induce immunogenic cell death and activate anti-tumor immune responses.

[0046] The triple targeting provided by this invention 10 B4C nanosheets offer a novel multimodal synergistic treatment strategy for tumor therapy. This strategy, through the synergistic effect of at least two of boron neutron capture therapy (BNCT), immunotherapy, and photothermal therapy (PTT), can not only effectively inhibit primary tumors but also induce systemic anti-tumor immune responses, inhibiting tumor metastasis and recurrence. It has significant application value and clinical translation potential in the field of tumor therapy technology.

[0047] The beneficial effects of this invention are:

[0048] This invention provides a triple targeting 10 B4C nanosheets. Through the synergistic effect of NGR peptide (targeting tumor angiogenesis) and NLS peptide (targeting the cell nucleus), [the following was achieved / achieved / etc.]. 10 B4C nanosheets enable precise delivery of tumor cell nuclei. This design holds promise for significantly improving... 10 The enrichment concentration of B near the tumor cell nucleus satisfies the requirements of BNCT. 10 High requirements for B concentration and T / N and T / B ratios. Combining BNCT, immune checkpoint blockade therapy (achieved through PD-L1 targeted peptides) with photothermal therapy (based on...) 10 The B4C nanosheet integrates three functions: photothermal properties, catalytic activity, and immunogenicity. Its synergistic mechanism lies in: the high-LET particles generated by BNCT can precisely kill tumor cells; the PD-L1 targeting peptide can block immune checkpoint pathways and reverse immunosuppression; simultaneously, the photothermal effect generated by the nanosheet under near-infrared laser irradiation can directly ablate tumors. This synergistic effect promises to achieve an organic combination of physical killing and immune activation. This triple targeting... 10 B4C nanosheets with 10 Using B4C nanosheets as a carrier, the surface is co-modified with NGR peptide, PD-L1 targeting peptide, and NLS peptide, achieving hierarchical targeting of tumor tissue, PD-L1, and tumor cell nuclei. Based on this design, the nanosheets can not only precisely target boron neutron capture therapy (BNCT) to the cell nucleus, but also simultaneously possess immune checkpoint blockade and photothermal therapy functions, realizing multimodal synergistic therapy. In summary, this triple-targeting... 10B4C nanosheets integrate tumor tissue targeting, immune checkpoint targeting, and cell nucleus targeting into a single nanoplatform, possessing triple targeting capabilities. This enables the efficient enrichment of boron drugs in the tumor cell nucleus, simultaneously exerting synergistic therapeutic effects of immunomodulation and photothermal killing.

[0049] This invention provides a triple targeting 10 A method for preparing B4C nanosheets mainly includes three steps: ball milling, surface functionalization, and coupling reaction. This method is simple and requires no complicated procedures. The ball-milled nanosheets are then processed. 10 B4C powder was prepared into 10 B4C nanosheets are beneficial 10 Surface functionalization and coupling with other substances on B4C nanosheets. NGR peptides, NLS peptides, and PD-L1 targeting peptides were integrated into the nanosheets via coupling reactions. 10 On the surface of B4C-PG nanosheets, a triple-targeted structure was finally prepared. 10 B4C nanosheets have a triple targeting function, enabling precise identification and enrichment of tumor cells. While improving the therapeutic effect, they also reduce damage to normal tissues, solving the problem of high toxicity to normal tissues of existing BNCT boron delivery agents.

[0050] The preparation method of this invention has outstanding advantages such as simple preparation process, mild conditions, and reliable purification. Under the presence of a coupling agent and in combination with a reaction solvent, a coupling reaction is carried out to efficiently integrate NGR peptide (targeting tumor angiogenesis), NLS peptide (targeting the cell nucleus), and PD-L1 targeting peptide (immune checkpoint blockade therapy) into a single product. 10 On the surface of B4C-PG nanosheets, surface-modified with targeting peptides was successfully constructed. 10 B4C nanosheets (triple targeting) 10 B4C nanosheets avoid the complex process of multi-step modification, significantly improving preparation efficiency. Furthermore, the entire reaction is carried out under mild solution conditions, effectively protecting the nanosheet structure and the bioactivity of each peptide. Combined with a repeated centrifugation-resuspending washing strategy, unreacted peptides, coupling agents, and byproducts are thoroughly removed, ensuring the high purity of the final product.

[0051] Further analysis of the embodiments reveals that this triple targeting method of the present invention... 10 B4C nanosheets possess excellent biocompatibility (low hemolysis rate, low cytotoxicity), stable dispersion in physiological environments, and efficient and stable photothermal cycling performance, providing a foundation for their clinical translation. This invention's triple targeting... 10 B4C nanosheets can also effectively induce tumor cell death and apoptosis and inhibit their proliferation.

[0052] The triple targeting provided by this invention 10B4C nanosheets offer a novel multimodal synergistic treatment strategy for tumor therapy. This strategy, through the synergistic effect of at least two of boron neutron capture therapy (BNCT), immunotherapy, and photothermal therapy (PTT), can not only effectively inhibit primary tumors but also induce systemic anti-tumor immune responses, inhibiting tumor metastasis and recurrence. It has significant application value and clinical translation potential in the field of tumor therapy technology. Attached Figure Description

[0053] Figure 1 for 10 Hydrated particle size distribution of B4C nanosheets;

[0054] Figure 2 Triple targeting 10 Hydrated particle size distribution of B4C nanosheets;

[0055] Figure 3 for 10 B4C nanosheets and triple targeting 10 Comparison of Zeta potentials of B4C nanosheets;

[0056] Figure 4 Triple targeting 10 Transmission electron microscope image of B4C nanosheets;

[0057] Figure 5 for 10 B4C nanosheets and triple targeting 10 Fine X-ray photoelectron spectroscopy analysis of B4C nanosheets;

[0058] Figure 6 for 10 B4C nanosheets and triple targeting 10 Infrared spectrum comparison of B4C nanosheets;

[0059] Figure 7 Triple targeting 10 Graph showing the change in hydrated particle size of B4C nanosheets over time in phosphate buffer solution from 1 to 7 days.

[0060] Figure 8 Triple targeting 10 Photothermal heating curves of B4C nanosheets in phosphate buffer, showing triple targeting. 10 The concentrations of B4C nanosheets in phosphate buffer were 0 mg / mL, 2 mg / mL, and 5 mg / mL;

[0061] Figure 9 Triple targeting 10 Photothermal cycling stability of B4C nanosheets in phosphate buffer solution;

[0062] Figure 10 Triple targeting at different concentrations 10 Infrared thermal imaging of B4C nanosheets in phosphate buffer solution;

[0063] Figure 11 Triple targeting at different concentrations 10 Schematic diagram of hemolysis experiment results for B4C nanosheets;

[0064] Figure 12 The graph shows the hemolysis rate results for the experimental group, PBS group, and H2O group.

[0065] Figure 13 For 0.1 M calcium chloride aqueous solution, PBS aqueous solution, and triple-targeted solutions of different concentrations 10 Comparison of the coagulation effect of B4C nanosheets at different time points;

[0066] Figure 14 Scc25 cells were subjected to triple targeting at different concentrations 10 Cell viability after B4C nanosheet treatment for 24 h, 48 h, and 72 h;

[0067] Figure 15 Scc25 cells were subjected to triple targeting at different concentrations 10 Live and dead staining fluorescence images of B4C nanosheets after 48 h of treatment;

[0068] Figure 16 For NC group, NCT group, PTT group, BNCT+ 10 B4C Group and BNCT+PTT+ 10 Cell viability of B4C group after 24 h, 48 h and 72 h of cell treatment;

[0069] Figure 17 For NC group, NCT group, PTT group, BNCT+ 10 B4C Group and BNCT+PTT+ 10 Live / dead staining fluorescence image of cells treated in group B4C;

[0070] Figure 18 For NC group, NCT group, PTT group, BNCT+ 10 B4C Group and BNCT+PTT+ 10 Apoptosis diagram of cells treated with B4C group;

[0071] Figure 19 For NC group, NCT group, PTT group, BNCT+ 10 B4C Group and BNCT+PTT+ 10 Cell clone formation diagram of cells treated with group B4C. Detailed Implementation

[0072] The present invention will be further described in detail below through embodiments, but these embodiments are not intended to limit the invention in any way. All other embodiments obtained by those skilled in the art based on the specific embodiments in this application without inventive effort are within the scope of protection of this application.

[0073] Unless otherwise specified in the following embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual shall apply.

[0074] Unless otherwise specified, all reagents or instruments used in the following examples are commercially available products.

[0075] The Chinese name for NGR peptide is asparagine-glycine-arginine peptide; the Chinese name for NLS peptide is nuclear localization signal peptide; and the Chinese name for PD-L1 targeting peptide is PD-L1 programmed death ligand-1 targeting peptide.

[0076] Example 1 Triple Targeting 10 Preparation of B4C nanosheets

[0077] Triple Targeting 10 The specific steps for preparing B4C nanosheets are as follows:

[0078] (1) 10 Preparation of B4C nanosheets:

[0079] Weigh out 3.0 g 10 B4C powder was dispersed in 10 mL of ultrapure water, and several agate beads with a diameter of approximately 2 mm were added. The mixture was then transferred to a 50 mL agate ball mill jar, with a filling density of approximately 50%. The agate ball mill jar was then fixed in a planetary ball mill and milled at 500 r / min for 24 h. The milled dispersion was centrifuged at 3000 r / min for 10 min, the supernatant was collected, and filtered through a 220 nm microporous membrane. The resulting filtrate was freeze-dried to obtain... 10 B4C nanosheets.

[0080] (2) 10 Preparation of B4C-PG nanosheets:

[0081] Add 0.1 g of the product obtained in step (1) to a 50 mL round-bottom flask. 10 B4C nanosheets were uniformly dispersed with 20 mL of glycidol (epoxypropanol) in a water bath using ultrasonication, and then reacted in an oil bath at 140 °C for 24 h. After the reaction was completed, the system was cooled to room temperature, and 20 mL of ultrapure water was added, followed by ultrasonic dispersion again. The dispersion was centrifuged at 3000 r / min for 30 min, and the supernatant, i.e., the glycidol-functionalized solution, was collected. 10Crude B4C nanosheets were obtained. The crude product was redispersed in ultrapure water and centrifuged at 14000 r / min for 2 h. The supernatant was discarded, the precipitate was collected, and the precipitate was washed three times to completely remove free polyglycerol. Finally, the product was freeze-dried to obtain... 10 B4C-PG nanosheets were stored at 4 °C for later use.

[0082] (3) Triple targeting 10 Preparation of B4C nanosheets:

[0083] Weigh 100 mg of the product obtained in step (2). 10 B4C-PG nanosheets, along with 10 mg NGR peptide, 10 mg NLS peptide, 10 mg PD-L1 targeting peptide, 12 mg EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride), and 12 mg DMAP (4-dimethylaminopyridine), were dissolved in 8 mL of anhydrous N,N-dimethylformamide (DMF). The mixture was reacted under nitrogen protection at 37°C with shaking for 24 hours; this was a coupling reaction. After the coupling reaction, the reaction solution was transferred to an ultrafiltration centrifuge tube (molecular cutoff: 100 kDa) and centrifuged at 8000 rpm for 15 minutes. The filtrate in the collection tube was discarded, and the precipitate (i.e., the product) was retained. To remove unreacted peptides, coupling agents, and byproducts, approximately 8 mL of ultrapure water (equal volume to the initial reaction system) was added to the precipitate for resuspending, followed by another centrifugation at 8000 rpm for 15 minutes. This washing process (including centrifugation and resuspension) was repeated 5 times. Finally, the resulting product was freeze-dried to obtain the triple-targeted product. 10 B4C nanosheets.

[0084] Example 2 Triple Targeting 10 Preparation of B4C nanosheets

[0085] Triple Targeting 10 The specific steps for preparing B4C nanosheets are as follows:

[0086] (1) 10 Preparation of B4C nanosheets:

[0087] Weigh out 3.0 g 10 B4C powder was dispersed in 10 mL of ultrapure water, and several agate beads with a diameter of approximately 2 mm were added. The mixture was then transferred to a 50 mL agate ball mill jar, with a filling density of approximately 50%. The agate ball mill jar was then fixed in a planetary ball mill and milled at 500 r / min for 24 h. The milled dispersion was centrifuged at 3000 r / min for 10 min, the supernatant was collected, and filtered through a 220 nm microporous membrane. The resulting filtrate was freeze-dried to obtain...10 B4C nanosheets.

[0088] (2) 10 Preparation of B4C-PG nanosheets:

[0089] Add 0.1 g of the product obtained in step (1) to a 50 mL round-bottom flask. 10 B4C nanosheets were uniformly dispersed with 20 mL of glycidol (epoxypropanol) in a water bath using ultrasonication, and then reacted in an oil bath at 150 °C for 48 h. After the reaction was completed, the system was cooled to room temperature, and 20 mL of ultrapure water was added, followed by ultrasonic dispersion again. The dispersion was centrifuged at 3000 r / min for 30 min, and the supernatant, i.e., the glycidol-functionalized solution, was collected. 10 Crude B4C nanosheets were obtained. The crude product was redispersed in ultrapure water and centrifuged at 14000 r / min for 2 h. The supernatant was discarded, the precipitate was collected, and the precipitate was washed three times to completely remove free polyglycerol. Finally, the product was freeze-dried to obtain... 10 B4C-PG nanosheets were stored at 4 °C for later use.

[0090] (3) Triple targeting 10 Preparation of B4C nanosheets:

[0091] Weigh 100 mg of the product obtained in step (2). 10 B4C-PG nanosheets, along with 10 mg NGR peptide, 10 mg NLS peptide, 10 mg PD-L1 targeting peptide, 12 mg EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride), and 12 mg DMAP (4-dimethylaminopyridine), were dissolved in 8 mL of anhydrous N,N-dimethylformamide (DMF). The mixture was reacted under nitrogen protection at 40 °C with shaking for 24 hours; this was a coupling reaction. After the coupling reaction, the reaction solution was transferred to an ultrafiltration centrifuge tube (molecular cutoff: 100 kDa) and centrifuged at 8000 rpm for 15 minutes. The filtrate in the collection tube was discarded, and the precipitate (i.e., the product) was retained. To remove unreacted peptides, coupling agents, and byproducts, approximately 8 mL of ultrapure water (equal volume to the initial reaction system) was added to the precipitate for resuspending, followed by another centrifugation at 8000 rpm for 15 minutes. This washing process (including centrifugation and resuspension) was repeated 5 times. Finally, the resulting product was freeze-dried to obtain the triple-targeted product. 10 B4C nanosheets.

[0092] Example 3 Triple Targeting 10 Preparation of B4C nanosheets

[0093] Triple Targeting 10The specific steps for preparing B4C nanosheets are as follows:

[0094] (1) 10 Preparation of B4C nanosheets:

[0095] Weigh out 3.0 g 10 B4C powder was dispersed in 10 mL of ultrapure water, and several agate beads with a diameter of approximately 2 mm were added. The mixture was then transferred to a 50 mL agate ball mill jar, with a filling density of approximately 50%. The agate ball mill jar was then fixed in a planetary ball mill and milled at 500 r / min for 24 h. The milled dispersion was centrifuged at 3000 r / min for 10 min, the supernatant was collected, and filtered through a 220 nm microporous membrane. The resulting filtrate was freeze-dried to obtain... 10 B4C nanosheets.

[0096] (2) 10 Preparation of B4C-PG nanosheets:

[0097] Add 0.1 g of the product obtained in step (1) to a 50 mL round-bottom flask. 10 B4C nanosheets were uniformly dispersed with 20 mL of glycidol (epoxypropanol) in a water bath using ultrasonication, and then reacted in an oil bath at 140 °C for 24 h. After the reaction was completed, the system was cooled to room temperature, and 20 mL of ultrapure water was added, followed by ultrasonic dispersion again. The dispersion was centrifuged at 3000 r / min for 30 min, and the supernatant, i.e., the glycidol-functionalized solution, was collected. 10 Crude B4C nanosheets were obtained. The crude product was redispersed in ultrapure water and centrifuged at 14000 r / min for 2 h. The supernatant was discarded, the precipitate was collected, and the precipitate was washed three times to completely remove free polyglycerol. Finally, the product was freeze-dried to obtain... 10 B4C-PG nanosheets were stored at 4 °C for later use.

[0098] (3) Triple targeting 10 Preparation of B4C nanosheets:

[0099] Weigh 100 mg of the product obtained in step (2). 10B4C-PG nanosheets, along with 10 mg NGR peptide, 10 mg NLS peptide, 10 mg PD-L1 targeting peptide, 13 mg DCC (N,N'-dicyclohexylcarbodiimide), and 8.5 mg HOBt (1-hydroxybenzotriazole), were dissolved in 8 mL of anhydrous N,N-dimethylformamide (DMF). The mixture was reacted under nitrogen protection at 37°C with shaking for 24 hours; this was a coupling reaction. After the coupling reaction, the reaction solution was transferred to an ultrafiltration centrifuge tube (molecular cutoff: 100 kDa) and centrifuged at 8000 rpm for 15 minutes. The filtrate in the collection tube was discarded, and the precipitate (i.e., the product) was retained. To remove unreacted peptides, coupling agents, and byproducts, approximately 8 mL of ultrapure water (equal volume to the initial reaction system) was added to the precipitate for resuspending, followed by another centrifugation at 8000 rpm for 15 minutes. This washing process (including centrifugation and resuspending) was repeated 5 times. Finally, the obtained product was freeze-dried to obtain the triple-targeted product. 10 B4C nanosheets.

[0100] Detection and Analysis

[0101] The triple-targeted preparation obtained in Example 1 10 B4C nanosheets were detected and analyzed.

[0102] 1. For triple targeting 10 B4C nanosheets were characterized in a series of ways.

[0103] (1) Particle size and Zeta potential:

[0104] The raw material was measured by dynamic light scattering method. 10 B4C nanosheets (such as Figure 1 (As shown) and the triple target prepared in Example 1 10 B4C nanosheets (such as Figure 2 The average hydrated particle sizes (shown) were 140 nm and 194 nm, respectively. These were measured using a Zeta potential analyzer. 10 B4C nanosheets and the triple-targeted membrane prepared in Example 1 10 The zeta potential of B4C nanosheets, such as Figure 3 As shown, raw materials 10 The zeta potential of B4C nanosheets is -0.15 mV, while after triple-targeting modification, its zeta potential becomes -53.17 mV, i.e., triple-targeting... 10 The Zeta potential of the B4C nanosheets is -53.17 mV, indicating that peptide molecules have been successfully modified onto the nanosheet surface.

[0105] (2) Morphology and structure:

[0106] Transmission electron microscopy (TEM) results, such as Figure 4 As shown, triple targeting 10 B4C nanosheets exhibit a near-spherical shape, good dispersibility, and uniform particle size distribution. Fine X-ray photoelectron spectroscopy (XPS) analysis results, such as... Figure 5 As shown, the results indicate that triple targeting 10 The B4C nanosheets showed significantly enhanced characteristic peak intensities in the N 1s and O 1s regions, further confirming the successful grafting of the peptide. FT-IR spectroscopy analysis, such as... Figure 6 As shown, triple targeting 10 B4C nanosheets at 1650 cm -1 (Amide I band) and 1540 cm -1 A characteristic absorption peak appears at the (amide II band), which is a typical signal of peptide bonds.

[0107] (3) Stability and photothermal properties:

[0108] Dispersion stability: The triple target prepared in Example 1 10 B4C nanosheets were dispersed in PBS (phosphate-buffered saline) solution to prepare a stock solution of 1 mg / mL. Samples were taken periodically over 1–7 days, and the hydrated particle size was determined using a dynamic light scattering nanoparticle size analyzer. Results are as follows: Figure 7 As shown in the figure, the results indicate that the particle size did not change significantly, indicating that it has good dispersion stability.

[0109] The photothermal performance test results are as follows:

[0110] The triple target prepared in Example 1 10 B4C nanosheets were prepared into solutions with concentrations of 0 mg / mL, 2 mg / mL, and 5 mg / mL using PBS and placed in quartz cuvettes. Subsequently, an 808 nm near-infrared laser (power density 1.5 W / cm²) was used. 2 The solution was vertically irradiated for 11 minutes, and the temperature change of the solution was monitored and recorded in real time using a thermal imager. The results are as follows: Figure 8 As shown, compared with the 0 mg / mL control group, the triple targeting at 2 mg / mL and 5 mg / mL... 10 The temperature of the B4C nanosheet solution increased significantly, and the higher the concentration, the greater the temperature rise, indicating triple targeting. 10 B4C nanosheets exhibit excellent photothermal conversion properties.

[0111] To evaluate photothermal stability, the triple-targeted [material] prepared in Example 1 was [used]. 10B4C nanosheets were prepared into a 2 mg / mL solution using PBS and placed in a quartz cuvette. Subsequently, the solution was vertically irradiated with an 808 nm near-infrared laser (power density 1.5 W / cm²) for three laser-on-off cycles (each cycle consisting of 15 min of laser irradiation followed by natural cooling to room temperature). The results are as follows: Figure 9 As shown, the heating curves of the three laser switch cycle tests basically overlap, indicating that the triple targeting... 10 B4C nanosheets exhibit excellent photothermal cycling stability.

[0112] In addition, the triple target prepared in Example 1 10 B4C nanosheets were dispersed in PBS solution to prepare triple-targeted formulations at different concentrations (0 mg / mL, 2 mg / mL, 5 mg / mL). 10 A B4C nanosheet solution was irradiated under the same laser parameters, and infrared thermal images were captured using a thermal imager. The results are as follows: Figure 10 As shown, the concentration-dependent photothermal effect is intuitively demonstrated, that is, the solution temperature rises more significantly with increasing nanosheet concentration. Among them, Figure 10 In this context, PBS refers to a triple-targeted formulation with a concentration of 0 mg / mL. 10 B4C nanosheet solution, 2 mg / mL refers to a triple-targeting concentration of 2 mg / mL. 10 B4C nanosheet solution, 5 mg / mL refers to a triple-targeting concentration of 5 mg / mL. 10 B4C nanosheet solution.

[0113] 2. The triple-targeted [treatment] prepared in Example 1 10 Biocompatibility evaluation of B4C nanosheets

[0114] The triple target prepared in Example 1 10 B4C nanosheets were dispersed in PBS solution to prepare triple-targeted formulations at different concentrations. 10 B4C nanosheet solution (0~1000 μg / mL).

[0115] (1) Hemolysis test:

[0116] Experimental group: Fresh red blood cells were reacted with different concentrations (0 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 500 μg / mL, 1000 μg / mL) of triple-targeting agents. 10 B4C nanosheet solution was co-incubated for 2 h; 3 duplicate wells were set for each concentration.

[0117] Negative control group (PBS group): Fresh red blood cells were co-incubated with PBS buffer for 2 h, and 3 replicates were set up to establish a baseline hemolysis standard under physiological conditions.

[0118] Positive control group (H2O group): Fresh red blood cells were incubated with ultrapure water for 2 h, and three replicates were set up to induce complete rupture of red blood cells to determine the 100% hemolysis value.

[0119] The hemolysis results of the experimental group, PBS group, and H2O group are as follows: Figure 11 As shown, from Figure 11 As can be seen from the results, the appearance of the supernatant in all concentration experimental groups was not significantly different from that in the PBS group; all were clear and transparent with trace amounts of red blood cell sediment at the bottom, indicating that the triple-targeting... 10 B4C nanosheets exhibit good blood compatibility and biocompatibility.

[0120] The absorbance (OD) values ​​of the supernatants of the experimental group, PBS group, and H2O group were measured in the hemolysis experiment, and the hemolysis rate was calculated according to the formula shown in equation (i):

[0121] Hemolysis rate = [(OD test - OD negative control) / (mean of OD positive control - mean of OD negative control)] × 100% (i)

[0122] The OD experiment uses the absorbance value of a single well of the supernatant from the experimental group, H2O group, or PBS group (the absorbance value of the single well in the experimental group is used to calculate the hemolysis rate, the absorbance value of the H2O group is used to calculate the hemolysis rate, and the absorbance value of the PBS group is used to calculate the hemolysis rate); the OD negative control is the absorbance value of the supernatant from the PBS group; the OD positive control is the absorbance value of the supernatant from the H2O group; the average value of the OD positive control is the average of the absorbance values ​​of the three replicates of the supernatant from the H2O group, and the average value of the OD negative control is the average of the absorbance values ​​of the three replicates of the supernatant from the PBS group.

[0123] The hemolysis rates of the experimental group, PBS group, and H2O group are as follows: Figure 12 As shown; triple targeting at all tested concentrations 10 The hemolysis rate of the B4C nanosheet group was less than 1% (see details). Figure 12 This meets the biosafety requirements for medical materials, indicating triple targeting. 10 B4C nanosheets have no significant hemolytic toxicity.

[0124] (2) Coagulation test:

[0125] Fresh rabbit blood was collected, anticoagulated with 3.2% sodium citrate, and centrifuged to obtain anemic platelet-rich plasma (PPP). 0.5 mL of triple-targeted plasma at different concentrations (0–1000 μg / mL) was then applied. 10 B4C nanosheet solutions were mixed with equal volumes of PPP as experimental samples; a mixture of 0.5 mL PBS aqueous solution and an equal volume of PPP served as a negative control, and a mixture of 0.5 mL 0.1 M calcium chloride aqueous solution and an equal volume of PPP served as a positive control. All samples were incubated at 37°C for different times (5 min, 15 min, 30 min, and 60 min). The formation and relative size of clots in each group were observed and recorded at different time points using a gentle rinsing method. Results are as follows: Figure 13 As shown, compared with the negative control group (PBS aqueous solution), none of the nanosheet groups at any concentration promoted the formation of significant blood clots; and compared with the significant blood clots induced by the positive control group (0.1 M calcium chloride), the nanosheet groups also did not show a similar procoagulant effect. The results indicate that triple targeting... 10 B4C nanosheets did not induce significant activation of the coagulation system within the testing range, demonstrating good blood compatibility.

[0126] Cytotoxicity:

[0127] ① CCK-8 assay for cell viability:

[0128] Scc25 cells were seeded at a density of 1000 cells per well in 96-well plates, and different concentrations (0–1000 μg / mL) of triple-targeting agents were added to each well. 10 Fresh B4C nanosheets were cultured in 5 replicates for each concentration, and a cell-free culture medium was set up as a blank control group, also with 5 replicates. The plates were cultured for 24 h, 48 h, and 72 h, respectively. At 24 h, 48 h, and 72 h, the 96-well plates were removed, and 10 μL of CCK-8 solution was added to each well. The plates were then incubated in the dark for 2 h. The absorbance (OD) of each well was then measured using a microplate reader at 450 nm. Cell viability was calculated according to formula (ii).

[0129] Cell viability = [(OD experiment - OD blank) / (mean of OD control - mean of OD blank)] × 100% (ii)

[0130] OD experiments showed triple targeting at different concentrations. 10 Single-pore absorbance of the B4C nanosheet-treated group; OD control is without triple targeting. 10 The absorbance values ​​of a single well in the cell group of B4C nanosheets; the OD blank is the absorbance value of a single well in the blank control group, and the average value of the OD control is the absorbance value without triple targeting.10 The average absorbance values ​​of the five duplicate wells of the B4C nanosheet cell group were used, while the OD blank was the average absorbance values ​​of the five duplicate wells of the blank control group.

[0131] The results are as follows Figure 14 As shown, triple targeting at different concentrations (0~1000 μg / mL) 10 Under the action of B4C nanosheets, the cell viability of Scc25 cells remained above 90% after culturing for 24 h, 48 h, and 72 h, respectively, indicating triple targeting. 10 B4C nanosheets showed no significant toxicity to Scc25 cells within the tested concentration range and exhibited good biocompatibility.

[0132] ② Staining of live and dead cells:

[0133] Scc25 cells were planted at a rate of 1 × 10⁶ cells per well. 5 Individual samples were seeded at a density of [number] cells per well in 6-well plates, and different concentrations (0–1000 μg / mL) of triple-targeted [product / treatment] were added to each well. 10 Fresh culture medium containing B4C nanosheets was used. After incubation for 48 h, the culture medium was discarded, and the cells were gently rinsed twice with PBS. Subsequently, a mixed staining solution of Calcein-AM (working concentration 2 μM) and propidium iodide (PI, working concentration 4.5 μM) was added to each dish, and the cells were incubated at 37 °C in the dark for 30 min. After incubation, the cells were rinsed again with PBS to remove residual staining solution. Immediately, the cells were observed and images were acquired using an inverted fluorescence microscope. Live cells labeled with Calcein-AM showed green fluorescence, while dead cells labeled with PI showed red fluorescence. Finally, the green and red fluorescence channel images from the same field of view were overlaid to obtain a merge image, which visually showed the spatial distribution of live and dead cells.

[0134] The results are as follows Figure 15 As shown, triple targeting at different concentrations (0~1000 μg / mL) 10 Under the action of B4C nanosheets, Scc25 cells exhibited uniform green fluorescence with only a very small amount of red fluorescence. These results indicate that this triple-targeting... 10 B4C nanosheets showed no significant toxicity to Scc25 cells within the tested concentration range and exhibited good biocompatibility.

[0135] 3. Using human oral squamous cell carcinoma Scc25 cells as a model, evaluate the triple-targeted therapy. 10 In vitro antitumor effects of B4C nanosheets

[0136] The triple target prepared in Example 1 10 B4C nanosheets were dissolved in PBS culture medium to prepare a triple-targeted solution at a concentration of 500 μg / mL.10 B4C nanosheet solution.

[0137] (1) The following groups were set up: ①NC group (negative control group): containing only cell culture medium, without any nanomaterial treatment or physical irradiation (neutron or laser), used to assess the basic viability of cells. ②NCT group (neutron irradiation control group): containing no boron, receiving only neutron irradiation at a dose of 2600 μm, this group was designed to exclude the non-specific biological effects of the neutron beam itself on cells. ③PTT group (photothermal therapy group): the culture medium contained 500 μg / mL of triple-targeted [agent / material]. 10 B4C nanosheets, with a power density of 1.5 W / cm². 2 The group was irradiated with 808 nm near-infrared laser for 10 minutes (10 min) to evaluate the efficacy of nanomaterial-mediated photothermal therapy alone. ④BNCT+ 10 Group B4C (boron neutron capture therapy group): The culture medium contained 500 μg / mL of triple-targeted [therapies]. 10 B4C nanosheets were subjected to neutron irradiation at a dose of 2600 mu, and this group was used to evaluate the effects of B4C nanosheets on neutrons. 10 The boron neutron capture therapy effect produced by the (n, α) nuclear reaction of B. ⑤BNCT+PTT+ 10 Group B4C (synergistic treatment group): The culture medium contained 500 μg / mL of triple-targeted therapy. 10 B4C nanosheets, with a power density of 1.5 W / cm². 2 The study involved 10 minutes of 808nm laser irradiation followed by neutron irradiation at a dose of 2600 μm. The aim of this study was to investigate the synergistic antitumor effect of combining photothermal therapy with boron neutron capture therapy.

[0138] The specific steps are as follows: Take Scc25 cells in the logarithmic growth phase, digest the cells at the bottom of the culture dish with trypsin, centrifuge after digestion, and resuspend the Scc25 cells in fresh complete culture medium to prepare a cell suspension with a concentration of 1×10⁻⁶. 4 Scc25 cells were seeded at a density of 1000 cells per well in 96-well plates and then incubated in an air incubator at 37°C with 5% CO2 for 24 hours to allow cell adhesion. After cell adhesion, the complete culture medium was discarded. PTT group, BNCT+ 10 B4C Group and BNCT+PTT+ 10 The B4C group was supplemented with a triple-targeted formulation at a concentration of 500 μg / mL. 10 B4C nanosheet solution, NC group and NCT group were added to PBS medium, with 5 replicates for each group, and cell-free medium was set up as a blank control. Incubation continued for 4-6 hours to ensure triple targeting.10 B4C nanosheets were fully taken up by cells. After incubation, the NCT group and BNCT+ were compared. 10 The B4C group received neutron irradiation at a dose of 2600 mu; the PTT group received neutron irradiation at a power density of 1.5 W / cm². 2 Irradiation with 808 nm near-infrared laser for 10 minutes; for BNCT+PTT+ 10 Group B4C first underwent a 10-minute test at a power density of 1.5 W / cm². 2 It was irradiated with an 808 nm laser, followed by neutron irradiation at a dose of 2600 mu.

[0139] (2) Evaluation of in vitro antitumor effects:

[0140] Cell viability was assessed using the CCK-8 assay to evaluate the triple-targeted inhibitors prepared in Example 1 after irradiation. 10 The killing effect of B4C nanosheets on Scc25 cells.

[0141] The specific operating steps are as follows: Cells from each group treated in step (1) are placed in an incubator and cultured for 24 h, 48 h, and 72 h respectively. At 24 h, 48 h, and 72 h, the 96-well plates are removed, the original culture medium is discarded, and 10 μL of CCK-8 solution is added to each well. The plates are then incubated in the dark for 2 h. The absorbance (OD value) of each well is measured at 450 nm using a microplate reader. Cell viability is calculated according to the formula shown in equation (iii):

[0142] Cell viability = [(OD experiment - OD blank) / (mean of OD control - mean of OD blank)] × 100% (iii)

[0143] The OD experiment was conducted for each experimental treatment group (i.e., NCT, PTT, BNCT+). 10 B4C, BNCT+PTT+ 10 The absorbance values ​​of a single well in the B4C group were used to calculate the cell viability of the NC group. The OD experiment used the absorbance values ​​of a single well in the NC group, the OD control used the absorbance values ​​of a single well in the cell group without drugs (NC group), and the OD blank used the absorbance values ​​of a single well containing only culture medium (blank control) without cells. The average value of the OD control was the average of the absorbance values ​​of 5 replicates of the cell group without drugs (NC group), and the average value of the OD blank was the average of the absorbance values ​​of 5 replicates of the cell group containing only culture medium (blank control) without cells.

[0144] The results are as follows Figure 16 As shown, compared with the NC group, BNCT+PTT+ 10The cell viability in the B4C experimental group decreased significantly to below 20% (p<0.01). The results indicate that this triple-targeting... 10 B4C nanosheets exhibit a significant synergistic killing effect on Scc25 cells under combined near-infrared laser and neutron irradiation, demonstrating a good combined therapeutic effect of photothermal therapy and boron neutron capture therapy.

[0145] (3) Live / dead staining and apoptosis analysis:

[0146] ① Staining of live and dead cells:

[0147] Cell viability was detected using a Calcein-AM / PI double staining kit.

[0148] The specific steps are as follows: After each group of cells was treated according to step (1), it was incubated in an incubator for 48 h. The culture medium was then discarded, and the cells were gently rinsed twice with PBS. Staining working solution containing Calcein-AM and PI was added to each well, and the cells were incubated at 37 ℃ in the dark for 30 min. After incubation, the cells were rinsed again with PBS to remove residual staining solution. Immediately, an inverted fluorescence microscope was used to observe and acquire images (where Calcein-AM labeled live cells showed green fluorescence, and PI labeled dead cells showed red fluorescence). Finally, the green and red fluorescence channel images in the same field of view were superimposed to obtain a Merge image to visually show the spatial distribution of live and dead cells.

[0149] The results are as follows Figure 17 As shown, cells in the NC, NCT, and PTT groups exhibited uniform green fluorescence (live cells), while BNCT+ cells showed uniform green fluorescence. 10 B4C Group and BNCT+PTT+ 10 The presence of abundant red fluorescence (dead cells) in the B4C group indicates that nanosheet treatment can effectively kill tumor cells.

[0150] ② Apoptosis analysis:

[0151] Apoptosis was detected by flow cytometry.

[0152] The specific steps were as follows: Cells from each group treated in step (1) were placed in an incubator and cultured for 48 h. Cells were then collected by trypsin digestion and resuspended in PBS for washing. Annexin V-FITC and PI staining solution were added, and the mixture was incubated in the dark for 15 min. Flow cytometry was then used to analyze the ratio of early to late apoptotic cells. Results are as follows: Figure 18 As shown, BNCT+ 10 B4C Group and BNCT+PTT+ 10The total apoptosis rate (early apoptosis + late apoptosis) of the B4C group was significantly higher than that of the NC group, NCT group and PTT group, indicating that the nanosheet can effectively induce tumor cell apoptosis.

[0153] (4) Cloning experiment:

[0154] To evaluate triple targeting 10 The effect of B4C nanosheets on the long-term proliferation ability of Scc25 cells was verified using a clonogenic assay.

[0155] Scc25 cells were divided into two groups: a control group (NC group, NCT group, PTT group) and an experimental group (BNCT+). 10 B4C Group, BNCT+PTT+ 10 (B4C group). The simplified steps are as follows: Cells from each group treated in step (1) were placed in an incubator and cultured under standard conditions for a certain period (usually 7-14 days), with the culture medium changed regularly to maintain nutrient supply. When visible cell colonies appeared in the control group, the culture was stopped, the culture medium was discarded, and the cells were gently washed twice with PBS. Each well was fixed with 4% paraformaldehyde solution for 15 minutes, and after discarding the fixative, stained with 0.1% crystal violet solution for 20 minutes. Excess staining was removed by slowly rinsing with running water, and the cells were air-dried at room temperature. The resulting cell colonies were photographed and recorded, and the number of colonies was counted using ImageJ software. The results are as follows: Figure 19 As shown in the figure, the results indicate that the number of clones formed in the experimental group was significantly reduced compared with the control group, suggesting that the nanosheets can effectively inhibit the long-term proliferation and clone formation ability of Scc25 cells.

[0156] The above experimental results (see details) Figure 16 , Figure 17 , Figure 18 and Figure 19 Together, they indicate that this triple targeting... 10 B4C nanosheets can effectively induce tumor cell death and apoptosis and inhibit their proliferation. Based on the known research that both BNCT and radiotherapy can induce ICD, the triple targeting of this invention... 10 B4C nanosheets are expected to further induce immunogenic cell death during BNCT and PTT, potentially activating an anti-tumor immune response.

[0157] In summary, the triple targeting provided by this invention... 10 B4C nanosheets and their preparation methods have the following advantages:

[0158] This invention provides a triple targeting 10 B4C nanosheets. Through the synergistic effect of NGR peptide (targeting tumor angiogenesis) and NLS peptide (targeting the cell nucleus), [the following was achieved / achieved / etc.]. 10B4C nanosheets enable precise delivery of tumor cell nuclei. This design holds promise for significantly improving... 10 The enrichment concentration of B near the tumor cell nucleus satisfies the requirements of BNCT. 10 High requirements for B concentration and T / N and T / B ratios. Combining BNCT, immune checkpoint blockade therapy (achieved through PD-L1 targeted peptides) with photothermal therapy (based on...) 10 The B4C nanosheet integrates three functions: photothermal properties, catalytic activity, and immunogenicity. Its synergistic mechanism lies in: the high-LET particles generated by BNCT can precisely kill tumor cells; the PD-L1 targeting peptide can block immune checkpoint pathways and reverse immunosuppression; simultaneously, the photothermal effect generated by the nanosheet under near-infrared laser irradiation can directly ablate tumors. This synergistic effect promises to achieve an organic combination of physical killing and immune activation. This triple targeting... 10 B4C nanosheets with 10 Using B4C nanosheets as a carrier, the surface is co-modified with NGR peptide, PD-L1 targeting peptide, and NLS peptide, achieving hierarchical targeting of tumor tissue, PD-L1, and tumor cell nuclei. Based on this design, the nanosheets can not only precisely target boron neutron capture therapy (BNCT) to the cell nucleus, but also simultaneously possess immune checkpoint blockade and photothermal therapy functions, realizing multimodal synergistic therapy. In summary, this triple-targeting... 10 B4C nanosheets integrate tumor tissue targeting, immune checkpoint targeting, and cell nucleus targeting into a single nanoplatform, possessing triple targeting capabilities. This enables the efficient enrichment of boron drugs in the tumor cell nucleus, simultaneously exerting synergistic therapeutic effects of immunomodulation and photothermal killing.

[0159] This invention provides a triple targeting 10 A method for preparing B4C nanosheets mainly includes three steps: ball milling, surface functionalization, and coupling reaction. This method is simple and requires no complicated procedures. The ball-milled nanosheets are then processed. 10 B4C powder was prepared into 10 B4C nanosheets are beneficial 10 Surface functionalization and coupling with other substances on B4C nanosheets. NGR peptides, NLS peptides, and PD-L1 targeting peptides were integrated into the nanosheets via coupling reactions. 10 On the surface of B4C-PG nanosheets, a triple-targeted structure was finally prepared. 10 B4C nanosheets have a triple targeting function, enabling precise identification and enrichment of tumor cells. While improving the therapeutic effect, they also reduce damage to normal tissues, solving the problem of high toxicity to normal tissues of existing BNCT boron delivery agents.

[0160] The preparation method of this invention has outstanding advantages such as simple preparation process, mild conditions, and reliable purification. Under the presence of a coupling agent and in combination with a reaction solvent, a coupling reaction is carried out to efficiently integrate NGR peptide (targeting tumor angiogenesis), NLS peptide (targeting the cell nucleus), and PD-L1 targeting peptide (immune checkpoint blockade therapy) into a single product. 10 On the surface of B4C-PG nanosheets, surface-modified with targeting peptides was successfully constructed. 10 B4C nanosheets (triple targeting) 10 B4C nanosheets avoid the complex process of multi-step modification, significantly improving preparation efficiency. Furthermore, the entire reaction is carried out under mild solution conditions, effectively protecting the nanosheet structure and the bioactivity of each peptide. Combined with a repeated centrifugation-resuspending washing strategy, unreacted peptides, coupling agents, and byproducts are thoroughly removed, ensuring the high purity of the final product.

[0161] This invention's triple targeting 10 B4C nanosheets possess excellent biocompatibility (low hemolysis rate, low cytotoxicity), stable dispersion in physiological environments, and efficient and stable photothermal cycling performance, providing a foundation for their clinical translation. This invention's triple targeting... 10 B4C nanosheets can also effectively induce tumor cell death and apoptosis and inhibit their proliferation.

[0162] The triple targeting provided by this invention 10 B4C nanosheets offer a novel multimodal synergistic treatment strategy for tumor therapy. This strategy, through the synergistic effect of at least two of boron neutron capture therapy (BNCT), immunotherapy, and photothermal therapy (PTT), can not only effectively inhibit primary tumors but also induce systemic anti-tumor immune responses, inhibiting tumor metastasis and recurrence. It has significant application value and clinical translation potential in the field of tumor therapy technology.

Claims

1. A triple-targeting 10 B4C nanosheets, characterized in that, include 10 B4C nanosheets and modifications in 10 Targeted peptides on the surface of B4C nanosheets; The targeted peptides include NGR peptide, NLS peptide, and PD-L1 targeted peptide; The 10 The surface of B4C nanosheets was functionalized with glycidol to obtain glycidol-functionalized nanosheets. 10 B4C nanosheets; The glycidol functionalized 10 Surface modification of the target peptide with B4C nanosheets yields triple-targeted peptides. 10 B4C nanosheets; The triple targeting 10 The preparation method of B4C nanosheets includes the following steps: S1, to 10 B4C powder was ball-milled to obtain 10 B4C nanosheets; S2, Using glycidol on the above 10 B4C nanosheets were surface functionalized to obtain glycidol-functionalized products. 10 B4C nanosheets, i.e. 10 B4C-PG nanosheets; S3, the above 10 B4C-PG nanosheets, NGR peptide, NLS peptide, and PD-L1 targeting peptide undergo a coupling reaction in the presence of a coupling agent to obtain the triple targeting peptide. 10 B4C nanosheets.

2. The triple targeting according to claim 1 10 B4C nanosheets, characterized in that, The triple targeting 10 The average hydrated particle size of B4C nanosheets is 194 nm.

3. The triple targeting as described in claim 1 10 The method for preparing B4C nanosheets is characterized by, S1 includes: 10 B4C powder was added to ultrapure water, ball-milled, centrifuged once, the supernatant was collected, filtered to obtain the filtrate, and then dried once to obtain... 10 B4C nanosheets; And / or, S2 includes: to 10 B4C nanosheets were mixed with glycidol, dispersed by ultrasonication in a water bath, and then subjected to a functionalization reaction. After cooling to room temperature, ultrapure water was added, and the mixture was ultrasonically dispersed again. A second centrifugation was performed, and the supernatant was collected, which contained the glycidol-functionalized nanosheets. 10 The crude product of B4C nanosheets was dispersed, centrifuged a third time, the precipitate was collected, washed, and dried to obtain the final product. 10 B4C-PG nanosheets; And / or, S3 includes: taking 10 B4C-PG nanosheets, NGR peptide, NLS peptide, PD-L1 targeting peptide, coupling agent and catalyst are dissolved in reaction solvent to carry out coupling reaction; After the coupling reaction was completed, a fourth centrifugation was performed, the precipitate was collected, resuspended, and repeated 5 times. The product was collected and then dried to obtain the triple-targeted product. 10 B4C nanosheets.

4. The preparation method according to claim 3, characterized in that, In step S1, the ball milling is performed using a planetary ball mill at a speed of 400-600 r / min for 20-28 h; the first centrifugation is performed at a speed of 3000 r / min for 10 min; the filtration uses a 200-250 nm microporous filter membrane; and the first drying is freeze drying. And / or, in S2, the functionalization reaction conditions are: reaction in an oil bath at 120~160 °C for 20~48 h; the second centrifugation speed is 3000 r / min for 30 min; the dispersion solvent is ultrapure water; the third centrifugation speed is 12000~16000 r / min for 2 h; and the second drying is freeze drying. And / or, in S3, the coupling reaction is carried out under inert conditions, the temperature of the coupling reaction is 35℃~40℃, and the time is 20 h~28 h; the speed of the fourth centrifugation is 8000 rpm, the time is 15 min, and the molecular cutoff of the ultrafiltration centrifuge tube used is 100 kDa; the solvent for resuspension is ultrapure water; and the third drying is freeze drying.

5. The preparation method according to claim 4, characterized in that, In S1, the 10 The ratio of B4C powder to ultrapure water is 3 g: 10 mL; And / or, in S2, the 10 The ratio of B4C nanosheets to glycidol is 0.1 g: 20 mL; And / or, in S3, the 10 The ratio of B4C-PG nanosheets, NGR peptide, NLS peptide, PD-L1 targeting peptide, coupling agent, catalyst and reaction solvent is 100 mg: 10 mg: 10 mg: 10 mg: 12 mg~13 mg: 8.5 mg~12 mg: 8 mL.

6. The preparation method according to claim 5, characterized in that, The coupling agent is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride or N,N'-dicyclohexylcarbodiimide; the catalyst is 4-dimethylaminopyridine or 1-hydroxybenzotriazole; and the reaction solvent is anhydrous N,N-dimethylformamide.

7. The triple targeting as described in any one of claims 1 to 2 10 B4C nanosheets or the triple-targeted preparation method according to any one of claims 3 to 6 10 Application of B4C nanosheets in the preparation of boron drugs.

8. The application according to claim 7, characterized in that, The boron drug is used to treat tumors, and the treatment includes at least one of boron neutron capture therapy, immunotherapy, and photothermal therapy.

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